Plant Physiology Preview. Published on March 22, 2017, as DOI:10.1104/pp.17.00220
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Short title: The role of era1 in stomatal responses
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Corresponding author: Mikael Brosché
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Division of Plant Biology, Department of Biosciences, Viikki Plant Science Centre, University of
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Helsinki, P.O. Box 65 (Viikinkaari 1), FI-00014 Helsinki, Finland
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The Role of ENHANCED RESPONSES TO ABA1 (ERA1) in Arabidopsis
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Stomatal Responses Is Beyond ABA Signaling
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Pirko Jalakas1, Yi-Chun Huang2, Yu-Hung Yeh2, Laurent Zimmerli2, Ebe Merilo1, Hannes Kollist1
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and Mikael Brosché1,3
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1 Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia
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2 Department of Life Science and Institute of Plant Biology, National Taiwan University, Taipei 106,
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Taiwan
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3 Division of Plant Biology, Department of Biosciences, Viikki Plant Science Centre, University of
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Helsinki, P.O. Box 65 (Viikinkaari 1), FI-00014 Helsinki, Finland
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One sentence summary:
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Analysis of single and double mutants shows an ABA-independent role for ERA1 in stomatal
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opening.
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Footnotes:
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Author contributions:
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M.B. conceived the project with help from H.K. and E.M. P.J. performed most of the experiments
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with assistance from E.M. and M.B. E.M. and M.B. supervised the experiments. Y.-C.H. and Y.-
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H.Y. performed the pathogen experiments under supervision from L.Z. P.J., E.M., H.K. and M.B.
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analyzed the data. M.B. wrote the article with input from all authors.
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Copyright 2017 by the American Society of Plant Biologists
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Funding information:
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This work was funded by the Estonian Ministry of Science and Education (IUT2-21 to H.K. and
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PUT-1133 to E.M.), the European Regional Development Fund (Center of Excellence in Molecular
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Cell Engineering CEMCE to H.K.), the Academy of Finland (grant number #307335, Center of
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Excellence in Primary Producers 2014-2019 to M.B.) and by the National Science Council of
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Taiwan (grant 105-2311-B-002-032-MY3 to L.Z.).
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Corresponding author: e-mail: [email protected]
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ABSTRACT
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Proper stomatal responses are essential for plant function in an altered environment. The core
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signaling pathway for abscisic acid (ABA)-induced stomatal closure involves perception of the
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hormone that leads to the activation of guard cell anion channels by the protein kinase OPEN
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STOMATA1 (OST1). Several other regulators are suggested to modulate the ABA signaling
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pathway, including the protein ENHANCED RESPONSE TO ABA 1 (ERA1), that encodes the
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farnesyl transferase beta subunit. The era1 mutant is hypersensitive to ABA during seed
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germination and shows a more closed stomata phenotype. Using a genetics approach with the
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double mutants era1 abi1-1 and era1 ost1, we show that while era1 suppressed the high stomatal
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conductance of abi1-1 and ost1, the ERA1 function was not required for stomatal closure in
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response to ABA and environmental factors. Further experiments indicated a role for ERA1 in blue
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light induced stomatal opening. In addition, we show that ERA1 function in disease resistance was
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independent of its role in stomatal regulation. Our results indicate a function for ERA1 in stomatal
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opening and pathogen immunity.
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INTRODUCTION
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Plants need to monitor their environment and precisely respond when conditions around them
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change. At the frontline are plant stomata, formed by a pair of guard cells, which regulate CO2
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uptake and simultaneously control water release. Guard cell function is regulated by a multitude of
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signals including light, humidity, CO2 concentration, abscisic acid (ABA) and secondary signals
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such as reactive oxygen species, nitric oxide and Ca2+ (Kollist et al., 2014; Sierla et al., 2016).
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The plant hormone ABA plays a central role in the regulation of guard cell function. ABA signaling
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is initiated by binding of the hormone to PYR/RCAR receptors that leads to inactivation of type 2C
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protein phosphatases (PP2Cs) (Ma et al., 2009; Park et al., 2009). This releases SNF1-related
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protein kinases (SnRK2s) such as OPEN STOMATA1 to activate multiple signaling pathways
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including activation of guard cell anion channels that leads to extrusion of water, loss of turgor, and
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concomitant stomatal closure (Kollist et al., 2014). Regulation of stomatal closure is coordinated
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with the regulation of stomatal opening. The driving force for stomatal opening is the
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phosphorylation-dependent activation of plasma membrane H+-ATPases. Blue light-induced
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stomatal opening is mediated through phototropins PHOT1 and PHOT2, BLUE LIGHT
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SIGNALING1 (BLUS1) kinase, and activation of H+-ATPases (Shimazaki et al., 2007; Takemiya
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and Shimazaki, 2016). ABA is involved in both stomatal closure and opening, promoting closure
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and inhibition of opening via OST1 (Hayashi et al., 2011; Yin et al., 2013).
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Another regulator of stomatal function is ENHANCED RESPONSE TO ABA1. ERA1 encodes the
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beta subunit of farnesyl-transferase (Cutler et al., 1996). Farnesylation is a post-transcriptional
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protein modification where 15-carbon isoprenoid units are attached to target proteins at the
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sequence CaaX (C = cysteine, a = aliphatic amino acid, X = typically alanine, cysteine, glutamine,
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methionine or serine). The addition of farnesyl groups facilitates protein association with
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membranes (Galichet and Gruissem, 2003). The era1 mutant was initially identified through its
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hypersensitivity to ABA inhibition of seed germination (Cutler et al., 1996). Furthermore, era1
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mutant plants have more closed stomata, enhanced ABA activation of anion channels and
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increased drought tolerance (Pei et al., 1998). While around 700 Arabidopsis proteins were
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identified as potential targets of ERA1-induced farnesylation, the underlying mechanisms have
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been characterized only for ALTERED SEED GERMINATION 2 (ASG2) and the cytochrome P450
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enzyme CYP85A2 that executes the last step in brassinosteroid biosynthesis (Dutilleul et al., 2016;
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Northey et al., 2016). Consistent with ASG2 being an ERA1 target, the asg2 mutant has a similar
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ABA hypersensitive seed germination phenotype as era1 (Dutilleul et al., 2016). CYP85A2 was
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identified as a potential ERA1 target due to similar developmental phenotypes (including shorter
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petioles and flowers with protruding carpels) between era1 and cyp85a2 mutants (Northey et al.,
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2016). In addition to regulation of stomatal responses, seed germination and developmental
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responses, ERA1 also regulates pathogen and heat stress responses (Goritschnig et al., 2008; Wu
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et al., 2017). The era1 mutant has enhanced susceptibility to the virulent pathogens Pseudomonas
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syringae pv maculicola and Hyaloperonospora parasitica (Goritschnig et al., 2008). However,
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despite some progress in decoding the function of ERA1-induced farnesylation in plants, its role in
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stomatal and immune functions remains an enigma.
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Here, we used double mutant analysis to better understand the role of ERA1 in stomatal signaling.
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This revealed that ERA1 function in guard cells is not required for stomatal closure in response to
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ABA and a change in the environment. Instead ERA1 is required for proper stomatal opening to
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blue light and to maintain overall plant stomatal openness. In pathogen infections, ERA1 regulated
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disease resistance independently from stomatal function. Collectively our data suggest that guard
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cell signaling output is the sum of multiple signaling pathways and that ERA1 regulates the basal
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level of stomatal openness.
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RESULTS AND DISCUSSION
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Steady-state stomatal conductance of era1 single and double mutants
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Genetic analysis is a powerful method to identify regulators of signaling pathways. Furthermore,
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through the use of double mutants it becomes possible to investigate whether a given mutant acts
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in the same or separate signaling pathways based on epistasis or additive effects between
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mutations. We crossed era1-2 that has low stomatal conductance, with ost1-3 and abi1-1 that have
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high stomatal conductance and measured stomatal conductance and rapid stomatal responses to
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different abiotic stimuli using a custom-made gas exchange device as described before (Kollist et
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al., 2007). Consistent with previous results for era1 abi1 (Pei et al., 1998), the era1 abi1-1 double
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mutant had lower stomatal conductance compared to the single mutant abi1-1 (Fig. 1). Similarly,
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the era1 mutation significantly lowered the high stomatal conductance of ost1 in the double mutant
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era1 ost1 (Fig. 1). One way to explain the steady-state stomatal conductance data would assign a
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role for ERA1 in the regulation of the ABA signaling pathway, where ABI1 and OST1 are key
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regulators. However, ABI1, OST1, but also other significant proteins of the ABA signaling pathway
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including ABA receptors, ABI2 and the ion channel SLAC1 (SLOW ANION CHANNEL1) do not
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have the CaaX motif, and thus are unlikely direct targets of ERA1. Another option would be that
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ERA1 functions in a different signaling cascade, which affects stomatal conductance, but is not the
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ABA signaling pathway.
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ERA1 does not affect fast stomatal closure in response to external ABA or environmental
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stimuli
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Several factors induce fast stomatal closure, including external ABA application, decreased air
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humidity, darkness and elevated CO2 concentration. All these treatments require OST1 for normal
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stomatal closure to take place (Mustilli et al., 2002; Merilo et al., 2013; Merilo et al., 2015). Since
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era1 suppressed the high stomatal conductance in ost1 (Fig. 1), we tested the response of era1
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ost1 to these stimuli (Fig. 2). The era1 ost1 double mutant behaved similarly to the single ost1
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mutant and showed reduced stimuli-induced stomatal closures (Figs. 2A-D); with the exception of
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small non-significant responsiveness to ABA re-gained in era1 ost1.
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ABI1 belongs to the PP2Cs that inhibit OST1 function (Fujii et al., 2009). While the abi1-1 mutation
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led to very high stomatal conductance (Fig. 1) and reduced response to ABA (Fig 2D and 2H), the
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initial changes in stomatal conductance induced by reduced air humidity, darkness and elevated
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CO2 were similar in abi1-1 and Col-0 due to nearly 3 times higher conductance of abi1-1. The era1
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abi1-1 double mutant behaved similarly to the single abi1-1 mutant and showed reduced ABA-
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induced stomatal closure (Fig. 2). No major differences between era1 and Col-0 to the applied
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treatments were detected, suggesting that ERA1-dependent farnesylation does not regulate fast
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stomatal closure. We conclude that while era1 can suppress the high stomatal conductance of
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abi1-1 and ost1, the function of ERA1 is not related to stomatal closure in response to ABA and
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abiotic factors. Recently it was demonstrated that protein farnesylation by ERA1 plays an important
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role in the regulation of plant heat stress responses, in an ABA-independent manner (Wu et al.,
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2017). These results further support that ERA1-dependent protein farnesylation also functions
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outside of ABA signaling.
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Taken together, OST1 was required for fast stomatal closure, while ERA1 was more important for
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the basal openness of the stomata. One challenge in building a proper model of stomatal behavior
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is the heterogeneity of assays used to investigate stomatal function. One of the most popular
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assays to study guard cell function is to measure stomatal aperture in epidermal peels or from leaf
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photos after a treatment (e.g. ABA) which is frequently done only at a single time point rather late
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after the treatment (Pei et al., 1998; Mustilli et al., 2002; Acharya et al., 2013; Yin et al., 2013). This
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type of assay is likely to miss the early dynamics of the stomatal response. While characterizing
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the function of a particular stomatal regulator, it is thus important to address its role in fast
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responses triggering stomatal movements as well as the role for overall stomatal opening.
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ERA1 targets ASG2 and CYP85A2 do not regulate stomatal closure
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ERA1 mediates farnesylation of target proteins, of which the best characterized are ASG2
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(Dutilleul et al., 2016) and CYP85A2 (Northey et al., 2016). Furthermore, the small GTPase
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ROP11 is a proposed regulator of ABA signaling downstream from the receptor kinase FERONIA
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(Li and Liu, 2012; Li et al., 2012; Yu et al., 2012), and ROP10 is proposed to be farnesylated by
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ERA1 (Zheng et al., 2002). We tested asg2 and cyp85a2 responses to various treatments that lead
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to stomatal closure (Supplemental Figure 1). Steady-state stomatal conductance and stomatal
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responsiveness to stimuli of asg2 and cyp85a2 were completely wildtype-like. As a next step, we
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tested stomatal responses of a rop10 rop11 double mutant, which were also similar to the wildtype
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(Supplemental Figure 1). Further mutant analysis could lead to more ERA1 targets identified, but
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this might be hampered by genetic redundancy. A protein purification strategy, similar to the one
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used by Dutilleul et al. (2016), but starting from isolated guard cells might more directly identify the
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relevant proteins farnesylated by ERA1 in guard cells.
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Gene expression analysis in era1
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One potential explanation for era1 phenotypes could be a higher accumulation of ABA in this
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mutant. However, direct ABA measurements in Col-0 and era1-2 showed that ERA1 does not
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regulate the ABA concentration (Ghassemian et al., 2000). Altered guard cell expression levels of
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key genes in ABA biosynthesis, ABA catabolism, ABA signaling or stomatal signaling could be
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another explanation for ERA1-dependent stomatal phenotypes. We isolated RNA from guard cell-
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enriched epidermal fragments, obtained with the ice-blender method (Bauer et al., 2013).
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Comparing the guard cell RNA and a corresponding whole-leaf RNA samples for two guard cell
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expressed genes (HT1 - HIGH LEAF TEMPERATURE1, GORK - GATED OUTWARDLY-
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RECTIFYING K+ CHANNEL), at least four-fold enrichment of guard cell gene expression was
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detected (Supplemental Figure 2). We tested the expression of 12 genes representing different
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steps of ABA homeostasis, ABA signaling and key stomatal ion transporters. No significant
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differences compared to Col-0 were observed except for slightly increased expression of AAO3
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(ABSCISIC ALDEHYDE OXIDASE3), GORK, HAB1 (HYPERSENSITIVE TO ABA1), HAI1
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(HIGHLY ABA-INDUCED PP2C GENE 1) and SLAC1 in the ost1 background (Supplemental
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Figure 2). Thus, ERA1 is unlikely to be a regulator of ABA-related gene expression in guard cells.
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The role of ERA1 in stomatal opening
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The stimuli studied above, decreased air humidity, darkness, increased CO2 and ABA, all induce
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stomatal closure. While traditionally signaling pathways in guard cells are broadly divided into the
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closure and opening pathways (Kollist et al., 2014), these pathways have extensive interactions
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(Lawson and Blatt, 2014). As previously mentioned, the ABA signaling pathway participates in both
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stomatal closure and opening. Another example is the ion channel SLAC1, whose loss of function
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mutant slac1 is not only impaired in stomatal closure, but also stomatal opening through a
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feedback change in pH, cytosolic [Ca2+] and the activity of K+ channels (Wang et al., 2012;
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Laanemets et al., 2013). Since the era1 mutation did not have any influence on fast stomatal
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closure either in single or double mutants, we tested whether ERA1 might be part of the stomatal
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opening pathway. Plants were first kept in darkness for 90 min, which ensured that stomatal
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conductances of era1 and Col-0 were similar. After application of white light the initial stomatal
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opening kinetics of dark-adapted era1 was similar to that of Col-0, however after 20 min in light the
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stomatal conductances of wildtype and era1 plants departed (Fig. 3). As a result, the era1 stomatal
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conductance was significantly lower than in Col-0 at the end of the opening experiment (Fig. 3A).
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ABA can also inhibit light-induced stomatal opening. This response was similar in Col-0 and era1,
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though stomatal conductance was again lower in era1 at the end of the experiment Fig. 3A).
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Stomatal opening in response to light is largely driven by blue light (Hayashi et al., 2011). Next, we
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compared the stomatal opening induced by blue and red light (Figs. 3B and 3C). This revealed that
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stomatal opening induced by blue light was impaired in era1 plants and suggests a potential
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function for ERA1 farnesylation in a biological process related to stomatal opening under blue light.
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PROTON ATPase TRANSLOCATION CONTROL 1 (PATROL1) regulates intracellular membrane
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traffic including the transport of the H+-ATPase AHA1 to the plasma membrane (Hashimoto-
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Sugimoto et al., 2013). The patrol1 mutant is impaired in light induced stomatal opening, similar to
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era1 (Fig. 3. (Hashimoto-Sugimoto et al., 2013)), however, PATROL1 does not have the CaaX
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motif and thus it is unlikely that this stomatal regulator is a direct target of ERA1. The vesicle-
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trafficking protein SYP121 is another regulator of stomatal opening and transport of ion channels,
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especially K+ channels (Eisenach et al., 2012). Possibly, the protein farnesylated by ERA1 is
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associated with some aspect, for example vesicle transport, of the proper translocation of H+-
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ATPases or other ion channels involved in stomatal opening to the plasma membrane.
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ERA1 regulates pathogen responses independently of its stomatal function
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The likely role of ERA1 farnesylation of target proteins in multiple biological processes makes it a
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challenge to pinpoint the precise function of ERA1 in any of the many phenotypes attributed to
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era1. Stomata also regulate entry of pathogens into leaves (Melotto et al., 2006). To investigate
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whether the ERA1 stomatal function is related to its role in basal pathogen resistance, we
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inoculated Col-0, era1, ost1 and era1 ost1 with virulent P. syringae pv. tomato DC3000 (Pst) and
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the coronatine deficient strain Pst DC3118 (Pst cor-) (Fig. 4). In pathogen and stomatal responses,
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coronatine activates JA signaling to suppress salicylic acid mediated defenses (Brooks et al.,
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2005) and reopens closed stomata during bacterial infection (Melotto et al., 2006).
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For the pathogen assays, dip–inoculation was used to favor the entry of Pst bacteria into leaves
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through stomata, thus allowing stomatal immunity to take place (Melotto et al., 2006). Importantly,
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the coronatine deficient strain, Pst cor-, is less virulent than Pst when surface inoculation is used
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as this strain cannot reopen stomata upon bacterial infection (Brooks et al., 2005; Melotto et al.,
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2006). Consistent with previous results, the ost1 mutant was susceptible to Pst cor- infection
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(Melotto et al., 2006). Both Pst and Pst cor- were strongly virulent in the era1 single mutant. This
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implies that the ERA1 function in stomata and its role in basal immunity are not related, since era1
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has constitutively closed stomata (Fig. 1), predicted to provide some level of resistance. Similarly,
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the era1 ost1 double mutant was susceptible to pathogen infection to the same level as era1,
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further suggesting that ERA1 regulation of disease resistance is not directly associated with its
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stomatal function (Fig. 4). However, since the exact target of ERA1 farnesylation in pathogen
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responses is currently not known (Goritschnig et al., 2008), further research is required to entangle
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the role of ERA1 and stomatal function in the response to pathogens.
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CONCLUSIONS
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Proper timing of stomatal movements is crucial to maintain overall plant water status. In this study,
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we are able to dissect the kinetics of stomatal conductance following a sudden change in the
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surrounding environment (Fig. 2). This made it clear that OST1 is required for fast responses,
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while ERA1 controls basal whole-plant stomatal conductance. Further phenotypic characterization
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suggests that ERA1 function in stomatal signaling is related to blue light induced opening, although
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the exact target protein that gets farnesylated by ERA1 remains elusive.
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MATERIALS AND METHODS
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Plant Material and growth conditions
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Col-0, era1-2, ost1-3 (srk2e, SALK_008068), rop10 (SALK_018747), rop11 (SALK_063154C),
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cyp85a2-2 (SALK_ 129352), asg2-1 (SALK_040151) and asg2-2 (SALK_113565) were from the
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European Arabidopsis Stock Centre (www.arabidopsis.info). The abi1-1 allele used was in the Col-
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0 accession and was a gift from Julian Schroeder. Double mutants and other crosses were made
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through standard techniques and genotyped with PCR-based markers (Supplemental Table 1).
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Plants for gas-exchange measurements were sown into 2:1 (v:v) peat:vermiculite mixture and
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grown through a hole in a glass plate covering the pot as described previously (Kollist et al., 2007).
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Plants were grown in growth chambers (AR-66LX, Percival Scientific, IA, USA and Snijders
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Scientific, Drogenbos, Belgia) with 12 h photoperiod, 23/18 °C day/night temperature, 100-150
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μmol m-2 s-1 light and 70% relative humidity. Plants were 24-32 days old during gas-exchange
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experiments. For guard cell isolation, seeds were sown into 8 x 8 cm pots with four plants per pot
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and grown in the conditions described above.
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Gas-exchange measurements
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Stomatal conductance of intact plants was measured using a rapid-response gas exchange
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measurement device consisting of eight flow-through whole-rosette cuvettes (Kollist et al., 2014).
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Representative photos of plants used for gas exchange measurements are presented in
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Supplemental Figure 3. Plants were inserted into measurement chambers and after stomatal
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conductance had stabilized, the following stimuli were applied: reduction in air humidity (decreased
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from 60-80% to 30-40%), darkness, CO2 (increase from 400 ppm to 800 ppm) and ABA. ABA-
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induced stomatal closure experiments were carried out as described previously (Merilo et al.,
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2015). Initial changes in stomatal conductance were calculated as gs18-gs0 (stomatal conductance
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value 18 min after factor application; 16min in case of ABA spraying). Opening experiments were
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performed with the application of either white light, blue light, red light or white light+ABA on dark-
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adapted plants kept in the measurement cuvettes at 0 light for at least 90 min. At timepoint 0,
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different light bulbs were switched on, light intensities were adjusted so that they were around 150
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µmol m-2 s-1 irrespective of light spectral characteristics. ABA-induced inhibition of stomatal
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opening experiments were carried out as described previously (Horak et al., 2016).
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RNA isolation and qPCR
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Samples enriched with guard cells were isolated from 5-7 week old plants, starting from 17-18
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plants and 4-5 leaves per plant, using the ice-blender method (Bauer et al., 2013). RNA was
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extracted with the Spectrum Plant RNA isolation kit (Sigma-Aldrich). Total RNA was DNAseI
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treated and cDNA was synthesized with Maxima H Minus reverse Transcriptase (Thermo Fischer
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Scientific). qPCR was performed in triplicate with 5 x HOT FIREPol EvaGreen qPCR Mix Plus
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ROX (Soils Biodyne) on an Applied Biosystems 7900HT Fast Real-Time PCR System (Foster
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City, CA, USA). Primer sequences and primer efficiencies are listed in Supplemental Table 1.
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Analysis of the quantitative PCR data was performed with qBase+ 3.0 (Biogazelle). The reference
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genes used for normalization were SAND, TIP41 and YLS8. Statistical analysis was performed on
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log2 transformed data.
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Pathogen assays
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Plants were grown in commercial potting soil/perlite (3:2) at 22–24◦ C day and 17–19◦ C night
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temperature under a 9-h-light/15-h-dark photoperiod. The lighting was supplied at an intensity of
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∼100 μE m−2 s−1 by fluorescence tubes. Bacterial strains Pst DC3000 and Pst DC3118 (cor-) were
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provided by Barbara Kunkel (Washington University, St. Louis, Missouri, USA). Bacteria were
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cultivated at 28◦C and 220 rpm in King’s B medium containing 50mg/mL rifampicin (DC3000) or
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rifampicin/kanamycin/spectinomycin (DC3118).
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Five-week-old Arabidopsis plants were dipped in a bacterial suspension of 106 colony-forming units
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(CFU)/mL Pst DC3000 and 107 (CFU)/mL Pst DC3118 in 10mM MgSO4 containing 0.01% Silwet L-
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77 (Lehle Seeds) for 15min. After dipping, plants were kept at 100% relative humidity overnight.
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For bacterial titers, leaf discs collected at 2 dpi were washed twice with sterile water and
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homogenized in 10 mM MgSO4. Quantification of bacterial growth was done as previously
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described (Zimmerli et al., 2000). Each biological repeat represents nine leaf discs from three
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different plants.
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Statistical analysis
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Statistical analysis were performed with Statistica v. 7.1 (StatSoft Inc., Tulsa, OK, USA). ANOVA
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with Dunnett’s, Tukey or Tukey unequal N HSD post hoc tests were used as indicated in figure
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legends. All effects were considered significant at p < 0.05.
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Acknowledgments
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We thank Irina Rasulova for technical assistance.
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SUPPLEMENTAL DATA
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Supplemental Figure 1. Stomatal responses of asg2-1, asg2-2, cyp85a2-2 and rop10 rop11.
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Supplemental Figure 2. Gene expression in guard cell enriched samples.
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Supplemental Figure 3. Representative photos of mutants and Col-0 wild type used for whole-
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plant gas exchange experiments.
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Supplemental Table 1. Primers used for genotyping and qPCR.
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Supplemental Figure 1. Stomatal responses of asg2-1, asg2-2, cyp85a2-2 and rop10 rop11.
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(A-D) Time courses of stomatal conductances in response to reduced air humidity, darkness,
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elevated CO2 and ABA treatment (n=5-7), respectively. (E-H) Changes in stomatal conductance
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values.
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Supplemental Figure 2. (A) Gene expression in guard cell enriched samples. RNA was isolated
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from Col-0 wildtype, era1, ost1, and era1 ost1 guard cells extracted from 5-7 week old plants using
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an ice blender method (Bauer et al., 2013). Relative expression of selected marker genes were
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determined with qPCR and is depicted relative to Col-0. (B) Relative expression of two guard cell
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expressed genes (HT1 and GORK) in a comparison of the guard cell enriched RNA versus RNA
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extracted from a Col-0 whole leaf sample. (C) Relative expression of a mesophyll expressed gene
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(At5g54250) in a comparison of the guard cell enriched RNA versus RNA extracted from a Col-0
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whole leaf sample.
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The mean of three biological replicates are shown; error bars depict ± SEM. Letters denote
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statistically significant differences between mutants, * denotes statistically significant difference
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from Col-0 in A (ANOVA with Tukey HSD post hoc test, p < 0.05) or from Col-0 leaf sample in B
334
and C (ANOVA with Dunnett’s test, p < 0.05).
335
Supplemental Figure 3. Representative photos of mutants and Col-0 wild type used for whole-
336
plant gas exchange experiments. Plants are 26-30 days old. Scale bar indicates 2 cm.
337
Supplemental Table 1. Primers used for genotyping and qPCR.
338
339
FIGURE LEGENDS
340
Fig. 1. Whole-plant stomatal conductances of single and double mutants of era1 with ost1 and
341
abi1-1. Stomatal conductance was measured from intact plants (Kollist et al., 2007). Letters denote
342
statistically significant differences (ANOVA with Tukey unequal N HSD post hoc test, p < 0.05; n
343
=8-15).
344
Fig. 2. Stomatal responses of single and double mutants of era1 with ost1 and abi1-1. (A-D) Time
345
courses of stomatal conductances in response to reduced air humidity, darkness, elevated CO2
346
and ABA treatment, respectively. (E-H) Changes in stomatal conductance during the first 18
347
minutes (except for ABA where 16 min was measured). Letters denote statistically significant
348
differences between the studied genotypes (ANOVA with Tukey unequal N HSD post hoc test, p <
349
0.05; n =6-15).
12
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
350
Fig. 3. Stomatal opening of dark-adapted Col-0 and era1 plants in response to (A) white light
351
added as a single factor or simultaneously with 2.5 μM ABA (n=8), (B) blue and (C) red light (n=7-
352
8). Before application of light, plants were adapted to full darkness for 90 minutes.
353
Fig. 4. Bacterial growth in Col-0, era1, ost1 and era1 ost1 plants. Pst titers were evaluated at 2 dpi.
354
Five-week-old plants were dip-inoculated with 106 CFU/mL Pst DC3000 (A) or 107 CFU/mL Pst
355
DC3118 (cor-) (B). Results are average ± SEM of 3 biological replicates each consisting of nine
356
leaf discs. Letters denote statistically significant differences (ANOVA with Tukey HSD post hoc
357
test, p < 0.05).
358
359
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abscisic acid signal transduction in Arabidopsis. Science 273: 1239-1241
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Labas V, Giglioli-Guivarc'h N, Ducos E (2016) ASG2 is a farnesylated DWD protein that acts as ABA
negative regulator in Arabidopsis. Plant Cell and Environment 39: 185-198
Eisenach C, Chen ZH, Grefen C, Blatt MR (2012) The trafficking protein SYP121 of Arabidopsis connects
programmed stomatal closure and K(+) channel activity with vegetative growth. Plant J 69: 241-251
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Galichet A, Gruissem W (2003) Protein farnesylation in plants - conserved mechanisms but different
targets. Current Opinion in Plant Biology 6: 530-535
Ghassemian M, Nambara E, Cutler S, Kawaide H, Kamiya Y, McCourt P (2000) Regulation of abscisic acid
signaling by the ethylene response pathway in Arabidopsis. Plant Cell 12: 1117-1126
Goritschnig S, Weihmann T, Zhang Y, Fobert P, McCourt P, Li X (2008) A novel role for protein
farnesylation in plant innate immunity. Plant Physiol 148: 348-357
Hayashi M, Inoue S, Takahashi K, Kinoshita T (2011) Immunohistochemical Detection of Blue Light-Induced
Phosphorylation of the Plasma Membrane H+-ATPase in Stomatal Guard Cells. Plant and Cell
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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 M, Brosche M, Schroeder JI, Kangasjarvi J, Kollist H (2016) A Dominant
Mutation in the HT1 Kinase Uncovers Roles of MAP Kinases and GHR1 in CO2-Induced Stomatal
Closure. Plant Cell 28: 2493-2509
Kollist H, Nuhkat M, Roelfsema MRG (2014) Closing gaps: linking elements that control stomatal
movement. New Phytologist 203: 44-62
Kollist T, Moldau H, Rasulov B, Oja V, Ramma H, Huve K, Jaspers P, Kangasjarvi J, Kollist H (2007) A novel
device detects a rapid ozone-induced transient stomatal closure in intact Arabidopsis and its
absence in abi2 mutant. Physiologia Plantarum 129: 796-803
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Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
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401
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403
404
405
406
407
408
409
410
411
412
413
414
415
416
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423
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433
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Laanemets K, Wang YF, Lindgren O, Wu J, Nishimura N, Lee S, Caddell D, Merilo E, Brosche M, Kilk K,
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stomatal opening and severely reduce K+ uptake channel activity via enhanced cytosolic [Ca2+] and
increased Ca2+ sensitivity of K+ uptake channels. New Phytol 197: 88-98
Lawson T, Blatt MR (2014) Stomatal size, speed, and responsiveness impact on photosynthesis and water
use efficiency. Plant Physiol 164: 1556-1570
Li Z, Liu D (2012) ROPGEF1 and ROPGEF4 are functional regulators of ROP11 GTPase in ABA-mediated
stomatal closure in Arabidopsis. FEBS Lett 586: 1253-1258
Li ZX, Kang J, Sui N, Liu D (2012) ROP11 GTPase is a Negative Regulator of Multiple ABA Responses in
Arabidopsis. Journal of Integrative Plant Biology 54: 169-179
Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, Grill E (2009) Regulators of PP2C
Phosphatase Activity Function as Abscisic Acid Sensors. Science 324: 1064-1068
Melotto M, Underwood W, Koczan J, Nomura K, He SY (2006) Plant stomata function in innate immunity
against bacterial invasion. Cell 126: 969-980
Merilo E, Jalakas P, Kollist H, Brosche M (2015) The Role of ABA Recycling and Transporter Proteins in
Rapid Stomatal Responses to Reduced Air Humidity, Elevated CO2, and Exogenous ABA. Molecular
Plant 8: 657-659
Merilo E, Laanemets K, Hu HH, Xue SW, Jakobson L, Tulva I, Gonzalez-Guzman M, Rodriguez PL,
Schroeder JI, Brosche M, Kollist H (2013) PYR/RCAR Receptors Contribute to Ozone-, Reduced Air
Humidity-, Darkness-, and CO2-Induced Stomatal Regulation. Plant Physiology 162: 1652-1668
Mustilli AC, Merlot S, Vavasseur A, Fenzi F, Giraudat J (2002) Arabidopsis OST1 protein kinase mediates
the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species
production. Plant Cell 14: 3089-3099
Northey JGB, Liang S, Jamshed M, Deb S, Foo E, Reid JB, McCourt P, Samuel MA (2016) Farnesylation
mediates brassinosteroid biosynthesis to regulate abscisic acid responses. Nature Plants 2
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the PYR/PYL Family of START Proteins. Science 324: 1068-1071
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Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Stomatal conductance mmol m-2 s-1
450
d
400
e
350
300
c
250
a
200
a
150
b
100
50
0
Co
l-0
a1
er
ab
-1
i1
os
t1
a1
er
os
t1
ab
i1
-1
a1
er
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 statistically significant differences (ANOVA with Tukey
unequal N HSD post hoc test, p < 0.05; n=8-15)
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Realtive air humidity
30-40
600 60-80
%
550
500
450
400
350
300
250
200
150
100
50
0
-25
0
25
50
Time, min
B
E
F
Change in stomatal conductance
(mmol m-2 s-1)
Stomatal conductance
(mmol m-2 s-1)
A
gs18-gs0
10
a
0
a
ab
c
bc
a
500
C
Light
darkness
light
450
400
400
350
350
D
CO2
400
450
800
μmol mol-1
450
400
150
100
100
100
50
50
50
0
0
0
20
Time, min
a
b
ab
c
c
ab
-25
0
25
Time, min
50
a
a
a
0
-25
b
b
a
a
0
-10
-10
-10
-20
-20
-30
-20
-20
-30
-40
-40
-50
-50
-60
-60
-50
-70
-70
-60
ab
bc
c
bc
50
bc
-30
-30
-40
-40
l-0 ra1 1-1 st1 st1 i1-1
o o b
Co e abi
a
a1
er ra1
e
0
25
Time, min
gs16-gs0
-10
l-0 ra1 1-1 st1 st1 i1-1
o
o
Co e abi
ab
a1
er ra1
e
era1 abi1-1
H
gs18-gs0
0
era1 ost1
150
G
gs18-gs0
0
40
ost1
200
150
-20
abi1-1
250
200
200
era1
300
250
250
Col-0
350
300
300
ABA
5 μM ABA
500
-50
-60
-70
l-0 ra1 1-1 st1 st1 i1-1
o
o
Co e abi
ab
a1
er ra1
e
Co
l-0 ra1 i1-1 st1 st1 i1-1
o o
e
ab
ab
a1
er ra1
e
Fig. 2. Stomatal responses of single and double mutants of era1 with ost1 and abi1-1. (A-D) Time courses of stomatal conductances 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 < 0.05; n=6-15)
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
160 darkness
Stomatal conductance mmol m-2 s-1
A
white light with +/- 2.5 μM ABA
140
120
100
60
40
20
0
-20
300
Stomatal conductance mmol m-2 s-1
B
80
Col-0 light
Col-0 light+ABA
era1 light
era1 light+ABA
0
20
Time, min
40
60
blue light
darkness
250
200
150
100
50
0
-20
160
Stomatal conductance mmol m-2 s-1
C
Col-0
era1
0
20
Time, min
40
60
red light
darkness
140
120
100
80
60
Col-0
era1
40
20
0
-20
0
20
Time, min
40
60
Fig. 3. Stomatal opening of darkadapted Col-0 and era1 plants in
response to (A) white light added as a
single factor or simultaneously with 2.5
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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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Bacterial number log(CFU/cm2)
A
8
7
b
a
b
a
8
6
5
5
4
4
3
3
2
2
1
1
Col-0 era1
ost1 era1 ost1
b
b
b
7
6
0
Pst cor -
B
Pst
0
a
Col-0
era1
ost1 era1 ost1
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
CFU/mL Pst DC3000 (A) or 107 CFU/mL Pst
DC3118 (cor-) (B). Results are average ± SEM of 3
biological replicates each consisting of nine leaf
discs. Letters denote statistically significant differences (ANOVA with Tukey HSD post hoc test, p <
0.05).
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Parsed Citations
Acharya BR, Jeon BW, Zhang W, Assmann SM (2013) Open Stomata 1 (OST1) is limiting in abscisic acid responses of Arabidopsis
guard cells. New Phytologist 200: 1049-1063
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Bauer H, Ache P, Lautner S, Fromm J, Hartung W, Al-Rasheid KA, Sonnewald S, Sonnewald U, Kneitz S, Lachmann N, Mendel RR,
Bittner F, Hetherington AM, Hedrich R (2013) The stomatal response to reduced relative humidity requires guard cell-autonomous
ABA synthesis. Curr Biol 23: 53-57
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Brooks DM, Bender CL, Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcoming
salicylic acid-dependent defences in Arabidopsis thaliana. Mol Plant Pathol 6: 629-639
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Cutler S, Ghassemian M, Bonetta D, Cooney S, McCourt P (1996) A protein farnesyl transferase involved in abscisic acid signal
transduction in Arabidopsis. Science 273: 1239-1241
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Dutilleul C, Ribeiro I, Blanc N, Nezames CD, Deng XW, Zglobicki P, Barrera AMP, Atehortua L, Courtois M, Labas V, GiglioliGuivarc'h N, Ducos E (2016) ASG2 is a farnesylated DWD protein that acts as ABA negative regulator in Arabidopsis. Plant Cell and
Environment 39: 185-198
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Eisenach C, Chen ZH, Grefen C, Blatt MR (2012) The trafficking protein SYP121 of Arabidopsis connects programmed stomatal
closure and K(+) channel activity with vegetative growth. Plant J 69: 241-251
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Fujii H, Chinnusamy V, Rodrigues A, Rubio S, Antoni R, Park SY, Cutler SR, Sheen J, Rodriguez PL, Zhu JK (2009) In vitro
reconstitution of an abscisic acid signalling pathway. Nature 462: 660-U138
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Galichet A, Gruissem W (2003) Protein farnesylation in plants - conserved mechanisms but different targets. Current Opinion in
Plant Biology 6: 530-535
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ghassemian M, Nambara E, Cutler S, Kawaide H, Kamiya Y, McCourt P (2000) Regulation of abscisic acid signaling by the ethylene
response pathway in Arabidopsis. Plant Cell 12: 1117-1126
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Goritschnig S, Weihmann T, Zhang Y, Fobert P, McCourt P, Li X (2008) A novel role for protein farnesylation in plant innate
immunity. Plant Physiol 148: 348-357
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hayashi M, Inoue S, Takahashi K, Kinoshita T (2011) Immunohistochemical Detection of Blue Light-Induced Phosphorylation of the
Plasma Membrane H+-ATPase in Stomatal Guard Cells. Plant and Cell Physiology 52: 1238-1248
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Horak H, Sierla M, Toldsepp K, Wang C, Wang YS, Nuhkat M, Valk E, Pechter P, Merilo E, Salojarvi J, Overmyer K, Loog M,
Brosche M, Schroeder JI, Kangasjarvi J, Kollist H (2016) A Dominant Mutation in the HT1 Kinase Uncovers Roles of MAP Kinases
and GHR1 in CO2-Induced Stomatal Closure. Plant Cell 28: 2493-2509
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kollist H, Nuhkat M, Roelfsema MRG (2014) Closing gaps: linking elements that control stomatal movement. New Phytologist 203:
44-62
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kollist T, Moldau H, Rasulov B, Oja V, Ramma H, Huve K, Jaspers P, Kangasjarvi J, Kollist H (2007) A novel device detects a rapid
ozone-induced transient stomatal closure in intact Arabidopsis and its absence in abi2 mutant. Physiologia Plantarum 129: 796-803
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Laanemets K, Wang YF, Lindgren O, Wu J, Nishimura N, Lee S, Caddell D, Merilo E, Brosche M, Kilk K, Soomets U, Kangasjarvi J,
Schroeder JI, Kollist H (2013) Mutations in the SLAC1 anion channel slow stomatal opening and severely reduce K+ uptake
channel activity via enhanced cytosolic [Ca2+] and increased Ca2+ sensitivity of K+ uptake channels. New Phytol 197: 88-98
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lawson T, Blatt MR (2014) Stomatal size, speed, and responsiveness impact on photosynthesis and water use efficiency. Plant
Physiol 164: 1556-1570
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Li Z, Liu D (2012) ROPGEF1 and ROPGEF4 are functional regulators of ROP11 GTPase in ABA-mediated stomatal closure in
Arabidopsis. FEBS Lett 586: 1253-1258
Pubmed: Author and Title
CrossRef: Author and Title
Google 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 of
Integrative Plant Biology 54: 169-179
Pubmed: Author and Title
CrossRef: Author and Title
Google 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 as
Abscisic Acid Sensors. Science 324: 1064-1068
Pubmed: Author and Title
CrossRef: Author and Title
Google 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 invasion.
Cell 126: 969-980
Pubmed: Author and Title
CrossRef: Author and Title
Google 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 Stomatal
Responses to Reduced Air Humidity, Elevated CO2, and Exogenous ABA. Molecular Plant 8: 657-659
Pubmed: Author and Title
CrossRef: Author and Title
Google 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, Kollist
H (2013) PYR/RCAR Receptors Contribute to Ozone-, Reduced Air Humidity-, Darkness-, and CO2-Induced Stomatal Regulation.
Plant Physiology 162: 1652-1668
Pubmed: Author and Title
CrossRef: Author and Title
Google 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 stomatal
aperture by abscisic acid and acts upstream of reactive oxygen species production. Plant Cell 14: 3089-3099
Pubmed: Author and Title
CrossRef: Author and Title
Google 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 brassinosteroid
biosynthesis to regulate abscisic acid responses. Nature Plants 2
Pubmed: Author and Title
CrossRef: Author and Title
Google 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 D,
Finkelstein R, Provart NJ, Desveaux D, Rodriguez PL, McCourt P, Zhu JK, Schroeder JI, Volkman BF, Cutler SR (2009) Abscisic
Acid Inhibits Type 2C Protein Phosphatases via the PYR/PYL Family of START Proteins. Science 324: 1068-1071
Pubmed: Author and Title
CrossRef: Author and Title
Google 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 cell
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
anion channels and plant water loss. Science 282: 287-290
Pubmed: Author and Title
CrossRef: Author and Title
Google 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: 219-247
Pubmed: Author and Title
CrossRef: Author and Title
Google 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. Plant
Physiology 171: 1569-1580
Pubmed: Author and Title
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