Ozone Quenching Properties of Isoprene and Its Antioxidant Role in Leaves

Isoprene is formed in and emitted by plants and the reason for this apparent carbon waste is still unclear. It has been proposed that isoprene stabilizes cell and particularly chloroplast thylakoid membranes. We tested if membrane stabilization or isoprene reactivity with ozone induces protection against acute ozone exposures. The reduction of visible, physiological, anatomical, and ultrastructural (chloroplast) damage shows that clones of plants sensitive to ozone and unable to emit isoprene become resistant to acute and short exposure to ozone if they are fumigated with exogenous isoprene, and that isoprene-emitting plants that are sensitive to ozone do not suffer damage when exposed to ozone. Isoprene-induced ozone resistance is associated with the maintenance of photochemical efficiency and with a low energy dissipation, as indicated by fluorescence quenching. This suggests that isoprene effectively stabilizes thylakoid membranes. However, when isoprene reacts with ozone within the leaves or in a humid atmosphere, it quenches the ozone concentration to levels that are less or non-toxic for plants. Thus, protection from ozone in plants fumigated with isoprene may be due to a direct ozone quenching rather than to an induced resistance at membrane level. Irrespective of the mechanism, isoprene is one of the most effective antioxidants in plants.     

Why only some plants emit isoprene

Isoprene (2‐methyl‐1,3‐butadiene) is emitted from many plants and it appears to have an adaptive role in protecting leaves from abiotic stress. However, only some species emit isoprene. Isoprene emission has appeared and been lost many times independently during the evolution of plants. As an example, our phylogenetic analysis shows that isoprene emission is likely ancestral within the family Fabaceae (= Leguminosae), but that it has been lost at least 16 times and secondarily gained at least 10 times through independent evolutionary events. Within the division Pteridophyta (ferns), we conservatively estimate that isoprene emissions have been gained five times and lost two times through independent evolutionary events. Within the genus Quercus (oaks), isoprene emissions have been lost from one clade, but replaced by a novel type of light‐dependent monoterpene emissions that uses the same metabolic pathways and substrates as isoprene emissions. This novel type of monoterpene emissions has appeared at least twice independently within Quercus, and has been lost from 9% of the individuals within a single population of Quercus suber. Gain and loss of gene function for isoprene synthase is possible through relatively few mutations. Thus, this trait appears frequently in lineages; but, once it appears, the time available for evolutionary radiation into environments that select for the trait is short relative to the time required for mutations capable of producing a non‐functional isoprene synthase gene. The high frequency of gains and losses of the trait and its heterogeneous taxonomic distribution in plants may be explained by the relatively few mutations necessary to produce or lose the isoprene synthase gene combined with the assumption that isoprene emission is advantageous in a narrow range of environments and phenotypes.     

On the relationship between isoprene emission and thermotolerance in Phragmites australis leaves exposed to high temperatures and during the recovery from a heat stress

Thermotolerance induced by isoprene has been assessed during heat bursts but there is little information on the ability of endogenous isoprene to confer thermotolerance under naturally elevated temperature, on the interaction between isoprene-induced thermotolerance and light stress, and on the persistence of this protection in leaves recovering at lower temperatures. Moderately high temperature treatment (38 °C for 1.5 h) reduced photosynthesis, stomatal conductance, and photochemical efficiency of photosystem II in isoprene-emitting, but to a significantly lower extent than in isoprene-inhibited Phragmites australis leaves. Isoprene inhibition and high temperature independently, as well as together, induced lipid peroxidation, increased level of H₂O₂, and increased catalase and peroxidase activities. However, leaves in which isoprene emission was previously inhibited developed stronger oxidative stress under high temperature with respect to isoprene-emitting leaves. The heaviest photosynthetic stress was observed in isoprene-inhibited leaves exposed to the brightest illumination (1500 µmol m⁻² s⁻¹) and, in general, there was also a clear additive effect of light excess on the formation of reactive oxygen species, antioxidant enzymes, and membrane damage. The increased thermotolerance capability of isoprene-emitting leaves may be due to isoprene ability to stabilize membranes or to scavenge reactive oxygen species. Irrespective of the mechanism by which isoprene reduces thermal stress, isoprene-emitting leaves are able to quickly recover after the stress. This may be an important feature for plants coping with frequent and transient temperature changes in nature.     

Evolutionary significance of isopreneemission from mosses

Isoprene emission has been documented and characterized from species in all major groups of vascular plants. We report in our survey that isoprene emission is much more common in mosses and ferns than later divergent land plants but is absent in liverworts and hornworts. The light and temperature responses of isoprene emission from Sphagnum capillifolium (Ehrh.) Hedw. are similar to those of other land plants. Isoprene increases thermotolerance of S. capillifolium to the same extent seen in higher plants as measured by chlorophyll fluorescence. Sphagnum species in a northern Wisconsin bog experienced large temperature fluctuations similar to those reported in tree canopies. Since isoprene has been shown to help plants cope with large, rapid temperature fluctuations, we hypothesize the thermal and correlated dessication stress experienced by early land plants provided the selective pressure for the evolution of light-dependent isoprene emission in the ancestors of modern mosses. As plants radiated into different habitats, this capacity was lost multiple times in favor of other thermal protective mechanisms.     

Isoprene Produced by Leaves Protects the Photosynthetic Apparatus against Ozone Damage, Quenches Ozone Products, and Reduces Lipid Peroxidation of Cellular Membranes

Many plants invest carbon to form isoprene. The role of isoprene in plants is unclear, but many experiments showed that isoprene may have a role in protecting plants from thermal damage. A more general antioxidant action has been recently hypothesized on the basis of the protection offered by exogenous isoprene in nonemitting plants exposed to acute ozone doses. We inhibited the synthesis of endogenous isoprene by feeding fosmidomycin and observed that Phragmites australis leaves became more sensitive to ozone than those leaves forming isoprene. Photosynthesis, stomatal conductance, and fluorescence parameters were significantly affected by ozone only in leaves on which isoprene was not formed. The protective effect of isoprene was more evident when the leaves were exposed for a long time (8 h) to relatively low (100 nL L(-1)) ozone levels than when the exposure was short and acute (3 h at 300 nL L(-1)). Isoprene quenched the amount of H(2)O(2) formed in leaves and reduced lipid peroxidation of cellular membranes caused by ozone. These results indicate that isoprene may exert its protective action at the membrane level, although a similar effect could be obtained if isoprene reacted with ozone before forming active oxygen species. Irrespective of the mechanism, our results suggest that endogenous isoprene has an important antioxidant role in plants.     

ISOPRENE SYNTHASE GENES FORM A MONOPHYLETIC CLADE OF ACYCLIC TERPENE SYNTHASES IN THE TPS‐B TERPENE SYNTHASE FAMILY

Many plants emit significant amounts of isoprene, which is hypothesized to help leaves tolerate short episodes of high temperature. Isoprene emission is found in all major groups of land plants including mosses, ferns, gymnosperms, and angiosperms; however, within these groups isoprene emission is variable. The patchy distribution of isoprene emission implies an evolutionary pattern characterized by many origins or many losses. To better understand the evolution of isoprene emission, we examine the phylogenetic relationships among isoprene synthase and monoterpene synthase genes in the angiosperms. In this study we identify nine new isoprene synthases within the rosid angiosperms. We also document the capacity of a myrcene synthase in Humulus lupulus to produce isoprene. Isoprene synthases and (E)‐β‐ocimene synthases form a monophyletic group within the Tps‐b clade of terpene synthases. No asterid genes fall within this clade. The chemistry of isoprene synthase and ocimene synthase is similar and likely affects the apparent relationships among Tps‐b enzymes. The chronology of rosid evolution suggests a Cretaceous origin followed by many losses of isoprene synthase over the course of evolutionary history. The phylogenetic pattern of Tps‐b genes indicates that isoprene emission from non‐rosid angiosperms likely arose independently.     

Stress-induced changes in carbon sources for isoprene production in Populus deltoides

Isoprene is emitted from leaves of numerous plant species and has important implications for plant metabolism and atmospheric chemistry. The ability to use stored carbon (alternative carbon sources), as opposed to recently assimilated photosynthate, for isoprene production may be important as plants routinely experience photosynthetic depression in response to environmental stress. A CO₂‐labelling study was performed and stable isotopes of carbon were used to examine the role of alternative carbon sources in isoprene production in Populus deltoides during conditions of water stress and high leaf temperature. Isotopic fractionation during isoprene production was higher in heat‐ and water‐stressed leaves (?8.5 and ?9.3‰, respectively) than in unstressed controls (?2.5 to ?3.2‰). In unstressed plants, 84–88% of the carbon in isoprene was derived from recently assimilated photosynthate. A significant shift in the isoprene carbon composition from photosynthate to alternative carbon sources was observed only under severe photosynthetic limitation (stomatal conductance < 0.05 mol m^?2 s^?1). The contribution of photosynthate to isoprene production decreased to 77 and 61% in heat‐ and water‐stressed leaves, respectively. Across water‐ and heat‐stress experiments, allocation of photosynthate was negatively correlated to the ratio of isoprene emission to photosynthesis. In water‐stressed plants, the use of alternative carbon was also related to stomatal conductance. It has been proposed that isoprene emission may be regulated by substrate availability. Thus, understanding carbon partitioning to isoprene production from multiple sources is essential for building predictive models of isoprene emission.     

Plants utilize isoprene emission as a thermotolerance mechanism

Isoprene is a volatile compound emitted from leaves of many plant species in large quantities, which has an impact on atmospheric chemistry due to its massive global emission rate (5 x 10(14) carbon g year(-1)) and its high reactivity with the OH radical, resulting in an increase in the half-life of methane. Isoprene emission is strongly induced by the increase in isoprene synthase activity in plastids at high temperature in the day time, which is regulated at its gene expression level in leaves, while the physiological meaning of isoprene emission for plants has not been clearly demonstrated. In this study, we have functionally overexpressed Populus alba isoprene synthase in Arabidopsis to observe isoprene emission from transgenic plants. A striking difference was observed when both transgenic and wild-type plants were treated with heat at 60 degrees C for 2.5 h, i.e. transformants revealed clear heat tolerance compared with the wild type. High isoprene emission and a decrease in the leaf surface temperature were observed in transgenic plants under heat stress treatment. In contrast, neither strong light nor drought treatments showed an apparent difference. These data suggest that isoprene emission plays a crucial role in a heat protection mechanism in plants.     

Isoprene and nitric oxide reduce damages in leaves exposed to oxidative stress

Isoprene and nitric oxide (NO) are two volatile molecules that are produced in leaves. Both compounds were suggested to have an important protective role against stresses. We tested, in two isoprene-emitting species, Populus nigra and Phragmites australis, whether: (1) NO emission outside leaves is measurable and is affected by oxidative stresses; and (2) isoprene and NO protect leaves against oxidative stresses, both singularly and in combination. The emission of NO was undetectable, and the compensation point was very low in control poplar leaves. Both emission and compensation point increased dramatically in stressed leaves. NO emission was inversely associated with stomatal conductance. More NO was emitted in leaves that were isoprene-inhibited, and more isoprene was emitted when NO was reduced by NO scavenger c-PTIO. Both isoprene and NO reduced oxidative damages. Isoprene-emitting leaves which were also fumigated with NO, or treated with NO donor, showed low damage to photosynthesis, a reduced accumulation of H(2)O(2) and a reduced membrane denaturation. We conclude that measurable amounts of NO are only produced and emitted by stressed leaves, that both isoprene and NO are effective antioxidant molecules and that an additional protection is achieved when both molecules are released.     

Within‐plant isoprene oxidation confirmed by direct emissions of oxidation products methyl vinyl ketone and methacrolein

Isoprene is emitted from many terrestrial plants at high rates, accounting for an estimated 1/3 of annual global volatile organic compound emissions from all anthropogenic and biogenic sources combined. Through rapid photooxidation reactions in the atmosphere, isoprene is converted to a variety of oxidized hydrocarbons, providing higher order reactants for the production of organic nitrates and tropospheric ozone, reducing the availability of oxidants for the breakdown of radiatively active trace gases such as methane, and potentially producing hygroscopic particles that act as effective cloud condensation nuclei. However, the functional basis for plant production of isoprene remains elusive. It has been hypothesized that in the cell isoprene mitigates oxidative damage during the stress‐induced accumulation of reactive oxygen species (ROS), but the products of isoprene‐ROS reactions in plants have not been detected. Using pyruvate‐2‐¹³C leaf and branch feeding and individual branch and whole mesocosm flux studies, we present evidence that isoprene (i) is oxidized to methyl vinyl ketone and methacrolein (i_ox) in leaves and that i_ox/i emission ratios increase with temperature, possibly due to an increase in ROS production under high temperature and light stress. In a primary rainforest in Amazonia, we inferred significant in plant isoprene oxidation (despite the strong masking effect of simultaneous atmospheric oxidation), from its influence on the vertical distribution of i_ox uptake fluxes, which were shifted to low isoprene emitting regions of the canopy. These observations suggest that carbon investment in isoprene production is larger than that inferred from emissions alone and that models of tropospheric chemistry and biota–chemistry–climate interactions should incorporate isoprene oxidation within both the biosphere and the atmosphere with potential implications for better understanding both the oxidizing power of the troposphere and forest response to climate change.     

Root isoprene formation alters lateral root development

Isoprene is a C5 volatile organic compound, which can protect aboveground plant tissue from abiotic stress such as short-term high temperatures and accumulation of reactive oxygen species (ROS). Here, we uncover new roles for isoprene in the plant belowground tissues. By analysing Populus x canescens isoprene synthase (PcISPS) promoter reporter plants, we discovered PcISPS promoter activity in certain regions of the roots including the vascular tissue, the differentiation zone and the root cap. Treatment of roots with auxin or salt increased PcISPS promoter activity at these sites, especially in the developing lateral roots (LR). Transgenic, isoprene non-emitting poplar roots revealed an accumulation of O₂⁻ in the same root regions where PcISPS promoter activity was localized. Absence of isoprene emission, moreover, increased the formation of LRs. Inhibition of NAD(P)H oxidase activity suppressed LR development, suggesting the involvement of ROS in this process. The analysis of the fine root proteome revealed a constitutive shift in the amount of several redox balance, signalling and development related proteins, such as superoxide dismutase, various peroxidases and linoleate 9S-lipoxygenase, in isoprene non-emitting poplar roots. Together our results indicate for isoprene a ROS-related function, eventually co-regulating the plant-internal signalling network and development processes in root tissue.     

Isoprene synthesis protects transgenic tobacco plants from oxidative stress

Isoprene emission represents a significant loss of carbon to those plant species that synthesize this highly volatile and reactive compound. As a tool for studying the role of isoprene in plant physiology and biochemistry, we developed transgenic tobacco plants capable of emitting isoprene in a similar manner to and at rates comparable to a naturally emitting species. Thermotolerance of photosynthesis against transient high-temperature episodes could only be observed in lines emitting high levels of isoprene; the effect was very mild and could only be identified over repetitive stress events. However, isoprene-emitting plants were highly resistant to ozone-induced oxidative damage compared with their non-emitting azygous controls. In ozone-treated plants, accumulation of toxic reactive oxygen species (ROS) was inhibited, and antioxidant levels were higher. Isoprene-emitting plants showed remarkably decreased foliar damage and higher rates of photosynthesis compared to non-emitting plants immediately following oxidative stress events. An inhibition of hydrogen peroxide accumulation in isoprene-emitting plants may stall the programmed cell death response which would otherwise lead to foliar necrosis. These results demonstrate that endogenously produced isoprene provides protection from oxidative damage.     

The Effect of Isoprene on the Properties of Spinach Thylakoids and Phosphatidyicholine Liposomes

Abstract: Isoprene is emitted from the leaves of some plants. It was recently reported that exogenous isoprene delays the onset of leaf damage during controlled increases in leaf temperature (Singsaas et al. Plant Physiology 115: 1413–1420 [1997^[17). Thylakoid membranes are presumed to be the site of action based upon isoprene's hydrophobicity, production in chloroplasts, and effect upon chlorophyll fluorescence at high temperatures. In an attempt to discern the mechanistic basis for isoprene's thermoprotective role, we studied the effect of exogenous isoprene on the peroxidation, permeability, and stability of spinach thylakoids and phosphatidylcholine liposomes. Isoprene, supplied at either 18 or 21 μ1 L¹, had no effect upon the rate of liposome peroxidation in the presence of a hydroxyl radical-generating system. Isoprene also did not affect liposome peroxidation at high temperatures. Neither the proton permeability of thylakoids nor the leakage of a fluorescent probe from liposomes was influenced by exogenous isoprene, when measured at several temperatures. Isoprene did not affect the stability of thylakoid membrane proteins during a temperature increase, as shown by differential scanning calorimetry. Therefore, despite the use of a variety of techniques to investigate fundamental membrane parameters, we were unable to demonstrate an effect of isoprene.     

Photosynthetic limitations and volatile and non‐volatile isoprenoids in the poikilochlorophyllous resurrection plant Xerophyta humilis during dehydration and rehydration

We investigated the photosynthetic limitations occurring during dehydration and rehydration of Xerophyta humilis, a poikilochlorophyllous resurrection plant, and whether volatile and non‐volatile isoprenoids might be involved in desiccation tolerance. Photosynthesis declined rapidly after dehydration below 85% relative water content (RWC). Raising intercellular CO₂ concentrations during desiccation suggest that the main photosynthetic limitation was photochemical, affecting energy‐dependent RuBP regeneration. Imaging fluorescence confirmed that both the number of photosystem II (PSII) functional reaction centres and their efficiency were impaired under progressive dehydration, and revealed the occurrence of heterogeneous photosynthesis during desiccation, being the basal leaf area more resistant to the stress. Full recovery in photosynthetic parameters occurred on rehydration, confirming that photosynthetic limitations were fully reversible and that no permanent damage occurred. During desiccation, zeaxanthin and lutein increased only when photosynthesis had ceased, implying that these isoprenoids do not directly scavenge reactive oxygen species, but rather protect photosynthetic membranes from damage and consequent denaturation. X. humilis was found to emit isoprene, a volatile isoprenoid that acts as a membrane strengthener in plants. Isoprene emission was stimulated by drought and peaked at 80% RWC. We surmise that isoprene and non‐volatile isoprenoids cooperate in reducing membrane damage in X. humilis, isoprene being effective when desiccation is moderate while non‐volatile isoprenoids operate when water deficit is more extreme.     

Effect of growth conditions on isoprene emission and other thermotolerance‐enhancing compounds

Isoprene is emitted from the leaves of many plants in a light‐dependent and temperature‐sensitive manner. Plants lose a large fraction of photo‐assimilated carbon as isoprene but may benefit from improved recovery of photosynthesis following high‐temperature episodes. The capacity for isoprene emission of plants in natural conditions (assessed as the rate of isoprene emission under standard conditions) varies with weather. Temperature‐controlled greenhouses were used to study the role of temperature and light in influencing the capacity of oak leaves for isoprene synthesis. A comparison was made between the capacity for isoprene emission and the accumulation of other compounds suggested to increase thermotolerance of photosynthesis under two growth temperatures and two growth light intensities. It was found that the capacity for isoprene emission was increased by high temperature or high light. Xanthophyll cycle intermediates increased in high light, but not in high temperature, and the chloroplast small heat‐shock protein was not expressed in any of the growth conditions. Thus, of the three thermotolerance‐enhancing compounds studied, isoprene was the only one induced by the temperature used in this study.