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More from less: plant growth under limited water
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More from less: plant growth under limited water Aleksandra Skirycz1,2 and Dirk Inze´1,2 When subjected to abiotic stresses, plants actively re-program their growth by modulating both cell division and cell expansion. Growth decreases rapidly upon stress onset but it recovers and adapts once stress conditions become stable. Here, we review recent advances in understanding the mechanisms underlying both stress-induced growth repression and adaptation with an emphasis on drought and leaf growth and we briefly discuss how this knowledge can be translated into crops. It is now clear that stress response of growing and mature leaves is distinct and should be studied separately. Both cell proliferation and expansion are regulated by common signaling pathways involving gibberellins and DELLA proteins while down stream effector genes are stage specific. Addresses 1 Department of Plant Systems Biology, VIB, Technologiepark 927, B-9052 Gent, Belgium 2 Department of Plant Biotechnology and Genetics, Ghent University, Technologiepark 927, B-9052 Gent, Belgium Corresponding author: Inze´, Dirk ([email protected])
Current Opinion in Biotechnology 2010, 21:197–203 This review comes from a themed issue on Plant biotechnology Edited by Antonio Molina and Jim Haseloff Available online 2nd April 2010 0958-1669/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2010.03.002
Introduction To minimize the deleterious effects of stress and to complete their life cycle under adverse conditions, plants have evolved different adaptive responses that are summarized in the avoidance/tolerance model [1]. For example, during drought, plants close their stomata and accumulate compatible solutes to maintain a low water potential and to avoid dehydration. Simultaneously newly synthesized protective proteins, such as dehydrins and antioxidants, restrict damage to other proteins and cellular membranes and lower the levels of harmful reactive oxygen species (ROS) (see article by J.M. Pardo in this issue). Plants also reduce their growth as a way to save and redistribute resources that can become limited under extreme stress. Accordingly, growth reduction occurs rapidly after stress onset and independently of photosynthetic rates and plant carbon status [2,3], arguing that growth reduction is not simply a secondary effect of www.sciencedirect.com
resource limitation, but an important adaptive response. Counter-intuitively, plants exposed to mild drought stress even accumulate sugars and starch [3]. Although growth retardation increases the survival rate, during moderate stress episodes when survival is not threatened, it can be seen as counter-productive with unnecessary yield losses and major consequences for agriculture. Therefore, new crop varieties in which growth would be less inhibited during sporadic spells of moderate stress would be extremely advantageous for plant productivity, particularly in mild climates. We might even speculate that in modern varieties, breeders have already selected for reduced growth sensitivity under mild stress conditions. Knowledge of the mechanisms underlying growth reduction under stress is an important prerequisite to further improve crop productivity. Whereas an enormous amount of information is available on the molecular mechanism controlling stress tolerance in mature leaves, our current understanding of stress-regulated growth is still very fragmentary, partly because studies combining detailed growth analysis and molecular characterization of growing organs are relatively scarce [3,4,5]. Most of the available stress data were obtained from mature tissues or whole plants, in spite of the fact that developmental stage and cell identity have a major influence on the observed stress responses [3,6]. Hundreds of genes and many metabolites were shown to change specifically in growing versus mature leaves and in different root tissues under osmotic and salt stress, respectively [3,6]. Additionally, in most studies the applied stresses are rather extreme, leading to plant death, a situation that is rare in modern agricultural practice. Related, while numerous screens identified mutants that cope better with extreme stress [7–9], often because of their inherent dwarfed stature [10,11], only a very limited number of studies report on plants that grow better under mild stress conditions that do not threaten plant survival although they restrict growth [12,13]. This review will mainly focus on drought stress and its impact on Arabidopsis thaliana leaf growth, but key findings on other stress conditions, plant species, and organs will also be highlighted.
‘Acute and adaptation’ growth responses Response of plant growth to stress onset is often characterized by a rapid and acute (‘acute response’) inhibition, followed by recovery and adaptation to the new condition (‘adaptation response’) (Figure 1A). In barley (Hordeum vulgare) leaves, the leaf elongation rate (LER) decreased close to zero within seconds after salt addition to the roots, followed by recovery of LER to approximately 46% and 70% of the non-stressed plants within minutes and days, respectively [14]. Similarly, in the shoot apical meristem Current Opinion in Biotechnology 2010, 21:197–203
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Figure 1
Growth response to stress. (a) Schematic representation of ‘acute’ (yellow arrow) and ‘adaptative’ (green arrow) growth response of plants subjected to stress. Red arrow marks stress onset. (b) Schematic representation of Arabidopsis leaf growth. Leaves initiate at the flank of the meristem (SAM). The number of recruited cells might influence the final cell number. Initially leaf growth is driven exclusively by cell proliferation (P, red shading). Within several days and starting from the leaf tip, cells exit the mitotic cell cycle and start to expand (E, green shading). After a few days, leaf growth is steered solely by cell expansion. Again starting at the tip, cells become mature (no shading), coinciding with growth cessation [63]. The final cell number and size might therefore depend on the length of the developmental windows of proliferation and expansion (white and light-green arrows), respectively, and/or the duration of single cell cycle (the shorter the cycle, the higher the cell production per unit of time) and rate of expansion.
(SAM) of salt-treated Arabidopsis plants [15], the cell cycle activity was reduced transiently (12 h and 36 h after stress onset) and subsequently recovered (4 days after stress onset). While the acute response prepares plants for possibly more severe conditions, the adaptive response can be seen as the establishment of a new steady state to prolonged and stable stress. This two-phase (‘acute’ and ‘adaptation’) growth response indicates that the mechanisms that govern growth repression upon stress sensing and those that allow growth adaptation are distinct and will be discussed in more detail below.
Drought reduces leaf growth by affecting cell division and expansion In dicotyledonous plant species, such as Arabidopsis, leaf growth results from the proliferation and subsequent expansion of founder cells, recruited from the SAM. Consequently, the final leaf size depends on the number of recruited cells as well as the rates and developmental windows of cell division and expansion [16] (Figure 1B). Soil water deficit and relatively mild osmotic stress affect the leaf area by reducing both cell number and size, as shown in Arabidopsis and sunflower (Helianthus annuus) [3,17,18]. Importantly, the initial growth reduction can be compensated by a longer proliferation and/or expansion time, a phenomenon denominated ‘growth extension’. In an extreme case, the final leaf sizes of the Arabidopsis accession An-1 grown under control and soil water deficit conditions were identical, but were reached in stressed plants only after an additional two weeks of growth [17,18]. Interestingly, in Arabidopsis and sunflower leaves, exposed to a long water restriction period, epidermal cells retained some ability to expand, even Current Opinion in Biotechnology 2010, 21:197–203
though the leaf apparently had reached its final size [19] (Figure 1).
Root-to-shoot signaling in leaf growth regulation In partial root drying experiments in which only part of the root system is subjected to drying, reduced leaf growth and stomatal closure occurred even when the remaining roots had enough moisture to fully supply leaves with water [20], leading to the conclusion that fast chemical signal(s) are generated in the drying roots mediating both leaf responses. Whereas the plant hormone abscisic acid (ABA) is a key regulator of stomatal conductance [21], results on leaf growth-regulating hormonal signals are still under debate and contradictory. For instance, because externally applied ABA represses growth, it is commonly viewed as a growth inhibitor [22], but a positive role of ABA through the inhibition of ethylene production has also been proposed on the basis of results obtained with ABA and ethylene mutants and ABA and ethylene inhibitors in maize (Zea mays), Arabidopsis, or poplar (Populus trichocarpa) [23–25]. Similarly to ABA, exogenously applied ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) inhibit growth [26] and accumulate under stress [27,28], and ACC can be transported by the xylem from root to shoot [29]. Moreover, ethylene-deficient tomato (Solanum esculentum) plants subjected to soil drying were characterized by stomatal closure, without effect on their leaf growth, in contrast to wild-type plants [28]. However, in maize plants grown under drought stress neither endogenous levels of ethylene nor ABA could be correlated with reduced leaf elongation [30]. Short-term www.sciencedirect.com
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Figure 2
Schematic representation of processes involved in growth regulation upon drought. Soil drying is sensed in roots activating a combination of hydraulic and chemical signals that are transported through the xylem to leaves where they initiate a number of tolerance mechanisms. Response of mature leaves can be described by the avoidance/tolerance model [1]. In growing leaves stress leads to acute growth inhibition followed by growth adaptation both mediated by DELLA signaling. Ethylene promotes DELLA stabilization and growth inhibition while ABA is possibly involved in growth recovery. Negative ABA and ethylene cross-talk was demonstrated. While hormonal signaling is common between expanding (E) and proliferating (P) leaves, effector genes are distinct. In P leaves, inhibitors of CDKA might play a role in acute response while alternative respiration in growth adaptation. In expanding leaves, cell wall tightening and changes in cell turgor lead to growth cessation, while osmotic adjustment and cell wall loosening are important for growth adaptation.
growth inhibition was rather attributed to aquaporinmediated changes in the root hydraulic conductivity (Lpr) and consequent decrease in cell turgor [31,32]. While phosphorylation-dependent internalization of aquaporins triggered by ROS provides a fast mechanism to reduce Lpr upon stress onset [33], ABA positively affects aquaporin transcript and protein levels, hinting at a role in growth recovery [31]. Interestingly, ethylene negatively influenced petal expansion of rose (Rosa sp.), at least partly, by suppressing expression of the Rh-PIP2;1 aquaporin [34]. Interplay between hormonal and hydraulic signals thus appears essential to mediate both acute and adaptation growth responses under stress conditions (Fig. 2). Other unknown or poorly characterized signals may also be involved in long-range stress signaling. A recent example is the bypass1-derived signal that is rootborne and inhibits shoot growth by affecting both leaves www.sciencedirect.com
and SAMs, although its exact biological function and chemical nature are unknown [35].
GA and DELLA signaling are of key importance for organ growth While the exact roles of ABA and ethylene in growth repression are still incompletely clear, gibberellins (GAs) have been convincingly shown to play a prominent role in growth regulation under optimal, but also, under stress conditions [13,36–38], by repressing the levels of DELLA proteins, known to inhibit growth (Fig. 2). Stress-activated production of the catabolic enzymes GA 2-oxidases (GA2ox) reduces GA levels, in turn, stabilizing DELLA proteins and leading to growth repression [39,40]. Two Arabidopsis DREB transcription factors (TFs) AtCBF1 and AtDDF1 are important for the GA2ox regulation under cold and salt stress, respectively [39,40]. Current Opinion in Biotechnology 2010, 21:197–203
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Concomitantly, growth of Arabidopsis plants lacking all DELLA proteins is less reduced by relatively mild salt stress, but also upon treatment with the pathogen attackmimicking elicitor flagellin [13,36]. Importantly, besides their involvement in growth repression, DELLA proteins activate tolerance mechanisms by diminishing ROS levels and, hence, are also essential for plant adaptation to stress [13,41]. DELLA-deficient plants are more sensitive to severe and prolonged stress [13], suggesting that growth retardation under such conditions is important for plant survival. Accordingly, our work on the molecular responses of fully proliferating, fully expanding, and mature Arabidopsis leaves to mild osmotic stress showed that both cell proliferation and expansion were regulated by common regulatory circuits, involving ethylene and GAs, but not necessarily ABA signaling [3]. Remarkably, DELLA-mediated growth regulation is conserved across different stresses [13,36,38] and provides a convergence point for classical stress hormones because ABA, ethylene, and jasmonate signaling all positively affect DELLA stability, primarily via changes in GA concentrations [13,36,42,43] (Fig. 2). Moreover, GAs and DELLA proteins are important for both cell expansion and cell proliferation in leaves and roots [37,38]. DELLA proteins regulate transcription, but because they have no DNA-binding domain, they were proposed to act in concert with other TFs. Light control experiments of hypocotyl elongation revealed that DELLA proteins sequester the Basic-Helix-Loop TFs, PIF3 and PIF4, restricting cell elongation [44]. Whether a similar mechanism operates for DELLA-mediated stress growth repression and which are the exact protein partners and target genes remains to be established.
Drought effects on the cell cycle Exposure of plants to mild stress reduces cell number [3,17]. This decrease in cell number might result from either a longer duration of single cell division cycli and/or a shorter developmental window for cell proliferation. In leaves of both Arabidopsis and sunflower, water deficit had no effect on the developmental window of cell division and the reduced cell number measured in sunflower leaves was attributed to the impact of drought on the G1-to-S transition and, thus, on reduced division rates [2,3]. Progression into the S-phase is mediated by the activity of the PSTAIRE-containing cyclin-dependent kinase A (CDKA) and can be arrested by the interaction of CDKA with inhibitory proteins, such as Kip-related proteins (KRPs) or SIAMESE (SIM/EL2) and SIAMESE-RELATED proteins [45–47]. Because of their inhibitory role and the stress-induced expression of several KRP and SIM/EL2 genes, these are considered good candidates to link cell cycle activity and environmental stimuli [4,47] (Fig. 2). Providing a link to GA signaling, DELLA proteins restrain cell production in roots by enhancing the levels of KRP2 and SIAMESE genes [37]. The G1/S block can be released easily allowing cell Current Opinion in Biotechnology 2010, 21:197–203
division rates to recover once cells have adapted to stress. Part of such adaptation involves re-programming of the mitochondrial metabolism. As stress compromises mitochondrial electron transport chain, alternative respiration is activated protecting proliferating cells against ROS and supplying them with sufficient energy and nucleotides [3,48]. For leaves that initiate under stress it is also possible that fewer cells are incorporated into leaf primordia. Salt stress and ethylene reduce the size of the root apical meristem but no data are available on stressinduced changes in activity and size of the SAM [49,50].
Cell expansion—changes in cell wall properties Changes in cell wall rheology are important for cell expansion under stress conditions. While tightening contributes to growth cessation, cell wall relaxation maintains growth under low turgor pressures. Both processes have been studied mainly in maize roots subjected to progressive water deficit. In such an experimental system, the cell walls of the apical region are flexible and tissues continue to expand, while the cell walls of the elongation region are rigid, resulting in growth inhibition [51]. On the one hand, tightening of cell walls is primarily associated with accumulation of phenolics and lignin monomers that are covalently cross-linked to wall polysaccharides through the activity of peroxidases and oxidases [52] (Fig. 2). On the other hand, an increase in cell wall extensibility can be achieved by different mechanisms: changes in pH and activity of cell wall-loosening enzymes (expansins and xyloglucan endotransglycosylase [XET]) contribute to acidic growth and loosening of connections between cellulose microfibrils [5,53,54] (Fig. 2). Moreover, apoplastic ROS production in the apical region of water-stressed roots contributes to cell wall extensibility by non-enzymatic cleavage of cell wall polysaccharides [55,56]. Importantly, evidence is emerging for similar mechanisms acting in leaves growing under low turgor pressure. Superoxide levels and the expression of a number of expansins and XETs were exclusively affected in the expanding, but not in the proliferating or mature, Arabidopsis leaves growing under osmotic stress [3]. Furthermore expansion rates of maize leaves was shown to correlate well with the expression of three expansin genes under a range of developmental, genetic, and environmental cues, including drought [57].
Perspectives and conclusions Abiotic stress is responsible for more than 50% yield loss worldwide [58]. As the world population is rising exponentially, this problem needs to be dealt with, especially taking into account the deleterious effects of global warming. Ultimately, developing new stress-tolerant crop varieties will require further understanding and modulation of the molecular processes underlying plant stress responses. Thus far, most studies have concentrated on transgenic modifications that create plants that survive www.sciencedirect.com
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extreme drought stress, usually at the expense of biomass accumulation [59]. Unfortunately, a number of these transgenic plants also suffer from a growth penalty already under optimal conditions, albeit this problem can be overcome by the use of stress-inducible promoters [60]. A more promising strategy will be to optimize plant growth so as to minimize its inhibition by mild stress. As long as the stress is moderate under mild climates, such plants would accumulate a maximum of biomass during their life cycle. For this, a systems understanding of the mechanisms underlying growth regulation is essential and some important pre-requisites have to be fulfilled. (1) High-throughput phenotyping platforms that allow reliable, non-destructive monitoring of growth dynamics under a range of environmental conditions have to be established to identify even relatively small differences in a trait as variable as growth. A published example of such a platform is PHENOPSIS [61], while a large phenotyping facility has just been opened at the University of Adelaide, Australia (http://www.plantaccelerator.org.au/). A conceptual framework for drought phenotyping has recently been reviewed [62]. (2) Molecular and physiological data obtained using in vitro osmotic stress conditions have to be confirmed in more realistic soil systems. (3) Molecular characterization of the stress responses of growing organs/tissues has to be performed with sufficient temporal resolution [3,6]. By bringing together sufficient physiological, genetic, and molecular information, ultimately gene-to-phenotype prediction might be used to direct efforts to engineer plants for enhanced productivity. As apparently many, often redundant, mechanisms are involved in growth regulation under stress conditions, the simultaneous engineering of superior alleles and multiple genes under the control of appropriate (artificial) promoters will allow the fine tuning of growth responses under stress. Such an approach, resembling that in the synthetic biology field, will probably contribute to coping with complex traits, including drought stress.
Acknowledgments We apologize to all colleagues whose work was, owing to space limitations, not cited. We thank Hannes Claeys, Dr Wim Verelst and Dr Matthew Hannah for useful discussion and Dr Martine De Cock for help in preparing the manuscript. This work was supported by grants from Ghent University (‘Bijzonder Onderzoeksfonds Methusalem project’ no. BOF08/01M00408 and ‘Geconcerteerde Onderzoeksacties’ no. 12051403), the Interuniversity Attraction Poles Programme (IUAP VI/33), initiated by the Belgian State, Science Policy Office.
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55. Liszkay A, Kenk B, Schopfer P: Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth. Planta 2003, 217:658-667. 56. Zhu J, Alvarez S, Marsh EL, LeNoble ME, Cho I-J, Sivaguru M, Chen S, Nguyen HT, Wu Y, Schachtman DP et al.: Cell wall proteome in the maize primary root elongation zone. II. Regionspecific changes in water soluble and lightly ionically bound proteins under water deficit. Plant Physiol 2007, 145:1533-1548.
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51. Sharp RE, Kuhn Silk W, Hsiao TC: Growth of the maize primary root at low water potentials. I. Spatial distribution of expansive growth. Plant Physiol 1988, 87:50-57. 52. Fan L, Linker R, Gepstein S, Tanimoto E, Yamamoto R, Neumann PM: Progressive inhibition by water deficit of cell wall extensibility and growth along the elongation zone of maize roots is related to increased lignin metabolism and progressive stelar accumulation of wall phenolics. Plant Physiol 2006, 140:603-612. 53. Fan L, Neumann PM: The spatially variable inhibition by water deficit of maize root growth correlates with altered profiles of proton flux and cell wall pH. Plant Physiol 2004, 135:2291-2300. 54. Wu Y, Thorne ET, Sharp RE, Cosgrove DJ: Modification of expansin transcript levels in the maize primary root at low water potentials. Plant Physiol 2001, 126:1471-1479.
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Current Opinion in Biotechnology 2010, 21:197–203
More from less: plant growth under limited water Aleksandra Skirycz1,2 and Dirk Inze´1,2 When subjected to abiotic stresses, plants actively re-program their growth by modulating both cell division and cell expansion. Growth decreases rapidly upon stress onset but it recovers and adapts once stress conditions become stable. Here, we review recent advances in understanding the mechanisms underlying both stress-induced growth repression and adaptation with an emphasis on drought and leaf growth and we briefly discuss how this knowledge can be translated into crops. It is now clear that stress response of growing and mature leaves is distinct and should be studied separately. Both cell proliferation and expansion are regulated by common signaling pathways involving gibberellins and DELLA proteins while down stream effector genes are stage specific. Addresses 1 Department of Plant Systems Biology, VIB, Technologiepark 927, B-9052 Gent, Belgium 2 Department of Plant Biotechnology and Genetics, Ghent University, Technologiepark 927, B-9052 Gent, Belgium Corresponding author: Inze´, Dirk ([email protected])
Current Opinion in Biotechnology 2010, 21:197–203 This review comes from a themed issue on Plant biotechnology Edited by Antonio Molina and Jim Haseloff Available online 2nd April 2010 0958-1669/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2010.03.002
Introduction To minimize the deleterious effects of stress and to complete their life cycle under adverse conditions, plants have evolved different adaptive responses that are summarized in the avoidance/tolerance model [1]. For example, during drought, plants close their stomata and accumulate compatible solutes to maintain a low water potential and to avoid dehydration. Simultaneously newly synthesized protective proteins, such as dehydrins and antioxidants, restrict damage to other proteins and cellular membranes and lower the levels of harmful reactive oxygen species (ROS) (see article by J.M. Pardo in this issue). Plants also reduce their growth as a way to save and redistribute resources that can become limited under extreme stress. Accordingly, growth reduction occurs rapidly after stress onset and independently of photosynthetic rates and plant carbon status [2,3], arguing that growth reduction is not simply a secondary effect of www.sciencedirect.com
resource limitation, but an important adaptive response. Counter-intuitively, plants exposed to mild drought stress even accumulate sugars and starch [3]. Although growth retardation increases the survival rate, during moderate stress episodes when survival is not threatened, it can be seen as counter-productive with unnecessary yield losses and major consequences for agriculture. Therefore, new crop varieties in which growth would be less inhibited during sporadic spells of moderate stress would be extremely advantageous for plant productivity, particularly in mild climates. We might even speculate that in modern varieties, breeders have already selected for reduced growth sensitivity under mild stress conditions. Knowledge of the mechanisms underlying growth reduction under stress is an important prerequisite to further improve crop productivity. Whereas an enormous amount of information is available on the molecular mechanism controlling stress tolerance in mature leaves, our current understanding of stress-regulated growth is still very fragmentary, partly because studies combining detailed growth analysis and molecular characterization of growing organs are relatively scarce [3,4,5]. Most of the available stress data were obtained from mature tissues or whole plants, in spite of the fact that developmental stage and cell identity have a major influence on the observed stress responses [3,6]. Hundreds of genes and many metabolites were shown to change specifically in growing versus mature leaves and in different root tissues under osmotic and salt stress, respectively [3,6]. Additionally, in most studies the applied stresses are rather extreme, leading to plant death, a situation that is rare in modern agricultural practice. Related, while numerous screens identified mutants that cope better with extreme stress [7–9], often because of their inherent dwarfed stature [10,11], only a very limited number of studies report on plants that grow better under mild stress conditions that do not threaten plant survival although they restrict growth [12,13]. This review will mainly focus on drought stress and its impact on Arabidopsis thaliana leaf growth, but key findings on other stress conditions, plant species, and organs will also be highlighted.
‘Acute and adaptation’ growth responses Response of plant growth to stress onset is often characterized by a rapid and acute (‘acute response’) inhibition, followed by recovery and adaptation to the new condition (‘adaptation response’) (Figure 1A). In barley (Hordeum vulgare) leaves, the leaf elongation rate (LER) decreased close to zero within seconds after salt addition to the roots, followed by recovery of LER to approximately 46% and 70% of the non-stressed plants within minutes and days, respectively [14]. Similarly, in the shoot apical meristem Current Opinion in Biotechnology 2010, 21:197–203
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Figure 1
Growth response to stress. (a) Schematic representation of ‘acute’ (yellow arrow) and ‘adaptative’ (green arrow) growth response of plants subjected to stress. Red arrow marks stress onset. (b) Schematic representation of Arabidopsis leaf growth. Leaves initiate at the flank of the meristem (SAM). The number of recruited cells might influence the final cell number. Initially leaf growth is driven exclusively by cell proliferation (P, red shading). Within several days and starting from the leaf tip, cells exit the mitotic cell cycle and start to expand (E, green shading). After a few days, leaf growth is steered solely by cell expansion. Again starting at the tip, cells become mature (no shading), coinciding with growth cessation [63]. The final cell number and size might therefore depend on the length of the developmental windows of proliferation and expansion (white and light-green arrows), respectively, and/or the duration of single cell cycle (the shorter the cycle, the higher the cell production per unit of time) and rate of expansion.
(SAM) of salt-treated Arabidopsis plants [15], the cell cycle activity was reduced transiently (12 h and 36 h after stress onset) and subsequently recovered (4 days after stress onset). While the acute response prepares plants for possibly more severe conditions, the adaptive response can be seen as the establishment of a new steady state to prolonged and stable stress. This two-phase (‘acute’ and ‘adaptation’) growth response indicates that the mechanisms that govern growth repression upon stress sensing and those that allow growth adaptation are distinct and will be discussed in more detail below.
Drought reduces leaf growth by affecting cell division and expansion In dicotyledonous plant species, such as Arabidopsis, leaf growth results from the proliferation and subsequent expansion of founder cells, recruited from the SAM. Consequently, the final leaf size depends on the number of recruited cells as well as the rates and developmental windows of cell division and expansion [16] (Figure 1B). Soil water deficit and relatively mild osmotic stress affect the leaf area by reducing both cell number and size, as shown in Arabidopsis and sunflower (Helianthus annuus) [3,17,18]. Importantly, the initial growth reduction can be compensated by a longer proliferation and/or expansion time, a phenomenon denominated ‘growth extension’. In an extreme case, the final leaf sizes of the Arabidopsis accession An-1 grown under control and soil water deficit conditions were identical, but were reached in stressed plants only after an additional two weeks of growth [17,18]. Interestingly, in Arabidopsis and sunflower leaves, exposed to a long water restriction period, epidermal cells retained some ability to expand, even Current Opinion in Biotechnology 2010, 21:197–203
though the leaf apparently had reached its final size [19] (Figure 1).
Root-to-shoot signaling in leaf growth regulation In partial root drying experiments in which only part of the root system is subjected to drying, reduced leaf growth and stomatal closure occurred even when the remaining roots had enough moisture to fully supply leaves with water [20], leading to the conclusion that fast chemical signal(s) are generated in the drying roots mediating both leaf responses. Whereas the plant hormone abscisic acid (ABA) is a key regulator of stomatal conductance [21], results on leaf growth-regulating hormonal signals are still under debate and contradictory. For instance, because externally applied ABA represses growth, it is commonly viewed as a growth inhibitor [22], but a positive role of ABA through the inhibition of ethylene production has also been proposed on the basis of results obtained with ABA and ethylene mutants and ABA and ethylene inhibitors in maize (Zea mays), Arabidopsis, or poplar (Populus trichocarpa) [23–25]. Similarly to ABA, exogenously applied ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) inhibit growth [26] and accumulate under stress [27,28], and ACC can be transported by the xylem from root to shoot [29]. Moreover, ethylene-deficient tomato (Solanum esculentum) plants subjected to soil drying were characterized by stomatal closure, without effect on their leaf growth, in contrast to wild-type plants [28]. However, in maize plants grown under drought stress neither endogenous levels of ethylene nor ABA could be correlated with reduced leaf elongation [30]. Short-term www.sciencedirect.com
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Figure 2
Schematic representation of processes involved in growth regulation upon drought. Soil drying is sensed in roots activating a combination of hydraulic and chemical signals that are transported through the xylem to leaves where they initiate a number of tolerance mechanisms. Response of mature leaves can be described by the avoidance/tolerance model [1]. In growing leaves stress leads to acute growth inhibition followed by growth adaptation both mediated by DELLA signaling. Ethylene promotes DELLA stabilization and growth inhibition while ABA is possibly involved in growth recovery. Negative ABA and ethylene cross-talk was demonstrated. While hormonal signaling is common between expanding (E) and proliferating (P) leaves, effector genes are distinct. In P leaves, inhibitors of CDKA might play a role in acute response while alternative respiration in growth adaptation. In expanding leaves, cell wall tightening and changes in cell turgor lead to growth cessation, while osmotic adjustment and cell wall loosening are important for growth adaptation.
growth inhibition was rather attributed to aquaporinmediated changes in the root hydraulic conductivity (Lpr) and consequent decrease in cell turgor [31,32]. While phosphorylation-dependent internalization of aquaporins triggered by ROS provides a fast mechanism to reduce Lpr upon stress onset [33], ABA positively affects aquaporin transcript and protein levels, hinting at a role in growth recovery [31]. Interestingly, ethylene negatively influenced petal expansion of rose (Rosa sp.), at least partly, by suppressing expression of the Rh-PIP2;1 aquaporin [34]. Interplay between hormonal and hydraulic signals thus appears essential to mediate both acute and adaptation growth responses under stress conditions (Fig. 2). Other unknown or poorly characterized signals may also be involved in long-range stress signaling. A recent example is the bypass1-derived signal that is rootborne and inhibits shoot growth by affecting both leaves www.sciencedirect.com
and SAMs, although its exact biological function and chemical nature are unknown [35].
GA and DELLA signaling are of key importance for organ growth While the exact roles of ABA and ethylene in growth repression are still incompletely clear, gibberellins (GAs) have been convincingly shown to play a prominent role in growth regulation under optimal, but also, under stress conditions [13,36–38], by repressing the levels of DELLA proteins, known to inhibit growth (Fig. 2). Stress-activated production of the catabolic enzymes GA 2-oxidases (GA2ox) reduces GA levels, in turn, stabilizing DELLA proteins and leading to growth repression [39,40]. Two Arabidopsis DREB transcription factors (TFs) AtCBF1 and AtDDF1 are important for the GA2ox regulation under cold and salt stress, respectively [39,40]. Current Opinion in Biotechnology 2010, 21:197–203
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Concomitantly, growth of Arabidopsis plants lacking all DELLA proteins is less reduced by relatively mild salt stress, but also upon treatment with the pathogen attackmimicking elicitor flagellin [13,36]. Importantly, besides their involvement in growth repression, DELLA proteins activate tolerance mechanisms by diminishing ROS levels and, hence, are also essential for plant adaptation to stress [13,41]. DELLA-deficient plants are more sensitive to severe and prolonged stress [13], suggesting that growth retardation under such conditions is important for plant survival. Accordingly, our work on the molecular responses of fully proliferating, fully expanding, and mature Arabidopsis leaves to mild osmotic stress showed that both cell proliferation and expansion were regulated by common regulatory circuits, involving ethylene and GAs, but not necessarily ABA signaling [3]. Remarkably, DELLA-mediated growth regulation is conserved across different stresses [13,36,38] and provides a convergence point for classical stress hormones because ABA, ethylene, and jasmonate signaling all positively affect DELLA stability, primarily via changes in GA concentrations [13,36,42,43] (Fig. 2). Moreover, GAs and DELLA proteins are important for both cell expansion and cell proliferation in leaves and roots [37,38]. DELLA proteins regulate transcription, but because they have no DNA-binding domain, they were proposed to act in concert with other TFs. Light control experiments of hypocotyl elongation revealed that DELLA proteins sequester the Basic-Helix-Loop TFs, PIF3 and PIF4, restricting cell elongation [44]. Whether a similar mechanism operates for DELLA-mediated stress growth repression and which are the exact protein partners and target genes remains to be established.
Drought effects on the cell cycle Exposure of plants to mild stress reduces cell number [3,17]. This decrease in cell number might result from either a longer duration of single cell division cycli and/or a shorter developmental window for cell proliferation. In leaves of both Arabidopsis and sunflower, water deficit had no effect on the developmental window of cell division and the reduced cell number measured in sunflower leaves was attributed to the impact of drought on the G1-to-S transition and, thus, on reduced division rates [2,3]. Progression into the S-phase is mediated by the activity of the PSTAIRE-containing cyclin-dependent kinase A (CDKA) and can be arrested by the interaction of CDKA with inhibitory proteins, such as Kip-related proteins (KRPs) or SIAMESE (SIM/EL2) and SIAMESE-RELATED proteins [45–47]. Because of their inhibitory role and the stress-induced expression of several KRP and SIM/EL2 genes, these are considered good candidates to link cell cycle activity and environmental stimuli [4,47] (Fig. 2). Providing a link to GA signaling, DELLA proteins restrain cell production in roots by enhancing the levels of KRP2 and SIAMESE genes [37]. The G1/S block can be released easily allowing cell Current Opinion in Biotechnology 2010, 21:197–203
division rates to recover once cells have adapted to stress. Part of such adaptation involves re-programming of the mitochondrial metabolism. As stress compromises mitochondrial electron transport chain, alternative respiration is activated protecting proliferating cells against ROS and supplying them with sufficient energy and nucleotides [3,48]. For leaves that initiate under stress it is also possible that fewer cells are incorporated into leaf primordia. Salt stress and ethylene reduce the size of the root apical meristem but no data are available on stressinduced changes in activity and size of the SAM [49,50].
Cell expansion—changes in cell wall properties Changes in cell wall rheology are important for cell expansion under stress conditions. While tightening contributes to growth cessation, cell wall relaxation maintains growth under low turgor pressures. Both processes have been studied mainly in maize roots subjected to progressive water deficit. In such an experimental system, the cell walls of the apical region are flexible and tissues continue to expand, while the cell walls of the elongation region are rigid, resulting in growth inhibition [51]. On the one hand, tightening of cell walls is primarily associated with accumulation of phenolics and lignin monomers that are covalently cross-linked to wall polysaccharides through the activity of peroxidases and oxidases [52] (Fig. 2). On the other hand, an increase in cell wall extensibility can be achieved by different mechanisms: changes in pH and activity of cell wall-loosening enzymes (expansins and xyloglucan endotransglycosylase [XET]) contribute to acidic growth and loosening of connections between cellulose microfibrils [5,53,54] (Fig. 2). Moreover, apoplastic ROS production in the apical region of water-stressed roots contributes to cell wall extensibility by non-enzymatic cleavage of cell wall polysaccharides [55,56]. Importantly, evidence is emerging for similar mechanisms acting in leaves growing under low turgor pressure. Superoxide levels and the expression of a number of expansins and XETs were exclusively affected in the expanding, but not in the proliferating or mature, Arabidopsis leaves growing under osmotic stress [3]. Furthermore expansion rates of maize leaves was shown to correlate well with the expression of three expansin genes under a range of developmental, genetic, and environmental cues, including drought [57].
Perspectives and conclusions Abiotic stress is responsible for more than 50% yield loss worldwide [58]. As the world population is rising exponentially, this problem needs to be dealt with, especially taking into account the deleterious effects of global warming. Ultimately, developing new stress-tolerant crop varieties will require further understanding and modulation of the molecular processes underlying plant stress responses. Thus far, most studies have concentrated on transgenic modifications that create plants that survive www.sciencedirect.com
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extreme drought stress, usually at the expense of biomass accumulation [59]. Unfortunately, a number of these transgenic plants also suffer from a growth penalty already under optimal conditions, albeit this problem can be overcome by the use of stress-inducible promoters [60]. A more promising strategy will be to optimize plant growth so as to minimize its inhibition by mild stress. As long as the stress is moderate under mild climates, such plants would accumulate a maximum of biomass during their life cycle. For this, a systems understanding of the mechanisms underlying growth regulation is essential and some important pre-requisites have to be fulfilled. (1) High-throughput phenotyping platforms that allow reliable, non-destructive monitoring of growth dynamics under a range of environmental conditions have to be established to identify even relatively small differences in a trait as variable as growth. A published example of such a platform is PHENOPSIS [61], while a large phenotyping facility has just been opened at the University of Adelaide, Australia (http://www.plantaccelerator.org.au/). A conceptual framework for drought phenotyping has recently been reviewed [62]. (2) Molecular and physiological data obtained using in vitro osmotic stress conditions have to be confirmed in more realistic soil systems. (3) Molecular characterization of the stress responses of growing organs/tissues has to be performed with sufficient temporal resolution [3,6]. By bringing together sufficient physiological, genetic, and molecular information, ultimately gene-to-phenotype prediction might be used to direct efforts to engineer plants for enhanced productivity. As apparently many, often redundant, mechanisms are involved in growth regulation under stress conditions, the simultaneous engineering of superior alleles and multiple genes under the control of appropriate (artificial) promoters will allow the fine tuning of growth responses under stress. Such an approach, resembling that in the synthetic biology field, will probably contribute to coping with complex traits, including drought stress.
Acknowledgments We apologize to all colleagues whose work was, owing to space limitations, not cited. We thank Hannes Claeys, Dr Wim Verelst and Dr Matthew Hannah for useful discussion and Dr Martine De Cock for help in preparing the manuscript. This work was supported by grants from Ghent University (‘Bijzonder Onderzoeksfonds Methusalem project’ no. BOF08/01M00408 and ‘Geconcerteerde Onderzoeksacties’ no. 12051403), the Interuniversity Attraction Poles Programme (IUAP VI/33), initiated by the Belgian State, Science Policy Office.
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