Reactive oxygen species signaling and stomatal movement in ... · Reactive oxygen species signaling and stomatal movement in plant responses to drought stress and pathogen attackFA

Reactive oxygen species signaling and stomatalmovement in plant responses to drought stressand pathogen attackFA

Junsheng Qi1, Chun-Peng Song2, Baoshan Wang3, Jianmin Zhou4, Jaakko Kangasj€arvi5, Jian-Kang Zhu6,7

and Zhizhong Gong1*

1. State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China2. Collaborative Innovation Center of Crop Stress Biology, Henan Province, Institute of Plant Stress Biology, Henan University, Kaifeng 475001,China3. Key Lab of Plant Stress Research, College of Life Science, Shandong Normal University, Ji’nan 250000, China4. State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101,China5. Division of Plant Biology, Viikki Plant Science Centre, Department of Biosciences, University of Helsinki, 00014 Helsinki, Finland6. Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China7. Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USAdoi: 10.1111/jipb.12654

Zhizhong Gong*Correspondence:[email protected]

Abstract Stomata, the pores formed by a pair of guardcells, are the main gateways for water transpiration andphotosynthetic CO2 exchange, as well as pathogen invasionin land plants. Guard cell movement is regulated by acombination of environmental factors, including waterstatus, light, CO2 levels and pathogen attack, as well asendogenous signals, such as abscisic acid and apoplasticreactive oxygen species (ROS). Under abiotic and biotic

stress conditions, extracellular ROS are mainly produced byplasma membrane-localized NADPH oxidases, whereasintracellular ROS are produced in multiple organelles. TheseROS forma sophisticated cellular signalingnetwork,with theaccumulationofapoplasticROSanearlyhallmarkof stomatalmovement. Here, we review recent progress in understand-ing themolecularmechanismsof the ROS signaling network,primarily during drought stress and pathogen attack. Wesummarize the roles of apoplastic ROS in regulating stomatalmovement, ABA and CO2 signaling, and immunity responses.Finally, we discuss ROS accumulation and communicationbetween organelles and cells. This information provides aconceptual framework for understanding howROS signalingis integrated with various signaling pathways during plantresponses to abiotic and biotic stress stimuli.

Edited by: Jia Li, Lanzhou University, ChinaReceived Feb. 20, 2018; Accepted Apr. 8, 2018; Online on Apr. 16,2018FA: Free Access

INTRODUCTION

Plants face fluctuating abiotic and biotic hazardsthroughout their sessile lives. Drought stress andpathogen infection are two of the most importantfactors that cause extensive loss to agriculturalproductivity. Increased global warming exacerbates

the frequency of extreme weather, which furtherthreatens agricultural productivity and world foodsecurity. Understanding the molecular mechanisms ofplant response and adaptation to adverse environmen-tal stimuli is urgently required for improving crop stressresistance and increasing yields through genetic anddesigned molecular breeding. Accordingly, plants have

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evolved sophisticated surveillance systems to perceiveand cope with these challenges (Baxter et al. 2014;Camejo et al. 2016).

The emergence of guard cells was a major landmarkin the evolution of land plants and their adaptation todry environmental conditions (Umezawa et al. 2010).Stomata, which are surrounded by a pair of guard cells,are the main gateways plants use to efficiently take upCO2 for photosynthesis while simultaneously regulatingwater transpiration. Controlling water transpirationthrough stomata is one of themain strategies for plantsto increase drought tolerance (Schroeder et al. 2001).

The opening and closing of the stomatal aperture(i.e., stomatal movements) is regulated by turgorpressure generated by ion and water channel proteinson the plasma membranes of guard cells (Schroederet al. 2001; Kim et al. 2010; Engineer et al. 2016). Thesemovements are tightly regulated by environmentalstimuli, such as water status, light and CO2, as well asendogenous factors, such as abscisic acid (ABA), Ca2þ

and reactive oxygen species (ROS) levels under variousconditions (Schroeder et al. 2001; Young et al. 2006).

Notably, the respiratory burst oxidase homologs(RBOHs) NADPH oxidases, and the core ABA signalingcomponents, emerged at almost the same time withinguard cells, in mosses during land plant evolution; thesecomponents are not found in algae (Umezawa et al. 2010;Mittler etal. 2011; Lindetal. 2015; Sussmilchetal. 2017).Thispatternofemergencesuggests thatapoplasticROS,guardcells andABAsignalingmechanismare the co-evolutionaryresults of the adaptation of land plants to dry environ-mental conditions (Umezawa et al. 2010;Mittler et al. 2011;Lind et al. 2015; Sussmilch et al. 2017). However, theevolution of stomata also opened the door for pathogeninvasion (Schroeder et al. 2001; Melotto et al. 2006).

When facing different biotic and abiotic stresses,plants quickly accumulate the common ROS as the firstlayer for defense (Kocsy et al. 2013; Wrzaczek et al. 2013;Baxter et al. 2014). The term “ROS” generally refers toincompletely reduced oxygen species, including thewell-studied singlet oxygen (1O2), superoxide anions (O2�

� ),hydrogen peroxide (H2O2) and hydroxyl radicals (�OH)(Mittler et al. 2004; Camejo et al. 2016; Sierla et al. 2016).ROS have high chemical activity and a relatively shorthalf-life. Due to their inherent features, all types of ROScan damagemacromolecules such as proteins, lipids andnucleic acids, eventually leading to cell death (Petrovet al. 2015). On the other hand, ROS act as signaling

molecules that modify protein properties through theformation of covalent bonds, and they also function asmajor inducers of programmed cell death (PCD) (Neillet al. 2002; Suzuki et al. 2012; Baxter et al. 2014;Choudhury et al. 2017; Mittler 2017). H2O2, which has arelatively long half-life (approximately 1ms in cells) and ismore stable than other ROS, often acts as a transducingsignal in both cell-to-cell communication and intracellularsignaling to trigger downstream responses (Wrzaczeket al. 2013; Baxter et al. 2014; Camejo et al. 2016).

In addition to ROS, reactive nitrogen species (RNS),comprising nitric oxide (NO) and its oxidized deriva-tives, including NO2, N2O3, peroxynitrite (ONOO� ), S-nitrosothiols and S-nitrosoglutathione (GSNO), areharmful to cells, but like ROS, they can also serve askey regulatory signals during various cellular responsesunder stress conditions (Qiao and Fan 2008; Kocsy et al.2013; Hu et al. 2015). Both NO and GSNO modify theirtarget proteins, through the S-nitrosylation of reactivecysteines. The modification of proteins by either ROS orRNS can alter their activity, stability, subcellular locationor interactions with other molecules (Qiao and Fan2008; Kocsy et al. 2013).

As by-products of both abiotic and biotic stress,intracellular ROS are produced in organelles such aschloroplasts, peroxisomes/glyoxysomes and mitochon-dria, whereas apoplastic ROS are produced by plasmamembrane-localized NADPH oxidases, cell wall perox-idases and amine oxidases (Kadota et al. 2014; Kadotaet al. 2015). These ROS activate anti-oxidative systems,which maintain proper ROS homeostasis in the cell;however, when too many ROS are produced, ROS-associated injury or cell death cannot be avoided (Milleret al. 2010; Suzuki et al. 2012; Baxter et al. 2014;Choudhury et al. 2017).

Major ROS-scavenging enzymes, including ascor-bate peroxidases (APX), catalases (CAT), thylakoidalascorbate peroxidase (tAPX), copper-zinc superoxidedismutases (SOD) and glutathione peroxidases (GPX),provide a highly efficient system for maintaining ROShomeostasis in various sites of a cell, under normal orstress conditions (Mittler et al. 2004). Alternativeoxidases (AOXs) are activated in response to ROSformation in mitochondrial electron transport chain(ETC) Complex III, where they indirectly prevent excessROS formation (Jacoby et al. 2012).

Low molecular weight antioxidants such as ascorbicacid and glutathione, which are present in almost all

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organelles, are involved in ROS quenching, primarily byproviding reducing equivalents to ROS scavengingenzymes. This process can keep ROS concentrationslow, even when they are produced at a high rate, andthese enzymes can also react directly with ROS (Milleret al. 2010; Kocsy et al. 2013; Singh et al. 2016). Plantshave evolved various mechanisms to perceive, trans-duce and respond to ROS signals to protect themselvesfrom pathogen attack and from damage caused byabiotic stresses such as drought, salinity and high andlow temperatures (Miller et al. 2010; Suzuki et al. 2012;Baxter et al. 2014; Choudhury et al. 2017).

Plant cells have developed fine-tuned regulatorymechanisms to orchestrate stomatal movement underdrought stress and pathogen attack (Schroeder et al.2001; Kim et al. 2010; Arnaud and Hwang 2015), and inresponse to CO2 levels (Young et al. 2006; Engineer et al.2016). Both drought stress and pathogen attack cantrigger the rapid production of ROS in the apoplast,which is essential for stomatal closure (Qi et al. 2017).Furthermore, ROS signaling can occur between cells(Mittler et al. 2011).

These ROS signaling pathways form a complicatednetwork that functions in plant responses to abiotic andbiotic stress (Wrzaczek et al. 2013; Zhu 2016; Qi et al.2017). In this review, we will summarize recent progressin our understanding of ROS signaling and discuss themolecular mechanisms underlying apoplastic H2O2-medi-ated stomatalmovement and theROS signaling network,which function primarily during plant responses todrought stress and pathogen attack.

APOPLASTIC H2O2 MEDIATES STOMATALMOVEMENT DURING ABA SIGNALING

The phytohormone ABA is synthesized in differenttissues and plays various roles in seed maturation, seeddormancy, seed germination, seedling growth anddevelopment, and the regulation of gene expressionand stomatal movement in response to abiotic stress(Finkelstein et al. 2002; Zhu 2016). Drought stresspromotes the production of ABA, which is perceivedby the PYR1/PYL/RCAR family of ABA receptors(Figure 1). ABA-bound PYLs interact with clade A type2C phosphatases (PP2Cs), which are core negativeregulators of ABA signaling, releasing their inhibition ofdownstream targets and, thus, activating protein

kinases, including SnRK2.2/2.3/2.6 (OPEN STOMATA1,OST1) and GUARD CELL HYDROGEN PEROXIDE-RESIS-TANT1 (GHR1) (Ma et al. 2009; Park et al. 2009; Hua et al.2012). The activated protein kinases phosphorylateand activate their targets, via their own activity orby interacting with downstream targets, such asSLOW ANION CHANNEL-ASSOCIATED1 (SLAC1), SLAH3(SLAC1 HOMOLOGUE3) and ALUMINUM-ACTIVATEDMALATE TRANSPORTER 12/QUICKLY ACTIVATED ANIONCHANNEL1 (ALMT12/QUAC1). At the same time, theseenzymes inhibit the inward rectifier potassium channelKAT1, resulting in stomatal closure (Vahisalu et al. 2008;Geiger et al. 2009; Sato et al. 2009; Geiger et al.2010; Meyer et al. 2010; Geiger et al. 2011; Imes et al.2013; Brandt et al. 2015).

Reactive oxygen species production is induced bydrought stress andABA signaling (Sierla et al. 2016). ABA-induced H2O2 accumulation was first reported in Viciafaba and Arabidopsis thaliana guard cells (Miao et al.2000; Pei et al. 2000). The apoplastic ROS produced byplasma membrane-localized NADPH oxidases have beenwell studied (Kwak et al. 2003; Miller et al. 2009; Kadotaet al. 2014; Li et al. 2014; Kadota et al. 2015). TheArabidopsis genome encodes 10 NADPH oxidasesbelonging to the RBOH family (Kwak et al. 2003). NADPHoxidases have NADPH or NADH binding sites (Suzukiet al. 2011; Kadota et al. 2015), which transfer electronsfrom cytosolic NADPH or NADH to apoplastic oxygen tocatalyze the production ofO2,which can be converted toH2O2, either spontaneously or via the activity ofsuperoxide dismutases (SODs). RBOHD and RBOHF areboth responsible for ABA-mediated ROS production inguard cells (Kwak et al. 2003).

NADPH oxidase activity is tightly regulated byprotein phosphorylation. ABA-activated OST1 can di-rectly phosphorylate serine (Ser) 13 and Ser174 at the N-terminus of RBOHF, in vitro (Sirichandra et al. 2009).OST1 interacts with BRI1-associated kinase 1 (BAK1),both of which function upstream of ROS production inguard cells, and are inhibited by ABI1 (Shang et al. 2016).BAK1 usually forms a heterodimer with other receptor-like protein kinases. However, The BAK1 partner, andthe molecular mechanism for how BAK1 regulates ROSproduction and stomatal movement, are not yet known(Shang et al. 2016).

Besides OST1, the calcineurin B-like (CBL)-interactingprotein kinase CIPK26 interacts with and phosphor-ylates the cytoplasmic N-terminus of RBOHF, but the

ROS signaling and stomatal movement 3


phosphorylation sites are currently unknown (Drerupet al. 2013). Co-expression of CIPK26 with CBL1 or CBL9enhances the Ca2þ-dependent activity of RBOHF inHEK293T cells (Drerup et al. 2013). However, whetherthese phosphorylation events have any effect onRBOHF activity remains unknown.

NADPH oxidases are regulated by other factorsbesides protein phosphorylation. Numerous stressescausephospholipaseDa1 (PLDa1) tohydrolyzemembranephospholipids into phosphatidic acid (PA) (Zhang et al.2009). The depletion of PLDa1 reduces ABA-mediated

ROS production in guard cells as well as stomatal closure,and ROS production can be recovered by the addition ofPA in plda1mutants, suggesting that PA is crucial for ROSproduction (Sang et al. 2001a, 2001b; Zhang et al. 2009).The loss of the PA binding motif in RBOHD compromisesABA-mediated ROS production (Zhang et al. 2009). PAalso binds to ABI1 (ABA INSENSITIVE 1) and inhibits itsphosphatase activity, thereby increasing ABA-promotedstomatal closure (Zhang et al. 2004).

Heterotrimeric G proteins have Ga, Gb and Gg

subunits. PLDa1 interacts with the Ga subunit GPA1

Figure 1. The roles of reactive oxygen species (ROS) in abscisic acid (ABA) signaling in stomatal movementDrought stress induces the biosynthesis and/or accumulation of ABA in guard cells, which is perceived by PYR1/PYL/RCAR ABA receptors. ABA-bound PYL interacts with and inhibits clade A PP2Cs, such as ABA INSENSTIVE1 (ABI1) andABI2, activating protein kinases, such as OPEN STOMATA1 (OST1) and GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1). After interacting with PYLs, ABI1 is degraded by PUB12/13 E3 ligases. ABI1 dephosphorylatesSLOW ANION CHANNEL-ASSOCIATED1 (SLAC1) and inhibits its activity. OST1 phosphorylates respiratory burstoxidase homolog protein F (RBOHF) to produce apoplastic O2�

� , which is converted to H2O2 by superoxidedismutase (SOD). The apoplastic H2O2 signal is transduced into the cytosol by GHR1 through an unidentifiedmechanism to activate a putative, unidentified calcium channel on the plasma membrane. After binding to Ca2þ,CIPK26 can phosphorylate RBOHF. Ca2þ-activated CPK4/6/21/23 can phosphorylate and activate SLAC1. ApoplasticH2O2 enters the cell through the aquaporin protein PIP2;1. CPK8 phosphorylates and enhances the activity ofCATALASE3 (CAT3). H2O2 in the cytosol may inhibit the activity of ABI1, ABI2 or GLUTATHIONE PEROXIDASE3 (GPX3)or induce NO production via nitrate reductases (NRs). Oxidized GPX3 inhibits ABI2 activity. Nitric oxide (NO)mediates the S-nitrosylation of OST1 and inhibits its activity. RBOHF can also be regulated by the G-proteina-subunitGPA1 and phosphatidic acid (PA) produced by phospholipase Da1 (PLDa1). AHAs, Arabidopsis P-ATPases; GORK,guard cell outward potassium channel; KAT1, the inward-rectifying K(þ) channel. Blue arrows indicate positiveregulation; red bars indicate inhibition; dotted lines indicate uncertain regulation.

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(Zhao and Wang 2004). GPA1 positively regulates ABA-induced ROS production (Zhang et al. 2011). In gpa1mutants, both ABA-induced ROS production and Ca2þ-channel activation are impaired (Zhang et al. 2011).However, exogenously applied H2O2 rescues thedefects in stomatal response to ABA and Ca2þ-channelactivation in gpa1, indicating that GPA1 functionssomewhere between ABA perception and ROS produc-tion (Zhang et al. 2011). These findings indicate thatPLDa1, its product PA and GPA1 are all required for ABAsignaling and ROS production.

Genetically speaking, during ABA-mediated stomatalregulation, ABI1 acts upstream of ROS production, whileABI2 (another clade A PP2C) acts downstream (Murataet al. 2001). H2O2 accumulation is not altered in the ghr1or abi2-1mutants, but it is greatly reduced in the ost1 andabi1-1mutants under ABA treatment (Murata et al. 2001;Mustilli et al. 2002; Hua et al. 2012). Furthermore, ost1 andabi1-1 respond normally to H2O2-promoted stomatalclosure, but ghr1 and abi2-1 are insensitive to this stimulus(Murata et al. 2001; Mustilli et al. 2002; Hua et al. 2012).

ABI1 interacts with two U-box ubiquitin E3 ligases,PUB12 and PUB13, but it is degraded by these enzymesonly after interacting with ABA receptors in thepresence or absence of ABA. The pub12 pub13 doublemutant accumulates more ABI1 protein and less H2O2 inits guard cells than wild type (Kong et al. 2015), which isconsistent with the role of ABI1 in negatively regulatingROS production. ABI1 physically interacts with, dephos-phorylates and directly inhibits SLAC1 activity inXenopus oocytes, but its in planta function is stillunresolved (Brandt et al. 2015).

Unlike pathogen-associated molecular pattern(PAMP)-triggered ROS production, in which calciumsignaling functions upstream of ROS during thisprocess, ABA signaling triggers ROS production. UponABA signaling, ROS, in turn, induce increased cytosolCa2þ levels, likely via GHR1, as no Ca2þ channel activitywas detected in the ghr1 mutant (Hua et al. 2012).Notably, flg22-induced stomatal closure is also impairedin the ghr1 mutant, suggesting that PAMP-triggeredROS regulate stomatal closure through the activity ofGHR1 (Hua et al. 2012).

NO is another signalingmolecule involved in stomatalmovement (Qiao and Fan 2008; Asgher et al. 2017). Inguard cells, ABA and H2O2 induce NO production, whichrequires nitrate reductase (NR). Indeed, the NR doublemutant nia1 nia2 fails to produce NO in guard cells under

exogenous ABA or H2O2 treatment (Bright et al. 2006).ABA-inducedNOproduction is alsogreatly reduced in theatrbohD/F double mutant (Bright et al. 2006) and in theguard cells of plda1 (Zhang et al. 2009). These findingsindicate that ABA-induced apoplastic H2O2 production isrequired for NO biosynthesis. ABA-induced NO canmodify the reactive cysteine thiol in a protein to form S-nitrosothiol. Protein S-nitrosylation is important forprotein activity and stability (Hu et al. 2015). OST1 activityis abolishedby S-nitrosylation at cysteine (Cys) 137,whichis adjacent to the kinase catalytic site (Wang et al. 2015a),suggesting that NO regulates ABA signaling via afeedback circuit.

As mentioned above, the production of extracellularROS, by NADPH oxidases, represents an evolutionaryachievement for land plants (Mittler et al. 2011). Moreimportantly, apoplastic ROS may act as signalingmolecules to transduce extracellular signals into cells,which protect the inner components of the cells fromROS damage. The plasma membrane-localized LRRreceptor-like protein kinase GHR1 is a key component inapoplastic ROS signal transduction (Hua et al. 2012).GHR1 directly interacts with and activates the slow-typeanion channel SLAC1 (Hua et al. 2012). It is likely thatOST1 phosphorylates and activates RBOHF to produceapoplastic ROS, a process inhibited by ABI1 (Yoshidaet al. 2006). Meanwhile GHR1 transduces apoplasticROS signals into guard cells via an unknownmechanismthat is specifically inhibited by ABI2 (Hua et al. 2012).

The N-terminal extracellular domain of GHR1 con-tains three Cys residues, including a Cys pair (Cys-57,Cys-66) that is well conserved in most RLKs and isrequired for their normal functioning (Hua et al. 2012).However, the other Cys residue, Cys-381, does notfunction in ROS sensing, as its mutation to Ala does notaffect the role of GHR1 (Hua et al. 2012). Thus, whetherGHR1 or other plasma membrane receptor-like proteinkinases can perceive apoplastic ROS and transducethem inside the cell remains unknown. Perhaps GHR1interacts with oxidized, ROS-induced peptide ligands oranother RLK that can be induced or oxidized by ROS totransduce apoplastic ROS signals.

Recent studies suggest that members of the largefamily of cysteine-rich receptor like kinases (CRKs) areimportant candidates that may achieve extracellular ROSsignal transduction to downstream targets (Idanheimoet al. 2014; Bourdais et al. 2015). Some CRKs play generaland/or specific roles in pathogen- andbiotic stress-induced



stomatal closure (Idanheimo et al. 2014; Bourdais et al.2015). Given the high similarity among these CRKs and thelarge family size, additional studies examining their roles inH2O2 signal transduction are required using the newlydeveloped CRISPR/Cas9 technique to create multiplemutants (Wang et al. 2015b).

As we discussed above, apoplastic ROS might besensed by some plasma membrane proteins totransduce signals into cells. Given that RBOHD is amembrane protein, it undergoes endocytosis, which iscooperatively regulated by clathrin- and microdomain-dependent endocytic pathways (Hao et al. 2014). ABAand flg22 treatments increase monomer-dimer tran-sitions and the diffusion efficiency of RBOHD (Haoet al. 2014). This process allows ROS to be generatedwithin endosomes for signal transduction (Hao et al.2014).

On the other hand, apoplastic H2O2 can be trans-ported into the cytosol by aquaporin proteins tomediatestomatal movement. ABA-activated OST1 phosphory-lates Ser121 of the aquaporin protein PIP2;1, whichmediates water transport and also likely moves H2O2from the apoplast to the cytosol (Grondin et al. 2015;Rodrigues et al. 2017). The pip2;1 mutant is defective inbothABA-inducedROSaccumulationandABA-promotedstomatal closure (Grondin et al. 2015). As an oxidativechemical, cytosolic H2O2 can directly oxidize someproteins to affect their activity. For example, H2O2oxidizes the general ROS-scavenging protein ATGPX3,which physically interacts with ABI2, thereby convertingit from the reduced to the oxidized form, whichsignificantly decreases ABI2 activity (Miao et al. 2006).As a ROS scavenger, in turn, ATGPX3 can scavenge ABA-or drought-inducedH2O2. Thus, ATGPX3may act as a ROSsensor to transduce oxidative signals during ABA anddrought stress signaling (Miao et al. 2006). Furthermore,cytosolic H2O2 may in turn inhibit ABA signaling, as bothABI1 andABI2 are sensitive toH2O2 in vitro (Meinhard andGrill 2001; Meinhard et al. 2002).

It is also reported that CPK8 interacts with cytosolicCAT3 and positively regulates its activity by phosphory-lating Ser261 during ABA-mediated stomatal regulation(Zou et al. 2015). Both the cpk8 and cat3 mutants havelower catalase activity, accumulate more H2O2 and losewater more rapidly than wild type (Zou et al. 2015).These findings suggest that apoplastic ROS communi-cate with the cytosol to regulate ABA signaling andstomatal movement.

CROSSTALK AMONG CO2, ABA AND ROSSIGNALING DURING STOMATALMOVEMENT

Elevated atmospheric CO2 levels are causing globalwarming, which increases the severity of drought stress(Xu et al. 2015). However, high concentrations of CO2provide more carbon for photosynthesis and stimulateplant growth under favorable water and nutrientconditions (Xu et al. 2015). Plants have evolved waysto optimize stomatal activity to efficiently acquire CO2for photosynthesis, while reducing water loss viatranspiration (Chater et al. 2013). Thus, guard cellshave developed a fine regulatory system for respondingto atmospheric CO2 levels and integrating informationabout environmental CO2 levels with endogenous ABAsignaling (Kim et al. 2010).

Short-term exposure to elevated CO2 levels pro-vokes stomatal closure, and long-term exposurereduces stomatal density, both of which improve wateruse efficiency and protect plants against drought stress.However, these changes can lead to increased leaftemperature, which can damage cells (Medlyn et al.2001). Nevertheless, treatment with high concentra-tions of bicarbonate (the product of CO2 catalyzed bycarbonic anhydrase, CA) induces H2O2 production andpromotes plasma membrane-localized NADPH oxidase-dependent stomatal closure, whereas low CO2 levelspromote stomatal opening (Kolla et al. 2007), suggest-ing there is crosstalk between CO2 signaling and ROS.

In order to identify the components in CO2 signaling, agenetic screen using thermal imaging to detect thetemperature change based on water transpiration hasbeen established (Hashimoto et al. 2006). The firstidentified mutant, by this thermal imaging screen, is thehigh leaf temperature1 (ht1), which showed higher leaftemperatures and smaller stomatal apertures comparedto wild type (Hashimoto et al. 2006). HT1 encodes aputative MAPKKK kinase that negatively regulates stoma-tal responses to changes in CO2 levels, but does not affectABA or blue light signaling (Hashimoto et al. 2006).

A recent study indicates that bCA1 and bCA4function in the early CO2 response; mutations in thesegenes strongly impair the stomatal CO2 response andincrease stomatal density, but like HT1, they do notaffect responses to ABA or blue light (Hu et al. 2010).The ca1 ca4 ht1-2 triple mutants exhibit a similar CO2response as ht1-2, suggesting that HT1 acts downstream

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of bCA1 and bCA4 in CO2mediated stomatal movement.bCA1 is localized to the chloroplast (Fabre et al. 2007; Huet al. 2010). bCA4 is targeted to the plasma membraneand interacts with PIP2;1, which might transportapoplastic CO2 and efficiently channel it to bCA4(Wang et al. 2016).

The anion channel SLAC1 plays a crucial role instomatal movement during CO2 signaling (Negi et al.2008). SLAC1 can be activated by elevated intracellularbicarbonate levels in guard cells (Xue et al. 2011). Arecent study showed that the SLAC1 transmembranedomain, but not the N-terminus or C-terminus, respondsto CO2, but not to ABA, whereas the N-terminus isimportant for ABA signaling (Yamamoto et al. 2016).

Both OST1 and GHR1 can activate SLAC1 in oocytes(Geiger et al. 2009; Hua et al. 2012), which can beinhibited by HT1 (Horak et al. 2016). Interestingly, theinhibition of OST1-, GHR1-activated SLAC1 by HT1 can becounteracted by MPK12 (Horak et al. 2016). In a recentnatural variation study, MPK12 was also identified as akey player in Arabidopsis Cvi-0 accession for guard cellCO2 signaling (Jakobson et al. 2016). These findingssuggest that MPK12 inhibits HT1, and HT1 inhibitsthe OST1/GHR1-activated SLAC1. Different from HT1that mainly functions in CO2 signaling, MPK12 is alsoactivated by ABA or H2O2 (Jammes et al. 2009).Interestingly, co-expressing bCA4 and PIP2;1 with OST1-SLAC1 or CPK6/23-SLAC1, in oocytes, activates SLAC1 viaextracellular CO2 (Wang et al. 2016). These findingssuggest that, in the presence of various protein kinases,SLAC1 directly senses cytosolic CO2/HCO3

� that has beentransported by PIP2;1 and converted by bCA4 in oocytes.

The crosstalk between ABA and CO2 signaling isfurther supported by the finding that ABA-responsemutants, such as abi1-1, abi2-1, gca2, ost1 and ghr1 and theABA receptor mutant pyr/acars, exhibit reduced guardcell responses to elevated CO2 (Webb and Hetherington1997; Young et al. 2006; Xue et al. 2011; Merilo et al. 2013;Horak et al. 2016). ABA biosynthesis is also required forCO2 signaling (Chater et al. 2015). These findings suggestthat full CO2 responses require components of the ABAsignalosome. It is likely that the ABA and CO2 signalingpathways converge to mediate stomatal movement,while OST1 and GHR1 act downstream of this conver-gence site (Engineer et al. 2016; Horak et al. 2016)(Figure 2).

CO2-induced stomatal closure and reduced guardcell density require ROS production by the NADPH

oxidases RBOHF and RBOHD. Both CO2-induced stoma-tal closure and reduced guard cell density are impairedin the rbohD rbohF double mutant, ABA deficiencymutants and high-order ABA receptor pyl mutants(Chater et al. 2015). Silencing of OST1 and RBOH1 intomato reduces H2O2 and NO accumulation in responseto elevated CO2 levels, whereas silencing NR onlyreduces the accumulation of NO. These findings indicatethat OST1 and NADPH oxidase-dependent H2O2 produc-tion act upstream of NO production during CO2signaling in guard cells (Shi et al. 2015). Together, thesefindings suggest that ABA and ROS signaling arerequired to enhance high CO2-induced stomatal closure.

APOPLASTIC H2O2 MEDIATES STOMATALMOVEMENT IN PLANT RESISTANCE TODISEASES

As mentioned above, land plants have evolved stomatafor H2O transpiration and gas exchange, which hassimultaneously opened the door for pathogen invasion(Melotto et al. 2006). Plants have evolved functionalimmune systems for defenses against pathogens. Thefirst layer of plant innate immunity is the recognition ofPAMPs by pattern recognition receptors (PRRs), whichinitiates PAMP-triggered immunity (PTI) to activatedownstream immune responses (the second layer),leading to a transient increase in cytosolic Ca2þ levels, aROS burst, stomatal closure and increased expressionof pathogen-related genes, thus restricting pathogenentry (Lu et al. 2010; Zhang et al. 2010).

ROS bursts produced by NADPH oxidases or apo-plastic enzymes can lead to rapid PCD in a few distinctcells at the infection sites, a process known as thehypersensitive response (HR). This process can impedethe invasion of biotrophic pathogens because theyobtain nutrients from living cells, while the neighboringcells acquire the ability to prevent cell death through thespreading of ROS (Wu et al. 2014). Apoplastic ROS playcrucial roles in mediating callose deposition and cell wallcross-linking, which reinforce the cell wall to impede thepenetration of pathogens (O’Brien et al. 2012).

Numerous PRRs localized on the plasma membraneinclude receptor-like kinases (RLKs) and receptor-likeproteins (RLPs) (Dou and Zhou 2012). In the modelplant Arabidopsis, the best-characterized RLKs includeFLAGELLIN SENSING2 (FLS2) and elongation factor-Tu



receptor (EFR), which recognize the bacterial flagellinepitope flg22 and bacterial elongation factor-Tu (EF-Tu)epitope elf18, respectively. The perception of flg22 byFLS2, or elf18 by EFR, induces their rapid associationwith the co-receptor BAK1. FLS2/EFR and BAK1 repre-sent the first layer of pathogen signal perceptionrequired to trigger an oxidative burst within the plant(Figure 3). By promoting stomatal closure, FLS2 plays animportant role in restricting bacterial entry into theplant (Melotto et al. 2006).

In the FLS2 signaling pathway, BOTRYTIS-INDUCEDKINASE1 (BIK1) plays an important role inmediating ROSproduction from ROBHD (Kadota et al. 2014; Li et al.2014). FLS2/EFR and BAK1 constitutively associatewith their downstream target, BOTRYTIS-INDUCEDKINASE1 (BIK1), which is rapidly phosphorylated byBAK1 and released from the FLS2/EFR-BAK1 complex

after flg22/EF-Tu is perceived by FLS2/EFR (Lu et al. 2010;Zhang et al. 2010). BIK1 belongs to the RLCK-VIIsubfamily, which consists of 46 members (Zhanget al. 2010). Both FLS2 and BIK1 interact with RBOHD(Li et al. 2014). After FLS2 perceives flg22, the activatedBIK1 directly phosphorylates RbohD at Ser39 andSer343, in a calcium-independentmanner, and regulatesRBOHD (Kadota et al. 2014; Li et al. 2014).

Consistently, bik1 mutants are compromised in theirability to generate a rapid ROS burst and immuneresponses (Lu et al. 2010; Zhang et al. 2010). However,BIK1-mediated phosphorylation is required but notsufficient for RBOHD activation (Li et al. 2014). The BIK1homologs PBL1, PBL2 and PBL5 are also involved in thegeneration of a pathogen-triggered ROS burst (Zhanget al. 2010; Liu et al. 2013). CPK28 was shown to interactwith and phosphorylates BIK1 and contributes to its

Figure 2. Crosstalk among CO2, abscisic acid (ABA) and reactive oxygen species (ROS) signaling during stomatalregulationElevated CO2 levels promote stomatal closure, which is enhanced by abscisic acid (ABA)-mediated ROS production.CO2 in the apoplast is transported into the cytosol by the aquaporin PIP2;1. PIP2;1 directly interacts with carbonicanhydrase bCA4 and might channel CO2 to bCA4 to produce bicarbonate (HCO3. HCO3

� signaling might activateMPK12/4 to repress HT1. Repressed HT1 would then release its phosphorylation and the inhibition of GUARD CELLHYDROGEN PEROXIDE-RESISTANT1 (GHR1) or OPEN STOMATA1 (OST1), resulting in the activation of SLOW ANIONCHANNEL-ASSOCIATED1 (SLAC1) or releasing its direct inhibition on SLAC1. HCO3

– might also activate ABA signalingto enhance SLAC1 activity by GHR1 and OST1 or Ca2þ-activated CPK3/23 or directly regulate SLAC1 via an unknownmechanism. ABA signaling induces H2O2 production through respiratory burst oxidase homolog protein D or F(RBOHD/F). H2O2 mediates nitric oxide (NO) production through the action of nitrate reductases (NRs). Blue arrowsindicate positive regulation; red bars indicate inhibition; dotted lines indicate uncertain regulation.

8 Qi et al.


turnover, resulting in decreased RBOHD-dependent ROSproduction (Monaghan et al. 2014).

The mutation of CPK28 protein increases BIK1stability and enhances PAMP-triggered signaling andantibacterial immunity in Arabidopsis (Monaghan et al.2014). Furthermore, a recent study indicated that thenon-canonical heterotrimeric Ga protein XLG2 interactswith both FLS2 and BIK1 and functions together withthe Gb protein AGB1 and the Gg proteins AGG1/2 toattenuate proteasome-mediated turnover of BIK1 priorto flg22 perception (Liang et al. 2016). After theperception of flg22 by FLS2, XLG2 dissociates fromAGB1; its N-terminus is phosphorylated by BIK1, whichenhances flg22-induced ROS production, likely throughRBOHD (Liang et al. 2016). Similar with ABA signaling,

the plasma membrane intrinsic proteins AtPIP1;4 andAtPIP2;1 transport apoplastic H2O2 to the cytoplasm,thus activating systemic acquired resistance and PTI(Tian et al. 2016; Rodrigues et al. 2017).

Besides BIK1 and its homologues, the calcium-dependent protein kinases CPK4, CPK5, CPK6 andCPK11 are thought to be involved in the generation of aPAMP-induced ROS bursts in Arabidopsis (Boudsocqet al. 2010; Dubiella et al. 2013). It is likely that CPK5specifically phosphorylates RBOHD at Ser148 andregulates its activity (Dubiella et al. 2013; Li et al.2014). BR-SIGNALING KINASE1 (BSK1) is a positiveregulator of brassinosteroid signaling and a substrateof the brassinosteroid receptor kinase BRASSINOSTE-ROID INSENSITIVE1 (BRI1) (Tang et al. 2008). Some

Figure 3. The roles of reactive oxygen species (ROS) in stomatal immunityDuring the stomatal immunity response, the flg22 peptide is perceived by the FLAGELLIN-SENSING2 (FLS2)–BRI1-ASSOCIATED RECEPTOR KINASE (BAK1) co-receptor complex, which activates BOTRYTIS-INDUCED KINASE1 (BIK1).BIK1 phosphorylates the N-terminus of respiratory burst oxidase homolog protein D, thereby activating it toproduce apoplastic H2O2. BIK1 might also activate an unknown Ca

2þ channel to increase cytosolic Ca2þ levels, whichwould in turn activate CPK28 to inhibit BIK1 or other calcium-dependent protein kinases (CPKs) such as CPK5 tophosphorylate and activate RBOHD.Without flg22 stimulation, the heterotrimeric G proteins composed of the non-canonical Ga proteins XLG2/3, Gb protein AGB1 and Gg proteins AGG1/2 interacts with FLS2-BIK1 complex. Afterperceiving flg22, the activated BIK1 phosphorylates the N-terminus of XLG2, which leads to G proteins to dissociatefrom receptor complex and increase RBOHD activity in an unknown manner. BR-SIGNALING KINASE1 (BSK1)interacts with FLS2 to generate a flg22-induced ROS burst. In the apoplast, PRX33/34, which are regulated by thecytokinin pathway, produce apoplastic O2�

–. Apoplastic H2O2 is transported into the cytosol by PIP1;4, which furtherinduces the production of nitric oxide (NO). NO mediates the S-nitrosylation of RBOHD and ASCORBATEPEROXIDASE1 (APX1), thereby inhibiting RBOHD activity but increasing APX1 activity. Blue arrows indicate positiveregulation; red bars indicate inhibition; dotted lines indicate uncertain regulation.



other factors, such as BSK1, are known to interact withFLS2 and required for flg22-induced ROS bursts (Shiet al. 2013).

Besides apoplastic ROS, pathogen infection alsoinduces the production of NO, which is required forplant resistance to pathogen invasion (Delledonne et al.2001). NO production likely acts downstream of H2O2 inthe plant pathogen resistance response (Arnaud andHwang 2015). NO modifies the Cys890 of RbohDthrough S-nitrosylation, which impairs its ability tobind the cofactor FAD, thus blunting NADPH oxidaseactivity (Yun et al. 2011). By contrast, S-nitrosylation atthe Cys32 of cytosolic APX1 enhances its enzymaticactivity for scavenging H2O2 (Yang et al. 2015). Thus,H2O2 and NO form a feedback regulation circuit in plantdisease resistance.

In addition to producing extracellular ROS viaNADPH oxidases, plants can produce these compoundsvia apoplastic peroxidases (Torres et al. 2002;Bindschedler et al. 2006). In Arabidopsis, knockdownof the class III apoplastic peroxidase genes PRX33 andPRX34 reduces ROS production and callose depositionduring various PTI responses (Daudi et al. 2012). Thecytokinin RESPONSE REGULATOR2 (ARR2) directlyregulates PRX33 and PRX34 expression, thereby medi-ating ROS accumulation, stomatal closure and PTI,which are independent of ABA signaling (Arnaud et al.2017). As NADPH oxidases are activated by ROS, NADPHoxidases and apoplastic peroxidases likely affect eachother in the modulation of ROS bursts during the plantimmunity response.

Not surprisingly, pathogens have developed anefficient evasion system to perturb host immunitythrough secreting various effector proteins, whichhijack plant proteins, as well as toxins (such ascoronatine produced by Pseudomonas syringae), caus-ing stomata to reopen (Melotto et al. 2006; Qi et al.2017). Under dry environmental conditions, mostphyllosphere microbes maintain low populations, andonly few bacterial pathogens enter through stomatalpores. Under high-humidity conditions, the phyllo-sphere pathogens aggressively propagate and invadeplants through the stomata, causing disease breakout(Xin et al. 2016). ABA can enhance disease resistance byclosing stomatal pores (Melotto et al. 2008), but it canalso increase a plant’s susceptibility to some powderymildew, fungal and bacterial diseases (Bostock et al.2014). For example, abi1-1 dominant mutants and

plants overexpressing HYPERSENSITIVE TO ABA1 exhibitincreased callose deposition and resistance to bacteriaP. syringae (de Torres-Zabala et al. 2007).

The ABA-deficient tomato mutant sitiens accumu-lates more H2O2 at the site of infection and is moreresistant to the necrotrophic fungus Botrytis cinereathan wild type (Asselbergh et al. 2007). By contrast, theABA biosynthetic mutants aba1-3 and ABA insensitivemutant abi1-1 exhibit reduced callose deposition anddisease resistance in response to another necrotrophicfungus, Leptosphaeria maculans (Kaliff et al. 2007).Some common signaling proteins, such as OST1, GHR1and SLAC1, are involved in both ABA- and pathogen-mediated stomatal movement (Melotto et al. 2008; Huaet al. 2012; Deger et al. 2015). These findings suggestthat ABA plays multifaceted roles in resistance tovarious diseases (Ton et al. 2009).

ROS SIGNALING IN CHLOROPLASTS,MITOCHONDRIA AND PEROXISOMESUNDER DROUGHT STRESS ANDPATHOGEN ATTACK

Under both abiotic and biotic stress, ROS are producedin various organelles due to a metabolic imbalance. Likeapoplastic ROS, these ROS can serve as stress signals tomodify some proteins and activate the expression ofstress-associated genes, which in turn counteractsstress-associated oxidative stress (Mittler et al. 2004;Miller et al. 2010; Shapiguzov et al. 2012; Suzuki et al.2012; Singh et al. 2016). The chloroplast is a key organellefor sensing environmental signals such as high light, lowor high temperature, salt and drought stress, as well aspathogen invasion. This organelle produces most ofthe ROS present in leaf cells (Figure 4).

Increased ROS production in chloroplasts negativelyaffects photosynthetic electron transport, impairs theassembly and repair of photosystem II (PSII) and affectschloroplast development (Dietz et al. 2016). Chloroplastsmainly produce singlet oxygen (1O2) in PSII and its light-harvesting antennae (Shapiguzov et al. 2012). Although1O2 is a ROS, it is unusual in that it is produced under high-intensity light when the excitation energy of tripletchlorophyll molecules is transferred to triplet-statemolecular oxygen (Triantaphylides and Havaux 2009).

The Arabidopsis fluorescent (flu) and chlorina1 (ch1)mutants specifically accumulate 1O2 without significant

10 Qi et al.


Figure 4. Crosstalk of reactive oxygen species (ROS) between organellesHigh levels of ROS are produced in chloroplasts under high-light conditions and drought stress. FLUORESCENTIN BLUE LIGHT (FLU) and CHLORINA1 (CHL) specifically regulate the accumulation of 1O2 in photosystem II(PSII). The chloroplast-localized proteins EXECUTER1 (EX1) and EX2 transduce 1O2 signals to the nucleus toregulate cell death-related genes. PSI mainly produces O2 �

� /H2O2 under stress conditions, which can bedecomposed by thylakoid-bound ascorbate peroxidase (tAPX). H2O2 has an antagonistic effect on

1O2 signalingin chloroplasts. H2O2 diffuses into the cytosol to activate OXIDATIVE SIGNAL-INDUCIBLE1 (OXI1), which isrequired to fully activate MPK3/6. Singlet oxygen may also go through the OXI1 and jasmonate pathways toregulate cell death. The pathogen cysteine protease effector HopN1 directly mediates the degradation of PsbQin PSII. The detection of pathogen-associated molecular patterns (PAMPs) leads to an increase in transient Ca2þ

levels, which is mediated by calcium-sensing receptors (CAS) in chloroplasts, likely via the MAPK cascade.Carotenoids quench 1O2 to become b-cyclocitral, which induces the expression of 1O2-responsive genes.Plastidial methylerythritol cyclodiphosphate (MEcPP) and triose phosphate from chloroplasts can also induceselected stress-responsive nucleus-encoded plastidial proteins. SAL1 dephosphorylates 30-phosphoadenosine50-phosphate (PAP) to adenosine monophosphate (AMP), which is negatively regulated by H2O2. PAP is boundby and inhibits nuclear 50-to-30 exoribonucleases (XRNs), resulting in the upregulation of various abscisic acid(ABA)-responsive genes. Chloroplasts communicate with the mitochondria and peroxisomes through MOSAICDEATH1 (MOD1)-mediated fatty acid biosynthesis. In the mitochondria, ABA signaling negatively regulatesComplex I activity through negatively regulating the ABA OVERLY SENSITIVE6 (ABO6) and ABO8 in Complex I.In peroxisomes, glycolate oxidase (GOX) and acyl-CoA oxidase (ACX) are the main enzymes that function inH2O2 production. Blue arrows indicate positive regulation or production; red bars indicate inhibition; dottedlines indicate uncertain regulation.



coproduction of other ROS under certain conditions(Meskauskiene et al. 2001; Ramel et al. 2013), makingthem particularly useful for studying 1O2 activity. Asdemonstrated using these mutants, 1O2 is crucial forregulating the expression of nucleus-encoded stress-responsive genes, and it also promotes cell death(Wagner et al. 2004; Lee et al. 2007; Ramel et al. 2013).

Carotenoids are the main 1O2 quenchers inthe chloroplast. The oxidized carotenoid product, b-cyclocitral, specifically induces the expression of genesin the 1O2 signaling pathway, but has little effect onthe expression of H2O2-mediated genes (Ramel et al.2012). The executer1 (ex1) mutation suppresses the1O2-induced cell death phenotype of the flu mutant(Wagner et al. 2004) but does not affect the accumula-tion of 1O2. Furthermore, crossing flu with the ex1 ex2double mutant fully suppressed the upregulation ofalmost all 1O2-responsive nuclear genes in this mutant(Lee et al. 2007). These findings suggest that chloro-plast-localized EX1 and EX2 are crucial transducers orsensors for 1O2-induced gene expression and cell death.

The application of flg22 to Arabidopsis quicklyinduces specific Ca2þ transients in the chloroplaststroma; this process relies on the presence of thethylakoid-associated calcium-sensing receptor (CAS)(Nomura et al. 2012). CAS regulates the PAMP-inducedexpression of defense genes and inhibits chloroplast-mediated transcriptional reprogramming, likely throughan 1O2-mediated pathway (Nomura et al. 2012). Thesefindings suggest that Ca2þ signaling communicates withROS signaling in the chloroplast to mediate nucleargene expression.

Pathogens can hijack plant immune signaling bysecreting various effector proteins. Pseudomonassyringae DC3000 effectors rapidly inhibit photosynthesisby reprogramming the expression of nucleus-encodedchloroplast-targeted genes, thus preventing the chlo-roplastic ROS burst. This phenomenon coincides withpathogen-induced ABA accumulation; exogenous appli-cation of ABA suppresses plant immunity and PSIIactivity (Zabala et al. 2015). The cysteine proteaseeffector HopN1 directly targets PsbQ in PSII, therebymediating the degradation of PsbQ and interferingwith PSII activity, thus promoting 1O2 production(Rodriguez-Herva et al. 2012).

PSI mainly produces O2�� /H2O2when photosynthetic

electron transfer and CO2 fixation rates are altered,especially under water-stress conditions that reduce

CO2 uptake due to stomatal closure (Shapiguzov et al.2012). Overexpressing tAPX reduces H2O2 levels butincreases 1O2-mediated stress responses in the flumutant, suggesting that H2O2 has an antagonistic effecton 1O2 signaling in chloroplasts (Laloi et al. 2007). H2O2accumulates in the chloroplasts of bundle sheath cellsunder high light and induces the ABA biosynthesis-dependent expression of ROS-responsive genes(Galvez-Valdivieso et al. 2009). H2O2 activates thekinase OXIDATIVE SIGNAL-INDUCIBLE1 (OXI1), which isrequired to fully activate MPK3 and MPK6 for ROSproduction under various conditions (Rentel et al. 2004;Petersen et al. 2009; Liu et al. 2010). The oxi1 mutant ismore tolerant to photo-induced oxidative damage andcell death than wild type (Shumbe et al. 2016). OXI1 alsosuppresses ch1-mediated 1O2 production and PCD underhigh-light conditions independently of EX1 and EX2, butlikely through jasmonate signaling. These findingssuggest that FLU- and CHL-mediated 1O2 productionregulate cell death via different pathways (Shumbeet al. 2016).

The metabolites produced in chloroplasts underhigh light and various stress conditions play importantroles in ROS signaling and the regulation of nucleargene expression (Zhu 2016). Phosphonucleotide 30-phosphoadenosine 50-phosphate (PAP) accumulates incells under drought and high-light stress and is regulatedby the phosphatase SAL1, which dephosphorylates PAPto adenosine monophosphate (AMP) (Estavillo et al.2011). SAL1 activity is inhibited by oxidative stressthrough oxidation-mediated dimerization, the forma-tion of intramolecular disulfide bridges and glutathio-nylation, resulting in the accumulation of PAP (Chanet al. 2016). PAP from chloroplasts to cytosol is thoughtto inhibit nuclear 50-to-30 exoribonucleases (XRNs),whichmight alter the levels of stress-responsive mRNAsvia RNA cleavage or affect transcription termination(Estavillo et al. 2011). Thus, SAL1 might be a conserved,general ROS sensor in chloroplasts that functions inplants under both drought stress and high-lightconditions (Chan et al. 2016).

Notably, the sal1mutation, exogenous manipulationof PAP and the xrn2 xrn3 double mutations all restoreABA responses in the ABA-hyposensitive mutantsost1, the snrk2.2/2.3/2.6 (ost1) triple mutant and abi1-1during stomatal movement and/or seed germination(Pornsiriwong et al. 2017). ABA-activated SnRK2.2/2.3/2.6 kinases phosphorylate XRN2 and XRN3 (Wang et al.

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2013b), suggesting that SAL1-PAP-XRN retrogradesignaling can bypass the ABA pathway (Pornsiriwonget al. 2017), but is regulated by ABA signaling (Wanget al. 2013b). SAL1-PAP-XRN signaling positively regu-lates the expression of many genes involved in the ABAand Ca2þ signaling pathways. These observationsemphasize the tight communication and complemen-tary interactions between ROS-mediated SAL1-PAP-XRNsignaling and ABA signaling under drought and high-light conditions (Pornsiriwong et al. 2017).

Many stress treatments can quickly activate MAPkinase cascades, including MAP3 and 6 (Meng andZhang 2013). MPK3/6 interact with and phosphorylateERF6 at Ser266 and Ser269, making ERF6 more stable,allowing it to directly upregulate the expression ofROS-responsive and defensin genes and conferringfungal disease resistance (Wang et al. 2010; Meng et al.2013). These responses are abolished in the tpt1 and tpt2mutants, which lack chloroplast membrane-localizedtriose phosphate translocators (TPT) (Vogel et al. 2014).Consistent with this role, the erf6 mutant accumulatesmore H2O2 and is more sensitive to photoinhibition thanwild type (Wang et al. 2013a). These observationssuggest that ROS are involved in regulating thesignaling of metabolites in chloroplasts that serve asretrograde signals to coordinate stress-response path-ways in the nucleus.

Mitochondria generate a major portion of ROS inroots but only a small portion in leaves. While ETCcomplexes I–IV in plants are similar to those in animalmitochondria, these complexes also contain five uniqueenzymes, including an alternative oxidase (AOX) andfour NAD(P)H dehydrogenases (Moller 2001; Jacobyet al. 2012). Complex I and III are major sites for ROSproduction in plants. AOX helps minimize ROS accumu-lation by the ETC (Moller 2001; Jacoby et al. 2012).

ABA and drought treatment promote ROS produc-tion in mitochondria (Rhoads et al. 2006; He et al. 2012;Yang et al. 2014). ABA OVERLY SENSITIVE6 (ABO6)encodes a DEXH box RNA helicase that regulates thesplicing of several genes in Complex I (He et al. 2012).ABO8 encodes a pentatricopeptide repeat (PPR) proteininvolved in mediating the splicing of NAD4 intron 3 inmitochondrial complex I (Yang et al. 2014). Both abo6and abo8mutants are hypersensitive to ABA in terms ofseed germination and primary root growth (He et al.2012; Yang et al. 2014). Interestingly, the expression ofABO6 and ABO8 is reduced by ABA treatment (He et al.

2012; Yang et al. 2014). Both ABA treatment and droughtstress promote greater ROS accumulation in the abo6and abo8mutants compared towild type (He et al. 2012;Yang et al. 2014), indicating that ABA signaling regulatesROS production in mitochondria.

The abo8 mutant exhibits increased ROS accumula-tion in the root tip, resulting in delayed differentiation ofdistal stem cells (DSC) (Yang et al. 2014). Another studyby Yu et al. showed that mutations in a P-loop NTPaselocalized to themitochondria in Arabidopsis reduce bothH2O2 and O2.

– levels in roots and promote DSCdifferentiation (Yu et al. 2016). UPBEAT1 (UPB1) encodesa bHLH transcriptional factor that negatively regulatesthe expression of several peroxidase genes in the rootelongation zone (Tsukagoshi et al. 2010). The upb1mutant exhibits increased O2.

– levels in the rootmeristem and decrease apoplastic H2O2 content in theelongation zone, which delays the transition from cellproliferation to differentiation, resulting in enlarged rootapical meristems (Tsukagoshi et al. 2010). ROS levelsmust also be maintained at moderate levels in animalcells in order to maintain stem cell homeostasis (Le Belleet al. 2011; Morimoto et al. 2013; Paul et al. 2014). Thesestudies indicate that ROS homeostasis is essential for celldivision and differentiation (Mittler 2017).

Peroxisomes mainly produce H2O2 and O2– throughseveral metabolic pathways. Water stress promotesstomatal closure and reduces CO2 assimilation, thusincreasing photorespiration and glycolate production.Glycolate from chloroplasts is oxidized by glycolateoxidase (GOX) to produce glyoxylate and most of theH2O2 in leaf peroxisomes, indicating that ROS signalsare communicated between chloroplasts and perox-isomes through various metabolites. Additionally, acyl-CoA oxidase (ACX) catalyzes the production of H2O2during peroxisomal fatty acid b-oxidation, a processthat occurs more actively in germinating seeds than inother tissues (Sandalio and Romero-Puertas 2015). Thematrix xanthine oxidase (XOD) in leaf peroxisomesgenerates O2

-, which is converted to H2O2 by SOD. H2O2is mainly detoxified by peroxisome-localized CATs inleaves (Miller et al. 2010). Furthermore, NO and itsderivative, RNS, are also produced in peroxisomes,which can influence many aspects of cellular function inresponse to environmental stress (Sandalio andRomero-Puertas 2015).

Reactive oxygen species produced from differentorganelles may diffuse into the cytosol. Whether these



ROS have similar effects in the cytosol is not yet clear.However, ROS produced from different organellescould form a specific signature to trigger unique nucleargene expression responses (Rosenwasser et al. 2011;Sewelam et al. 2014). For example, chloroplastic H2O2has a significant bias to induce the expression of genesrelated to wounding and pathogen attack, while H2O2from peroxisomes regulates more genes involved inprotein repair responses (Sewelam et al. 2014). As itmight not be possible for cellular components todetermine which ROS comes from which organelle, thisfinding suggests that ROS, with or without othercomponents, are involved in regulating the expressionof specific genes.

Although ROS signals from different organelles cancommunicate with each other, little is known about thiscommunication process (Shapiguzov et al. 2012).Mutations in Arabidopsis MOSAIC DEATH1 (MOD1),encoding an enoyl-acyl carrier protein (ACP) reductasefor fatty acid biosynthesis in chloroplasts, lead to highH2O2 and O2�

� production in mitochondria (Mou et al.2000; Wu et al. 2015), suggesting that chloroplastsignals might be transmitted to mitochondria tomodulate ROS production. A screening for suppressorsof mod1 identified several nuclear genes encodingproteins involved in mitochondrial Complex I (Wu et al.2015). In fact, mutations in these of Complex I genesusually result in higher ROS levels than wild type (Leeet al. 2002; Meyer et al. 2009; He et al. 2012). Signalsfrom mod1 chloroplasts might enhance relative mito-chondrial ETC activity, causing greater electron leakageand more ROS to be produced in Complex I. This effectis compromised by mutations in Complex I genes, asmod1 suppressor mutations reduce ETC activity (Wuet al. 2015). The mod1 mutant and its suppressor linesrepresent valuable materials for further studying ROS-related communication between chloroplasts andmitochondria.

THE ROLE OF ROS SIGNALING INCELL-TO-CELL AND LONG-DISTANCECOMMUNICATION DURING STRESS

Besides inside cells, ROS communicationoccurs betweencells. ROS, Ca2þ and electric signals can be transmittedfrom the tissue of origin to distant tissues withinminutes, allowing plants to achieve systemic acquired

acclimation (SAA) under abiotic stress (Figure 5)(Mittler et al. 2011; Gilroy et al. 2016). Various abioticstresses, such as wounding, heat, cold, high intensitylight and high salinity, trigger the production ofRBOHD-mediated apoplastic ROS, which move inrelay-type fashion in the apoplast from the localtissue throughout the entire plant at speeds of up to8.4 cm/min (Miller et al. 2009).

RBOHD-mediated ROS enter neighboring cells totrigger an increase in transient Ca2þ levels. This, in turn,activates protein kinases such as CPK5 to phosphorylateand activate RBOHD, thus forming a wave of ROS andCa2þ that functions in cell-to-cell and long-distancecommunication (Dubiella et al. 2013). Heat stress alsotransiently increases the accumulation of ABA insystemic tissues in an RBOHD-dependent manner.Blocking ABA biosynthesis or signaling impairs theSAA response to heat stress, indicating the importanceof coordination between ABA and the ROS wave in SAA(Suzuki et al. 2013).

Osmotic stress quickly induces increases in [Ca2þ]ilevels through the action of OSCA1 (REDUCED HYPER-OSMOLARITY, INDUCED CA2þ INCREASE1), a Ca2þ

channel and osmosensor localized on the plasmamembrane. However, while the osca1 mutant shows areduced Ca2þ response to osmotic stress, it stillresponds normally to ABA and H2O2 treatment,suggesting that OSCA1 functions upstream of ABAand H2O2 signaling (Yuan et al. 2014). Further studies arerequired to determine whether OSCA1 is involvedproducing the Ca2þ wave.

The long distance, salt-stress-induced Ca2þ wave(which travels at a speed of 2.4 cmmin� 1) requiresthe vacuolar cation-permeable channel TWO PORECHANNEL1 (TPC1) (Choi et al. 2014). TPC1 is sensitiveto Ca2þ levels in both the cytosol and vacuole lumenand is likely involved in calcium-induced calciumrelease during Ca2þ wave propagation (Choi et al.2014). Glutamate receptor-like proteins (GLRs), local-ized on the plasma membrane, might also function asamino acid-gated Ca2þchannels (Kong et al. 2016).L-methionine (L-Met) activates GLR3.1 and GLR3.5Ca2þ channels to increase cytosolic Ca2þ levels, whichis required for NADPH oxidase-mediated ROS pro-duction and stomatal closure but functions indepen-dently of ABA (Kong et al. 2016).

Wounding rapidly induces distal systemic electricalsignals, a process that requires the participation of

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GLR3.2, GLR3.3 and GLR3.6 (Mousavi et al. 2013). GLR3.3and GLR3.6 are required for propagating wound-induced electrical signals beyond the wounded leaf,while GLR3.5 is required to prevent the formation ofwound-induced electrical potentials in distal, non-neighboring leaves (Salvador-Recatal�a 2016). However,the molecular connections among ROS/Ca2þ andelectrical signals require further exploration.

FUTURE PROSPECTS

In this review, we summarize the molecular mecha-nisms that regulate the ROS signaling network in plants,primarily under drought stress and pathogen attack.Althoughwedivided our review into several sections forthe sake of discussion, ROS production and signaling,under different conditions, cannot be separated withina cell. Even under very strict experimental conditions,ROS production cannot be regulated in only a specificsite in the cell. Once a stress is applied, different types

of ROS will be produced, immediately, in variouscompartments and different sites of a cell.

These ROS form a complicated network, making itquite difficult to study ROS signaling. Therefore, wheninvestigating ROS signaling, we should take the wholepicture into account for their roles in plant responses toabiotic and biotic stress. While ROS are metabolic by-products, high ROS levels are toxic to cells, and thustheir concentrations are kept low by the action ofvarious antioxidants. At the same time, ROS areabsolutely required for many plant developmentalprocesses and responses to biotic and abiotic stress(Mittler 2017). For example, in rice dst (drought and salttolerance) mutant, it has shown that increasing ROS toan appropriate level does not affect the plant growthand yield, but greatly increase plant tolerance todrought and salt stress (Huang et al. 2009). DSTencodes a zinc finger transcriptional factor that likelydirectly regulates peroxidase 24 precursor for scaveng-ing H2O2 (Huang et al. 2009).Wheat seedlings carrying amutated Ta-sro1 gene encoding a poly(ADP ribose)

Figure 5. Reactive oxygen species (ROS) and Ca2+ waves function in cell-to-cell communicationApoplastic ROS mediated by respiratory burst oxidase homolog protein D (RBOHD) triggers the generation ofCa2þ transients via the activity of unknown receptor-like kinases, or they enter neighboring cells via water channels.Osmotic stress activates OSCA1 (REDUCED HYPEROSMOLARITY, INDUCED CA2þ INCREASE1) to increase Ca2þ levels inthe cytosol. In turn, Ca2þ activates protein kinases such as CPK5 to phosphorylate and activate RBOHD, thus formingROS and Ca2þ waves that function in cell-to-cell and long-distance communication. The vacuolarcation-permeable channel TWO PORE CHANNEL1 (TPC1) and plasma membrane-localized glutamate receptor-likeproteins (GLRs) may be involved in increasing the levels of Ca2þ. Ca2þ can diffuse into neighboring cells throughplasmodesmata (PD). Blue arrows indicate positive regulationor production; dotted lines indicate uncertain regulation.



polymerase (PARP) domain protein with high PARPactivity increase ROS production and yield under salinitystress (Liu et al. 2014). These studies point out a newstrategy for breeding of stress resistant crops bymodifying the ROS production in the future. Althoughmuch exciting progress has been made, thanks largelyto newly developed techniques and rapid progress ingenome and protein analysis methods, a completeunderstanding of the exact nature of ROS signalingremains elusive.

Some gaps in our knowledge are summarized bythe following questions: How do we measure thelevels of different types of ROS and their dynamicchanges at various sites in the cell? How are ROSsignals that are produced as a result of normaldevelopmental processes physically interconnectedand integrated with those produced as a result ofbiotic and abiotic stress? How are ROS differentiallyrecognized as second messengers in a cell or inneighboring cells during processes such as PCD?Different ROS sensors must exist, but how do weidentify these sensors? Given the diversity of receptor-like protein kinases in plants (Shiu and Bleecker 2001),do they directly sense and transmit apoplastic ROSsignals? If so, how? Do they act alone or together withother components to transduce a ROS wave?

It is well known that pretreatment with lowconcentration of exogenous H2O2 (called H2O2-priming)can increase plant tolerance to various abiotic stresses(Hossain et al. 2015), but the molecular mechanisms forthis are not well understood. Right now, ROS level canbe controlled in crops by genetically manipulating theexpression of ROS-related genes. But which ROS levelshould be maintained in order to increase stresstolerance without apparent yield penalty needs furtherexploration in various crops with different geneticbackgrounds.

Most studies on ROS tend to focus on theirproduction and signal transduction in the cytosol,organelles and plasmamembrane. However, the effectsof ROS on chromatin structure and epigenetic mod-ifications in plants requires further exploration.

ACKNOWLEDGEMENTS

J.Z. and Z.G. are supported by theNational Key ScientificResearch Project (2011CB915400). Z.G. is supported by

the National Natural Science Foundation of China(31730007).

REFERENCES

Arnaud D, Hwang I (2015) A sophisticated network of signalingpathways regulates stomatal defenses to bacterialpathogens. Mol Plant 8: 566–581

Arnaud D, Lee S, Takebayashi Y, Choi D, Choi J, Sakakibara H,Hwang I (2017) Cytokinin-mediated regulation of reactiveoxygen species homeostasis modulates stomatal immu-nity in Arabidopsis. Plant Cell 29: 543–559

Asgher M, Per TS, Masood A, Fatma M, Freschi L, Corpas FJ,Khan NA (2017) Nitric oxide signaling and its crosstalk withother plant growth regulators in plant responses to abioticstress. Environ Sci Pollut Res 24: 2273–2285

Asselbergh B, Curvers K, Franca SC, Audenaert K, VuylstekeM,Van Breusegem F, Hofte M (2007) Resistance to Botrytiscinerea in sitiens, an absci acid-deficient tomato mutant,involves timely production of hydrogen peroxide and cellwall modifications in the epidermis. Plant Physiol 144:1863–1877

Baxter A, Mittler R, Suzuki N (2014) ROS as key players in plantstress signalling. J Exp Bot 65: 1229–1240

Bindschedler LV, Dewdney J, Blee KA, Stone JM, Asai T,Plotnikov J, Denoux C, Hayes T, Gerrish C, Davies DR,Ausubel FM, Bolwell GP (2006) Peroxidase-dependentapoplastic oxidative burst in Arabidopsis required forpathogen resistance. Plant J 47: 851–863

Bostock RM, Pye MF, Roubtsova TV (2014) Predisposition inplant disease: Exploiting the nexus in abiotic and bioticstress perception and response. Annu Rev Phytopathol52: 517–549

Boudsocq M, Willmann MR, McCormack M, Lee H, Shan LB,He P, Bush J, Cheng SH, Sheen J (2010) Differential innateimmune signalling via Ca2þ sensor protein kinases. Nature464: 418–U116

Bourdais G, Burdiak P, Gauthier A, Nitsch L, Salojarvi J,Rayapuram C, Idanheimo N, Hunter K, Kimura S, Merilo E,Vaattovaara A, Oracz K, Kaufholdt D, Pallon A, AnggoroDT, Glow D, Lowe J, Zhou J, Mohammadi O, Puukko T,Albert A, Lang H, Ernst D, Kollist H, Brosche M, Durner J,Borst JW, Collinge DB, Karpinski S, Lyngkjaer MF,Robatzek S, Wrzaczek M, Kangasjarvi J, Consortium C(2015) Large-scale phenomics identifies primary and fine-tuning roles for CRKs in responses related to oxidativestress. PLoS Genet 11: e1005373

Brandt B,Munemasa S,Wang C, Nguyen D, Yong TM, Yang PG,Poretsky E, Belknap TF, Waadt R, Aleman F, Schroeder JI(2015) Calcium specificity signaling mechanisms in abscisicacid signal transduction in Arabidopsis guard cells. eLife 4

Bright J, Desikan R, Hancock JT, Weir IS, Neill SJ (2006) ABA-induced NO generation and stomatal closure in Arabidopsisare dependent on H2O2 synthesis. Plant J 45: 113–122

16 Qi et al.


Camejo D, Guzman-Cedeno A, Moreno A (2016) Reactiveoxygen species, essential molecules, during plant-patho-gen interactions. Plant Physiol Biochem 103: 10–23

Chan KX, Mabbitt PD, Phua SY, Mueller JW, Nisar N,Gigolashvili T, Stroeher E, Grassl J, Arlt W, Estavillo GM,Jackson CJ, Pogson BJ (2016) Sensing and signaling ofoxidative stress in chloroplasts by inactivation of the SAL1phosphoadenosine phosphatase. Proc Natl Acad Sci USA113: E4567–E4576

Chater C, Gray JE, Beerling DJ (2013) Early evolutionaryacquisition of stomatal control and development genesignalling networks. Curr Opin Plant Biol 16: 638–646

Chater C, Peng K, Movahedi M, Dunn JA, Walker HJ, Liang YK,McLachlan DH, Casson S, Isner JC, Wilson I, Neill SJ,Hedrich R, Gray JE, Hetherington AM (2015) Elevated CO2-induced responses in stomata require ABA and ABAsignaling. Curr Biol 25: 2709–2716

Choi WG, Toyota M, Kim SH, Hilleary R, Gilroy S (2014) Saltstress-induced Ca2þwaves are associated with rapid, long-distance root-to-shoot signaling in plants. Proc Natl AcadSci USA 111: 6497–6502

Choudhury FK, Rivero RM, Blumwald E, Mittler R (2017)Reactive oxygen species, abiotic stress and stresscombination. Plant J 90: 856–867

Daudi A, Cheng ZY, O’Brien JA, Mammarella N, Khan S,Ausubel FM, Bolwell GP (2012) The apoplastic oxidativeburst peroxidase in Arabidopsis is a major component ofpattern-triggered immunity. Plant Cell 24: 275–287

de Torres-Zabala M, Truman W, Bennett MH, Lafforgue G,Mansfield JW, Egea PR, Bogre L, Grant M (2007)Pseudomonas syringae pv. tomato hijacks the Arabidopsisabscisic acid signalling pathway to cause disease. EMBO J26: 1434–1443

Deger AG, Scherzer S, Nuhkat M, Kedzierska J, Kollist H,BroscheM, Unyayar S, BoudsocqM, Hedrich R, RoelfsemaMRG (2015) Guard cell SLAC1-type anion channels mediateflagellin-induced stomatal closure. New Phytol 208:162–173

Delledonne M, Zeier J, Marocco A, Lamb C (2001) Signalinteractions between nitric oxide and reactive oxygenintermediates in the plant hypersensitive disease resis-tance response. Proc Natl Acad Sci USA 98: 13454–13459

Dietz KJ, Turkan I, Krieger-Liszkay A (2016) Redox- andreactive oxygen species-dependent signaling into and outof the photosynthesizing chloroplast. Plant Physiol 171:1541–1550

Dou DL, Zhou JM (2012) Phytopathogen effectors subvertinghost immunity: Different foes, similar battleground. CellHost Microbe 12: 484–495

Drerup MM, Schlucking K, Hashimoto K, Manishankar P,Steinhorst L, Kuchitsu K, Kudla J (2013) The calcineurin B-like calcium sensors CBL1 and CBL9 together with theirinteracting protein kinase CIPK26 regulate the ArabidopsisNADPH oxidase RBOHF. Mol Plant 6: 559–569

Dubiella U, Seybold H, Durian G, Komander E, Lassig R, WitteCP, Schulze WX, Romeis T (2013) Calcium-dependentprotein kinase/NADPH oxidase activation circuit is

required for rapid defense signal propagation. Proc NatlAcad Sci USA 110: 8744–8749

Engineer CB, Hashimoto-Sugimoto M, Negi J, Israelsson-Nordstrom M, Azoulay-Shemer T, Rappel WJ, Iba K,Schroeder JI (2016) CO2 Sensing and CO2 regulation ofstomatal conductance: Advances and open questions.Trends Plant Sci 21: 16–30

Estavillo GM, Crisp PA, Pornsiriwong W, Wirtz M, Collinge D,Carrie C, Giraud E, Whelan J, David P, Javot H, Brearley C,Hell R, Marin E, Pogson BJ (2011) Evidence for a SAL1-PAPChloroplast retrograde pathway that functions in droughtand high light signaling in Arabidopsis. Plant Cell 23:3992–4012

Fabre N, Reiter IM, Becuwe-Linka N, Genty B, Rumeau D(2007) Characterization and expression analysis ofgenes encoding alpha and beta carbonic anhydrases inArabidopsis. Plant Cell Environ 30: 617–629

Finkelstein RR, Gampala SSL, Rock CD (2002) Abscisic acidsignaling in seeds and seedlings. Plant Cell 14: S15–S45

Galvez-Valdivieso G, FryerMJ, Lawson T, Slattery K, TrumanW,Smirnoff N, Asami T, Davies WJ, Jones AM, Baker NR,Mullineaux PM (2009) The high light response inArabidopsis involves ABA signaling between vascularand bundle sheath cells. Plant Cell 21: 2143–2162

Geiger D, Maierhofer T, AL-Rasheid KAS, Scherzer S, Mumm P,Liese A, Ache P, Wellmann C, Marten I, Grill E, Romeis T,Hedrich R (2011) Stomatal closure by fast abscisic acidsignaling is mediated by the guard cell anion channelSLAH3 and the receptor RCAR1. Sci Signal 4: ra32

Geiger D, Scherzer S, Mumm P, Marten I, Ache P, Matschi S,Liese A, Wellmann C, Al-Rasheid KAS, Grill E, Romeis T,Hedrich R (2010) Guard cell anion channel SLAC1 isregulated by CDPK protein kinases with distinct Ca2þ

affinities. Proc Natl Acad Sci USA 107: 8023–8028

Geiger D, Scherzer S, Mumm P, Stange A, Marten I, Bauer H,Ache P, Matschi S, Liese A, Al-Rasheid KAS, Romeis T,Hedrich R (2009) Activity of guard cell anion channelSLAC1 is controlled by drought-stress signaling kinase-phosphatase pair. Proc Natl Acad Sci USA 106:21425–21430

Gilroy S, Bialasek M, Suzuki N, Gorecka M, Devireddy AR,Karpinski S, Mittler R (2016) ROS, calcium, and electricsignals: Key mediators of rapid systemic signaling inplants. Plant Physiol 171: 1606–1615

Grondin A, Rodrigues O, Verdoucq L, Merlot S, Leonhardt N,Maurel C (2015) Aquaporins contribute to ABA-triggeredstomatal closure through OST1-mediated phosphoryla-tion. Plant Cell 27: 1945–1954

Hao HQ, Fan LS, Chen T, Li RL, Li XJ, He QH, Botella MA, Lin JX(2014) Clathrin and membrane microdomains coopera-tively regulate RbohD dynamics and activity inArabidopsis.Plant Cell 26: 1729–1745

Hashimoto M, Negi J, Young J, Israelsson M, Schroeder JI, IbaK (2006) Arabidopsis HT1 kinase controls stomatal move-ments in response to CO2. Nat Cell Biol 8: 391–U352

He JN, Duan Y, Hua DP, Fan GJ,Wang L, Liu Y, Chen ZZ, Han LH,Qu LJ, Gong ZZ (2012) DEXH box RNA helicase-mediated



mitochondrial reactive oxygen species production inArabidopsis mediates crosstalk between abscisic acidand auxin signaling. Plant Cell 24: 1815–1833

Horak H, Sierla M, Toldsepp K, Wang C, Wang YS, Nuhkat M,Valk E, Pechter P,Merilo E, Salojarvi J, Overmyer K, Loog B,Brosche A, Schroeder JI, Kangasjarvi J, Kollist H (2016)A dominant mutation in the HT1 kinase uncovers roles ofMAP kinases and GHR1 in CO2-induced stomatal closure.Plant Cell 28: 2493–2509

Hossain MA, Bhattacharjee S, Armin SM, Qian PP, XinW, Li HY,Burritt DJ, Fujita M, Tran LSP (2015) Hydrogen peroxidepriming modulates abiotic oxidative stress tolerance:Insights from ROS detoxification and scavenging. FrontPlant Sci 6: 420

Hu HH, Boisson-Dernier A, Israelsson-Nordstrom M, BohmerM, Xue SW, Ries A, Godoski J, Kuhn JM, Schroeder JI (2010)Carbonic anhydrases are upstream regulators of CO2-controlled stomatal movements in guard cells. Nat CellBiol 12: 87–U234

Hu JL, Huang XH, Chen LC, Sun XW, Lu CM, Zhang LX,Wang YC,Zuo JR (2015) Site-specific nitrosoproteomic identificationof endogenously S-nitrosylated proteins in Arabidopsis.Plant Physiol 167: 1731–1746

Hua DP, Wang C, He JN, Liao H, Duan Y, Zhu ZQ, Guo Y, ChenZZ, Gong ZZ (2012) A plasma membrane receptor kinase,GHR1, mediates abscisic acid- and hydrogen peroxide-regulated stomatal movement in Arabidopsis. Plant Cell24: 2546–2561

Huang XY, Chao DY, Gao JP, Zhu MZ, Shi M, Lin HX (2009)A previously unknown zinc finger protein, DST, regulatesdrought and salt tolerance in rice via stomatal aperturecontrol. Genes Dev 23: 1805–1817

Idanheimo N, Gauthier A, Salojarvi J, Siligato R, Brosche M,Kollist H, Mahonen AP, Kangasjarvi J, Wrzaczek M (2014)The Arabidopsis thaliana cysteine-rich receptor-like kinasesCRK6 and CRK7 protect against apoplastic oxidativestress. Biochem Biophys Res Commun 445: 457–462

Imes D, Mumm P, Bohm J, Al-Rasheid KAS, Marten I, Geiger D,Hedrich R (2013) Open stomata 1 (OST1) kinase controlsR-type anion channel QUAC1 in Arabidopsis guard cells.Plant J 74: 372–382

Jacoby RP, Li L, Huang SB, Lee C, Millar AH, Taylor NL (2012)Mitochondrial composition, function and stress responsein plants. J Integr Plant Biol 54: 887–906

Jakobson L, Vaahtera L, Toldsepp K, Nuhkat M,Wang C, WangYS, Horak H, Valk E, Pechter P, Sindarovska Y, Tang J, XiaoCL, Xu Y, Talas UG, Garcia-Sosa AT, Kangasjarvi S, Maran U,Remm M, Roelfsema MRG, Hu HH, Kangasjarvi J, Loog M,Schroeder JI, Kollist H, Brosche M (2016) Natural variationin Arabidopsis Cvi-0 accession reveals an important role ofMPK12 in guard cell CO2 signaling. PLoS Biol 14: e2000322

Jammes F, Song C, Shin DJ, Munemasa S, Takeda K, Gu D,Cho D, Lee S, Giordo R, Sritubtim S, Leonhardt N, Ellis BE,Murata Y, Kwak JM (2009) MAP kinases MPK9 and MPK12are preferentially expressed in guard cells and positivelyregulate ROS-mediated ABA signaling. Proc Natl Acad SciUSA 106: 20520–20525

Kadota Y, Shirasu K, Zipfel C (2015) Regulation of the NADPHOxidase RBOHD during plant immunity. Plant Cell Physiol56: 1472–1480

Kadota Y, Sklenar J, Derbyshire P, Stransfeld L, Asai S,Ntoukakis V, Jones JDG, Shirasu K, Menke F, Jones A,Zipfel C (2014) Direct Regulation of the NADPH OxidaseRBOHD by the PRR-associated kinase BIK1 during plantimmunity. Mol Cell 54: 43–55

Kaliff M, Staal J, Myrenas M, Dixelius C (2007) ABA is requiredfor Leptosphaeria maculans resistance via ABI1- and ABI4-dependent signaling. Mol Plant Microbe Interact 20:335–345

Kim TH, Bohmer M, Hu HH, Nishimura N, Schroeder JI (2010)Guard cell signal transduction network: Advances inunderstanding abscisic acid, CO2, and Ca2þ signaling.Annu Rev Plant Biol 61: 561–591

Kocsy G, Tari I, Vankova R, Zechmann B, Gulyas Z, Poor P,Galiba G (2013) Redox control of plant growth anddevelopment. Plant Sci 211: 77–91

Kolla VA, Vavasseur A, Raghavendra AS (2007) Hydrogenperoxide production is an early event during bicarbonateinduced stomatal closure in abaxial epidermis of Arabi-dopsis. Planta 225: 1421–1429

Kong D, Hu HC, Okuma E, Lee Y, Lee HS, Munemasa S, Cho D,Ju C, Pedoeim L, Rodriguez B, Wang J, ImW, Murata Y, PeiZM, Kwak JM (2016) L-met activates Arabidopsis GLR Ca2þ

channels upstream of ROS production and regulatesstomatal movement. Cell Rep 17: 2553–2561

Kong L, Cheng J, Zhu Y, Ding Y, Meng J, Chen Z, Xie Q, Guo Y, LiJ, Yang S, Gong Z (2015) Degradation of the ABA co-receptor ABI1 by PUB12/13 U-box E3 ligases. Nat Commun6: 8630

Kwak JM, Mori IC, Pei ZM, Leonhardt N, Torres MA, Dangl JL,Bloom RE, Bodde S, Jones JDG, Schroeder JI (2003)NADPH oxidase AtrbohD and AtrbohF genes function inROS-dependent ABA signaling in Arabidopsis. EMBO J 22:2623–2633

Laloi C, Stachowiak M, Pers-Kamczyc E, Warzych E, Murgia I,Apel K (2007) Cross-talk between singlet oxygen- andhydrogen peroxide-dependent signaling of stress re-sponses in Arabidopsis thaliana. Proc Natl Acad Sci USA104: 672–677

Le Belle JE, Orozco NM, Paucar AA, Saxe JP, Mottahedeh J,Pyle AD, Wu H, Kornblum HI (2011) Proliferative neuralstem cells have high endogenous ROS levels that regulateself-renewal and neurogenesis in a PI3K/Akt-dependantmanner. Cell Stem Cell 8: 59–71

Lee BH, Lee HJ, Xiong LM, Zhu JK (2002) A mitochondrialcomplex I defect impairs cold-regulated nuclear geneexpression. Plant Cell 14: 1235–1251

Lee KP, Kim C, Landgraf F, Apel K (2007) EXECUTER1- andEXECUTER2-dependent transfer of stress-related signalsfrom the plastid to the nucleus of Arabidopsis thaliana.Proc Natl Acad Sci USA 104: 10270–10275

Li L, Li M, Yu LP, Zhou ZY, Liang XX, Liu ZX, Cai GH, Gao LY,Zhang XJ, Wang YC, Chen S, Zhou JM (2014) The FLS2-associated kinase BIK1 directly phosphorylates the NADPH

18 Qi et al.


oxidase RbohD to control plant immunity. Cell HostMicrobe 15: 329–338

Liang XX, Ding PT, Liang KH, Wang JL, Ma MM, Li L, Li L, Li M,Zhang XJ, Chen S, Zhang YL, Zhou JM (2016) Arabidopsisheterotrimeric G proteins regulate immunity by directlycoupling to the FLS2 receptor. eLife 5: e13568

Lind C, Dreyer I, Lopez-Sanjurjo EJ, von Meyer K, Ishizaki K,Kohchi T, Lang D, Zhao Y, Kreuzer I, Al-Rasheid KAS, RonneH, Reski R, Zhu JK, Geiger D, Hedrich R (2015) Stomatalguard cells co-opted an ancient ABA-dependent desicca-tion survival system to regulate stomatal closure. Curr Biol25: 928–935

Liu ST, Liu SW, Wang M, Wei TD, Meng C, Wang M, Xia GM(2014) A wheat SIMILAR TO RCD-ONE gene enhancesseedling growth and abiotic stress resistance by modulat-ing redox homeostasis and maintaining genomic integrity.Plant Cell 26: 164–180

Liu Y, He JN, Chen ZZ, Ren XZ, Hong XH, Gong ZZ (2010) ABAoverly-sensitive 5 (ABO5), encoding a pentatricopeptiderepeat protein required for cis-splicing of mitochondrialnad2 intron 3, is involved in the abscisic acid response inArabidopsis. Plant J 63: 749–765

Liu ZX,Wu Y, Yang F, Zhang YY, Chen S, Xie Q, Tian XJ, Zhou JM(2013) BIK1 interacts with PEPRs to mediate ethylene-induced immunity. Proc Natl Acad Sci USA 110: 6205–6210

Lu DP, Wu SJ, Gao XQ, Zhang YL, Shan LB, He P (2010)A receptor-like cytoplasmic kinase, BIK1, associates with aflagellin receptor complex to initiate plant innateimmunity. Proc Natl Acad Sci USA 107: 496–501

Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A,Grill E (2009) Regulators of PP2C phosphatase activityfunction as abscisic acid sensors. Science 324: 1064–1068

Medlyn BE, Barton CVM, Broadmeadow MSJ, Ceulemans R,De Angelis P, Forstreuter M, Freeman M, Jackson SB,Kellomaki S, Laitat E, Rey A, Roberntz P, Sigurdsson BD,Strassemeyer J, Wang K, Curtis PS, Jarvis PG (2001)Stomatal conductance of forest species after long-termexposure to elevated CO2 concentration: A synthesis.NewPhytol 149: 247–264

Meinhard M, Grill E (2001) Hydrogen peroxide is a regulator ofABI1, a protein phosphatase 2C from Arabidopsis. FEBSLett 508: 443–446

Meinhard M, Rodriguez PL, Grill E (2002) The sensitivity ofABI2 to hydrogen peroxide links the abscisic acid-responseregulator to redox signalling. Planta 214: 775–782

Melotto M, Underwood W, He SY (2008) Role of stomata inplant innate immunity and foliar bacterial diseases. AnnuRev Phytopathol 46: 101–122

Melotto M, UnderwoodW, Koczan J, Nomura K, He SY (2006)Plant stomata function in innate immunity againstbacterial invasion. Cell 126: 969–980

Meng XZ, Xu J, He YX, Yang KY, Mordorski B, Liu YD, Zhang SQ(2013) Phosphorylation of an ERF transcription factor byArabidopsis MPK3/MPK6 regulates plant defense geneinduction and fungal resistance. Plant Cell 25: 1126–1142

Meng XZ, Zhang SQ (2013) MAPK cascades in plant diseaseresistance signaling. Annu Rev Phytopathol 51: 245–266

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 receptorscontribute to ozone-, reduced air humidity-, darkness-,and CO2-induced stomatal regulation. Plant Physiol 162:1652–1668

Meskauskiene R, NaterM, Goslings D, Kessler F, den CampRO,Apel K (2001) FLU: A negative regulator of chlorophyllbiosynthesis in Arabidopsis thaliana. Proc Natl Acad SciUSA 98: 12826–12831

Meyer EH, Tomaz T, Carroll AJ, Estavillo G, Delannoy E,Tanz SK, Small ID, Pogson BJ, Millar AH (2009)Remodeled respiration in ndufs4 with low phosphoryla-tion efficiency suppresses Arabidopsis germination andgrowth and alters control of metabolism at night. PlantPhysiol 151: 603–619

Meyer S, MummP, Imes D, Endler A, Weder B, Al-Rasheid KAS,Geiger D, Marten I, Martinoia E, Hedrich R (2010)AtALMT12 represents an R-type anion channel requiredfor stomatal movement in Arabidopsis guard cells. Plant J63: 1054–1062

Miao YC, Song CP, Dong FC, XC W (2000) ABA-inducedhydrogen peroxide generation in guard cells of Vicia faba.Acta Phytophysiol Sin (In Chinese) 26: 53–58

Miao YC, Lv D, Wang PC, Wang XC, Chen J, Miao C, Song CP(2006) An Arabidopsis glutathione peroxidase functionsas both a redox transducer and a scavenger in abscisicacid and drought stress responses. Plant Cell 18:2749–2766

Miller G, Schlauch K, Tam R, Cortes D, Torres MA, Shulaev V,Dangl JL, Mittler R (2009) The plant NADPH oxidaseRBOHD mediates rapid systemic signaling in response todiverse stimuli. Sci Signal 2: ra45

Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R (2010) Reactiveoxygen species homeostasis and signalling during droughtand salinity stresses. Plant Cell Environ 33: 453–467

Mittler R (2017) ROS are good. Trends Plant Sci 22: 11–19

Mittler R, Vanderauwera S, GolleryM, Van BreusegemF (2004)Reactive oxygen gene network of plants. Trends Plant Sci9: 490–498

Mittler R, Vanderauwera S, Suzuki N, Miller G, Tognetti VB,Vandepoele K, Gollery M, Shulaev V, Van Breusegem F(2011) ROS signaling: The new wave? Trends Plant Sci 16:300–309

Moller IM (2001) PLANT MITOCHONDRIA AND OXIDATIVESTRESS: Electron transport, NADPH turnover, and metab-olism of reactive oxygen species. Annu Rev Plant PhysiolPlant Mol Biol 52: 561–591

Monaghan J, Matschi S, Shorinola O, Rovenich H, Matei A,Segonzac C, Malinovsky FG, Rathjen JP, MacLean D,Romeis T, Zipfel C (2014) The calcium-dependent proteinkinase CPK28 buffers plant immunity and regulates BIK1turnover. Cell Host Microbe 16: 605–615

Morimoto H, Iwata K, Ogonuki N, Inoue K, Atsuo O, Kanatsu-Shinohara M, Morimoto T, Yabe-Nishimura C, Shinohara T(2013) ROS are required for mouse spermatogonial stemcell self-renewal. Cell Stem Cell 12: 774–786



Mou ZL, He YK, Dai Y, Liu XF, Li JY (2000) Deficiency in fattyacid synthase leads to premature cell death and dramaticalterations in plant morphology. Plant Cell 12: 405–417

Mousavi SA, Chauvin A, Pascaud F, Kellenberger S, Farmer EE(2013) GLUTAMATE RECEPTOR-LIKE genes mediate leaf-to-leaf wound signalling. Nature 500: 422–426

Murata Y, Pei ZM, Mori IC, Schroeder J (2001) Abscisic acidactivation of plasma membrane Ca2þ channels in guardcells requires cytosolic NAD(P)H and is differentiallydisrupted upstream and downstream of reactive oxygenspecies production in abi1-1 and abi2-1 protein phospha-tase 2C mutants. Plant Cell 13: 2513–2523

Mustilli AC, Merlot S, Vavasseur A, Fenzi F, Giraudat J (2002)Arabidopsis OST1 protein kinase mediates the regulationof stomatal aperture by abscisic acid and acts upstreamof reactive oxygen species production. Plant Cell 14:3089–3099

Negi J, Matsuda O, Nagasawa T, Oba Y, Takahashi H, Kawai-Yamada M, Uchimiya H, Hashimoto M, Iba K (2008) CO2regulator SLAC1 and its homologues are essential for anionhomeostasis in plant cells. Nature 452: 483–486

Neill S, Desikan R, Hancock J (2002) Hydrogen peroxidesignalling. Curr Opin Plant Biol 5: 388–395

Nomura H, Komori T, Uemura S, Kanda Y, Shimotani K, NakaiK, Furuichi T, Takebayashi K, Sugimoto T, Sano S,Suwastika IN, Fukusaki E, Yoshioka H, Nakahira Y, ShiinaT (2012) Chloroplast-mediated activation of plant immunesignalling in Arabidopsis. Nat Commun 3: 926

O’Brien JA, Daudi A, Butt VS, Bolwell GP (2012) Reactiveoxygen species and their role in plant defence and cell wallmetabolism. Planta 236: 765–779

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, VolkmanBF, Cutler SR (2009) Abscisic acid inhibits type 2C proteinphosphatases via the PYR/PYL family of START proteins.Science 324: 1068–1071

Paul MK, Bisht B, Darmawan DO, Chiou R, Ha VL, Wallace WD,Chon AT, Hegab AE, Grogan T, Elashoff DA, Alva-OrnelasJA, Gomperts BN (2014) Dynamic changes in intracellularROS levels regulate airway basal stem cell homeostasisthrough Nrf2-dependent notch signaling. Cell Stem Cell 15:199–214

Pei ZM, Murata Y, Benning G, Thomine S, Klusener B, Allen GJ,Grill E, Schroeder JI (2000) Calcium channels activated byhydrogen peroxide mediate abscisic acid signalling inguard cells. Nature 406: 731–734

Petersen LN, Ingle RA, Knight MR, Denby KJ (2009) OXI1protein kinase is required for plant immunity againstPseudomonas syringae in Arabidopsis. J Exp Bot 60:3727–3735

Petrov V, Hille J, Mueller-Roeber B, Gechev TS (2015) ROS-mediated abiotic stress-induced programmed cell death inplants. Front Plant Sci 6: 69

Pornsiriwong W, Estavillo GM, Chan KX, Tee EE, Ganguly D,Crisp PA, Phua SY, Zhao C, Qiu J, Park J, Yong MT, Nisar N,

Yadav AK, Schwessinger B, Rathjen J, Cazzonelli CI, WilsonPB, Gilliham M, Chen ZH, Pogson BJ (2017) A chloroplastretrograde signal, 30-phosphoadenosine 50-phosphate,acts as a secondary messenger in abscisic acid signalingin stomatal closure and germination. eLife 6: pii: e23361

Qi J, Wang J, Gong Z, Zhou JM (2017) Apoplastic ROS signalingin plant immunity. Curr Opin Plant Biol 38: 92–100

Qiao WH, Fan LM (2008) Nitric oxide signaling in plantresponses to abiotic stresses. J Integr Plant Biol 50:1238–1246

Ramel F, Birtic S, Ginies C, Soubigou-Taconnat L, Triantaphy-lides C, Havaux M (2012) Carotenoid oxidation productsare stress signals that mediate gene responses to singletoxygen in plants. Proc Natl Acad Sci USA 109: 5535–5540

Ramel F, Ksas B, Akkari E,MialoundamaAS,Monnet F, Krieger-Liszkay A, Ravanat JL, Mueller MJ, Bouvier F, Havaux M(2013) Light-induced acclimation of the Arabidopsischlorina1 mutant to singlet oxygen. Plant Cell 25:1445–1462

Rentel MC, Lecourieux D, Ouaked F, Usher SL, Petersen L,Okamoto H, Knight H, Peck SC, Grierson CS, Hirt H,Knight MR (2004) OXI1 kinase is necessary for oxidativeburst-mediated signalling in Arabidopsis. Nature 427:858–861

Rhoads DM, Umbach AL, Subbaiah CC, Siedow JN (2006)Mitochondrial reactive oxygen species. Contribution tooxidative stress and interorganellar signaling. PlantPhysiol 141: 357–366

Rodrigues O, Reshetnyak G, Grondin A, Saijo Y, Leonhardt N,Maurel C, Verdoucq L (2017) Aquaporins facilitate hydro-gen peroxide entry into guard cells to mediate ABA- andpathogen-triggered stomatal closure. Proc Natl Acad SciUSA 114: 9200–9205

Rodriguez-Herva JJ, Gonzalez-Melendi P, Cuartas-Lanza R,Antunez-Lamas M, Rio-Alvarez I, Li Z, Lopez-Torrejon G,Diaz I, del Pozo JC, Chakravarthy S, Collmer A, Rodriguez-Palenzuela P, Lopez-Solanilla E (2012) A bacterial cysteineprotease effector protein interferes with photosynthesisto suppress plant innate immune responses. Cell Micro-biol 14: 669–681

Rosenwasser S, Rot I, Sollner E, Meyer AJ, Smith Y, Leviatan N,Fluhr R, Friedman H (2011) Organelles contribute differen-tially to reactive oxygen species-related events duringextended darkness. Plant Physiol 156: 185–201

Salvador-Recatal�a V (2016) New roles for the GLUTAMATERECEPTOR-LIKE 3.3, 3.5, and 3.6 genes as on/off switchesof wound-induced systemic electrical signals. Plant SignalBehav 11: e1161879

Sandalio LM, Romero-Puertas MC (2015) Peroxisomes senseand respond to environmental cues by regulating ROS andRNS signalling networks. Ann Bot 116: 475–485

Sang YM, Cui DC, Wang XM (2001a) Phospholipase D andphosphatidic acid-mediated generation of superoxide inArabidopsis. Plant Physiol 126: 1449–1458

Sang YM, Zheng SQ, Li WQ, Huang BR, Wang XM (2001b)Regulation of plant water loss by manipulating theexpression of phospholipase D alpha. Plant J 28: 135–144

20 Qi et al.


Sato A, Sato Y, Fukao Y, Fujiwara M, Umezawa T, Shinozaki K,Hibi T, Taniguchi M, Miyake H, Goto DB, Uozumi N (2009)Threonine at position 306 of the KAT1 potassium channel isessential for channel activity and is a target site for ABA-activated SnRK2/OST1/SnRK2.6 protein kinase. Biochem J424: 439–448

Schroeder JI, Kwak JM, Allen GJ (2001) Guard cell abscisic acidsignalling and engineering drought hardiness in plants.Nature 410: 327–330

SewelamN, Jaspert N, Van der Kelen K, Tognetti VB, Schmitz J,Frerigmann H, Stahl E, Zeier J, Van Breusegem F,Maurino VG (2014) Spatial H2O2 signaling specificity:H2O2 from chloroplasts and peroxisomes modulates theplant transcriptome differentially. Mol Plant 7: 1191–1210

Shang Y, Dai C, Lee MM, Kwak JM, Nam KH (2016) BRI1-associated receptor kinase 1 regulates guard cell ABAsignaling mediated by open stomata 1 in Arabidopsis.MolPlant 9: 447–460

Shapiguzov A, Vainonen JP, Wrzaczek M, Kangasjarvi J (2012)ROS-talk - how the apoplast, the chloroplast, and thenucleus get the message through. Front Plant Sci 3: 292

ShiH, ShenQJ,Qi YP, YanHJ,NieHZ, ChenYF, ZhaoT,Katagiri F,Tang DZ (2013) BR-SIGNALING KINASE1 physically asso-ciateswith FLAGELLINSENSING2and regulatesplant innateimmunity in Arabidopsis. Plant Cell 25: 1143–1157

Shi K, Li X, Zhang H, Zhang GQ, Liu YR, Zhou YH, Xia XJ, ChenZX, Yu JQ (2015) Guard cell hydrogen peroxide and nitricoxide mediate elevated CO2-induced stomatal movementin tomato. New Phytol 208: 342–353

Shiu SH, Bleecker AB (2001) Plant receptor-like kinase genefamily: Diversity, function, and signaling. Sci STKE 2001:re22

Shumbe L, Chevalier A, Legeret B, Taconnat L, Monnet F,Havaux M (2016) Singlet oxygen-induced cell death inArabidopsis under high-light stress is controlled by OXI1kinase. Plant Physiol 170: 1757–1771

Sierla M, Waszczak C, Vahisalu T, Kangasjarvi J (2016) Reactiveoxygen species in the regulation of stomatal movements.Plant Physiol 171: 1569–1580

Singh R, Singh S, Parihar P, Mishra RK, Tripathi DK, Singh VP,Chauhan DK, Prasad SM (2016) Reactive oxygen species(ROS): Beneficial companions of plants’ developmentalprocesses. Front Plant Sci 7: 1–19

Sirichandra C, Gu D, Hu HC, Davanture M, Lee S, Djaoui M,Valot B, Zivy M, Leung J, Merlot S, Kwak JM (2009)Phosphorylation of the Arabidopsis AtrbohF NADPHoxidase by OST1 protein kinase. FEBS Lett 583: 2982–2986

Sussmilch FC, Brodribb TJ, McAdam SAM (2017) What are theevolutionary origins of stomatal responses to abscisic acidin land plants? J Integr Plant Biol 59: 240–260

Suzuki N, Koussevitzky S, Mittler R, Miller G (2012) ROS andredox signalling in the response of plants to abiotic stress.Plant Cell Environ 35: 259–270

Suzuki N, Miller G, Morales J, Shulaev V, Torres MA, Mittler R(2011) Respiratory burst oxidases: The engines of ROSsignaling. Curr Opin Plant Biol 14: 691–699

Suzuki N, Miller G, Salazar C, Mondal HA, Shulaev E, CortesDF, Shuman JL, Luo XZ, Shah J, Schlauch K, Shulaev V,Mittler R (2013) Temporal-spatial interaction betweenreactive oxygen species and abscisic acid regulatesrapid systemic acclimation in plants. Plant Cell 25:3553–3569

Tang WQ, Kim TW, Oses-Prieto JA, Sun Y, Deng ZP, Zhu SW,Wang RJ, Burlingame AL, Wang ZY (2008) BSKs mediatesignal transduction from the receptor kinase BRI1 inArabidopsis. Science 321: 557–560

Tian S, Wang XB, Li P, Wang H, Ji HT, Xie JY, Qiu QL, Shen D,Dong HS (2016) Plant aquaporin AtPIP1;4 links apoplasticH2O2 induction to disease immunity pathways. PlantPhysiol 171: 1635–1650

Ton J, Flors V, Mauch-Mani B (2009) The multifaceted role ofABA in disease resistance. Trends Plant Sci 14: 310–317

TorresMA, Dangl JL, Jones JDG (2002)Arabidopsis gp91(phox)homologues AtrbohD and AtrbohF are required foraccumulation of reactive oxygen intermediates in theplant defense response. Proc Natl Acad Sci USA 99:517–522

Triantaphylides C, Havaux M (2009) Singlet oxygen in plants:Production, detoxification and signaling. Trends Plant Sci14: 219–228

Tsukagoshi H, Busch W, Benfey PN (2010) Transcriptionalregulation of ROS controls transition from proliferation todifferentiation in the root. Cell 143: 606–616

Umezawa T, Nakashima K, Miyakawa T, Kuromori T, TanokuraM, Shinozaki K, Yamaguchi-Shinozaki K (2010) Molecularbasis of the core regulatory network in ABA responses:Sensing, signaling and transport. Plant Cell Physiol 51:1821–1839

Vahisalu T, Kollist H, Wang YF, Nishimura N, Chan WY, ValerioG, Lamminmaki A, Brosche M, Moldau H, Desikan R,Schroeder JI, Kangasjarvi J (2008) SLAC1 is required forplant guard cell S-type anion channel function in stomatalsignalling. Nature 452: 487–U415

Vogel MO, Moore M, Konig K, Pecher P, Alsharafa K, Lee J,Dietz KJ (2014) Fast retrograde signaling in response tohigh light involves metabolite export, MITOGEN-ACTI-VATED PROTEIN KINASE6, and AP2/ERF transcriptionfactors in Arabidopsis. Plant Cell 26: 1151–1165

Wagner D, Przybyla D, Camp ROD, Kim C, Landgraf F, Lee KP,Wursch M, Laloi C, Nater M, Hideg E, Apel K (2004) Thegenetic basis of singlet oxygen-induced stress responsesof Arabidopsis thaliana. Science 306: 1183–1185

Wang C, Hu HH, Qin X, Zeise B, Xu DY, Rappel WJ, Boron WF,Schroeder JI (2016) Reconstitution of CO2 regulation ofSLAC1 anion channel and function of CO2-permeable PIP2;1aquaporin as CARBONIC ANHYDRASE4 interactor. PlantCell 28: 568–582

Wang PC, Du YY, Li YA, Ren DT, Song CP (2010) Hydrogenperoxide-mediated activation of MAP kinase 6 modulatesnitric oxide biosynthesis and signal transduction inArabidopsis. Plant Cell 22: 2981–2998

Wang PC, Du YY, Zhao XL, Miao YC, Song CP (2013a) The MPK6-ERF6-ROS-responsive cis-acting element7/GCC box complex



modulates oxidative gene transcription and the oxidativeresponse in Arabidopsis. Plant Physiol 161: 1392–1408

Wang PC, Dua YY, Hou YJ, Zhao Y, Hsu CC, Yuan FJ, Zhu XH,Tao WA, Song CP, Zhu JK (2015a) Nitric oxide negativelyregulates abscisic acid signaling in guard cells byS-nitrosylation of OST1. Proc Natl Acad Sci USA 112:613–618

Wang PC, Xue L, Batelli G, Lee S, Hou YJ, VanOostenMJ, ZhangHM, Tao WA, Zhu JK (2013b) Quantitative phosphopro-teomics identifies SnRK2 protein kinase substrates andreveals the effectors of abscisic acid action. ProcNatl AcadSci USA 110: 11205–11210

Wang ZP, Xing HL, Dong L, Zhang HY, Han CY, Wang XC, ChenQJ (2015b) Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants formultiple target genes in Arabidopsis in a single generation.Genome Biol 16: 144

Webb AAR, Hetherington AM (1997) Convergence of theabscisic acid, CO2, and extracellular calcium signaltransduction pathways in stomatal guard cells. PlantPhysiol 114: 1557–1560

Wrzaczek M, Brosche M, Kangasjarvi J (2013) ROS signalingloops - production, perception, regulation. Curr Opin PlantBiol 16: 575–582

Wu J, Sun YF, Zhao YN, Zhang J, Luo LL, Li M, Wang JL, Yu H,Liu GF, Yang LS, Xiong GS, Zhou JM, Zuo JR,Wang YH, Li JY(2015) Deficient plastidic fatty acid synthesis triggers celldeath by modulating mitochondrial reactive oxygenspecies. Cell Res 25: 621–633

Wu L, Chen H, Curtis C, Fu ZQ (2014) Go in for the kill howplants deploy effector-triggered immunity to combatpathogens. Virulence 5: 710–721

Xin XF, Nomura K, Aung K, Velasquez AC, Yao J, Boutrot F,Chang JH, Zipfel C, He SY (2016) Bacteria establish anaqueous living space in plants crucial for virulence. Nature539: 524–529

Xu ZZ, Jiang YL, Zhou GS (2015) Response and adaptation ofphotosynthesis, respiration, and antioxidant systems toelevated CO2 with environmental stress in plants. FrontPlant Sci 6: 701

Xue SW, Hu HH, Ries A, Merilo E, Kollist H, Schroeder JI (2011)Central functions of bicarbonate in S-type anion channelactivation and OST1 protein kinase in CO2 signal transduc-tion in guard cell. EMBO J 30: 1645–1658

Yamamoto Y, Negi J, Wang C, Isogai Y, Schroeder JI, Iba K(2016) The transmembrane region of guard cell SLAC1channels perceives CO2 signals via an ABA-independentpathway in Arabidopsis. Plant Cell 28: 557–567

Yang HJ, Mu JY, Chen LC, Feng J, Hu JL, Li L, Zhou JM, Zuo JR(2015) S-nitrosylation positively regulates ascorbate per-oxidase activity during plant stress responses. PlantPhysiol 167: 1604–U1753

Yang L, Zhang J, He JN, Qin YY, Hua DP, Duan Y, Chen ZZ, GongZZ (2014) ABA-mediated ROS in mitochondria regulateroot meristem activity by controlling PLETHORA expres-sion in Arabidopsis. PLoS Genet 10: e1004791

Yoshida R, Umezawa T, Mizoguchi T, Takahashi S, Takahashi F,Shinozaki K (2006) The regulatory domain of SRK2E/OST1/SnRK2.6 interacts with ABI1 and integrates abscisic acid(ABA) and osmotic stress signals controlling stomatalclosure in Arabidopsis. J Biol Chem 281: 5310–5318

Young JJ, Mehta S, Israelsson M, Godoski J, Grill E,Schroeder JI (2006) CO2 signaling in guard cells: Calciumsensitivity response modulation, a Ca2þ-independentphase, and CO2 insensitivity of the gca2 mutant. ProcNatl Acad Sci USA 103: 7506–7511

Yu QQ, Tian HY, Yue K, Liu JJ, Zhang B, Li XG, Ding ZJ (2016)A P-loop NTPase regulates quiescent center cell divisionand distal stem cell identity through the regulation of ROShomeostasis in Arabidopsis root. PLoS Genet 12: e1006175

Yuan F, Yang HM, Xue Y, Kong DD, Ye R, Li CJ, Zhang JY,Theprungsirikul L, Shrift T, Krichilsky B, Johnson DM,Swift GB, He YK, Siedow JN, Pei ZM (2014) OSCA1mediatesosmotic-stress-evoked Ca2þ increases vital for osmosens-ing in Arabidopsis. Nature 514: 367

Yun BW, Feechan A, Yin MH, Saidi NBB, Le Bihan T, Yu M,Moore JW, Kang JG, Kwon E, Spoel SH, Pallas JA, Loake GJ(2011) S-nitrosylation of NADPH oxidase regulates celldeath in plant immunity. Nature 478: 264–268

Zabala MDT, Littlejohn G, Jayaraman S, Studholme D, Bailey T,Lawson T, Tillich M, Licht D, Bolter B, Delfino L, TrumanW,Mansfield J, Smirnoff N, GrantM (2015) Chloroplasts play acentral role in plant defence and are targeted by pathogeneffectors. Nat Plants 1: 15074

Zhang J, Li W, Xiang TT, Liu ZX, Laluk K, Ding XJ, Zou Y, Gao MH,Zhang XJ, Chen S, Mengiste T, Zhang YL, Zhou JM (2010)Receptor-like cytoplasmic kinases integrate signaling frommultiple plant immune receptors and are targeted by aPseudomonas syringaeeffector.CellHostMicrobe 7: 290–301

Zhang W, Jeon BW, Assmann SM (2011) HeterotrimericG-protein regulation of ROS signalling and calciumcurrents in Arabidopsis guard cells. J Exp Bot 62: 2371–2379

Zhang WH, Qin CB, Zhao J, Wang XM (2004) Phospholipase Dalpha 1-derived phosphatidic acid interacts with ABI1phosphatase 2C and regulates abscisic acid signaling. ProcNatl Acad Sci USA 101: 9508–9513

Zhang YY, Zhu HY, Zhang Q, Li MY, Yan M, Wang R, Wang LL,Welti R, Zhang WH, Wang XM (2009) Phospholipase Dalpha 1 and phosphatidic acid regulate NADPH oxidaseactivity and production of reactive oxygen species in ABA-mediated stomatal closure in Arabidopsis. Plant Cell 21:2357–2377

Zhao J, Wang XM (2004) Arabidopsis phospholipase D alpha 1interacts with the heterotrimeric G-protein alpha-subunitthrough a motif analogous to the DRY motif in G-protein-coupled receptors. J Biol Chem 279: 1794–1800

Zhu JK (2016) Abiotic stress signaling and responses in plants.Cell 167: 313–324

Zou JJ, Li XD, Ratnasekera D, Wang C, Liu WX, Song LF, ZhangWZ, Wu WH (2015) Arabidopsis CALCIUM-DEPENDENTPROTEIN KINASE8 and CATALASE3 function in abscisicacid-mediated signaling and H2O2 homeostasis in stomatalguard cells under drought stress. Plant Cell 27: 1445–1460

22 Qi et al.


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