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Invited Expert Review

ROS signaling and stomatal movement in plant responses to drought stress and

pathogen attack

Running title: ROS signaling and stomatal movement

Junsheng Qi1, Chun-Peng Song2, Baoshan Wang3, Jianmin Zhou4, Jaakko Kangasjärvi

5, Jian-Kang Zhu 6, 7 and Zhizhong Gong1*

1State Key Laboratory of Plant Physiology and Biochemistry, College of Biological

Sciences, China Agricultural University, Beijing 100193, China

2Collaborative Innovation Center of Crop Stress Biology, Henan Province, Institute of

Plant Stress Biology, Henan University, Kaifeng 475001, China

3Key Lab of Plant Stress Research, College of Life Science, Shandong Normal

University, Ji'nan, China

4State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of

Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101,

China

5Division of Plant Biology, Viikki Plant Science Centre, Department of Biosciences,

University of Helsinki, 00014 Helsinki, Finland

6Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological

Sciences, Chinese Academy of Sciences, Shanghai 200032, China

7Department of Horticulture and Landscape Architecture, Purdue University, West

Lafayette, IN 47907, USA

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1111/jipb.12654]

This article is protected by copyright. All rights reserved. Received: February 20, 2018; Accepted: April 8, 2018

 

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*Correspondence: Zhizhong Gong (gongzz@cau.edu.cn)

Special Issue: Cell Signaling

Edited by: Jia Li, Lanzhou University, China

Abstract:

Stomata, the pores formed by a pair of guard cells, are the main gateways for water

transpiration and photosynthetic CO2 exchange, as well as pathogen invasion in land

plants. Guard cell movement is regulated by a combination of environmental factors

including water status, light, CO2 levels and pathogen attack, as well as endogenous

signals such as abscisic acid and apoplastic reactive oxygen species (ROS). Under

abiotic and biotic stress conditions, extracellular ROS are mainly produced by plasma

membrane-localized NADPH oxidases, whereas intracellular ROS are produced in

multiple organelles. These ROS form a sophisticated cellular signaling network, with

the accumulation of apoplastic ROS an early hallmark of stomatal movement. Here,

we review recent progress in understanding the molecular mechanisms of the ROS

signaling network, primarily during drought stress and pathogen attack. We

summarize the roles of apoplastic ROS in regulating stomatal movement, ABA and

CO2 signaling, and immunity responses. Finally, we discuss ROS accumulation and

communication between organelles and cells. This information provides a conceptual

framework for understanding how ROS signaling is integrated with various signaling

pathways during plant responses to abiotic and biotic stress stimuli.

 

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INTRODUCTION

Plants face fluctuating abiotic and biotic hazards throughout their sessile lives.

Drought stress and pathogen infection are two of the most important factors that cause

extensive loss to the agricultural productivity. Increasing global warming exacerbates

the frequency of extreme weather, which further threatens the agricultural

productivity and world food security. Understanding the molecular mechanisms of

plant response and adaptation to adverse environmental stimuli is urgently required

for improving crop stress resistance and increasing yields through genetic and

molecular designing breeding. Accordingly, plants have evolved sophisticated

surveillance systems to perceive and cope with these challenges (Baxter et al. 2014;

Camejo et al. 2016).

The emergence of guard cells is a major landmark in the evolution of land plants

and their adaptation to dry 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 up CO2 for photosynthesis while simultaneously regulating water

transpiration. Controlling water transpiration through stomata is one of the main

strategies for plants to increase drought tolerance (Schroeder et al. 2001). The opening

and closing of stomatal aperture (i.e., stomatal movements) are regulated by turgor

pressure generated by ion and water channel proteins on the plasma membranes of

guard cells (Schroeder et al. 2001; Kim et al. 2010; Engineer et al. 2016). These

movements are tightly regulated by environmental stimuli such as water status, light

 

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and CO2, as well as endogenous factors such as abscisic acid (ABA), Ca2+ and

reactive oxygen species (ROS) levels under various conditions (Schroeder et al. 2001;

Young et al. 2006). Notably, the respiratory burst oxidase homologs (RBOHs)

NADPH oxidases and the core ABA signaling components emerged at almost the

same time as guard cells in mosses during land plant evolution; these components are

not found in algae (Umezawa et al. 2010; Mittler et al. 2011; Lind et al. 2015;

Sussmilch et al. 2017). This pattern of emergence suggests that apoplastic ROS, guard

cells and ABA signaling mechanism are the co-evolutionary results of the adaptation

of land plants to dry environmental conditions (Umezawa et al. 2010; Mittler et al.

2011; Lind et al. 2015; Sussmilch et al. 2017). However, the evolution of stomata has

also opened the door for pathogen invasion (Schroeder et al. 2001; Melotto et al.

2006).

When facing different biotic and abiotic stresses, plants quickly accumulate the

common ROS as the first layer for defense (Kocsy et al. 2013; Wrzaczek et al. 2013;

Baxter et al. 2014). The term “ROS” generally refers to incompletely reduced oxygen

species, including the well-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 short

half-life. Due to their inherent features, all types of ROS can damage macromolecules

such as proteins, lipids and nucleic acids, eventually leading to cell death (Petrov et al.

2015). On the other hand, ROS act as signaling molecules that modify protein

properties through the formation of covalent bonds, and they also function as major

 

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inducers of programmed cell death (PCD) (Neill et al. 2002; Suzuki et al. 2012;

Baxter et al. 2014; Choudhury et al. 2017; Mittler 2017). H2O2, which has a relatively

long half-life (~1 ms in cells) and is more stable than other ROS, often acts as a

transducing signal in both cell-to-cell communication and intracellular signaling to

trigger downstream responses (Wrzaczek et 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 derivatives including NO2, N2O3, peroxynitrite (ONOO−),

S-nitrosothiols and S-nitrosoglutathione (GSNO), are harmful to cells, but like ROS,

they can also serve as key regulatory signals during various cellular responses under

stress conditions (Qiao and Fan 2008; Kocsy et al. 2013; Hu et al. 2015). Both NO

and GSNO modify their target proteins through the S-nitrosylation of reactive

cysteines. The modification of proteins by either ROS or RNS can alter their activity,

stability, subcellular location or interactions with other molecules (Qiao and Fan 2008;

Kocsy et al. 2013).

As by-products of both abiotic and biotic stress, intracellular ROS are produced

in organelles such as chloroplasts, peroxisomes/glyoxysomes and mitochondria,

whereas apoplastic ROS are produced by plasma membrane-localized NADPH

oxidases, cell wall peroxidases and amine oxidases (Kadota et al. 2014; Kadota et 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 (Miller et al. 2010; Suzuki et al. 2012; Baxter et

al. 2014; Choudhury et al. 2017). Major ROS-scavenging enzymes including

 

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ascorbate peroxidases (APX), catalases (CAT), thylakoidal ascorbate peroxidase

(tAPX), copper-zinc superoxide dismutases (SOD) and glutathione peroxidases (GPX)

provide a highly efficient system for maintaining ROS homeostasis in various sites of

a cell under normal or stress conditions (Mittler et al. 2004). Alternative oxidases

(AOXs) are activated in response to ROS formation in mitochondrial electron

transport chain (ETC) Complex III, where they indirectly prevent excess ROS

formation (Jacoby et al. 2012). Low molecular weight antioxidants such as ascorbic

acid and glutathione, which are present in almost all organelles, are involved in ROS

quenching, primarily by providing reducing equivalents to ROS scavenging enzymes.

This process can keep ROS concentrations low, even when they are produced at a

high rate, and these enzymes can also react directly with ROS (Miller et al. 2010;

Kocsy et al. 2013; Singh et al. 2016). Plants have evolved various mechanisms to

perceive, transduce and respond to ROS signals to protect themselves from pathogen

attack and from damage caused by abiotic stresses such as drought, salinity and high

and low temperatures (Miller et al. 2010; Suzuki et al. 2012; Baxter et al. 2014;

Choudhury et al. 2017).

Plant cells have developed fine-tuned regulatory mechanisms to orchestrate

stomatal movement under drought stress and pathogen attack (Schroeder et al. 2001;

Kim et al. 2010; Arnaud and Hwang 2015), and in response CO2 levels (Young et al.

2006; Engineer et al. 2016). Both drought stress and pathogen attack can trigger 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).

 

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These ROS signaling pathways form a complicated network that functions in plant

responses to abiotic and biotic stress (Wrzaczek et al. 2013; Zhu 2016; Qi et al. 2017).

In this review, we will summarize recent progress in our understanding of ROS

signaling and discuss the molecular mechanisms underlying apoplastic

H2O2-mediated stomatal movement and the ROS signaling network, which function

primarily during plant responses to drought stress and pathogen attack.

APOPLASTIC H2O2 MEDIATES STOMATAL MOVEMENT DURING ABA

SIGNALING

The phytohormone ABA is synthesized in different tissues and plays various roles in

seed maturation, seed dormancy, seed germination, seedling growth and development

and the regulation of gene expression and stomatal movement in response to abiotic

stress (Finkelstein et al. 2002; Zhu 2016). Drought stress promotes the production of

ABA, which is perceived by the PYR1/PYL/RCAR family of ABA receptors (Figure

1). ABA-bound PYLs interact with clade A type 2C phosphatases (PP2Cs), which are

core negative regulators of ABA signaling, releasing their inhibition of downstream

targets and thus activating protein kinases including SnRK2.2/2.3/2.6 (OPEN

STOMATA1, OST1) and GUARD CELL HYDROGEN PEROXIDE-RESISTANT1

(GHR1) (Ma et al. 2009; Park et al. 2009; Hua et al. 2012). The activated protein

kinases phosphorylate and activate their targets via their own activity or by interacting

with downstream targets such as SLOW ANION CHANNEL-ASSOCIATED1

(SLAC1), SLAH3 (SLAC1 HOMOLOGUE3) and ALUMINUM-ACTIVATED

MALATE TRANSPORTER 12/QUICKLY ACTIVATED ANION CHANNEL1

 

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(ALMT12/QUAC1). At the same time, these enzymes inhibit the inward rectifier

potassium channel KAT1, 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).

ROS production is induced by drought stress and ABA signaling (Sierla et al.

2016). ABA-induced H2O2 accumulation was first reported in Vicia faba and

Arabidopsis thaliana guard cells (Miao YC et al. 2000; Pei et al. 2000). The

apoplastic ROS produced by plasma membrane-localized NADPH oxidases have

been well studied (Kwak et al. 2003; Miller et al. 2009; Kadota et al. 2014; Li et al.

2014; Kadota et al. 2015). The Arabidopsis genome encodes 10 NADPH oxidases

belonging to the RBOH family (Kwak et al. 2003). NADPH oxidases have NADPH

or NADH binding sites (Suzuki et al. 2011; Kadota et al. 2015). These binding sites

transfer electrons from cytosolic NADPH or NADH to apoplastic oxygen to catalyze

the production of O2∙−, which can be converted to H2O2, either spontaneously or via

the activity of superoxide dismutases (SODs). RBOHD and RBOHF are both

responsible for ABA-mediated ROS production in guard cells (Kwak et al. 2003).

NADPH oxidase activity is tightly regulated by protein phosphorylation.

ABA-activated OST1 can directly phosphorylate serine (Ser) 13 and Ser174 at the

N-terminus of RBOHF in vitro (Sirichandra et al. 2009). OST1 interacts with BAK1

(BRI1-associated kinase 1), both of which function upstream of ROS production in

guard cells, and are inhibited by ABI1 (Shang et al. 2016). BAK1 usually forms a

heterodimer with other receptor-like protein kinases. However, The BAK1’s patterner

 

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and the molecular mechanism for how BAK1 regulates ROS production and stomatal

movement are not known yet (Shang et al. 2016). Besides OST1, the calcineurin

B-like (CBL)-interacting protein kinase CIPK26 interacts with and phosphorylates the

cytoplasmic N-terminus of RBOHF, but the phosphorylation sites are currently

unknown (Drerup et al. 2013). Co-expression of CIPK26 with CBL1 or CBL9

enhances the Ca2+-dependent activity of RBOHF in HEK293T cells (Drerup et al.

2013). However, whether these phosphorylation events have any effect on RBOHF

activity remains unknown.

NADPH oxidases are regulated by other factors besides protein phosphorylation.

Numerous stresses cause phospholipase Dα1 (PLDα1) to hydrolyze membrane

phospholipids into phosphatidic acid (PA) (Zhang et al. 2009). The depletion of

PLDα1 reduces ABA-mediated ROS production in guard cells as well as stomatal

closure, and ROS production can be recovered by the addition of PA in pldα1 mutants,

suggesting that PA is crucial for ROS production (Sang et al. 2001a, 2001b; Zhang et

al. 2009). The loss of the PA binding motif in RBOHD compromises ABA-mediated

ROS production (Zhang et al. 2009). PA also binds to ABI1 (ABA INSENSITIVE 1)

and inhibits its phosphatase activity, thereby increasing ABA-promoted stomatal

closure (Zhang et al. 2004). Heterotrimeric G proteins have Gα, Gβ and Gγ subunits.

PLDα1 interacts with the Gα subunit GPA1 (Zhao and Wang 2004). GPA1 positively

regulates ABA-induced ROS production (Zhang et al. 2011). In gpa1 mutants, both

ABA-induced ROS production and Ca2+-channel activation are impaired (Zhang et al.

2011). However, exogenously applied H2O2 rescues the defects in stomatal response

 

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to ABA and Ca2+-channel activation in gpa1, indicating that GPA1 functions

somewhere between ABA perception and ROS production (Zhang et al. 2011). These

findings indicate that PLDα1, its product PA and GPA1 are all required for ABA

signaling and ROS production.

Genetically speaking, during ABA-mediated stomatal regulation, ABI1 acts

upstream of ROS production, while ABI2 (another clade A PP2C) acts downstream

(Murata et al. 2001). H2O2 accumulation is not altered in the ghr1 or abi2-1 mutants,

but it is greatly reduced in the ost1 and abi1-1 mutants under ABA treatment (Murata

et al. 2001; Mustilli et al. 2002; Hua et al. 2012). Furthermore, ost1 and abi1-1

respond normally to H2O2-promoted stomatal closure, 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 enzymes only after interacting with ABA receptors in the presence

or absence of ABA. The pub12 pub13 double mutant accumulates more ABI1 protein

and less H2O2 in its guard cells than wild type (Kong et al. 2015), which is consistent

with the role of ABI1 in negatively regulating ROS production. ABI1 physically

interacts with, dephosphorylates and directly inhibits SLAC1 activity in Xenopus

oocytes, but its in planta function is still unresolved (Brandt et al. 2015). Unlike

pathogen-associated molecular pattern (PAMP)-triggered ROS production, in which

calcium signaling functions upstream of ROS during this process, ABA signaling

triggers ROS production. Upon ABA signaling, ROS, in turn, induce increased

cytosol Ca2+ levels, likely via GHR1, as no Ca2+ channel activity was detected in the

 

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ghr1 mutant (Hua et al. 2012). Notably, flg22-induced stomatal closure is also

impaired in the ghr1 mutant, suggesting that PAMP-triggered ROS regulate stomatal

closure through the activity of GHR1 (Hua et al. 2012).

NO is another signaling molecule involved in stomatal movement (Qiao and Fan

2008; Asgher et al. 2017). In guard cells, ABA and H2O2 induce NO production,

which requires nitrate reductase (NR). Indeed, the NR double mutant nia1 nia2 fails

to produce NO in guard cells under exogenous ABA or H2O2 treatment (Bright et al.

2006). ABA-induced NO production is also greatly reduced in the atrbohD/F double

mutant (Bright et al. 2006) and in the guard cells of pldα1 (Zhang et al. 2009). These

findings indicate that ABA-induced apoplastic H2O2 production is required for NO

biosynthesis. ABA-induced NO can modify the reactive cysteine thiol in a protein to

form S-nitrosothiol. Protein S-nitrosylation is important for protein activity and

stability (Hu et al. 2015). OST1 activity is abolished by S-nitrosylation at cysteine

(Cys) 137, which is adjacent to the kinase catalytic site (Wang et al. 2015a),

suggesting that NO regulates ABA signaling via a feedback circuit.

As mentioned above, the production of extracellular ROS by NADPH oxidases

represents an evolutionary achievement for land plants (Mittler et al. 2011). More

importantly, apoplastic ROS may act as signaling molecules to transduce extracellular

signals into cells, which protect the inner components of the cells from ROS damage.

The plasma membrane-localized LRR receptor-like protein kinase GHR1 is a key

component in apoplastic ROS signal transduction (Hua et al. 2012). GHR1 directly

interacts with and activates the slow-type anion channel SLAC1 (Hua et al. 2012). It

 

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is likely that OST1 phosphorylates and activates RBOHF to produce apoplastic ROS,

a process inhibited by ABI1 (Yoshida et al. 2006). Meanwhile GHR1 transduces

apoplastic ROS signals into guard cells via an unknown mechanism that is

specifically inhibited by ABI2 (Hua et al. 2012). The N-terminal extracellular domain

of GHR1 contains three Cys residues, including a Cys pair (Cys-57, Cys-66) that is

well conserved in most RLKs and is required for their normal functioning (Hua et al.

2012). However, the other Cys residue, Cys-381, does not function in ROS sensing, as

its mutation to Ala does not affect the role of GHR1 (Hua et al. 2012). Thus, whether

GHR1 or other plasma membrane receptor-like protein kinases can perceive

apoplastic ROS and transduce them inside the cell remains unknown. Perhaps GHR1

interacts with oxidized, ROS induced peptide ligands or another RLK that can be

induced or oxidized by ROS to transduce apoplastic ROS signals. Recent studies

suggest that members of the large family of cysteine-rich receptor like kinases (CRKs)

are important candidates that may achieve extracellular ROS signal transduction to

downstream targets (Idanheimo et al. 2014; Bourdais et al. 2015). Some CRKs play

general and/or specific roles in pathogen- and biotic stress-induced stomatal closure

(Idanheimo et al. 2014; Bourdais et al. 2015). Given the high similarity among these

CRKs and the large family size, additional studies examining their roles in H2O2

signal transduction are required using the newly developed CRISPR/Cas9 technique

to create multiple mutants (Wang et al. 2015b).

As we discussed above, apoplastic ROS might be sensed by some plasma

membrane proteins to transduce signals into cells. Given that RBOHD is a membrane

 

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protein, it undergoes endocytosis, which is cooperatively regulated by clathrin- and

microdomain-dependent endocytic pathways (Hao et al. 2014). ABA and flg22

treatments increase monomer-dimer transitions and the diffusion efficiency of

RBOHD (Hao et al. 2014). This process allows ROS to be generated within

endosomes for signal transduction (Hao et al. 2014). On the other hand, apoplastic

H2O2 can be transported into the cytosol by aquaporin proteins to mediate stomatal

movement. ABA-activated OST1 phosphorylates Ser121 of the aquaporin protein

PIP2;1, which mediates water transport and also likely moves H2O2 from the apoplast

to the cytosol (Grondin et al. 2015; Rodrigues et al. 2017). The pip2;1 mutant is

defective in both ABA-induced ROS accumulation and ABA-promoted stomatal

closure (Grondin et al. 2015). As an oxidative chemical, cytosolic H2O2 can directly

oxidize some proteins to affect their activity. For examples, H2O2 oxidizes the general

ROS-scavenging protein ATGPX3, which physically interacts with ABI2, thereby

converting it from the reduced form to the oxidized form, which significantly

decreases ABI2 activity (Miao et al. 2006). As a ROS scavenger, in turn, ATGPX3

can scavenge ABA- or drought-induced H2O2. Thus, ATGPX3 may act as a ROS

sensor to transduce oxidative signals during ABA and drought stress signaling (Miao

et al. 2006). Furthermore, cytosolic H2O2 may in turn inhibit ABA signaling as both

ABI1 and ABI2 are sensitive to H2O2 in vitro (Meinhard and Grill 2001; Meinhard et

al. 2002). It is also found that CPK8 interacts with cytosolic CAT3 and positively

regulates its activity by phosphorylating Ser261 during ABA-mediated stomatal

regulation (Zou et al. 2015). Both the cpk8 and cat3 mutants have lower catalase

 

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activity, accumulate more H2O2 and lose water more rapidly than wild type (Zou et al.

2015). These findings suggest that apoplastic ROS communicate with the cytosol to

regulate ABA signaling and stomatal movement.

CROSSTALK AMONG CO2, ABA AND ROS SIGNALING DURING

STOMATAL MOVEMENT

Elevated atmospheric CO2 levels on Earth are causing global warming, which

increases the severity of drought stress (Xu et al. 2015). However, high concentrations

of CO2 provide more carbon for photosynthesis and stimulate plant growth under

favorable water and nutrient conditions (Xu et al. 2015). Plants have evolved ways to

optimize stomatal activity to efficiently acquire CO2 for photosynthesis while

reducing water loss via transpiration (Chater et al. 2013). Thus, guard cells have

developed a fine regulatory system for responding to atmospheric CO2 levels and

integrating information about environmental CO2 levels with endogenous ABA

signaling (Kim et al. 2010). Short-term exposure to elevated CO2 levels provokes

stomatal closure, and long-term exposure reduces stomatal density, both of which

improve water use efficiency and protect plants against drought stress. However,

these changes can lead to increased leaf temperature, which can damage cells

(Medlyn et al. 2001). Nevertheless, treatment with high concentrations of bicarbonate

(the product of CO2 catalyzed by carbonic anhydrase, CA) induces H2O2 production

and promotes plasma membrane-localized NADPH oxidase-dependent stomatal

closure, while low CO2 levels promote stomatal opening (Kolla et al. 2007),

suggesting there is crosstalk between CO2 signaling and ROS.

 

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In order to identify the components in CO2 signaling, a genetic screening using

thermal imaging to detect the temperature change based on water transpiration has

been established (Hashimoto et al. 2006). The first identified mutant by this thermal

imaging screening is the high leaf temperature1 (ht1), which showed higher leaf

temperatures and smaller stomatal apertures compared to wild type (Hashimoto et al.

2006). HT1 encodes a putative MAPKKK kinase that negatively regulates stomatal

responses to changes in CO2 levels but does not affect ABA or blue light signaling

(Hashimoto et al. 2006). A recent study indicates that βCA1 and βCA4 function in the

early CO2 response; mutations in these genes strongly impair the stomatal CO2

response and increase stomatal density, but like HT1, they do not affect responses to

ABA or blue light (Hu et al. 2010). The ca1 ca4 ht1-2 triple mutants exhibit a similar

CO2 response as ht1-2, suggesting that HT1 acts downstream of βCA1 and βCA4 in

CO2 mediated stomatal movement. βCA1 is localized to the chloroplast (Fabre et al.

2007; Hu et al. 2010). βCA4 is targeted to the plasma membrane and interacts with

PIP2;1, which might transport apoplastic CO2 and efficiently channel it to βCA4

(Wang et al. 2016).

The anion channel SLAC1 plays a crucial role in stomatal movement during CO2

signaling (Negi et al. 2008). SLAC1 can be activated by elevated intracellular

bicarbonate levels in guard cells (Xue et al. 2011). A recent study found that the

SLAC1 transmembrane domain, but not the N-terminus or C-terminus, responds to

CO2, but not to ABA, while the N-terminus is important for ABA signaling

(Yamamoto et al. 2016). Both OST1 and GHR1 can activate SLAC1 in oocytes

 

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(Geiger et al. 2009; Hua et al. 2012), which can be inhibited by HT1 (Horak et al.

2016). Interestingly, the inhibition of OST1-, GHR1-activated SLAC1 by HT1 can be

counteracted by MPK12 (Horak et al. 2016). In a recent natural variation study,

MPK12 was also identified as a key player in Arabidopsis Cvi-0 Accession for guard

cell CO2 signaling (Jakobson et al. 2016). These findings suggest that MPK12 inhibits

HT1, and HT1 inhibit the OST1/GHR1-activated SLAC1. Different from HT1 that

mainly functions in CO2 signaling, MPK12 is also activated by ABA or H2O2

(Jammes et al. 2009). Interestingly, co-expressing βCA4 and PIP2;1 with

OST1-SLAC1 or CPK6/23-SLAC1 in oocytes activates SLAC1 via extracellular CO2

(Wang et al. 2016). These findings suggest that in the presence of various protein

kinases, SLAC1 directly senses cytosolic CO2/HCO3− that has been transported by

PIP2;1 and converted by βCA4 in oocytes.

The crosstalk between ABA and CO2 signaling is further supported by the

finding that ABA response mutants such as abi1-1, abi2-1, gca2, ost1 and ghr1 and

the ABA receptor mutant pyr/acars exhibit reduced guard cell responses to elevated

CO2 (Webb and Hetherington 1997; Young et al. 2006; Xue et al. 2011; Merilo et al.

2013; Horak et al. 2016). ABA biosynthesis is also required for CO2 signaling (Chater

et al. 2015). These findings suggest that full CO2 responses require components of the

ABA signalosome. It is likely that the ABA and CO2 signaling pathways converge to

mediate stomatal movement, while OST1 and GHR1 act downstream of this

convergence site (Engineer et al. 2016; Horak et al. 2016) (Figure 2). CO2-induced

stomatal closure and reduced guard cell density require ROS production by the

 

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NADPH oxidases RBOHF and RBOHD. Both CO2-induced stomatal closure and

reduced guard cell density are impaired in the rbohD rbohF double mutant, ABA

deficiency mutants and high-order ABA receptor pyl mutants (Chater et al. 2015).

Silencing of OST1 and RBOH1 in tomato reduces H2O2 and NO accumulation in

response to elevated CO2 levels, while silencing NR only reduces the accumulation of

NO. These findings indicate that OST1 and NADPH oxidase-dependent H2O2

production act upstream of NO production during CO2 signaling in guard cells (Shi et

al. 2015). Together, these findings suggest that ABA and ROS signaling are required

to enhance high CO2-induced stomatal closure.

APOPLASTIC H2O2 MEDIATES STOMATAL MOVEMENT IN PLANT

RESISTANCE TO DISEASES

As mentioned above, land plants have evolved stomata for H2O transpiration and gas

exchange, which has simultaneously opened the door for pathogen invasion (Melotto

et al. 2006). Plants have evolved functional immune systems for defenses against

pathogens. The first layer of plant innate immunity is the recognition of PAMPs by

pattern recognition receptors (PRRs), which initiates PAMP-triggered immunity (PTI)

to activate downstream immune responses (the second layer), leading to a transient

increase in cytosolic Ca2+ levels, a ROS burst, stomatal closure and increased

expression of pathogen-related genes, thus restricting pathogen entry (Lu et al. 2010;

Zhang et al. 2010). ROS bursts produced by NADPH oxidases or apoplastic enzymes

can lead to rapid PCD in a few distinct cells at the infection sites, a process known as

the hypersensitive response (HR). This process can impede the invasion of biotrophic

 

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pathogens because they obtain nutrients from living cells, while the neighboring cells

acquire the ability to prevent cell death through the spreading of ROS (Wu et al.

2014). Apoplastic ROS play crucial roles in mediating callose deposition and cell wall

crossing-linking, which reinforce the cell wall to impede the penetration of pathogens

(O'Brien et al. 2012).

Numerous PRRs localized on the plasma membrane include receptor-like kinases

(RLKs) and receptor-like proteins (RLPs) (Dou and Zhou 2012). In the model plant

Arabidopsis, the best-characterized RLKs include FLAGELLIN SENSING2 (FLS2)

and elongation factor-Tu receptor (EFR), which recognize the bacterial flagellin

epitope flg22 and bacterial elongation factor-Tu (EF-Tu) epitope elf18, respectively.

The perception of flg22 by FLS2 or elf18 by EFR induces their rapid association with

the co-receptor BAK1. FLS2/EFR and BAK1 represent the first layer of pathogen

signal perception required to trigger an oxidative burst within the plant (Figure 3). By

promoting stomatal closure, FLS2 plays an important role in restricting bacterial entry

into the plant (Melotto et al. 2006).

In the FLS2 signaling pathway, BOTRYTIS-INDUCED KINASE1 (BIK1) play

important role in mediating ROS production from ROBHD (Kadota et al. 2014; Li et

al. 2014). FLS2/EFR and BAK1 constitutively associate with their downstream target,

BOTRYTIS-INDUCED KINASE1 (BIK1), which is rapidly phosphorylated by

BAK1 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-VII subfamily, which consists of 46 members (Zhang et al. 2010). Both FLS2

 

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and BIK1 interact with RBOHD (Li et al. 2014). After FLS2 perceives flg22, the

activated BIK1 directly phosphorylates RbohD at Ser39 and Ser343 in a

calcium-independent manner and regulates RBOHD (Kadota et al. 2014; Li et al.

2014). Consistently, bik1 mutants are compromised in their ability to generate a rapid

ROS burst and immune responses (Lu et al. 2010; Zhang et al. 2010). However,

BIK1-mediated phosphorylation is required but not sufficient for RBOHD activation

(Li et al. 2014). The BIK1 homologs PBL1, PBL2 and PBL5 are also involved in the

generation of a pathogen-triggered ROS burst (Zhang et al. 2010; Liu et al. 2013).

CPK28 was found to interact with and phosphorylates BIK1 and contributes to its

turnover, resulting in decreased RBOHD-dependent ROS production (Monaghan et al.

2014). The mutation of CPK28 protein increases BIK1 stability and enhances

PAMP-triggered signaling and antibacterial immunity in Arabidopsis (Monaghan et al.

2014). Furthermore, a recent study indicated that the non-canonical heterotrimeric Gα

protein XLG2 interacts with both FLS2 and BIK1 and functions together with the Gβ

protein AGB1 and the Gγ proteins AGG1/2 to attenuate proteasome-mediated

turnover of BIK1 prior to flg22 perception (Liang et al. 2016). After the perception of

flg22 by FLS2, XLG2 dissociates from AGB1; its N-terminus is phosphorylated by

BIK1, which enhances flg22-induced ROS production, likely through RBOHD (Liang

et al. 2016). Similar with ABA signaling, the plasma membrane intrinsic proteins

AtPIP1;4 and AtPIP2;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,

 

20

CPK5, CPK6 and CPK11 are thought to be involved in the generation of a

PAMP-induced ROS bursts in Arabidopsis (Boudsocq et al. 2010; Dubiella et al.

2013). It is likely that CPK5 specifically phosphorylates RBOHD at Ser148 and

regulates its activity (Dubiella et al. 2013; Li et al. 2014). BR-SIGNALING

KINASE1 (BSK1) is a positive regulator of brassinosteroid signaling and a substrate

of the brassinosteroid receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1)

(Tang et al. 2008). Some other factors such as BSK1 are found to interact with FLS2

and required for flg22-induced ROS bursts (Shi et al. 2013).

Besides apoplastic ROS, pathogen infection also induces the production of NO,

which is required for plant resistance to pathogen invasion (Delledonne et al. 2001).

NO production likely acts downstream of H2O2 in the plant pathogen resistance

response (Arnaud and Hwang 2015). NO modifies the Cys890 of RbohD through

S-nitrosylation, which impairs its ability to bind the cofactor FAD, thus blunting

NADPH oxidase activity (Yun et al. 2011). By contrast, S-nitrosylation at the Cys32

of cytosolic APX1 enhances its enzymatic activity for scavenging H2O2 (Yang et al.

2015). Thus, H2O2 and NO form a feedback regulation circuit in plant disease

resistance.

In addition to producing extracellular ROS via NADPH oxidases, plants can

produce these compounds via apoplastic peroxidases (Torres et al. 2002; Bindschedler

et al. 2006). In Arabidopsis, knockdown of the class III apoplastic peroxidase genes

PRX33 and PRX34 reduces ROS production and callose deposition during various

PTI responses (Daudi et al. 2012). The cytokinin RESPONSE REGULATOR2

 

21

(ARR2) directly regulates PRX33 and PRX34 expression, thereby mediating ROS

accumulation, stomatal closure and PTI, which are independent of ABA signaling

(Arnaud et al. 2017). As NADPH oxidases are activated by ROS, NADPH oxidases

and apoplastic peroxidases likely affect each other in the modulation of ROS bursts

during the plant immunity response.

Not surprisingly, pathogens have developed an efficient evasion system to disturb

host immunity through secreting various effector proteins, which hijack plant proteins,

as well as toxins (such as coronatine produced by Pseudomonas syringae), causing

stomata to reopen (Melotto et al. 2006; Qi et al. 2017). Under dry environmental

conditions, most phyllosphere microbes maintain low populations, and only few

bacterial pathogens enter through stomatal pores. Under high-humidity conditions, the

phyllosphere pathogens aggressively propagate and invade plants through the stomata,

causing disease breakout (Xin et al. 2016). ABA can enhance disease resistance by

closing stomatal pores (Melotto et al. 2008), but it can also increase a plant’s

susceptibility to some powdery mildew, fungal and bacterial diseases (Bostock et al.

2014). For example, abi1-1 dominant mutants and plants overexpressing

HYPERSENSITIVE TO ABA1 exhibit increased callose deposition and resistance to

bacteria P. syringae (de Torres-Zabala et al. 2007). The ABA-deficient tomato mutant

sitiens accumulates more H2O2 at the site of infection and is more resistant to the

necrotrophic fungus Botrytis cinerea than wild type (Asselbergh et al. 2007). By

contrast, the ABA biosynthetic mutants aba1-3 and ABA insensitive mutant abi1-1

exhibit reduced callose deposition and disease resistance in response to another

 

22

necrotrophic fungus, Leptosphaeria maculans (Kaliff et al. 2007). Some common

signaling proteins, such as OST1, GHR1 and SLAC1, are involved in both ABA- and

pathogen-mediated stomatal movement (Melotto et al. 2008; Hua et al. 2012; Deger et

al. 2015). These findings suggest that ABA plays multifaceted roles in resistance to

various diseases (Ton et al. 2009).

ROS SIGNALING IN CHLOROPLASTS, MITOCHONDRIA AND

PEROXISOMES UNDER DROUGHT STRESS AND PATHOGEN ATTACK

Under both abiotic and biotic stress, ROS are produced in various organelles due to a

metabolic imbalance. Like apoplastic ROS, these ROS can serve as stress signals to

modify some proteins and activate the expression of stress-associated genes, which in

turn counteracts stress-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 organelle for sensing environmental signals such as high light, low or high

temperature, salt and drought stress, as well as pathogen invasion. This organelle

produces most of the ROS found in leaf cells (Figure 4). Increased ROS production in

chloroplasts negatively affects photosynthetic electron transport, impairs the assembly

and repair of photosystem II (PSII) and affects chloroplast development (Dietz et al.

2016). Chloroplasts mainly produce singlet oxygen (1O2) in PSII and its

light-harvesting antennae (Shapiguzov et al. 2012). Although 1O2 is a ROS, it is

unusual in that it is produced under high-intensity light when the excitation energy of

triplet chlorophyll molecules is transferred to triplet-state molecular oxygen

(Triantaphylides and Havaux 2009). The Arabidopsis fluorescent (flu) and chlorina1

 

23

(ch1) mutants specifically accumulate 1O2 without significant coproduction of other

ROS under certain conditions (Meskauskiene et al. 2001; Ramel et al. 2013), making

them particularly useful for studying 1O2 activity. As demonstrated using these

mutants, 1O2 is crucial for regulating 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 in the chloroplast.

The oxidized carotenoid product, β-cyclocitral, specifically induces the expression of

genes in the 1O2 signaling pathway but has little effect on the expression of

H2O2-mediated genes (Ramel et al. 2012). The executer1 (ex1) mutation suppresses

the 1O2-induced cell death phenotype of the flu mutant (Wagner et al. 2004) but does

not affect the accumulation of 1O2. Furthermore, crossing flu with the ex1 ex2 double

mutant fully suppressed the upregulation of almost all 1O2-responsive nuclear genes in

this mutant (Lee et al. 2007). These findings suggest that chloroplast-localized EX1

and EX2 are crucial transducers or sensors for 1O2-induced gene expression and cell

death.

The application of flg22 to Arabidopsis quickly induces specific Ca2+ transients

in the chloroplast stroma; this process relies on the presence of the

thylakoid-associated calcium-sensing receptor (CAS) (Nomura et al. 2012). CAS

regulates the PAMP-induced expression of defense genes and inhibits

chloroplast-mediated transcriptional reprogramming, likely through an 1O2-mediated

pathway (Nomura et al. 2012). These findings suggest that Ca2+ signaling

communicates with ROS signaling in the chloroplast to mediate nuclear gene

 

24

expression.

Pathogens can hijack plant immune signaling by secreting various effector

proteins. Pseudomonas syringae DC3000 effectors rapidly inhibit photosynthesis by

reprogramming the expression of nucleus-encoded chloroplast-targeted genes, thus

preventing the chloroplastic ROS burst. This phenomenon coincides with

pathogen-induced ABA accumulation; exogenous application of ABA suppresses

plant immunity and PSII activity (Zabala et al. 2015). The cysteine protease effector

HopN1 directly targets PsbQ in PSII, thereby mediating the degradation of PsbQ and

interfering with PSII activity, thus promoting 1O2 production (Rodriguez-Herva et al.

2012).

PSI mainly produces O2∙−/H2O2 when 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 but increases 1O2-mediated stress responses in the flu mutant,

suggesting that H2O2 has an antagonistic effect on 1O2 signaling in chloroplasts (Laloi

et al. 2007). H2O2 accumulates in the chloroplasts of bundle sheath cells under high

light and induces the ABA biosynthesis-dependent expression of ROS-responsive

genes (Galvez-Valdivieso et al. 2009). H2O2 activates the kinase OXIDATIVE

SIGNAL-INDUCIBLE1 (OXI1), which is required to fully activate MPK3 and MPK6

for ROS production under various conditions (Rentel et al. 2004; Petersen et al. 2009;

Liu et al. 2010). The oxi1 mutant is more tolerant to photo-induced oxidative damage

and cell death than wild type (Shumbe et al. 2016). OXI1 also suppresses

 

25

ch1-mediated 1O2 production and PCD under high-light conditions independently of

EX1 and EX2, but likely through jasmonate signaling. These findings suggest that

FLU- and CHL-mediated 1O2 production regulate cell death via different pathways

(Shumbe et al. 2016).

The metabolites produced in chloroplasts under high light and various stress

conditions play important roles in ROS signaling and the regulation of nuclear gene

expression (Zhu 2016). Phosphonucleotide 3′-phosphoadenosine 5′-phosphate (PAP)

accumulates in cells under drought and high-light stress and is regulated by the

phosphatase SAL1, which dephosphorylates PAP to adenosine monophosphate (AMP)

(Estavillo et al. 2011). SAL1 activity is inhibited by oxidative stress through

oxidation-mediated dimerization, the formation of intramolecular disulfide bridges

and glutathionylation, resulting in the accumulation of PAP (Chan et al. 2016). PAP

from chloroplasts to cytosol is thought to inhibit nuclear 5′-to-3′ exoribonucleases

(XRNs), which might alter the levels of stress-responsive mRNAs via RNA cleavage

or affect transcription termination (Estavillo et al. 2011). Thus, SAL1 might be a

conserved, general ROS sensor in chloroplasts that functions in plants under both

drought stress and high-light conditions (Chan et al. 2016).

Notably, the sal1 mutation, exogenous manipulation of PAP and the xrn2 xrn3

double mutations all restore ABA responses in the ABA-hyposensitive mutants ost1,

the snrk2.2/2.3/2.6 (ost1) triple mutant and abi1-1 during 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. 2013b), suggesting that

 

26

SAL1-PAP-XRN retrograde signaling can bypass the ABA pathway (Pornsiriwong et

al. 2017), but is regulated by ABA signaling (Wang et al. 2013b). SAL1-PAP-XRN

signaling positively regulates the expression of many genes involved in the ABA and

Ca2+ signaling pathways. These observations emphasize the tight communication and

complementary interactions between ROS-mediated SAL1-PAP-XRN signaling and

ABA signaling under drought and high-light conditions (Pornsiriwong et al. 2017).

Many stress treatments can quickly activate MAP kinase cascades, including

MAP3 and 6 (Meng and Zhang 2013). MPK3/6 interact with and phosphorylate ERF6

at Ser266 and Ser269, making ERF6 more stable, allowing it to directly upregulate

the expression of ROS-responsive and defensin genes and conferring fungal disease

resistance (Wang et al. 2010; Meng et al. 2013). These responses are abolished in the

tpt1 and tpt2 mutants, which lack chloroplast membrane-localized triose phosphate

translocators (TPT) (Vogel et al. 2014). Consistent with this role, the erf6 mutant

accumulates more H2O2 and is more sensitive to photoinhibition than wild type

(Wang et al. 2013a). These observations suggest that ROS are involved in regulating

the signaling of metabolites in chloroplasts that serve as retrograde signals to

coordinate stress-response pathways in the nucleus.

Mitochondria generate a major portion of ROS in roots but only a small portion

in leaves. While ETC complexes I–IV in plants are similar to those in animal

mitochondria, these complexes also contain five unique enzymes, including an

alternative oxidase (AOX) and four NAD(P)H dehydrogenases (Moller 2001; Jacoby

et al. 2012). Complex I and III are major sites for ROS production in plants. AOX

 

27

helps minimize ROS accumulation by the ETC (Moller 2001; Jacoby et al. 2012).

ABA and drought treatment promote ROS production 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 the splicing of several genes in Complex I

(He et al. 2012). ABO8 encodes a pentatricopeptide repeat (PPR) protein involved in

mediating the splicing of NAD4 intron 3 in mitochondrial complex I (Yang et al.

2014). Both abo6 and abo8 mutants are hypersensitive to ABA in terms of seed

germination and primary root growth (He et al. 2012; Yang et al. 2014). Interestingly,

the expression of ABO6 and ABO8 is reduced by ABA treatment (He et al. 2012;

Yang et al. 2014). Both ABA treatment and drought stress promote greater ROS

accumulation in the abo6 and abo8 mutants compared to wild type (He et al. 2012;

Yang et al. 2014), indicating that ABA signaling regulates ROS production in

mitochondria. The abo8 mutant exhibits increased ROS accumulation in the root tip,

resulting in delayed differentiation of distal stem cells (DSC) (Yang et al. 2014).

Another study by Yu et al. showed that mutations in a P-loop NTPase localized to the

mitochondria in Arabidopsis reduce both H2O2 and O2.- levels in roots and promote

DSC differentiation (Yu et al. 2016). UPBEAT1 (UPB1) encodes a bHLH

transcriptional factor that negatively regulates the expression of several peroxidase

genes in the root elongation zone (Tsukagoshi et al. 2010). The upb1 mutant exhibits

increased O2.- levels in the root meristem and decrease apoplastic H2O2 content in the

elongation zone, which delays the transition from cell proliferation to differentiation,

resulting in enlarged root apical meristems (Tsukagoshi et al. 2010). ROS levels must

 

28

also be maintained at moderate levels in animal cells in order to maintain stem cell

homeostasis (Le Belle et al. 2011; Morimoto et al. 2013; Paul et al. 2014). These

studies indicate that ROS homeostasis is essential for cell division and differentiation

(Mittler 2017).

Peroxisomes mainly produce H2O2 and O2- through several metabolic pathways.

Water stress promotes stomatal closure and reduces CO2 assimilation, thus increasing

photorespiration and glycolate production. Glycolate from chloroplasts is oxidized by

glycolate oxidase (GOX) to produce glyoxylate and most of the H2O2 in leaf

peroxisomes, indicating that ROS signals are communicated between chloroplasts and

peroxisomes through various metabolites. Additionally, acyl-CoA oxidase (ACX)

catalyzes the production of H2O2 during peroxisomal fatty acid β-oxidation, a process

that occurs more actively in germinating seeds than in other tissues (Sandalio and

Romero-Puertas 2015). The matrix xanthine oxidase (XOD) in leaf peroxisomes

generates O2-, which is converted to H2O2 by SOD. H2O2 is mainly detoxified by

peroxisome-localized CATs in leaves (Miller et al. 2010). Furthermore, NO and its

derivative, RNS, are also produced in peroxisomes, which can influence many aspects

of cellular function in response to environmental stress (Sandalio and Romero-Puertas

2015).

ROS produced from different organelles may diffuse into the cytosol. Whether

these ROS have similar effects in the cytosol is not yet clear. However, ROS

produced from different organelles could form a specific signature to trigger unique

nuclear gene expression responses (Rosenwasser et al. 2011; Sewelam et al. 2014).

 

29

For example, chloroplastic H2O2 has a significant bias to induce the expression of

genes related to wounding and pathogen attack, while H2O2 from peroxisomes

regulates more genes involved in protein repair responses (Sewelam et al. 2014). As it

might not be possible for cellular components to determine which ROS comes from

which organelle, this finding suggests that ROS, with or without other components,

are involved in regulating the expression of specific genes.

Although ROS signals from different organelles can communicate with each

other, little is known about this communication process (Shapiguzov et al. 2012).

Mutations in Arabidopsis MOSAIC DEATH1 (MOD1), encoding an enoyl-acyl carrier

protein (ACP) reductase for fatty acid biosynthesis in chloroplasts, lead to high H2O2

and O2∙− production in mitochondria (Mou et al. 2000; Wu et al. 2015), suggesting

that chloroplast signals might be transmitted to mitochondria to modulate ROS

production. A screening for suppressors of mod1 identified several nuclear genes

encoding proteins involved in mitochondrial Complex I (Wu et al. 2015). In fact,

mutations in these of Complex I genes usually result in higher ROS levels than wild

type (Lee et al. 2002; Meyer et al. 2009; He et al. 2012). Signals from mod1

chloroplasts might enhance relative mitochondrial ETC activity, causing greater

electron leakage and more ROS to be produced in Complex I. This effect is

compromised by mutations in Complex I genes, as mod1 suppressor mutations reduce

ETC activity (Wu et al. 2015). The mod1 mutant and its suppressor lines represent

valuable materials for further studying ROS-related communication between

chloroplasts and mitochondria.

 

30

THE ROLE OF ROS SIGNALING IN CELL-TO-CELL AND

LONG-DISTANCE COMMUNICATION DURING STRESS

Besides inside cells, ROS communication occurs between cells. ROS, Ca2+ and

electric signals can be transmitted from the tissue of origin to distant tissues within

minutes, allowing plants to achieve systemic acquired acclimation (SAA) under

abiotic stress (Figure 5) (Mittler et al. 2011; Gilroy et al. 2016). Various abiotic

stresses, such as wounding, heat, cold, high intensity light and high salinity, trigger

the production of RBOHD-mediated apoplastic ROS, which move in relay-type

fashion in the apoplast from the local tissue throughout the entire plant at speeds of up

to 8.4 cm min–1 (Miller et al. 2009).

RBOHD-mediated ROS enter neighboring cells to trigger an increase in transient

Ca2+ levels. This, in turn, activates protein kinases such as CPK5 to phosphorylate and

activate RBOHD, thus forming a wave of ROS and Ca2+ that functions in cell-to-cell

and long-distance communication (Dubiella et al. 2013). Heat stress also transiently

increases the accumulation of ABA in systemic tissues in an RBOHD-dependent

manner. Blocking ABA biosynthesis or signaling impairs the SAA response to heat

stress, indicating the importance of coordination between ABA and the ROS wave in

SAA (Suzuki et al. 2013).

Osmotic stress quickly induces increases in [Ca2+]i levels through the action of

OSCA1 (REDUCED HYPEROSMOLARITY, INDUCED CA2+ INCREASE1), a

Ca2+ channel and osmosensor localized on the plasma membrane. However, while the

 

31

osca1 mutant shows a reduced Ca2+ response to osmotic stress, it still responds

normally to ABA and H2O2 treatment, suggesting that OSCA1 functions upstream of

ABA and H2O2 signaling (Yuan et al. 2014). Further studies are required to determine

whether OSCA1 is involved producing the Ca2+ wave.

The long distance, salt-stress-induced Ca2+ wave (which travels at a speed of 2.4

cm min–1) requires the vacuolar cation-permeable channel TWO PORE CHANNEL1

(TPC1) (Choi et al. 2014). TPC1 is sensitive to Ca2+ levels in both the cytosol and

vacuole lumen and is likely involved in calcium-induced calcium release during Ca2+

wave propagation (Choi et al. 2014). Glutamate receptor-like proteins (GLRs)

localized on the plasma membrane might also function as amino acid-gated

Ca²⁺channels (Kong et al. 2016). L-methionine (L-Met) activates GLR3.1 and

GLR3.5 Ca2+ channels to increase cytosolic Ca2+ levels, which is required for

NADPH oxidase-mediated ROS production and stomatal closure but functions

independently of ABA (Kong et al. 2016). Wounding rapidly induces distal systemic

electrical signals, a process that requires the participation of GLR3.2, GLR3.3 and

GLR3.6 (Mousavi et al. 2013). GLR3.3 and GLR3.6 are required for propagating

wound-induced electrical signals beyond the wounded leaf, while GLR3.5 is required

to prevent the formation of wound-induced electrical potentials in distal,

non-neighboring leaves (Salvador-Recatalà 2016). However, the molecular

connections among ROS/Ca2+ and electrical signals require further exploration.

FUTURE PROSPECTS

 

32

In this review, we summarized the molecular mechanisms that regulate the ROS

signaling network in plants, primarily under drought stress and pathogen attack.

Although we divided our review into several sections for the sake of discussion, ROS

production and signaling under different conditions cannot be separated within a cell.

Even under very strict experimental conditions, ROS production cannot be regulated

in only a specific site in the cell. Once a stress is applied, different types of ROS will

be produced immediately in different compartments and different sites of a cell. These

ROS form a complicated network, making it quite difficult to study ROS signaling.

Therefore, when investigating ROS signaling, we should take the whole picture into

account for their roles in plant responses to abiotic and biotic stress. While ROS are

metabolic by-products, high ROS levels are toxic to cells, and thus their

concentrations are kept low by the action of various antioxidants. At the same time,

ROS are absolutely required for many plant developmental processes and responses to

biotic and abiotic stress (Mittler 2017). For example, in rice dst (drought and salt

tolerance) mutant, it has shown that increasing ROS to an appropriate level does not

affect the plant growth and yield, but greatly increase plant tolerance to drought and

salt stress (Huang et al. 2009). DST encodes a zinc finger transcriptional factor that

likely directly regulates peroxidase 24 precursor for scavenging H2O2 (Huang et al.

2009). Wheat seedlings carrying a mutated Ta-sro1 gene encoding a poly(ADP ribose)

polymerase (PARP) domain protein with high PARP activity increase ROS

production and yield under salinity stress (Liu et al. 2014). These studies point out a

new strategy for breeding of stress resistant crops by modifying the ROS production

 

33

in the future. Although much exciting progress has been made, thanks largely to

newly developed techniques and rapid progress in genome and protein analysis

methods, a complete understanding of the exact nature of ROS signaling remains

elusive.

Some gaps in our knowledge are summarized by the following questions: How do

we measure the levels of different types of ROS and their dynamic changes at various

sites in the cell? How are ROS signals that are produced as a result of normal

developmental processes physically interconnected and integrated with those

produced as a result of biotic and abiotic stress? How are ROS differentially

recognized as second messengers in a cell or in neighboring cells during processes

such as PCD? Different ROS sensors must exist, but how do we identify these sensors?

Given the diversity of receptor-like protein kinases in plants (Shiu and Bleecker 2001),

do they directly sense and transmit apoplastic ROS signals? If so, how? Do they act

alone or together with other components to transduce a ROS wave? It is well known

that pretreatment with low concentration of exogenous H2O2 (called H2O2-priming)

can increase plant tolerance to various abiotic stresses (Hossain et al. 2015), but the

molecular mechanisms for this are not well understood. Right now, ROS level can be

controlled in crops by genetically manipulating the expression of ROS related genes.

But which ROS level should be maintained in order to increase stress tolerance

without apparent yield penalty needs further explored in various crops with different

genetic backgrounds.

Most studies on ROS tend to focus on their production and signal transduction in

 

34

the cytosol, organelles and plasma membrane. However, the effects of ROS on

chromatin structure and epigenetic modifications in plants require further exploration.

ACKNOWLEDGEMENTS

J.Z. and Z.G. are supported by the National Key Scientific Research Project

(2011CB915400). Z.G. is supported by the National Natural Science Foundation of

China (31730007).

 

35

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Figure legends

Figure 1. The roles of reactive oxygen species (ROS) in abscisic acid (ABA)

signaling in stomatal movement

Drought stress induces the biosynthesis and/or accumulation of ABA in guard cells,

 

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which is perceived by PYR1/PYL/RCAR ABA receptors. ABA-bound PYL interacts

with and inhibits clade A PP2Cs such as ABA INSENSTIVE1 (ABI1) and ABI2,

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 dephosphorylates SLOW ANION

CHANNEL-ASSOCIATED1 (SLAC1) and inhibits its activity. OST1 phosphorylates

respiratory burst oxidase homolog protein F (RBOHF) to produce apoplastic O2∙-,

which is converted to H2O2 by superoxide dismutase (SOD). The apoplastic H2O2

signal is transduced into the cytosol by GHR1 through an unidentified mechanism to

activate a putative, unidentified calcium channel on the plasma membrane. After

binding to Ca2+, CIPK26 can phosphorylate RBOHF. Ca2+-activated CPK4/6/21/23

can phosphorylate and activate SLAC1. Apoplastic H2O2 enters the cell through the

aquaporin protein PIP2;1. CPK8 phosphorylates and enhances the activity of

CATALASE3 (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-protein α-subunit GPA1 and phosphatidic acid (PA) produced by phospholipase

Dα1 (PLDα1). KAT1, the inward-rectifying K(+) channel; GORK, guard cell outward

potassium channel; AHAs, Arabidopsis P-ATPases. Blue arrows indicate positive

regulation; red bars indicate inhibition; dotted lines indicate uncertain regulation.

Figure 2. Crosstalk among CO2, ABA and reactive oxygen species (ROS)

signaling during stomatal regulation

 

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Elevated 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 carbonic anhydrase βCA4 and

might channel CO2 to βCA4 to produce bicarbonate (HCO3. HCO3- signaling might

activate MPK12/4 to repress HT1. Repressed HT1 would then release its

phosphorylation and the inhibition of GUARD CELL HYDROGEN

PEROXIDE-RESISTANT1 (GHR1) or OPEN STOMATA1 (OST1), resulting in the

activation of SLOW ANION CHANNEL-ASSOCIATED1 (SLAC1) or releasing its

direct inhibition on SLAC1. HCO3- might also activate ABA signaling to enhance

SLAC1 activity by GHR1 and OST1 or Ca2+-activated CPK3/23 or directly regulate

SLAC1 via an unknown mechanism. 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 arrows indicate positive regulation; red bars indicate inhibition; dotted lines

indicate uncertain regulation.

Figure 3. The roles of reactive oxygen species (ROS) in stomatal immunity

During 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 to produce apoplastic H2O2. BIK1 might also activate

an unknown Ca2+ channel to increase cytosolic Ca2+ levels, which would in turn

 

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activate CPK28 to inhibit BIK1 or other calcium-dependent protein kinases (CPKs)

such as CPK5 to phosphorylate and activate RBOHD. Without flg22 stimulation, the

heterotrimeric G proteins composed of the non-canonical Gα proteins XLG2/3, Gβ

protein AGB1 and Gγ proteins AGG1/2 interacts with FLS2-BIK1 complex. After

perceiving flg22, the activated BIK1 phosphorylates the N-terminus of XLG2, which

leads to G proteins to dissociate from 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 the cytokinin pathway, produce apoplastic O2∙-. Apoplastic H2O2 is transported into

the cytosol by PIP1;4, which further induces the production of nitric oxide (NO). NO

mediates the S-nitrosylation of RBOHD and ASCORBATE PEROXIDASE1 (APX1),

thereby inhibiting RBOHD activity but increasing APX1 activity. Blue arrows

indicate positive regulation; red bars indicate inhibition; dotted lines indicate

uncertain regulation.

Figure 4. Crosstalk of reactive oxygen species (ROS) between organelles

High levels of ROS are produced in chloroplasts under high-light conditions and

drought stress. FLUORESCENT IN 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 to regulate cell death-related genes. PSI mainly produces O2∙−/H2O2 under

stress conditions, which can be decomposed by thylakoid-bound ascorbate peroxidase

(tAPX). H2O2 has an antagonistic effect on 1O2 signaling in chloroplasts. H2O2

 

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diffuses into the cytosol to activate OXIDATIVE SIGNAL-INDUCIBLE1 (OXI1),

which is required to fully activate MPK3/6. Singlet oxygen may also go through the

OXI1 and jasmonate pathways to regulate cell death. The pathogen cysteine protease

effector HopN1 directly mediates the degradation of PsbQ in PSII. The detection of

pathogen-associated molecular patterns (PAMPs) leads to an increase in transient

Ca2+ levels, which is mediated by calcium-sensing receptors (CAS) in chloroplasts,

likely via the MAPK cascade. Carotenoids quench 1O2 to become β-cyclocitral, which

induces the expression of 1O2-responsive genes. Plastidial methylerythritol

cyclodiphosphate (MEcPP) and triose phosphate from chloroplasts can also induce

selected stress-responsive nucleus-encoded plastidial proteins. SAL1

dephosphorylates 3′-phosphoadenosine 5′-phosphate (PAP) to adenosine

monophosphate (AMP), which is negatively regulated by H2O2. PAP is bound by and

inhibits nuclear 5′-to-3′ exoribonucleases (XRNs), resulting in the upregulation of

various abscisic acid (ABA)-responsive genes. Chloroplasts communicate with the

mitochondria and peroxisomes through MOSAIC DEATH1 (MOD1)-mediated fatty

acid biosynthesis. In the mitochondria, ABA signaling negatively regulates Complex 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 in H2O2 production. Blue arrows indicate

positive regulation or production; red bars indicate inhibition; dotted lines indicate

uncertain regulation.

Figure 5. Reactive oxygen species (ROS) and Ca2+ waves function in cell-cell

 

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communication

Apoplastic ROS mediated by respiratory burst oxidase homolog protein D (RBOHD)

triggers the generation of Ca2+ transients via the activity of unknown receptor-like

kinases, or they enter neighboring cells via water channels. Osmotic stress activates

OSCA1 (REDUCED HYPEROSMOLARITY, INDUCED CA2+ INCREASE1) to

increase Ca2+ levels in the cytosol. In turn, Ca2+ activates protein kinases such as

CPK5 to phosphorylate and activate RBOHD, thus forming ROS and Ca2+ waves that

function in cell-to-cell and long-distance communication. The vacuolar

cation-permeable channel TWO PORE CHANNEL1 (TPC1) and plasma

membrane-localized glutamate receptor-like proteins (GLRs) may be involved in

increasing the levels of Ca2+. Ca2+ can diffuse into neighboring cells through

plasmodesmata (PD). Blue arrows indicate positive regulation or production; dotted

lines indicate uncertain regulation.

 

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Figure. 1

Figure. 2

 

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Figure. 3

Figure. 4

 

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Figure. 5