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.     

Isoprene emission from plants: why and how

BACKGROUND: Some, but not all, plants emit isoprene. Emission of the related monoterpenes is more universal among plants, but the amount of isoprene emitted from plants dominates the biosphere-atmosphere hydrocarbon exchange. SCOPE: The emission of isoprene from plants affects atmospheric chemistry. Isoprene reacts very rapidly with hydroxyl radicals in the atmosphere making hydroperoxides that can enhance ozone formation. Aerosol formation in the atmosphere may also be influenced by biogenic isoprene. Plants that emit isoprene are better able to tolerate sunlight-induced rapid heating of leaves (heat flecks). They also tolerate ozone and other reactive oxygen species better than non-emitting plants. Expression of the isoprene synthase gene can account for control of isoprene emission capacity as leaves expand. The emission capacity of fully expanded leaves varies through the season but the biochemical control of capacity of mature leaves appears to be at several different points in isoprene metabolism. CONCLUSIONS: The capacity for isoprene emission evolved many times in plants, probably as a mechanism for coping with heat flecks. It also confers tolerance of reactive oxygen species. It is an example of isoprenoids enhancing membrane function, although the mechanism is likely to be different from that of sterols. Understanding the regulation of isoprene emission is advancing rapidly now that the pathway that provides the substrate is known.     

Precise RNAi-mediated silencing of metabolically active proteins in the defence secretions of juvenile leaf beetles

Allomones are widely used by insects to impede predation. Frequently these chemical stimuli are released from specialized glands. The larvae of Chrysomelina leaf beetles produce allomones in gland reservoirs into which the required precursors and also the enzymes are secreted from attached gland cells. Hence, the reservoirs can be considered as closed bio-reactors for producing defensive secretions. We used RNA interference (RNAi) to analyse in vivo functions of proteins in biosynthetic pathways occurring in insect secretions. After a salicyl alcohol oxidase was silenced in juveniles of the poplar leaf beetles, Chrysomela populi, the precursor salicyl alcohol increased to 98 per cent, while salicyl aldehyde was reduced to 2 per cent within 5 days. By analogy, we have silenced a novel protein annotated as a member of the juvenile hormone-binding protein superfamily in the juvenile defensive glands of the related mustard leaf beetle, Phaedon cochleariae. The protein is associated with the cyclization of 8-oxogeranial to iridoids (methylcyclopentanoid monoterpenes) in the larval exudates made clear by the accumulation of the acylic precursor 5 days after RNAi triggering. A similar cyclization reaction produces the secologanin part of indole alkaloids in plants.     

<Emphasis Type="Italic">Catalpa bignonioides</Emphasis> alters extrafloral nectar production after herbivory and attracts ant bodyguards

Inducible anti-herbivore defenses are found within many plant taxa, but there are fewer examples of inducible indirect defenses that incorporate the third trophic level. This study links caterpillar foraging, herbivore-induced changes in extrafloral nectar production, and the attraction of ants to vulnerable leaves and plants. Catalpa bignonioides Walter (Bignoniaceae) uses extrafloral nectar to attract ant (Forelius pruinosus(Roger)) bodyguards in response to Ceratomia catalpae (Boisduval)(Lepidoptera: Sphingidae) herbivory. Ant density per leaf increased with the sugar content of extrafloral nectar excreted by sampled leaves, suggesting that increased nectar production could attract or retain beneficial arthropods. The masses of sucrose, fructose, glucose and all three sugars combined in the extrafloral nectar increased two- to three-fold on attacked leaves within 36 h of the experimental addition of caterpillars. Production rates for neighboring non-attacked leaves and non-attacked leaves on adjacent plants did not differ over the same time period. Ant attendance at caterpillar-attacked leaves increased two- to three-fold within 24 h of herbivory, relative to attendance at neighboring, undamaged leaves. These attacked leaves attracted the fewest ants prior to the onset of herbivory, suggesting the specialist caterpillar may avoid or be excluded from leaves with more bodyguards. The removal of leaf tissue with scissors did not alter ant attendance at damaged leaves. Mean ant attendance per leaf on attacked plants increased 6- to 10-fold after caterpillar introduction, relative to adjacent unattacked plants. The plant's biotic defense thus operates at two scales; the number of bodyguards (ant workers) on the plant increases after attack, and this increased workforce is biased towards attacked leaves within plants. Fewer caterpillars remained on plants that attracted greater numbers of ants, suggesting these bodyguards benefit the plant.     

Genetic structure and regulation of isoprene synthase in Poplar (Populus spp.)

Isoprene is a volatile 5-carbon hydrocarbon derived from the chloroplastic methylerythritol 2-C-methyl-d-erythritol 4-phosphate isoprenoid pathway. In plants, isoprene emission is controlled by the enzyme isoprene synthase; however, there is still relatively little known about the genetics and regulation of this enzyme. Isoprene synthase gene structure was analysed in three poplar species. It was found that genes encoding stromal isoprene synthase exist as a small gene family, the members of which encode virtually identical proteins and are differentially regulated. Accumulation of isoprene synthase protein is developmentally regulated, but does not differ between sun and shade leaves and does not increase when heat stress is applied. Our data suggest that, in mature leaves, isoprene emission rates are primarily determined by substrate (dimethylallyl diphosphate, DMADP) availability. In immature leaves, where isoprene synthase levels are variable, emission levels are also influenced by the amount of isoprene synthase protein. No thylakoid isoforms could be identified in Populus alba or in Salix babylonica. Together, these data show that control of isoprene emission at the genetic level is far more complicated than previously assumed.     

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.     

Strong correlation between isoprene emission and gross photosynthetic capacity during leaf phenology of the tropical tree species Hymenaea courbaril with fundamental changes in volatile organic compounds emission composition during early leaf development

Changes of the volatile organic compounds (VOC) emission capacity and composition of different developmental stages of the tropical tree species Hymenaea courbaril were investigated under field conditions at a remote Amazonian rainforest site. The basal emission capacity of isoprene changed considerably over the course of leaf development, from young to mature and to senescent leaves, ultimately spanning a wide range of observed isoprene basal emission capacities from 0.7 to 111.5 µg C g^?1 h^?1 during the course of the year. By adjusting the standard emission factors for individual days, the diel courses of instantaneous isoprene emission rates could nevertheless adequately be modelled by a current isoprene algorithm. The results demonstrate the inadequacy of using one single standard emission factor to represent the VOC emission capacity of tropical vegetation for an entire seasonal cycle. A strong linear correlation between the isoprene emission capacity and the gross photosynthetic capacity (GP_max) covering all developmental stages and seasons was observed. The present results provide evidence that leaf photosynthetic properties may confer a valuable basis to model the seasonal variation of isoprenoid emission capacity; especially in tropical regions where the environmental conditions vary less than in temperate regions. In addition to induction and variability of isoprene emission during early leaf development, considerable amounts of monoterpenes were emitted in a light‐dependent manner exclusively in the period between bud break and leaf maturity. The fundamental change in emission composition during this stage as a consequence of resource availability (supply side control) or as a plant's response to the higher defence demand of young emerging leaves (demand‐side control) is discussed. The finding of a temporary emergence of monoterpene emission may be of general interest in understanding both the ecological functions of isoprenoid production and the regulatory processes involved.     

Genetics of resistance to transgenic Bacillus thuringiensis poplars in Chrysomela tremulae (Coleoptera: Chrysomelidae)

The area under genetically engineered plants producing Bacillus thuringiensis (Bt) toxins is steadily increasing. This increase has magnified the risk of alleles conferring resistance to these toxins being selected in natural populations of target insect pests. The speed at which this selection is likely to occur depends on the genetic characteristics of Bt resistance. We selected a strain of the beetle Chrysomela tremulae Fabricius on a transgenic Bt poplar clone Populus tremula L. x Populus tremuloides Michx producing high levels of B. thuringiensis Cry3Aa toxin. This strain was derived from an isofemale line that generated some F2 offspring that actively fed on this Bt poplar clone. The resistance ratio of the strain was >6400. Susceptibility had decreased to such an extent that the mortality of beetles of the strain fed Bt poplar leaves was similar to that of beetles fed nontransgenic poplar leaves. Genetic crosses between susceptible, resistant, and F1 hybrids showed that resistance to the Cry3Aa toxin was almost completely recessive (D(LC) = 0.07) and conferred by a single autosomal gene. The concentration of Cry3Aa produced in the transgenic Bt poplar used in this study was 6.34 times higher than the LC99 of the F1 hybrids, accounting for the complete recessivity (D(ML) = 0) of survival on Bt poplar leaves. Overall, the genetic characteristics of the resistance of C. tremulae to the Cry3Aa toxin are consistent with the assumptions underlying the high-dose refuge strategy, which aims to decrease the selection of Bt resistance alleles in natural target pest populations.     

小猿叶甲对寄主植物衰老和缺乏的适应

小猿叶甲Phaedon brassicae Baly是十字花科蔬菜上的一种重要害虫,具有复杂的生活史。测定小猿叶甲取食幼嫩和衰老小白菜叶片后的滞育比率和食料缺乏条件下小猿叶甲成虫的存活时间。研究结果表明,在25℃配合光周期12L∶12D条件下,小猿叶甲取食衰老期小白菜叶片后的滞育比率显著高于取食幼期小白菜叶片后的滞育比率。在25℃、12L∶12D条件下以幼期小白菜叶片饲养小猿叶甲,其成虫羽化后取食0～7d,再分别转入30℃、14L∶10D,不供食的条件下,其结果表明,雌雄成虫存活时间随取食时间延长而延长;羽化成虫取食5d以上,成虫存活时间显著延长,雌雄成虫平均存活时间均在75d以上。最后,讨论小猿叶甲对寄主植物衰老和缺乏的可能适应机制。     

Carbon balance in leaves of young poplar trees

In the present study, important components of carbon metabolism of mature leaves of young poplar trees (Populus x canescens) were determined. Carbohydrate concentrations in leaves and xylem sap were quantified at five different times during the day and compared with photosynthetic gas exchange measurements (net assimilation, transpiration and rates of isoprene emission). Continuously measured xylem sap flow rates, with a time resolution of 15 min, were used to calculate diurnal balances of carbon metabolism of whole mature poplar leaves on different days. Loss of photosynthetically fixed carbon by isoprene emission and dark respiration amounted to 1% and 20%. The most abundant soluble carbohydrates in leaves and xylem sap were glucose, fructose and sucrose, with amounts of approx. 2 to 12 mmol m(-2) leaf area in leaves and about 0.2 to 15 mM in xylem sap. Clear diurnal patterns of carbohydrate concentration in xylem sap and leaves, however, were not observed. Calculations of the carbon transport rates in the xylem to the leaves were based on carbohydrate concentrations in xylem sap and xylem sap flow rates. This carbon delivery amounted to about 3 micromol C m(-2) s(-1) during the day and approx. 1 micromol C m(-2) s(-1) at night. The data demonstrated that between 9 and 28 % of total carbon delivered to poplar leaves during 24 h resulted from xylem transport and, hence, provide a strong indication for a significant rate of carbon cycling within young trees.     

Controls of the quantum yield and saturation light of isoprene emission in different‐aged aspen leaves

Leaf age alters the balance between the use of end‐product of plastidic isoprenoid synthesis pathway, dimethylallyl diphosphate (DMADP), in prenyltransferase reactions leading to synthesis of pigments of photosynthetic machinery and in isoprene synthesis, but the implications of such changes on environmental responses of isoprene emission have not been studied. Because under light‐limited conditions, isoprene emission rate is controlled by DMADP pool size (S_DMADP), shifts in the share of different processes are expected to particularly strongly alter the light dependency of isoprene emission. We examined light responses of isoprene emission in young fully expanded, mature and old non‐senescent leaves of hybrid aspen (Populus tremula x P. tremuloides) and estimated in vivo S_DMADP and isoprene synthase activity from post‐illumination isoprene release. Isoprene emission capacity was 1.5‐fold larger in mature than in young and old leaves. The initial quantum yield of isoprene emission (α_I) increased by 2.5‐fold with increasing leaf age primarily as the result of increasing S_DMADP. The saturating light intensity (Q_I90) decreased by 2.3‐fold with increasing leaf age, and this mainly reflected limited light‐dependent increase of S_DMADP possibly due to feedback inhibition by DMADP. These major age‐dependent changes in the shape of the light response need consideration in modelling canopy isoprene emission.     

Alterations of the resin canal system of <Emphasis Type="Italic">Pinus pinaster</Emphasis> seedlings after fertilization of a healthy and of a <Emphasis Type="Italic">Hylobius abietis</Emphasis> attacked stand

Changes in resource availability and biotic and abiotic stress may alter the defensive mechanisms of pine trees. The effect of fertilisation on the resin canal structure of Pinus pinaster seedlings established in two trials in NW Spain, one attacked by Hylobius abietis and the other non-attacked, was studied. The leaders of 50 plants were destructively sampled and the resin canal density, the canal area and its relative conductive area in the phloem and xylem were assessed. Experimentally increased nutrient availability significantly decreased resin canal density in the phloem of the seedlings in the two analysed trials, where unfertilised seedlings presented up to 30% more resin canal density than the fertilised seedlings (mean value ± SEM = 0.32 ± 0.02 resin canals mm⁻² in the fertilised plants versus 0.45 ± 0.04 resin canals mm⁻² in the control plants). Fertilisation had no effect on the resin canal system in the xylem, but significantly increased tracheid size. Significant differences of resin canals among sites were observed mainly in the xylem; the resin canal density was 1.7-fold greater in the attacked site than in the non-attacked site. The similar structure of phloem resin canals in both sites supports that phloem resin canals are constitutive mechanisms of defence in P. pinaster, whereas xylem resin canals would be constitutive mechanisms but also inducible mechanisms of resistance following the attack of pine weevils or bark beetles. XM and LS equally contributed to this paper.     

The relationship between the amount of insect-repelling phenolic compounds in pennywort tea and the number of infesting cigarette beetles in the tea bag during one-year storage

Cigarette beetle, Lasioderma serricorne (F.), is a common food-product pest widely found in homes. This paper investigated the relationship between the number of infesting cigarette beetles and the phenolic content of pennywort tea made from young and mature leaves during one-year storage. It was found that cigarette beetles started to appear in tea bags after six months of storage. More adults were found than larvae and pupae. In addition, the number of cigarette beetles infesting pennywort tea made from mature leaves was higher than when it was made from young leaves. The amount of phenolic compounds in pennywort tea was analyzed by Folin–Ciocalteau assay. The results of a regression analysis indicated that the number of adult cigarette beetles correlated negatively with the amount of phenolic compounds in pennywort tea made from young leaves – when the total phenolic content decreased, the number of infesting cigarette beetles increased (r² = 0.80). The number of larvae and pupae showed a weak negative correlation with the amount of phenolic compounds (r² = 0.40 and r² = 0.56, respectively). Similar correlations were found in pennywort tea made from mature leaves, but the number of infesting beetles was higher due to the lower phenolic content of the mature leaves.     

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.     

Bionomics of <Emphasis Type="Italic">Momordica cochinchinensis</Emphasis> Fed <Emphasis Type="Italic">Aulacophora foveicollis</Emphasis> (Coleoptera: Chrysomelidae)

The effects of feeding on root by the larvae and three types of Momordica cochinchinensis Spreng (Cucubitaceae) leaves (young, mature and senescent) by the adults of Aulacophora foveicollis Lucas (Coleoptera: Chrysomelidae) were studied under laboratory conditions. Total larval developmental time was 19.7 ± 0.2 days by feeding on young roots. Adult males lived for 28.4 ± 1, 65.7 ± 1.1 and 22.8 ± 1.3 days on young, mature and senescent leaves, respectively; whilst adult females lived for 34.3 ± 1.2, 68.5 ± 0.9 and 26.4 ± 1.4 days on young, mature and senescent leaves, respectively. Fecundity was highest in mature leaves fed insects (202.2 ± 10.6). Total carbohydrate, protein, lipid, nitrogen and amino acid were much higher in root followed by mature leaves than young and senescent leaves. Moisture content was highest in mature leaves than the roots, young and senescent leaves. Phenols were greatest in young leaves followed by mature leaves and least in senescent leaves and roots of the said plant. Flavonols were higher in young leaves and least in root. These results suggest that A. foveicollis adults perform better on mature leaves than young and senescent leaves for their nutrition.     

Novel evidence for systemic induction of silicon defences in cucumber following attack by a global insect herbivore

1. Silicon (Si) is a beneficial nutrient that has been reported to ameliorate many abiotic and biotic stresses in plants, including insect herbivory. Insect herbivory has been shown to induce Si defences in plants, although the magnitude and nature of induction remain largely ambiguous. In particular, it is unclear whether herbivore induction of Si defences is confined to attacked tissues (local) or occurs elsewhere in the plant (systemic). 2. We grew cucumber, Cucumis sativus L. plants (var. Burpless F1 and Beit Alpha), an intermediate Si accumulator, hydroponically under Si-supplemented or Si-free conditions and measured the level of Si induction caused by a polyphagous chewing insect, the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). We also examined the impacts of Si on insect performance by conducting in vitro feeding assays on excised leaves (ex situ) and intact leaves on plants (in situ). 3. Herbivory significantly increased Si accumulation both locally in attacked leaves (21% increase in Beit Alpha and 17% in Burpless F1) and systemically in non-attacked leaves (19% increase in Beit Alpha and 10% in Burpless F1). Si supplementation significantly increased % foliar Si and C:N ratio, while significantly decreasing larval relative consumption (RC) and relative growth rate (RGR) in the in situ assays. In ex situ assays, however, Si only reduced larval RGR when fed on Beit Alpha plants. 4. Our results confirm that Si-based defences can also operate in moderate Si-accumulating plants and, for the first time, that insect herbivory induces systemic Si accumulation equivalently between plant varieties.     

Hybridization of European oaks (Quercus ilex × Q. robur) results in a mixed isoprenoid emitter type

European oaks have been reported to emit isoprene or monoterpenes derived from recently fixed photosynthetic carbon. The emission type is plant species specific and can be used as chemo‐taxonomic marker. In the present article the isoprenoid biochemical properties of mature Quercus × turneri‘Pseudoturneri’ hybrids resulting from a crossing of a Mediterranean evergreen monoterpene‐emitting species (subgenus Sclerophyllodrys; Quercus ilex L.) and an isoprene‐emitting deciduous oak species (subgenus Lepidobalanus; Quercus robur L.) are described. Both species are compared with respect to the capacity for isoprenoid synthesis and the actual isoprenoid emission pattern of different tree‐types. The analysis showed that the oak hybrid combines properties of both parental species. Furthermore, it could be shown that the enzyme activities of isoprene synthase and monoterpene synthases are reflected in the isoprenoid emission pattern of the hybrids as well as in the observed emission rates.     

Occurrence of Euphoria lurida (Fabricius) (Coleoptera: Scarabaeidae) in the corn cultivated in Lowland at Rio Grande do Sul State

Euphoria lurida adults (Fabricius) is registered as pests of some fruits and flowers. However, this is the first record of this insect in maize fields. In march, 2006 adults attacked about 15% of maize plants in an area with almost 1 ha. The damage occurred in plants in the reproductive stage. Transversal and longitudinal holes were found in the stalks just above the ear insertion. The attack to the ear occurred more intensively in the upper part of the plant, where the insect fed on the silk and the filling grains.     

Regulation of isoprene emission in Populus trichocarpa leaves subjected to changing growth temperature

The hydrocarbon isoprene is emitted in large quantities from numerous plant species, and has a substantial impact on atmospheric chemistry. Temperature affects isoprene emission at several levels: the temperature at which emission is measured, the temperature at which leaves develop, and the temperatures to which a mature leaf is exposed in the days prior to emission measurement. The molecular regulation of the response to the last of these factors was investigated in this study. When plants were grown at 20 degrees C and moved from 20 to 30 degrees C and back, or grown at 30 degrees C and moved from 30 to 20 degrees C and back, their isoprene emission peaked within 3 h of the move and stabilized over the following 3 d. Trees that developed at 20 degrees C and experienced 30 degrees C episodes had higher isoprene emission capacities than did leaves grown exclusively at 20 degrees C, even 2 weeks after the last 30 degrees C episode. The levels and extractable activities of isoprene synthase protein, which catalyses the synthesis of isoprene, and those of dimethylallyl diphosphate (DMADP), its substrate, alone could not explain observed variations in isoprene emission. Therefore, we conclude that control of isoprene emission in mature leaves is shared between isoprene synthase protein and DMADP supply.     

沙蒿大粒象的生物学特性

沙蒿大粒象Adosomus sp.,是近几年在我国宁夏、内蒙古、陕西等地沙蒿灌木林中大面积发生的一种钻蛀性害虫。在宁夏盐池,该虫1年1代,以成虫和老熟幼虫在沙蒿根部越冬。越冬成虫始见于4月中下旬,终见于6月中旬;越冬老熟幼虫5月中旬开始化蛹,成虫始见于6月下旬,终见于8月上旬。成虫产卵于沙蒿根茎部至地下2cm之间,外被一层1~2mm厚的沙壳。幼虫主要分布在地下根部12cm以内,主要钻蛀沙蒿根部,在根部作蛹室化蛹;成虫取食沙蒿叶片作为营养补充。