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.
; Cornic, 2000
; Lawlor and
; Flexas et al.
), 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 Chaveset al.
, for reviews). Although early biochemical
effects of water deficits that involve alterations in photophosphorylation
were described by Tezara et al.
, it is not widely accepted
that this is the most sensitive water-stress component of photosynthesis
(Flexas et al.
). Recent work by Bota et al.
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 transportcapacity are very resilient to drought (Cornic et al., 1989;Epron and Dreyer, 1992) and variations in PSII photochemistrycan 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 electronchain is finely tuned to that of CO2 uptake (Genty et al., 1989;Loreto et al., 1995). Meyer and Genty (1998) found out thatthe decrease observed in photochemical efficiency in dehydratedor ABA-treated leaves could be almost completely reversed aftera fast transition of the leaves to an atmosphere enriched inCO2. This is an indication that photosynthetic capacity remainedhigh during dehydration and the limitation by CO2 was the mainfactor responsible for the decrease in the net photosyntheticcarbon uptake rate. A de-activation of the carboxylating enzymeRubisco by low intercellular CO2 (Ci) could account for themetabolic component of photosynthetic inhibition that was notreversed after the fast transition to an elevated CO2 atmosphere(Meyer and Genty, 1998). Other types of evidence suggest thatdecreased intercellular CO2 can play a pivotal role as mediatorof biochemical alterations in photosynthesis (Ort et al., 1994)(Fig. 1). According to Vassey and Sharkey (1989), sucrose-phosphatesynthase (SPS), a highly regulated enzyme that plays a key rolein plant source–sink relationships, seems to be a maintarget for the biochemical effects of water stress. Followingstomatal closure and the fall in CO2 concentration in the intercellularairspaces of the leaves, a decrease in SPS activity was observed.This effect may lead to a limitation of carbon assimilationby Pi under water deficits, as was observed by Maroco et al.(2002) in grapevines, by using the A/Ci analysis for estimatingthe limitation of A by triose phosphate utilization. However,increasing CO2 in the surrounding atmosphere can reverse thiseffect (Sharkey, 1990). Speer et al. (1988) also found out thatwhen stomata closed under mild dehydration (RWC 90–95%)nitrate reduction in spinach leaves was also inhibited. Whenthose leaves were illuminated in an atmosphere of 15% CO2, thisinhibition was reversed, nitrate reduction occurring then ata normal rate.
Figure 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.
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
) (Medrano et al.
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
in the mesophyll under drought has not deserved enough attention
(Centritto et al.
). In fact, these authors argue that
stomatal resistance is not the only diffusive limitation encountered
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
) 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 Centrittoet al.
and by Ethier and Livingston (2004)
Under field conditions plants are commonly subjected to multiplestresses in addition to drought, such as high light and heat.The combination of high irradiance (and/or heat) with CO2 deprivationat the chloroplast (driven by stomatal closure) predisposesthe plants for a down-regulation of photosynthesis or for photoinhibition.In fact, under conditions that limit CO2 fixation, the rateof reducing power production can overcome the rate of its useby the Calvin cycle. Protection mechanisms that prevent theproduction of excess reducing power are thus an important strategyunder water stress. Such protection may be achieved by the regulatedthermal 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 photoprotectivemechanisms compete with photochemistry for the absorbed energy,leading to a down-regulation of photosynthesis which is shownby the decrease in quantum yield of PSII (Genty et al., 1989).If the limitation of the rate of CO2 assimilation is accompaniedby an increase in the activity of another sink for the absorbedenergy, for example, photorespiration (Genty et al., 1990; Harbinsonet al., 1990; Wingler et al., 1999) or Mehler-peroxidase reaction(Biehler and Fock, 1996), the decline in non-cyclic electrontransport will be proportionally less than the decrease observedin the rate of CO2 assimilation. This type of response has mainlybeen documented in plants native to semi-arid regions. Muchless is known about how crop plants cope with excessive light,conditions that may arise even in irrigated field-grown plantsduring the summer period.
Oxidative stress or redox signalling under drought?
In agriculture, crop survival of a stress episode, such as droughtplus high temperature is vital. Protective responses at theleaf level must be triggered quickly to prevent the photosyntheticmachinery from being irreversibly damaged. Therefore, signalsare key players in plant resistance to stress.
As already mentioned, the over-reduction of components withinthe electron transport chain, following a drastic decrease inintercellular CO2 under drought results in electrons being transferredto oxygen at PSI or via the Mehler reaction. This generatesreactive oxygen species (ROS), such as superoxide, hydrogenperoxide (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 photosyntheticelectron components and the redox-active molecules synthesizedalso act as regulatory agents of metabolism (Neill et al., 2002;Foyer and Noctor, 2003).
Redox signals are early warnings, exerting control over theenergy balance of a leaf. Alterations in the redox state ofredox-active compounds regulate the expression of several geneslinked to photosynthesis (both in the chloroplast and in thenucleus), thus providing the basis for the feedback responseof photosynthesis to the environment, or in other words, theadjustment of energy production to consumption. It must be pointedout that the data on the redox regulation of photosynthesisgenes is still contradictory, suggesting a highly complex signallingnetwork (see the review by Pfannschmidt, 2003). Redox signallingmolecules include some key electron carriers, such as the plastoquinonepool (PQ), or electron acceptors (e.g. ferredoxin/thioredoxinsystem) as well as ROS (e.g. H2O2). The PQ redox state was shownto control gene transcription of photosystem reaction centresof cyanobacteria and chloroplasts (Allen, 1993). In particular,a reduced PQ pool activates the transcription of the PSI reactioncentre, whereas an oxidized pool activates the transcriptionof the PSII reaction centre (Li and Sherman, 2000).
The intracellular concentrations of ROS are controlled by theplant detoxifying system, which includes ascorbate and glutathionepools. Accumulating evidence suggests that these compounds areimplicated in redox signal transduction, acting as secondarymessengers 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 heatshock proteins (Karpinska et al., 2000); see also the reviewby Pastori and Foyer, 2002). The effects of H2O2 on guard cellswere first reported in Vicia faba by McAinsh et al. (1996),who found that exogenous applications of H2O2 induced an increasein cytosolic calcium as well as stomatal closure. On the otherhand, ABA applied to guard cells of Arabidopsis was shown toinduce a burst of H2O2 that resulted in stomatal closure (Peiet al., 2000; Desikan et al., 2004). However, when the productionof H2O2 exceeds a threshold, programmed cell death might follow.
H2O2 and other redox compounds play an important role in thestress perception of the apoplast, which acts as a bridge betweenthe environment and the symplast. Recently it was observed thatH2O2 is transported from the apoplast to the cytosol throughthe aquaporins, suggesting that the regulation of signal transductioncan also occur via the modulation of transport systems (Pastoriand Foyer, 2002). The interplay between the signalling oxidantsand their antioxidants counterparts, in particular ascorbicacid (AA), the most important buffer of the redox state in theapoplast, are key factors in the regulation of plant growthand defence in relation to biotic and abiotic stresses, as recentlypointed out by Pignocchi and Foyer (2003). These authors proposethat the modulation of the apoplast redox state modifies thereceptor activity and the signal transduction, leading to thestress response. It was also suggested recently that AA in theapoplast and the enzyme responsible for its redox state, theascorbate oxidase (AO), are involved in cell division and expansion,processes that are generally affected by diverse stresses, namelydrought. For example, the inhibition of cell division was observedwhen 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 signallingmolecule, in particular by mediating the effects of hormonesand other primary signalling molecules in response to environmentalstimuli. It may act by increasing cell sensitivity to thesemolecules (Neill et al., 2003). Recently, NO was shown to playa role as an intermediate of ABA effects on guard cells (Hetherington,2001; Neill et al., 2003). Likewise H2O2, NO may be also involvedin stress perception by the apoplast, since this compartmentcan be a major site of its synthesis. It is also likely thatboth NO and H2O2 are synthesized in parallel and act in a concertedway in a number of physiological responses, including stomatalresponses to the environmental stresses. Although the linksbetween dehydration and NO are not yet fully resolved, it seemsthat 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 anantioxidant by interacting with ROS produced under differentstresses, such as superoxide, and by inhibiting lipid peroxidation.However, if NO is produced in excess it may result in nitrosativestress (see Neill et al., 2003, for a review). The balance betweenNO and H2O2 also seems to play a role in some critical cellularresponses, 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, cytosolicpH) this may be reflected in NO production and regulatory functions,such as those exerted on stomatal aperture (Garcia-Mata andLamattina, 2003). It was also suggested that NO might operateover long distances, acting for example as root signal via nitritecoming from the roots to the shoot via the xylem stream. Itwould then produce NO in the guard cells. This evidence suggeststhat besides the role of NR in the co-ordination of C to N metabolism,this enzyme might also participate in the regulation of stomatalresponse to ABA and other stress factors.
Finally, NO also seems to play a role in the root response todrought and other stresses, namely by inducing adventitiousroot development (Pagnussat et al., 2002).
The carbohydrate status of the leaf, which is altered in quantityand quality by water deficits, may act as a metabolic signalin the response to stress (Koch, 1996; Jang and Sheen, 1997;Chaves et al., 2003). The signalling role of sugars under thiscontext is not totally clear. In general, drought can lead eitherto increased (under moderate stress) or to constant (under intensestress) concentration of soluble sugars in leaves, in spiteof lowered carbon assimilation, because growth and export arealso inhibited. Under very severe dehydration soluble sugarsmay decrease (Pinheiro et al., 2001). However, starch synthesisis, in general, strongly depressed, even under moderate waterdeficits (Chaves, 1991).
An increase in acid invertase activity was observed in leavesof droughted plants, coinciding with the rapid accumulationof 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 leafpetioles is anti-parallel to the changes in leaf blades, suggestingthat, under severe stress, leaves are increasing export (Pinheiroet al., 2001). Interestingly, the activity of acid vacuolarinvertase was highly correlated with xylem sap ABA concentration(Trouverie et al., 2003). Recent molecular analysis indicatedthat ABA is a powerful enhancer of the IVR2 vacuolar invertaseactivity and expression (Trouverie et al., 2003). There is alsothe indication of a direct glucose control of ABA biosynthesis.An increase in the transcription of several genes of ABA synthesisby glucose was observed in Arabidopsis seedlings (Cheng et al.,2002). Modulation of the expression of ABA signalling genesby glucose and ABA was also reported. Other evidence indicatesthat CO2, light, water, and other environmental signals canbe integrated and perceived as sugar signals (Pego et al., 2000),suggesting that different signal types may be perceived by thesame receptor or that the signal pathways converge downstream(Ho et al., 2001). On the other hand, sugars travelling in thexylem of droughted plants or sugars that might increase dramaticallyin the apoplast of guard cells under high light are likely toexert an important influence on stomatal sensitivity to ABA(Wilkinson and Davies, 2002).
Crosstalk between the sugar and plant hormone pathways, namelythose of ABA and ethylene (Pego et al., 2000; see also the reviewby Leon and Sheen, 2003) was also revealed. It was shown, forexample, that glucose and ABA at high concentrations act insynergy to inhibit growth, whereas at low concentrations theycan promote growth. On the other hand, it was demonstrated thatthe 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 tobe both dependent on concentrations and on the particular tissue;an example of the latter is the opposite effect of ABA on growthof shoot and root (Sharp, 2002).
Sugars are also involved in the control of the expression ofdifferent genes related to biotic stress, and lipid and nitrogenmetabolism (Koch, 1996; Jang and Sheen, 1997). They also affectthe expression of genes encoding photosynthesis via a complexand branched pathway. Depletion of sugars triggers an increasein photosynthetic activity, presumably due to a de-repressionof sugar controls on transcription, and an accumulation of sugars,due to a lower consumption of photoassimilates, have the oppositeeffect (Pego et al., 2000).
Chloroplast resistance to dehydration and rehydration: the importance of membrane stability
Contrary to poikilohydrous plants that change their tissue waterpotential in parallel with that of the soil and/or air, quicklyrecovering from dehydration, higher plants can buffer to a certainextent the variations in plant water status. As already discussed,this can be achieved by preventing water loss through stomatalclosure or by improving water acquisition from drying soil,either via a process of root osmotic adjustment or via an additionalinvestment in the root system.
When water deficits become too intense (generally agreed tobe in the range of leaf RWC lower than 70% (Kaiser, 1987; Chaves,1991) or too prolonged, leaves can wilt, cells shrink, and mechanicalstress on membranes may follow. Because membranes play a centralrole in various cellular functions, in particular those membraneswith embedded enzymes and water/ion transporters, the strainon membranes is one of the most important effects of severedrought and survival. Recovery under these conditions is closelylinked to plant capacity to avoid or to repair membrane damage,maintaining membrane stability during dehydration and rehydrationprocesses. Speer et al. (1988) found out that photosyntheticmembranes from spinach leaves wilted slowly under natural conditionsand were damaged earlier (i.e. become transiently permeable)than the plasma membrane. Chloroplastic membranes, and theirmembrane bound-structures, are especially susceptible to oxidativestress because large amounts of ROS can be produced in thesemembranes. ROS can cause an extensive peroxidation and de-esterificationof membrane lipids, as well as protein denaturation and DNAmutation (Bowler et al., 1992). On the other hand, intense shrinkageleads to an increased concentration of internal solutes thatmay reach toxic concentrations for certain proteins/enzymes(Speer et al., 1988), thereby intensifying detrimental effectson photosynthetic machinery, the cytosol, and other organelles.Upon the decrease in cellular volume, cell contents become viscous,increasing the probability of molecular interactions that canlead to protein denaturation and membrane fusion (Hoekstra etal., 2001).
Interestingly, studies of oxidative stress have shown that someantioxidants or their transcripts (e.g. glutathione reductase,GR or ascorbate peroxidase, APX) may be higher during recoverythan during the drought period, as observed, for example, incotton (Ratnayaka et al., 2003) or in pea plants (Mittler andZilinskas, 1994). This might suggest that either the stresshad induced an antioxidant response that ‘hardens’the plants for future stressful conditions (Ratnayaka et al.,2003) or/and that antioxidant protection is pivotal under therecovery phase. A broad range of compounds has been identifiedas playing a protective role on membranes and macromolecules.They comprise proline, glutamate, glycine-betaine, carnitine,mannitol, sorbitol, fructans, polyols, trehalose, sucrose, andoligosaccharides. All these compounds enable the proteins tomaintain their hydration state (Hoekstra et al., 2001). Uponfurther drying, sugars may replace the water associated withthe membrane macromolecules, therefore maintaining their structuralintegrity. In particular, the hydroxyl groups substitute waterin the maintenance of hydrophilic interactions with membranelipids and proteins. Dehydrins are supposed to protect proteinsagainst denaturating agents, therefore stabilizing membranes,through ion sequestration and replacement of hydrogen bonding(Close, 1996). Small heatshock proteins (HSPs) might act asmolecular chaperones, both during dehydration and rehydrationprocesses. Generally, HSPs are able to maintain partner proteinsin a folded-competent state, minimizing the aggregation of non-nativeproteins and degrading and removing them from the cell (Federand Hofmann, 1999). Among compatible solutes, sugars, especiallythe non-reducing disaccharides but also tri- and tetrasaccharidesand fructans, are the most effective for preserving proteinsand membranes under low water content (below 0.3 g H2O g–1DW). At this water content, water dissipates from the watershell of macromolecules and therefore, the hydrophobic effectresponsible for structure and function is lost (Hoekstra etal., 2001).
In the work done by Speer et al. (1988) it is also inferredthat membrane damage (namely the chloroplast envelope) was morepronounced during rapid rehydration than during the precedingdehydration process. During rehydration, water replaces thesugar (or other compatible compound) at the membrane surfaceand, during this process, a transient membrane leakage takesplace (Hoekstra et al., 2001). When dehydration is too intense,giving rise to some rigidification of membranes, an irreversibleleakage happens, followed by lethal injury. It seems that membranefluidity is an important factor in resistance to injury. Theeffects of rehydration on membranes might explain the retardationof recovery after rewatering, often observed after prolongedand/or intense drought. It was also suggested that the degreeof reversibility of the effects of dehydration is more speciesspecific than the effects of dehydration itself, which mightreflect differences in leaf structure rather than biochemicaldifferences among species (Speer et al., 1988).
Long-distance signalling: the root chemical signals
The importance of the chemical signals synthesized in the rootsfor the plant feedforward response to water stress has beenunder debate for some time (Wilkinson and Davies, 2002). Root-to-shootsignalling requires that chemical compounds travel through theplant in response to stress sensed in the roots. These signalsmay either be positive, in the sense that something is addedto the xylem flow, or negative, if something is taken away (ornot produced) from the xylem stream.
Hormones may become important controllers of plant metabolismunder poor growth conditions, such as imbalances in light, nutrients,and water availability (Weyers and Paterson, 2001), where developmentalplasticity could provide benefits through altered growth, optimizingthe 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, eitheracting in isolation or concomitantly. This long-distance signallingby hormones may be mediated by reactive oxygen species (Lakeet al., 2002). One example of the combined action of hormonesin root–shoot communication is that increased cytokininsconcentration in the xylem sap was shown to promote stomatalopening directly as well as to decrease stomatal sensitivityto ABA (see the review by (Wilkinson and Davies, 2002). Thecentral role of ABA in this process has been extensively reviewedrecently, covering aspects as different as biosynthesis, compartmentationwithin the cell/tissue, modulation by different factors andco-ordination of the responses at the whole plant level (seethe reviews by Hartung et al., 2002; Wilkinson and Davies, 2002).Since the mid-1980s chemical compounds synthesized in dryingroots, namely ABA or its conjugates (glucose esters), were shownto act as long-distance signals inducing leaf stomatal closure(Blackman and Davies, 1985) or restricting leaf growth, by arrestingmeristematic development (Gowing et al., 1990, see also Daviesand Zhang, 1991, for a review). Such knowledge has enabled itto be understood how some plant responses to soil drying canoccur without significant changes in the shoot water status.This is the case of ‘isohydric’ plants that areable to buffer their leaf water potential by controlling stomatalaperture via feed-forward mechanisms.
Further work has shown that ABA transport into the root xylemcan be modulated by the environment, namely through xylem pH,and also that the sensitivity of guard cells to ABA and changesin pH seem to be dependent on the time of the day (Wilkinsonand Davies, 2002). Under water deficits an increase in xylempH can occur, enhancing ABA loading to the root xylem (Hartungand Radin, 1989; Hartung et al., 2002). Water stress may alsoreduce ABA catabolism and prevent rhizosphere- and phloem ABAfrom entering the symplast, thus enhancing the ABA root signal(Wilkinson and Davies, 2002). Environmental conditions thatstimulate transpiration (e.g. VPD) also increase leaf sap pH,such increases in sap pH being correlated with reductions instomatal conductance. Davies et al. (2002) and Wilkinson andDavies (2002) speculated that differences in species in relationto stomatal sensitivity to ABA may be related with differentdegrees of alkalinization in response to soil drying. On theother hand, an increase in xylem sap pH may act alone as a droughtsignal to reduce leaf expansion via an ABA-mediated mechanism,as found in barley ABA-deficient mutants and in tomato (Baconet 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’