Mechanisms underlying plant resilience to water deficits: prospects for water-saving agriculture

Abstract

Mechanisms underlying plant resilience to water deficits: prospects for water-saving agriculture

M. M. Chaves1,3,* and M. M. Oliveira2,3

1Departamento Botânica e Engenharia Biológica, Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal
2Faculdade de Ciência, Universidade de Lisboa, Lisboa, Portugal
3Instituto de Tecnologia Química e Biológica, Oeiras, Portugal

 

Journal of Experimental Botany 2004 55(407):2365-2384.

 

Abstract 

 

Drought is one of the greatest limitations to crop expansionoutside the present-day agricultural areas. It will become increasinglyimportant in regions of the globe where, in the past, the problemwas negligible, due to the recognized changes in global climate.Today the concern is with improving cultural practices and cropgenotypes for drought-prone areas; therefore, understandingthe mechanisms behind drought resistance and the efficient useof water by the plants is fundamental for the achievement ofthose goals. In this paper, the major constraints to carbonassimilation and the metabolic regulations that play a rolein plant responses to water deficits, acting in isolation orin conjunction with other stresses, is reviewed. The effectson carbon assimilation include increased resistance to diffusionby stomata and the mesophyll, as well as biochemical and photochemicaladjustments. Oxidative stress is critical for crops that experiencedrought episodes. The role of detoxifying systems in preventingirreversible damage to photosynthetic machinery and of redoxmolecules as local or systemic signals is revised. Plant capacityto avoid or repair membrane damage during dehydration and rehydrationprocesses is pivotal for the maintenance of membrane integrity,especially for those that embed functional proteins. Among suchproteins are water transporters, whose role in the regulationof plant water status and transport of other metabolites isthe subject of intense investigation. Long-distance chemicalsignalling, as an early response to drought, started to be unravelledmore than a decade ago. The effects of those signals on carbonassimilation and partitioning of assimilates between reproductiveand non-reproductive structures are revised and discussed inthe context of novel management techniques. These applicationsare designed to combine increased crop water-use efficiencywith sustained yield and improved quality of the products. Throughan understanding of the mechanisms leading to successful adaptationto dehydration and rehydration, it has already been possibleto identify key genes able to alter metabolism and increaseplant tolerance to drought. An overview of the most importantdata on this topic, including engineering for osmotic adjustmentor protection, water transporters, and C4 traits is presentedin this paper. Emphasis is given to the most successful or promisingcases of genetic engineering in crops, using functional or regulatorygenes. as well as to promising technologies, such as the transferof transcription factors.

Key words: Diffusional and metabolic limitations, genetic engineering, photosynthesis, water deficits, water-saving irrigation

 


Introduction

Water scarcity imposes huge reductions in crop yield and isone of the greatest limitations to crop expansion outside present-dayagriculture areas. Because the scenarios for global environmentalchange suggest a future increase in aridity and in the frequencyof extreme events in many areas of the earth (IPCC, 2001Go), irrigationand the use of appropriate crops is an important issue worldwide.Nowadays, approximately 70% of the global available water isemployed in agriculture and 40% of the world food is producedin irrigated soils. Some irrigation (around 10%) uses waterfrom aquifers, leading to many underground water tables beingexploited unsustainably (Somerville and Briscoe, 2001Go).

It is now recognized that fine-tuning irrigation can improvecrop water-use efficiency, allowing a more precise use of waterand, at the same time, having a positive impact on the qualityof the products. Similarly, modern biotechnology offers newtools for agricultural improvement and sustainability. Whereasthe main advances in agriculture during the 1960s were designedfor favourable environments, today, crop performance for sub-optimalenvironments and marginal lands which were bypassed by the ‘greenrevolution’ are also being addressed. In recent decades,physiological and molecular bases for plant responses to drought,and concurrent stresses, such as high temperature and irradiance,have been the subject of intense research (see reviews by Chaveset al., 2003Go; Flexas et al., 2004aGo).

Plant water deficits may occur as a consequence of a seasonaldecline in soil water availability, developing in the long term,or may result from drought spells. An increased evaporativedemand of the atmosphere, occurring mostly on a daily basis,affects total carbon gain by the crops, even irrigated ones.The timing, intensity and duration of stress episodes are pivotalto determine the effects produced by drought. Plant strategiesto control water status and resist drought are numerous (Schulze,1986Go). In general, genotypes native from climates with markedseasonality are able to acclimate to the fluctuating environmentalconditions, enhancing their efficiency for those conditions(Pereira and Chaves, 1993Go, 1995Go). In the case of slowly developingwater deficits, plants may also escape dehydration by shorteningtheir life cycle. In the case of rapid dehydration, oxidativestress developing as a secondary effect is potentially verydamaging to the photosynthetic machinery (Ort, 2001Go). The capacityfor energy dissipation (Flexas et al., 2002Go) and metabolic protection(induced or constitutive) against the damaging effects of reactiveoxygen species (Foyer and Noctor, 2003Go) is a key element forthe success of plants under drought. Tissue tolerance to severedehydration is not common in most higher plants, including crops,but do arise in species native from extremely dry environments(Ingram and Bartels, 1996Go). Understanding the mechanisms underlyingthose different responses can support the design of new managementtools and genotypes for modern precision agriculture.

It is well known that a major effect of decreased water availabilityis diminished leaf carbon fixation (A) due to stomatal closure,which may start at moderate plant water deficits. At the wholeplant level, total carbon uptake is further reduced due to theconcomitant or even earlier inhibition of growth. It has beenshown that cell division and expansion are directly inhibitedby water stress (Zhu, 2001aGo). Slower growth has been suggestedas an adaptive feature for plant survival under stress, becauseit allows plants to divert assimilates and energy, otherwiseused for shoot growth, into protective molecules to fight stress(Zhu, 2002Go) and/or to maintain root growth, improving wateracquisition (Chaves et al., 2003Go). This feature may be relevantfor crops intended for drought-prone areas, but inconvenientfor regions where only mild and sporadic stress is likely tooccur. On the other hand, the ability to accumulate (and lateron remobilize) stem reserves is likely to be an important characteristicto maintain reproductive growth under water deficits in variousspecies, like cereals and some legumes (Blum et al., 1994Go).

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 waterdeficits impose on leaf carbon assimilation have again beenunder debate (Tezara et al., 1999Go; Cornic, 2000Go; Lawlor andCornic, 2002Go; Flexas et al., 2004bGo), namely in what concernsstomatal constraints versus non-stomatal limitations, it isgenerally accepted that, under field conditions, the decreasein photosynthesis observed in response to moderate soil and/oratmospheric water deficits (leaf relative water contents downto 70–75%) is primarily due to stomatal closure (see Chaveset al., 2002Go, 2003Go, for reviews). Although early biochemicaleffects of water deficits that involve alterations in photophosphorylationwere described by Tezara et al. (1999)Go, it is not widely acceptedthat this is the most sensitive water-stress component of photosynthesis(Flexas et al., 2004bGo). Recent work by Bota et al. (2004)Go showedthat limitation of photosynthesis by decreased Rubisco activityand 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., 1989Go;Epron and Dreyer, 1992Go) and variations in PSII photochemistrycan be explained by changes in substrate availability. In fact,{phi}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., 1989Go;Loreto et al., 1995Go). Meyer and Genty (1998)Go 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, 1998Go). Other types of evidence suggest thatdecreased intercellular CO2 can play a pivotal role as mediatorof biochemical alterations in photosynthesis (Ort et al., 1994Go)(Fig. 1). According to Vassey and Sharkey (1989)Go, 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)Go 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, 1990Go). Speer et al. (1988)Go 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 suggeststhat metabolic impairment of photosynthesis does not occur untilmaximum light-saturated stomatal conductance is very low (generallylower than 50 mmol m–2 s–1) (Medrano et al., 2002Go).This agrees with the hypothesis of a CO2-scarcity mediated effecton metabolism under drought. On the other hand, the limitationto photosynthesis by an increased resistance to CO2 diffusionin the mesophyll under drought has not deserved enough attention(Centritto et al., 2003Go). In fact, these authors argue thatstomatal resistance is not the only diffusive limitation encounteredby CO2 in its route from the atmosphere to the chloroplasts.The mesophyll resistance to CO2 transfer can be sufficientlylarge to decrease the CO2 concentration from the intercellularspaces (Ci) to the site of carboxylation (Cc) and when not takeninto account, can lead to an overeestimation of the metaboliclimitations to carbon assimilation as discussed by Centrittoet al. (2003)Go and by Ethier and Livingston (2004)Go.

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, 1996Go;Horton et al., 1996Go; Ort, 2001Go) and presumably the lutein cycle(Bungard et al., 1999Go; Matsubara et al., 2001Go). 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., 1989Go).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., 1990Go; Harbinsonet al., 1990Go; Wingler et al., 1999Go) or Mehler-peroxidase reaction(Biehler and Fock, 1996Go), 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., 2002Go;Foyer and Noctor, 2003Go).

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, 2003Go). 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, 1993Go). 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, 2000Go).

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, 2003Go),namely stomatal movements (Pei et al., 2000Go).

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., 2000Go); see also the reviewby Pastori and Foyer, 2002Go). The effects of H2O2 on guard cellswere first reported in Vicia faba by McAinsh et al. (1996)Go,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., 2000Go; Desikan et al., 2004Go). 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, 2002Go). 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)Go. 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., 2000Go; Foyer and Noctor, 2003Go).

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., 2003Go). Recently, NO was shown to playa role as an intermediate of ABA effects on guard cells (Hetherington,2001Go; Neill et al., 2003Go). 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., 2004Go). 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., 2003Go, 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, 2003Go). 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., 2002Go).

Sugar signalling
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, 1996Go; Jang and Sheen, 1997Go;Chaves et al., 2003Go). 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., 2001Go). However, starch synthesisis, in general, strongly depressed, even under moderate waterdeficits (Chaves, 1991Go).

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., 2003Go)and with the accumulation of glucose, fructose, and sucrose,in both leaf blades and petiole of lupins (Pinheiro et al.,2001Go). 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., 2001Go). Interestingly, the activity of acid vacuolarinvertase was highly correlated with xylem sap ABA concentration(Trouverie et al., 2003Go). Recent molecular analysis indicatedthat ABA is a powerful enhancer of the IVR2 vacuolar invertaseactivity and expression (Trouverie et al., 2003Go). 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.,2002Go). 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., 2000Go),suggesting that different signal types may be perceived by thesame receptor or that the signal pathways converge downstream(Ho et al., 2001Go). 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, 2002Go).

Crosstalk between the sugar and plant hormone pathways, namelythose of ABA and ethylene (Pego et al., 2000Go; see also the reviewby Leon and Sheen, 2003Go) 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, 2003Go). 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, 2002Go).

Sugars are also involved in the control of the expression ofdifferent genes related to biotic stress, and lipid and nitrogenmetabolism (Koch, 1996Go; Jang and Sheen, 1997Go). 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., 2000Go).

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, 1987Go; Chaves,1991Go) 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)Go 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., 1992Go). On the other hand, intense shrinkageleads to an increased concentration of internal solutes thatmay reach toxic concentrations for certain proteins/enzymes(Speer et al., 1988Go), 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., 2001Go).

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., 2003Go) or in pea plants (Mittler andZilinskas, 1994Go). This might suggest that either the stresshad induced an antioxidant response that ‘hardens’the plants for future stressful conditions (Ratnayaka et al.,2003Go) 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., 2001Go). 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, 1996Go). 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, 1999Go). 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., 2001Go).

In the work done by Speer et al. (1988)Go 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., 2001Go). 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., 1988Go).

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, 2002Go). 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, 2001Go), where developmentalplasticity could provide benefits through altered growth, optimizingthe response to the environment (Trewavas, 1986Go). 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., 2002Go). 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, 2002Go). 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., 2002Go; Wilkinson and Davies, 2002Go).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, 1985Go) or restricting leaf growth, by arrestingmeristematic development (Gowing et al., 1990Go, see also Daviesand Zhang, 1991Go, 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, 2002Go). Under water deficits an increase in xylempH can occur, enhancing ABA loading to the root xylem (Hartungand Radin, 1989Go; Hartung et al., 2002Go). Water stress may alsoreduce ABA catabolism and prevent rhizosphere- and phloem ABAfrom entering the symplast, thus enhancing the ABA root signal(Wilkinson and Davies, 2002Go). 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)Go and Wilkinson andDavies (2002)Go 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., 1998Go).

In a recent review Sharp (2002)Go proposed that the role of ABAin the control of shoot and root growth under water stress isan indirect one, resulting from the inhibitory effect of ABAon the synthesis of ethylene. Because ethylene inhibits growth,an insufficient ABA accumulation would result in an ethyleneinhibition of shoot growth, whereas, in roots, the higher accumulationof ABA would prevent the ethylene-mediated inhibition of growth.Translocation of ABA from roots to shoots, in addition to producingstomatal closure and therefore turgor maintenance would, tosome extent, counter-balance the inhibition of shoot growthby ethylene (Sharp, 2002Go). Considering that ABA ultimately co-ordinateswhole plant performance, by regulating the partition of assimilatesbetween the shoot and root, this ABA long-distance signallingcould be described as a typical ‘resource allocation’hormonal action.

Applications to water-saving agriculture

Improving plant trade-off between assimilated carbon and water by using controlled irrigation
The understanding of the factors that regulate the trade-offbetween carbon assimilation and water loss, and those that drivepartitioning of assimilates between reproductive and non-reproductivestructures in relation to water availability are essential toidentify the technologies for matching water input with plantrequirements. Irrigation strategies that exploit the knowledgeof a plant's long-distance signalling system are increasinglybeing used to get improved crop water use efficiency under sustainedor improved quality of the product (Davies et al., 2002Go; Loveysand Ping, 2002Go). Indeed, it was demonstrated that large unregulatedfluxes of water are not essential to plant functioning and thatwater can be saved by manipulating stomatal functioning (Loveysand Davies, 2004Go). A measure of successful regulation of carbonassimilation under variable water availability is the plantability to maintain an equilibrium among the intervening processes,namely CO2 diffusion, light harvesting, photochemistry, andbiochemistry (Geiger and Servaites, 1994Go), so that the fluxthrough each component of the process is in balance with theothers, except for brief periods of transition. When water deficitsstart to build up, leaf stomatal conductance usually decreasesfaster than carbon assimilation, leading to increased wateruse efficiency, WUE (Chaves et al., 2004Go). It is also well knownthat when irrigation is above the optimum, an excessive shootgrowth can occur at the expense of roots and fruits (Zhang,2004Go). Manipulation of pre- and post-flowering water use incrops can be used to increase harvest index (HI) and by usingmethods of controlled irrigation the optimized water use bystomata can lead to an increase in WUE, without a significantdecrease in production and eventually with beneficial effectsin quality.

Closure of stomata under dehydrating conditions is the resulteither from a feedback response to the generation of water deficitsin the leaf itself that is transmitted to the guard cells, orfrom a feed-forward control before any alteration in leaf tissuewater status takes place (Schulze, 1986Go). These feed-forwardresponses of guard cells comprise the responses to high vapourpressure deficit, whose mechanisms area still under debate (Franksand Farquhar, 1999Go) and dehydration taking place elsewhere inthe plant, namely in the roots (Davies and Zhang, 1991Go). Inaddition to stomatal closure, shoot growth is slowed down ata very early stage of water stress (Hsiao, 1973Go; Kramer, 1983Go).As discussed in the previous section, strong evidence has accumulatedsuggesting that this kind of response to decreasing soil watermay be mediated by long-distance signals produced in dryingroots, namely of chemical origin (such as the hormone ABA orcytokinins) and transported to the shoot in the transpirationstream (Wilkinson and Davies, 2002Go). They will provide to theshoot a measure of the water available in the soil. However,ABA signalling is a complex process which involves not onlythe up-regulation of ABA biosynthesis and transport via thexylem to the leaf, but ultimately depends on homeostasis ofxylem sap along the length of the transport system and on thevariable role of anion trapping (Wilkinson and Davies, 2002Go).In fact, a large proportion of ABA transported from the rootsis catabolized in the cells of the leaf in a process termedABA filtration (Wilkinson, 2004Go). The pH of the xylem sap andof the leaf apoplast was shown to prevent ABA from enteringthe apoplast via the xylem. This is based on the ‘aniontrap’ concept (Wilkinson and Davies, 2002Go), which establishesthat ABA accumulates in the most alkaline compartments of thecells. The arrival of these signals at the guard cells (Alvimet al., 2001Go) or the growing tissues (Wilkinson, 2004Go) is thereforeultimately governed by the apoplastic pH,. Environmental factors(such as PPFD, temperature or VPD) that influence shoot physiologicalprocesses will interact with factors that affect the rhizosphere,determining the final apoplastic pH. As a consequence, plantWUE will reflect the multiple environmental stimuli perceivedand the ability of the particular genotype to sense the onsetof changes in moisture availability and therefore fine-tuneits water status in response to the environment (Wilkinson,2004Go).

This knowledge has inspired a special kind of deficit irrigation,the so-called partial root-zone drying (PRD), where each sideof the root system is irrigated during alternate periods. InPRD the maintenance of the plant water status is insured bythe wet part of the root system, whereas the decrease in wateruse derives from the closure of stomata promoted by dehydratingroots (Davies et al., 2000Go). Large-scale implementation of PRDirrigation in vineyards has already taken place in Australia(Loveys and Ping, 2002Go). This irrigation type has been furtherstudied in grapevines (Souza et al., 2003Go; Santos et al., 2003Go)and in other crops, such as tomato (Davies et al., 2000Go; Mingoet al., 2003Go), raspberries (Grant et al., 2004Go), orange trees(Loveys and Davies, 2004Go) or olive trees (Mentritto et al.,unpublished data). Although the nature of the signals is nottotally clear, it is recognized that stomatal closure and growthinhibition are likely to be responding simultaneously to differentstimuli, some of which may operate through common signal transductionsystems (Webb and Hetherington, 1997Go; Shinozaki and Yamaguchi-Shinozaki,2000Go). Physiological data that are being accumulated (e.g. ingrapevines under PRD) point to subtle differences between PRDand the deficit irrigation (DI), where the same amount of wateris distributed by the two sides of the root system (Souza etal., 2003Go; Santos et al., 2003Go). These differences include somereduction of stomatal aperture in PRD (more apparent when measurementsof stomatal conductance are done under constant light and temperature,rather than under the fluctuating conditions prevailing in thefield), a depression of vegetative growth, and an increase incluster exposure to solar radiation, with some potential toimprove fruit quality (Table 1). An interesting finding is thelink found between the intensity of the PRD stomatal responseand VPD, high VPD intensifying PRD stomatal closure comparedwith the controls (Loveys and Davies, 2004Go). These authors suggestthat the enhanced response of stomata to VPD in PRD irrigationcould be related to an increased ability of the xylem to supplyABA.

Table 1. Effect of controlled irrigation on physiological responses of field-grown grapevines

There is also evidence that PRD can increase fruit quality intomato, presumably as a result of differential effects on vegetativeand reproductive production (Davies et al., 2000Go). The rootsystem also seems to be significantly altered in response topartial dehydration, not only in respect to total extensionand biomass but also in architecture (Dry et al., 2000Go; TPdSantos et al., unpublished results; MA Bacon and WJ Davies,personal communication). It is likely that this alteration inthe root characteristics and in the source/sink balance playsan important role in plant performance under PRD.

In some crops, such as cereals (Blum et al., 1994Go; Gent, 1994Go)and some legumes (Chaves et al., 2002Go), reserves accumulatedin the stem before anthesis can be utilized for grain fillingin addition to current assimilates, therefore contributing toimportant gains in HI. Under stress conditions (Blum et al.,1994Go) or high respiration rates (for example, high temperatures)stem reserves are essential to complete grain filling (Gent,1994Go). The potential for storing reserves in the stem is dependenton stem length and weight density, although these characteristicsper se are not sufficient to ensure that those reserves wouldbe translocated to the fruit. Mobilization of reserves is dependenton sink strength, which varies with the genotype and is affectedby the environment (e.g. water availability). On the other hand,the stem (in particular, the stem stele, which is associatedwith the vascular tissue) is especially well protected againstenvironmental stress. In fact, studies in lupin subjected todrought indicated that the stem stele never dropped its relativewater content (RWC) below 83%, whereas the other organs in theplant exhibited values below 60%, namely the leaves 57%, theroots 58%, and the stem cortex 58% (Pinheiro et al., 2004Go).It can be speculated that this response is associated with theprotection given by the accumulation of assimilates, mainlyglucose, fructose, and sucrose whose concentration in the stemstele doubles under water deficits (Pinheiro et al., 2001Go).These sugars could also act as signals for the observed inductionof protective proteins such as late embryogenesis abundant (LEA)proteins, much more pronounced in the stele than in the cortex(C Pinheiro et al., unpublished data).

Controlled soil drying was shown to promote the remobilizationof carbon reserves during late grain filling in wheat and improveHI, especially when the crop is grown under high nitrogen (Yanget al., 2000Go, 2001Go). In fact, under such conditions, a mildsoil drying counteracts the delay in senescence of vegetativetissues that usually accompanies the heavy use of N, and improvesremobilization of stem reserves to the grains. Stay-green fortoo long results in the non-remobilization of pre-anthesis reservesin leaves, glumes, and stems, which may account for 30–47%of the carbon in protein and 8–27% of the carbon in carbohydratesdeposited in the grain (Gebbing and Schnyder, 1999Go). In China,if crop maturation is delayed, dry winds at the end of the growingseason can dehydrate wheat very rapidly and reduce grain yield.Yang et al. (2001)Go showed that, by applying a moderate soildrying and thus inducing an earlier senescence, they could accelerategrain filling and therefore improve yield.

However, in regions without the constraints described aboveextending the grain filling period, and therefore delaying leafsenescence, could benefit yield by allowing more time for thetranslocation of assimilates to the grain (Richards et al.,2001Go). This can be achieved either by controlling irrigationand/or by selecting genotypes for stay-green capability.

Genetic engineering for improved plant response to water deficit: recent advances
In the past decade most of the genetic engineering work thathas been successful in agricultural terms was directed towardscrop resistance to biotic stresses or to technological properties(see the review by Sonnewald, 2003Go). The studies addressingplant resistance to abiotic stress, namely in relation to drought,have been confined so far to experimental laboratory work andto single gene approaches, which has led to marginal stressimprovement (Ramanjulu and Bartels, 2002Go). However, recent advancessuggest that rapid progress will be possible in the near future,with large economical impact in many areas of the globe (Dunwell,2000Go; Garg et al., 2002Go; Wang et al., 2003Go) (Table 2). In fact,even modest improvements in crop resistance to water deficitsand in water use efficiency will increase yield and save water.One of the major challenges of this technology is to developplants not only able to survive stress, but also able to growunder adverse conditions with reasonable biomass production,overcoming the negative correlation between drought resistanttraits and productivity, which was often present in past breedingprogrammes (Mitra, 2001Go). Such a compromise requires improvedefficiency in maintaining homeostasis, detoxifying cells fromharmful elements (like ROS), and recovering growth that is arrestedupon acute osmotic stress (Xiong and Zhu, 2002Go). This also meansthat there is the need to introduce sets of genes that governquantitative traits, a technological approach that has alreadyproved to be successful, for example, in the case of transgenicrice with introduced provitamin A (Ye et al., 2000Go). The progressivecloning of many stress-related genes and responsive elements,and the proof of their association to stress-tolerant QTLs (QuantitativeTrait Loci), suggests that these genes may represent the molecularbasis of stress tolerance (Cattivell et al., 2002Go). On the otherhand, the identification of QTLs associated with drought toleranceis also an important tool for marker-assisted selection (MAS)of tolerant plants. These studies have been conducted on a broadvariety of species (see for instance Casasoli et al., 2004Go;Lanceras et al., 2004Go; Tuberosa et al., 2002Go). A lot of workhas been done on this topic and will not be covered here; however,it is clear that the combination of traditional and molecularbreeding (MAS and genetic engineering) will allow a more rapidway to improve abiotic stress tolerance in agricultural crops.

Table 2. Recent achievements in improving drought tolerance in crops through genetic engineering

The increasing knowledge of stress adaptation processes andthe identification of key pathways and interactions involvedin the plant response to the stress conditions is being exploitedto engineer plants with higher tissue tolerance to dehydrationor with drought avoidance characteristics (Laporte et al., 2002Go).The latter is, of course, more difficult to achieve, becauseit is linked to whole-plant morphological and physiologicalcharacteristics (Altman, 2003Go).

The recent progress in gene discovery and knowledge of signaltransduction pathways is raising the possibility of engineeringimportant traits by manipulation of one single gene, downstreamof signalling cascades, with putative impact on more than onestress type. Moreover, in genetic engineering, it is importantto mimic nature and activate, at the correct time, only thegenes that are necessary to protect the plants against stresseffects. This may be achieved by using appropriate stress-induciblepromoters and will minimize effects on growth under non-stressingconditions, which is essential for agricultural crops. It isalso desirable to target the desired tissue/cellular location,to control the intensity and time of expression, and to ensurethat all the metabolic intermediates are available, so thatno negative effects will arise (Holmberg and Bulow, 1998Go). Finally,to be able to prove that a transgenic plant is more resistantto water stress than the wild type, one needs a rigorous evaluationof the physiological performance as well as the water statusof transformed plants. This will avoid ambiguous interpretationsof the gene effects on plant drought resistance, such as thoseoften appearing in the literature (see, for example, the commentby Blum in www.plantstress.com/admin/Files/Hsieh_PlantPhysiol_130.htmand Hsieh et al., 2002Go). In other words, the impact of the introducedgenes must be separated in their direct versus indirect effects(for example, increased resistance of the photosynthetic apparatusversus effects on plant or leaf size, phenology etc.).

Among the genes that are known to respond to drought stressand which are being manipulated by genetic engineering, someencode enzymes involved in metabolism (for example, linked todetoxification or osmotic response), others are active in signalling,or in the transport of metabolites (for example, the prolinetransporter) or in regulating plant energy status. Some genesdo not have a well-established function, such as those encodingthe LEA proteins, but result in protection of the cellular machineryagainst various stresses (Bray, 1997Go; Xu et al., 1996Go).

Engineering for osmotic adjustment and/or protection of macromolecules:
Engineering for increasing osmolytes, such as mannitol, fructans,trehalose, ononitol, proline, or glycinebetaine, among othersmay increase resistance to drought, although the protectionmechanisms are still not fully understood (Ramanjulu and Bartels,2002Go). If the osmolyte accumulation is sufficient to decreasecell osmotic potential thereby enabling the maintenance of waterabsorption and cell turgor at lower water potentials (Morgan,1984Go), one can talk of osmotic adjustment. When the accumulationis low, it is reasonable to ascribe osmolytes a function inprotecting macromolecules (such as, for example, enzymes) eitherby stabilizing proteins or by scavenging reactive oxygen speciesproduced under drought (Shen et al., 1997aGo; Zhu, 2001bGo). Althoughthe benefits of osmolyte accumulation for crop yield are thesubject of some controversy (Serraj and Sinclair, 2002Go), someresults of genetic transformation point to advantages for plantperformance under drought, which may open avenues for the future.Still, transgenic plants that have been engineered to overproduceosmolytes often exhibit impaired growth in the absence of stress.This is probably due to the involvement of osmolytes in signalling/regulatingplant responses to multiple stresses, including reduced growththat may be part of the plant adaptation strategy against stress,as suggested by Maggio et al. (2002)Go.

The raffinose family oligosaccharides, such as raffinose andgalactinol, are among the sugars involved in desiccation tolerance.Taji et al. (2002)Go engineered Arabidopsis plants for over-expressionof AtGolS 1, 2, or 3, all genes coding for galactinol synthasefrom A. thaliana. The overexpression of AtGolS2 did increaseendogenous galactinol and raffinose in transgenic plants andwas found to reduce transpiration from leaves and to improvedrought tolerance. These compounds seem to act as osmoprotectants,rather than by providing osmotic adjustment (Taji et al., 2002Go).

Expression of bacterial fructan in tobacco and sugar beet ledto an improved growth under water deficits in transgenic plantsthan in the wild type (Pilon-Smits et al., 1995Go, 1998Go).

Mannitol, the most widely distributed sugar alcohol in nature(Stoop et al., 1996Go), was demonstrated to scavenge hydroxylradicals and stabilize macromolecular structures, such as phosphoribulokinase(a thiol-regulated enzyme), thioredoxin, ferredoxin, and glutathione(see for example, Shen et al., 1997aGo, bGo). The protective effectseems to result from the formation of hydrogen bonds betweenmacromolecules and osmolytes under limited water availability,thus preventing the formation of intramolecular H-bonds thatcould irreversibly modify the three-dimensional molecular structures.Recently, Abebe et al. (2003)Go achieved a significant improvementof wheat tolerance to water and salt stress through the ectopicexpression of the mtlD gene (mannitol-1-phosphate dehydrogenase)from E. coli. The authors found that the amount of mannitolaccumulated (0.6–2.0 µmol g–1 FW) was toolow to ensure protection through osmotic adjustment, but waseffective in improving stress tolerance. Lines containing over0.7 µmol g–1 FW in the flag leaf, started showingside effects of mannitol accumulation and lines with over 1.6µmol g–1 FW in the flag leaf showed severe abnormalities,including sterility. This was accompanied by an exceptionallylow sucrose content. The plants with the lower mannitol contents(up to 0.7 µmol g–1 FW), however, did not sufferfrom the adverse effects of excess mannitol, which would depletethe sucrose pool and negatively impact the growth of wheat plants.Because mannitol is a naturally occurring sugar-alcohol andis used as an additive in many processed foods, its overexpressionmay prove to be a useful tool to enhance crop resistance todrought and salt. The overexpression of IMTI1 (inositol methyltransferase) gene, from the ice plant Mesembryanthemum crystallinuminto tobacco, led to the accumulation of another sugar-alcohol,the methylated form of inositol, D-ononitol, leading to an increasedtolerance to drought and salt stress (Sheveleva et al., 1997Go).

Trehalose, a non-reducing disaccharide of glucose, has beenshown to stabilize biological structures and macromolecules(proteins, membrane lipids) in different organisms during dehydration(Crowe et al., 1992Go). Through the regulated over-expressionof a fusion gene containing the coding regions of both otsAand otsB (trehalose-6-P synthase and trehalose 6-P-phosphatase)of E. coli, Garg et al. (2002)Go showed that trehalose has a primarypositive effect in transformed plants under abiotic stress conditions.This effect was linked to the maintenance of an elevated capacityfor photosynthesis under stress. The positive effect of trehaloseaccumulation (an increase in 3–9-fold compared with thewild type) was observed under salt, drought, and low-temperatureconditions. Under drought, trehalose accumulation accountedfor an increased protection of Photosystem II against photo-oxidativedamage, as assessed by in vivo chlorophyll fluorescence ({phi}PSIIand Fv/Fm). These effects were observed both when the fusiongene was directed to the chloroplast (with a transit peptideand under the control of the promoter of the small subunit ofrbcS) or to the cytosol (under the control of an ABA-induciblepromoter). The reason why photosynthetic capacity was preservedin drought-stressed transgenic rice is, however, not clear;is it because shoot water status was improved, or is it simplybecause, under a dehydration intensity similar to that affectingthe wild-type plants, the photosynthetic apparatus is protectedagainst oxidative stress? It may be speculated that becausethe transgenic lines with gene expression in the chloroplastshowed protection against drought at lower trehalose concentrationsthan those with cytosolic expression, the second hypothesisis the most likely.

Garg et al. (2002)Go also found an increase in other soluble carbohydratesafter exposure to abiotic stress (20% higher concentrationsin transformed than in wild-type plants). These results areconsistent with the hypothesis raised by Paul et al. (2001)Go,working with tobacco plants expressing E. coli trehalose biosyntheticgenes, that trehalose may play a role in the modulation of carbonmetabolism in response to external factors, through sugar-sensingmechanisms. The work by Garg et al. (2002)Go confirmed some beneficialeffects observed in earlier transformation work done by Pilon-Smitset al. (1998)Go in tobacco. However, very significant progresswas achieved by comparison with previous studies, where undesirablepleiotropic effects, including stunted growth and the formationof abnormal leaves, occurred in plants where the two enzymesinvolved in the trehalose biosynthesis were overexpressed (Goddijnet al., 1997Go; Holmstrom et al., 1996Go). If these studies areconfirmed by field trials, they increase the possibility forcultivating rice, a major staple crop worldwide, in rainfedconditions or in saline soils (Penna, 2003Go).

Betaines, ectoine, and proline are among the compatible solutesthat also accumulate in plants as a widespread response againstenvironmental stress (Chen and Murata, 2002Go; Rontein et al.,2002Go). Some crop plants have low levels of these compounds,and engineering their biosynthetic pathways is a potential wayto improve stress tolerance. For instance, in wheat, the accumulationand mobilization of proline was found to correlate with thelevel of tolerance towards water stress (Nayyar and Walia, 2003Go),the tolerant genotype being more responsive to ABA. Overexpressingthe gene P5CS from Vigna aconitifolia in tobacco led to a 2-foldincrease in proline and a better growth under water and saltstress (Kavi Kishor et al., 1995Go). A number of genes involvedin the biosynthetic pathways of such compounds, such as choline-oxidaseor sorbitol-6-phosphate dehydrogenase, have been tested in transgenicplants with positive results in increasing stress tolerance(Chen and Murata, 2002Go). In some cases, the accumulation ofthese solutes is marginal, implying that they were not actingthrough an effect of osmotic adjustment (Holmstrom et al., 1996Go).

A group of proteins commonly involved in the enhancement ofstress tolerance are the LEA proteins. The role of LEA proteinswas suggested as chaperones, in binding water, in protein ormembrane stabilization, and in ion sequestration (Cushman andBohnert, 2000Go). Rice and wheat plants expressing the barleygroup 3 LEA gene HVA1 in leaves and roots showed improved osmoticstress tolerance and improved recovery after drought and salinitystress (Xu et al., 1996Go; Sivamani et al., 2000Go). Group 2 ofthe LEA proteins, the dehydrins (also known as the Lea D11 family)has been commonly observed accumulating in response to dehydrationor low temperature (Close, 1997Go). With one or more copies ofa putative amphipathic {alpha}-helix-forming domain (the K-segment),dehydrins are the best-studied LEA proteins. They have beenconsidered as having a role as surfactants, preventing the coagulationof numerous macromolecules (Close, 1997Go).

Other proteins may also play a role in protection against drought.This is the case of some heat shock (HS) proteins, includingsmall HS (smHS) such as the At-HSP17.6A class from Arabidopsisthaliana, which, upon over-expression, could increase salt anddrought tolerance, presumably due to its chaperone activitydemonstrated in vitro (Sun et al., 2001Go). Their action includespreventing protein degradation and assisting the refolding ofproteins denaturated during stress. In transgenic tobacco plants,the enhanced accumulation of the chaperone-binding protein BiP,of the endoplasmic reticulum (shown to be induced by a varietyof environmental stresses), conferred tolerance to water stress(Alvim et al., 2001Go). Under progressive drought, leaf BiPs concentrationwas correlated with shoot water content and photosynthetic rateswere maintained in stressed transgenic plants to values similarto those measured in wild-type well-watered plants.

NtC7, a gene encoding a membrane-located receptor-like protein,with transmembrane domains, was also found to induce, in transgenictobacco plants, a marked increase in tolerance to mannitol-inducedosmotic stress, with rapid recovery from severe wilting, whereaswild-type plants showed leaf necrosis (Tamura et al., 2003Go).The authors suggested that the NtC7 gene is involved in thesignalling pathway that activates genes responsive to osmoticstress (independently of ion homeostasis), presumably as partof the osmosensor system. Osmotic adaptation may occur throughmechanic-sensitive signalling, in which alterations in turgorcould be the starting point for a signalling cascade, by generatinga signal eventually triggering conformational changes in membraneproteins. In potato, mechanical stress has an early cellularresponse of the significant and rapid synthesis of superoxideradicals (Johnson et al., 2003Go).

Protection against excessive accumulation of ROS has been achievedby overexpressing a stress-inducible aldehyde dehydrogenasegene, already present in Arabidopsis thaliana (Sunkar et al.,2003Go). The function of this enzyme is to catalyse the oxidationof various toxic aldehydes, accumulated as a result of sidereactions of ROS with lipids and proteins. Transgenic linesshowed improved tolerance when exposed to dehydration, as wellas to other types of stress (salt, heavy metals, H2O2) and thiswas accompanied by a decreased accumulation of lipid peroxidation-derivedtoxic aldehydes. Transgenics also survived for longer periodsof drought than wild-type plants. The authors claim that thesefindings may lead to applications in crop plants, such as maize,wheat or soybean. In addition, the ectopic expression, in tobacco,of the alfalfa aldose/aldehyde reductase MsALR, provided toleranceto multiple stresses, including drought stress, with reducedamounts of reactive aldehydes generated from lipid peroxidation(Oberschall et al., 2000Go). Manipulation of ROS scavenging enzymes,yielding the effective reduction of ROS concentration, however,may lead to increased susceptibility to biotic stress, sincecell wall fortification, as a barrier to pathogen penetration,is increased by ROS (Xiong et al., 2002Go). On the other hand,manipulation of ROS scavenging enzymes aiming to reduce oxidativedamage is limited by the high number of isoforms and by theirlocation in different sub-compartments and membranes (Bohnertand Sheveleva, 1998Go).

Engineering for water transporters:
Water transport in plants uses both the apoplastic and the symplasticroutes. This means that a high number of water molecules haveto cross numerous cell membranes. This process is facilitatedby aquaporins, membrane-intrinsic proteins found in all livingorganisms and forming water-permeable complexes (Uehlein etal., 2003Go). The apoplastic water potential influences the phosphorylationstatus of aquaporins, so that its ability to transport waterincreases when phosphorylated. Therefore, aquaporins are likelyto play an important role in the control of cellular water statusin response to water deficits (Assmann and Haubrick, 1996Go; Bray,1997Go). Differential expression of genes that encode differentaquaporin isoforms during plant development were shown to beassociated with different physiological processes, includingstomatal opening (Chrispeels and Agre, 1994Go). However, the relationshipbetween the role of aquaporins in the regulation of plant waterstatus and the regulation of aquaporin gene expression is stillunclear (Aharon et al., 2003Go). For example, the over-expressionin tobacco of the Arabidopsis aquaporin AthH2, which encodesPIP1b aquaporin, improved growth performance under non-stressconditions, but it was not effective under drought or salt stress(Aharon et al., 2003Go).

Aquaporins may also transport other small molecules such asglycerol, solutes and ions (Tyerman et al., 2002Go) and they showcytosolic pH-dependent gating (changes in the conductance ofindividual water channels), a feature providing a mechanismof co-ordinated inhibition of plasma membrane aquaporins uponcytosol acidosis (Tournaire-Roux et al., 2003Go). This behaviourjustifies the reduced ability of roots to absorb water underflooding conditions, as a consequence of anoxia.

Recently it was found that the tobacco aquaporin NtAQP1 actsas a CO2 membrane-transport-facilitating protein, playing asignificant role in photosynthesis and in stomatal opening (Uehleinet al., 2003Go). The overexpression of NtAQP1 in tobacco raisedmembrane permeability for CO2 and water, and increased leafgrowth (Uehlein et al., 2003Go), a feature that may have an impactin plant performance under drought. Photosynthesis increasedin these transgenic plants by 36% under ambient CO2 (380 ppm)and by 81% at elevated CO2 (810 ppm). This was accompanied byan increase in stomatal conductance in both situations. Therefore,the increase in photosynthesis may result from a combinationof more open stomata and a higher mesophyll conductance, resultingfrom the decreased membrane resistance to CO2. Both effectsled to an increase in CO2 availability to the cells.

Engineering for C4 traits:
The ability to optimize net carbon gain and therefore increaseWUE under reduced water availability is critical for plant survival(Chaves et al., 2004Go). In species with C4 photosynthesis highphotosynthetic rates can be associated with low stomatal conductance,leading to high WUE (Cowan and Farquhar, 1977Go; Schulze and Hall,1982Go). Manipulating WUE is a highly complex desideratum, becauseit implies co-ordinated changes relating to stomatal apertureand photosynthesis. Following various attempts to use conventionalhybridization to get C3–C4 hybrids, several groups havesuccessfully transformed C3 plants to acquire C4 characteristics(see the review by Matsuoka et al., 2001Go). Ku et al. (1999)Go,for example, introduced in rice the phosphoenolpyruvate carboxylase(PEPC) from maize, achieving a high-level expression of thePEPC protein (1–3-fold that of maize leaves). Althoughno significant effects were observed in the rates of photosynthesis,the transformed rice plants exhibited a reduction in the O2inhibition of photosynthesis characteristic of C3 plants thatmay attain 40% of potential photosynthesis. These transgenicplants may theoretically have some advantage over the wild type,especially under low CO2 conditions, prevalent for example underwater deficits, when carbon loss associated with photorespirationbecomes maximal. Some beneficial effects of the introductionof PEPC were observed under supra-optimal temperatures in transgenictobacco and potato (see Matsuoka et al., 2001Go). The hypothesisunderlying this response is that PEPC participates in the initialCO2 fixation or it increases CO2 in the vicinity of Rubisco.

A recent paper by von Caemmerer (2003)Go suggests, based on amodelling exercise, that C4 photosynthesis in a single C3 cell,although theoretically inefficient due to the absence of appropriatestructural features of C4 plants (see the review by Leegood,2002Go), may ameliorate the CO2-diffusion limitations of C3 leaves.Again, this could be beneficial under water-limited conditions,when stomata close and intercellular CO2 decreases drastically.

An alternative strategy to improve WUE would be to enhance photosyntheticcapacity in C3 crop plants by expressing improved forms of Rubisco,exhibiting higher relative specificity for CO2 compared withO2, such as those encountered in rodophyte algae, or to increasethe catalytic rate of Rubisco (Spreitzer and Salvucci, 2002Go;Parry et al., 2003Go). There is also scope for over-expressingRubisco activase, which seems to be more susceptible to extremeenvironments, namely high temperatures (Feller et al., 1998Go;Rokka et al., 2001Go).

Engineering via signal components and transcription factors:
In spite of the complex nature of the physiological adaptationof plants to the stress conditions and the difficulty of understandingthe regulatory mechanisms behind adaptation, there are alreadya number of genes that have been found to be involved in thesignal transduction pathways. They play important roles downstreamof signalling cascades, which could be used to engineer a higherability for plant protection from abiotic stress (Iba, 2002Go;Zhu, 2002Go). The modulation of these genes has been reportedto improve abiotic stress tolerance in a number of plant specieswith positive effects, sometimes regarding more than one stresstype (Dubouzet et al., 2003Go).

Multiple stress stimuli lead to Ca2+ influx in the cell andto its increased concentration in the cytoplasm. A number oftransport proteins such as the aquaporins, H+-ATPases and ionchannels, responsible for cytosolic osmoregulation and involvedin stress adaptation, are regulated by calcium-dependent proteinkinases (CDPKs). Saijo et al. (2000)Go investigated the functionof the rice cold- and salt-inducible OsCDPK7, and found thatits over-expression in transgenic rice plants conferred saltand drought-tolerance, apparently through the induced expressionof LEA proteins, namely rab16A (group 2 LEA protein), salT (aglycine-rich protein) and wsi18 (group 3 LEA protein). Thiseffect, however, was only observed in the rice cells after stressstimuli, pointing to a strong post-translational control andOsCDPK7 activation after the stress-induced calcium influx.The over-expression of OsCDPK7 did not significantly affectplant development and fertility.

The transfer of individual genes to plants, for acquiring higherstress tolerance, has so far only had a limited impact; however,the simultaneous transcriptional activation of a subset of thosegenes, by transferring transcription factors, has been revealedas a promising strategy (Jaglo-Ottosen et al., 1998Go; Liu etal., 1998Go).

There are several classes of transcription factors (TFs) playingmajor roles in dehydration and desiccation (Ramanjulu and Bartels,2002Go). In Arabidopsis, the TFs DREBs/CBFs specifically interactwith the dehydration responsive element/C repeat (DRE/CRT) cis-activeelement, controlling the expression of many stress-induciblegenes. DREB/CBF proteins are encoded by AP2/EREBP multigenefamilies and mediate the transcription of a number of genes,such as rd29A, rd17, cor6.6, cor15a, erd10, kin1, kin2, andothers, in response to cold and water stress (Ingram and Bartels,1996Go; Liu et al., 1998Go; Seki et al., 2001Go; Thomashow et al.,2001Go). A novel transcriptional regulator of the DRE/CRT classof genes, FIERY2 (FRY2), acts by repressing stress inductionof the upstream DREBs/CBFs TFs (Xiong et al., 2002Go). Recessivemutations in FRY2 result in super-induction of the DRE/CRT classof stress-responsive genes. Because FRY2/CPL1 contains dsRNA-bindingdomains, Xiong and Zhu (2002)Go speculated that dsRNA could bea regulator of the phosphatase enzymatic activity of FRY2/CPL1.RNA could then regulate hormone and stress responses in plants,as it does in animals. As cited by Xiong and Zhu (2002)Go, somecomponents in mRNA processing (such as the cap-binding proteinABH1 and Sm-like snRNP protein SADI) are specifically involvedin ABA and stress responses.

The over-expression in Arabidopsis of DREB1 and DREB2 improvedtolerance to dehydration (Liu et al., 1998Go). Under the controlof a constitutive promoter, DREB1A was, however, detrimentalwhen stress was not applied, although it had a positive effectfor plants under stress. The use of the stress-inducible promoterrd29A, instead of the CaMV 35S promoter, to over-express DREB1Aminimized the negative effects on plant growth (Kasuga et al.,1999Go). DREB genes under the control of rd29A are presently beingtested on tropical rice (Datta, 2002Go).

The Arabidopsis CBF1 (DREB1B) ectopically expressed in tomato,resulted in enhanced resistance to water-deficit, although growthretardation was observed as well as reduced fruit and seed numberswhen under the control of the 35S promoter (Hsieh et al., 2002Go).An ABA-inducible promoter did not affect plant morphology orgrowth, but was less effective under stress conditions. In transgenicCBF1 tomato under water-deficit, stomata closed faster thanin wild-type plants and proline concentration was higher, whilecatalase activity increased and H2O2 decreased compared withwild plants. Another gene, CBF4, found to be up-regulated onlyby drought (and not cold) when over-expressed in transgenicArabidopsis was able to activate genes involved in both droughtadaptation and cold acclimation (Haake et al., 2002Go). The authorsproposed that plant responses to cold and drought evolved froma common CBF-like transcription factor, first through gene duplicationand then through promoter evolution.

An homologous gene isolated from rice, OsDREB1A, and testedin Arabidopsis indicated a functional similarity to the ArabidopsisDREB1A, although in microarray and RNA blot analyses some differenceswere observed regarding the induced target genes (Dubouzet etal., 2003Go). The authors suggested that OsDREB1A is potentiallyuseful for producing transgenic monocots tolerant to drought,high-salt, and/or cold stresses.

Improved osmotic stress tolerance was achieved by 35S:AtMYC2/AtMYB2in transgenic plants, as assessed by electrolyte-leakage tests(Abe et al., 2003Go). Constitutive expression of TFs, however,usually leads to growth retardation (Abe et al., 2003Go; Hsiehet al., 2002Go; Kasuga et al., 1999Go). The Arabidopsis MYB TF proteinsAtMYC2 and AtMYB2 were found to function as transcriptionalactivators in ABA-inducible gene expression (Abe et al., 2003Go).This role points to a novel regulatory system for gene expressionin response to ABA, other than the ABRE (abscisic acid responsiveelement)-ZIP regulatory system (Wang et al., 2003Go).

The over-expression of bZIP (basic region leucine zipper) TFs,binding to ABRE cis-elements (e.g. ABF3 and AREB2/ABF4) werefound to cause ABA hypersensitivity, reduced transpiration rate,and enhanced drought tolerance in transgenic plants (Kang etal., 2002Go).

 

 


Conclusions

Most of the terrestrial plants have evolved either to escapedrought by appropriate phenology or to avoid drought, by developingstrategies that conserve water or optimize its acquisition.This requires early warning systems and different types of signalling.In general, plants also have to cope with the interaction ofother stresses that often arise concomitantly with drought,and ultimately involve oxidative stress. Protective responsesat the leaf level must then be triggered quickly in responseto the stress effectors to prevent the photosynthetic machinerybeing irreversibly damaged. Therefore, signals are key playersin plant resistance to stress. It is now apparent that redoxsignals are early warnings, exerting control over the energybalance of a leaf, and alterations in the redox state of redox-activecompounds regulate the expression of several genes linked tophotosynthesis and other metabolic pathways. It is also knownthat plant responses to stresses arise from the interplay betweendifferent signalling pathways.

The importance of the long-distance signalling for the plantfeed-forward response to water stress is acknowledged, namelythe role played by chemical signals synthesized in the rootsand transported to the shoot via the xylem sap. Novel managementtechniques that exploit the knowledge of plant's long-distancesignalling are increasingly being applied to get improved planttrade-off between carbon assimilated and water used, while sustainingyield and improving the quality of the crop products.

On the other hand, because drought-tolerance traits, ‘dryingwithout dying’ as described by Alpert and Oliver (2002)Go,are not common in higher plants, genetic engineering to introducethese traits may be a way forward for marginal environments,complementing the breeding work and marker-assisted selectionfor tolerance that explores the natural allelic variation atgenetically identifiable loci. Moreover, QTL mapping alliedwith comparative mapping and map-based cloning in plants maybe used to screen genes important in the response to stress.The molecular understanding of stress perception, signal transduction,and transcriptional regulation of these genes, may help to engineertolerance to multiple stresses. Engineering a single gene, suchas a Group 3 LEA gene or one affecting sugar metabolism, orplaying a role as an anti-oxidant, proved to alter metabolism,but in most cases only led to marginal stress improvement. However,recent advances suggest that rapid progress will be possiblein the near future. It may be possible to achieve multiple tolerancemechanisms for one or more abiotic stresses, with sufficientsuccess for commercial exploitation through co-transformationor gene pyramiding. Moreover, the upstream targeting of regulatorynetworks may have a more consistent role in providing tolerance,either through protection or repair mechanisms. Advances inthe molecular biology of stress response in tolerant organismsare raising a number of possibilities concerning regulatorygenes that may be used in agricultural programmes, not onlyto ensure survival under water deficit but also to guaranteea reasonable productivity under reduced water availability.

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Table 1

Table 1. Effect of controlled irrigation on physiological responses of field-grown grapevines





Full irrigation (FI)


Deficit irrigation (DI)


Partial root-zone drying (PRD)


Non-irrigated (NI)

Afield (µmol m–2 s–1) 16.3–10.0 13.3–11.3 14.6–8.3 12.5–3.3
gs field (mol m–2 s–1) 0.30–0.28 0.23–0.19 0.19–0.15 0.13–0.07
Acontrolled (µmol m–2 s–1) 13.3±0.5 12.0±1.1 11.5±0.9 9.5±0.9
gs controlled (mol m–2 s–1) 0.35±0.04 0.33±0.05 0.25±0.05 0.14±0.01
{Psi}pd (MPa) –0.10 to –0.18 –0.14 to –0.44 –0.14 to –0.30 –0.22 to –0.64
Sap flow (g h–1 m–2) 402–356 275–196 145–130 109–101
Berries {delta}13 C ({per thousand}) –26.3±0.17 –25.9±0.28 –23.7±0.07 –22.4±0.69
Leaf area (m2 per vine) 6.3±0.26 4.9±0.15 4.3±0.21 3.6±0.18
Exposed clusters (%)

9.8±2.8

12.9±3.2

16.8±3.5

22.8±3.8

Maximum and minimum values of leaf net photosynthetic rates (Afield) and stomatal conductance (gs field) measured at midday, from mid-June to mid-September 2000, in the grapevine cultivar Moscatel, under different irrigation treatments, FI, DI, PRD, and NI. Acontrolled and gs controlled measured under controlled conditions of light (1200 µmol m–2 s–1) and temperature (25 °C) at the end of August (mean values ± SE). Maximum and minimum values of leaf predawn water potential ({Psi}pd) for the same period as above and for sap flow measurements done during August. Discrimination of 13C in the berries, measured at harvest, in September (mean values ± SE). Leaf area per vine measured at harvest and percentage of sun-exposed cluster at maturation (mean values ± SE). (Data from Souza et al., 2003; Santos et al., 2003).


Table 2

Table 2. Recent achievements in improving drought tolerance in crops through genetic engineering


Gene/enzyme


Organism of origin


Target plant


Effect


Author

Functional proteins        
Superoxide dismutase (MnSOD) Nicotiana plumbaginifolia Alfalfa Better performance in the field under drought McKersie et al. (1996)
HVA1 (group 3 Lea gene) Barley Rice Constitutive expression leads to protein accumulation in leaves and roots and improved recovery after drought and salt stress Xu et al. (1996)
Myo-inositol O-methyltransferase (IMT1) Mesembryanthemum crystallinum Tobacco Enhanced photosynthesis protection and increased recovery under drought, through the accumulation of D-ononitol. Sheveleva et al. (1997)
Trehalose-6-P synthase, Trehalose-6-P phosphatase Bacteria Tobacco Better photosynthetic efficiency and higher dry weight under drought stress Pilon-Smiths et al. (1998)
HVA1 (group 3 Lea gene) Barley Wheat Constitutive expression (ubiP) improved biomass productivity and water use efficiency under water-stress Sivamani et al. (2000)
Aldose/aldehyde reductase (MsALR) Alfalfa Tobacco Detoxification effect (reduced amounts of reactive aldehydes derived from lipid peroxidation) leading to tolerance to multiple stresses, including drought Oberschall et al. (2000)
NADP-malic enzyme Maize Tobacco Drought avoidance phenotype through decreased stomatal conductance and increased fresh weight per unit water consumed. Growth and rate of development similar to wild type Laporte et al. (2002)
Fusion gene with Trehalose-6-P synthase and Trehalose-6-P phosphatase (TPSP) regulated by ABA inducible promoter or small subunit rbcS promoter E. coli Rice Sustained plant growth and reduced photo-oxidative damage under drought and other abiotic stresses. Improved photosynthetic activity also under non-stress conditions. Garg et al. (2002)
Mannitol-1-phosphate dehydrogenase (mtlD) E. coli Wheat Improved drought tolerance with mannitol accumulation at a concentration insufficient for osmotic adjustment Abebe et al. (2003)
Aquaporin NtAQP1 Tobacco Tobacco Over-expression of NtAQP increased membrane permeability for CO2 and water, and increased leaf growth Uehlein et al. (2003)
Regulatory proteins        
Calcium dependent protein kinase (OsCDPK7) Rice Rice Over-expression of OsCDPK7 led to induced expression of a glycine rich protein (salT) and LEA proteins (rab16A, wsi18) under stress. Increased salt and drought-tolerance. Saijo et al. (2000)
CBF1 (DREB1B) (driven by P35SCaMV)

Arabidopsis

Tomato

Increased resistance to water-stress, but dwarf phenotype. Higher levels of proline than controls, and faster closure of stomata under water stress. Higher catalase activity and lower (McAinsh et al., 1996), with or without stress

Hsieh et al. (2002)

The genes used were originated from plants or bacteria and accounted for various cellular responses ending up in increased drought tolerance.


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