mittler oxidative

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TRENDSi n Plant Science Vol.7 No.9 September 2002

http://plants.trends.com 1360-1385/02/$ see front matter 2002 Elsevier Science Ltd. All ri ghts r eserved. PII: S1360-1385(02)02312-9

405Review

Ron Mitt ler

Dept of Bot any, Plant

Sciences Institute,

353 Bessey Hall, Iowa

State University, Ames,

IA 50011, USA.

e-mail: rmittler@

iastate.edu

Reactive oxygen int ermediat es (ROIs) ar e par tially

redu ced forms of at mospheric oxygen (O2). They

typically result from t he excitation of O2

to form singlet

oxygen (O2

1) or from t he tr ans fer of one, two or t hree

electrons t o O2

to form , respectively, a su peroxide

ra dical (O2), hydrogen per oxide (H

2O

2) or a hydr oxyl

ra dical (HO). In contrast to atm ospheric oxygen, ROIs

ar e capable of un rest ricted oxidat ion of var ious cellular

componen ts and can lead t o the oxidative destr uction of

th e cell [14].

Production of ROIs in cells

There ar e ma ny potential sources of ROIs in plants

(Table 1). Some a re r eactions involved in norm al

metabolism, such as photosynthesis an d r espiration.

These ar e in line with th e tra ditional concept,

considerin g ROIs as una voidable byproducts of

aer obic meta bolism [1]. Other sources of ROIs belong

to pathways enhan ced dur ing abiotic stresses,

such a s glycolat e oxidase in per oxisomes dur ing

photorespira tion. However, in recent year s, new

sources of ROIs ha ve been identified in plant s,

including NADPH oxidases, amine oxidases an d

cell-wall-bound per oxidases. Th ese a re tightly

regulat ed and par ticipate in the production of ROIs

dur ing processes such as pr ogra mm ed cell deat h

(PCD) and pat hogen defense [2,4,5].

Wherea s, under norm al growth conditions, the

production of ROIs in cells is low (240 M s1 O2 and a

stea dy-state level of 0.5M H2O

2in chloroplast s) [6],

man y stresses tha t disrupt the cellular homeostasis of

cells enh an ce the production of ROIs (240720M s1

O2 an d a st eady-stat e level of 515 M H

2O

2) [6].

These include drought s tr ess an d desiccation, salt

str ess, chilling, heat sh ock, heavy met als, ultr aviolet

radiation, air pollutant s such a s ozone an d SO2,

mechanical st ress, nu trient deprivation, pathogen

at ta ck and h igh light str ess [2,710]. The production

of ROIs dur ing these str esses results from pathways

such as photorespira tion, from the photosynth etic

appara tus a nd from mitochondrial respiration. In

addition, pathogens and woun ding or environment al

str esses (e.g. drought or osmotic str ess) have been

shown to tr igger t he a ctive production of ROIs by

NADPH oxidases [4,1113]. The en ha nced pr oduction

of ROIs dur ing str ess can pose a thr eat t o cells but

it is also thought t hat ROIs act as signals for t heactivation of str ess-response and defense pat hways

[9,14]. Thus , ROIs can be viewed as cellular indicators

of str ess an d as seconda ry mess engers involved in the

stress-response signal tra nsduction pathway.

Although t he st eady-stat e level of ROIs can be

used by plant s to monitor their int ra cellular level of

stress, th is level has t o be kept under tight contr ol

becau se over-accumulat ion of ROIs can r esult in cell

deat h [14]. ROI-induced cell death can r esult from

oxidative processes such as m embr an e lipid

peroxidation, protein oxidation, enzyme inhibition

and DNA and RNA dama ge (the t raditional concept).

Alter na tively, enha nced levels of ROIs can a ctivate aPCD path way, as was recently demonstrat ed by the

inhibition of oxidative st ress (para quat )-induced cell

deat h in tobacco by ant i-apoptotic genes [15].

Because ROIs ar e toxic but also part icipate in

signaling event s, plant cells require a t least t wo

different mechanisms t o regulate their intracellular

ROI concentr at ions by scavenging of ROIs: one t ha t

will enable t he fine m odulat ion of low levels of ROIs

for signaling pur poses, an d one th at will enable the

detoxificat ion of excess ROIs, especially durin g str ess.

In addition, the types of ROIs produced and th e balance

between th e stea dy-sta te levels of different ROIs can

also be importa nt. These ar e determined by the

interplay between different ROI-producing a nd ROI-

scavenging mechan isms, and can cha nge drast ically

depend ing upon the physiological condit ion of th e

plant and the integration of different environmenta l,

developmen ta l and biochem ical st imuli.

Scavenging of ROIs in cells

Major ROI-scavenging mechanism s of plan ts in clude

super oxide dismut ase (SOD), ascorba te per oxidase

(APX) an d cat ala se (CAT) [1,7,16] (Table 1). The

balan ce between SOD a nd APX or CAT a ctivities in

cells is crucial for determ ining th e stea dy-sta te level of

Traditionally, reactive oxygen intermediates (ROIs) were considered to be toxic

by-products of aerobic metabolism, which were disposed of using antioxidants.

However, in recent years, it has become apparent that plants actively produce

ROIs as signaling molecules to control processes such as programmed cell

death, abiotic stress responses,pathogen defense and systemic signaling.

Recent advances including microarray studies and the development of m utants

with altered ROI-scavenging mechanisms provide new insights into how the

steady-state level of ROIs are controlled in cells. In addition, key steps of the

signal transduction pathway that senses ROIs in plants have been identified.

These raise several intriguing questions about the relationships betw een

ROI signaling,ROI stress and the production and scavenging of ROIs in the

different cellular compartments.

Published online: 12 August 2002

Oxidative stress, antioxidants andstress toleranceRon Mittler


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superoxide radicals an d hydrogen per oxide [17]. This

balance, together with sequest ering of meta l ions, isthought t o be importa nt t o prevent the form ation of the

highly toxic hydroxyl radical via t he m etal-dependent

HaberWeiss or the F enton reactions [1]. The different

affinities of APX (M ra nge) and CAT (mM ra nge) for

H2O

2suggest t ha t th ey belong to two different

classes of H2O

2-scavenging en zymes: APX might be

responsible for th e fine modulat ion of ROIs for

signaling, wherea s CAT might be responsible for or

the r emoval of excess ROIs dur ing str ess.

The ma jor ROI-scavenging pat hways of plant s

(Fig. 1) include SOD, found in a lmost a ll cellula r

compa rt ment s, the water water cycle in

chloroplasts (Fig. 1a), the as corba tegluta th ione

cycle in chloroplas ts , cytosol, mit ochondr ia, apoplas t

and peroxisomes (Fig. 1b), gluta th ione per oxidase

(GPX; Fig. 1c), and CAT in peroxisomes (Fig. 1d). The

finding of the a scorba tegluta th ione cycle in alm ost

all cellular compar tment s test ed to date, as well as

th e high affinity of APX for H2O

2, suggests tha t th is

cycle plays a crucial r ole in contr olling t he level of

ROIs in th ese compa rt ment s. By contra st, CAT is only

presen t in per oxisomes, but it is indispensa ble for

ROI detoxificat ion du ring st ress, wh en h igh levels of

ROIs ar e produced [16]. In a ddition, oxidative str ess

causes t he prolifera tion of peroxisomes [18]. Drawing

upon th e model for bacteria [19], a dens e population of

peroxisomes m ight be h ighly efficient in scavenging

of ROIs, especially H2O

2, which diffuses int o

peroxisomes from the cytosol.

The wat erwat er cycle (Fig. 1a) dra ws its redu cing

energy directly from the photosynthet ic appa ra tu s

[3]. Thus, t his cycle appear s to be au tonomous with

respect t o its en ergy sup ply. However, the source of

redu cing ener gy for ROI scavenging by the

ascorbategluta th ione cycle (Fig. 1b) during norm al

metabolism and pa rticularly during stress, when th e

photosynthetic apparat us m ight be suppressed or

damaged, is not entirely clear. In anima ls and yeast,

the pent ose-phosphate pathway is t he ma in source of

NADPH for ROI removal [20,21]. Becau se CAT does

not requ ire a su pply of reducing equivalent s for its

function, it might be insens itive to th e redox sta tu s of

cells and it s function might n ot be affected during

str ess, unlike the oth er mechan isms (Fig. 1).

Antioxidants s uch as ascorbic acid an d glutat hione,

which a re found at high concentra tions in chloroplasts

an d oth er cellular compa rt ment s (520 mM ascorbicacid and 15 mM glutat hione) ar e cru cial for plan t

defense agains t oxidative str ess [8]. Consequent ly,

both mut ant s with su ppressed ascorbic acid

levels [22] and tra nsgenic plants with alter ed

cont ent of gluta th ione [23] ar e hypersen sitive to

str ess conditions. It is genera lly believed tha t

ma inta ining a high redu ced per oxidized ratio of

ascorbic acid and gluta th ione is essent ial for t he

proper scavenging of ROIs in cells. This r at io is

ma inta ined by glutat hione redu ctase (GR),

monodehydroascorba te r eductase (MDAR) and

dehydroascorbate r eductase (DHAR) using NADPH

as r educing power (Fig. 1) [3,8]. In a ddition, th e overa llbalance between different a ntioxidant s ha s to be

tightly contr olled. Enh anced gluta thione biosynth esis

in chloroplasts can r esult in oxidative dam age to cells

rat her t han their pr otection, possibly by altering the

overa ll redox state of chloroplasts [23]. It h as a lso

been suggested t ha t t he oxidized:redu ced ratio of the

different an tioxidan ts can ser ve as a signal for the

modulat ion of ROI-scavenging m echanism s [24].

Avoiding ROI production

Avoiding ROI production m ight be as imp ortan t a s

active scavenging of ROIs. Becau se m an y abiotic

str ess conditions ar e accompanied by an enha nced

ra te of ROI pr oduction, avoiding or alleviating th e

effects of stress es such as drought or high light on

plant met abolism will reduce the r isk of ROI

production. Mecha nisms t ha t might r educe ROI

production durin g str ess (Table 1) include:

(1) ana tomical adapt ations such as leaf movement

an d curling, developmen t of a refracting epiderm is

an d hiding of stomat a in specialized str uctur es;

(2) physiological ada pta tions such as C4

an d CAM

meta bolism; an d (3) molecular m echanism s tha t

rear ran ge the photosynthetic appara tus an d its

ant ennae in accordan ce with light quality and


http://plants.trends.com

406 Review

Table 1. Producing, scavenging and avoiding reactive oxygen

intermediates in plantsa

Mechanism Localization Primary ROI Refs

Production

Photosynthesis ET and PSI or II Chl O2

[1,3]

Respiration ET Mit O2

[2,25]

Glycolate oxidase Per H2O2 [31]

Excited chlorophyll Chl O2

1[1]

NADPH oxidase PM O2

[4,5]

Fatty acid -oxidation Per H2O2 [31]

Oxalate oxidase Apo H2O2 [2

Xanthine oxidase Per O2

-[31]

Peroxi dases, Mn2+

and NADH CW H2O

2, O

2

[4,5]

Amine oxidase Apo H2O2 [46]

Scavenging

Superoxide dismutase Chl, Cyt, Mit, Per, Apo O2

[7]

Ascorbate peroxidase Chl, Cyt, Mit, Per, Apo H2O2 [1,3]

Catalase Per H2O2 [16]

Glutathione peroxidase Cyt, H2O

2, ROOH [47]

Peroxidases CW, Cyt, Vac H2O

2[1]

Thioredoxin peroxidase Chl, Cyt, Mit H2O2 [48]

Ascorbic acid Chl, Cyt, Mit, Per, Apo H2O2, O2

[3,8]

Glutathione Chl, Cyt, Mit, Per, Apo H2O

2[3,8]

-Tocopherol Membranes ROOH, O21

[1]

Caretenoids Chl O21

[1]

Avoidance

Anatom ical adaptations Leaf structure, epiderm is O2

, H2O2, O21

[44,49]

C4 or CAM metabolism Chl, Cyt, Vac O2

, H2O2 [49]

Chl movement Cyt O2

, H2O2, O21

[44]

Suppression of photosynthesis Chl O2

, H2O

2[49]

PS and antenna modulations Chl O2

, O21

[44]

Alternative oxidases Chl, Mit O2

[25,50]

aAbbrevi ations: Apo, apopl ast; Chl, chloro plast; CW, cell wall; Cyt, cytosol; ET, electron t ransport;

Mit, mitochondria; O21, singlet oxygen; Per, peroxisome; PM, plasma membrane; PS,

photosystem; ROI, reactive oxygen intermediate; Vac, vacuole.


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inten sity or completely suppr ess photosynthesis. By

balan cing the a mount of light en ergy absorbed by th e

plant with t he ava ilability of CO2, these mechanisms

might repr esent a n a ttem pt to avoid th e over-reduction

of the photosynthetic apparatu s and t he tr ansfer of

electr ons to O2

rather than for CO2

fixation.

ROI production can a lso be decrea sed by the

alter na tive cha nn eling of electrons in th e electr on-

tr an sport chains of th e chloroplast s and mitochondria

by a group of enzymes called alter na tive oxidases

(AOXs). AOXs can divert electrons flowing t hr ough

electron-transport chains and u se them to reduce O2

to water (Fig. 2). Thu s, they decreas e ROI production

by two mechan isms: they pr event electrons from

redu cing O2

into O2 an d they redu ce th e overall level

of O2, the subst ra te for ROI production, in the

organelle. Decreasing th e am ount of mitochondria l

AOX increases the sensitivity of plan ts to oxidative

str ess [25]. In a ddition, chloroplast AOX is indu ced in

tr an sgenic plan ts t hat lack APX and/or CAT, and in

norma l plants in r esponse to high light [50].

Production and scavenging of ROIs in different cellular

compartments

Recent man ipulat ions of ROI-scavenging pathways in

different cellular compar tmen ts suggest some

intr iguing possibilities. For year s, the chloroplast was

consider ed to be the ma in source of ROI production in

cells and consequen tly one of the ma in ta rgets for ROI

dama ge during st ress. However, it has recently been

suggested that the chloroplast is n ot as sensitive to ROI

dam age as pr eviously thought [26]. The mit ochondr ion

is another cellular site of ROI production. However,

recent stu dies suggest t hat the m itochondrion is a lso a

key regulator of PCD in plants a nd th at enh anced ROIs

levels at t he mitochondr ia can tr igger PCD [27].

Both the mitochondrion a nd t he chloroplast conta in

ROI-scavenging mecha nism s. By cont ra st, little is

known about the ROI-scavenging properties of the

nucleus, which might cont ain redox-sens itive

tr an scription factors [28]. Because H2O

2can diffuse

thr ough aqua porins [29], ROIs pr oduced at a specific

cellular site (e.g. the chloroplast du ring st ress or

the a poplast during pa thogen att ack) can affectother cellular compart ment s, overwhelm their

ROI-scavenging capabilities and alter the pa tter n of

gene expression during str ess, pat hogen infection or

PCD. In support of this assu mption, stresses th at

result in t he enha nced production of ROIs at t he

chloroplast induce cytosolic and n ot chloroplast ic ROI-

scavenging mechanism s [24,30], an d ROI production

at the apoplast induces th e production of pathogenesis-

response proteins [4]. Because the plant mitochondria

an d nuclei ar e involved in th e activat ion of PCD [27],

the level of ROIs tha t r eaches these compar tmen ts

during st ress or pa thogen challenge needs to be tightly

contr olled to prevent abnorma l PCD a ctivation. Thecytosol, with its a scorba teglut at hione cycle, and th e

peroxisomes, with CAT, might t her efore a ct as a buffer

zone t o control th e overa ll level of ROIs tha t r eaches

different cellular compart ment s during stress a nd

normal meta bolism.

The import an ce of peroxisomes in ROI met abolism

is beginning t o gain r ecognition [31]. Per oxisomes ar e

not only th e site of ROI detoxificat ion by CAT but also

th e site of ROI pr oduction by glycolate oxidase a nd

fatty a cid -oxidation. In addit ion, per oxisomes might

be one of the cellular sites for n itr ic oxide (NO)

biosynth esis [31]. In a nima l cells, NO activat es fatt y

acid -oxidation and enha nces the production of ROIs

in cells. However, although NO h as been shown to be

involved in ROI-induced cell death in plan ts [32] and

NO is known to be a key regulat or of pat hogen

responses [5], little is kn own a bout how NO is

involved in the r esponse of plant s to abiotic stresses.

Redundancy in ROI-scavenging mechanisms

Some of the complex relat ionsh ips between th e

different ROI-scavenging a nd ROI-producing

mechanisms ha ve been revealed in tran sgenic plant s

with suppr essed pr oduction of ROI-detoxifying

mechanisms. Thus, plants with suppressed APX



407Review

TRENDS in Plant Science

(a)

(b)

(c)

(d)

SOD

Fd tAPX

APX MDAR

AsA + DHA GSSG NAD(P)+

DHAR GR

AsA GSH NAD(P)H

SOD

CAT

H2O2 GSSG NAD(P)+

GPX GR

H2O

H2O2

O2

GSH NAD(P)H

H2O O2

O2 H2O2

e PSI

H2O2

H2O2 MDA NAD(P)+

MDA

H2O

H2O NAD(P)HAsA

AsA

Fig. 1. Pathways for

reactive oxygen

interm ediate (ROI)

scavenging in plants.

(a) The waterwater cycle.

(b) The ascorbate

glutathione cycle. (c) The

glutathione peroxidase

(GPX) cycle. (d) Catalase

(CAT). Superoxide

dismutase (SOD) acts asthe first line of d efense

converting O2 into H

2O

2.

Ascorbate peroxidases

(APX), GPX and CAT then

detoxify H2O

2. In contrast

to CAT (d), APX and GPX

requir e an ascorbate (AsA)

and/or a glutathione (GSH)

regenerating cycle (ac).

This cycle uses electrons

directly from the

photosynthetic apparatus

(a) or NAD(P)H (b,c) as

reducing pow er. ROIs are

indicated in red,

antioxidants in blue and

ROI-scavenging enzym esin green. Abbreviations:

DHA, dehydroascorbate;

DHAR, DHA reductase;

Fd, ferredoxin; GR,

glutathione reductase;

GSSG, oxidized

glutathione; MDA,

monodehydroascorbate;

MDAR, MDA reductase;

PSI, photosystem I; tAPX,

thylakoid-bound APX.


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ROI signal transduction pathway

Recent st udies ha ve ident ified several components

involved in the signal tr an sduction path way of plant s

tha t senses ROIs. These include the m itogen-activat ed

protein (MAP) kinase kin ase kina ses AtANP1 an d

NtNPK1, and t he MAP kinases AtMPK3/6 and

Ntp46MAPK [39,40]. In addition, calmodulin ha s

been implicated in ROI signaling [9,41]. A hypothet ical

model depicting some of the players in volved in th is

pat hway is sh own in F ig. 4. H2O

2is sensed by a sen sor

tha t might be a two-component histidine kina se,

as in yeast [9]. Calmodulin and a MAP-kinase

cascade ar e th en a ctivated, resulting in t he a ctivation

or suppr ession of severa l tra nscription factors.

These regula te th e response of plant s to oxidative

str ess [9,42]. Cross-ta lk with th e path ogen-response

signal t ran sduction pa thway a lso occurs and might

involve intera ctions between different MAP-kinase

pat hways, feedback loops and th e action of NO and

SA as key horm onal regulat ors. This model (Fig. 4) is

simplified an d is likely to cha nge as r esear ch

advances our underst anding of this path way.

ROIs act as signals th at m ediate the systemic

activation of gene expression in r esponse to pathogen

at ta ck [43], wounding [11] and high light [44]. They

were suggested to act in conjunction with a compound

tha t t ravels systemically and activates their production

in distal parts of the plant, where they mediate the

induction of gene expr ession [11]. The in volvement of

ROIs in the regulat ion of stoma ta l closur e [13] and in

other cellular responses involving auxin [39,45] might

suggest tha t more signaling pathways involving ROIs

as in ducers of systemic signals a wait d iscovery. It is

unlikely that ROIs can t ra vel systemically because

they are h ighly rea ctive an d would be scavenged along

the way by the ma ny ant ioxidative mechan isms an d

ant ioxidant s present in th e apoplast. However, it is

possible th at a wave of activity similar to the oxidative

burstis activated in cells along the systemic path and

in distal t issues, resulting in t he a ccumu lation of ROIs.

Fut ure st udies using plants with alter ed levels of

ROI-scavenging an d/or ROI-producing mechanism s

might r esolve this question.

Future challenges and questions

The cause of cell death induced in plan ts by oxidat ivestr ess is not k nown. Is it sim ply the t oxicity of ROIs

th at da ma ges cells or is it th e activation of a PCD

pat hway by ROIs? It is possible tha t t he level of H2O

2

th at is cur ren tly thought to kill cells by direct cellular

dam age actua lly induces PCD [15,27], an d it might

requir e a higher level of ROIs to kill cells by dir ect

oxidation. Perh aps futu re stu dies applying oxidative

stress to muta nts deficient in different PCD path ways

will an swer th is question.

Many questions related t o ROI m etabolism rem ain

una nswer ed (Box 1). We are cur ren tly at a n exciting

time, when m ost of th e technologies required t o

answer t hese questions a re in place. Thus, acompr ehens ive analysis of gene express ion u sing

microarr ays an d chips, coupled with pr oteomics and



409Review

Acknowledgements

I apologize to all colleagues

whose work could not be

reviewed here b ecause of

space limitation. I thank

Barbara A. Zilinskas and

EveSyrkin Wurtele for

critical reading of the

manuscrip t. Research at

my laboratory is supported

by funding fr om the Israeli

Academy o f Sciences and

the Biotechnology Council

of Iowa State University.

TRENDS in Plant Science

SensorH2O2

WRKYEREBPDREBAmybAP-1Ocs/AS-1HSF

AtANP1AtMPK3/6p46MAPK

Calmodulin

Ca2+

Gene-for-gene

?

?

Photo-synthesis

NADPHsupply

HSPs

ROI

scavenging

ROIproduction

Pathogenresponses

PCD

MAPKcascade(s)

Ca/Calmodulinkinase(s)

SA NO

Tyr

phosphataseTwo-component

His-kinase?

Ras/Rac

?

Transcriptionfactors

Fig. 4. A suggested model for the activation of signal transduction events during oxidative stress.

H2O

2is detected by a cellular receptor or sensor. Its detection result s in the activation of a mito gen-

activated-prot ein kinase (MAPK) cascade and a group of transcript ion factors that contr ol different

cellular pathway s. H2O

2sensing is also lin ked to changes in the lev els of Ca2+ and calmodulin, and to

the activation or induction of a Ca2+calmodu lin kinase that can also activate or suppress the activity

of transcription factors. The regulation of gene expression by the different transcription factors results

in the inducti on of variou s defense pathways, such as reactive oxygen int ermedi ate (ROI) scavenging

and heat-shock proteins (HSPs), and in th e suppression of som e ROI-producing m echanisms and

photo synthesis. There is also cross-talk with the plant pathogen signal transduction p athway, which

might depend on pathogen recognition by the gene-for-gene mechanism and can result in an inverse

effect on the regulatio n of ROI-produ ction and ROI-scavenging m echanisms, as well as on the

activation o f progr amm ed cell death (PCD). The plant horm ones nitric oxid e (NO) and salicylic acid

(SA) are key regulato rs of thi s response.

How t oxic are reactive oxygen i ntermediates (ROIs)to plant cells?

What are the sensors for ROIs in pl ants? What regulatory networks control the production

and scavenging of ROI in plants? How are theycoordinated? Is nitric oxide involved in abiot icstress signaling?

How is ROI metabolism regulated during acombinati on of abio tic and bioti c stresses, andduring m ultipl e abioti c stresses?

Are there un discovered or neglected ROI-producingand/or ROI-scavenging mechanisms [a]?

What is the chloroplast to nucleus signal involved i nsensing ROI stress?

What is t he source of r educing energy [ i.e. NAD(P)H]for ROI removal during norm al metabolism and stress?

Is the diffusion of H2O

2through aquaporins regulated?

Can we develop imaging t ools to study thesubcellular location of ROIs in plants?

Reference

a Oberschall, A.et al. (2000) An ovel aldose/aldehyde redu ctase

protects tran sgenic plants against lipid peroxidation under

chemical and dr ought stresses. Plant J. 24, 437446

Box 1.Questions and future challenges


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