Annals of Botany 96: 507518, 2005

doi:10.1093/aob/mci206, available online at www.aob.oupjournals.org

INVITED REVIEW

Sensing and Signalling in Response to Oxygen Deprivation in

Plants and Other Organisms
J U L I A B A I L E Y - S E R R E S * and R U T H C H A N G
Department of Botany and Plant Sciences, University of California, Riverside, CA 92521-0124, USA

Received: 14 January 2005 Returned for revision: 11 March 2005 Accepted: 19 April 2005 Published electronically: 28 July 2005

Aims and Scope All aerobic organisms require molecular di-oxygen (O2) for efficient production of ATP though
oxidative phosphorylation. Cellular depletion of oxygen results in rapid molecular and physiological acclimation.
The purpose of this review is to consider the processes of low oxygen sensing and response in diverse organisms,
with special consideration of plant cells.

Conclusions The sensing of oxygen deprivation in bacteria, fungi, metazoa and plants involves multiple sensors
and signal transduction pathways. Cellular responses result in a reprogramming of gene expression and metabolic
processes that enhance transient survival and can enable long-term tolerance to sub-optimal oxygen levels. The
mechanism of sensing can involve molecules that directly bind or react with oxygen (direct sensing), or recognition
of altered cellular homeostasis (indirect sensing). The growing knowledge of the activation of genes in response to
oxygen deprivation has provided additional information on the response and acclimation processes. Conservation of
calcium fluxes and reactive oxygen species as second messengers in signal transduction pathways in metazoa
and plants may reflect the elemental importance of rapid sensing of cellular restriction in oxygen by aerobic
organisms.

Key words: Oxygen sensing, gene expression, hypoxia, anoxia, alcohol dehydrogenase, reactive oxygen species, cytosolic
calcium, second messenger, G-protein, ethylene.

INTRODUCTION plants. Progress has been made toward the understanding of

the role of activation or de-repression of transcription fac-
Molecular di-oxygen (O2) is an absolute requirement for
tors and alterations in oxygen-regulated gene expression in
efficient production of ATP though oxidative phosphoryla-
several model prokaryotes and eukaryotes. The processes
tion in aerobic organisms. In eukaryotes, oxygen is the final
that determine these responses include multiple direct or
electron acceptor in the mitochondrial electron transport
indirect sensors and signal transduction pathways that
chain. A depletion of oxygen has rapid and profound
are independent or interacting (Fig. 1). Direct sensing
consequences on cell physiology. This condition alters
mechanisms might involve proteins or ligands that bind
gene expression, energy consumption, cellular metabolism,
or react with oxygen. Perturbations in cellular homeostasis,
growth and development. Plant cells are frequently chal-
such as altered energy levels, redox status or calcium levels,
lenged with limited levels of oxygen due to changes in the
may underlie indirect sensing mechanisms. Paradoxically,
external environment or high rates of cellular metabolism
eukaryotic responses to oxygen deprivation appear to
(reviewed by Drew, 1997; Geigenberger, 2003; Gibbs and
involve the formation of reactive oxygen species (ROS)
Greenway, 2003; Greenway and Gibbs, 2003). Natural
including the superoxide anion (O2), hydrogen peroxide
conditions such as spring floods, excess rainfall, winter
(H2O2) and nitric oxide (NO). This review summarizes
ice encasement, submergence, soil compaction and micro-
mechanisms of oxygen sensing in model bacterial and
organism activity can lead to oxygen deficiency. During
eukaryotic systems and discusses our nascent understanding
seed imbibtion and germination, microspore production
of low oxygen sensing and response mechanisms in plants.
or fruit development, the availability of oxygen for energy
production can become limited. This condition can also be
due to restricted diffusion of oxygen into internal tissues or
high rates of cellular metabolism, as in actively dividing OXYGEN SENSING MECHANISMS
cells of meristems. IN BACTERIA
Cells of aerobic organisms have evolved adaptive The most efficient pathway for the production of ATP in
responses to compensate for the energy crisis caused by Escherichia coli is the aerobic respiratory pathway that
oxygen deprivation. The responses at the cellular to utilizes cytochrome bo oxidase as the terminal electron
whole plant level are varied and include alterations in meta- acceptor. The activity of the cytochrome bd oxidase,
bolism and development that in some cases confer long- which has a higher affinity for oxygen, increases when
term tolerance. The mechanisms that underlie the sensing oxygen levels are limiting. Under anaerobic conditions,
and response to oxygen deprivation have not been fully less energy-efficient alternative oxidoreductases are
unravelled in any aerobic organism, let alone in higher utilized for ATP production. Escherichia coli adjusts gene
regulation as a consequence of oxygen deprivation through
* For correspondence. E-mail serres@ucr.edu multiple direct or indirect sensing mechanisms. These
The Author 2005. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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508 Bailey-Serres and Chang Initiation of Responses to Low Oxygen
O2 deprivation
DIRECT
O2 Homeostasis INDIRECT
SENSING sensor SENSING
sensor
Adaptive responses
F I G . 1. Sensing of oxygen deprivation through direct and indirect
mechanisms. Oxygen deprivation can result in rapid changes in cell
physiology, transient changes in gene transcription or long-term
alterations in physiology and development. These adaptive responses are
promoted by rapid alteration in the accumulation, location or activity of
transcription factors or activation of signal transduction pathways. The
sensing of oxygen deprivation involves molecules which bind or
consume oxygen or that are altered by oxidation state. Indirect sensing
occurs as a result of a change in cellular homeostasis, possibly driven by
flux in cytosolic calcium levels, adenylate charge, ratio of reduced to
oxidized glutathione and carbohydrate availability. Diversity in
responses may result from cross-talk between one or more sensing and
signalling pathways.
adjustments modulate the transcription of genes that encode
the various oxidoreductases and other machinery of cellular
metabolism. One of these oxygen sensing regulatory sys-
tems is the fumarate and nitrate reduction (FNR) transcrip-
tional regulator. FNR is regulated directly by molecular
oxygen concentration. Under fully aerobic conditions,
FNR is inactive, whereas under low oxygen conditions
FNR forms homodimers that repress the transcription of
genes required for aerobic metabolism and promote expres-
sion of genes involved in anaerobic electron transport
and metabolism (Kiley and Beinert, 2003). The underlying
mechanism of FNR regulation is the presence of a struc-
turally dynamic cluster of iron molecules, the [4Fe4S]
cluster. The active homodimeric factor has a [4Fe4S]2+
cluster in each subunit. In the presence of oxygen,
the redox state of this cluster is rapidly converted to
[2Fe2S]2+, promoting a change in conformation that
disrupts FNR dimerization and DNA binding activity.
A second system in E. coli that controls gene expression
under conditions of reduced oxygen availability is the
ArcAB system. This two-component system includes the
membrane-bound histidine kinase ArcB and its phos-
phorylation target ArcA. Oxygen deficiency promotes the
auto-phosphorylation of ArcB that activates phosphoryla-
tion of ArcA and results in regulation of numerous operons
that provide control of carbon catabolism and cellular redox
status (Unden et al., 1997; Alexeeva et al., 2003). ArcB
auto-phosphorylation is not regulated by direct oxygen
sensing but is a response to a reduction in electron flow
through the respiratory chain (Alexeeva et al., 2000). Thus,
the ArcAB system mediates fine-tuned responses to the
reduction in cellular ATP levels that occur as oxygen avail-
ability diminishes (Alexeeva et al., 2003).
Root nodule-forming Rhizobia and Bradyrhizobium
species adjust metabolism in response to oxygen levels
by use of another two-component system (Fischer, 1994).
The direct sensing of oxygen tension occurs through
an oxygen- and haem-binding histidine kinase, FixL.
When oxygen tension is severely reduced during root
nodule formation, oxygen is released from the haem-
binding site of membrane-bound FixL and triggers its auto-
phosphorylation. The kinase then transfers a phosphate to
the transcription factor FixJ, causing a change in conforma-
tion that results in activation of transcription of genes
required for symbiotic nitrogenase reactions (Fischer,
1994; Gong et al., 2000). The activity of FixJ regulation
increases as oxygen levels fall, providing tight environ-
mental regulation of gene expression during symbiont
invasion (Sciotti et al., 2003).
OXYGEN SENSORS AND SIGNALLING
PATHWAYS IN MODEL METAZOANS
AND FUNGI
Depletion of cellular oxygen levels promotes changes in
gene regulation in yeast and metazoans through varying
direct or indirect sensing mechanisms. There is considerable
knowledge of the transcription factors that mediate altera-
tions in gene expression. In contrast, the signal transduc-
tion pathways activated in response to hypoxia in model
eukaryotes are complex and wrought with opposing models,
especially those that propose modulation of ROS as a
second messenger (Lopez-Borneo et al., 2001; Bruick,
2003; Giaccia et al., 2004; Waypa and Schumaker,
2005). Interest in oxygen sensing in mammals is intense
due to its importance in erythropoiesis, tissue vasculariza-
tion, trachea development, closure of the ductus arterious in
newborns, progression of tumour development, and control
of blood oxygen levels by the carotid and aortic bodies
of the lung, heart and vascular smooth muscle cells. The
emerging view is that a cellular oxygen crisis promotes
multiple sensing and physiological response pathways.
The HIF1a story: transcriptional regulation by direct
oxygen sensing
A key regulator of transcription in response to hypoxia in
animals is the hypoxia-inducible heterodimeric transcrip-
tion factor (HIF) (reviewed by Bruick, 2003; Acker and
Acker, 2004; Giaccia et al., 2004; Semenza, 2004). This
complex is highly conserved in metazoa including
mammals, Drosophila melanogaster and Caenorhabditis
elegans. HIF is composed of the constitutively synthesized
HIF1a and HIF1b subunits, and its activation promotes
transcription of genes required for anaerobic metabolism,
iron homeostasis, vascularization and erythropoiesis in
mammals. HIF activity also indirectly inhibits the expres-
sion of genes through the activation of a transcriptional
repressor, Dec1 (Yun et al., 2002). HIF is controlled by
multiple parameters, including mRNA accumulation and
alternative splicing, protein turnover, nuclear localization
and transactivation. Primary control is through turnover of
the HIF1a subunit when oxygen is available. Oxygen-
dependent HIF prolyl hydroxylases modify specific proline
residues within HIF1a in a reaction that is dependent upon
 Bailey-Serres and Chang Initiation of Responses to Low Oxygen
2-oxogluturate and ascorbate. This proline hydroxylation
targets HIF1a for rapid ubiquitination and proteosomal
degradation (Ivan et al., 2001; Jaakkola, 2001). The three
human HIF1a prolyl hydroxylases have a high Km for O2
(230250 mM) (Hirsila et al., 2003). Therefore, their
activity is proportional to oxygen availability. This oxygen
concentration-dependent activity qualifies the prolyl hydro-
xylases as a bone fide oxygen sensor (Bruick, 2003).
An additional level of HIF regulation is mediated by a
second 2-oxyglutarate-dependent hydroxylase, an aspar-
aginyl hydroxylase. This enzyme modifies an asparagine
residue within the N-terminal trans-activation domain of
HIF1a (Bruick, 2003). This change reduces the interaction
of HIF with transcriptional co-activators, providing another
level of control through direct oxygen sensing. Additional
enhancement of HIF activity, via indirect sensing of oxygen
deprivation, involves elevation of HIF1a mRNA via
production of ROS that promotes a G-protein signalling
cascade (Turcotte et al., 2003, 2004).
Transcriptional regulation by haem-binding
proteins in yeast
Saccharomyces cerevisiae can grow anaerobically if
provided with a fermentable carbon source. Yeast cells
respond to oxygen deprivation via multiple low oxygen
sensing and transduction pathways (Kwast et al., 1998;
Poyton, 1999). A predominant pathway involves haem,
an oxygen-binding molecule, in the adjustment of transcrip-
tion by haem-containing factors. Unlike the direct oxygen
sensing mediated by haem-containing transcription factors
in bacteria, transcriptional regulation by the yeast factors is
governed by rates of de novo synthesis of haem, in a redox-
insensitive manner. The activity of the haem biosynthesis
pathway is proportional to oxygen concentration at levels
above 01 mM O2, due to properties of several haem biosyn-
thesis enzymes within the mitochondrion (Hon et al., 2003).
Consequentially, oxygen depletion dramatically influences
the production of haem, which is necessary for the tran-
scriptional activator Hap1 to drive expression of genes
required for aerobic metabolism (Poyton, 1999; Zhang
and Hach, 1999). The reduction in free haem levels
under hypoxia also compromises the stability of Hap1
(Hon et al., 2003). In turn, reduced Hap1 activity limits
the production of two transcriptional repressors, Rox1
and Mot3, and thereby de-represses transcription of a
number of genes required for anaerobic metabolism.
Another haem-binding transcription factor, the tetrameric
Hap2/3/4/5, is also regulated by haem levels and controls
the expression of aerobic genes in a manner similar to Hap1.
The enigmatic role of ROS in oxygen sensing and
response mechanisms
There is considerable evidence that ROS production, by
either a plasma membrane (PM) NAD(P)H oxidase and/or
mitochondria, regulates responses to oxygen deprivation.
There are at least four models that propose a role for
ROS produced by one or both of these sources (Sham,
2002). One model proposes that O2 produced by the PM
509
NAD(P)H oxidase functions to constitutively repress low
oxygen signalling pathways (Lopez-Barneo et al., 2001,
2004). In this scenario, H2O2 generated by enzymatic or
spontaneous dismutation of O2 maintains a cellular oxid-
ization state that inhibits signal transduction in cell types
such as arterial and airway chemoreceptors. Hypoxia
reduces this ROS production leading to a cascade of events:
alteration of cytosolic redox state, closure of oxygen-
sensitive voltage-dependent potassium channels, activation
of voltage-gated calcium channels and an elevation of
cytosolic calcium (Archer et al., 2000; Lopez-Barneo
et al., 2001, 2003). Although there is considerable support
for this PM NAD(P)H oxidase model, it is unlikely to reflect
oxygen sensing in all cell types. Examination of PM
NAD(P)H oxidase-deficient mice revealed that pulmonary
vascular and neuroepithelial cells differ in the requirement
for this enzyme in the response to hypoxia (Archer et al.,
1999; Fu et al., 2000). Moreover, diphenyl iodonium (DPI),
a non-specific inhibitor of flavin-binding proteins including
the PM NAD(P)H oxidase, nitric oxide synthase (NOS)
and mitochondrial NADH dehydrogenase (complex I), is
an effective inhibitor of hypoxia responses in mammalian
cells, leading to the suggestion that ROS evolution may
actually be promoted under conditions of oxygen deficiency
(Chandel et al., 2000).
Another model proposes that the mitochondrion, the
major consumer of cellular oxygen, is a sensor of oxygen
availability (Chandel et al., 1998, 2000; Chandel and
Schumaker, 2000; Schumaker, 2003). In this scenario,
hypoxic conditions promote production of ROS within
mitochondria, leading to a release of calcium and other
molecules, such as cytochrome c. In support of the mito-
chondrial sensor hypothesis, mammalian and yeast cell
lines deficient in mitochondrial electron transport chain
components or treated with compounds that block electron
transport upstream of centre N of complex III are impaired
in hypoxia responses (Chandel et al., 1998, 2000; Chandel
and Schumaker, 2000; Dirmeier et al., 2002; Schroedl
et al., 2002; Waypa et al., 2002; Waypa and Schumaker,
2005). In contrast, antimycin A, which blocks electron
transport at centre N of complex III, mimics hypoxia
responses including the elevation of HIF1a mRNA.
These studies attribute mitochondrial ROS production to
the elevation of ubisemiquinone ion, which donates an elec-
tron to oxygen to produce O2. The effective production of
O2 is purportedly stimulated by a decrease in the Vmax
of cytochrome c oxidase (Chandel and Schumaker, 2000).
Arguments against the mitochondrial sensor model cite the
failure of cytochrome c oxidase inhibitors to mimic the low
oxygen response in mammals and the inconsistency in the
effect of mitochondrial inhibitors on different cell types.
However, cytochrome c oxidase-deficient strains of yeast
display altered expression of a sub-set of the hypoxia-
induced genes (Kwast et al., 1999). A current challenge
is to determine if the proposed oxygen concentration sens-
ing by mitochondria is mediated directly by oxygen
(i.e. through regulation of haem) or a change in cellular
homeostasis (i.e. ATP levels or adenylate charge ratio
([ATP] + 05[ADP])/([ATP] + [ADP] + [AMP]),[NAD(P)+]/
[NAD(P)H], [GSSG]/[GSH]). If the latter is correct, the
510 Bailey-Serres and Chang Initiation of Responses to Low Oxygen
mitochondrial response may be similar to the indirect
sensing paradigm of ArcAB in E. coli.
NO, a gaseous second messenger, is implicated in oxygen
sensing and the responses leading to vasodilatation in
mammals (Shiva et al., 2005). In hypoxic cells, NO
binds competitively to the haem group of cytochrome c
oxidase and inhibits its activity (Hagen et al., 2003).
This interaction was proposed to enhance the maintenance
of HIF1a prolyl hydroxylase activity, thereby delaying the
increase in HIF activity. It was also suggested that NO
controls the production of O2 by mitochondria and allows
establishment of oxygen gradients within organs (Shiva
et al., 2005). Confirmation of a role for NO in the response
to hypoxia in Drosophila embryos indicates that this ROS
may be involved in evolutionarily conserved response
mechanisms.
There may be cross-talk between ROS produced at the
PM and within mitochondria in low oxygen response mech-
anisms. Both the PM NADPH oxidase and mitochondrial
sensor models involve the modulation of production of ROS
and flux in cytosolic calcium. It can be predicted that these
processes involve positive and negative feedback systems
that are controlled by the spatial and temporal location
of these second messengers. It seems likely that the presence
of multiple interacting sensory circuits would enhance
the diversity and fine-tuning of the response to oxygen
deprivation (Fig. 1).
LOW OXYGEN SENSING AND SIGNAL
TRANSDUCTION MECHANISMS IN
PLANT CELLS
Evidence for indirect low oxygen sensing
mechanisms in plants
To date, there is no clear understanding of the sensor(s)
of oxygen deprivation in plant cells (Drew, 1997;
Geigenberger, 2003; Gibbs and Greenway, 2003). Analyses
of the molecular responses of maize (Zea mays), rice
(Oryza sativa) and arabidopsis (Arabidopsis thaliana) to
oxygen deprivation have focused on the regulation of
expression of genes and activation of enzymes involved
in acclimation of metabolism and alteration of development.
These include enzymes involved in the breakdown of
starch, catabolism of soluble sugars and fermentation,
aerenchyma formation, cell and organ elongation and
adventitious root formation. As in other multicellular
eukaryotes, the plant response depends upon the severity
of the stress, cell type and developmental stage. Cells may
endure different degrees of oxygen deficiency within a plant
tissue or organ. As the external oxygen concentration
declines, the rate of oxygen uptake into internal cells
must keep up with the rate of oxygen consumption. A
zone of anoxia may occur in internal tissues, as observed
in the stele of maize roots or the centre of fruits or tubers,
even when surrounding cells have sufficient oxygen to
maintain aerobic respiration (Geigenberger, 2003; Gibbs
and Greenway, 2003). It is well established that adaptive
responses, such as increased alcohol dehydrogenase (ADH)
gene expression and fermentative metabolism, are initiated
under conditions of hypoxia, as well as anoxia (Paul and
Ferl, 1991; Drew, 1997; Koch et al., 2000). Plant cells have
evolved adaptive mechanisms that allow for avoidance of
anoxia by reducing aerobic respiration and energetic
processes as ATP levels decline (Geigenberger, 2003;
van Dongen et al., 2004). The sensing and signalling that
lead to modification of gene expression may be triggered
by a change in cellular homeostasis and not necessarily
by a direct sensing of oxygen concentration (Figs 1, 2).
As revealed by the ArcAB system of E. coli and
indicated by the mitochondrial sensing model of metazoa,
a universal indirect response to hypoxia may be curtailment
of the consumption of limited ATP and other limited
resources such as carbohydrates and lipids. It remains to
be determined if the plant response is mediated exclusively
by indirect sensing mechanisms or also involves direct
oxygen sensing.
An oxygen sensor is capable of directly detecting oxygen
availability and subsequently triggering a signalling cas-
cade. It is still unclear if such sensors exist in plants. The
plant oxygen-binding protein, haemoglobin, has been ruled
out as a potential oxygen sensor or carrier because of its
extremely low dissociation constant for oxygen (Dordas
et al., 2003). Although haem-containing transcription
factors have not been reported in plants, there is evidence
of redox-sensitive transcription factors, at least one of which
might be involved in the adaptive response to low oxygen.
ZAT12, a putative zinc finger-containing transcription
factor, is recognized as a component in the oxidative stress
response signalling network of arabidopsis (Rizhsky et al.,
2004). During oxidative stress, ZAT12 promotes expression
of other transcription factors and the upregulation of cyto-
solic ascorbate peroxidase 1, a key enzyme in the removal of
H2O2. Accumulation of ROS is a common consequence of
biotic and abiotic stresses, including oxygen deprivation
and re-oxygenation (discussed below). ZAT12 transcript
levels were significantly elevated in response to hypoxia
and anoxia in several independent analyses (Branco-Price
et al., 2005), indicating involvement of this potentially
redox-regulated factor.
Another mechanism of indirect oxygen sensing in plant
cells may be manifested through the rapid reduction in
cytosolic pH (Fig. 2). A 0206 unit decline in cytoplasmic
pH occurs within 15 min of oxygen deprivation and
is associated with changes in cell physiology (reviewed
by Wilkinson, 1999; Greenway and Gibbs, 2003). For
example, the decrease in cytosolic pH is proposed to inhibit
water transport by water channel proteins (aquaporins) of
the PM intrinsic protein subgroup under anoxia (Tournaire-
Roux et al., 2003). Greenway and Gibbs (2003) suggested
that the decrease in cytosolic pH, typically from about 75
to 71 in anoxia-tolerant tissues, aids in the acclimation to
the cellular energy crisis. This drop in pH may contribute to
the reduction in the energy consumptive process of protein
synthesis and stimulation of ethanolic fermentation
(Roberts et al., 1984a, b; Webster et al., 1991).
Davies biochemical pH-stat hypothesis predicts that
anaerobic metabolism is modulated by the activities
of pH-sensitive enzymes, but has been highly debated
(Davies, 1986; Drew, 1997; Tadege et al., 1999;
 Bailey-Serres and Chang Initiation of Responses to Low Oxygen 511
O2 deprivation

ATP
DIRECT INDIRECT [pH]cyt
O2 SENSING SENSING [Ca2+]cyt
ROS

Initiation of signal transduction

GTP GDP
Other Growth
pathways? ROP-GDP ROP-GTP regulators

Pi ROPGAP

Antioxidant Elevated
Carbohydrate Developmental
defence ethanolic
conservation adaptations
Limited ROS fermentation

F I G . 2. Sensing and signalling in response to oxygen deprivation in plant cells. Depression of cellular oxygen concentration leads to changes in the cellular
milieu that promote altered gene expression, metabolism and development. To date, there is no known mechanism of direct oxygen sensing in plant cells.
Indirect sensing may be regulated by the changes in cytosolic pH, local and temporal fluxes in calcium concentration, reduction in adenylate charge or
production of ROS, including NO and H2O2. The accumulation of the active form of the ROP GTPase, ROP-GTP, stimulates the induction of ADH expression
and ethanolic fermentation, at least in Arabidopsis. The increase in cytosolic calcium, released from the mitochondria and influx from the apoplast, contributes
to this gene regulation. Paradoxically, the activation of ROP signalling is associated with increased levels of H2O2; genotypes that are unable to regulate ROP
signalling negatively are sensitive to oxygen deprivation despite strong induction of ADH gene expression. ROP GTPase signalling is attenuated by a
ROPGAP, which promotes hydrolysis of GTP that is bound to ROP. This negative regulation limits ROS production and most probably conserves
carbohydrates. H2O2 is a signalling molecule in plant cells as well as a damaging agent; antioxidants and antioxidant enzymes ameliorate its
accumulation. NO is also a signalling molecule; its levels may be regulated by increases in haemoglobin in response to oxygen deprivation. The
growth regulators ethylene, gibberellin, auxin and ABA control developmental adaptations including aerenchyma formation, cell, stem and petiole
elongation, petiole hyponasty and adventitious root formation. There is evidence of cross-talk between the response pathways.

Geigenberger, 2003; Greenway and Gibbs, 2003). Both
lactate- and ethanol-producing fermentations yield NAD+.
Under conditions of oxygen deprivation, the pyruvate pro-
duced by glycolysis may initially be converted to lactate
in a reaction catalysed by lactate dehydrogenase (LDH).
Lactate accumulation, however, contributes to cytosolic
acidosis and may ultimately cause cell death if not con-
trolled. As the cytosolic pH falls near to 70, pyruvate
decarboxylase (PDC) activity increases, promoting the con-
version of pyruvate to acetaldehyde, which is reduced to
ethanol by ADH. Ethanol, an uncharged molecule, can
cross the PM, whereas lactate efflux can require ATP con-
sumption. The root tips of maize seedlings that are deficient
in ADH specific activity succumb more rapidly to oxygen
deprivation due to an inability to limit lactate production
and the decline in cytosolic pH (Roberts et al., 1984b),
whereas those root tips acclimated to low oxygen condi-
tions are better equipped to extrude protons from the cyto-
sol (Xia and Roberts, 1996). Thus, avoidance of cytosolic
acidosis is likely to involve the mode of fermentation as
well as the activity of PM H+ ATPases. Tadege et al. (1999)
challenged the hypothesis that lactate-induced acidification
triggers the onset of ethanol production, citing that the
reduction in cytosolic pH and onset of lactate fermentation
were not well correlated in studies of several species. These
authors proposed that flux of pyruvate to ethanol is not
regulated by lactate production but rather by pyruvate
availability, noting that the Km for pyruvate of pyruvate
dehydrogenase (PDH) is in the mM range whereas that of
PDC is in the mM range. An important observation is
that fermentation can occur under aerobic conditions and
may not necessarily be driven by a decrease in cytosolic
pH. Nonetheless, alteration of cytosolic pH is likely to
impact specific metabolic pathways and could play a
role in indirect oxygen sensing.
Evaluation of oxygen deprivation-induced changes in
mRNA accumulation and recognition of signalling
machinery and response mechanisms
It is well established that gene regulation is altered in
response to oxygen deprivation in plants. Both early
molecular investigations and recent mRNA profiling
studies have demonstrated that regulation occurs at the
level of mRNA accumulation and translation in A. thaliana
and other model species (Sachs et al., 1980; Bailey-Serres,
1999; Klok et al., 2002; Paul et al., 2003; Branco-Price et al.,
2005; Liu et al., 2005; Loreti et al., 2005). Profiling of the
mRNAs in large polyribosome complexes confirmed that
selective mRNA translation is a significant regulatory
mechanism under hypoxia (Branco-Price et al., 2005).
The strong impairment of protein synthesis is most
512 Bailey-Serres and Chang Initiation of Responses to Low Oxygen
probably a mechanism of energy conservation since a large
proportion of cellular mRNAs show no reduction in steady-
state abundance but are poorly translated under hypoxia.
Alterations in mRNA accumulation in response to oxygen
deprivation are regulated in a temporal manner and depend-
ent upon multiple parameters including the severity of the
stress, light and nutrient availability, tissue, organ and
developmental age. The genes that are induced by anoxia
and hypoxia in arabidopsis are extremely diverse and
include proteins involved in carbohydrate catabolism, gly-
colysis, ethanolic and other fermentation pathways, lipid
metabolism, ethylene synthesis, auxin-mediated processes,
amelioration of ROS, calcium and ROS-mediated signal
transduction and gene transcription (Klok et al., 2002;
Paul et al., 2003; Branco-Price et al., 2005; Liu et al.,
2005; Loreti et al., 2005). A comparison of the results
from several of these experiments is presented by
Branco-Price et al. (2005). Although these experiments
involved different organs, developmental ages and treat-
ment conditions, increases in a number of gene transcripts
were observed in multiple studies and are consistent with
known physiological and developmental processes that are
affected by low oxygen stress. Interestingly, a significant
proportion of the hypoxia-induced genes encode proteins of
unknown function, providing a challenge for future invest-
igations. Despite the complex nature of RNA profiling data,
such data are useful for the development of hypotheses
on signal transduction events because genes that encode
signalling components are frequently upregulated when
the pathway is active (Leonhardt et al., 2004; Davletova
et al., 2005).
The DNA microarray reports have identified transcription
factor/activator mRNAs that increase in response to various
regimes of oxygen deprivation in arabidopsis (Klok et al.,
2002; Branco-Price et al., 2005; Liu et al., 2005; Loreti
et al., 2005). These include heat shock factors, ethylene
response-binding proteins, MADS-box proteins, AP2
domain, leucine zipper, zinc finger and WRKY factors.
Plants lack orthologues of mammalian HIF1a or yeast
Hap1, Rox1 or Mot3; other factors must control the
transcriptional reprogramming that occurs. A transcription
factor shown to regulate gene expression under hypoxia in
plants is the MYB-class transcription factor AtMYB2 of
arabidopsis (Hoeren et al., 1998; Dolferus et al., 2003).
This factor binds a GT-motif sequence element present in
the 50 -flanking region of ADH1 and a number of hypoxia-
induced genes of diverse species (Hoeren et al., 1998; Klok
et al., 2002; Dolferus et al., 2003; Liu et al., 2005). Other
sequence motifs present at a significant frequency in hyp-
oxia-induced genes include a SURE-a-like element that
interacts with WRKY factors and a G-box-like element
that interacts with bZIP factors (Liu et al., 2005).
The regulation of AtMYB2 appears to be governed by
differential turnover of AtMYB2 mRNA (Hoeren et al.,
1998; Dolferus et al., 2003). Treatment of cultured ara-
bidopsis seedlings with cycloheximide, an effective inhib-
itor of protein synthesis, promoted an increase in AtMYB2
mRNA under non-stress conditions (Hoeren et al., 1998).
A change in mRNA stability in response to this drug is
typical of mRNAs that are destabilized by constitutively
synthesized proteins (Gutierrez et al., 1999). AtMYB2
mRNA may be stabilized under hypoxia as a consequence
of the depletion of a factor with a short half-life (Dolferus
et al., 2003). This could be regulated through direct or
indirect oxygen sensing by proteins involved in mRNA
degradation. Alternatively, AtMYB2 mRNA stability may
be regulated by differential mRNA translation when oxygen
or nucleotide triphosphate levels are limiting. AtMYB2
mRNA accumulation is promoted by cold temperature,
dehydration stress and wounding (Dolferus et al., 2003),
all of which reduce global levels of protein synthesis and
increase differential mRNA translation (Bailey-Serres,
1999; Kawaguchi and Bailey-Serres, 2002; Kawaguchi
et al., 2004).
The independent DNA microarray analyses have identi-
fied a number of genes that encode putative components of
signal transduction pathways (Klok et al., 2002; Branco-
Price et al., 2005; Liu et al., 2005; Loreti et al., 2005). These
include calcium-binding proteins, protein-modifying
enzymes [i.e. receptor-like kinases and mitogen-activated
protein (MAP) kinase], and known signalling pathway com-
ponents such as the gp91-phox subunit of the respiratory
burst oxidase NAD(P)H oxidase. Several of these RNA
profiling studies reported the significant induction of the
non-symbiotic haemoglobin mRNA, which participates in
regulation of cellular redox and energy status under hypoxia
in an NO- and nitrate-dependent manner and has been
shown to enhance survival of hypoxia (Dordas et al.,
2003; Igamberdiev and Hill, 2004). It is of particular interest
to know whether the low oxygen sensing mechanisms and
signalling processes in plants and other eukaryotes are
evolutionarily conserved, as implicated for NO in diverse
eukaryotes.
Calcium flux is necessary for ADH induction
under oxygen deprivation
Hypoxia promotes an activation of voltage-gated PM
channels and influx of calcium as well as flux in mito-
chondrial calcium levels in mammalian cells (Archer et al.,
2000; Lopez-Barneo, 2001, 2004). In plants, including
maize, rice and arabidopsis, movement of calcium is a
prerequisite for certain alterations in gene expression in
response to anoxia and hypoxia (Subbaiah and Sachs,
2003) (Fig. 2). Maize cells require an increase in cytosolic
calcium for the induction of expression of Adh1 and other
genes (Subbaiah et al., 1994a, b; He et al., 1996). Fluor-
escent imaging analyses demonstrated that a reversible
increase in cytosolic calcium occurs immediately follow-
ing transfer of maize cells to anoxia (Subbaiah et al.,
1994a, 1998). Treatment of cells with caffeine, which
also promotes an increase in cytosolic calcium, was suf-
ficient to induce Adh1 (Subbaiah et al., 1994a). However,
anoxia further stimulated an increase in cytosolic calcium
in caffeine-treated cells, suggesting that calcium is
released from both caffeine-sensitive and -insensitive
stores in response to anoxia. Reversible changes in cal-
cium levels inside and on the periphery of mitochondria
were observed upon transfer to anoxia and following
re-oxygenation, leading to the conclusion that anoxia
 Bailey-Serres and Chang Initiation of Responses to Low Oxygen 513
stimulates calcium release from mitochondria (Subbaiah A GTPase rheostat regulates ADH1 induction
et al., 1998). The increase in cytosolic calcium and release in response to hypoxia in arabidopsis
of calcium from mitochondria under anoxia were dramat-
A screen for transposon insertions into genes induced by
ically inhibited by ruthenium red, which blocks mito-
hypoxia that influence the induction of ADH1 expression
chondrial and endoplasmic reticulum calcium channels
revealed that the RHO-like GTPases of plants (ROP)
(Subbaiah et al., 1994a, 1998). Further evidence for a role
monomeric GTPase modulates the induction of ADH1 in
for mitochondria in the induction of Adh1 was revealed in
arabidopsis seedlings (Baxter-Burrell et al., 2002, 2003).
a study of gene regulation in the T-cytoplasm mitochon-
Based on findings with arabidopsis, it was proposed that
drial genotype of maize. Methomyl, which uncouples the
a ROP GTPase rheostat promotes ethanolic fermentation
mitochondrial inner membrane potential and promotes
when active and conserves carbohydrates when inactive
calcium release from the organelle, engendered a strong
(Fukao and Bailey-Serres, 2004) (Fig. 2). The ROP family
induction in Adh1 transcript accumulation in the absence
of proteins activates and represses signalling cascades that
of oxygen deprivation (Kuzmin et al., 2004). These data
control diverse mechanisms in plant cells (Gu et al., 2004).
suggest that dynamic alterations in mitochondrial and
Constitutive active and dominant-negative mutants of
cytosolic calcium, which are likely to play a role as
ROP2, one of the ten ROP proteins of arabidopsis, showed
second messengers, are important in the response of
increased sensitivity to hypoxia and altered induction of
oxygen-deprived maize cells.
ADH1 at the seedling stage. The importance of regulation
Calcium modulation is also necessary for responses to
of ROP-GTP and ROP-GTP levels in ADH induction was
hypoxia in arabidopsis and rice. Sedbrook et al. (1996)
implicated further by the phenotype of a loss-of-function
monitored arabidopsis seedlings expressing a calcium-
mutant of a negative regulator of ROP signalling,
sensitive luminescent protein, jellyfish AEQUORIN, and
ROPGAP4. ropgap4-1 seedlings showed increased sensit-
confirmed three oscillations in cytosolic calcium in response
ivity to hypoxia and uncontrolled induction of ADH1. The
to anoxia. Transfer to anoxia caused a rapid transient spike
examination of these mutants led to the finding that the
(10 min) in calcium followed by a prolonged increase
levels of the active form of ROP, ROP-GTP, increased
(154 h), whereas re-oxygenation also elicited a transient
within 15 h of oxygen deprivation and declined after pro-
spike. The initial spike was less prominent in seedlings
longed stress (>12 h oxygen deprivation under low light).
repeatedly exposed to 10 min of anoxia or pre-treated
These observations indicate that ROP activation is
with ruthenium red, the calcium chelator EGTA or the
necessary to promote ADH1 induction, but moderation
PM calcium channel blockers lanthanum and gadolinium
and reversibility of ROP signalling is an additional pre-
chloride. In contrast, the prolonged increase in cytosolic
requisite for survival of transient hypoxia.
calcium was greatly enhanced in the presence of gadolinium
Further studies revealed that a consequence of ROP
or lanthanum chloride. These observations led to the pro-
activation was the induction of H2O2 accumulation. The
posal that the initial calcium increase results from an influx
elevation of H2O2 in crude cell extracts was inhibited by
of calcium from the apoplast and release of calcium from
DPI, an inhibitor of flavin-binding proteins. DPI also dra-
an internal store(s). The secondary calcium transient most
matically inhibited increases in ADH activity, leading to the
probably involves the activity of PM channels.
suggestion that ROS production is a necessary component
Curiously, the calcium signatures recorded in these seed-
of a low oxygen signalling pathway. However, the inability
lings were primarily the response of the stems and cotyle-
of the ropgap4-1 mutant to control the elevation in H2O2
dons; roots showed no reproducible increase in cytosolic
indicated that negative regulation of the signalling is neces-
calcium (Sedbrook et al., 1996). These investigators con-
sary to avoid oxidative stress (Baxter-Burrell et al., 2002;
cluded that either root and aerial tissues have distinct
Fukao and Bailey-Serres, 2004). These findings support the
responses or the AEQUORIN of their system lacked sens-
hypothesis that rheostat-like regulation of ROP activity
itivity to record calcium modulation in roots. Most prob-
mediates temporal activation of an H2O2-dependent signal-
ably, the latter is correct since increased cytosolic calcium
ling pathway that leads to ADH1 expression. The import-
was required to drive expression of the ADH1 promoter in
ance of the production and amelioration of ROS in the
the roots and shoot apex of arabidopsis (Chung and Ferl,
response of plant cells to oxygen deprivation was recog-
1999). The presence of ruthenium red, EGTA and gadolin-
nized prior to the elucidation of the ROP rheostat (Blokhina
ium chloride effectively blocked the increase in reporter
et al., 2003). A DPI-sensitive increase in H2O2 was reported
enzyme activity observed in the absence of these com-
in response to hypoxia in rhizomes of two iris species (Iris
pounds. In rice, treatment of seedlings with ruthenium
pseudoacorus and Iris germanica). By use of electron
red inhibited the induction of ADH1 and alternative
microscopy, these studies detected H2O2 accumulation in
oxidase1a (AOX1a) mRNA, but did not influence the
the apoplast and in association with the PM in hypoxic
decrease in abundance of several transcripts (Tsuji et al.,
tissues. An intriguing possibility is that the source of this
2000). Together, these studies indicate that low oxygen-
ROS is a PM NAD(P)H oxidase.
induced alterations in gene expression in plants can be
There may be a connection between ROP-promoted H2O2
dependent upon calcium flux(es). Further research is needed
production and the hypoxia-induced increase in cytosolic
to resolve the biological relevance of the spatial and
calcium. Caffeine treatment mimics anoxia in maize, at
temporal distinctions in calcium transients and to determine
least in part, by increasing cytosolic calcium (Subbaiah
the specific role(s) of calcium in signal transduction under
et al., 1994a, 1998). When arabidopsis seedlings were
oxygen deprivation.
514 Bailey-Serres and Chang Initiation of Responses to Low Oxygen
transferred to solid medium that contained caffeine,
significant increases in ROP-GTP levels and ADH specific
activity were observed (Baxter-Burrell et al., 2002). An
interaction between ROP signalling and calcium is suppor-
ted by the demonstration that EDTA and the calcium
channel antagonists ruthenium red and lanthanum chloride
affect the accumulation of ROP-GTP in response to hypoxia
(A. Baxter-Burrell and J. Bailey-Serres, unpubl. res.).
Interestingly, an increase in cytosolic calcium is a likely
prerequisite for the activation of apoplastic H2O2 produc-
tion by the calcium-dependent NAD(P)H oxidase of plants
(Keller et al., 1998; Sagi and Fluhr, 1999). In arabidopsis
roots, analysis of mutants of the gene family encoding the
gp91-phox subunit of the NAD(P)H oxidase revealed that
ROS produced by this oxidase activates calcium channels
and facilitates calcium flux(es) necessary for root growth
(Foreman et al., 2003; Mori et al., 2004). Moreover, ara-
bidopsis ROP1 is responsible for an intracellular calcium
gradient at the tip of pollen tubes (Gu et al., 2004) and root
hairs (Jones et al., 2002). These observations lead to the
speculation that ROP may further promote an increase in
cytosolic calcium under hypoxic conditions, which could be
mediated by an NAD(P)H oxidase, possibly located at the
PM. Thus, calcium dynamics may provide a balance
between production of H2O2 as a signalling molecule and
the damage it can cause as an ROS. This is consistent with
the finding that DPI treatment prolonged the tolerance of
ropgap4-1 seedlings (Baxter-Burrell et al., 2002). More-
over, it is of interest to note that members of the gp91-phox
subunit of the PM NAD(P)H oxidase gene family were
identified as significantly induced by low oxygen stress
in several DNA microarray analyses (Klok et al., 2002;
Branco-Price et al., 2005). Additional experimentation is
needed to determine if a PM NAD(P)H oxidase, and cal-
cium channels it may regulate, plays a role in low oxygen
signal transduction and whether this mechanism is evolu-
tionarily related to the roles during hypoxia of the PM
NAD(P)H oxidase and G-proteins in mammalian cells.
Ethylene perception enhances responses to
oxygen deprivation
One growth regulator that has received significant atten-
tion in the studies of low oxygen responses is ethylene.
Ethylene biosynthesis increases within 4 h of transfer to
hypoxic conditions in several species (Drew et al., 1979;
Lorbiecke and Sauter, 1999). In arabidopsis, phosphoryla-
tion of ACC synthase (ACS) by the stress-responsive MAP
kinase (MAPK) 6 leads to the accumulation of ACS protein
(Liu and Zhang, 2004). Consequently, levels of cellular
ACS activity and ethylene production are elevated. It is
not yet known if a MAPK signalling pathway is activated
by oxygen deprivation. The biosynthesis of ethylene is not
likely to occur under strict cellular anoxia because the con-
version of 1-aminocyclopropane-1-carboxylic acid (ACC)
to ethylene by ACC oxidase (ACO) requires consumption of
oxygen (Yang and Hoffman, 1984; Kende, 1993). Possibly
to compensate for the ACC to ethylene rate-limiting step
during hypoxia, cellular ACC levels and ACO enzyme
activity are elevated in submerged Rumex palustris plants
(Banga et al., 1996; Vriezen et al., 1999). Ethylene mediates
internode elongation and adventitious root initiation in
deepwater rice (Lorbiecke and Sauter, 1999), and petiole
elongation and leaf hyponasty in R. palustris (Voesenek
et al., 2003). An increase in intracellular calcium is involved
in the transduction of the ethylene signal that leads to the
formation of aerenchyma in hypoxic maize roots (He et al.,
1996). Ethylene also enhanced the hypoxic induction of
ADH1 in arabidopsis (Peng et al., 2001).
Several studies have examined the interplay between
ethylene and other hormones during acclimation of plants
to flooding stress. When gibberellic acid (GA) inhibitors are
applied to seedlings of deepwater rice, the growth induced
by ethylene and submergence is inhibited (Raskin and
Kende, 1984). Abscisic acid (ABA) is a potent inhibitor
of GA action in rice, but ethylene application can reduce
endogenous ABA levels (Hoffmann-Benning and Kende,
1992). Ethylene, GA and indole-3-acetic acid (IAA) act
in consort to promote differential petiole elongation and
hyponastic upward curvature in submerged R. palustris
(Voesenek et al., 2003; Cox et al., 2004). In contrast,
ABA appears to be responsible for the negative regulation
of the submergence-induced hyponastic growth. In addition,
there appears to be a synergism between ethylene and
IAA during adventitious root formation in submerged
R. palustris (Visser et al., 1996).
Differential responses of aerial organs and roots
to oxygen deprivation
There is clear evidence that aerial organs and roots
respond differently to oxygen deprivation. In a pioneering
characterization of the molecular response, Okimoto et al.
(1980) observed that anaerobic polypeptides (ANPs) were
synthesized in anaerobic maize roots, but leaves exhibited
no detectable protein synthesis and died after a short anaer-
obic treatment. In arabidopsis, the induction of ADH1 by
hypoxia was reported to occur primarily in the roots, and
only at low levels in the aerial portion of the plant (Dolferus
et al., 1994). Ellis et al. (1999) found that ethanol fermenta-
tion was essential for tolerance to hypoxia in roots but not
in shoots of arabidopsis. Moreover, ABA treatment pro-
moted tolerance to oxygen deprivation only in the roots
of arabidopsis. These observations, as well as organ-specific
differences in calcium signatures mentioned earlier
(Sedbrook et al., 1996), indicate that shoots and roots
differ in mechanisms of sensing and response to oxygen
deprivation.
Another aspect of whole plant response is long-distance
transport of oxygen, mobilization of carbohydrate reserves
and long-distance signalling. Survival of oxygen depriva-
tion is extended by the transport of oxygen from aerial
organs through aerenchyma to roots (Drew, 1997). On
the other hand, roots of flooded plants transmit signals
to aerial organs. For example, in flooded tomato plants,
ACC is rapidly synthesized in the roots and transported
to the shoot (Shiu et al., 1998). There, in the presence
of oxygen, ACC is converted to ethylene, resulting in
leaf curvature. Chung and Ferl (1999) observed that the
growth of arabidopsis seedling roots through an agar
 Bailey-Serres and Chang Initiation of Responses to Low Oxygen
medium triggered ADH1 expression in the root as well as
in the shoot apical meristem. The signal transmission to
aerial parts of the plant was mediated by a mechanism that
uses calcium as a second messenger. These observations
emphasize that response to oxygen deprivation occurs at
the whole plant level.
Oxygen sensing mechanisms in Chlamydomonas reinhardtii
and responses involving chloroplast function
Results from the genetic dissection of sensing
and response to hypoxia in the single-celled algae
Chlamydomonas reinhardtii provide additional insights. It
was noted that a sub-set of hypoxia-induced genes were
activated in cells deficient in copper and in cells that
were oxygen deprived in the presence of mercuric chloride,
an antagonist of the copper-deficiency response (Quinn
et al., 2002). The co-regulation of these responses appears
to involve the common use of a cis-acting GTAC core,
present in one copy for copper-regulated expression and
in two copies for hypoxic induction, and a trans-acting
factor, copper resistance response 1 (CRR1). Gene induc-
tion under hypoxia is regulated by both CRR1-dependent
and -independent pathways. The CRR1-dependent pathway
governs enzymes involved in plastid function, indicating
that plastids contribute to the acclimative response to oxy-
gen deprivation. The importance of this organelle may be
reflected by the differential response to oxygen deprivation
observed in root and aerial tissue of arabidopsis (Sedbrook
et al., 1996; Ellis et al., 1999). The observation that light
levels affect submergence tolerance in rice and gene induc-
tion in oxygen-deprived arabidopsis and Nicotiana tabacum
is also consistent with a role for chloroplasts in the whole
plant response programme (Hansch et al., 2003; Mohanty
and Ong, 2003). The influence of chloroplasts may be com-
plex due to the role of this organelle in oxygen evolution,
carbon dioxide consumption, sucrose production, redox
regulation, and ROS generation and amelioration.
CONCLUSIONS AND PERSPECTIVES
Investigation of sensory mechanisms and signal
transduction cascades responsible for triggering responses
to sub-optimal oxygen levels in plants cells is an exciting
area of research. The sensing and signalling mechanisms
that are known to contribute to responses to oxygen depriva-
tion in plant cells are generally integrated into a model in
Fig. 2. Advances in genome biology, genetic resources and
high throughput technologies provide excellent resources
for the exploration of oxygen sensing mechanisms in
plant cells. Of particular interest is whether a sensing mech-
anism, perhaps initiated by mitochondria and/or involving
PM NAD(P)H oxidases, is present in plants and evolution-
arily conserved in eukaryotes. It is imperative to identify
sensors and dissect the signalling pathways that occur at the
cellular, tissue, organ and whole plant level. Comparative
analyses of near isogenic genotypes that differ in the adapt-
ive response to oxygen deprivation are likely to yield critical
information on regulatory mechanisms. It is anticipated that
515
distinctions in the location, timing, magnitude, duration and
frequency of signalling cascades will contribute to the
responses of individual cells. It is also likely that alterations
in cytosolic pH and calcium are involved in the signalling
processes. Although paradoxical, ROS may prove to be
second messengers in the response mechanism. The import-
ance of changes in adenylate charge, redox status and car-
bohydrate levels must also be considered. Many questions
remain to be answered about the response of individual
cells. How do cellular signalling and response mechanisms
differ between stress-tolerant and intolerant organs and spe-
cies? What signalling transduction pathway(s) are activated
or inhibited? How do multiple and interacting pathways
control adaptive responses? Another inviting area for
research is the consideration of cellcell and long-distance
signalling mechanisms that determine the organ and whole
plant response to oxygen deprivation, such as regulation
of leaf and internode elongation, petiole curvature, aeren-
chyma formation and adventitious root growth. It is likely
that these responses involve growth regulators such as ethyl-
ene, auxin, gibberellins and ABA. At the organ and whole
plant level, there are also many pertinent questions. How
might cells that surround an oxygen-deficient core, such as
the stele of a root, respond to benefit the survival of the
organ or the whole plant? How are the energetic needs of
meristematic cells safeguarded and how is programmed cell
death promoted or avoided? How do cells in roots and aerial
organs communicate over a long distance when there is an
oxygen crisis in the roots? Success at answering these ques-
tions will be of relevance to agriculture and will provide
knowledge of the fundamental nature of aerobic life.
ACKNOWLEDGEMENTS
This work was supported by grants from the National
Science Foundation (MCB-0-131486) and USDA Cooper-
ative State Research, Education and Extension Service
(2003-35100-13359). We are indebted to members of
the Bailey-Serres lab for many productive discussions,
especially Takeshi Fukao.
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