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.     

Interaction of drought and ozone exposure on isoprene emission from extensively cultivated poplar

The combined effects of ozone (O₃) and drought on isoprene emission were studied for the first time. Young hybrid poplars (clone 546, Populus deltoides cv. 55/56 x P. deltoides cv. Imperial) were exposed to O₃ (charcoal‐filtered air, CF, and non‐filtered air +40 ppb, E‐O₃) and soil water stress (well‐watered, WW, and mild drought, MD, one‐third irrigation) for 96 days. Consistent with light‐saturated photosynthesis (A_sat), intercellular CO₂ concentration (C_i) and chlorophyll content, isoprene emission depended on drought, O₃, leaf position and sampling time. Drought stimulated emission (+38.4%), and O₃ decreased it (?40.4%). Ozone increased the carbon cost per unit of isoprene emission. Ozone and drought effects were stronger in middle leaves (13th–15th from the apex) than in upper leaves (6th–8th). Only A_sat showed a significant interaction between O₃ and drought. When the responses were up‐scaled to the entire‐plant level, however, drought effects on total leaf area translated into around twice higher emission from WW plants in clean air than in E‐O₃. Our results suggest that direct effects on plant emission rates and changes in total leaf area may affect isoprene emission from intensively cultivated hybrid poplar under combined MD and O₃ exposure, with important feedbacks for air quality.     

Regulation of isoprene synthase promoter by environmental and internal factors

Isoprene synthase (ISPS) catalyzes the formation of isoprene, an important volatile terpenoid with strong effects on global atmospheric chemistry and protective physiological functions in plant leaves. Many terpene synthase genes including isoprene synthase, a member of the TPS-b cluster of this numerous gene family, were already functionally analysed but much less is known about regulation of their promoters. To study regulation of the PcISPS gene in detail we developed transgenic Grey poplar (Populus × canescens) and Arabidopsis thaliana plants in which the PcISPS promoter is fused to enhanced green fluorescent protein (E-GFP) and β-glucuronidase (GUS) reporter genes. We analysed these reporters during plant development, for organ specificity and in plants subjected to different light and temperature regimes. We observed low promoter activity in non-isoprene emitting tissue like roots where ISPS gene is transcribed but no active enzyme is detectable. In leaves we demonstrate that light and temperature directly modulate ISPS promoter activity. Moreover, with confocal laser scanning microscopy we show a cell specific gradient of ISPS promoter activity within the leaf parenchyma depending on light direction. Our results indicate that ISPS promoter activity, which correlates with basal isoprene emission capacity, is not uniformly distributed within leaf tissue and that it can adapt rapidly towards internal as well as external environmental stimuli.     

Light-dependent modulation of foliar glutathione synthesis and associated amino acid metabolism in poplar overexpressing γ-glutamylcysteine synthetase

Glutathione (GSH), γ-glutamylcysteine (γ-EC) and major free amino acids were measured in darkened and illuminated leaves from untransformed poplars (Populus tremula × P. alba) and poplars expressing Escherichia coli genes for γ-glutamylcysteine synthetase (γ-ECS; EC 3.2.3.3) and glutathione reductase (GR; EC 1.6.4.2). In poplars overexpressing γ-ECS, foliar γ-EC contents and GSH contents were markedly enhanced compared to poplars lacking the bacterial gene for the enzyme. However, the quantitative relationship between the foliar pools of γ-EC and GSH in these transformants was markedly dependent on light. In the dark, GSH content was relatively low and γ-EC content high, the latter being higher than the foliar GSH contents of untransformed poplars in all conditions. Hence, this transformation appears to elevate γ-EC from the ranks of a trace metabolite to one of major quantitative importance. On illumination, however, γ-EC content decreased fourfold whereas GSH content doubled. Glutathione was also higher in the light in untransformed poplars and in those overexpressing GR. In these plants, γ-EC was negligible in the light but increased in the dark. Cysteine content was little affected by light in any of the poplar types. No light-dependent changes in the extractable activities of γ-ECS, glutathione synthetase (EC 3.2.3.2) or GR were observed. In contrast, both the activation state and the maximum extractable activity of nitrate reductase (EC 1.6.6.1) were increased by illumination. In all poplar types, glutamate and aspartate were the major amino acids. The most marked light-induced increases in individual amino acids were observed in the glutamine, asparagine, serine and glycine pools. Illumination of leaves from poplars overexpressing γ-ECS at elevated CO₂ or low O₂ largely abolished the inverse light-dependent changes in γ-EC and GSH. Low O₂ did not affect foliar contents of cysteine or glutamate but prevented the light-induced increase in the glycine pool. It is concluded that light-dependent glycine formation through the photorespiratory pathway is required to support maximal rates of GSH synthesis, particularly under conditions where the capacity for γ-EC synthesis is augmented. Received: 17 December 1996 / Accepted: 28 January 1997     

Analysis of 1-deoxy-d-xylulose 5-phosphate synthase activity in Grey poplar leaves using isotope ratio mass spectrometry

Deoxy-xylulose phosphate synthase (DXS) catalyzes the first step of the methylerythritol phosphate (MEP) pathway and it might regulate the metabolic flux in plastidic isoprenoid biosynthesis. We developed a sensitive assay suitable for plant extracts that is based on the decarboxylation of labeled pyruvate (1-¹³C)-PYR and detection of ¹³CO₂ by isotope ratio mass spectrometry. We tested our method investigating the DXS activity in poplar leaves. Apparent DXS activity showed Michaelis constants of 111 and 158 μM for glyceraldehyde phosphate and pyruvate, respectively; pH and temperature optima were found at pH 8.6 and 45 °C. DXS activity was inhibited when the competitive inhibitor β-fluoropyruvate was added to the reaction mixture. DXS activity strongly depended on leaf development with higher activity in young leaves and correlated fairly well with leaf isoprene emission potential. In mature poplar leaves, isoprene emission is the main metabolic sink of plastidic isoprenoid intermediates. Consequently, we found lower DXS activity in non-isoprene-emitting lines of poplar than in emitting plants as indicator of a lower demand of metabolic flux within the MEP pathway.     

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.     

Constitutive and herbivore-induced monoterpenes emitted by Populus × euroamericana leaves are key volatiles that orient Chrysomela populi beetles

Chrysomela populi beetles feed on poplar leaves and extensively damage plantations. We investigated whether olfactory cues orientate landing and feeding. Young, unexpanded leaves of hybrid poplar emit constitutively a blend of monoterpenes, primarily ( E )- β -ocimene and linalool. This blend attracts inexperienced adults of C. populi that were not previously fed with poplar leaves. In mature leaves constitutively emitting isoprene, insect attack induces biosynthesis and emission of the same blend of monoterpenes, but in larger amount than in young leaves. The olfactometric test indicates that inexperienced beetles are more attracted by adult than by young attacked leaves, suggesting that attraction by induced monoterpenes is dose dependent. The blend does not attract adults that previously fed on poplar leaves. Insect-induced emission of monoterpenes peaks 4 d after the attack, and is also detected in non-attacked leaves. Induced monoterpene emission is associated in mature leaves with a larger decrease of isoprene emission. The reduction of isoprene emission is faster than photosynthesis reduction in attacked leaves, and also occurs in non-attacked leaves. Insect-induced monoterpenes are quickly and completely labelled by ¹³C. It is speculated that photosynthetic carbon preferentially allocated to constitutive isoprene in healthy leaves is in part diverted to induced monoterpenes after the insect attack.     

Productivity of Populus nigra L. in two different soils over five rotations

This study describes growth performance and productivity of poplars (Populus nigra L.) on a 5-year rotation in two soil types. Two adjacent and even-aged stands of poplar (PS₁ and PS₂) with different productivity were selected in poplar plantations of Tehran, Iran. On a 5-year rotation, growth parameters of poplar trees were measured and productivity was computed. A transect sampling method with 10% intensity was used for measurement of the growth parameters. A composite soil sample was taken for all transects, and soil physico-chemical properties were determined. Texture, EC, organic matter percent, the contents of some macro and micro nutrients and some heavy metal concentrations of soil had significant differences between two stands; as a consequence growth performance and productivity of poplars were significantly difference between PS₁ and PS₂. In PS₁, productivity of 5-year trees was 2.5 times higher than in PS₂. Soil with Clay Loam texture, more contents of organic matter and nutrients, lower EC and lower contents of Ca, Mg, S and heavy metals provided more suitable conditions for growth of poplars. The growth parameters were positively correlated with the contents of nutrients, clay and organic matter in the soil, but they were negatively related to EC and the contents of silt, Ca, Mg, S and heavy metals of soil. Careful selecting sites based on soil properties before planting can be useful for increasing productivity of poplars.     

Enhanced tolerance of transgenic poplar plants overexpressing gamma-glutamylcysteine synthetase towards chloroacetanilide herbicides.

A wild-type poplar hybrid and two transgenic clones overexpressing a bacterial gamma-glutamylcysteine synthetase in the cytosol or in the chloroplasts were exposed to the chloroacetanilide herbicides acetochlor and metolachlor dispersed in the soil. The transformed poplars contained higher gamma-glutamylcysteine and glutathione (GSH) levels than wild-type plants and therefore it was supposed that they would have an elevated tolerance towards these herbicides, which are detoxified in GSH-dependent reactions. Phenotypically, the transgenic and wild-type plants did not differ. The growth and the biomass of all poplar lines were markedly reduced by the two chloroacetanilide herbicides. However, the decrease of shoot and root fresh weights caused by the herbicides was significantly smaller in the transgenic than in wild-type plants. In addition, the growth rate of poplars transformed in the cytosol was reduced to a significantly lesser extent than that of wild-type plants following herbicide treatments. The effects of the two herbicides were similar. Herbicide exposures markedly increased the levels of gamma-glutamylcysteine and GSH in leaves of each poplar line. The increase in the foliar amounts of these thiols was stronger in the transgenic lines than in the wild type, particularly in the upper leaves. Considerable GST activities were detected in leaves of all poplar plants. Exposure of poplars to chloroacetanilide herbicides resulted in a marked induction of GST activity in upper leaf positions but not in middle and lower leaves. The extent of enzyme induction did not differ significantly between transgenic and wild-type poplars. Although the results show that the transgenic poplar lines are good candidates for phytoremediation purposes, the further improvement of their detoxification capacity, preferably by transformation using genes encoding herbicide-specific GST isoenzymes, seems to be the most promising way to obtain plants suitable for practical application.     

Effects of heat and drought stress on post‐illumination bursts of volatile organic compounds in isoprene‐emitting and non‐emitting poplar

Over the last decades, post‐illumination bursts (PIBs) of isoprene, acetaldehyde and green leaf volatiles (GLVs) following rapid light‐to‐dark transitions have been reported for a variety of different plant species. However, the mechanisms triggering their release still remain unclear. Here we measured PIBs of isoprene‐emitting (IE) and isoprene non‐emitting (NE) grey poplar plants grown under different climate scenarios (ambient control and three scenarios with elevated CO₂ concentrations: elevated control, periodic heat and temperature stress, chronic heat and temperature stress, followed by recovery periods). PIBs of isoprene were unaffected by elevated CO₂ and heat and drought stress in IE, while they were absent in NE plants. On the other hand, PIBs of acetaldehyde and also GLVs were strongly reduced in stress‐affected plants of all genotypes. After recovery from stress, distinct differences in PIB emissions in both genotypes confirmed different precursor pools for acetaldehyde and GLV emissions. Changes in PIBs of GLVs, almost absent in stressed plants and enhanced after recovery, could be mainly attributed to changes in lipoxygenase activity. Our results indicate that acetaldehyde PIBs, which recovered only partly, derive from a new mechanism in which acetaldehyde is produced from methylerythritol phosphate pathway intermediates, driven by deoxyxylulose phosphate synthase activity.     

Over-expression of gsh1 in the cytosol affects the photosynthetic apparatus and improves the performance of transgenic poplars on heavy metal-contaminated soil

Recent studies of transgenic poplars over‐expressing the genes gsh1 and gsh2 encoding γ‐glutamylcysteine synthetase (γ‐ECS) and glutathione synthetase, respectively, provided detailed information on regulation of GSH synthesis, enzymes activities and mRNA expression. In this experiment, we studied quantitative parameters of leaves, assimilating tissues, cells and chloroplasts, mesophyll resistance for CO₂ diffusion, chlorophyll and carbohydrate content in wild‐type poplar and transgenic plants over‐expressing gsh1 in the cytosol after 3 years of growth in relatively clean (control) or heavy metal‐contaminated soil in the field. Over‐expression of gsh1 in the cytosol led to a twofold increase of intrafoliar GSH concentration and influenced the photosynthetic apparatus at different levels of organisation, i.e., leaves, photosynthetic cells and chloroplasts. At the control site, transgenic poplars had a twofold smaller total leaf area per plant and a 1.6‐fold leaf area per leaf compared to wild‐type controls. Annual aboveground biomass gain was reduced by 50% in the transgenic plants. The reduction of leaf area of the transformants was accompanied by a significant decline in total cell number per leaf, indicating suppression of cell division. Over‐expression of γ‐ECS in the cytosol also caused changes in mesophyll structure, i.e., a 20% decrease in cell and chloroplast number per leaf area, but also an enhanced volume share of chloroplasts and intercellular airspaces in the leaves. Transgenic and wild poplars did not exhibit differences in chlorophyll and carotenoid content of leaves, but transformants had 1.3‐fold fewer soluble carbohydrates. Cultivation on contaminated soil caused a reduction of palisade cell volume and chloroplast number, both per cell and leaf area, in wild‐type plants but not in transformants. Biomass accumulation of wild‐type poplars decreased in contaminated soil by more than 30‐fold, whereas transformants showed a twofold decrease compared to the control site. Thus, poplars over‐expressing γ‐ECS in the cytosol were more tolerant to heavy metal stress under field conditions than wild‐type plants according to the parameters analysed. Correlation analysis revealed strong dependence of cell number per leaf area unit, chloroplast parameters and mesophyll resistance with the GSH level in poplar leaves.     

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.     

杨树叶中Na^＋的积累与其耐盐能力的关系

以受体杨树品种‘107号’和转基因杨树‘18—1’的一年生枝条为材料,采用Hoagland营养液水培方法,检测二者植株生长以及根、茎、叶中Na^＋和K^＋含量变化的结果表明,在含100mmol·L^-1NaCl的Hoagland营养液中,二者的生长速度都受到明显抑制,但后者的受抑程度较小;叶中Na^＋含量呈持续增加趋势,9d后其叶中的Na’含量显著高于前者。在二者叶片枯萎程度相同的情况下,‘18—1’叶中的Na^＋含量是‘107号’的1．6倍左右。在4mol．L^-1NaCl溶液中,它们的表皮细胞死亡率分别为42％和97％。说明后者比前者有更高的耐盐能力。     

Emission of isoprene from salt-stressed Eucalyptus globulus leaves

Eucalyptus spp. are among the highest isoprene emitting plants. In the Mediterranean area these plants are often cultivated along the seashore and cope with recurrent salt stress. Transient salinity may severely but reversibly reduce photosynthesis and stomatal conductance of Eucalyptus globulus leaves but the effect on isoprene emission is not significant. When the stress is relieved, a burst of isoprene emission occurs, simultaneously with the recovery of photosynthetic performance. Later on, photosynthesis, stomatal conductance, and isoprene emission decay, probably because of the onset of leaf senescence. Isoprene emission is not remarkably affected by the stress at different light intensities, CO(2) concentrations, and leaf temperatures. When CO(2) was removed and O(2) was lowered to inhibit both photosynthesis and photorespiration, we found that the residual emission is actually higher in salt-stressed leaves than in controls. This stimulation is particularly evident at high-light intensities and high temperatures. The maximum emission occurs at 40 degrees C in both salt-stressed and control leaves sampled in ambient air and in control leaves sampled in CO(2)-free and low-O(2) air. However, the maximum emission occurs at 45 degrees C in salt-stressed leaves sampled in CO(2)-free and low-O(2) air. Our results suggest the activation of alternative non-photosynthetic pathways of isoprene synthesis in salt-stressed leaves and perhaps in general in leaves exposed to stress conditions. The temperature dependence indicates that this alternative synthesis is also under enzymatic control. If this alternative synthesis still occurs in the chloroplasts, it may involve a thylakoid-bound isoprene synthase.     

转基因杨树林下种植转基因棉花对转基因杨棉复合系统内节肢动物群落多样性的影响

【目的】转Bt基因棉花和转Bt基因杨树在我国已推广使用。本研究的目的是调查和分析不同转基因杨棉复合系统内地上节肢动物群落多样性变化,为转基因杨树大规模应用提供生态安全方面的数据。【方法】2019年4-10月,在河北任丘采取欧洲黑杨Populus nigra林下种植转基因棉花Gossypium hirsutum的模式,设置转基因杨树 转基因棉花(复合生态系统1)和非转基因杨树-转基因棉花(复合生态系统2)两种杨棉复合系统,调查杨树和棉花地上部植株节肢动物种类和个体数量,比较节肢动物各功能群的物种数量和个体数量以及香农指数、优势集中性指数、均匀度指数等群落多样性指数,并测定了转基因杨树和棉花叶片Bt蛋白含量。【结果】复合生态系统1中转基因杨树和棉花各自植株上鳞翅目种群累计个体数均显著低于复合生态系统2相应植株上的个体数量。转基因杨树上叶甲类累计个体数量显著高于非转基因杨树上的,寄生蜂类累计个体数量则显著低于非转基因杨树上的。在6月5日、6月21日和9月7日的调查中,转基因杨树上的节肢动物群落香农指数显著高于非转基因杨树上的。除鳞翅目害虫外,2个复合系统中棉株上节肢动物各功能群累计个体数量无显著性差异,香农指数、优势集中性指数和均匀度指数随时间变化的趋势基本一致且同一调查时间无显著性差异,仅在8月17调查中复合生态系统1中棉株上节肢动物香农指数显著高于复合生态系统2棉株上的。将杨树和棉花上节肢动物各类群累计数量相加作为系统整体,复合生态系统1和复合生态系统2在香农指数、优势集中性指数和均匀度指数数值上无显著差异。在调查后期,2个复合系统中节肢动物种群均向杨树叶片聚集。转基因棉花叶片Bt蛋白含量在各调查期均显著高于转基因杨树叶片的。【结论】转基因杨树对靶标害虫种群数量有较好的控制作用,对同系统内棉花上鳞翅目害虫数量亦有协同控制作用。转基因杨棉复合系统中地上节肢动物群落结构稳定,对系统内棉花地上部节肢动物群落多样性无显著影响。靶标害虫对转Bt基因杨树的抗性发展需持续监测。     

粒肩天牛危害对杨树单宁含量的影响

粒肩天牛是福建省杨树的一种新蛀干性害虫,经对杨树受害与未受害、不同危害期、不同品系、不同危害部位单宁含量测定。结果表明：受害植株与健康植株间单宁含量差异显著;不同危害期单宁含量差异极显著,在天牛危害末期和越冬期,受害植株单宁的含量比健康植株增加;不同杨树品种单宁含量变化不同,意大利杨健康植株和受害植株的单宁含量均比欧关杨高;受害植株中远离蛀孔与蛀孔周边的单宁含量未表现出显著差异。杨树受害引起单宁含量的变化,可能是杨树受到危害后的一种应激反应的表现,具有诱导抗虫性,即通过增加单宁的含量来抵御害虫的取食。     

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.