2003_The role of stomata in sensing and driving environmental change

The role of stomata in sensing and driving environmental change Alistair M. Hetherington1 & F. Ian Woodward2 1Department of Biological Sciences, The Lancaster Environment Centre, University of Lancaster, Lancaster LA1 4YQ, UK 2Department of Animal & Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK ............................................................................................................................................................................................................................................................... Stomata, the small pores on the surfaces of leaves and stalks, regulate the flow of gases in and out of leaves and thus plants as a whole. They adapt to local and global changes on all timescales from minutes to millennia. Recent data from diverse fields are establishing their central importance to plant physiology, evolution and global ecology. Stomatal morphology, distribution and behaviour respond to a spectrum of signals, from intracellular signalling to global climatic change. Such concerted adaptation results from a web of control systems, reminiscent of a ‘scale-free’ network, whose untangling requires integrated approaches beyond those currently used. S tomata (Fig. 1) are small pores on the surfaces of leaves and stems, bounded by a pair of guard cells, that control the exchange of gases—most importantly water vapour and CO2—between the interior of the leaf and the atmosphere. In this capacity they make major contrib- utions to the ability of the plant to control its water relations and to gain carbon. Gas exchange is regulated by controlling the aperture of the stomatal pore and the number of stomata that form on the epidermis. Environmental signals such as light intensity, the con- centration of atmospheric carbon dioxide and endogenous plant hormones control stomatal aperture and development. The acqui- sition of stomata and an impervious leaf cuticle are considered to be key elements in the evolution of advanced terrestrial plants1, allowing the plant to inhabit a range of different, often fluctuating environments but still control water content. Here, we describe how the application of knowledge from cognate disciplines is providing new insights into how stomata evolve and are able to process information from simultaneous, often conflicting and sometimes rapidly changing signals. Although it is too early to say whether these recent advances will result in paradigm shifts in our under- standing of how plants both respond to and drive environmental change, it is quite clear that stomata are a key experimental tool to investigate these phenomena. Before considering specific aspects of stomatal biology it is important to reflect on the impact of stomata at the global level. Although the total stomatal pore area may be only 5% of a leaf surface2, the rate of water vapour loss may reach as high as 70% of a similar structure without a cuticle. Stomata exert major controls on both the water and carbon cycles of the world. Annual precipitation over the land is about 110,000 km3, or 110 £ 1015 kg (ref. 3) and evaporation and transpiration total about 70 £ 1015 kg. The con- tribution of stomatal transpiration alone to the global water cycle can be determined by using a dynamic vegetation model4 (Fig. 2). The greatest rates of transpiration occur in the uniform and warm forested areas between the tropics with 32 £ 1015 kg yr21 of water vapour passing through stomata. This is double the water vapour content of the atmosphere (15 £ 1015 kg yr21). Terrestrial gross photosynthesis annually fixes about 120 £ 1015 g C (440 £ 1015 g CO2) from the atmosphere’s 730 £ 1015 g C (ref. 5). The global distribution of this flux parallels the distribution of transpiration, indicating the closely coupled controls of stomata on CO2 and water vapour diffusion. Stomatal evolution Stomatal control of water loss allows plants to occupy habitats with fluctuating environmental conditions and so it can be predicted that stomata must be important contributors to speciation and evolu- tionary change. Stomata first appeared in terrestrial land plants over 400 million years ago (Myr)6 and since then have changed markedly in size and density on plant surfaces. There are two broad morpho- logical types of stomata, the dumb-bell-shaped stomata typical of the grasses and the kidney-shaped form found in other species (Fig. 1). Is there any evidence that stomata are involved in speciation? Figure 1 Dumb-bell-shaped stoma of rice typical of the grasses (left) and the kidney-shaped stoma typical of other species such as Arabidopsis and Commelina (right). review article NATURE | VOL 424 | 21 AUGUST 2003 | www.nature.com/nature 901© 2003 Nature Publishing Group Intriguingly, over the 400 million years of the Phanerozoic era, periods of low atmospheric CO2 concentrations are associated not only with high stomatal densities7 but also the emergence of new plant groups such as the ferns, pteridosperms and angiosperms8. Whether this involves some interaction and indeed causation requires further investigation. Correlations between changes in global environmental conditions and stomatal evolution can be demonstrated for species in the Proteaceae and for the evolution of dumb-bell stomata in the Poaceae. These changes occurred in the Cenozoic era of the last 66 million years when there were profound changes in global climate and terrestrial flora and fauna9. Chloroplast DNA has been used to derive a phylogeny for species of Banksia and Dryandra in the Proteaceae of Australia10. Two clades of species are currently recognized by differences in stomatal distributions. In the clade Cryptostomata, stomata occur in shallow pits or in crypts, whereas they have a more superficial distribution in the clade Phanerostomata. The superficial occurrence appears to be the primitive state and species in the clade Phanerostomata occur in moist climates (such as the most recent common ancestor). Species of the clade Cryptostomata occur in much drier climates and probably diverged from the Phanerostomata clade 55–35 Myr, at a time when the climate was becoming more arid11. Although stomatal differences are not the only differences between the clades, the marked differences in stomatal location would have exerted differential capabilities for the spread and survival of the two clades in moist and arid climates11. The environmental correlates of the differences in stomatal distribution seen for the Proteaceae are nicely demonstrated in Cistus incanus, for which similar differences in stomatal distribution occur, but between the summer and winter of a Mediterranean climate12. Leaves produced in the cool and wet winter are large and flat with frequent stomata on the abaxial leaf surface; however, leaves developed in the hot and dry summer are crimped and partially rolled, forming a crypt on the lower surface, the only location of stomata. The Poaceae consists of about 10,000 species for which the macro-evolutionary history has recently become established by the analysis of chloroplast and nuclear DNA13. The linear dumb- bell-shaped stomata of grasses (Fig. 1) are generally believed to represent a more evolutionary advanced form than their kidney- shaped counterparts. This is supported by the observation14 that during development, Timothy grass guard cells adopt a transient kidney-shaped phase before assuming their typical (mature) dumb- bell shape. The linear dumb-bell design magnifies small changes in width to cause large openings, and maximizes the potential of the stomata to track changes in environmental conditions, probably with little energetic cost. Smaller changes in guard and subsidiary cell turgor lead to greater increases in stomatal aperture15 in the dumb-bell-shaped stomata than occur for kidney-shaped stomata. This efficiency and speed of stomatal opening in grasses enhances photosynthesis and water use efficiency compared with non-grass species16. A rapid stomatal response to blue light augments photo- synthesis in early morning and in intermittent sunlight, in which light has an enhanced blue light content16 and which would have characterized the understorey environment during the early evolu- tion of grasses. The low aerodynamic conductance of a grassland canopy could reduce the impact of changes in stomatal aperture on gas exchange8. However, field observations17 demonstrate a limited impact of aerodynamic conductance on stomatal dynamics. Grasses originated between about 55 and 70 Myr (ref. 13), leading to lineages that were understorey plants of tropical forests. Their spread and diversification, during global aridification 30–45 Myr, would have been enhanced by the dumb-bell stoma, capable of responding quickly and efficiently to the enhanced light conditions of newly open habitats, but with the capacity to avoid the increased likelihood of drought. This period just preceded the diversification of grazing animals9,18 and was well before the origin of the C4 pathway of photosynthesis. Animal grazing and browsing of grazing-intolerant shrubs and trees would have enhanced the spread of grazing-tolerant grasses, particularly into areas of open wood- land, whereas the higher albedo of the grasslands may have enhanced regional aridity18. Environmental control of stomatal development We shall not discuss here the details of stomatal development but rather the control of stomatal distribution and size by environmen- tal factors. Depending on the species and the environmental conditions stomata range in size from about 10 to 80 mm in length and occur at densities between 5 and 1,000 mm22 of epidermis2 (Fig. 3a). In spite of this wide variability there is a strong and general relationship between density and size (Fig. 3a) for different plant Figure 2 Transpiration (mm yr21) from terrestrial vegetation simulated with the Sheffield Dynamic Global Vegetation Model (SDGVM)4 and averaged for the 1990s. review article NATURE | VOL 424 | 21 AUGUST 2003 | www.nature.com/nature902 © 2003 Nature Publishing Group groups (grasses and non-grasses), fossil leaves and for different stomatal distributions on either one or both leaf surfaces. Simu- lations with a stomatal model of gaseous exchange7 indicate that the average relationship in Fig. 3a has virtually the same trend to one in which changes in density and size are exactly compensatory, in terms of CO2 and water vapour exchange. Apart from the earliest land plants of the Silurian and Devonian periods, with very low stomatal densities and small sizes, it also seems that this relationship has existed for the last 300 million years. On this basis it is not obvious how selection favours any particular species’ characteristic of stomatal density and size. The plant must maintain movement of water from the soil to the leaf, and rapid stomatal responses to environmental change are a major feature of this maintenance1. One study19 has demonstrated that stomatal size has a key role in this control and for six forest trees there is a clear negative relationship between the length of the stomatal pore and sensitivity to increasing drought. In these species larger stomata (species with kidney-shaped stomata) were slower to close and demonstrated a greater potential for hydraulic dysfunc- tion under drought. Ferns from deep shade possess large stomata at low densities20 and in this natural environment, which may be cool and humid, it is found that truly shade-tolerant species often retain open stomata, even in deep shade, at least for early parts of the day21. The constancy of the open stomata will minimize the impact of what would otherwise be slow opening limitations to photosynthesis during short-lived periods of sunlight, which are critical for enhancing photosynthesis in this light-limited environment. There- fore the limited available information suggests that large kidney- shaped stomata seem to be an important feature for plants of humid and deep shade conditions but their slow dynamic behaviour could lead to problems under dry conditions. Small stomata can open and close more rapidly and their general association with high densities (Fig. 3a) provides the capacity for rapid increases in the stomatal conductance of a leaf, maximizing CO2 diffusion into the leaf during favourable conditions for photosynthesis19. The effect of growth at elevated concentrations of CO2 on stomatal density and stomatal index (the fraction of epidermal cells that are stomata) is one of the most intensively studied environmental controls on stomatal development. CO2 enrichment changes the stomatal density of different species and different accessions (ecotypes) of Arabidopsis thaliana (Fig. 3b and ref. 22). With stomatal densities ranging from 45 to 720 mm22 the mean response is an 11% reduction in density with a doubling of the CO2 concentration (Fig. 3b), and which is insensitive to the basal Figure 3 Control of stomata by the environment. a, Relationship between stomatal width and density. Data from amphistomatous species, hypostomatous species, grasses and fossil leaves are shown. The data are from refs 2, 7, 67–69 and F.I.W. (personal observations). The solid curve is log-normal, y ˆ 228.75 ‡ 162x 20.2086, r 2 ˆ 0.5; the dashed line shows equal stomatal conductance with variable stomatal density. b, Responses (per cent change) of stomatal density to CO2 doubling for 125 species and 63 accessions of A. thaliana (from ref. 22 and new observations). Smoothed contours indicate fraction of genotypes (%) with particular values of stomatal density and per cent response to enrichment. c, Variations in stomatal characteristics with different genotypes. Curve as for a. Birch polyploids (2n, 5n, 6n)70, apple polyploids (3n, 4n)71, different races of pinyon pine72, drought-selected lines in barley73 and different polyploids in coffee74 are shown. d, Stomatal responses to different environmental conditions. Curves as for a. CO2 enrichment experiments, prairies 75; Arabidopsis (our own unpublished data) where adaxial surface (filled triangles) and abaxial surface (inverted filled triangles) are indicted; tulip–poplar changes through a canopy76; and drought responses of cotton77 are shown. Different ecotypes are represented by different colours within groups. review article NATURE | VOL 424 | 21 AUGUST 2003 | www.nature.com/nature 903© 2003 Nature Publishing Group stomatal density. The reduction in stomatal density with CO2 enrichment leads generally to a decrease in maximum stomatal conductance but an increase in the maximum rate of photosyn- thesis, at the elevated CO2 concentration 7,8,22. Royer23 established that the reductions of stomatal density and stomatal index associated with growth under increased concen- trations of atmospheric CO2 were recorded more frequently in observations from longer-term measurements of stomatal responses (herbarium material with a decade to century timescale and from fossil leaves with a millennial timescale) than in short- term field or growth chamber seasonal experiments. Royer suggested that this increased frequency of response was due to the transition from a variable plastic response by species in short-term experiments to a genetic response, as a result of selection on longer timescales. Figure 3c supports the suggestion that genetic change can alter the stomatal density–size relationship (Fig. 3a), whereas environmental changes seem primarily to move the position of a species along the stomatal size–density curve (Fig. 3d). Changes in the degree of ploidy can also change stomatal density and size (Fig. 3c). Notably, in Arabidopsis, an unresponsive accession (C24) for the stomatal density response to CO2 enrichment is actually regulated to show this minimum response because the mutation of a single gene leads to a very significant response to CO2 (ref. 24). Currently we know rather little about the signalling pathways by which environmental signals control stomatal development. Recent work has shown that in Arabidopsis the HIC gene, which encodes a putative 3-ketoacyl CoA synthase (KCS), is involved in the control of stomatal development by elevated concentrations of CO2 (ref. 24). As KCS is involved in the synthesis of wax components found in the cuticle it has been suggested that in hic an alteration to cuticular structure and properties interferes with the diffusion of an endogenous inhibitor that controls stomatal development24. This suggestion receives support from the observation that some Arabi- dopsis wax-deficient mutants display abnormal stomatal pattern- ing24,25. Analyses of other Arabidopsis mutants26 indicate that the CO2 response of stomatal index is absent in fad-4, a jasmonic acid mutant, whereas in ein-2, an ethylene-insensitive mutant, the CO2 response is absent only from the adaxial leaf surface. The jasmonate and ethylene transduction pathways are also involved in defence responses to pathogens27 and the ein-2 mutant is susceptible to attack by pathogens. It is remarkable that mechanisms for addres- sing pathogen attack are also central in the responses of stomatal development to CO2 concentration. Accessions of Arabidopsis differ widely in resistance both to powdery mildew disease28 and to CO2 concentration22, and it is tempting to link these two major responses, but experimental support is still wanting. Any connec- tion between the two processes would influence strongly the processes of selection for the stomatal response to CO2, in the long term, supporting the notion that the response is more reliable the longer the period of study23. More recent work29 shows that, similar to responses to pathogen attack30 there is also systemic control of stomatal development during growth under elevated CO2 as mature leaves both detect CO2 and produce a signal to influence development of younger expanding leaves. The work on the ein-2 mutant of Arabidopsis26 suggests that ethylene differentially controls stomatal development on the upper and lower leaf surfaces. Comparing stomatal densities among Solanum pennellii, Lycoperscicon esculentum and a graft-induced periclinal chimaera, having an S. pennellii epidermis and an L. esculentum mesophyll, showed31 that the two surfaces could behave independently. Both donors had more stomata on the lower than on the upper surface and L. esculentum had a greater stomatal density than S. pennellii. The stomatal density on the upper epidermis of the chimaera was similar to that of the epidermal donor S. pennellii. However, the density on the chimaeral abaxial epidermis was significantly lower than its donor. These data suggest that stomatal differentiation is subject to different controls on each surface, and at least in the case of the lower epidermis a role for the mesophyll seems possible. Control of stomatal aperture by environmental signals Recent work shows that the control of stomatal aperture by envi- ronmental signals depends on coordinated alterations to guard cell turgor (ionic fluxes and sugars), cytoskeleton organization, membrane transport and gene expression (see refs 32 and 33 and references therein). A number of lines of evidence suggest that Figure 4 Model of guard cell signalling. a, Guard cell ABA and blue light signalling represented by a network-based model. In this model the nodes represent modules or groups of functionally related processes or second messengers. Modules linked by blue connections indicate that these links exist in guard cells. Red connections indicate that these links occur in other plant cells but their existence has not been investigated in guard cells. Yellow font indicates that an ABA receptor has not been isolated in guard cells. BLR, blue light receptor; pmc, plasma membrane ion channels; traffic, membrane trafficking; cytosk., cytoskeleton; vc, vacuolar ion channels; metab., metabolism of