JXB Advance Access originally published online on April 29, 2008
Journal of Experimental Botany 2008 59(8):2221-2232; doi:10.1093/jxb/ern087
RESEARCH PAPER |
Genetic variability for leaf growth rate and duration under water deficit in sunflower: analysis of responses at cell, organ, and plant level
Unidad Integrada Balcarce, Facultad de Ciencias Agrarias, Universidad Nacional de Mar del Plata, Instituto Nacional de Tecnología Agropecuaria, CC 276, 7620 Balcarce, Argentina
* To whom correspondence should be addressed. E-mail: laguirre{at}mdp.edu.ar
Received 6 December 2007; Revised 3 March 2008 Revised 4 March 2008 Accepted 4 March 2008
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Abstract |
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Plants under water deficit reduce leaf growth, thereby reducing transpiration rate at the expense of reduced photosynthesis. The objective of this work was to analyse the response of leaf growth to water deficit in several sunflower genotypes in order to identify and quantitatively describe sources of genetic variability for this trait that could be used to develop crop varieties adapted to specific scenarios. The genetic variability of the response of leaf growth to water deficit was assessed among 18 sunflower (Helianthus annuus L.) inbred lines representing a broad range of genetic diversity. Plants were subjected to long-term, constant-level, water-deficit treatments, and the response to water deficit quantified by means of growth models at cell-, leaf-, and plant-scale. Significant variation among lines was found for the response of leaf expansion rate and of leaf growth duration, with an equal contribution of these responses to the variability in the reduction of leaf area. Increased leaf growth duration under water deficit is usually suggested to be caused by changes in the activity of cell-wall enzymes, but the present results suggest that the duration of epidermal cell division plays a key role in this response. Intrinsic genotypic responses of rate and duration at a cellular scale were linked to genotypic differences in whole-plant leaf area profile to water deficit. The results suggest that rate and duration responses are the result of different physiological mechanisms, and therefore capable of being combined to increase the variability in leaf area response to water deficit.
Key words: Cell division, leaf expansion, leaf growth duration, leaf growth rate, sunflower (Helianthus annuus L.), water deficit
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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Plants generally respond to water deficits by reducing their transpiration rate. Depending on the species and genotype, this can be achieved by stomatal closure (Tardieu and Davies, 1993), wilting, or leaf rolling (O'Toole and Cruz, 1980). However, one of the earliest water-saving mechanisms, present in a great majority of plants, is reduced leaf growth. Changes in leaf area can be a consequence of reduced leaf number (although it is not affected to a great extent; Aguirrezábal et al., 2006) and/or reduced area of individual leaves. Reduced leaf expansion rates under water deficit have been found to be related to different mechanisms, such as decreased cell division rate (Schuppler et al., 1998; Granier et al., 2000), to cell wall hardening (Matthews et al., 1984; Neumann, 1995), or to decreased turgor (Hsiao et al., 1998; Bouchabké et al., 2006).
The extent of leaf growth reduction caused by water deficit is very important in determining the adaptation of a certain crop variety to a climate scenario. In a scenario where long-term droughts are expected, a genotype which reduces its leaf growth is more likely to reach maturity with a certain amount of available water. On the other hand, in a scenario where short-term water deficits are expected, a genotype which maintains leaf growth is likely to have higher yields (Reymond et al., 2003). It has been shown that a genotype can maintain its leaf area by maintaining growth rate (Reymond et al., 2003) or by increasing the duration of leaf growth (Aguirrezábal et al., 2006). Moreover, an increased duration of growth could have the benefit of increasing the opportunity for recovery after rainfall (Alves and Setter, 2004). The natural genetic variability for these traits could be used to develop crop varieties adapted to specific scenarios. Despite this, breeding for these traits is not a common approach for obtaining drought resistance in crop species, probably because of a lack of well-characterized sources of genetic variability.
The genetic variability of leaf growth response to water deficit has not been studied in many crop species. In maize, the response of leaf extension rate to water deficit has been genetically analysed (Reymond et al., 2003). This analysis, however, disregards the effect of water deficit on the duration of leaf growth, which has been shown to be significant in the model species Arabidopsis thaliana (Aguirrezábal et al., 2006). In sunflower, reports about the response of leaf growth duration to water deficit have been contradictory (Takami et al., 1981; Granier and Tardieu, 1999). The analysis of responses to water deficit should also take into account how processes at cell- and leaf-level affect the leaf area of the whole plant, since radiation interception, and hence photosynthesis and transpiration, are largely determined at this scale. In sunflower, a whole-plant model of leaf growth has been developed (Dosio et al., 2003), which uses a set of parameters to describe the co-ordinated growth of different leaves of the plant, and could be used as a framework for quantitatively analysing the response of leaf area to water deficit.
The objective of this work was to analyse the response of leaf growth to water deficit in several sunflower genotypes in order to identify and quantitatively describe sources of genetic variability for this trait. Plants were subjected to stable, long-term, mild water deficits, and leaf growth kinetics was analysed. Genotypes with contrasting response in terms of leaf growth rate and leaf growth duration were identified. The response of these genotypes was further characterized at cell-level (cell division and cell growth kinetics), and at plant-level (using the whole-plant model of Dosio et al., 2003). The response of each genotype to water deficit was quantified in terms of the changes in the parameters of this model. The relationships between responses at cell, organ, and plant level were analysed.
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Materials and methods |
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Eighteen non-branching sunflower inbred lines were selected, based on pedigree data (Korell et al., 1992), so as to comprise most of the genetic variability of cultivated sunflower. This group included two lines showing high and low osmotic adjustment (lines ‘OA+’ and ‘OA–’; Chimenti et al., 2002). Four experiments were carried out to investigate the response of leaf growth to a water stress in these genotypes. The first experiment was conducted with the complete set of genotypes. Based on the results from this experiment, four genotypes were selected for the second experiment. Experiments 3 and 4 were conducted with the two most contrasting genotypes in terms of the response of leaf growth rate and duration.
Culture methods and growth conditions
Seeds were sown in 35-cm-deep, 10-cm-wide containers made of PVC pipe, filled with soil (Typic Argiudoll, horizon A). Soil water content was measured initially by oven-drying soil samples at 105 °C for 48 h, and then monitored daily by weighing the container. Plants were grown without water limitations (–0.05 MPa) until 
100 degree-days (base temperature = 4.8 °C; Granier and Tardieu, 1998) after leaf 8 initiation in each genotype. At this moment, soil water content was decreased until –0.65 MPa was reached, using the method described in Pereyra-Irujo et al. (2007). The method consists of sowing maize (Zea mays L.) plants earlier, in the same pots as sunflower plants. This method allows the desired soil water content to be reached quickly, the resulting soil water content in the pot is uniform, the rate of soil water depletion is independent of the sunflower genotype being evaluated (Pereyra-Irujo et al., 2007). Maize plants did not affect the growth of the sunflower plant (Pereyra-Irujo et al., 2007). The specified water content in each treatment was maintained by daily irrigation as in Granier et al. (2005).
Experiments were carried out under controlled (growth chamber; Refrimax SRL, Mar del Plata, Argentina) or semi-controlled (heated greenhouse) environmental conditions. Air temperature, relative humidity, incident radiation, and leaf temperature were measured every 15 min, and averaged and recorded every 1 h, with dataloggers (Cavadevices, Buenos Aires, Argentina). Thermal time was calculated as the daily integral of the difference between temperature and base temperature (4.8 °C; Granier and Tardieu, 1998). The base temperature for leaf initiation and leaf expansion did not differ significantly among the evaluated inbred lines in a preliminary experiment (not shown).
Leaf growth measurements
Non-destructive measurements of length and width (in the greenhouse experiments) or area (by means of a digital photograph, in the growth chamber experiment) of the 8th leaf were made at least three times a week until the end of its expansion, in four (experiment 1) or five (experiments 2, 3, and 4) plants per genotype and treatment. A highly significant linear relationship (R2 > 0.99, n=1755, P < 0.0001) was established between lengthxwidth and leaf area, which was applicable to any leaf regardless of leaf number, leaf age, genotype, or treatment. In addition, all leaves of the plant were measured every 7 d.
In experiments 3 and 4, destructive measurements of leaf area were made in leaves larger than 0.005 mm2 (average area at initiation; Dosio et al., 2003) from three plants harvested every 2–4 d (experiment 3) or 6–12 d (experiment 4). Leaves were scanned or photographed under a microscope and measured with image analysis software (Matrox Inspector; Matrox Electronic Systems Ltd, Quebec, QC, Canada).
In addition, the number of initiated leaves was determined under a microscope in two plants harvested every 1–2 d, until the initiation of leaf 12 (experiments 1, 2, and 4) or leaf 20 (experiment 3).
Absolute leaf expansion rate (LER) was calculated as the slope of the relationship between leaf area (L) and thermal time (t) in two consecutive measurements, and leaf relative expansion rate (RER) as the slope of the relationship between the logarithm of leaf area and thermal time:
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Cell growth measurements
At five different moments during experiment 4, leaf 8 was divided into two to four transects (depending on the size of the leaf). The area of each transect was measured, and imprints (obtained after evaporation of a varnish spread on the leaf) of the adaxial epidermis were taken halfway between the mid-rib and the leaf margin. The size of 25 cells/imprint was measured under a microscope coupled to an image analyser. Mean cell area of the leaf was calculated as the sum of the products of average cell area and transect area for each transect, divided by the sum of all transect areas. Epidermal cell number was calculated as the ratio between leaf area and mean cell area. Relative cell expansion rate (RCR) was calculated as the slope of the relationship between the logarithm of cell area (C) and thermal time (t) in two consecutive measurements:
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Growth curves fit
In experiments 1 and 2, growth of leaf 8 was described by means of a sigmoidal curve as described in Aguirrezábal et al. (2006):
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Final leaf area was calculated as the upper asymptote (A) of the sigmoidal curve. The duration of leaf expansion was calculated as the time elapsed between leaf initiation and the moment the leaf reaches 95% of its final area as calculated from the fitted curve. The mean expansion rate was calculated as the ratio between the final leaf area and the duration of leaf expansion.
In experiments 3 and 4, growth of different leaves of the plant was described by means of the model proposed by Dosio et al. (2003). This model divides the growth of each leaf into three distinct phases (Fig. 1). During phase 1 growth is initially exponential, at a high constant relative rate. During phase 2, growth continues to be exponential but at a lower rate (Fig. 1B). During phase 3 most of the growth of the leaf occurs (high absolute growth rate), in an almost linear fashion (i.e. with declining relative growth rate, Fig. 1B), until the end of expansion. Leaf area in each phase is defined by these equations:
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Parameters for these equations are the moments of leaf initiation (t0) and end of phases 1 and 2 (t1 and t2), relative leaf expansion rates (RER) during phases 1 and 2 (R1 and R2) and at the beginning of phase 3 (R3), and a parameter describing the decline in RER during phase 3 (a). The values used for leaf area at initiation (A0), and R1 in well-watered conditions were those reported by Dosio et al. (2003), which were confirmed to be the same in the genotypes used in this study, in a preliminary experiment (not shown). Leaf area at the beginning of phases 2 and 3 (A1 and A2) are calculated from equations 5 and 6. The duration of each phase was determined as in Dosio et al. (2003). The total duration of leaf expansion was calculated as in experiments 1 and 2.
A log-normal three-parameter curve was fitted to describe the time-course of relative cell expansion rate (RCR) (Cookson and Granier, 2006):
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Relative cell division rate (RDR) was estimated by subtracting RCR from RER, as in Cookson and Granier (2006).
All curves were fitted using least-squares (Marquardt–Levenberg algorithm; Sigma-Plot software version 8.0, SPSS Science, Chicago, IL, USA). Analysis of variance was carried out using SAS (SAS Institute Inc., Cary, NC, USA)
Relative contributions of rate and duration to final leaf area
In experiments 1 and 2, the relative contributions of the changes in expansion rate and duration of growth to the changes in final leaf area under water deficit were quantified for each genotype, in order to select the most contrasting genotypes for further analyses. The impact of a reduced expansion rate was quantified, independently of changes in duration, as the reduction in leaf area observed at the end of leaf growth in the control treatment (‘rate effect’). This measure equals the reduction in final leaf area in the case of no increase in the duration of growth (Fig. 2A). The impact of an increased duration of growth was quantified by the compensatory expansion that takes place between the end of leaf growth in the control treatment and the end of leaf growth in the water-deficit treatment (‘duration effect’). In the case that duration is increased, the sum of both indicators equals the reduction in final leaf area (Fig. 2B).
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Results |
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Water deficit reduced leaf area to a different extent depending on the genotype
In the group of 18 genotypes evaluated in experiment 1, total leaf area after 28 d of the beginning of the treatments ranged between 716 cm2 and 1245 cm2 in the control plants (Fig. 3A), and between 312 cm2 and 569 cm2 in the water-deficit treatment. This represents a reduction between 49% and 62% (Fig. 3B). The response of total leaf area is a complex combination of the responses of different leaves of the plant, which were at different developmental stages at the onset of water deficit. The response of leaf 8 was chosen for comparison between genotypes, since the initiation of this leaf was taken as reference for the imposition of treatments. The final area of leaf 8 ranged between 50 cm2 and 156 cm2 in control conditions (Fig. 3C), and between 30 cm2 and 86 cm2 under water deficit (reduction between 38% and 57%; Fig. 3D).
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A two-factor (genotype and treatment) analysis of variance was performed, showing significant genetic variability for the area of leaf 8 (P < 0.0001) as well as a significant response to water deficit (P < 0.0001). The interaction between both factors, which represents the genetic variability for the response of leaf area to water deficit, was also highly significant (P < 0.0001). The response of leaf 8 area to water deficit was not related to the total leaf area of each genotype (compare D and A in Fig. 3).
Responses of leaf expansion rate and duration were variable between genotypes
The growth of leaf 8 under well-watered conditions followed the typical sigmoidal shape, with rates and durations that differed between genotypes (Fig. 4). Water deficit altered the kinetics of leaf growth by reducing expansion rate and, in most cases, increasing the duration of leaf growth. The reduction in expansion rate in the water deficit ranged between 44% and 67%. The duration of expansion was increased between 2% and 26%.
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The relative contributions of the change in leaf expansion rate and the change in the duration of growth to the final reduction in the area of leaf 8 are plotted in Fig. 5 for each genotype. As explained in Materials and methods, the contribution of the reduction in expansion rate was quantified as the reduction of leaf area observed at the end of leaf growth in the control treatment (‘rate effect’, Fig. 2). The effect caused by an increased duration of growth was quantified by the compensatory expansion that takes place between the end of leaf growth in the control treatment and the end of leaf growth in the water-deficit treatment (‘duration effect’, Fig. 2). The sum of both indicators equals the reduction in final leaf area (i.e. the reduction in final leaf area increases downward and to the left of the graph).
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Differences between genotypes in the reduction of final leaf area were caused either by differences in the reduction of expansion rate (‘HA64’ versus ‘OA+’) or differences in the compensatory growth during the increase in the duration of growth (‘HAR2’ versus ‘OA+’). The genotypes showing high and low osmotic adjustment (‘OA–’ and ‘OA+’) differed mainly in the response of their expansion rate to water deficit. Relative responses of the four genotypes analysed in both experiments were conserved.
The genetic variability for both effects was quantitatively similar. Average (experiments 1 and 2) differences between genotypes for the relative contribution of reduced rate were 15.6 percentage points, while differences for the relative contribution of increased duration were 15.3 percentage points.
Water deficit affected the rate and duration of different phases of leaf growth
Two genotypes, ‘HAR2’ and ‘HA64’, were selected for further analysis based on their contrasting response to water deficit, both in terms of rate and duration of growth (Fig. 5). In experiments 3 and 4, leaf growth dynamics showed the same three-phase pattern described by Dosio et al. (2003), consisting of two phases of exponential growth followed by a phase of quasi-linear growth. Water deficit altered the duration of the phases and the relative expansion rates in each of them, as can be observed for leaf 8 in Fig. 6.
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In leaf 8, growth rates were reduced to a greater extent in genotype ‘HAR2’ than in ‘HA64’, both during phase 2 (30% versus 12%, average of experiments 3 and 4) and phase 3 (57% versus 38%). The increase in the duration of growth was greater for ‘HAR2’ than ‘HA64’, both for phase 2 (40% versus 6%) and for phase 3 (48% versus 8%). The effect of water deficit on phase 1 could not be analysed in leaf 8 since the treatments began approximately at the end of this phase, but in upper leaves it was found that its response was similar in both exponential phases (phases 1 and 2; not shown).
The whole-plant framework of analysis described by Dosio et al. (2003) was applied to leaf growth data from experiment 3. Figure 7A and E shows the beginning of the water-deficit treatment relative to the leaf development in each genotype. These figures also show that durations of exponential growth and total growth were increased in genotype ‘HAR2’ and were similar to the controls in ‘HA64’. Leaves of genotype ‘HAR2’ that were already at phase 3 at the onset of water deficit (up to leaf 4) showed less response than leaves that were at phase 1 or 2 (e.g. leaves 8 and 10). In both genotypes, the rates of expansion during the exponential phases of growth (Fig. 7B, F) were increasingly affected from leaf 6 onwards, while the expansion rates during phase 3 were affected in all the measured leaves. Water deficit reduced the final area of leaves 8 onwards in genotype ‘HAR2’ (Fig. 7D), while in ‘HA64’ leaves 4 onwards were affected (Fig. 7H).
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Water deficit affected cell division and expansion
In experiment 4, mean adaxial epidermal cell area and cell number were measured at different moments during growth of leaf 8. Water deficit reduced the relative rates of leaf expansion (Fig. 8A, D), cell expansion (Fig. 8B, E), and cell division (Fig. 8C, F), in both genotypes. In genotype ‘HAR2’ the period of cell division was extended by 80 degree-days (Fig. 8C), and the end of cell (and leaf) expansion was delayed by an additional 70 degree-days (Fig. 8B). In this genotype, the increase in the duration of cell division under water deficit is similar to the increase in the duration of exponential leaf growth (Fig. 8A). In genotype ‘HA64’, the duration of the phases of exponential and linear growth were only slightly modified (Fig. 8B). The duration of cell division and expansion also remained almost unchanged (Fig. 8E, F).
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Discussion |
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A water-deficit treatment was imposed on 18 sunflower genotypes by maintaining a reduced soil water content for a long period. A method specially designed for sunflower (Pereyra-Irujo et al., 2007) was used to apply this treatment independently of the leaf area of the sunflower plant, thus allowing a fair comparison between genotypes. This kind of water-deficit (constant intensity) allowed intrinsic genotypic differences in leaf growth responses to be identified independently of differences in total plant leaf area (Fig. 3) or water depletion rate (not shown).
It has been generally assumed that the effect of water deficit on leaf growth consists solely of a reduction in expansion rate, without any change in the duration of growth, as evidenced by the assumptions included in crop models, both in specific (e.g. for sunflower; Villalobos et al., 1996) and general (Connor and Fereres, 1999) crop models. In the case of sunflower, this assumption was later supported by experimental evidence that showed that, in one sunflower hybrid, water deficit did not increase leaf growth duration (Granier and Tardieu, 1999). On the other hand, it was recently reported that several A. thaliana genotypes showed increased leaf growth duration under water deficit, with significant genetic variability (Aguirrezábal et al., 2006). The present results showed that there are sunflower genotypes that respond to water deficit by considerably increasing leaf growth duration. This variability in the responses of leaf growth to water deficit could be incorporated into existing sunflower simulation models, in order to improve the estimations of the performance of different genotypes under water stress.
Changes in rate and duration contributed in a similar amount to the variability in the response of final leaf area
The analysis of the growth dynamics of individual leaves permitted the dissection of the response of each genotype into its components, leaf growth rate and duration. A novel approach that quantifies the relative contributions of the changes in rate and duration on final leaf area was used to select genotypes of contrasting response to water deficit. The effect of an increased duration on final leaf area was smaller than the effect of a reduced growth rate, since expansion rate is usually low during the final stage of leaf expansion. Nevertheless, the average (experiments 1 and 2) range of variation between genotypes in the effect of increased duration (15.3%) was similar to that of reduced rate (15.6%), rendering both components of the response equally interesting from a breeding point of view.
Correlation between the response of rate and duration was weak, as was previously found in A. thaliana (Aguirezábal et al., 2006). However, of the four possible combinations of high or low rate response and high or low duration response, only three of them were identified (i.e. responses of ‘HAR2’, ‘HA64’, and ‘OA+’). No genotype was found combining a relatively low reduction in rate and a relatively large increase in duration. In A. thaliana, genotype ‘An-1’ showed this kind of response, reaching a similar leaf area under well-watered or water-deficit conditions (Aguirrezábal et al., 2006). Under the hypothesis that rate and duration are governed by different physiological mechanisms, a cross between two genotypes showing a differential response in terms of rate and duration (e.g. ‘HAR2’ and ‘HA64’) could theoretically produce an offspring with a response to water deficit ranging from that of ‘AO+’ (high rate response, low duration response) to a final leaf area response smaller than that of the parents (low rate response, high duration response). This hypothesis is supported by the low correlation between both components of the response. This is currently being tested by means of a genetic analysis of a segregating population derived from the cross between ‘HAR2’ and ‘HA64’.
The rate of tissue expansion is proportional to cell wall extensibility and turgor pressure above a yield threshold (Lockhart, 1965). The genotypes differing in osmotic adjustment capacity differed mainly in the response of their leaf expansion rate (Fig. 4), with the genotype with high osmotic adjustment showing the greatest reduction in expansion rate. This is in agreement with previous findings which showed that sunflower genotypes having a high degree of osmotic adjustment showed relatively lower leaf expansion and tissue elasticity (increased bulk modulus of elasticity) under water stress (Chimenti and Hall, 1994). These results suggest that, in this case, differences in growth rate could be mainly determined by cell wall characteristics, with osmotic adjustment being a consequence of reduced growth decreasing the rate of solute dilution, as has been previously suggested (Van Volkenburgh and Boyer, 1985; Munns, 1988).
Using a whole-plant framework to describe the response of the leaf area profile to water deficit
Granier and Tardieu (1999) showed that the effect of water deficit depended largely on the timing of the treatment, relative to the development of the leaf. A similar behaviour was observed in the present experiments, where young leaves generally showed larger reductions in their area under water deficit. In spite of this, the whole-plant framework used allowed the genetic differences in the response of leaf growth to water deficit to be highlighted, and changes in the leaf area profile to be understood.
Irrespective of leaf position, genotype ‘HAR2’ showed a higher sensitivity to water deficit, both in terms of expansion rate and growth duration. In ‘HAR2’, the increase in duration of leaves stressed at phase 3 (e.g. leaf 4) was able to compensate the reduction in relative growth rate, allowing them to reach the same area as in the control treatment. In ‘HA64’, these leaves showed a similar reduction in relative growth rate but no increased duration, leading to a reduced final area. In younger leaves (e.g. leaf 10), the more marked reduction in relative growth rate shown by ‘HAR2’ prevails over the compensatory effect of increased duration, and the reductions in final leaf area become larger than in ‘HA64’. These differences in the response to water deficit result in the plant's leaf area profile being altered in a different fashion on each genotype (Fig. 7D, H). On the other hand, the relatively high expansion rate of ‘HA64’ under water deficit results in a leaf area profile similar to that of control plants (Fig. 7H).
To allow a fair comparison of genotypes in the experiments where only leaf 8 was analysed (experiments 1 and 2), care was taken to start the water-deficit treatment at a fixed developmental stage of this leaf, and not at a fixed date after emergence. The whole-plant analysis of the two contrasting genotypes confirmed that their differential response was not due to differences in timing of the treatment relative to leaf age, since each genotype's specific responses could be observed in different leaves of the plant.
The increase in the duration of exponential growth was correlated to an extended period of epidermal cell division
In the present work, it was found that, in one of the two genotypes analysed in detail (‘HAR2’), the increase in the duration of the phase of exponential growth coincided with an increase in the duration of epidermal cell division, while in the other genotype (‘HA64’) neither of these durations were appreciably affected. An increase in the duration of cell division in response to water stress was also observed in wheat leaves (Schuppler et al., 1998). In sunflower, however, it had been reported that neither water nor light stresses caused any increases in the period of cell division in leaves (Granier et al., 2000). These results, obtained with a different genotype (cv. ‘Albena’), support the existence of genetic variability for this trait.
Increased leaf growth duration under water deficit is usually suggested to be caused by changes in the activity of cell-wall enzymes (Aguirrezábal et al., 2006), such as peroxidases (which increases drastically at the end of cell expansion; MacAdam and Grabber, 2002). The present results, however, suggest that the duration of cell division in the epidermis plays a key role in this response. During the early stages of leaf development, proliferative growth is highly energy-requiring (Fleming, 2006). Maintaining cells under a proliferative growth state in the epidermis could allow the leaf to maintain the high relative expansion rates which occur during the first two phases of leaf growth, by acting as a strong sink for carbon and nutrients (as suggested previously by Van Volkenburgh, 1999). This would be in agreement with the fact that the epidermis has been shown to be the leaf tissue which drives and restricts leaf growth (Savaldi-Goldstein et al., 2007). Subsequently, a delayed entry of cells into a non-proliferative, vacuole-associated growth state could lead to delayed leaf growth cessation at leaf level. A similar increase in the duration of epidermal cell division, accompanied by an increase in total leaf growth duration has been reported in A. thaliana plants subjected to light stress (Cookson and Granier, 2006). Based on these results, the genetic variability for the response of the duration of cell division can be considered a likely cause of the apparently contradictory responses of leaf growth duration to water deficit in sunflower (Takami et al., 1981; Granier and Tardieu, 1999).
In the genotypes analysed in detail, the largest increase in duration of cell division coincided with the largest decrease in cell division rate. Genetic evidence in A. thaliana, however, suggests that rate and duration of cell division are not necessarily linked. Inactivation or overexpression of different genes in this species were able independently to alter cell division rate (cyclin-dependent-kinase inhibitors KRP1 and KRP2: Wang et al., 2000; de Veylder et al., 2001) or the duration of the period of cell division (AINTEGUMENTA: Mizukami and Fischer, 2000; STRUWWELPETER: Autran et al., 2002). The genotypes of contrasting response described in the present paper could be useful for further studying the relationship between duration and rate of epidermal cell division under changing environmental conditions.
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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Significant variation among lines was found for the response of leaf expansion rate and for leaf growth duration. The experimental and statistical methods applied allowed the identification of intrinsic genotypic differences in leaf growth responses at cell-, leaf-, and plant-level, that were not due to differences in timing or intensity of the treatment, or differences in plant leaf or water depletion rate.
One of the objectives of this work was to identify the mechanisms underlying genetic differences in the response of leaf growth to water deficit, as has been done previously for different environmental factors (Harrison et al., 1998; Masle, 2000; Stiles and Van Volkenburgh, 2002). The present results suggest that genetic differences in leaf growth rate under water deficit could be determined by cell wall properties, while increased duration of leaf growth is partly due to a prolonged phase of epidermal cell division. This implies that rate and duration responses could be the result of different physiological mechanisms, and are therefore capable of being combined to increase the variability in leaf area response to water deficit in sunflower.
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Acknowledgements |
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We are grateful to Luis Méndez, for his technical assistance, and to Carlos Sala, José Bruniard, Claudio Chimenti, Advanta Semillas, and the North Central Regional Plant Introduction Station (USDA) for providing the seeds of the inbred lines used in this study. This research was supported by Asociación Argentina de Girasol (ASAGIR) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), grant PICT-O 13168, and by Instituto Nacional de Tecnología Agropecuaria (INTA), grant PNCER1336. Luis Aguirrezábal is a member of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Gustavo Pereyra-Irujo and Leandra Lechner hold scholarships form CONICET. This work is part of a thesis submitted by Gustavo Pereyra-Irujo in partial fulfilment for the requirements for a Doctor's degree.
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References |
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