How does stomata density affect transpiration

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A negative correlation has frequently been suggested between these two stomatal traits. This inverse relationship has been observed in plastic developmental responses to changes in environment and also during long-term evolutionary adaptation Dilcher et al. Analysis of herbarium and fossilized plant remains suggest that SS and SD have changed in response to atmospheric CO 2 concentration over evolutionary time, probably to enable adjustments to g smax and CO 2 diffusion into the leaf Woodward, ; Dilcher et al.

In samples from periods when CO 2 concentrations were low, a reduction in SS and an increase in SD have been observed. Such adaptive responses to CO 2 are also found in many extant lineages; however, this is not always the case in all species surveyed Casson and Gray, ; Haworth et al.

Although various combinations of SS and SD can result in similar alteration to g smax , there are limitations as to how much of the epidermis can be patterned by stomata.

First, other functionally important leaf structures such as veins and trichomes are also absolutely required. Second, stomata need to be spaced by at least one epidermal cell to function efficiently Franks and Farquhar, ; Dow et al. In general, plants optimize g smax through investing in increases in SD coupled with reductions in SS Franks and Beerling, ; de Boer et al. According to Franks and Beerling and Franks and Farquhar , changes toward increased SD in combination with reduced SS could maintain or improve total pore area due to increased SD but can also provide a shorter diffusion path due to the smaller pore depth , potentially resulting in improved gas exchange.

While small SS coupled with high SD often leads to a higher g smax , it is also possible for g smax to be reduced by a smaller SS alone.

Decreases in g smax due to a smaller SS have been associated with higher water conservation, as reported for plants exposed to drought Doheny-Adams et al. Growth under low soil moisture conditions has been shown to cause a decrease in SS in several species Xu and Zhou, ; Doheny-Adams et al. Stomatal size and density responses to vapor pressure deficit VPD are also variable. Miyazawa et al. Nutrient availability can also affect plant development. However, as described for soil moisture and VPD responses, adjustments in stomatal development in response to nutrient availability appear to be variable with no consistent response emerging Gao et al.

Small stomatal size can provide a reduction in total leaf pore area and might also facilitate faster aperture response Franks and Beerling, ; Drake et al. The higher cell surface area to volume ratio of smaller cells is believed to permit faster ion fluxes, leading to faster guard cell turgor changes and a more rapid g s response Lawson and Vialet-Chabrand, This faster stomatal behavior in plants with smaller SS has been observed in response to changes in light intensity across species of Banksia , rainforest trees, and in cereal species with dumbbell-shaped guard cells Drake et al.

However, although rapid stomatal movements might help to maximize WUE under fluctuating light environments, this is unlikely to have much impact on water loss over long periods of water stress under field conditions.

SS is clearly not the only anatomical trait influencing stomatal behavior. In addition, the distribution of stomata between leaf abaxial and adaxial surfaces may also affect plant responses to environmental stresses. For example, stomata on the abaxial surfaces of wheat leaves show a stronger decrease in g s than adaxial stomata when they are exposed to water stress Lu, , and abaxial and adaxial stomata of cotton show differing responses to light quality Lu et al.

Moreover, as shown by Elliott-Kingston et al. Comparisons of cultivars or mutants of the same species with altered SS , but similar SD , would improve our understanding of the effect of guard cell size on speed of stomatal movement and explore if there is potential for SS as trait for improving WUE. Although a correlation between SS and genome size has been documented Beaulieu et al. Reductions in SD also have the potential to constrain g s and transpiration E , representing a shift towards a more conservative use of water.

If not limiting A or evaporative cooling, this reduction in water loss should represent an advantage under low water availability scenarios. In comparison to SS , significant advances have been made in understanding the molecular signals regulating stomatal density and patterning, which allow the study of the physiological effects of altering SD. The large reduction in SD resulted in plants with improved tolerance to drought, without detrimental effects to uptake of nitrogen or phosphate Hepworth et al.

Improved plant drought responses were also achieved by Wang et al. WUE and drought tolerance were improved in poplar plants with lower SD , which also showed relatively lower decreases in levels of A and biomass under water restricted conditions Wang et al. A more encouraging result was achieved by Yoo et al. GTL1 loss-of-function Arabidopsis mutants had higher SDD1 expression resulting in lower SD and g s , without detrimental effects to the photosynthetic rates over a range of light levels.

The lower water loss observed in the gtl1 mutants significantly improved WUE, when water loss versus shoot dry weight was assessed. Taken together, these data indicate that it is possible to improve WUE by altering g smax and g s using genetic engineering tools. It is not fully understood, however, how severe reductions in g smax may limit short-term stomatal responses or whether adjustments to stomatal development in response to changes in environmental conditions would be affected in these genetically modified plants.

Although stomatal development in grasses differs from that of eudicots in various aspects, recent findings demonstrate that several components of the stomatal signaling pathway, including bHLH transcription factors Liu et al. This has allowed researchers to begin to test the implications of targeted manipulations in stomatal density in grasses, a family of plants that comprises many important food crops.

Research on barley and rice further discussed below shows that the overexpression of EPF1 can result in improved WUE without yield penalty, despite in some cases small reductions in photosynthetic rate under well-watered conditions Hughes et al.

Interestingly, in both crops, an increase in guard cell size was not observed in plants with reduced SD , contrasting with that described for poplar and Arabidopsis above. These observations suggest that the response of SS to altered SD may be differentially regulated between monocots and eudicots. The shape of guard cells and the presence or absence of subsidiary cells have implications for the mechanics and responsiveness of stomatal movement Franks and Farquhar, Diversity in stomatal morphology is commonly observed across species and can be linked to adaptability to certain environments Chen et al.

In the grass family, for example, stomatal morphology has often been hypothesized to have contributed to successful diversification, particularly in habitats with fluctuating water availability Hetherington and Woodward, ; Cai et al. In contrast to the two kidney-shaped guard cells observed in many species, grass species develop stomatal complexes formed by a pair of dumbbell-shaped guard cells, which are flanked by two paracytic subsidiary cells Stebbins and Shah, ; Sack, ; Rudall et al.

Several studies comparing stomatal opening and closing responses, between grasses and species with kidney-shaped stomata, suggest that grasses exhibit faster and more efficient stomatal regulation Grantz and Zeiger, ; Vico et al.

The linear dumbbell-shaped guard cells require only small changes in volume to bring about stomatal opening and, consequently, to achieve a higher diffusible pore area Hetherington and Woodward, The large and rapid responses of grass stomata are also related to the physical interaction between dumbbell-shaped guard cells and flanking subsidiary cells.

Subsidiary cells are not only able to limit but also to accommodate guard cell movement, providing a mechanical advantage Franks and Farquhar, This efficient osmotic flux aids rapid stomatal movement and therefore is believed to confer adaptive advantages to grasses.

Slow stomatal responses are proposed to lead to less efficient uptake of CO 2 during stomatal opening and unnecessary water loss during stomatal closure McAusland et al. Under particular environmental conditions e. Mutant plants lacking subsidiary cells failed to open guard cells as widely as control plants and also showed slower stomatal responses to changes in light intensity, further suggesting that subsidiary cells are integral for efficient stomatal functioning in grasses Raissig et al.

Despite the relatively recent discoveries of MUTE and PAN proteins in grasses, there are still many unanswered questions in relation to subsidiary and guard cell interactions, especially in non-grass species, which show a diversity of stomatal complex morphologies, with different numbers and positions of subsidiary cells Rudall et al. Further study of how the diversity of stomatal complex morphologies affects plant physiology could improve our understanding of how these features might contribute to improved WUE.

An increasing number of genetic resources are enabling researchers to test whether targeted alterations in stomatal development can improve WUE and drought tolerance in crop species Winter et al. Although results are yet to be demonstrated in the field, in overexpressing orthologs of Arabidopsis SDD1 in maize and tomato, respectively, Liu et al. These plants were able to avoid water stress for longer periods, showing drops in photosystem II activity 4—5 days later than the control plants.

Carbon isotope analysis suggested that plants with reduced SD had improved WUE under the water stress treatment, and despite small reductions in A , no detrimental effects on plant growth or yield were observed Hughes et al. Despite the changes in stomatal properties, neither A nor g s was significantly different from controls suggesting that increased SD neither positively or negatively impacted on gaseous exchange.

In this particular study, WUE was not reported, but based on A and g s values, alterations seem unlikely. Given the predicted temperature increases for the coming century, however, crop plants with more stomata and potentially increased gas exchange capacity may be important in mitigating the effects of heat stress through increased transpiration-mediated cooling.

While most of the crop studies discussed above have characterized drought and photosynthetic performance, to better understand how crops with altered SD , SS , or function might perform under future climate scenarios, it is important to consider the combinatory effects of multiple abiotic factors.

Of particular importance are the predicted reductions in water availability, increasing atmospheric CO 2 , concentration, and increasing temperature. While reduced water availability and elevated CO 2 often result in stomatal closure leading to reduced g s , increased temperature might have the opposite effect, forcing stomata to open to mitigate the effects of overheating Zhou et al.

This essentially means that in future climates, if plants are going to conserve water, they may be less able to prevent overheating, possibly leading to photoinhibition, leaf damage, and reduction in yields. The increase in gas exchange rates were seemingly achieved through regulation of stomatal apertures Figure 2 , with the trade-off being a loss of superior WUE relative to control plants Caine et al.

These results raise a number of questions regarding the physiological behavior of these reduced SD plants. Firstly, will plants with fewer, smaller stomata be capable of continuing to increase g s to maintain water flow and A at extreme temperatures, and will this be at the expense of WUE?

If so, will plants with the lowest SD be less water-use efficient than plants with higher SD at very high temperatures in order to maintain cooling? The answers to such questions are critical to understand if targeted SD reductions are to be an effective tool to improve rice production in areas where drought and high temperatures are predicted to become more prevalent.

Figure 2. OsEPF1oe rice plants with reduced stomatal density and size are able to maintain high rates of gas exchange under heat stress conditions by opening their stomatal pores adapted from Caine et al. Control plants show increases in stomatal density and in maximum leaf stomatal conductance average values under high temperature conditions. The central position of stomata in the gas exchange process makes them an obvious target for improving WUE; nonetheless, the manipulation of other processes with potential for improving plant carbon and water relations has also been investigated Lefebvre et al.

Alterations in mesophyll conductance g m , for example, can have a great impact in A , and its coregulation with g s is essential for plant WUE. Indeed, it has been suggested that increases in g m coupled with decreases in g s could improve WUE, without the potential detrimental impacts in A and yield Flexas et al.

Moreover, stomata are not the only structures on the epidermis to prevent water loss — trichomes, the cuticle, and cuticular waxes are also important Guo et al. While research into crop plant stomata is of long-standing Teare et al. For example, by crossing the wild drought tolerant tomato relative, Solanum pennellii which has abundant trichomes , with the cultivated species Solanum lycopersicum , Galdon-Armero et al.

It was found that the plants with the best WUE were those with the highest ratio of trichomes to stomata. One possible explanation for this is that plants with fewer stomata and abundant trichomes have a more significant boundary layer, thus creating a greater resistance to diffusion of water from the leaf Galdon-Armero et al. In this study, wax chimneys were detected on the cuticle that encircled stomata, which like trichomes, prevented excessive water loss, thereby potentially improving WUE.

Further investigations exploring how stomata and other epidermal structures jointly contribute to regulate WUE may be a critical piece in the jigsaw of preserving water and negating drought. Indeed, the recent discovery of the Fused Outer Cuticular Ledge1 stomata gene in Arabidopsis may help facilitate such studies Hunt et al. The knowledge relating to the genetics underpinning stomatal development and physiology in both Arabidopsis and crop species has advanced substantially, with noticeable advancements made in improving WUE.

However, there are still many questions to answer, of particular importance is how SS is regulated at the genetic level and why do SS-SD responses vary so much between species. In addition to this, understanding how stomatal complex architecture is modified and how ion fluxes are directed between guard and subsidiary cells at the genetic level is the key area where further advances in knowledge are required.

In crops, recent studies are showing that engineering plants to reduce stomatal number may be an effective tool to improve plant WUE and drought tolerance without yield reductions. Of course, as modified plants have typically been evaluated in laboratory conditions, it is still necessary to answer how such plants might perform in the real world. In a field context, many other environmental variables and stressors will impact on performance.

Additional studies are necessary to understand how plants with altered stomatal development will respond to multiple stresses in different developmental phases. Moreover, combining changes in stomatal traits with other alterations associated with improved water relations, such as modifications to the leaf epidermis, photosynthesis, g m , and root growth, among others, could further benefit plant WUE and drought tolerance under future predicted climate scenarios.

LB and RC wrote the paper. LB designed the figures. JG provided advice and comments. All authors read, commented on, and approved this version of the manuscript. University of Sheffield library—to fund journal fees. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Assmann, S. Guard cell sensory systems: recent insights on stomatal responses to light, abscisic acid, and CO 2. Plant Biol. Bacon, M. Water use efficiency in plant biology. Oxford, UK: Blackwell. Google Scholar. Bakker, J. Effects of humidity on stomatal density and its relation to leaf conductance. Bartlett, M. The correlations and sequence of plant stomatal, hydraulic, and wilting responses to drought. Beaulieu, J. Genome size is a strong predictor of cell size and stomatal density in angiosperms.

New Phytol. Bergmann, D. It has previously been shown that the application of exogenous BA cannot mimic the effect of endogenous CKs on tomato leaf development Fleishon et al.

Next, we analyzed transpiration in the transgenic plants under normal and drought conditions. To measure whole-plant transpiration, we used an array of load cells lysimeters; see Materials and Methods placed in the greenhouse and simultaneously measured the daily weight loss of each plant.

The reduced endogenous level of CKs did not, however, affect the pattern of daily transpiration, suggesting a similar pattern of stomatal movement Fig. The transgenic plants maintained their low and stable transpiration rate throughout the drought treatment Fig. AtCKX3 overexpression reduces whole-plant transpiration under irrigation and during drought stress in tomato. A Transpiration rate under normal irrigation normalized to total leaf area.

B Stomatal conductance under normal irrigation normalized to total leaf area. Pot weight was measured every 10 s and readings were averaged over 3 min. For clarity, SE bars are shown for only three time points during each 24 h cycle. AtCKX3 overexpression reduces water loss in tomato. Phenotype and RWC were recorded after 4 days. B RWC in leaf number 3 counted from the bottom of the plant up.

This figure is available in colour at JXB online. S1B was probably a major cause of the lower whole-plant transpiration expressed as relative weight change in Fig. Thus, factors in addition to plant size probably contributed to the reduced transpiration rate. To determine which of these possibilities was the case, we calculated the stomatal index the ratio between stomata and epidermal pavement cells in a given leaf area.

This suggested that CK deficiency affects epidermal cell division but not stomatal patterning. Taken together, the results from both experiments indicated that changes in CK levels affect plant transpiration indirectly through developmental mechanisms and not through changes in stomatal movement.

AtCKX3 overexpression reduces stomatal density in tomato. A Stomatal aperture measured at B Number of stomata per 0. C Stomatal index stomata:epidermal cell ratio. AtCKX3 overexpression accelerates leaf senescence and reduces transpiration in tomato. A Chlorophyll content. B Stomatal aperture measured on imprints of abaxial epidermis taken at C Transpiration rate calculated from stomatal conductance measured at In A to C , all measurements were taken from leaves number 2 and 4 from the bottom up in plants with seven leaves.

Stomatal opening has been shown to be suppressed in senescing leaves Wardle and Short, We therefore examined stomatal aperture in leaves number 2 older and 4 younger in transgenic and M82 plants both at the seven-leaf stage. Reduced photosynthetic activity in senescing leaves has been suggested to increase CO 2 levels, leading to stomatal closure Thimann and Satler, a , b ; Wardle and Short, ; Willmer et al.

It is possible that the accelerated senescence reduces photosynthesis and increases CO 2 levels, which in turn promotes stomatal closure. The role of CK in the plant drought response has been linked to its antagonistic interaction with ABA Nishiyama et al. ABA had a similar effect on stomatal closure in the two genetic backgrounds Fig. Next, we examined whether the effect of increased levels of CK is the opposite of that of CKX3 overexpression on plant transpiration.

Three days after the last BA treatment, daily transpiration was measured using an array of load cells lysimeters. Treated plants displayed a small but significant increase in transpiration rate and stomatal conductance g s Fig. The daily pattern of stomatal conductance was unaffected Fig. This suggested that the BA treatments had no effect on stomatal movement.

Microscopic analysis of imprints taken from the abaxial epidermis of young leaves which unfurled during the treatment revealed that CK treatment had no effect on stomatal aperture Fig. This analysis also showed that CK increases stomatal density the number of stomata per given leaf area; Fig.

However, despite finding more and smaller pavement cells and more stomata in a given leaf area, the ratio between stomata and pavement cells was not affected by the BA treatment Fig. These results suggest that BA increased stomatal density by promoting epidermal cell division. It should be noted that while CK treatment increased the total number of cells per leaf area and reduced pavement cell size, it had no effect on the size of the guard cells Fig.

Since BA treatment had no effect on leaf size or total leaf area Supplementary Fig. S7 , the higher stomatal density should increase the total number of stomata per plant. Taken together, these results suggested that CK promotes whole-plant transpiration by enhancing epidermal cell division, which increases the total number of stomata. The relatively small effect of CK on transpiration Fig. Application of the cytokinin 6-benzylaminopurine BA to tomato M82 plants increased plant transpiration and stomatal density.

Plants were grown in the greenhouse on lysimeters and daily transpiration was measured 3 days after the last BA treatment. A Transpiration rate normalized to total leaf area. B Stomatal conductance g s normalized to total leaf area. D Stomatal aperture measured at E Number of stomata per 0.

F Stomatal index stomata:epidermal cell ratio. For C to F , epidermis imprints were taken from leaf number 3 from the apex down. The lack of effect of CK on stomatal movement raised the question of whether guard cells are sensitive to this hormone. S8 , suggesting that guard cells are sensitive to CK. This strong response in the guard cells raised the possibility that CK has a rapid but transient effect on stomatal movement that cannot be detected after several days.

We did not find any significant effect of the BA treatment on stomatal aperture Supplementary Fig. A recent study in Arabidopsis has suggested that CK has a role in the circadian regulation of stomatal movement Marchadier and Hetherington, To examine the possible role of CK in daily stomatal movement in tomato leaves, we used transgenic tomato plants expressing the YFP reporter gene under the regulation of the TCSv2 promoter and quantified stomatal aperture and YFP signal in the guard cells, twice a day morning and afternoon.

While the stomata closed toward the afternoon Fig. Stomatal aperture and TCS activity in guard cells of tomato during the day and in response to drought. A Stomatal aperture. B Percentage of stomata showing YFP signal. C YFP signal intensity.

In A to C , measurements were taken at D Stomatal aperture. E Percentage of stomata with YFP signal. F YFP signal intensity. In D to F , measurements were taken at G YFP signal in guard cell nuclei of irrigated plants Irig and plants exposed to mild drought stress D.

It is also possible that transient changes in CK levels play a role in the regulation of stomatal closure in response to water deficiency. Since not all guard cells showed YFP signal, we also compared stomatal aperture in YFP-expressing versus non-expressing stomata, in irrigated and drought-treated plants. We did not find differences in stomatal aperture between YFP-expressing and non-expressing stomata, in either treatment Supplementary Fig.

Taken together, we did not find any link between CK activity and stomatal movement in tomato plants. Plants adjust their transpiration rate to changes in the environment through a range of physiological and molecular mechanisms Osakabe et al. The rate of transpiration is regulated mainly by stomatal movement but is also affected by stomatal size and density Jones, ; Kramer and Boyer, ; Nir et al.

Previous studies in several plant species have shown that exogenous treatment with CK regulates stomatal aperture Dodd, ; Incoll and Jewer, In this study, neither endogenous nor exogenous manipulation of CK levels in tomato leaves had any effect on stomatal movement.

Taken together, our results do not support a role for CK in the regulation of diurnal stomatal activity or in the stomatal response to drought or ABA in tomato plants.

These leaves also exhibited reduced transpiration. Thus, reduced levels of CK promoted stomatal closure in mature leaves. Our results imply that this effect of CK is indirect, probably caused by premature leaf senescence. The effect of CK on leaf senescence is well documented, and transgenic plants with increased CK levels exhibit delayed senescence Gan and Amasino, Reduced photosynthesis during senescence Wingler et al.

Although previous studies in Arabidopsis and tobacco did not find premature senescence in transgenic plants overexpressing CKX Werner et al. Although CK did not affect stomatal movement, it promoted transpiration. Our results suggest that CK affects transpiration by promoting stomatal density through its general effect on cell division Miller et al. CK treatment of M82 leaves promoted cell division in the abaxial epidermis and, as a result, CK-treated leaves contained more epidermal pavement cells and more stomata, leading to a higher transpiration rate.

It should be noted that CK reduced pavement cell size but not guard cell or stomatal pore size. Taken together, these results suggest that increased CK levels increase the transpiration rate indirectly through a general effect on cell division.

Overexpression of CKX3 strongly reduced whole-plant transpiration. Leaf size and shape are strongly regulated by environmental conditions and hormones, including CK, and affect transpiration Bar and Ori, ; Fleishon et al.

Previous studies have suggested a role for leaf shape and size in plant adaptation to stress conditions Farris, ; Schurr et al. Under drought conditions, plant and leaf growth are strongly inhibited, in part by increased levels of ABA Creelman et al.

Several studies have shown that CK levels are also reduced under drought conditions Davies et al. Indeed, previous studies have found reduced stomatal density under drought conditions Xu and Zhou, Schmidt, R. Afriandi, unpublished. In the first trial, eight of the late successional species were planted in a degraded forest area, with microhabitats ranging from open areas dominated by grasses to shaded habitats under secondary forest cover.

The design was a randomized block design with species randomized within blocks. The average height growth per year of surviving plants was calculated based on height recorded at planting and 2 years after planting. For this analysis, we use only the six species where the number of surviving plants was more than four in each category of light.

Stomatal and gas exchange traits were analyzed using a General Linear Model GLM with two way interactions between species 11 levels and light treatment two levels , using the statistical software R version 3. The validity of the statistical models was evaluated through residuals plots, q-q plots, histograms of the residuals and the Shapiro—Wilk test. Tests were considered significant when the critical values P were less than 0.

Where results indicated significant effects of light treatment or interactions between light and species, differences within species were analyzed with t -tests. Associations between anatomical traits, physiological measurements and growth in the two associated trials were analyzed using mean values across species and treatments. We applied analyses of co-variance with the dependent variables e. Since the plants were measured species by species, there is a possible confoundation between species and the time of measurement.

The effect of this is likely to be small as anatomical traits tend to change little over short time periods. Furthermore, during the two and half months when the measurements took place, the weather conditions were relatively stable, making changes in the physiology of trees less likely. In any case, in the analyses of associations between stomatal characters, physiological traits and growth, such confoundation will add to the residual error and will not bias conclusions.

All anatomical parameters of stomata were significantly different between species. While a significant effect of light was found for stomatal density, stomatal size showed significant interactions between species and light. For length and width of guard cells, these interactions were marginally significant Table 1. The variation in guard cell form resulted in circular stomata in Eusideroxylon zwageri and Irvingia malayana , while other species tended to have elliptic stomata Table 1.

Within-species differences between shade and sun were significant only in Aquilaria malaccensis , where stomata in the shade were larger than in the sun. The significant effect of light indicated that the stomatal density tended to increase in the sun. Within species, large differences of stomatal density between shade and sun were found in I. Macaranga triloba was the only species with stomata on both leaf surfaces.

All gas exchange parameters were significantly different between species, and all parameters except g s-op had a significant interaction between species and light Table 2.

Within species, significant light effects were found in A. However, speed of stomatal opening varied considerably and ranged from 0. Three species were significantly affected by light, as M. The two included pioneer species were strongly different in stomata and gas exchange parameters, with small-sized stomata and fast stomatal opening in M.

This also means that there were no significant differences between the group of late successional species and the two pioneers considered as a group not shown. Table 2. The lower part of the table shows results from analysis of variance of the parameters as a function of species and light treatments. Result from analysis of co-variance of biomass in response to physiological traits of six late successional species. Numbers in parenthesis indicate the numbers of degrees of freedom.

Analyses of co-variance showed that smaller stomata opened faster than larger stomata Figure 1 A. Large stomata hence tended to open more slowly in the shade than in the sun Figure 1. Closed circles and the solid line indicate the shade plants, while open circles and the dashed line indicate sun plants.

Each point represents the mean of a species. Note the logarithmic scales on the Y -axis. Symbols in red represent the two early successional species. As use of stomatal imprints may lead to underestimation of the true size of stomata if epidermal cells overlap with guard cells, we made cross-sections of leaves of M.

These sections showed that the guard cells were fully exposed and the outlier tendency in this species is unexplained. The relationships between stomatal density and stomatal size A , and between speed of opening and g s-op across species B.

Symbols in red represent the two pioneer species. In A , one species M. Differences between the annual height growth of the six species tested in Harapan Rainforest were highly significant and showed significant interactions with the light environment. Height growth was not related to any of the stomatal or gas exchange parameters. However, biomass showed a significant association with g s-dark , g s-op and speed of opening Table 3 , Figure 3 , generally being larger for species with larger gas exchange and faster speed of opening.

Furthermore the light environment had a significant influence, biomass being larger under shaded conditions. The relationships between dry biomass and g s-dark A , g s-op B and speed of opening C.

Closed and open circles represent shade and open treatments, respectively. Our results confirmed a relation between size of stomata and the speed of opening as previously observed by Drake et al. Whereas the study of Drake et al. Hence, there is reason to believe that the association between stomatal size and the speed of opening applies more generally. Similar relationships seem to apply within species, as studies of transpiration following leaf detachment in Rosa hybrid and Solanum introgression lines have shown that varieties and lines with small stomata tend to be more responsive than varieties and lines with large stomata Giday et al.

Conversely, Elliott-Kingston et al. However, while pore length may be an appropriate proxy for size in closely related plants where the dimensions of stomata scale with the pore lengths, it is likely less robust when species with different shapes of stomata are compared. If the limiting factor for stomatal speed of opening is the rate of change in osmotic potential in the guard cells Raven , volume or a related factor such as projected area as in our case are likely to be better proxies.

In angiosperms, stomatal opening appears to involve a displacement of the subsidiary cells. This has consequences for the speed of opening, as lycopod and fern species without subsidiary cells had less responsive stomata compared with a dicot and a grass, despite having close-to-similar sizes of stomata Franks and Farquhar Likewise, Elliott-Kingston et al.

Generalizations beyond the group of angiosperm trees that we studied should therefore be treated with care. The light treatment influenced both stomatal size and density, and our results show that the relationship between size and speed of stomatal opening also varied with the light climate, as the stomatal size had a larger influence on the speed in plants grown in shade compared with sun plants Figure 1. In the absence of studies on this topic we speculate that this may be caused by changed stomatal micromorphology or biochemistry in response to light conditions.

Possible candidates could be changed activities of enzymes responsible for building up high levels of osmotic potential or changes in the concentrations of substrates needed for this Raven While stomatal density generally was higher in plants in the sun compared with shade plants, some species had larger and some species smaller stomata in response to the light treatment Table 1.