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. 2011 May 17;108(1):159–168. doi: 10.1093/aob/mcr091

The ERECTA gene controls spatial and temporal patterns of epidermal cell number and size in successive developing leaves of Arabidopsis thaliana

Sébastien Tisné 1,2,†, François Barbier 1,3,†, Christine Granier 1,*
PMCID: PMC3119605  PMID: 21586531

Abstract

Background and Aims

ERECTA has been identified as a pleiotropic regulator of developmental and physiological processes in Arabidopsis thaliana. Previous work demonstrated a role for ERECTA in the control of compensation between epidermal cell expansion and division in leaves

Methods

In this work, spatial and temporal analyses of epidermal cell division and expansion were performed on successive developing vegetative leaves of Arabidopsis thaliana in both ERECTA and erecta lines, LER and Ler, respectively, to understand how the ERECTA gene regulates compensation between these two processes.

Key Results

The loss of ERECTA function leads to a low cell expansion rate in all zones of a leaf and in all successive leaves of a plant. This low cell expansion rate is counterbalanced by an increase in the duration of cell division. As a consequence, the ERECTA mutation eliminates the tip to base cellular gradient generally observed in the leaf epidermis and also flattens the heteroblastic changes in epidermal cell area and number within a rosette. Ablation of floral buds eliminates the heteroblastic changes in cellular patterns in an ERECTA-dependent manner.

Conclusions

The results provide a detailed description of changes in leaf growth dynamics and cellular variables in both LER and Ler. Altogether they suggest that ERECTA influences leaf cellular development in relation to whole plant ontogeny.

Keywords: Arabidopsis, leaf growth, cell division, cell expansion, heteroblasty, epidermis, ERECTA

INTRODUCTION

One of the difficulties in the elucidation of gene function is that a single gene can influence multiple phenotypic traits. One can explain this phenomenon, called pleiotropy, when a gene codes for a product that is used by various cell types, has a signalling function on various targets or affects a process which affects others in a cascade. One example of pleiotropy in plant literature is the numerous functions that have been reported for the ERECTA gene (for a review, see van Zanten et al., 2009). This gene encodes a signalling molecule, from the family of the cell surface receptor kinase with leucine-rich repeat extracellular domains (the LRR-LRK protein family; Torii et al., 1996). It is expressed in the shoot apical meristem, in young and growing aerial parts of the plants including growing leaves, and developing floral organs, but not in mature organs or in roots (Yokoyama et al., 1998). Its expression in the shoot apical meristem is weak early in plant development and increases at the transition from the vegetative to the reproductive growth phase (Yokoyama et al., 1998). In Arabidopsis thaliana, erecta mutants have characteristic compact rosettes and inflorescences (Rédei, 1992). This modified morphology is the consequence of rounder cotyledons and leaves, and shorter petioles, flowers, pedicels and siliques (Shpak et al., 2003, 2004).

ERECTA expression has been analysed in many plant organs, but detailed morphological and cellular analyses of erecta mutants have mainly been performed on reproductive organs (Shpak et al., 2003, 2004; Woodward et al., 2005; Pillitteri et al., 2007). Pedicels and petals have reduced cell number, but these cells are more expanded than the wild type (Shpak et al., 2003, 2004). In leaves, erecta mutation affects stomatal patterning (Shpak et al., 2005) and stomatal density (Masle et al., 2005). It also causes an increase in epidermal cell number but a decrease in cell size resulting in final leaf area comparable with the wild type (Tisné et al., 2008). ERECTA has also been shown to control physiological processes such as the Rubisco carboxylation rate, electron transport capacity, whole plant transpiration and water use efficiency (Masle et al., 2005). It is also involved in plant responses to biotic and abiotic environments as shown for heat stresses (Qi et al., 2004), drought (Masle et al., 2005) and infection by bacteria (Godiard et al., 2003).

One striking feature of the erecta mutation is that it leads to opposite phenotypes at the cellular level in vegetative and reproductive organs. Cell number is decreased in reproductive organs, whereas it is increased in vegetative leaves. Cell area is increased in reproductive organs, whereas it is decreased in vegetative leaves. Combining quantitative genetics and statistical modelling in a population of recombinant inbred lines (RILs) derived from a cross between Landsberg erecta (Ler) and Antwerp-1 (An-1), it was shown that the relative contribution of epidermal cell number and size in the epidermis of mature leaves was functionally related to processes associated with reproductive transition (Tisné et al., 2008). Late flowering RILs had large epidermal cell area but low epidermal cell number in their rosette leaves, whereas early flowering lines had the opposite phenotype. This was also consistent with the effect of daylength on epidermal cell number and size in Col-0 leaves (Cookson et al., 2007).

The aim of the work performed here was to better understand the role of ERECTA in the balance between epidermal cell division and expansion in leaves. For that purpose, spatial and temporal analyses of epidermal cell division and expansion were performed on successive developing rosette leaves of A. thaliana in both ERECTA and erecta, LER and Ler lines. The effect of reproductive transition on these cellular patterns was analysed by comparing plants grown in short and long days and by removing floral buds to mimic a delay in reproductive transition (Cookson et al., 2007). All results presented here indicate that ERECTA is an important mediator of the relationships between whole plant ontogeny and cellular patterns in leaf epidermis.

MATERIALS AND METHODS

Plant material and growth conditions

The 120 RILs of Arabidopsis thaliana previously generated from a cross between Ler and An-1 (El-Lithy et al., 2006) were grown in two independent experiments at two different photoperiods (experiments 1 and 2, respectively, under a 12 and a 16 h daylength) in the PHENOPSIS automated phenotyping platform (Granier et al., 2006). As the Ler parental line carries a spontaneous erecta mutation that segregates in the mapping population, the whole population was divided into two subpopulations depending on the allele at the ER marker: RILs with Ler alleles at ER (58 RILs) and RILs with An-1 alleles at the same marker (57 RILs).

The parental Ler line and the same line complemented by the Columbia wild allele of ERECTA (LER) were grown in three experiments, one in the PHENOPSIS platform under a 12 h daylength (experiment 3), and the two others in a growth chamber under a 16 and a 12 h daylength, respectively (experiments 4 and 5).

In all five experiments, all micro-meteorological conditions were controlled to remain constant during the whole growing period and homogeneous within the growth chambers (Granier et al., 2006). Light was provided by HQi lamps with additional cool white fluorescent tubes. It was measured continuously at the plant level, using a photosynthetic photon flux density (PPFD) sensor (LI-190SB, LICOR, Lincoln, NE, USA). Daily incident PPFD was calculated by multiplying daylength and mean instantaneous incident PPFD. Air temperature and relative humidity were measured every 20 s (HMP35A Vaisala Oy, Helsinki, Finland). All measurements of temperature, PPFD and relative humidity were averaged and stored every 600 s in a datalogger (Campbell Scientific, LTD-CR10 Wiring Panel, Shepshed, Leicestershire, UK). Mean air vapour pressure deficit (VPD) was calculated during the light period. For each experiment, mean micro-meteorological conditions are presented in Table 1.

Table 1.

Growth conditions in the five experiments

Exp. Soil water content (g H2O g−1 dry soil) Daylength (h) Temperature (°C) Daily incident light (mol m−2 d−1) Air vapour pressure deficit (kPa)
1 0·40 12 20·54 10·8 0·80
2 0·40 16 19·14 8·31 1·13
3 0·40 12 20·92 6·07 0·65
4 0·40 16 19·64 10·24 0·47
5 0·40 12 20·83 7·38 0·59
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For each experiment, mean micrometeorological conditions were calculated from germination to the end of rosette leaf development.

Seeds were sown in 200 mL conical pots (9 cm height and 4·5 cm diameter) filled with a mixture (1:1, v/v) of a loamy soil and organic compost. Soil water content was determined before planting and, subsequent changes in pot weight were attributed to a change in soil water humidity. Soil water content was adjusted daily to 0·40 g H2O g−1 dry soil from germination until the end of rosette leaf development with a modified one-tenth-strength Hoagland solution (Hoagland and Arnon, 1950). This was done automatically with the automaton in the PHENOPSIS platform during experiments 1, 2 and 3 and manually during experiments 4 and 5. This value of soil water content corresponds to optimal soil humidity for the soil used in this study (Granier et al., 2006).

During experiment 5, when plants reached stage 5·01 (flower bud emergence; Boyes et al., 2001), the flower bud was removed from half of the plants being cultured.

Measurements of leaf growth variables

Digital pictures of all individual pots were taken automatically on a daily basis during the three PHENOPSIS experiments (experiments 1, 2 and 3) or each 2 or 3 d for the two growth chamber experiments (experiments 4 and 5). Three times a week, stages of leaf development were scored on these pictures, for each individual plant as described in Boyes et al. (2001).

Final rosette area, number of rosette leaves, individual leaf area, epidermal cell area and epidermal cell number

When plants reached stage 6·00 (first flower open; Boyes et al., 2001), the rosettes were cut, leaves were detached, laminas were separated from the petiole, and stuck with double-sided adhesive on a sheet of paper in the order of their emergence on the rosette. The leaves were then scanned for further measurements. In addition, a transparent negative film of the adaxial epidermis of leaf 6 (for experiments 1, 2 and 3) or all leaves (for experiments 4 and 5) was obtained after evaporation of a varnish spread on the leaf surface. Individual leaf area (LAi, cm2) was measured on the scans with image analysis software (Bioscan-Optimas V 4·10, Edmonds, WA, USA), and rosette area (RA, cm2) was determined as the sum of the individual leaf blade areas. Rosette leaf number (LN, leaves) was estimated by counting the number of leaves formed after the two cotyledons. Epidermal cell area (CA, μm2) was estimated on epidermal imprints placed under a microscope (Leica, Leitz DM RB, Wetzlar, Germany) by measuring 25 pavement cell areas at four different zones on each leaf, near the base, near the tip and one on each side of the leaf, with the aforementioned image analysis software. Mean epidermal cell area is the mean of these 100 pavement cells. Epidermal cell number (CN) was calculated from the ratio between leaf area and mean epidermal cell area.

Temporal development of leaf area, epidermal cell area and epidermal cell number for each successive rosette leaves

During experiment 4, five plants of each of the two lines, Ler and LER, were harvested each 2–3 d from plant germination until the end of rosette development (at stage 6·00). At each date, individual leaves were scanned and epidermal imprints were done for each rosette leaves, as described before. Leaf area, epidermal cell area and epidermal cell number were measured on the scans and imprints, as described before. Sigmoidal curves were fitted to the curves relating these three variables (y) to time (X) using the R software (R development Core Team, 2007):

graphic file with name mcr091eqn1.jpg (1)

where B represents the slope or curvature of the sigmoid curve and X0 represents the inflection point, i.e. the time when the variable reaches half of its final value. The final value of each variable was calculated as the upper asymptote (A, the plateau) of the sigmoidal curve (1).

The maximum absolute expansion rate of each process (LERmax), namely leaf expansion, cell expansion and cell division, was calculated as the point of inflection of the fitted sigmoidal curve by the equation (adapted from Torres and Frutos, 1989):

graphic file with name mcr091eqn2.jpg (2)

The duration of each process was calculated as the time elapsed between the date at which the leaf reached 0·05 cm2 (x0·05) and the date when the variable reached 95 % of its final value as calculated from the sigmoidal curve (1).

graphic file with name mcr091eqn3.jpg (3)

Spatial analysis of epidermal cell density during leaf 6 development

During experiment 4, epidermal imprints of leaf 6 were used to analyse the spatial distribution of epidermal cell density in the leaf and its variation over time. At each date, epidermal cells were counted in four zones of the leaf, one at the tip, one at the base and two intermediary zones.

RESULTS

Effects of the erecta mutation on leaf area, epidermal cell area and epidermal cell number in an individual leaf of different lines grown at two daylengths

To analyse the effect of erecta mutation in the RIL population derived from the cross between Ler and An-1, the whole population was subdivided into two subpopulations: one including all RILs with Ler alleles at ER and the other RILs with An-1 alleles at the same marker. Mean final leaf 6 area was slightly lower in RILs with Ler alleles at ER than those with An-1 alleles at the same marker (Fig. 1A). In contrast to the slight change in leaf area, epidermal cell area was halved and epidermal cell number was doubled in the subpopulation of RILs with Ler alleles at ER compared with those with An-1 alleles at the same marker (Fig. 1C, E). Results were consistent at both daylengths (Fig. 1A, C, E).

Fig. 1.

Fig. 1.

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Leaf 6 area (A, B), epidermal cell area in leaf 6 (C, D) and epidermal cell number in leaf 6 (E, F) measured in lines with or without the erecta mutation (white and black bars, respectively). Plants were grown under a 12 or 16 h daylength, as indicated. Data shown in (A), (C) and (E) were measured on RILs from the Ler × An-1 population: 57 RILs with An-1 and 58 with Ler alleles at the ER marker, respectively (n = 4). Data shown in (B), (D) and (F) were measured on LER and Ler (n = 5). Asterisks indicate significant difference between the two sets of RILs (A, C, E) or the two lines (B, D, F) with *P < 0·05, **P < 0·01 and ***P < 0·001, respectively. For each variable, the mean and s.e. are presented.

Results obtained on the two subpopulations of RILs were also consistent with results obtained when the Ler accession was compared with the same line complemented with the Columbia allele of the ERECTA gene (LER) under both short and long days. The erecta mutation caused a slight reduction in final leaf 6 area but the difference was not significant (Fig. 1B). Epidermal cell area in leaf 6 was halved in Ler when compared with LER, but this reduction was totally compensated for by a strong increase in epidermal cell number (Fig. 1D, F).

Effects of the erecta mutation on the dynamics of leaf expansion, epidermal cell expansion and epidermal cell division in an individual rosette leaf

Similar final leaf 6 area between LER and Ler was reached by common dynamics patterns as shown by the non-significant differences in rate and duration of expansion (Fig. 2A). Dynamics of epidermal cell area and number revealed that the lower epidermal cell area observed in Ler lines compared with LER lines resulted from a lower cell expansion rate (Fig. 2B), whereas the higher epidermal cell number was due to an increase in the duration of the cell division (Fig. 2C). Neither duration of epidermal cell expansion nor maximal cell division rate differed between the two lines (Fig. 2B, C).

Fig. 2.

Fig. 2.

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Changes over time of leaf 6 area (A), epidermal cell area (B) and epidermal cell number (C) in LER and Ler, as indicated. A logistic function was fitted to the data and sigmoid curves are presented for both LER and Ler. Plants were grown under a 16-h daylength.

Effect of the erecta mutation on the tip to base gradient in epidermal cell density in an individual rosette leaf

The cellular dynamics analyses presented in Fig. 2 were performed by considering a mean cell in the middle of the leaf. However, it is known that cell division and expansion are not uniformly distributed within the leaf, and both exhibit a strong tip to base gradient during leaf development (Granier and Tardieu, 1998). To analyse further the effect of the erecta mutation on the spatial distributions of cell division and expansion, changes in epidermal cell density over time were measured in four zones of leaf 6 of Ler and LER plants grown in long days (Fig. 3).

Fig. 3.

Fig. 3.

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Spatial distribution of epidermal cell density in leaf 6 of LER (left) and Ler (right) at different dates during their development. Epidermal cell density was measured at four locations in the leaf from the base to the tip. For each date and in each zone, mean values of epidermal cell density are expressed as a percentage of the mean value measured at the tip of the leaf for the same date. For both lines, leaf 6 emerged 9 d after stage 1·02 and stopped growing 20 d after stage 1·02. Plants were grown under a 16-h daylength.

Nine days after stage 1·02, just after leaf 6 emergence, epidermal cell density was homogeneous in the whole leaf from tip to base as it did not differ significantly between the different zones of the leaf both in LER and Ler (Fig. 3). Then, 2 d after, a gradient in epidermal cell density set up from the tip to the base in the leaf of LER with higher cell density at the base of the leaf, indicating that cell division was still high at the base whereas cell expansion was predominant at the tip. This gradient was still visible 17 d after stage 1·02, i.e. 8 d after leaf 6 emergence (Fig. 3). It disappeared at the end of leaf expansion, when both cell division and expansion stopped in the entire leaf (Fig. 3). In contrast, epidermal cell density remained homogeneous in the whole leaf during the whole growing period in Ler, indicating that both cell division and cell expansion coexisted in the entire leaf during its whole expansion (Fig. 3).

Effects of the erecta mutation on the heteroblastic-associated changes in the dynamics of leaf expansion, epidermal cell expansion and division for plants grown in long days

Both Ler and LER lines had eight rosette leaves at flowering when grown in long days (Fig. 4A). In both lines, final leaf area increased with leaf position on the rosette (Fig. 4A). This was due to an increase in leaf expansion rate without a clear tendency of changes in duration of expansion between successive leaves (Fig. 4B, C). In agreement with results presented for leaf 6 (Fig. 2A), final individual leaf area was reduced in Ler lines compared with LER lines for all successive leaves, but this was not significant and corresponded to a non-significant decrease in leaf expansion rate for each leaf (Fig. 4B).

Fig. 4.

Fig. 4.

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Leaf area (A), maximal expansion rate (B) and duration of leaf expansion (C) at each leaf position for LER and Ler, as indicated. Plants were grown under a 16-h daylength.

In both lines, final epidermal cell area decreased gradually with leaf position on the rosette (Fig. 5A). For all eight rosette leaves, epidermal cell area was lower in Ler than in LER (Fig. 5A). In LER, the maximal cell expansion rate decreased drastically from one leaf to the next successive leaf after leaf 3 (Fig. 5B). However, this was not the case in Ler where the maximal cell expansion rate was low and at similar values in all successive leaves (Fig. 5B). As a consequence, the maximal cell expansion rate was reduced in all successive leaves of Ler compared with LER, but it was more reduced for the first leaves than the last leaves (Fig. 5B). The duration of cell expansion increased slightly with leaf position in both Ler and LER plants (Fig. 5C). It was slightly increased or decreased by the erecta mutation depending on the position of the leaf, without a clear tendency (Fig. 5C).

Fig. 5.

Fig. 5.

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Epidermal cell area (A), maximal cell expansion rate (B) and duration of cell expansion (C) at each leaf position for LER and Ler, as indicated. Plants were grown under a 16-h daylength.

In both lines, the final number of epidermal cells increased considerably from one leaf to the next successive leaf (Fig. 6A). It did not differ significantly between the lines for the first two leaves but then it was systematically higher for Ler than for LER (Fig. 6A). The maximal cell division rate increased with increasing leaf position on the rosette in both Ler and LER (Fig. 6B). It was slightly increased or decreased by the erecta mutation depending on the position of the leaf, without a clear tendency (Fig. 6B). The duration of cell division did not vary with a clear tendency between successive leaves of the rosette in both lines but it was significantly increased in all successive leaves of Ler compared with LER (Fig. 6C).

Fig. 6.

Fig. 6.

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Epidermal cell number (A), maximal cell division rate (B) and duration of cell division (C) at each leaf position for LER and Ler, as indicated. Plants were grown under a 16-h daylength.

In summary, the erecta mutation caused a decrease in cell expansion rate balanced by an increase in duration of cell division for all successive rosette leaves and it flattened the gradient in cell expansion rate observed between successive leaves of a plant.

Effects of the erecta mutation on the heteroblastic-associated changes in leaf area, epidermal cell area and epidermal cell number for plants grown in short days

Final leaf area, epidermal cell area and epidermal cell number were measured on successive leaves of both Ler and LER plants grown under a 12 h daylength. Both lines had 15 leaves formed on the rosette at flowering (Fig. 7A). Leaf area increased with increasing leaf position without a significant difference between the two lines (Fig. 7A).

Fig. 7.

Fig. 7.

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Leaf area (A), epidermal cell area (B) and epidermal cell number (C) at each leaf position for LER and Ler; at bolting, half of the plants was subjected to successive flower bud ablations whereas the other half were left intact, as indicated. Plants were grown under a 12-h daylength. For each variable, the mean and s.e. are presented.

In LER, epidermal cell area was around 2800 µm2 in the first five leaves, whereas in Ler it was around 1700 µm2 for the same leaves (Fig. 7B). In both lines, epidermal cell area declined afterwards with increasing leaf position (Fig. 7B). The decrease in epidermal cell area from one leaf to another was steeper in LER than in Ler and, finally, epidermal cell area was similar in the last leaves of the two lines (Fig. 7B).

In both lines, epidermal cell number increased in leaves with increasing leaf position (Fig. 7C). At each position, it was higher in Ler than LER, but the increase in cell number was more pronounced in the last leaves of LER plants. Finally, epidermal cell number was similar in the last leaves of the two lines (Fig. 7B)

In conclusion, heteroblastic changes resulted in an erecta-like cellular phenotype, i.e. high number of cells with small areas, in the epidermis of the last leaves of LER rosettes.

Interaction between the effects of the erecta mutation and flower bud removal

Final leaf area, epidermal cell area and epidermal cell number were measured on successive leaves of both Ler and LER plants grown under a 12 h daylength without floral buds. Floral bud ablation did not cause any changes in final individual leaf area of the successive leaves formed on the rosette, either in LER or in Ler (Fig. 7A). It did not affect the epidermal cell number and area of successive leaves in Ler (Fig. 7B, C). In contrast, it affected both epidermal cell area and number in the last leaves of the rosette in LER. (Fig. 7B, C). Floral bud ablation in LER eliminated the decrease in epidermal cell area observed from leaf 10 to the next successive five leaves. It also flattened the increase in epidermal cell number for the last four leaves on the rosette (Fig. 7B, C).

In summary, the erecta-like cellular phenotype in the epidermis of the last leaves of LER (i.e. a high number of cells with small areas) could be eliminated by removing floral buds. In addition, Ler plants with or without floral buds did not differ in terms of epidermal cell area and number, indicating that ERECTA may have a role in cellular patterns in vegetative leaves in relation to the whole plant phenology.

DISCUSSION

Dynamics of cell expansion and division are affected in Ler leaves and lead to a total ‘compensation’ phenotype, opposite to the usually described ‘compensation syndrome’

Leaf expansion is the result of the production of a number of cells which then expand in particular directions to lead to a genetically and environmentally determined organ size and shape. This suggests that leaf development is controlled at the cellular scale and, consequently, suggests that modifications of either cell division or cell expansion would cause changes in leaf development. However, results of cellular analyses of mutants affected in positive regulators of cell cycle were contrary to this theoretical framework (Hemerly et al., 1995; Beemster et al., 2003; Horiguchi et al., 2005). Many mutants with a reduced cell number in leaves actually have larger cells (Hemerly et al., 1995; Mizukami and Fischer, 2000; Autran et al., 2002; Ferjani et al., 2007). This phenomenon was called the compensation syndrome (Tsukaya, 2003, 2006). In a few studies, these behaviours were used to illustrate that organ growth was controlled at the organ and not at the cellular scale because cell division could be uncoupled from organ expansion (Hemerly et al., 1995). However, the compensation theory is not sufficient to explain all data. First, because in most of these lines, the compensation was not total and leaves with a smaller number of cells were also slightly smaller even if their cell size was increased (Ferjani et al., 2007). Secondly, because a few mutants with fewer cells and unchanged cell size were also identified (Horiguchi et al., 2006a, b). Incomplete compensation between a reduced number of cells and larger cells in leaves has also been reported in response to environmental changes such as soil water deficit, reduced light or a decrease in daylength (Aguirrezabal et al., 2006; Cookson and Granier, 2006; Cookson et al., 2007).

In our study, the compensation between cell number and size reported for Ler when compared with its wild type is opposite to the compensation syndrome phenotype. The erecta mutation confers smaller cells with more cells per leaf. Such a phenotype is, to some extent, similar to the phenotype reported for a new class of mutants called ‘the more and smaller cells’ mutants with increased cell number and decreased cell size in leaves (Usami et al., 2009). However, Ler differs from this class of mutants as compensation is total, i.e., the increased cell number does not lead to an increase in leaf area. The unchanged leaf area in Ler could have masked differences in dynamics of whole leaf expansion. For example, unchanged leaf area in An-1, one of the A. thaliana accessions, subjected to water deficit masked a decrease in leaf expansion rate compensated for by an increase in the duration of expansion (Aguirrezabal et al., 2006). However, this was not the case; final leaf size and its underlying dynamics components, leaf expansion rate and whole duration of leaf expansion were unchanged. In contrast, the dynamics of cellular processes were affected as the cell expansion rate was decreased in Ler whereas cell division occurred later during leaf development. The duration of cell division was so increased that epidermal cell division features were observed for pavement cells until the end of leaf expansion, which is not usual in broad-leaved plants in which cell division occurs generally during the first two-thirds of the leaf expansion period (Granier and Tardieu, 1998; Beemster et al., 2005). This is consistent with observations made on the formation of stomatal complexes in the epidermis of erecta mutants (Shpak et al., 2005). Stomatal precursors were observed until the end of epidermis development (Shpak et al., 2005), suggesting that the lengthening of the cell division period was not specific to the pavement cells observed in our study but a general behaviour in the epidermis.

The erecta mutation eliminates the tip to base gradient in epidermal cell density within a rosette leaf

In broad-leaved plants, temporal and spatial gradients of cell division and cell expansion within the leaf epidermis lead to a particular cellular pattern in the developing leaf. During the first phase, from leaf initiation on the apical bud until leaf emergence, epidermal cells divide and enlarge at similar rates in the entire lamina, and epidermal cell density is then uniform within the leaf. Then, during a second phase, epidermal cell division stops gradually from the tip to the base of the lamina and this coincides temporally and spatially with a rapid increase in cell size. The boundary where the switch from proliferation to expansion occurs has been called the cell cycle arrest front and is promoted by a diffusive signal produced through the action of KLUH encoding CYP78A5 (Donnelly et al., 1999; Kazama et al., 2010). Finally, during a third phase, epidermal cell expansion stops following the same spatial gradient from the tip to the base of the leaf and, at the end of leaf expansion, epidermal cell density is again uniform within the whole lamina (Granier and Tardieu, 1998; Donnelly et al., 1999). As a consequence, the distribution of epidermal cell density is not homogenous within the whole leaf as cells with large areas at the tip of the leaf coexist with smaller ones at the base during a part of leaf development, from leaf emergence until cessation of leaf expansion (Granier and Tardieu, 1998; Donnelly et al., 1999). In our study, a gradient in epidermal cell density is reported within the lamina during a large part of leaf development in LER. In contrast, the spatial gradient in epidermal cell density never occurs in Ler, suggesting that cell expansion and division are coordinated in all zones of the leaf during whole leaf expansion and that there is no progression of the cell cycle arrest front within leaves of this mutant (Kazama et al., 2010).

The erecta mutation eliminates the heteroblastic changes in epidermal cell area between successive rosette leaves

In addition to gradients observed within the leaf, there are also gradients within the plant, from one leaf to the next successive leaf. This pattern, commonly called heteroblasty, includes gradients in leaf shape, size and cell densities (Allsopp, 1967). In most species, final leaf area generally increases with leaf rank and then decreases distally, prior to transition to flowering, resulting in a bell-shaped curve when leaf area is plotted against phytomer position (Kobayashi, 1975; Dosio et al., 2003; Cookson et al., 2007; Gonzalez et al., 2010). This pattern can be altered depending on the genotype or the growing condition. For example, final area increased from one leaf to the successive leaf until the last leaf formed in A. thaliana plants grown in long days, as also reported here for Ler and LER (e.g. Cookson et al., 2007). The steep increase in final leaf area between successive leaves coincides with a steep increase in epidermal cell number (Cookson et al., 2007; Usami et al., 2009). In contrast, epidermal cell area is fairly uniform between leaves or it decreases gradually with increasing phytomer position and this is more obvious when plants are grown in short days, because they have an increased number of leaves (Cookson et al., 2007; Usami et al., 2009). Therefore, the last leaves formed on the rosette have many more epidermal cells but of smaller size when compared with the first leaves.

Epidermal cell number increased more rapidly from one leaf to another in Ler than in LER, and epidermal cell area decreased more gradually. As a consequence, heteroblastic changes in cell number and size were accelerated in Ler, consistent with results reported for the more and smaller cells mutants (Usami et al., 2009). In other words, Ler rosette leaves acquired the cellular characteristics of late leaves at a more basal phytomer position. Heteroblastic cellular changes could be reverted in wild-type plants by removing floral buds, but this treatment did not affect heteroblasty in Ler. The 15th leaf formed on LER rosettes without floral buds had the cellular characteristics of the 12th leaf on intact LER rosettes suggesting that heteroblastic cellular changes in vegetative leaves are a consequence of the transition from a vegetative shoot apical meristem to a reproductive one. This transition is consistent with the expression pattern of ERECTA, showing that it is weakly expressed in the shoot apical meristem in early plant development but that its expression increases during the reproductive transition (Yokoyama et al., 1998). All this suggest that ERECTA could mediate a mobile signal from the shoot apical meristem to the developing leaves to modulate patterns of cell expansion between zones of a leaf and between successive leaves of a plant.

Which developmental process is the target of the ERECTA signalling function?

ERECTA could have a signalling function on both cell division and cell expansion independently or on one of them which would affect the other in a cascade. With the second hypothesis, two scenarios could lead to the same phenotype.

  1. The erecta mutation could prevent cell cycle arrest, which in turn would preclude the increase in cell expansion rate. In other words, as cells, or at least some cells, continue dividing, they do not enter the phase of rapid expansion. The low expansion rate in all zones of the leaf and in successive leaves of the rosette of Ler would be the consequence of the lengthening of the cell division phase.

  2. The erecta mutation could limit the increase in cell expansion which in turn would allow cells to divide for a longer period. In this case, arrest in cell division would be driven by a high cell expansion rate which never occurs in Ler. The lengthening of the cell division phase in all zones of the leaf and in successive leaves of Ler would be the consequence of the low cell expansion rate which is consistent with the theory of a cell size control for cell division progression (Gardner et al., 1998).

However, even if it is often assumed that cell division and cell expansion are not independent (Gardner et al., 1998; Roeder et al., 2010), results obtained in a quantitative trait locus (QTL) analysis combined with statistical modelling in Tisné et al. (2008) suggested that the first hypothesis is the most probable. It was reported that a QTL detected at the ER marker controlled the decrease in cell area and increase in cell number in epistatic interaction with another QTL on chromosome IV. The decrease in cell area depended on alleles at the ER QTL, but the increase in cell number was strictly conditioned by alleles at the second QTL. Then, the decrease in cell area and the increase in cell number were to some extent genetically separable and this result suggested that ERECTA had a proper effect on cell expansion as a first target but the effect on cell number was controlled epistatically by at least one additional gene (Tisné et al., 2008). Such genetically separable effects on cell expansion and cell division have also been reported in mutants defective in cell proliferation but with increased cell size, such as an3 (Fujikura et al., 2007).

A theoretical model is proposed to describe the multiscale effect of the erecta mutation on leaf development from epidermal cells to the whole rosette (Fig. 8). As suggested by our results, the only direct effects of the erecta mutation are a decrease in cell expansion rate and an increase in the duration of cell division. Also, as suggested in Tisné et al. (2008), these two effects are independent. They trigger a cascade of other variables in the leaf, namely the cell cycle arrest front progression, epidermal cell number, epidermal cell area and epidermal cell density. Leaf expansion rate, leaf area and rosette area that depend (more or less directly) on the cell expansion rate or the duration of cell division are not affected by the mutation because of compensation processes. The dissection of leaf growth in underlying cellular processes with spatial and temporal resolutions allowed us to identify processes directly affected by the mutation and distinguish them from those indirectly affected.

Fig. 8.

Fig. 8.

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Schematic representation of the effect of the erecta mutation on variables underlying leaf area development. All variables that are affected by the erecta mutation are surrounded by boxes. Variables that are increased in Ler compared with LER are surrounded by solid lines and shown in bold, whereas variables that are decreased are surrounded by dotted lines and shown in italics. All other variables (not surrounded) are unaffected by the mutation. In this model, erecta mutation limits the cell expansion rate and increases the duration of cell division, independently. Combination of these two independent effects eliminates the progression of the cell cycle arrest front. Both the cell division rate and the duration of cell expansion are not affected by the mutation. Combination of an increased duration of cell division and an unchanged cell division rate causes an increase in cell number. Combination of a decreased cell expansion rate and an unchanged duration of cell expansion causes a decrease in cell area. The leaf expansion rate is unchanged because it depends on cell number, which is increased, and cell expansion rate, which is decreased. The duration of leaf expansion is not altered because it coincides with the duration of cell expansion. Leaf area is unchanged for two reasons: first, because the leaf expansion rate and duration of expansion are not affected; and secondly, because at the cellular scale, cell number is increased whereas cell area is decreased. Cell density is increased because of the increase in cell number in the same leaf area (see pictures in the top left corner). Finally, the whole rosette area is unchanged because both individual leaf area and the number of rosette leaves are not affected.

ACKNOWLEDGEMENTS

We thank Myriam Dauzat for assistance using the PHENOPSIS platform, and Jean-Jacques Thioux, Crispulo Balsera and Alexis Bédiée for help with plant culture and phenotypic measurements.

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