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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
TRENDSi n Plant Science Vol.7 No.9 September 2002
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
TRENDSi n Plant Science Vol.7 No.9 September 2002
http://plants.trends.com
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
TRENDSi n Plant Science Vol.7 No.9 September 2002
http://plants.trends.com
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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