2.1 Gas Exchange
Under mild to moderate water deficits, stomatal closure is one of the earliest plant responses, concomitant with the reduced water potential and turgor associated with even a small decrease in relative water content (Chaves et al. 2003, Lawlor and Tezara 2009). Reduced stomatal conductance limits water loss and CO2 diffusion, and hence photosynthetic assimilation. Intercellular partial pressure of CO2 (pi) is determined both by the supply of CO2 to the chloroplasts and the demand for CO2 for photosynthesis by the chloroplasts. If photosynthetic capacity is uninhibited, then the demand for CO2 by the chloroplasts remains unchanged, but the supply of CO2 to the chloro-plasts will be reduced in parallel with stomatal closure and reduced transpiration. Ultimately, reduced photosynthetic assimilation rates result in
Fig. 5.1 The three main topics covered by articles published on "drought" in 2010 (a), and the main topics which overlap with each of those three topics (b-d), with overlapping circles indicating that both topics are combined in some of the articles. Topics were determined by assessing the number of articles containing relevant keywords in the 745 articles in the "Plant Sciences" category of the Science Citation Index for that year. ABA abscisic acid, g stomatal conductance, A photosynthesis
Fig. 5.1 The three main topics covered by articles published on "drought" in 2010 (a), and the main topics which overlap with each of those three topics (b-d), with overlapping circles indicating that both topics are combined in some of the articles. Topics were determined by assessing the number of articles containing relevant keywords in the 745 articles in the "Plant Sciences" category of the Science Citation Index for that year. ABA abscisic acid, g stomatal conductance, A photosynthesis reduced vegetative growth, and for many crops even mild drought stress results in reduced yield. Nonetheless, in some crops both stomatal conductance and carbon assimilation can be maintained until water potential falls to relatively low levels (Flexas and Medrano 2002). On the other hand, in species which are adapted to dry conditions, sto-matal closure can be seen even where tissue water content is still high - in response to very mild soil moisture reduction or at midday on hot days (Pinheiro and Chaves 2011) . Such stomatal closure helps to prevent xylem pressure exceeding cavitation thresholds (David et al. 2007).
Both hydraulic and chemical signals sent from drying roots to the shoot are involved in the regulation of stomatal closure and decreased growth during soil drying (Tardieu et al. 2010). Whether hydraulic limitation or chemical signalling dominates, appears to depend on the species and growing conditions. The importance of abscisic acid (ABA) as a root-sourced signal transported via the xylem and involved in stomatal regulation during drought has been highlighted in several studies (e.g., Dodd et al. 2006), but other compounds such as the precursors of ABA or cytokinins also play a role, as do changes in mineral composition or pH of the xylem (e.g., Wilkinson and Davies 2008). Where xylem sap is acidic, ABAH is partitioned into alkaline components in the symplast of the leaf cells, away from the sites of action of ABA on the stomata. As pH increases, the proportion of ionised ABA transported in the xylem sap increases, and has a greater impact on stomatal behaviour (Hartung et al. 1998). Sharp and Davies (2009) found that changes in xylem sap pH induced by drought are more common in herbaceous species than in woody perennials.
The main cause of reduced photosynthetic rate under mild to moderate water deficits is a reduction in the diffusion of atmospheric CO2 to the site of carboxylation (Chaves et al. 2009) . This is as a result of both stomatal closure and a reduction in mesophyll conductance - although the extent of the influence of mesophyll conductance is still debated (Pinheiro and Chaves 2011). Water stress also directly impacts on internal transport of CO2 and on enzyme activity and hence photosynthetic capacity (Lawlor and Tezara 2009), and these metabolic and diffusive limitations become predominant relative to stomatal limitation as water stress becomes more severe (Flexas and Medrano 2002).
It should be noted that despite the importance of reduced photosynthetic assimilation rates on growth and yield, slowly developing water deficit can result in a small leaf area index. This will impact on productivity even though assimilation rates may be close to those of well-watered plants (see Lawlor and Tezara 2009) .
Instantaneous water use efficiency (WUE.), the ratio of photosynthetic assimilation (A) to evapotranspiration (E), is a key component of both crop water use efficiency (WUE) and yield potential. WUEi is often increased when water availability is limited, as a result of stomatal closure and hence reduced E (Farquhar et al. 1982). This can ultimately lead to an increase in WUE at the whole plant scale (biomass/water transpired) or even crop scale (yield of harvestable product/ water used or applied).
Aquaporin (AQP) proteins are a class of membrane proteins that are now understood to play an important role in water transport, both under optimal and stress conditions (Kjellbom et al. 1999). These proteins consist of six membrane-spanning domains linked by three extracellular loops and two intercellular loops. They occur as tetramers, each monomer forming a functionally independent pore (Luu and Maurel 2005) . Aquaporins in plants occur as multiple isoforms, for example, with 35 homologues in Arabidopsis. There are multiple subfamilies of aquaporins in plant genomes, two of which have been shown to be particularly involved in water transport. Tonoplast intrinsic proteins (TIPs) are predominantly found in the vacuolar membranes, while plasma membrane intrinsic proteins (PIPs) are predominantly located in the plasma membranes. The PIPs can be subdivided into PIP1 and PIP2 homology subgroups and are considered to be exclusive transporters of water as shown by the highly conserved topology of the aromatic/argin-ine selectivity filter (Wallace and Roberts 2004). They are particularly important in controlling transcellular water transport, and are especially abundant in roots, where they play a substantial role in the uptake of water from soil (Javot and Maurel 2002) .
The naturally occurring gradient in water potential between the environment of plant roots and the environment of the shoots drives the uptake of water (Knipfer and Fricke 2011). Hydraulic resistances both in the root and shoot can limit the flow of water through the plant. The main hydraulic barrier to water uptake by roots is the radial transport pathway between root epidermis and xylem, rather than the axial path along xylem conduits (Steudle and Peterson 1998). The radial resistance to water flow can be divided into an apoplastic component (cell wall, middle lamella, and intercellular air space) and a cell-to-cell component (through plasmodesmata and across membranes) (Knipfer and Fricke 2011).
Regulation of AQP genes can be positively correlated with hydraulic conductance (Secchi et al. 2007, Secchi and Zwieniecki 2010) and transpiration rate (Aroca et al. 2006). Thus increased aquaporin expression during drought is often seen as a mechanism of maintaining hydraulic conductance and transpiration (Sade et al. 2010). Considerable variation, however, exists between species and even between cultivars in expression of AQP isoforms in response to drought. Cocozza et al. (2010) found that a Populus nigra clone which reduced stomatal conductance (as indicated by enrichment in carbon isotope composition in leaves) under drought also showed enhanced expression in the roots of two aquaporin genes, during drought. Another clone, which showed proline accumulation under drought in old leaves (this may allow osmotic adjustment - see later section - and hence maintenance of stomatal conductance), showed down-regulation of the same genes. The authors deduced that the clone that reduced stomatal conductance under drought increased the permeability of vascular tissue by overexpressing aquaporin genes, in order to facilitate water transport, whereas the proline-accumulating clone increased water conservation in root cells by down-regulating aquaporins. Drought was found to increase the expression of one of three PIPs examined in the leaves of Phaseolus vulgaris, and of all three PIPs in the roots (Aroca et al. 2006). PvPIP2;1 gene expression and PIP1 protein abundance were increased in the leaves also when ABA or methotrexate (MTX), an inhibitor of stomatal opening, were applied. None of the treatments in that study changed the leaf water status, suggesting that rapid stomatal closure allowed leaf water status to be maintained. Almeida-Rodriguez et al. (2010) also found up-regulation of six out ofll studied aquaporin genes in response to drought in the poplar Populus simonii x balsamifera, which they found to be drought avoiding, rapidly reducing stomatal conductance in response to stress. In contrast, no up-regulation of these genes was found in response to stress in the less drought-avoiding P. balsamifera. Secchi et al. (2006) consider that the trunk diameter fluctuations found in drought-sensitive Olea europaea require rapid water transport, involving aquaporin expression and gating. They found that expression of a PIPl, a PIP2, and a TIP1 was drastically decreased in response to 3-4 weeks of drought stress. Twig water potential and hydraulic conductivity were also decreased by the stress. Up-regulation of a PIP isoform occurred in the anisohydric grapevine "Chardonnay" during drought stress, but not in "Grenache", which shows far stronger stomatal control of water loss during drought (Vandeleur et al. 2009), indicating that there is no consistent correlation between the relative stomatal control in different species and aquaporin expression during drought. PIP isoforms in leaves of the model plant Arabidopsis thaliana were generally down-regulated upon gradual drought stress (which was reflected at the protein level) (Alexandersson et al. 2005).
Up-regulation and down-regulation of genes during drought stress has been found to be dependent on the method of water withdrawal employed (Bray 2004). In leaves of Richter-110 (a Vitis hybrid), expression levels were strongly decreased under short-term moderate water stress but maintained or increased under short-term severe water stress. After maintenance for 7 days under water stress, however, transcript abundance was approximately 50% of that in well-watered plants, regardless of whether stress was mild or severe (Galmes et al. 2007).
Some of the contrasting behaviour of different aquaporins (up- vs. down-regulation) may be explained by the hypothesis that two "classes" of AQP exist: (1) AQPs involved in the maintenance of cellular water (osmotic) status, which are not involved in controlling large stress-related variations in water potential, but which buffer local variations at the cell level or allow the movement of some gases of small non-electrolytes through membranes, and (2) AQPs that are specifically expressed and/or regulated following stresses in appropriate organs to compensate for the altered water potential (Hachez et al. 2006). In the stem parenchyma of Populus trichocarpa, PIP1 aqua-porins were up-regulated in response to drought stress, but PIP2 aquaporins were not (Secchi and Zwieniecki 2010). Interestingly, in that study, two PIP1 aquaporin genes which were not found to be up-regulated in response to drought were found to be up-regulated in response to xylem vessel embolism artificially induced without the presence of water stress. In addition, those two genes were down-regulated after embolism removal, suggesting a local role of these particular channels in refilling of embolisms. Expression profiles can also differ for the same aquaporin gene in different organs (Aroca et al. 2006; Guo et al. 2006; Galmes et al. 2007). Zhang et al. (2008) compared the expression of a TIP in seedlings of a cultivar of wheat growing in water and growing in polyethylene glycol (PEG). Expression in shoots was up-regulated while that in roots was down-regulated in the PEG treatment.
Expression in shoots was also up-regulated when salt was added to the water. When ABA was added to the water, no effect on transcript abundance was detected. This may suggest a direct effect of the osmotic potential of the solution on the aquaporin expression, as opposed to the expression being regulated by the transpirational demand. Plants were also grown with split roots, where half the roots were in PEG and the expression in these roots was down-regulated, while that in the other half of the roots was up-regulated. In this case, there was no change in transcript abundance in shoots compared to control - and osmotic potential of the shoots of the split-root plants was very similar to that of control plants. The authors suggest that this aquaporin must be involved in distribution of water from where there is enough water to where there is less.
Comprehensive studies using macro or microarrays are required to determine with certainty whether AQP genes are expressed in a coordinated fashion (Javot and Maurel 2002) . Such a study would ideally be undertaken in combination with assessment of hydraulic conductivity, to determine whether expression parallels water transport. It should be noted that there have been several reports showing that the respective abundance in aquaporin transcripts and in the encoded proteins are not necessarily correlated (see discussion in Luu and Maurel 2005) . While much of the assessment of the role of aquaporins in water transport during drought stress has been based on studies of levels of aqua-porin transcription. Water stress also acts on aquaporin protein relocalisation and on gating via reversible phosphorylation or via direct effects of osmotic or hydrostatic gradients (Luu and Maurel 2005) .
Stomatal closure as a result of drought coincides with exposure to high photosynthetically active radiation. When leaves are subjected to excess incident radiation relative to the available intracellular CO2, the rate of electron production exceeds the rate of electron use in the Calvin cycle. Reactive oxygen species (ROS), such as the superoxide anion (O^-), hydrogen peroxide (H2O2) , the hydroxyl radical (HO') , and singlet oxygen ('O2), are therefore produced, particularly in the chloroplasts, which are both the main producers as well as targets of ROS (Sofo et al. 2005) . The ROS react with proteins and lipids, causing damage to cellular structures and metabolism, particularly those associated with photosynthesis (Lawlor and Tezara 2009). This situation will ultimately damage the photosynthetic apparatus, unless either photoprotective mechanisms are available to down-regulate photosynthesis, or the decline in CO2 assimilation coincides with an increase in the strength of another sink for the absorbed radiation. Photoprotective mechanisms include thermal dissipation in the xanthophylls or lutein cycles (Demmig-Adams and Adams III 1996, Garcia-Plazaola et al. 2003), while alternative sinks include photorespiration (Harbinson et al. 1990) or the Mehler peroxidase reaction, in which electrons are transferred from reduced ferredoxin to O2". ROS accumulation under such conditions depends on the balance between ROS synthesis and ROS dissipation (discussed below).
Unfortunately, ROS synthesis, dissipation, and damage associated with ROS accumulation has not been quantified under clearly defined levels/ duration of irradiance, and levels of water deficit (Lawlor and Tezara 2009), leaving the impact of this system poorly understood as yet. Also, ROS are generally considered to lead to photodamage, but Nishiyama et al. (2011) argue that the impact of ROS is more related to inactivation of repair of photodamaged PSII than to the photodamage itself. Another complication in understanding the role of ROS in drought-stressed plants is that in addition to causing damage, ROS also act as signal molecules that activate multiple defence responses: Increased ROS production and the high redox state of the electron transport chain during water deficit induce expression of genes coding for components of energy-dissipating and regulation systems in chloroplasts, which assists in acclimation (Pfannschmidt et al. 2009).
Plants use both enzymatic and non-enzymatic antioxidant defence mechanisms to scavenge ROS. The enzymatic system includes superoxide dismutases (SOD), which act as the first line of defence against superoxide radicals as they catalyse the dismutation of superoxide radicals to H2O2 and O2 (Fridovich 1995) . The subsequent defences are mostly concerned with depleting the resulting H2 O2 before it can be converted to the highly reactive (and extremely damaging) hydroxyl radical (HO') by the Fenton reaction, in the presence of ferrous (Fe2+) ions (Mittler 2002). The enzymes involved include catalase (CAT), guaiacol-type peroxidases (POD), and enzymes of the ascorbate-glutathione cycle (Mittler 2002), such as ascorbate peroxidase (APX). In the process of converting O2'- to H2O2 and O2, ascorbic acid (AsA) is oxidised to form the monohy-droascorbate radical (MDA) that is reduced back to AsA by either reduced ferredoxin or by NADPH, catalysed by MDA reductase (MDAR). Dehydroascorbate (DHA) is produced when MDAR fails to reduce MDA to AsA, and is reduced to As A by DHA reductase (DHAR). Alternatively, in the glutathione peroxidase (GPX) cycle, glutathione (GSH) is required to restore AsA as the electron donor (Pfannschmidt et al. 2009); glutathione reductase (GR) catalyses the NADPH-dependent reduction of oxidised glutathione to its reduced form (Ahmad et al. 2010) . Polyphenol oxidase isoenzymes, located mainly in the thylakoid lumen, oxidise o-diphenolic substrates to o-quinones, and are therefore involved in the metabolism of phenols, which have a non-enzymatic antioxidant action. In another cycle, the catalase cycle (Mittler 2002) , catalases - heme-containing enzymes particularly abundant in the glyoxysomes - destroy the H2O2 generated by oxidases involved in the b-oxidation of fatty acids, and in the peroxisomes of green leaves, where they scavenge the H2 O2 arising from the oxidation of the photorespiratory-produced glycolate.
Changes of expression and activities of antioxidant enzymes have been detected in many species of plants in response to adverse environmental conditions, such as water deficit and other abiotic, biotic, and developmental stimuli (Ahmad et al. 2010) . Sofo et al. (2005) found that the activities of SOD, APX, CAT, and POD increased in relation to the severity of drought stress in both leaves and roots of olive (grown under high temperature and irradiance). In particular, a marked increase in APX activity was found in leaves of plants during severe drought stress. The authors suggested that up-regulation of the antioxi-dant system might be an important attribute linked to drought tolerance, which could limit cellular damage caused by ROS during water deficit. APX in the roots, in contrast, showed reduced levels of activity, possibly indicating that APX activity could be attributed mainly to the chloroplast-located enzyme (chlAPX) of leaf tissues. The huge increase in APX activity in the leaves under drought could explain how the chloroplasts were sufficiently protected against reactive oxygen species to maintain high electron transport rates.
Over-expression of one or more ROS-scavenging enzymes in various compartments has been shown to relieve oxidative stress (Miller et al. 2010). Eltayeb et al. (2007) found that overexpression of a MDAR gene in tobacco resulted in enhanced tolerance of PEG-induced water stress; the authors suggested this may be due to increased levels of AsA which mainly resulted from the enhanced activity of MDAR.
Accurate characterisation of the complex stress tolerance phenotypes of transgenic plants (over-)expressing a variety of antioxidant enzymes has been identified as a significant challenge in understanding antioxidant defences (Allen et al. 1997I . To date, assessment of the behaviour of mutants with altered ROS-scavenging capacity has focussed on stress factors other than drought. Thus much work is still needed to better understand the role of ROS and ROS-scavenging in drought tolerance.
Osmotic adjustment relates to the lowering of osmotic potential due to the net accumulation of solutes in response to water deficits (Zhang et al. 1999).
Osmotic adjustment is often induced during drought (Chaves et al. 2009), with solutes accumulating, resulting in the maintenance of a higher turgor potential at a given leaf water potential (Zhang et al. 1999). Different types of compatible solutes can be responsible e.g. various sugars, organic acids, amino acids, sugar alcohols, and ions. Concentrations of soluble sugars (sucrose, glucose, and fructose) are altered by drought - in general concentrations increase (Chaves and Oliveira 2004) - although under severe dehydration they may decrease (Pinheiro et al. 2001). Soluble sugars act as signalling molecules under stress (Chaves and Oliveira 2004) , interact with hormones, and modify the expression of genes involved in photosynthetic metabolism - generally resulting in a reduction in source activity such as photoassimilate export and an increase in sink activity such as production of lipids and proteins (Chaves et al. 2009).
Osmotic adjustment in plant cells can aid the maintenance of water uptake and cell turgor during stress, and therefore can allow a plant to continue growth during water deficit, since zero turgor occurs at a lower water potential in osmot-ically adjusted leaf tissue. However, where water supply is not replenished, continued abstraction of water will ultimately be detrimental - and thus osmotic adjustment is not always advantageous (Sinclair and Purcell 2005). Engineering osmo-protectant synthesis pathways into model plant species has led to significant (albeit modest) improvements in stress tolerance; adding multiple genes to increase osmoprotectant flux in response to stress may be more beneficial (Rathinasabapathi 2000) .
Developments in molecular biology have opened up the possibility of exploring the role of diverse molecules in drought tolerance. Many molecular adjustments have been found during drought stress, and comparison of drought-tolerant and non-drought tolerant lines has been used to indicate whether or not the extent of such adjustments may in some way be related to drought tolerance.
It has been suggested, for example, that microRNAs may play a role in drought tolerance in maize (Kong et al. 2010) , MicroRNAs are small RNA molecules that are important regulators of gene expression at the post-transcriptional level by repressing mRNA expression (Covarrubias and Reyes 2010) , The expression of a wide range of genes is altered during drought. Dehydrins are among the most frequently observed proteins induced by dehydration and may help in stabilizing membranes or proteins during stress (Egerton-Warburton et al. 1997) , A relationship has been suggested between both water-soluble inositol-polyphophates and membrane lipid polyphos-phoinositides and drought stress (Munnik and Vermeer 2010). The activity of phospholipase D (PLD), which regulates the production of phos-phatidic acid - a key class of lipid mediators in plant response to environmental stress, increases under drought (Hong et al. 2010). PLDa1 is particularly interesting with respect to drought tolerance, since it promotes stomatal closure and reduces water loss.
The method of imposing drought in many molecular papers, however, limits their application to "real" drought situations. For example, in the above-mentioned microRNA publication (Kong et al. 2010), "drought" actually involved dehydration by removing plants from soil and leaving them on filter paper - and Pinheiro and Chaves (2011) found that results from such experiments are very different to those where plants are subjected to drought conditions in soil/growing media. The lack of measurement of plant water relations in many molecular studies also means comparisons cannot be made across studies. A specific disadvantage of transcriptomic analysis is that in most comparisons protein abundance correlated very poorly with gene expression (Deyholos 2010), which can be particularly problematic in stress physiology, where sometimes only a small portion of the transcripts representing a specific subset of genes are actively translated. Deyholos (2010) pointed out another problem with many transcrip-tomic studies: they tend to focus on young tissue, which may not be the most relevant tissue in "real" crops in the field. Nonetheless, collaboration between ecophysiologists, agronomists, and molecular biologists in improving these investigations should be encouraged and is essential to optimise our understanding of plant responses to drought. In particular, the generation of mapping populations of contrasting cultivars or ecotypes has provided powerful new resources for dissecting the genetic basis of differences in drought tolerance and/or water use efficiency (e.g. Juenger et al. 2010). "Model" plants, however, are still not necessarily well understood in relation to physiological responses to water stress (Secchi and Zwieniecki 2010) - this needs rapid correction in order to fully exploit the wealth of genetic and genomic data available for such plants.
As highlighted in the introduction, the response of plants to drought is a huge topic. Water stress has an impact on many processes e.g. inflorescence development (Setter et al. 2011) that are outside the scope of this chapter. Several different processes interact. To give some examples, Kadioglu and Terzi (2007) highlight links between ROS-scavenging, osmolyte accumulation, and leaf rolling in dehydration avoidance; AQP down-regulation during drought stress may be a response to a cascade of events triggered initially by ROS accumulation (Luu and Maurel 2005) ; while ABA induces transcription factors that regulate the expression of PIP AQPs (Kaldenhoff et al. 1996).
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