Molecular biological and (eco)physiological studies on isoprene emission in Arabidopsis and Grey poplar
Year: 2009
Type: dissertation
Abstract: Plants interact with their environment with a great variety of volatile organic compounds (VOCs), isoprenoids (≡ terpenes), i.e. isoprene, mono-, homo-, di- and sesquiterpenes, being the most prominent group. Isoprene, a hemiterpene, is the simplest isoprenoid compound whose main source, woody plant species, comprises 75% of the 500 Tg C isoprene emitted to the atmosphere per year. Due to the significant influence of isoprene in atmospheric chemistry, growing research interests have focused to investigate this C5 compound. However, physiological function(s) of isoprene emission in planta is not elucidated to date. Actual studies indicate that isoprene can enhance thermotolerance or quench oxidative stress, but the underlying mechanisms are widely unknown. The work presented here significantly contributes to the understanding of physiological function of isoprene and regulation of isoprene biosynthesis by exploiting transgenic Arabidopsis thaliana and Populus x canescens as model systems. The first part of the work aimed to elucidate whether isoprene biosynthesis in plants is triggered by endogenous regulatory mechanisms like the circadian clock. Isoprene emission varies diurnally in several species, also in the natural isoprene emitter Grey poplar (P. x canescens). Moreover it was recently proved that the poplar isoprene synthase gene (PcISPS) displays diurnal variation in its expression. Working on shoot cultures of Grey poplar, placed under different light regime in climate chambers, it was possible to show that under continuous light PcISPS expression, measured by quantitative reverse transcriptase PCR, oscillated with amplitude of approximately 24 hours testifying for endogenous clock regulation. Furthermore, circadian rhythms were not only limited to the level of gene expression. Isoprene emission rates also displayed circadian changes. In contrast, on the protein level circadian changes could not be detected. It, however, appeared that PcISPS activity and protein content became reduced under constant darkness, while under constant light activity and protein content were higher than under day/night regime. Measurement of additional selected isoprenoid genes revealed that phytoene synthase (PcPSY; carotenoids pathway) also displays circadian fluctuations of gene expression whereas 1-deoxy-D-xylulose 5-phosphate reductoisomerase (PcDXR), the first committed enzyme of the methylerythritol phosphate (MEP)-pathway only shows a light regulation of its expression. In the second part of the work Arabidopsis thaliana (ecotype Columbia-0), a natural non-emitter of isoprene, has been constitutively transformed with PcISPS from Grey poplar. Over-expression of poplar ISPS in Arabidopsis resulted in isoprene emitting rosettes that showed enhanced growth rates compared to wild type under moderate thermal stress. The fact that highest growth rates, higher DMADP levels and ISPS enzyme activities were detected in young developing plants indicates that enhanced growth of the transgenic plants under thermal stress is due to the introduced PcISPS gene. However, the emission rates did not reach the level of natural isoprene emitters, like poplars, suggesting possible different regulation of the isoprene biosynthesis and/or lack of substrate for that. To study the physiology of these plants a dynamic gas exchange system was developed allowing miming the natural rapid fluctuations of leaf temperature and light intensity (‘sun- or lightflecks’) in order to study isoprene emission. The results showed that wild type Arabidopsis is already well enough thermotolerant against transient and moderate light and/or heat stress. In contrast, when the same conditions, combining light and heat flecks, were applied to wild type and transgenic Grey poplar lines in which gene-expression of PcISPS was knock-down by RNA interference technology, assimilation was impaired in the non-isoprene emitting lines compared to wild type. Thus, for poplar the ability to emit isoprene can be detrimental. Transgenic Arabidopsis lines were further applied in ecophysiological studies to investigate the role of isoprene in plant-insect interactions. Feeding of herbivores on plants makes plants release volatile compounds that attract herbivore enemies. In all the performed studies the parasitic wasp Diadegma semiclausum searching for its host Plutella xylostella (Diamondback moth) preferred the volatiles emitted by wild type Arabidopsis to those from transgenic, isoprene emitting Arabidopsis plants. Furthermore low external isoprene concentration in the volatile blend of either Arabidopsis or herbivore-infested Brassica oleracea, the natural host of the Diamondback moth, repelled the parasitic wasps. The behaviors of the two examined herbivores (Pl. xylostella and Pieris rapae (Small White Cabbage butterfly)) were not affected by isoprene emission, and GC-MS detection showed despite of isoprene no other differences in the VOC blends of wild type and transgenic plants. These findings suggest that isoprene emission of plants plays a complex ecophysiological role, influencing biotic interactions between plants and insects.
DOI: 10.5445/ir/200075844
Author: Loivamäki, M.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2484620544