Revising the constraints to photosynthesis and the regulatory systems operating under water deficits
Diffusive and metabolic limitations: the role of intercellular CO2 as mediator of metabolic alterations
Although the nature and timing of the limitations that water deficits impose on leaf carbon assimilation have again been under debate (Tezara et al., 1999
; Cornic, 2000; Lawlor and Cornic, 2002; Flexas et al., 2004b), namely in what concerns stomatal constraints versus non-stomatal limitations, it is generally accepted that, under field conditions, the decrease in photosynthesis observed in response to moderate soil and/or atmospheric water deficits (leaf relative water contents down to 70–75%) is primarily due to stomatal closure (see Chaves et al., 2002, 2003, for reviews). Although early biochemical effects of water deficits that involve alterations in photophosphorylation were described by Tezara et al. (1999), it is not widely accepted that this is the most sensitive water-stress component of photosynthesis (Flexas et al., 2004b). Recent work by Bota et al. (2004) showed that limitation of photosynthesis by decreased Rubisco activity and RuBP content does not occur until drought is very severe.
Primary events of photosynthesis such as the electron transport capacity are very resilient to drought (Cornic et al., 1989; Epron and Dreyer, 1992) and variations in PSII photochemistry can be explained by changes in substrate availability. In fact, 
PSII often declines concomitantly with A under water stress, suggesting that the activity of the photosynthetic electron chain is finely tuned to that of CO2 uptake (Genty et al., 1989; Loreto et al., 1995). Meyer and Genty (1998) found out that the decrease observed in photochemical efficiency in dehydrated or ABA-treated leaves could be almost completely reversed after a fast transition of the leaves to an atmosphere enriched in CO2. This is an indication that photosynthetic capacity remained high during dehydration and the limitation by CO2 was the main factor responsible for the decrease in the net photosynthetic carbon uptake rate. A de-activation of the carboxylating enzyme Rubisco by low intercellular CO2 (Ci) could account for the metabolic component of photosynthetic inhibition that was not reversed after the fast transition to an elevated CO2 atmosphere (Meyer and Genty, 1998). Other types of evidence suggest that decreased intercellular CO2 can play a pivotal role as mediator of biochemical alterations in photosynthesis (Ort et al., 1994) (Fig. 1). According to Vassey and Sharkey (1989), sucrose-phosphate synthase (SPS), a highly regulated enzyme that plays a key role in plant source–sink relationships, seems to be a main target for the biochemical effects of water stress. Following stomatal closure and the fall in CO2 concentration in the intercellular airspaces of the leaves, a decrease in SPS activity was observed. This effect may lead to a limitation of carbon assimilation by Pi under water deficits, as was observed by Maroco et al. (2002) in grapevines, by using the A/Ci analysis for estimating the limitation of A by triose phosphate utilization. However, increasing CO2 in the surrounding atmosphere can reverse this effect (Sharkey, 1990). Speer et al. (1988) also found out that when stomata closed under mild dehydration (RWC 
90–95%) nitrate reduction in spinach leaves was also inhibited. When those leaves were illuminated in an atmosphere of 15% CO2, this inhibition was reversed, nitrate reduction occurring then at a normal rate.
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Fig. 1. Under moderate water deficits intercellular CO2 (Ci) decreases due to stomatal closure, while photosynthetic capacity is maintained. This decrease in Ci may induce reversible inhibition of some enzymes (e.g. SPS). At the same time, starch content decreases and reducing sugars are maintained or even increase. This change in the carbohydrate status can lead to alterations of gene expression.
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A recent survey in different species under drought suggests that metabolic impairment of photosynthesis does not occur until maximum light-saturated stomatal conductance is very low (generally lower than 50 mmol m–2 s–1) (Medrano et al., 2002). This agrees with the hypothesis of a CO2-scarcity mediated effect on metabolism under drought. On the other hand, the limitation to photosynthesis by an increased resistance to CO2 diffusion in the mesophyll under drought has not deserved enough attention (Centritto et al., 2003). In fact, these authors argue that stomatal resistance is not the only diffusive limitation encountered by CO2 in its route from the atmosphere to the chloroplasts. The mesophyll resistance to CO2 transfer can be sufficiently large to decrease the CO2 concentration from the intercellular spaces (Ci) to the site of carboxylation (Cc) and when not taken into account, can lead to an overeestimation of the metabolic limitations to carbon assimilation as discussed by Centritto et al. (2003) and by Ethier and Livingston (2004).
Under field conditions plants are commonly subjected to multiple stresses in addition to drought, such as high light and heat. The combination of high irradiance (and/or heat) with CO2 deprivation at the chloroplast (driven by stomatal closure) predisposes the plants for a down-regulation of photosynthesis or for photoinhibition. In fact, under conditions that limit CO2 fixation, the rate of reducing power production can overcome the rate of its use by the Calvin cycle. Protection mechanisms that prevent the production of excess reducing power are thus an important strategy under water stress. Such protection may be achieved by the regulated thermal dissipation occurring in the light-harvesting complexes, involving the xanthophyll cycle (Demmig-Adams and Adams, 1996; Horton et al., 1996; Ort, 2001) and presumably the lutein cycle (Bungard et al., 1999; Matsubara et al., 2001). These photoprotective mechanisms compete with photochemistry for the absorbed energy, leading to a down-regulation of photosynthesis which is shown by the decrease in quantum yield of PSII (Genty et al., 1989). If the limitation of the rate of CO2 assimilation is accompanied by an increase in the activity of another sink for the absorbed energy, for example, photorespiration (Genty et al., 1990; Harbinson et al., 1990; Wingler et al., 1999) or Mehler-peroxidase reaction (Biehler and Fock, 1996), the decline in non-cyclic electron transport will be proportionally less than the decrease observed in the rate of CO2 assimilation. This type of response has mainly been documented in plants native to semi-arid regions. Much less is known about how crop plants cope with excessive light, conditions that may arise even in irrigated field-grown plants during the summer period.
Oxidative stress or redox signalling under drought?
In agriculture, crop survival of a stress episode, such as drought plus high temperature is vital. Protective responses at the leaf level must be triggered quickly to prevent the photosynthetic machinery from being irreversibly damaged. Therefore, signals are key players in plant resistance to stress.
As already mentioned, the over-reduction of components within the electron transport chain, following a drastic decrease in intercellular CO2 under drought results in electrons being transferred to oxygen at PSI or via the Mehler reaction. This generates reactive oxygen species (ROS), such as superoxide, hydrogen peroxide (H2O2) and the hydroxyl radical, that may lead to photo-oxidation, if the plant is not efficient in scavenging these molecules. It is now acknowledged that the redox-state of the photosynthetic electron components and the redox-active molecules synthesized also act as regulatory agents of metabolism (Neill et al., 2002; Foyer and Noctor, 2003).
Redox signals are early warnings, exerting control over the energy balance of a leaf. Alterations in the redox state of redox-active compounds regulate the expression of several genes linked to photosynthesis (both in the chloroplast and in the nucleus), thus providing the basis for the feedback response of photosynthesis to the environment, or in other words, the adjustment of energy production to consumption. It must be pointed out that the data on the redox regulation of photosynthesis genes is still contradictory, suggesting a highly complex signalling network (see the review by Pfannschmidt, 2003). Redox signalling molecules include some key electron carriers, such as the plastoquinone pool (PQ), or electron acceptors (e.g. ferredoxin/thioredoxin system) as well as ROS (e.g. H2O2). The PQ redox state was shown to control gene transcription of photosystem reaction centres of cyanobacteria and chloroplasts (Allen, 1993). In particular, a reduced PQ pool activates the transcription of the PSI reaction centre, whereas an oxidized pool activates the transcription of the PSII reaction centre (Li and Sherman, 2000).
The intracellular concentrations of ROS are controlled by the plant detoxifying system, which includes ascorbate and glutathione pools. Accumulating evidence suggests that these compounds are implicated in redox signal transduction, acting as secondary messengers in hormonal-mediated events (Foyer and Noctor, 2003), namely stomatal movements (Pei et al., 2000).
H2O2 acts as a local or systemic signal for leaf stomata closure, leaf acclimation to high irradiance, and the induction of heat shock proteins (Karpinska et al., 2000); see also the review by Pastori and Foyer, 2002). The effects of H2O2 on guard cells were first reported in Vicia faba by McAinsh et al. (1996), who found that exogenous applications of H2O2 induced an increase in cytosolic calcium as well as stomatal closure. On the other hand, ABA applied to guard cells of Arabidopsis was shown to induce a burst of H2O2 that resulted in stomatal closure (Pei et al., 2000; Desikan et al., 2004). However, when the production of H2O2 exceeds a threshold, programmed cell death might follow.
H2O2 and other redox compounds play an important role in the stress perception of the apoplast, which acts as a bridge between the environment and the symplast. Recently it was observed that H2O2 is transported from the apoplast to the cytosol through the aquaporins, suggesting that the regulation of signal transduction can also occur via the modulation of transport systems (Pastori and Foyer, 2002). The interplay between the signalling oxidants and their antioxidants counterparts, in particular ascorbic acid (AA), the most important buffer of the redox state in the apoplast, are key factors in the regulation of plant growth and defence in relation to biotic and abiotic stresses, as recently pointed out by Pignocchi and Foyer (2003). These authors propose that the modulation of the apoplast redox state modifies the receptor activity and the signal transduction, leading to the stress response. It was also suggested recently that AA in the apoplast and the enzyme responsible for its redox state, the ascorbate oxidase (AO), are involved in cell division and expansion, processes that are generally affected by diverse stresses, namely drought. For example, the inhibition of cell division was observed when DHA (an oxidized form of AA) accumulates in the apoplast (Potters et al., 2000; Foyer and Noctor, 2003).
Nitric oxide (NO), a reactive nitrogen species, acts as a signalling molecule, in particular by mediating the effects of hormones and other primary signalling molecules in response to environmental stimuli. It may act by increasing cell sensitivity to these molecules (Neill et al., 2003). Recently, NO was shown to play a role as an intermediate of ABA effects on guard cells (Hetherington, 2001; Neill et al., 2003). Likewise H2O2, NO may be also involved in stress perception by the apoplast, since this compartment can be a major site of its synthesis. It is also likely that both NO and H2O2 are synthesized in parallel and act in a concerted way in a number of physiological responses, including stomatal responses to the environmental stresses. Although the links between dehydration and NO are not yet fully resolved, it seems that some of signalling components down-stream of NO (and H2O2) in the ABA-induced stomatal closure are calcium, protein kinases, and cyclic GMP (Desikan et al., 2004). NO also serves as an antioxidant by interacting with ROS produced under different stresses, such as superoxide, and by inhibiting lipid peroxidation. However, if NO is produced in excess it may result in nitrosative stress (see Neill et al., 2003, for a review). The balance between NO and H2O2 also seems to play a role in some critical cellular responses, including programmed cell death.
Because nitrite can act as a precursor of NO, nitrate reductase (NR)-dependent NO production is now receiving much attention. Since the activity of NR is highly regulated by the environment (including nitrate supply, light, temperature, CO2, cytosolic pH) this may be reflected in NO production and regulatory functions, such as those exerted on stomatal aperture (Garcia-Mata and Lamattina, 2003). It was also suggested that NO might operate over long distances, acting for example as root signal via nitrite coming from the roots to the shoot via the xylem stream. It would then produce NO in the guard cells. This evidence suggests that besides the role of NR in the co-ordination of C to N metabolism, this enzyme might also participate in the regulation of stomatal response to ABA and other stress factors.
Finally, NO also seems to play a role in the root response to drought and other stresses, namely by inducing adventitious root development (Pagnussat et al., 2002).
Sugar signalling
The carbohydrate status of the leaf, which is altered in quantity and quality by water deficits, may act as a metabolic signal in the response to stress (Koch, 1996; Jang and Sheen, 1997; Chaves et al., 2003). The signalling role of sugars under this context is not totally clear. In general, drought can lead either to increased (under moderate stress) or to constant (under intense stress) concentration of soluble sugars in leaves, in spite of lowered carbon assimilation, because growth and export are also inhibited. Under very severe dehydration soluble sugars may decrease (Pinheiro et al., 2001). However, starch synthesis is, in general, strongly depressed, even under moderate water deficits (Chaves, 1991).
An increase in acid invertase activity was observed in leaves of droughted plants, coinciding with the rapid accumulation of glucose and fructose in maize leaves (Trouverie et al., 2003) and with the accumulation of glucose, fructose, and sucrose, in both leaf blades and petiole of lupins (Pinheiro et al., 2001). The trend of changes observed in sucrose of the leaf petioles is anti-parallel to the changes in leaf blades, suggesting that, under severe stress, leaves are increasing export (Pinheiro et al., 2001). Interestingly, the activity of acid vacuolar invertase was highly correlated with xylem sap ABA concentration (Trouverie et al., 2003). Recent molecular analysis indicated that ABA is a powerful enhancer of the IVR2 vacuolar invertase activity and expression (Trouverie et al., 2003). There is also the indication of a direct glucose control of ABA biosynthesis. An increase in the transcription of several genes of ABA synthesis by glucose was observed in Arabidopsis seedlings (Cheng et al., 2002). Modulation of the expression of ABA signalling genes by glucose and ABA was also reported. Other evidence indicates that CO2, light, water, and other environmental signals can be integrated and perceived as sugar signals (Pego et al., 2000), suggesting that different signal types may be perceived by the same receptor or that the signal pathways converge downstream (Ho et al., 2001). On the other hand, sugars travelling in the xylem of droughted plants or sugars that might increase dramatically in the apoplast of guard cells under high light are likely to exert an important influence on stomatal sensitivity to ABA (Wilkinson and Davies, 2002).
Crosstalk between the sugar and plant hormone pathways, namely those of ABA and ethylene (Pego et al., 2000; see also the review by Leon and Sheen, 2003) was also revealed. It was shown, for example, that glucose and ABA at high concentrations act in synergy to inhibit growth, whereas at low concentrations they can promote growth. On the other hand, it was demonstrated that the glucose inhibition of growth could be overcome by ethylene, although, in general, this hormone acts as a growth inhibitor (Leon and Sheen, 2003). Responses and interactions appear to be both dependent on concentrations and on the particular tissue; an example of the latter is the opposite effect of ABA on growth of shoot and root (Sharp, 2002).
Sugars are also involved in the control of the expression of different genes related to biotic stress, and lipid and nitrogen metabolism (Koch, 1996; Jang and Sheen, 1997). They also affect the expression of genes encoding photosynthesis via a complex and branched pathway. Depletion of sugars triggers an increase in photosynthetic activity, presumably due to a de-repression of sugar controls on transcription, and an accumulation of sugars, due to a lower consumption of photoassimilates, have the opposite effect (Pego et al., 2000).
Chloroplast resistance to dehydration and rehydration: the importance of membrane stability
Contrary to poikilohydrous plants that change their tissue water potential in parallel with that of the soil and/or air, quickly recovering from dehydration, higher plants can buffer to a certain extent the variations in plant water status. As already discussed, this can be achieved by preventing water loss through stomatal closure or by improving water acquisition from drying soil, either via a process of root osmotic adjustment or via an additional investment in the root system.
When water deficits become too intense (generally agreed to be in the range of leaf RWC lower than 70% (Kaiser, 1987; Chaves, 1991) or too prolonged, leaves can wilt, cells shrink, and mechanical stress on membranes may follow. Because membranes play a central role in various cellular functions, in particular those membranes with embedded enzymes and water/ion transporters, the strain on membranes is one of the most important effects of severe drought and survival. Recovery under these conditions is closely linked to plant capacity to avoid or to repair membrane damage, maintaining membrane stability during dehydration and rehydration processes. Speer et al. (1988) found out that photosynthetic membranes from spinach leaves wilted slowly under natural conditions and were damaged earlier (i.e. become transiently permeable) than the plasma membrane. Chloroplastic membranes, and their membrane bound-structures, are especially susceptible to oxidative stress because large amounts of ROS can be produced in these membranes. ROS can cause an extensive peroxidation and de-esterification of membrane lipids, as well as protein denaturation and DNA mutation (Bowler et al., 1992). On the other hand, intense shrinkage leads to an increased concentration of internal solutes that may reach toxic concentrations for certain proteins/enzymes (Speer et al., 1988), thereby intensifying detrimental effects on photosynthetic machinery, the cytosol, and other organelles. Upon the decrease in cellular volume, cell contents become viscous, increasing the probability of molecular interactions that can lead to protein denaturation and membrane fusion (Hoekstra et al., 2001).
Interestingly, studies of oxidative stress have shown that some antioxidants or their transcripts (e.g. glutathione reductase, GR or ascorbate peroxidase, APX) may be higher during recovery than during the drought period, as observed, for example, in cotton (Ratnayaka et al., 2003) or in pea plants (Mittler and Zilinskas, 1994). This might suggest that either the stress had induced an antioxidant response that ‘hardens’ the plants for future stressful conditions (Ratnayaka et al., 2003) or/and that antioxidant protection is pivotal under the recovery phase. A broad range of compounds has been identified as playing a protective role on membranes and macromolecules. They comprise proline, glutamate, glycine-betaine, carnitine, mannitol, sorbitol, fructans, polyols, trehalose, sucrose, and oligosaccharides. All these compounds enable the proteins to maintain their hydration state (Hoekstra et al., 2001). Upon further drying, sugars may replace the water associated with the membrane macromolecules, therefore maintaining their structural integrity. In particular, the hydroxyl groups substitute water in the maintenance of hydrophilic interactions with membrane lipids and proteins. Dehydrins are supposed to protect proteins against denaturating agents, therefore stabilizing membranes, through ion sequestration and replacement of hydrogen bonding (Close, 1996). Small heatshock proteins (HSPs) might act as molecular chaperones, both during dehydration and rehydration processes. Generally, HSPs are able to maintain partner proteins in a folded-competent state, minimizing the aggregation of non-native proteins and degrading and removing them from the cell (Feder and Hofmann, 1999). Among compatible solutes, sugars, especially the non-reducing disaccharides but also tri- and tetrasaccharides and fructans, are the most effective for preserving proteins and membranes under low water content (below 0.3 g H2O g–1 DW). At this water content, water dissipates from the water shell of macromolecules and therefore, the hydrophobic effect responsible for structure and function is lost (Hoekstra et al., 2001).
In the work done by Speer et al. (1988) it is also inferred that membrane damage (namely the chloroplast envelope) was more pronounced during rapid rehydration than during the preceding dehydration process. During rehydration, water replaces the sugar (or other compatible compound) at the membrane surface and, during this process, a transient membrane leakage takes place (Hoekstra et al., 2001). When dehydration is too intense, giving rise to some rigidification of membranes, an irreversible leakage happens, followed by lethal injury. It seems that membrane fluidity is an important factor in resistance to injury. The effects of rehydration on membranes might explain the retardation of recovery after rewatering, often observed after prolonged and/or intense drought. It was also suggested that the degree of reversibility of the effects of dehydration is more species specific than the effects of dehydration itself, which might reflect differences in leaf structure rather than biochemical differences among species (Speer et al., 1988).
Long-distance signalling: the root chemical signals
The importance of the chemical signals synthesized in the roots for the plant feedforward response to water stress has been under debate for some time (Wilkinson and Davies, 2002). Root-to-shoot signalling requires that chemical compounds travel through the plant in response to stress sensed in the roots. These signals may either be positive, in the sense that something is added to the xylem flow, or negative, if something is taken away (or not produced) from the xylem stream.
Hormones may become important controllers of plant metabolism under poor growth conditions, such as imbalances in light, nutrients, and water availability (Weyers and Paterson, 2001), where developmental plasticity could provide benefits through altered growth, optimizing the response to the environment (Trewavas, 1986). Hormones, with particular relevance to ABA, but also cytokinins and ethylene, have been implicated in the root–shoot signalling, either acting in isolation or concomitantly. This long-distance signalling by hormones may be mediated by reactive oxygen species (Lake et al., 2002). One example of the combined action of hormones in root–shoot communication is that increased cytokinins concentration in the xylem sap was shown to promote stomatal opening directly as well as to decrease stomatal sensitivity to ABA (see the review by (Wilkinson and Davies, 2002). The central role of ABA in this process has been extensively reviewed recently, covering aspects as different as biosynthesis, compartmentation within the cell/tissue, modulation by different factors and co-ordination of the responses at the whole plant level (see the reviews by Hartung et al., 2002; Wilkinson and Davies, 2002). Since the mid-1980s chemical compounds synthesized in drying roots, namely ABA or its conjugates (glucose esters), were shown to act as long-distance signals inducing leaf stomatal closure (Blackman and Davies, 1985) or restricting leaf growth, by arresting meristematic development (Gowing et al., 1990, see also Davies and Zhang, 1991, for a review). Such knowledge has enabled it to be understood how some plant responses to soil drying can occur without significant changes in the shoot water status. This is the case of ‘isohydric’ plants that are able to buffer their leaf water potential by controlling stomatal aperture via feed-forward mechanisms.
Further work has shown that ABA transport into the root xylem can be modulated by the environment, namely through xylem pH, and also that the sensitivity of guard cells to ABA and changes in pH seem to be dependent on the time of the day (Wilkinson and Davies, 2002). Under water deficits an increase in xylem pH can occur, enhancing ABA loading to the root xylem (Hartung and Radin, 1989; Hartung et al., 2002). Water stress may also reduce ABA catabolism and prevent rhizosphere- and phloem ABA from entering the symplast, thus enhancing the ABA root signal (Wilkinson and Davies, 2002). Environmental conditions that stimulate transpiration (e.g. VPD) also increase leaf sap pH, such increases in sap pH being correlated with reductions in stomatal conductance. Davies et al. (2002) and Wilkinson and Davies (2002) speculated that differences in species in relation to stomatal sensitivity to ABA may be related with different degrees of alkalinization in response to soil drying. On the other hand, an increase in xylem sap pH may act alone as a drought signal to reduce leaf expansion via an ABA-mediated mechanism, as found in barley ABA-deficient mutants and in tomato (Bacon et al., 1998).
In a recent review Sharp (2002) proposed that the role of ABA in the control of shoot and root growth under water stress is an indirect one, resulting from the inhibitory effect of ABA on the synthesis of ethylene. Because ethylene inhibits growth, an insufficient ABA accumulation would result in an ethylene inhibition of shoot growth, whereas, in roots, the higher accumulation of ABA would prevent the ethylene-mediated inhibition of growth. Translocation of ABA from roots to shoots, in addition to producing stomatal closure and therefore turgor maintenance would, to some extent, counter-balance the inhibition of shoot growth by ethylene (Sharp, 2002). Considering that ABA ultimately co-ordinates whole plant performance, by regulating the partition of assimilates between the shoot and root, this ABA long-distance signalling could be described as a typical ‘resource allocation’ hormonal action.
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