recent advances in photosynthesis under drought and salinitysaibo/ownpub/abr2011.pdfciency has been...

Recent Advances in Photosynthesis Under Drought and Salinity

MARIA M. CHAVES,*,{,1 J. MIGUEL COSTA*,{ AND

NELSON J. MADEIRA SAIBO*

*Instituto de Tecnologia Quımica e Biologica, Universidade Nova de

Lisboa, Av. da Republica, Oeiras, Portugal{CBAA, Instituto Superior de Agronomia, Universidade Tecnica de

Lisboa, Tapada da Ajuda, Lisboa, Portugal

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50II. Studying Drought and Salinity Effects on Photosynthesis . . . . . . . . . . . . . . . . 52

A. From Controlled Conditions to the Field ...... .. .. .. .. .. .. .. .. ... .. .. .. .. 52B. The Relevance of Studying Recovery Responses .... .. .. .. .. .. ... .. .. .. .. 53C. The Use of Model Plants ..... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. ... .. .. .. .. 54

III. Photosynthetic Limitations Under Water and Saline Stress . . . . . . . . . . . . . . . 56A. Diffusive Limitations (Stomatal and Mesophyll).. .. .. .. .. .. .. ... .. .. .. .. 56B. Biochemical and Metabolic Limitations ..... ... .. .. .. .. .. .. .. .. ... .. .. .. .. 64

IV. Is Water Use Optimized by the Leaves Under Water Deficits? . . . . . . . . . . . . 66V. How are Stomata and Photosynthetic Genes Regulated? . . . . . . . . . . . . . . . . . 67

A. Regulation of Stomatal Aperture ..... .. .. .. .. ... .. .. .. .. .. .. .. .. ... .. .. .. .. 67B. Stomatal Development .... ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. ... .. .. .. .. 75C. Expression of Photosynthetic Related Genes .... .. .. .. .. .. .. .. ... .. .. .. .. 77

VI. Improving Carbon Fixation Under Environmental Stress?. . . . . . . . . . . . . . . . 79VII. Conclusions and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

1Corresponding author: E-mail: [email protected]

Advances in Botanical Research, Vol. 57 0065-2296/11 $35.00Copyright 2011, Elsevier Ltd. All rights reserved. DOI: 10.1016/B978-0-12-387692-8.00003-5

ABSTRACT

Fast increase in world population, scarcer water resources and climate change areputting pressure on the maximization of crop yield, while optimizing the use of waterand soil. Salinity causes tremendous yield losses at world scale, especially in dry areas.We revise the current understanding of the impact of drought and salinity on photo-synthesis, a highly sensitive process to these stresses and a major determinant ofplant’s growth and yield. The CO2 diffusive limitations (stomatal and mesophyll) tophotosynthesis under water deficits and the underlying regulatory mechanisms ofstomatal behaviour and photosynthetic metabolism are presented. Recent molecularadvances are described, in particular those related to stomatal development and guardcell signalling. Special emphasis is given to the effects of ABA signalling on stomatalregulation under water deficits. The role of transcription factors controlling guard cellmovement and photosynthetic activity under drought and high salinity is discussed.Coordination of stomatal conductance with the CO2 requirements of leaf mesophyllthat may allow constant water use efficiency (WUE) in different environments isanalysed on the basis of recent data from transformed plants. The improvement ofWUE by optimizing Rubisco carboxylase capability to increase photosynthetic effi-ciency has been ineffective. Therefore, we stress the importance of knowledge on leafgas-exchange limitations caused by drought and high salinity for future breedingstrategies. The direct transfer of the knowledge gathered from model plants intocrops needs to be carefully considered.

I. INTRODUCTION

Soil water deficit and soil salinity are major limiting factors of plant growth

and agricultural productivity worldwide. Water scarcity and poor water

management increasingly affect large agricultural areas in Asia, Europe,

America and Australia (Aldaya et al., 2009; Brown, 2008; Collins et al.,

2009; World Economic Forum, 2009). Overall, the occurrence of extreme

climate events has been increasing in recent years and is expected to keep

rising in the near future (IPCC, 2007; Tin, 2008). Major concern exists in

countries like China or India where the combination of increasing domestic/

industrial water consumption and deficient management of soil and water

resources endanger food security (Beddington, 2010; Brown, 2008; Costa and

Heuvelink, 2004; Morison et al., 2008). In Europe, water scarcity may

produce relevant socio-economic and environmental impacts, in particular

in the dry South Mediterranean areas or the Sub-Saharan Africa, considered

to be amongst the most altered regions in the world by different climate

change scenarios (Beddington, 2010; Chaves and Davies, 2010; Tin, 2008).

As for salinity, it is known to affect more than 6% of the arable land and

about 30–50% of the irrigated area worldwide (Unesco Water Portal, 2007).

Salinity risks are larger near coastal areas due to saline water intrusion,

which is directly related to the over-exploitation of underground water for

50 MARIA M. CHAVES ET AL.

either domestic or agricultural purposes, causing a decrease in the ground-

water levels (Brown, 2008; Carvalho, 2000; Collins et al., 2009; http://www.

abc.net.au/learn/silentflood/stats.htm).

The ability of plants to adapt and/or acclimate to different environments

is directly or indirectly related with the plasticity and resilience of photosynthe-

sis, in combination with other processes, determining plant growth and devel-

opment, namely reproduction. Evolutionary success depends on integrative

and effective regulation of these processes at the whole-plant level (Lawlor,

2009). Regular drought-tolerant plants can withstand moderate tissue dehy-

dration of about 30% water loss. By contrast, desiccation-tolerant plants

(generally referred to as resurrection plants) are tolerant to further cell dehy-

dration (around 90% water loss) and keep the ability to rehydrate successfully.

Molecular studies suggest that desiccation tolerance in the vegetative tissues of

resurrection plants is unlikely to result from the presence of genes that are

unique to these plants, since the relevant genes are also present in the genome of

non-tolerant plants, but may reside in the expression patterns of those genes,

therefore being largely a quantitative characteristic (Ramanjulu and Bartels,

2002).When confronted by salinity, plants can adapt to it by compartmentaliz-

ing Naþ and Cl� in the vacuole and to keep cellular water status and growth

(Blumwald et al., 2000). These mechanisms are particularly developed in halo-

phytes, but over-expression of Naþ/Hþ antiporter has already shown to im-

prove salt and drought tolerance in transgenic Arabidopsis (Shi et al., 2003).

Identifying limitations to photosynthesis and the regulatory processes

under water deficits and salinity is essential to minimize (or to improve

through breeding) the negative impact of such stresses on agricultural crops

and to protect ecosystem functioning. Recent reviews have dealt with this

subject at various levels of complexity, from the cell to the whole plant

(Chaves et al., 2003, 2009; Flexas et al., 2004; Lawlor and Tezara, 2009;

Munns, 2002; Munns and Tester, 2008; Zhu, 2001). The reinforcement of

research funding in this thematic area was recently proposed by the EU that

also recommended increasing research coordination amongmember states so

that plant/crop adaptation to changing climate may be promoted (EU, 2010;

The Royal Society, 2009).

In this chapter, we aim revising the current status of our understanding of

the impact of water scarcity and salinity on photosynthesis. Besides referring

to the different systems (model plants, crops) and approaches (physiological

and molecular) that are enabling advances in this scientific area, we will

revise the controlling factors of carbon uptake in different species and

different conditions, with particular emphasis on the regulatory mechanisms

that make possible to coordinate carbon assimilation and water loss. Indeed,

optimization of water use by plants has and will have major impact in crop

PHOTOSYNTHESIS UNDER DROUGHT AND SALINITY 51

breeding for arid and semi-arid regions. Major molecular advances related

with guard cell signalling and transcriptional regulation of photosynthetic

responses will also be reviewed.

II. STUDYING DROUGHT AND SALINITY EFFECTSON PHOTOSYNTHESIS

A. FROM CONTROLLED CONDITIONS TO THE FIELD

In nature, abiotic stress conditions like drought and salinity rarely occur in

isolation and combination of different stresses is usually not predictable by

single-factor analyses because synergistic, antagonistic, or overlapping effects

can occur (Valladares and Pearcy, 1997). An example of an antagonistic

response to stresses is the observation that critical temperatures for photo-

synthesis can increase in leaves of water-stressed plants as compared to well-

watered ones, as it was reported in lupins (Chaves et al., 2002) and in various

solanaceae (Havaux, 1992). This suggests that water deficits may provide

protection against heat stress, presumably by increasing membrane stability.

It is also factual that one stressmay originate a second one. This is the case of

drought- and salinity-induced stomatal closure that prevents leaf water loss.

However, stomatal closure diminishes leaf cooling due to transpiration, gen-

erating a superimposed heat stress with leaf temperatures often rising up to 5 or

6 8C in relation to air temperature. Regarding high salinity, the detrimental

effects on leaf physiology were shown to depend on exposure to sun light, with

more negative impacts being found for sunny than for the shade sites of the

canopy, as reported for Olea europea trees (Remorini et al., 2009).

Under multiple stresses plants elicit unique and complex responses regard-

ing photosynthesis, respiration (Mittler, 2006; Rizhsky et al., 2002, 2004) and

signalling (Okamoto et al., 2009). Therefore, the need to study such interac-

tions under conditions mimicking nature is emphasized (Cimato et al., 2010).

At molecular level, co-occurrence of different stresses can generate

the co-activation of different response signalling pathways (abscisic acid,

ethylene, jasmonic acid, etc.) (Mittler, 2006). For example, in Arabidopsis

thaliana plants subjected to both heat and drought about 10% of the regu-

lated genes under such conditions were overlapping with cohort genes regu-

lated by both type of stresses, when they were applied separately (Rizhsky

et al., 2004; Voesenek and Pierik, 2008).

It is recognized that the velocity of stress imposition dictates plant

responses—immediate responses to a short-term stress aiding in plant sur-

vival, whereas acclimation responses to slowly imposed stress contribute to


an improvement of plant performance under adverse conditions. Indeed,

these acclimation responses are not only due to direct effects of resource

deprivation or hostile conditions, but also to physiological adjustments that

minimize disturbances in plant metabolism (Chaves and Oliveira, 2004).

Many studies addressing stress effects rely on short-term experiments,

which will not be directly applicable to field situation since they are far

from the natural growing environment (Cai et al., 2010; Vinocur and

Altman, 2005). Although setting up complex design field experiments is a

difficult task, the testing under laboratory conditions of newly developed

stress-tolerant genotypes to multiple stresses needs to be rechecked with field

studies and in different years, to allow the analysis crop performance under a

variety of conditions (Mittler, 2006; Richards et al., 2010).

In the case of tolerance to salinity, many studies are carried out using

hydroponics. However, complex interactions between the soil solution and

the soil matrix were shown to affect plant response to salt, as recently shown

in Hordeum by Tavakkoli et al. (2010). This implies that field tests are

essential, although the confounding effects from several environmental fac-

tors changing concurrently have to be taken into account (Cuin et al., 2010;

Travers et al., 2010).

B. THE RELEVANCE OF STUDYING RECOVERY RESPONSES

Cycles of stress and recovery from stress are prevalent processes occurring

under natural conditions during different seasons and under agricultural

practices such as irrigation (Vinocur and Altman, 2005). Recovery of photo-

synthesis following stress relief largely determines plant resilience to water

deficits and salinity. For perennial plant species, it is undoubtedly one of the

most relevant survival strategies (Hu et al., 2010a). Recovery depends on the

intensity of photosynthesis decline under stress (Chaves et al., 2009; Flexas

et al., 2006) and is closely linked to plant capacity to avoid or to repair

membrane damage when stress intensifies (Chaves and Oliveira, 2004).

Moreover, instability of photosynthetic membranes under water deficits,

which means becoming transiently permeable, was shown to occur at an

early stage of dehydration than in plasma membranes (Speer et al., 1988).

In spite of such importance, studies on the capacity of photosynthetic recov-

ery from different stresses have only been conducted in few species, for

example, tobacco (Galle et al., 2009), beans (Miyashita et al., 2005), grape-

vine (Flexas et al., 2009; Galmes et al., 2007a), beech (Galle et al., 2007), and

several Mediterranean species (Galmes et al., 2007b).

Recovery is generally characterized by a rapid increase (recovery) of leaf

water potential (within 2 days or earlier) followed by a later recovery of


stomatal conductance, which may be associated with hormonal balance

being re-established. This will allow the plants to limit transpirational

water losses and regain full turgor after rewatering, as shown in citrus plants

by Ruiz-Sanchez et al. (1997).

The intensity and/or duration of stresses have particular effect on both the

velocity and the extent of recovery after stress relief (Chaves et al., 2009). In

general, when a severe water stress is imposed, recovery is partial and can reach

only 40–60% of the maximum photosynthesis rate during the day after irriga-

tion restarts. Nevertheless, recovery process may continue in the following

days but the maximum photosynthetic rates are sometimes never reached

again (Bogeat-Triboulot et al., 2007; Galle et al., 2007; Grzesiak et al., 2006;

Kirschbaum and Pearcy, 1988; Miyashita et al., 2005; Sofo et al., 2004).

When a severe water stress is imposed, recovery is much slower and may

require de novo synthesis of photosynthetic proteins. It is possible that water

stress (and in general all stresses) irreversibly affect photosynthetic capacity

and accelerates leaf senescence (Chaves et al., 2011). Slow and/or incomplete

photosynthetic recovery has also been linked to sustained oxidative stress

(Galmes et al., 2007a,b). Incomplete recovery of photosynthesis following

stress seems to be more frequent in fast growing (e.g. herbaceous) than in

slow-growing species. Besides the duration and intensity of the stress, recent

findings also suggest a positive effect of a pre-drought treatment on the

future recovering after re-watering (Xu et al., 2009).

Physiological mechanisms involved in the recovery of plants subjected to

high salinity are poorly understood. It is known that the time period required

for photosynthesis recovery after salinity stress is generally much longer (up

to 15–20 days) than that following drought, even when the average stomatal

conductance (gs) before the onset of recovery was threefold that observed in

drought studies (Chaves et al., 2011). This likely reflects the metabolic nature

of a large proportion of photosynthesis limitations occurring under salinity.

Despite the slower recovery, reports on full recovery are more numerous than

in plants subjected to severe drought. However, the interaction of salt and

water stress strongly reduces plant’s capacity to recover photosynthesis after

stress alleviation, as compared with plants subjected to a single stress (Perez-

Perez et al., 2007).

C. THE USE OF MODEL PLANTS

1. The model plants

Several model plants have been used in stress physiology studies to allow

molecular dissection of ‘‘stress-tolerance mechanisms’’ in important crop

plants. One of the most well known is A. thaliana, which is considered a


model for dicotyledonous species. Genetic screens—applied in both forward

and reverse modes—have permitted isolation of numerous Arabidopsis

mutants, which are important tools to identify genes acting in signalling

networks and controlling plant response to environmental stress (Feng and

Mundy, 2006; Papdi et al., 2009; Sirichandra et al., 2009a; Vinocur and

Altman, 2005). Mutants with abnormal stomatal characteristics (Badger

et al., 2009; Hashimoto et al., 2006a,b; Merlot et al., 2002, 2007; Xie et al.,

2006) or photosynthetic behaviour (Niyogi et al., 1998; Overmyer et al., 2008;

Shikanai et al., 1999; Varotto et al., 2000) have been isolated in large scale

screens and used to study different aspects of stomatal regulation and/or

photosynthetic mechanisms as well as to identify genes implicated in relevant

signalling networks, for example, of abscisic acid (ABA) (Wasilewska et al.,

2008). Studies by Koiwa et al. (2006) have shown that T-DNA-tagging

followed by phenotypic screens (forward genetics) allow the identification of

genes involved in cold, osmotic and salinity stress as well as ABA-mediated

gene expression. More recently, Koiwa et al. (2006) isolated mutations caus-

ing NaCl hypersensitivity of Arabidopsis seedlings and identified the major

components of the SOS pathway which controls ion homeostasis and salt

tolerance.

Arabidopsis is sensitive to moderate levels of NaCl and has provided much

information about both Naþ transport processes and Naþ tolerance (Møller

and Tester, 2007). Many of known transcriptional elements of environmental

stress-responsive genes in higher plants regulating and controlling stress reac-

tions related to drought or salinity were isolated inArabidopsis (Ni et al., 2009).

More recently, the species Thellungiella halophyla emerged as a new model

plant for studies on plant responses to salinity (Amtmann, 2009; Amtmann

et al., 2005; Bressan et al., 2001; Zhu et al., 2004). T. halophyla is a close

relative of Arabidopsis but tolerates extreme salinity and drought (Amtmann,

2009; Bressan et al., 2001; Inan et al., 2004). An important aspect of salt

tolerance is the accumulation of compatible solutes in the cytoplasm to

osmotically balance ions accumulation in the vacuole during salt adaptation

(Inan et al., 2004). Thellungiella is a dramatic accumulator of proline and can

be used to study unique aspects of proline-related signalling pathways in

determining fitness under environmental stress conditions (Amtmann, 2009).

Rice (Oryza sativa) is probably the most important genetic model species

for monocotyledonous (Xu et al., 2009). Research on salt tolerance in legume

crops is conducted in model legumes, such as Medicago (Eckardt, 2009;

Nunes et al., 2008) or Lotus (Udvardi et al., 2007; Varshney and Koebner,

2006). Regarding woody species, poplar has been used as a model plant

(Cronk, 2005; Jansson and Douglas, 2007), including research on stress

responses (Bradshaw et al., 2000). Grapevine (Vitis vinifera) also became a


model system for fruit trees following the fully sequence of its genome

(Troggio et al., 2008). The large phenotypic and genetic variation character-

izing grapevine is an advantage for comparative physiology and molecular

biology studies and/or studies on resistance to water stress (Chaves et al.,

2010; Vandeleur et al., 2009).

2. Limitations of model plants

The use of Arabidopsis in water deficit stress studies was first limited by

methodological difficulties (e.g. gas-exchangemeasurements).However, specif-

ic growing conditions (e.g. short days) and progress in the available equipment

for leaf gas-exchange (e.g. development of specific leaf chambers for Arabidop-

sis plants) or the use of imaging techniques (e.g. IR thermal and Chl a imaging)

helped to overcome the limitations regarding physiological characterization of

mutants (Badger et al., 2009; Merlot et al., 2002, 2007; Oxborough, 2004).

Other limitation of model plants relates to the fact that the knowledge

gathered with model species might not be directly transposable to distantly

related crop species, namely concerning long-term strategies for improved

stress tolerance in the field (e.g. to cereal crops) (Møller and Tester, 2007).

For example, the relationship between Naþ tolerance and Naþ accumulation

is different in Arabidopsis and cereals, with an inverse relationship often

found within cereal species that is not that evident in Arabidopsis ecotypes

(Møller and Tester, 2007). Therefore, the results on salinity tolerance

obtained in Arabidopsis should be extrapolated to cereal crops with caution.

Regarding the root system, root architecture is another trait influencing

resistance to water and nutrient stress. The A. thaliana is a Brassica char-

acterized by a tap root system, which represents a limitation for studies of

crop root systems that are typically deep (Watt et al., 2008). Therefore, a

better understanding of the mechanisms underlying crop species response to

environmental stress should include the use of non-model species, grown

under field conditions (Travers et al., 2010) and observed over long periods in

order to more closely mimic crop’s life span (Vinocur and Altman, 2005).

III. PHOTOSYNTHETIC LIMITATIONS UNDERWATER AND SALINE STRESS

A. DIFFUSIVE LIMITATIONS (STOMATAL AND MESOPHYLL)

In steady state photosynthesis, CO2 diffusion fromatmosphere to the active site

of ribulose 1–5 biphosphate carboxylase/oxygenase (Rubisco) in the chloro-

plast follows a complex pathway that involves three major conductance


components: (1) the boundary layer, (2) stomatal conductance and (3) meso-

phyll conductance (gm) (Farquhar and Sharkey, 1982).

Stomata guard cells respond to multiple exogenous and endogenous sig-

nals, including light, CO2, leaf to air VPD, ozone, hormones (ABA, auxin) or

ions and reactive oxygen species, like hydrogen peroxide (Assmann, 1993;

Gray and Hetheringthon, 2004; Schroeder et al., 2001; Wasilewska et al.,

2008a,b). This enables stomata to adjust their aperture very fast (within

minutes) in response to changes in the surrounding environment and con-

tributes to optimize the balance between water vapour loss and CO2 uptake

(Chaves and Oliveira, 2004). By controlling transpiration, stomata also

influence leaf temperature and the fluxes of metabolites and long-distance

chemical signalling (Brownlee, 2001; Lake et al., 2001).

In C3 and C4 plants, stomatal closure is generally the main cause of

reduced photosynthesis under mild to moderate drought stress (Chaves

et al., 2009; Erismann et al., 2008; Flexas et al., 2004; Grassi and Magnani,

2005) and high salinity (Chaves et al., 2009; Delfine et al., 1999; Ghannoum,

2009; Hura et al., 2006). Because photosynthesis of C4 plants saturates at

much lower CO2 concentrations than that of C3 plants, it is unlikely that C4

assimilation is affected by stomatal closure like C3 photosynthesis. However,

the limited capacity for photorespiration or for the Water–Water cycle

(Mehler reaction) to emerge as alternative sinks of electrons under drought

may explain why C4 photosynthesis can be as sensitive or even more sensitive

to water stress than C3 photosynthesis, in spite of the larger photosynthetic

capacity and water use efficiency (WUE) of C4 plants (Ghannoum, 2009).

Once inside the leaf, CO2 has to diffuse from the intercellular air spaces to the

chloroplast. However, CO2 diffusion is limited by resistances in both gaseous

and liquid phases in the cytosol and by several diffusion barriers like intercellu-

lar spaces, the cell wall, the plasmalemma and chloroplast’s envelope. As a

whole this represents the so-called mesophyll resistance (or the inverse of the

biophysical diffusion resistance, the mesophyll conductance, gm) (Evans and

Loreto, 2000; Flexas et al., 2008; Pons et al., 2009). The gm emerged as an

important CO2 diffusive limitation of photosynthesis (Barbour et al., 2010;

Evans and Loreto, 2000; Flexas et al., 2008; Pons et al., 2009; Warren, 2006).

Low mesophyll conductance can reduce the partial pressure of CO2 at the site

of carboxylation, limit photosynthesis and affect carbon isotope discrimination

(�) (Niinemets et al., 2009).The relative contribution of stomatal and non-

stomatal limitations to photosynthesis depends on the severity, velocity and

type of stress being imposed (short term vs. long term). When water deficit is

intensified, limitations of non-stomatal processes become more important, in

particular, due to lower gm and impaired photobiochemistry (Chaves et al.,

2003; Flexas and Medrano, 2002; Flexas et al., 2004, 2008).


Studies by Flexas et al. (2004, 2008), Niinemets et al. (2005, 2009) and

Warren and Adams (2006) showed that both water and salinity stress

resulted in decreased gm in many species. Mesophyll conductance also

seems to respond very fast to external stimulus such as light, temperature

or CO2 (Flexas et al., 2008). This led to the hypothesis that stomatal and

mesophyll conductance could be interacting and/or being intrinsically co-

regulated (Galmes et al., 2007a,b; Peeva and Cornic, 2009; Warren, 2008a,b).

However, recent findings showed that the supposed co-regulation of gs and

gm depends to some extent on external signals, such as environmental

conditions. Flexas et al. (2009) showed that gm reduction caused by water

stress was fully reversed under conditions of cloudy days. In turn, Galle et al.

(2009) showed that the effects of water stress on gm, and the delayed recovery

after re-watering, were dependent on the prevailing irradiance, being more

marked under high light.

The relevance of leaf structure on gm is also emphasized in the literature

(Niinemets et al., 2009). Mesophyll conductance has physical characteristics

that relate to surface area of the intercellular spaces, walls and cytosol and

dimensions of the intercellular spaces (Lawlor and Tezara, 2009; Niinemets

et al., 2009). These physical characteristics change as a function of the

shrinking response of tissues to drought (Lawlor and Tezara, 2009). It is

known that prolonged water stress or salinity, especially during plant devel-

opment, may cause profound modifications in leaf anatomy, such as thick-

ened cell walls and smaller and more densely packed leaf cells (Bongi and

Loreto, 1989; Qiu et al., 2007). This may explain the long-term reduction of

mesophyll conductance in salt stressed plants (Niinemets et al., 2009). In a

recent paper, Perez-Martin et al. (2009) showed that leaf structural para-

meters (mass per unit leaf area—which means mesophyll porosity) influence

gm values. Although scattered, the relationship between leaf mass per unit

area (MA) and gm revealed that MA imposes a limitation to the maximum

values of gm Perez-Martin et al. (2009), an effect that seems to be species

dependent. MA decreased under water stress inO. europea but it increased in

V. vinifera, resulting in a negative relationship between MA and CO2 be-

tween sub-stomatal cavities and chloroplasts in O. europea, and in a positive

relationship in grapevine Perez-Martin et al. (2009).

We must also have in account the metabolic component of gm, which is

possibly related to the activity of proteins like carbonic anhydrase (CA) and

aquaporins (as channels for CO2), which together or individually can facili-

tate CO2 diffusion from the sub-stomatal cavity to the active sites of Rubisco

in the chloroplast (Niinemets et al., 2009). The fact that gm is affected by

environmental variables and changes rapidly in response to, for example,

CO2 concentration, leaf temperature and drought suggests that these


biochemical factors may indeed be involved in gm response to the environ-

ment (Warren, 2008a,b). Mesophyll conductance has also been shown to

depend on the genotype (Barbour et al., 2010).

1. The role of carbonic anhydrase

Carbonic anhydrase may have a relevant role in CO2 exchange by influencing

the metabolic component of gm (Warren, 2008a,b), especially under limiting

conditions of CO2 supply, as it happens in severe water and saline stress. It is

estimated that CA activity facilitates CO2 diffusion across the chloroplasts

by reducing diffusion resistance by about 1/3 (Evans and Von Caemmerer,

1996; Gillon and Yakir, 2000). Although CA is inhibited by progressive

water stress, its activity has been considered large enough not to pose limita-

tions on net assimilation (Flexas et al., 2004).

In leaves, CA is a Zn-containing enzyme that catalyzes the reversible

conversion of CO2 to HCO3�. Recently, it was shown that other CA isoforms

besides the well-characterized �CA1 may also contribute to CO2 transfer in

the cell to the catalytic site of Rubisco (Fabre et al., 2007). CA is the second

most abundant protein following Rubisco (Fabre et al., 2007; Tiwari et al.,

2005) and participates in a broad range of biochemical processes, like car-

boxylation and decarboxylation reactions, pH regulation, inorganic carbon

transport, ion transport, and water and electrolyte balance (Fabre et al.,

2007; Moroney et al., 2001; Smith and Ferry, 2000).

C4 plants have generally lower amount of CA than C3 plants, which

probably explains the low conductance to CO2 across the bundle sheath of

C4 plants (Brown and Byrd, 1993; Gillon and Yakir, 2000). Besides the

potential role on CO2 diffusion at the leaf mesophyll, CAs were also shown

to be upstream regulators of CO2 controlled stomatal movements in guard

cells (Hu et al., 2010b). Transcriptome analyses showed that � CA genes

�CA1, �CA4, �CA6 are highly expressed in both stomatal guard cells and

mesophyll cells (Leonhardt et al., 2004; Yang et al., 2008). In rice seedlings,

expression of the gene (OsCA1) coding for CA in leaves and roots was

induced by salt and osmotic stress (Yu et al., 2007). In Arabidopsis, over-

expression of the OsCA1 gene improved growth under NaCl as compared to

the wild type, suggesting a beneficial effect of CA activity on growth under

saline conditions.

Considering the role of CA in CO2 diffusion, future breeding strategies

(either of C3 or C4), aiming at crop improvement with regards to both water

and saline stress, may involve changes in the expression of CA in guard (and

mesophyll) cells.


2. The role of aquaporins

Aquaporins are abundant in the vacuolar and plasma membranes, including

mesophyll cells of higher plants (Terashima and Ono, 2002). There is increas-

ing evidence that aquaporins besides facilitating water transport, may also

facilitate CO2 diffusion across plasma membrane (Hanba et al., 2004;

Katsuhara and Hanba, 2008; Martre et al., 2002; Maurel and Chrispeels,

2001; Terashima and Ono, 2002; Tyerman et al., 2002). This characteristic

might be of interest under reduced CO2 availability, for example, under mild

to severe water and saline stress. Nevertheless, the relationship between

aquaporins and water stress resistance is still elusive (Aharon et al., 2003).

In Arabidopsis, when the expression of plasma membrane aquaporins

(PIPs) was reduced, there was an increase in the time needed for recovery

of hydraulic conductance and transpiration compared to plants normally

expressing PIPs (Martre et al., 2002). Aquaporins were also shown to be

involved in the recovery of winter embolism in walnut trees (Sakr et al., 2003)

and associated to changes in hydraulic conductance of olive tree leaves

(Cochard et al., 2007).

Several studies show upregulation of aquaporins in response to water

stress in species like A. thaliana (Jang et al., 2007), Phaseolus vulgaris

(Aroca et al., 2006), V. vinifera (Galmes et al., 2007a,b; Vandeleur et al.,

2009) and tobacco (Aharon et al., 2003; Jang et al., 2007; Siefritz et al., 2002).

This is in line with the suggestion by Vandeleur et al. (2009) that increased

aquaporin activity is part of an adaptation strategy to water stress. However,

and in contrast with this, gene expression studies in various species adapted

to desert climate showed a downregulation of aquaporins that reduced water

loss. This is the case of Opuntia acanthocarpa (Martre et al., 2001) or Agave

deserti (North et al., 2004). Identical behaviour was observed in Populus spp.

(Martre et al., 2001; Siemens and Zwiazek, 2003).

Although the reduction of gm under water stress may be partly linked to

physical changes in the structure of intercellular spaces due to leaf shrinkage

(Lawlor and Cornic, 2002), the fine and rapid regulation of gm in response to

varying environmental conditions are likely to be explained by alterations in

the expression and/or regulation of PIPs (Flexas et al., 2006; Hanba et al.,

2004; Uehlein et al., 2003).

3. Stomatal and photosynthetic patchiness under stress

Stomatal distribution and/or mean stomatal conductance can vary signifi-

cantly among adjacent micro-areas, often corresponding to the surface of

areoles. Such non-random, spatially arranged variation in gs (and in the

dimensions of stomatal aperture) is called ‘‘stomatal patchiness’’ (Mott and

Peak, 2007; Pospısolova and Santrucek, 1994). The phenomenon occurs in a


large number of families and species and is induced by multiple factors

(Table I). It characterizes by formation of patches of different stomatal

opening, with diverse size, shape, movement and is often transient in nature

(Mott and Peak, 2007).

Stomatal patchiness is relevant for plant physiology because suggests the

existence of still unrecognized mechanisms of stomatal functioning (Mott

and Buckley, 1998). However, the physiological consequences of the

‘‘patchy’’ stomatal behaviour remain unclear. Patchiness of stomatal con-

ductance is almost always detrimental to instantaneous WUE (Mott and

Peak, 2007) and is not consistent with an optimal behaviour of stomata

(Buckley et al., 1999). In turn, several authors emphasize that patchiness

may have a minor effect on Ci (Buckley et al., 1997) and that it can have a

much less important effect on plant assimilation and A/Ci response than once

thought (Lawlor and Cornic, 2002; Lawlor and Tezara, 2009). Flexas and

Medrano (2002) also concluded that for conductance to water vapour above

30–40 mmol H2O m� 2 s� 1 the effects of patchy stomatal closure on Ci

calculation were small.

Patchy stomatal closure is usually more common in pot experiments,

where plants experience a rapid dehydration, than under field conditions,

where drought is imposed slowly (Grassi and Magnani, 2005). Patchy sto-

matal conductance cannot be detected unequivocally with standard gas-

exchange techniques. It has been monitored by using techniques such as

starch staining, pressure infiltration and autoradiography and imaging (see

Table I). The different interpretation on the impact of patchiness on plant

physiology may result not only from the use of different species but also from

different methodologies (Table I).

Stomatal patchiness can occur in any plant leaf but mostly in heterobaric

ones (Beyschlag and Eckstein, 2001). These leaves are characterized by exten-

sions of the vascular bundles that span across the leaf, forming a physical

barrier to lateral gaseous diffusion and resulting in the discrete compartmen-

tation of the leaf and no lateral CO2 diffusion between neighbouring areoles.

This is more evident in response to water stress (Meyer and Genty, 1999) or

ABA treatment (Meyer and Genty, 1998). Homobaric leaves are deprived of

those bundle sheath extensions and have large interconnected intercellular air

space with few or no barriers for lateral gas fluxes. They should be therefore

less prone to patchiness than heterobaric leaves. However, according to

Morison et al. (2007), there is no dichotomy between species considered

homobaric and heterobaric, but rather a gradation which depends on

extension and size of bundle sheath extensions. According to Morison et al.

(2007), the relevance of lateral CO2 diffusion depends not only on stomatal

conductance and net assimilation but also on lateral permeability. Lateral


TABLE INon-Exaustive List of Literature Describing the Stomatal/Photosynthetic Patchiness in Leaves of Different Species and Anatomy (Homobaric, H;

Heterobaric, Het) in Response to Different Factors

Species Leaf anatomyFactor inducing

patchiness Detection method Reference

Vicia Faba H ABA in xylem Starch staining Terashima et al. (1988)Arbutos unedo Het No treatment (diurnal

variation)Liquid infiltration Beyschlag and Pfanz

(1990)Olea europea H Low humidity 14CO2 autoradiography Loreto and Sharkey

(1990)Quercus coccifera L. Het No treatment (diurnal

variation)Liquid Infiltration Beyschlag et al. (1992)

Gossypium hirsutum Het Drought 14CO2 autoradiography Wise et al. (1992)Xanthium strumarium Het Low humidity Chl a imaging Mott et al. (1993)Rosa rubiginosa L. Het Darkness to high PPFD

transitionChl a imaging Bro et al. (1996)

Nicotiana plumbaginifolia Het Exogenous ABA Chl a imaging Eckstein et al. (1996)Nicotiana plumbaginifolia Het Changes CO2 concentration,

light intensity and leafdetachment

Chl a imaging Eckstein et al. (1998)

Rosa rubiginosa Het Water stress and ABA Chl a imaging Meyer and Genty(1999)

Vitis vinifera, Nerium Oleander Het Water stress 14CO2 autoradiography Downton et al. (1988)Rosa rubiginosa Het Exogenous ABA Chl a imaging Meyer and Genty

(1998)Phaseolus vulgaris Het Exogenous ABA IR imaging Jones (1999)

Rosa hybrida Het Leaves from excised cuttings Chl imaging Costa (2002)Avena sativa No treatment (attached leaves) IR imaging Prytz et al. (2003)Xanthium strumarium L. Het Decrease in air humidity Chl imaging þ IR imaging West et al. (2005)Gossypium hirsutum L. Het No treatment (attached leaves) Chl imaging Marenco et al. (2006)Tradescantia virginiana Rapid desiccation of excised

well-watered leavesChl a imaging Nejad et al. (2006)

Nicotiana tabacum H CO2 concentration Chl a imaging Morison et al. (2007)Helianthus annus Het Light intensity Chl a imaging Morison et al. (2007)Phaseolus vulgarisVicia faba

HetH

Light intensity Chl a imaging Pieruschka et al. (2008)

Glycine maxVicia faba

HetH

Light intensity and water stress Chl a imaging Pieruschka et al. (2010)

diffusion of CO2, in addition to vertical CO2 diffusion through stomata, has

been shown to promote CO2 assimilation in both homo- and heterobaric

leaves, in particular when photosynthesis rates were low and stomata were

closed (Morison et al., 2007). Pieruschka et al. (2008, 2010) concluded that

lateral CO2 diffusion supports photosynthesis in some species whereas in

others like Glycine max, which is heterobaric, it did not.

Stomatal patchiness has been considered a resultant of heterogeneous

water status in different parts of the leaf (Beyschlag and Eckstein, 2001).

Hydraulic interactions among stomata were suggested as one of the possible

mechanisms involved in formation and movement of stomatal conductance

patches. Stomata can interact locally via hydraulic interactions of epidermal

cells (Mott and Franks, 2001; Mott et al., 1997) and these hydraulic interac-

tions can serve to coordinate the movements of adjacent stomata (Mott et al.,

1999). In a recent paper, Nardini et al. (2008) suggest that spatial stomatal

heterogeneity may arise from heterogeneous distribution of local hydraulic

resistances, which would maintain local water potential above critical values,

possibly by triggering vein cavitation.

B. BIOCHEMICAL AND METABOLIC LIMITATIONS

Metabolic potential of photosynthesis is determined by the amount and

activity of light harvesting components, electron transport components and

energy transduction processes, as well as by carbon metabolism, namely the

Calvin cycle enzymes involved in carboxylation (Rubisco) and in the regen-

eration processes of ribulose 1–5 bisphosphate (RuBP).

The photosynthetic apparatus is generally considered very resistant to mild

and moderate water stress (Chaves et al., 2002, 2009; Cornic, 2000; Flexas

et al., 2004; Warren et al., 2004), with stomata being the main limiting factor

to carbon uptake under such conditions (Angelopoulos et al., 1996; Cornic

et al., 1992; Kaiser, 1987). However, as water deficit progresses biochemical

limitations become apparent. The most frequently reported biochemical

mechanisms involved in drought-related downregulation of photosynthesis

are: limitations of phosphorylation (Lawlor and Tezara, 2009; Tezara et al.,

1999), RuBP regeneration (Medrano et al., 2002) and Rubisco carboxylation

(Parry et al., 2002; Zhou et al., 2007). All enzymes related to these main

mechanisms may experience a decrease in activity and/or amount under

stress.

Flexas and Medrano (2002) suggested that in C3 plants alterations in

Rubisco activity had a minor role in the drought induced limitation of

photosynthesis. However, literature describes discrepant results on the nega-

tive effects of drought on Rubisco activity and/or quantity, which can vary


from major reductions (Maroco et al., 2002; Parry et al., 2002) to minor

decrease (Flexas et al., 2006) or no effect at all (Delfine et al., 2001; Pankovic

et al., 1999). Such conflicting results can be related to the use of different

species and/or different experimental conditions (long-term vs. short-term

experiments) (Damour et al., 2008; Flexas et al., 2006; Parry et al., 2002).

At present atmospheric CO2 conditions (well below saturating values for

photosynthesis) the carboxylation rate by Rubisco is the major factor limit-

ing photosynthesis and not the electron transport nor the regeneration of the

RuBP (Long et al., 2004).

Under drought, stomatal closure will reduce CO2 availability, which leads

to low CO2 operating range and the carboxylation by Rubisco will arise as a

major rate limiting factor of net assimilation (Flexas et al., 2004; Grassi and

Magnani, 2005).

Under severe, long-term drought together with high temperatures and

irradiance will result in limited net assimilation and an unbalance between

PSII photochemical activity and the electron requirement for photosynthesis,

leading to an over-excitation and, subsequently, photoinhibitory phenomena

(Epron et al., 1992). The damage caused to PSII is associated with light-

induced oxidative stress. Indeed, active oxygen species (H2O2, OH� and O2�)are produced when photon absorption exceeds the rate of photon utilization

(Foyer and Noctor, 2000; Quartacci et al., 2002).

Drought stress usually leads to an increase of starch hydrolysis and

reduced sucrose translocation, with the maintenance and/or even the build-

up of the concentration of reducing sugars in leaf tissue (Bunce, 1982;

Chaves, 1991; Campos et al., 1999; de Souza et al., 2005). Such build-up of

sugars in leaves may have a protective effect (osmotic protection). The

osmotic contribution of sugars may also be essential to the build-up of a

driving force for roots to uptake water from the soil (Silveira et al., 2010).

Moreover, and although there is no clear evidence for feedback inhibition of

photosynthesis by sugars under drought, carbohydrate accumulation may be

associated with lower Rubisco activation state and repression of photosyn-

thetic genes expression (Paul and Foyer, 2001; Sheen, 1990). Proline accu-

mulation is another common metabolic response of higher plants to both

drought and salinity stress (Taylor, 1996; Hare et al., 1999, for a review).

Metabolic limitations occurring under high salinity are related with the

high concentrations of Naþ and Cl� in leaf tissue (in general above 250 mM)

(Munns et al., 2006). The depletion of organic acids accompanies stomatal

closure and decreased assimilation. Such a reduction of the organic acids

under high salinity may work as a compensatory mechanism for ionic imbal-

ance (Chaves et al., 2009; Sanchez et al., 2007). At higher concentrations,

NaCl may directly inhibit photosynthesis due to oxidative stress (Chaves


et al., 2009). In Sorghum under salinity, Netondo et al. (2004) reported a

significant decrease in maximum quantum yield of photosystem II, photo-

chemical quenching coefficient and electron transport rate and an increase in

the non-photochemical quenching (qN). However, in studies with different

rice cultivars with contrasting salinity tolerance, the potential quantum yield

of PSII (Fv/Fm) was almost not affected by salt stress, whereas qN increased

in sensitive cultivars with increasing salt stress (Dionisio-Sese and Tobita,

2000). It is possible that sensitivity to salt stress in cereals might be related

with a reduction in PSII photochemical efficiency associated with an

enhanced qN, as means to dissipate excess energy (Moradi and Ismail, 2007).

IV. IS WATER USE OPTIMIZED BY THE LEAVESUNDER WATER DEFICITS?

In leaves under water deficits, caused either by drought or by salinity, the

coordinated regulation of gas exchange (water vapour vs. carbon dioxide)

takes place. Such a tight coordination between photosynthetic CO2 assimila-

tion and leaf water loss determines land plant survival by preventing

desiccation, while allowing some CO2 to enter into the leaf (Nilson and

Assmann, 2007).

Since the early work by Cowan and co-workers (Wong et al., 1979, 1985),

Sharkey and Raschke (1981) and Schulze and Hall (1982) that the parallel

response of stomata and photosynthesis observed in the long-term response

to different environmental variables has inspired the hypothesis of an ‘‘opti-

mization’’ theory of stomatal opening that may have accompanied plant

evolution (Cowan and Farquhar, 1977). In other words, stomatal conduc-

tance would be coordinated with the CO2 requirement of the mesophyll,

leading to the maintenance of the ratio intercellular CO2 partial pressure/

external CO2 partial pressure (pi/pa) for variable conditions of CO2, light or

N nutrition, except when changes were too fast to produce a decrease in

photosynthesis with stomata still open (Wong et al., 1979). This assumption

had long supported empirical models of photosynthesis (Ball et al., 1987;

Leuning, 1995). The question still not fully clarified concerns the mechanism

by which guard cells responds to internal CO2 thus creating this correlation

between photosynthetic capacity and stomatal conductance. Propositions

included several metabolites – ATP, NADPH or RuBP (Farquhar and

Wong, 1984) that would act as signals from the mesophyll to stomata or

from the guard cells (zeaxanthin) to stomata (Zeiger and Zhu, 1998).

Later work using transgenic plants with reduced Rubisco showed that the

correlation between photosynthetic capacity and stomatal conductance can


be broken—indeed stomatal conductance was not affected in the plants with

reduced amounts of Rubisco and lower photosynthetic capacity, when they

were growing under high light (Krapp et al., 1994; Quick et al., 1991; von

Caemmerer et al., 1997). More recently, von Caemmerer et al. (2004), also

working with transgenic tobacco plants with reduced Rubisco, confirmed

that stomatal conductance is not tightly linked to the photosynthetic capacity

of guard cells or the mesophyll and further suggested that either pa (partial

pressure of atmospheric CO2) or CO2 in the stomatal pore may be sensed by

stomata, instead of pi.

Under well-watered conditions and large stomatal conductance, pi is gen-

erally around 70–80% of the pa. Under mild to moderate water deficits

stomata limit CO2 access to the mesophyll but the photosynthetic demand

for CO2 keeps the same, and pi values will decrease to 60–70% of pa (Chaves

et al., 2004). This explains why under mild to moderate water deficits an

increase in the ratio of carbon assimilated (A) by the leaf and the

corresponding water transpired (termed intrinsic water use efficiency—

WUEi, for the ratio between A and gs), is commonly observed (Chaves and

Oliveira, 2004). This is more evident in C4 plants, because their CO2 uptake is

less sensitive to the initial decline in gs than C3 plants (Ghannoum et al., 2002;

Long, 1999). In contrast, when leaf water deficits becomes severe an increase

in pi is observed due to the decline of net assimilation induced by non-

diffusive limitations, with a consequent decrease in WUEi.

Summarizing, pi tends to be maintained constant in leaves of plants kept in

absence of water or salinity stress. When tissue water deficits is installed,

stomata respond to leaf water potential, and both respond to and control the

supply and loss of water by leaves (Leuning et al, 2003). Under these

circumstances, the balance between the supply of CO2 to the chloroplast

(a function of the diffusion from the air to the site of carboxylation) and the

demand for CO2 by photosynthesis, governed by chloroplast biochemistry,

irradiance or sink strength (Chaves et al., 2004) will dictate the pi and WUEi.

V. HOW ARE STOMATA AND PHOTOSYNTHETICGENES REGULATED?

A. REGULATION OF STOMATAL APERTURE

As discussed above, environmental signals, such as drought and high salinity,

regulate stomatal pore opening and closure. By controlling CO2 uptake and

transpired water vapour, stomata play a crucial role in abiotic stress toler-

ance. The phytohormone ABA is the primary signal controlling the reactions


of guard cells to either drought or high salinity (Chaerle et al., 2005;

Wasilewska et al., 2008). In response to these stresses, plant ABA concentra-

tion increases, thus modulating the expression of target genes and controlling

adaptive physiological responses, both leading to stomata closure

(Christmann et al., 2007; Rabbani et al., 2003; Seki et al., 2002a). It is

therefore essential to better understand the molecular mechanisms underl-

ying stomata responses to water deficit.

During many years, ABA was assigned as the long-distance signal that

communicates water stress from the root to the shoot (Wilkinson andDavies,

2002). However, it was recently observed in Arabidopsis that, although the

water deficit response does require ABA biosynthesis and signalling in the

shoot, it is not affected by the capacity to generate ABA in the root. Instead,

water deficit seems to elicit a root-to-shoot communication by a hydraulic

signal, which precedes ABA biosynthesis in the shoot and consequent signal-

ling, followed by stomatal closure (Christmann et al., 2007). ABA-induced

stomatal closure requires the coordinate control of several cellular processes,

such as guard cell turgor, cytoskeleton organization, membrane trafficking

and gene expression (Hetherington, 2001) and is mediated by a complex,

guard cell signalling network of kinases/phosphatases, secondary messen-

gers, and ion channel regulation (Kim et al., 2010; Wasilewska et al., 2008).

An enormous number of signalling components that affect ABA-dependent

stomatal closing have been identified by forward and reverse genetic

approaches. Most of the Arabidopsis mutants with impaired ABA-mediated

stomatal responses correspond to guard cell signalling components

(Table II).

1. ABA perception

Upon perception of a stress signal ABA biosynthesis is induced primarily

in vascular tissues and ABA is exported into other cells by specific ATP-

dependent transporters (Kang et al., 2010; Kuromori et al., 2010). This

mechanism allows the rapid distribution of ABA to the guard cells, where

it triggers stomatal closure through changes of ion fluxes (Fig. 1). ABA

perception by the guard cells is undertaken by ABA receptors, whose identity

has remained under debate until recently (McCourt and Creelman, 2008).

Before the discovery of the RCAR/PYR1/PYL-PP2C complexes (Ma et al.,

2009; Park et al., 2009), a number of ABA-binding proteins, such as ABAR/

CHLH/GUN5 (Shen et al., 2006), the plasma membrane-localized GCR2

(Liu et al., 2007) and GTG1/GTG2 (Pandey et al., 2009) had already been

reported as ABA receptors. They affect ABA responses and are likely

involved in the network of ABA responses. However, how these ABA-

binding proteins are placed into the molecular events governing the main


TABLE IINon-Exhaustive List of Arabidopsis Mutants Showing Alterations in Stomatal

Response to Environmental Cues

Mutant Phenotype Reference

abi1 Stomata of abi1-1 are more openthan are those of the wild type

Koornneef et al. (1984)

los5/aba3 Reduced accumulation of ABAunder drought stress andconsequent increasedtranspirational water loss

Xiong et al. (2001)

atmpr4 Stomatal aperture in atmrp4mutant alleles was larger than inwild-type plants, resulting inincreased water loss

Klein et al. (2004)

atmpr5 Limited stomatal opening inducedby light

Klein et al. (2003)

atmyb60-1 Constitutive reduction in stomatalopening and decreased wiltingunder water stress conditions

Cominelli et al. (2005)

atmyb61 atmyb61 mutants do not closetheir stomata to as great anextent as wild-type plants inresponse to darkness

Liang et al. (2005)

atabcb14 Guard cells close more rapidly inresponse to high CO2

Lee et al. (2008)

nced3 Stomata are less responsive todrought due to reduced ABAbiosynthesis

Iuchi et al. (2001)

atabcg40 Mutant stomata closed moreslowly in response to ABA,resulting in reduced droughttolerance

Kang et al. (2010)

ost1 ost1 mutants showed reducedABA responsiveness in guardcells

Merlot et al. (2002)

ost2 ost2 responds to CO2 anddarkness, but not to ABA

Merlot et al. (2002,2007)

slac1 The slac1 mutations impair CO2-,ABA- and dark-inducedstomatal closure

Negi et al. (2008)

gork Impaired stomatal closure inresponse to darkness or thestress hormone abscisic acid

Hosy et al. (2003)

gpa1 Reduced stomatal density andindex. More sensitive to low-CO2-induced stomatal opening.Insensitivity in aspects of guardcell ABA responses

Nilson and Assmann(2010)

Pandey and Assmann(2004)

gtg1gtg2 gtg1 gtg2 mutants arehyposensitive to ABA

Pandey et al. (2009)

(continues)


TABLE II (continued )

Mutant Phenotype Reference

mpk9-1/12-1 ABA and calcium failed to activateanion channels in guard cells ofmpk9-1/12-1. Mutation in only 1of these genes did not show anyaltered phenotype.

Jammes et al. (2009)

pyr1pyl1pyl2pyl4 ABA-induced stomatal closureand ABA-inhibition of stomatalopening are impaired inquadruple mutant plants.

Nishimura et al. (2010)

dst Increased stomatal closure andreduced stomatal density,consequently resulting inenhanced drought and salttolerance in rice

Huang et al. (2009)

AtrbohD Impaired ABA-induced stomatalclosure

Kwak et al. (2003)

AtrbohF Impaired ABA-induced stomatalclosure

Kwak et al. (2003)

era1 ABA hypersensitivity of guard cellanion-channel activation and ofstomatal closure

Pei et al. (1998)

abh1 BA-hypersensitive stomatalclosure and reduced wiltingduring drought

Hugouvieux et al.(2001)

gcr1 Hypersensitivity to ABA in assaysof stomatal response

Pandey and Assmann(2004)

pp2ca-2 Stomatal closure is ABAhypersensitive

Kuhn et al. (2006)

tpk1 ABA-induced stomatal closure isslowed

Gobert et al. (2007)

abo1-1 The closure of abo1-1 stomata inresponse to ABA treatment wasgreatly enhanced compared tothat of the wild type

Chen et al. (2006)

rdc3 gs is significantly higher in controlconditions and exhibited noO3-induced closure

Overmyer et al. (2008)

ozs1 gs levels and the size of stomatalapertures are greater than in thewild type. Mutant and wild-typeplants responded similarly toenvironmental stimuli

Saji et al. (2008)

dri1 Reduced stomatal response toH2O2, CO2 and ABA

Song et al. (2006)

hit1 Impaired CO2 response, but showsfunctional responses to bluelight, fusicoccin and ABA

Hashimoto et al.(2006a,b)


ABA responses remains to be clearly determined and their role as ABA

receptors is largely controversial. Thus, the recent discovery of RCAR/

PYR1/PYL-PP2C, which was shown to control the main ABA responses

(Ma et al., 2009), has paved the way to understand the main ABA signalling

events leading to gene regulation and ion channel control. The identification

of RCAR/PYR1/PYL was obtained by two independent groups using

Fig. 1. Guard cell signalling and ion channel regulation leading to stomatalclosure in plants under water deficit conditions. Water shortage induces ABA accu-mulation, which is perceived by the ABA receptor. The receptor is formed by theheteromeric complex of a PP2C such as ABI1 and an ABA-binding RCAR/PYR1member. ABA receptor is present in both cytosol and nucleus. Without ABA, thephosphatase activity of the PP2C inhibits the activity of the protein kinase OST1 andrelated SnRKs. In the presence of ABA, the phosphatase activity of the receptor isblocked and the protein kinases released from inhibition. OST1 and related SnRKs, ina Ca2þ-dependent or Ca2þ-independent way, regulate several events leading to sto-matal closure. Dashed lines represent regulation by ABA through SnRKs or otherproteins (??). Abbreviations: ABA, abscisic acid; ABI1, ABA insensitive 1; PP2C,protein phosphatase 2C; RCAR/PYR1, regulatory component of ABA receptor1/pyrabactin resistante 1; OST1, open stomata 1; RBOHF, respiratory burst oxidasehomologue F; AHA1/OST2, Arabidopsis Hþ ATPase 1/open stomata 2; SLAC1,slow anion channel associated 1; GAP1, G protein alpha 1; KAT1/2, Kþ transporterof Arabidopsis thaliana 1/2; GORK1, guard cell outward rectifying Kþ-channel 1;CDPK, calcium-dependent protein kinase; S-type, slow-type; R-type, rapid-type; VK,vacuolar Kþ selective; TPK1, two pore Kþ channel; CE, cis-element.


different approaches. Using an Arabidopsis screening to identify pyrabactin

(selective ABA agonist)—insensitive mutants, Sean Cutler’s group identified

PYRABACTIN RESISTANTE 1 (PYR1) (Park et al., 2009). PYR1 and

several PYR1-Like (PYL) homologues were then characterized as ABA-

dependent inhibitors of group A type 2C protein phosphatases (PP2C),

such as ABI1 (ABA INSENSITIVE 1) ABI2, HAB1 (HYPERSENSITIVE

TO ABA 1), and HAB2, which are known to negatively regulate the ABA

signalling at an early step in the pathway (Merlot et al., 2001; Moes et al.,

2008; Saez et al., 2006).

On the other hand, Erwin Grill’s group, using a Yeast two Hybrid

approach, identified the REGULATORY COMPONENT OF ABA RE-

CEPTOR 1 (RCAR1), identical to PYL9, as an ABI1 and ABI2—interacting

proteins (Ma et al., 2009). They also showed that RCAR1 and its homo-

logues bind ABA with strong affinity and with stereoselectivity. In addition,

the RCAR1–ABA complex inhibits certain PP2C, such as ABI1, ABI2,

HAB1, and HAB2. Given that six PP2C proteins from clade A are related

with ABA responses and there are 14 RCARs, an enormous number of

combinatorial interactions would be possible if all RCAR members can

regulate the same PP2C. It is believed that different RCAR/PP2C complexes

may vary in their affinity to the hormone and address different downstream

signalling elements, thus allowing the adjustment of the ABA signalling to

the broad range of ABA levels (Raghavendra et al., 2010), induced by

different intensities of water deficit. Interestingly, it was shown in Arabidop-

sis that ABA stimulates PYR1/ABI1 interactions within 5 min. (Nishimura

et al., 2010).

2. ABA signalling transduction

ABA signalling transduction is another important aspect of stomatal regula-

tion. Recently, a series of crystallographic studies have clearly demonstrated

that interaction of PP2C with the hydrophobic surface of ABA-bound

receptors inhibits PP2C activity (Melcher et al., 2009; Miyazono et al.,

2009; Yin et al., 2009). This will then activate the SnRK2 protein kinase

SnRK2.6/OST1 (OPEN STOMATA 1), which functions as positive regula-

tor of ABA-induced stomatal closure (Figure 1) (Mustilli et al., 2002). Thus,

in non-stress conditions, the PP2C protein ABI1 interacts with OST1 in vitro

and negatively regulates ABA-induced OST1 kinase activity (Yoshida et al.,

2006). In response to water deficit, ABA increase will avoid inactivation of

OST1, which thereby activates the S-type anion channel SLAC1 (SLOW

ANION CHANNEL ASSOCIATED 1) (Geiger et al., 2009; Lee et al.,

2009) and inhibits the Kþ inward channel KAT1 by phosphorylation (Sato

et al., 2009) (Figure 1).


The SLAC1 is also regulated by calcium-dependent protein kinases

(CDPK) (Geiger et al., 2010). Interestingly, the guard cell outward rectifying

Kþ-channel GORK is up regulated by drought and salt and its regulation is

mediated by ABI1 and ABI2 (Becker et al., 2003); however, whether OST1

(or other protein downstream PP2C) is also involved in GORK regulation is

not yet known. OST1 also interacts with and activates the AtRBOHF

(RESPIRATORY BURST OXIDASE HOMOLOGUE F), a plasma mem-

brane localized NADPH oxidase that generates H2O2 (Sirichandra et al.,

2009a). H2O2 increases is known to mediate stomatal closure through activa-

tion of calcium channels (Pei et al., 2000).

The ABA-induced signalling transduction mechanisms in guard cells in-

clude two pathways, a Ca2þ-dependent and a Ca2þ elevation-independent

pathway, which might cross talk to regulate stomata movements (Fig. 1)

(Kim et al., 2010). For instance, KAT1 and SLAC1 channels are reciprocally

regulated by both pathways (Siegel et al., 2009). Other signals also known to

be involved in ABA-induced stomatal closure are: cytoplasmic pH; lipid-

based signalling molecules such as inositol 1,4,5-triphosphate (IP3),

sphingosine-1-phosphate (SP1), inositol hexakisphosphate (IP6), and phos-

phatidic acid (PA); and production of signal compounds such as nitric oxide

(NO) and malate catabolism (Kim et al., 2010). Besides the regulation of

other pumps/channels, these signals have been proposed to inhibit the Hþ

pump AHA1/OST2 (ARABIDOPSIS Hþ-ATPASE/OPEN STOMATA 2)

activity, in a Ca2þ-dependent way, thus contributing to stomatal closure

(Sirichandra et al., 2009b).

ABA-induced stomatal closure is also mediated by the vacuolar potassium

channel TPK (TWO PORE Kþ CHANNEL1) (Gobert et al., 2007) and the

ABC transporter AtMRP5 (MULTIDRUG RESISTANT PROTEIN 5),

which is a high affinity IP6 transporter (Nagy et al., 2009). The �-subunit

of the Arabidopsis heterotrimeric G protein, GPA1, is a regulator of trans-

piration efficiency. GPA1 regulates stomata density via the control of epi-

dermal cell size and stomata formation (Nilson and Assmann, 2010), but also

regulates potassium and anion channels in guard cells (Wang et al., 2001).

3. Transcription factors

The control of stomatal aperture by ABA signalling involves the transcrip-

tional regulation of many genes, however, little is known about the transcrip-

tion factors (TFs) involved. Although the role of the bZIP domain proteins in

ABA signalling (through their interaction with gene promoters containing

ABRE motifs) is well established, none of these TFs have yet been shown to

influence stomatal movements under abiotic stress conditions. On the other

hand, three MYB TFs from the subclass R2R3 were already reported to be


involved in stomatal movements in response to environmental stimuli.

AtMYB60 was shown to be specifically expressed in guard cells, and its

expression regulated by light conditions, ABA and water stress (Cominelli

et al., 2005). Its expression is negatively regulated under drought, concomi-

tantly with stomatal closure. However, elevated CO2 concentrations, which

are known to induce stomatal closure, do not modulate AtMYB60 expres-

sion. The atmyb60-1 null mutant shows a constitutive reduction in stomatal

opening and decreased wilting under water stress conditions. Interestingly,

many genes altered in atmyb60-1 (e.g. Aquaporin, ERD10, ERD13 and ERF)

are known to be involved in plant response to water stress (Cominelli et al.,

2005). AtMYB61 gene is also specifically expressed in guard cells in a manner

consistent with its involvement in the regulation of stomatal aperture (Liang

et al., 2005). While AtMYB60 gene expression is induced by light, leading to

an increased stomatal aperture, AtMYB61 transcription is repressed by light,

although it has the same effect on stomatal movement. AtMYB61 gene

expression was not altered in plants treated with ABA, salt, and drought,

known to induce stomatal closure. Although AtMYB61 seems to act in a

mechanism parallel to that responsible for closing stomata in response to

water deficit, a post transcriptional/translational regulation of AtMYB61

cannot be ruled out. The AtMYB44 gene is expressed in the vasculature

and leaf epidermal guard cells and is rapidly activated (within 30 min)

under dehydration, salinity, low temperature and ABA treatment (Jung

et al., 2008). When AtMYB44 is over-expressed in Arabidopsis, plants

show a higher sensitivity to ABA and a more rapid ABA-induced stomatal

closure then wild type. In addition, these transgenic plants exhibit a reduced

rate of water loss and an enhanced abiotic stress tolerance, mediated by the

reduced activation of PP2C genes, which are known as negative regulators of

ABA signalling. DROUGHT AND SALT TOLERANCE (DST) is a zinc-

finger TF that regulates drought and salt tolerance via stomatal aperture

control (Huang et al., 2009). It negatively regulates stomatal closure by direct

modulation of genes related to H2O2 homeostasis. Interestingly, loss of DST

function, besides increasing stomatal closure, also reduces stomatal density,

thus indicating a putative function in the abiotic stress control of stomata

development. The control of stomata aperture may also involve the activity

of the TF SNAC1. Transgenic rice plants over-expressing SNAC1 are more

sensitive to ABA and lose water more slowly by closing more stomata, thus

showing an enhanced tolerance to drought and high salinity (Hu et al., 2006).

Although we have focused our review on the ABA-mediated stomatal

responses, it is likely that other signals can control stomatal movement

independently of ABA. This is suggested, for instance, by the ABA-insensi-

tive mutant mrp5, which shows limited stomatal opening induced by light. In


addition, in the ABA insensitive mutant abi1-1 stomatal closure can be

induced by Cd2þ (Perfus-Barbeoch et al., 2002). However, this is an area

still largely unknown.

B. STOMATAL DEVELOPMENT

Significant advances have been made in Arabidopsis to understand the basic

genetic pathways controlling stomatal development, which requires a strict

control of a series of asymmetric and symmetric cell divisions in a specialized

epidermal cell lineage, followed by cell differentiation. Stomatal development

is initiated by an asymmetric division of a meristemoid mother cell (MMC)

producing a small meristemoid and a larger sister cell. This division is

regulated by the bHLH TF SPEECHLESS (SPCH) (MacAlister et al.,

2007) and requires the novel plant specific protein BASL (Dong et al.,

2009). The meristemoid has limited self-renewing capabilities and after one

to three divisions differentiates into a guard mother cell (GMC). This transi-

tion is controlled by MUTE (MacAlister et al., 2007; Pillitteri et al., 2007),

while the final differentiation step is regulated by FAMA (Ohashi-Ito and

Bergmann, 2006). Both MUTE and FAMA are also bHLH TFs. It was

recently shown that despite the different stomata morphologies in monocots

and in dicots, FAMA function is conserved, whereas the roles of MUTE and

two SPCH paralogs are somewhat divergent (Liu et al., 2009). In Arabidop-

sis, two further bHLH TFs, ICE1/SCRM1 and SCRM2, directly interact

with and specify the sequential actions of SPCH, MUTE and FAMA

(Kanaoka et al., 2008). In addition, the TOO MANY MOUTHS (TMM)

leucine rich repeat receptor-like protein (Nadeau and Sack, 2002), which is

involved in the spacing mechanism that inhibits the development of adjacent

stomata (the one-cell spacing rule), was proposed to associate with members

of the ERECTA family of LRR-receptor-like kinases (ER, ERL1 and ERL2)

and to negatively regulate several aspects of stomatal differentiation (Shpak

et al., 2005). Interestingly, the ERECTA protein is known to regulate plant

transpiration efficiency in Arabidopsis through modulation of stomatal den-

sity (Masle et al., 2005). The �-subunit of the heterodimeric G protein,

GPA1, is another regulator of the plant transpiration efficiency and similarly

to ERECTA was also shown to have a function in stomatal development

(Nilson and Assmann, 2010). Two related secreted peptides, EPIDERMAL

PATTERNING FACTOR 1 and 2, were also found to negatively regulate

stomatal development and their function is dependent on TMM and

ER-family function (Hara et al., 2007; Hunt and Gray, 2009). If EPF1 and/

or EPF2 prove to be peptide ligands for those receptors, and particularly for


ERECTA, it would be important to investigate whether they also play a role

in plant transpiration efficiency.

Recently, another secreted peptide STOMAGEN was reported as a meso-

phyll-derived positive regulator of stomatal density (Sugano et al., 2010).

This intercellular signalling factor suggests that inner photosynthetic tissues

may optimize their uptake of CO2 by regulating epidermal stomatal density.

Whether STOMAGEN expression mediates stomatal development in plants

subjected to abiotic stress has not yet been directly addressed. However, in

Arabidopsis, the expression of the STOMAGEN (At4g12970) seems to be

differentially regulated by ABA, drought, cold, high salinity, and heat treat-

ments (https://www.genevestigator.com).

Although the basic genetic mechanisms regulating stomatal development

in Arabidopsis have been rather well characterized (as seen above), how

environmental cues are influencing stomatal development is still an unre-

solved question. We know that to optimize leaf gas exchange under prevail-

ing environmental conditions, plants modulate stomata aperture, but they

can also regulate stomata number. Changes in environment, such as

increased CO2 in the atmosphere, increased temperatures and changes in

light quality, all have impact on stomatal density (Casson and Gray, 2008;

Casson et al., 2009). It has also been reported that water availability (Wu

et al., 2009) as well as exogenous applied ABA (Franks and Farquhar, 2001)

can modulate stomatal development. However, only the HIGH CARBON

DIOXIDE (HIC) protein has been shown so far to regulate stomatal devel-

opment in response to CO2 (Gray et al., 2000) and the PHYTOCROME

INTERACTING FACTOR 4 (PIF4) has been suggested to regulate stoma-

tal development in response to light (Casson et al., 2009). It is however

predictable that environmental stresses, such as water deficit or high salinity,

when perceived by plant cells, are transduced through a signalling pathway

that will converge on the proteins involved in stomatal development (e.g.

SPCH, MUTE, FAMA, ICE1/SCRM1). Curiously, ICE1/SCRM1 was first

identified as an abiotic stress-responsive TF (Chinnusamy et al., 2003), thus

showing a potential link between environmental adaptation and stomatal

development. In Arabidopsis, the MITOGEN-ACTIVATED PROTEIN

KINASE3 (MPK3) and MPK6, two environmentally responsive mitogen-

activated protein kinases (MAPKs), and their upstream MAPK kinases,

MKK4 and MKK5, compose the MKK4/MKK5-MPK3/MPK6 module.

This module is downstream YODA, a MAPKKK described as key regulator

of stomatal development and patterning (Wang et al., 2007). Recently, it

was shown that SPCH, which controls entry into the stomatal lineage, is

a substrate of AtMPK3 and AtMPK6, suggesting that SPCH activity may

be directly affected by adverse environmental conditions thereby enabling


the plant to modify stomatal development in response to abiotic stress

(Lampard et al., 2008). Curiously, the ABA-overly sensitive mutant abo1

shows a drought-resistant phenotype. The abo1 mutation enhances ABA-

induced stomatal closing and also influences development of guard

cells, resulting in stomata reduced to half the number compared to the

wild type (Chen et al., 2006). This suggests that ABA signalling must be

also involved in modulation of stomatal development in response to water

deficit.

C. EXPRESSION OF PHOTOSYNTHETIC RELATED GENES

In addition to stomatal regulation, adverse environmental conditionsmodulate

photosynthetic rate by controlling expressionof genes involved innon-stomatal

processes associated with photosynthesis and carbohydrate metabolism. Most

genes associated with photosynthesis are under control of a transcriptional

regulatory network evolved to control plant response to external stimuli. Tran-

scriptional profiling studies have shown that although some are upregulated,

most photosynthesis-related genes and the genes for carbohydrate metabolism

are downregulated under drought and high salinity (Chaves et al., 2009; Seki

et al., 2002b; Wong et al., 2006).

1. Transcription factors regulating photosynthetic related genes

TFs are usually defined as proteins that show sequence-specific DNA binding

and are capable of activating and/or repressing gene expression. Regulation

of gene expression controls many biological processes, such as cell cycle,

metabolic and physiological balance, and responses to the environment

(Riechmann et al., 2000). In the last decade, many TFs, belonging to different

TF families and sub-families, were shown to be involved in plant responses to

adverse environmental conditions, such as high salinity, drought, heat, and

low temperatures (Saibo et al., 2009; Yamaguchi-Shinozaki and Shinozaki,

2006). Among the TF families present in plants, AP2/EREBP (APETALA2/

ethylene responsive element binding protein), NAC (NAM, ATAF and

CUC), ZF-HD (zinc-finger homeodomain), AREB/ABF (ABA-responsive

element binding protein/ABA-binding factor), and MYC (myelocytomatosis

oncogene)/MYB (myeloblastosis oncogene) have been the most related with

abiotic stress responses. Interestingly, AP2/ERF and NAC proteins are

widely present in land plant genomes but no homologue has been identified

so far in other eukaryotes (Riechmann et al., 2000).

Although the differential expression of photosynthesis-related genes in

plants subjected to various abiotic stresses has been demonstrated (Chaves

et al., 2009; Seki et al., 2002b; Wong et al., 2006), only few TFs have been


associated with this process. Among the few examples is the regulation of

genes encoding CHLOROPHYLL A/B-BINDING (CAB) proteins of PSII.

Two MYB-like TFs from barley, HvMCB1 and HvMCB2, bind specifically

to defined regions of CAB promoters derived from barley and wheat. These

TFs have characteristic features of transcriptional activators and are

required for maximal CAB gene expression, but are not necessary for expres-

sion related to light and circadian clock. Interestingly, the transcription level

of both genesHvMCB1 andHvMCB2 is downregulated by salt, osmotic, and

oxidative stress (Churin et al., 2003). GLK1 andGLK2 areMYB TFs known

to regulate genes involved in chlorophyll biosynthesis and light harvesting

(Fitter et al., 2002; Waters et al., 2009), and their gene expression in Brassica

napus is altered by cold stress (Savitch et al., 2005). Whether these TFs also

respond to other abiotic stresses, such as drought or high salinity, is yet to be

investigated. Remarkably, MYB TFs seem to be involved in regulation of

photosynthetic related gene expression upon abiotic stress. The transcript

level of Ppcl and Gapl genes encoding a CAM (Crassulacean Acid Metabo-

lism)-specific isozyme of phosphoenolpyruvate carboxylase and NAD-de-

pendent glyceraldehyde-3-phosphate dehydrogenase, respectively, is

upregulated under high salinity stress. The promoters of both genes include

several common sequence motifs resembling consensus binding sites for the

MYB class of TFs (Schaeffer et al., 1995). The regions where these motives

are located were shown to be essential for regulation of transcription by

salinity, thus suggesting that MYB-type TFs control expression of Ppcl and

Gapl in plants under salt stress conditions. CAM is an adaptation of photo-

synthetic carbon fixation to water-limited environments that results in im-

proved WUE. Upon water-deficit or salt stress CAM adaptation requires

that CAM-specific genes are regulated at transcription level.

In addition toMYBs, other TF families may be involved in photosynthesis

response to stress. The LONG HYPOCOTYL 5 (HY5) is a bZIP-type TF

that regulates transcription of several photosynthesis-related genes, such as

CAB2 and RIBULOSE BISPHOSPHATE CARBOXYLASE SMALL

SUBUNIT (RBCS1A) and also regulates several stress-responsive genes

(e.g. CBF1, DREB2A, RD20 and MYB59) (Lee et al., 2007; Maxwell et al.,

2003). It has long been known that ABA controls the transcription of CAB

and RBCS genes, as observed in tomato (Bartholomew et al., 1991). Hence,

although there is no direct evidence showing that expression of photosynthe-

sis-related genes is regulated by HY5 in response to abiotic stress, this is

a likely hypothesis worthwhile being investigated. HAHB4 encodes a

sunflower TF belonging to the HD-Zip (sub-family I) and is positively

regulated by drought and ABA. The over-expression of this TF in sunflower

led to the downregulation of a large group of photosynthesis-related genes


(e.g. genes encoding components of photosystem I (LHCa) and photosystem

II (PSBx), related to chlorophyll biosynthesis, and others that comprise the

Calvin cycle, such as PRK and Rubisco) (Manavella et al., 2008). Although

the interaction between the TF and the promoters of these genes was not

investigated, there is strong evidence that HAHB4 regulates expression of

numerous photosynthesis-related genes and, besides light signalling, this

regulation may also be triggered by ABA/drought signalling.

Recently, it was reported in rice that over-expression of TSRF1, which

encodes an ethylene response factor (ERF) involved in drought and osmotic

stress responses, induces expression of OsRBCS (Quan et al., 2010). The

C2/H2-type zinc-finger proteins STZ and AZF2 from Arabidopsis have been

shown to function as transcription repressors under drought, cold, and high-

salinity stress conditions and expression of both STZ and AZF2 genes is

induced mainly in leaves under drought stress (Sakamoto et al., 2004). It has

been suggested that they play a role in the regulation of photosynthesis-

related genes. This hypothesis agrees with the fact that transgenic plants

over-expressing STZ show growth retardation, which might be explained

by STZ repression of genes related with photosynthesis and carbohydrate

metabolism. Phytochrome interacting factors (PIFs) are bHLH TFs acting

as negative regulators in light responses. In Arabidopsis, various PIFs were

shown to negatively regulate chlorophyll and photosynthesis genes in etio-

lated seedlings (Shin et al., 2009). In rice, there is at least one PIF, OsPIF,

regulated by cold and drought (Figueiredo et al., unpublished results). This

suggests that PIFs may also be involved in the regulation of photosynthetic

responses to different abiotic stresses.

VI. IMPROVING CARBON FIXATION UNDERENVIRONMENTAL STRESS?

Under low water availability or salinity photosynthesis is predominantly

limited by the available CO2 to the catalytic site of Rubisco (as a result of

stomatal closure) and therefore by the carboxylation rate. Under restricted

CO2 and high temperature the use of O2 as substrate for Rubisco (and

therefore photorespiration) will increase. This leads to losses of more than

50% of the carbon fixed by photosynthesis, as demonstrated with transgenic

antisense plants with reduced Rubisco, where growth under high light and

temperature decreased photosynthesis dramatically (Krapp et al., 1994). This

means that the activity of Rubisco will play an important role in determining

carbon assimilation by the leaves in stress conditions. However, Calvin cycle

enzymes involved in RuBP regeneration may also influence the rate of carbon


uptake, particularly when CO2 is saturating (Raines, 2003), but also under

severe drought conditions (Maroco et al., 2002).

These limitations have been successfully overcome through evolution with

the appearance of C4 plants that are able to concentrate CO2 in the vicinity of

Rubisco and therefore to decrease photorespiration, being more efficient

under lower stomatal conductance, high light and high temperatures. Indeed,

C4 photosynthesis, contrary to C3, is CO2 saturated in the present atmo-

sphere (Ghannoum et al., 2001) and therefore can cope with restricted

intercellular CO2 induced by stomatal closure. Interestingly, C4 plants have

evolved when atmospheric CO2 was lowest in the history of the planet earth

and occurred several times independently, suggesting that this pathway is a

strong ‘‘solution’’ to overcome photorespiration in spite of its complexity

(Long et al., 2006; Sage, 2004).

By growing plants in an enriched CO2 atmosphere limitations by stomata

and carboxylation due to water deficits may also be partially overcome, as

demonstrated by a large number of experiments in the last two decades

(Chaves and Pereira, 1992; Long et al., 2004; Wullschleger et al., 2002).

Indeed, by exposing plants to long-term CO2 enrichment a number of growth

and physiological alterations do occur, including some that ameliorate the

negative impacts of drought. They include (i) an increase in leaf net photo-

synthetic rates due to higher carbon substrate availability and the competi-

tive inhibition of the oxygenase activity of Rubisco (decreased

photorespiration), (ii) stomatal conductance generally reduced and (iii)

increased intrinsic WUE, resulting from higher A and lower gs than under

present atmospheric CO2 (Chaves et al., 2001; Gunderson and Wullschleger,

1994). Also observed increases in the root system, whole-plant hydraulic

conductance and osmotic adjustment may be important in this context.

Herbaceous crops and grasslands are reported to be the most responsive to

growth at elevated CO2 (Wullschleger et al., 2002).

In addition to using CO2 and O2 as substrates, Rubisco is known to have

low efficiency. An improvement in the Rubisco specificity factor for CO2 (�)

by genetic modification has been considered by researchers to improve car-

boxylation efficiency and ultimately plant performance under water deficits

and salinity (Long et al., 2006; Zhu et al., 2004). In a survey with 24 species, it

was shown that � tends to be higher in plants native from drier, hotter and

saline environments with respect to those from more mesophytic climates

(Galmes et al., 2005). The goal of increasing the efficiency photosynthesis of

plants by improvements on the kinetics of Rubisco has been a long-term goal

scoring modest successes (Mueller-Cajar and Whitney, 2008). Attempts to

manipulate Rubisco have not been successful so far and resulted in even a less

efficient enzyme (Parry et al., 2003; Spreitzer and Salvucci, 2002).


Reduced photorespiratory rates could be achieved by introducing enzymes

of the C4 pathway into C3 plants, through the combined use of genetic

engineering and transgenic technologies (Raines, 2006). Indeed, genes encod-

ing for different genes of the C4 pathway have already been successfully

transferred to C3 plants such as rice, and tobacco (Hausler et al., 2001; Ku

et al., 1999; Leegood, 2002). Although some interesting results have already

been reported, such as an increase synthesis of C4 acids or a decrease in CO2

compensation point, still no clear picture of alterations was obtained. As

pointed out by Raines (2006), a way forward in this investigation may also be

related to exploring C4 native genes in C3 plants, as pioneered by Hibberd

and Quick (2002). In a theoretical analysis of C4 photosynthesis in a C3 cell

von Caemmerer (2003) concluded that although energetically inefficient it

may ameliorate CO2 diffusion limitation in the mesophyll and therefore have

positive impact in C3 photosynthetic response to water deficits.

In a future atmosphere with saturating CO2, limitation of photosynthesis

will be shifted from carboxylation efficiency to the capacity for regenerating

RuBP, the CO2 acceptor of the C3 cycle, which depends on the electron

transport capacity and the enzymes of the Calvin cycle (Long et al., 2004).

Under such circumstances it would be possible to improve photosynthesis in

C3 plants by over-expressing the enzymes of the regenerative phase of the C3

cycle, as revised by Raines (2003).

VII. CONCLUSIONS AND FUTURE PROSPECTS

Abiotic stresses, such as drought and high salinity, are amongst the primary

causes of crops loss worldwide. Fast increase in world population, scarcer

soil and water resources, climate change and more environmentally aware

consumers are putting pressure on the agricultural sector to maximize crop

yield while guaranteeing a more efficient use of resources (Beddington, 2010;

Stefanelli et al., 2010). In this context, breeding to improve crop stress

tolerance remains a major goal to guarantee food security and sustainable

production. Consequently, increased knowledge on the factors limiting rele-

vant plant processes like photosynthesis under abiotic stress emerged as a

major topic of research in plant sciences.

Under water stress and high salinity, photosynthesis can be restricted by

CO2 diffusion (stomatal and mesophyll related) as well as by photochemistry

and carbon metabolism. Considering the inefficiency of Rubisco activity and

the limitation in its improvements, possible photosynthetic gains under

unfavourable environmental conditions may derive preferentially from opti-

mization of leaf gas exchange, via stomatal and mesophyll control.


Therefore, it is essential to fully understand the mechanisms regulating

stomatal aperture and mesophyll conductance under stress conditions. In

the plasma membrane of guard cells several ion transporters involved in

stomatal movement have been characterized, but it is still unknown how

they are globally regulated. In addition, genetic screens may unveil novel ion

transporters. The recent identification of the missing ABA receptor, helped

to better understand ABA signalling that controls stomatal aperture under

water deficits. Transcriptional regulation of genes involved in guard cell

movements may also play an important role in stomatal responses to the

environment. These findings can be used in breeding programmes to obtain

plants with more responsive guard cells to water scarcity or high salinity.

Research on the effects of abiotic stress on development of stomata is

required. Genetic screens to isolate mutants with abnormal stomatal devel-

opment (e.g. stomatal index, size) under moderate water deficit, may help to

identify new players involved in this regulation.

Photosynthetic limitations due to mesophyll conductance are currently

under research and, giving the large variation among species, it is expected

to find plants with higher gm (Warren, 2008). Stomatal mutants are being

used to decouple stomatal from mesophyll response, which could clarify

the possible coordination between stomata and mesophyll regarding gas-

exchange and plant water relations. The role of different CA on stomatal

regulation and of different CA isoforms on mesophyll conductance is being

investigated (Fabre et al., 2007; Hu et al., 2010a,b). Future research direc-

tions may include as well the clarification of the functional role of aquaporins

on leaf gas exchange.

Many photosynthetic related genes are regulated by either drought or salini-

ty at the transcription level. Given that only few TFs have been reported as

being associatedwith this regulation, further investigation on the transcription-

al network underlying photosynthetic responses to abiotic stress should receive

attention. A possible approach may include the identification of TFs that bind

to promoters of photosynthetic genes transcriptionally regulated by drought or

salinity. On the other hand, identification of targets for stress-responsive TFs

will make possible to spot photosynthesis-related genes.

ACKNOWLEDGEMENTS

Miguel Costa is supported by a fellowship granted by Fundacao para a

Ciencia e Tecnologia (FCT). Our research work is supported by FCT

through the projects POCI/AGR/59079/2004, PPCDT/AGR/61980/2004

and PTDC/BIA-BCM/099836/2008.


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