plant physiology preview. published on january 30, 2015, as doi:10.1104/pp… · 2015. 1. 30. · 0...

Running head: Evolution of stomatal responses to VPD 1

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Corresponding author: 3

Scott McAdam 4

School of Biological Sciences 5

University of Tasmania 6

Private Bag 55 7

Hobart, TAS, 7005 8

AUSTRALIA 9

Ph: +61362262248 10

[email protected] 11

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Research area: Ecophysiology and Sustainability 13

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Plant Physiology Preview. Published on January 30, 2015, as DOI:10.1104/pp.114.252940

Copyright 2015 by the American Society of Plant Biologists

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Title: The evolution of mechanisms driving the stomatal response to vapour pressure deficit 15

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Authors: 17

Scott A. M. McAdam 18

Timothy J. Brodribb 19

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Institution address: 21

School of Biological Sciences 22

University of Tasmania 23

Hobart, TAS, 7001 24

AUSTRALIA 25

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One-sentence summary: The mechanism for a stomatal response to vapour pressure deficit evolved 27

from a passive regulation in basal vascular plants to mediation by ABA in the earliest angiosperms. 28



Footnotes: 29

Financial Source: This work was funded by Australian Research Council grants DP120101868 and 30

FT100100237 (to TJB) and DE140100946 (to SAMM). 31

Corresponding Author: Scott A. M. McAdam ([email protected]) 32

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Keywords: stomata, humidity, vapour pressure deficit (VPD), abscisic acid (ABA), hysteresis, leaf gas 34

exchange 35



Abstract 36

Stomatal responses to vapour pressure deficit (VPD) are a principal means by which vascular land 37

plants regulate daytime transpiration. While much work has focused on characterising and 38

modelling this response, there remains no consensus as to the mechanism that drives it. 39

Explanations range from passive regulation by leaf hydration, to biochemical regulation by the 40

phytohormone abscisic acid (ABA). We monitored ABA levels, leaf gas exchange and water status in 41

a diversity of vascular land plants exposed to a symmetrical, mild transition in VPD. The stomata in 42

basal lineages of vascular plants, including gymnosperms, appeared to respond passively to changes 43

in leaf water status induced by VPD perturbation, with minimal changes in foliar ABA levels and no 44

hysteresis in stomatal action. In contrast, foliar ABA appeared to drive the stomatal response to VPD 45

in our angiosperm sample. Increased foliar ABA level at high VPD in angiosperm species resulted in 46

hysteresis in the recovery of stomatal conductance (gs); this was most pronounced in herbaceous 47

species. Increased levels of ABA in the leaf epidermis were found to originate from sites of synthesis 48

in other parts of the leaf, rather than from the guard cells themselves. The transition from a passive, 49

to ABA-regulation of the stomatal response to VPD, in the earliest angiosperms is likely to have had 50

critical implications for the ecological success of this lineage. 51

52



Introduction 53

Plants continuously regulate transpiration by controlling the aperture of the stomatal pores on the 54

surface of the leaf. The principal atmospheric determinant of stomatal aperture is the humidity of 55

the air, which can be expressed as the vapour pressure difference between the leaf and the 56

atmosphere. Stomatal responses to atmospheric vapour pressure deficit (VPD) have been well 57

characterised across the diversity of vascular plant species (Darwin, 1898; Lange et al., 1971; Turner 58

et al., 1984; Franks and Farquhar, 1999; Oren et al., 1999; Brodribb and McAdam, 2011; Mott and 59

Peak, 2013), with stomata typically closing at high VPD and opening at low VPD. This 60

comprehensive characterisation has allowed for the development of highly effective empirical and 61

mechanistic models of leaf gas exchange which provide robust predictions of the responses of 62

transpiration to changes in VPD (Buckley et al., 2003; Katul et al., 2009; Damour et al., 2010; Medlyn 63

et al., 2011). Despite the success of this modelling, the mechanism for the stomatal response to VPD 64

remains poorly understood (Damour et al., 2010). Different hypotheses range from one extreme 65

whereby stomata respond passively through changes in leaf water content induced by the VPD or 66

humidity perturbation (Lange et al., 1971; Mott and Peak, 2013) to the other extreme whereby 67

stomata close uniquely in response to the phytohormone abscisic acid (ABA) (Xie et al., 2006; Bauer 68

et al., 2013). 69

From the earliest recognition that stomata open and close by changes in guard cell turgor (Heath, 70

1938), there have been many attempts to link the passive changes in water status that occur during 71

VPD or humidity transitions with stomatal responses to VPD or humidity (Lange et al., 1971; Mott 72

and Peak, 2013). Studies have suggested that changes in atmospheric water content passively drives 73

stomatal responses by changing bulk leaf water status which in turn changes guard cell turgor (Oren 74

et al., 1999) or alternatively by changing guard cell turgor directly (Mott and Peak, 2013). Models 75

based on these entirely passive processes are highly effective in predicting steady-state stomatal 76

conductance (gs) in response to changes in VPD or humidity in angiosperms (Mott and Peak, 2013). 77



While hydraulic models provide robust predictions of steady-state gs, they are less effective at 78

predicting the dynamic responses of stomata to short term perturbations, particularly with respect 79

to the “wrong-way responses” that typically occur as transients (Buckley, 2005), as well as feed-80

forward behaviour (Farquhar, 1978; Bunce, 1997; Franks et al., 1997; Tardieu and Simonneau, 1998; 81

Ocheltree et al., 2014; although cf. Mott & Peak, 2013). Although some of these models provide a 82

pathway for incorporating the effect of ABA (Buckley, 2005), a lack of knowledge of ABA dynamics or 83

action makes it difficult to integrate the influence of this active regulator of guard cell aperture into 84

models. Stomatal behaviour of single gene mutants (most notably the ABA synthesis and signalling 85

mutants of Arabidopsis); strongly support a role for ABA in mediating standard stomatal responses 86

to changes in VPD. The stomata of these mutants are known to have less pronounced responses to 87

a reduction in relatively humidity compared to wild-type plants (Xie et al., 2006). Recently, 88

molecular work has shown that guard cells express many of the genes required to synthesise ABA 89

(Okamoto et al., 2009; Bauer et al., 2013), with molecular proxies for ABA level also indicating that 90

the biochemical activity of ABA in guard cell may increase following short-term exposure of leaves to 91

a reduction in relative humidity (Waadt et al., 2014). These findings suggest a role for ABA in 92

regulating stomatal responses to VPD; and have led to the conclusion that ABA synthesised 93

autonomously by the guard cells is the predominant mechanism for stomatal responses to increased 94

VPD (Bauer et al., 2013). 95

Although the experimental evidence from molecular studies presents an argument for the role of 96

ABA in the responses of stomata to changes in VPD, very few studies have quantified changes in ABA 97

level in response to VPD. It is well established that ABA levels in leaves and guard cells can increase 98

rapidly following the imposition of turgor loss or water stress (Pierce and Raschke, 1980; Harris et 99

al., 1988; Harris and Outlaw, 1991). However, only a few studies have reported increases in foliar 100

ABA level in response to high VPD (Bauerle et al., 2004; Giday et al., 2013), and none have 101

investigated whether these observed dynamic changes or differences in ABA level were functionally 102



relevant for stomatal control. In addition, no study has quantified the levels of ABA in guard cells 103

during a transition in VPD. 104

Here we investigate the relative importance of ABA for the stomatal response to VPD in whole 105

plants, sampled from across the vascular land plant lineage. We provide the first functional 106

assessment of changes in ABA levels driving stomatal responses to VPD; as well as critically 107

investigate the recent suggestion that stomatal responses to VPD are driven by an autonomous 108

guard cell synthesis of ABA. 109

Results 110

Hysteresis in the response of angiosperm stomata to VPD 111

During a reversible sequence of VPD transitions (from 0.7 to 1.5 to 0.7 kPa) all species showed a 112

pronounced reduction in gs in response to the increase in VPD to 1.5 kPa (Figs 1, 2 and 3). In all 113

species gs declined by between 50% and 70% when VPD was increased from 0.7 kPa to 1.5 kPa 114

(Supplementary Table S1). While stomatal closure at increased VPD was relatively similar across 115

species there were large differences between species in the recovery of gs on returning to 0.7 kPa, 116

with only angiosperm species displaying hysteresis in the recovery of gs (Fig. 4). In the six diverse 117

fern and conifer species, the rate of stomatal closure during a VPD transition from 0.7 to 1.5 kPa was 118

the same as that for stomatal opening when the original VPD was restored (Fig. 4) indicating a rapid, 119

symmetrical stomatal response to VPD in these basal lineages. In contrast in the two herbaceous 120

angiosperm species the rate of stomatal closure at high VPD was much greater than the rate of 121

stomatal opening on returning to low VPD (Fig. 4). Stomatal responses to VPD in the woody 122

angiosperm species (including the most basal angiosperm A. trichopoda) were intermediate in terms 123

of difference between the rate of stomatal closure at high VPD and opening at low VPD, between 124

the species from the basal vascular land plant groups and herbaceous angiosperms (Fig. 4). 125

The role of foliar ABA levels during a VPD transition 126



In the angiosperm species (Fig. 1) and two of the three conifers (Fig. 2), a concomitant increase in 127

foliar ABA level was observed as gs declined when plants were exposed to a moderate increase in 128

VPD from 0.7 kPa to 1.5 kPa. In the two herbaceous angiosperm species, P. sativum and D. hybrida, 129

increasing foliar ABA levels alone were sufficient to account for the observed stomatal closure on 130

exposure to a VPD of 1.5 kPa (Fig. 1; Fig. 5). This functional level of foliar ABA is shown by the red 131

horizontal line and represents the ABA level required to produce the equivalent response of stomata 132

assuming ABA was uniquely driving the closure of stomata. This level was determined from the 133

sensitivity of gs to exogenously applied ABA in the absence of water stress (Fig. 5). 134

In the two herbaceous species returning to low VPD was accompanied by a very slow decline in foliar 135

ABA levels, as well as a similar, very slow, hysteretic recovery of gs (Fig. 1). The very slow recovery in 136

gs at low VPD in the two herbaceous species was reflected in higher Ψ l following the final transition 137

in VPD to 0.7 kPa, compared with values measured at 0.7 kPa prior to the VPD transitions (Table 1). 138

In the most basal woody angiosperm species, A. trichopoda, rising foliar ABA levels also explained 139

the observed decline in gs following a transition to high VPD as predicted by the relationship 140

between gs and endogenously applied ABA (Figs 1 and 5). When VPD was returned to 0.7 kPa foliar 141

ABA levels returned slowly to their original levels leading to significant hysteresis in the recovery of 142

gs (Figs 1 and 4). In the other woody angiosperm species, Q. robur, exposure to high VPD resulted in 143

a rapid increase in foliar ABA almost to a level sufficient to explain the depression in gs (Figs 1 and 5). 144

In Q. robur declining ABA levels upon restoration of the initial VPD were also relatively rapid and 145

were accompanied by no significant hysteresis in the response of gs (P = 0.052, Fig. 4). 146

To test whether the increase in foliar ABA levels in the leaves of angiosperm species at high VPD was 147

due to an enhanced delivery of ABA from the xylem sap, driven by increased evaporation, or 148

endogenous synthesis of foliar ABA, we measured ABA levels and gs in stems of A. trichopoda that 149

were excised under water and exposed to a reversible transition in VPD. In branches of A. 150

trichopoda excised under water, foliar ABA levels increased on exposure to a step increase in VPD 151



after 20 min and this increase in foliar ABA level corresponded with a reduction in gs (Fig. 6). The 152

functional increase in foliar ABA level, in these excised stems, occurred despite xylem sap ABA 153

concentrations being 90 times lower than levels in an intact plant under low VPD conditions, 154

approaching the detection limits of the ultra-performance liquid chromatograph tandem mass 155

spectrometer (UPLC-MS) (Fig. 6). 156

Unlike the angiosperm representatives, foliar ABA levels in the conifer species remained an order of 157

magnitude too low to explain the significant stomatal closure observed in response to increased VPD 158

this was based on the sensitivity of gs to exogenously applied ABA (Figs 2 and 4) and in M. 159

glyptostroboides the sensitivity of gs to ABA determined by three independent methods (including 160

isolated epidermis, exogenous feeding and rehydration of branches with different levels of 161

endogenously synthesised ABA (McAdam and Brodribb, 2014)). In addition, changes in Ψ l in all 162

conifer species were consistent with a passive control of stomatal aperture by leaf water status 163

(Table 1). All of the representative fern species showed rapid and pronounced stomatal closure in 164

response to the imposed VPD transition, but no significant change in foliar ABA level was observed 165

during the VPD transition (Fig. 3). All differences in foliar ABA level observed in the fern species 166

were due to differences between leaves sampled rather than due to the VPD treatment imposed on 167

the plants (Supplementary Fig. S1). 168

Distribution of ABA in angiosperm leaves 169

At the same time as ABA levels, in the leaf of the herbaceous angiosperm P. sativum, doubled during 170

an increasing transition in VPD from 0.7 kPa to 1.5 kPa (Fig. 1), the proportion of ABA in the abaxial 171

epidermis, declined by more than half, from 40% to just under 15% of the total ABA found the in 172

whole leaf (Fig. 7a). When ABA levels in the epidermis were expressed relative to fresh weight of 173

the sample, the increases in abaxial epidermal ABA during an increase in VPD significantly lagged 174

behind the more pronounced increase in ABA levels that occurred in the remainder of the leaf (Fig. 175

7b, Supplementary Fig. S2). To test whether this increase in foliar ABA could reach the guard cells in 176



this mutant with an extreme morphology (having an epidermis that is only connected to the leaf 177

along major veins) we fed labelled [2H6]ABA into the transpiration stream. During the feeding of 178

labelled [2H6]ABA we monitored leaf gas exchange and performed UPLC-MS analysis to investigate 179

the presence of [2H6]ABA in the abaxial epidermis. Following feeding into the transpiration stream 180

the stomata of the Argenteum mutant did indeed close and [2H6]ABA could be readily identified in 181

the epidermis immediately upon closure of stomata (Fig. 5d) despite the significant separation of the 182

epidermis from the mesophyll. 183

Discussion 184

Ancestral, passive-hydraulic regulation of stomatal responses to VPD 185

In monitoring both foliar ABA levels and leaf gas exchange during VPD transitions we show evidence 186

for systematic differences between plant groups in the mechanisms that regulate the stomatal 187

response to VPD in vascular land plants. Similar to previously described patterns in the responses of 188

stomata to water stress across diverse species (Brodribb and McAdam, 2011; McAdam and Brodribb, 189

2014), we show that stomatal responses to VPD are characterised by passive-hydraulic regulation in 190

ferns and conifers, whereby stomata respond as simple, hydraulic valves opening in response to an 191

increase in leaf turgor and closing in response to a decrease in leaf turgor (we refer to this as the 192

ancestral, passive-hydraulic regulation of stomatal aperture. Unlike in lycophytes, ferns and 193

conifers, when angiosperm species are exposed to an increase in VPD initially stomata open, this 194

wrong-way response is also passive but driven by the epidermis loosing turgor faster than the guard 195

cells (Mott et al., 1997; Franks and Farquhar, 2007)). In this study we pinpoint the origin of ABA-196

mediated stomatal responses to VPD to the earliest angiosperms. 197

Lycophytes, ferns and conifers have stomatal responses that conform to a passive-hydraulic model 198

for gs which is independent of foliar ABA (Brodribb and McAdam, 2011; McAdam and Brodribb, 199

2014) and we show here that no functional increase in ABA level occurs in the leaves of fern and 200



conifer species in response to changes in VPD. These results contrast the with conclusions reached 201

by Ruszala et al. (2011) and Chater et al. (2011) who suggested that the stomatal response to ABA 202

(and presumably, although not tested, the regulation of stomatal responses to VPD by ABA) had an 203

ancient evolutionary origin at the base of the mosses. Interestingly, while we have suggested that 204

the conifers represent the first extant lineage of vascular plants to possess a functional stomatal 205

response to ABA (Brodribb and McAdam, 2011; McAdam and Brodribb, 2012; cf. Ruszala et al., 206

2011), the earliest role for ABA in the regulation of stomatal aperture can been attributed solely to 207

enhancing stomatal closure during drought stress, and probably not functional stomatal responses 208

to VPD, at least in well watered plants (Brodribb and McAdam, 2013). 209

Foliar ABA regulates the stomatal response to VPD in angiosperms 210

While the passive, hydraulic regulation of the stomatal response to VPD can be readily observed in 211

the basal lineages of vascular land plants (Brodribb and McAdam, 2011; McAdam and Brodribb, 212

2014), angiosperm stomata are characteristically distinct from the stomata of other land plant 213

groups. During reversible transitions in VPD we identified a functionally and statistically significant 214

increase in foliar ABA levels in three of the four angiosperm species examined (Fig. 1), and show that 215

this increase in ABA level is likely due to synthesis in the leaf rather than increased delivery of ABA 216

from the xylem sap (Fig. 5). This provides the first quantitative evidence that increases in foliar ABA 217

levels in response to changes in VPD in angiosperms can exceed levels that will cause a similar 218

degree of stomatal closure in the absence of water stress, and complements conclusions reached by 219

the qualitative assessments of ABA signalling and synthesis mutants; namely that ABA levels play a 220

major role in delivering stomatal responses to VPD in angiosperms (Xie et al., 2006). Many of the 221

past methods, especially the popular immuno-assay methods, used to quantify foliar ABA levels 222

(Walker-Simmons et al., 2000) and other hormones, from foliar auxin levels (Cohen et al., 1987) to 223

steroids in baboon faeces (Gesquiere et al., 2014), unless rigorously standardised against 224

physiocochemical methods, do not possess the resolution required to detect small but functionally 225



significant increases in hormone levels similar to what we show occur in angiosperm leaves during 226

exposure to high VPD. This limitation in resolution may explain previous observations that ABA 227

levels only noticeably increase when plants are exposed to water stress sufficient enough to cause 228

turgor loss (Pierce and Raschke, 1980; Trejo and Davies, 1991; Wilkinson and Davies, 1997) and the 229

paucity of data showing rapid ABA accumulation in leaves during an increasing VPD transition. While 230

the increase in foliar ABA levels that we observed in angiosperm species with increasing VPD were 231

functionally and statistically significant, there was a degree of variation between leaves in the 232

canopy which may have been due to heterogeneity in foliar morphology or hydraulics. 233

Interestingly, while we show here that foliar ABA appears to drive stomatal responses to changes in 234

VPD in angiosperms, a number of studies have shown that the exposure of angiosperm leaves to low 235

humidity is a critical prerequisite for delivering normal stomatal responses to ABA and that high 236

humidity can desensitise stomata to ABA (Arve et al., 2013; Pantin et al., 2013; Aliniaeifard et al., 237

2014). This interaction is like due to low humidity being a strong up regulator of both ABA synthetic 238

and signalling genes (Pantin et al., 2013) and not due to changes in stomatal or leaf anatomy at high 239

humidity (Aliniaeifard et al., 2014). 240

Predominance of ABA from the leaf driving the stomatal responses to VPD in angiosperms 241

Qualitative molecular studies of ABA synthesis mutants have led to the hypothesis that ABA 242

originating in guard cells is solely responsible for driving the stomatal response to VPD (Bauer et al., 243

2013). We tested this hypothesis using the Argenteum mutant of the angiosperm herb Pisum 244

sativum, which has fully functional stomata yet an epidermis that is substantially isolated from the 245

mesophyll (Jewer et al., 1982). We observed a significant decline in the proportion of ABA in the 246

adaxial epidermis when plants were exposed to increased VPD, suggesting that guard cell ABA 247

synthesis in response to increased VPD is substantially dwarfed by the accumulation of ABA 248

occurring in the remainder of the leaf (see Supplementary Fig. S2 for mass normalised ABA levels in 249

the leaf and epidermis). This concurs with a number of studies which show that the vascular tissue, 250



particularly the phloem cells, have the genetic capacity to synthesise ABA, as well as proteins 251

capable of transporting ABA to the guard cells (Koiwai et al., 2004; Okamoto et al., 2009; Kuromori 252

et al., 2010; Seo and Koshiba, 2011; Kuromori et al., 2014). We also found evidence that ABA can be 253

transported from major sites of synthesis in the leaf to the epidermis even in a pea mutant where 254

much of the normal liquid pathway for ABA flow between the vasculature and stomata is air-filled 255

(Fig. 7d). In this mutant we observed a delayed increase in ABA accumulation in the abaxial 256

epidermis in response to an increase in VPD suggesting that the source of the epidermal ABA was 257

probably derived from other tissue in the leaf. While there is an abundance of molecular evidence 258

illustrating the biochemical capacity of guard cells to synthesise ABA (Christmann et al., 2005; 259

Okamoto et al., 2009; Bauer et al., 2013), ABA accumulation in the leaf during a VPD transition 260

occurs before levels increase in the epidermis or guard cells. These findings suggest that ABA 261

synthesised by the leaf only, yet outside of the guard cells, may be the dominant signal for stomatal 262

closure in response to changes in VPD. Using ABA signalling mutants it has been suggested that 263

increases in ABA level in the xylem sap can cause a reduction in leaf hydraulic conductivity (Kleaf) and 264

stomatal closure in the absence of stomatal sensitivity to ABA (Pantin et al., 2013). This study 265

suggests that foliar increases in ABA in response to increased VPD may lead to a hydraulically driven 266

reduction in gs as well as hysteresis in the stomatal response to VPD. We found only a very small 267

(<10%) mean reduction in Kleaf (from evaporation and Ψl data) in the two angiosperm herb species 268

after VPD was returned to 0.7 kPa that would not be sufficient to cause the >50% reduction in gs. 269

Contribution of an ancestral, passive-hydraulic regulation of stomatal responses to VPD in 270

angiosperms 271

Foliar ABA level was able to account for the stomatal response to VPD, including the reduced rate of 272

stomatal re-opening after returning to low VPD in three angiosperm species; however, this was not 273

the case in the woody angiosperm species Q. robur. The small increase in ABA level in response to 274

the VPD transition, as well as the similar rate of the recovery of gs in this species suggests that the 275



ancestral, passive-hydraulic regulation of stomatal response to VPD remains a significant influence 276

over stomatal aperture in some angiosperm species. Whether this is also the case in angiosperm 277

species during stomatal responses to very gradual transitions in VPD, which may not be severe 278

enough to trigger ABA biosynthesis, remains to be tested. However, the presence of the ancestral, 279

passive-hydraulic regulation of stomatal responses to VPD, is supported by gas exchange studies in 280

the single-gene ABA signalling and synthesis mutants of Arabidopsis all of which still show a degree 281

of stomatal response to both increasing and decreasing VPD (Assmann et al., 2000; Xie et al., 2006). 282

This had been attributed to an as yet unknown metabolic stomatal signal (Xie et al., 2006); however 283

it appears more likely that this partial response in the absence of a stomatal ABA signal is also due to 284

passive-hydraulic regulation of stomatal response to VPD in Arabidopsis. 285

Interestingly, a significant efflux of potassium ions from the guard cells of the ABA-biosynthetic 286

mutant, aba3, has been observed following a decrease in relative humidity (Bauer et al., 2013). This 287

efflux of potassium ions in mutant plants exceeded that of wild-type plants; however neither 288

stomatal aperture nor gs was measured in this study. This observation further suggests that ABA 289

alone is not entirely responsible for stomatal responses to VPD in angiosperms, and highlights the 290

importance of further investigation into the stomatal responses of ABA signalling and synthesis 291

mutants to understand the relative importance of both mechanisms in delivering normal stomatal 292

responses to changes in VPD in angiosperms. 293

A fundamental unanswered question remains as to what the selective advantage for evolving ABA-294

dependent stomatal responses to VPD is. This may lie in the similar selective advantages obtained 295

by the evolution of additional active stomatal control mechanisms across the vascular land plant 296

lineages (including a response to blue light (Doi et al., 2008), photosynthetic signalling from the 297

mesophyll (McAdam and Brodribb, 2012) and ABA (Brodribb and McAdam, 2011; cf. Ruszala et al., 298

2011) in seed plants, as well as a response to low CO2 in the dark (Doi and Shimazaki, 2008) and high 299

CO2 in the light (Brodribb et al., 2009), possibly due to calcium signalling in the stomata, which 300



appears to be unique to angiosperms (Brodribb and McAdam, 2013). The evolution of these 301

additional stomatal control mechanisms over the course of the last 400 million years has greatly 302

enhanced the environmental signals that stomata can perceive and respond to (McAdam and 303

Brodribb, 2012). This increased the complexity in the regulation of stomatal control mirrors the 304

increasing ecological success of progressive lineages of vascular land plants. 305

Conclusion 306

We show that the earliest response of stomata to VPD was a passive-hydraulic response that does 307

not require foliar ABA, this response is currently found in the extant relatives of basal vascular land 308

plants, including the lycophytes, ferns and gymnosperms. However, a transition to an ABA 309

regulation of the VPD response appears to have evolved very early in the angiosperm clade, as 310

evidenced by the ABA dependence of the stomatal response to VPD in the most basal extant 311

angiosperm, Amborella trichopoda. We provide evidence that bulk leaf ABA provides the signal for 312

active stomatal responses to VPD in angiosperms. 313

Materials and Methods 314

Plant material and experimental conditions 315

Potted individuals of three morphologically and ecologically diverse fern species including the 316

terrestrial species Pteridium esculentum [Dennsteadiatace], semi-aquatic Marsilea hirsuta 317

[Marsileaceae] and epiphyte Pyrrosia lingua [Polypodiaceae], the conifers Metasequoia 318

glyptostroboides [Cupressaceae], Pinus caribaea var. hondurensis [Pinaceae] and Prumnopitys ladei 319

[Podocarpaceae], woody angiosperms Amborella trichopoda [Amborellaceae] and Quercus robur 320

[Fagaceae] and herbaceous angiosperms Pisum sativum ‘Argenteum’ [Fabaceae] and Dahlia hybrida 321

‘Cinderella’ [Asteraceae] were used in this study. Plants were grown under controlled glasshouse 322

conditions of 25°C/16°C day/night temperatures and 16 h photoperiod, with natural light 323

supplemented by sodium vapour lamps to ensure a minimum 300 μmol quanta m -2 s-1 at the pot 324

surface. All plants received weekly applications of liquid fertilizer (Aquasol, Hortico Ltd). Single 325



individuals of the fern, conifer and woody angiosperm species, or five even-aged individuals of the 326

herbaceous angiosperm species (due to a limited number of leaves per plant), were placed in a 327

growth cabinet (PGC-105, Percival Scientific Inc.) to acclimate for a week prior to experimentation. 328

The temperature and photoperiod conditions in the growth cabinet were maintained the same as in 329

the glasshouse (25°C/16°C day/night temperatures and 16 h photoperiod provided by mixed 330

incandescent and fluorescent lights ensuring a minimum 300 μmol quanta m-2

s-1

at the pot surface). 331

During this acclimation period, however, a daytime VPD of 0.7 kPa (77% relative humidity) was 332

sustained by the presence of containers of water and a 1 m2 surface of wet hessian; temperature 333

and relative humidity were monitored every five min during this period by a data logger (HOBO Pro 334

Series, Onset). Following a week of acclimation, the simultaneous monitoring of leaf gas exchange, 335

water potential (Ψl) and ABA levels were carried out approximately every 15 min (as described 336

below), during a relatively fast, step change in VPD. After an initial simultaneous measurement of 337

leaf gas exchange, Ψl, and ABA level, VPD was increased to 1.5 kPa (42% relative humidity) using a 338

condensing dehumidifier (SeccoUltra 00563, Olimpia-Splendid) in the growth cabinet. Temperature 339

and relative humidity were monitored every 30 s during the experimental period by a humidity 340

probe (HMP45AC, Vaisala) and temperature thermocouple connected to a data logger (CR10X, 341

Campbell Scientific). A VPD of 1.5 kPa was maintained until leaf gas exchange had stabilised (60-90 342

minutes), VPD was then returned to 0.7 kPa and maintained until leaf gas exchange had again 343

stabilised. The relatively small and contained volume of air in the growth cabinet (3 m3) resulted in a 344

relatively fast half time for the VPD transition of 150 s following transitions between 0.7 and 1.5 kPa 345

(with no hysteresis when VPD was returned to 0.7 kPa). 346

Leaf gas exchange and water potential measurements 347

During transitions in VPD, leaf gas exchange in three even-aged, fully irradiated leaves was measured 348

approximately every 15 mins using an infrared gas analyser (LI-6400, LI-COR Biosciences). Conditions 349

in the leaf cuvette were maintained as close as possible to the conditions in the growth cabinet, with 350



VPD regulated by a portable dew point generator (LI-610, LI-COR Biosciences). Leaves were enclosed 351

in the cuvette and instantaneous gas exchange was logged following stability in cuvette conditions 352

(after approximately 30 s). Following gas exchange measurements, the same leaf was then excised 353

and immediately sampled for ABA quantification (see below). In the fern species P. esculentum, 6 354

cm2 of leaf material could not be contained in the leaf cuvette due to the pinnate nature of the 355

leaves; the same three pinnules were, therefore, measured over the course of the experiment and 356

neighbouring tissue was sampled for foliar ABA quantification, after which leaf gas exchange was 357

adjusted for leaf area in the cuvette. 358

Leaf water potentials were assessed in three leaves at each equilibrium point before and after VPD 359

transitions. Leaves were harvested and immediately wrapped first in damp paper towel, then 360

aluminium foil, and bagged for assessment of Ψ l using a Scholander pressure chamber and 361

microscope to precisely measure the balance pressure. 362

ABA collection, extraction, purification and quantification 363

In all species, except the angiosperm herb P. sativum, samples harvested for ABA quantification 364

were immediately weighed (±0.0001 g, MS204S, Mettler-Toledo) into 50 ml tubes, covered in 365

approximately 15 ml of cold (0°C) 80% methanol in water (v v -1) with 250 g/L (m v-1) of added 366

butylated hydroxytoluene (BHT) and transferred to -20°C. Foliar ABA levels in these samples were 367

extracted, purified and quantified by physicochemical methods using an added internal standard and 368

UPLC-MS according to the methods of McAdam and Brodribb (2014). 369

Testing the origin of the ABA increase in the leaf 370

To test whether the increase in foliar ABA level observed in angiosperm species was due to 371

endogenous synthesis of ABA in the leaf or an enhanced delivery of ABA from the xylem sap because 372

of an increase in evaporation we measured foliar ABA level and gs in branches of A. trichopoda 373

excised under water. Three branches (with approximately 7 leaves) were selected from the same 374



plant used in the earlier experiments and excised under filtered, degassed water. Branches were 375

acclimated to the growth chamber in which experiments were conducted (described above) for 24 376

hours to ensure that ABA levels in the xylem sap were reduced. After this time the branches were 377

exposed to the above described transition in VPD. Foliar ABA and gs were measured simultaneously 378

in a single leaf from each branch, after 20 mins of exposure to a VPD of 1.5 kPa, then again after the 379

VPD was lowered to 0.7 kPa after a further 20 mins. On completion of this experiment branches 380

were removed from the water and three branches of similar size were excised from the original 381

plant. The remaining leaves on each branch were excised and the branch was enclosed in a 382

Scholander pressure chamber and 1.5 MPa of pressure was applied to the branch. This pressure 383

exuded xylem sap and approximately 200 μl of sap was collected from each branch and ABA levels 384

were quantified as described above, after determining the exact volume of sap expressed. 385

Fractional distribution of ABA in the leaf 386

Both foliar ABA and abaxial epidermal ABA levels were measured in P. sativum. For these 387

experiments we used the Argenteum mutant of P. sativum as it has fully functional stomata, yet an 388

epidermis that is only attached to the mesophyll along major veins (Jewer et al., 1982). The isolated 389

epidermis of this mutant allowed for a rapid sampling of epidermal tissue from the same leaves in 390

which leaf gas exchange was measured. Abaxial epidermes were entirely removed in a single peel 391

and briskly placed in a 1.5 ml tube, weighed (±0.0001 g), covered in cold 80% methanol with added 392

BHT and transferred to -20°C (no more than 20 s elapsed from leaf excision to the covering of 393

epidermes in cold methanol solution). ABA level was quantified from the remainder of the leaf, 394

which was enclosed briefly (less than 30 s) in damp paper towel. This tissue was weighed in a similar 395

fashion, covered in cold 80% methanol with added BHT, and also transferred to -20°C. To each 1.5 396

ml tube, a single stainless steel ball bearing was added, along with 15 ng of internal standard 397

[2H6]ABA. Samples were then homogenised using a cell lyses machine (TissueLyser II, Qiagen) and 398

ABA extracted by storing samples overnight at 4°C, centrifuging for 5 min at 13000 rpm and drying 399



the supernatant to completeness under vacuum. For all samples, endogenous ABA was quantified 400

by UPLC-MS, according to the methods of McAdam and Brodribb (2014). Both mass normalised ABA 401

levels were determined from each sample (as described above) and, as the entire extract from each 402

sample was used to quantify ABA levels, the proportion of total foliar ABA found in the abaxial 403

epidermis was also determined from the raw UPLC-MS peak areas. Total foliar ABA was calculated 404

as the sum of peak areas from both the abaxial epidermal sample and remaining leaf tissue sample. 405

Transfer of [2H6]ABA from the transpiration stream to the epidermis in the Argenteum mutant of 406

Pisum sativum 407

To assess whether ABA synthesised in the leaf could be transported to the epidermis and influence 408

stomatal aperture, again we used the Argenteum mutant of P. sativum. Labelled [2H6]ABA was fed 409

into the transpiration stream of an Argenteum leaf through the stem excised under water while leaf 410

gas exchange was continuously monitored. The experimental plant was 15 days old with three fully 411

expanded leaves. The night prior to this experiment, the stem of the plant was excised under water 412

and maintained in the dark until the following morning. At 0830 h on the day of the experiment, the 413

most recent fully expanded leaf was enclosed in the cuvette of an infrared gas analyser (LI-6400, LI-414

COR Biosciences), environmental conditions in the cuvette were sustained for the duration of the 415

experiment under the following conditions: air temperature 22°C, light intensity 1000 μmol quanta 416

m-2

s-1

, and VPD regulated at 1.2 kPa (using a portable dew-point generator (LI-610, LI-COR 417

Biosciences)). When gs had reached a steady-state maximum for at least six min, background levels 418

of [2H6]ABA in the abaxial epidermis and in the leaf tissue were assessed in a neighbouring leaf, by 419

harvesting both tissues as described above. After this initial collection, [2H6]ABA was added to the 420

transpiration stream at a concentration of 1000 ng ml -1 (the cut stem was approximately 5 cm from 421

the lamina of the leaf in the cuvette). Leaf gas exchange was monitored until gs had declined and 422

reached stability after 35 min, following which the leaf in the cuvette was harvested for [2H6]ABA 423



quantification. [2H6]ABA levels were assessed in both the abaxial epidermis and remaining leaf 424

tissue, using the harvest, extraction, purification and quantification methods described above. 425

Stomatal sensitivities to exogenous ABA 426

In all species for which there was a systematic change in foliar ABA level during transitions in VPD, 427

one of two methods were used to assess the sensitivity of stomata to exogenously applied ABA. In 428

species that did not have pronounced hydropassive, wrong-way responses after excision under 429

water and in which epidermal peels or assessment of stomatal apertures by microscopy were not 430

possible in (the conifer species P. ladei and the woody angiosperm species Q. robur) stomatal 431

sensitivity to ABA was assessed in branches that were fed exogenous ABA as described by McAdam 432

and Brodribb (2014). Namely leaf gas exchange was measured (as described above) in three 433

branches that were excised under resin-filtered, deionized water. Leaves were equilibrated in the 434

leaf cuvette until maximum and stable gs was reached; then, an aliquot of concentrated exogenous 435

ABA, prepared by dissolving 90% crystalline ABA of mixed isomers (Sigma Chemical Co.) in 1 mL 436

methanol and then diluting up to 250 mL with resin-filtered, deionized water, was added to the leaf 437

water supply, increasing the concentration of ABA in the water to 2,500 ng mL. Periodically, as 438

stomata closed, short shoots (of P. ladei) or neighbouring leaves (of Q. robur) were taken from the 439

branch for foliar ABA quantification (see above). Each branch contributed at least five paired gs and 440

foliar ABA level data points. In the remaining species: the woody angiosperm A. trichopoda, and 441

herbaceous angiosperms D. hybrida and P. sativum, stomatal sensitivity to ABA was determined by 442

measuring the dose-dependent response of stomatal apertures (in viable guard cells from isolated 443

epidermis) to exogenous ABA. Both of these methods are known to accurately reflect stomatal 444

sensitivities ABA, having been both tested against each other (and found to correspond) as well 445

against the sensitivity of stomata to endogenously synthesised ABA (McAdam and Brodribb, 2014). 446

Epidermes were prepared as described by (Brodribb and McAdam, 2013). Following incubation in 447

the light (200 µmol quanta m-2 s-1) for 1 h in control buffer solution (50 mM KCl, 10 mM MES, 0.1 448



mM CaCl2, pH 6.15, rendered nominally CO2 free following 1 h of bubbling with N2 gas), at least two 449

epidermes were further incubated for 2 h in one of five increasing concentrations (0, 2, 20, 80 or 200 450

ng ml-1

) of exogenous ABA dissolved in the same buffer solution. Stomatal apertures (n=50 451

minimum) were then observed. Brodribb and McAdam (2013) describe the methods that were used 452

to prepare epidermes, assess guard cell viability and perform the double-blind measurements of 453

stomatal apertures. Stomatal conductance from measured stomatal apertures at each 454

concentration of ABA was calculated according to the formula given by Parlange and Waggoner 455

(1970). Stomatal length was determined from the same images used to quantify stomatal apertures. 456

Stomatal density was quantified from ten images taken at random at a magnification of x20 from the 457

epidermes used to quantify stomatal apertures. Stomatal pore depth was measured from leaf cross-458

sections of each species. To determine the relative importance of ABA-induced stomatal closure 459

during VPD transitions, empirical relationships between gs and exogenously applied foliar ABA level 460

were established for each species (Supplementary Figure S1), exponential decay functions were 461

fitted to these relationships to determine the foliar ABA level in the absence of water stress that 462

would be sufficient to result in the observed decline in gs during the VPD transition. For the conifer 463

species M. glyptostroboides the empirical relationship between gs and foliar ABA level was taken 464

from McAdam and Brodribb (2014). 465

466

Determining changes in the rates of stomatal responses to VPD 467

To provide a quantification of hysteresis observed during the response of stomata to a reversible 468

transition in VPD, comparisons were made between the slopes of linear regressions fitted to the 469

dynamic gs data collected from 15, 30 and 45 minutes following a transition in VPD. The percentage 470

change between the slope of stomatal closure compared to the slope of stomatal opening was 471

calculated for each species. Analysis of covariance was then used to compare the two linear 472

regressions using the AOV function in R (version 2.15.1). 473



474

Acknowledgements 475

We thank John Ross for his sage advice on the quantification of ABA levels; Jim Weller for the gift of 476

Argenteum seeds; Forestry Plantations Queensland for the gift of Pinus caribaea seeds; Noel Davies 477

and David Nichols for performing UPLC-MS analyses; Erin McAdam for statistical analyses; Samuel 478

Martins and Rachael Berry for technical assistance; Frances Sussmilch for photography; Chris Cooper 479

for helpful discussion; and three reviewers for insightful comments. 480

481

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625

626



Figure Legends 627

628

Figure 1. Trajectories of stomatal conductance (gs, black circles, n=3 leaves, ±SE) and foliar ABA level 629

(white circles, n=3, ±SE) taken from the same leaves, as plants from four angiosperm species were 630

exposed to a reversible sequence of VPD transitions from 0.7 kPa (blue) to 1.5 kPa (green) and 631

returning to 0.7 kPa (blue). In all species the exogenously applied ABA level known to induce a 632

similar reduction in gs as what was observed at 1.5 kPa is presented as a red horizontal line (see Fig. 633

3). 634


(white circles, n=3, ±SE) taken from the same leaves, as plants from three conifer species were 636

exposed to a reversible sequence of VPD transitions from 0.7 kPa (blue) to 1.5 kPa (green) and 637

returning to 0.7 kPa (blue). In all species for which an increase in foliar ABA level was observed at 638

1.5 kPa, the exogenously applied ABA level known to induce a similar reduction in gs as what was 639

observed at 1.5 kPa is presented as a red horizontal line (see Fig. 3). 640


(white circles, n=3, ±SE) taken from the same leaves (except in Pteridium esculentum), as plants from 642

three fern species were exposed to a reversible sequence of VPD transitions from 0.7 kPa (blue) to 643

1.5 kPa (green) and returning to 0.7 kPa (blue). 644

Figure 4. The percentage change in the rate of stomatal response to VPD between the closing 645

response of stomata following a transition from 0.7 to 1.5 kPa with stomatal opening on returning to 646

0.7 kPa. Stars denote significant differences between the slopes of linear regressions (n.s. not 647

significant, ** 0.02 > P > 0.01, *** 0.01 > P). The percentage change in the rate of the stomatal 648

response to VPD in Quercus robur was almost significant (P=0.052) reflecting the increase in foliar 649

ABA at a VPD of 1.5 kPa to a level that did not quite reach the threshold for stomatal closure (see 650

Fig. 1). 651



Figure 5. The response of stomatal conductance (gs) to exogenously applied ABA with fitted three 652

parameter exponential decay functions (black lines). In three species gs values were calculated from 653

stomatal aperture dimensions of isolated epidermis incubated at various ABA concentrations (A to C, 654

insert depicts stomatal aperture measurements of n=50 viable stomata, ±SE), while in two species 655

the sensitivity of stomata to ABA was assessed in hydrated branches in which gs was monitored 656

following the feeding of exogenous ABA into the transpiration stream (D and E). 657

658

Figure 6. Mean foliar ABA levels (A) and stomatal conductance (B) as three stems (± SE) of Amborella 659

trichopoda were exposed to a reversible transition in VPD from 0.7 kPa (blue, right bar) to 1.5 kPa 660

(green) and returning to 0.7 kPa (blue, left bar) for 20 min at each VPD. (C) The mean concentration 661

of ABA in the xylem sap of the three branches (± SE) taken from a plant grown under 0.7 kPa and the 662

three branches excised under water on completion of the experiment. 663

664

Figure 7. (A) The proportion of ABA from the whole leaf that is found in the abaxial epidermis (n=3, 665

±SE) and stomatal conductance (gs, red line taken from Fig. 1) during a sequence of VPD transitions 666

from 0.7 kPa (blue) to 1.5 kPa (green) and returning to 0.7 kPa (blue) in the Argenteum mutant of 667

the herbaceous angiosperm Pisum sativum. (B) A comparison between the mass normalised foliar 668

ABA level and abaxial epidermal ABA level from the same leaves (taken from Supplementary Fig. S2) 669

following a step-increase in VPD from 0.7 kPa to 1.5 kPa, times of sampling are shown, the red line 670

represents a fitted linear regression while the black line a 1:1 relationship between the two mass 671

normalised ABA levels (the two regressions are significantly different, P=0.0018). See 672

Supplementary Fig. S1 for ABA levels plotted over time. (C) The distinctive foliar morphology of the 673

Argenteum mutant (right) in comparison to a wild-type leaflet (left), the silvery colouring is due to a 674

large air space between the epidermis and mesophyll (scale bar = 1 cm). (d) The response of gs in a 675

leaf of the Argenteum mutant excised under water and fed labelled [2H6]ABA into the transpiration 676

stream (dashed red line). Chromatograms from UPLC-MS analyses of the [2H6]ABA channel (m/z 677



269.2 to 159.1) with peaks from leaf tissue (green) and abaxial epidermal tissue (blue) prior to and 678

after feeding (denoted by arrows, peaks are relative to sample mass, red stars indicate the retention 679

time for [2H6]ABA). 680

681

Table 682

683

Table 1. The leaf water potentials (MPa, n=3, ±SE) at each equilibrium point before and after VPD 684

transitions in all sampled species. 685

686 Group Species Ψl Initial 0.7 kPa Ψl

1.5 kPa

Ψl Final 0.7

kPa

Fern Pteridium esculentum -0.32 ±0.04 -0.67 ±0.02 -0.32 ±0.05

Fern Marsilea hirsuta -0.17 ±0.04 -0.58 ±0.04 -0.26 ±0.06

Fern Pyrrosia lingua -0.61 ±0.06 -0.95 ±0.02 -0.71 ±0.03

Conifer Metasequoia glyptostroboides -0.4 ±0.04 -0.64 ±0.04 -0.41 ±0.03

Conifer Prumnopitys ladei -0.63 ±0.04 -1.05 ±0.1 -0.67 ±0.01

Conifer Pinus caribaea -0.36 ±0.05 -0.56 ±0.03 -0.37 ±0.04

Woody angiosperm Amborella trichopoda -0.38 ±0.02 -0.7 ±0.01 -0.36 ±0.03

Woody angiosperm Quercus robur -0.36 ±0.02 -0.39 ±0.02 -0.35 ±0.01

Herbaceous angiosperm Dahlia hydrida -0.4 ±0.01 -0.7 ±0.04 -0.3 ±0.04

Herbaceous angiosperm Pisum sativum -0.3 ±0.02 -0.42 ±0.01 -0.12 ±0.01

687



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