Land Plants Acquired Active Stomatal Control Early in Their Evolutionary History

Home , Cooksonia, Selaginella tamariscina, Selaginella uncinata

Current Biology 21, 1030–1035, June 21, 2011 ª2011 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2011.04.044 Report Land Plants Acquired Active Stomatal Control Early in Their Evolutionary History

Elizabeth M. Ruszala,1 David J. Beerling,2 Peter J. Franks,2,4 the basis of a major evolutionary hypothesis proposing that Caspar Chater,2 Stuart A. Casson,1 Julie E. Gray,3 as plants diversiﬁed, increasingly complex stomatal control and Alistair M. Hetherington1,* responses evolved to improve the economics of photosyn- 1School of Biological Sciences, University of Bristol, Bristol thesis and facilitate colonization of new terrestrial environ- BS8 1UG, UK ments [7, 8]. The alternative hypothesis is that the ﬁrst stomata, 2Department of Animal and Plant Sciences appearing on early land plants some 420 million years ago, 3Department of Molecular Biology and Biotechnology possessed a suite of active aperture control responses to envi-

University of Sheffield, Sheffield S10 2TN, UK ronmental stimuli, including ABA and CO2. 4Faculty of Agriculture, Food & Natural Resources, The Here we evaluate these hypotheses with comprehensive University of Sydney, Sydney, NSW 2006, Australia mechanistic analyses of guard cell physiology using the most primitive extant vascular plant division, the lycophytes [10](Figure 1). Lycophytes first appeared about 420 million Summary years ago and dominated the landscape as forests in the Carboniferous period, reaching heights of up to 30 m [9, 11, Stomata are pores that regulate plant gas exchange [1]. They 14]. Our approach represents a significant departure from evolved more than 400 million years ago [2, 3], but the origin that of recent investigations [7, 8] in that we focus on the extant of their active physiological responses to endogenous and lycophyte Selaginella uncinata, a member of a model genus environmental cues is unclear [2–6]. Recent research that is tractable to detailed in-depth physiological experimen- suggests that the stomata of lycophytes and ferns lack tation [9]. We use this species and exploit detailed mechanistic pore closure responses to abscisic acid (ABA) and CO2. knowledge of how stomata open and close gleaned from the This evidence led to the hypothesis that a fundamental tran- model angiosperm Arabidopsis[10] to show that the active sition from passive to active control of plant water balance control of guard cell function is present and well developed occurred after the divergence of ferns 360 million years in a lineage predating the evolution of ferns. ago [7, 8]. Here we show that stomatal responses of the lycophyte Selaginella [9] to ABA and CO2 are directly comparable Selaginella Stomata Respond to ABA and CO2 to those of the flowering plant Arabidopsis [10]. Further- We focused first on stomatal responses to the drought more, we show that the underlying intracellular signaling hormone ABA. In flowering plants, this phytohormone builds pathways responsible for stomatal aperture control are up under conditions of reduced water availability and, among similar in both basal and modern vascular plant lineages. other responses, promotes reductions in stomatal aperture, Our evidence challenges the hypothesis that acquisition of thereby allowing the plant to conserve water [10]. In Selagi- active stomatal control of plant carbon and water balance nella, ABA also accumulates during periods of reduced water represents a critical turning point in land plant evolution availability and is involved in desiccation tolerance [15]. Inves- [7, 8]. Instead, we suggest that the critical evolutionary tigating whether ABA promotes reductions in the aperture of development is represented by the innovation of stomata S. uncinata is a key experiment in determining whether lyco- themselves and that physiologically active stomatal control phyte stomata exhibit active control of stomatal function. originated at least as far back as the emergence of the lyco- Our fully replicated results indicate that the addition of ABA phytes (circa 420 million years ago) [11]. from 0 to 25 mM induced a dose-dependent reduction in stomatal aperture and a dose-dependent inhibition of light- Results and Discussion induced stomatal opening (Figures 2A and 2B). These data indicating that the stomata of S. uncinata exhibit similar Molecular phylogenies indicate that extant land plants include responses to ABA as those of higher plants were comple- three ‘‘nonvascular’’ lineages: liverworts, mosses, and horn- mented by detailed gas-exchange analysis on isolated shoot worts, collectively known as bryophytes (Figure 1)[12, 13]. material. When isolated shoots of S. uncinata were fed Although these lineages possess tissue specialized for internal increasing concentrations of ABA, stomatal conductance to transport of water, they lack xylem and phloem (the vascular water vapor, gw, markedly declined and then recovered system) found in more advanced lineages. Within the bryo- when ABA was withdrawn (Figure 2C). Our physiological phytes, stomata are found in hornworts and mosses but not in results provide three lines of evidence that S. uncinata stomata liverworts [12]. The remaining extant plants, all of which respond to ABA and are supported by observations that horn- possess stomata, are known as vascular plants because they wort stomata are sensitive to ABA [16]. Hornworts are a group contain internal water-conducting tissues (Figure 1). Among of nonvascular land plants that occupy an even more evolu- vascular plant lineages, lycophytes and ferns are the most basal tionarily ancient phylogenetic position than lycophytes (Fig- (Figure 1), and recent gas-exchange-based analyses [7, 8] indi- ure 1). Taken together, this evidence directly challenges the cate that the stomata of both groups lack strong responses to evolutionary hypothesis of Brodribb and McAdam [8]. the drought hormone abscisic acid (ABA) and changes in The concentration of CO2 in the atmosphere is a further important environmental signal to which stomata in flowering concentration of atmospheric CO2. These observations form plants are known to respond actively [10]. We investigated whether S. uncinata stomata are capable of responding phys- *Correspondence: [email protected] iologically to changes in the concentration of atmospheric Stomatal Evolution 1031

Figure 1. Phylogenetic Tree Showing Relation- ships between Major Groups of Extant Land Plants Land plants are divided into vascular (green) and nonvascular (bryophyte, light blue) groups, with the sister group to the land plants (charo- phycean algae) shown in dark blue. Approxi- mate timings of divergence (millions of years ago) are displayed [11–14]. The red asterisk marks the acquisition of stomata; the upper black asterisk marks the acquisition of active stomatal control reported by Brodribb and McAdam [8], whereas the lower black asterisk marks the acquisition of active stomatal control demonstrated in the current study. An image of Selaginella uncinata, the model lycophyte used for these physiological experiments, is shown at right.

These responses directly mirror those of many flowering plants [18] and were further confirmed by results obtained from intact plants using gas exchange, which showed a clear non- linear decline in gw with an increase in CO2 and therefore play an important role in allowing stomata CO2 concentration from 180 ppm to 700 ppm (Figure 3B). to optimize water-use efficiency under a changing CO2 The results of our CO2 investigations in a lycophyte and those regime [17]. Our results (Figure 3A) demonstrate that stomatal reported for the fern species Phyllitis scolopendrium [19] apertures decrease in response to elevated CO2 (700 ppm) build further support for the early acquisition of active and open in response to reduced concentrations of CO2. stomatal control of plant carbon and water balance.

Figure 2. Stomata of the Basal Vascular Plant Selaginella uncinata Respond to Abscisic Acid (A) Stomatal aperture measurements show abscisic acid (ABA) inhibition of light-induced opening of preclosed stomata. (B) ABA promotion of stomatal closure in preopened stomata. Data are mean values 6 standard error of the mean (SEM) of three independent experiments (n = 120). All ABA treatments in (A) and (B) are signiﬁcantly different from control treatment (p < 0.005; Student’s t test).

(C) S. uncinata steady-state stomatal conductance to water vapor (gw) is reduced in the presence of ABA and returns to higher gw after ABA treatment ceases (indicated by dashed vertical line). Data are mean gw values 6 SEM (n = 3) from three independent experiments. Current Biology Vol 21 No 12 1032

Figure 3. Stomata of the Basal Vascular Plant Selaginella uncinata Respond to External Concentration of CO2

(A) Stomatal aperture measurements following incubation in CO2-free air, ambient air ([CO2] 425 ppm), or 700 ppm CO2. Data are mean values 6 SEM of three independent experiments (n = 120); 425 ppm and 700 ppm treatments are signiﬁcantly different from 0 ppm (p < 0.005; Student’s t test).

(B) Steady-state stomatal conductance to water vapor (gw) is increased at subambient CO2 and reduced at above-ambient CO2 concentrations. Data are mean gw values 6 SEM (n = 3) from three independent experiments.

The Mechanism of Guard Cell ABA Signal Transduction manner consistent with the light-induced increase in potas- in Selaginella and Arabidopsis Is Highly Similar sium concentration seen in higher plants [24](Figures 4C Having established physiological evidence for active stomatal and 4D). To investigate whether light-induced stomatal opening control of water balance in a lineage predating the divergence in S. uncinata involves activation of the plasma membrane of ferns (Figure 2; Figure 3), we next investigated whether the H+-ATPase, known to be responsible for plasma membrane underlying intracellular signaling network responsible for hyperpolarization during stomatal opening in higher plants controlling stomatal movements in S. uncinata is similar to [24, 26], we used fusicoccin (FC), a compound that irreversibly that found in flowering-plant guard cells. In flowering plants, activates the plasma membrane H+-ATPase by stabilizing the typified by Arabidopsis, ABA-induced changes in stomatal interaction between the H+-ATPase and 14-3-3 proteins [27]. aperture involve increases in the intracellular second messen- The results in Figure 4E show, just as we would expect from gers Ca2+ and reactive oxygen species (ROS) [20, 21]. We there- the flowering-plant stomatal opening paradigm [24], that FC fore sought evidence for a similar mechanism in S. uncinata promotes stomatal opening in S. uncinata. These results by making use of EGTA and verapamil, two widely used strongly suggest that, as in Arabidopsis,K+, the plasma compounds known to interfere with ABA-induced increases membrane H+-ATPase, and 14-3-3 proteins are all part of the in the concentration of guard cell cytosolic calcium ions underlying cellular machinery responsible for stomatal opening [22, 23]. Our results indicate that application of each of these in S. uncinata. compounds partially interfered with ABA-induced stomatal We extended the evidence from these physiological data closure, just as in higher plants (Figure 4A). To investigate sets with a cross-species genetic complementation investiga- whether ABA induces an increase in ROS in S. uncinata guard tion to establish whether a key gateway gene (OST1) involved cells, we used the fluorescent ROS reporter H2DCF-DA [21]. in Arabidopsis guard cell ABA signaling is functionally identical The results show that, as in Arabidopsis, ABA addition to in S. moellendorffii, a genetically tractable species related to

S. uncinata resulted in increases in H2DCF-DA fluorescence S. uncinata.InArabidopsis, the OPEN STOMATA 1 (AtOST1) in a manner consistent with an increase in guard cell ROS (Fig- gene encodes a serine/threonine protein kinase involved in ure 4B). We also showed that (2)ABA, which in flowering the signaling network responsible for stomatal closure in plants is biologically inactive, failed to induce increases in response to ABA [28, 29]. In S. tamariscina, the homolog of H2DCF-DA fluorescence (data not shown). Taken together, Arabidopsis OST1 is known as StOST1 (85% identical to our results show that not only do lycophyte stomata respond AtOST1 at the amino acid sequence level). It is upregulated in the same way to ABA and CO2 as flowering plants do, but by ABA, and transcripts of this gene accumulate during desic- Ca2+ and ROS, key intracellular messengers in the Arabidopsis cation [15]. To investigate whether a lycophyte OST1 gene is guard cell ABA signaling pathway [8], are also required in capable of playing a role in guard cell ABA signaling similar S. uncinata guard cell ABA signaling. However, direct mea- to that of AtOST1, we cloned the S. moellendorffii homolog surement of stimulus-induced Ca2+ elevations in Selaginella of AtOST1. This gene product (S. moellendorffii JGI database guard cells will be required to fully verify the involvement of protein ID 158991) is >75% identical to AtOST1 at the amino this intracellular second messenger. acid level. We used SmOST1 to rescue ABA-responsive The experiments described so far focused on stimulus- stomatal behavior in the Arabidopsis ost1-4 mutant by genetic induced reductions in stomatal aperture. It is well known that complementation (Figures 4F and 4G), indicating conservation light-induced stomatal opening in flowering plants is driven of function in a key gene that plays a regulatory role within the by an increase in guard cell turgor and that underlying this is ABA signaling network across 400 million years of land plant accumulation of K+ ions in the guard cell vacuole [24]. Using evolution. These results suggest that an array of components a histochemical approach [25], we found that light-induced (H+-ATPase, 14-3-3 proteins, OST1, Ca2+, and ROS) known to stomatal opening in S. uncinata is marked by the accumulation be involved in flowering-plant guard cell intracellular signaling of sodium potassium cobaltinitrite in the guard cells in a are also involved in the stomatal responses of a lycophyte. Stomatal Evolution 1033

Figure 4. Stomatal Response Mechanisms Are Conserved between a Basal Vascular Plant and Flowering Plants (A) Treatment with the Ca2+ chelator EGTA (+EGTA, 10 mM) or the Ca2+ channel blocker verapamil (+Ver, 25 mM) partially inhibits ABA-induced reductions in S. uncinata stomatal aperture. Gray bars show controls, white bars show 25 mM ABA. Data are mean values 6 SEM of three independent experiments (n = 120). Stomata are signiﬁcantly more closed with ABA alone than in combination with EGTA or verapamil (p < 0.005; Student’s t test). (B) Following ABA treatment (white bar, 25 mM ABA), reactive oxygen species (ROS) levels in S. uncinata stomata increase signiﬁcantly compared to controls

(gray bar, 2ABA) (p < 0.005; Student’s t test) as detected by fluorescence intensity of H2DCF-DA. Data shown are mean fluorescence intensity 6 SEM of three independent experiments (n = 150). (C and D) Accumulation of vacuolar K+ deposits in guard cells of S. uncinata incubated in light (C) or dark (D) conditions. White arrows highlight sodium potassium cobaltinitrite deposit (n = 3). (E) 10 mM fusicoccin (FC, hatched bar) promotes significant stomatal opening in preclosed stomata of S. uncinata compared to control (black bar) (p < 0.005; Student’s t test). Data shown are mean values 6 SEM from three independent experiments (n = 100). (F) ABA-induced promotion of stomatal closure in Arabidopsis thaliana lines wild-type (WT; Col-2), ost1-4, and three T2 independent transgenic lines (#1–#3) expressing SmOST1 in the ost1-4 background (ost1-4;AtOST1PRO::SmOST1). Transgenic lines are indicated by brackets. Data are mean values 6 SEM (n = 90) for peels incubated with 1 mM ABA (light gray bars) and 10 mM ABA (white bars) or controls (dark gray bars); ABA induces significant closure in the WT line and transgenic lines #1, #2, and #3 (p < 0.001; Student’s t test). ABA and control treatments do not differ significantly in ost1-4 (p = 0.125; Student’s t test). (G) Detection of the SmOST1 transgene in complemented lines #1, #2, and #3 by RT-PCR. Actin was amplified as a control. Transgenic lines expressing

SmOST1 in the ost1-4 background (ost1-4;AtOST1PRO::SmOST1) are indicated by the bracket. Current Biology Vol 21 No 12 1034

Conclusions was measured throughout the experiment using a CARBOCAP hand-held We conclude that the stomata of the lycophyte S. uncinata carbon dioxide meter (Vaisala). Elevated CO2, (700 ppm) was obtained respond to ABA and CO like the model flowering plant from a cylinder (BOC), and CO2-free air was obtained by passing ambient 2 air through a bell jar containing soda-lime crystals. All gases were bubbled Arabidopsis thaliana and furthermore employ similar under- into Petri dishes containing epidermal peels at 1000 cm3/min. Peels lying signaling mechanisms. We therefore reject the hypothesis were incubated for 2 hr in CO2-free air conditions and then transferred that active stomatal control appeared after the evolutionary for 2 hr to either CO2-free air, ambient air ([CO2] 425 ppm), or 700 ppm appearance of ferns some 60 million years later [8]. Our data CO2 conditions. Apertures were measured on the monitor of an inverted (Figure 2; Figure 3; Figure 4) and the data for other basal land microscope. + plant lineages [16, 19] provide strong support for the alternative Staining for K ions was carried out using the Macullum procedure, repeated three times, and images were compiled as in [25]. For ROS detec- hypothesis that active stomatal regulation is a phenomenon tion, S. uncinata peels were incubated in 40–50 mmol m22 s21 light at 22C that first appeared at least 420 million years ago and likely for 2 hr in 10 mM MES/50 mM KCl (pH 6.15) buffer. Peels were then trans- conferred substantial physiological advantages to land plants. ferred to buffer + 50 mMH2DCF-DA for 10 min, washed for 20 min in buffer, Early vascular land plants with stomata and limited root or transferred to buffer + 25 mM ABA or 0.25% ethanol (v/v) for 10 min, and rhizoid systems appeared before arborescent life forms examined using a fluorescence microscope with a GFP filter set. Z stacks casting shade and so would have been exposed periodically were taken, and the mean fluorescence intensity of 50 guard cells per experiment was recorded (maximum of 10 per peel). Each experiment was carried to full sunlight and drying of early skeletal soils lacking substan- out three times. tial water-holding capacity. Possessing stomata with active control mechanisms would have aided these plants in avoiding Gas-Exchange Measurements catastrophic dehydration under such circumstances. For CO2 response, leaves were placed in a gas-exchange cuvette (LI-COR The results in the present study and indeed others cited here 6400, LI-COR Biosciences) and brought to steady-state stomatal conduc- [16, 19] are clearly at odds with the conclusions described in tance at 350 ppm leaf external CO2 concentration (ambient CO2), with other m 22 21 the recent paper from Brodribb and McAdam [8]. At this stage, environmental variables controlled at 200 mol m s light, 1.0 kilopascal (kPa) leaf-to-air water vapor pressure difference (VPD), and 23C leaf it is not possible to fully explain this disparity. Methodological temperature. Plants were kept well watered at all times. Stomatal conduc- differences seem an unlikely explanation because both papers tance to water vapor, gw, was logged every minute until steady (less than used gas-exchange techniques to address the question of 1% change for 20 min). Step changes in external CO2 concentration were stomatal responsiveness yet reach opposite conclusions. applied to obtain steady-state gw at 150 ppm CO2 (‘‘subambient CO2’’) However, it is worth pointing out that close inspection of the and then 700 ppm CO2 (elevated CO2). For ABA response, a fresh shoot data in the Brodribb and McAdam paper (Figure 2B) [8] reveals was severed from the plant while keeping the severed end immersed in distilled water. Maintaining the shoot base in a vial of distilled water, that, in the case of Lycopodiella inundata, one of three lyco- a section of shoot was clamped into the gas-exchange cuvette, and gw phytes investigated, ABA induced an approximately 20% was allowed to reach steady state at 350 ppm external CO2 concentration, reduction in stomatal conductance. One explanation for the 200 mmol m22 s21 light, 23C leaf temperature, and 1.4 kPa VPD. Distilled difference in the conclusions between the two papers could water was then replaced with 0.1 mM ABA, followed by 1.0 mM ABA and be that the other two lycophytes investigated by Brodribb then distilled water, with steady-state gw measured after each change. and McAdam [8] either have lost the ability to respond to For both experiments, the last ten measurements for each of three separate leaves on different plants were averaged to obtain mean g . ABA during evolution or were grown under conditions that w resulted in stomatal insensitivity to ABA. Further work will be Molecular Procedures needed to resolve this question. To identify SmOST1-1 (JGI database protein ID 158991), we used the amino acid sequence of A. thaliana OST1 (At4G33950) to screen the available Experimental Procedures S. moellendorffii database (http://genome.jgi-psf.org/Selmo1/Selmo1. home.html) by TBLASTN. Selaginella and Arabidopsis tissue was homoge- Plant Material and Growth Conditions nized in liquid nitrogen, and total RNA was isolated using a QIAGEN RNeasy S. uncinata was kindly provided by Nigel Rothwell at the Royal Botanic Plant Mini Kit and treated with DNase1. cDNA was synthesized using a Fer- Gardens, Kew (UK) and S. moellendorffii by the Royal Botanic Garden mentas Life Sciences kit, and the full coding region of the SmOST1 gene was Edinburgh (UK). Arabidopsis was grown in controlled environment cabinets amplified using primers Ost1_forward (50-CACCATGGATAGATTCGAATT 0 0 0 as described in [30]. S. uncinata and S. moellendorffii were grown from CG-3 ) and Ost1_reverse (5 -TCAGTACACACCAGACACGAAC-3 ) from cuttings in multipurpose sand-peat compost in 7 3 7 cm Teku plant pots S. moellendorffii cDNA and verified by DNA sequencing. SmOST1-1 (LS Systems) in a Sanyo Versatile Environmental Test Chamber at a temper- RT-PCR product was cloned into p$POHA [32] (kindly provided by Christo- ature of 23C (day) and 19C (night) in a 14 hr day in the light (40–50 mmol phe Belin, Institut des Sciences du Ve´ ge´ tal, CNRS, France) using the m22 s21) and a 10 hr night at 90% relative humidity (RH) for 3 weeks and pENTRO/D-Topo system (Invitrogen). The direction of cloning in the gene then at 70% RH for at least 2 weeks prior to experimental use. Plants construct was checked by PCR with primers Ost1_forward and Ost1_re- were watered daily, with water fed directly into the soil and the plants misted verse in combination with standard M13 and M13 reverse before introduc- with a pressure sprayer. tion into Agrobacterium tumefaciens GV3101 pMP90 by electroporation and then into A. thaliana ost1-4 mutant [33] by floral dipping [34]. Trans- Stomatal Aperture Measurements and K+ and ROS Determinations formed seeds were selected by GFP fluorescence using a Leica DMI6000 Stomatal bioassays were based on a previously described method [31], with CS inverted microscope with EL6000 fluorescence illumination. Presence the exception of the light intensity, which was 40–50 mmol m22 s21 for and expression of the transgene was verified by PCR and RT-PCR, and S. uncinata and 100 mmol m22 s21 for Arabidopsis, and the FC treatment, stomatal responses were analyzed in the T2 generation. RT-PCR was per- 0 0 0 which was carried out in darkness. Forty stomatal pores were measured formed using actin primers (5 -GATCCTAACCGAGCGTGGTTAC-3 and 5 - 0 after 2 hr treatment (or 1 hr for FC and verapamil) in each of three separate GACCTGACTCGTCATACTCTGC-3 ) and Ost1_forward and Ost1_reverse. experiments (n = 100–120) for each treatment. S. uncinata bioassays were carried out using abaxial epidermal strips from the ventral leaves of 5- Acknowledgments week-old plants and A. thaliana stomatal bioassays using epidermal strips from 4- to 5-week-old plant leaves. ABA, EGTA (Ca2+ chelating agent), We thank the Gatsby Charitable Foundation and the Biotechnology and verapamil (Ca2+ channel blocker), and FC treatments were carried out Biological Sciences Research Council for funding PhD studentships to alongside control treatments containing an equivalent concentration of E.M.R. and C.C., respectively. D.J.B. and P.J.F. gratefully acknowledge solvent (0.25% ethanol [v/v] for ABA, EGTA, and verapamil and 0.1% ethanol funding from the University of Sheffield and the Australian Research

[v/v] for FC). All epidermal bioassays were carried out in CO2-free air, Council, and D.J.B. also gratefully acknowledges support from a Royal except for the experiment in Figure 2A. For this experiment, ambient CO2 Society Wolfson Research Merit Award. Stomatal Evolution 1035

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