PERSPECTIVE
A unified mechanism of action for volatile isoprenoids
in plant abiotic stress
© 2009 Nature America, Inc. All rights reserved.
Claudia E Vickers, Jonathan Gershenzon, Manuel T Lerdau & Francesco Loreto
The sessile nature of plants has resulted in the evolution of an
extraordinarily diverse suite of protective mechanisms against
biotic and abiotic stresses. Though volatile isoprenoids are
known to be involved in many types of biotic interactions, they
also play important but relatively unappreciated roles in abiotic
stress responses. We review those roles, discuss the proposed
mechanistic explanations and examine the evolutionary
significance of volatile isoprenoid emission. We note that abiotic
stress responses generically involve production of reactive
oxygen species in plant cells, and volatile isoprenoids mitigate
the effects of oxidative stress by mediating the oxidative status
of the plant. On the basis of these observations, we propose a
‘single biochemical mechanism for multiple physiological
stressors’ model, whereby the protective effect against abiotic
stress is exerted through direct or indirect improvement in
resistance to damage by reactive oxygen species.
Owing to their sedentary lifestyle, plants must be capable of coping
with a variety of changes in light intensity, temperature, moisture
and other abiotic factors in their environments (Fig. 1). When
these factors shift out of a certain range, plants are subjected to
stress; this can lead to decreased growth rate, reduced reproduction
and even death. Beyond the single-plant level, changes in environmental stress can also select for short- and long-term shifts in
ecological traits, potentially affecting biological diversity, ecosystem
functioning and carbon sequestration. Abiotic stress also results
in significant losses in crop yields, the magnitude of which
fluctuates from year to year. Sources of abiotic stress include
extremes of temperature (including freezing), high light intensity,
drought, air pollutants, salinity and mechanical damage. These
stresses are rarely experienced singularly: they often occur in
combination. The simultaneous occurrences of rapidly rising temperatures, drought and pollutants are among the most striking
phenomena associated with global change, and they threaten plants
that have not adapted and are not able to rapidly acclimate to
these factors1.
Claudia E. Vickers is at the University of Queensland, Australian Institute for
Bioengineering and Nanotechnology, St. Lucia, Australia. Jonathan Gershenzon
is at the Max Planck Institute for Chemical Ecology, Jena, Germany. Manuel T.
Lerdau is in the Environmental Sciences and Biology Departments, University
of Virginia, Charlottesville, Virginia, USA. Francesco Loreto is at Consiglio
Nazionale delle Ricerche, Istituto di Biologia Agroambientale e Forestale,
Monterotondo Scalo (Roma), Italy. e-mail: [email protected].
Published online 17 April 2009; doi:10.1038/nchembio.158
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Few responses of plants are stress-specific. Most often, stresses elicit
generic responses—in particular, production of excess reactive oxygen
species (ROS) such as singlet oxygen (1O2), superoxide (O2 ),
hydrogen peroxide (H2O2) and hydroxyl radicals (OH). ROS are
important signaling molecules and also serve to initiate defense
responses2. The cellular balance of ROS is normally kept under tight
control3; however, when this control is lost, damage occurs. ROS cause
direct damage to plant cells through oxidation of biological components (nucleic acids, proteins and lipids) and can instigate chain
reactions resulting in accumulation of more ROS and initiation of
programmed cell death2. Plants have a complex response network of
lipid-phase and aqueous-phase antioxidant compounds and enzymes
that defend against conditions of excess ROS. Direct reactions to
quench and remove ROS occur (for example, Fig. 2), as well as
indirect responses including hormone-mediated signaling to upregulate primary defense genes and activate secondary defense genes
(reviewed previously2–4). When the antioxidant defense network is
overloaded, oxidative stress results. Measuring abiotic stress responses
and attributing mechanistic behavior can be problematic because of
the complexity of the ROS response network, which makes it
difficult to distinguish cause-and-effect relationships. The network is
linked between lipid and aqueous phases, so understanding physical
compartmentalization is fraught. Many ROS are short-lived, so
direct measurements are also difficult; further, many molecules
involved in signaling cascades have not been identified and thus
cannot be measured.
Although most research on plant antioxidants has focused on
nonvolatile compounds, certain volatiles belonging to the isoprenoid
family have also been implicated in protection against oxidative and
other abiotic stresses. The isoprenoids are a very large and extremely
diverse group of organic compounds. Isoprenoid carbon skeletons are
composed of five-carbon building blocks that may be assembled in a
variety of formations and contain many different modifications. In
plants, two separate metabolic pathways are responsible for the
production of the C5 building block of isoprenoids: the cytosolic
mevalonic acid (MVA) pathway, and the plastidic 2-C-methyl-Derythritol 4-phosphate (MEP) pathway5–7 (Fig. 3). The two pathways
can be linked through exchange of metabolic precursors across the
plastid membrane8–11. It is generally assumed that later steps in the
formation of hemiterpenes (C5), monoterpenes (C10), diterpenes (C20)
and tetraterpenes (C40) are present in the plastids, whereas the
formation of sesquiterpenes (C15) and triterpenes (C30) takes
place in the cytosol. Volatile isoprenoids are generally lipophilic, lowmolecular-weight compounds with masses under 300 (see Fig. 4).
Only hemiterpenes (isoprene and methylbutenol), monoterpenes,
283
PERSPECTIVE
conditions, particularly under heat and
light stress13,15,19,20. Stressed plants can maintain high isoprenoid emissions, and the
emission can be transiently enhanced in
SO2
High temperature stress denatures
Mechanical damage—both biotic
plants recovering from stresses, particularly
O3
proteins and causes lipid peroxidation.
(e.g., from insect feeding) and abiotic
(e.g., from wind damage)—triggers
in drought-stressed plants14,21,22. Application
expression of defense-related genes.
Water deficit, or drought, interferes
of jasmonates, which trigger defense response
with metabolism. ROS produced
under drought conditions trigger
pathways in both biotic and abiotic stresses,
signaling pathways that generate
stimulates production of volatile isopredefense responses.
noids23,24. Stored carbon can even be mobiCold
stress
interferes
with
metabolic
Soil salinity is usually caused by
processes (particularly enzyme
excess salts of chloride and sulfate.
lized to maintain volatile production under
activity) and alters membrane
Salinity results in ion cytotoxicity and
properties. Frosting can severely
stress conditions14,25,26. Subsequent experiosmotic stress, and decreases uptake
damage tissues when ice forms.
of nutrients. Resulting metabolic
ments have demonstrated that volatile isopreExtracellular ice formation also
imbalances lead to oxidative stress.
causes intracellular water deficit.
noids play a protective role under thermal,
Na+ K+
Cl–
radiative, oxidative, drought and salt stress.
2+
2+
Mg Ca
Here we will examine the evidence showing
that volatile isoprenoids confer protection
Figure 1 Plants are exposed to a variety of abiotic stresses. Complex response and protective systems
against abiotic stress, and we will argue that
are triggered under these stress conditions (reviewed previously86–90). All of these abiotic stresses
the common mechanism driving abiotic
result in production of reactive oxygen species (ROS). Excess light and heat, as well as exposure to
stress protection is an antioxidant effect of
oxidizing air pollutants, cause direct accumulation of ROS (see Fig. 2). High temperatures are often
these compounds.
coincident with high light stress. Drought results in osmotic stress and intracellular water deficit; soil
It should be noted that other functional
salinity and cold stress (particularly frosting) also result in water deficit, and the molecular responses to
these three stresses are similar (though not identical). When stresses are combined, responses are often
possibilities, including balancing of subamplified; for example, high light/low temperature stress and high light/low water stress can result in
cellular supplies of phosphoenolpyruvate27
very high production of ROS. ROS are particularly important for initiating signal cascades that trigger
and dissipation of excess carbon28 and
defense gene transcription and adaptive responses. Phytohormones are also important in these
energy29 from photosynthesis, have been
responses and are involved in signaling pathways. ABA is particularly important in water deficit
put forward to explain isoprene emission;
responses, and ethylene, salicylic acid and jasmonic acid are often involved in wound responses.
however, there are stoichiometric and biochemical arguments against these proposed
sesquiterpenes and some diterpenes have sufficient vapor pressure to functions30, and they have not been extended to volatile isoprenoids in
volatilize at ordinary biological temperatures12. Volatility bestows general. Isoprene emissions have also been shown to affect biotic
particular properties on these compounds—for example, the ability interactions31,32. The roles of volatile isoprenoids may be multifaceted,
to carry chemical messages away from the sites of synthesis in the plant. and different functional hypotheses are not necessarily mutually
Production of volatile isoprenoids represents a substantial invest- exclusive. Here we will focus on the behavior and mechanism of
ment for the plant in terms of carbon (loss of which in the form of action of volatile isoprenoids in abiotic stress conditions.
volatiles is irretrievable) and energy. Constitutive emissions in isoprene
emitters and strong monoterpene emitters are generally in the range Volatile isoprenoids protect against abiotic stress
of 1–100 nmol m 2 s 1 (http://www.es.lancs.ac.uk/cnhgroup/down Experimental evidence has shown that volatile isoprenoids confer a
load.html). This amount is equivalent to 1–2% of photosynthetic protective effect to photosynthesis under thermal and oxidative
carbon fixation13. Even when the carbon budget becomes negative stress conditions. This evidence comes from three types of experiunder stress conditions and photosynthesis is severely or completely ment: (i) the use of fosmidomycin, an antibiotic/herbicide that
inhibited, isoprenoid emission is often sustained14–16. This large cost, selectively inhibits the activity of 1-deoxy-D-xylulose 5-phosphate
especially under stressful conditions, suggests the strong possibility that reductoisomerase (DXR), thereby inhibiting the MEP pathway33
isoprenoid emission also confers benefits to the plant. Though the role (see Fig. 3), (ii) fumigation of non-emitting species with exogenous
of some volatiles in biotic interactions is well established and has gaseous isoprenoids, also followed by reconstitution experiments in
been well reviewed12,17,18, there are many volatiles for which the leaves in which endogenous emission was previously inhibited
benefit remains obscure. Higher order (nonvolatile) isoprenoids have chemically, and (iii) the use of transgenic plants in which isoa variety of roles in plant cells, including in abiotic stress defense. The prenoid synthesis has been either engineered by insertion of the
mechanisms by which they act are diverse; some are hormonal signals appropriate terpene synthase genes, or repressed by silencing the
that can carry messages throughout the plant and elicit systemic same genes.
responses (for example, abscisic acid), whereas others act directly as
antioxidants (for example, carotenoids and tocopherols; see Fig. 4). Thermal (high temperature) stress. The role of volatiles in protecRecent research, which we will highlight here, has revealed that certain tion against thermal stress has been relatively well studied. Sharkey
volatile isoprenoids also play an important role in abiotic stress and co-workers have been investigating the isoprene effect for over
responses. Though the bulk of this research has focused historically a decade13,30,34. Under sun-flecking conditions, leaf temperature
on isoprene (2-methyl-1,3-butadiene), there is increasing evidence that can fluctuate dramatically, with variations of up to 20 1C occurring
many (and perhaps all) volatile isoprenoids are involved in abiotic in very short time periods; in this scenario, co-incident light
and heat stress are experienced in a punctuated fashion. Recovery
stress responses.
Changes in volatile emission patterns under stress conditions origi- from temperature-induced decreases in photosynthetic efficiency
nally supplied circumstantial evidence that volatiles are linked is poorer in isoprene-emitting plants that are treated with
with stress responses. Emissions often increase under abiotic stress fosmidomycin, and fumigation with isoprene can partially restore
© 2009 Nature America, Inc. All rights reserved.
High light causes production of excess
excitation energy in the photosynthetic
reaction centers, resulting in direct
accumulation of a variety of reactive
oxygen species.
284
Air pollution with oxidizing species
(including ozone and sulfuric acid)
causes direct oxidative damage to
tissues. Local and systemic signaling
responses also occur.
VOLUME 5
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PERSPECTIVE
recovery in fosmidomycin-poisoned leaves. These results were
confirmed in an independent laboratory35–37. However, given
that fosmidomycin inhibition of the MEP pathway acts by inhibition
of one of the early pathway enzymes, compounds besides isoprene
might also be affected (for example, fosmidomycin can affect abscisic
acid biosynthesis38). The inhibition studies were followed up using
transgenic Arabidopsis thaliana plants bearing heterologously expressed
isoprene synthase genes39,40. Prolonged heat stress experiments
confirmed that isoprene emission could confer a thermotolerant
growth phenotype relative to non-emitting plants, but protection of
photosynthesis could not be tested in this system because control
plants did not show inhibition of photosynthesis after heat stress
episodes39. However, these transgenic plants emitted only low levels
of isoprene relative to a naturally emitting plant. A second approach
was the use of transgenic poplar plants with silenced isoprene
synthase expression41. These plants showed little or no isoprene
emission, and demonstrated greater sensitivity of photosynthesis to
repeated heat/light stress events. However, interpretation of these
experiments is complex because stress sensitivity often occurs in
primary-generation transgenic plants due to collateral damage
during transformation and tissue culture processes, and can be
carried through several transgenic generations42. Consequently, analysis
of stress responses in transgenic plants in species with long generation times is fraught with difficulties.
To address these issues, transgenic tobacco plants heterologously
expressing isoprene synthase have been engineered43. These lines
produced high levels of isoprene, and azygous plants were generated
for use as controls. When fourth-generation plants were placed under
a repeated heat/light stress regime, photosynthesis recovered better in
homozygous emitting plants compared to azygous non-emitting
controls. The effect was only distinguishable by longitudinal analysis
and was not as strong as that observed in fosmidomycin-poisoned
leaves, which suggests that (i) inhibition of other MEP products might
play a role in the phenotype observed using fosmidomycin treatments,
and/or (ii) species-dependent variation exists. However, these results
demonstrated that endogenously emitted isoprene plays a role in
thermotolerance of photosynthesis. The subtlety of the effect might
explain why it was not observed in the low-emitting transgenic
A. thaliana plants.
Protection of photosynthesis against high temperature stress is also
observed in monoterpene-emitting plants, when emission of monoterpenes inhibited by fosmidomycin is restored with exogenous
monoterpene fumigation19. Monoterpene fumigation can also
confer thermotolerance on low-emitting species that are not treated
3
Thylakoid lumen
1
APX
T(OOH) αT(OH)
1
O2
PSII
SOD
1
O2
O2.–
.OH
PL
PSI
O2.–
2 H+
O2
1
PL-OH
APX
H2O2
NAD(P)H
MDAR
Asc
+
APX
αT(OH)
PSII
.
PL-OO
PL-OOH
4
DHA
2
H2O2
αC(O.)
H2O
MDA
Asc
PHGPX
O2
H2O2
PRX
NTR
DHAR GSSG
NAD(P)H
H2O2
GSSG
GR
GPX
GSH
NAD(P) MDA
H2O2
GSH
H2O
GR
H2O
TRX-SH
TRX-SS
NAD(P)+
NAD(P)H
NAD(P)+ NTR
TRX-SH
TRX-SS PRX
H2O
H2O
Chloroplast stroma
H2O2
Figure 2 The chloroplastic antioxidant/enzyme defense network reactions in response to increased light and temperature. When absorbed energy is in excess
of that used in photosynthesis, various different ROS are formed at the photosystems in the thylakoid membranes of the chloroplast, and a complex
scavenging system activates2,70,91. At photosystem II (PSII), O2 , H2O2, singlet oxygen (1O2) and hydroxyl radicals (OH) are produced from molecular
oxygen (1). Superoxide ions are produced during the Mehler reaction by Fd-NADPH oxidase at photosystem I (PSI) (1O2 may also be produced at PSI) (2).
O2 is dismutated by superoxide dismutase (SOD) to hydrogen peroxide (H2O2). 1O2 is highly reactive and has an extremely short half-life, reacting very
quickly with molecules close to the site of synthesis (proteins, pigments and lipids), and recent research in fact suggests that 1O2 is the major cause of
photo-oxidative damage under high light stress71. It is quenched by b-carotene in the PSII reaction center and a-tocopherol (aT(OH)) in the thylakoid
membranes; excess 1O2 reacts with D1 protein, resulting in degradation of D1 and loss of PSII activity (photoinhibition). Unquenched 1O2 also causes lipid
peroxidation (3) and can trigger defense gene expression via signal transduction (1O2 reacts with free radical nitric oxide (NO) to produce peroxynitrite
(ONO2 ), which acts as a signaling molecule). 1O2 and OH can be scavenged by ascorbate (Asc), tocopherols and glutathione (GSH). H2O2 is produced
during a variety of different reactions under stress conditions, often from detoxification of other, more dangerous ROS. It is scavenged by three different
antioxidant/enzyme reactions: the Asc, GSH and peroxiredoxin (PRX) cycles. These cycles are interlinked through shared metabolites and reducing
equivalents. Increased temperature, which is often co-incident with high light stress, also causes lipid peroxidation and results in phospholipid peroxy
radicals (PL-OO) (4). PL-OO is converted to phospholipid hydroperoxide (PL-OOH) by oxidation of a-T(OH); PL-OOH is reduced to phospholipid alcohol
(PL-OH) by the action of phospholipid hydroperoxide–dependent glutathione peroxidase (PHGPX). Enzymes controlling regeneration of metabolites can be
found in the aqueous environment (stroma) or bound to the thylakoid lipid membrane. Lipid- and aqueous-phase antioxidant networks are linked through
Asc-mediated regeneration of a-T(OH) and activity of PHGPX. Not all chloroplast redox reactions are shown here. Similar antioxidant reaction networks can
be found in the cytosol and in other organelles; cross-talk between these compartments also occurs. Additional abbreviations: aC(O), a-chromanoxyl radical;
APX, ascorbate peroxidase; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; GR, glutathione reductase; GSH, reduced glutathione; GSSG,
glutathione disulfide; GPX, glutathione peroxidase; MDA, monodehydroascorbate; MDAR, monodehydroascorbate free radical reductase; NTR, NADPHthioredoxin reductase; T(OOH), hydroperoxytocopherone; TRX, thioredoxin; TRX-SH, reduced thioredoxin; TRX-SS, thioredoxin disulphide.
NATURE CHEMICAL BIOLOGY
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© 2009 Nature America, Inc. All rights reserved.
PERSPECTIVE
Figure 3 Isoprenoid biosynthetic pathways
MVA
MEP
(simplified). Two linked isoprenoid biosynthetic
GA3P
GA3P
pathways are found in plant cells: the cytosolic
MVA pathway and the chloroplastic MEP
PEP
PEP
Pyruvate
pathway. The universal five-carbon building
blocks produced by these pathways are isopentyl
Chloroplast
DXP
Pyruvate
pyrophosphate (IPP) and its isomer dimethylallyl
DXR
diphosphate (DMADP); conversion between
MEP
Acetyl-CoA
isoforms is catalyzed by isopentyl diphosphate
isomerase (IDI). The cytosolic MVA pathway
HMG-CoA
produces sesquiterpenes, triterpenes,
HMGR
homoterpenes and precursors for sterols and
Isoprene (C5)
DMAPP(C5)
MVA
IPP
ubiquinone via farnesyl diphosphate (FPP);
IDI
IPP
the chloroplastic MEP pathway produces the
DMAPP
IPP
hemiterpene isoprene, monoterpenes (via geranyl
Monoterpenes (C10)
GPP (C10)
2x
diphosphate, GPP), diterpenes, tetraterpenes and
2 x IPP
PQ-9 (C45)
higher order isoprenoids (via geranylgeranyl
(prenyl chain)
diphosphate, GGPP). Fosmidomycin inhibits
+5 IPP
GGPP (C20)
FPP (C15)
Diterpenes (C20)
1-deoxy-D-xylulose 5-phosphate reductoisomerase
2x
2x
Phytyl chains
(DXR), thereby inhibiting formation of IPP and
Chlorophylls
Squalene
Tetraterpenes (C40)
DMADP from the MEP pathway. Members of the
Tocopherols
Sesquiterpenes (C15)
Carotenoids
Phylloquinones
isoprenoid group have an extraordinarily wide
Triterpenes (C30)
Abscisic acid
Phytoalexins
Sterols
Homoterpenes (C11, C30)
range of roles in the plant; these include wellGibberellins
Polyprenols
characterized physiological functions in primary
Taxol
metabolism such as growth regulation (for
example, hormones), photosynthetic components (phytol side chains of chlorophylls, prenyl side chains of plastoquinones) and structural (for example, sterol
membrane components) roles, and also secondary functions such as defense (for example, some phytoalexins) and antioxidants (for example, tocopherols
and carotenoids). In addition to this, isoprenoids fulfill a variety of roles in secondary metabolism; these roles are also very diverse, and many remain to be
fully characterized. ABA, abscisic acid; DXP,1-deoxy-D-xylulose 5-phosphate; GAs, gibberellins; GA3P, glyceraldehyde 3-phosphate; HMG-CoA, 3-hydroxy-3methylglutaryl-CoA; HMGR, HMG-CoA reductase; PEP, phosphoenolpyruvate; PPi, pyrophosphate; PQ, plastoquinone.
with fosmidomycin44. Transgenics that over- or underexpress
higher order volatile isoprenoids have not yet been studied for
resistance to abiotic stress.
Oxidative stress. Several studies show that isoprene protects leaf
tissues against oxidative stress. Leaves show lower accumulation of
ROS, less cellular damage and less damage to photosynthetic processes
in response to ozone (O3) fumigation when isoprene is also applied45.
Similarly, fosmidomycin-poisoned leaves show more photosynthetic
damage from ozone fumigation than leaves that are producing
isoprene46. Under photo-oxidative stress, singlet oxygen is produced
at the photosynthetic membranes, and photosynthetic assimilation
Figure 4 Chemical structures of isoprenoids
with antioxidant properties. Many higher order
nonvolatile isoprenoids have been shown to have
antioxidant functions (left panel). Tocopherols
and tocotrienols are lipid-phase antioxidants.
They scavenge lipid peroxy radicals and react
with and physically quench singlet oxygen;
R groups are methyl (-CH3) or hydrogen (-H).
Carotenoids are photosynthetic pigments that
provide photoprotection through antioxidant
activity in addition to absorbing light energy
for photosynthesis. There are two classes of
carotenoids: unoxygenated (carotenes) and
oxygenated (xanthophylls). Carotenes quench
triplet chlorophyll, and xanthophylls such as
violoxantin participate in the xanthophyll cycle,
which is responsible for quenching singlet
chlorophyll. The volatile isoprenoids shown
in the right panel have either been shown to
have antioxidant properties (for example,
isoprene) or have chemical properties
conducive to antioxidant activities.
286
is inhibited47. Isoprene fumigation of non-emitting leaves results
in protection of photosynthetic processes when singlet oxygen is
produced by addition of the photosensitizer Rose Bengal48; fosmidomycin-based inhibition studies support this49. Experiments using
transgenic tobacco plants heterologously expressing isoprene synthase
(described above) confirmed that isoprene-emitting plants show
much greater resistance to ozone-induced oxidative stress compared
to azygous control plants43. This showed that the protective effect was
associated with endogenous isoprene production.
As is the case for thermal stress, much less evidence has been
collected from monoterpene-emitting plants than from isoprene
emitters. However, it has been demonstrated clearly that the
Hemiterpenes
Tocopherols
Monoterpenes
R1
O
R2
HO
R3
Isoprene
Tocotrienols
α-Pinene
β-Pinene
Myrcene
R1
R2
O
HO
R3
Limonene
Carotenoids
Sabinene (E )-β-Ocimene (Z )-β-Ocimene
Sesquiterpenes
α-Carotene
α-Humulene
(E )-β-Farnesene
(E ,E)-α-Farnesene
β-Carotene
OH
O
O
HO
Violoxanthin
(E )-β-Caryophyllene
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δ-Cadinene
NATURE CHEMICAL BIOLOGY
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PERSPECTIVE
Environmental
Figure 5 The ‘single biochemical mechanism for multiple physiological
stress
stressors’ model. The model shows how oxidative damage resulting from
1
+ VIP
environmental stress occurs (in gray), and how volatile isoprenoids (VIPs)
may exert protective effects through antioxidant activity (in black). Solid
Ox
2
lines represent direct reactions, and broken lines represent indirect
+ VIP
reactions. Environmental stress (high light, temperature, ozone exposure)
2
causes oxidative stress (Ox), which results in production of ROS (for
+ VIP
ROS
RES
example, hydrogen peroxide, singlet oxygen and superoxide) and reactive
3
RNS
3
nitrogen species (RNS; for example, nitric oxide, peroxynitrite). These
+ VIP
+ VIP
3
compounds initiate cell signaling directly and also through interactions with
Defense+
VIP
associated
the hormonal response network, as well as causing further direct oxidative
gene
Hormones
damage. Different stresses trigger different response pathways. For example,
Oxidative
expression
(SA,
JA,
ET)
damage
ozone exposure also triggers a response that overlaps with biotic stress
responses though the plant hormone network; salicylic acid (SA), jasmonic
Signal
acid (JA) and ethylene (ET) trigger signal cascades that initiate programmed
transduction
cell death (PCD), resulting in accelerated senescence via an inappropriate
Tissue
necrosis
hypersensitive response (HR). VIPs may act at several different levels to
PCD-associated
arrest oxidative stress response processes. (1) Because it is lipophilic, VIP
gene expression
may physically stabilize hydrophobic interactions in membranes, minimizing
Accelerated
lipid peroxidation and reducing oxidative stress and downstream buildup of
senescence
HR
ROS/RNS. (2) VIP may react with ROS/RNS to produce reactive electrophile
species (RES) such as methacrolein and methylvinylketone (products of
isoprene/ozone reaction), which are known to induce antioxidant and other defenses. If the stressor is itself an ROS (for example, ozone), VIP may react
directly with the stressor. (3) Direct antioxidant behavior (scavenging ROS/RNS) also prevents accumulation to damaging levels, thus preventing further
oxidative damage. As a consequence, ROS/RNS-activated signal cascades and PCD pathways that normally result in tissue necrosis are prevented. Figure
prepared with assistance from P. Mullineaux (Essex University).
photosynthesis of monoterpene-emitting plants becomes more
sensitive to ozone if the emission is inhibited by fosmidomycin, and
that, in contrast, photosynthesis becomes less sensitive to ozone in
non-emitting plants that are exogenously fumigated with volatile
isoprenoids50. Like isoprene and monoterpenes, many volatile plant
sesquiterpenes combine rapidly with ROS51, and their emission is
stimulated by high light and temperature conditions52; thus, these
compounds might also be involved in resistance to abiotic stress.
Unfortunately, sesquiterpenes have not been well studied owing to
difficulties in accurately quantifying emission rates as a consequence
of their high reactivity and sensitivity to disturbance53.
Mode of action
The mechanisms by which isoprenoids exert their protective effect are
as yet undetermined, though two primary mechanistic hypotheses
have been put forward in the context of thermal54 and oxidative46
stress tolerance. Mechanistic separation in this way assumes a complex
‘multiple mechanisms for multiple stressors’ model. However, as all
environmental stress responses are characterized by the release of
dangerous oxidative species, it is parsimonious to argue that the
abiotic stress tolerance enhancement conferred by these compounds
can be grouped under a common rubric of oxidant protection. Here
we propose a ‘single biochemical mechanism for multiple physiological stressors’ model (Fig. 5) that attempts to unify the diverse
empirical studies of volatile isoprenoid compounds and their role in
abiotic stress tolerance.
Membrane stabilization. Membrane stabilization as a mechanistic
explanation for isoprene action was first proposed by Sharkey and coworkers34. Owing to its lipophilic properties and the site of synthesis
(the chloroplast), isoprene is likely to partition into lipid phases of
thylakoid membranes. When heat stress occurs, membranes become
more fluid, and photosynthetic processes (which are membraneassociated) exhibit a decrease in efficiency. It was therefore proposed
that the mechanism of the protective effect is through physical
stabilization of hydrophobic interactions (lipid-lipid, lipid-protein
NATURE CHEMICAL BIOLOGY
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and/or protein-protein) under increasing temperatures. According to
theoretical modeling of molecular dynamics, isoprene does indeed
partition into the center of phospholipid membranes54. This enhances
membrane order without a significant change in the dynamic properties of the membrane. Decreased rates of electron transport are also
observed in photosynthetic membranes after inhibition by fosmidomycin, which also suggests that the presence of isoprene facilitates
photosynthetic processes under heat stress36. Given that other volatile
isoprenoids tend to also be hydrophobic, this mechanism might
be generic. However, direct testing of this mechanistic theory in an
in vivo system is difficult.
Despite this indirect empirical support, there are certain physicochemical problems with membrane stabilization as a mechanism for
the isoprene effect. Given that it is a hydrocarbon, isoprene is highly
hydrophobic; it is virtually insoluble in pure water, and the Henry’s
Law constant even in seawater has been estimated at KH B 3.1
(ref. 55). As the site of isoprene production is the chloroplast, it seems
reasonable to assume that, even at low emission rates, lipid membranes (particularly chloroplast membranes) must be saturated with
isoprene by the time isoprene is measured at the leaf surface. At any
given temperature, once membranes are saturated, increasing the
production of isoprene must increase the rate that isoprene travels
through the membrane rather than the concentration of isoprene in
the membrane. If membranes are always isoprene-saturated in emitting species, it follows that fumigation by isoprene should not confer
further protection—a result that has been observed56. Interestingly,
exogenously supplied isoprene also does not supply protection to
isolated membranes under thermal stress57; such membranes presumably have no endogenous isoprene. On the other hand, fumigation
with isoprene can partially complement the effects of fosmidomycininduced inhibition of the MEP pathway on photosynthesis under high
temperature30,35. These results are somewhat contradictory and can
only be reconciled if we assume that isolated membranes are not
physiologically the same as intact plant membranes and do not
respond the same way to the presence of isoprene. Partial complementation also suggests that other products from the MEP pathway
287
PERSPECTIVE
© 2009 Nature America, Inc. All rights reserved.
(apart from isoprene) contribute to protection of photosynthesis
under thermal stress. Finally, if the isoprene effect were purely a
physical stabilization of membranes, it should be relatively speciesindependent. However, a species-dependent effect is observed: in
species that do not normally emit isoprene, fumigation with isoprene
can confer protection in some cases but not in others30,40,56,58. This
variability may arise from differences in membrane properties among
species, but such differences have not been identified.
The direct antioxidant hypothesis. An alternative mechanistic
hypothesis is that isoprene behaves directly as an antioxidant, scavenging ROS by reactions through the conjugated double bond system45,46,48,49. In the atmosphere, highly reduced isoprenoids react with
reactive oxygen and nitrogen species, including ozone, singlet oxygen,
hydroxyl radicals and nitrous oxides (NOx)59–61. However, liquidphase chemistry may be different from gas-phase chemistry. In leaves,
these reactions potentially occur either in the aqueous environment
within the cell (probably at membrane surfaces) or in the humid
environment in intercellular spaces and at the boundary layer of the
leaf lamina. Lipid-phase reactions may also occur.
Under stress conditions, a variety of ROS are produced in plant
cells. These ROS cause oxidative damage. The plant responds to the
presence of excess ROS through an antioxidant defense system that
consists of antioxidant compounds that are either lipophilic (for
example, tocopherols and carotenoids) or hydrophilic (for example,
ascorbate and glutathione) and enzymes that either mediate regeneration of antioxidants (for example, monodehydroascorbate reductase,
dehydroascorbate reductase and glutathione reductase) or dismutate
toxic ROS to water or less reactive compounds (for example, superoxide dismutase, catalase and peroxidase)2 (example shown in Fig. 2
for chloroplast ROS response). Lipid- and aqueous-phase oxidative
states are closely linked through action of antioxidant enzymes
(Fig. 2). Hydrogen peroxide is produced as a byproduct of detoxification of the more dangerous ROS and is involved in initiating stress
signaling networks. ROS are also constitutively present in plant cells
under nonstress conditions and are involved in a number of cellular
responses. For example, production of ROS at the photosynthetic
membranes is integral to photosynthetic processes, and ROS signaling
is involved in regulation of those processes2,62,63. The regulation
of ROS is normally tightly controlled, but under stress conditions
the cellular balance of ROS is often perturbed2,3. The cellular network
of antioxidants and antioxidant enzymes is then required to scavenge
the excess ROS and prevent cytotoxic effects. It is well known
that nonvolatile isoprenoids such as tocopherols, zeaxanthin and
carnosic acid can scavenge ROS directly by reactions through hydroxyl
radicals64. Volatile isoprenoids are typically olefins with reactive
conjugated and/or terminal double bonds. We propose that volatile
isoprenoids also form part of the non-enzymatic oxidative defense
system. This hypothesis rests on the following evidence.
Isoprene affects the oxidative status of plants under stress. Inhibition of isoprene emission by fosmidomycin results in greater accumulation of H2O2, increased lipid peroxidation levels and increases in
antioxidant enzyme activities when plants are placed under thermal
stress35–37,46. Fumigation of fosmidomycin-inhibited leaves with exogenous isoprene partially restores H2O2 and lipid peroxidation levels
to those found in non-inhibited leaves. These results were confirmed
in a transgenic tobacco system using plants azygous and heterozygous
for isoprene synthase43. Further, pools of reduced antioxidant (ascorbate) were higher in transgenic isoprene-emitting plants relative to
non-emitting plants, which suggests that the emitting plants had a
288
reduced requirement for antioxidant capacity compared to nonemitting plants. These findings all indicate that isoprene plays some
role in reducing the oxidizing load under stress conditions.
Direct reactions can occur between volatile isoprenoids and oxidizing species. It has been suggested that volatile isoprenoids, especially
isoprene, can react directly with ozone, either in planta or at the leaf
surface, thus decreasing ozone levels and potentially mitigating oxidative damage caused to the leaf45,46. In highly oxidizing, humid
atmospheric environments, isoprene does react with ozone65; similar
conditions might occur at the leaf boundary layer and in the intercellular spaces of the mesophyll tissue when ozone is present. However, only monoterpenes have been experimentally demonstrated to
scavenge a significant amount of ozone in the boundary layer66. The
reaction between ozone and isoprene is relatively slow, and direct
removal of ozone in this way is insufficient to result in the observed
protection of fumigated tissues66. Furthermore, ozonolysis of isoprene
in humid environments results in production of hydrogen peroxide65;
this is inconsistent with the lowered hydrogen peroxide levels observed
in tissue extracts from ozone-fumigated leaves, which emit isoprene46.
Methacrolein (MACR) and methylvinylketone (MVK) are the first
products of isoprene ozonolysis65; reactive electrophile species (RES)
such as these are known to activate expression of defense genes67.
Fares et al. observed a surprisingly low production of MACR and
MVK from isoprene-emitting leaves that were fumigated with
ozone66; this implies that secondary reactions either with ozone or
with other metabolites inside the mesophyll remove these RES.
When ozone enters the leaf, it is degraded to other ROS: superoxide,
singlet oxygen, hydroxyl radicals and hydrogen peroxide. Scavenging
of some or all of these ROS by volatile isoprenoids might help explain
the protective effect observed. In aqueous solution, isoprene reacts
with hydroxyl radicals to produce 2-methyltetrols68, and it has been
suggested that isoprene might act as a hydroxyl radical scavenger to
protect from oxidative damage69. The presence of conjugated double
bounds (delocalized p-electrons) in the isoprene molecule may serve
to mediate electron and energy transfers, conferring a ROS-scavenging
ability to the molecule46,48,49. There is strong evidence that isoprene
scavenges singlet oxygen. Singlet oxygen is produced (in addition to
other ROS) at the thylakoid membranes when absorbed energy is in
excess of that used in photosynthesis70 (see Fig. 2). This may occur
because excess light is present (at high light intensities) and/or because
use of excitation energy is retarded (for example, under various abiotic
stress conditions). Recent research suggests that singlet oxygen is the
major cause of photo-oxidative damage under high light stress71.
When production of singlet oxygen at the photosynthetic membranes
is exacerbated under light stress using chemical treatments, young
non-emitting leaves of isoprene-emitting species that are fumigated
with isoprene show higher net assimilation rates than non-fumigated
leaves48. Similar results are observed in mature leaves when isoprene
emission is inhibited by application of fosmidomycin49. In isopreneinhibited leaves, H2O2 and malonyldialdehyde (MDA) levels were
increased compared to uninhibited leaves. MDA is an indicator of
lipid peroxidation and is produced at the thylakoid membranes under
oxidative stress (see Fig. 2).
Changes in signaling responses may also contribute to protective
effects. Recent studies show that isoprene may also have indirect
effects on oxidative state. Ozone damage is typified by accumulation
of hydrogen peroxide followed by biochemical and transcriptional
responses similar to those observed during the hypersensitive response
in an incompatible plant-pathogen interaction72. Nitric oxide (NO) is
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PERSPECTIVE
involved in the signal cascade that initiates this cellular response; in
addition, NO is involved in a number of physiological processes, many
of which are stress-related73,74. NO may also directly scavenge ROS,
attenuating the effects of photo-oxidative stress75. Isoprene-emitting
leaves produce less NO compared to fosmidomycin-treated leaves
when fumigated with ozone, which leads to the suggestion that
isoprene can also quench NO58. Isoprene can react with oxygenated
nitrogen species in the troposphere, but one reaction product is ozone.
If this happened also in planta, it would obviously result in further
oxidative damage. It therefore seems unlikely that this reaction is
occurring in plant cells. The observed decrease in relative NO levels
could also be explained if there was a decreased requirement for NO in
the presence of isoprene. NO and isoprene may indeed cooperate in
protecting leaves against oxidative stresses when they are present at
physiological levels76. Although the mechanism for this effect is
unknown, a reduction in the amount of NO might attenuate the
induction of stress-induced hypersensitive response, thus avoiding the
signal cascade that results in accumulation of H2O2 and subsequent
foliar necrosis. This is supported by the decreased levels of H2O2 and
lipid peroxidation markers observed in isoprene-emitting leaves
compared to non-emitting leaves after ozone fumigation35. If isoprene
interacts with NO, then a number of other processes that are initiated
by the hypersensitive responses are also likely to be affected, principally the MAPK cascade and the elicitation of jasmonate and salicylate
stress signaling pathways. This has yet to be elucidated, and may
become a major field of study in stress physiology.
Evolution of the stress tolerance function
Enzymatic production and emission of volatile isoprenoids occurs in
many plants, from bryophytes to highly derived angiosperms. The
DOX-MEP pathway appears to be primitive to green plants, and the
terpene synthase enzyme that converts the substrate dimethylallyl
diphosphate (DMADP) to isoprene has evolved multiple times in
mosses, ferns, gymnosperms and angiosperms77,78. Isoprene biosynthesis appears not to have evolved (or to have been lost) in several
groups defined taxonomically or physiologically, most notably in two
ancient divisions—hornworts (anthocerotophytes) and liverworts
(marchantiophytes)—and in two physiologically defined groups
whose members are much more recently evolved—C4 and crassulation acid metabolism (CAM) plants. In the oaks (Quercus spp.), where
isoprene emission appears to be a primitive trait, the subgenus that
has lost the trait, the European live oaks, has replaced enzymatically
controlled light-dependent isoprene emission with enzymatically controlled light-dependent monoterpene emission79. This offers the
strongest phylogenetic evidence of an adaptive significance for volatile
diene production and emission by plants, as monoterpenes appear to
be more effective in scavenging antioxidants in the gas phase than
isoprene66 and, because of their lower volatility, form larger pools in
membranes and intercellular spaces80,81. In other groups of nonisoprene-emitting taxa, monoterpenes or sesquiterpenes may play
the same role as isoprene in protection against abiotic stress. However,
much more information about the phylogenetic occurrence of foliar
monoterpene and sesquiterpene emission in the plant kingdom is
needed to support or reject this conjecture.
Analysis of online emission databases (http://www.es.lancs.ac.uk/
cnhgroup/download.html) reveals very few patterns between isoprene
emission and particular growth forms, ecologies, habitats or phylogenies. However, some strong correlations have been noted. Nearly all
isoprene emitters are woody; within taxa that contain both woody and
herbaceous groups (for example, the grasses), isoprene emission is far
more common in woody groups (for example, bamboos and reeds).
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Based on the phylogenetic distribution of isoprene emission among
nonvascular plants, one may speculate that isoprene emission first
developed when plants abandoned the aquatic environment to
conquer the land82. Terrestrial plants may have developed isoprene
as a quick and primitive method to cope with rapid, short-term
changes of temperatures that occur on land but that do not occur in
the more thermally buffered aquatic environment. In support of this,
desert ecosystems tend to be lacking in isoprene-emitting taxa; this
can be considered as evidence in favor of a role for isoprene in plant
tolerance against short-term heat stress (a rare event in desert
systems). Exposure to the oxygen-rich terrestrial atmosphere may
also have selected for isoprene biosynthesis as an antioxidant mechanism, though this selective pressure does not account for the phylogenetic distribution of the trait or for the sensitivity of isoprene synthesis
to light and temperature83.
There is no clear evidence that isoprenoid-emitting species cope
better in the long term than non-emitting species in the presence of
environmental stresses. However, it should be noted that a wide
variety of complex and efficient mechanisms for protection against
abiotic stresses can be found in plants. In species where isoprenoid
emission has not evolved or has been lost, these mechanisms may be
an alternative response. Nonetheless, the potential exists for changes in
population structure to occur under global warming conditions, given
that (i) emissions specifically increase with increasing temperature,
and (ii) oxidative stress (in particular the occurrence of increased
ozone pollution) is increasing in parallel83. On the other hand, rising
CO2, which drives the temperature increase globally, is expected to
enhance photosynthesis, thus reducing the oxidative stress in plants.
This might negatively feed back on isoprenoid emission. An independent, negative feedback of rising CO2 on isoprene emission has
been observed84 and may be explained by the insufficient supply of
phosphoenolpyruvate (PEP) to isoprene, as PEP is increasingly
diverted to oxaloacetate production for anabolic support of mitochondrial respiration under rising CO2 (ref. 85).
Summary and conclusions
It is now clear that volatile isoprenoids play an important role in
protection against a variety of abiotic stresses, including high light,
temperature, drought and oxidizing conditions of the atmosphere.
These stresses all result in oxidative stress, and the presence of
isoprenoids improves the ability of plants to deal with internal
oxidative changes regardless of the nature of the external (physiological) stressor. Our ‘single biochemical mechanism for multiple
physiological stressors’ model provides a unified mechanistic explanation for the protection provided by volatile isoprenoids under diverse
stress events. Carbon is redirected to volatile production under stress
conditions, and the presence of these compounds results in protection,
thus justifying the metabolic expense of production. The importance
of this defense mechanism is further evidenced by the breadth of taxa
that emit volatiles and the apparent repeated evolution of this trait.
The mechanism of volatile isoprenoid action is difficult to test
directly in planta because current techniques do not allow discrimination between cause and effect. Activity may be mediated through
(i) direct reactions of isoprenoids with oxidizing species, (ii) indirect
alteration of ROS signaling, and/or (iii) membrane stabilization.
Stabilization of lipid membranes also presumably decreases lipid
peroxidation, thus directly impacting the oxidative state of the cell;
this mechanism might explain a generic oxidative protection that is
not necessarily due to direct reactions. The antioxidant behavior of
isoprene and other volatiles might be further investigated by searching
for specific reaction products from isoprenoid oxidation.
289
PERSPECTIVE
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