JIPB - Protein S-Nitrosylation in plants

Protein S-Nitrosylation in plants: Currentprogresses and challengesJian Feng1, Lichao Chen2,3 and Jianru Zuo2,3*

1. Sainsbury Laboratory, University of Cambridge, Cambridge CB2 1LR, UK2. State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, CAS Center for Excellence in Molecular Plant Sciences,Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China3. The University of Chinese Academy of Sciences, Beijing 100049, Chinadoi: 10.1111/jipb.12780

[email protected]

Abstract Nitric oxide (NO) is an important signalingmolecule regulating diverse biological processes in allliving organisms. A major physiological function of NOis executed via protein S-nitrosylation, a redox-based

the past decade, significant progress has been madein functional characterization of S-nitrosylated proteins

in plants. Emerging evidence indicates that protein S-nitrosylation is ubiquitously involved in the regulation ofplant development and stress responses. Here we reviewcurrent understanding on the regulatory mechanisms ofprotein S-nitrosylation in various biological processes inplants and highlight key challenges in this field.

Edited by: Zhizhong Gong, China Agricultural University, ChinaReceived Dec. 25, 2018; Accepted Jan. 14, 2019; Online on Jan. 21,2019

INTRODUCTION

In 1774, Joseph Priestly published his book “Experimentsand Observations on Different Kinds of Air” (Volume 1), inwhich he described the discovery of several airs,including ‘nitrous air’ (Priestly 1774), currently knownas ‘nitric oxide’ (NO), which simply consists of oneoxygen atom and one nitrogen atom. For a long timesince its discovery, NO has been known as a colorless,relatively unstable gaseous molecule with a free radicalthat makes it relatively reactive and is thought as an airpollutant. In the 1980s, the importance of NO wasrecognized as a signaling molecule in mammals widelypresent in different tissues and organs with diversephysiological roles in the nervous system, immunesystem, cardiovascular system and endocrine system(Schmidt and Walter 1994; Stamler et al. 1997). Later

studies identified NO as a highly conserved signalingmolecule found in all living organisms. In higher plants,

NO has been found to play important regulatory roles inalmost all aspects of the life cycle as well as responsesto biotic and abiotic stresses with diverse, yet not fullyunderstood, mechanisms (Freschi 2013; Puyaubert andBaudouin 2014; Yu et al. 2014; Domingos et al. 2015;Trapet et al. 2015; Asgher et al. 2017; Fancy et al. 2017;Begara-Morales et al. 2018).

Based on our current understanding, NO executesits physiological roles mainly through covalent post-translational modifications, including tyrosine nitration,metal nitrosylation, and S-nitrosylation. Tyrosine nitra-tion involves the addition of a nitro group at the orthoposition of the phenolic hydroxyl group of tyrosine toproduce 3-nitrotyrosine (Radi 2013). Peroxynitrite(OONO

-

) and nitrogen dioxide (NO2), two reactive

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*Correspondence:

posttranslational modification by covalently adding a NOmolecule to a reactive cysteine thiol of a target protein.S-nitrosylation is an evolutionarily conserved mechanismmodulating multiple aspects of cellular signaling. During

*Correspondence: J ianru Z uo ( [email protected])doi: 10.1111/jipb.12780

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nitrogen species, generated by reactions of NO withsuperoxide radicals (O2

.-) and O2, respectively, aremajorNO donors to mediate tyrosine nitration (Liu et al. 1998Radi 2013; Bartesaghi and Radi 2018). Tyrosine nitration

High-Im

pact

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has been used as a marker of oxidant burden in human

diseases (Ischiropoulos 2009). In plants, a number of

proteins modified by tyrosine nitration has been

identified in proteomic studies (Romero-Puertas et al.

2007; Chaki et al. 2011; Lozano-Juste et al. 2011; Tanou

et al. 2012; Begara-Morales et al. 2013) and several

tyrosine nitration-modified proteins have been func-

tionally characterized (Melo et al. 2011; Begara-Morales

et al. 2015; Castillo et al. 2015; Holzmeister et al. 2015;

Takahashi et al. 2017a, 2017b).

More attention has been given to S-nitrosylation, a

redox-based protein posttranslational modification by

covalently forming an S-linked NO group to the reactive

thiols of a cysteine residue. A cyclic GMP (cGMP)-

dependent NO signaling mechanism has been pro-

posed, of which NO enhances the activity of soluble

guanylate cyclase (sGC) to boost the intracellular level

of cGMP, eventually activating downstream Ca2þ-modulated signaling (Wendehenne et al. 2001; Ahern

et al. 2002; Besson-Bard et al. 2008). A key step of this

signaling pathway is NO-mediatedmetal nitrosylation of

sGC (Ahern et al. 2002; Besson-Bard et al. 2008), a

regulatory mechanism similar to that of protein

S-nitrosylation.

Similar to other posttranslational modifications,

S-nitrosylation regulates protein activities by various

mechanisms, including stability, biochemical activity,

conformation change, subcellular localization, and

protein–protein interaction (Hess et al. 2005; Astier

et al. 2011; Astier et al. 2012b; Lamotte et al. 2014). In

plants, a number of S-nitrosylated proteins involved in

various physiological processes have been functionally

characterized (Table 1). In this review, we will outline

the current progress in NO metabolism and functional

characterization of representative S-nitrosylated pro-

teins in plants.

METABOLISM OF NITRIC OXIDE

In mammalian cells, biosynthesis of NO is mainlydependent on nitric oxide synthase (NOS), whichcatalyzes the generation of NO by converting L-arginineto L-citrulline (Nathan and Xie 1994; Mayer and

Hemmens 1997; Wendehenne et al. 2001). Althoughthe production of NO in plants appears to be sensitive

to animal NOS inhibitors, structural and functional

analogues of NOS-like proteins have not been found inany land plants (Besson-Bard et al. 2008; Gas et al. 2009;

Frohlich and Durner 2011; Jeandroz et al. 2016; Astier

et al. 2018). Instead, currently available evidence

suggests that NO in land plants is produced from

nitrite via nonenzymatic or enzymatic pathways, of

which the latter is catalyzed by nitrate reductase (NR),

nitrite-NO reductase (Ni-NOR) and xanthine oxidore-

ductase (XOR) (Besson-Bard et al. 2008; Gupta et al.2011; Mur et al. 2013; Fancy et al. 2017).

In higher plants, NR is considered as a key enzyme of

nitrate assimilation (Wendehenne et al. 2004; Besson-

Bard et al. 2008). Cytosolic NR reduces nitrate (NO3-)

to nitrite (NO2-) in an NAD(P)H-dependent manner.

NR contains an N-terminal molybdenum-containing

domain, a b-type cytochrome domain and a C-terminal

flavin adenine dinucleotide (FAD) domain, each con-

nected by a hinge region (Figure 1A). All of these

domains function as redox centers to transfer electrons

from NAD(P)H to nitrate (Dean and Harper 1988;Campbell and Kinghorn 1990; Campbell 1996; Yamasakiand Sakihama 2000; Rockel et al. 2002). Evidenceobtained from both genetic and pharmacological

studies suggests that NR plays an important role inNO production (Desikan et al. 2002; Bright et al. 2006;Yamamoto-Katou et al. 2006; Kolbert et al. 2008;Seligman et al. 2008; Zhao et al. 2009;Wang et al. 2010).NR is capable of reducing NO2

- to NO and ONOO- (Deanand Harper 1988; Rockel et al. 2002). In Chlamydomonas,a different mechanism has been proposed that theamidoxime reducing component (renamed as nitricoxide-forming nitrite reductase, NOFNiR) catalyzes NOproduction from nitrite using NAD(P)H-NR as electrondonors. This dual NR-NOFNiR system catalyzes theproduction of NO from nitrite in the presence of arelatively high concentration of nitrate, which inhibitsthe activity of NR (Chamizo-Ampudia et al. 2016). Intobacco (Nicotiana tabacum L. cv. Samsun) roots, theplasma membrane-bound NR is associated with anitrite:NO oxidoreductase (Ni:NOR), which may reduceapoplastic nitrite and contribute to NO production(Stohr et al. 2001). Moreover, a peroxisomal enzymeXOR is also involved in the production of NO (Corpaset al. 2001; Harrison 2002; Corpas et al. 2008). Similar toNR, XOR is an oxidoreductase using molybdenum as a

2 Feng et al.

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Table1.

Function

alcharacterizedS-nitrosylated

proteins

inplan

ts

Protein

Accession

numbe

rAGIco

deOrgan

ism

S-nitrosylated

residu

eFu

nction

Referen

ce

ABI5

NP_

5658

40AT2G36

270

Arabido

psisthaliana

Cys-153

Prom

oteABI5

degrad

ationan

dseed

germ

ination

Albertoset

al.20

15

AHP1

NP_

1887

88AT3G21510

Arabido

psisthaliana

Cys-115

Rep

ress

AHP1

phosph

orylationan

dcytokinin

respon

ses

Feng

etal.20

13

APX

1NP_

00131894

9AT1G07

890

Arabido

psisthaliana

Cys-32

Enha

nceAPX

1activity

andresistan

ceto

oxidativestress

Yang

etal.20

15

ASK

1NP_

565123

AT1G7595

0Arabido

psisthaliana

Cys-37,Cy

s-118

Enha

nceASK

1bind

ingto

CUL1-TIR1an

dau

xin

sign

aling

Iglesias

etal.20

18

CDC4

8NP_

00131246

2NA

Nicotiana

taba

cum

Cys-526

InhibitATP

aseactivity

Astieret

al.20

12a

cFBP1

CAB39

759

NA

Pisum

sativum

Cys-153

Inhibiten

zymatic

activity

Serratoet

al.20

18

GAPC

1P2

5858

AT3G04

120

Arabido

psisthaliana

Cys-149

Red

uceGAPD

Hactivity

Zaffag

nini

etal.20

13

GSN

OR1

NP_

1992

07AT5

G43

940

Arabido

psisthaliana

Cys-10

Indu

ceau

toph

agicde

grad

ationof

GSN

OR1an

dhy

poxiarespon

ses

Zhan

etal.20

18;Gue

rra

etal.20

16

Hop

AI1

NP_

7907

45NA

Pseudo

mon

assyring

aeCy

s-138

Inhibitph

osph

othreo

nine

lyaseactivity

and

activate

cellde

ath

Ling

etal.20

17

MAT1

P236

86AT1G02

500

Arabido

psisthaliana

Cys-114

Inhibiten

zymatic

activity

Lind

ermayret

al.20

06

MC9

Q9F

YE1

AT5

G04

200

Arabido

psisthaliana

Cys-147

Supp

ress

autoproc

essing

andproteo

lytic

activity

Belen

ghie

tal.20

07

NAB1

XP_0

0169

6518

NA

Chlamydom

onas

reinha

rdtii

Cys-226

InhibitNAB1activity

anden

hancelight

capture

Berge

ret

al.20

16

NPR

1P9

3002

AT1G64

280

Arabido

psisthaliana

Cys-156

FacilitateNPR

1oligom

erizationan

dim

mun

erespon

ses

Tada

etal.20

08

PDK1

NP_

001234

519

NA

Solanu

mlycope

rsicum

Cys-128

Inhibiten

zymatic

activity

Liuet

al.20

17

PRMT5

Q8G

WT4

AT4

G31120

Arabido

psisthaliana

Cys-125

Enha

ncemethy

ltransferase

activity

andstress

tolerance

Huet

al.20

17

PrxIIE

Q94

9U7

AT3G5296

0Arabido

psisthaliana

Cys-121

Inhibiten

zymatic

activity

Rom

ero-Pu

ertaset

al.

2007

RBOHD

Q9F

IJ0

AT5

G47

910

Arabido

psisthaliana

Cys-89

0Inhibiten

zymatic

activity

andpe

rturbcell

death

Yunet

al.20

11

SABP3

P27140

AT3G0150

0Arabido

psisthaliana

Cys-28

0Inhibitbind

ingof

SAan

den

zymatic

activity

Wan

get

al.20

09 (Con

tinu

ed)

Protein S-nitrosylation in plants 3

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co-factor. Under anaerobic conditions, XOR catalyzesthe conversion of nitrite to NO, using NADH or xanthineas reducing agents (Godber et al. 2000a; Godber et al.2000b) (Figure 1B).

Under certain conditions, NO can be producedthrough non-enzymatic pathways (Figure 1B). In thealeurone layers of barley (Hordeum vulgare), non-enzymatic synthesis of NO is facilitated by reductionof apoplastic nitrite under acidic conditions (Bethkeet al. 2004a). In tobacco leaves, the mitochondrialelectron transport is proposed to contribute to thenitrite-dependent NO production, whereas other non-enzymatic sources of NO include reduction of nitrite toNOby carotenoids, ascorbic acid or polyamines (Cooneyet al. 1994; Crawford 2006; Tun et al. 2006). Currently,the mechanisms and the physiological significance ofnon-enzymatic production of NO remain largely unclear.

The homeostasis of NO is tightly controlled by itsbiosynthesis and turnover. Although the biosynthesisof NO is likely using different mechanisms in animaland plant cells, the turnover of bioactive NO speciesis controlled by a conserved mechanism. In allliving organisms, S-nitrosoglutathione (GSNO), derivedfrom the reaction of NO and glutathione (GSH), is amajor reservoir of bioactive NO species (Hess et al.2005; Gupta 2011), which is irreversibly degraded byS-nitrosoglutathione reductase (GSNOR). GSNORs, aclass of highly conserved NADH-dependent reductasespresented in all living organisms, catalyze the reduc-tion of GSNO to glutathione disulfide (GSSG) andammonia (NH3) in the presence of GSH (Liu et al. 2001;Sakamoto et al. 2002) (Figure 1C). Mutations in GSNORgenes cause severe defects in development and stressresponses in both animals and plants (Liu et al. 2004;Feechan et al. 2005; Que et al. 2005; Lee et al. 2008;Chen et al. 2009; Kwon et al. 2012), illustrating thecritical role of NO homeostasis.

In Arabidopsis, the single-copied GSNOR1 gene andGSNOR1 protein have been biochemically and geneti-cally characterized. Purified GSNOR1 recombinantprotein catalyzes the reduction of GSNO in a NADH-dependentmanner (Sakamoto et al. 2002). Mutations inGSNOR1 cause the accumulation of excessive amountsof NO and S-nitrosothiol (SNO), accompanied withvarious developmental defects and altered responsesto both biotic and abiotic stresses (Feechan et al. 2005;Lee et al. 2008; Chen et al. 2009; Kwon et al. 2012).Notably, GSNOR1 has been identified in genetic screensTa

ble1.

Continue

d

Protein

Accession

numbe

rAGIco

deOrgan

ism

S-nitrosylated

residu

eFu

nction

Referen

ce

SnRK2.6

Q94

0H6

AT4

G3395

0Arabido

psisthaliana

Cys-137

Inhibiten

zymatic

activity

andABA-in

duced

stom

atal

closure

Wan

get

al.20

15

SRG1

Q39

224

AT1G1702

0Arabido

psisthaliana

Cys-87

RelievesDNAbind

ingan

dtran

scriptiona

lrepression

activity

Cuie

tal.20

18

TGA1

Q39

237

AT5

G65

210

Arabido

psisthaliana

Cys-26

0Cy

s-26

6En

hancetheDNAbind

ingactivity

Lind

ermayret

al.20

10

TIR1

Q570C

0AT3G62

980

Arabido

psisthaliana

Cys-140

Enha

nceTIR1-Aux

/IAAinteraction

Terrile

etal.20

12

VND7

Q9C

8W9

AT1G7193

0Arabido

psisthaliana

Cys-26

4Cy

s-34

0Inhibittran

scriptiona

lactivity

andxylem

cell

differen

tiation

Kaw

abeet

al.20

18

4 Feng et al.


1209

for thermotolerance-defective and oxidative-stress-resistant mutants, designated as SENSITIVE TO HOTTEMPERATURE5 (HOT5) and PARAQUAT RESISTANT2(PAR2), respectively (Lee et al. 2008; Chen et al.2009), implying a tight link of NO with abiotic stressresponses (see also below).

As mentioned above, the nitrogen assimilationenzyme NR reduces NO2

- to NO, hence playing a criticalrole in NO production in higher plants. SeveralArabidopsis mutants have been characterized to showan increased NO or SNO level, including gsnor1/hot5/par2, nox1 (for NO overproducer1) (He et al. 2004), andnia1 nia2, of which NIA1 and NIA2 are two closely relatedgenes encoding NRs (Wilkinson and Crawford 1991).Analysis of these mutants revealed that nitrogenassimilation is regulated by SNO via suppressing nitratetransport and reduction (Frungillo et al. 2014). More-over, GSNOR1 itself is S-nitrosylated (Frungillo et al.2014; Guerra et al. 2016; Zhan et al. 2018), correlatedto the (S)NO-induced inhibition of the enzymaticactivity, suggesting that this negative regulatorymechanism plays an important role for the homeostasisof S-nitrosothiols via nitrogen assimilation (Frungilloet al. 2014). While multiple cysteine residues of GSNOR1are found to be S-nitrosylated, none of the singlesubstitutional mutations in these cysteine residuesshows an apparent effect on the enzymatic activity(Guerra et al. 2016; Zhan et al. 2018). A triple mutation inthree highly conserved cysteine residues abolishes theresponsiveness to NO in the inhibition of the GSNOR1enzymatic activity in an in vitro assay (Guerra et al.2016). Moreover, NO induces autophagic degradationof GSNOR1 (Zhan et al. 2018). Therefore, both theenzymatic activity and stability of GSNOR1 are nega-tively regulated by NO, indicating that a positivefeedback regulatory loop modulates the accumulationNO (Figure 1C), which is likely a key during earlysignaling for the burst of NO in response to variousstimuli.

In addition to the GSNOR-mediated NO turnover,other NO scavenging mechanisms have also beendescribed. In a genetic screen for Arabidopsis NO-insensitive mutants, continuous no-unstressed1 wasfound to be allelic to altered meristem program1 (Liuet al. 2013), a mutant showing a pleiotropic phenotypewith an increased cytokinin level (Helliwell et al. 2001).Subsequent analyses found that ONOO-, a reactive NOintermediate NO, directly interacts with the bioactive

Figure 1. Nitric oxide synthesis and metabolism inplants(A) Schematic illustration of plant nitrate reductase(NR). Mo-MPT, Mo-molybdopterin domain; Dimer,dimerization domain; Cyt B, cytochrome b domain;FAD, flavin adenine dinucleotide (FAD) bindingdomain; NADH, NADH binding domain. (B) Majorroutes of nitric oxide (NO) biosynthesis and turnoverin plants. NR catalyzes the reduction of nitrate (NO3

-)to nitrite (NO2

-) using reducing equivalents providedby NADH. Nitrite is further reduced to NO by NR, nitricoxide-forming nitrite reductase (NOFNiR), plasmamembrane bound nitrite:NO oxidoreductase (Ni:NOR) or xanthine oxidoreductase (XOR). NO canalso be produced through non-enzymatic pathwaysunder certain conditions. NO reacts with O2, O2

-, andglutathione (GSH) to produce nitrogen oxides (NOx),peroxynitrite (OONO-), and S-nitrosoglutathione(GSNO), respectively. Reversible interaction of NOwith transition metal of metalloprotein (M) leads tonitrosylated metalloprotein (M-NO). NO reacts withthe cysteine thiol group of target protein (RSH) toform S-nitrosylated protein (RSNO). (C) S-nitrosoglu-tathione reductase (GSNOR) catalyzes the reductionGSNO to glutathione disulphide (GSSG) and ammonia(NH3) in the presence of NADH and GSH. Glutathione-disulfide reductase (GSR) catalyzes the reduction ofGSSG to GSH in the presence of NADPH. Theenzymatic activity and stability of GSNOR are nega-tively regulated by GSNO.



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cytokinin compound zeatin, resulting in the reduced NOlevel, thereby modulating NO homeostasis (Liu et al.2013). In Chlamydomonas reinhardtii, two truncatedhemoglobins, THB1 and THB2, function with NR toconvert NO into nitrate (Sanz-Luque et al. 2015).Moreover, the NO-derived compounds react withunsaturated fatty acids to produce nitro-fatty acids(NO2-FAs), which act as NO donors in both animals andplants (Villacorta et al. 2007; Kelley et al. 2008; Bakeret al. 2009; Sanchez-Calvo et al. 2013; Mata-Perez et al.2016a). The presence of NO2-FAs [in particular, nitro-oleic acid (NO2-OA), nitro-linoleic acids (NO2-LA) andnitro-linolenic acid (NO2-Ln)] has been confirmed inArabidopsis, pea and rice by mass spectrometry (Mata-Perez et al. 2016b). NO2-Ln is involved in regulatingresponses to abiotic stresses and its level is induced bysalt, heavy metal, low temperature and mechanicalwounding, suggesting an important role of NO2-FAs inthe adaption of abiotic stresses in plants (Mata-Perezet al. 2016b).

OVERVIEW OF S-NITROSYLATION,TRANSNITROSYLATION ANDDENITROSYLATION

A major physiological activity of NO is mediated byS-nitrosylation, a redox-based posttranslational modifi-cation that covalently links a NO group to the reactiveCys thiol (-SH) to form S-nitrosothiol (-SNO) (Figure 2A).This highly conserved posttranslational mechanismplays crucial roles in diverse physiological and patho-logical processes by modulating protein activities.

S-nitrosylation is a dynamic and reversible process,involving nitrosylation, denitrosylation and transnitro-sylation (Hess et al. 2005; Benhar et al. 2009; Stomberskiet al. 2019).

S-nitrosylation has been thought mainly as a nonen-

zymatic process. The formation of an S-nitrosothiol can

be directly mediated by NO, or indirectly mediated by

various higher nitrogen oxides (NOx), metal-NO inter-

mediates, SNOs or ONOO-(Wang et al. 2006). The

addition of NO donors (usually GSNO) to purified

recombinant proteins causes the S-nitrosylation of the

target in the absence of any co-factors or enzyme-like

proteins, a process similar to that of auto-phosphor-

ylation. More critically, yet no highly specific enzymes

catalyzing S-nitrosylation have been identified (but, see

below, for transnitrosylation). This possible nonenzy-

matic-reaction feature is unique to S-nitrosylation, which

is possibly attributed to the fact that S-nitrosylation (and

otherNO-mediatedmodifications) is largelydrivenby the

redox potentials between a NO donor molecule and a

modified target (Stamler et al. 2001; Hess et al. 2005;

Benhar et al. 2009), whereas most, if not all, other

posttranslational modifications, are energy-consuming

processes. Possiblybecauseof itsnonenzymatic-reaction

nature, at least in part, the redox-based S-nitrosylation

has been recently proposed to act predominantly as

transient intermediates prior to the formation of

disulfide bonds (Wolhuter et al. 2018).A key question in this newly emerged research field

is the specificity or selectivity of the nonenzymatic-reaction modification, i.e., how to select and determinea modified target protein. Currently available evidence

Figure 2. Biochemistry of protein S-nitrosylation, denitrosylation and transnitrosylation(A) Schematic representation of S-nitrosylation and denitrosylation. S-nitrosylation is the covalent incorporation ofa NO group to the thiol group of a cysteine residue to form S-nitrosothiol (SNO). Thioredoxin (Trx) denitrosylatesS-nitrosylated proteins through its dithiol moiety, forming a reduced protein thiol (-SH) and oxidized Trx; oxidizedTrx is reduced by NADPH-dependent Trx reductase (NTR). (B) Schematic representation of transnitrosylation. Atransnitrosylase carrying an SNO group transfers the NO moiety to a target protein.

6 Feng et al.


1211

suggests that the specificity of protein S-nitrosylation ismainly determined by the structure of a target and thelocal NO concentration. Several motifs have beenproposed by analysis of known S-nitrosylated proteinsand peptides, largely identified by the proteomicapproach in various organisms (Lindermayr et al.2005; Hao et al. 2006; Romero-Puertas et al. 2008;Palmieri et al. 2010; Fares et al. 2011; Puyaubert et al.2014; Chen et al. 2015; Hu et al. 2015; Ib�a~nez-Vea et al.2018). One of the commonly known motifs is (H/K/R)CX(D/E) (X as any amino acid residue) in mammals andcharacterized as a modified cysteine residue flanking byacidic and/or basic amino acid residues in a hydrophobicmicroenvironment (Hess et al. 2005; Hao et al. 2006;Seth and Stamler 2011). However, several motifs thatcontain acidic, but not basic, amino acid residuesflanking the S-nitrosylated cysteine residues have beenproposed, based on a large-scale proteomic study inArabidopsis (Hu et al. 2015). It remains unclear whetherthese variations are caused by differences betweenanimal and plant models or other unknown reasons.

The second factor for the determination ofS-nitrosylation is the local NO concentration near themodified target, and a higher local NO concentration isconsidered to induce S-nitrosylation of a target (Hesset al. 2005; Iwakiri et al. 2006; Benhar et al. 2009). Inanimal cells, the S-nitrosylation of a target protein istemporally and spatially linked to the NOS activity.Under certain circumstances, a target protein physicallyinteracts with NOS, which directly transfers the de novosynthesized NO group to the modified cysteine residue(Kim et al. 2005; Benhar et al. 2009; Martinez-Ruiz et al.2013; Jia et al. 2014). Similar selective mechanisms havenot been discovered in plants.

Although S-nitrosylation has long considered as anonenzymatic reaction, recent studies suggest thepresenceof transnitrosylation, inwhich a transitionmetal(e.g., Fe2þ, Cu2þ) complex or an S-nitrosylated proteinmediates the transfer of its bearingNOmoiety to anotherprotein and nitrosylates the latter (Figure 2B). During thisprocess, a nitroso group is transferred from a transitionmetal nitrosyl to a Cys residue or between two Cysresidues, whereas the protein that carries and transfersthe NO group to its target is termed as a transnitrosylase(Anand and Stamler 2012; Stomberski et al. 2019). Severaltransnitrosylases have been identified and functionallycharacterized in bacterial and mammalian cells, includinghemoglobins, cytoglobin, neuroglobin, ceruloplasmin,

cytochrome c, cyclin-dependent kinase 5 (CDK5), caspase3, thioredoxin, and glycerol-3-phosphatase-dehydrase(GAPDH) (Anand and Stamler 2012). Two transnitrosylasecomplexes have been recently characterized. In humancells, a heterotrimeric S-nitrosylase complex consists ofthree subunits, inducible NO synthase (iNOS), S100A8,and transnitrosylase S100A9. During the transnitrosyla-tion, S100A9 is first S-nitrosylated by NO released fromiNOS and the S-nitrosylated S100A9 subsequently trans-nitrosylates the substrates containing a conservedmotif that is coordinately recognized by S100A8 andS100A9 (Jia et al. 2014). More recently, a multiplexenzymatic S-nitrosylation machinery was identified inE. coli, which includes NO synthase, SNO-proteins (SNOsynthase), and transnitrosylases to regulate a subset ofphysiological activities under anaerobic conditions (Sethet al. 2018). In both cases, the transnitrosylase complexescatalyze both de novo S-nitrosylation and transnitrosyla-tion. In general, all transnitrosylases characterized thusfar show distinctive structural features and no commonor conserved domains/motifs specific to this class ofproteins have been identified, a case different fromthat for the enzymes that catalyze other types ofposttranslational modifications, such as phosphorylation.It remains largely elusive for the mechanisms of selectiveS-nitrosylation and transnitrosylation. Nevertheless,transnitrosylation is an important enzymatic mechanismto determine the specificity of S-nitrosylation. To date,no transnitrosylase has been found in plants.

Similar to other posttranslational modifications, thedecoration of cysteine residues with NO is a reversibleprocess, of which the removal of NO from the modifiedcysteine residue of a protein is termed as denitrosyla-tion. Denitrosylation is mediated by the thioredoxin(Trx) system consisting of Trx and Trx reductase (TrxR).Trx denitrosylates SNO proteins through its dithiolmoiety, forming a reduced protein thiol and oxidizedTrx. The oxidized Trx in turn is reduced by TrxRand NADPH (Benhar et al. 2009) (Figure 2A). InArabidopsis, TRXh3 and TRXh5 selectively denitrosylatethe S-nitrosylated proteins to regulate plant immunity(Kneeshaw et al. 2014) (see also below).

During the past decade, a number of S-nitrosylatedproteins have been functionally characterized in plants,mostly in Arabidopsis (Table 1). Progresses on thestudies of the representative members of theseproteins involved in various physiological or signalingprocesses are summarized below.



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GROWTH AND DEVELOPMENT

The important role of NO in plant growth anddevelopment has been well documented. NO is widelyinvolved in regulating multiple aspects of development,including seed germination, root development, flower-ing time, pollen tube guidance, and leaf senescence(Besson-Bard et al. 2008; Astier et al. 2011; Mur et al.2013; Yu et al. 2014). In Arabidopsis, loss-of-functionmutations in the GSNOR1 gene cause severe develop-mental defects, including dramatically altered root andshoot architecture, reduced hypocotyl elongation, andreduced fertility (Lee et al. 2008; Chen et al. 2009; Kwonet al. 2012). These defects are closely associated withthe impaired auxin signaling and polar auxin transport ingsnor1, correlating with reduced levels of PIN proteins(Shi et al. 2015; Ni et al. 2017).

VASCULAR-RELATED NAC-DOMAIN (VND), a smallfamily of NAM/ATAF/CUC (NAC) transcription factors,are important regulators of xylem vessel differentia-tion. Overexpression of VND6 and VND7 induces trans-differentiation into xylem vessels, and this phenotype issuppressed by a gsnor1 loss-of-function mutation, whichin turn affects xylem vessel development. Correlated tothese observations, Cys-264 and Cys-320 of VND7 areS-nitrosylated analyzed in an in vitro biotin-switchassay and these two residues are directly related tothe transactivation activity of VND7, implying thatS-nitrosylation plays an importance role in modulatingxylem vessel cell differentiation (Kawabe et al. 2018). Anumber of VND6/7-regulated genes have been identi-fied, including METACASPASE9 (MC9), which is specifi-cally expressed in developing xylem and plays animportant role in xylem cell death (Bollhoner et al.2013). S-nitrosylation of MC9 at Cys-147 suppresses theMC9 autoprocessing and proteolytic activity, therebydirectly regulating the activity of MC9 (Belenghi et al.2007). Together, these findings suggest a tight linkbetween NO signaling and xylem development.

PHYTOHORMONE SIGNALING

S-nitrosylation has been found as an important mecha-nism to regulate signaling of phytohormones, includingauxin and cytokinin. Auxin signaling is activated byproteasomal degradation of the transcriptional repress-ors Aux/IAAs via the auxin-dependent interaction withreceptors TIR1/AFBs, leading to the alterations of

downstream auxin-responsive genes. While auxin

induces the accumulation of NO, the combined

treatment with sodium nitroprusside (SNP), a NO

donor, and auxin enhances the degradation of AUX/

IAA proteins and the expression of auxin-responsive

genes (Terrile et al. 2012). Further studies indicate that

the S-nitrosylation of the auxin receptor TIR1 at Cys-140

and Arabidopsis SKP1-like (ASK1) protein at Cys-37 and

Cys-118 enhances the formation of the SCFTIR1/AFB-Aux/

IAAs complex, thereby facilitating proteasomal degra-

dation of Aux/IAA repressors (Iglesias et al. 2018).

Cytokinin signaling is mediated by a two-component

system-based multiple-step phosphorelay involved in

sequential transferring of phosphoryl groups from the

receptors to the downstream components histidine

phosphotransfer proteins (AHPs) and response regula-

tors (ARRs). Of those components, AHP1 is S-nitrosylated

at Cys-115, which represses its phosphorylation and

subsequent transferring of the phosphoryl group to

ARR1. The NO-inhibited phosphorylation of AHP1 and

ARR1 causes a compromised cytokinin response, indicat-

ing that redox signaling and cytokinin signaling coordi-

nate plant growth and development (Feng et al. 2013).

Notably, ONOO- and zeatin directly interact, which in

principle causes the reduction of both bioactive NO and

cytokinin species (Liu et al. 2013), further supporting a

negative regulatory role of NOon the cytokinin pathway.

The observation that NO-mediated S-nitrosylation posi-

tively and negatively regulates auxin signaling and

cytokinin signaling, respectively, is consistent with the

notion that these two phytohormones are considered to

play agonistic roles in regulating plant growth and

development.

IMMUNE RESPONSES

Plant immune responses are often associated withredox status changes and pathogen infection triggers arapid burst of NO and reactive oxygen species (ROS).The regulatory roles of NO on plant immunity have beencomprehensively reviewed and, in particular, theinteractions between ROS and NO signaling havebeen long thought of as an important means toregulate stress responses (Delledonne et al. 1998;Delledonne et al. 2001; Wendehenne et al. 2004;Zaninotto et al. 2006; Besson-Bard et al. 2008; Leitneret al. 2009; Wang et al. 2013; Wendehenne et al. 2014;

8 Feng et al.


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Yu et al. 2014; Qi et al. 2018). The Arabidopsis gsnor1mutants and the tomato GSNOR-silenced plantsshow altered immune responses, indicating the directinvolvement of NO in response to pathogens (Feechanet al. 2005; Liu et al. 2017). However, a GSNOR1transgene rescues the SNO-dependent phenotype ofpar2 (an allele of gsnor1), but not the nox1 mutantphenotype, whereas a gsnor1-3 nox1-1 double mutantshows an additive phenotype, indicating that GSNO andNO may have distinctive molecular targets duringimmune responses and development (Yun et al.2016). It remains unknown how plants distinguish thesetwo types of signals.

The burst of ROS is a key event during the onsetof immune responses. However, a higher intracellularlevel of ROS is also toxic to cells. To minimize its toxiceffects, the intracellular ROS level is tightly controlledby various mechanisms, of which ROS-scavengingenzymes, including ascorbate peroxidase (APX), cata-lase, glutathione peroxidase, peroxidase and superox-ide dismutase, play a dominant role in regulating thehomeostasis of ROS (Mittler et al. 2004; Mittler et al.2011). The chloroplast-localized peroxiredoxin II E (PrxIIE), a member of the peroxiredoxin family, is aperoxidase that efficiently removes H2O2. PrxII E isS-nitrosylated at Cys-121, leading to the inhibition ofboth the peroxidase and ONOO- reductase activities.During pathogen infection, S-nitrosylation also inhibitsthe ONOO- detoxification activity of PrxII E, resulting inan increase of ONOO--mediated tyrosine nitration andlipid peroxidation (Romero-Puertas et al. 2007). Nota-bly, cytosolic PrxII B and mitochondria localized PrxII Fare also found to be S-nitrosylated in Arabidopsis cellculture extracts treated with GSNO in a proteomicstudy, suggesting that this family of proteins issubjected to the regulation by NO (Lindermayr et al.2005).

When challenged by pathogens, gsnor1-3 and nox1,two mutants accumulating excessive amount ofintracellular NO, show increased cell death but reducedROS accumulation. This phenotype is directly associatedwith NO-regulated activity of the NADPH oxidaseRBOHD. The S-nitrosylation of RBOHD at Cys-890inhibits its enzyme activity, resulting in reduced ROSbiosynthesis. Conversely, the non-nitrosylatable mutantRBOHDC890A shows the increased ROS production andcell death (Yun et al. 2011). In the ROS-scavengingsystem, the S-nitrosylation of APX1 at Cys-32 positively

regulates its enzymatic activity to reduce H2O2 into H2O,resulting in enhanced resistance to oxidative stress andcompromised immune signaling (Yang et al. 2015).These observations indicate that the biosynthesisand turnover of ROS are coordinated by NO viaS-nitrosylation.

The mitogen-activated protein kinase (MAPK) cas-cades play central roles in plant immune signalingagainst pathogens. Multiple effectors have beenidentified to target MAPK cascades, including HopAI1that dephosphorylates MPK3 and MPK6 by thephosphothreonine lyase activity to suppress immunesignaling (Zhang et al. 2007). HopAI1 is S-nitrosylated atCys-138 during the onset of the hypersensitive responseto inhibit its phosphothreonine lyase activity, whichrelieves the suppressing effect on MAPK signaling toactivate immune responses (Ling et al. 2017). TheS-nitrosylation of tobacco CDC48 at Cys-526 and tomatoPDK1 at Cys-128 has also been shown to be involved inregulating defense responses and cell death (Astieret al. 2012a; Liu et al. 2017; Rosnoblet et al. 2017).

Salicylic acid (SA) is an important signaling moleculeregulating plant immune responses and the accumula-tion of SA is rapidly induced by pathogen infection. Ingsnor1-3, SA-induced gene expression is compromised,correlated to the reduced SA level, suggestive of acritical role of S-nitrosylation in SA biosynthesis andsignaling (Feechan et al. 2005). In the SA signalingpathway, a number of proteins have been shown to beregulated by S-nitrosylation. The S-nitrosylation of SA-binding protein3 at Cys-280 inhibits its binding affinity toSA and its carbonic anhydrase activity, of which thelatter is required for the pathogen resistance (Wanget al. 2009). NON-EXPRESSOR OF PATHOGENESIS-RELATED 1 (NPR1) is a master regulator of SA-induceddefense responses, which, together with its paralogsNPR3 and NPR4, function as the SA receptors. It hasbeen reported that NPR1 is localized in the cytoplasm asan oligomer connected by intermolecular disulphidebonds in the absence of pathogen challenge. Uponpathogen attack, the NPR1 oligomer is reduced tomonomer in response to the increased SA level and thealtered cellular redox status and subsequently trans-located into the nucleus to activate the expression ofdownstream genes (Mou et al. 2003). During theprocess, S-nitrosylation at Cys-156 is reported tofacilitate NPR1 oligomer assembly and the substitutionof Cys-156 with Ser results in constitutive nuclear



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localization of NPR1 monomer and enhanced resis-

tance (Tada et al. 2008). The S-nitrosylated NPR1 and

other S-nitrosylated proteins are selectively denitrosy-

lated by the oxidoreductase thioredoxin TRX-h5,

thereby resetting SA signaling. As a consequence,

overexpression of TRX-h5 selectively rescues the

immune deficiencies in nox1, but not in gsnor1, through

selective protein denitrosylation (Kneeshaw et al.

2014).

Downstream of the SA receptors, two important

components have been found being regulated by

S-nitrosylation. Upon translocating into the nucleus,

NPR1 and NPR3/4 interact with and positively or

negatively regulate a group of transcription factors

TGAs. The S-nitrosylation and S-glutathionylation of

TGA1 at Cys-260 and Cys-266, respectively, inhibit

intramolecular disulfide bond formation between these

two cysteine residues, leading to the enhanced DNA-

binding activity of TGA1 (Lindermayr et al. 2010). A

recent study shows that SRG1, a zinc finger transcription

factor, interacts with the general transcriptional

corepressor TOPLESS to repress the expression of

target genes. The S-nitrosylation of SRG1 at Cys-87

decreases its DNA-binding affinity and transcriptional

repression activity, thereby allowing a higher expres-

sion of downstream target genes (Cui et al. 2018).

In both the TGA1 and SRG1 cases, S-nitrosylation acts

to positively and negatively regulate the activity of

a transcriptional activator and a transcriptional repres-

sor, respectively, which collectively boosts immune

responses, illustrating the importance of NO-mediated

S-nitrosylation in SA signaling.

ABIOTIC STRESS RESPONSES

Similar to that of immune responses, when chal-lenged by various abiotic stresses, plants rapidlyrespond by the burst of ROS and NO, which playcritical roles in modulating growth and developmentunder adverse environmental conditions. To date, NO-mediated S-nitrosylation has been found to be largelyinvolved in regulation of biotic and abiotic stressresponses. As discussed above, the homeostasis ofROS is regulated by the S-nitrosylation of RBOHD,PrxII E and APX1, which modulates the biosynthesis orturnover of ROS (Romero-Puertas et al. 2007; Yunet al. 2011; Yang et al. 2015).

The homeostasis of NO is regulated by GSNOR,a master regulator of NO signaling, whose enzymaticactivity and stability are in turn regulated by S-nitrosylation (Guerra et al. 2016; Zhan et al. 2018).Among the several identified S-nitrosylated cysteineresidues in ArabidopsisGSNOR1 (Guerra et al. 2016; Zhanet al. 2018), the regulatory role of Cys-10 has beenstudied in detail (Zhan et al. 2018). Analysis of the crystalstructure of GSNOR1 shows that Cys-10 is located in ab-sheet covering an ATG8-interacting motif (AIM)(Guerra et al. 2016; Zhan et al. 2018). The S-nitrosylationof Cys-10 causes local conformational changes, render-ing the AIM accessible by ATG8 and subsequently beingrecruited into autophagosome for degradation in anATG8-dependent manner. Whereas NO induces auto-phagic degradation of GSNOR1, the substitution ofCys-10 with Ser abolishes the responsiveness to NO andtherefore stabilizes the GSNOR1C10S mutant protein. AGSNOR1C10S transgene rescues all abnormalities of thegsnor1-3 mutant, but with an impaired response to NOunder low oxygen conditions during seed germination,suggesting that S-nitrosylation-mediated autophagicdegradation of GSNOR1 is involved in hypoxia responses(Zhan et al. 2018).

S-methionine adenosyltransferase (MAT) catalyzesthe biosynthesis of S-adenosylmethionine, a methyldonor for thebiosynthesis of ethylene.TheS-nitrosylationof MAT1 at Cys-114 results in reversible inhibition of itsenzymatic activity. Cys-114 is absent in MAT2 and MAT3,which do not show an inhibitory effect of NO on theirenzymatic activities (Lindermayr et al. 2006). Because ofpossible functional redundancy, it remains unclearwhether or not a mutation in Cys-114 will affect theintracellular concentration of S-adenosylmethionine.Nevertheless, as NO regulates ethylene production, thisstudy highlights a linkbetweenethylene andNOsignaling(Lindermayr et al. 2006).

Abscisic acid (ABA) plays important roles in regulat-ing plant development and stress responses. Uponbinding by ABA, the ABA receptors interact with andsuppress the activity of protein phosphatase 2C,resulting in the activation of the SnRK2-type proteinkinases, which subsequently phosphorylate and acti-vate downstream components. NO inhibits the kinaseactivity of SnRK2.6/OST1 by the S-nitrosylation at Cys-137that is adjacent to the kinase catalytic site, therebynegatively regulating ABA signaling (Wang et al. 2015).Interestingly, ABA enhances the S-nitrosylation of

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SnRK2.6/OST1, correlated to the observation that ABAinduces the burst of NO, suggesting that ABA and NOform a negative regulatory loop to modulate ABAsignaling. Consistently, an increased S-nitrosylation ofSnRK2.6/OST1 has also been found in gsnor1 thatshows the impaired ABA-induced stomatal closure.The S-nitrosylated cysteine residue is highly conservedin land plants, mammals and several yeast species,suggesting that S-nitrosylation-mediated inhibition mayrepresent an evolutionarily conserved mechanism forprotein kinase regulation (Wang et al. 2015).

Seed germination and subsequent seedling establish-ment are critical processes regulated by ABA and NO. Ingeneral, whereas ABA inhibits, NO promotes, seedgermination and seedling establishment. The promotiverole of NO on seed germination have been reported in anumber of species (Beligni and Lamattina 2000; Bethkeet al. 2004b; Lozano-Juste and Leon2010). InArabidopsis,the ABI5 transcription factor is a key regulator ofseed germination and seedling establishment, and theexpression of ABI5 is directly regulated by the ERF VIItranscription factors (Gibbs et al. 2014). The increasedNOlevel is sensed by ERF VII, which are then targeted forproteasomal degradation via the N-end rule pathway,leading to the reduced expression of ABI5, therebypromoting seed germination (Gibbs et al. 2014). Thestability of ABI5 is in turn regulated by NO throughS-nitrosylation. S-nitrosylation of ABI5 at Cys-153 pro-motes its proteasomal degradation, resulting in theenhancement of seed germination and seedling estab-lishment (Albertos et al. 2015). Therefore, boththe expression of ABI5 and the stability of ABI5 proteinare negatively regulated by NO, thus revealing themolecular basis of NO-promoted seed germination.In both cases as highlighted above, NO-mediatedS-nitrosylationof SnRK2.6/OST1 andABI5plays a negativeregulatory role in ABA signaling, thus enhancing thestress resistance (Albertos et al. 2015; Wang et al. 2015).

Crosstalks between different types of proteinposttranslational modifications play an importantrole in regulating cellular signaling. A recent studyrevealed that S-nitrosylationmodulates protein argininemethylation during stress responses (Hu et al. 2017).Protein arginine methylation is catalyzed by a group ofhighly conserved protein arginine methyltransferases(PRMTs), of which PRMT5 has been well characterizedin higher eukaryotes. Mutations in the ArabidopsisPRMT5 gene causes a pleiotropic phenotype, including

hypersensitivity to salt stress, associated with reduced

arginine methylation of small nuclear ribonucleoprotein

Sm-like4 and altered pre-mRNA splicing of a subset

of stress-related genes (Zhang et al. 2011). PRMT5 is

S-nitrosylated at Cys-125, leading to the increased

methyltransferase activity. A PRMT5C125S transgene,

encoding a non-nitrosylatable mutant protein, fully

rescues the developmental defects of prmt5 under

normal growth conditions. However, the PRMT5C125S

transgenic plants are hypersensitive to NaCl and ABA,

but insensitive to NO that enhances resistance to stress,

indicating that S-nitrosylation at Cys-125 is essential for

the PRMT5-regulated stress responses. Moreover, salt

stress-induced arginine symmetric demethylation is also

abolished in PRMT5C125S transgenic plants, correlated to

aberrant pre-mRNA splicing of stress-related genes (Hu

et al. 2017). This study illustrates the importance of the

crosstalk between S-nitrosylation and arginine methyl-

ation in modulating stress responses.

CHLOROPHYLL METABOLISM ANDPHOTOSYNTHESIS

Chlorophyll metabolism and photosynthesis are largely

oxidation-reduction reaction. Emerging evidence sug-

gests that these two processes are extensively

regulated by the redox-based S-nitrosylation. In a site-

specific nitrosoproteomic studies, a large number of

proteins related to chlorophyll metabolism and photo-

synthesis have been identified as S-nitrosylated proteins

in Arabidopsis (Hu et al. 2015). Nearly one third of

proteins annotated as related to chlorophyll metabo-

lism were identified as S-nitrosylated proteins in the

nitrosoproteomic analysis and the S-nitrosylation of

several of these proteins has been further confirmed by

the biotin-switch assay (Hu et al. 2015). Whereas several

proteins are S-nitrosylated at a higher level in gsnor1

than in wild-type plants, the allelic gsnor1/hot5/par2

mutants show a reduced chlorophyll level and yellowing

leaves, suggesting that NO plays an important role in

chlorophyll metabolism (Lee et al. 2008; Hu et al. 2015).Again, nitrosoproteomic studies have revealed the

S-nitrosylated proteins significantly enriched in thephotosynthesis process, correlated to the alteredphotosynthetic properties in gsnor1 (Lindermayr et al.2005; Fares et al. 2011; Hu et al. 2015). In particular, alarge number of key components of both light and dark



1216

reactions of photosynthesis is S-nitrosylated (Hu et al.

2015). In the dark reaction (also known as the Calvin

cycle), the identified S-nitrosylated proteins cover in all

three phases of the Calvin cycle (carbon fixation,

reduction, and the regeneration of ribulose), including

the Rubisco complex [ribulose bisphosphate carboxyl-

ase, phosphoglycerate kinase, and glyceraldehyde-3-

phosphate dehydrogenase (GAPDH)]. These results

suggest that key components of photosynthesis are

extensively regulated by S-nitrosylation (Hu et al. 2015).Several S-nitrosylated proteins in chlorophyll metab-

olism and photosynthesis have been further character-ized by the biotin-switch assay (Abat et al. 2008; Abatand Deswal 2009; Hu et al. 2015). In the green algaeChlamydomonas reinhardtii, the translation of LHCBM, amajor light-harvesting protein at PSII, is controlledby light conditions. NAB1, a cytosolic RNA-bindingprotein, represses the translation of LHCBM in responseto high light exposure. Under low light conditions,S-nitrosylation of NAB1 at Cys-226 causes the decreasedrepressor activity, resulting in the accumulation of ahigher level of LHCBM and efficient light capture.Conversely, high light triggers denitrosylation of NAB1mediated by TRX-h1. This light-modulated nitrosylation-denitrosylation identifies NAB1 as a key elementfor redox-regulated photosynthetic light harvesting(Berger et al. 2016).

FUTURE CHALLENGES

In recent years, significant progress has been made inunderstanding the biochemistry and biological func-tionality of protein S-nitrosylation in plants. However, akey technical bottleneck is the lack of methods that arehighly sensitive, low noise/signal ratio, and easy-handling for the analysis of protein S-nitrosylation.Currently, the most often-used method for the analysisof S-nitrosylation is the biotin-switch assay (Jaffrey andSnyder 2001). This method involves several key steps,including the block of free thiol groups of cysteineresidues by methylation, followed by reducing themodified thiols with a strong reductant, usually ascorbicacid, and then decorating the reduced thiol with a biotinmolecule. The biotin-labelled protein or peptides arethen detected by immunoblotting using anti-biotinantibodies or by mass spectrometry. While the biotin-switch assay has been improved in some degrees

(Huang and Chen 2010), this original method is time-consuming and tedious, especially unsuitable formonitoring dynamic changes of this important post-translational modification in vivo. The development ofhigh sensitivity and user-friendly new methods istherefore technically demanding and emergent.

In addition to the methodological requirements,there are several challenging questions that need to befocused on in plant NO biology. First, do land plantshave a NOS-like system for NO biosynthesis? This hasbeen a long-time puzzle in NO biology. Until very recent,genomics studies indicate that the NOS-like genes arepresented in algae but disappeared since plantsconquered land (Rensing et al. 2008; Jeandroz et al.2016; Bowman et al. 2017; Nishiyama et al. 2018). Inresponse to this enigma, it has been proposed that landplants may have evolved and utilized a nitrateassimilation and reduction mechanism for NO biosyn-thesis, rather than using the conserved NOS enzymesfound in animals (Jeandroz et al. 2016). Perhaps, thehunt for NOS-like proteins or the unambiguousdemonstration of the absence of NOS-like genes inland plants will still be hard tasks.

Second, whereas S-nitrosylation has been consid-ered mainly as a non-enzymatic reaction, the recentlydiscovered transnitrosylation in animals has revealed animportant mechanism to determine the specificity ofS-nitrosylation. However, no transnitrosylase has beenfound in plants. Identification and characterization ofpotential transnitrosylases in plants will be criticalfor understanding the biochemical basis of selectiveS-nitrosylation.

Third, GSNOR is a master regulator of NO signaling.Both the enzymatic activity and the stability ofArabidopsis GSNOR1 are negatively regulated by NO(Guerra et al. 2016; Zhan et al. 2018). While this positiveregulatory feedback mechanism is important foramplifying the NO signal during early signaling, itremains completely unknown about the mechanismsthat turn down NO signaling. Addressing any of thesethree questions will be challenging and undoubtedlymajor breakthroughs in plant NO biology.

In response to these challenges, we expect thatbreakthrough progress will be achieved within the next5–10 years in addressing some key questions in plant NObiology. It may be optimistic to reveal, at least partially,the molecular mechanisms of selective S-nitrosylationandnegative regulationofNOsignaling in thenear future.

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ACKNOWLEDGEMENTS

We apologize to colleagues whose work could not be

cited owing to space limitations. This work was

supported by grants from the National Natural Science

Foundation of China (31830017 and 31521001), Chinese

Academy of Sciences (XDB27030207), and State Key

Laboratory of Plant Genomics (SKLPG2016-22).

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