Enhanced isoprene-related tolerance of heat- and light-stressed photosynthesis at low, but not high, CO2 concentrations

The principal function of isoprene biosynthesis in plants remains unclear, but emission rates are positively correlated with temperature and light, supporting a role for isoprene in maintaining photosynthesis under transient heat and light stress from sunflecks. Isoprene production is also inversely correlated with CO(2) concentrations, implying that rising CO(2) may reduce the functional importance of isoprene. To understand the importance of isoprene in maintaining photosynthesis during sunflecks, we used RNAi technology to suppress isoprene production in poplar seedlings and compared the responses of these transgenic plants to wild-type and empty-vector control plants. We grew isoprene-emitting and non-emitting trees at low (190 ppm) and high (590 ppm) CO(2) concentrations and compared their photosynthetic responses to short, transient periods of high light and temperature, as well as their photosynthetic thermal response at constant light. While there was little difference between emitting and non-emitting plants in their photosynthetic responses to simulated sunflecks at high CO(2), isoprene-emitting trees grown at low CO(2) had significantly greater photosynthetic sunfleck tolerance than non-emitting plants. Net photosynthesis at 42°C was 50% lower in non-emitters than in isoprene-emitting trees at low CO(2), but only 22% lower at high CO(2). Dark respiration rates were significantly higher in non-emitting poplar from low CO(2), but there was no difference between isoprene-emitting and non-emitting lines at high CO(2). We propose that isoprene biosynthesis may have evolved at low CO(2) concentrations, where its physiological effect is greatest, and that rising CO(2) will reduce the functional benefit of isoprene in the near future.     

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.     

Stabilization of thylakoid membranes in isoprene-emitting plants reduces formation of reactive oxygen species

Isoprene is emitted by a significant fraction of the world''s vegetation. Isoprene makes leaves more thermotolerant, yet we do not fully understand how. We have recently shown that isoprene stabilizes thylakoid membranes under heat stress. Here we show that heat-stressed, isoprene-emitting transgenic Arabidopsis plants also produce a lower pool of reactive oxygen and reactive nitrogen species, and that this was especially due to a lower accumulation of H₂O₂ in isoprene emitting plants. It remains difficult to disentangle whether in heat stressed plants isoprene also directly reacts with and quenches reactive oxygen species (ROS), or reduces ROS formation by stabilizing thylakoids. We present considerations that make the latter a more likely mechanism, under our experimental circumstances.          

Arabidopsis, a model to study biological functions of isoprene emission?

The volatile hemiterpene isoprene is emitted from plants and can affect atmospheric chemistry. Although recent studies indicate that isoprene can enhance thermotolerance or quench oxidative stress, the underlying physiological mechanisms are largely unknown. In this work, Arabidopsis (Arabidopsis thaliana), a natural nonemitter of isoprene and the model plant for functional plant analyses, has been constitutively transformed with the isoprene synthase gene (PcISPS) from Grey poplar (Populus x canescens). Overexpression of poplar ISPS in Arabidopsis resulted in isoprene-emitting rosettes that showed transiently enhanced growth rates compared to the wild type under moderate thermal stress. The findings that highest growth rates, higher dimethylallyl diphosphate levels, and enzyme activity were detected in young plants during their vegetative growth phase indicate that enhanced growth of transgenic plants under moderate thermal stress is due to introduced PcISPS. Dynamic gas-exchange studies applying transient cycles of heat stress to the wild type demonstrate clearly that the prime physiological role of isoprene formation in Arabidopsis is not to protect net assimilation from damage against thermal stress, but may instead be to retain the growth potential or coordinated vegetative development of the plant. Hence, this study demonstrates the enormous potential but also the pitfalls of transgenic Arabidopsis (or other nonnatural isoprenoid emitters) in studying isoprene biosynthesis and its biological function(s).     

Effects of growth under elevated UV-B on photosynthesis and isoprene emission in Quercus gambelii and Mucuna pruriens

Increasing surface levels of UV-B resulting from stratospheric ozone reduction directly affect tropospheric photochemistry. There may also be indirect tropospheric effects due to changes in emission of organic compounds from vegetation. We treated woody and herbaceous isoprene-emitting species in the field with supplemental UV-B simulating 30% ozone depletion. For Quercus gambelii, photosynthesis and isoprene emission were significantly greater in elevated UV-B treatments when expressed on a leaf area basis, but not on a leaf mass basis. Leaves of Mucuna pruriens, however, showed no significant differences in photosynthesis or isoprene emission between treatments, nor when exposed for 45 min to acute high levels of UV-B. Elevated UV-B during growth did not elicit significant isoprene emission from Acer platanoides, a non-emitting species. Other potential UV-B effects, such as changes in leaf area or species composition, which may influence regional isoprene emissions, should be examined.     

Isoprene emissions influence herbivore feeding decisions

Isoprene (C(5)H(8), 2-methyl 1,3-butadiene) is synthesized and emitted by many, but not all, plants. Unlike other related volatile organic compounds (monoterpenes and sesquiterpenes), isoprene has not been shown to mediate plant-herbivore interactions. Here, for the first time, we show, in feeding choice tests using isoprene-emitting transgenic tobacco plants (Nicotiana tabacum cv. Samsun) and non-emitting azygous control plants, that isoprene deters Manduca sexta caterpillars from feeding. This avoidance behaviour was confirmed using an artificial (isoprene-emitting and non-emitting control) diet. Both in vivo and in vitro experiments showed that isoprene can activate feeding avoidance behaviour in this system with a dose-response effect on caterpillar behaviour and an isoprene emission threshold level of <6 nmol m(-2) s(-1).     

Isoprene decreases the concentration of nitric oxide in leaves exposed to elevated ozone

Isoprene reduces visible damage (necrosis) of leaves caused by exposure to ozone but the mechanism is not known. Here we show that in Phragmites leaves isoprene emission was stimulated after a 3-h exposure to high ozone levels. The photosynthetic apparatus of leaves in which isoprene emission was inhibited by fosmidomycin became more susceptible to damage by ozone than in isoprene-emitting leaves. Three days after ozone fumigation, the necrotic leaf area was significantly higher in isoprene-inhibited leaves than in isoprene-emitting leaves. Isoprene-inhibited leaves also accumulated high amounts of nitric oxide (NO), as detected by epifluorescence light microscopy. Our results confirm that oxidative stresses activate biosynthesis and emission of chloroplastic isoprenoid, bringing further evidence in support of an antioxidant role for these compounds. It is suggested that, in nature, the simultaneous quenching of NO and reactive oxygen species by isoprene may be a very effective mechanism to control dangerous compounds formed under abiotic stress conditions, while simultaneously attenuating the induction of the hypersensitive response leading to cellular damage and death.     

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.     

The capacity to emit isoprene differentiates the photosynthetic temperature responses of tropical plant species

Experimental research shows that isoprene emission by plants can improve photosynthetic performance at high temperatures. But whether species that emit isoprene have higher thermal limits than non‐emitting species remains largely untested. Tropical plants are adapted to narrow temperature ranges and global warming could result in significant ecosystem restructuring due to small variations in species' thermal tolerances. We compared photosynthetic temperature responses of 26 co‐occurring tropical tree and liana species to test whether isoprene‐emitting species are more tolerant to high temperatures. We classified species as isoprene emitters versus non‐emitters based on published datasets. Maximum temperatures for net photosynthesis were ~1.8°C higher for isoprene‐emitting species than for non‐emitters, and thermal response curves were 24% wider; differences in optimum temperatures (T_opt) or photosynthetic rates at T_opt were not significant. Modelling the carbon cost of isoprene emission, we show that even strong emission rates cause little reduction in the net carbon assimilation advantage over non‐emitters at supraoptimal temperatures. Isoprene emissions may alleviate biochemical limitations, which together with stomatal conductance, co‐limit photosynthesis above T_opt. Our findings provide evidence that isoprene emission may be an adaptation to warmer thermal niches, and that emitting species may fare better under global warming than co‐occurring non‐emitting species.     

Study of the effects of foliar application of ABA during acclimatization

This study aims to elucidate the effects of abscisic acid (ABA) foliar application on plant hardening during ex vitro acclimatization using a forest model species Ulmus minor L. Plant leaves were sprayed with ABA (0, 50 and 100 μM) immediately after ex vitro transfer and twice a week for the first 3 weeks of acclimatization. After this period, parameters related with photosynthesis, hormone levels and oxidative stress were measured to assess plant performance. The results demonstrated that ABA foliar application alleviates the negative shock of ex vitro acclimatization since it reduces the water loss through transpiration, relieving the risk of wilting. Moreover, ABA promoted net CO₂ assimilation rate (P _N ) and plant dry mater accumulation. ABA treatment increased the antioxidant battery during acclimatization, with more effective results at the concentration of 50 μM ABA. Also, flow cytometry data support that cytosolic compounds, which may increase in response to 50 μM ABA, could also protect DNA from oxidative damage. We propose here that ABA foliar application (immediately after ex vitro transfer), by preventing water loss, enhancing photosynthesis efficiency and the activity of antioxidant enzymes, improves the plants hardening and ability to deal with the ex vitro stresses.     

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.     

Sustained accumulation of methyl salicylate alters antioxidant protection and reduces tolerance of holm oak to heat stress

Methyl salicylate (MeSA) is thought to have a major role in biotic and abiotic stresses by acting as a signal to trigger the oxidative burst, which is needed to activate gene expression in plant stress responses. To assess the potential effects of sustained foliar accumulation of MeSA on plant stress tolerance, the extent of photo- and antioxidant protection, lipid peroxidation and visual leaf area damage were evaluated in MeSA-treated ( c. 60 nl l⁻¹ in air) and control holm oak ( Quercus ilex L.) plants exposed to heat stress. Control plants showed an increase in foliar MeSA levels up to 1.8 nmol [gDW]⁻¹ as temperature increased and they displayed tolerance to temperatures as high as 45°C, which might be attributed, at least in part, to enhanced xanthophyll de-epoxidation and increases in ascorbate and α-tocopherol. MeSA-treated plants showed a sustained foliar accumulation of this compound, with values ranging from 10 to 23 nmol [gDW]⁻¹ throughout the experiment. These plants showed lower ascorbate and tocopherol levels and higher oxidative damage at 50°C than controls, as indicated by enhanced malondialdehyde levels and leaf area damage and lower maximum efficiency of PSII photochemistry ( F _v/ F _m ratio). These results demonstrate that a sustained foliar accumulation of MeSA is detrimental to plant function and that it can reduce thermotolerance in holm oak by altering antioxidant defences.     

Endogenous isoprene protects Phragmites australis leaves against singlet oxygen

The possible protective role of endogenous isoprene against oxidative stress caused by singlet oxygen (¹O₂) was studied in the isoprene‐emitting plant Phragmites australis. Leaves emitting isoprene and leaves in which isoprene synthesis was inhibited by fosmidomycin were exposed to increasing concentrations of ¹O₂ generated by Rose Bengal (RB) sensitizer at different light intensities. In isoprene‐emitting leaves, photosynthesis and H₂O₂ and malonyldialdehyde (MDA) contents were not affected by low to moderate ¹O₂ concentrations generated at light intensities of 800 and 1240 µmol m^?2 s^?1, but symptoms of damage and reactive oxygen accumulation started to be observed when high levels of ¹O₂ were generated by very high light intensity (1810 µmol m^?2 s^?1). A dramatic decrease in photosynthetic performance and an increase in H₂O₂ and MDA levels were measured in isoprene‐inhibited RB‐fed leaves, but photosynthesis was not significantly inhibited in leaves in which the isoprene leaf pool was reconstituted by fumigating exogenous isoprene. The inhibition of photosynthesis in isoprene‐inhibited leaves was linearly associated with the light intensity and with the consequently formed ¹O₂. Hence, physiological levels of endogenous isoprene may supply protection against ¹O₂. The protection mechanisms may involve a direct reaction of isoprene with ¹O₂. Moreover, as it is a small lipophilic molecule, it may assist hydrophobic interactions in membranes, resulting in their stabilization. The isoprene‐conjugated double bond structure may also quench ¹O₂ by facilitating energy transfer and heat dissipation. This action is typical of other isoprenoids, but we speculate that isoprene may provide a more dynamic protection mechanism as it is synthesized promptly when high light intensity produces ¹O₂.     

Overexpression of an Arabidopsis peroxisomal ascorbate peroxidase gene in tobacco increases protection against oxidative stress.

The Arabidopsis gene APX3 that encodes a putative peroxisomal membrane-bound ascorbate peroxidase was expressed in transgenic tobacco plants. APX3-expressing lines had substantial levels of APX3 mRNA and protein. The H2O2 can be converted to more reactive toxic molecules, e.g. .OH, if it is not quickly removed from plant cells. The expression of APX3 in tobacco could protect leaves from oxidative stress damage caused by aminotriazole which inhibits catalase activity that is found mainly in glyoxysomes and peroxisomes and leads to accumulation of H2O2 in those organelles. However, these plants did not show increased protection from oxidative damage caused by paraquat which leads to the production of reactive oxygen species in chloroplasts. Therefore, protection provided by the expression of APX3 seems to be specific against oxidative stress originated from peroxisomes, not from chloroplasts, which is consistent with the hypothesis that APX3 is a peroxisomal membrane-bound antioxidant enzyme.     

Sodium hydrosulfide priming improves the response of photosynthesis to overnight frost and day high light in avocado (Persea americana Mill,cv. ‘Hass’)

Radiation frost events, which have become more common in the Mediterranean Basin in recent years, inflict extensive damage to tropical/subtropical fruit crops. During radiation frost, sub-zero temperatures are encountered in the dark, followed by high light during the subsequent clear-sky day. One of the key processes affected by these conditions is photosynthesis, which, when significantly inhibited, leads to the enhanced accumulation of reactive oxygen species (ROS) and damage. The use of ‘chemical priming’ treatments that induce plants' endogenous stress responses is a possible strategy to improve their coping with stress conditions. Herein, we studied the effects of priming with sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H₂S), on the response of photosynthesis to overnight frost and day high-light conditions in ‘Hass’ avocado (Persea americana Mill). We found that priming with a single foliar application of NaHS had positive effects on the response of grafted ‘Hass’ plants. Primed plants exhibited significantly reduced inhibition of CO₂ assimilation, a lower accumulation of hydrogen peroxide as well as lower photoinhibition, as compared to untreated plants. The ability to maintain a high CO₂ assimilation capacity after the frost was attained on the background of considerable inhibition in stomatal conductance. Thus, it was likely related to the lower accumulation of ROS and photodamage observed in primed ‘Hass’ plants. This work contributes toward the understanding of the response of photosynthesis in a subtropical crop species to frost conditions and provides a prospect for chemical priming as a potential practice in orchards during cold winters.     

Isoprene synthesis in plants: lessons from a transgenic tobacco model

Isoprene is a highly reactive gas, and is emitted in such large quantities from the biosphere that it substantially affects the oxidizing potential of the atmosphere. Relatively little is known about the control of isoprene emission at the molecular level. Using transgenic tobacco lines harbouring a poplar isoprene synthase gene, we examined control of isoprene emission. Isoprene synthase required chloroplastic localization for catalytic activity, and isoprene was produced via the methyl erythritol (MEP) pathway from recently assimilated carbon. Emission patterns in transgenic tobacco plants were remarkably similar to naturally emitting plants under a wide variety of conditions. Emissions correlated with photosynthetic rates in developing and mature leaves, and with the amount of isoprene synthase protein in mature leaves. Isoprene synthase protein levels did not change under short-term increase in heat/light, despite an increase in emissions under these conditions. A robust circadian pattern could be observed in emissions from long-day plants. The data support the idea that substrate supply and changes in enzyme kinetics (rather than changes in isoprene synthase levels or post-translational regulation of activity) are the primary controls on isoprene emission in mature transgenic tobacco leaves.     

Exploring the impact of wounding and jasmonates on ascorbate metabolism

Vitamin C (ascorbate, AsA) is the most abundant water-soluble antioxidant in plants. Ascorbate provides the first line of defense against damaging reactive oxygen species (ROS), and helps protect plant cells from many factors that induce oxidative stress, including wounding, ozone, high salinity, and pathogen attack. Plant defenses against these stresses are also dependent upon jasmonates (JAs), a class of plant hormones that promote ROS accumulation. Here, we review evidence showing that wounding and JAs influence AsA accumulation in various plant species, and we report new data from Arabidopsis and tomato testing the influence of JAs on AsA levels in wounded and unwounded plants. In both species, certain mutations that impair JA metabolism and signaling influence foliar AsA levels, suggesting that endogenous JAs may regulate steady-state AsA. However, the impact of wounding on AsA accumulation was similar in JA mutants and wild type controls, indicating that this wound response does not require JAs. Our findings also indicate that the effects of wounding and JAs on AsA accumulation differ between species; these factors both enhanced AsA accumulation in Arabidopsis, but depressed AsA levels in tomato. These results underscore the importance of obtaining data from more than one model species, and demonstrate the complexity of AsA regulation.