Reactive Oxygen Species in Plant Signalingbiologia.ucr.ac.cr/profesores/Garcia Elmer/reactive oxygen species in... · This classical model has been recently extended by the discovery

PP69CH08_Kangasjarvi ARI 4 April 2018 11:1

Annual Review of Plant Biology

Reactive Oxygen Speciesin Plant SignalingCezary Waszczak, Melanie Carmody,and Jaakko KangasjarviOrganismal and Evolutionary Biology Research Programme, Faculty of Biological andEnvironmental Sciences, and Viikki Plant Science Centre, University of Helsinki,00014 Helsinki, Finland; email: [email protected]

Annu. Rev. Plant Biol. 2018. 69:209–36

First published as a Review in Advance onFebruary 28, 2018

The Annual Review of Plant Biology is online atplant.annualreviews.org

https://doi.org/10.1146/annurev-arplant-042817-040322

Copyright c© 2018 by Annual Reviews.All rights reserved

Keywords

abiotic stress, plant–pathogen interactions, signal perception, signaltransduction, long-distance signaling, stomatal closure, plant development

Abstract

As fixed organisms, plants are especially affected by changes in their en-vironment and have consequently evolved extensive mechanisms for ac-climation and adaptation. Initially considered by-products from aerobicmetabolism, reactive oxygen species (ROS) have emerged as major regulatorymolecules in plants and their roles in early signaling events initiated by cel-lular metabolic perturbation and environmental stimuli are now established.Here, we review recent advances in ROS signaling. Compartment-specificand cross-compartmental signaling pathways initiated by the presence ofROS are discussed. Special attention is dedicated to established and hypo-thetical ROS-sensing events. The roles of ROS in long-distance signaling,immune responses, and plant development are evaluated. Finally, we outlinethe most challenging contemporary questions in the field of plant ROS biol-ogy and the need to further elucidate mechanisms allowing sensing, signalingspecificity, and coordination of multiple signals.

209

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ANNUAL REVIEWS Further

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https://www.annualreviews.org/doi/full/10.1146/annurev-arplant-042817-040322


Singlet oxygen(1O2): oxygenmolecule in which thetwo highest-energyelectrons haveopposite spin and arepaired, in contrast totriplet oxygen (3O2)

Contents

1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2102. PRODUCTION, SCAVENGING, AND SIGNALING OF ROS . . . . . . . . . . . . . . . . 210

2.1. Chloroplasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2112.2. Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2152.3. Peroxisomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2162.4. Apoplast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2172.5. Transport of ROS Through Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

3. ROS-SENSING MECHANISMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2193.1. Oxidative Posttranslational Modifications of Cysteine Residues . . . . . . . . . . . . . . . 2203.2. Oxidative Posttranslational Modifications of Methionine Residues . . . . . . . . . . . . 221

4. INTRACELLULAR INTERACTIONS BETWEEN ORGANELLE ROSAND REDOX SIGNALING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2224.1. How Is Signaling Specificity Retained in the Cytosol? . . . . . . . . . . . . . . . . . . . . . . . . 2224.2. Chloroplast-Mitochondrion Crosstalk, Signaling, and PAP . . . . . . . . . . . . . . . . . . . 223

5. APOPLASTIC AND ORGANELLE ROS INTERACTIONSDURING STRESS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2245.1. Cell-to-Cell ROS Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2245.2. ROS in Plant Immunity and Stomatal Closure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

6. ROS IN PLANT DEVELOPMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2277. CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

1. INTRODUCTION

The evolution of multicellular life forms has been shaped by the oxygen-rich atmosphere (113). Inthe presence of oxygen, cellular processes characterized by high rates of electron or energy transferinevitably lead to the formation of reactive oxygen species (ROS) by electron or energy leakageto molecular oxygen (O2). Additionally, multiple enzymatic reactions have evolved to produceROS either as a primary product or as a by-product. ROS are defined as oxygen-containingmolecules exhibiting higher chemical reactivity than O2. In plants, the major forms of ROS aresinglet oxygen (1O2), superoxide anion (O2

·−), hydrogen peroxide (H2O2), and hydroxyl radical(HO·) (Figure 1). The potential for cellular damage from enhanced production of these moleculeshas been alleviated through evolutionary pressure to develop and expand a range of enzymaticand nonenzymatic ROS scavengers (Figure 2). Rapid changes in compartmental redox balanceand ROS homeostasis are among the earliest symptoms following fluctuations in environmentalconditions. Plants monitor these parameters and utilize them as signals in multiple processes thatserve to adjust metabolism or physiology either at the whole plant or tissue level or in specificsubcellular compartments.

2. PRODUCTION, SCAVENGING, AND SIGNALING OF ROS

Compartmentalization of production and scavenging determines the biological functions of ROSin plants. In particular, O2

·−, H2O2, and HO· can be produced in nearly every subcellular com-partment. Consequently, with the exception of the apoplast, which has a low antioxidant capacity,each production site is equipped with an array of antioxidant systems to buffer the local redoxenvironment (reviewed in 49, 103). The concentration and longevity of ROS are determined by

210 Waszczak · Carmody · Kangasjarvi

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Superoxideanion

Tripletoxygen

Singletoxygen

Hydrogenperoxide

Hydroxylradical

SOD1O2

3O2 H2O2•–O2

•HO

•OHH

HO

O

3P680*

P680

e– e– Fe2+/Cu+

Fe3+/Cu2+

O–O•O O O O• •

Lifetime µs ms ms to s ns

Reactivity Reactive Moderatelyreactive

Moderatelyreactive

Highlyreactive

Figure 1Generation and chemical structures of reactive oxygen species. Electrons are depicted as dots. Abbreviations: 3P680

∗, triplet state of the

primary electron donor of photosystem II; SOD, superoxide dismutase.

Carotenoids: organicpigments produced byplants and algae

the composition and availability of antioxidant systems. The estimated lifetime for HO·, the mostreactive form of ROS, is of the order of nanoseconds, and for 1O2 microseconds. The lifetimes ofH2O2—the most stable form of ROS—and O2

·− are considerably longer (milliseconds to seconds)and depend largely on the presence and activity of dedicated ROS scavengers (87).

During the past few decades the concept of oxidative stress, in which ROS were regarded asharmful substances that indiscriminately oxidize various molecules and structures, has changed tothe concept of ROS signaling (48, 66). According to current understanding, effective antioxidativesystems in the symplastic compartments keep ROS concentrations low even under increased ROSproduction rates (49, 103). Therefore, any elevation in ROS concentration in different subcellularcompartments appears to be transient, reflecting the efficiency of scavenging systems. The higherproduction rates provoke a shift in the compartmental redox balance toward a more oxidized state,which can be sensed by various compartment-specific systems to regulate gene expression. In thefollowing sections we review and give selected examples and concepts of how ROS are formed,scavenged, and act as signaling molecules in the major cellular compartments.

2.1. Chloroplasts

Chloroplastic ROS production is tightly associated with light-dependent photosynthetic reactions,and increased ROS production serves as a marker of changing internal or external conditions thatrequire the acclimation or adjustment of metabolism. Below we review the most important chloro-plastic ROS sources and the signal transduction events that mediate these plant–environmentinteractions.

2.1.1. Chloroplastic ROS production. Unique to chloroplasts is the formation of nonradical,highly reactive 1O2 (Figure 2). The singlet state of oxygen is generated within thylakoid mem-branes mainly by energy transfer from the triplet state of the primary electron donor (3P680

∗ )of photosystem II to ground state molecular oxygen (3O2) (Figure 1) (46). No direct enzymaticscavengers of 1O2 have evolved. Instead, scavenging of 1O2 occurs primarily through reactionswith other molecules, particularly carotenoids (111), tocopherols (76), and membrane lipids (44).Recent studies have extended our knowledge of 1O2-induced cleavage of β-carotene, in which β-cyclocitral and other breakdown products are involved in 1O2-related chloroplast retrograde sig-naling (112). For more details on 1O2 signaling, readers are directed to recent reviews (17, 29, 81).

www.annualreviews.org • Reactive Oxygen Species 211

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Stroma

Thylakoid lumen

Chloroplast

PSIIFdCu/Zn

SODtAPX

ASCMDA

sAPX

H+

FeSOD

Carotenoids,tocopherols

DHA

MDAR

GSH

GSSG

NADPH

NADPH

FTR

NTRNTR Trx

NTRC

Peroxisome

II

III

IV VMitochondrion

?

AOX

Glycolate

Glyoxylate

GOXCAT

Prxr

APX

MnSOD

Matrix

IMS

LIGHT

NADPH

ASCASC

ASCASC

GPXL

GPXL

APX

APX

XO

MDA ASC

SA

OPPP

NADPH

OPPP

PRXNADPH

RBOH

SOD?

Apoplast

Neighboringcells

ASCASC

Nucleus

GSH

GSH

?

PAO

ASCoxidase

Cytoplasm

Trxred

Trxox

PrxroxPrxrred

GPXLoxGPXLred

H2OH2O2

NADP+

NADP+

NADP+

H2O

H2O

H2O

H2O

H2O

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2H2O2

H2O2

H2O2

H2O2

H2O2

O2

O2

O2

O2

O2

O2 O2

•–O2

•–O2

•–O2

•–O2

•–O2

•–O2

O3

e–

e–

e–

e–

e–

e–e–

e–e–e–

e–1O2

Cu/ZnSOD

Cu/ZnSOD

PSIFNR

GRDHAR

I

Activation Inhibition Hypothetical

Figure 2Production and scavenging of ROS. Formation of singlet oxygen occurs mainly by the transfer of energy from the triplet state of theprimary electron donor (3P680

∗) of PSII to the ground state triplet oxygen. Superoxide anions arise during processes characterized by

high rates of electron flow, such as mitochondrial and chloroplastic electron transport chains, and as a result of multiple enzymaticreactions. Production of hydrogen peroxide results from enzymatic and spontaneous dismutation of superoxide anions and activity ofglycolate oxidases. With the exception of the apoplast, subcellular compartments possess various nonenzymatic and enzymatic ROSscavengers that under optimal growth conditions keep ROS concentrations very low. Hydrogen peroxide might cross biologicalmembranes via aquaporins. In the inner membrane, boxes I–V represent complexes of the mitochondrial electron transport chain.Abbreviations: 3P680

∗, triplet state of the primary electron donor of photosystem II; AOX, alternative oxidase; APX, ascorbate

peroxidase; ASC, ascorbate; CAT, catalase; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; Fd, ferredoxin; FNR,ferredoxin-NADP+ reductase; FTR, ferredoxin-dependent thioredoxin reductase; GOX, glycolate oxidase; GR, glutathione reductase;GPXL, glutathione peroxidase-like; IMS, intermembrane space; MDA, monodehydroascorbate; MDAR, monodehydroascorbatereductase; NTR, NADPH-dependent thioredoxin reductase; NTRC, NADPH-dependent thioredoxin reductase C; OPPP, oxidativepentose phosphate pathway; PAO, polyamine oxidase; PRX, peroxidase; Prxr, peroxiredoxin; PSI/II, photosystem I/II; RBOH,respiratory burst oxidase homolog; ROS, reactive oxygen species; SA, salicylic acid; SOD, superoxide dismutase; TRX, thioredoxin;XO, xanthine oxidase.


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APX: ascorbateperoxidase

GPXL: glutathioneperoxidase-like

Prxr: peroxiredoxin

TRX: thioredoxin

GRX: glutaredoxin

SA: salicylic acid

Hypersensitiveresponse: controlleddeath of cellssurrounding the placeof infection to limit thespread of pathogens

One-electron reduction of oxygen at PSI produces the moderately reactive O2·− (Figure 1,

Figure 2), which is dismutated into H2O2 on the stromal side of the thylakoid membranespontaneously or enzymatically via superoxide dismutases (SODs) (3). Chloroplast SODs includeiron-SODs (FeSODs) and copper/zinc-SODs (Cu/ZnSODs), and they are crucial for chloroplastfunction and development. In chloroplast stroma H2O2 is detoxified by ascorbate peroxidases(APXs), glutathione peroxidase-like (GPXLs) enzymes, and peroxiredoxins (Prxrs) (3).

The flow of electrons from water through the photosynthetic electron transport chain and viaO2

·− to H2O2 and then back to water serves as a sink for excess electrons (water-water cycle).This classical model has been recently extended by the discovery of the photoprotective role of2-Cys Prxrs acting synergistically with thylakoid APX under high-light conditions (5). Prxrs usethiol-based catalytic mechanisms to reduce H2O2 (35). Regeneration of Prxrs can be catalyzedby thioredoxins (TRXs), glutaredoxins (GRXs), and NADPH-dependent thioredoxin reductaseC, a two-domain enzyme exhibiting both thioredoxin reductase and TRX activity (35). TRXs arereduced by NADPH-dependent thioredoxin reductases and ferredoxin-dependent thioredoxinreductases (118).

2.1.2. Chloroplastic ROS and redox signaling. Chloroplastic formation of H2O2 and con-sequent alterations in the status of chloroplast antioxidant systems have been recognized as pa-rameters providing information about environmental and metabolic changes to the nucleus inchloroplast retrograde signaling (reviewed in 17, 29, 81). As a simple and ubiquitous molecule,H2O2 does not carry any information about its origin. Therefore, an outstanding problem in therole and significance of H2O2 as a retrograde signal has been the question of specificity—how canthe nucleus differentiate H2O2 produced in different subcellular compartments, all of which po-tentially affect the concentration of H2O2 in the cytoplasm? This key issue is discussed further inSection 4.1. Recent reports of chloroplastic ROS transport and signal transduction to the nucleushave presented evidence for (a) direct stromule-mediated delivery of ROS and proteins to thenucleus; (b) fast regulation of nuclear H2O2 concentration by a population of companion chloro-plasts localized around the nucleus; and (c) signaling via accumulation of chloroplast metabolites,their oxidative derivatives, or both (Figure 3).

Stromules are dynamic plastid projections thought to maintain direct links between a smallnumber of plastids and nuclei (40, 53). Stromule formation in vivo was rapidly induced withchemicals that cause chloroplastic ROS generation and have been linked to the light-dependentchloroplast redox status (14). Regulation can be independent from the cellular environment, asisolated chloroplasts can also form stromules (14). Whereas rapid stromule induction was ob-served 1 h after exogenous H2O2 and salicylic acid (SA) treatments, slower reactions have beenobserved after 22–48 h in response to bacterial effectors (15), most likely related to the onset ofthe hypersensitive response. Importantly, stromules are capable of protein (15, 53) and H2O2 (15)transport—bypassing a long transit through the cytosol.

In addition, H2O2 produced in chloroplasts in a light-dependent manner can be transferredto nuclei with no apparent stromule formation from a subpopulation of chloroplasts closely sur-rounding the nucleus (43). This finding agrees with an earlier report showing that an increase inlocalized H2O2 production induced by application of a 405-nm laser to chloroplasts adjacent tonuclei resulted in the transfer of H2O2 to the nucleus (15). Such a transfer of ROS is believedto regulate the expression of high-light-responsive genes and could be incorporated into existingsignaling models to explain how ROS signals can reach the nucleus.

Chloroplastic ROS production also affects nuclear gene expression indirectly. ROS-dependent oxidative posttranslational modifications of chloroplast catabolic enzymes increase the


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concentration of metabolites that have a signaling function. The chloroplastic 3′-phosphoadenosine 5′-phosphate (PAP) phosphatase SAL1 undergoes redox- or H2O2-dependentoxidative inactivation (16). This leads to the accumulation of PAP, which is suggested to act as asecond messenger conveying the information about chloroplast redox status to the nucleus (41). Inthe context of signaling specificity, it would be of interest to investigate whether signaling eventsinitiated by direct H2O2 translocation from chloroplasts to nucleus, which most likely modifythe activity of nuclear redox-regulated transcription factors (36), interact with PAP-dependentsignaling. Both PAP-dependent signaling and transcription regulation would be downstream ofchloroplastic H2O2 production. The delivery of chloroplastic H2O2 directly to the nucleus doesnot seem to provide sufficient information because from the nuclear perspective a specificityfactor that could differentiate, e.g., chloroplastic and peroxisomal H2O2, would be missing. CouldPAP be such a specificity factor? This is discussed further in Section 4.2.

AOX

ANAC017 ANAC013

Nuclear "companion" chloroplasts

CAT

SA

ASCGSH

JA, IAA

β-cyclocitral

SAL1 PAP

Stromule

MEcPP

Proteins

ASC

GSH

NADPH

L

L

Lred ox

red ox ox

Downstreamsignaling components

ANAC017

ANAC013

PPPPPP

?

GRI

MC9

PRK5

SAL1PAPPAPS PAPSOT12

PAP

2CPA

PAPS

ROS- and redox-regulated TFs

PAPPAP XRN 5' 3' RNA decay

H2O2H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

1O2β-carotene

Ca2+

Ca2+

Ca2+Ca2+

RCD1

RAP2.4a

H2O2

2CPA

Apoplast

Peroxisome

Mitochondrion

Nucleus

ER

Chloroplast


CASMDS

LIGHT

LIGHT

(Caption appears on following page)


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Figure 3 (Figure appears on preceding page)

ROS signaling processes. Information from elevated ROS concentrations or altered compartmental redox balance resulting fromactivity of ROS-scavenging systems is rapidly transferred to the nucleus and triggers adaptation or acclimation mechanisms. The initialROS-sensing events are mediated by ROS- and redox-sensitive proteins or direct oxidative damage of cellular metabolites, e.g.,β-carotene. Accumulation of ROS within the apoplast triggers a rapid increase in cytosolic Ca2+. The apoplastic ROS-sensingmechanisms as well as the identity of ROS-dependent Ca2+ channels are currently not known. Elevated production of ROS in thechloroplast leads to accumulation of PAP, which acts as a secondary messenger conveying the information about the redox status withinthe organelle. In consideration of the dual localization of the PAP catabolism enzyme SAL1, it can be assumed that a similar processcan occur in the mitochondria. Perturbed mETC triggers nuclear import of mobile transcription factors located in the ER that arenegatively regulated by the RCD1 transcriptional coregulator. RCD1 is necessary for part of the transcriptomic responses to alteredchloroplast and mitochondrial redox balance. SA-mediated inactivation of peroxisomal CAT leads to inhibition of JA and IAAbiosynthesis and alters the cytoplasmic glutathione redox status. H2O2 might be transported to the nucleus via stromules and fromsubpopulations of chloroplasts surrounding the nucleus. Nuclear ROS accumulation likely changes the activity of multiple ROS- andredox-sensitive transcription factors. Abbreviations: 2CPA, 2-CYS PEROXIREDOXIN A; ANAC, ARABIDOPSIS NAC DOMAINCONTAINING PROTEIN; AOX, alternative oxidase; ASC, ascorbate; CAS, CALCIUM SENSOR; CAT, catalase; ER, endoplasmicreticulum; GRI, GRIM REAPER; GSH, glutathione; IAA, indole-3-acetic acid; JA, jasmonic acid; MC9, METACASPASE9; MDS,mitochondrial dysfunction stimulon; MEcPP, methylerythritol cyclodiphosphate; mETC, mitochondrial electron transport chain;PAP, 3′-phosphoadenosine 5′-phosphosulfate; PRK5, POLLEN RECEPTOR LIKE KINASE5; RAP2.4a, RELATED TOAPETALA-2.4a; RCD1, RADICAL-INDUCED CELL DEATH1; ROS, reactive oxygen species; SA, salicylic acid; SOT12,SULFOTRANSFERASE12; XRN, exoribonuclease.

2.2. Mitochondria

In photosynthetic tissues, the contribution of mitochondria to total cellular ROS productionis relatively low. However, mitochondrial redox balance can serve as an indicator of multipleenvironmental cues and is intrinsically linked to the chloroplast function. In the following sectionwe analyze how mitochondrial redox status contributes to regulation of gene expression.

2.2.1. Mitochondrial ROS production. Mitochondrial ROS production is tightly associatedwith the mitochondrial electron transport chain (mETC), located in the inner mitochondrialmembrane. Mitochondrial complexes I, II, and III are believed to be the major sources of O2

·−

production; however, the relative importance of these production sites is difficult to assess andmost information is derived from animal mitochondria (55). Complexes I and II produce O2

·− atthe matrix side of the inner mitochondrial membrane (Figure 2). In animal mitochondria, complexIII produces O2

·− on both sides of the inner mitochondrial membrane (8). Because there are nomajor differences between plant and animal complex III, it can be assumed that O2

·− productionon both sides of the inner mitochondrial membrane occurs in plants (Figure 2). Thus, ROS inthe intermembrane space (IMS) could have a signaling function in plants similar to that in animals(8). It is not clear whether the IMS has enzymatic ROS scavengers; however, in consideration ofthe permeability of the outer mitochondrial membrane, O2

·− dismutation and H2O2 quenchingcould depend on cytoplasmic components. Additionally, because the last step of ascorbate (ASC)biosynthesis takes place in the mitochondrial IMS, O2

·− could be scavenged directly by reducedASC and thus alter the ASC redox state, which could act as a signal. Within the mitochondrialmatrix O2

·− is dismutated to H2O2 either spontaneously or via the mitochondrial manganese-SOD; subsequently, H2O2 is scavenged by Prxrs (45) and APX, because a full set of enzymesnecessary for completion of the ascorbate-glutathione (ASC-GSH) cycle have been localized toplant mitochondria (22).

2.2.2. Mitochondrial ROS and redox signaling. Plants have strategies to avoid mitochon-drial ROS formation. Electron flow is redirected through an alternate pathway bypassing the


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complexes III and IV (55). Alternative oxidases (AOXs) have a major role in this redirectionupstream of complex III when, for example, functioning of complex III is suboptimal. Althoughno major impacts on development, physiology, or metabolism have been reported for AOXmutants grown under favorable conditions (131), AOX is indispensable under conditions thatcause mitochondrial redox imbalance (50). Stress conditions perturbing mETC activate AOX byreducing the AOX dimer and by increasing the expression of genes that encode AOX and severalother mitochondria-targeted proteins (mitochondrial dysfunction stimulon, MDS) throughmitochondrial retrograde signaling, most likely in response to complex III–derived ROS (28,102). A subsequent increase in AOX activity directs electrons to O2 before complex III to alleviateROS formation and counteract the altered redox imbalance. The execution of this processinvolves two endoplasmic reticulum (ER)-localized transcription factors ARABIDOPSIS NACDOMAIN CONTAINING PROTEIN13 (ANAC013) (28) and ANAC017 (102), which arethought to translocalize to the nucleus in response to organelle-dependent stress signals. Recentresults on chloroplast-mitochondrion crosstalk are discussed further in Section 4.2.

2.3. Peroxisomes

Peroxisomes host multiple metabolic processes, many of which lead to formation of ROS. In thissection we review factors shaping the peroxisomal ROS pool and present the most recent findingsin peroxisomal ROS signaling.

2.3.1. Peroxisomal ROS production. Under the current concentration of CO2 in the atmo-sphere a significant proportion of RuBisCO catalytic cycles result in oxygenation rather thancarboxylation of ribulose-1,5-bisphosphate. The resulting 2-phosphoglycolate is metabolized toglycolate and transported to peroxisomes, where it undergoes glycolate oxidase–dependent oxida-tion to glyoxylate with concomitant production of H2O2 (Figure 2). In photosynthetically activetissues, peroxisomal ROS production is tightly linked to photosynthesis and accounts for most ofthe ROS produced (49). However, because peroxisomes have a potent H2O2-scavenging system,flux through the photorespiratory pathway does not necessarily translate into an increase in H2O2

content. Catalases (CATs) are major peroxisomal H2O2 scavengers, and mutant plants deficientin CAT activity exhibit severe growth abnormalities dependent on photoperiod, light intensity,and CO2 concentration (110). Despite the relatively low affinity for H2O2, CATs can efficientlyremove photorespiratory H2O2 and keep the estimated peroxisomal H2O2 concentrations be-low 10 μM (49). Whereas the existence of peroxisomal APX and ASC-GSH cycle enzymes hasbeen demonstrated for multiple species (31), no phenotypes have been observed in Arabidopsisplants deficient in peroxisomal APX3 (101), possibly indicating a relatively low contribution tothe peroxisomal antioxidant system.

2.3.2. New concepts in peroxisomal ROS and redox signaling. The paradigm of a purelymetabolic function for peroxisomal H2O2 scavenging is slowly changing. Peroxisomal ROS levels,closely regulated by CAT activity, are now emerging as a source of signals, with most informationoriginating from the analysis of Arabidopsis plants deficient in CAT2, the primary peroxisomalH2O2 scavenger (110). Disruption of photorespiratory H2O2 scavenging combined with exposureto photorespiration-promoting conditions shifts the redox status of the cellular GSH and ASCpool toward a more oxidized state (110), coinciding with rapid transcriptome reprogramming (70,110). However, under steady-state conditions little or no increases in H2O2 concentration could beobserved in cat2 mutants (92), possibly owing to low glycolate oxidase activity (70) combined withthe antioxidant function of cytoplasmic H2O2-scavenging enzymes (137). This finding suggests


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JA: jasmonic acid

that some of the phenotypes observed in cat2 plants might be related to perturbed glycolatemetabolism or altered cytoplasmic redox balance rather than H2O2 buildup.

The efficiency of CATs raises a crucial question about the signaling role of photorespiratoryH2O2. Can CAT activity decrease to allow transient H2O2 accumulation? Recent results suggestthat this could take place at least during SA signaling in plant-pathogen interactions (Figure 3).Following the seminal study demonstrating that binding of SA to CAT decreased its activity (20),Yuan et al. (153) showed that SA-mediated inhibition of peroxisomal H2O2 scavenging inhibitsauxin and jasmonic acid (JA) biosynthesis to increase the resistance to biotrophic pathogens. Thisfinding agrees with earlier reports that describe increased resistance of cat2 plants to biotrophicbacteria (18). At the molecular level, inhibition of auxin biosynthesis was related to sulfenylationand deactivation of TRYPTOPHAN SYNTHETASE β SUBUNIT1 (TSB1), which deliversprecursors for auxin biosynthesis, and inhibition of JA synthesis was attributed to the requirementof CAT for the activity of peroxisomal acyl-CoA oxidases ACX2 and ACX3 (153). TSB1 is locatedin the chloroplast stroma; therefore, the regulation of TSB1 activity is likely related to secondaryeffects following the decrease in CAT activity, e.g., altered chloroplast redox status. Furthermore,this could mean that any treatment resulting in chloroplastic redox imbalance might affect TSB1activity, as from the chloroplast perspective it is difficult to differentiate between internal andexternal ROS and redox signals.

In addition to inhibition by SA, CAT activity can be regulated by many other factors. In-creases in cytoplasmic Ca2+ were rapidly followed by a Ca2+ rise in peroxisomes (25), whichpromoted CAT activity possibly via Ca2+-dependent interactions between CAT and calmodulin(150). Optimal CAT function was maintained by the chaperone NO CATALASE ACTIVITY1(82), providing another mode of regulation for its activity. NUCLEOREDOXIN1, a pathogen-inducible oxidoreductase, was found to be a crucial determinant of CAT function (74). Anotherfactor contributing to an increase in the rate of peroxisomal ROS production could be the ac-tivity of cytosolic glyoxylate reductase (GLYR1 in Arabidopsis), which reduces glyoxylate back toglycolate, potentially enabling its peroxisomal uptake for reoxidation. Recently, Denecker et al.(33) demonstrated that introduction of the glyr1 mutation into the cat2 background improved thesurvival of the double mutant under conditions promoting photorespiration.

2.4. Apoplast

The apoplast serves as an interface for the exchange of nutrients and signals between plant cellsand the environment. In many cases, plant responses to environmental and endogenous stimuliinvolve the accumulation of ROS within this compartment. Here, we review the most prominentproduction mechanisms and signaling roles of apoplastic ROS.

2.4.1. Apoplastic ROS production. In contrast to intracellular sources, apoplastic ROS produc-tion results from active stimulation of ROS-producing enzymes such as apoplastic peroxidases,polyamine oxidases (PAOs), and plasma membrane-localized NADPH oxidases (respiratory burstoxidase homologs, RBOHs) (Figure 2) (64, 73, 119). The role of apoplastic peroxidases as ROSproducers was initially shown pharmacologically (86) and later by silencing (7) or generating stablemutant lines (27), which established two Arabidopsis peroxidases, PRX33 and PRX34, as key en-zymes contributing to the apoplastic ROS burst in biotic stress. However, although accumulatingevidence supports the importance of apoplastic peroxidases especially in plant immunity, little isknown about the molecular mechanisms that regulate the activity of these enzymes.

PAOs catalyze catabolism of spermidine and spermine with concomitant production of H2O2.The PAO-generated H2O2 plays a role under biotic (151, 152) and abiotic stress conditions (98),


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Microbe-associatedmolecular patterns(MAMPs): moleculesof microbial originthat elicit immuneresponses

as well as in plant development (126). Multiple lines of evidence suggest that polyamine availabilityis a major factor contributing to the regulation of H2O2 production by PAOs.

NADPH oxidases, which are perhaps the most important class of apoplastic ROS producers,transfer electrons across the plasma membrane from cytoplasmic NADPH to molecular oxygento produce O2

·−. The Arabidopsis genome contains 10 genes for the RBOH isoforms (A–J) (123).Most ROS signaling during abiotic and biotic stress relies on the activity of two partially redundantisoforms, RBOHD and RBOHF (64, 77, 94, 129, 130); some of the other isoforms are involved inthe regulation of plant development (discussed in Section 6). In contrast to the activity of apoplasticperoxidases, the molecular mechanisms regulating the activity of NADPH oxidases are relativelywell understood and involve transcriptional (97) and posttranslational regulation (64, 73, 119).

The O2·− formed in the apoplast can be dismutated to H2O2 either spontaneously or by

apoplastic SODs. In Arabidopsis, the identity of apoplastic SODs remains unknown, even thoughindirect evidence suggests their existence. None of the seven canonical SODs possess a secretorysignal peptide, but some are found in the extracellular space following activation of a SA-inducedsecretory pathway (21). Two putative SODs, AT3G56350 and AT4G00651, are predicted to besecreted; however, the function of these proteins remains to be determined. Intriguingly, comparedwith the intracellular environment, the apoplast is maintained in a relatively oxidized state and isestimated to contain most of the leaf H2O2 (see 49 and 103 for the estimates) while containinglow concentrations of ASC and GSH. The apoplastic metabolism of these compounds clearly hasimplications for cellular metabolism, including acclimation of photosynthesis to fluctuating light(68); however, it is not clear whether this is related to their antioxidant functions.

2.4.2. Ozone as a tool for investigating apoplastic ROS. Whereas the accumulation of ROS inthe apoplast serves to coordinate local responses, such as execution of cell death, in plant-microbeinteractions, early apoplastic ROS accumulation probably serves as a signal to neighboring ordistal cells. Therefore, it is crucial to recognize and separate the signaling events triggered by ROSaccumulation within the apoplast from those dependent on local recognition of microbe-associatedmolecular patterns (MAMPs), although these processes might not be completely independent (73).Such approaches might focus on signaling events initiated by perception of MAMPs in rboh mutants(148) that identify ROS-independent responses, or utilize apoplastic ROS donors such as ozone(O3) to investigate ROS-dependent responses (134). O3 enters plant tissues through stomata,decomposes in the cell walls to various ROS, and triggers active ROS generation—ultimatelyleading to the formation of hypersensitive response-like cell death (67, 134).

Application of O3 as an experimental tool effectively mimics the intrinsic ROS burst specific tothe apoplast. Unlike other methods, it can be applied noninvasively at a precise concentration andduration (67, 134) and has been described as maybe the most environmentally relevant way to probethe function of ROS in signaling processes (103). Among the first events following accumulationof apoplastic ROS is the activation of H2O2-dependent Ca2+ channels, which mediate the influxof apoplastic Ca2+ into the cytoplasm (107). The identity of these channels remains elusive stilland presents a major challenge in understanding apoplastic ROS signaling. Other early responsesinclude accumulation of ethylene (134); however, the regulation of most O3 responsive transcriptsis independent of ethylene, as well as SA and JA, signaling (147), suggesting the existence ofROS-dependent apoplast-to-nucleus signaling pathways independent from hormonal signaling.

2.4.3. Sensing of ROS within the apoplast. The signaling function of apoplastic ROS was sug-gested over 20 years ago (67) and is now well established (64, 73, 119, 123). However, the initialprocesses in apoplastic ROS perception, as well as in other subcellular compartments, are still un-resolved. In view of compartment-specific ROS signaling, mechanisms for apoplastic ROS sensing


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Ectodomain: theapoplastic domain ofplasmamembrane–localizedprotein

Dipole moment:a measure of moleculepolarity

HyPer: geneticallyencoded H2O2-sensing probe

proposed thus far (73, 119) assume the existence of local signaling components that monitor theapoplastic redox status. These mechanisms include sensing of apoplastic H2O2 or redox status viaspecialized sensors that could detect ROS, e.g., directly via oxidative posttranslational modifica-tions (PTMs) or indirectly through oxidized apoplastic proteins, metabolites, or both (Figure 3;discussed in Section 3). The apoplast contains a number of cysteine-rich peptides (127) thatcould participate in ROS sensing. Another group of proteins that function in processes relatedto apoplastic ROS accumulation are cysteine-rich receptor-like kinases (CRKs) (11). Evidencesupporting their possible role in apoplastic ROS sensing includes (a) the presence of conservedcysteines in the ectodomain, (b) transcriptional regulation in response to apoplastic ROS (145),and (c) a functional analysis of crk mutant plants (11). Additionally, according to publicly avail-able interactome data (62), a set of CRKs interact with RBOHs. Furthermore, emerging evidenceindicates cysteine-dependent function of CRKs (149); however, it remains unclear whether theseresidues are involved in signaling processes or have structural functions.

The existence of local perception mechanisms has been recently supported by the discovery ofdownstream processes involved in the regulation and progression of O3-induced cell death, whichrelies on RBOHD, an apoplastic cysteine-rich protein GRIM REAPER (GRI), and a receptor-likekinase (RLK), POLLEN RECEPTOR LIKE KINASE5 (PRK5), which together may form anapoplastic signaling module (144, 146). Cleavage of the GRI preprotein by METACASPASE9resulted in a 12-amino-acid GRI peptide that bound to its receptor PRK5 to initiate the onsetof ROS-dependent cell death (146). The whole process was dependent on ROS produced byRBOHD, but whether ROS were acting on GRI, involved in the activation of METACASPASE9,or required for the interaction of the short peptide ligand with PRK5 is unknown.

2.5. Transport of ROS Through Membranes

Another line of evidence supports intracellular perception of apoplastic ROS. The dipole momentof H2O2 is larger than that of H2O, preventing free diffusion through membranes. However, ac-cording to studies of yeast survival, multiple plant aquaporins can transport H2O2 (reviewed in6). Following these initial discoveries, Costa et al. (25) demonstrated that extracellular supple-mentation of H2O2 to plant cells expressing a cytoplasmic HyPer probe provoked accumulationof H2O2 within the cytoplasm with no apparent delay, indicating rapid transfer through theplasma membrane. The H2O2 permeability of biological membranes was also demonstrated inchloroplastic ROS signaling, because even under low-light intensities a fraction of chloroplasticROS leaked or was transported from the chloroplast to the cytosol (100). Finally, the aquaporinPLASMA MEMBRANE INTRINSIC PROTEIN1;4 (PIP1;4) imported apoplastic ROS duringplant-pathogen interactions (128) and PIP2;1 was suggested to mediate H2O2 transport in guardcell signaling (51). Mechanisms for apoplastic and cytosolic perception of apoplastic ROS likelycoexist to fine-tune cellular responses. However, intracellular sensing of apoplastic ROS raisesquestions related to ROS signaling specificity (see Section 4.1 and the sidebar titled Are Tissue-and Organ-Resolution ROS Concentration Measurements Biologically Relevant?).

3. ROS-SENSING MECHANISMS

At the molecular level the biological roles of ROS are dictated largely by their ability to react with abroad range of proteins and metabolites as well as by their consumption of reducing equivalents bythe ROS-scavenging machinery. Most ROS-sensing mechanisms are thought to use the oxidizingproperties of ROS. The initial ROS-sensing events include oxidative PTMs of sensory proteins andoxidation of metabolites. Additionally, the activity of ROS-scavenging enzymes shifts the redox


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ARE TISSUE- AND ORGAN-RESOLUTION ROS CONCENTRATIONMEASUREMENTS BIOLOGICALLY RELEVANT?

In many published articles, ROS concentration from whole leaf and organ samples has been measured by severaldifferent methods. However, recent results indicate that such measurements, apart from being highly dependent onthe techniques (see 103 for a detailed description of critical issues in ROS measurements), might not be biologicallyrelevant in the context of ROS signaling. Tissue-level ROS measurements average the steady-state ROS levels ofthe whole cell, neglecting compartment-specific differences (49); thus, these measurements cannot give meaningfulinformation about the changes at the subcellular level, which can have important implications for signaling function.Furthermore, experiments that use the membrane-anchored HyPer genetically encoded H2O2 sensor indicate theexistence of discrete ROS maxima and a limited ROS diffusion distance (95). Such spatial distribution of ROS mightbe explained by a local downregulation of cellular antioxidant mechanisms that enables controlled accumulation ofROS, triggering downstream signaling processes (142, 143). Therefore, in order to gain more information about thephysiological relevance of ROS signaling, the application of nondiffusible ROS sensors at a subcellular resolutionis necessary (139). The question about localized ROS sensing gains additional importance in the context of theheterogeneous environment within subcellular compartments, e.g., the plasma membrane (60).

pKa: logarithmicderivative of the aciddissociation constant;negatively correlateswith the ability of acidto dissociate

status of electron donors such as NADPH, GSH, or ASC toward a more oxidized state. Impor-tantly, not all responses mentioned above can be attributed to signaling, especially if ROS accumu-lation has been artificially increased in mutant backgrounds or by the application of external stimuli.

3.1. Oxidative Posttranslational Modifications of Cysteine Residues

PTMs of proteins can alter the function or stability of proteins by adding covalent functionalgroups or by other modifications that affect protein properties. Phosphorylation, glycosylation,and ubiquitinylation are well-known examples of such PTMs. ROS and altered cellular redoxbalance can also directly affect a number of proteins which have signaling function that leads tochanges in cellular acclimation as a response to changes in the surrounding environment. Sulfuratoms in cysteine and methionine residues are the primary targets for H2O2-dependent oxidativePTMs of ROS-sensitive proteins.

3.1.1. Factors determining reactivity of cysteine residues. Reaction of H2O2 with the cysteinethiolate anion (-S−) leads to the formation of cysteine sulfenic acid (-SOH). Unless stabilized withinthe protein environment, -SOH reacts with either GSH, leading to protein S-glutathionylation(-SSG), or other thiol groups, resulting in the formation of intra- or intermolecular disulfide bonds(-S-S-). Under high concentrations of H2O2, or when the protective thiols are not available, -SOHcan undergo further oxidation to sulfinic (-SO2H) and sulfonic (-SO3H) acid, which in most casesleads to protein damage (117). Waszczak et al. (140) identified approximately 100 sulfenylatedcytosolic proteins in plant cells treated with H2O2; therefore, it can be assumed that sulfenylationis a widespread PTM. With the exception of sulfonylation, oxidative PTMs of cysteine residuesare reversible. Deglutathionylation and reduction of disulfide bonds are catalyzed by GRXs andTRXs, which form large multigene families (91). Reduction of sulfinic acid can be catalyzed bysulfiredoxins; however, in Arabidopsis this mechanism has been described only for chloroplasticand mitochondrial Prxrs (56, 114). Within ROS-sensitive proteins the susceptibility of cysteineresidues to react with H2O2 is not equal and is determined largely by the local electrostaticenvironment affecting cysteine residue pKa. For example, relatively nonreactive cysteine thiol


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Green fluorescentprotein (GFP): emitsfluorescence whenexposed to light ofcertain wavelengths

Rate constant:measure of chemicalreaction rate

groups (-SH) are more prone to oxidation in their activated thiolate anion (-S−) form; therefore,the local pH largely determines the susceptibility of cysteine residues to oxidation (116).

3.1.2. ROS or redox signaling? GRXs and TRXs, which reduce multiple forms of cysteine ox-idative PTMs, are selective toward their target proteins (118) and their reactivity is determinedlargely by the availability of reducing equivalents, GSH and reduced ferredoxin (Fdred) or NADPH.Therefore, conditions that limit the abundance of electron donors, e.g., darkness for Fdred or ac-tivity of the GSH-ASC cycle for GSH, ultimately limit the function of GRXs and TRXs. Thus,the status of oxidative PTMs of cysteine residues is a result of H2O2 concentration (usually termedROS signaling) and the availability of reducing equivalents (usually termed redox signaling). Al-though these two mechanisms might be connected, redox signaling is a much broader processbecause it does not necessarily result from increased ROS concentrations at the site of signalperception. An example of such signaling is the light-dependent reducing activation of Calvincycle enzymes by the ferredoxin-TRX redox relay (118). Another example was recently demon-strated with a redox-sensitive roGFP2 probe, constructed by introducing cysteine residues into thenascent green fluorescent protein (GFP) sequence to enable the formation of a redox-dependentdisulfide bond that influences the GFP emission spectra (37). Later, it was discovered that thein vitro oxidation of roGFP2 by H2O2 is a slow process, and in plants the fast in vivo responseresults from GRX-mediated reduction or formation of disulfide bonds within roGFP2. Therefore,the roGFP2 probe responds to cellular redox changes by monitoring the GSH/GSSG ratio (90).Results obtained with this system suggest that GRXs might serve as a sensor of the GSH redoxstatus and target host proteins for signaling purposes. The two examples listed above indicate thataltered compartmental redox balance might change the activity of multiple enzymes and signalingproteins, even when the target proteins are distant from the site of ROS formation.

3.1.3. Two-step ROS-sensing mechanisms. In the context of ROS signaling, emerging evi-dence indicates that oxidation of cysteine residues within the target proteins might be catalyzedby specialized ROS-scavenging enzymes such as Prxrs or GPXs characterized by extremely highrate constants for reactions with H2O2, enabling perception of physiological ROS concentrations.Upon oxidation, these sensor proteins interact with and oxidize effector proteins, forming a redoxrelay. Thus far, the only example of such a redox relay in plants has been described for the GLU-TATHIONE PEROXIDASE-LIKE3 (GPXL3)–ABA-INSENSITIVE2 (ABI2) H2O2-sensingsystem, which has been suggested to control stomatal closure (93). In vitro, GPXL3 underwentH2O2-dependent oxidation and interacted with and oxidized ABI2, ultimately inhibiting its ac-tivity (93). However, recent data suggest that such interaction is unlikely in planta as GPXL3localizes to the ER membrane with its catalytic domain facing the ER lumen (4), whereas ABI2 isa cytoplasmic protein. It remains to be elucidated whether ABI2 interacts with other GPXLs orother specialized H2O2 sensors and whether it is able to directly sense H2O2 as demonstrated invitro (88). Analogous redox relay mechanisms in yeast have been described, in which the thiol per-oxidase GPX3 oxidizes the transcription factor YAP1, ultimately leading to its nuclear import andtranscriptional activity (32). In humans, Prxr-2 oxidizes the transcription factor STAT3, leadingto its inactivation (120). Because cysteine thiols in most proteins are less susceptible to oxidationthan those of thiol peroxidases, it is likely that many direct ROS-sensing mechanisms can utilizesimilar signaling principles.

3.2. Oxidative Posttranslational Modifications of Methionine Residues

In addition to cysteine thiols, methionine residues can be subject to oxidative PTMs. Re-action of H2O2 with methionine residues (-S-CH3) leads to the formation of methionine


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sulfoxide (-(S = O)-CH3), which can be further irreversibly oxidized to methionine sulfone(-(SO2)-CH3). Methionine sulfoxide can be reduced by a large group of methionine sulfoxidereductases, which, along with target substrate specificity, exhibit stereoselectivity toward S- andR- isomers of methionine sulfoxide (125). As part of the catalytic cycle, methionine sulfoxidereductases utilize TRX as the electron donor; therefore, reduction of methionine sulfoxide issubject to limitations described in Section 3.1.2. Exposure of Arabidopsis to photorespiratorystress resulted in oxidation of methionine residues in approximately 400 proteins (59), indicatingthat methionine oxidation is a common PTM. However, compared with mechanisms involvingcysteine oxidative PTMs, signaling events triggered by methionine oxidation remain largelyuncharacterized. Most methionine oxidation events inactivate protein function (59, 79); however,recent data from bacterial models indicate that it might also have the opposite effect (38).

4. INTRACELLULAR INTERACTIONS BETWEEN ORGANELLE ROSAND REDOX SIGNALING

It is common to compare gene expression data from chemical, genetic, and conditional alterationsin ROS and redox homeostasis; however, the concept of a specific transcriptional marker geneis inaccurate (132). In a more recent cross-study comparison of microarrays, there did not seemto be direct correlation with the subcellular production site or ROS type, but rather with thetemporal production of ROS and many later responses appeared to converge with the RBOHF-mediated ROS burst (141). However, an outstanding question in ROS signaling is how signalorigin information is retained or transmitted from the same ROS molecule produced in differentsubcellular compartments once the signal reaches the nucleus. This question is not likely to beanswered by transcriptional analyses but rather from consideration of the biochemical nature andlocation of these molecules.

4.1. How Is Signaling Specificity Retained in the Cytosol?

ROS alone are unable to transmit information about their site of origin. Initial predictions (96)therefore correctly suggested that (a) ROS might rely on site-specific sensors for signal perception,for which recently characterized and hypothetical mechanisms are described in Section 3, and that(b) signal specificity could be achieved through local production of secondary ROS messengersin the form of source-specific metabolites such as PAP (Sections 2.1.2 and 4.2), their oxidativederivatives, e.g., β-cyclocitral, or both (Section 2.1.1). Recent findings suggest that specificitycould also be achieved by direct positioning of H2O2 near the nuclear membrane from companionchloroplasts or stromule projections (Section 2.1.2).

Other mechanisms might require the presence of diverse, spatially distinct internal receptorssituated next to aquaporins within each compartment. The movement of ROS, e.g., throughchannels, could be sensed at the location of entry. Woo et al. (142) addressed this question us-ing an animal system and demonstrated that activation of a plasma membrane receptor led to adecrease in local cytoplasmic ROS-scavenging, allowing the formation of local ROS maxima thatmight be sensed by proximal sensor proteins. This finding highlights the need to further char-acterize transporters or aquaporins in different compartment membranes in the context of ROSsignal specificity (6). A seemingly analogous process is the regulation of Ca2+ stores, in whichCa2+ transport mechanisms involve diverse membrane- or compartment-specific transportersand internal or external sensing and activation mechanisms. For example, 10 calcineurin B-likeCa2+ sensor proteins (CBLs) and 26 CBL-interacting protein kinases (CIPKs) with cell type andsubcellular spatial diversity have been identified in Arabidopsis (121), suggesting that they mightcontribute significantly to Ca2+ specificity. Although both ROS and Ca2+ molecules are tightly


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Methyl viologen:a herbicide whosemajor mode of actioninvolves enhancedproduction ofsuperoxide anions inthe chloroplasts

controlled to reduce damage, Ca2+ signatures are thought to confer specificity in the cytosol in theform of concentration fluctuations that are reproducibly distinct from particular stimuli. H2O2 incontrast would not last long in the cytosol owing to scavenging, and compartment-specific locationinformation would be rapidly lost owing to cytoplasmic streaming (96, 138).

4.2. Chloroplast-Mitochondrion Crosstalk, Signaling, and PAP

As described in Section 2.1.2, the accumulation of PAP in chloroplasts has been suggested to actas a retrograde signal that relays information of chloroplastic redox state to the nucleus. However,there is evidence that this process involves additional interorganelle crosstalk. The specific seriesof events leading to activation of the PAP retrograde signal are as follows: 3′-phosphoadenosine-5′-phosphosulfate (PAPS) is synthesized in the chloroplast and, to a lesser extent, in the cytoplasm.Following export from chloroplasts, PAPS is used in the cytoplasm as a sulfate donor for sulfo-transferases (SOTs) such as SOT12 (9). The resulting PAP is transported back to the chloroplastvia the PAPS/PAP antiporter PAPST1 according to a concentration gradient (9). Normally, thedual chloroplast-mitochondrion-localized PAP phosphatase, SAL1, then detoxifies PAP in chloro-plasts and maybe also in mitochondria. However, changes in the chloroplastic redox state or inH2O2 production can inactivate SAL1 (16), causing PAP accumulation. This correlates with theexpression of specific nuclear genes, proposedly by affecting nuclear 5′-to-3′ exoribonuclease ac-tivity. This promotes acclimation to high light, drought (16, 41), and plant immunity throughregulation of SA- and JA-mediated signaling pathways (58).

Although SAL1 is dual-targeted to both chloroplasts and mitochondria and is likely to be in-volved in mitochondrial retrograde signaling, it is at present only a putative sensor of mitochondrialredox status (135). The role and function of PAP in interorganellar signaling also appear increas-ingly complex by the likely involvement of the nuclear protein RADICAL-INDUCED CELLDEATH1 (RCD1), originally identified as an O3-induced lesion mutant (1). RCD1 is a nucleus-localized transcriptional hub protein with a specific domain for protein-protein interactions (61,105). RCD1 has been identified in multiple screens, including as one of five redox imbalance (rimb)mutants in a screen for mutants lacking chloroplastic redox-sensitive 2-Cys Prxr gene reporter ac-tivity, where it was suggested to be responsible for chloroplastic redox change–induced activationof antioxidant genes via interaction with the redox-regulated transcription factor RAP2.4a (54).

A functional interaction between mitochondrial and chloroplastic retrograde signaling involv-ing ANAC017 and PAP has been proposed (136). RCD1 has a role in mitochondrial ANAC013and ANAC017 signaling (described in Section 2.1.2) owing to its direct interaction with ANAC013(61, 105). ANAC017 is involved in the regulation of chloroplastic redox imbalance induced bymethyl viologen (135), to which rcd1 is resistant (106). ANAC017 regulates the expression ofANAC013 (135), which, in addition to a self-amplifying loop, upregulates the MDS (28), and PAPinduces the same genes (136). Because the rcd1 mutant has elevated expression of ANAC013 andother MDS genes (13, 61), RCD1 appears to be a negative regulator of PAP signaling and of thenuclear function of ANAC013.

Although RCD1 may be involved in coordinating PAP-derived chloroplastic and mitochondrialredox states in the nucleus, how PAP retains specific information from organelles when it issynthesized in the cytosol is still unclear. A possible mechanism is through SOT12, which makesPAP from PAPS in the cytosol. Expression of SOT12 is negatively regulated by RCD1 (13,61) and positively regulated by ANAC013 (28), ANAC017 (135, 136), and PAP (136), whichsuggests that the ROS- or redox-dependent inactivation of SAL1 in chloroplasts (16) will lead to anincrease in cytoplasmic PAP concentration also by a positive feedback loop. This also implies eitherthat a second organelle-specific signal is required for organelle specificity, or that chloroplast-mitochondrion PAP responses are linked (102).


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5. APOPLASTIC AND ORGANELLE ROS INTERACTIONSDURING STRESS

As potent signaling molecules, ROS control many aspects of plant-environment interactions, aswell as growth and development. In the following section we review major functions of ROS inlong-distance signaling, plant-pathogen interactions, and stomatal closure.

5.1. Cell-to-Cell ROS Signaling

Single-cell models do not adequately illustrate how ROS signals can affect the surrounding tissueand whole plant (Figure 4). A pivotal study by Miller et al. (94) identified a self-propagating

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Figure 4ROS in long-distance signaling. Perception of local stimulus triggers rapid activation of NADPH oxidases that produce superoxideanions on the apoplastic side of the plasma membrane. Superoxide anions are dismutated to H2O2 and diffuse within the apoplast to theneighboring cells. Apoplastic H2O2 activates plasma membrane–located Ca2+ channels, ultimately leading to an increase in theconcentration of Ca2+ in the cytoplasm resulting from an influx from the apoplast and other intracellular Ca2+ stores. Ca2+ activatesRBOHD directly by binding to the EF-hands (Ca2+-binding motifs) and indirectly by activating multiple CDPKs, which in turnresults in accumulation of ROS within the apoplast of neighboring cells. These processes are most likely accompanied by signalingevents triggered by apoplastic or cytoplasmic ROS sensors. Such a sequence of events allows the systemic spread of information in theform of a self-propelling wave. Abbreviations: CDPK, calcium-dependent protein kinase; ER, endoplasmic reticulum; GLR, glutamatereceptor-like; RBOH, respiratory burst oxidase homolog; ROS, reactive oxygen species; TPC1, TWO-PORE CHANNEL1.


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mechanism for cell-to-cell signaling via RBOHD-derived apoplastic ROS—a central responseto multiple internal ROS signaling mechanisms outlined above (141). The relatively oxidizedstate of the apoplast and low redox regulation and antioxidant capacity during stress would allowfaster diffusion of H2O2 in the apoplastic space than in the cytosol (49). Individual cell-to-cellROS-relaying stations in the apoplast could also more rapidly diffuse signals than symplasticmovement via plasmodesmata (138). Current gaps in these cell-to-cell models are (a) how do ROSsignals in the apoplast reach and affect the cytosolic signaling components, (b) how do these initialcompartment-specific ROS-sensing and ROS-signaling mechanisms retain specificity, and (c) howdo they then relate to downstream hormone signaling.

Since the discovery of the central role of RBOHD and the apoplastic movement of H2O2, morecomplex internal mechanisms for phosphorylation of NADPH oxidases have been elucidated invivo in plants. Particularly important discoveries include MAMP and ROS-induced phosphoryla-tion of RBOHD via the Ca2+ sensor CALCIUM-DEPENDENT PROTEIN KINASE5 (CPK5)(39); phosphorylation of RBOHD and RBOHF by CIPK26, CBL1, and CBL9 (122); and the dis-covery of the ROS-activated tonoplastic channel TWO-PORE CHANNEL 1 (TPC1), whichreleases Ca2+ into the cytosol during stress (24). This evidence suggests that coordination of Ca2+

and ROS signals occurs via Ca2+-dependent phosphorylation of NADPH oxidases; however, otherintersections such as the inclusion of vacuolar TPC1 in these models are still speculative (42, 122).

In a similar way, long-distance electrochemical wound-activated surface potential changesrequire functional putative Ca2+-permeable glutamate receptor-like cation channels, GLR3.3 andGLR3.6, which are activated cell autonomously almost immediately in the absence of a locallyinitiated ROS or Ca2+ wave (23, 99, 122). Given that GLR3.1 and GLR3.5 channels providethe basal cytoplasmic Ca2+ levels required to activate NADPH oxidases (75), it is tempting tospeculate that this is also the case for GLR3.3 and GLR3.6. Electrochemical activation of GLRchannels could also suggest analogous activation of specific electrochemically activated receptorsrequired for opening aquaporins.

5.2. ROS in Plant Immunity and Stomatal Closure

The phenomenon of enhanced disease resistance triggered by increased production of apoplasticROS was demonstrated over 20 years ago. ROS have a direct antimicrobial effect; they are involvedin cell wall stiffening and, most importantly, act as local and systemic signal molecules that areinvolved in the activation of antimicrobial defenses.

5.2.1. Regulation of apoplastic ROS sources and activity during plant-microbe interac-tions. With the use of specific inhibitors and mutant plants, multiple studies have contributed toour understanding of apoplastic ROS in plant immunity, and the role of NADPH oxidases (64,73), apoplastic peroxidases (27), and PAOs (151, 152) is now established. However, it is difficultto determine the relative contributions of apoplastic ROS sources to the development of plantimmune responses, because currently available data indicate that all three contribute to a success-ful defense. Their roles are most likely separated temporally, with activation of NADPH oxidasesamong the first processes following perception of MAMPs and apoplastic peroxidases and PAOsperhaps playing a role in later stages.

Although multiple studies demonstrate partially overlapping functions of RBOHD andRBOHF, RBOHD appears to be a major isoform involved in plant immune responses. Thepredominant role of RBOHD can be observed at the level of promoter activity, as the RBOHDpromoter is highly responsive to MAMP or pathogen treatments, whereas the RBOHF promoteris largely unresponsive (97). The primary role of RBOHD is also evident from the molecular


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Chitosan:chitin-derivedmicrobe-associatedmolecular pattern

Cytokinins: a groupof plant hormones

mechanisms following the perception of MAMPs by RLKs (64). In Arabidopsis, perception ofMAMPs such as flagellin (epitope flg22) and elongation factor Tu (EF-Tu; epitopes elf18 andelf26) by their receptors leads to the activation of receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE1 (26), which further phosphorylates and activates RBOHD (65, 83). Signal-ing events leading to RBOH activation downstream of MAMP perception and those initiated byRLKs not directly involved in plant pathogen responses have been recently reviewed (26, 64, 73).

5.2.2. Apoplast-to-chloroplast signaling. Among the most interesting events following the ini-tial apoplastic ROS burst is the accumulation of ROS in the chloroplasts. The light dependencyof the hypersensitive response suggests that chloroplasts have a crucial role in coordinating ROSproduction during plant immune responses (84). Similar conclusions can be drawn from a reportdemonstrating the negative influence of chloroplast 2-Cys Prxr on pathogen-induced cell deathprogression (57). Finally, multiple pathogen effectors target the chloroplastic electron transportchain to limit ROS production (30). However, it remains unknown how the apoplastic ROS burstis involved in the regulation of chloroplastic ROS production. Furthermore, whereas the functionof chloroplastic ROS is established, it remains to be investigated how plants decrease their exten-sive chloroplast antioxidant capacity to allow ROS to accumulate. Results obtained by Nomuraet al. (104) indicated that Ca2+ signaling mediated by chloroplastic CALCIUM SENSOR (CAS)is a major determinant of chloroplast function related to plant immunity. Recognition of MAMPstriggers plasma membrane Ca2+ influx channels to open, resulting in transient Ca2+ concentrationfluctuations in the cytoplasm, followed by a prolonged CAS-dependent Ca2+ concentration in-crease in chloroplast stroma. Both early and late Ca2+ peaks were sensitive to inhibitors of serine/threonine protein kinase and MAPKKs but were largely unaffected by an NADPH oxidase in-hibitor. Further, cas plants exhibited partial reduction of late pathogen-induced ROS accumu-lation and impaired resistance to virulent and avirulent bacteria. Interestingly, CAS-dependenttranscriptomic responses appear to significantly overlap with those triggered by the accumulationof 1O2 (104), suggesting that 1O2 has a role in executing a CAS-dependent pathogen-inducedhypersensitive response.

5.2.3. ROS in the regulation of stomatal closure. Both RBOHD and RBOHF are necessaryfor executing stomatal closure in response to abscisic acid (ABA) and high levels of CO2 (19, 77).In contrast, stomatal movements initiated by perception of MAMPs rely mostly on RBOHD (85,89). Successful activation of RBOH-mediated ROS production requires basal level of cytoplasmicCa2+, which is provided by the H2O2-independent plasma membrane GLUTAMATE RECEP-TOR GLR3.1 and GLR3.5 Ca2+ influx channels (75). However, treatments with fungal elicitorssuch as yeast elicitor (71) and chitosan (72) or with cytokinins (2) rely mostly on apoplastic peroxi-dases as ROS sources. Furthermore, treatments with O3 (133) as well as H2O2 (109) are sufficient toinduce stomatal closure, indicating the existence of mechanisms for rapid perception of apoplasticROS and execution of further signaling events required for stomatal closure. However, the actualperception mechanisms remain elusive (119). Recent data indicate that the entry of apoplastic ROSinto the cytoplasm of guard cells is facilitated by aquaporin PIP2;1 as pip2;1 guard cells failed to ac-cumulate H2O2 in response to ABA and flg22 (115). Accumulation of ROS in the apoplast activatesthe still unidentified H2O2-dependent Ca2+ influx channels (107), and the increase in cytoplasmicCa2+ concentration triggers secondary Ca2+ transients in chloroplasts as well as activation of multi-ple Ca2+-dependent protein kinases that further stimulate the activity of RBOHs and activate anionchannels (119). At the same time, ROS accumulation in the guard cell chloroplasts is increased(133).


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Casparian strip:a structure locatedwithin the rootendodermis thatprevents free passageof solutes

Most signaling pathways leading to stomatal closure involve the function of OPEN STOM-ATA1 (OST1), which serves as a hub of information flow (119). The only stimulus identified thusfar that triggers stomatal closure in ost1 mutant plants is PAP (108), indicating that the activity ofCPKs is sufficient to execute stomatal closure. This recent finding agrees with a previous reportdemonstrating reduced stomatal response to abscisic acid in the cpk5 cpk6 cpk11 cpk23 quadru-ple mutant despite intact OST1 function (12). Thus, it was hypothesized that OST1 might actprimarily upstream of apoplastic ROS production and that part of the phenotypes observed inost1 mutants could be related to impaired activation of the ROS-regulated Ca2+ channels andconsequential compromised activation of CPKs (119). Further, the activity of OST1 is negativelyregulated by protein phosphatases type 2C, some of which in turn are inhibited by H2O2 (88),suggesting the existence of a self-amplifying loop linking OST1 and H2O2 in stomatal closure.

6. ROS IN PLANT DEVELOPMENT

As evident from the previous sections, ROS are crucial regulators of metabolism and plant stressresponses. However, in addition to these canonical functions, ROS are also inherent to multipleprocesses regulating plant development. In this context, most of the available functional dataestablish apoplastic ROS as the primary regulator of these processes. Here, we review selecteddevelopmental processes that require active ROS production.

Well-characterized developmental processes involving apoplastic ROS production are pollentube formation and root hair growth. RBOHC is the primary ROS-producing enzyme requiredfor the root hair elongation, as rbohC mutants are deficient in this process (47). RBOHC exhibitspolar subcellular localization associated mainly with the tip apex. Stimulation of apoplastic ROSproduction triggers an influx of extracellular Ca2+, which is required for cell elongation. Analo-gous to RBOH regulation in guard cell signaling, an influx of Ca2+ further activates RBOHC,creating a positive feedback loop (124). Following these initial discoveries, Jones et al. (63) foundthat production of O2

·− at the root hair tip depended on RHO-RELATED PROTEIN FROMPLANTS2, a GTPase acting upstream of RBOHC controlling ROS production. Conceptuallysimilar sequences of events control the polarized pollen tube elongation. Two apparently redun-dant RBOH isoforms, RBOHH and RBOHJ, served as sources of apoplastic ROS and were crucialfor proper pollen tube growth (10, 69, 78). Although the precise mechanisms regulating RBOHHand RBOHJ activity remain to be elucidated, two Catharanthus roseus RLK1-like (CrRLK1L)family kinases, ANXUR1 and ANXUR2, appear to be involved in this process, as anx1 anx2double mutants exhibited pollen tube growth deficiency and phenotypes observed in ANXUR1overexpressors were dependent on functional RBOHH and RBOHJ (10).

In addition to establishing the primary cell wall structure, apoplastic ROS are instrumental inregulating the development of secondary cell wall architecture. Cell wall lignification is amongthe secondary events following the perception of cell wall damage (CWD), and ROS producedby RBOHD are necessary for the execution of this response (52). Mutants deficient in RBOHDexhibit reduced lignin deposition and this effect is further intensified in the rbohD rbohF doublemutant (34). In the context of CWD signaling, the activity of RBOHD and RBOHF appears tobe controlled by the THESEUS1 CrRLK1L kinase, as the1 mutants accumulated less apoplasticROS following the induction of CWD (34).

In contrast to lignification induced by CWD, the developmentally regulated formation of theCasparian strip within the endodermis relies solely on the function of RBOHF (80). Interestingly,this production of precisely localized ROS appears to be determined by recruitment of RBOHFto the site of lignin deposition by Casparian strip domain proteins as well as specific activation ofRBOHF activity, as rbohF plants expressing the RBOHF catalytic domain under the control of the


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RBOHB regulatory domain failed to develop the Casparian strip (80). This observation impliesthe existence of a specific mechanism for regulating RBOHF activity that is not yet understood.

7. CONCLUSIONS

ROS are ubiquitous metabolites in all aerobic organisms and were originally regarded only asunwanted substances damaging to cells. However, studies performed over the course of the past twodecades and earlier have elucidated cellular and molecular mechanisms involved in the adaptationand acclimation of plants to their environment, highlighting the important signaling function forROS in these processes, and the concept of ROS as signaling substances has been established.Now, it is clearly recognized that controlled production of ROS enables these critical signals toact in response to stress and in development. This implies that there must be coordinated functionof signaling networks that govern ROS-responses, although detailed descriptions of how suchinteractions work are still mostly lacking. Furthermore, the perception of ROS and the immediateprocesses downstream of perception are still almost completely unknown in any species. Studiesaddressing these questions will result in discoveries of new, previously uncharacterized ROS-related regulatory mechanisms involved in the coordination of plant gene expression as well as inthe adaptation and acclimation of plants to their surrounding conditions.

SUMMARY POINTS

1. ROS levels are tightly controlled; increased ROS production rates do not necessarilytranslate into elevated steady-state concentrations of ROS.

2. Increased ROS production or levels often serve as initiation signals for multiple signalingpathways.

3. Plants respond to ROS in a ROS molecule type- and localization-dependent manner.

4. ROS signaling specificity is likely determined by local ROS sensors and the productionof metabolites, their derivatives, or both.

5. The accumulation of ROS is necessary for multiple metabolic, physiological, and devel-opmental processes that function at the cellular and whole-organism levels.

FUTURE ISSUES

1. Multiple ROS-sensing mechanisms have been recently documented or suggested. How-ever, although indirect evidence suggests their existence, some mechanisms, e.g., sensingof apoplastic ROS, remain unidentified.

2. In many cases, ROS accumulation is connected with Ca2+ signals. However, it is notclear how these stimuli are connected. In this context, the most outstanding questionsrelate to the identity of Ca2+ channels and their activation mechanisms.

3. Multiple lines of evidence support the concept of locality in ROS signaling. Therefore,special emphasis should be dedicated to the spatial distribution of potential ROS sensors.In this context, the use of up-to-date tools for monitoring local ROS maxima shouldprovide better information than approaches in which the whole leaf or whole cell isanalyzed.


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4. Recent documentation of chloroplast-to-nucleus protein and H2O2 transport means thatthe previously proposed retrograde signaling pathways based on ROS diffusion to thecytoplasm should be reevaluated, given that cytoplasmic ROS can arise from multiplesources.

5. Although the signaling function of metabolites and their oxidative derivatives begins toemerge, there are probably many more such signaling molecules that await identification.

6. The relationship between compartment-specific perturbations in redox balance and ROSformation, and how they affect each other, is not always clear and will need to be addressedto determine exactly how they contribute to different signaling pathways.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We thank Estee Tee and Kai Xun Chan from the Pogson laboratory, and Ian Max Møller for usefulcomments and discussions during the preparation of the manuscript. All authors are members ofthe Centre of Excellence in the Molecular Biology of Primary Producers (2014–2019). C.W. isfunded by the Academy of Finland (decision #294580).

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PP69_FrontMatter ARI 5 April 2018 9:11

Annual Review ofPlant Biology

Volume 69, 2018

Contents

My Secret LifeMary-Dell Chilton � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Diversity of Chlorophototrophic Bacteria Revealed in the Omics EraVera Thiel, Marcus Tank, and Donald A. Bryant � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �21

Genomics-Informed Insights into Endosymbiotic Organelle Evolutionin Photosynthetic EukaryotesEva C.M. Nowack and Andreas P.M. Weber � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �51

Nitrate Transport, Signaling, and Use EfficiencyYa-Yun Wang, Yu-Hsuan Cheng, Kuo-En Chen, and Yi-Fang Tsay � � � � � � � � � � � � � � � � � � � � �85

Plant VacuolesTomoo Shimada, Junpei Takagi, Takuji Ichino, Makoto Shirakawa,

and Ikuko Hara-Nishimura � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 123

Protein Quality Control in the Endoplasmic Reticulum of PlantsRichard Strasser � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 147

Autophagy: The Master of Bulk and Selective RecyclingRichard S. Marshall and Richard D. Vierstra � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 173

Reactive Oxygen Species in Plant SignalingCezary Waszczak, Melanie Carmody, and Jaakko Kangasjarvi � � � � � � � � � � � � � � � � � � � � � � � � � � 209

Cell and Developmental Biology of Plant Mitogen-Activated ProteinKinasesGeorge Komis, Olga Samajova, Miroslav Ovecka, and Jozef Samaj � � � � � � � � � � � � � � � � � � � � � 237

Receptor-Like Cytoplasmic Kinases: Central Players in Plant ReceptorKinase–Mediated SignalingXiangxiu Liang and Jian-Min Zhou � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 267

Plant Malectin-Like Receptor Kinases: From Cell Wall Integrity toImmunity and BeyondChristina Maria Franck, Jens Westermann, and Aurelien Boisson-Dernier � � � � � � � � � � � � 301

Kinesins and Myosins: Molecular Motors that Coordinate CellularFunctions in PlantsAndreas Nebenfuhr and Ram Dixit � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 329

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The Oxylipin Pathways: Biochemistry and FunctionClaus Wasternack and Ivo Feussner � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 363

Modularity in Jasmonate Signaling for Multistress ResilienceGregg A. Howe, Ian T. Major, and Abraham J. Koo � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 387

Essential Roles of Local Auxin Biosynthesis in Plant Developmentand in Adaptation to Environmental ChangesYunde Zhao � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 417

Genetic Regulation of Shoot ArchitectureBing Wang, Steven M. Smith, and Jiayang Li � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 437

Heterogeneity and Robustness in Plant Morphogenesis: From Cellsto OrgansLilan Hong, Mathilde Dumond, Mingyuan Zhu, Satoru Tsugawa,

Chun-Biu Li, Arezki Boudaoud, Olivier Hamant, and Adrienne H.K. Roeder � � � � � � 469

Genetically Encoded Biosensors in Plants: Pathways to DiscoveryAnkit Walia, Rainer Waadt, and Alexander M. Jones � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 497

Exploring the Spatiotemporal Organization of Membrane Proteins inLiving Plant CellsLi Wang, Yiqun Xue, Jingjing Xing, Kai Song, and Jinxing Lin � � � � � � � � � � � � � � � � � � � � � � � 525

One Hundred Ways to Invent the Sexes: Theoretical and ObservedPaths to Dioecy in PlantsIsabelle M. Henry, Takashi Akagi, Ryutaro Tao, and Luca Comai � � � � � � � � � � � � � � � � � � � � � � 553

Meiotic Recombination: Mixing It Up in PlantsYingxiang Wang and Gregory P. Copenhaver � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 577

Population Genomics of Herbicide Resistance: Adaptation viaEvolutionary RescueJulia M. Kreiner, John R. Stinchcombe, and Stephen I. Wright � � � � � � � � � � � � � � � � � � � � � � � � � 611

Strategies for Enhanced Crop Resistance to Insect PestsAngela E. Douglas � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 637

Preadaptation and Naturalization of Nonnative Species: Darwin’s TwoFundamental Insights into Species InvasionMarc W. Cadotte, Sara E. Campbell, Shao-peng Li, Darwin S. Sodhi,

and Nicholas E. Mandrak � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 661

Macroevolutionary Patterns of Flowering Plant Speciationand ExtinctionJana C. Vamosi, Susana Magallon, Itay Mayrose, Sarah P. Otto,

and Herve Sauquet � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 685

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When Two Rights Make a Wrong: The Evolutionary Genetics ofPlant Hybrid IncompatibilitiesLila Fishman and Andrea L. Sweigart � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 707

The Physiological Basis of Drought Tolerance in Crop Plants:A Scenario-Dependent Probabilistic ApproachFrancois Tardieu, Thierry Simonneau, and Bertrand Muller � � � � � � � � � � � � � � � � � � � � � � � � � � � � 733

Paleobotany and Global Change: Important Lessons for Species toBiomes from Vegetation Responses to Past Global ChangeJennifer C. McElwain � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 761

Trends in Global Agricultural Land Use: Implications forEnvironmental Health and Food SecurityNavin Ramankutty, Zia Mehrabi, Katharina Waha, Larissa Jarvis,

Claire Kremen, Mario Herrero, and Loren H. Rieseberg � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 789

Errata

An online log of corrections to Annual Review of Plant Biology articles may be found athttp://www.annualreviews.org/errata/arplant

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Documents

Reactive Oxygen Species in Plant Signalingbiologia.ucr.ac.cr/profesores/Garcia Elmer/reactive oxygen species in... · This classical model has been recently extended by the discovery