African Journal of Agricultural Research Vol. 7(13), pp.

1976-1981, 5 April, 2012

Available online at http://www.academicjournals.org/AJAR
DOI: 10.5897/AJARX11.084
ISSN 1991-637X © 2012 Academic Journals

Review

Waterlogging stress in plants: A review

Muhammad Arslan Ashraf
Department of Botany, University of Agriculture, Faisalabad. E-mail: arsilpk@gmail.com. Tel: 041-9200161-67 ext. 3306.
Fax: 041-9200312.
Accepted 17 January, 2012

Waterlogging is the major obstacle for sustainable agriculture. Plants subjected to waterlogging suffer
from substantial yield losses. Under natural environmental conditions, plants often get exposed to
transient or permanent waterlogging. Flooding induces a number of alterations in important soil physio-
chemical properties like soil pH, redox potential and oxygen level. Thus, the plants growing on the
waterlogged soil face the stressful environment in terms of hypoxia (deficiency of O2) or anoxia
(absence of O2). These oxygen deficient conditions substantially hamper plant growth, development
and survival. Plants under O2-restrictive environment exhibit metabolic switch from aerobic respiration
to anaerobic fermentation. It is evident from the available literature that most of the genes expressed
under flooding stress are potentially involved in the synthesis of enzymes known to play active role in
the establishment of this fermentative pathway. Plants undergo this metabolic change in order to get
continuous supply of Adenosine triphosphate (ATP). Under waterlogged conditions, plants exhibit
several responses including hampered stomata conductance, net CO2-assimilation rate and root
hydraulic conductivity. Furthermore, plants grown under waterlogged conditions often face the
oxidative damage induced by the generation of reactive oxygen species. These reactive oxygen species
in turn affects the integrity of membranes and induce damage to the efficiency of photosystem II,
thereby, causing considerable decrease in net photosynthetic rates. Moreover, these perturbations in
physiological mechanisms may affect the carbohydrate reserves and translocations. In fact,
waterlogging tolerant and sensitive plant species could be discriminated on the basis of their efficient
carbohydrate utilization. Waterlogging is also known to induce adverse effects on several physiological
and biochemical processes of plants by creating deficiency of essential nutrients like nitrogen,
magnesium, potassium, calcium. Apart from these waterlogging-induced alterations in physiological
mechanisms, plants growing under flooded conditions also exhibit certain morphological changes
entailing the formation of adventitious roots, initiation of hypertrophied lenticels and/or establishment
of aerenchyma. Therefore, the aim of this review is to highlight the major morphological, physiological
and biochemical adaptations of plants to tolerate the flooding stress.

Key words: Hypoxia, anoxia, fermentation, Adenosine triphosphate (ATP), reactive oxygen species (ROS),
antioxidants.

INTRODUCTION

In tropical and subtropical regions, excessive rainfall is
the major constraint for crop production. Elevated levels
of water in soil create hypoxic conditions (decrease in the
level of oxygen) within a short period of time. As a result
plant roots suffer from anoxia, complete absence of
oxygen (Gambrell and Patrick, 1978). However, plants
tolerant to waterlogging (flooding) stress exhibit certain
adaptations, for example, formation of aerenchyma and
adventitious roots. Furthermore, the formation of
adventitious roots is due to the interaction of plant
hormones, auxin and ethylene (McNamara and Mitchell,
1989). Oxygen deficiency inhibits the root respiration of
plants which results in substantial reduction in energy
status of root cells. Since oxygen is a terminal electron
acceptor in aerobic respiration, in its absence, Kreb’s
cycle and electron-transport system are blocked.
Therefore, plants under waterlogged conditions use
alternate pathway for energy extraction. This alternate
pathway uses fermentative metabolism to produce
Adenosine triphosphate (ATP), thereby, resulting in

enhanced accumulation of ethanol.
Moreover, the activity of alcohol dehydrogenase (ADH)
is also increased (Davies, 1980; Vartapetian, 1991).
In fermentation, plants could get only two ATP per
glucose molecule, whereas, 36 ATP molecules are
produced per glucose molecule in aerobic respiration.
Flood-tolerant plants are able to maintain their energy
status using fermentation. In addition, the maintenance of
cytosolic pH is of prime importance. In waterlogged
plants, initial decline in cytosolic pH has been observed
and this decline is attributed to the production of lactic
acid during fermentation. This initial decrease in pH helps
the plant to switch from lactate to ethanol fermentation by
activation of alcohol dehydrogenase and inhibition of
lactate dehydrogenase (Chang et al., 2000). As under
hypoxic or anoxic conditions oxygen is lacking, therefore,
alternative electron acceptor is required. For example,
nitrate has been considered as terminal electron acceptor
of plant mitochondria under anoxic or hypoxic conditions
(Vartapetien et al., 2003). It has also been suggested that
nitrate reduction is an alternate respiratory pathway and
is important for the maintenance and energy homeostasis
of the cell in the oxygen deficient environment
(Igamberdiev and Hill, 2004).
WATERLOGGING-INDUCED ALTERATIONS IN
PHYSIOLOGICAL MECHANISMS
One of the first plant responses to waterlogging is the
reduction in stomata conductance (Folzer et al., 2006).
Plants exposed to flooding stress exhibit increased
stomata resistance as well as, limited water uptake
leading to internal water deficit (Parent et al., 2008). In
addition, low levels of O2 may decrease hydraulic
conductivity due to hampered root permeability (Else et
al., 2001). Oxygen deficiency generally leads to the
substantial decline in net photosynthetic rate (Ashraf et
al., 2011). This decrease in transpiration and
photosynthesis is attributed to stomata closure (Ashraf
and Arfan, 2005). However, other factors such as
reduced chlorophyll contents, leaf senescence and
reduced leaf area are also held responsible for
decreased rates of photosynthesis (Malik et al., 2001). In
this context, Yordanova et al. (2005) reported fast
stomata closure in barley plants when subjected to
flooding conditions. Similarly, when pea plants were
subjected to flooding conditions, a prompt closure of
stomata was recorded (Zang and Zang, 1994). This
stomata closure of pea plants was attributed to the
abscisic acid (ABA) transport from older to younger
leaves or denovo synthesis of this hormone.
Furthermore, prolonged exposure of plants to flooding
conditions could result in root injuries which in turn
restrict photosynthetic capacity by inducing certain
alterations in biochemical reactions of photosynthesis.
These biochemical alterations include restricted activity of
Ashraf 1977
ribulose bisphosphate carboxylase (RuBPC),
phosphoglycollate and glycollate oxidase (Yordanova and
Popova, 2001), demolition of chloroplast membrane
inhibiting photosynthetic electron transport and efficiency
of photosystem II (Titarenko, 2000). It is evident from the
literature that flooding causes a marked reduction in
photosynthetic capacity of a number of plants, for
example, Lolium perenne (McFarlane et al., 2003),
Lycopersicon esculentum (Bradford, 1983; Jackson,
1990) Pisum sativum (Jackson and Kowalewska, 1983,
Zhang and Davies, 1987), and Triticum aestivum
(Trought and Drew, 1980). However, plants exhibit
certain adaptation under waterlogging stress to maintain
photosynthetic capacity (Li et al., 2004). Moreover, flood-
induced destruction of chlorophyll has been investigated
widely by a number of researchers (Jackson et al., 1991;
Huang et al., 1994; Ashraf et al., 2011). This decrease in
chlorophyll directly or indirectly affects the photosynthetic
capacity of plants under waterlogged conditions (Ashraf
et al., 2011).
The adverse effects of waterlogging on different gas
exchange attributes of plants have been reported in some
earlier studies. For example, Ashraf and Arfan (2005)
reported decrease in photosynthetic rate, water use
efficiency and intrinsic water use efficiency of 32-day okra
plants when subjected to waterlogged conditions. It is a
general consensus that stomata regulation controls the
CO2 exchange rate of plants under waterlogged
conditions (Ashraf and Arfan, 2005; Ashraf et al., 2011).
Furthermore, water potential of plants is also controlled to
some extent by stomata regulations (Liao and Lin, 1996).
However, there are contrasting reports on the
involvement of stomatal regulation in maintenance of
water potential. For example, waterlogging caused a
marked reduction in stomata conductance of bitter melon.
This reduction in gs resulted in increased leaf water
potential (Liao and Lin, 1994). In contrast, Ashraf and
Arfan (2005) found no significant correlation between
stomata conductance and water potential of okra plants
under waterlogged conditions. In fact, these authors were
of the view that osmotic potential and pressure potential
are the main factors that determine water potential.
Waterlogging stress is also known to cause marked
perturbation in different chlorophyll fluorescence
attributes of plants. Since chlorophyll fluorescence is an
excellent physiological marker that determine the primary
processes involved in photosynthesis such as energy
transfer due to excitation, absorption of light and
photochemical reactions occurring in the PSII
(photosystem II) (DeEll et al., 1999; Saleem et al., 2011).
Therefore, changes in chlorophyll fluorescence
parameters determine the function and stability of
photosystem II (Jimenez et al., 1997; Abdeshahian et al.,
2010). The plants subjected to waterlogged conditions
exhibit certain alterations in this physiological marker. For
example, when Cork oak (Quercus variabilis) and China
wingnut (Pterocarya stenoptera) were subjected to
1978 Afr. J. Agric. Res.
waterlogging stress, a prominent decrease in maximum
quantum efficiency (Fv/Fm) was recorded (Hua et al.,
2006). Likewise, decrease in the maximum quantum yield
of PS II photochemistry (Fv/Fm) was also recorded in
flied beans when subjected to varying days of
waterlogging stress (Pociecha et al., 2008). PSII
photochemistry was also impaired due to waterlogging in
Medicago sativa. The decrease in Fv/Fm indicated the
sensitivity of photosynthetic apparatus to abiotic stress
and also inability of the plants to regenerate rubisco
under stressful conditions (Smethurst et al., 2005).
OXIDATIVE DAMAGE INDUCED BY REACTIVE
OXYGEN SPECIES (ROS)
Despite the fact that oxygen is important for life on earth,
its reduction by any means could result in the production
of ROS perturbing several cellular metabolic processes of
plants (Ashraf, 2009; Ashraf et al., 2010). Lethal reactive
− photosynthetic machinery of plants under waterlogged
oxygen species include superoxide (O2 ), hydrogen
peroxide (H2O2) and the hydroxyl radical (OH). Singlet
oxygen generated due to the reaction of oxygen with
excited chlorophyll, is also considered as potential ROS
(Ashraf and Akram, 2009). These ROS are extremely
reactive in nature and induce damage to a number of
cellular molecules and metabolites such as proteins,
lipids, pigments, DNA etc (Ashraf, 2009). ROS are also
produced in plants under normal conditions or non-
stressed conditions but their concentration is very low.
However, when plants are facing some environmental
stress like waterlogging stress, the concentration of ROS
is elevated to a level that is damaging for several cellular
metabolic reactions of plants such as photosynthesis,
efficiency of PS II (Ashraf, 2009). For example, elevated
cellular levels of hydrogen peroxide result in inhibition of
calvin cycle (Ashraf and Akram, 2009).
ROS are free radicals possessing one or more
unpaired electrons. This is not a stable configuration;
therefore, the radicals react with other cellular molecules
to produce more free radicals (Foyer and Halliwell, 1976;
Hideg, 1997). Generation of reactive oxygen species
occurs via different mechanisms, for example, when
molecules of aerobic system come in contact with the
ionizing radiations, this interaction results in the
production of ROS. It is now a well established fact that
electrons flowing through electron transport chain may
leak from their proper rout and in the absence of any
electron acceptor, these electrons react with oxygen to
produce reactive oxygen species (Ashraf, 2009). Different
celluar organelles such as mitochondria, chloroplasts and
peroxisomes are considered as the sites for production of
reactive oxygen species (Sairam and Srivastva, 2002).
ANTIOXIDANT DEFENSE MECHANISM OF PLANTS
UNDER WATERLOGGED CONDITIONS
All the plants have the ability to detoxify the adverse
effects of ROS by producing different types of
antioxidants. Generally, antioxidants are categorized into
enzymatic and non-enzymatic antioxidants. Enzymatic
antioxidants include ascorbate peroxidase (APX),
superoxide dismutase (SOD), peroxidase (POD),
catalase (CAT), glutathione reductase (GR), whereas,
ascorbic acid, glutathione, tocopherols and carotenoids
are included in non-enzymatic antioxidants (Gupta et al.,
2005).
A marked alteration in the endogenous levels of
different enzymatic and non-enzymatic antioxidants has
been recorded in a number of studies. For example,
when mungbean plants were subjected to waterlogging
stress, the activities of various enzymatic antioxidants
such as glutathione reducatse (GR), superoxide
dismutase (SOD), catalase (CAT), and ascorbate
peroxidase (APX) decreased markedly (Ahmed et al.,
2002). These authors also stated that oxidative damage
was not directly involved in the impairment of
conditions. Likewise, waterlogging-induced reduction in
the activity of one of oxygen processing enzyme SOD
has also been reported in corn (Yan et al., 1996). In
contrast, increase in the activities of different enzymatic
antioxidants was recorded in maize seedlings when
subjected to varying degree of waterlogging stress (Tang
et al., 2010). Similarly, when pigeon pea genotypes were
exposed to waterlogging stress, the activities of
superoxide dismutase (SOD), catalase (CAT), peroxidase
(POD) and ascorbate peroxidase (APX) increased
markedly (Kumutha et al., 2009). From these reports, it
is amply clear that plants when exposed to waterlogged
conditions employ antioxidant defense system to get
through the damaging effects of oxidative stress induced
by ROS.
EFFECT OF WATERLOGGING ON NUTRIENT
COMPOSITION
Waterlogging reduces the endogenous levels of nutrient
in different parts of plants (Ashraf et al., 2011). Oxygen
deficiency in the root zone causes a marked decline in
+ +
the selectivity of K /Na uptake and impedes the transport
+
of K to the shoots (Armstrong and Drew, 2002). It has
also been reported in the literature that hypoxic
conditions cause decrease in the permeability of root
membranes to Na+ (Barrett-Lennard et al., 1999).
Generally, waterlogging causes acute deficiencies of
essential nutrients such as nitrogen, phosphorous,
potassium, magnesium and calcium (Smethurst et al.,
2005). In this context, Boem et al. (1996) reported a
marked decline in the uptake of N, P, K and Ca in canola
when exposed to short period of waterlogging stress.
Likewise, reduced endogenous levels of N, P and K have
been reported in maize (Atwell and Steer, 1990). When
M. sativa was subjected to flooding stress, a marked
reduction in leaf and root nutrient composition (P, K, Ca,

Mg, B, Cu and Zn) was recorded in plants (Smethurst et
al., 2005). Similarly, Stieger and Feller (1994) reported
reduced concentrations of P, K and Mg in wheat shoots
due to waterlogging. In contrast, the endogenous levels
of calcium remained unaffected in wheat under
waterlogged conditions. However, decrease in calcium
contents along with other nutrients (N, P, K and Mg) were
also recorded in different organs of wheat under
waterlogged conditions (Sharma and Swarup, 1989).
Similarly, Tarekegne et al. (2000) recorded a marked
reduction in Cu, Zn, P and K uptake in waterlogging
susceptible wheat genotype when compared with the
tolerant genotypes. These researchers were of the view
that genotypes that possess the ability to avoid
waterlogging-induced nutrient deficiency, particularly Zn
and P deficiency should be selected. Moreover, the
hampered efficiency of PS II is attributed to the
deficiencies of N, P, K, Mg and Ca (Smethurst et al.,
2005). It is evident from the literature that adverse effects
of waterlogging are not due to the toxic levels of Na and
Fe but reduced concentrations of N, P, K, Ca and Mg are
the major contributors (Sharma and Swarup, 1989;
Smethurst et al., 2005).
MORPHOLOGICAL AND ANATOMICAL CHANGES
Waterlogging stress is also known to cause a number of
morphological and anatomical changes in plants. For
example, the presence of hypertrophied lenticels is a
common anatomical change observed in different woody
species under flooding stress (Yamamoto et al., 1995).
Radical cell division and expansion near stem base
results in hypertrophic growth. In addition, it is also
believed to be associated with ethylene and auxin
production (Kozlowski, 1997). The lenticels are thought to
be involved in the downward diffusion of O2 as well as,
the compounds produced as by-products of anaerobic
metabolism (ethanol, CO2 and CH4). Although, the actual
physiological role of lenticels is still unclear, their
presence is often linked to waterlogging tolerance in
plants (Parelle et al., 2006). Moreover, the number of
hypertrophied lenticels is more under the water surface
that supports the argument stating their involvement in
maintenance of plant water homeostasis and deviating
from the argument that dictates their role as important
facilitators of oxygen entry toward the root system. Their
potential role in the plant water homeostasis is evident
from their active involvement in partially replacing the
decaying roots and facilitating water intake for the shoot
(Parent et al., 2008).
Formation of adventitious roots potentially replacing the
basal roots is considered as one of the potential
morphological adaptations depicted by plants under
waterlogging stress (Malik et al., 2001). These
specialized roots maintain the continuous supply of water
and minerals when the basal root system fails to do so
Ashraf 1979
(Mergemann and Sauter, 2008). Furthermore, the
deterioration of the main root system is taken as the
sacrifice providing energy for the development of well
adapted root system (Dat et al., 2006). In addition, the
formation of adventitious roots is associated with
waterlogging tolerance of plants (Steffens et al., 2006).
Another important morphological response of plant is
the development of lacunae gas spaces (aerenchyma) in
the root cortex. The formation of aerenchyma is
considered as an adaptive response of the plant under
flooding stress (Evans, 2004). There are two types of
processes involved in the development of aerenchyma.
The first is constitutive development of aerenchyma as it
is not linked with the abiotic stress. It is formed by the
cells separated during tissue development. This type of
cell death occurring as a result of cell separation is
termed as shizogeny, regulated developmentally and
independent of external stimulus. It is formed as a result
of highly regulated tissue specific pattern of cell
separation. The second type of aerenchyma development
is known as Isogeny since it is formed due to partial
breakdown of the cortex that resembles programmed cell
death and its formation depends on the external stimulus
like abiotic stress (Pellinen et al., 1999).
GENETIC VARIATION FOR WATERLOGGING
TOLERANCE
Plants under waterlogged conditions exhibit marked up
and/or down-regulation of a number of genes. By
investigating the induced expression of these genes in
low oxygen environment, it is possible to identify certain
gene products. Then these potential genes involved in
conferring waterlogging tolerance can be isolated and
introduced into the transgenic plants in order to identify
their possible contribution in stress tolerance. Early
studies performed by isotopic labeling of maize roots with
35
S-methionine clearly indicated the synthesis of
anaerobic polypeptides when plants were subjected to
low oxygen environment (Sachs et al., 1980). The
anaerobic polypeptides include the enzymes involved in
fermentation, that is, pyruvate decarboxylate, alcohol
dehydrogenase and lactate dehydrogenase.
Moreover, there exists a marked variation in genetic
resources of potential crops for flooding tolerance. For
example, it has been widely reported in the literature that
genetic differences exists in wheat for waterlogging
tolerance (Gradner and Flood, 1993; Ding and Musgrave,
1995). Setter et al. (1999) showed that there exists a
significant genetic diversity among 14 wheat varieties
when exposed to flooding stress under glasshouse
conditions. Similarly, genetic variation has also been
reported in many other plant species, for example, oat
(Lemons e Silva et al., 2003), cucumber (Yeboah et al.,
2008), Soybean (VanToai et al., 1994) and maize(Anjus e
Silva et al., 2005).
1980 Afr. J. Agric. Res.
SHORTGUN APPROACHES TO INDUCE
WATERLOGGING TOLERANCE
Scientists from different geographical regions of the world
are actively involved in making the plants tolerant to
flooding stress by the use of exogenous application of
nutrient and plant hormones. For example, recently,
Ashraf et al. (2011) reported that exogenous application
of potassium in soil and as foliar spray alleviated the
adverse effects of waterlogging on cotton plants.
Likewise, Ashraf and Rehman (1999) reported that
application of nitrate in soil proved useful in mitigating the
harmful effects of waterlogging on different physiological
attributes of maize. Likewise, Yiu et al. (2009) found that
exogenous application of spermidine and spermine
provoked several biochemical and physiological
adaptations in onion when exposed to flooding stress. In
this context, exogenous application of uniconazole was
also helpful in circumventing the damaging effects of
waterlogging in wheat and oil seed rape plants (Webb
and Fletcher, 1996; Zhou et al., 1997). Therefore, the use
of these organic and inorganic compounds offers an
excellent platform for inducing tolerance to flooding
stress.
CONCLUSION
It can be inferred from the aforesaid discussion that
waterlogging is one of the major constraints for
sustainable agriculture. Its effects are evident on the
entire plant as well as, cellular levels. There is the need
to screen available germplasm for waterlogging tolerance
and use the genes responsible for inducing tolerance in
other potential crops so as to make them resistant as
well. Waterlogging causes deficiency of several essential
nutrients. Therefore, exogenous application of these
nutrient or other plant hormones could be used so as to
alleviate the adverse effects of waterlogging.
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 89

~'cPicbl~S

Strategies for engineering water-

stress tolerance in plants
Hans J. Bohnert and Richard G. Jensen

Water deficit is the commonest environmental stress factor limiting plant

productivity. The ability of plants to tolerate water deficit is determined by multiple
biochemical pathways that facilitate retention and/or acquisition of water, protect
chloroplast functions, and maintain ion homeostasis. Essential pathways include
those that lead to synthesis of osmotically active metabolites and specific proteins
that control ion and water flux, support scavenging of oxygen radicals, or may
act as chaperones. The ability of plants to detoxify radicals under conditions of
water deficit is probably the most critical requirement. Many stress-tolerant
species accumulate methylated metabolites, which play a crucial dual role as
osmoprotectants, and as radical scavengers, Their synthesis is correlated with
stress-induced enhancement of photorespiration. However, transfer of individual
genes from tolerant plants only confers marginally increased water-stress tolerance
to stress-sensitive species: tolerance engineering will probably require the transfer
of multiple genes.

Plants are continuously exposed to environmental
stimuli that influence development and growth, and
determine productivity. At species-specific thresholds,
such stimuli become 'stressors' (Ref. 1); high and low
temperature, mineral imbalance, excess or insufficient
light, and lack of water can all compromise produc-
tivity. Water deficit is intrinsic to most abiotic forms
of stress - not only during drought, but also at low
temperature 2 and when the soil contains a high con-
centration of ions. Changes in gene expression are
involved in physiological responses to water stress, but
the method by which specific genes might play a part
in stress is unknown. Such lack of progress is mainly
due to the multigenic nature of sensitive and tolerant
phenotypes. In addition, different plant species have a
variety of mechanisms that have evolved in a family-
specific or order-specific manner to confer tolerance.
The stress response can also vary depending on the
developmental stage during which a plant is subjected
to stress 3.
Drought and conditions of high salinity have a major
adverse effect on the productivity, of plants4,-% Apart
from the 'lack of water', high salinity leads to a toxic
sodium effect following long-term irrigation; this
H.J. Bohnert and R. G.Jensen are at the Department of Biochemistry,
The University of Arivona, Biosciences l+'est, Tucson, A Z 85721,
USA.
results in a decline in the productivity of arid land.
Breeding programs to improve salt and drought toler-
ance are particularly important in countries that would
benefit most from stress-tolerant crops s,(~. Conven-
tional breeding approaches alone will be valuable% but
it would be more beneficial to include as molecular
markers the genes for the mechanisms associated with
stress tolerance.
Differences in the expression of specific genes
between stress-sensitive and stress-tolerant plants indi-
cate that tolerance is conferred by genetically encod6d
mechanisms 3,7. Studies have modeled the overex-
pression of specific proteins, and have analyzed their
potential for reducing the effects of stress using physio-
logical measurements in transgenic plants ~ m. Such
studies have revealed two promising strategies for
increasing water-stress tolerance: (1) engineering
increased content of osmolytes (mannitol s, fructans 9,
proline 1°,11 and glycine betaine12), and (2) overex-
pressing enzymes to increase oxygen-radical scaveng-
ing in chloroplasts .3.
While increased osmolyte concentration may
intuitively be recognized as helpful for water retention,
the rationale for radical scavenging may be less ob-
vious. However, radical scavenging may be the more
crucial requirement, because water stress disrupts
cellular redox homeostasis and, therefore, chloroplast
functions; this inevitably leads to the generation o f

Copyright © 1996, Elsevier Science Ltd, All rights reserved. 0167 - 7799/96/$15.00 TIBTECH MARCH 1996 (VOL 14)
 9O

reviews

Table 1. Responses to water stress - accumulating products a and their function(s) in conferring
tolerance

Product Specific Suggested

group compound function(s) Refs

Ions Potassium Osmotic adjustment 15,25

Macro-nutrient requisition 25
Sodium exclusion/export 42-44

Proteins LEA/dehydrins b Osmoprotection 3,7

Osmotin b Pathogenesis-related proteins 7
SOD/catalase Radical detoxification 13,14,40,48

Amino acids Proline Osmotic adjustment 10,11

Ectoine Osmoprotection 50

Sugars Sucrose Osmotic adjustment 9

Fructans Osmoprotection, carbon storage 9

Polyols Acyclic (e.g. mannitol) Carbon storage, osmotic adjustment 8,15

Cyclic (e.g. pinitol) Osmoprotection, osmotic adjustment 15,33,34
Radical scavenging 29,41

Polyamines Spermine, spermidine Ion balance, chromatin protection 51

Quaternary amines Glycine betaine Osmoprotection 24

18-Alanine betaine Osmoprotection 28
Dimethyl-sylfonio propionate Osmoprotection 12

Pigments and carotenoids Carotenoids, anthocyanins, Protection against photoinhibition 40,52

betalaines

aNot all accumulating products are found in all species, but biochemical pathwaysare specificfor orders or families of plants2,12,15,24.
b LEAs(late-embryogenesis-abundantproteins) and dehydrins,as well as pathogenesis-related~roteins,are examples demonstrating
the recruitment of 'other-functionproteins' in abiotic stress responses2,3,7.

oxygen-radical species 14. In this review, we briefly dis-
cuss strategies based on other mechanisms that are less-
well understood; these include controlled ion-uptake,
the ability to exclude or compartmentalize sodium,
and the control of water flux1-%
Table 1 provides examples of plant products whose
action correlates with metabolic functions that are
known or assumed to enable plants to cope with water
deficit. Differences in the complexity ofbiochenhcal
pathways may distinguish xerophytes (drought-toler-
ant) and halophytes (salt-tolerant) from glycophytic
(stress-sensitive) species. Despite a lack of mechanistic
models for the stress-induced regulation o f gene
expression, available data t6Jv indicate that all plants
may respond to ubiquitous signals and possess similar
response mechanisms. However, data also point
towards species-specific differences in sensitivity to
growth-regulators that determine how stress signals
are processed u~ 18.
Plant r e s p o n s e s to water deficit
The initial reaction to stress-induced water deficit is
to modify stomata] conductance by reducing stomata]
aperture; this minimizes the loss of water by tran-
spiration 19. However, stomatal closure has complex
consequences, reqmring adjustments in the levels of
photosynthesis and respiration, altering ion, nutrient
and water fluxes, and changing the allocation of
carbon and nitrogen 2°. Reducing or abolishing
transpiration affects the chloroplast light-harvesting
and energy-conversion systems, because C O 2 may
become limiting and because water-splitting reactions
become less effective. The resulting condition, called
photoinhibition, may lead to long-lasting damage or
irreversible destruction of the chloroplast compart-
ment. However, as plants regulate stomata] conduc-
tance according to a diurnal cycle and in response to
normal, low-stress environments 21, tolerant and sen-
sitive plants must possess the machinery to perceive
and respond to changes in water supply.
What are the mechanisms by which tolerance or
resistance are conferred, and that distinguish water-
stress-sensitive glycophytes from xerophytes or halo-
phytes? While there is little information about stress
perception and signaling, many genes that are tran-
scribed in response to stress have been analyzed. As
yet, it is not possible to generalize about the function
of cis-acting sequences and their binding proteins,
but there are specific promoter elements involved
in transcriptional induction as a result of water

IBTECHMARCH1996 (VOL 14)

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reviews

stress 2,3,v,16,17,22. Signaling pathways have been de-
tected that are both dependent and independent of the
action of the plant growth regulator (PGI<) abscisic
acid, which accumulates in plants experiencing water
deficit 2,3a6,1v. Changes in the post-transcriptional
modification of transcripts may affect the half-life o f
m R N A and/or the regulation of translation during
stress 18,23. Consequently, it seems that specific re-
sponses occur downstream from sensing, signal
transduction and general control o f gene ex-
pression; glycophytes may lack components o f
these pathways. Table 1 provides a list o f responses
to water deficit, emphasizing those apparently
correlated with increased tolerance: whether
such responses equate with tolerance requires study
o f mutants or transgenic plants remains to be
determined.
Osmotic adjustment, compatible solutes or
both?
The response that distinguishes tolerant and sensi-
tive plants most clearly is a dramatic accumulation of
solutes in tolerant species (Fig. 1 and Table 1); sensi-
tive plants are less able to accumulate solutes, but
increases in proline can be found in most organisms
(including animals) following water stress >. Proline
accumulation may serve as a means of storing carbon
and nitrogen as stress leads to slower growth. Engi-
neered overexpression o f proline in tobacco has re-
cently been demonstrated to confer some tolerance to
water stress 11, although the mechanism by which this
is achieved is unknown. Some metabolites that accu-
mulate are specific to phylae, subphylae, orders or fam-
ilies of plants 12,24. (The structures of some common
osmolytes are shown in Fig. 1.) Plants may be able to

CH2OH + +
OH OH I NH3(CH2)3NH(CH2)4NH(CH2)3NH3
~ ? ] I
HOCH
Spermine
CH 3 HOCH
I
HO I OH HCOHI
OH HCOH
I HOCH 2
D-Pinitol CH2OH
__.joH

o-Mannitol
OH I
OH
CH3 0
I CH3 HOCH2 I
CH3~+CH2 COO- I
CH3S+CH2CH2COO -
CH3
I~-Dimethylsulfoniopropionate
Glycine betaine OH iH20 H

O
HOCH2

COO-
/ coo-
/ \ [ CH2OH
CH3 H H H OH

Ectoine Proline Isokestose

Figure 1
Structures of common osmolytes. >Pinitol and other methylated inositols, e.g. bornesitol, quebrachitol, 1-methyl-muco-inositol
and liriodendritol, are cyclic polyols33,34. Acyclic polyols used as osmolytes in higher plants and eukaryotic algae include mannitol,
sorbitol and galactitol 8. Proline commonly accumulates lo. Examples of quaternary ammonium osmolytes include glycine betaine,
6-alanine betaine and proline betainel2,24. I~-Dimethylsulfoniopropionate,a tertiary sulfonium osmolyte, and choline O-sulfate are found
in some higher plants and in many algae~2.24. Many osmolytes are zwitterions, and the unusual zwitterion ectoine, which is found in
halophilic eubacteria, is an exceptionally efficient stabilizer of enzyme activity in the presence of active oxygen species 50. Polyamines,
such as spermine, are not strictly considered osmolytes, but the concentration of potyamines increases during water deficit in many
species5L

TIBTECHMARCH1996(VOL14)
 92
reviews
accumulate potassium, if sufficient is available, in a
'salt-stress remedy' similar to that observed in halo-
phytic bacteria 25. Quaternary ammonium and tertiary
sulfonium compounds, or cyclic polyols and fructans,
are, like proline, synthesized from ubiquitous, abun-
dant metabolites.
Two hypotheses have been suggested to explain the
functions of accumulating solutes 24,26. In the first,
accumulation leads to 'osmotic adjustment' through
mass action, which results in increased water retention
and/or sodium exclusion. Evidence to support this
view is that proline, glycine betaine or pinitol in dif-
ferent species may exceed a 1 M concentration. The
second hypothesis considers accumulating compounds
as 'compatible solutes'. In this function, they could
replace water as a solute in biochemical reactions.
Compatible solutes could also, or as an alternative,
associate with lipids or proteins and thus prevent
membrane disintegration, the dissociation of protein
complexes, or the inactivation of enzymes (see below).
In this sense, they may be considered to be low mol-
ecular weight chaperones. Similar functions might be
provided by the various classes of late-embryogen-
esis-abundant (LEA) and dehydrin proteins that are
expressed primarily during the desiccation phase of
seed maturation 3,7. The synthesis of these proteins is
also frequently induced under conditions of drought
and low-temperature stressT,27; this provides an exam-
pie of the 'recruitment' of a developmental process to
serve a function in the adaptation to water deficit.
Compatible with the osmotic-adjustment concept is
the observation that high amounts of pinitol, proline
and potassium (where present) protect against water
stress in the halophytic ice plant (A1esembryanthemum
crystallinum18). Sodium that is taken up is confined to
the vacuole, where its concentration may exceed 1 M.
The osmotic potential provided by the high concen-
tration of sodium in the vacuole facilitates water
influx, which may explain why the ice plant grows
faster at elevated concentrations of sodium than in
the absence of sodium. There is a linear correlation
between the concentrations of potassium and
osmolytes; plants grown under potassium deficiency
accumulate more pinitol, but when potassium is in
excess, less pinitol synthesis is observed. A similar
relationship between sodium and metabolite concen-
trations has been reported for a number ofhalotolerant
species and also for salt-adapted cell-suspension
cultures 24,2s. These observations support the feasibility
of a strategy for engineering osmolyte synthesis and
accumulation.
Further experiments have indicated that osmolytes
may have a different function. Expressing foreign
genes in transgenic tobacco has afforded some pro-
tection through the accumulation of mannitol 8 at
cytosolic concentrations of 100raM, or fructans at
even lower concentrations 9. These concentrations
seem too low to act osmotically by mass action. Sinfi-
larly, data from in vitro experiments indicate that some
osmolytes act at low concentration, indicating that
the protective role resulting from a protein-osmolyte
IBTECHMARCH1996 (VOL 14)
association leads to the exclusion of ions, solutes
and/or radicals from the protein surface26,>,3°.
Biochemical differences - a m o d e l pathway
The inositol biosynthesis pathway (Fig. 2) indicates
the ways in which glycophytic and halophytic bio-
chemical reactions differ in response to water stress.
This pathway31, in which inositol is derived from the
glucose 6-phosphate pool, is the source of products
essential for the biosynthesis of plasma-membrane
phospholipids, phosphoinositides, phytate (phosphate
storage in seeds), galactinol, cell wall gums, mucilages
and glycoproteins32. Myo-inositol and its naturally
occurring stereoisomers can be methylated to form a
variety of products, such as D-ononitol and D-pinitol
(Fig. 1). In various families of species that are tolerant
to drought or high salt conditions, such as mangroves,
pines, and some legumes 33, the presence of other
enzymes that catalyze epimerization or methylation
reactions extends this pathway and results in the syn-
thesis and accumulation of methylated inositols.
The halophytic ice plant provides evidence of a
difference in metabolic regulation, compared with
Arabidopsis thaliana, which distinguishes the former as
halotolerant and the latter as salt-stress-sensitive. Stress
conditions induce the synthesis of D-pinitol from
inositol through the transcriptional activation of the
Imtl gene, which encodes my0-inositol O-methyl-
transferase (Fig. 2). This enzyme is only found after
plants are subjected to conditions of stress. Transcrip-
tion of Inpsl, which encodes inositol 1-phosphate
synthase, the first enzyme in the pathway (Fig. 2), is
also induced. As a consequence, the concentrations of
my0-inositol increase, and the end-product, D-pinitol,
accumulates to levels that are osmotically significant34.
This upregulation of inositol biosynthesis has not
been observed in A. thaliana; there is no induction
of Inpsl, no accumulation of Inps i mRNA, and no
significant increase in the level of myo-inositol. In ad-
dition, genes encoding enzymes that would extend
the pathway to pinitol synthesis seem to be absent in
Arabidopsis (P,,ef. 34).
Synthesis of methyl-ammonium and -sulfonium
compounds, such as glycine betaine and dimethyl-
sulfoniopropionate, is catalyzed by specific methyl-
transferases 12,24, and they accumulate in some plant
families subjected to water deficit. Below, we outline
a hypothesis that correlates methylations with protec-
tion against water stress.
Key players in water-stress tolerance
Methylation reactions and photorespiration
Osmotic adjustment may be only one function of
accumulating metabolites. There may be an additional
role in water-stress tolerance for metabolites that are
methylated by methyltransferases that use S-adenosyl-
methionine (SAM) as the methyl donor; examples of
such metabolites include inositol derivatives, glycine
betaine, dimethylsulfonium compounds, polyamines
and ectoine (Fig. 1). But, why are many metab-
olites that accunmlate methylated? SAM-dependent
 93

ret, ieu,s

D-Glucose 6-phosphate pool

Inositol
Inps I 1-phosphate synthase
•~ %

..Q Phosphoinositides,
E
myo-lnositol 1-phosphate pool phospholipids,
"O phytate

"No,~ l Inositol
if) ~ c- E Imp 1 monophosphatase
e-- ~ D-Glucuronate
1-phosphate,
_E myo-lnositol cell-wall synthesis,
._o ~ galactinol for
raffinose sugars
Inositol
Imtl O-methyltransferase

>,.----.
D-Ononitol

a_
Oep I T Ononitolepimerase

D-PinitoI

Figure 2
The pathway of synthesis of D-pinitoIfrom glucose 6-phosphate (G6P). This pathway is inducible by drought and salinity in a variety of plant
families, and demonstrates how a ubiquitous pathway (G6P ~ myc-inositoP4), which is closely connected to the main flux of carbon, has
been modified by the addition of two enzymes that lead to the accumulation of a metabolite suitable for osmotic adjustment in halophytes.
An appropriate flux through the pathway must be present for accumulation to occur. In the ice plant (Mesembryanthemumcrystallinum),
co-ordinate induction of Inpsl and lint1 transcription and enzyme activity have been shown, indicating that the entire pathway is induced in
this halophyte34. By contrast, Inpsl is not induced under stress in the glycophyte Arabidopsistha#ana,and the gene encoding the IMT1
enzyme appears to be missing34. If the Imtl gene is present, it is not expressed, because Arabidopsisdoes not contain any product of the
methylation reaction catalyzed by IMTI. A complex spectrum of mono- and di-methylated inositol derivatives has been observed in other
plant familiesa~m.

reactions connect accumulating metabolites to the
activated methyl cycle 3B - the primary, metabolic
source of single carbons (Fig. 3). SAM-accepting
methyltransferase form methyl-esters, -ethers,
-thioethers, -sulfonides, -amines and -amides with
amino acids, nucleic acid bases, sugars, polysaccharides
and lipids. The biosynthesis of ethylene, cysteine and
polyamines is also linked to the activated methyl cycle.
Increased levels of transcript for SAM synthetase in
salt-stressed tomato suggest that the synthesis of SAM
is enhanced as a result of stress36. SAM is regenerated
by homocysteine S-methyltransferase and SAM syn-
thetase, with NS-methyltetrahydrofolate (NSMeTHF)
as the methyl donor. We suggest that the origin of
these methyl groups is the photorespiratory cycle37,
which operates at enhanced levels under conditions of
water stress 14.
With stomatal aperture decreased during periods of
stress, photosynthesis must rely increasingly on oxy-
gen as the substrate for ribulose bisphosphate car-
boxylase (P, ubisco). One of the resulting products,
glyoxylate, is catabolized to CO 2 and methylene-THE
which can be reduced to NSMeTHF; this is then used
to make SAM, and to supply substrate for methyl-
ations (Fig. 3). Such a pathway would be energetically
favorable, especially under photoinhibitory condi-
tions, because the pathway assists in oxidation of the
photosystems. Another metabolite connected to
photorespiration, e~-ketoglutaric acid, can be chan-
neled into the biosynthesis of the osmolyte proline.
It is important to determine whether photorespi-
ratory flux is sufficient to satisfy the demand for
methylated inositols or to stimulate the accumulation
of glycine betaine at times of stress. If photorespira-
tory rates under non-stress conditions37 are compared
with the amounts ofmethylated products that are syn-
thesized under stress 14,15,24, the potential supply of
methyl groups far exceeds demand. If the activity, of

TIBTECHMARCH1996 (VOL 14)

 94

rcPic~l~'$

Glucose 6-phosphate
Ethylene Polyamines

CO 2 InositOlsynthasel-phosphate] 1
\
ACC
Inositol 1- phosphate
Inositol
monophosphatase
S-Adenosylmethionine I ....,- S-Aden°syl- Myo-inositol
methionine ~ / [ Myo-inositol ]
O-methyltransferase
[ synthetase
~_~/ATP (SAM) [ ~ ~ --Ononitol - ]
/ Ononitol
Pinitol

~ Activated ~ epimerase
Methionine methyl S-Adenosylhomocysteine
cycle , (SAH)

Homocysteine ~ J S-Adenosylhomocysteine
S-methyltransferase I"--~ ?o?$CnYSts~ne ~ hydrolase

NS-Methyl THF
NADP + ~ Serine

NADPH

N5,Nm-Methylene THF ~

Figure 3
The activated methyl cycle. The entry of methyl groups into the activated methyl cycle35,36, and the conversion of glycine into serine
could occur as a result of increased levels of NS-methyltetrahydrofolate(NS-MethylTHF) and glycine derived from photorespiratory
metabolism3738,40.The activated methyl cycle supplies many methylation reactions leading to the production of lignin and the synthesisof
a variety of methylated osmolytes (Fig. 1). The cycle is also involved in the production of polyaminesand ethylene [via ACC(1-aminocyclo-
propane-1 carboxylic acid)], and provides substrate, for example, for DNA methylations and various methylations in the synthesis of
secondary products in plants. Three enzymes(boxed) have been shownto be transcriptionally induced in the ice plant and in tomato34,36.
Myo-inositol O-methyltransferase production can be induced in previously unstressed ice plants by salt stress and low temperature
(G. Rammesmayer, R. G. Jensen and H. J. Bohnert, unpublished).

the activated methyl cycle is increased, it is possible
that increased photorespiratory flux supports the syn-
thesis of methylated compounds, which thus repre-
sent a carbon sink under stress conditions.
Hydroxyl-radical scavenging
The photosynthetic electron-transport system is the
major source of active oxygen species in plant tissues3s,
and its assembly and disassembly are intimately con-
nected to the regulation of photosynthetic electron-
flow. The superoxide radical dismutates to 0 2 and
H20 2 in the presence of superoxide dismutases
(SODs); this can directly inhibit CO 2 fixation. In turn,
superoxide and H20, can react to generate the
hydroxwl radical, the most potent oxidant39; this
attacks most cellular macromolecules, generating
lesions in DNA, and affecting protein synthesis and
stability, leading to metabolic dysfunction and cell
death. While superoxide and H202 can be broken
down by SOD, catalase and various peroxidases, there
are no known direct scavengers of the hydroxyl radi-
cal. The extent to which production of active oxygen
species increases under water stress4" depends on many
factors.
The hydroxyl-radical scavenging activities of some
biocompatible solutes have been assessed in vitro by

IBTECHMARCH1996 (VOL 14)

 95

rg12igws

testing their ability to protect enzyme activity, in the
presence of two radical-generating systems2~ (using
either an ascorbate-H20 2 system or a xanthine
oxidase-hypoxanthine-H20 2 system). Radicals were
detected by monitoring salicylate hydroxylation, or
the loss of activity of malate dehydrogenase. O f the
solutes tested, myo-inositolwas the most effective scav-
enger, closely followed by sorbitol, mannitol and pro-
line; glycine betaine proved ineffective in this system.
In similar tests using isolated cyclitols, O-methyl-
muco-inositol, pinitol, ononitol and quebrachitol
proved to be even better hydro,'~q-radical scavengers
than my0-inositol<. Although generation of hydroxyl
radicals in vivo appears to be enhanced during water
deficit and temperature extremes, there is, as yet, no
clear evidence for the role o f polyols in protec-
ting intact cells and tissues against internally generated
radicals.
Engineering must target multiple mechanisms
The most likely mechanisms for improving toler-
ance to water and salt stress are osmotic adjustment
and oxygen-radical scavengingy,S,>, 4~, but ion uptake
and compartmentation, and the control of water flux
should also be considered ~5.
Exclusion would seem to be the most obvious strat-
egy for dealing with excess sodium. However, pro-
longed salinity stress is likely to ovelnvhelm any such
system, and efficient exclusion can result in drought
stress unless the internal osmotic pressure can be con-
comitantly increased. In addition, changes in sodium
concentration also affect ion homeostasis 42. In particu-
lar, sodium interferes with the uptake of potassium, an
essential macro-ion. Subjecting cells that maintain a
physiological membrane potential to salt stress results
in the passive influx o f sodium through potassium
channels and transporters 42,43. A recent study in wheat
has indicated that sodium may enter predominantly
through the high-affinity potassium-uptake transport
(HKT) system 43. Mutagenesis of the homologous
yeast H K T gene resulted in yeast that discriminated
better than the wild type against sodimn; this indicates
a possible avenue for stress engineering 43. Reducing
passive entry, and influx through other cation chan-
nels, would need to be supported by the addition of
energy-dependent efflux mechanisms, presumably
sodium-proton antiporters 44. Another strategy would
be to allow sodium into the plant, but then to deposit
it in vacuoles or excrete it through glands. Under con-
ditions of salt stress, the tonoplast membrane poten-
tial, counterions and/or osmotically active metabolites
must be stringently maintained to facilitate effective
compartmentation.
Another class of proteins that may be important
for maintaining osmotic balance are the water-
channel proteins (aquaporins) that are present in all

f
Extracellular and cell-wall space • Ion exclusion
• Ion export
• Cell-wall modification

)~ Cytosol and organelle space ..... ....

• Osmotic adjustment • Plant growth regulator

• Radical scavenging sensitivity adjustment ....?
• Ion-selectivity changes • Osmoprotection
• Enhancement of proton pumping • Ion partitioning
• Aquaporin-activity control
~iiiiii!il

Vacuolar space

• Ion (sodium, calcium) storage

• Ion (potassium) export :
~ , ~ • Osmotic adjustment j ....
'"{ , • Proton-gradient maintenance
.....<~i~ii~<~
........ ~ J .......~!iii'iiii~ii?~!~
i .......

Figure 4
Biochemicalfunctions associated with tolerance to plant water-deficit. The schematic presentation of a plant cell includesthree compart-
ments that are defined by the plasma membrane and tonoplast. The results of the actions of proteins that have been shown to be related
to water-deficit tolerance are depicted (see also Refs 15,24,42). The scheme focuses on biochemical principles, and does not include
signaling events and pathwaysthat lead to altered gene expression; these have been discussed elsewhere2,3,7.

TIBTECHMARCH1996(VOL14)

reviews
organisms 45. They facilitate water movement along an
existing osmotic gradient, and their activity (i.e. as a
result of an open or closed conformation) appears to
be regulated by protein phosphorylation 46. Regulat-
ing aquaporin concentration and activity may be an
additional mechanism for sensing the presence of
sodium or the lack of water in the external medium.
I~egulation of aquaporin activity and local or tissue-
specific osmolyte production might act synergistical]y
to facilitate water uptake and transport in the absence
of transpiration.
Strategies for engineering tolerance to water stress
are, therefore (Fig. 4): (1) control over the expression,
activity and ion-discrimination properties of potassium
transport systems (and possibly aquaporin activity);
(2) capabilities for enhanced sodium/proton antiport
in the plasma membrane of root ceils (sodium export),
and in the tonoplast of mesophyll or other sink ceils
(sodium compartmentalization); (3) osmotic adjust-
ment as a result of metabolite accumulation to
counteract photoinhibitory damage; and (4) protection
of sensitive metabolic reactions by protecting either
individual enzymes, or protein complexes or mem-
brane structures, by an increased capacity for hydroxyl-
radical scavenging.
Prospects for tolerance engineering
We are beginning to understand the molecular and
biochemical basis of multigenic tolerance to water
stress. Modeling biochemical mechanisms in trans-
genic plants is underway, but can we also begin to
engineer crop plants?
Plants have already been transformed with single
genes, but it is obvious that increases in stress toler-
ance will be marginal for several reasons, the most seri-
ous of which is that engineering strategies are still too
primitive. If the enzymes involved in osmolyte pro-
duction are expressed in the cytosol, for example,
there will be effects on other compartments that may
negate the protective effect, especially when these
osmolytes are strictly confined to the compartment in
which they are synthesized. In addition, engineering
the increased scavenging of oxygen radicals, which at
present seems to be a straightforward strategy, and the
most likely to be successful, may interfere with nor-
mal processes that require local increases in H 2 0 2 as a
signal, for example, in pathogen responses47, 48. Most
studies have used readily available promoters for
'constitutive' gene expression whereas tissue-specific
expression or expression in a developmental or stress-
inducible context would be preferable. Final]y, intro-
ducing a metabolic pathway into a species that does
not normal]y have the enzymatic activities involved
may affect flux through other, resident pathways.
Genes that encode proteins for the functions dis-
cussed above seem to be the most promising candi-
dates for conferring stress resistance, as their intro-
duction would provide new physiological mechanisms
for counteracting the effects o f stress. However, little
is known about how expression o f genes would be
controlled through a stress-perception and signal
IBTECHMARCH1996 (VOL 14)
96
transduction pathway; but abscisic acid is known to be
involved in some stress responses 2,3,1(',17. Plants natu-
rally tolerant or resistant to stress may control stress-
induced proteins using a simple response mechanism,
i.e. a few genes could act as 'masters' that elicit the
entire response. If such genes exist, they will probably
not elicit the same co-ordinate response in glycophytic
species. Engineering stress-sensitive plants will require
either the constitutive expression oftransgenes, or the
introduction of a pathway enabling stress-responsive
control of transgenes. The latter would be preferable,
as constitutive expression of a stress protection
mechanism would probably compromise productiv-
ity. While the prospect of transferring, expressing and
stably maintaining ten or more genes seems daunting,
it can be achieved with present technology. Future
alternative routes may be provided by developments
in technology, such as the construction of transgenic
chromosomes, the improvement of chromosome
microsurgery, and the construction of master 'switch
genes', which would enhance glycophytic responses
to stress.
The goal should not be to modify crop plants so that
they are resistant to the stress created by seawater and
still produce seeds, like the ice plant. Rather than aim-
ing for stress resistance, which inevitably results in
yield penalties s, the goal should be to increase toler-
ance. However, we do not yet know to what degree
increased tolerance would affect yield. A realistic strat-
egy might be to engineer plants to tolerate transient
drought longer before meristem growth declines, or
to be productive at slightly higher salinity levels. The
ice plant is still productive at sodium levels higher than
30 parts per thousand (ppt), although it is most pro-
ductive in the 10-15 ppt range; however, cultivated
tomato, for example, ceases to grow at a sodium
concentration of approximately 4ppt (Refs 4,49).
Increasing the sodium tolerance of crop plants to
10 ppt seems achievable with present knowledge and
technology.
The transfer of multiple gene systems and the
accumulation o f engineered mechanisms through
breeding should result in increased tolerance to water
deficit.
Acknowledgements
Different aspects of our work have been, or are
being, supported by the US Department of Energy,
the US Department of Agriculture, the National Sci-
ence Foundation, the Rockefeller Foundation and the
Arizona Agricultural Experiment Station. We apolo-
gize to the many authors whose work we were not
able to quote directly:
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TIBTECHMARCH1996 (VOL141
 D ROU G HT

REVIEW (8). In most instances, crops experience mod-

erate droughts caused by prolonged precipi-
The physiology of plant responses to drought tation deficits, reduced groundwater levels,
and/or limited access to water supplies, lead-
Aditi Gupta, Andrés Rico-Medina, Ana I. Caño-Delgado* ing to substantial losses in overall yield. There-
fore, investigating the mechanisms of how a
Drought alone causes more annual loss in crop yield than all pathogens combined. To adapt to moisture plant sustains its growth during moderate
gradients in soil, plants alter their physiology, modify root growth and architecture, and close stomata drought and devising strategies to improve
on their aboveground segments. These tissue-specific responses modify the flux of cellular signals, resulting plant health during such periods can provide
in early flowering or stunted growth and, often, reduced yield. Physiological and molecular analyses of the solutions for future food security. Understand-
model plant Arabidopsis thaliana have identified phytohormone signaling as key for regulating the response ing the response of cellular signaling to water
to drought or water insufficiency. Here we discuss how engineering hormone signaling in specific cells and shortage is key for shedding light on these
cellular domains can facilitate improved plant responses to drought. We explore current knowledge and modern agricultural problems (12). Here we
future questions central to the quest to produce high-yield, drought-resistant crops. explore how water availability cues cell and
tissue growth patterns and how these patterns

D
rought is a misfortune for agriculture,
humanity, and livestock alike (1). Climate
change is leading us toward a hotter,
more parched world (2). There is an
urgent need to produce high-yielding
referred to as the drought survival rate. From
the perspective of molecular biology, cellular
water loss marks the first event of drought
stress. At the cellular level, drought signals
promote production of stress-protectant metab-
are coordinated in the whole plant to improve
drought resistance without loss of yield. Over-
expression of drought-responsive genes often
results in growth deficits and yield loss. Tissue-
or time-specific expression of drought-response
traits may improve drought response without

Downloaded from http://science.sciencemag.org/ on April 16, 2020

plants that use water more efficiently than
their present-day counterparts (Fig. 1A). In the
past decade, global losses in crop production
due to drought totaled ~$30 billion. Global pop-
ulation rose from 5 billion inhabitants in 1990
to more than 7.5 billion presently and is pre-
dicted to rise to 9.7 billion to 10 billion by 2050
(3), at which time 5 billion people are projected
to be living in water-scarce regions (Fig. 1B) (4).
Despite the moderate increase in global arable
land, an additional 1 million ha will be needed
to ensure food security (Fig. 1C) (5). In addition,
water demand for agriculture could double
by 2050, whereas the availability of fresh water
is predicted to drop by 50%, owing to climate
change (Fig. 1D) (6). Certainly, plant biotech-
nology holds one of the promises to meet
the societal demand for increased global crop
production.
Water is crucial for plant survival, and water
deficits limit plant growth. However, plants
have strategies to prevent water loss, balance
optimal water supply to vital organs, maintain
cellular water content, and persevere through
periods of drought. The ability of a plant to
olites such as proline and trehalose, trigger the
antioxidant system to maintain redox homeosta-
sis, and deploy peroxidase enzymes to prevent
acute cellular damage and membrane integrity.
Factors such as the extent of water stress and
the plant organ in which the stress is sensed also
trigger specific signaling responses, including but
not limited to abscisic acid, brassinosteroids, and
ethylene phytohormone pathways (8–11).
Drought’s impact on agriculture depends
on the degree and duration of the reduced
precipitation and soil water gradients, as well
as on plant species and developmental stages
A
Year 2020
B C
10
depressing yield. A combination of strategies
may boost agricultural yields despite increased
water insecurity.
Traits for improving drought resistance
During drought spells, plant systems actively
maintain physiological water balance by (i) in-
creasing root water uptake from the soil, (ii)
reducing water loss by closing stomata, and
(iii) adjusting osmotic processes within tissues
(13). Activated stress response pathways include
phytohormone signaling as well as antioxidant
and metabolite production and mobilization (11).
Duplicate
crop production
Year 2050
D
2.5 5
Billions of cubic meters

sense the water-deficiency signal and initi-

Billions of hectares
Billions of people

8 2 4
ate coping strategies in response is defined as
drought resistance. Drought resistance is a 6 1.5 3
complex trait that proceeds through several
mechanisms: (i) escape (acceleration of plant 4 1 2
reproductive phase before stress that could 2 0.5 1
hinder its survival), (ii) avoidance (endurance
with increased internal water content and 0 0 0
1990 2020 2050 1990 2020 2050 1990 2020 2050
prevention of tissue damage), and (iii) tolerance
(endurance with low internal water content Fig. 1. Past, present, and future of global climate, agriculture, and food security. (A) Most scenarios
while sustaining growth over the drought predict that water scarcity will increase in the coming years. With the world’s population continuously
period) (7). After a period of drought, the per- growing, crop production must also increase to fulfill civilization’s basic needs. For this purpose, plants must
centage of viable plants upon rewatering is become more water efficient. (B) Estimated world population for the 1990–2050 time period. The arrow
indicates the estimated number of people living in water-scarce areas. (C) Global arable land for agriculture
for the 1990–2050 time period. The arrow indicates the predicted demand for arable land to ensure food
Department of Molecular Genetics, Centre for Research in security, given the current rates of crop production per hectare. (D) Global freshwater demand for agriculture
Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus
UAB (Cerdanyola del Vallès), 08193 Barcelona, Spain. for the 1990–2050 time period. The arrow indicates the predicted decline in freshwater availability for
*Corresponding author. Email: ana.cano@cragenomica.es agriculture, given the current trends for climate change and precipitation.

Gupta et al., Science 368, 266–269 (2020) 17 April 2020 1 of 4

 Roots respond to changes in soil moisture
at the cellular scale and with the entire root
system architecture. The root stem cell niche,
meristem, and vasculature each coordinate re-
sponses to drought (Fig. 2, A and B). During
periods of water scarcity, the root system ar-
chitecture undergoes morphological changes
to enhance its ability to absorb water and nu-
trients (9, 10). These modifications can be
traced to coordinated cell division, elongation,
and differentiation events in the root apex. In
the pursuit of moisture, root systems grow
differentially and adapt their architecture to
be either deep or shallow (Fig. 2C). Longer and
deeper roots with reduced branching angles
can efficiently capture water from soil that is
dry at the surface but retains moisture in deep
layers. By contrast, shallower root architec-
tures are more beneficial for maximizing water
capture from the soil surface in regions of low
precipitation (9). Roots that encounter a soil
ways (Fig. 3) (21). Many existing schemes to
improve water use efficiency and drought re-
sistance engage the ABA pathway.
Genetic engineering to improve the function
of PYR/PYL/RCAR (Pyrabactin Resistance 1/
PYR1-Like/Regulatory Component of ABA Re-
ceptors) and SnRK2 (SNF1-related protein
kinase 2) and repress the negative regulator
PP2C (clade A type 2C protein phosphatase)
has resulted in improved water use efficiency in
plants such as A. thaliana and wheat under
controlled laboratory growth conditions and
greenhouses (22–25). A regulatory network
of ABA pathway genes, a hierarchy of ABA-
related transcription factors, and signaling
A C
ciency and drought resistance in A. thaliana,
tomato, and wheat (27). Thus, computational
design combined with experimental biology
led to identification of a small molecule that
can mitigate the effects of drought on crop
yields.
Brassinosteroid hormones also regulate
drought response through signaling com-
ponents linked to the ABA response pathway
(Fig. 3) (28, 29). Brassinosteroid signaling
negative regulator BRASSINOSTEROID-
INSENSITIVE 2 (BIN2) is dephosphorylated
by ABA INSENSITIVE1 (ABI1) and ABI2. ABA
activates BIN2 by inhibiting the activity of
ABI1 and ABI2 (30). BIN2 phosphorylates
E

Downloaded from http://science.sciencemag.org/ on April 16, 2020

environment with nonhomogeneous water dis-
tribution display hydropatterning by favoring
lateral root emergence toward soil patches
with higher water content, a process that is also
mediated by auxin signaling (9, 14). Another
adaptive response to nonhomogeneous distribu-
tion of moisture through soil is hydrotropism
(Fig. 2D), in which root tips grow toward zones 50 µm 100 µm
with higher water content to optimize the root
system architecture for water acquisition (15). B D F
Stomatal closure is a more rapid defense
against dehydration (Fig. 2, D and E). Stomatal
pores on leaf surfaces open or close accord-
ing to the turgidity of the surrounding guard
cells. The turgor-driven shape changes of guard
cells are affected by the cell wall structure,
plasma membrane, tonoplast properties, and
ia
ed
m

cytoskeletal dynamics (16). Plant vascular tis-

ic
ot

sues, the xylem and phloem, transmit water

m
os

availability signals from roots to shoots and

gh
Hi

transmit photoassimilates from shoots to roots, 50 µm 1 cm 20 µm

respectively (17). Development of these inner
vasculature tissues also affects drought re-
sistance. Crop yield becomes most vulnerable
if the drought occurs during a plant’s repro-
ductive phase. In Arabidopsis thaliana, early
flowering associated with drought escape is
linked to phloem loading and transport of the
photoperiod-dependent protein FLOWERING
LOCUS T (FT) from leaves to the shoot apical
meristem (18).
Fig. 2. Root and shoot traits that account for drought resistance. (A and B) Plants initially sense
drought through their roots, where particular cell types (shown in blue)—such as stem cells, cortical cells,
and vascular cells—mediate adaptive responses toward water limitations. Roots can modulate their
system architecture to (C) maximize access to superficial humidity or delve into deep humid soil layers,
as well as to (D) bend toward more humid soil zones (hydrotropism). (E and F) In aboveground plant organs
such as leaves and stems, stomata work actively against dehydration. In water-limiting conditions,
stomata remain closed to reduce water loss.

Phytohormones to combat drought
The hormone abscisic acid (ABA) regulates
plant responses to dehydration and optimizes
water use. Dehydration signals stimulate local
production of ABA in different plant organs.
However, ABA production is more efficient
in the leaf mesophyll cells than in the root
tissues (19). The accumulated ABA then ac-
tivates downstream signaling components
(20). ABA executes its function during stress
by mediating signal cross-talk with other path-
feedback were identified among ABA-mediated
stress responses to drought (26). Engineering
the ABA receptor PYR1 for heightened sen-
sitivity toward the preexisting agrochemical
mandipropamid resulted in improved drought
resistance in A. thaliana and tomato (22). Vir-
tual screening for ABA receptor agonists led to
the identification of a bioactive ABA mimic
called opabactin. This small molecule can
enhance ABA receptor activation and down-
stream signaling to improve water use effi-
SnRK2s and activates the downstream path-
way (31). ABA signals can also converge with
the brassinosteroid pathway at the level of
downstream transcription factors (Fig. 3).
BRI1-EMS-SUPPRESSOR 1 (BES1) inhibited
ABA induction of a drought-related transcrip-
tion factor RESPONSIVE TO DESICCATION 26
(RD26) (32). RD26 shows reciprocal antag-
onism with brassinosteroid by modulating
BES1-regulated transcription and inhibiting
brassinosteroid-regulated growth (33). WRKY46,

Gupta et al., Science 368, 266–269 (2020) 17 April 2020 2 of 4

 D ROU G HT

-54, and -70 belong to another class of tran-
scription factors that interact with BES1 to pro-
mote plant growth while repressing drought
responses (34). BIN2 can phosphorylate and
destabilize WRKY54 to negatively regulate its
effect on the BES1-mediated brassinosteroid
response (35). BIN2 phosphorylates and acti-
vates the ubiquitin receptor protein DSK2,
which leads to BES1 degradation via autoph-
agy and coordinates plant growth and
survival under drought conditions. (36).
An AP2/ERF transcription factor called
TINY is another candidate that balances
brassinosteroid-mediated stress adap-
tation with growth. TINY interacts with
BES1 and antagonizes brassinosteroid-
regulated growth. BIN2, on the other
hand, phosphorylates and stabilizes
TINY to promote ABA-induced stomatal
closure and drought resistance (37).
Thus, brassinosteroids as well as ABA
ulates local auxin transport by affecting the
homeostasis of the auxin efflux carrier PIN-
FORMED 4 in root columella cells (Fig. 3).
Natural variation in EXO70A3 was correlated
with seasonal precipitation and conferred dif-
ferent adaptive root system architecture con-
figurations under different rainfall patterns.
In areas with high temperatures and irrigated
soils, deeper root architectures proved better
ABA
PYR/PYL
RCAR Stomata
closure
SnRK2 ABA-responding
genes
for drought adaptation. In rice, the auxin-
inducible gene DEEPER ROOTING1 provides
drought resistance by promoting a more ver-
tical and deeper root system architecture (41).
Although auxin modulates root architecture
under stress (40, 41), hydrotropic root re-
sponses function relatively independent of
auxin and involve ABA signaling in root elon-
gation zones. Coordinated activity of ABA-
inducible MIZU-KUSSEI1 (MIZ1) and
SNF1-RELATED KINASE 2 (SnRK2.2)
in root elongation zone cortical cells
interprets water potential gradients in
soil environments (15, 42).
Brassinosteroid receptors regulate
root hydrotropic responses (Fig. 3).
Overexpression of the vascular-enriched
brassinosteroid receptor BRI1-Like3
(BRL3) promotes root hydrotropic bend-
ing. The brl1brl3bak1 triple mutant of
the BRL3 signalosome shows a reduced

CL

Downloaded from http://science.sciencemag.org/ on April 16, 2020

E2
aid drought resistance. hydrotropic response, suggesting a role
BIN

5
2
for the vascular BRL3 receptor complex
Tissue-specific responses BRASSINOSTEROIDS
in regulating hydrotropic responses (43)
for drought resistance (Fig. 3). Activation of the BRL3 pathway
BIN2 BRL3
Stomatal closure preserves water in the Hydrotropism, in vasculature triggers accumulation of
plant. ABA content in leaves regulates osmoprotectant osmoprotectant metabolites such as
stomatal movement in response to metabolites proline, trehalose, and raffinose family
accumulation
water availability (25) (Fig. 3). Because oligosaccharides in plant roots in re-
BES1 WRKYs/ RD26
stomatal movements control CO2 in- TINY
sponse to water withdrawal, which
flux and transpiration, efforts to reduce improves drought resistance without
water loss via stomatal closure occur at penalizing growth (43) (Fig. 3). Phloem-
the cost of photosynthesis, growth, specific localization of BRL3 is likely to
5

AUXIN
E2

and yield (13). Therefore, most strat- be the determining factor for promot-
CL

egies to improve water efficiency and
drought resistance in plants focus on
fine-tuning stomatal conductance
and manipulating ABA signaling via
stomata-specific promoters (38). With
optogenetics, scientists have improved
the responsiveness of the stomata and
overcome the coupling of CO2 uptake
with water vapor loss. Upon introduc-
ing BLINK1 (a light-activated synthetic
K+ ion channel) into guard cells, sto-
mata became more synchronized with
fluctuating light conditions (39). This
manipulation improved the performance
of the stomata and, consequently, growth
and productivity of the plant. Thus,
water use efficiency was improved by
engineering the stomata to maximize
the amount of carbon fixed per unit of
water lost.
Improving water acquisition by roots
can also improve plant performance
upon drought. In A. thaliana, the auxin
pathway modulator EXOCYST SUB-
UNIT EXO70 FAMILY PROTEIN A3
(EXO70A3), which regulates root system
depth, was identified through genome-
wide association mapping (40). EXO70A3, a
component of the exocytosis system, is
expressed in root tips. EXO70A3 reg-
EXO70A3
IAA
Root system
architecture
PIN4
CLE25
Drought
sensing
Fig. 3. Hormone signaling events underpinning drought. Sche-
matic representation of hormone signaling modules that control
drought adaptation. Plants work against dehydration in organs
such as leaves, vasculature, and roots. ABA, through SnRK2,
activates a variety of genes that trigger stomata closure and
improve water balance. When roots sense drought, the CLE25
peptide moves through the vasculature to the leaves, where it
locally controls ABA biosynthesis and stomata closure. Brassinosteroids
also play roles in regulating plant drought response. Brassinosteroid signals.
pathways converge with ABA by activating SnRK2 through
downstream pathway component BIN2 and vice versa. Indepen-
dently of ABA, brassinosteroid receptors (BRI1, BRL1, and BRL3)
modulate hydrotropic responses in the roots. The vascular BRL3
receptor coordinates plant growth and survival under drought
stress by promoting the accumulation of osmoprotectant metabo-
lites in the root tissues. Furthermore, noncanonical auxin responses
via EXO70A3 and PIN4 can modulate root architecture patterning
and depth to boost water absorption from the soil, thereby
improving drought tolerance.
ing drought resistance without impair-
ing yield (29, 43).
In drought conditions, roots sense
water scarcity from soil. The above-
ground segments of plants respond by
closing stomata in leaves, implicating
a systemic communication system. In
times of drought, the CLE25 peptide is
produced in the roots and moves through
the vasculature to plant leaves to drive
ABA production by activating the bio-
synthetic enzyme NCED3. This burst of
ABA synthesis leads to stomatal closure
and improved water balance, thereby
promoting drought survival (44) (Fig. 3).
This insight into small-peptide signaling
in A. thaliana may help with identifica-
tion of similar mechanisms in crop plants
for root-to-shoot mobilization of stress
A view to the future
Genetic traits that sustain crop plant
growth under moderate drought may
come from multiple sources, including
natural genetic variation in wild relatives
or bioengineering. Traditional breeding
has been the main strategy for exploiting
the genetic diversity of adaptive traits
in natural alleles. The advent of genomic

Gupta et al., Science 368, 266–269 (2020) 17 April 2020 3 of 4

technologies and gene mapping tools such as egies to combat water scarcity. Discovering
genome-wide association study (GWAS) and ways to ameliorate agriculture’s “thirst” will
precision genome editing with the CRISPR- ease competition for freshwater resources,
Cas9 system has been instrumental for the even as the world’s population grows.
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AC KNOWLED GME NTS
Funding: A.I.C.-D. has received funding from the European Research
Council (ERC) under the European Union’s Horizon 2020 research
and innovation programme (grant agreement 683163). A.I.C.-D. is a
recipient of a BIO2016-78150-P grant funded by the Spanish Ministry
of Economy and Competitiveness and Agencia Estatal de Investigación
(MINECO/AEI) and Fondo Europeo de Desarrollo Regional (FEDER). A.G.
has received funding by a postdoctoral fellowship from the “Severo
Ochoa Programme for Centers of Excellence in R&D” 2016–2019 from
the Ministerio de Ciencia e Innovación (SEV-2015-0533). A.G. and A.R.-M.
have received funding from ERC-2015-CoG–683163 granted to the
A.I.C.-D. laboratory. A.R.-M. is a predoctoral fellow from Fundación
Tatiana Pérez de Guzmán el Bueno. This work was supported by the
CERCA Programme/Generalitat de Catalunya. Competing interests: The
authors declare no competing or financial interests.
10.1126/science.aaz7614
4 of 4
The physiology of plant responses to drought
Aditi Gupta, Andrés Rico-Medina and Ana I. Caño-Delgado

Science 368 (6488), 266-269.

DOI: 10.1126/science.aaz7614

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Review

ABA Transport and Plant Water Stress

Responses
Takashi Kuromori,1,* Mitsunori Seo,2,* and Kazuo Shinozaki1

To understand the integrative networks of signaling molecules, the sites of their Highlights
biosynthesis and action must be clarified, particularly for phytohormones such Plants close stomata to limit water loss
on water deficit. It is evident that ABA is
as abscisic acid (ABA). The relationship between the sites of ABA biosynthesis
required in this process based on the
and transport has been discussed extensively in the context of guard cells and phenotypes of mutants defective in ABA
stomatal regulation. However, guard cells are not the only site of ABA action. biosynthesis or signal transduction.

Recent studies have reported multiple sites of ABA biosynthesis and multiple ABA may be translocated from the sites
ABA transporters, indicating that ABA transport regulation is not unidirectional of biosynthesis, such as roots and leaf
but rather forms complex networks. Therefore, it is important to determine vascular tissues, to the guard cells.
Recent identification of multiple trans-
how multiple ABA sources coordinately contribute to individual biological membrane ABA transporters indicates
processes under various physiological conditions. that the movement of this hormone
within a plant is actively regulated in
an intercellular network.
Plant Hormone Transport
Hormones are endogenous chemicals that induce various physiological responses at low ABA regulates various molecular events
concentrations. In animals, hormones are secreted from specific cell types (e.g., endocrine in different organs and tissues as well as
cells) and transported to distant target sites through the circulatory system within the body. guard cells to tolerate water stress
dependent on environmental condi-
Similar to hormones in animals, most plant hormones are mobile. However, the sites of their tions. However, the dynamics of ABA
synthesis and actions are often not clearly defined, and it is not always clear whether movement transport within a plant must be ana-
or transport of plant hormones is a prerequisite of their biological function. This is possibly lyzed and discussed in a more spatio-
temporally physiological context.
because cells in a relatively wide range of tissues and organs can synthesize hormones as well
as respond to them.

The plant hormone ABA regulates various physiological processes throughout plant life cycles
[1–4]. For example, in response to water deficit ABA induces stomatal closure and the
expression of numerous stress-responsive genes. By contrast, ABA promotes the accumu-
lation of seed storage compounds during seed development and is also required for the
induction and maintenance of seed dormancy. It has been also reported that ABA is involved
in plant pathogen responses. Here we try to determine how the biosynthesis and transport of
ABA are coordinately regulated within a plant. Especially, we focus on plant water relations,
since multiple sites of ABA biosynthesis, namely vascular cells and guard cells, have been
suggested in this context. The recent identification of several classes of ABA transporters
indicates that plants are equipped with a highly sophisticated system to sense and respond to 1
RIKEN Center for Sustainable
water availability under diversely fluctuating environments. Resource Science, 2-1 Hirosawa,
Wako, Saitama 351-0198, Japan
2
Sites of ABA Biosynthesis and Transport between Roots and Shoots RIKEN Center for Sustainable
Resource Science, 1-7-22 Suehiro-cho,
Higher plants absorb water in soil from roots and transpire water from aboveground organs Tsurumi-ku, Yokohama, Kanagawa
such as leaves and stems through stomata located on their epidermal tissue. In addition, 230-0045, Japan
stomata mediate the uptake and release of carbon dioxide and oxygen for photosynthesis and
respiration. Therefore, plants must integrate multiple environmental signals to optimize the
*Correspondence:
stomatal aperture under diverse environmental conditions. Since the discovery of ABA as a takashi.kuromori@riken.jp (T. Kuromori)
chemical that induces stomatal closure, many studies have examined its role in root-to-shoot and mitsunori.seo@riken.jp (M. Seo).

Trends in Plant Science, June 2018, Vol. 23, No. 6 https://doi.org/10.1016/j.tplants.2018.04.001 513
© 2018 Elsevier Ltd. All rights reserved.
signaling under drought stress [5]. Under mild stress conditions when soil drying begins, ABA
accumulation in root tissues is closely correlated with a decrease in leaf stomatal conductance
[6]. In addition, several studies have found that drought stress can induce ABA accumulation in
xylem sap [7,8]. Although the substance associated with root-to-shoot signaling under drought
stress has not been fully elucidated, these observations suggest that ABA synthesized in root
tissue is released to xylem vessels and transported to shoots. Because ABA is a weak acid
containing a carboxyl group (pKa = 4.7), it exists in solution in the protonated or deprotonated
form, with the proportion of each determined by the pH. An increase in xylem sap pH in
response to soil drying has been observed in many plant species, which could enable ABA to
move more freely through extracellular space toward guard cells according to the transpiration
stream, because a smaller amount of ABA would be trapped by surrounding cells during
transport than under lower pH conditions [6,9–11].

Although the significance of root-derived ABA in stomatal closure has been widely
accepted and documented in many textbooks, ABA is synthesized not only in roots
but also in shoots (leaves). Moreover, several studies have shown that ABA accumulates
at much higher concentrations in leaves than in roots on water deficit and that ABA
accumulation in roots is sometimes dependent on basipetal ABA transport from aerial
organs [12–17]. In addition, reciprocal grafting between ABA-deficient mutants and wild-
type plants demonstrates that stomatal closure is affected by leaf genotype but not root
genotype [18–22]. Several candidates involved in root-to-shoot signaling under drought
stress have been proposed, such as hydraulic signals, pH, a small peptide, or some
chemical agent other than ABA [23,24].

Site of ABA Biosynthesis and Transport between Vascular Cells and Guard
Cells
In addition to ABA biosynthesis in roots and leaves, tissue-specific expression of ABA biosyn-
thesis genes and enzymes in arabidopsis (Arabidopsis thaliana) offers another level of com-
plexity. Experiments using antisense RNAs, antibodies, or promoter-driven expression of
fluorescent proteins have suggested that ABA biosynthesis enzymes (e.g., NCED3, ABA2,
AAO3) are preferentially localized in vascular bundles in leaves [25–27]. Guard cells, the main
ABA target sites in terms of stomatal closure, are located on the surface or epidermal layer of
plants, whereas vascular tissue, which is possibly the primary site of ABA biosynthesis in leaves,
is located inside the plant body. This suggests that ABA is transmitted between distant tissues
within leaves. Moreover, a study showed that leaf surface temperature increased when NCED3
was expressed under the control of a promoter specific to phloem companion cells in a wild-
type background, possibly due to the induction of stomatal closure [27].

Guard cells do not have direct connections with surrounding mesophyll or epidermal cells
through plasmodesmata. The major ABA receptors responsible for stomatal closure (PYR/PYL/
RCAR) are soluble proteins. Therefore, if ABA synthesized in vascular tissues were transported
to guard cells, at least two transmembrane ABA transport steps would be required for stomatal
closure. Supporting this hypothesis, several ABA transporters have been identified [11,28]
(Figure 1, Box 1, and Table 1). Two arabidopsis ATP-binding cassette (ABC) transporters,
AtABCG25 and AtABCG40, have been identified as ABA transporters involved in ABA sensi-
tivity or stomatal closure based on the phenotypes of mutants defective in the respective
proteins [29,30]. Biochemical characterization using heterologous expression systems has
shown that AtABCG25 and AtABCG40 exhibit ABA export and import activity, respectively
[27,29,30]. For example, the Km values of AtABCG25 and AtABCG40 for ABA were relatively
low (260 nM and 1 mM, respectively) compared with ABA levels in water-stressed guard cells of

514 Trends in Plant Science, June 2018, Vol. 23, No. 6

 ABCG40

•Stomatal closure DTX50

•Water resistance
(bundle sheath cells)

ABCG25 NPF4.6
DTX50

ABCG25
DTX50
•Root architecture
•Water resistance
NPF4.6
•Suberization (endodermis)

Figure 1. Abscisic Acid (ABA) Transport and ABA Regional Functions in Plant Water Stress Responses.
This schematic diagram shows root and leaf cross-sections, including three possible sites of ABA biosynthesis: root
(vascular tissue), leaf vascular tissue, and guard cells. Tissues and cells expressing ABA transporters are shaded yellow
(Box 1). The blue arrows indicate transmembrane ABA transport mediated by transporters. Purple indicates the possible
movement of ABA through xylem and phloem. The physiological functions of ABA in roots, leaf vascular tissues, and guard
cells discussed in the text are also shown.

Box 1. ABA Transporters

The designation of the four membrane factors AtABCG25, AtABCG40, AtNPF4.6, and AtDTX50 as typical ABA
transporters is supported by several lines of evidence (summarized in Table 1). All four proteins are localized to plasma
membranes, implying the involvement of ABA transfer between the inside and outside of cells. All gene-disruption
mutants showed aberrant sensitivity to ABA accompanied by biochemical evidence of ABA transport activity. Inter-
estingly, these transporter genes are predominantly expressed in vascular tissues or guard cells, strongly indicating that
ABA transport is regulated in a network spanning distant tissues related to the site of biosynthesis and the site of
physiological actions (see main text). Although this review focuses on ABA transporters related to water stress
response, additional ABA transporters have been reported (e.g., those related to seed germination) [66].

Trends in Plant Science, June 2018, Vol. 23, No. 6 515

516
Trends in Plant Science, June 2018, Vol. 23, No. 6

Table 1. Characteristics of the Four Typical ABA Transporters Reported to Date

Gene name Gene Gene identification Tissue Subcellular Transport assay KO phenotype KO phenotype OE phenotype OE phenotype Suggested Refs
family expression localization of germinative of adult stage of germinative of adult stage function
growth growth

AtABCG25a ABCG Mutant screen based Vascular Plasma Vesicles and insect ABA (N.D.) ABA Higher surface Exporter [30]
half-size on ABA sensitivity tissues membrane cells hypersensitive insensitive temperature of
type during seed leaves, less
(WBC) germination/early water loss
seedling growth
stages from
transposon-tagged
lines

AtABCG40 ABCG Characterization of Broad, Plasma Yeast and BY2 cells ABA Drought (N.D.) (N.D.) Importer [29]
full-size mutants defective in highest in membrane insensitive sensitive
type PDR members guard cells
(PDR)

AtNPF4.6/ NPF Screening of proteins Vascular Plasma Yeast and insect ABA Lower surface ABA Lower surface Importer [34]
NRT1.2/ capable of tissues membrane cells insensitive temperature of hypersensitive temperature of
AIT1 transporting ABA in inflorescence leaves and
yeast stems inflorescence
stems

AtDTX50 MATE Characterization of Guard cells Plasma Escherichia coli and ABA Growth arrest, (N.D.) Rapid wilting Exporter [35]
mutants defective in and vascular membrane Xenopus oocyte hypersensitive drought under drought
MATE members tissues tolerant stress

a
Abbreviations: ABCG, ABC G subfamily; AIT, ABA-importing transporter; DTX, detoxification efflux carrier; KO, knockout mutant; MATE, Multidrug and toxin efflux; NPF, nitrate transporter 1/peptide
transporter family; NRT, nitrate transporter; OE, overexpressed plant; PDR, pleiotropic drug resistance; WBC, white–brown complex; Y2H, yeast two-hybrid system.
Vicia faba (
15 mM) [31]. Both AtABCG25 and AtABCG40 are localized to the plasma
membrane when expressed as fluorescent fusion proteins in plant cells [29,30]. Interestingly,
one study showed that plasma membrane localization of AtABCG25 was regulated by abiotic
stress and ABA [32]. In another remark, the AtABCG25 promoter was active in vascular tissue,
where ABA biosynthesis enzymes are preferentially expressed, whereas higher AtABCG40
expression determined by a promoter–reporter system was observed in guard cells. In addition,
AtABCG25 expression under the control of the CaMV 35S promoter enhanced stomatal
closure on activation of the ABA-inducible RD29B promoter [33]. These findings are consistent
with the hypothesis that vascular tissue is the primary site of ABA synthesis, from which ABA is
transported to guard cells to elicit stomatal response, possibly mediated by these ABA
transporters (Figure 1). However, mutants defective in either AtABCG25 or AtABCG40 exhibit
relatively mild phenotypes compared with typical ABA-deficient mutants, suggesting that
passive-diffusion ABA transport mechanisms could compensate for the functions of
AtABCG25 and AtABCG40 or that active ABA transport mediated by transporters is highly
redundant. Subsequent studies have identified additional ABA transporters and support the
latter scenario, as described below.

Functional screening using a yeast system identified an arabidopsis NITRATE TRANSPORTER

1/PEPTIDE TRANSPORTER FAMILY (NPF) member, NPF4.6 (originally named AIT1), as a
protein that could mediate ABA uptake into cells [34]. NPF4.6 was previously characterized as a
low-affinity nitrate transporter NRT1.2 (Km = 5.9 mM); however, the same protein exhibited
much higher affinity for ABA (Km = 5 mM). Fluorescent protein-fused NPF4.6 is predominantly
localized to the plasma membrane, suggesting that this protein could also have a role in
intercellular ABA transport. Expression of NPF4.6 under the control of the CaMV 35S promoter
results in a reduction in leaf surface temperature, possibly due to defective stomatal closure.
Constitutive ABA import activity could inhibit ABA movement within plant tissues. This suggests
that ABA transport is necessary for proper stomatal function. Conversely, loss-of-function
npf4.6 mutants have shown only limited phenotypes in terms of stomatal closure, as observed
for abcg25 and abcg40, suggesting that ABA transport systems are highly redundant. Inter-
estingly, while both NPF4.6 and AtABCG40 mediate cellular ABA uptake in heterologous
systems, their spatial expression patterns differ; NPF4.6 is expressed in vascular cells whereas
AtABCG40 is expressed in guard cells. NPF4.6 could regulate the amount of ABA transported
from vascular tissues toward guard cells in association with AtABCG25 (Figure 1). Moreover,
NPF4.6-mediated ABA uptake into vascular tissues could be related to ABA distribution within
plants via xylem and phloem (Figure 1).

A member of the multidrug and toxin efflux (MATE) transporter family, AtDTX50, has also been
identified as an ABA exporter [35]. A comprehensive reverse genetic approach in MATE
transporters found that a mutant defective in AtDTX50 (dtx50) exhibited growth defects,
possibly due to its higher sensitivity to ABA or higher endogenous ABA levels. Fluores-
cence-tagged AtDTX50 proteins were localized predominantly to the plasma membrane
and AtDTX50 facilitated ABA efflux in both Escherichia coli and Xenopus oocyte cells.
DTX50 was preferentially expressed in vascular tissues, indicating that it regulates ABA export
from vascular tissues in association with AtABCG25 and NPF4.6 (Figure 1). AtDTX50 expres-
sion in guard cells suggests that ABA levels in these cells, and hence stomatal aperture, are
regulated not only by ABA influx but also by ABA extrusion (Figure 1).

Leaf vascular tissue appears to largely contribute to ABA accumulation in response to

drought under many experimental conditions. However, this does not mean that this tissue
is the only source of ABA. For example, guard cells themselves autonomously support the

Trends in Plant Science, June 2018, Vol. 23, No. 6 517

ABA synthesis required for the stomatal response to leaf water status [36,37]. This system
would be beneficial for plants to respond to rapid changes in aerial humidity before the root-
derived root-to-shoot signals on soil drying induce ABA biosynthesis in vascular tissues,
whereas a large quantity of ABA produced in vascular tissues might be required for plants to
more comprehensively respond to severe water deficit (discussed more in detail below). In
addition, it is possible that ABA synthesized in guard cells could be exported outside by
AtDTX50 and affects some ABA responses in the surrounding cells. Interestingly, it has been
shown that ABA catabolism is differentially regulated in vascular tissues and guard cells by
distinct CYP707As. CYP707A3 is predominantly expressed in vascular tissues and regulates
the total amount of ABA accumulated in leaves. Conversely, CYP707A1, which is preferen-
tially expressed in guard cells, makes a minor contribution to the bulk ABA content in leaves.
However, CYP707A1 could regulate the stomatal aperture similarly to CYP707A3 [38]. These
results indicate that site-specific ABA levels, for example in guard cells, are determined
based on a combination of biosynthesis, catabolism, and transport in vascular cells and
guard cells [39].

ABA Action in Guard Cells for Stomatal Aperture Regulation

One of the major causes of stomatal closure is the activation of outward K+ channels, which
leads to a reduction in the water potential inside guard cells. As a result, water is exported
from guard cells according to the potential, resulting in a reduction in turgor and finally
stomatal closure [40]. ABA-induced stomatal closure is generally considered to be a
relatively rapid response that occurs within a few seconds or minutes [41] and does not
require de novo transcription. Nevertheless, microarray analysis using isolated guard cell
protoplasts has identified genes that are up- or downregulated by exogenous ABA treat-
ment [36,42,43]. Interestingly, three basic helix–loop–helix (bHLH) transcription factors,
AKS1, 2, and 3, have been identified as possible SnRK2 kinase substrates [44,45].
Unphosphorylated AKSs form homomultimers that transactivate the expression of
shaker-type inward K+ channel genes, including KAT1, to promote stomatal opening.
Meanwhile, phosphorylation results in the monomerization of AKSs, which inhibits DNA
binding and transcription of target genes. This suggests that ABA-mediated transcriptional
regulation inside guard cells is important for stomatal aperture regulation. It is important to
further understand how the rapid and long-term stomatal responses are regulated by
multiple ABA sources and ABA transporters.

Involvement of guard cell aquaporins in stomatal aperture regulation has also been sug-
gested. Plasma membrane-intrinsic proteins (PIPs) are the major aquaporins that facilitate
diffusion of water across the plasma membrane [46]. It was recently shown that ABA
regulates transmembrane water transport in guard cells [47]. Mutants defective in PIP2;1
(pip2;1) were less sensitive to exogenously applied ABA than the wild type in terms of
stomatal closure, while mutants exhibited high carbon dioxide-induced stomatal closure,
similar to the wild type. In guard cell protoplasts prepared from pip2;1, ABA-induced
increases in water permeability and reactive oxygen species, which normally occur in
wild-type guard cell protoplasts, were not observed. OST1/SRK2E/SnRK2.6 enhanced
PIP2;1-mediated water transport when coexpressed in Xenopus oocytes and phosphory-
lation of PIP2;1 by OST1/SRK2E/SnRK2.6 was required for the response. These results
suggest that ABA-mediated activation of PIP2;1 in guard cells could help induce stomatal
closure by facilitating water transport, although the stomatal aperture of pip2;1 is similar to
that of the wild type in the absence of exogenously applied ABA. The lack of a stomatal
phenotype in pip2;1 in the absence of exogenous ABA might be due to the complex roles of
PIP2;1 in different tissues and organs.

518 Trends in Plant Science, June 2018, Vol. 23, No. 6

Physiological Roles of ABA in Vascular Tissues
If the total amount of ABA synthesized in a plant predominantly depends on vascular tissues,
why is ABA biosynthesized in vascular tissues far from guard cells located on epidermal tissue?
The simplest answer is that vascular tissues have an important role in distributing ABA
throughout the plant body to tolerate water stress by regulating physiological processes
beyond direct induction of stomatal closure (Figure 1).

Leaf transpiration rates are regulated not only by stomatal aperture (stomatal conductance) but
also by several factors such as hydraulic resistance and water flow rates within plant tissues. In
principle, water transported to leaves via xylem can move through cells (via cell-to-cell transport
across the biological membrane or plasmodesmata) and the apoplastic space toward stomata
[48]. The density of cells in mesophyll tissues is often low and these tissues often contain a large
proportion of air space, suggesting that water travels via apoplastic movement in such tissues
in leaves. Conversely, vascular bundles comprise tightly associated cell layers. Bundle sheath
cells (BSCs), which form a layer surrounding vascular tissues [49], are connected to neighbor-
ing mesophyll cells via plasmodesmata and the cells do not share symplastic connections with
xylem and phloem [50]. When a water-soluble cell-wall-specific dye was loaded via the cut
surface of a leaf petiole, staining was observed along veins but not in mesophyll cells [51],
indicative of a tight apoplastic barrier surrounding the vascular bundle. In addition, in some plant
species, outer tangential cell walls of BSCs are preferentially suberized [52]. These results
suggest the importance of water uptake into BSCs and subsequent transcellular water flow.

In one study, feeding ABA to detached Arabidopsis leaves via xylem reduced leaf hydraulic
conductance, whereas ABA application to the leaf surface did not affect leaf hydraulic
conductance [51]. The water permeability of BSC protoplasts was reduced by ABA treatment;
however, the same treatment did not affect water permeability in protoplasts prepared from
mesophyll cells. Meanwhile, BSC-specific silencing of the PIP1 aquaporin subfamily by miRNA
resulted in reduced water permeability of mesophyll cells and BSCs and decreased leaf
hydraulic conductance [53]. These data suggest that ABA-mediated inhibition of aquaporin
in BSCs has a role in reducing leaf hydraulic conductance and transpiration independent of the
stomatal aperture. In addition, the stomata of an arabidopsis ost2 mutant, which is defective in
an H+-ATPase, were insensitive to ABA; however, ABA application to ost2 via xylem induced
stomatal closure [54]. It is possible that ABA synthesized in vascular tissue induces stomatal
closure indirectly by reducing the water permeability of BSCs and leaf hydraulic conductance.

Physiological Role of ABA in Roots

Soil drying can result in enhanced or reduced hydraulic conductivity in root cells depending on
plant species, variety, and natural variations [55]. ABA can influence root hydraulic conduc-
tance, possibly by regulating aquaporin activity, although the effects can differ depending on
the plant materials and experimental conditions. Such complex regulation might be required for
plant adaptation to different environmental conditions. For example, increased water perme-
ability is required for efficient water uptake during the early phase of water deficit. However, after
prolonged water deficit, higher water permeability could result in the loss of water from root
tissue to soil. Inhibiting root hydraulic conductance by 50% resulted in a greater than 50%
reduction in leaf hydraulic conductance. This suggests that root hydraulic conductivity could
affect leaf-water relationships [56]. However, it is unknown which cells in roots are responsible
for ABA-mediated responses.

In roots, ABA regulates not only hydraulic conductance but also growth. Salt stress regulates
the expression of numerous ABA-dependent genes in roots, some of which are regulated

Trends in Plant Science, June 2018, Vol. 23, No. 6 519

globally in root tissues and others that are regulated in specific cell types [57]. It has been Outstanding Questions
reported that ABA signaling in endodermis promotes lateral root quiescence during salt stress How do plants sense water deficit at
[58]. Arabidopsis root growth is also inhibited by osmotic stress, which is dependent on different sites?

endogenous ABA because ABA-deficient mutants are less sensitive to osmotic stress [59].
How does ABA synthesized at different
Similarly, in most cases ABA inhibits root growth when applied exogenously. However, sites contribute to a given physiologi-
endogenous ABA has also been observed to promote root growth [21,60]. The effects of cal response?
ABA on root growth could differ depending on the strength and duration of stress, as discussed
with respect to the role of aquaporin. How do transporters regulate the mul-
tidirectional movement of ABA within a
plant?
The Casparian strip is a ring-like cell-wall modification in root endodermis that surrounds
vascular tissues (e.g., BSCs in leaves). It is primarily composed of lignin and serves as a barrier How do ABA synthesis and transport
against apoplastic diffusion of minerals and nutrients from vascular bundles [61]. A recent differ between the initial and later stages
report indicated that endodermis is reversibly coated with hydrophobic suberin in response to of water stress?
various stresses such as nutrients and salinity, which might restrict apoplastic movement of
How do various types of cells in shoots
water, minerals, and nutrients [62]. Interestingly, suberization was induced by the enhanced
or roots orchestrate ABA synthesis
expression of a suberin synthesis gene in response to exogenous ABA treatment. Furthermore, and transport?
in several mutants defective for ABA biosynthesis and signaling (aba2, abi3, abi4, and abi5),
suberin deposition in endodermis was significantly delayed. ABA-mediated suberization
requires ABA signaling in endodermis, because the expression of the dominant-negative
aba1-1 protein in endodermis under the control of CASP1 and ELTP1 inhibits suberization
under non-stressed conditions and in response to exogenous ABA treatment. ABA synthe-
sized in aboveground tissues is reportedly translocated to roots, possibly via the vasculature
[14,16,17]. In addition, some ABA biosynthesis and transporter genes are expressed in root
vascular tissues [25,30,34,35,63]. This suggests that ABA synthesized in or translocated to
root vascular tissues might be associated with endodermis suberization.

Concluding Remarks
This review examined the relationship between ABA biosynthesis and transport, with a focus on
water stress. First, we discussed the possible sites of ABA biosynthesis in relation to stomatal
regulation. Leaf vascular tissue is an active site of ABA biosynthesis in response to water stress;
however, guard cells also serve as a source of ABA. ABA synthesized in roots could also induce
stomatal closure. Overall, ABA biosynthesis is not restricted to one site; however, it should be
noted that the contribution of ABA synthesized at different sites to stomatal closure sometimes
differs among reports, possibly due to differences in plant materials and/or experimental
conditions. To understand how ABA derived from multiple sources coordinately regulates
stomatal closure, the molecular mechanisms by which plants sense water deficit at different
sites under various conditions (e.g., rapid, gradual, temporal, or successive water deficit in soil
and/or atmosphere) must be clarified and the largely unknown signal transduction pathways
upstream of ABA biosynthesis must be identified. At least four ABA transporters involved in
stomatal regulation have been identified. Interestingly, both ABA exporters and importers are
expressed in vascular tissues and guard cells, indicating that ABA can be transported bidirec-
tionally across the plasma membrane and that ABA transport does not follow a simple one-way
route. Since plants tolerate water deficit under the control of complex regulatory networks,
understanding the physiological processes regulated by ABA in different organs, tissues, and
cells will help create an overview of plant water stress responses. ABA is likely to regulate
stomatal closure within and outside guard cells through combinational mechanisms. Further-
more, ABA functions are related not only to stomatal closure but more completely to water
penetration and hydraulic control, to systemically cope with water stress. More detailed
analyses of the spatiotemporal expression/localization of ABA transporters, as well as ABA
biosynthesis and catabolism enzymes are required to determine the dynamics of ABA transport

520 Trends in Plant Science, June 2018, Vol. 23, No. 6

within plants. For example, we do not know the exact route of ABA transport in vascular tissue
and guard cells. Some transporters of the plant hormone auxin and nutrients are distributed
unevenly within cells. Thus, it would be interesting to determine whether polar distribution of
ABA transporters within specific cell types can be observed. In addition, the visualization of ABA
by recently developed systems [64,65] could be used to contribute to monitoring ABA
movement within plants. Given that the phenotypes of mutants defective in the ABA trans-
porters identified to date are relatively mild compared with typical severely ABA-deficient
mutants, the identification of additional transporters is required to better understand the highly
redundant ABA transport system (see Outstanding Questions).

Acknowledgments
The preparation of this review was supported by JSPS KAKENHI Grant Number 17K07458 (to T.K.).

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 MINI REVIEW ARTICLE
published: 13 March 2014
doi: 10.3389/fpls.2014.00086

Response of plants to water stress

Yuriko Osakabe1 *, Keishi Osakabe 2 , Kazuo Shinozaki 1 and Lam-Son P. Tran 3 *
1
Gene Discovery Research Group, RIKEN Center for Sustainable Resource Science, Tsukuba, Japan
2
Center for Collaboration among Agriculture, Industry and Commerce, The University of Tokushima, Tokushima, Japan
3
Signaling Pathway Research Unit, RIKEN Center for Sustainable Resource Science, Yokohoma, Japan

Edited by: Water stress adversely impacts many aspects of the physiology of plants, especially
Nobuyuki Uozumi, Tohoku University, photosynthetic capacity. If the stress is prolonged, plant growth, and productivity
Japan
are severely diminished. Plants have evolved complex physiological and biochemical
Reviewed by:
Christa Testerink, University of
adaptations to adjust and adapt to a variety of environmental stresses. The molecular and
Amsterdam, Netherlands physiological mechanisms associated with water-stress tolerance and water-use efficiency
Vasileios Fotopoulos, Cyprus have been extensively studied. The systems that regulate plant adaptation to water stress
University of Technology, Cyprus through a sophisticated regulatory network are the subject of the current review. Molecular
*Correspondence: mechanisms that plants use to increase stress tolerance, maintain appropriate hormone
Yuriko Osakabe, Gene Discovery
Research Group, RIKEN Center for
homeostasis and responses and prevent excess light damage, are also discussed. An
Sustainable Resource Science, 3-1-1 understanding of how these systems are regulated and ameliorate the impact of water
Koyadai, Tsukuba, Ibaraki 305-0074, stress on plant productivity will provide the information needed to improve plant stress
Japan tolerance using biotechnology, while maintaining the yield and quality of crops.
e-mail: yuriko.osakabe@riken.jp;
Lam-Son P. Tran, Signaling Pathway Keywords: abiotic stress, biomass, drought stress, photosynthesis, reactive oxygen species, stomatal closure
Research Unit, RIKEN Center for
Sustainable Resource Science,
1-7-22 Suehiro-cho, Tsurumi,
Yokohama 230-0045, Japan
e-mail: son.tran@riken.jp

INTRODUCTION factors (TFs) involved in the regulation of stomatal responses,

Plant growth and productivity are adversely affected by water
stress. Therefore, the development of plants with increased sur-
vivability and growth during water stress is a major objective
in the breeding crops. Water use efficiency (WUE), a parame-
ter of crop quality and performance under water deficit is an
important selection trait. In fact, plants have evolved various
molecular mechanisms to reduce their consumption of resources
and adjust their growth to adapt to adverse environmental con-
ditions (Yamaguchi-Shinozaki and Shinozaki, 2006; Ahuja et al.,
2010; Skirycz and Inze, 2010; Osakabe et al., 2011; Nishiyama et al.,
2013; Ha et al., 2014).
Plant growth is anchored by photosynthesis; however, excess
light (EL) can cause severe damage to plants. EL induces pho-
tooxidation, which results in the increased production of highly
reactive oxygen intermediates that negatively affect biological
molecules and, if severe, a significant decrease in plant produc-
tivity (Li et al., 2009). Water stress that induces a decrease in leaf
water potential and in stomatal opening (Figure 1), leading to
the down-regulation of photosynthesis-related genes and reduced
availability of CO2 , has been known as one of the major factors in
the EL stress (Osakabe and Osakabe, 2012).
Various molecular networks, including signal transduction,
are involved in stress responses (Osakabe et al., 2011, 2013b;
Nishiyama et al., 2013). The elucidation of these networks is essen-
tial to improve the stress tolerance of crops. In this review, plant
responses to water stress are summarized, revealing that they are
controlled by complex regulatory events mediated by abscisic acid
(ABA) signaling, ion transport, and the activities of transcription
all of which are integrated into orchestrated molecular networks,
enabling plants to adapt and survive. Furthermore, recent findings
on molecular mechanisms involved in protecting photosynthesis
in order to adjust plant growth during water stress are discussed.
STOMATAL SIGNALING DURING WATER STRESS
MEMBRANE TRANSPORT AND ABA SIGNALING IN STOMATAL
RESPONSES
Stomatal activity, which is affected by environmental stresses,
can influence CO2 absorption and thus impact photosynthesis
and plant growth. In response to a water deficit stress, ion- and
water-transport systems across membranes function to control
turgor pressure changes in guard cells and stimulate stomatal
closure. Endogenous ABA is rapidly produced during drought,
triggering a cascade of physiological responses, including stomatal
closure, which is regulated by a signal transduction network. 9-cis-
epoxycarotenoid dioxygenase 3 (NCED3) in Arabidopsis catalyzes
a key step in ABA biosynthesis, and NCED3 expression is rapidly
induced by drought stress in a vascular tissue-specific manner
(Iuchi et al., 2001; Endo et al., 2008; Behnam et al., 2013; Figure 2).
Mutations in nced3 reduced, while the overexpression of NCED3
enhanced drought tolerance and/or increased WUE in several
plant species (Iuchi et al., 2001; Tung et al., 2008). During drought
stress, the accumulated ABA in the vascular tissue is transported
to guard cells via passive diffusion in response to pH changes
and by specific transporters. Two members of the membrane-
localized ABC transporter family, ABCG25 and ABCG40, and one
member from a nitrate transporter family, AIT1/NRT1.2/NPF4.6,

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Osakabe et al.
FIGURE 1 | Illustration of the response of plants to water stress.
Stomatal response, ROS scavenging, metabolic changes, and
photosynthesis are all affected when plants are subjected to water stress.
These collective responses lead to an adjustment in the growth rate of
plants as an adaptive response for survival.
have been independently isolated from Arabidopsis and reported
as ABA transporters (Kang et al., 2010; Kuromori et al., 2010;
Kanno et al., 2012; Figure 2). ABCG25 has a role in ABA export,
whereas ABCG40 and AIT1 are involved in the import of ABA.
ABA-induced stomatal closure and gene expression are reduced
in the atabcg40 mutation, resulting in reduced drought tolerance
(Kang et al., 2010). These data indicate that the ABA transport sys-
tem plays a significant role in water deficit tolerance and growth
adjustment. Transcription of ABCG25 was induced by ABA and
drought stress, and exhibited vascular tissue-specificity (Kuromori
et al., 2010). In contrast, ABCG40 was expressed in guard cells
(Kang et al., 2010), suggesting the possibility that the ABA syn-
thesized in the vasculature during drought stress can be imported
into the guard cells via these transporters. The expression pattern
of AIT1/NRT1.2/NPF4.6 was similar to ABCG25 and also showed
vascular tissue-specificity (Kanno et al., 2012). This finding sug-
gests that ABA import systems in vascular tissues may also play an
important role in the regulation of water stress responses.
In response to drought stress, ABA stimulates a signaling path-
way that triggers the production of reactive oxygen species (ROS),
which in turn induces an increase in cytosolic Ca2+ . Subsequently,
two distinct types of anion channels, a slow-activating sustained
(S-type), and a rapid-transient (R-type), are activated and the
anion efflux results in a depolarization of the plasma membrane.
This leads to a decrease in the inward K+ channels (KAT1/KAT2)
and H+ -ATPase, which are involved in stomatal opening, and
the activation of outward K+ channels, including GUARD CELL
OUTWARD RECTIFYING K+ CHANNEL (GORK) that has a
Frontiers in Plant Science | Plant Physiology
Plant water stress response
role in K+ efflux. The anion and K+ efflux from guard cells
results in a reduction of guard cell turgor which causes stom-
atal closure (Schroeder and Hagiwara, 1989; Pei et al., 1997; Kwak
et al., 2003; Negi et al., 2008; Vahisalu et al., 2008). SLAC1 (SLOW
ANION CHANNEL-ASSOCIATED 1) functions as a major S-type
anion channel in guard cells (Negi et al., 2008; Vahisalu et al.,
2008), and is activated directly by a Snf1-related protein kinase
2 (SRK2E/OST1/SnRK2.6). This kinase is involved in the ABA-
signaling complex of the ABA receptor, PYR family and PP2Cs
(Geiger et al., 2009; Lee et al., 2009). S-type anion channels are
also activated by the calcium-dependent protein kinases CPK3,
CPK6, CPK21, and CPK23 (Geiger et al., 2010; Brandt et al., 2012).
KAT1 has also been shown to be a direct target of regulation by
ABA, since its activity is directly inhibited via phosphorylation by
an ABA-activated SRK2E (Sato et al., 2009). Recently, the activity
of KUP6, a KUP/HAK/KT family K+ transporter, has also been
shown to be involved in the direct regulation during drought stress
via phosphorylation by an ABA-activated SRK2E (Osakabe et al.,
2013a). These results suggest that the complicated, but direct, con-
trol of ion transport systems by ABA may play an important role
in stomatal responses that impact the tolerance of plants to water
stress and influence plant growth (Figure 2).
TRANSCRIPTION FACTORS
The expression of various genes with functions in the water deficit
responses, are specifically induced during the stress. Transcrip-
tomic and proteomic analyses in various species have identified
the involvement of general physiological processes associated
with drought-responsive gene expression (Molina et al., 2008;
Aprile et al., 2009; Walia et al., 2009; Abebe et al., 2010; Dugas
et al., 2011; Jogaiah et al., 2012; Le et al., 2012; Utsumi et al.,
2012). These studies have identified the conserved, as well
as, species-specific regulatory and functional drought-responsive
genes, including osmoprotectants and ABA biosynthesis, late
embryogenesis abundant (LEA) and chaperone, ROS-related, ion
homeostasis, and signaling genes. Additionally, key TFs reg-
ulating drought-responsive gene transcription have also been
identified, such as MYB, MYC, DREB/CBF (drought-responsive
cis-element binding protein/C-repeat-binding factor), ABF/AREB,
NAC, and WRKY TFs (Stockinger et al., 1997; Sakuma et al.,
2006; Tran et al., 2007b; Nakashima et al., 2009; Ishida et al.,
2012; Figure 2). Corresponding cis-motifs, DRE/CRT and ABRE
(ABA-responsive cis-element), have also been discovered in the
promoters of many stress-responsive genes (Yamaguchi-Shinozaki
and Shinozaki, 2006).
ABA-responsive cis-element-mediated transcription via ABF/
AREB is directly regulated by an ABA receptor complex involving
SnRK2 that activate ABF/AREBs by phosphorylation (Umezawa
et al., 2010). The action of SnRK2 represents one of the impor-
tant mechanisms regulating the rapid, adaptive response of plants
to drought. DREB and AREB activate the transcription of var-
ious genes that are expressed in variety tissues. Additionally,
novel types of TFs, with critical functions in stomatal responses,
have also been identified. DST (drought and salt tolerance),
a C2 H2 -type TF, controls the expression of genes involved in
H2 O2 homeostasis, and mediates ROS-induced stomatal clo-
sure and abiotic stress tolerance in rice (Huang et al., 2009).
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Osakabe et al.
FIGURE 2 | Model for the role of signaling factors in stomatal closure and retrograde signaling during water stress.
Drought-inducible nuclear TF, NFYA5, was reported to control
stomatal aperture and play a role in drought tolerance in Ara-
bidopsis (Li et al., 2008). SNAC1 (STRESS-RESPONSIVE NAC1)
is expressed in rice guard cells, and overexpression of this gene
enhanced ABA sensitivity, stomatal closure, and both DST in rice
(Hu et al., 2006). AtMYB60 and AtMYB61 are expressed mainly in
guard cells, and important TFs regulating stomatal aperture and
drought tolerance in plants (Cominelli et al., 2005). AtMYB60 is
a negative regulator of stomatal closure (Cominelli et al., 2005;
Liang et al., 2005). Further studies to determine the molecu-
lar targets and signaling systems associated with these TFs in
stomatal responses will increase our understanding of the regu-
latory networks controlling plant drought responses and growth
adjustment.
EARLY WATER STRESS RESPONSE AND SIGNAL
TRANSDUCTION PATHWAYS
Receptor and sensor proteins localized to membranes play impor-
tant roles in various signaling pathways, conveying information
to their cytoplasmic target proteins via catalytic processes, such as
phosphorylation. Plasma membrane signaling has been hypoth-
esized to be involved in the initial process of water status
perception outside the cell (Maathuis, 2013). AHK1, an Ara-
bidopsis histidine kinase (HK) localized to the plasma membrane
www.frontiersin.org
Plant water stress response
mediates osmotic-stress signaling in prokaryotes and has been
shown to function as an osmosensor. Overexpression of AHK1
enhanced drought tolerance in Arabidopsis (Urao et al., 1999; Tran
et al., 2007a). ahk1 mutants exhibited decreased sensitivity to
ABA and the downregulation of ABA- and/or stress-responsive
genes, indicating that AHK1 acts as an osmosensor and func-
tions as a positive regulator of osmotic-stress signaling (Tran
et al., 2007a; Wohlbach et al., 2008). Downstream AHK1 cascades
appear to be controlled by AHPs and ARRs as part of a mul-
tiple His-Asp phosphorelay. However, the factors that receive
signals from AHK1, and also the precise composition of the
signaling cascades, remain to be determined. In contrast, in
Arabidopsis, the cytokinin (CK) receptor HKs, AHK2, AHK3,
and AHK4, have been shown to negatively regulate ABA and
drought signaling (Tran et al., 2007a, 2010). Multiple mutants of
ahk2, ahk3, and ahk4 display increased sensitivity to ABA and
enhanced tolerance to drought (Tran et al., 2007a; Jeon et al.,
2010). These findings indicate the existence of crosstalk among
ABA, CK, and stress-signaling pathways (Nishiyama et al., 2011;
Ha et al., 2012).
In Arabidopsis, the receptor-like kinase (RLK) family includes
more than 600 members, with the leucine rich-repeat (LRR)-RLKs
constituting the largest subgroup (Gish and Clark, 2011). Sev-
eral RLKs localized to the plasma membrane are known to be
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Osakabe et al.
involved in the early steps of osmotic-stress signaling in a variety
of plant species (Osakabe et al., 2013b). These stress-related RLKs
possess a number of different extracellular domains (e.g., LRR,
an extensin-like domain, or a cysteine-rich domain; Bai et al.,
2009; de Lorenzo et al., 2009; Osakabe et al., 2010a; Yang et al.,
2010; Tanaka et al., 2012), indicating that different environmen-
tal stimuli may activate RLK-mediated signaling pathways and
convey the osmotic conditions outside of the cells. RLKs that
bind to cell-walls, such as cell wall-associated kinases (WAKs),
the proline-rich extensin-like receptor kinase (PERKs; Osakabe
et al., 2013b), and the CrRLKs (Catharanthus roseus RLK1-like
family; Schulze-Muth et al., 1996) have recently been predicted to
be involved in the perception of turgor pressure (Steinwand and
Kieber, 2010; Christmann et al., 2013). A potential link between
the RLKs in cell-wall binding, ABA biosynthesis and water stress
response could be determined by analyzing their roles in signal-
ing systems associated with specific mechanosensing pathways
activated in response to water stress. This would shed light on
the early signaling system controlling water stress tolerance and
growth adjustment.
PROTECTING PHOTOSYNTHESIS DURING WATER STRESS
Water stress directly affects rates of photosynthesis due to the
decreased CO2 availability resulted from stomatal closure (Flexas
et al., 2006; Chaves et al., 2009), and/or from changes in pho-
tosynthetic metabolism (Lawlor, 2002). EL has a negative effect
on photosynthesis when the rates of photosynthesis are reduced
by water stress (Li et al., 2009; Osakabe and Osakabe, 2012). A
strong interconnection between the responses to EL and drought
stresses has been suggested, and around 70% genes induced
by EL are also induced by drought (Kimura et al., 2002; Chan
et al., 2010; Estavillo et al., 2011). EL also stimulates the pro-
duction of ROS, such as H2 O2 , superoxide (O2 − ) and singlet
oxygen (1 O2 ), by specific photochemical and biochemical pro-
cesses, which also exerts deleterious effects on photosynthesis
(Li et al., 2009). H2 O2 induces the up-regulation of a vari-
ety of genes that overlap with genes up-regulated by various
chemical and environmental stresses, such as methyl viologen,
heat, cold, and drought (Vandenabeele et al., 2004; Vander-
auwera et al., 2005). The transcription of cytosolic ascorbate
peroxidase encoding genes (APXs), which have important roles
in the scavenging of cytosolic H2 O2 , responds positively to
EL stress and the redox state of plastoquinone (PQ; Karpin-
ski et al., 1997). APX loss-of-function mutants exhibited an
accumulation of degraded chloroplast proteins, indicating that
APXs play a protective role as ROS scavengers for chloroplast
proteins under EL conditions (Davletova et al., 2005; Li et al.,
2009). AtAPX2 was also induced by drought stress and ABA
(Rossel et al., 2006), suggesting that APX mediates ROS scaveng-
ing in response to both EL and water stress. A gain-of-function
mutant, altered apx2 expression 8 (alx8), which has consti-
tutively higher levels of APX2 expression, exhibited improved
WUE and drought tolerance (Rossel et al., 2006; Wilson et al.,
2009; Estavillo et al., 2011). In Arabidopsis, the zinc-finger TFs,
ZAT10 and ZAT12, are induced in plants acclimated to EL or
ROS treatment. The overexpression of ZAT10 and ZAT12 highly
induced expression of various stress-related genes, including
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Plant water stress response
APXs (Rizhsky et al., 2004; Davletova et al., 2005; Rossel et al.,
2007). Several transgenic lines that overexpressed ZAT10 exhib-
ited enhanced drought stress tolerance (Sakamoto et al., 2004).
ZAT10 and ZAT12 regulate the responses to EL and drought
stresses, which are mediated by ROS (Davletova et al., 2005;
Mittler et al., 2006), suggesting their potential roles in pro-
tecting photosynthesis from the injury during water stress
(Figure 2).
Plants can monitor chloroplast status by plastid-to-nucleus
signals, as plastid-to-nucleus retrograde signaling. This signal-
ing system can regulate the expression of genes that function in
the chloroplast. The retrograde signaling plays an important role
in regulating the chloroplastic processes and also in the adaptive
responses to environmental stresses (Chan et al., 2010). Chloro-
phyll intermediates, such as Mg-protoporphyrin IX (Mg-Proto),
control the expression of nuclear genes in plants exposed to EL
conditions, acting as a retrograde signal. The genomes uncou-
pled (gun) mutants, gun4 and gun5, exhibit impaired generation
of Mg-Proto that has been shown to act as a signal to repress
LHCB gene expression in Arabidopsis (Mochizuki et al., 2001;
Strand et al., 2003; Pontier et al., 2007). LHCB expression is also
controlled by GUN1 and ABI4 (ABSCISIC ACID-INSENSITIVE
4) that encodes a TF involved in ABA signaling (Koussevitzky
et al., 2007). Collectively, these factors are thought to be involved
in multiple retrograde signaling pathways. Moulin et al. (2008)
re-examined the proposed role of Mg-Proto and other chloro-
phyll intermediates as signaling molecules and reported that none
of the intermediates could be detected in ROS-induced plants
under conditions where nuclear gene expression was repressed.
The authors hypothesized that Mg-Proto (which accumulates in a
light-dependent manner) is extremely short-lived and may gener-
ate 1 O2 under EL conditions; however, a much more complex ROS
signal may be generated during chloroplast degradation. There is
increasing evidence for the regulation of nuclear gene expression
by 1 O2 (op den Camp et al., 2003) and H2 O2 (Kimura et al., 2003).
However, a clear role for these ROS molecules, either individually
or in combination, requires further investigation.
Recently, several novel retrograde signaling pathways have
been identified, including the 3 -phosphoadenosine 5 -phosphate
(PAP) pathway, which is regulated by SAL1/ALX8/FRY1, and the
methylerythritol cyclodiphosphate (MEcPP) pathway (Estavillo
et al., 2011; Xiao et al., 2012). PAP has been described as a chloro-
plast to nuclear mobile signal that regulates gene expression.
ALX8 encodes a phosphatase that converts PAP to AMP and
regulates PAP levels (Wilson et al., 2009; Estavillo et al., 2011).
alx8 mutant exhibited drought-tolerant phenotypes and consti-
tutive upregulation of approximately 25% of the EL-regulated
transcriptome, suggesting that SAL1/ALX8/FRY1 can act as a
component of both EL and drought signaling networks, and
that the SAL1-PAP retrograde pathway can alter nuclear gene
expression during EL and drought (Rossel et al., 2006; Wilson
et al., 2009; Estavillo et al., 2011; Figure 2). MEcPP is a precur-
sor of isoprenoids generated by the methylerythritol phosphate
(MEP) pathway, and can induce expression of nuclear encoded
stress-responsive genes (Xiao et al., 2012). MEcPP is induced by
various abiotic stresses, such as high light and wounding, and
has been proposed to act as a retrograde signal in response to
March 2014 | Volume 5 | Article 86 | 4
Osakabe et al.
these stresses (Xiao et al., 2012). Evidence from the above stud-
ies suggests that metabolite signals, whose levels are influenced
by environmental conditions, are used to establish an interaction
between plastids and the nucleus and regulate chloroplast function
to adjust plant growth in response to various stresses, including
drought.
CONCLUSION AND FUTURE PERSPECTIVE
Due to the sessile life cycle, plants have evolved mechanisms to
respond and adapt to adverse environmental stresses during their
development and growth. Plant growth is impaired by severe
drought stress due to a decrease in stomatal opening, which limits
CO2 uptake and hence reduces photosynthetic activity. In order to
develop strategies to maintain plant productivity, it is essential to
understand the various regulatory mechanisms that control and
enhance adaptive responses to stress in different plant species. In
this review, we focused on the molecular mechanisms involved in
the plant responses to water stress and the concomitant growth
adjustment. These mechanisms include stomatal responses, ion
transport, activation of stress signaling pathways, and responses
to protect photosynthesis from injury. Understanding these key
factors will enable us to improve plant productivity during water
stress.
In parallel with the identification of the key molecular factors
involved in these mechanisms, new technologies to bioengineer
superior plants will also enable the development of plants with
improved plant productivity. Although transgenic approaches
have been effectively used to develop plant genotypes with
improved stress tolerance under field conditions, estimation of
the desired effects and their stability over many generations is
required. Mutagenesis has also been used in plant breeding for
a long time to create genetic variation; however, it takes con-
siderable resources and effort to generate genotypes with the
desired phenotype due to the random nature of the introduc-
tion of mutations. Recently, genome editing technology has
made remarkable advances in the ability to modify the genome
in a site-specific manner. Genome editing technology utilizes
custom-designed restriction endonucleases, such as zinc finger
nucleases (ZFN) or TAL-effector nucleases (TALEN; Shukla et al.,
2009; Osakabe et al., 2010b; Zhang et al., 2010; Cermak et al.,
2011), and more recently, the CRISPR/CAS system (Li et al., 2013;
Nekrasov et al., 2013; Shan et al., 2013). Utilization of this tech-
nology will make it possible to modify the regulation of key
genes that will convey improved stress tolerance while maintain-
ing productivity. Further studies using new molecular approaches,
including the identification of gene variants associated with the
significant agronomic traits, will facilitate the molecular engineer-
ing of plants with increased tolerance to severe environmental
stresses.
ACKNOWLEDGMENTS
This work was supported by the Programme for Promotion
of Basic and Applied Researches for Innovations in the Bio-
Oriented Industry of Japan (Yuriko Osakabe and Kazuo Shi-
nozaki). Research in Lam-Son P. Tran’s lab was supported by
the Grant (No. AP24-1-0076) from RIKEN Strategic Research
Program for R & D.
www.frontiersin.org
Plant water stress response
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Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Received: 27 November 2013; accepted: 24 February; published online: 13 March 2014.
Frontiers in Plant Science | Plant Physiology
Plant water stress response
Citation: Osakabe Y, Osakabe K, Shinozaki K and Tran L-SP (2014) Response of plants
to water stress. Front. Plant Sci. 5:86. doi: 10.3389/fpls.2014.00086
This article was submitted to Plant Physiology, a section of the journal Frontiers in
Plant Science.
Copyright © 2014 Osakabe, Osakabe, Shinozaki and Tran. This is an open-access article
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 BIOLOGIA PLANTARUM 52 (3): 401-412, 2008

REVIEW

Physiology and biochemistry of waterlogging tolerance in plants

R.K. SAIRAM*, D. KUMUTHA, K. EZHILMATHI, P.S. DESHMUKH and G.C. SRIVASTAVA

Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110012, India

Abstract

Waterlogging is a serious problem, which affects crop growth and yield in low lying rainfed areas. The main cause of
damage under waterlogging is oxygen deprivation, which affect nutrient and water uptake, so the plants show wilting
even when surrounded by excess of water. Lack of oxygen shift the energy metabolism from aerobic mode to anaerobic
mode. Plants adapted to waterlogged conditions, have mechanisms to cope with this stress such as aerenchyma
formation, increased availability of soluble sugars, greater activity of glycolytic pathway and fermentation enzymes and
involvement of antioxidant defence mechanism to cope with the post hypoxia/anoxia oxidative stress. Gaseous plant
hormone ethylene plays an important role in modifying plant response to oxygen deficiency. It has been reported to
induce genes of enzymes associated with aerenchyma formation, glycolysis and fermentation pathway. Besides, non-
symbiotic-haemoglobins and nitric oxide have also been suggested as an alternative to fermentation for maintaining
lower redox potential (low NADH/NAD ratio), and thereby playing an important role in anaerobic stress tolerance and
signaling.
Additional key words: anoxia, antioxidative enzymes, ethylene, fermentation, flooding, glycolysis, hypoxia, nitric oxide, non-
symbiotic haemoglobin, oxidative stress, sugars.

Introduction

Although all higher plants require access to free water,
excess water in the root environment of land plants can
be injurious or even lethal because it blocks the transfer
of oxygen and other gases between the soil and the
atmosphere. Crop plants require a free exchange of
atmospheric gases for photosynthesis and respiration.
Like animals, plants can be easily suffocated if this gas
exchange is impeded. Oxygen status of cells and tissues
varies significantly during ontogenesis, and depends on
environmental oxygen supply. The most common
impediment to gas diffusion is water that saturates the
root environment in poorly drained soils or that
accumulates above soil capacity as a result of the
overflow of rivers, excessive rainfall or excessive
irrigation. Flooding and submergence are major abiotic
stresses and rank alongside water shortage, salinity and
extreme temperatures as major determinants of species
distribution worldwide. During waterlogging or
submergence plants are exposed to a reduction in oxygen
supply because of the slow diffusion rate of oxygen in
water and its limited solubility (Armstrong 1978). Turbid
flood-water can become anaerobic, especially during the
night (Setter et al. 1987). Growth is greatly inhibited in
the deficiency (hypoxia) or complete absence (anoxia) of
oxygen. In water-saturated soils roots grow only in a
small region near the surface and do not exploit a large
soil volume as they would under aerated conditions.
Plants invariably wilt within few hours to 2 - 4 d of
imposing a flooding stress (Jackson and Drew 1984).
This is a consequence of higher resistance to mass flow
of water through the root. Wilting is caused by the
inhibition of respiration and loss of ATP synthesis in the
roots. This blocks the ion transport systems that normally
create the gradient in water potential across the root
endodermis. Plants react to an absence of oxygen by
switching from an oxidative to a solely substrate-level

⎯⎯⎯⎯

Received 5 November 2007, accepted 3 May 2008.

Abbreviations: AA - ascorbate; ACC - 1-aminocyclopropane-1-carboxylic acid; ADH - alcohol dehydrogenase; AEC - adenylate
energy charge; ANP - anaerobic protein; APX - ascorbate peroxidase; CAT - catalase; DHAR - dihydroascorbate peroxidase;
GR - glutathione reductase; GSH - glutathione; Hb - haemoglobin; cNR - cytosolic nitrate reductase; LDH - lactate dehydrogenase;
MDHAR - monodihzdroascorbate peroxidase; PM-NR - plasma membrane nitrate reductase; PM-Ni-NOR - plasma membrane nitrite
nitric oxide reductase; NAD(P)H - nicotine amide adenine dinucleotide phosphate-reduced; NiR - nitrite reductase; NO - nitric oxide;
PDC - pyruvate decarboxylase; SOD - superoxide dismutase; SuSy - sucrose synthase; XET - xyloglucan endo-trans-glucosylase
* Corresponding author; e-mail: rks_ppl@yahoo.co.uk

401
R.K. SAIRAM et al.
phosphorylation of ADP to ATP, the latter reactions
predominantly involve glycolysis and fermentation. An
important adaptive response is formation of aerenchyma,
specialized tissues in roots, which allow diffusion of
gases like O2 from aerobic shoot to hypoxic/anoxic roots.
It has been suggested that physiological and molecular
studies of the mechanisms of anoxia tolerance in wild
Hypoxia and anoxia
Normally plant roots are in contact with oxygen at a
partial pressure equivalent to the gaseous atmosphere.
The reduction of oxygen below optimal levels, termed
hypoxia, is the most common form of stress in wet soils
and occurs during short-term flooding when the roots are
submerged under water but the shoot remains in the
atmosphere. Hypoxia will also occur in roots near the
surface of longer-term flood water. The complete lack of
oxygen, termed anoxia, occurs in soils that experience
Flooding and ethylene production
Many of the adaptive growth response in hypoxic roots
and shoots occur in response to ethylene. Ethylene
accumulates in flooded soils and in submerged plant parts
to concentrations of 10 cm3 dm-3 (Musgrave et al. 1972).
This is accomplished by two mechanisms. First the
diffusion of ethylene from the root into the water is
10 times slower than its diffusion into air (Stünzi and
Kende 1989). This ethylene may be released into the
internal aerenchyma channels and diffuse from the root to
the shoot. Secondly, the synthesis of ethylene in the
hypoxic root and in the aerobic shoot is increased
(Jackson 1985). The immediate precursor of ethylene is
1-amino cyclopropane 1-carboxylic acid (ACC), which is
synthesized to a large extent in roots (Bradford and Yang
1980). Because the conversion of ACC to ethylene has an
obligate requirement for oxygen, this reaction is blocked
in an anaerobic root cell. The ACC is therefore,
translocated from the anaerobic root cells towards the
more aerobic portions of the root or to the shoot. The
previous biochemical steps in the synthesis of ACC do
not require oxygen, and in fact, ACC synthase activity is
stimulated in roots under flooding conditions (Cohen and
Kende 1987). As a consequence the quantity of ACC
transported to the shoot increases. The lower portions of
the stems are usually the site of highest ACC
accumulation and in the presence of oxygen ethylene is
released.
Van Der Straeten et al. (2001) isolated a genomic
Ethylene and aerenchyma formation
Ethylene initiates and regulates many adaptive molecular,
chemical and morphological responses that allow the
plant to avoid anaerobiosis by increasing oxygen
402
plants that still possess long-term anoxia-tolerance are
more likely to provide evidence of physiological
mechanisms than studies of crop plants (Crawford and
Braendle 1996). Therefore, reference is made to some of
these studies, but the emphasis of the following is on crop
plants.
long-term flooding, in plants completely submerged by
water, in deep roots below flood waters. Long-term
flooding shifts the microbial flora in the soil in favour of
anaerobic micro-organisms that use alternative electron
acceptors to oxygen. As a consequence the soil tends to
accumulate more reduced and phytotoxic forms of
mineral ions such as nitrite (from nitrate) and ferrous
(from ferric) ions and few plants are adapted to growth in
these soils (Ponnamperuma 1972).
cDNA clone (OS-ACS5) encoding ACC synthase from
rice, which catalyzes a regulatory step in ethylene
biosynthesis upon short- and long-term submergence.
Rieu et al. (2005) reported cloning of a Rumex palustris
cDNA corresponding to an ACC synthase gene
(RP-ACS1), whose expression is induced by long term
submergence. Vriezen et al. (1999) suggested a more
prominent role of ACC oxidase in ethylene synthesis and
anoxia tolerance during flooding. They isolated a cDNAs
from R. palustris corresponding to a ACC oxidase gene
(RP-ACO1) in submerged Rumex palustris. An increase
in RP-ACO1 mRNA was observed 2 h after the start of
submergence, and ACC oxidase enzyme activity was
highest after 24 h. It was earlier reported by these workers
that the ethylene production rate of submerged shoots
does not increase significantly during the first 24 h of
submergence (Voesenek et al. 1993), suggesting that
under these conditions ACC oxidase activity is inhibited
in vivo. They suggested that this inhibition is caused by a
reduction of oxygen levels, and hypothesized that an
increased ACC oxidase enzyme concentration
counterbalances the reduced enzyme activity caused by
low oxygen concentration during submergence, thus
sustaining ethylene production under these conditions.
Therefore, ethylene biosynthesis seems to be limited at
the level of ACC oxidase activity rather than by ACC
synthase in R. palustris during submergence (Voesenek
et al. 1993).
availability to the roots in a flooded or waterlogged soil,
such as development of aerenchyma. Aerenchyma are
soft tissues with large intercellular spaces to provide low

resistance internal pathway for the exchange of gases
between aerobic shoot to the anaerobic root (Jackson and
Armstrong 1999).
Aerenchyma formation under waterlogged condition
have been reported in a wide range of crop species such
as, Trifolium subterraneum (Aschi-Smiti et al. 2004),
soybean (Bacanamwo and Purcell 1999), wheat (Watkin
et al. 1998), barley (Arikado and Adichi 1955), rice
(Justin and Armstrong 1991), maize (Gunawardena et al.
2001), Carex spp. (Visser et al. 2000) and Vigna luteola
(Sairam et al. - unpublished).
Some of the oxygen transported through the
aerenchyma to plant root tips leaks out of pores in the
root and into the surrounding soil. This can result in a
small zone of oxygenated soil around individual roots
providing an aerobic environment for microorganisms
that can prevent the influx of potentially toxic soil
components (Armstrong and Armstrong 1988) such as
nitrites and sulphides of Fe, Cu and Mn.
Ethylene synthesis was strongly enhanced in roots
under hypoxia, coincident with the development of
aerenchyma (Visser et al. 1997). Aerenchyma formation
in maize roots was also stimulated by exogenous
application of ethylene at rates as low as 0.1 cm3 dm-3
and was inhibited in the presence of Ag+ ions, an
inhibitor of ethylene action (Drew et al. 1981). In the
Ethylene accumulation and adventitious roots formation
Adventitious roots emerge from the submerged part of
the stem in flooded plants and grow horizontally
(diageotropism). Presumably, this is also an adaptive
mechanism allowing these new roots to replace the
function of the original root system (Jackson and Drew
1984). Since these roots emerge and grow close to the
water surface, and since they are connected to the stem
close to the site of aerenchyma formation, oxygen is more
available to these roots than the original root system. The
flood tolerant Rumex species R. crispus and R. palustris
developed new flood-resistant roots in the upper 10 cm of
the waterlogged soil, whereas the flood susceptible
R. acetosa did not change its vertical root distribution
(Voesenek et al. 1989).
Visser et al. (1996) reported that accumulation of
ethylene has a role in the formation of flooding-induced
adventitious roots formation. The large air-spaces in these
roots enable diffusion of gases between shoots and roots.
Application of ethylene to non-flooded Rumex plants
resulted in the formation of adventitious roots. Inhibition
of ethylene production in R. palustris by L-α-(2-amino-
ethoxyvinyl)-glycine (AVG) or α-iminobutyric acid
(AIB) decreased the number of adventitious roots induced
Shift in energy metabolism and anaerobiosis induced proteins (ANP)
The anaerobic response of plant cells was first studied in
maize roots. Using two-dimensional electrophoresis, it
WATERLOGGING TOLERANCE IN PLANTS
presence of ethylene synthesis inhibitors, flooding failed
to induce aerenchyma formation (Konings 1982). The
presence of a growing root tip is also essential for the
formation of aerenchyma. The root tip may serve as the
site of ACC synthesis, with subsequent ACC transport to
more mature, better aerated tissue leading to ethylene
synthesis and aerenchyma formation (Drew et al. 1981).
However, there is little information to date on the
molecular regulation of aerenchyma formation or the
identity of other enzymes involved. In maize, a flooding-
induced gene (xet1) encodes a xyloglucan endotrans-
glycosylase (XET1), a putative cell wall loosening
enzyme (Peschke and Sachs 1994, Saab and Sachs 1996).
O2 deprivation induces expression of XET1 in the
primary root, mesocotyl, and coleoptile of maize
seedlings. The induction of xet1 appears to be specific to
O2 deprivation, since other stresses do not induce the gene
(Peschke and Sachs 1994). The xet1 gene appears to be a
member of a large multigene family in which only xet1 is
inducible by oxygen deprivation. The induction of xet1 by
hypoxia was associated with aerenchyma development.
Hypoxic induction of XET1 mRNA is repressed by
ethylene antagonists [(aminooxy)acetic acid, 2-amino-
ethoxyvinyl-glycine, AgNO3]. XET1 is also induced
under aerobic conditions by exogenous ethylene, as is
aerenchyma.
by flooding, indicating that high ethylene concentrations
may be a prerequisite for the flooding-induced formation
of adventitious roots in Rumex species. Bragina et al.
(2003) reported in maize seedlings that hypoxia resulted
in the accelerated ethylene production and the activation
of enzymes destroying cell walls in the adventitious roots
and changed their growth pattern. The conclusion is that
the interrelated responses are adaptive ones and the
adventitious roots play a key role in plant adaptation
Mergemann and Sauter (2000) reported that in deep-
water rice, adventitious root primordia initiate at the
nodes as part of normal development but emergence of
the root is dependent on flooding induced ethylene
mediated death of nodal epidermal cell covering the tip of
the primordia. This facilitates adventitious root emergence
and prevents injury to the growing root. Cell death was
inducible not only by submergence but also by
application of ACC precursor of ethylene, and it was
suppressed in the presence of 2,5-norbornadiene, an
inhibitor of ethylene action. Adventitious root growth and
epidermal cell death are therefore, linked to the ethylene
signaling pathway, which is activated in response to low
oxygen stress.
was shown that a set of about 20 anaerobic proteins were
synthesized during low oxygen treatment, while synthesis
403
R.K. SAIRAM et al.
of the normal aerobic proteins was drastically repressed
(Sachs et al. 1980). Many of these induced proteins were
subsequently identified as enzymes of the glycolytic and
fermentation pathways (Dolferus et al. 2003). The
identified ANP include sucrose synthase, phosphohexose
isomerase, fructose-1,6-diphosphate aldolase, pyruvate
decarboxylase (PDC), lactate dehydrogenase (LDH), and
alcohol dehydrogenase (ADH) (Chung and Ferl 1999,
Zeng et al. 1999).
The second major effect of oxygen depletion in
flooded roots occurs because oxygen serves as the
terminal electron acceptor of mitochondrial electron
transport. Lack of oxygen thus effectively blocks ATP
synthesis in the mitochondria (Pradet and Bomsel 1978).
In the absence of an electron acceptor, NADH oxidation
is blocked. Once the mitochondrial respiration stops, the
adenylate energy charge of the cell (ratio of ATP to ADP
and AMP) declines. In the absence of an adaptive
response, the flooded root cell rapidly depletes its
available supply of ATP. One supplemental source of
ATP for the cell is accessed through a stimulation of
glycolysis and fermentation, known as the Pasteur effect.
However, glycolysis is relatively inefficient at energy
production compared to mitochondrial respiration. It also
generates large quantities of pyruvate as an end-product
that must be converted to alternative products to recycle
NADH to NAD. These end-products of glycolysis and
fermentation pathway, such as ethanol, lactic acid and
carbon dioxide pose an additional hazard to the cell.
Since many of the enzymes of the Krebs cycle are
regulated allosterically by the NADH/NAD ratio, the
entire Krebs cycle is blocked and glycolysis is stimulated
by an increase in redox potential [NAD(P)H/NAD(P)].
The pyruvate that accumulates from glycolysis is
converted initially by LDH to lactic acid. Cytoplasmic pH
consequently declines as a result of lactic acid
accumulation, a process known as cytoplasmic acidosis.
Low pH inactivates LDH, which has a pH optimum
above 7.0 and activates PDC and ADH that are normally
inactive above pH 7. Therefore, pyruvate is shunted to the
production of either ethanol or lactic acid in a pH
dependent manner that allows tight regulation of
cytoplasmic pH around 6.8.
ADH activity is critical for the recycling of NADH
and thus for the continuation of glycolytic pathway
(Johnson et al. 1994). Peng et al. (2001) demonstrated
that ADH gene induction is linked to ethylene production.
They demonstrated that hypoxic induction of ADH could
be partially inhibited by aminooxyacetic acid, an inhibitor
of ethylene biosynthesis. This partial inhibition can be
reversed by the addition of ACC. In addition, the hypoxic
induction of the ADH is also reduced in etr1-1 and ein2-1,
two ethylene insensitive mutants (Peng et al. 2001).
The importance of ADH in flooding tolerance has
been emphasized in the study of a maize mutant deficient
in one of its ADH genes, and therefore, unable to produce
a functional ADH enzyme. When this mutant was
flooded, LDH synthesized lactic acid, pH declined, but
ADH was not able to synthesize ethanol. As there was no
404
counterbalance to LDH and the pH continued to decline
to very low levels, consequently this mutant was more
sensitive to flooding injury than the wild type plant and
died after 3 d of submergence (Roberts et al. 1984a,b).
Other enzymes of glycolytic pathway are also over-
expressed under waterlogged condition and reported to
confer tolerance to hypoxic/anoxic condition. Hänsch
et al. (2003) reported maize glyceraldehyde-3-phosphate
dehydrogenase 4 (GapC4) gene expression in tobacco
(Nicotiana tabacum) and potato (Solanum tuberosum).
They showed that the gene is also anaerobically induced
in Arabidopsis thaliana roots, leaves, stems and flower
organs.
Due to shift in energy metabolism from aerobic to
anaerobic mode the energy requirements of tissue is
greatly restricted because of very few ATPs generated per
molecule of glucose. This necessitates availability of
comparatively higher amount of readily metabolizable
sugar pool. Recard et al. (1998) provided evidence for the
critical role of sucrose synthase, an enzyme involved in
sucrose hydrolysis, in anoxic tolerance of maize roots
using sucrose synthase double mutants, sh1sus1. Zeng
et al. (1999) reported in case of hypoxic maize seedlings
that of the two enzymes involved in sucrose hydrolysis,
the activity of invertase is down-regulated, while that of
sucrose synthase is up-regulated. Aschi-Smiti et al.
(2004) reported that in case of 30-d-old plants of
T. subterraneum 15 d of hypoxia showed induction of
sucrose synthase, fructose kinase, lactate dehydrogenase
and alcohol dehydrogenase, enhanced ethanol production,
and improved energy charge in association with
haemoglobin induction. Our studies in pigeon pea have
revealed that roots of comparatively tolerant (ICPL 84023
and ICP 301) genotypes have greater total, reducing and
non-reducing sugars content than susceptible genotypes
(ICP 7035 and Pusa 207). Waterlogging resulted in
decline in total and non-reducing sugars in all the
genotypes, while the content of reducing sugar increased
only in ICPL 84023 and ICP 301. The variation in
reducing sugar content was parallel to sucrose synthase
activity. The tolerant genotypes also showed lower
decline in total and non-reducing sugars and greater
increase in reducing sugar and sucrose synthase (SuSy)
activity than susceptible genotypes. Susceptible genotype
(ICP 7035) showed very little SuSy mRNA expression in
control and waterlogged condition, while the gene
expression increased significantly in tolerant genotype
under waterlogged condition (Sairam et al. - unpublished
data). The results further confirm the significance of root
sugar reserve and sucrose hydrolyzing enzyme in
waterlogging tolerance of crop plants.
Chang et al. (2000) reported expression of 48 anoxia
induced proteins in maize root tips by using two-
dimensional gel electrophoresis. Proteomic studies have
identified 46 oxygen stress induced proteins, which are
associated with processes other than sugar breakdown,
glycolysis and fermentation, suggesting that many other
biochemical and metabolic processes are modulated
during low oxygen stress. For instance, xyloglucan

endotransglycosylase, a cell wall loosening enzyme
(Sachs et al. 1996) is induced, as is the ethylene
biosynthetic enzyme ACC synthase (Olson et al. 1995),
Hypoxia and non-symbiotic haemoglobins
It is now known that there are several classes of
haemoglobins in plants. Non-symbiotic haemoglobins, as
the name implies, are not involved in symbiotic nitrogen
fixation (Hill 1998). There are two classes of
nonsymbiotic haemoglobins, one has oxygen-binding
properties similar to symbiotic haemoglobins (class 2),
the second with dramatically different oxygen-binding
properties (class 1). Class 1 haemoglobins are induced by
hypoxic stress and oversupply of some nutrients, and are
generally referred as stress-induced haemoglobin.
Expression of stress-induced haemoglobins has also
been reported in callus, cell suspension, seed, root and
stem tissue of both dicot and monocot plants (Hill 1998,
Dordas et al. 2003a, 2004). They are generally found at
low concentrations [1 - 20 nmol g-1(f.m.)] in plant organs.
In addition to their induction by hypoxic stress (Taylor
et al. 1994), these are also induced by elevated sucrose
content in Arabidopsis (Trevaskis et al. 1997), nitrate
ions in barley aleurone layer and Arabidopsis roots
(Wang et al. 2000) and have also been reported in rapidly
growing root tips of germinating seeds (Hill 1998). There
is reason to believe that their appearance in rapidly
growing tissues may be due to hypoxic stress as well
(Guy et al. 2002). Respiratory chain inhibitors (e.g.
cyanide, dinitrophenol and oligomycin) that inhibit ATP
Haemoglobin and nitric oxide interaction
While hypoxic stress-induced haemoglobins (Hb) are
widespread in the plant kingdom, their function has not
been elucidated. Dordas et al. (2003b) proposed that
nitric oxide is an important metabolite in hypoxic plant
cells and that at least one of the functions of hypoxic
stress-induced Hb is to modulate nitric oxide levels in the
cell. Dordas et al. (2003b) demonstrated the presence of
NO/haem complexes in both hypoxic maize cell cultures
and alfalfa root cultures using electron paramagnetic
resonance (EPR) spectroscopy. The characteristic signal
for the complex is not evident in aerobic systems, even
though Hb is present. Furthermore, using NO traps,
Dordas et al. (2003b) showed that significant amounts of
NO are formed in hypoxic maize cells during the first
24 h of hypoxic treatment. Transformed lines with
reduced stress-induced Hb expression produced greater
amounts of NO than wild-type or over expressing-Hb
lines, suggesting that Hb may be involved in turnover of
the NO. There is also the possibility that NO may be
activating guanylate cyclase, as it is reputed to do in
defence gene induction (Durner et al. 1998). The
induction of stress-induced Hb in Arabidopsis in the
presence of elevated nitrate may also relate to a
WATERLOGGING TOLERANCE IN PLANTS
antioxidant enzyme superoxide dismutase, SOD (Monk
et al. 1987), and non-symbiotic haemoglobin (Hill 1998).
production also induce haemoglobin expression,
suggesting that expression is not directly influenced by
the O2 content, but is influenced by content of ATP or
some consequence of ATP action (Nie and Hill 1997).
Constitutive expression of barley haemoglobin in
wild-type and transformed maize cells lines maintained
cell adenine nucleotide levels and energy charge under
hypoxic conditions, whereas wild-type cells and cells in
which haemoglobin expression is suppressed had lowered
adenine nucleotide levels and energy charge (Sowa et al.
1998). In similarly transformed alfalfa root cultures, lines
constitutively expressing barley haemoglobin maintained
root growth during hypoxic treatment, whereas wild-type
and lines with suppressed stress-induced haemoglobin
expression had slower root growth (Dordas et al. 2003a,
2004). The low dissociation constant of the oxyhaemo-
globin complex (Duff et al. 1997) may preclude functions
for stress-induced haemoglobins as oxygen stores,
carriers or signal molecules, however, the molecule is a
highly efficient scavenger of oxygen at low oxygen
tensions. There is, thus, the possibility that it may act in a
metabolic reaction involving oxygen, where it could
potentially interact with other enzyme proteins or
molecules in an oxygen-consuming reaction in low
oxygen environments.
requirement to modulate NO levels (Wang et al. 2000).
Stress-induced Hb have also been implicated in
regeneration of NAD+ during hypoxia (Hill 1998) based
on the observations that alcohol dehydrogenase activity
and CO2 production is reduced under hypoxia in maize
cells constitutively expressing barley Hb (Sowa et al.
1998).
Nitric oxide synthesis in roots may involve nitrate
reductase (NR) and nitrite oxide synthase or nitrite-nitric
oxide reductase (NiR-NOR). In roots, two distinct types
of nitrate reductase are present, one located in the cytosol
(cNR) and the other attached to the plasma membrane
and facing the apoplast (PM-NR) (Stöhr and Ullrich
1997, Stöhr and Mäck 2001). There is a 2.5-fold
activation of cNR during exposure of plant roots to
hypoxia (Botrel and Kaiser 1997), with nitrite reduction
being suppressed at the nitrite reductase step (Botrel et al.
1996). The limitation of nitrite reduction is connected
both with cellular acidification and with increased flux
through nitrate reductase (Botrel and Kaiser 1997, Botrel
et al. 1996). The potential maximum activity of activated
nitrate reductase, although lower than alcohol
dehydrogenase, exceeds the rate of hypoxic ethanol
405
R.K. SAIRAM et al.
formation by more than 3-fold (Botrel and Kaiser 1997).
In Arabidopsis root cultures, two nitrate reductase genes
were induced under low-oxygen (5 %). The NR1 gene
showed moderate induction after 0.5 - 4 h of hypoxia and
strong induction after 20 h. The NR2 gene was strongly
activated in 2 - 4 h and even more after 20 h (Klok et al.
2002).
Yamasaki et al. (1999) using purified cNR from
maize showed that a side-reaction of cNR is the reduction
of nitrite to NO with NADH as an electron donor,
probably catalyzed by the same molybdenum cofactor-
containing domain as in nitrate reduction. NO formation
by cNR requires high nitrite concentration, as the Km of
cNR for nitrite is about 300 mM (Yamasaki and
Sakihama 2000). Yet the accumulation of nitrite to high
concentration within the cell seems unlikely under natural
conditions, since nitrite as well as its acid form, nitrous
acid, are highly toxic (Sinclair 1987), and nitrite is
rapidly reduced by nitrite reductase (NiR). However,
under anaerobic conditions nitrite does accumulate
in vivo (Botrel et al. 1996). This may suggest that NO can
be formed by cNR and accumulates only when the cells
are in transition to the unfavourable anaerobic conditions.
PM-NR activity was initially demonstrated by Ward
et al. (1989) and Meyerhoff et al. (1994). It is present
only in root tissue where it exceeds the activity of cNR,
particularly during the night (Stöhr and Mäck 2001). It
can use both succinate and NADH, but succinate is the
preferred electron donor. Taking into account succinate
accumulation during hypoxia (Fan et al. 2003) and the
possibility of fumarate reduction back to succinate by
succinate dehydrogenase in co-operation with complex I
under the accumulation of reduced ubiquinone (Cecchini
2003), there is the possibility that the plasma membrane
may have an important role in nitrate reduction during
hypoxic conditions. Plasma membrane-bound nitrite-NO
reductase (Ni-NOR) is the likely enzyme that converts
nitrite to NO rather than PM-NR. Ni-NOR faces the
apoplast and has an activity sufficient to convert all of the
nitrite formed by PM-NR to NO (Stöhr and Ulrich 2002).
Ni-NOR uses reduced cytochrome c for nitrite conversion
to NO (Stöhr et al. 2001). Since participation of
cytochrome c at the plasma membrane is unlikely, it is
possible that the physiological electron donor for this
reaction could be either another cytochrome or Hb,
induced under hypoxic condition. A haem protein
oxidized during this reaction can be reduced by a protein
possessing cytochrome reductase activity. The pH
optimum of Ni-NOR is favourable for hypoxic conditions
Waterlogging, ROS production and antioxidant activity
Excessive generation of reactive oxygen species (ROS) or
oxidative stress is an integral part of many stress
situations, including hypoxia. Hypoxic tissues exhibit
enhanced mitochondria-dependent ROS generation,
acetaldehyde dependent superoxide formation via
xanthine oxidase, lipoxygenase action on membrane
406
(pH 6.1), and it can utilize even low amounts of nitrite
(Vmax is reached at a nitrite concentration of
100 µM) (Stöhr et al. 2001).
Regeneration of nitrate is essential under nitrate
limiting conditions of anaerobic roots for the continuation
of Hb-NO cycle. It has been suggested that
oxyhaemoglobin would donate negatively charged
dioxygen to NO, forming nitrate and methaemoglobin, a
known reaction of oxyhaemoglobin (Di Iorio 1981). The
reduction of methaemoglobin to Hb can occur in a
number of ways. A methaemoglobin reductase has been
demonstrated in nodules of leguminous plants (Topunov
et al. 1980). A number of diaphorase-type enzymes, such
as cytochrome b5 reductase of the endoplasmic reticulum
(Hagler et al. 1979) or dihydrolipoamide dehydrogenase
(Moran et al. 2002, Igamberdiev and Hill 2004) have
methaemoglobin reductase activity. Another possibility is
the presence of this reaction in the Hb molecule itself.
Hb may be pivotal in the short-term survival of plant
root cells by regulating the levels of NO. Plant roots that
express sufficient Hb soon after exposure to hypoxic
stress may modulate levels of NO, produced as a result of
the stress, either through reaction of the NO with
oxyhaemoglobin or through formation of nitrosyl-
haemoglobin. This would prevent the onset of cell death,
maintaining ATP levels and energy charge, as has been
observed in hypoxic maize cells overexpressing Hb
(Sowa et al. 1998). In primary roots, this may provide
sufficient time for the plant to develop adventitious roots,
needed for prolonged survival under hypoxia.
NO is also an attractive candidate for involvement in
aerenchyma formation. It has been suggested that NO
may interact with reactive oxygen species to produce
peroxynitrite (ONOO-), which may directly kill plant
pathogens (Durner and Klessig 1999). There is an
abundance of literature on NO and programmed cell death
in many mammalian tissues. Depending on NO
concentration and other factors, NO may either accelerate
or inhibit apoptosis (Kim et al. 2001). The effect may be
either direct, through cell necrosis, or through regulatory
pathways and it may also be selective in relation to the
cells that do respond. A similar type of reaction could be
responsible for selected cell death during aerenchyma
formation in roots exposed to waterlogging (Drew 1997).
Similarly, NO has been implicated in programmed cell
death of Arabidopsis cell suspension cultures through its
action on signal transduction pathways involving
guanylate cyclase (Clarke et al. 2000).
lipids and finally lipolytic acyl hydrolase-catalyzed
liberation of FFA, which underpins a burst in lipid
peroxidation on return to normoxia. The main cellular
components susceptible to damage by free radicals are
lipids (peroxidation of unsaturated fatty acids in
membranes), proteins (denaturation), sugars and nucleic

acids. Consequences of hypoxia-induced oxidative stress
depend on tissue and/or species (i.e. their tolerance to
anoxia), on membrane properties, on endogenous
antioxidant content and on the ability to induce the
response in the antioxidant system.
Anaerobic tissue has a very high redox potential and
the soil environment surrounding the roots contains
highly reduced forms of metal ions such as Fe2+, which
can readily reduce atmospheric oxygen to superoxide.
Therefore, in the interim between return to high oxygen
partial pressures and reactivation of the mitochondrial
electron transport system, conditions are ideal for
activation of oxygen.
It was observed in a study of soybean roots that a
short anoxic stress (1 - 2 h) increased the potential for
superoxide production (Van Toai and Bolles 1991). With
longer durations of anoxia (3 - 5 h) the roots developed
an increased ability to cope with oxygen free radicals,
and therefore exhibited less post-anoxic injury.
Incubation in the presence of exogenous ascorbic acid
alleviated post-anoxic injury in these roots. Hydrogen
peroxide accumulation under hypoxic conditions has
been shown in the roots and leaves of Hordeum vulgare
(Kalashnikov et al. 1994) and in wheat roots (Biemelt
et al. 2000). The presence of H2O2 in the apoplast and in
association with the plasma membrane has been
visualized by transmission electron microscopy under
hypoxic conditions in four plant species (Blokhina et al.
2001). Indirect evidence of ROS formation (i.e. lipid
peroxidation products) under low oxygen has also been
reported (Blokhina et al. 1999). Using diphenylene
iodonium chloride, a specific inhibitor of membrane
bound NADPH oxidase, and gene expression studies we
have shown increase in NADPH oxidase activity and
increased NADPH oxidase-mRNA expression under
hypoxic condition in pigeon pea genotypes (Sairam et al.-
unpublished data). These studies suggest that there is
ROS production even during the hypoxia, which probably
is due to the induction of membrane bound NADPH
oxidase.
Hypoxic signaling and gene regulation
Hypoxia/anoxia is one of the most important abiotic
stresses encountered by most of the higher organisms.
Studies are required to isolate and characterize the genes
involved in tolerance to anaerobic stress and to determine
the molecular mechanisms and analysis of genes that
confer increased flooding and anaerobic tolerance in crop
plants. Genetic analysis indicates that tolerance is a fairly
simple dominant trait. A recessive factor that increases
anaerobic tolerance in plants that are null for ADH
activity has been reported in maize (Lemke-Keyes and
Sachs 1989a,b). Subbaiah and Sachs (2003) have also
demonstrated how a simple post-translational
modification of sucrose synthase by the addition/removal
of phosphate can lead to potent changes in the tolerance
of seedlings to anoxia. Discovery of genes and proteins
WATERLOGGING TOLERANCE IN PLANTS
There are only a few reports of investigations of
changes in activities of components of antioxidative
system in response to anoxic or hypoxic conditions.
Monk et al. (1987) reported increase in superoxide
dismutase (SOD) activity when the rhizomes of Iris
pseudacorus were flooded. Van Toai and Bolles (1991)
suggested that it probably has a critical role in the
survival of the plant when oxygen levels increase as the
flooding stress abates. Induction of enzymes involved in
the ascorbate-dependent antioxidative system (ascorbate
peroxidase − APX, monodehydroascorbate reductase −
MDHAR, and dehydroascorbate reductase − DHAR) has
been shown for anaerobically germinated rice seedlings
after transfer to air (Ushimaru et al. 1997). Roots of
wheat (Triticum aestivum) seedlings could cope with the
deleterious effects of oxygen radical generation due to
post-hypoxia by increasing glutathione reductase (GR)
activity and the content of glutathione (Albrecht and
Wiedenroth 1994).
Investigations involving 11 species with contrasting
tolerance to anoxia have revealed an increase in MDHAR
and/or DHAR in the anoxia-tolerant plants after several
days of anoxic treatment. In the intolerant plants activities
were very low or without any changes. Glutathione
(GSH) content decreased significantly during the post-
anoxic period, while ascorbate (AA) showed increased
values in the tolerant species (Wollenweber-Ratzer and
Crawford 1994). Biemelt et al. (1998) reported a slight
decrease in the activities of MDHAR, DHAR and GR or
no change in the roots of wheat seedlings under hypoxia,
while anoxia caused a significant inhibition of enzyme
activities, and a significant increase in the reduced forms
of AA and GSH. Nevertheless, a rapid decrease in the
redox state of both antioxidants was observed during
reaeration. Inhibition of GR, APX, catalase (CAT) and
superoxide dismutase (SOD) activities has been shown
by Yan et al. (1996) in maize leaves under prolonged
flooding, while a short-term treatment led to an increase
in the activities.
likely to be involved in structural modifications
(aerenchyma formation and root tip death) indicate
further that these mechanisms are multi-pronged and
multi-component, perhaps tailored to adapt to different
levels of stress.
The co-ordinated expression of the anaerobic protein
(ANP) is accomplished by a common trans-acting factor
that interacts with an anaerobic responsive element
(ARE) in the promoter region of each gene (Olive et al.
1991). Oxygen deficiency changes its conformation, or
the natures of its binding to ARE, and thereby promotes
transcription. Aerobic mRNA is not translated under
anaerobic condition in maize roots, whereas those for
ANP are translated, presumably reflecting the recognition
of a specific anaerobic signal on the mRNA. Finally, the
407
R.K. SAIRAM et al.
anaerobic mRNA is much more stable and has a longer
half-life under oxygen deficiency (Drew 1990).
The anaerobic stress-response of maize offers an
opportunity to characterize the regulatory components of
a family of 20 genes that are coordinately expressed. The
anaerobically induced proteins appear to be encoded by a
set of genes, whose expression is stimulated by a
deprivation of oxygen, a condition that would occur in
nature during flooding. Regulation of protein synthesis
under anaerobiosis appears to occur at multiple levels.
Subbaiah and Sachs (2003) have characterized several
genes involved in the anaerobic response and provided
some insight into a few components of the signal
transduction pathway to understand how maize perceives
the changes in external O2 concentration and adapts its
growth and metabolism over the short- and long-term.
They demonstrated that Ca2+ acts as a key transducer of
changes in O2 availability. An additional aim should be to
characterize the promotor elements of the anaerobically
induced genes as well as the signalling components
down-stream to calcium that trigger gene induction.
Though the molecular basis of the adaptation to
transient low oxygen conditions has not been completely
characterized, but progress has been made towards
identifying genes and gene products induced during low
oxygen conditions. Promoter elements and transcription
factors involved in the regulation of anaerobically
induced genes have been characterized. Transgenic plants
may clarify the physiological role of the fermentation
pathways, and their contribution to flooding tolerance
(Dennis et al. 2000). Dennis et al. (2000) reported first
results with the inducible AtMYB2 transcription factor.
Sequencing of the Arabidopsis anaerobically-induced root
cDNA library may identify novel genes concerned with
the low oxygen response.
Lee et al. (2007) investigated the transcriptional
expression in vitro for low-oxygen treatment. Dramatic
increases in the transcripts of a TaMyb1 (Triticum
aestivum Myb transcription factor 1) gene occurred under
hypoxia. The transcriptional expression of TaMyb1 was
enhanced by light under hypoxia. The TaMyb1
expression was high in the epidermis, endodermis and the
cortex adjacent to the endodermis under hypoxia but
undetectable in the vascular tissues or cortex, which
contained aerenchyma. TaMyb1 transcription levels in
roots also gradually increased as the result of treatment
with abscisic acid (ABA), polyethylene glycol (PEG) and
NaCl. They suggested that the expression of TaMyb1 in
roots could be strongly related to the oxygen
concentration in root environment.
In plants, the involvement of Rop signaling in
response to hypoxia was revealed by a screen of
Arabidopsis seedlings that possess a Ds-GUS transposon
gene-trap element for genes induced by low oxygen
levels (Baxter-Burrell et al. 2003). The screen yielded
ropgap4-1, a loss-of-function mutant generated by a
Ds-GUS insertion in the first exon of ROPGAP4. The
ropgap4-1 seedlings induced significantly higher levels
of ADH mRNA and specific activity than wild-type
408
seedlings, hinting that ROPGAP4 negatively regulates
ADH induction. Rop activation is a prerequisite for
hypoxic induction of ADH because a line overexpressing
a dominant negative mutant of ROP2 (35S:DN-rop2) that
stably binds GDP showed no detectable increase in ADH
mRNA and enzymatic activity in response to the stress.
By contrast, a line producing a mutant ROP2 that
constitutively binds GTP (35S:CA-rop2) had elevated
ADH activity under control growth conditions (Baxter-
Burrell et al. 2002). Consistent with this hypothesis, Rop-
GTP levels increased dramatically within 1.5 h of
hypoxia and then decreased between 12 and 24 h of
oxygen deprivation in wild-type seedlings. The ropgap4-1
seedlings had a brownish, water-soaked appearance after
oxygen deprivation, similar to that caused by oxidative
stress. This led to the unexpected finding that hypoxia
promoted an increase in H2O2. Significantly higher levels
of H2O2 were measured in extracts from ropgap4-1
mutants, whereas no rise in H2O2 was detected in
35S:DN-rop2 seedlings. In wild-type, ropgap4-1 and
35S:CA-rop2 seedlings, increases in H2O2 and ADH
activity were inhibited by diphenylene iodonium chloride
(DPI), an inhibitor of flavin-binding proteins. By
contrast, ADH activity was induced under aerobic
conditions when H2O2 was enzymatically generated on
the surface of seedlings (Baxter-Burrell et al. 2003).
These observations support the conclusion that ROS
production is a component of the pathway that induces
ADH expression under low oxygen (Fukao and Bailey-
Serres 2004). Role of ROS signaling in the induction of
transcription factors associated with induction of genes of
antioxidant enzymes has also been reported by various
workers (Pastori and Foyer 2002, Agarwal et al. 2005). It
is thus possible that hypoxia generated ROS also induce
the genes of antioxidant enzymes, resulting in the observed
increase in the activity of various antioxidative enzymes.
Nitric oxide has been reported as a signaling molecule
in a wide range of responses in animals and plants. The
accumulated evidence suggests that a metabolic pathway
involving NO and Hb provides an alternative type of
respiration to mitochondrial electron transport under
limited oxygen. Hb in hypoxic plants acts as part of a
soluble terminal NO dioxygenase system, yielding nitrate
ion from the reaction of oxyHb with NO. NO is mainly
formed due to anaerobic accumulation of nitrite. The
overall reaction sequence, referred to as the Hb/NO cycle,
consumes NADH and maintains ATP levels via an as yet
unknown mechanism (Igamberdiev et al. 2005). Hb gene
expression appears to influence signal transduction
pathways, possibly through its effect on NO, as evidenced
by phenotypic changes in normoxic Hb-varying transgenic
plants. Ethylene levels are elevated when Hb gene
expression is suppressed, which could be a factor leading
to root aerenchyma formation during hypoxic stress.
Interestingly, both NO and H2O2 have recently been
found to function as localized and long-range root-
derived signals capable of rapidly communicating the
redox status and indirectly activating MAP kinase-like
activity in the shoots of A. thaliana (Capone et al. 2004).

Conclusions and perspectives
Examination of regulatory mechanisms and signaling
events responsible for triggering responses to oxygen
deficient conditions in plants is an interesting area of
research. Advances in genome biology, genetic resources
and high throughput technologies provide excellent
resources for the exploration of oxygen sensing
mechanisms in plant cells. It is imperative to identify
sensors and dissect the signaling pathways that occur at
the cellular, tissue, organ and whole plant level. It will be
interesting to investigate the participation of an ROS
sensing mechanism involving PM-NADPH oxidase in
different plants species. Again the paradoxical ROS may
prove to be second messenger in the response
mechanism. Further it will be interesting to determine
whether observed increases in NO evolution under
flooding condition from roots or soils can contribute as a
positive message in root-to-shoot communication.
Analyses of near isogenic genotypes that differ in the
adaptive response to oxygen deprivation are likely to
yield critical information on regulatory mechanisms.
Alterations in cytosolic pH and calcium may also have a
role in the signaling processes. The importance of
changes in adenylate charge, redox status and
carbohydrate levels must also be considered. Many
questions remain to be answered about the response of
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 review REVIEW
Plant Signaling & Behavior 5:6, 649-654; June 2010; © 2010 Landes Bioscience

Plant responses to drought and rewatering

Zhenzhu Xu,1,3,* Guangsheng Zhou1,2 and Hideyuki Shimizu3
1
State Key Laboratory of Vegetation and Environmental Change; Institute of Botany; Chinese Academy of Science; Xiangshan; Haidian District, Beijing PR China;
2
Chinese Academy of Meteorological Sciences; China Meteorological Administration; Haidian District, Beijing PR China; 3Asian Environment Research Group;
National Institute for Environmental Studies (NIES); Tsukuba, Ibaraki Japan

Key words: drought stress, peroxidation, photosynthesis, relative growth rate, pre-drought limitation, rewatering, signals,
stomatal conductance

Abbreviations: ABA, abscisic acid; Anet, net photosynthetic rate; Fv/Fm, maximum efficiency of PSII photochemistry; gs, stomatal
conductance; MDA, malondialdehyde; PDL, pre-drought limitation; PSII, photosystem II; RGR, relative growth rate;
ROS, reactive oxygen species; RWC, relative water content; SOD, superoxide dismutase; WUE, water use efficiency

Plants would be more vulnerable to water stress and thereafter
rewatering or a cycled water environmental change, which
occur more frequently under climatic change conditions in
terms of the prediction scenarios. Effects of water stress on
plants alone have been well-documented in many reports.
However, the combined responses to drought and rewatering
and its mechanism are relatively scant. As we know, plant
growth, photosynthesis and stomatal aperture may be limited
under water deficit, which would be regulated by physical and
chemical signals. Under severe drought, while peroxidation
may be provoked, the relevant antioxidant metabolism would
be involved to annihilate the damage of reactive oxygen
species. As rewatering, the recoveries of plant growth and
photosynthesis would appear immediately through growing
new plant parts, re-opening the stomata, and decreasing
peroxidation; the recovery extents (reversely: pre-drought
limitation) due to rewatering strongly depend on pre-drought
intensity, duration and species. Understanding how plants
respond to episodic drought and watering pulse and the
underlying mechanism is remarkably helpful to implement
vegetation management practices in climatic changing.
Under the climatic changing context, drought has been, and is
becoming an acute problem most constraining plant growth,
terrestrial ecosystem productivity, in many regions all over the
world, particularly in arid and semi-arid area.1-3 Based on the
fourth assessment report by IPCC, global surface average temper-
ature will have a 1.1–6.4°C range increase by the end of this cen-
tury.3 It is indicated that a warming above 3°C would eliminate
thoroughly fixed carbon function of global terrestrial vegetation,
shift a net carbon source. With global warming, it is expected
that water deficit would be escalated by increasing evapotranspi-
ration, increasing the frequency and intensity of drought with an
increase from 1% to 30% in extreme drought land area by 2100;3
which would offset the beneficial effect from the elevated CO2
*Correspondence to: Zhenzhu Xu; Email: xuzz@ibcas.ac.cn
Submitted: 02/01/10; Accepted: 02/02/10
Previously published online:
www.landesbioscience.com/journals/psb/article/11398
concentration, further limiting the structure and function of the
terrestrial ecosystem. The global climate models may forecast
the precipitation regimes including its distribution and amount,
but the complicated responses of terrestrial ecosystem to climate
change may adversely affect the predict accuracy.1,4
Plant would response to water stress by dramatically complex
mechanisms from genetic molecular express, biochemical metab-
olism through individual plant physiological processes to ecosys-
tem levels2,5,6 which may mainly includes six aspects: (1) drought
escape via completing plant life cycle before severe water deficit.
E.g., earlier flowering in annuals species before the onset of severe
drough;7 (2) drought avoidance via enhancing capacity of get-
ting water. E.g., developing root systems or conserving it such as
reduction of stomata and leaf area/canopy cover; 8,9 (3) drought
tolerance mainly via improving osmotic adjustment ability and
increasing cell wall elasticity to maintain tissue turgidity;10
(4) drought resistance via altering metabolic path for life survives
under severe stress (e.g., increased antioxidant metabolism);11,12
(5) drought abandon by removing a part of individual, e.g.,
shedding elder leaves under water stress;2 (6) drought-prone bio-
chemical-physiological traits for plant evolution under long-term
drought condition via genetic mutation and genetic modifica-
tion.13-15 The processes may be involved in multi-aspects simul-
taneously in responses of plants to drought stress and thereafter
rewatering.
In the field context, there is always interval occurrence in
drought and/or rewetting events, particular under climatic
change conditions predicting more frequent drought and flooding
events.3 The water cycle change may greatly impact plant growth,
photosynthesis and many key metabolic functions, thereby eco-
system productivity and agricultural achievement.5,16-18 Actually,
sporadic precipitation would become a critical issue for maintain-
ing ecosystem structural stability and even it’s surviving in arid
and semi-arid area. For example, a small rainfall pulse can induce
a rapid response in a desert ecosystem, which quickly triggers
plant growth so that the plants can survive.19 Thus, to highlight
how plant and terrestrial ecosystem cope with adverse abnormal
climatic change variables is, and always will be crucial research
issue in practical management of plant growth and vegetation
productivity. Here, we try to provide a brief insight into how

www.landesbioscience.com Plant Signaling & Behavior 649

plant responses to the pre-drought and rewatering in terms of
the plant growth, gas exchange and key related-physiological pro-
cesses such as reactive oxygen species (ROS) metabolism. Finally
a regulation path schematic is presented to try to explain the
involved processes.
Plant Growth and its Allocation
Plant growth and its biomass allocation are two most funda-
mental processes of vegetable kingdom, being remarkably influ-
enced by environmental variables including water changing
factor.2,20 Plant growth and vegetation production were obviously
restricted during 2003 European drought with hot summer, a
remarkable recovery seem to appear following the 2004 wetted
year.3,21 The over-compensation for plant growth upon rewater-
ing after drought also has been confirmed by many experimental
investigations.22-24 For example, as plant individuals of Leymus
chinensis have been subjected to a short-term drought, its leaf area
was significantly stimulated by rewatering, which might tran-
scend levels of control pants always in well-watered conditions.24
However, in the Patagonian steppe, when water was applied again
after pre-drought, the vegetation density (as an indicator of eco-
system structure) still not fully recovered, which would lead to
annual net primary productivity lags of the response to rewetting
following dry year,25 indicating so-called pre-drought limitation
(PDL) could exist. Our reports also showed that the final biomass
or leaf area in plants subjected to long-term or severe drought can
not reached the level of the control treatment, highlighting that
whether plant growth have the complete recovery following rewa-
tering may depend on the pre-drought intensity or duration.17,24
This pre-drought constraint after rewetting may ascribe to mer-
istem limitation in plant tissues.25,26 Our recent study indicated
that tillers’ numbers of Leymus chinensis still was lower relative
to those control treated within 50 d after rewatering, but a full,
even over-compensatory recovery occurred 70 after rewatering,
implying the tiller meristems’ occurrence and development may
play a critical role in the response to rewatering after an experi-
enced pre-drought.17 Plant growth of Koeleria macrantha had a
great response to rewatering after a 20-d-long-term drought with
lower tiller relative water content (RWC) of 13%.27 However,
the response to rewatering could not be observed for Briza media
plants. This may explained why Koeleria macrantha can be eas-
ily found in xeric calcicolous grasslands where severe episodic
drought often occur, but Briza media did not exist in the same
extreme dry conditions,27 even both Briza media and Koeleria
macrantha grasses bear a similar and high drought-tolerant abil-
ity which was confirmed by that titters’ RWC can maintain a
similar level even as soil moisture had been decreased by less than
9%;27 it is suggested that the different responses of the two spe-
cies to recycle watering may play a critical role in their evolution-
ary processes under a certain environmental pressures, possibly
involving a physiological plasticity to drought.15
Many investigators have proposed using carryover and/or
memory effects to describe the response of production to current
environmental constraint from previous years as additional inde-
pendent variables.28,29 In a semiarid grassland of South Africa,
650 Plant Signaling & Behavior Volume 5 Issue 6
for instance, Wiegand et al.29 found that 33–68% of the unex-
plained variation is related to a memory index combining mean
monthly temperature and a memory of past precipitations. The
limitation to growth due to pre-drought may be portrayed one
of plant memory behavior on past drought stress. In our work,
PDL to plant biomass occurred under severe drought. However,
PDL of relative growth rate (RGR) was not found, in contrast,
pre-drought stimulation appeared.17 It is strongly suggested that
the pre-stresses’ memory may play the central role in new parts’
growth rather than final production, which may derive from the
re-trigger of the store resources such as soil nutrition besides the
water reapplication.
Plants may try to obtain water in soil by enhancing its root
system under soil drought. Many reports indicated that the
increases in biomass ratio of root and shoot under water stress
confirm this conclusion.9 However, under extreme soil drought,
no root: shoot ratio increase was observed, implying that there
would be a threshold of soil moisture in response to plant biomass
allocation to water stress.30 When plant is subjected to extreme
soil drought, the regulation capacity through asymmetric growth
approach may be also lost abruptly.
ROS and Peroxidation
Under environmental stresses such as drought, a rapid ROS
accumulation including singlet oxygen (O2-), superoxide (•O2),
hydroxyl (OH-1) and hydrogen peroxide (H2O2) may occur, lead-
ing to negative impact on antioxidant metabolism, and conse-
quently cell peroxidation damage.31-33 Generally, a ROS increase
during pre-drought can be reversed following refilling water.34,35
There was a sharp decrease in H2O2 with a better recovery of net
photosynthetic rate (Anet) and net transpiration rate as Prunus
plants were rewatered after a 70 d long-term drought stress, to
which the degreed reached severe water stress level of -4 MPa leaf
water potential.34 When a tee plant was drought-treated for 20 d,
leaf RWC fell to a severe drought status of 41–53%, H2O2 con-
tents significantly increased, thereafter they drastically decreased
by rewetting.35 H2O2 contents in bluegrass leaves increased by
67% with a low RWC of 68% under drought, but it could return
to control level only one day after rewatering.33 In roots, however,
both drought and rewatering led to remarkably H2O2 accumula-
tion,33 indicating the response mechanism would differ in differ-
ent plant parts. As well reported, antioxidative enzymes such as
superoxide dismutase (SOD) might play an central role in anti-
oxidant metabolism in plants subjected to environmental stresses
including drought possibly through regulating their gene expres-
sions and/or activities.32,36 In the bluegrass leaves, although SOD
activities did not change obviously under moderate drought and
following rewatering, the gene expressions of FeSOD and Cu/
ZnSOD were remarkably downregulated by drought, but can
recover to the control level after refilling soil water.33 In Alfalfa
nodules, however, FeSOD and CuZnSODc was upregulated by
moderate drought, while the upregulation only in CuZnSODp
was observed following rewatering relative to control level, but
the expression of MnSOD for any of the treatments maintained
relatively constant, implicating that the responses may differ
because of species and tissues.36 Thus, in fact, the ROS and their
regulated metabolism might depend on species, cultivars/variet-
ies, tissues, stress intensity and its durations.34,35
In field-grown evergreen Phillyrea angustifolia (Oleaceae)
plants, even when leaf had a low RWC of 50% after 48-d
drought treatment, malondialdehyde (MDA) contents did not
significantly change, but the photoprotector zeaxanthin and the
antioxidant α-tocopherol accumulated more 200% compared to
always well-irrigated plants, and leaf reflectance also had a cor-
responding change, indicating higher capacity of the photo- and
antioxidative protection of this species through involvement of
xanthophyll cycle and antioxidant promotions.12,37 In bentgrass
species, Bian and Jiang33 reported that moderate drought and
recovery only have a marginal effect on lipid peroxidation in blue-
grass leaves. However, it was obviously provoked by a prolonged
drought when plant leaves’ RWC fell to sever dehydration level
of less than 40%;38 and as tee plants were subjected to a severe
water deficit, MDA was significantly accumulated, which could
be reversed drastically by rewetting,39 indicating that whether
lipid peroxidation appears or recovers strongly depends on
drought intensity/duration,33,38 and species/cultivars.35,39 In our
research, cell ion leakage, an indicator of cell integration damage,
was drastically attenuated by rewatering after a relatively long-
term pre-drought (30 d), albeit no reaching the normal level in
well-watered plants; moreover, this occurrence was only in young
leaves of Leymus chinensis,24 implicating that the new parts of the
plants might have a highly metabolically repairing capacity upon
the rewatering following a pre-water-deficit stress.
Photosynthesis and Stomatal Behavior
Drought limitation to photosynthesis has been reported in many
studies,2 of which a few documents covered the photosynthetic
responses to cycled water deficit. As reported, full recovery of
net photosynthetic rate (Anet) has been observed as drought
stress is eliminated following rewatering.5,17 For example, after
15 d rewatering, Populus nigra L. leaf Anet can completely recover
to the level recorded following water deficit in the plants re-
watered to field capacity level of soil moisture after leaf Anet fell
to almost zero at lowest soil moisture (Fraction of available soil
water, FASW 25%) at 35 d after starting drought treatment.40
In our recent report,17 severe and extreme drought caused light-
saturated net photosynthetic rate (Asat) to significant drops of
22% and 75% compared to the ample moisture treatment as a
control, respectively. However, rewatering almost completely
negated the difference between drought-treated and the control
plants, indicating a complete resumption. The Anet and stomatal
conductance (gs) in summer drought-stressed Phillyrea angusti-
folia plants were decreased by about 90% with a low leaf RWC
of 50%, but the maximum efficiency of photosystem II (PSII)
photochemistry (Fv/Fm) was not affected under the same drought
condition,12 indicating the photosynthesis downregulation may
mainly derive from stomatal limitation for this species. Based
on recent study of Izanloo et al.5 for different wheat cultivars,
PSII activities demonstrated different change trends as exposed
to a cycle drought-treated regime even reaching wilting point of
www.landesbioscience.com Plant Signaling & Behavior 651
the soil moisture, that a drastic decline in Fv/Fm (from 0.82 to
0.65) was found in Kukri plants, with no significant change for
other two cultivars compared to well-watered treatment. Plants
of Lonicera japonica Thumb. with tetraploid chromosome have
a higher drought-resistance during water deficit and more rapid
recovery after rewatering in terms of gas exchange and chloro-
phyll fluorescence compared to those with diploid chromosome,39
showing the different responses from different genetic types. In
our experiment, there was an obvious decline in Fv/Fm of Leymus
chinensis leaves under severe soil drought, while a fully recovery
appeared following refilling water. This is indicated that, com-
pared to those gas exchange parameters, the functional activity
of PSII photochemistry may contribute to both higher drought-
tolerance and higher recovery capacity, which might not be inde-
pendent of species and/or cultivars.
Stomatal regulation plays a key role in gas exchange between
vegetation and atmosphere interface. Ninety percent loss of plants
is from transpiration thought stomatal opening.41 On the other
hand, stomatal limitation would be recognized a major factor for
photosynthetic reduction when available water become scant,
while non-stomatal limitation such as decreases in Rubisco activ-
ity, CO2 availability in the chloroplast and PSII photochemistry
efficiency is progressive gradually with water stress intensity and
persistence duration.17,42,43 However, stomatal conductance would
not be always associated with Anet in some cases,44 which is still
under debate. It is interesting, from the report by Flexas et al.42
that Rubisco activity is directly and closely linked to decreased
gs and mesophyll conductance induced by abscisic acid (ABA)
rather than leaf RWC, indicating that some signals such as ABA
between stomatal behavior and CO2 fixation in mesophyll cell
might play a sensing role in regulating the relation between pho-
tosynthesis and stomatal movement. As reported, full recovery
always co-occurred in both photosynthetic function and sto-
matal aperture when rewatering after an episodic drought,5 even
a larger gs in pre-drought-treated plants appeared following rewa-
tering compared to the control plants not underwent pre-drought
experience, indicating a occurrence of over-compensation of gas
exchange.45 However, in Leymus chinensis leaves, gs only had a
partial recovery after rewatering, no reaching the level of the
well-watered treatment,46 being similar in hybrid Richter-110
(Vitis berlandierix, Vitis rupestris) plants.47,48 Flexas et al.48 fur-
ther indicated that the relative contribution of stomatal (SL) and
mesophyll conductance (MCL) limitations rather than biochem-
ical limitations would be responsible for the decreased photo-
synthesis during water stress and slow recovery after rewatering,
and the former could mostly limit photosynthesis recovery after
rewatering. The relative contributions from different limitation
components during drought and after rewatering may need to be
elucidated in future experiments using different species.
Moreover, based unequivocally on a recent report of Huang
et al.49 H2O2 could also be a regulator for stomatal behavior in
optimizing water use efficiency (WUE), which a drought and salt
tolerance (DST) protein has been confirmed to be involved in the
process by mediating H2O2 products. There is evidence that DST
function loss can lead to a decrease in stomatal aperture and its
density, increasing WUE and enhancing tolerance of rice plants
 of function processes such as growth rate, photosynthe-
sis and gs from drought-stressed limitation after rewa-
tering, depending on different genotypes.5 However,
there is evidence showing that some physical signals
such as hydraulic press from roots may induce leaf sto-
matal closure when soil water deficit occurres.52,55,56 No
ABA increase in some species was also observed as the
plants subjected to rapid dehydration.57 An ABA appli-
cation to leaves of Craterostigma plantagineum does
not increase drought-tolerance.58 Thus, whether and
how the stomatal aperture is controlled by ABA is still
under debate.
Recent works showed the Cytokinins (CKs) can
be as a regulator in response to water stress in tobacco
transgenic plants with an increase in catalase inside
peroxisomes and the CO2 compensation point, indi-
Figure 1. Simplified representation of some response routes in plants subjected to cating the cytokinin-mediated occurrence of pho-
drought and subsequent rewatering. Note: From bottom, stomatal conductance torespiration may play a beneficial role in protecting
and net photosynthetic rate are reduced as plants were subjected to different photosynthetic processes during severely restricted
extent drought stresses in which involve some signals such as ABA; photosyn- water condition.51 Water channel as an initial hydro-
thetic apparatus may be damaged under severe/extreme drought, e.g., leading
static signal also is a critical component for plants to
to declines in PSII photochemical efficiency and enhancing peroxidation. Plant
growth rate would decrease gradually with water deficit. Following rewatering, absorb water from soil and transport to shoot and up-
the gas changes and plant growth may be recovered, to which the extent depends leaf, undergoing a comprehensive biological process.53,59
obviously on intensities of drought stress. Thus, under severe drought stress, damage to tissue cell
may adversely affect the biochemical activities includ-
ing the water channel, and consequently the flexible
to water deficit.49 Actually, the H2O2 produced by photorespira- response of plants to water changing may be restrained.6 In
tion can act on the redox states of leaf antioxidant pools, impli- another recent observation, isoprene emission also had a partial
cating the possibility of photorespiratory H2O2 as a signal role recovery following the Anet complete recovery after rewatering
under drought.50 It is implicated that stomatal movement and in plants experienced 35 d pre-drought, suggesting its protein
photosynthesis may link to ROS metabolism, signal cascade and level or substrate supply constrained by re-drought may play an
photorespiration,50,51 which together needs to be investigated in important role in the isoprene biochemical synthetic processes.40
the future. It is noted that the limitations to pre-drought and recoveries by
subsequently rewatering may influence biochemical metabolism
Signals and Other Biochemical Process and signal cascade.

Drought-sensed signals and their roles including physical and Conclusions

chemical types have already been well documented.2,52 Of them,
a stress hormone, ABA usually is emphasized, being recognized
a key drought-sensed signal from root to shoot,2,41 although does
not to do so in all species.52 The ABA produced in root would be
transported to shoot which regulate the stomatal behavior.53,54
ABA may exist as an apoplastic component in the stem xylem
that comes from root through apoplastic route, which may
play a linkage signal role between shoot and roots.53 In Glycine
max L. Merr. plants, a decrease in gs and an increase in xylem
[ABA] may occur simultaneously before leaf turgor had a sig-
nificant change, implying that seemingly root-originated ABA
would regulate stomatal behavior at moderate drought.55 It is
also suggested that the ABA accumulation in leaves induced by
root physical or other chemical signals would directly regulate
the stomatal movement.2,54,55 An evidence indicated that high
ABA level can be eliminated when plant is rewatered to an opti-
mal water condition,41 perhaps leading to re-opening of the sto-
mata. Wheat plants with low ABA content, together with high
osmotic adjustment, would have a rapid and complete recovery
The responses of plants to water changing conditions cover many
aspects from genetic molecular level, biochemical and physiolog-
ical processes, through whole individual to community levels.
For example, as plant is exposed to moderate drought, a stomatal
aperture may be decreased possible through sensing physical or
chemical signals such as hydraulic press and ABA. A decline of
the stomatal conductance may limit the net photosynthetic rate
and water transpiration with progressive water stress, leading to
increased WUE because the transpiration is inhibited more than
photosynthesis. When drought intensity became severe even
extreme, photochemical efficiency and Rubisco activity would
be constrained, which may reduce Anet to zero; at the same time,
other adverse biochemical-physiological metabolisms including
peroxidation may be exacerbated, together consequently reduc-
ing plant growth rate. The photosynthesis and plant growth may
start to be stimulated immediately following an application of
watering pulse. However, the extent and magnitude of the stim-
ulation from rewatering may depend on pre-drought intensity

652 Plant Signaling & Behavior Volume 5 Issue 6

and duration (severe stress may irreversibly injury tissue appara-
tus), and species/varieties, of which a compensatory/full/partial
recovery may appear (Fig. 1). Additionally, plants could accli-
mate to episodic drought or watering pulse through abandoning
older parts and renewing younger their parts, and by promoting
re-allocation of biomass into roots. To highlight the complicated
processes of the responses to water cycled changing is of impor-
tant ecological significance because there is both aridifying and
warming trends in terrestrial ecosystems at global and regional
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Acknowledgements
This work was supported by the National Key Basic Research
Specific Foundation (2006CB400502), the National Natural
Science Foundation of China (40625015) and the Japan Society
for the Promotion of Science (P07622).
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Plant Cell Physiol. 38(10): 1095-1102 (1997)
JSPP © 1997

Mini Review

Regulation of Levels of Proline as an Osmolyte in Plants under Water Stress

Yoshu Yoshiba 1 , Tomohiro Kiyosue2, Kazuo Nakashima 3 , Kazuko Yamaguchi-Shinozaki3 and Kazuo
Shinozakj 2
1
Advanced Research Laboratory, Hitachi Ltd., Hatoyama, Saitama, 350-03 Japan
2
Laboratory of Plant Molecular Biology, The Institute of Physical and Chemical Research (RIKEN), Koyadai, Tsukuba, Ibaraki, 305
Japan
3
Biological Resources Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Ministry of Agriculture,
Forestry and Fisheries, Ohwashi, Tsukuba, Ibaraki, 305 Japan

Compatible osmolytes are potent osmoprotectants genase (oxidase) (EC 1.5.99.8) — A'-pyrroline-5-carboxy-
that play a role in counteracting the effects of osmotic late synthetase — Transcriptional regulation — Water stress.
stress. Proline (Pro) is one of the most common compatible

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osmolytes in water-stressed plants. The accumulation of
Pro in dehydrated plants is caused both by activation of the
biosynthesis of Pro and by inactivation of the degradation
of Pro. In plants, L-Pro is synthesized from L-glutamic acid
(L-G1U) via zl'-pyrroline-S-carboxylate (P5C) by two en-
zymes, P5C synthetase (P5CS) and P5C reductase (P5CR).
L-Pro is metabolized to L-G1U via P5C by two enzymes, pro-
line dehydrogenase (oxidase) (ProDH; EC 1.5.99.8) and
P5C dehydrogenase (P5CDH; EC 1.5.1.12). Such metabo-
lism of Pro is inhibited when Pro accumulates during de-
hydration and it is activated when rehydration occurs. Un-
der dehydration conditions, when expression of the gene
for P5CS is strongly induced, expression of the gene for
ProDH is inhibited. By contrast, under rehydration condi-
tions, when the expression of the gene for ProDH is strong-
ly induced, the expression of the gene for P5CS is in-
hibited. Thus, P5CS, which acts during the biosynthesis of
Pro, and ProDH, which acts during the metabolism of
Pro, appear to be the rate-limiting factors under water
stress. Therefore, it is suggested that levels of Pro are regu-
lated at the level of transcriptional the genes of these two
enzymes during dehydration and rehydration. Moreover, it
has been demonstrated that Pro acts as an osmoprotectant
and that overproduction of Pro results in increased toler-
ance to osmotic stress of transgenic tobacco plants. Geneti-
cally engineered crop plants that overproduce Pro might,
thus, acquire osmotolerance, namely, the ability to tolerate
environmental stresses such as drought and high salinity.
Key words: Abscisic acid — Proline — Proline dehydro-
Abbreviations: Glu, glutamic acid; Pro, proline; GSA, glu-
tamic-y-semialdehyde; GSADH, glutamic-y-semialdehyde dehy-
drogenase; P5C, A '-pyrroline-5-carboxylate; P5CS, /l'-pyrro-
line-5-carboxylate synthetase; P5CR, A '-pyrroline-5-carboxylate
reductase; ProDH, proline dehydrogenase; P5CDH, j'-pyrro-
line-5-carboxylate dehydrogenase; ProT, proline transporter;
ABRE, ABA-responsive element; DRE, dehydration-responsive
element.
Plants are exposed to many types of enviromental
stress. Among these stresses, osmotic stress, in particular
that due to drought and salinity, is the most serious pro-
blem that limits plant growth and crop productivity in
agriculture (Boyer 1982).
Many plants, including halophytes, accumulate com-
patible osmolytes, such as proline (Pro), glycine betaine
and sugar alcohols, when they are exposed to drought or
salinity stress (Hellebust 1976, Yancey et al. 1982, Csonka
1989, Delauney and Verma 1993). It has been suggested
that compatible osmolytes do not interfere with normal
biochemical reactions and act as osmoprotectants during
osmotic stress. Among known compatible solutes, Pro is
probably the most widely distributed osmolyte. T h e accu-
mulation of P r o has been observed not only in plants b u t
also in eubacteria, marine invertebrates, protozoa, and
algae (McCue and Hanson 1990, Delauney and Verma
1993).
Genes for enzymes involved in the biosynthesis and me-
tabolism of P r o have been isolated from various plants,
and their expression and the functions of their gene pro-
ducts have been characterized. Results of investigations of
the relationship between the expression of these genes and
the accumulation of P r o under water stress indicate that
the level of P r o in plants is mainly regulated at transcrip-
tional level during water stress. Moreover, the overproduc-
tion of Pro results in the increased tolerance of transgenic
tobacco plants to osmotic stress. In this review, we shall
discuss the transcriptional regulation of the level of Pro
during dehydration and rehydration, as well as the possibili-
ty of breeding of transgenic plants that can tolerate water
stress.
Pathways for the biosynthesis and metabolism of pro-
line in plants—The pathway for the biosynthesis of P r o in
plants was elucidated by reference to the pathway in Esche-
richia coli (Leisinger 1987). Figure 1 shows this pathway in

1095
 1096 Regulation of levels of proline in plants

A 1 -pyrroline-5-carboxylate
(P5C) synthetase P5C reductase
(P5CS) (P5CR)

spontaneous

•iiiltiain
nuniMimuii
C CHO C c
/ \ / \
NH2 COOH NH2 COOH N COOH N COOH
L-Glu glutamic-y-semialdehyde A -pyrroline-5-carboxylate L-Pro
(GSA) (P5C)

P5C dehydrogenase
(P5CDH)
t
i
proline dehydrogenase
(ProDH)

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h
i
._ Y-glutamyl kinase
(Y-GK)
GSA dehydrogenase
(GSADH)

i •uninmimaf

COPO3 C
/ \
NH2 COOH
L-glutamyl-vphosphate

Fig. 1 Pathways for the biosynthesis and metabolism of proline (L-Pro) in plants, and the pathway for the biosynthesis of proline in
bacteria.

b a c t e r i a ^ d the pathways for the biosynthesis and metab-
olism of Pro in plants. The pathway in bacteria begins
with the ATP-dependent phosphorylation of the y-carboxy
group of L-glutamic acid (L-G1U) by y-glutamyl kinase (y-
GK). The product of y-GK is reduced to glutamic-y-semi-
aldehyde (GSA) by GSA dehydrogenase (GSADH), with
which y-glutamyl kinase forms an obligatory enzyme com-
plex. GSA cyclizes spontaneously to form A '-pyrroline-5-
carboxylate (P5C), which is finally reduced to Pro by P5C
reductase (P5CR). It has been suggested that, in plants,
Pro is synthesized either from Glu or from ornithine and
that the pathway from Glu is the primary route for the syn-
thesis of Pro under conditions of osmotic stress and nitro-
gen limitation, while the pathway from ornithine predomi-
nates at high levels of available nitrogen (Delauney et
al. 1993). The biosynthetic pathway to Pro from Glu is
thought to involve conversion of Glu to Pro via the interme-
diates y-glutamyl phosphate, GSA and P5C, as is the case
in E. coli, because a cDNA clone for P5C synthetase
(P5CS) was isolated from mothbean (Vigna aconitifolia)
by complementation of a mutant of E. coli, and recombi-
nant P5CS protein, expressed in E. coli, had both y-GK
and GSA dehydrogenase activities (Hu et al. 1992). cDNAs
for plant P5CS have been isolated from mothbean (Hu
et al. 1992), Arabidopsis thaliana (Yoshiba et al. 1995,
Savoure et al. 1995), and rice (Igarashi et al. 1997). A
cDNA for a plant P5CR was also isolated by complementa-
tion of an E. coli mutant from soybean (Delauney and Ver-
ma 1990), and homolog of this cDNA were isolated from
pea (Williamson and Slocum 1992), and Arabidopsis (Ver-
bruggen et al. 1993).
The second important factor that controls levels of
Pro in plants is the degradation or metabolism of Pro. L-
Pro is oxidized to P5C in plant mitochondria by proline de-
hydrogenase (oxidase) (ProDH; EC 1.5.99.8), and P5C is
converted to L-G1U by P5C dehydrogenase (P5CDH; Bog-
gess et al. 1977, Elthon and Stewart 1981). Such oxidation
of Pro is inhibited during the accumulation of Pro under
water stress and is activated in rehydrated plants (Stewart
et al. 1977, Rayapati and Stewart 1991). ProDH and
P5CDH catalyze reactions that are the reverse of those cata-
lyzed by P5CS and P5CR, respectively, in the biosynthesis
of Pro. cDNA for plant ProDH was isolated from Arabi-
dopsis (Kiyosue et al. 1996, Verbruggen et al. 1996, Peng et
al. 1996). However, no gene for P5CDH has yet been isolat-
ed. Recently, the P5CDH (EC 1.5.1.12) protein was puri-
 Regulation of levels of proline in plants 1097

fied from cultured cells of potato (Forlani et al. 1997), and
we anticipate that the corresponding gene will be cloned in
the near future.
Water stress and the expression of genes for enzymes
involved in the biosynthesis of proline—In plants, L-Pro is
produced from L-G1U via P5C, in reactions catalyzed by
two enzymes, P5CS and P5CR. Many plants, when they
are exposed to water stress, drought or salinity stress, accu-
mulate Pro. The expression of genes for P5CS and P5CR
has been analyzed under dehydration conditions in moth-
bean and Arabidopsis (Hu et al. 1992, Verbruggen et al.
1993, Yoshiba et al. 1995, Savoure et al. 1995). In Arabi-
dopsis, mRNA for P5CS appeared within 2 h of the start of
dehydration, and its level increased for up to 10 h (Fig. 2).
When plants were dehydrated for 10 h and then rehydrated
by transfer to water, the level of the transcript decreased
within 5 h, returning eventually to the level in untreated
plants. Limited accumulation of mRNA for P5CS in re-
sponse to cold stress was also observed 24 h after the initia-
tion of low-temperature treatment. Western blotting analy-
sis, demonstrated that the level of P5CS protein increased
after the initiation of dehydration treatment in direct pro-
portion to the level of accumulated mRNA for P5CS
(Yoshiba et al. unpublished results).
The expression of the gene for P5CR appears not to be
enhanced to any significant extent by dehydration, high
salinity, or treatment with ABA (Yoshiba et al. 1995). Ver-
bruggen et al. (1993) reported that, in Arabidopsis under
salt stress, levels of mRNA for P5CR were 5-fold higher in
leaves and 2-fold higher in roots than in non-stressed

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1 plants. However, the level of expression of the gene for
1 1
P5CR was very low compared with that of the gene for
20 _ A - 400
P5CS in Arabidopsis. These results suggest that the gene
to
for P5CS might play a more important role than the gene
OC a.
E ~o for P5CR in the accumulation of Pro under osmotic stress.
300
E The levels of Pro in Arabidopsis under dehydration
"5 and rehydration stress were compared with the levels of
c
0)
§510
O U
- o 200 mRNAs for P5CS and P5CR. The level of Pro increased
— during dehydration and decreased during rehydration in
0) / / o proportion to the level of the mRNA for P5CS. It was
Pro a
JB
/ / 100 reported that transgenic tobacco plants that expressed soy-
- O - P5CS — o
4) ProDH bean P5CR had an elevated level of P5CR activity but did
OC
a.
"*" not accumulate Pro to any significant extent (Szoke et al.
n!2 1992). By contrast, transgenic tobacco plants expressing
o0- 20 mothbean P5CS produced a large amount of the enzyme
10
Time (h) and subsequently accumulated 10-fold more Pro than did
control plants (Kavi Kishor et al. 1995). Therefore, our
1 ' results and these reports suggest that P5CS is the principal
1L L I ' I ' 1 ' enzyme involved in the biosynthesis of Pro in plants under
v - 400
20
it)

Q water stress.
CC ^> It was reported recently that the expression of the gene
E —300 O for P5CS is independent of ABA upon exposure to cold
"o E and osmotic stress, even though expression of this gene can
^ ^ c be triggered by treatment with exogenous ABA (SavourS et
tent

|ioc ^_ 200 al. 1997). The expression of the gene for P5CS was induced
Pro
0) c by osmotic stress and exogenous application of ABA both
- O - P5CS o in wild-type and in ABA-deficient (abal) and ABA-insensi-
lati

o
"•" ProDH _ 100 o tive (abil and abi2) mutants of Arabidopsis. These observa-
a
/ V/t"
OC • — — 1 tions suggest that the expression of the gene for P5CS can
-A
1 —I r 1 ,-o be induced by two different pathways, an ABA-independ-
(T ent and an ABA-dependent pathway, under dehydration
0 10 20 30 40
conditions (Fig. 3).
Time (h) Water stress and the expression of genes for enzymes
involved in the metabolism ofproline—L-Proline is metabo-
Fig. 2 Time course of the accumulation and the degradation of
lized to Glu by two enzymes, ProDH and P5CDH, which
proline (Pro), the mRNA for A '-pyrroline-5-carboxylate syn-
thetase (P5CS), and the mRNA for proline dehydrogenase catalyze reactions that are the reverse of those catalyzed by
(ProDH) in Arabidopsis during dehydration (A) and rehydration P5CR and P5CS, respectively, in the biosynthesis of Pro
after 10-h dehydration (B). (Fig. 1). It was proposed initially that the metabolism of
1098 Regulation of levels of proline in plants

L-Pro

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Fig. 3 Regulation of genes for A '-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) under dehydration
and rehydration conditions in Arabidopsis. The gene for P5CS is induced via both ABA-dependent and ABA-independent pathways dur-
ing dehydration, but it is repressed by rehydration. By contrast, the gene for ProDH is induced by accumulated proline during rehydra-
tion, but it is repressed by dehydration. See also list of abbreviations.

Pro might be inhibited during the accumulation of Pro
under water stress and activated by rehydration (Stewart et
al. 1977, Rayapati and Stewart 1991). It was difficult in ear-
ly experiments to purify ProDH in substantial amounts be-
cause of the localization of the enzyme on the matrix side
of the inner membrane of mitochondria. ProDH seems to
donate electrons directly to the respiratory electron trans-
port system (Elthon and Stewart 1981, 1982). However,
cDNAs encoding ProDH were recently isolated from Arabi-
dopsis by three groups (Kiyosue et al. 1996, Verbruggen et
al. 1996, Peng et al. 1996). Kiyosue et al. isolated a cDNA
clone (ERD5) for ProDH from a cDNA library of one-
hour-dehydrated plants by differential screening. Verbrug-
gen et al. and Peng et al. found a conserved sequence of
ProDH in the Arabidopsis EST database by using the
amino acid sequences of ProDH from Saccharomyces cere-
visiae and from Drosophila melanogaster, and then they
isolated cDNA clones. Comparison of the deduced amino
acid sequence with other sequences in sequence databases
revealed that the ProDH protein of Arabidopsis is 34.5%
and 23.6% homologous to those of Drosophila (Hayward
et al. 1993) and S.cerevisiae (Wang and Brandriss 1987),
respectively. The amino acid sequence of ProDH protein
from Arabidopsis contains a putative signal for mitochon-
drial localization that is characteristic of proteins that are
imported into mitochondria. The ProDH protein appears
to be targeted to mitochondria and the gene product was de-
tected immunologically in a mitochondria] fraction from
Arabidopsis cells (Kiyosue et al. 1996).
The expression of the gene for ProDH was strongly in-
duced by rehydration after dehydration for 10 h, but not
by heat or cold stress (Kiyosue et al. 1996). A low level of
mRNA for ProDH was detected transiently for 1 h after
the start of dehydration and then the level decreased
(Fig. 2). When plants were dehydrated for 10 h and then re-
hydrated, accumulation of the mRNA for ProDH began to
increase within 2 h, and large amounts of the mRNA were
detectable after rehydration for up to 48 h. The level for
Pro decreased rapidly for 10 h from the beginning of rehy-
dration but then decreased gradually for the next 38 h.
Figure 2 shows the relationship between the accumulation
of Pro and the level of expression of the gene for ProDH
during dehydration and rehydration. Furthermore, the ex-
pression of the gene for ProDH was strongly induced by ex-
ogenous L-Pro and D-Pro. Thus, the expression of the gene
for ProDH in plants was induced by Pro but repressed by
 Regulation of levels of proline in plants
osmotic stress (Kiyosue et al. 1996). Dehydration of plants
caused osmotic stress and led subsequently to elevated
levels of Pro in plant cells. The Pro formed several hours
after the onset of osmotic stress as a result of biosynthesis
de novo could not induce the expression of the gene for
ProDH because of repression by osmotic stress. When
plants were rehydrated, the expression of this gene became
inducible by Pro because of the removal of osmotic stress
and absence of repression (Fig. 3).
In dehydrated plants, the accumulation of Pro occurs
as the result of both the activation of its biosynthesis and
the inactivation of its degradation, whereas a decrease in
the accumulation of Pro occurs as a result of both the inac-
tivation of biosynthesis and the activation of degradation
in rehydrated plants. The decrease in ProDH activity in
water-stressed plants and the increase in this activity in re-
hydrated plants were observed by Rayapati and Stewart
(1991). They postulated that the decrease in activity of
ProDH might have been caused by a specific change in the
inner membrane of mitochondria. Our results demonstrate
that both the inactivation of ProDH in dehydrated plants
and its activation in rehydrated plants are regulated at the
level of the accumulation of the mRNA (Fig. 3). Therefore,
the activity of ProDH is regulated not only by the level of
the enzyme but also by the level of gene expression in
plants.
Transcriptional regulation of the level of proline
under water stress—The transcriptional regulation of the
level of Pro under dehydration and rehydration conditions
in Arabidopsis is shown schematically in Figure 3. The
gene for P5CS is induced by dehydration but is repressed
by rehydration. Expression of the gene for P5CR is slightly
upregulated by dehydration. These results indicate that the
induction of the gene for P5CS plays a major role in the
biosynthesis of Pro. The plant hormone ABA accumulates
under environmental stresses such as drought, high salini-
ty, and low temperature and it is involved in responses and
tolerance to dehydration (Giraudat et al. 1994). Many
genes that respond to water stress are also induced by the
exogenous application of ABA (Bohnert et al. 1995, In-
gram and Bartels 1996, Shinozaki and Yamaguchi-Shino-
zaki 1996, Bray 1997). It appears that water stress triggers
the production of ABA which, in turn, induces various
genes. Because the gene for P5CS is also induced by the ex-
ogenous application of ABA, it seems likely that this gene
is also one of many ABA-inducible genes. However, evi-
dence for the ABA-independent expression of the gene for
P5CS under dehydration conditions was also suggested by
studies of an ABA-deficient mutant (Yoshiba et al. un-
published results). These observations indicate, therefore,
that the expression of the gene for P5CS is induced by two
different pathways, an ABA-independent and an ABA-de-
pendent pathway, under dehydration conditions (Fig. 3).
Four independent signal-transduction pathways that
1099
control dehydration-induced genes have been proposed.
Two pathways are ABA-dependent and two are ABA-inde-
pendent (Shinozaki and Yamaguchi-Shinozaki 1996).
Two different ds-acting elements appear to function in
ABA-dependent and ABA-independent gene expression
under water stress. The ABRE (ABA-responsive element;
PyACGTGGC) functions as a cis-acting element that is in-
volved in ABA-responsive transcription. DNA-binding pro.-
teins that contain the bZIP motif have been shown to bind
to the ABRE sequence (Chandler and Robertson 1994,
Giraudat et al. 1994). A coupling element is also required
in conjunction with the ABRE to generate the ABA-respon-
sive complex (Shen and Ho 1995). By contrast, the DRE
(dehydration-responsive element; TACCGACAT) has been
identified as a cis-acting element involved in ABA-inde-
pendent gene expression under dehydration, high-salinity,
Downloaded from http://pcp.oxfordjournals.org/ by guest on March 21, 2016
and low-temperature conditions (Yamaguchi-Shinozaki
and Shinozaki 1994). The CCGAC core motif has been
found in the promoter regions of many cold- and drought-
inducible genes (Nordin et al. 1993, Baker et al. 1994,
White et al. 1994, Wang et al. 1995). Analysis of the pro-
moter of the P5CS gene should provide further informa-
tion about the control of the expression of this gene under
dehydration conditions.
The gene for ProDH is induced by rehydration but is
repressed by dehydration. Moreover, its expression is in-
duced by Pro and repressed by osmotic stress. The expres-
sion of the gene for ProDH might be inducible by an
elevated level of intracellular Pro during rehydration, but it
might be repressed by osmotic stress under dehydration
conditions (Fig. 3). Analysis of the promoter of the gene
for ProDH is in progress in an attempt to identify cis-ac-
ting elements that are involved in the Pro-inducible expres-
sion and repression of this gene by osmotic stress.
Localization and transportation of proline under
water stress—The maintenance of an appropriate water
potential during a water deficit can be achieved by osmotic
adjustment. A reduction in the cellular water potential
to below the external water potential, resulting from a
decrease in osmotic potential, allows water to move into
the cell. The osmotic potential inside the cell is lowered by
the accumulation in the cytosol of compatible solutes, such
as Pro. Accumulation of compatible solutes occurs pre-
ferentially in the cytosol under water stress (Hall et al.
1978, Leigh et al. 1981, Pahlich et al. 1983, Matoh et al.
1987). Fricke and Pahlich (1990) demonstrated that 34% of
the total intracellular Pro was accumulated in vacuoles in
non-water-stressed cultured cells of potato. They also ob-
served that the total amount of Pro in cells increased and
the amount of Pro in vacuoles decreased when cultured
cells were exposed to water stress. Thus, it seems that the
plant vacuole plays an important role in the accumulation
and transportation of Pro during water stress.
Recently, two genes encoding a proline transporter
1100 Regulation of levels of proline in plants
(ProT) were isolated from Arabidopsis by use of a yeast
targeting mutant (Frommer et al. 1993, Rentsch et al.
1996). A yeast mutant lacking SHR3, a protein that is spe-
cifically required for the correct targeting of plasma mem-
brane-localized amino acid permeases, was used to isolate
novel SHR3-independent transporters of amino acids. The
SHR3 gene was isolated by complementation of the shr mu-
tant with a cDNA library from Arabidopsis and it was
shown to encode a membrane protein that is located in
the endoplasmic reticulum and seems to serve as a cargo
specifier for plasma membrane amino acid permeases in
vesicles of the endoplasmic reticulum (Ljungdahl et al.
1992). Genes encoding eight different transporters of amino
acids, including two genes for specific proline transporters
(ProTl and ProT2) were isolated by Rentsch et al. (1996).
ProTl mRNA was found in all organs and, in particular in
roots, stems, and flowers. ProT2 mRNA was found ubi-
quitously in almost all tissues, but its level was strongly en-
hanced under water or salt stress. Thus, it is suggested that,
under water stress, ProT2 might play an important role in
the distribution of Pro. Under water-stressed conditions,
massive changes in the partitioning of nitrogen and carbon
take place, for example, depression of the delivery of
nitrate to shoots (Shaner and Boyer 1976) and a reduction
in phloem transport (Tully et al. 1979).
The relationship between the accumulation of proline
and tolerance to water stress—The role of Pro as an
osmoprotectant was demonstrated in Salmonella oranien-
burg. Christian (1955a, 1955b) reported that exogenous
Pro could alleviate the inhibition of growth of S. oranien-
burg that was due to osmotic stress. It was reported subse-
quently that a wide variety of osmotically stressed bacteria
accumulate Pro (Measures 1975). Moreover, a mutation
(proB74) in E. coli that resulted in the overproduction of
Pro also endowed a resistance to osmotic stress (Csonka et
al. 1988). These observations indicated that Pro can act as
an osmoprotectant, in other words, it can protect bacteria
from osmotic stress.
Eubacteria, protozoa, marine invertebrates, and many
plants including algae (e.g., halophytes, tobacco, spinach,
potato, tomato, Arabidopsis, alfalfa, field bean, soybean,
wheat, barley and rice) can all accumulate Pro (McCue
and Hanson 1990, Delauney and Verma 1993). Therefore,
among compatible organic solutes, it is probable that Pro
is the most widely distributed osmolyte. Tomato cells cul-
tured under water stress rapidly accumulated about 300
times more Pro than non-water-stressed cells, and they
adapted to osmotic stress (Handa et al. 1983, 1986, Rhodes
et al. 1986). These observations indicate that many plants
have the ability to adapt to water stress at the cellular level
and that Pro is involved in tolerance to osmotic stress, ac-
ting as a compatible osmolyte.
Kavi Kishor et al. (1995) reported that transgenic
tobacco plants that expressed a cDNA for mothbean P5CS
under the control of the 35S promoter of cauliflower mo-
saic virus produced a high level of the enzyme and subse-
quently accumulated 10- to 18-fold more Pro than control
plants. They also reported that overproduction of Pro en-
hanced root biomass and the development of flower in
transgenic plants exposed to drought. Thus, overproduc-
tion of Pro resulted in the increased tolerance of plants to
osmotic stress and it seems likely that Pro-overproducing
crop plants obtained by genetic engineering, might acquire
osmotolerance, namely, the ability to tolerate environmen-
tal stresses such as high salinity.
Igarashi et al. (1997) recently isolated a cDNA for
P5CS from rice, and they compared the level of the mRNA
for P5CS and the level of accumulation of Pro in a salt-sen-
sitive rice, IR28, and a salt-tolerant rice, Dee-gee-woo-gen
(DGWG), under high-salinity conditions. The transcript of
Downloaded from http://pcp.oxfordjournals.org/ by guest on March 21, 2016
the rice gene for P5CS appeared within 10 h after the start
of salt treatment, and the level of the transcript increased
for up to 48 h in both kinds of rice, but the level of the tran-
script in salt-tolerant DGWG was higher than that in salt-
sensitive IR28. The level of Pro that accumulated in
DGWG was also higher than that in IR28. These observa-
tions suggest that expression of the gene for P5CS and the
accumulation of Pro might be correlated with salt toler-
ance in rice.
Future perspectives—Plants respond to various types
of water stress, such as drought, high salinity, and low tem-
perature, by a number of physiological and developmental
changes. During water stress, plant cells can undergo
changes in concentrations of solutes, in cell volume and in
the shape of cell membranes, as well as disruption of gra-
dients in water potential, loss of turgor, disruption of mem-
brane integrity and the denaturation of proteins. Three
genes for enzymes involved in the biosynthesis and metabo-
lism of Pro have been cloned to date, and their expression
has been analyzed. A gene for P5CDH, which catalyzes the
conversion of P5C to Glu, has not been cloned from
plants, but the P5CDH protein has recently been purified.
In the near future, all the genes involved in the synthesis
and degradation of Pro should be cloned. From an analysis
of these genes, the functions of their products and the regu-
lation of their gene expression, we should develop a better
understanding of the role of Pro in stress reponses and de-
velopment at the molecular level. Regulation of the expres-
sion of genes for P5CS, P5CR, and ProDH is now being an-
alyzed at the transcriptional level by use of transgenic
plants. Both cis- and f/ww-acting elements that are in-
volved in the regulation of expression of these genes should
also be identified in the near future. An understanding of
the transcriptional regulation of Pro-related genes should
give us better insight into the roles and regulation of level
of Pro in the stress responses of plant cells, where Pro acts
as an osmolyte and as a source of energy and nitrogen.
Moreover, details of the tissue-specific expression of Pro-re-
 Regulation of levels of proline in plants
lated genes should provide more information about the
role of Pro in plant development.
The synthesis of Pro represents one response of plant
cells to a water deficit. Other known compatible solutes are
sugar alcohols (e.g., pinitol), other sugars (e.g., fructans)
and quaternary ammonium compounds (e.g., glycine be-
taine), and they can accumulate at high levels without
disruption of protein functions. It was reported that when
Synechococcus was transformed with genes for enzymes re-
lated to the synthesis of glycine betaine, the cells exhibited
enhanced tolerance to salt stress (Nomura et al. 1995,
Deshnium et al. 1995). In the case of higher plants, trans-
genic tobacco harboring the gene from E. coli for mann-
itol-1 -phosphate dehydrogenase accumulated mannitol in
the cytosol and this accumulation increased the tolerance
of plants to high salinity (Tranczynski et al. 1993). The ac-
cumulation of fructan or trehalose has also been shown to
promote stress tolerance in transgenic tobacco (Pilon-Smits
et al. 1995, Holmstrom et al. 1996). In all such transfor-
mants, the demonstrated tolerance to water stress did not
appear to be due to osmotic adjustment because the
amount of osmolyte that accumulated was insufficient to ac-
count for an alteration in gradients in water potential at the
cellular level. However, the cited reports do suggest that
the application of gene-engineering techniques to many
crops might improve their tolerance to water stress. In the
future, application of genetic engineering to the breeding
of environmental stress-tolerant transgenic plants should
lead to improvements in crop production in unfavorable en-
vironments, such as those with high salinity or insufficient
water.
This work was supported by the Program for Promotion of
Basic Research Activities for Innovative Biosciences, the Special
Coordination Fund of STA and a Grant-in-Aid from MESC of
Japan.
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Wang, H., Datla, R., Georges, F., Leowen, M. and Cuter, A.J. (1995)
(Received June 13, 1997; Accepted September 10, 1997)