Light and soil water content affect leaf surface area expansion through modifications in epidermal cell numbers and area, while effects on leaf thickness and mesophyll cell volumes are far less documented. Here, three-dimensional imaging was applied in a study of Arabidopsis thaliana leaf growth to determine leaf thickness and the cellular organization of mesophyll tissues under moderate soil water deficit and two cumulative light conditions. In contrast to surface area, thickness was highly conserved in response to water deficit under both low and high cumulative light regimes. Unlike epidermal and palisade mesophyll tissues, no reductions in cell number were observed in the spongy mesophyll; cells had rather changed in volume and shape. Furthermore, leaf features of a selection of genotypes affected in leaf functioning were analysed. The low-starch mutant pgm had very thick leaves because of unusually large palisade mesophyll cells, together with high levels of photosynthesis and stomatal conductance. By means of an open stomata mutant and a 9-cis-epoxycarotenoid dioxygenase overexpressor, it was shown that stomatal conductance does not necessarily have a major impact on leaf dimensions and cellular organization, pointing to additional mechanisms for the control of CO2 diffusion under high and low stomatal conductance, respectively.
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Leaves are the main sites of energy capture, carbon conversion and water flux regulation in plants. Plant responses to environmental constraints such as drought include a reduction in transpiration by stomatal closure on a short timescale and adaptations in leaf development on a longer timescale; both processes affect leaf functioning and consequently whole plant biomass production and yield. Developmental responses to drought have been at the core of many studies in different plant species including the model plant Arabidopsis thaliana (Aguirrezabal et al. 2006; Granier et al. 2006; Granier & Tardieu 2009; Tisnéet al. 2010; Skirycz et al. 2011) and have gained interest in the context of climate change. The reduction in leaf surface area induced by water deficit is related to a reduced expansion rate, whereas the duration of expansion is often lengthened (Aguirrezabal et al. 2006). On the cellular level, the respective roles of epidermal cell size and number in leaf size plasticity have been determined (Granier & Tardieu 1999; Aguirrezabal et al. 2006; Tisnéet al. 2010).
Compelling evidence exists that at least part of the reduction in leaf surface area in response to drought is not caused by an effect on leaf functioning itself as reduced leaf expansion is often reported before any changes in stomatal conductance or net carbon assimilation rate on a surface area basis (Bogeat-Triboulot et al. 2007; Hummel et al. 2010). The reasons for the maintenance of leaf functioning, in terms of CO2 diffusion and photosynthesis, while surface area is reduced, could be strongly related to leaf thickness and the cellular organization and processes in mesophyll tissues. The latter contain the main actors in photosynthesis and direct the pathway inside leaves of intercepted light (mainly palisade mesophyll), CO2 diffusion and water transport (mainly spongy mesophyll). Leaf boundary layer and stomatal conductance act at the entrance of CO2 in the leaf, whereas the internal or mesophyll conductance determines CO2 diffusion to the sites of carbon fixation in chloroplasts. The latter consists of a gaseous phase, which is affected by the extent of intercellular airspaces, and a liquid phase across the mesophyll cell wall and plasma membrane, which may be influenced by the pH of the apoplast, by cell wall thickness, porosity and hydration levels and by the presence and functioning of cooporins, that is aquaporins which have a role in membrane permeability to CO2 (Terashima et al. 2006; Evans et al. 2009). Stomatal conductance (gs) and mesophyll conductance (gm) do not necessarily act in the same sense: gm is reduced upon high internal CO2 concentration (Ci), while reduced gs (low Ci) may have no effect on gm (Vrábl et al. 2009). Furthermore, mesophyll tissues participate in water transport from the xylem and bundle sheath cells towards the sites of evaporation; it remains unclear whether these are situated deep within or evenly across the mesophyll, or superficially near and in the epidermis and guard cells (Sack & Holbrook 2006). Light is an environmental factor known to induce developmental changes in mesophyll tissues: light intensity modulates the number of mesophyll cell layers and the dimensions of palisade mesophyll cells, while both short days and shade treatments negatively affect final leaf area and epidermal cell proliferation (Dengler 1980; Lee et al. 2000; Yano & Terashima 2001, 2004; Kim et al. 2005; Cookson & Granier 2006; Cookson, Chenu & Granier 2007).
In most studies on the link between leaf development, whole plant growth and unfavourable environments, stress-induced changes in leaf growth have been assessed in two dimensions, with quantification of leaf surface area and cellular variables in the adaxial epidermis only. The epidermis is generally considered as the tissue driving or controlling leaf expansion (Savaldi-Goldstein & Chory 2008; Marcotrigiano 2010); however, the effects of environmental stresses on leaf thickness and cell volumes in mesophyll cell layers are not well-documented in the recent literature. Direct measurement of leaf thickness is not straightforward, and, in many cases, it has been estimated indirectly (Vile et al. 2005). Two-dimensional images obtained by histological sectioning are limited by the number of sections that can be obtained and the information within. Then, protocols to assess the three-dimensional cellular organization within the leaf are recent and have not been used until now to explore the effects of environmental or genetic factors on cell number and volumes in the different tissue layers (Wuyts et al. 2010).
The aim of this work was to look beyond leaf surface area and epidermal tissues in the assessment of leaf growth responses to environmental constraints by considering effects on leaf thickness as well. Moreover, three-dimensional imaging allowed us to determine cell densities and cell volumes in all mesophyll tissue layers. As a first step, Col-4 plants were grown under two light regimes and subjected to a moderate constant water deficit applied early in rosette development, thus present from the cell proliferation stage in the 6th leaf of the arabidopsis rosette. The two light regimes, low and high cumulative light, differed in the amount of daily accumulated light by means of day length differences. So far, effects on leaf thickness of day length alone or in combination with drought stress have not been described.
Thickness and the internal cellular organization of the leaf were also scored in leaves of genotypes selected for certain functional properties which may impact on leaf thickness. The mutant pgm, deficient in chloroplast phosphoglucomutase activity, was chosen because of its particular carbon metabolism status, as soluble sucrose and hexose sugars accumulate instead of being metabolized into starch (Caspar, Huber & Somerville 1985; Streb et al. 2009). An accumulation of soluble sugars has already been associated with increased leaf thickness by increased height of palisade mesophyll cells in tobacco and potato (Hoffmann-Benning, Willmitzer & Fisahn 1997; Keller et al. 1998). Two other genotypes, ost2 and 35S::AtNCED6, were selected for their unfavourable and favourable leaf water status, respectively. The former was originally identified based on its ‘open stomata’ phenotype which is even conserved in response to abscisic acid (ABA) under drought stress due to the constitutive activity of a major plasma membrane proton pump (H+-ATPase) (Merlot et al. 2002, 2007). The latter was selected based on its high ABA content which is achieved by overexpression of a 9-cis-epoxycarotenoid dioxygenase (35S::AtNCED6), and the concomitant stomatal closure induced by ABA (Lefebvre et al. 2006). Its phenotype has been associated with the maintenance of a favourable plant water status and increased tolerance in response to drought stress (Lefebvre et al. 2006; Parent et al. 2009).
Overall, the study was organized based on a set of questions: (1) does leaf thickness respond to moderate soil water deficit in the same way and to the same extent as leaf surface area and does this depend on day length? (2) Do leaf mesophyll tissues differ in their responses from epidermal tissues? (3) To what extent is thickness affected in pgm, a small leaf mutant with high levels of soluble sugars? (4) Does stomatal conductance influence leaf dimensions and cellular organization by means of its effects on carbon conversion or is this counterbalanced by the plant's water status?
MATERIALS AND METHODS
Plant material and growth conditions
Arabidopsis Col-4 (N933, http://Arabidopsis.org.uk) was grown in a mixture (1:1) of a loamy soil and organic compost in the PHENOPSIS phenotyping platform (Granier et al. 2006) under controlled air temperature, air humidity and incident light intensity, provided by a bank of cool-white fluorescent tubes and HQi lamps (Table 1). Soil humidity was maintained at 40% or 23% for well-watered and moderate water deficit conditions, respectively, by automated irrigation from stage 1.02 (two visible leaves) to 6.00 (first-flower-open stage) (Boyes et al. 2001). Day length was set at 8 h for the low cumulative light experiment (5.9 mol m−2 d−1) and 16 h for the high cumulative light experiment (9.6 mol m−2 d−1). Mutants in the Col-0 background, pgm, ost2 and 35S::AtNCED6 were grown alongside Col-4 and Col-0 in the high cumulative light experiment (Table 1).
Table 1. Environmental conditions and genotypes studied under well-watered (40% soil humidity) and moderate water deficit (23% soil humidity) conditions
PPFD (µmol m−2 s−1)
Daily cumulative light (mol m−2 d−1)
Mean air temperature (°C)
Mean air humidity (%)
Mean VPD (kPa)
PFFD, photosynthetic photon flux density; VPD, air vapour pressure deficit.
Col-0, pgm, ost2, 35S::AtNCED6
Arabidopsis leaf growth assessment
Col-4 plants were collected every 2–3 days from stage 1.02 until 6.00. Surface area was measured on the 6th leaf harvested from five rosettes per time point, using a binocular microscope and dedicated image analysis software (Bioscan-Optimas V 4.10; Edmonds, WA, USA) until the emergence of the 6th leaf, and using an ImageJ macro on scans of dissected leaves afterwards (Rasband 1997–2011; Fabre et al. 2011). Six plants per time point were harvested for the measurement of leaf 6 thickness and cellular growth variables by three-dimensional imaging. Col-0 and mutant plants were collected at stage 6.00 for leaf 6 imaging.
Three-dimensional imaging and image analysis
Whole seedlings or leaves were fixed, conserved and subsequently cleared and stained using propidium iodide as described by Wuyts et al. (2010). Image stacks covering the complete leaf thickness were produced for the middle of the leaf along the longitudinal axis, and approximately midway between the leaf midvein and margin using multi-photon laser scanning microscopy (Wuyts et al. 2010). Visualization of image stacks, orthogonal and three-dimensional views was done in ImageJ (Supporting Information Fig. S1). For the quantitative analysis of tissue and cell dimensions in image stacks, specifically developed ImageJ macros and R scripts were used (R Development Core Team 2010; Wuyts et al. 2010). The following leaf variables were determined: surface area, thickness, epidermal and mesophyll tissue thickness and relative proportions, cell density, volume, height and width at maximum cell circumference in epidermal and mesophyll tissues, stomatal density and index in the adaxial and abaxial epidermis. Cell number was calculated by surface area × cell density. Mesophyll cell solidity was used as a shape descriptor giving the ratio of the area of a cell to the area of its convex hull, and was used to describe the deviation from a round shape by the number and arrangement of lobes in spongy mesophyll cells.
Photosynthesis and stomatal conductance were determined for the 6th leaf of Col-0 and leaf functional mutants grown under well-watered and water deficit conditions at stage 6.00, when the leaf was fully expanded. Measurements of the net CO2 assimilation rate were performed in a custom-made arabidopsis single-leaf chamber connected to a gas analyser (CIRAS-2, PP Systems, Hitchin, Hertfordshire, UK).
Kinematic and statistical analyses were performed in R. A sigmoid function was fitted to leaf surface area and thickness expansion profiles over time, defined as the number of days after the initiation of the 6th leaf, to extract kinematic growth variables (Cookson, Van Lijsebettens & Granier 2005). The first derivative of the sigmoid function represented the profile of the absolute expansion rate; the maximum of which and its position in time were determined. The period between the minimum and maximum of the second derivative of the sigmoid was considered as the time at which expansion was in its most intensive phase. The Kruskal–Wallis rank sum test was used to determine differences in leaf growth variables between genotypes and treatments (light regime, soil water content). To compare the characteristically large distributions of cell dimensions between treatments, beanplots were drawn in R for cell volume, height and width (‘Beanplot’ package, Kampstra 2008). Beanplots show the density trace of a distribution as a polygon shape which is asymmetric when two distributions (e.g. data from well-watered and soil water deficit treatments) are plotted as one bean. The distribution mean is equally shown. A beanplot is an alternative for boxplots when distributions are not normal and require visual comparison.
Different susceptibilities of leaf surface area and thickness to light and moderate water deficit
Under both well-watered and water deficit conditions, leaves were significantly larger (P < 0.01) and showed higher rates of expansion in the high cumulative light treatment compared to the low cumulative light treatment (Fig. 1a,b, Supporting Information Table S1). The response to water deficit of leaf expansion in surface area differed depending on the light regime. In low cumulative light, final leaf surface area was reduced by 49% in response to a constitutive soil water deficit (Fig. 1a). The kinematic analysis of leaf surface area expansion in water-stressed plants revealed a maximum expansion rate of only 30% of its value under well-watered conditions, and this was accompanied by a longer duration of expansion (until 24 instead of 17 days after leaf initiation). In the high cumulative light experiment as well, leaf expansion in surface area was substantially affected by soil water deficit: final leaf area and maximum expansion rate were reduced by 34% and 33%, respectively (Fig. 1a,b). In contrast to low cumulative light, the duration of leaf expansion was not increased under soil water deficit conditions when plants were grown in high cumulative light (Fig. 1a,b; Supporting Information Table S1).
Leaves were also significantly thicker (P < 0.01) in high compared to low cumulative light (Fig. 1c) under both well-watered and water deficit conditions. But, in contrast to surface area expansion, rates of expansion in thickness did not differ significantly between light regimes in well-watered conditions (Fig. 1d; Supporting Information Table S1). In response to water stress, the reduction in thickness expansion rate was more severe under low cumulative light (46% compared to 21% in high cumulative light), but this was compensated by a broader expansion period (Fig. 1d; Supporting Information Table S1). Thus, in contrast to leaf surface area and irrespective of the light regime, final leaf thickness was not significantly affected in plants grown under moderate soil water deficit.
Low cumulative light negatively affected cell number
In leaves of plants grown in high cumulative light, cell numbers in both the epidermis and mesophyll were significantly higher than in low cumulative light, while cell densities did not depend on the light regime (Fig. 2). The larger leaf surface areas in high cumulative light were thus obtained based on cell numbers, not individual cell expansion.
To assess differences in leaf thickness, tissue proportions and mesophyll cell dimensions were determined. Thick leaves in high cumulative light had a bigger proportion of spongy mesophyll (33% spongy and 54% palisade mesophyll) than thinner leaves in low cumulative light (24% spongy and 63% palisade mesophyll) (Fig. 3). Palisade mesophyll cells were organized in two layers, while two to three spongy mesophyll cell layers were encountered upon crossing of the mesophyll to the abaxial epidermis. In the high cumulative light treatment, it was more common to encounter three spongy mesophyll layers, in which cells also had a larger volume because of increased cell height, not width (Fig. 4).
In low cumulative light, moderate soil water deficit affected mainly cell number not volume, except for the spongy mesophyll
In fully expanded leaves of plants grown in low cumulative light, cell densities did not differ between well-watered and water deficit conditions for the adaxial and abaxial epidermis and the palisade mesophyll, while in the spongy mesophyll, cell densities were significantly higher under water stress (Fig. 2).
Leaf cell numbers were significantly lower in the soil water deficit treatment (P < 0.01), except for the spongy mesophyll (Fig. 2). This meant that the reduction in leaf surface area under soil water deficit in low cumulative light was caused by reduced cell numbers in the adaxial and abaxial epidermis and the palisade mesophyll. In the spongy mesophyll, however, cell number was not affected, while volumes were significantly reduced (mean volume of 36284 µm3 versus 47426 µm3 under water deficit and well-watered conditions, respectively, P < 0.05, Fig. 4a). Mesophyll cells were characterized by a large range of volumes, which had shifted towards lower values in the spongy mesophyll of water-stressed plants. These volume differences were mainly brought about by reduced cell width, not height, hence the absence of a significant effect on leaf thickness (Fig. 4). Moreover, the solidity shape descriptor for spongy mesophyll cells was lower in the water deficit treatment revealing a changed shape upon the reduction in volume from slightly lobed and inflated to slender, more lobed and deflated (Fig. 5).
In high cumulative light, moderate soil water deficit caused small reductions in both cell number and volume
The response to soil water deficit in terms of cell densities and numbers was far less clear-cut in the high cumulative light regime: cell numbers tended to be reduced, but far less than in short days (Fig. 2). Cell densities tended towards higher values under water stress, and, accordingly, the cell volume distribution had shifted towards lower values. Smaller surface areas were thus obtained by a combination of small reductions in both cell numbers and volumes. In the spongy mesophyll, cell volumes did become clearly concentrated round smaller values (Fig. 4). Moreover, as in low cumulative light, the solidity shape descriptor for spongy mesophyll cells was lower in the water deficit treatment (Fig. 5).
In the low-starch mutant pgm, leaf thickness is exceptionally high and conserved under soil water deficit
Leaf structure was analysed at the organ and cellular scale in the low-starch mutant pgm, which is characterized by an accumulation of soluble sugars instead of starch. Compared to Col-0, leaves were 50% smaller in surface area, while at the same time, they were 30% larger in thickness (Fig. 6). Despite of these differences, cell densities in pgm were not different from wild-type leaves for mesophyll and adaxial epidermal tissues (Fig. 6). Palisade mesophyll cell volumes were even bigger on average (Fig. 7), but this was because cells were significantly higher than in Col-0 palisade mesophyll: 71–76 µm versus 58–61 µm (P < 0.05), respectively, hence pgm's thick leaves (Fig. 2; Supporting Information Fig. S2).
In response to water deficit, surface area was reduced by 23% in pgm, compared to a reduction of 38% in Col-0. Cell density in the adaxial epidermis was increased under soil water deficit, while in the abaxial epidermis, it was independent of any treatment, but higher than in Col-0 (Fig. 6). Again, as for the other genotypes, leaf thickness was not significantly affected by water stress. As in Col-0, palisade cell volumes tended to be reduced under soil water deficit, but this was because of a reduction in cell width, not height (Fig. 7). Spongy mesophyll cells also changed shape as in the wild type, which was reflected in the solidity of cells (0.93 and 0.86 for well-watered and water deficit conditions, respectively, P < 0.05; Fig. 5).
The low-starch phenotype of pgm was thus associated with significant reductions in leaf surface area and whole rosette expansion, also under soil water deficit; while on the other hand, it showed thick leaves with unusually large palisade mesophyll cells. Leaves of pgm were also darker green compared to Col-0, suggesting a larger chlorophyll concentration, and had a higher stomatal density in the abaxial epidermis in well-watered and water deficit conditions (Fig. 8). These observations were associated with higher levels of photosynthesis and stomatal conductance in both conditions (Table 2).
Table 2. Photosynthesis and stomatal conductance measured in the 6th leaf of the Arabidopsis thaliana rosette in well-watered (WW) and soil water deficit (WD) conditions for the Col-0 wild type and leaf mutants
Data are means and standard deviations. Data within a row (treatment) followed by the same letter are not significantly different (P < 0.05) according to Kruskal–Wallis sum of ranks. Differences between genotypes (columns) are not significant (ns) or significant at P < 0.05 (*) or 0.01 (**) according to Kruskal–Wallis sum of ranks. Differences between treatments are not significant according to Kruskal–Wallis sum of ranks.
Constitutively high stomatal conductance under moderate water deficit did not imply major responses in leaf volume or cellular organization
To investigate the interaction between stomatal conductance and cellular organization, genotypes affected at the level of stomatal functioning, ost2 and 35S::AtNCED6, were compared to their wild-type Col-0. Neither leaf surface area nor leaf thickness differed between Col-0 and ost2 (Figs 2 & 6), despite fivefold higher stomatal conductance in the latter (Table 2). Under soil water deficit, leaf surface area was significantly reduced, but not more than in Col-0, whereas leaf thickness was conserved, as in Col-0. There were no significant differences in epidermal and mesophyll cell densities between ost2 and Col-0, and cell numbers were reduced in response to water stress in both genotypes for the adaxial and abaxial epidermis and palisade mesophyll. In contrast to Col-0 (and Col-4 as shown earlier), spongy mesophyll cell numbers did get affected by water deficit in ost2.
Under well-watered conditions, the ranges of cell volumes in the palisade and spongy mesophyll were concentrated around smaller values in ost2 compared to Col-0 (P < 0.05), by means of reductions in cell width, not height, and there was no further reduction upon soil water deficit (Fig. 7). The change in mesophyll cell shape observed in Col-4 under soil water deficit conditions was also found in Col-0. In ost2, however, this was not the case and cells remained round and inflated (Fig. 5).
In 35S::AtNCED6, there were no measurements on well-watered plants; their leaves were chlorotic and became vitrified when grown at 40% soil humidity (well-watered conditions) (Fig. 5), while the phenotype was absent at 23% soil humidity (moderate water deficit), suggesting that 40% soil humidity constituted a waterlogging treatment for this genotype. Under soil water deficit, photosynthesis was significantly lower compared to the other genotypes (Table 2). Stomatal conductance could not be reliably measured because of its low values. No differences were detected in surface area, thickness, cell densities or cell volumes between 35S::AtNCED6 and Col-0 water-stressed leaves, despite lower levels of photosynthesis and stomatal conductance (Fig. 6 & Table 2). Spongy mesophyll cells were, however, severely deflated and stomatal densities were significantly lower in the abaxial epidermis (Figs 5 & 8).
Low cumulative light treatment causes a reduction in leaf area and thickness together with reductions in cell number in all tissues and cell height in the spongy mesophyll
Daily cumulative light treatments combine the effects of incident light intensity and day length. Here, light quality was the same in all experiments. Effects of changes in light intensity on leaf expansion in area and thickness have been studied in different plant species. Reducing light intensity causes a reduction in final leaf area together with a reduction in epidermal cell number, whereas epidermal cell area is generally increased (Dengler 1980; Cookson & Granier 2006). Decreases in leaf thickness have also been reported when light intensity is reduced, and differences are then established with changes in the number of mesophyll cell layers while mesophyll cell numbers are conserved (Dengler 1980; Yano & Terashima 2004; Kim et al. 2005). In our experiment, the low cumulative light regime was imposed by shortening day length, whereas instantaneous light intensity did not change significantly. Short days have been shown to reduce leaf expansion rate and final leaf surface area together with a reduction in cell proliferation in the adaxial epidermis of arabidopsis plants (Cookson et al. 2007). Here, the reduction in surface area by 39% was accompanied by declined cell numbers in epidermal as well as in mesophyll tissues, whereas final cell densities were unchanged in all leaf tissues. References are missing on how thickness is affected by changes in day length, but for Heptacodium miconioides, short days had a tendency to increase leaf thickness, compared to prolonged days under low light conditions (Lee, Bilderback & Thomas 1991). In our case, short days caused a reduction in leaf thickness by 22% and this was established by a reduced height of spongy mesophyll cells without changes in their width and overall fewer cells in the third layer of this tissue.
Moderate soil water deficit did affect leaf surface area but in interaction with day length
Soil water deficit treatments imposed during the whole duration of leaf expansion have been shown to reduce leaf area expansion with concomitant reductions in both leaf expansion rate and epidermal cell number, whereas duration of leaf expansion and epidermal cell size can be increased, decreased or unchanged depending on the plant species and the stress intensity (Granier et al. 2006). It is shown here that these variables also have a day length-dependent response to water deficit. When plants were grown in short days, duration of leaf expansion was increased by the drought treatment and final epidermal cell size was unchanged (as shown previously in a collection of accessions grown in at a 12 h photoperiod in Aguirrezabal et al. 2006). In contrast, water deficit did not affect duration of expansion and caused a reduction in cell volume when plants were grown in long days. This may be related to arabidopsis being a quantitative long-day plant (Giakountis et al. 2010). Short days bring about a lengthening of the vegetative phase and the development of a significantly larger number of leaves (on average, 29 versus 10 leaves at 8 h and 16 h photoperiods, respectively). A role for floral transition in determining leaf cell area has already been shown in arabidopsis by manipulating flowering time and in a large population of recombinant inbred lines (Cookson et al. 2007; Tisnéet al. 2008). Here, under long-day conditions, floral transition had already occurred in the very early development of the 6th leaf, while in short days, it developed completely within the vegetative phase. It seemed that in the latter case, cell expansion rates, as well as the duration of expansion, were adjustable in response to the water limitation, whereas upon imminent flowering, both variables had become less yielding.
Leaf thickness and leaf functioning are conserved under moderate soil water deficit
In contrast to our findings, reductions in leaf thickness in response to water stress have been reported for many species, but with water deficit treatments far more severe than the constitutive moderate water deficit imposed here. Decreased leaf thickness when plants are subjected to a high level of dehydration is caused by decreased cell height in the mesophyll (Scippa et al. 2004; Sancho-Knapik et al. 2011). Stomatal conductance and rates of photosynthesis expressed on a unit leaf area basis did not differ between our well-watered and water deficit conditions for all genotypes tested. Thickness was highly conserved, which could indicate that a certain thickness is required and worth maintaining for leaf and whole plant functioning.
Pronounced palisade mesophyll elongation and increased stomatal density in pgm leaves were associated with high levels of photosynthesis and stomatal conductance, and unaffected by moderate water stress. The origin of pgm's thick leaves may lie in the large concentrations of free hexoses which could contribute to a high osmotic pressure for cell expansion, whereas in wild-type plants, excess sugars are stored in osmotically inactive starch during the day. Free hexoses or osmotic pressure have been related to oriented cell expansion (Hoffmann-Benning et al. 1997; Keller et al. 1998). An increase in cell wall thickness because of increased availability of cell wall material was not confirmed here, as pgm palisade mesophyll cells were only heavier than Col-0 cells because of their significantly larger volume (Supporting Information Fig. S3).
Leaf surface area can be maintained under constitutively high stomatal conductance
The higher rate of photosynthesis in ost2 leaves was most likely linked to its high levels of stomatal conductance due to constitutively open stomata. Although the basal stomatal aperture of the mutant in the Col-0 background has been described as not different from the wild type, in contrast to the Ler background mutant (Merlot et al. 2007), in the present experiment, a fivefold higher level of stomatal conductance was observed under well-watered and water-deficit conditions. Despite of constitutively high stomatal conductance, neither leaf surface area nor stomatal density was different in ost2 compared to the wild-type Col-0, even under soil water deficit. On the other hand, palisade mesophyll cells tended to be smaller in ost2, which could be related to higher water losses in these leaves and thus adaptations in terms of cell expansion to maintain cell turgor.
Lower stomatal conductance and photosynthesis in 35S::AtNCED6 with constitutively high concentrations of ABA did not cause any differences in terms of leaf surface area or thickness, or cellular numbers or densities compared to the wild-type Col-0 in moderate soil water conditions. However, the lower dry weight of these leaves did suggest adaptations in terms of cell wall thickness in response to lower internal CO2 diffusion (Evans et al. 2009), or signified the absence of any requirement for cell wall reinforcement in well-hydrated leaves with low water losses (Supporting Information Fig. S3). Vrábl et al. (2009) have shown in Helianthus annuus that reduced stomatal conductance induced by ABA does not affect mesophyll conductance. At 40% soil humidity, leaves of 35S::AtNCED6 became waterlogged. Flooding of intercellular spaces with sap at high relative humidity and low vapour pressure demand has already been observed in leaves of plants containing high ABA concentrations and is due to a combination of closed stomata and greater root hydraulic conductivity (Thompson et al. 2007; Tardieu, Parent & Simonneau 2010). ABA-induced activity of aquaporins and overall increased conductivity of mesophyll cells have also been put forward (Morillon & Chrispeels 2001).
The results obtained here indicate that levels of stomatal conductance do not necessarily have a large impact on overall leaf growth and cellular organization, which points to potentially efficient mechanisms to control and maintain optimal CO2 diffusion at the level of the chloroplasts.
ABA may be involved in spongy mesophyll adaptation to water stress and affect stomatal development
In contrast to epidermal tissues and palisade mesophyll, cell numbers in spongy mesophyll were not reduced in response to soil water deficit. Moreover, spongy mesophyll cells had changed shape from inflated and lobular to deflated and slender with higher curvature. These adaptations could be related to maintenance of intercellular spaces and an optimization for internal CO2 diffusion and mesophyll surface area exposed to intercellular air spaces (Flexas et al. 2004). The plasticity of the response needs further investigation, but in these experiments, soil water deficit was constitutive, which means that changes may have been initiated during cell expansion following signals originating in mature leaves (Jung & Wernicke 1990; Panteris & Galatis 2005). The mesophylic component of CO2 diffusion is known to be particularly resilient to water deficit (Kaiser 1987; Cornic 2000; Flexas & Medrano 2002). Ribulose 1·5-bisphosphate carboxylase/oxygenase activity is maintained even when leaf relative water content drops to 50% and stomata are already 75% closed (Kaiser 1987; Flexas et al. 2006). Changes in mesophyll cell shape (‘buckling’) have been observed in Quercus muehlenbergii, but only beyond the cell turgor loss point upon dehydration of cut leaves (Sancho-Knapik et al. 2011).
Remarkably and in contrast to the other genotypes studied, spongy mesophyll cells had not changed shape in ost2, which seemed to indicate that the process involves H+-ATPases, that is, proton pumps that bring about the acidification of the apoplast, and is regulated by ABA in a similar way as stomatal closure, that is, loss of turgor in guard cells (Van Volkenburgh 1999; Davies, Wilkinson & Loveys 2002). This is in agreement with observations in 35S::AtNCED6 which had high ABA concentrations, very low stomatal conductance and, at the same time, a very pronounced change in cell shape in spongy mesophyll.
In general, drought did not affect the development of stomata, and stomatal density kept in line with epidermal cell division and expansion. The influence of light intensity captured by mature leaves on the development of stomata of younger leaves has been described, whereas it is recognized that the effects of drought on stomatal numbers are unclear as both increases and decreases in stomatal density and indices have been reported (Casson & Gray 2008; Vile et al. 2012). In 35S::AtNCED6, stomatal indices were lower than in the other genotypes under moderate drought conditions, which suggests that ABA is not only involved in stomatal closure, but also in the development of stomata. It acts most likely as an indirect suppressor because of its influence on leaf and plant water status and the signalling events between mature and developing leaves (Casson & Hetherington 2010).
The present study has been funded by the Agropolis Fondation (RTRA 07047), Montpellier, France and Agron-Omics, a European sixth framework integrated project (LSHG-CT-2006-037704). The authors would like to thank Gaëlle Rolland, Alexis Bédiée and Crispulo Balsera for assistance during experiments.