Opening stomata agriculture as a proposal toward a sustainable tomato

production.

Authores: Almeida, V.S. PhD student of Federal University of Vicosa,

Brazil; Delazari, F.T. PhD student of Federal University of Vicosa, Brazil;
Gomez, C.N. Adjunt professor of Federal University of Vicosa, Brazil, Araújo,
W.L. Adjunt professor of Federal University of Vicosa; Silva, Derly José
Henriques. derly@ufv.br (Corresponding author). Full professor of Federal
University of Vicosa - Brazil;

Farmers and agronomists have been challenged to achieve sustainable

trade of tomatoes for decades. This objective is associated to maximum
efficiency in the use of natural resources [water, carbon dioxide (CO2), and
solar radiation], agricultural inputs (fertilizers, pesticides, etc.), and minimizing
the social and environmental impacts. Therefore, it is necessary to properly
understand both plant physiology and plant production system in order to use
those information as principle for sustainable tomato production.

Improvements in our understanding of crop production systems in

relation to yield might be acquired by knowledge of basic plant biochemist and
physiology. Thus, an ancient experiment assessing the contribution of plant
physiology to the yield, published in the seventeenth century, is noteworthy. The
Belgian physician and alchemist Jean Baptista Van Helmont carried out a test
by planting a seedling of willow tree weighing about five pounds (2.25 kg) in a
pot with 200 pounds of soil (90 kg). This plant was irrigated by rain water or
distilled water during five years. After that period, he cut and weighed all the
plant parts and noted that the tree weighed 169 pounds (77 kg) while the soil
had lost only two ounces (57 g). He concluded that 164 pounds of wood, bark,
and roots were produced from the water.

Notably, the principles of photosynthesis in Van Helmont’s time were not

known. Currently it is known that, on an average, 96% of the dry matter of a
plant is composed of carbon (45%), oxygen (about 45%), and hydrogen (about
6%). These elements are natural resources that go into the plant via stomata,
the main channel for gas flow into and of leaves. Remarkably, the operation of

1
stomata is strongly linked to the balance between photosynthesis, transpiration,
and respiration (Wong et al 1979).

The stomatal opening chamber need to be open for the operation of

these major biochemical mechanisms in order to produce vegetable dry matter
efficiently. Accordingly, this aperture is strongly affected by environmental
conditions (Hetherington and Woodward, 2003). Thus, to achieve sustainable
production, it must be ensured that the mechanism of stomata movement allows
the plant to acquire CO2 (from the air) and water and minerals from the soil
efficiently. This will allow that by using the light energy they produce 96% of
vegetable dry matter (Zelitch, I. 1975).

The efficient absorption of CO2 can be estimated by the liquid rate of CO2
absorbed by the leaf area. It is important to mention that the carbon absorbed
will be used not only for the production of dry matter of commercial interest, but
also to produce dry matter for all plant parts as well as serving as a substrate in
the respiratory process.

The harvest index indicates the ratio of the dry matter formed in
commercial parts in relation to the total dry matter of the plant. In this case, the
production of tomato fruits compared to the total production of dry matter by the
plant. Thus, during breeding varieties are usually selected to present greater
commercial productive plants, which mean a higher harvest index. Thus,
commercial tomato varieties have great efficiency in transforming CO2 in fruits.

In this context agronomists and farmers should use growing techniques

ensuring plants have maximum efficiency in absorbing and using CO 2, water,
and nutrients, which will in turn result, in maximum dry matter of the fruit. The
whole process of absorption and efficient use of CO2, water, and nutrients
begins when the stomata is open. Thus, it is reasonable to assume that the
development of a tomato production system, aiming to achieve a plant
population that ensures longer stomatal opening time in the plant canopy during
the light period, we will be able to reach higher yields. Notably, this increase in
yield will not require extra investment in agricultural inputs, which means a more
sustainable tomato production. We are calling this system as the "Opening
Stomata Agriculture" - "OSA".

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 The importance of the maintenance of the stomatal aperture for tomato
production can be analyzed by the composition of a tomato fruit. The tomato
fruit is composed, on average, 90% water and 10% dry matter. The dry matter
is 96% composed by carbon (C), hydrogen (H), and oxygen (O). Accordingly, in
it a 100 g tomato fruit, 90 g will be water and 10 g will be dry matter. In the 10 g
of dry matter, 9.6 g is carbon (C), hydrogen (H) e oxygen (O) and thus only 0.4g
is composed of mineral constituents. Therefore, the inflow of CO 2 and water as
well as the output of O2 (principles of "OSA") account for 99.6 g (99.6%) of the
100 g fruit that will be marketed. Accordingly, increments in stomatal
conductance might improve not only photosynthesis but also potentially
increase yield (Zheng et al 2011; Franks et al., 2009).

The opening stomata pore occurs through the movement of water into
the guard cells by a difference in osmotic potential. This difference occurs as a
result of solutes movement entering into the guard cells, reducing intracellular
osmotic potential and resulting in a water movement from the apoplast to the
within cell medium (Roelfsema et al., 2001).

The process of opening and closure of the stomata pore, which regulates
the photosynthetic process (Heuvelink et.al, 2005), occurs in response to
various biotic and abiotic factors as well as to internal and external stimulus to
the plant. The main goal of this process is to preserve the internal water status
and absorb atmospheric CO2. Importantly, stomatal conductance is positively
correlated with growth and yield, even in different environments (Condon et al,
2006).

Additionally to the control of water loss, stomata directly affect CO 2

fixation in response to environmental cues. It is important to mention that under
field conditions, this positive response between stomatal conductance and yield
might be modified by several factors including interaction with other plants and
or stresses in general. This fact apart, the main factors that affect opening and
closure of the stomata pore are: soil water availability, light, CO2, relative air
moisture, air temperature, and wind. By discussing these factors below, here we
provide novel insights for improving tomato yield based in the maintenance of
stomatal pores open during the light period. Therefore the main focus of this

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work is to consider the potential of stomatal movements in governing plant
growth and yield exploiting the several aspects related to stomatal behavior.

Soil water availability

The soil is a three-phase system consisting essentially of minerals,
organic matter, water, and air (Schjønning, et. al., 2002; Tuli et. al, 2005;
Deepagoda, et. al., 2011). The liquid phase corresponds to the soil solution that
contains water and several important ions for plant nutrition, and fills part of the
pore spaces in the soil.
In this environment, the soil solution is retained at different levels of
strength, depending on the existing amount of the water and pore soil size. The
plants can readily absorb water from the soil when the moisture content is
optimal for plant growth (field capacity) and most of this water is found in the
intermediate pore soil size. Notably, in the case of reduction in soil water
content, only the solution retained in smaller soil pores or as a thin film around
soil particles remains. There will be a competition between soil particles and
plant by water from this point until the point of remaining water soil level
reaches the permanent wilting point (where the plant is not able to absorbing
water and dies).
The amount of soil solution available to be used by the plant varies
according to soil characteristics (texture, porosity, etc.) and the plant (root
distribution and depth). The water available to the plants (WAP) is the amount
of water between the field capacity (θCC) and the permanent wilting point
(θPMP) (Kirkham, 2005; Veihmeyer and Hendrickson, 1949).
The gaseous phase in the soil occupies the pore space not filled by the
liquid phase. A well-aerated soil is very important for crop yield because the
roots of the plants require oxygen to carry out their metabolic processes. The
soil organisms also need to respire and under poor oxygen content, a
competition for oxygen between those organisms and plant roots might occur.
The transport of nitrogen and oxygen, which are indispensable for plants
growth, occurs from the gaseous phase of the soil (Seyfried; Murdock., 1997;
Smith et al., 2009; Roberts et al., 2011.).

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 Human activities interfere with the agricultural system through practical
acts, changing and affecting the fundamental physical properties of the soil for
the development of plants and ultimately the preservation of water resources
(Klein, 1998). Changes that occur in the soil structure, as evidenced by changes
in its density, affect the soil mechanical resistance to penetration, distribution of
pore soil size, water storage, and solution availability to the plants (Klein, 1998;
Camara & Klein, 2005a; Vieira, 2006).
Soils with physical characteristics suitable for cultivation have large
amounts of solution at water field capacity and appropriate air content for plant
growth. The soil becomes a limiting factor for crop production when subjected to
intensive cultivation and use of heavy machinery, which cause high density of
soil meaning compacted soil (Hamza and Anderson, 2005; Becerra et al., 2010
Tracy et al., 2011).
Soil compaction results in reduced soil porosity, aeration, total capacity of
water infiltration, and hydraulic conductivity (Silva et al., 2009). Increasing the
soil density and root penetration resistance (Moraes et al., 2014) is associated
with limitation of the soil depth and thus reduction in the effective volume
explored by the roots for water and nutrient uptake (Chen et al., 2014).
Therefore, soil compaction challenge root growth, resulting in the reduction of
biomass and yield (Tubeileh et al., 2003; Ma et al., 2013) and may interfere in
the opening and closing of the stomata.

The presence of water is detected by the roots, which need to develop

osmotic potential lower than to the soil allowing thus roots to absorb soil
solution, as it always moves up from a higher to a lower potential. The lower the
osmotic potential of the roots is the higher is the velocity of water entering in the
plant.

The absorption of water by the apoplast (xylem) accounts for

approximately 90% of the water that enters in the plant. The movement of water
through the symplast (phloem) has a complementary significance to this
phenomenon (Castro et al., 2005), allowing aerial parts to maintain hydrated.

The plant senses limited water supply through the root hair. When this
limitation approaches a drought status, there is a signaling toward the root–leaf.
Thus, by the transpiration flow, it triggers stomatal closure, to prevent water

5
plant limitation (Flexas et al., 2002; Anjum et al., 2011). As the stomata close,
the leaf transpiration and CO2 assimilation decrease, reducing the efficiency of
the mechanisms associated with photosynthesis, such as, electron transport,
chlorophyll content, and enzymatic processes (Farquhar & Sharkey, 1982).
The phytohormone abscisic acid (ABA) plays a pivotal role not only
during fruit development and ripening but also, during suboptimal conditions
(e.g. biotic and abiotic stresses such as drought or higher temperature), being
the main phytohormone responsible for stomatal closure (Bright et al., 2006,
Jiang & Hartung 2008), senescence (Finkelstein, 2013), and root elongation,
and shoot etiolation (Luo et al., 2014; Thole et al., 2014.).
ABA plays a central role in long distance signaling during water limitation
episodes controlling stomatal movements and thus retaining water within the
plant (Zhang & Davies, 1991; Davies et al., 2002). It also coordinates the
reduction of the water potential in the roots in response to low water soil
potential (Puertolas et al., 2013). Thus, under these limiting conditions ABA
maintains a small flow of water by roots xylem toward the leaves (Li et al., 2011;
Wang et al. 2012; Correia et al., 2014.).
There is a clear regulation of metabolism by production of ABA under
water stress conditions, where the production of ABA is triggered rapidly to
preserve water status in plants and thus avoid desiccation. On the other hand
ABA is rapidly degraded and deactivated when the water stress is reduced or
released that allows plants to reassume normal growth and development
activities (Zhang et al., 2006).
Although we have witnessed an increased knowledge regarding ABA
metabolism, signal transduction and transport over the last decades the precise
mechanisms by which ABA acts reducing the effects of water deficit remains to
be elucidated. There is a growing body of evidence that the radial K+ transport
through the membranes is mediated by ABA, which is extremely important for
normal physiological processes in plants (Roberts & Snowman, 2000).
ABA controls stomatal movement by mediating the input and output of
K+ in the guard cells. This hormone increases cytosolic Ca2+, which causes
depolarization of the plasma membrane and deactivates the K+ channels,
leading to K+ efflux from the guard cells and ultimately leading to stomatal
closure (Ilan et al., 1994; Miedema & Assmann, 1996; Schulz-Lessdorf et al.,

6
1996). ABA can also regulate the turgor of the guard cells by converting malate
into starch, which is osmotically inactive (MacRobbie, 1998). Further evidence
about the role of sucrose as an osmolyte mediating stomatal regulation has
been recently revisited (Daloso et al., 2016).
Although our understanding of the mechanisms by which plants control
and regulate their metabolism to face limited water supply has seen
considerable progress the mechanism associated with plant sense that
whenever there is limitation in the water supply and culminated with stomatal
closure in order to preserve internal water status remains rather unclear. This
fact apart it seems clear that in order to prevent reductions in photosynthesis
efficiency as a response to stomatal closure farmers should prioritize the
growth of tomatoes in deeper soils with enriched medium-diameter pores,
allowing thus that plants ensure greater and more regular water supply through
the entire plant and prevent stomatal closure.

Light
Light provides not only the energy source for photosynthesis, but also a
wealth of information to optimize plant growth (Chen et al., 2004). Additionally,
light is also one important environmental factor involved in the control of
stomata movements in the nature. Two signaling pathways, controlled by blue
and red lights are involved in stomatal movements (Shimazaki et al., 2007).
Blue light is the major wavelength to trigger stomatal opening, and two families
of blue light receptors, phototropins (phots) and cryptochromes (crys), regulate
this response additively (Chen et al., 2004; Shimazaki et al., 2007). This fact
apart, the main components in the signaling cascades that link the perception of
blue light to the opening of stomata remain largely unknown (Chen et al 2012).

During the morning, which is blue light enriched, a plasma membrane H+-
ATPase present in the guard cells is activated, resulting in hyperpolarization of
the membrane and activation of K+ channels that transport this ion into the cells
(Shimazaki et al., 2007). During the afternoon, sucrose accumulated in the
guard cells to replace K+, keeping the osmotic potential and the stomata open.
Although sucrose has long been proposed as an osmolyte involved in guard cell
movement (Tallman & Zeiger, 1988; Vavasseur, et al., 2005), it was only

7
recently that experimental evidences was provided for functional roles of
sucrose in guard cells others than the osmotic ones. Sucrose breakdown in
guard cells seems to be directly involved in mechanisms able to induce both
stomatal closure (Kelly et al., 2013) and opening (Daloso et al., 2015) providing
non-osmolytic functions for sucrose in the regulation of guard cell movements.
Further experimentation is still required to unequivocally figure out the function
of sucrose during blue light mediated response.

It is important to mention that experimental evidence has already

demonstrated that only a short period of exposure to blue light is sufficient for
stomata opening (Lino et al., 1985). Accordingly, it seems reasonable to
assume that during the morning is when the stomata can be open longer with
enhanced CO2 fixation capacity, considering that there is no further limitation by
other factors including water availability in the soil, relative humidity, CO2
concentration, hormones, temperature and wind, among others (Zeiger, 1984).

Although red light also influences the stomatal opening, it is worthy to

mention that it requires high intensity and duration (Roelfsema et al., 2002; Mott
et al., 2008). This opening process occurs mainly by reducing the intercellular
carbon concentration (Ci) as a result of the stimulation of photosynthesis by red
light in the mesophyll cells (Hanstein et al., 2001), leading to increased
consumption of CO2. Remarkably, the red light response of stomatal
conductance is independent of the photosynthetic activity of the guard cells or
the underlying mesophyll. Moreover, when leaves were treated under a
constant intracellular CO2 concentration, red light still stimulated the opening of
stomata (Messinger et al., 2006).

Compelling evidence has demonstrated a synergistic effect between blue

and red lights. It was demonstrated that in presence of both blue and red lights,
stomatal opening is larger compared to the sum of the effect of each light
separately (Shimazaki et al., 2007).

The components involved in such additive response remain largely

unknown. Although it is still not well understood how the light signals are
transduced from the various photoreceptors, several transcription factors in the
nucleus are involved in the regulation of the stomatal aperture under light

8
conditions. It is clear, however, that the process of stomatal opening and closing
is depending on both light supply and quality in the leaf blade. Therefore, light
should be promoted whether by plant population, staking or by selecting
cultivars with more upper and short leaves ensuring that the light interception is
maintaining stomata open along the plant canopy. After many decades of
studies involving light responses to stomata, we have increased our knowledge
on the photoreceptors and some downstream components involved in the
regulation of stomatal aperture in response to light signals.

We posit that much more needs to be explored in the years to come in

order to increase productivity and we strongly believe that the maintenance of
stomata open for long periods during the light time should help to increase crop
yield, particularly in plants such as tomato, where populations and crop systems
can be easily manipulated. In broad terms, light can be assumed as one of the
major environmental factors that might ultimately limit biomass production and
crop yield. Further studies aiming at the maximization of photosynthesis will
open new avenues to engineer stomata activity and to allow plants to enhance
yield.

Carbon dioxide

An annual increase, of approximately 2 µmol mol-1, has been noted in the

CO2 concentration, [CO22], in atmospheric air, which resulted in the [CO2]
exceeding 400 µmol mol-1 in the air, in 2014. Accordingly, in the case this
increment is persistent, the carbon dioxide concentration will exceed 700 ppm
at the end of the century (IPCC, 2013).

High [CO2] normally favors crops with C3 carbon fixation metabolism,

such as tomato. This favor is usually observed by an increase in the net
photosynthesis rate and significant enhancements in total dry biomass. The
enrichment of the atmospheric air with carbon dioxide can ensure a higher influx
of [CO2], increasing Ci, resulting in a better efficiency in liquid carbon
assimilation rates.

9
 Two main reasons are associated with the increased in both rate and
photosynthetic efficiency usually verified in C3 plants: (i) there is a substrate
limitation under the current CO2 conditions and an increase in this concentration
may result in higher rates of carboxylation reactions of Rubisco; and (ii) these
increases in [CO2] will lead to reduction in the Rubisco oxygenation reactions,
reducing losses of CO2 and energy cost associated with photorespiration
mechanisms (Ainsworth & Rogers, 2007). It is important however to stress that
photorespiration allows the recovery of carbon atoms lost during Rubisco
oxygenation, presenting itself as a highly efficient metabolic repair system
(Bauwe et al 2012; Linster et al 2013).

The photorespiratory carbon flow is exceptionally high in C3 plants and

thus about half of the photorespiratory CO2 is not reassimilated but lost to the
environment, resulting in a considerable difference between gross and net
photosynthesis. This is arguably the main reason why photorespiration has
been a prime target for crop improvement over decades (Ort et al., 2015).
Although increments in growth of Arabidopsis thaliana plants under well
controlled conditions were achieved by introducing two different
photorespiratory bypasses via metabolic engineering (for a review see
Peterhansel et al 2013), it remains to be tested whether this would result in
similar increments under field conditions.

Increments in CO2 are associated with increases in net photosynthetic

rates, biomass, sugars, and organic acids, firmness, seed production, efficiency
in the use of light, water and nutrients uptake, as well as in changes in stomatal
conductance (Moretti et al. 2010). In the same vein, under high CO2
concentrations (700 ppm CO2) there is an increase in the net carbon
assimilation rate , decrease in stomatal conductance, reduction in the osmotic
potential, and an increase in leaf water potential in the stages of flowering and
fruiting, as well as higher fruit production. However, there is a decrease in the
content of phenols, flavonoids, soluble solids, and titratable acidity (Mamatha et
al., 2014). High CO2 results also in a higher number of tubers per plant in
potatoes (Miglietta et al. 1998). In tomato plants, increased CO2 resulted in
lower respiratory rates, fruit ripening, citric and malic acid content, and higher
reduction in sugar concentrations (Da Matta et al., 2010).

10
 By contrast, elevated CO2 can reduce the photosynthetic capacity of
some species, a phenomenon called acclimatization, which is usually related to
nutrient limitation (Da Matta et al., 2010). In C3 species, such as the tomato, the
most pronounced and universally observed response is the accumulation of
carbohydrates in the leaves as the main cause of photosynthetic acclimation,
which is attributed to the lower carboxylation rate of Rubisco (Da Matta et al.,
2015). Collectively, this clearly indicated that changes observed under such
environment should be taken with caution and that further studies are still
required to fully understand the complexity beyond such metabolic and growth
responses.

Temperature

Temperature stress can be assumed as changes in the temperature

above or below an optimum range for a period, sufficient to cause damage to
the growth and development of the plant. This clearly depends on the intensity,
duration, and rate of temperature alterations (Wahid et al., 2007).

High temperature can totally inhibit the photosynthetic process, even

before other symptoms are detected (Berry & Björkman, 1980). Interestingly, it
has been suggested that higher temperatures reduce net carbon gain by
increasing plant respiration more than photosynthesis. In fact, the light-
saturated photosynthetic rate of C3 crops such as tomato and rice is at a
maximum for temperatures from about 20-32°C, whereas total crop respiration
shows a steep non-linear increase for temperatures from 15 to 40 °C, followed
by a rapid and approximately linear decline (Porter, 2005).

Is common knowledge that cultivation under higher temperatures can

reduce plant production (Zhang, 2010). Nevertheless, plant growth at high
temperatures can also increase the number of stomata on the leaves which can
be seen an one strategy used to increase leaf transpiration and lower the
temperature (Zhang et al., 2014). Furthermore, under temperature above the
optimum, there is an increase in stomatal conductance, transpiration, and
absorption of CO2. Even so, photosynthesis is reduced, because the

11
photosystem is damaged, causing a reduction in the activity of the Calvin cycle,
which may vary in intensity between different cultivars (Camejo et al., 2005).

This is most likely because the effects of high temperature are primarily
on photosynthetic function of higher plants. However, as a consequence of
global warming, plants have to face more severe and more frequently occurring
periods of high temperature stress. While this affects the whole plant, sexual
reproduction can be assumed as one of the most sensitive processes to heat
stress, affecting flower development and number, and pollen production and
viability leading to reduced seed set and yield (Prasad et al., 2006; Das et al.,
2014). It has been also recognized as the most heat stress susceptible phase in
cereals (Monterroso and Wien, 1990; Barnaba et al., 2008) and vegetables
(Erickson and Markhart, 2002), with male reproductive stages being more
sensitive to heat stress than female or vegetative stages of growth (Sakata and
Higashitani, 2008).

In tomato plants it has been demonstrated that the major effect of high
temperature on the pollen development process is associated with the
disruption of carbohydrate metabolism and proline translocation (Sato et al.,
2006). This will in turn lead to reduction in starch and sugar concentration in
mature pollen grains with a subsequent loss of pollen viability (Pressman et al.,
2002). Consequently it culminates with fruit set failure and crop losses at high
temperatures. Interestingly, it has been previously demonstrated that
differences between heat tolerant and heat sensitive tomato genotypes relays in
the ability to accumulate starch and soluble sugars by the former during pollen
development under heat stress conditions (Firon et al., 2006). As a
consequence, under high temperature, heat tolerant tomatoes produce viable
pollen and high fruit set (Firon et al., 2006). The precise mechanism by which
plants manage to maintain the appropriate levels of starch and soluble sugars
under heat stress allowing higher crop production is, however, still not yet
understood.

It is important to mention that whilst changes in temperature impacts

different components of all cells and require an exquisite and organized cellular
response and acclimation, high temperature effects and responses in sexual
organs are distinct from vegetative tissues at several points. This could be

12
related to specific physiological characteristics of developing pollen and
supporting fruit tissues which make them vulnerable to high temperature, or its
derived effects such as reactive oxygen species accumulation and carbohydrate
starvation. That being said, selection and breeding of plants with increased
tolerance to high temperature stress seems highly promising as a way to
mitigate adverse effects of increasing global temperatures and allowing
maintenance or increments in crop yield.

Relative Air Humidity

Plants grown under high relative air humidity (RH) can keep the stomata
open longer than plants in environments with moderate or low humidity (Torre et
al. 2003; Nejad & van Meeteren, 2005). There is evidence that plant hormones
play an important role in the response of the stomata under high humidity
conditions (Aliniaeifard & van Meeteren 2013) but the precise mechanism
remains to be elucidated.

In plants growing under high RH for long periods, the stomata become
less sensitive to signals that normally induce its closure such as darkness,
abscisic acid, high CO2 concentration, and water stress (Tower & Fjeld, 2001;
Arve et al. 2013).

Interestingly, in tomato plants, a short period of exposure to high RH is

sufficient to keep the stomata open, even during the night (Arve & Tower,
2015). Accordingly, high air humidity also leads to less transpiration even with
the stomata open (Arve & Tower, 2015) since the gradient allowing transpiration
is somehow reduced.

The stomatal conductance of tomato plants under high RH conditions is

directly related to the interaction between ABA and ethylene. While ethylene
stimulates stomatal opening and ABA display an antagonistic effect. Thus,
plants growing under high RH have a low ABA to ethylene ratio, while in those
growing under moderate or low RH, there is an increase in the proportion
between these hormones (Arve & Tower, 2015), directly impacting stomatal
responses.

13
 The rapid growing of internodes is another effect that is usually related to
high RH, resulting in greater plant height and development of adventitious roots,
as a result of ethylene formed in this condition (Arve & Tower, 2015).
Importantly, this is not directly associated with higher biomass production and
therefore caution must be taken when considering these results.

The reduction of transpiration in high RH also results in greater efficiency

of water use. However, this condition may reduce nutrient absorption leading to
nutritional deficiencies, especially nutrients of little mobility, such as Ca and Mg
(Suzuki et al., 2015).

Wind

Moderate winds can drag the accumulated moisture in the abaxial part of
the leaf, and ultimately increasing transpiration. By contrast, strong winds may
reduce transpiration by mechanical movement and thus help to cool the leaves
(Castro et al., 2005).

The wind also influences the stomatal conductance and transpiration

rate, as it can be directly related to the relative air humidity. In plants grown in
an environment with moderate RH, the wind increases the transpiration rate.
However, when the RH is high, the effect of the wind is virtually nil, as the air
present at the boundary layer is saturated with moisture and will be replaced by
another layer of air with high humidity (Carvalho et al., 2015).

Interaction of the factors associated with opening stomata agriculture

The process of opening and closing of stomata is highly complex and

results from the interaction of all the factors mentioned above. A favorable
condition for stomatal opening, high relative humidity for instance, has no effect
if the plant is under soil drought stress. In addition, the excess of one of these
factors can lead to stomatal closure. Accordingly, soaked soils may result in
anaerobic conditions causing the death of roots and consequently reducing the
water availability to the plant.

Stomata pores also constitute a natural entry site for potentially harmful
microbes. To prevent microbial invasion, stomata close upon perception of

14
microbe-associated molecular patterns presents itself as an important layer of
active immunity at the preinvasive level. The signaling pathways leading to
stomatal closure triggered by biotic and abiotic stresses employ several
common components, which have been expertly reviewed elsewhere (Sawinski
et al 2013). In this context, infestation of plant roots with endoparasitic
nematodes such as the root-knot nematodes, of the genus Meloidogyne, makes
such plants more prone to water stress and they also show symptoms of
nutrient deficiency and premature senescence (Bartlem et al, 2014), resulting in
water deficit even in soils with high water availability.

Due to global climate change, less availability of irrigation water for

crops, and rising fertilizer prices, it is needless to mention that a better
understanding can be gained of how nematode infestation impairs root function
and reduces yields and product quality. By applying the concept of sustainable
agriculture, where the objective is to maximize the efficiency of use of water,
light, and CO2, it is of pivotal importance to integrate all factors leading to
stomatal opening in order to optimize the environmental conditions for the
maximization of plant growth and production. Clearly, this is not an easy task
given that mostly, we have little or no control over environmental factors.
However, a deep understanding of the factors involved in stomatal regulation
may allow further advances in our understanding of how guard cells achieve
their fine-tuned integration to ensure growth optimization.

The soil needs to have high water-holding capacity easily accessible to

the plants. Deep soils with large amounts of soil pores, with medium diameter
allowing higher water and O2 retention are desired. Certainly, under these
conditions roots will be able to explore deeper layers and larger areas,
increasing their capacity to absorb water and nutrients.

Considering the climatic factors, the ideal solution would be an

environment with high availability of light and [CO2], moderate temperature (25-
30 °C), and a relative air humidity of approximately 70%. These would be the
optimal conditions for higher stomatal opening and assimilation of CO2, without
stimulating excessive transpiration, which can lead to low efficiency in water
use.

15
 We are aware that there is a wide variation in these factors under field
conditions, which can compromise the success of the crop. Certainly,
understanding genetic factors involved in the plant response to these factors will
allow plant breeder to find the most suitable genotype. Needless to mention that
this will take much more time and research efforts than the manipulation of
some cultural practices that can be used to cope with these conditions, such as,
plant population, and pruning and staking systems.

Plant Population

The availability of water, light, [CO2], temperature, and relative air

humidity are important factors in determining the final plant population.
Normally, the higher availability of these resources enables higher possible
number of plants per area.

The plant population directly affects the leaf area index (LAI), which is the
leaf area of a plant per unit of ground surface (m² / m²). Accordingly, the
interception of solar radiation is approximately 90% when the LAI is close to 3.
Compelling evidence has demonstrated that increase in light interception and
productivity gain is minimal and even difficult to experimentally detect above this
value (Heuvelink, 1996).

In greenhouses production it is common to allow the development of a

lateral bud in crops growing at the arrival of summer. This will increase the plant
density, since the light availability is higher during this season (Heuvelink,
2005). In this context, during winter, when the amount of light is lower, the
spacing between plants should be larger to increase light interception and thus
reduce self-shading inside the canopy.
Similarly, fewer plants per area are also recommended under conditions
of high relative humidity, which favors diseases. The lower planting density
allows more air renewal along the rows and greater light penetration through the
canopy, reducing the incidence of diseases and increasing transpiration, water
and nutrient uptake, and assimilation of CO2 or ultimately growth.

16
 Increasee number of plants per area results in higher yields, but reduces
the production per plant and fruit size (Ara et al., 2007; Maboko et al., 2011).
Thus, the optimization of plant spacing results in better use of water, light, and
nutrients without significantly affecting the fruit size (Ismail et al., 2014). With
the density of the tomato crop at 2.5 plants per square meter, the yield
increases, with a large fruit production by 227 and 212%, respectively (Almeida
et al., 2015).

Pruning

In indeterminate growth type tomato cultivation, it is common to cultivate

the tomato plant with one or two stems, eliminating the remaining side shoots.
The cultivation of plants with two stems increases the Leave Area Index (LAI)
and production per plant, but reduces the weight of the fruit (Ambroszczyk et al.,
2008; Maboko et al., 2011.).
Under high light conditions, it is possible to increase the number of stems
as an alternative to increase planting density, reducing the costs of seeds and
seedlings. Under these conditions, the largest number of stems reduces wind
among plants and increases the humidity along the canopy. This technique is
highly recommended to areas that receive high radiation and temperature,
given that higher temperatures normally reduce the relative air humidity.
However, in places with high air humidity, high plant density is not
recommended because besides reducing transpiration by higher humidity it can
also increase the incidence of diseases.
The removal of young leaves to increase dry matter partitioning to the
fruit is also a common practice, especially in greenhouses. However, this
technique can reduce the LAI and negatively influence the yield (Xiao et al.,
2004).
The removal of lower leaves is another technique commonly used,
especially in a greenhouse, to reduce the relative air humidity, increase
aeration, and reduce pests and diseases (Silva et al., 2011). The leaves are

17
removed below clusters where the fruits have been harvested. However, when
an excessive number of leaves are removed its enable lower leaf area and
reduction of the LAI may occur, with a consequent lower light absorption and
yield (Kim et al., 2014).

Staking methods
Staking tomato plants is common in cultivation for fresh market,
especially in countries like Brazil and India, and also under greenhouse
conditions. The staking aims to avoid contact of the fruit with the ground and
can directly influence the microclimate throughout the plant canopy.
The main staking methods used in Brazil are: Traditional or inverted "V"
and vertical. In the traditional method the plants are tied to stakes (bamboo)
arranged obliquely to the ground forming an inverted "V" between two
consecutive rows (Wanser et al., 2007). In this system, a chamber is formed
under the inverted "V", which reduces the incidence of radiation and wind and
increases the relative air humidity, creating an unfavorable environment for
photosynthesis.
In the vertical staking method, the plants are vertically tied to tutors
(bamboos or strings), increasing the interception of light and ventilation and
reducing the relative air humidity throughout the plant canopy (Wanser et al.,
2007).
A new growing tomato system in Brazil, named Viçosa System has been
recently proposed (Almeida et al., 2015). In this system, plants are transplanted
at 0.2 m and 2 m between plants and rows, respectively. The plants are staked
with strings, inclined at approximately 75º to the ground. They are inclined to
both sides alternately in a real "V" shape, from a top view. This ensures a
higher plant density once the light and photosynthesis interception conditions
are optimized, as there is greater light interception and higher exposure to
moderated wind, reducing the relative air humidity and renewing the
concentration of CO2. This method is suitable for places where there is no water
limitation in the soil and there is high relative air humidity.
In places where the relative air humidity is low, the ideal method is to
grow plants in conditions that increase the relative air humidity over the canopy,
such as the inverted “V” staking system. This increases the relative air humidity

18
among plant and leaves, favors the stomata opening, reduces transpiration,
water absorption, and enable assimilation of CO2.

Evaluation of the plants water status

Some methods can be used to evaluate the stomatal opening or

transpiration such as: Sap flow, water balance in the soil, stomatal
conductance, water potential in leaves, and leaf temperature (Trentin et al.,
2011).

Transpiration is one of the main factors that determines the leaf

temperature (Leuzinger et al., 2010). It is reduced when subjected to water
stress, leading to an increase in leaf temperature by the absorption of solar
radiation (Gontia & Tiwari, 2008; Wang & Gartung, 2010). Therefore, leaf or
canopy temperature can be a good indicator of water restriction and stomatal
conductance.

Several water stress indices were developed from transpiration and leaf
temperature, using the differences between the temperature of the culture and
air, to estimate the severity of water stress (Testi et al., 2008; Lebourgeois et
al., 2010).

The crop water stress index (CWSI) is one of the most used. This takes
into account higher and lower temperatures. The higher temperature is when
the crop is under water stress and not transpiring and the lower temperature
corresponds to a well-irrigated crop without limitation to transpiration. These are
determined from the canopy, air temperature, and vapor pressure deficit (Zia et
al., 2012).

The leaf or canopy temperature can be measured by infrared

thermometry, which detects the thermal radiation emitted by the surface of the
leaves (Mn and Mm, 2008). Thermal cameras provide the temperatures of
multiple leaves in only one image, which can be used as a non-destructive
method for estimating stomatal conductance (Jones et al., 2002).

19
 The temperature difference between the leaves in the canopy is an
alternative to using the canopy temperature to estimate water stress and
stomatal closure. The higher the temperature variation along the canopy, the
higher the closure of the stomata and lower the stomatal conductance (Jones et
al., 2002).

The leaf temperature may be 1 to 4°C lower than the ambient

temperature when transpiration occurs under optimal conditions. Under water-
stress conditions, transpiration is reduced and the leaf temperature may reach 4
to 6°C above ambient temperature. Ramirez et. al. (2015) reported that tomato
plants grown under water stress, the difference between leaf and ambient
temperature was -1.18 to 9.71°C, but when tomato grown under no water
limitation temperature ranged from -6.29 until - 2.98°C.

Concluding remarks

Despite of the vast knowledge accumulated during recent year following

both the generation of several genetic resources and the specific analyses of
guard cell metabolism, it seems that our understanding of stomata responses to
environmental factors still remains to be completely elucidated. Molecular and
genetics aspects involved in the regulation of this highly specialized cell type
are being demonstrated and it is clear that the translation of this knowledge
from model plants to crop growing under field conditions still remains a
challenge.

Yet, we cannot currently formally exclude that genotype x environment

interactions will impact our understanding. That being said, the application of
specific agronomical practices coupled with molecular and ecophysiological
tools may provide considerable information concerning the regulation of
stomata and allowing that “Opening Stomata Agriculture” become a regular
practice under field conditions. In this scenario, we posit that coupling the best
practice including plant population, irrigation system and growth system with
genetic material recommended for specific places will be highly pertinent to crop

20
science, providing exciting opportunities not only to researcher but also to
farmers by improving economic yields.

Altogether, the mounting evidence presented here correct choice of

agronomical practices coupled with the best genetic material should become
more frequently in the near future due to open mind farmers allowing significant
increases in crop yield, that are must needed nowadays.

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