Correlation between leaf growth variables suggest intrinsic and early controls of leaf size in Arabidopsis thaliana

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1 Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment Blackwell Science Ltd 2005? ? Original Article Plant, Cell and Environment (2005) 28, Kinematic analysis and leaf size control in Arabidopsis S. J. Cookson et al. Correlation between leaf growth variables suggest intrinsic and early controls of leaf size in Arabidopsis thaliana SARAH J. COOKSON 1, MIEKE VAN LIJSEBETTENS 2 & CHRISTINE GRANIER 1 1 Laboratoire d Ecophysiologie des Plantes sous Stress Environnementaux (LEPSE), INRA-AGROM, UMR 759, 2 Place Viala, Montpellier Cedex 1 France and 2 Department of Plant Systems Biology Ghent University/Flemish Interuniversity Institute for Biotechnology (VIB) Technology Park 927 B-9052 Gent (Zwijnaarde) Belgium ABSTRACT Leaf development is affected by both internal (genetic) and external (environmental) regulatory factors. The aim of this work was to investigate how leaf growth variables are related to one another in a range of environments. The leaf growth variables of wild-type Arabidopsis thaliana and leaf development mutants (,,, elo2 and elo4) were studied under different incident light treatments (light and shade). The leaves studied were altered in various leaf development variables, such as the duration of expansion, relative and absolute expansion rates, epidermal cell size, epidermal cell number and initiation rate. Final leaf area was correlated to maximal absolute leaf expansion rate and cell number, but not to duration of leaf expansion or cell size. These relationships were common to all studied genotypes and light conditions, suggesting that leaf size is determined early in development. In addition, the early variables involved in leaf development were correlated to one another, and initial relative expansion rate was negatively correlated to the duration of expansion. These relationships between the leaf development variables were used to construct a conceptual model of leaf size control. Key-words: Arabidopsis; conceptual model; epidermal cells; kinematic analysis; leaf development; shade. INTRODUCTION Leaf growth and development responds to hormone, nutritional and environmental conditions such as soil water content, incident light and leaf temperature. Combined with this regulation there are genotype-specific differences between species, subspecies and ecotypes of the same species. How this intrinsic genetic control functions, and which developmental regulators are involved, is not well understood. Mutational analysis and the study of transgenic lines have shown that the regulation of leaf size can be disrupted by alteration of genes involved in numerous plant processes Correspondence: Christine Granier. Tel: + 33 (0) ; fax: +33 (0) ; granier@ensam.inra.fr such as metabolism, hormone action, cell division or cell expansion (as reviewed by Tsukaya 2002). Both intra- and interspecific differences in the leaf size of plants grown in different environmental conditions are generally associated with differences in cell number and not cell size (Dale 1992; Tardieu & Granier 2000). Total cell number of an organ is determined by the number of divisions of undifferentiated cells. During shoot development, leaves are initiated as primordia in which most cells divide and proliferate. As the organ develops, nuclei of these cells are progressively blocked in the G1 phase of cell cycle with a well-defined developmental programme depending on the positions of the cells in the leaf (Granier & Tardieu 1998a) and environmental conditions (Powell, Davies & Francis 1986; Schuppler et al. 1998; Tardieu & Granier 2000). Thus, it is the time at which cells are progressively blocked in a phase of the cell cycle which defines total cell number in a leaf. The relationship between cell number and final leaf size (Granier, Turc & Tardieu 2000) suggests that cell division solely controls leaf size, and this is the basis of classical cell theory in which the leaf is made by the sum of the behaviour of each cell. However, there is contrasting evidence which indicates that the level of leaf size control is at the scale of the tissue (organismal theory) rather than at the scale of the cells (Green 1976). In this case, blocking cell division is to some extent compensated for by an increase in cell size (Haber & Foard 1963). Recently, a Neo cell theory has been proposed in which co-operative compensation between the parameters of cell behaviour has been added to classical cell theory (Tsukaya 2003). Thus, there is considerable controversy concerning the regulation of leaf size and numerous recent reviews are devoted to this subject (e.g. Fleming 2002, Tsukaya 2003). The wide genetic variability in Arabidopsis thaliana (a large number of accessions and mutants) provides a unique opportunity to identify how leaf growth variables are affected by internal regulatory factors. Ironically, despite the nearly iconic status of this species there has been little careful analysis of leaf growth. Generally, leaf growth mutants have been described phenotypically in terms of final measurements of leaf shape and area, cell number and cell size (e.g. Tsuge, Tsukaya & Uchimiya 1996; Autran et al. 2002; Nelissen et al. 2003, Cnops et al. 2004). These final measurements reflect the integrated out Blackwell Publishing Ltd 1355

2 1356 S. J. Cookson et al. put of various developmental processes, and, despite the fact that leaf growth is a dynamic process, quantification of the rates and duration of leaf development are rare in this species. The aim of the study presented here was to analyse the relationships between leaf growth variables in Arabidopsis thaliana. Thus a wide range of leaf growth curves was obtained by selecting leaf development mutants and manipulating the light conditions to which they were subjected to during their development. Leaf development mutants used in this study were selected from the 255 EMS-induced leaf mutants isolated by the laboratory of J. L. Micol (Berná, Robles & Micol 1999; Robles & Micol 2002) on the basis of reduced or increased final individual leaf area or altered leaf shape. was selected from the rotunda class, with the representative trait of broad and rounded lamina. The RON2 gene was shown to be identical to LEUNIG, a transcriptional corepressor with a role in flower development (Cnops et al. 2004). was selected from the angusta class, with the representative trait of narrow lamina, the ANG4 gene was isolated by map-based cloning according to Peters et al. (2004). Additionally,, elo2 and elo4 were selected from the elongata class, with the representative trait of narrow, elongated lamina and long petioles. There mutations affect components of the Elongator complex that is involved in RNA polymerase II transcription (Nelissen et al. 2003, 2005). Superimposed upon this regulation of leaf development by internal factors, shade treatments were used to further investigate how leaf growth variables are affected by external factors. Shade treatments are known to affect the development of leaves, producing smaller leaves and decreasing leaf epidermal cell number in other species (Granier & Tardieu 1999). Relationships between the leaf growth variables were investigated with the aim of identifying relationships common across different internal and external factors. These robust relationships were therefore considered as intrinsic properties of leaf development and were related in a conceptual model of leaf development. This model is the first of its kind as generally models of leaf size control are based upon the processes of cell division, cell expansion and tissue size control (as reviewed by Fleming 2002; Tsukaya 2003) rather than rates and durations of growth processes. MATERIALS AND METHODS Plant culture and experimental design Wild-type () seeds of Arabidopsis thaliana (L) Heynh. ecotype Landsberg erecta (Nottingham Arabidopsis Stock Centre) and mutants (,,, elo2 and elo4 produced in a Landsberg erecta background, Berná et al. 1999) were grown during three experiments (exp. 1, exp. 2 and exp. 3) in two growth-chambers with different light intensities ( light and shade treatments, see Table 1). The shading used was neutral, the light spectrum was unaffected and this was tested using a LI-1800 spectroradiometer (Li-Cor Inc., Lincoln, NE, USA) (data not shown). Photosynthetic photon flux density (PPFD) was measured continuously at plant level using a radiation sensor (LI-190SB; Li Cor Inc.). Air temperature and relative humidity were measured by sensors at 20 s intervals (HMP35A; Vaisala Oy, Helsinki, Finland). Leaf temperature was first estimated by measuring soil temperature with a copper constantan thermocouple (0.4 mm diameter) from leaf initiation until the emerged leaf was visible on the rosette. Then, the copper constantan thermocouple was positioned touching the lower side of the lamina after leaf emergence. All measurements of temperature, PPFD and relative humidity were averaged and stored every 600 s by a datalogger (Campbell Scientific, Ltd, CR10 Wiring Panel; Shepshed, Leicestershire, England). Seeds were stored at 4 C before sowing and then five seeds were sown in 200 cylindrical pots (53 mm diameter and 88 mm height) containing a 50 : 50 mixture (v/v) of loamy soil and organic compost. To avoid population density effects, young seedlings were thinned to one plant per pot 10 d after plant germination. The substrate was maintained at 80% of field capacity (corresponding to a soil water content of 0.50 g g -1 of dry soil) by weighing the pots once a day and watering them with a modified one-tenth strength Hoagland s solution with additional micronutrients (Hoagland & Arnon 1950). Light in the growth chamber was provided by a bank of cool-white fluorescent tubes and sodium lamps for a photoperiod of 10 h. Air humidity was maintained at approximately 70%. The shade treatments were applied 8 d after sowing when the cotyledons were fully opened and the first two leaves were starting to emerge, before the initiation of leaf 6. The Table 1. Mean environmental conditions and genotypes studied during experiments 1, 2 and 3 under the light (L) and shade (S) treatments Mean air temperature ( C) Mean relative air humidity (%) Mean leaf temperature ( C) Mean PPFD (mmol m -2 s -1 ) Genotypes studied Exp. 1 L ,,, S Exp. 2 L ,, S Exp. 3 L ,, elo2, elo4 S

3 Kinematic analysis and leaf size control in Arabidopsis 1357 daily mean PPFD in the light treatment was 9.4, 8.5 and 8.4 mol m -2 d -1 and in the shade treatments it was 2.7, 2.2 and 2.5 mol m -2 d -1, respectively, in exp. 1, exp. 2 and exp. 3 (Table 1). Growth measurements Number of leaves initiated Five plants per genotype were harvested at intervals of 2 3 d. Plants were dissected in a drop of water using a microscope (Leica stereomicroscope, Wild F8Z; Wetzlar, Germany) at magnification 160. The number of leaves and leaf primordia visible on the apex were counted (leaves were visible when the areas were approximately mm 2 ). Individual leaf development Areas of leaves at position 6 on the rosette were measured at intervals of 2 3 d from initiation to the end of expansion of the leaf. From leaf initiation to leaf emergence, this was done by dissecting the apex of five plants in a drop of water under the microscope, the area of the excised leaf 6 was measured with image analysis software (Bioscan-Optimas V 4.10; Edmonds, WA, USA). After leaf emergence, the leaf area of six plants was measured with the aforementioned image analysis software on digital photographs until the end of leaf expansion. Epidermal cell area A transparent negative film of the adaxial epidermis was obtained after evaporation of a varnish spread on the upper surface of the fully expanded leaf. Films were place under a microscope (Leica, Leitz DM RB; Wetzlar, Germany) coupled to an imager analyser (Granier & Tardieu 1998a). Twenty-five epidermal cell areas were measured at four different places on each leaf, near the base, near the tip and one on each side of the leaf. Epidermal cell number was calculated from the mean cell area and the final leaf area of each leaf. Calculations of maximum leaf initiation rate, date of leaf 6 initiation, absolute and relative leaf expansion rate, final leaf area and duration of expansion Maximum leaf initiation rate (IR max ) was calculated as the maximum slope of the relationship between leaf number (N) and time (t) during the initiation of the first 20 leaves. Ir max = d(n)/dt (1) The date of leaf 6 initiation was calculated by linear extrapolation to leaf 6 of the curve relating number of leaves initiated to time. Absolute leaf expansion rate at time j (LER j ) was calculated from initiation to the end of expansion as the local slope (at time j) of the relationship between leaf area (A) and time (Granier & Tardieu 1998a): LER j = [d(a)/dt] j (2) A sigmoidal curve was fitted to the curve relating leaf expansion to time: y = A/[1 + exp -((X-X0)/B) ] (3) Final leaf area was calculated as the upper assymptote (A, the plateau) of the sigmoidal curve (Eqn 3) relating the increase in leaf area with time. Leaf expansion was considered to begin at the time at which the leaf was initiated and to end when it reached 95% of its final area as calculated from the sigmoidal curve (Eqn 3). The maximum absolute expansion rate (LER max ) was calculated as the point of inflection of the fitted sigmoidal curve by the equation (Torres & Frutos 1989): LER max = [A (1/B)]/4 (4) Leaf relative expansion rate at time j (RER j ) was calculated from initiation to the end of expansion as the local slope (at time j) of the relationship between the logarithm of leaf area (A) and time (see Granier & Tardieu 1998a): RER j = [d(ln A)/dt] j (5) Initial relative leaf expansion rate (RER i ) was calculated as the mean slope of the relationship between leaf area and time on a logarithmic scale during the period that this relationship is near-linear. Statistical analysis of data Each analysis was set with a significance level of P = 0.05 and all statistical analysis was done using the computer package SPSS 11.0 for Windows (SPSS Inc., Chicago, IL, USA). The differences in final leaf area, maximal absolute leaf expansion rate, duration of expansion, initial relative leaf expansion rate and epidermal cell number were compared using a two-way ANOVA where the assumptions of the ANOVA were met (General Linear Model with genotype and treatment as factors (also termed fixed effects), a Tukey post-hoc test was done on the genotype factor and subgroups are indicated by letters on the results tables). Median epidermal cell area was analysed using a Mann Whitney U-test. The effect of the shade treatments on initial relative expansion rate was tested using a paired samples t-test in which measurements from each experiment were compared separately. Leaf initiation rates were compared using a t-test of the linear regressions relating the number of leaves initiated to time. Correlations were tested using the Pearson correlation coefficient and the associated statistical test in the computer package used to evaluate whether the correlation was significant. The presence of clusters containing only measurements from one genotype, treatment or experiment were determined (using a hierarchical cluster analysis with the nearest neighbour clustering method) within the scatter diagrams from which correlations were drawn. If such a population of measurements was identified then the correlation was not considered to be robust over all internal and external factors.

4 1358 S. J. Cookson et al. RESULTS Plants were grown in rigorously controlled conditions a Light b Shade Plants were grown in two growth chambers, one with light and the other with shade on three separate occasions with different light treatments in each case (experiments 1, 2 and 3; Table 1). Temperature and air humidity were similar between the two growth chambers and between experiments (Table 1). Owing to the large number of plants necessary for an experiment (150 plants per genotype and treatment) and the constraints of the chamber sizes, all selected mutants could not be grown together in all the experiments. As a consequence, different groups of mutants were selected for each experiment (Table 1). In each experiment, for each mutant and both light and shade, the expansion of the 6th leaf was studied and unless otherwise stated all data pertain to the 6th leaf. A non-inductive photoperiod was selected to reduce the possible effects of altered timing of floral transition on leaf development. Each experiment was analysed independently because of the differences in light conditions. c e d f Leaf growth variables were affected by internal (leaf development mutants) and external (shade treatments) factors Final leaf area A wide range of final leaf sizes was produced by growing leaf development mutants and wild-type plants under light and shade treatments (Fig. 1). Mean final leaf area varied from 16 to 198 mm 2 depending on the genotype and the light treatment (Table 2). The plants produced significantly larger leaf areas than the Landsberg erecta wild type () (Table 2) and all the other genotypes (data not shown). The and elo2 plants produced significantly smaller leaf areas than (Table 2) and all the other genotypes (data not shown). There was no statistically significant difference in final leaf area between, elo4 and. In all genotypes, the shade treatments caused a significant reduction in final leaf area (Table 2). Duration and rates of leaf expansion Leaf size changes continuously during development. Typically leaf area expansion forms a sigmoidal-shaped curve when plotted on a linear scale (Fig. 2a & c). This suggests that leaf expansion is slow during the early stage of leaf development and that it then accelerates with time until finally slowing down towards the end of leaf expansion (Fig. 2a & c). As a consequence, plotting absolute leaf expansion rate (the area formed per unit of time) against time produces a bell-shaped curve (Fig. 2b & d), the maximum of which can be quantified (maximum absolute leaf expansion rate). In contrast, plotting leaf area data on a natural log scale reveals that the young leaves are in fact rapidly expanding elo2 elo4 g i k Figure 1. Photographs of wild-type and leaf development mutants grown in light and shade treatments. Plants are shown at the end of leaf 6 expansion. a and b, ; c and d, ; e and f, ; g and j, elo2; i and j, elo4; k and l,. a, c, e, g, i and k, plants grown under light intensity; b, d, f, h, j and l, plants grown under the shade treatment.,, and were grown during exp. 1, and elo2 and elo4 grown during exp.3. Scale bars = 1.5 cm. j h l

5 Kinematic analysis and leaf size control in Arabidopsis 1359 Table 2. Mean leaf 6 area, maximal absolute leaf expansion rate (LER max ) and duration of leaf expansion of wild type () and leaf development mutants (,, elo2, elo4 and ) grown under the light (L) and shade (S) treatments during experiments 1, 2 and 3 Exp. 1 Exp. 2 Exp. 3 Final area (mm 2 ) LER max (mm 2 d -1 ) Duration (d) Final area (mm 2 ) LER max (mm 2 d -1 ) Duration (d) Final area (mm 2 ) LER max (mm 2 d -1 ) Duration (d) L S 117* 13.2* 23.4* 71* 8.0* 24.6* 59* 8.0* 24.8* L S 147 * 16.9 * 24.7* 102 * 12.2 * 23.1 * L S 130* 12.4* 31.7 * 62* 5.2* 33.7 * elo2 L S 33 * 3.0 * 33.2 * elo4 L S 99* 7.4* 33.7 * L S 20 * 3.1 * 25.0 * 16 * 2.2 * 21.9 * Indicates a significant difference (P < 0.05) between the mutant and wild-type Ler. *Shade treatment resulted in a significant difference (P < 0.05). during the early stage of leaf development (Fig. 3a & c). This rapid relative leaf expansion is clearly viewed by plotting relative expansion rate (the increase in unit area formed per unit area and per unit of time) against time (Fig. 3b & d). Relative expansion rate was high at the beginning of leaf expansion and then declined continuously until the end of expansion. The rapid expansion of young leaves can be quantified by estimating the slope of the change in leaf area on the natural log scale against time when this relationship is near-linear (initial relative expansion rate). Three leaf growth variables were considered in our kinematic analysis: maximal absolute leaf expansion rate, initial relative leaf expansion rate and the duration of leaf expansion. Under light conditions, maximal absolute leaf expansion rate (LER max ) was significantly increased in and (a) Light (c) Shade Leaf 6 area (mm 2 ) Absolute expansion rate (mm 2 d -1 ) (b) Time after leaf 6 initiation (d) (d) Time after leaf 6 initiation (d) Figure 2. Changes with time of leaf area and absolute leaf expansion rate of plants grown under light (open symbols) or shade treatment (closed symbols) during exp. 1. (a) and (c), mean leaf 6 area plotted on a linear scale; (b) and (d), mean absolute leaf 6 expansion rate. Key to genotypes: circles; triangles pointing downwards; squares, diamonds. n = 6.

6 1360 S. J. Cookson et al. Light Shade e 5 (a) (c) Leaf 6 area (mm 2 ) e 4 e 3 e 2 e 1 Relative expansion (b) Time after leaf 6 initiation (d) (d) Time after leaf 6 initiation (d) Figure 3. Changes with time of leaf area plotted on a natural log scale and relative leaf expansion rate of plants grown under light (open symbols) or shade treatment (closed symbols) during exp. 1. (a) and (c), mean leaf 6 area plotted on a natural log scale; (b) and (d), mean relative leaf 6 expansion rate. Key to genotypes: circles; triangles pointing downwards; squares, diamonds. n = 6. decreased in the elo2 and mutants when compared with the (Table 2). However, there were no significant differences among, elo4 and. It was decreased by the shade treatments in all genotypes (Fig. 2b & d; Table 2). Initial relative expansion rate varied among the genotypes and was significantly reduced by the shade treatments (Fig. 3b & d; Table 3). Duration of leaf expansion also varied among the genotypes (Table 2). Under light conditions, the duration of leaf expansion was significantly increased in the elongator mutants when compared with. In contrast, the duration of leaf expansion was not consistently affected in or (Table 2). The duration of leaf expansion was increased by the shade treatments in all genotypes (Table 2). Leaf formation Maximum leaf initiation rate (calculated as the maximum slope of the relationship relating the number of leaves visible on the apex with time) was affected in most of the leaf development mutants and by the shade treatments (Table 3). Table 3. Initial relative leaf expansion rate and maximal leaf initiation rate of wild type () and leaf development mutants (, and ) grown under the light (L) and shade (S) treatments during experiments 1 and 2 Exp. 1 Exp. 2 Initial relative expansion Maximum leaf initiation rate (leaves d -1 ) Initial relative expansion Maximum leaf initiation rate (leaves d -1 ) L S 0.75* 1.070* 0.70* 0.564* L S 0.90* * 0.71* * L S 0.61* * L S 0.63* 0.879* 0.51* * Indicates a significant difference (P < 0.05) between the mutant and wild-type Ler. *Shade treatment resulted in a significant difference (P < 0.05).

7 Kinematic analysis and leaf size control in Arabidopsis 1361 Table 4. Leaf 6 median epidermal cell area and cell number of wild type () and leaf development mutants (,, elo2, elo4 and ) grown under the light (L) and shade (S) treatments during experiments 1, 2 and 3 Exp. 1 Exp. 2 Exp. 3 Median cell area (mm 2 ) Mean cell number ( 00) Median cell area (mm 2 ) Mean cell number ( 00) Median cell area (mm 2 ) Mean cell number ( 00) L S * 3795* 268* 1688* 362* L S 3222 * 394* 4143 * 255* L S 3337 * 311* * elo2 L S 1964* 198 * elo4 L S 2200* 445* L S * * Indicates a significant difference (P < 0.05) between the mutant and wild-type Ler. *Shade treatment resulted in a significant difference (P < 0.05). Epidermal cell size and cell number The areas of one hundred epidermal cells were measured for each fully expanded leaf and epidermal cell number was calculated from the final leaf area and average cell size. In light conditions, produced significantly larger epidermal cells and produced significantly smaller epidermal cell areas when compared with (Table 4). Generally, there was no consistent difference between the elongator mutants (, elo2 and elo4) and (Table 4). The shade treatments caused an increase in the median of the distribution of cell area in all genotypes although this difference was not always significant (Table 4). In light conditions, epidermal cell number was not significantly different from the wild type in, and elo4 leaves but was significantly decreased in and elo2 (Table 4). The shade treatments resulted in significant and dramatic reductions in epidermal cell number in all genotypes (Table 4). Relationships between leaf growth variables Relationships between the leaf growth variables were investigated by determining which variables were correlated to one another. The robustness of these correlations was tested by using a hierarchical cluster analysis to identify whether one genotype, treatment or experiment could be resolved as a separate cluster. If such a population of measurements was identified, then the correlation was not considered to be robust over all internal and external factors. Whenever correlations between non-destructive measurements (final leaf area, maximum absolute leaf expansion rate, duration of leaf expansion, epidermal cell area and number) were studied the data from individual leaves were analysed. However, when correlations involving destructive measurements (maximum leaf initiation rate and initial relative expansion rate) were analysed the mean values were used. Final leaf area was robustly correlated to maximum absolute leaf expansion rate and cell number but not to the initial relative leaf expansion rate, the duration of expansion or cell size The variability in maximal absolute leaf expansion rate was positively correlated to final leaf area (Fig. 4a). This correlation was unique and took into account variability of final leaf area due to internal and external factors. In contrast, the significant correlation between initial relative expansion rate and final leaf area was not considered robust because the measurement arising from the genotype could be resolved using a hierarchical cluster analysis (Fig. 4b). There was no correlation between the duration of leaf expansion and final leaf area (Fig. 4c). A positive correlation was found between final epidermal cell area and leaf size (Fig. 5a), although this was mainly due to the measurements arising from the genotype which formed a separate cluster. Thus this relationship was not considered robust across all the internal and external factors. However, the variability in final leaf area was robustly positively correlated to variability in final cell number (Fig. 5b). Early events of leaf development are correlated to each other A significant positive correlation was obtained for the relationship between maximum absolute expansion rate and initial relative expansion rate (Fig. 6a); however, this was not considered robust as the measurements arising from the population formed a separate cluster. Correlations were found between the events of leaf development occurring earlier than the point of the maximum absolute expan-

8 1362 S. J. Cookson et al. Maximum absolute expansion rate (mm 2 d -1 ) Initial relative expansion Duration of expansion (d) (a) (b) (c) Final leaf area (mm 2 ) Figure 4. Relationships between final leaf 6 area and leaf 6 expansion variables. (a), maximum absolute expansion rate; (b), initial relative expansion rate; (c), the duration of expansion. Where statistically significant correlations were found in the distributions the Pearson correlation coefficient (P) and significance level have been indicated on the graphs. Each point represents the measurements arising from one individual leaf for panels (a) and (c), whereas the mean values are shown in panel (b) (n = 6). Key to genotypes: circles; triangles pointing downwards; squares; elo2 triangles pointing upwards; elo4 hexagons; diamonds. Plants grown under light (open symbols) or shade conditions (closed symbols). sion rate (leaf initiation rate, initial relative expansion rate and leaf cell number); three examples are presented (Fig. 6b d). Initial relative leaf expansion rate is negatively correlated to the duration of leaf expansion A negative correlation was found between initial relative leaf expansion rate and duration of expansion 2D P = P = elo2 elo (Fig. 7) which was robust across all internal and external factors. DISCUSSION The role of kinematic analysis in understanding organ growth and its regulation Kinematic analyses have been widely used to study organs growing in one-dimension such as roots or monocotyledonous leaves (Ben Haj Salah & Tardieu 1995, Beemster & Baskin 1998). It has also been used to study the effects of environmental conditions on growth (Sacks, Silk & Burman 1997) and to compare different genotypes (Beemster & Baskin 2000). It has revealed a high degree of co-ordination between cell division and tissue expansion in many cases (Muller, Stosser & Tardieu 1998). However, such analyses do not quantify the duration of expansion although this variable could be strongly involved in the control of organ final size. The leaf growth analysis presented here is the first of its kind in Arabidopsis thaliana and is ideally suited to the study of both the effects of environmental conditions and Median epidermal cell area (µm 2 ) Epidermal cell number (a) (b) elo2 elo Final leaf area (mm 2 ) P = P = Figure 5. Relationships between final leaf 6 area and cellular variables. (a), median final epidermal cell size; (b), epidermal cell number. Statistically significant correlations are indicated on the graphs by the Pearson correlation coefficient (P) and significance level. Each point represents the measurements from one individual leaf. Key to genotypes: circles; triangles pointing downwards; squares; elo2 triangles pointing upwards; elo4 hexagons; diamonds. Plants grown under light (open symbols) or shade conditions (closed symbols).

9 Kinematic analysis and leaf size control in Arabidopsis 1363 Maximum absolute expansion rate (mm 2 d -1 ) Maximum initiation rate (leaves d -1 ) (a) (b) P = P = Initial relative expansion Initial relative expansion Maximum initiation rate (leaves d -1 ) (c) (d) P = P = P < Epidermal cell number Figure 6. Relationships between the various early variables of leaf 6 expansion. (a), initial relative expansion rate and maximal absolute expansion rate; (b), initial relative expansion rate and maximal initiation rate; (c), epidermal cell number and initial relative expansion rate; (d), epidermal cell number and maximal initiation rate. Where statistically significant correlations were found in the distributions the Pearson correlation coefficient (P) and significance level have been indicated on the graphs. Each data point represents the mean value obtained for each experiment (n = 6). Key to genotypes: circles; triangles pointing downwards; squares; diamonds. Plants grown under light (open symbols) or shade conditions (closed symbols). Duration of expansion (d) P = P < Initial relative expansion Figure 7. Relationship between initial relative expansion rate and the duration of leaf expansion. The statistically significant correlation is indicated on the graphs by the Pearson correlation coefficient (P) and significance level. Each data point represents the mean value obtained for each experiment (n = 6). Key to genotypes: circles; triangles pointing downwards; squares; diamonds. Plants grown under light (open symbols) or shade conditions (closed symbols). the phenotypes of leaf development mutants. Kinematic analysis can reveal invisible phenotypes which cannot be detected if only final size measurements are made. Here, kinematic analysis revealed that both internal and external factors affect the rates and durations of leaf expansion. In certain circumstances, an increase in the duration of expansion can compensate for a reduction in leaf expansion rate, for example in plants with mutations in components of the Elongator complex. Additionally, only kinematic analysis could reveal the phenotypes of transgenic plants overexpressing the D-type cyclin, CycD2, which do not show visible phenotypic differences at maturity but have accelerated rates and reduced durations of growth (Cockcroft et al. 2000). Evidence that final leaf area is determined early in leaf development when plants are grown in stable environmental conditions The maximal absolute leaf expansion rate is related to final leaf area with a common relationship accounting for differences due to internal (genetic) or external (environmental) factors thus suggesting that final leaf area is determined early in development. In all the studied leaves, the values

10 1364 S. J. Cookson et al. of maximal absolute leaf expansion rate were reached during the first two-thirds of total duration of leaf expansion. In our standardized and stable environmental conditions, the last third of leaf development was completely defined at the time at which the maximal absolute leaf expansion rate was reached. This would not have been established in fluctuating conditions as it is known that differences in temperature or soil water content can affect the expansion of dicotyledon leaves. A similar linear correlation between absolute leaf expansion rate and final leaf area had been reported previously in Poa species (Fiorani et al. 2000). In Aegilops species this relationship was related to processes that occur even earlier in leaf development: the size of the elongation zone, the size of the zone with dividing cells and to cell division rate (Bultynck et al. 2003). In agreement with most studies of plant organ development in different genotypes and environmental conditions, final organ size is related to final cell number. This has been shown in leaves of sunflower or xanthium grown in different light intensities (Wilson 1966; Dengler 1980; Granier & Tardieu 1999), in pea leaves subjected to soil water deficit (Granier et al. 2000a) and in bonsai plants (Korner, Pelaez Mendez-Riedl & John 1989). Epidermal cell number is fixed during the first two-thirds of leaf development (Granier & Tardieu 1998a) further suggesting that the last third of leaf development is determined early. The early processes of leaf development are correlated to one another The relationship between maximum leaf initiation rate and initial relative leaf expansion rate suggests that processes of leaf formation and early growth are related to each other or regulated by common or similar processes. There is also a positive statistically significant relationship between the epidermal cell number and initial relative leaf expansion rate suggesting a relationship between cell division and the expansion of young leaf tissue. Initial relative leaf expansion occurs when the leaf is very small and its growth is quasi-constant (exponential). During this same period, the relative increase in cell number is also maximal and quasiconstant (Granier & Tardieu 1998a; Granier et al. 2000a). Perhaps these parameters are related to one another or regulated in a similar fashion which would explain the correlation between initial relative leaf expansion rate and cell number. Relative tissue expansion rate and relative cell division rate of different parts of a leaf are similar to each other and to the kinetics of leaf growth as a whole, further suggesting that a measure of initial relative leaf expansion rate when the leaf is very small can be compared with the final cell number of a leaf (Maksymowych 1963; Granier & Tardieu 1998a). Cell division rate and relative tissue expansion rate have been shown to be similarly affected by temperature changes (Granier & Tardieu 1998b), by reduction in incident light (Granier & Tardieu 1999) and by water deficit (Granier, Inzé & Tardieu 2000b). Additionally, epidermal cell number was correlated to the maximal leaf initiation rate suggesting common or similar regulatory processes for the early phases of leaf formation and cell division. In agreement with this correlation, disruption of the ANTEGUMENTA (Mizukami & Fischer 2000) and STRUWWELPETER (Autran et al. 2002) genes reduces cell number per organ and the number of organs produced. Initial relative leaf expansion rate is negatively correlated to the duration of leaf expansion The robust negative correlation between initial relative expansion rate and the duration of leaf expansion has also been reported in other studies of different environmental conditions. For example, in sunflower leaves grown under different light treatments the peak relative expansion rate and the inverse of the period of leaf expansion were positively correlated (Rawson & Dunstone 1986). Additionally, in sunflower and Arabidopsis thaliana leaves, initial relative expansion rate and the reciprocal of duration of expansion were both positively correlated to temperature suggesting a negative relationship between relative expansion rate and duration (Wilson & Ludlow 1968; Granier & Tardieu 1998b; Granier et al. 2002). Here, this relationship is also observed in different genotypes with contrasted leaf development. Because this relationship is robust across internal and external factors we suggest that it is an intrinsic property of leaf development. A kinematic-based conceptual model of leaf development The presence of robust relationships between the leaf development variables discussed above has led us to propose a kinematic-based conceptual model of leaf development in which the processes early and late in leaf development are interconnected (Fig. 8). In this model, the early processes include initiation rate, epidermal cell number, and initial relative expansion rate, which are all significantly correlated to one another and presumably related to the processes of the regulation of cell division. The late process in the model is the duration of leaf expansion. These early and late processes are linked through the negative correlation between initial relative expansion rate and the duration of leaf expansion to regulate overall leaf development. Therefore, internal and external factors affecting the ability of the young tissue to expand (initial relative leaf expansion rate) would regulate overall leaf size at the organ scale (through the regulation of the duration of leaf expansion) and provide the means for compensation of disruptions in cell division/expansion. Despite the strong correlation between cell number and final leaf size, plants altered in cell cycle control often show phenotypes which considerably deviate from this relationship. Transgenic plants over-expressing inhibitors of cell division tend to produce leaves with a smaller number of cells in which there can be a considerable degree of final leaf size compensation by an increase in cell size (Hemerly et al. 1995; Wang et al. 2000; De Veylder et al. 2001). This

11 Kinematic analysis and leaf size control in Arabidopsis 1365 Regulation of the late processes of leaf formation Duration of Leaf Expansion Epidermal Cell Size Epidermal Cell Number Cell Expansion Leaf Size Regulation of the early processes of leaf formation Leaf Initiation Rate Cell Division RER i LER max Figure 8. A conceptual model of leaf size control. Solid arrows refer to the correlated kinematic variables shown here and dashed arrows indicate theoretical means by which these variables could be related to one another. phenotype could be explained in the model presented here by a decrease in initial relative leaf expansion rate (related to a decrease in cell number) and an associated increase in the duration of expansion. Conversely, mature leaves of plant over-expressing the D-type cyclin CycD3 have 18-fold more cells than the wild type and yet the final leaf area is half that of the wild type (Dewitte et al. 2003). This phenotype could be explained by an increase in initial relative leaf expansion rate (related to an increase in cell number) and an associated decrease in the duration of expansion. The analysis of the relationships between various growth variables in leaf development mutants under different environmental conditions presented here is the first attempt at testing the robustness of these relationships in terms of regulation by internal (genetic) and external (environmental) factors. The relationships between the various growth variables could suggest the presence of intrinsic regulatory factor(s) governing the kinematics of leaf development. ACKNOWLEDGMENTS This work was funded by the The European Community Human Potential Program (HPRN-CT ) as part of the DAGOLIGN Research Training Network. We thank Professor J. L. Micol for allowing access to the seeds of his mutant collection, J. J. Thioux for technical assistance, Drs D. Fleury, A. Christophe and B. Muller for fruitful comments on the manuscript, and A Christophe and D Combes for determining the light quality. REFERENCES Autran D., Jonak C., Belcram K., Beemster G.T.S., Kronenberger J., Grandjean O., Inzé D. & Traas J. (2002) Cell numbers and leaf development in Arabidopsis: a functional analysis of the STRUWWELPETER gene. EMBO Journal 21, Beemster G.T.S. & Baskin T.I. (1998) Analysis of cell division and elongation underlying the developmental acceleration of root growth in Arabidopsis thaliana. Plant Physiology 116, Beemster G.T.S. & Baskin T.I. (2000) STUNTED PLANT 1 mediates effects of cytokinin, but not of auxin, on cell division and expansion in the root of Arabidopsis. Plant Physiology 124, Ben Haj Salah H. & Tardieu F. (1995) Temperature affects expansion rate of maize leaves without a change in spatial distribution of cell length. Analysis of the coordination between cell division and cell expansion. Plant Physiology 109, Berná G., Robles P. & Micol J.L. (1999) A mutational analysis of leaf morphogenesis in Arabidopsis thaliana. Genetics 152, Bultynck L., Fiorani F., Van Volkenburgh E. & Lambers H. (2003) Epidermal cell division and cell elongation in two Aegilops species with contrasting leaf elongation rates. Functional Plant Biology 30, Cnops G., Jover-Gil S., Peters J., Neyt P., De Block S., Robles P., Ponce M.R., Gerats T., Micol J.L. & Van Lijsebettens M. (2004) The rotunda2 mutants identity a role for the LEUNIG gene in vegetative leaf morphogenesis. Journal of Experimental Botany 55, Cockcroft C.E., Den Boer B.G.W., Healy J.M.S. & Murray J.A.H. (2000) Cyclin D control of growth rate in plants. Nature 405, Dale J.E. (1992) How do leaves grow? Advances in cell and molecular biology are unravelling some of the mysteries of leaf development. Bioscience 42, De Veylder L., Beeckman T., Beemster G.T.S., Krols L., Terras F., Landrieu I., Van Der Schueren E., Maes S., Naudts M. & Inzé D. (2001) Functional analysis of cyclin-dependent kinase inhibitors of Arabidopsis. Plant Cell 13, Dengler N.G. (1980) Comparative histological basis of sun and shade leaf dimorphism in Helianthus annuus. Canadian Journal of Botany 58, Dewitte W., Riou-Khamlichi C., Scofield S., Healy J.M.S., Jacqmard A., Kilby N.J. & Murray J.A.H. (2003) Altered cell cycle distribution, hyperplasia, and inhibited differentiation in Arabidopsis caused by the D-type cyclin CYCD3. Plant Cell 15, Fiorani F., Beemster G.T.S., Bultynck L. & Lambers H. (2000) Can meristematic activity determine variation in leaf size and

12 1366 S. J. Cookson et al. elongation rate among four Poa species? A kinematic study. Plant Physiology 124, Fleming A.J. (2002) The mechanism of leaf morphogenesis. Planta 216, Granier C. & Tardieu F. (1998a) Spatial and temporal analyses of expansion and cell cycle in sunflower leaves. A common pattern of development for all zones of a leaf and different leaves of a plant. Plant Physiology 116, Granier C. & Tardieu F. (1998b) Is thermal time adequate for expressing the effects of temperature on sunflower leaf development? Plant, Cell and Environment 21, Granier C. & Tardieu F. (1999) Leaf expansion and cell division are affected by reducing absorbed light before but not after the decline in cell division rate in the sunflower leaf. Plant, Cell and Environment 22, Granier C., Inzé D. & Tardieu F. (2000b) Spatial distribution of cell division rate can be deduced from that of p34 cdc2 kinase activity in maize leaves grown at contrasting temperatures and soil water conditions. Plant Physiology 124, Granier C., Massonnet C., Turc O., Muller B., Chenu K. & Tardieu F. (2002) Individual leaf development in Arabidopsis thaliana: a stable thermal-time-based programme. Annals of Botany 89, Granier C., Turc O. & Tardieu F. (2000a) Co-ordination of cell division and tissue expansion in sunflower, tobacco and pea leaves. Dependence or independence of both processes? Journal of Plant Growth Regulation 19, Green P. (1976) Growth and cell pattern formation on an axis: critique of concepts, terminology and modes of study. Botanical Gazette 137, Haber A.H. & Foard D.E. (1963) Nonessentiality of concurrent cell divisions for degree of polarization of leaf growth. II. Evidence from untreated plants and from chemically induced changes of the degree of polarization. American Journal of Botany 50, Hemerly A., de Almeida Engler J., Bergounioux C., Van Montagu M., Engler G., Inzé D. & Ferreira P. (1995) Dominant negative mutants of the Cdc2 kinase uncouple cell division from iterative plant development. EMBO Journal 14, Hoagland D.R. & Arnon D.I. (1950) The water culture method for growing plants without soil. Californian Agricultural Experimental Station Circular 347, Korner C., Pelaez Mendez-Riedl S. & John P.C.S. (1989) Why are bonsai plants small? A consideration of cell size. Australian Journal of Plant Physiology 16, Maksymowych R. (1963) Cell division and cell elongation in leaf development of Xanthium pennsylvanicum. American Journal of Botany 50, Mizukami T. & Fischer R.L. (2000) Plant organ size control: organogenesis. Proceedings of the National Academy of Sciences of the USA 97, Muller B., Stosser M. & Tardieu F. (1998) Spatial distributions of tissue expansion and cell division rates are related to irradiance and to sugar content in the growing zone of maize roots. Plant, Cell and Environment 21, Nelissen H., Clarke J.H., De Block M., De Block S., Vanderhaeghen R., Zielinski R.E., Dyer T., Lust S., Inzé D. & Van Lijsebettens M. (2003) DRL1, a homolog of the yeast TOT4/KTI12 protein, has a function in meristem activity and organ growth in plants. Plant Cell 15, Nelissen H., Fleury D., Bruno L., Robles P., De Veylder L., Traas J., Micol J.L., Van Montagu M., Inzé D. & Van Lijsebettens M. (2005) The elongata mutants identify the Elongator complex in plants with a function in cell proliferation during organ growth. Proceedings of the National Academy of Sciences of the USA 102, Peters J.L., Cnops G., Neyt P., Zethof J., Cornelis K., Van Lijsebettens M. & Gerats T. (2004) An AFLP-based genomewide mapping strategy. Theoretical and Applied Genetics 108, Powell M.J., Davies M.S. & Francis D. (1986) The influence of zinc on the cell cycle in the root meristem of a zinc-tolerant and nontolerant cultivar of Festuca rubra L. New Phytologist 102, Rawson H.M. & Dunstone R.L. (1986) Simple relationships describing the responses of leaf growth to temperature and radiation in sunflower. Australian Journal of Plant Physiology 13, Robles P. & Micol J.L. (2002) Genome-wide linkage analysis of Arabidopsis genes required for leaf development. Molecular Genetics and Genomics 226, Sacks M.M., Silk W.K. & Burman P. (1997) Effect of water stress on cortical cell division rates within the apical meristem of primary roots of maize. Plant Physiology 114, Schuppler U., He P.H., John P.C.L. & Munns R. (1998) Effect of water stress on cell division and cell-division-cycle 2-like cellcycle kinase activity in wheat leaves. Plant Physiology 117, Tardieu F. & Granier C. (2000) Quantitative analysis of cell division in leaves: methods, developmental patterns and effects of environmental conditions. Plant Molecular Biology 43, Torres M. & Frutos G. (1989) Analysis of germination curves of aged fennel seeds by mathematical models. Environmental and Experimental Botany 29, Tsuge T., Tsukaya H. & Uchimiya H. (1996) Two independent and polarized processes of cell elongation regulate leaf blade expansion in Arabidopsis thaliana (L.) Heynh. Development 122, Tsukaya H. (2002) Interpretation of mutants in leaf morphology: genetic evidence for a compensatory system in leaf morphogenesis that provides a new link between cell and organismal theories. International Review of Cytology 217, Tsukaya H. (2003) Organ shape and size: a lesson from studies of leaf morphogenesis. Current Opinion in Plant Biology 6, Wang H., Zhou Y., Gilmer S., Whitwell S. & Fowke C.L. (2000) Expression of the plant cyclin-dependent kinase inhibitor ICK1 affects cell division, plant growth and morphology. Plant Journal 24, Wilson G.L. (1966) Studies on the expansion of the leaf surface V. Cell division and expansion in a developing leaf as influenced by light and upper leaves. Journal of Experimental Botany 17, Wilson G.L. & Ludlow M.M. (1968) Bean leaf expansion in relation to temperature. Journal of Experimental Botany 19, Received 6 December 2004; received in revised form 17 March 2005; accepted for publication 18 April 2005