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Short title The role of era1 in stomatal responses 1

Corresponding author Mikael Broscheacute 2

Division of Plant Biology Department of Biosciences Viikki Plant Science Centre University of 3 Helsinki PO Box 65 (Viikinkaari 1) FI-00014 Helsinki Finland 4

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The Role of ENHANCED RESPONSES TO ABA1 (ERA1) in Arabidopsis 6

Stomatal Responses Is Beyond ABA Signaling 7

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Pirko Jalakas1 Yi-Chun Huang2 Yu-Hung Yeh2 Laurent Zimmerli2 Ebe Merilo1 Hannes Kollist1 10 and Mikael Broscheacute13 11

1 Institute of Technology University of Tartu Nooruse 1 50411 Tartu Estonia 12

2 Department of Life Science and Institute of Plant Biology National Taiwan University Taipei 106 13 Taiwan 14

3 Division of Plant Biology Department of Biosciences Viikki Plant Science Centre University of 15 Helsinki PO Box 65 (Viikinkaari 1) FI-00014 Helsinki Finland 16

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One sentence summary 18

Analysis of single and double mutants shows an ABA-independent role for ERA1 in stomatal 19 opening 20

21

Footnotes 22

Author contributions 23

MB conceived the project with help from HK and EM PJ performed most of the experiments 24 with assistance from EM and MB EM and MB supervised the experiments Y-CH and Y-25 HY performed the pathogen experiments under supervision from LZ PJ EM HK and MB 26 analyzed the data MB wrote the article with input from all authors 27

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Plant Physiology Preview Published on March 22 2017 as DOI101104pp1700220

Copyright 2017 by the American Society of Plant Biologists

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Funding information 29

This work was funded by the Estonian Ministry of Science and Education (IUT2-21 to HK and 30 PUT-1133 to EM) the European Regional Development Fund (Center of Excellence in Molecular 31 Cell Engineering CEMCE to HK) the Academy of Finland (grant number 307335 Center of 32 Excellence in Primary Producers 2014-2019 to MB) and by the National Science Council of 33 Taiwan (grant 105-2311-B-002-032-MY3 to LZ) 34

Corresponding author e-mail mikaelbroschehelsinkifi 35

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ABSTRACT 37

Proper stomatal responses are essential for plant function in an altered environment The core 38

signaling pathway for abscisic acid (ABA)-induced stomatal closure involves perception of the 39

hormone that leads to the activation of guard cell anion channels by the protein kinase OPEN 40

STOMATA1 (OST1) Several other regulators are suggested to modulate the ABA signaling 41

pathway including the protein ENHANCED RESPONSE TO ABA 1 (ERA1) that encodes the 42

farnesyl transferase beta subunit The era1 mutant is hypersensitive to ABA during seed 43

germination and shows a more closed stomata phenotype Using a genetics approach with the 44

double mutants era1 abi1-1 and era1 ost1 we show that while era1 suppressed the high stomatal 45

conductance of abi1-1 and ost1 the ERA1 function was not required for stomatal closure in 46

response to ABA and environmental factors Further experiments indicated a role for ERA1 in blue 47

light induced stomatal opening In addition we show that ERA1 function in disease resistance was 48

independent of its role in stomatal regulation Our results indicate a function for ERA1 in stomatal 49

opening and pathogen immunity 50

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INTRODUCTION 52

Plants need to monitor their environment and precisely respond when conditions around them 53 change At the frontline are plant stomata formed by a pair of guard cells which regulate CO2 54 uptake and simultaneously control water release Guard cell function is regulated by a multitude of 55 signals including light humidity CO2 concentration abscisic acid (ABA) and secondary signals 56 such as reactive oxygen species nitric oxide and Ca2+ (Kollist et al 2014 Sierla et al 2016) 57

The plant hormone ABA plays a central role in the regulation of guard cell function ABA signaling 58 is initiated by binding of the hormone to PYRRCAR receptors that leads to inactivation of type 2C 59 protein phosphatases (PP2Cs) (Ma et al 2009 Park et al 2009) This releases SNF1-related 60 protein kinases (SnRK2s) such as OPEN STOMATA1 to activate multiple signaling pathways 61 including activation of guard cell anion channels that leads to extrusion of water loss of turgor and 62 concomitant stomatal closure (Kollist et al 2014) Regulation of stomatal closure is coordinated 63 with the regulation of stomatal opening The driving force for stomatal opening is the 64 phosphorylation-dependent activation of plasma membrane H+-ATPases Blue light-induced 65 stomatal opening is mediated through phototropins PHOT1 and PHOT2 BLUE LIGHT 66 SIGNALING1 (BLUS1) kinase and activation of H+-ATPases (Shimazaki et al 2007 Takemiya 67 and Shimazaki 2016) ABA is involved in both stomatal closure and opening promoting closure 68 and inhibition of opening via OST1 (Hayashi et al 2011 Yin et al 2013) 69

Another regulator of stomatal function is ENHANCED RESPONSE TO ABA1 ERA1 encodes the 70 beta subunit of farnesyl-transferase (Cutler et al 1996) Farnesylation is a post-transcriptional 71 protein modification where 15-carbon isoprenoid units are attached to target proteins at the 72 sequence CaaX (C = cysteine a = aliphatic amino acid X = typically alanine cysteine glutamine 73 methionine or serine) The addition of farnesyl groups facilitates protein association with 74 membranes (Galichet and Gruissem 2003) The era1 mutant was initially identified through its 75 hypersensitivity to ABA inhibition of seed germination (Cutler et al 1996) Furthermore era1 76 mutant plants have more closed stomata enhanced ABA activation of anion channels and 77 increased drought tolerance (Pei et al 1998) While around 700 Arabidopsis proteins were 78 identified as potential targets of ERA1-induced farnesylation the underlying mechanisms have 79 been characterized only for ALTERED SEED GERMINATION 2 (ASG2) and the cytochrome P450 80 enzyme CYP85A2 that executes the last step in brassinosteroid biosynthesis (Dutilleul et al 2016 81 Northey et al 2016) Consistent with ASG2 being an ERA1 target the asg2 mutant has a similar 82 ABA hypersensitive seed germination phenotype as era1 (Dutilleul et al 2016) CYP85A2 was 83 identified as a potential ERA1 target due to similar developmental phenotypes (including shorter 84 petioles and flowers with protruding carpels) between era1 and cyp85a2 mutants (Northey et al 85 2016) In addition to regulation of stomatal responses seed germination and developmental 86

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responses ERA1 also regulates pathogen and heat stress responses (Goritschnig et al 2008 Wu 87 et al 2017) The era1 mutant has enhanced susceptibility to the virulent pathogens Pseudomonas 88 syringae pv maculicola and Hyaloperonospora parasitica (Goritschnig et al 2008) However 89 despite some progress in decoding the function of ERA1-induced farnesylation in plants its role in 90 stomatal and immune functions remains an enigma 91

Here we used double mutant analysis to better understand the role of ERA1 in stomatal signaling 92 This revealed that ERA1 function in guard cells is not required for stomatal closure in response to 93 ABA and a change in the environment Instead ERA1 is required for proper stomatal opening to 94 blue light and to maintain overall plant stomatal openness In pathogen infections ERA1 regulated 95 disease resistance independently from stomatal function Collectively our data suggest that guard 96 cell signaling output is the sum of multiple signaling pathways and that ERA1 regulates the basal 97 level of stomatal openness 98

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RESULTS AND DISCUSSION 100

101 Steady-state stomatal conductance of era1 single and double mutants 102

Genetic analysis is a powerful method to identify regulators of signaling pathways Furthermore 103 through the use of double mutants it becomes possible to investigate whether a given mutant acts 104 in the same or separate signaling pathways based on epistasis or additive effects between 105 mutations We crossed era1-2 that has low stomatal conductance with ost1-3 and abi1-1 that have 106 high stomatal conductance and measured stomatal conductance and rapid stomatal responses to 107 different abiotic stimuli using a custom-made gas exchange device as described before (Kollist et 108 al 2007) Consistent with previous results for era1 abi1 (Pei et al 1998) the era1 abi1-1 double 109 mutant had lower stomatal conductance compared to the single mutant abi1-1 (Fig 1) Similarly 110 the era1 mutation significantly lowered the high stomatal conductance of ost1 in the double mutant 111 era1 ost1 (Fig 1) One way to explain the steady-state stomatal conductance data would assign a 112 role for ERA1 in the regulation of the ABA signaling pathway where ABI1 and OST1 are key 113 regulators However ABI1 OST1 but also other significant proteins of the ABA signaling pathway 114 including ABA receptors ABI2 and the ion channel SLAC1 (SLOW ANION CHANNEL1) do not 115 have the CaaX motif and thus are unlikely direct targets of ERA1 Another option would be that 116 ERA1 functions in a different signaling cascade which affects stomatal conductance but is not the 117 ABA signaling pathway 118

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ERA1 does not affect fast stomatal closure in response to external ABA or environmental 120 stimuli 121

Several factors induce fast stomatal closure including external ABA application decreased air 122 humidity darkness and elevated CO2 concentration All these treatments require OST1 for normal 123 stomatal closure to take place (Mustilli et al 2002 Merilo et al 2013 Merilo et al 2015) Since 124 era1 suppressed the high stomatal conductance in ost1 (Fig 1) we tested the response of era1 125 ost1 to these stimuli (Fig 2) The era1 ost1 double mutant behaved similarly to the single ost1 126 mutant and showed reduced stimuli-induced stomatal closures (Figs 2A-D) with the exception of 127 small non-significant responsiveness to ABA re-gained in era1 ost1 128

ABI1 belongs to the PP2Cs that inhibit OST1 function (Fujii et al 2009) While the abi1-1 mutation 129 led to very high stomatal conductance (Fig 1) and reduced response to ABA (Fig 2D and 2H) the 130 initial changes in stomatal conductance induced by reduced air humidity darkness and elevated 131 CO2 were similar in abi1-1 and Col-0 due to nearly 3 times higher conductance of abi1-1 The era1 132 abi1-1 double mutant behaved similarly to the single abi1-1 mutant and showed reduced ABA-133 induced stomatal closure (Fig 2) No major differences between era1 and Col-0 to the applied 134 treatments were detected suggesting that ERA1-dependent farnesylation does not regulate fast 135 stomatal closure We conclude that while era1 can suppress the high stomatal conductance of 136 abi1-1 and ost1 the function of ERA1 is not related to stomatal closure in response to ABA and 137 abiotic factors Recently it was demonstrated that protein farnesylation by ERA1 plays an important 138 role in the regulation of plant heat stress responses in an ABA-independent manner (Wu et al 139 2017) These results further support that ERA1-dependent protein farnesylation also functions 140 outside of ABA signaling 141

Taken together OST1 was required for fast stomatal closure while ERA1 was more important for 142 the basal openness of the stomata One challenge in building a proper model of stomatal behavior 143 is the heterogeneity of assays used to investigate stomatal function One of the most popular 144 assays to study guard cell function is to measure stomatal aperture in epidermal peels or from leaf 145 photos after a treatment (eg ABA) which is frequently done only at a single time point rather late 146 after the treatment (Pei et al 1998 Mustilli et al 2002 Acharya et al 2013 Yin et al 2013) This 147 type of assay is likely to miss the early dynamics of the stomatal response While characterizing 148 the function of a particular stomatal regulator it is thus important to address its role in fast 149 responses triggering stomatal movements as well as the role for overall stomatal opening 150

151

ERA1 targets ASG2 and CYP85A2 do not regulate stomatal closure 152

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ERA1 mediates farnesylation of target proteins of which the best characterized are ASG2 153 (Dutilleul et al 2016) and CYP85A2 (Northey et al 2016) Furthermore the small GTPase 154 ROP11 is a proposed regulator of ABA signaling downstream from the receptor kinase FERONIA 155 (Li and Liu 2012 Li et al 2012 Yu et al 2012) and ROP10 is proposed to be farnesylated by 156 ERA1 (Zheng et al 2002) We tested asg2 and cyp85a2 responses to various treatments that lead 157 to stomatal closure (Supplemental Figure 1) Steady-state stomatal conductance and stomatal 158 responsiveness to stimuli of asg2 and cyp85a2 were completely wildtype-like As a next step we 159 tested stomatal responses of a rop10 rop11 double mutant which were also similar to the wildtype 160 (Supplemental Figure 1) Further mutant analysis could lead to more ERA1 targets identified but 161 this might be hampered by genetic redundancy A protein purification strategy similar to the one 162 used by Dutilleul et al (2016) but starting from isolated guard cells might more directly identify the 163 relevant proteins farnesylated by ERA1 in guard cells 164

165

Gene expression analysis in era1 166

One potential explanation for era1 phenotypes could be a higher accumulation of ABA in this 167 mutant However direct ABA measurements in Col-0 and era1-2 showed that ERA1 does not 168 regulate the ABA concentration (Ghassemian et al 2000) Altered guard cell expression levels of 169 key genes in ABA biosynthesis ABA catabolism ABA signaling or stomatal signaling could be 170 another explanation for ERA1-dependent stomatal phenotypes We isolated RNA from guard cell-171 enriched epidermal fragments obtained with the ice-blender method (Bauer et al 2013) 172 Comparing the guard cell RNA and a corresponding whole-leaf RNA samples for two guard cell 173 expressed genes (HT1 - HIGH LEAF TEMPERATURE1 GORK - GATED OUTWARDLY-174 RECTIFYING K+ CHANNEL) at least four-fold enrichment of guard cell gene expression was 175 detected (Supplemental Figure 2) We tested the expression of 12 genes representing different 176 steps of ABA homeostasis ABA signaling and key stomatal ion transporters No significant 177 differences compared to Col-0 were observed except for slightly increased expression of AAO3 178 (ABSCISIC ALDEHYDE OXIDASE3) GORK HAB1 (HYPERSENSITIVE TO ABA1) HAI1 179 (HIGHLY ABA-INDUCED PP2C GENE 1) and SLAC1 in the ost1 background (Supplemental 180 Figure 2) Thus ERA1 is unlikely to be a regulator of ABA-related gene expression in guard cells 181

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The role of ERA1 in stomatal opening 183

The stimuli studied above decreased air humidity darkness increased CO2 and ABA all induce 184 stomatal closure While traditionally signaling pathways in guard cells are broadly divided into the 185 closure and opening pathways (Kollist et al 2014) these pathways have extensive interactions 186

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(Lawson and Blatt 2014) As previously mentioned the ABA signaling pathway participates in both 187 stomatal closure and opening Another example is the ion channel SLAC1 whose loss of function 188 mutant slac1 is not only impaired in stomatal closure but also stomatal opening through a 189 feedback change in pH cytosolic [Ca2+] and the activity of K+ channels (Wang et al 2012 190 Laanemets et al 2013) Since the era1 mutation did not have any influence on fast stomatal 191 closure either in single or double mutants we tested whether ERA1 might be part of the stomatal 192 opening pathway Plants were first kept in darkness for 90 min which ensured that stomatal 193 conductances of era1 and Col-0 were similar After application of white light the initial stomatal 194 opening kinetics of dark-adapted era1 was similar to that of Col-0 however after 20 min in light the 195 stomatal conductances of wildtype and era1 plants departed (Fig 3) As a result the era1 stomatal 196 conductance was significantly lower than in Col-0 at the end of the opening experiment (Fig 3A) 197 ABA can also inhibit light-induced stomatal opening This response was similar in Col-0 and era1 198 though stomatal conductance was again lower in era1 at the end of the experiment Fig 3A) 199

Stomatal opening in response to light is largely driven by blue light (Hayashi et al 2011) Next we 200 compared the stomatal opening induced by blue and red light (Figs 3B and 3C) This revealed that 201 stomatal opening induced by blue light was impaired in era1 plants and suggests a potential 202 function for ERA1 farnesylation in a biological process related to stomatal opening under blue light 203 PROTON ATPase TRANSLOCATION CONTROL 1 (PATROL1) regulates intracellular membrane 204 traffic including the transport of the H+-ATPase AHA1 to the plasma membrane (Hashimoto-205 Sugimoto et al 2013) The patrol1 mutant is impaired in light induced stomatal opening similar to 206 era1 (Fig 3 (Hashimoto-Sugimoto et al 2013)) however PATROL1 does not have the CaaX 207 motif and thus it is unlikely that this stomatal regulator is a direct target of ERA1 The vesicle-208 trafficking protein SYP121 is another regulator of stomatal opening and transport of ion channels 209 especially K+ channels (Eisenach et al 2012) Possibly the protein farnesylated by ERA1 is 210 associated with some aspect for example vesicle transport of the proper translocation of H+-211 ATPases or other ion channels involved in stomatal opening to the plasma membrane 212

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ERA1 regulates pathogen responses independently of its stomatal function 214

The likely role of ERA1 farnesylation of target proteins in multiple biological processes makes it a 215 challenge to pinpoint the precise function of ERA1 in any of the many phenotypes attributed to 216 era1 Stomata also regulate entry of pathogens into leaves (Melotto et al 2006) To investigate 217 whether the ERA1 stomatal function is related to its role in basal pathogen resistance we 218 inoculated Col-0 era1 ost1 and era1 ost1 with virulent P syringae pv tomato DC3000 (Pst) and 219 the coronatine deficient strain Pst DC3118 (Pst cor-) (Fig 4) In pathogen and stomatal responses 220

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coronatine activates JA signaling to suppress salicylic acid mediated defenses (Brooks et al 221 2005) and reopens closed stomata during bacterial infection (Melotto et al 2006) 222

For the pathogen assays dipndashinoculation was used to favor the entry of Pst bacteria into leaves 223 through stomata thus allowing stomatal immunity to take place (Melotto et al 2006) Importantly 224 the coronatine deficient strain Pst cor- is less virulent than Pst when surface inoculation is used 225 as this strain cannot reopen stomata upon bacterial infection (Brooks et al 2005 Melotto et al 226 2006) Consistent with previous results the ost1 mutant was susceptible to Pst cor- infection 227 (Melotto et al 2006) Both Pst and Pst cor- were strongly virulent in the era1 single mutant This 228 implies that the ERA1 function in stomata and its role in basal immunity are not related since era1 229 has constitutively closed stomata (Fig 1) predicted to provide some level of resistance Similarly 230 the era1 ost1 double mutant was susceptible to pathogen infection to the same level as era1 231 further suggesting that ERA1 regulation of disease resistance is not directly associated with its 232 stomatal function (Fig 4) However since the exact target of ERA1 farnesylation in pathogen 233 responses is currently not known (Goritschnig et al 2008) further research is required to entangle 234 the role of ERA1 and stomatal function in the response to pathogens 235

236

CONCLUSIONS 237

Proper timing of stomatal movements is crucial to maintain overall plant water status In this study 238 we are able to dissect the kinetics of stomatal conductance following a sudden change in the 239 surrounding environment (Fig 2) This made it clear that OST1 is required for fast responses 240 while ERA1 controls basal whole-plant stomatal conductance Further phenotypic characterization 241 suggests that ERA1 function in stomatal signaling is related to blue light induced opening although 242 the exact target protein that gets farnesylated by ERA1 remains elusive 243

244

MATERIALS AND METHODS 245

Plant Material and growth conditions 246

Col-0 era1-2 ost1-3 (srk2e SALK_008068) rop10 (SALK_018747) rop11 (SALK_063154C) 247 cyp85a2-2 (SALK_ 129352) asg2-1 (SALK_040151) and asg2-2 (SALK_113565) were from the 248 European Arabidopsis Stock Centre (wwwarabidopsisinfo) The abi1-1 allele used was in the Col-249 0 accession and was a gift from Julian Schroeder Double mutants and other crosses were made 250 through standard techniques and genotyped with PCR-based markers (Supplemental Table 1) 251

Plants for gas-exchange measurements were sown into 21 (vv) peatvermiculite mixture and 252 grown through a hole in a glass plate covering the pot as described previously (Kollist et al 2007) 253

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Plants were grown in growth chambers (AR-66LX Percival Scientific IA USA and Snijders 254 Scientific Drogenbos Belgia) with 12 h photoperiod 2318 degC daynight temperature 100-150 255 μmol m-2 s-1 light and 70 relative humidity Plants were 24-32 days old during gas-exchange 256 experiments For guard cell isolation seeds were sown into 8 x 8 cm pots with four plants per pot 257 and grown in the conditions described above 258

259

Gas-exchange measurements 260

Stomatal conductance of intact plants was measured using a rapid-response gas exchange 261 measurement device consisting of eight flow-through whole-rosette cuvettes (Kollist et al 2014) 262 Representative photos of plants used for gas exchange measurements are presented in 263 Supplemental Figure 3 Plants were inserted into measurement chambers and after stomatal 264 conductance had stabilized the following stimuli were applied reduction in air humidity (decreased 265 from 60-80 to 30-40) darkness CO2 (increase from 400 ppm to 800 ppm) and ABA ABA-266 induced stomatal closure experiments were carried out as described previously (Merilo et al 267 2015) Initial changes in stomatal conductance were calculated as gs18-gs0 (stomatal conductance 268 value 18 min after factor application 16min in case of ABA spraying) Opening experiments were 269 performed with the application of either white light blue light red light or white light+ABA on dark-270 adapted plants kept in the measurement cuvettes at 0 light for at least 90 min At timepoint 0 271 different light bulbs were switched on light intensities were adjusted so that they were around 150 272 micromol m-2 s-1 irrespective of light spectral characteristics ABA-induced inhibition of stomatal 273 opening experiments were carried out as described previously (Horak et al 2016) 274

275

RNA isolation and qPCR 276

Samples enriched with guard cells were isolated from 5-7 week old plants starting from 17-18 277 plants and 4-5 leaves per plant using the ice-blender method (Bauer et al 2013) RNA was 278 extracted with the Spectrum Plant RNA isolation kit (Sigma-Aldrich) Total RNA was DNAseI 279 treated and cDNA was synthesized with Maxima H Minus reverse Transcriptase (Thermo Fischer 280 Scientific) qPCR was performed in triplicate with 5 x HOT FIREPol EvaGreen qPCR Mix Plus 281 ROX (Soils Biodyne) on an Applied Biosystems 7900HT Fast Real-Time PCR System (Foster 282 City CA USA) Primer sequences and primer efficiencies are listed in Supplemental Table 1 283 Analysis of the quantitative PCR data was performed with qBase+ 30 (Biogazelle) The reference 284 genes used for normalization were SAND TIP41 and YLS8 Statistical analysis was performed on 285 log2 transformed data 286

Pathogen assays 287

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Plants were grown in commercial potting soilperlite (32) at 22ndash24 C day and 17ndash19 C night 288 temperature under a 9-h-light15-h-dark photoperiod The lighting was supplied at an intensity of 289 sim100 μE mminus2 sminus1 by fluorescence tubes Bacterial strains Pst DC3000 and Pst DC3118 (cor-) were 290 provided by Barbara Kunkel (Washington University St Louis Missouri USA) Bacteria were 291 cultivated at 28C and 220 rpm in Kingrsquos B medium containing 50mgmL rifampicin (DC3000) or 292 rifampicinkanamycinspectinomycin (DC3118) 293

Five-week-old Arabidopsis plants were dipped in a bacterial suspension of 106 colony-forming units 294 (CFU)mL Pst DC3000 and 107 (CFU)mL Pst DC3118 in 10mM MgSO4 containing 001 Silwet L-295 77 (Lehle Seeds) for 15min After dipping plants were kept at 100 relative humidity overnight 296 For bacterial titers leaf discs collected at 2 dpi were washed twice with sterile water and 297 homogenized in 10 mM MgSO4 Quantification of bacterial growth was done as previously 298 described (Zimmerli et al 2000) Each biological repeat represents nine leaf discs from three 299 different plants 300

Statistical analysis 301

Statistical analysis were performed with Statistica v 71 (StatSoft Inc Tulsa OK USA) ANOVA 302 with Dunnettrsquos Tukey or Tukey unequal N HSD post hoc tests were used as indicated in figure 303 legends All effects were considered significant at p lt 005 304

Acknowledgments 305

We thank Irina Rasulova for technical assistance 306

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SUPPLEMENTAL DATA 308

Supplemental Figure 1 Stomatal responses of asg2-1 asg2-2 cyp85a2-2 and rop10 rop11 309

Supplemental Figure 2 Gene expression in guard cell enriched samples 310

Supplemental Figure 3 Representative photos of mutants and Col-0 wild type used for whole-311 plant gas exchange experiments 312

Supplemental Table 1 Primers used for genotyping and qPCR 313

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Supplemental Figure 1 Stomatal responses of asg2-1 asg2-2 cyp85a2-2 and rop10 rop11 319

(A-D) Time courses of stomatal conductances in response to reduced air humidity darkness 320 elevated CO2 and ABA treatment (n=5-7) respectively (E-H) Changes in stomatal conductance 321 values 322

Supplemental Figure 2 (A) Gene expression in guard cell enriched samples RNA was isolated 323 from Col-0 wildtype era1 ost1 and era1 ost1 guard cells extracted from 5-7 week old plants using 324 an ice blender method (Bauer et al 2013) Relative expression of selected marker genes were 325 determined with qPCR and is depicted relative to Col-0 (B) Relative expression of two guard cell 326 expressed genes (HT1 and GORK) in a comparison of the guard cell enriched RNA versus RNA 327 extracted from a Col-0 whole leaf sample (C) Relative expression of a mesophyll expressed gene 328 (At5g54250) in a comparison of the guard cell enriched RNA versus RNA extracted from a Col-0 329 whole leaf sample 330

The mean of three biological replicates are shown error bars depict plusmn SEM Letters denote 331 statistically significant differences between mutants denotes statistically significant difference 332 from Col-0 in A (ANOVA with Tukey HSD post hoc test p lt 005) or from Col-0 leaf sample in B 333 and C (ANOVA with Dunnettrsquos test p lt 005) 334

Supplemental Figure 3 Representative photos of mutants and Col-0 wild type used for whole-335 plant gas exchange experiments Plants are 26-30 days old Scale bar indicates 2 cm 336

Supplemental Table 1 Primers used for genotyping and qPCR 337

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FIGURE LEGENDS 339

Fig 1 Whole-plant stomatal conductances of single and double mutants of era1 with ost1 and 340 abi1-1 Stomatal conductance was measured from intact plants (Kollist et al 2007) Letters denote 341 statistically significant differences (ANOVA with Tukey unequal N HSD post hoc test p lt 005 n 342 =8-15) 343

Fig 2 Stomatal responses of single and double mutants of era1 with ost1 and abi1-1 (A-D) Time 344 courses of stomatal conductances in response to reduced air humidity darkness elevated CO2 345 and ABA treatment respectively (E-H) Changes in stomatal conductance during the first 18 346 minutes (except for ABA where 16 min was measured) Letters denote statistically significant 347 differences between the studied genotypes (ANOVA with Tukey unequal N HSD post hoc test p lt 348 005 n =6-15) 349

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Fig 3 Stomatal opening of dark-adapted Col-0 and era1 plants in response to (A) white light 350 added as a single factor or simultaneously with 25 μM ABA (n=8) (B) blue and (C) red light (n=7-351 8) Before application of light plants were adapted to full darkness for 90 minutes 352

Fig 4 Bacterial growth in Col-0 era1 ost1 and era1 ost1 plants Pst titers were evaluated at 2 dpi 353 Five-week-old plants were dip-inoculated with 106 CFUmL Pst DC3000 (A) or 107 CFUmL Pst 354 DC3118 (cor-) (B) Results are average plusmn SEM of 3 biological replicates each consisting of nine 355 leaf discs Letters denote statistically significant differences (ANOVA with Tukey HSD post hoc 356 test p lt 005) 357

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Li ZX Kang J Sui N Liu D (2012) ROP11 GTPase is a Negative Regulator of Multiple ABA Responses in 403 Arabidopsis Journal of Integrative Plant Biology 54 169-179 404

Ma Y Szostkiewicz I Korte A Moes D Yang Y Christmann A Grill E (2009) Regulators of PP2C 405 Phosphatase Activity Function as Abscisic Acid Sensors Science 324 1064-1068 406

Melotto M Underwood W Koczan J Nomura K He SY (2006) Plant stomata function in innate immunity 407 against bacterial invasion Cell 126 969-980 408

Merilo E Jalakas P Kollist H Brosche M (2015) The Role of ABA Recycling and Transporter Proteins in 409 Rapid Stomatal Responses to Reduced Air Humidity Elevated CO2 and Exogenous ABA Molecular 410 Plant 8 657-659 411

Merilo E Laanemets K Hu HH Xue SW Jakobson L Tulva I Gonzalez-Guzman M Rodriguez PL 412 Schroeder JI Brosche M Kollist H (2013) PYRRCAR Receptors Contribute to Ozone- Reduced Air 413 Humidity- Darkness- and CO2-Induced Stomatal Regulation Plant Physiology 162 1652-1668 414

Mustilli AC Merlot S Vavasseur A Fenzi F Giraudat J (2002) Arabidopsis OST1 protein kinase mediates 415 the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species 416 production Plant Cell 14 3089-3099 417

Northey JGB Liang S Jamshed M Deb S Foo E Reid JB McCourt P Samuel MA (2016) Farnesylation 418 mediates brassinosteroid biosynthesis to regulate abscisic acid responses Nature Plants 2 419

Park SY Fung P Nishimura N Jensen DR Fujii H Zhao Y Lumba S Santiago J Rodrigues A Chow TFF 420 Alfred SE Bonetta D Finkelstein R Provart NJ Desveaux D Rodriguez PL McCourt P Zhu JK 421 Schroeder JI Volkman BF Cutler SR (2009) Abscisic Acid Inhibits Type 2C Protein Phosphatases via 422 the PYRPYL Family of START Proteins Science 324 1068-1071 423

Pei ZM Ghassemian M Kwak CM McCourt P Schroeder JI (1998) Role of farnesyltransferase in ABA 424 regulation of guard cell anion channels and plant water loss Science 282 287-290 425

Shimazaki K Doi M Assmann SM Kinoshita T (2007) Light regulation of stomatal movement Annu Rev 426 Plant Biol 58 219-247 427

Sierla M Waszczak C Vahisalu T Kangasjarvi J (2016) Reactive Oxygen Species in the Regulation of 428 Stomatal Movements Plant Physiology 171 1569-1580 429

Takemiya A Shimazaki K (2016) Arabidopsis phot1 and phot2 phosphorylate BLUS1 kinase with different 430 efficiencies in stomatal opening J Plant Res 129 167-174 431

Wang Y Papanatsiou M Eisenach C Karnik R Williams M Hills A Lew VL Blatt MR (2012) Systems 432 dynamic modeling of a guard cell Cl- channel mutant uncovers an emergent homeostatic network 433 regulating stomatal transpiration Plant Physiol 160 1956-1967 434

Wu JR Wang LC Lin YR Weng CP Yeh CH Wu SJ (2017) The Arabidopsis heat-intolerant 5 (hit5)enhanced 435 response to aba 1 (era1) mutant reveals the crucial role of protein farnesylation in plant responses 436 to heat stress New Phytol 213 1181-1193 437

Yin Y Adachi Y Ye WX Hayashi M Nakamura Y Kinoshita T Mori IC Murata Y (2013) Difference in 438 Abscisic Acid Perception Mechanisms between Closure Induction and Opening Inhibition of 439 Stomata Plant Physiology 163 600-610 440

Yu F Qian L Nibau C Duan Q Kita D Levasseur K Li X Lu C Li H Hou C Li L Buchanan BB Chen L 441 Cheung AY Li D Luan S (2012) FERONIA receptor kinase pathway suppresses abscisic acid signaling 442 in Arabidopsis by activating ABI2 phosphatase Proc Natl Acad Sci U S A 109 14693-14698 443

Zheng ZL Nafisi M Tam A Li H Crowell DN Chary SN Schroeder JI Shen JJ Yang ZB (2002) Plasma 444 membrane-associated ROP10 small GTPase is a specific negative regulator of abscisic acid 445 responses in Arabidopsis Plant Cell 14 2787-2797 446

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15

Zimmerli L Jakab G Metraux JP Mauch-Mani B (2000) Potentiation of pathogen-specific defense 447 mechanisms in Arabidopsis by beta -aminobutyric acid Proc Natl Acad Sci U S A 97 12920-12925 448

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httpsplantphysiolorgDownloaded on February 14 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

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Fig 1 Whole-plant stomatal conductances of single and double mutants of era1 with ost1 and abi1-1 Stomatal conductance was measured from intact plants (Kollist et al 2007) Letters denote statisti-cally significant differences (ANOVA with Tukey unequal N HSD post hoc test p lt 005 n=8-15)

httpsplantphysiolorgDownloaded on February 14 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

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Fig 2 Stomatal responses of single and double mutants of era1 with ost1 and abi1-1 (A-D) Time courses of stomatal conduct-ances in response to reduced air humidity darkness elevated CO2 and ABA treatment respectively (E-H) Changes in stomatal conductance during the first 18 minutes (except for ABA where 16 min was measured) Letters denote statistically significant differences between the studied genotypes (ANOVA with Tukey unequal N HSD post hoc test p lt 005 n=6-15)

5 μM ABAABA

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c

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a a aa b b a ab bc bc bcc

gs16-gs0gs18-gs0gs18-gs0gs18-gs0

httpsplantphysiolorgDownloaded on February 14 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

0

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Fig 3 Stomatal opening of dark-adapted Col-0 and era1 plants in response to (A) white light added as a single factor or simultaneously with 25 μM ABA (n=8) (B) blue and (C) red light (n=7-8) Before application of light plants were adapted to full darkness for 90 minutes

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0

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teria

l num

ber l

og(C

FUc

m2 )

A B

Fig 4 Bacterial growth in Col-0 era1 ost1 and era1 ost1 plants Pst titers were evaluated at 2 dpi Five-week-old plants were dip-inoculated with 106 CFUmL Pst DC3000 (A) or 107 CFUmL Pst DC3118 (cor-) (B) Results are average plusmn SEM of 3 biological replicates each consisting of nine leaf discs Letters denote statistically significant differ-ences (ANOVA with Tukey HSD post hoc test p lt 005)

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Parsed CitationsAcharya BR Jeon BW Zhang W Assmann SM (2013) Open Stomata 1 (OST1) is limiting in abscisic acid responses of Arabidopsisguard cells New Phytologist 200 1049-1063

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Galichet A Gruissem W (2003) Protein farnesylation in plants - conserved mechanisms but different targets Current Opinion inPlant Biology 6 530-535

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Ghassemian M Nambara E Cutler S Kawaide H Kamiya Y McCourt P (2000) Regulation of abscisic acid signaling by the ethyleneresponse pathway in Arabidopsis Plant Cell 12 1117-1126

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Goritschnig S Weihmann T Zhang Y Fobert P McCourt P Li X (2008) A novel role for protein farnesylation in plant innateimmunity Plant Physiol 148 348-357

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Hayashi M Inoue S Takahashi K Kinoshita T (2011) Immunohistochemical Detection of Blue Light-Induced Phosphorylation of thePlasma Membrane H+-ATPase in Stomatal Guard Cells Plant and Cell Physiology 52 1238-1248

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Horak H Sierla M Toldsepp K Wang C Wang YS Nuhkat M Valk E Pechter P Merilo E Salojarvi J Overmyer K Loog MBrosche M Schroeder JI Kangasjarvi J Kollist H (2016) A Dominant Mutation in the HT1 Kinase Uncovers Roles of MAP Kinasesand GHR1 in CO2-Induced Stomatal Closure Plant Cell 28 2493-2509

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Kollist H Nuhkat M Roelfsema MRG (2014) Closing gaps linking elements that control stomatal movement New Phytologist 20344-62 httpsplantphysiolorgDownloaded on February 14 2021 - Published by

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Kollist T Moldau H Rasulov B Oja V Ramma H Huve K Jaspers P Kangasjarvi J Kollist H (2007) A novel device detects a rapidozone-induced transient stomatal closure in intact Arabidopsis and its absence in abi2 mutant Physiologia Plantarum 129 796-803

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Laanemets K Wang YF Lindgren O Wu J Nishimura N Lee S Caddell D Merilo E Brosche M Kilk K Soomets U Kangasjarvi JSchroeder JI Kollist H (2013) Mutations in the SLAC1 anion channel slow stomatal opening and severely reduce K+ uptakechannel activity via enhanced cytosolic [Ca2+] and increased Ca2+ sensitivity of K+ uptake channels New Phytol 197 88-98

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Lawson T Blatt MR (2014) Stomatal size speed and responsiveness impact on photosynthesis and water use efficiency PlantPhysiol 164 1556-1570

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Li Z Liu D (2012) ROPGEF1 and ROPGEF4 are functional regulators of ROP11 GTPase in ABA-mediated stomatal closure inArabidopsis FEBS Lett 586 1253-1258

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Li ZX Kang J Sui N Liu D (2012) ROP11 GTPase is a Negative Regulator of Multiple ABA Responses in Arabidopsis Journal ofIntegrative Plant Biology 54 169-179

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Ma Y Szostkiewicz I Korte A Moes D Yang Y Christmann A Grill E (2009) Regulators of PP2C Phosphatase Activity Function asAbscisic Acid Sensors Science 324 1064-1068

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Melotto M Underwood W Koczan J Nomura K He SY (2006) Plant stomata function in innate immunity against bacterial invasionCell 126 969-980

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Merilo E Jalakas P Kollist H Brosche M (2015) The Role of ABA Recycling and Transporter Proteins in Rapid StomatalResponses to Reduced Air Humidity Elevated CO2 and Exogenous ABA Molecular Plant 8 657-659

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Merilo E Laanemets K Hu HH Xue SW Jakobson L Tulva I Gonzalez-Guzman M Rodriguez PL Schroeder JI Brosche M KollistH (2013) PYRRCAR Receptors Contribute to Ozone- Reduced Air Humidity- Darkness- and CO2-Induced Stomatal RegulationPlant Physiology 162 1652-1668

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Mustilli AC Merlot S Vavasseur A Fenzi F Giraudat J (2002) Arabidopsis OST1 protein kinase mediates the regulation of stomatalaperture by abscisic acid and acts upstream of reactive oxygen species production Plant Cell 14 3089-3099

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Northey JGB Liang S Jamshed M Deb S Foo E Reid JB McCourt P Samuel MA (2016) Farnesylation mediates brassinosteroidbiosynthesis to regulate abscisic acid responses Nature Plants 2

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Park SY Fung P Nishimura N Jensen DR Fujii H Zhao Y Lumba S Santiago J Rodrigues A Chow TFF Alfred SE Bonetta DFinkelstein R Provart NJ Desveaux D Rodriguez PL McCourt P Zhu JK Schroeder JI Volkman BF Cutler SR (2009) AbscisicAcid Inhibits Type 2C Protein Phosphatases via the PYRPYL Family of START Proteins Science 324 1068-1071

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Pei ZM Ghassemian M Kwak CM McCourt P Schroeder JI (1998) Role of farnesyltransferase in ABA regulation of guard cellhttpsplantphysiolorgDownloaded on February 14 2021 - Published by

Copyright (c) 2020 American Society of Plant Biologists All rights reserved

anion channels and plant water loss Science 282 287-290Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Shimazaki K Doi M Assmann SM Kinoshita T (2007) Light regulation of stomatal movement Annu Rev Plant Biol 58 219-247Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Sierla M Waszczak C Vahisalu T Kangasjarvi J (2016) Reactive Oxygen Species in the Regulation of Stomatal Movements PlantPhysiology 171 1569-1580

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Takemiya A Shimazaki K (2016) Arabidopsis phot1 and phot2 phosphorylate BLUS1 kinase with different efficiencies in stomatalopening J Plant Res 129 167-174

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Wang Y Papanatsiou M Eisenach C Karnik R Williams M Hills A Lew VL Blatt MR (2012) Systems dynamic modeling of a guardcell Cl- channel mutant uncovers an emergent homeostatic network regulating stomatal transpiration Plant Physiol 160 1956-1967

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Wu JR Wang LC Lin YR Weng CP Yeh CH Wu SJ (2017) The Arabidopsis heat-intolerant 5 (hit5)enhanced response to aba 1(era1) mutant reveals the crucial role of protein farnesylation in plant responses to heat stress New Phytol 213 1181-1193

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Yin Y Adachi Y Ye WX Hayashi M Nakamura Y Kinoshita T Mori IC Murata Y (2013) Difference in Abscisic Acid PerceptionMechanisms between Closure Induction and Opening Inhibition of Stomata Plant Physiology 163 600-610

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Yu F Qian L Nibau C Duan Q Kita D Levasseur K Li X Lu C Li H Hou C Li L Buchanan BB Chen L Cheung AY Li D Luan S(2012) FERONIA receptor kinase pathway suppresses abscisic acid signaling in Arabidopsis by activating ABI2 phosphatase ProcNatl Acad Sci U S A 109 14693-14698

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Zheng ZL Nafisi M Tam A Li H Crowell DN Chary SN Schroeder JI Shen JJ Yang ZB (2002) Plasma membrane-associatedROP10 small GTPase is a specific negative regulator of abscisic acid responses in Arabidopsis Plant Cell 14 2787-2797

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Zimmerli L Jakab G Metraux JP Mauch-Mani B (2000) Potentiation of pathogen-specific defense mechanisms in Arabidopsis bybeta -aminobutyric acid Proc Natl Acad Sci U S A 97 12920-12925

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

httpsplantphysiolorgDownloaded on February 14 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved