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Passive Origins of Stomatal Control in Vascular Plants

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Science  04 Feb 2011:
Vol. 331, Issue 6017, pp. 582-585
DOI: 10.1126/science.1197985

Abstract

Carbon and water flow between plants and the atmosphere is regulated by the opening and closing of minute stomatal pores in surfaces of leaves. By changing the aperture of stomata, plants regulate water loss and photosynthetic carbon gain in response to many environmental stimuli, but stomatal movements cannot yet be reliably predicted. We found that the complexity that characterizes stomatal control in seed plants is absent in early-diverging vascular plant lineages. Lycophyte and fern stomata are shown to lack key responses to abscisic acid and epidermal cell turgor, making their behavior highly predictable. These results indicate that a fundamental transition from passive to active metabolic control of plant water balance occurred after the divergence of ferns about 360 million years ago.

The evolution of stomata at least 400 million years ago (1) enabled plants to transform their epidermis into a dynamically permeable layer that could be either water-tight under dry conditions or highly permeable to photosynthetic CO2 during favorable conditions. The combination of adjustable stomata with an internal water transport system was a turning point in plant evolution that enabled vascular plants to invade most terrestrial environments (2). Today, the leaves of vascular plants possess arrays of densely packed stomata, each one comprising a pair of adjacent guard cells (Fig. 1). High turgor pressure deforms the guard cells to form an open pore, which allows rapid diffusion of atmospheric CO2 through the epidermis into the photosynthetic tissues inside the leaf. Declining turgor causes the guard cells to close together, greatly reducing leaf water loss while also restricting entry of CO2 for photosynthesis. Despite the anatomical simplicity of the stomatal valve, there is little consensus on how angiosperm stomata sense and respond to their extrinsic and intrinsic environment. Because stomata are the gatekeepers of terrestrial photosynthetic gas exchange, understanding their dynamic control is imperative for predicting CO2 and water fluxes across all scales from leaf (3, 4) to globe (5, 6).

Fig. 1

Contrasting stomatal responses to exogenous ABA applied to diverse lineages of vascular plants. (A to D) Typical time courses of stomatal conductance (mol m−2 s−1) are shown after introduction of a relatively concentrated 5.67 × 104 μmol m−3 ABA solution into the xylem a short distance from sample leaves. Dotted red lines show the moment of ABA application. The stomata of the angiosperm Helianthus annuus (A) and conifer Callitris rhomboidea (B) closed rapidly in response to ABA. In contrast, neither the lycophyte Lycopodium deuterodensum (C) nor the fern species Pteridium esculentum (D) showed any response to high xylem ABA concentration, despite the accumulation of high levels of ABA in the leaves of both species. Only leaf excision (arrow) caused stomatal closure in these two ancestral lineages. Stomatal anatomy and dimensions were similar for each species (light microscope images taken at 100× magnification; cuticles stained with crystal violet).

Stomatal research has focused on the most abundant and successful group of vascular plants, the angiosperms. Within this plant group, various independent control “modules” have been proposed to explain guard cell responses to light (7, 8), the concentrations of phytohormones [most notably abscisic acid (ABA)] (9), and metabolic rate (10). Our understanding of the response of angiosperm stomata to environmental parameters remains imprecise because we know little about the mechanics of such stomatal control modules (1113). Even the most fundamental action of stomata as regulators of leaf water balance is poorly understood because of interactions between active and passive processes in the leaf epidermis (1416).

We sought to disentangle the complex stomatal regulation of water balance in angiosperms by reconstructing the earliest, hypothetically less complex stomatal control process that existed in the common ancestor of vascular plants. Recent studies indicate that fern stomata do not respond to blue light (17) or elevated ambient CO2 concentrations (18), both characteristic activators of stomatal movement in angiosperm leaves. The absence of key stomatal control components in ferns raises the hypothesis that early-diverging clades of vascular plants may preserve an ancestral stomatal behavior that predates much of the complexity present in angiosperm stomatal responses.

In vascular plants, the primary function of stomata is the control of plant hydration (2); in seed plants, stomatal control of leaf water balance is thought to occur primarily by active metabolic regulation of stomatal aperture rather than by passive responses of stomata to leaf hydration (11). Two lines of evidence support this view: (i) the induction of stomatal closure by the phytohormone ABA (19), and (ii) the transient “wrong-way” response of stomata to rapid changes in leaf hydration (20). We targeted these two key indicators of metabolic regulation of water balance in two basal lineages of vascular plants—ferns and lycophytes—to determine whether their stomata also regulate leaf water balance by metabolic rather than passive control.

We first examined whether fern and lycophyte stomata were sensitive to ABA by adding high concentrations of this phytohormone to the transpiration stream of a group of nine fern and lycophyte species as well as representative seed plants (angiosperm and gymnosperm species). In agreement with previous studies (21), we found that the addition of ABA to the transpiration stream of angiosperm leaves (Asteraceae, Helianthus annuus) and gymnosperm leaves (Ginkgoaeae, Ginkgo biloba; Cupressaceae, Callitris rhomboidea) led to rapid stomatal closure at stem xylem ABA concentrations of 15,000 ng ml−1 (5.67 × 104 μmol m−3) and leaf levels of 1500 to 2000 ng g−1 fresh weight (Fig. 1, A and B, and fig. S1). By contrast, we found no ABA sensitivity in any of the sampled fern and lycophyte species even when a 15,000 ng ml−1 solution was fed into the leaf transpiration stream for 90 min (Fig. 2). To ensure that ABA levels in the leaf epidermis were high, and to rule out the possibility that xylem-delivered ABA was catabolized before it reached the stomata, we tested a range of concentrated ABA solutions and found no effect of any concentration even when leaf ABA levels were >7000 ng g−1 (fig. S2). Furthermore, we found that control levels of ABA in the leaves of all fern and lycophyte species were extremely low (<10 ng g−1; table S1) relative to levels in the angiosperm (60 ng g−1) and gymnosperm species (90 to 272 ng g−1), which were within the normal range for unstressed seed plants (fig. S3). ABA levels in the leaves of seed plants rarely rise above 1200 ng g−1 fresh weight, even during water stress (table S2 and fig. S3), and the lack of a stomatal response in our fern or lycophyte species to foliar concentrations in excess of 7000 ng g−1 implies that ABA is not involved in stomatal closure in these plants. After 90 min without response, the ABA-fed fern and lycophyte leaves were severed, causing immediate and rapid stomatal closure (Fig. 1). This confirmed that the absence of a closing response to ABA in ferns and lycophytes was due to ABA insensitivity rather than an inability of stomata to close effectively.

Fig. 2

Absent ABA responses in the stomata of a diverse sample of ferns and lycophytes. (A) Changes in stomatal conductance 90 min after the introduction of ABA into the transpiration stream at an equivalent xylem sap concentration of 5.67 × 104 μmol m−3, in nine fern and lycophyte species and the representative angiosperm Helianthus annuus and gymnosperms Ginkgo biloba and Callitris rhomboidea. Values shown are percentages relative to an initial steady-state stomatal conductance established under optimal conditions. Means ± SE are shown for Dicksonia antarctica and Pteridium esculentum additionally fed both half and double the above-mentioned concentration. (B) Foliar ABA level (ng g−1) after 90 min of feeding an equivalent ABA xylem sap concentration of 5.67 × 104 μmol m−3 into the transpiration stream.

In the absence of an active ABA closure pathway in ferns and lycophytes, we hypothesized that the earliest evolved stomatal response to plant water deficit in vascular plants was a passive process, driven by the direct effect of leaf water content on the turgor of the guard cells. Such a relationship seems intuitively correct in terms of energetics, but in angiosperms, passive stomatal closure in response to desiccation has been ruled out by the role of subsidiary cells in the angiosperm stomatal complex (22). Water deficit in angiosperm leaves causes subsidiary cells to lose turgor before the guard cells, and this collapse causes the stomata to open “hydropassively” rather than closing during rapid dehydration (23). However, differences in stomatal development between seed plants and nonseed plants mean that the guard cells of ferns and lycophytes may not be as intimately related to neighboring epidermal pavement cells as in seed plants (24). Correspondingly, we found that none of the fern or lycophyte species examined showed evidence of hydropassive stomatal opening when leaves were severed while transpiring (fig. S4). The absence of either ABA-mediated or epidermal cell–mediated responses to water balance in these early-diverging vascular plant clades strongly supports the concept that the ancestral state for stomatal control of water balance is for stomata to act as passive hydraulic valves.

To test the passive hydraulic valve hypothesis in ferns and lycophytes, we determined whether observed stomatal responses to changing leaf water balance could be explained with an explicit model that predicted stomatal aperture as a function of leaf water content (25). After measuring the water transport and capacitance properties of two species of ferns and one lycophyte, we found that stomatal conductance to water vapor, measured in leaves during step transitions in ambient water vapor pressure, behaved exactly as predicted by the passive hydraulic valve model (Fig. 3). Step transitions in humidity caused the stomata of Pteridium, Dicksonia, and Lycopodium to respond according to exponential decay or rise functions very close to the modeled kinetics under passive hydraulic control (Fig. 3, B to D). Furthermore, we found that steady-state stomatal conductances after humidity transitions were almost identical to the absolute values predicted by the hydraulic model (Fig. 3A). Rapid dehydration of leaves produced by leaf excision also led to rapid stomatal closure, which likewise followed closely the kinetics predicted by the hydraulic model (Fig. 3, E to G).

Fig. 3

The transient and steady-state responses of fern and lycophyte stomata conform strongly to a passive hydraulic model. (A) Equilibrium stomatal conductance before and after step changes in vapor pressure deficit showed very close agreement with predictions from a passive hydraulic model of stomatal control in two fern species, Dicksonia antarctica and Pteridium esculentum, and the lycophyte Lycopodium deuterodensum. None of the species showed any significant deviation from a 1:1 relationship between observed and predicted stomatal conductance. Observed transient data between equilibrium states and after leaf excision also conformed strongly to predicted values in all species. (B to G) Representative dynamic responses of stomata to step increases and decreases in vapor pressure [(B) to (D); red line] and leaf excision [(E) to (G); arrows] show clear agreement between observed stomatal conductance time courses (blue circles) and modeled conductances (thick black line) derived from a passive hydraulic model. Despite large differences in the half times of the stomatal response kinetics between species, all showed minimal deviation from predicted kinetics.

Our data demonstrate that daytime stomatal aperture in ferns and lycophytes can be precisely predicted by leaf water content because stomata conform to a passive hydraulic valve model of aperture regulation. The only evidence of active stomatal control in these plants is the response to red light, apparently via the induction of photosynthesis in guard cell chloroplasts (17) (fig. S5). Beyond this, we found no evidence for the active control of plant hydration in fern and lycophyte species. Ancestral stomatal function in vascular plants is therefore reconstructed as being free of ion-pumping processes in response to dehydration, requiring only equilibrium in turgor between guard cells and the rest of the leaf tissue for stomatal movement. Under these conditions, declining leaf water content has a direct effect on guard cell turgor, which in turn directly reduces stomatal aperture and transpiration. On the basis of this reconstruction, our data indicate a major shift in the stomatal control process between early-diverging spore-bearing vascular plant clades (lycophytes and ferns) and later-diverging seed plants (gymnosperms and angiosperms).

Our findings suggest that the complexity of stomatal water balance control in seed plants is due to features derived after the divergence of ferns, about 360 million years ago. Key derived features in seed plants, including the mechanical advantage of epidermal cells over guard cells, and guard cell sensitivity to ABA, are likely to be linked because both invoke the active transport of ions. In angiosperms, active ion exchange between guard cells and epidermal cells is required for stomata to close in response to decreasing leaf water content (22), and ABA appears to activate ion exchange between guard cells and epidermal cells (26). However, the absence of ABA responses in ferns and lycophytes is not due to absent ABA synthetic genes, given that most of the ABA synthetic and sensory genes sequenced in Arabidopsis are present in the genome of the moss Physcomitrella (27). The presence of ABA-associated genes in ferns and lycophytes is consistent with the demonstrated role of ABA in mosses and ferns in both the sexual determination of gametophytes (28) as a promoter of dehydration resistance (29) and in vegetative phase change (30). It seems likely that seed plants have co-opted the existing ABA stress-signaling pathway as a means of modifying guard cell responses to leaf water content.

Further work on bryophyte stomata is required to find the functional root of the stomatal lineage in land plants (31), although this group will need to be approached with caution because the function of stomata in mosses is often not related to the maintenance of plant hydration (32). It is possible that early stomatal evolution may mirror early vascular evolution, whereby a diversity of morphotypes characterized early-branching clades before form and function became canalized in the tracheophyte lineage (2, 33).

Complexity in seed plant stomatal behavior has evolved from a common ancestor with a simple passive stomatal control process, found to operate today in ferns and lycophytes. A derived system of active rather than passive stomatal control of water balance in seed plants provides an evolutionary mechanism that allows great adaptability in the regulation of plant water content and response to drought. The evolution of active stomatal regulation of water balance in angiosperm and gymnosperm clades likely contributes to their great success relative to ferns and lycophytes, particularly in water-limited environments.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1197985/DC1

Materials and Methods

Figs. S1 to S6

Tables S1 to S3

References

References and Notes

  1. See supporting material on Science Online.
  2. We thank K. Mott and G. Jordan for discussion and comments on the manuscript, and J. Ross and N. Davies for assistance with ABA quantification techniques. Supported by Australian Research Council grants DP0878177 and DP0559266 (T.J.B.) and an APA award (S.A.M.M.).
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