Biochemische, physiologische und klimatische Einflüsse auf die Isoprenoidemission der Graupappel (Populus x canescens (Aiton) Sm.) und der Steineiche (Quercus ilex L.)

Because of their important role for the atmospheric chemistry, global daily and seasonal emission rates of isoprene and monoterpenes have to be estimated with accuracy. Therefore, detailed knowledge of biochemical and physiological processes within the plant metabolism has to be gathered. Afterwards the gained cognitions are used as information for process-based model calculations. The major scope of the work was therefore to enlarge basic knowledge of the regulation of isoprenoid emission, which is known to be dependent on several environmental factors, especially light and temperature. Measurements of diurnal isoprene emission have been performed in parallel on physiological, translational and transcriptional level on leaves of Grey Poplar (Populus x canescens), a strong isoprene emitting species. Additionally, examinations of diurnal monoterpene emission in connection to physiologic and enzymatic processes was conducted in leaves of Holm Oak (Quercus ilex), which emits a large spectrum of monoterpenes. Furthermore a hypothesis was tested, whether isoprene emission may serve the plant as antioxidative protection mechanism in order to overcome oxidative stress. In main parts, the following results have been reached: 1. In the first part of this work, isolation of PcDXR (DXR of Grey Poplar) from a cDNA-Genbank and heterologous expression of the isolated gene was accomplished. The whole PcDXR-sequence consists of 1724 bp and encodes for a proteine sequence of 472 aminoacids. The molecular weight was approximately 51,3 kDa. Comparing the PcDXR sequence with the DXR sequences from 15 different plants, highest similarity to the DXR of A. thaliana (86,23%) has been found. With help of the photometrical measurement of the NADPH implementation mediated through the PcDXR, its enzyme activity has been determined. Specific inhibition of the enzyme by Fosmidomycin and thus decreased NADPH implementation rate demonstrated functionality of the isolated PcDXR enzyme. 2. Daytime variation of physiological and biochemical parameters of the isoprene emission of Grey Poplar was measured twice on 2 following days in 2 years. All together, measurements have been performed on 8 representative plants. Photosynthetic gas exchange (netto-assimilation, C i , transpiration and stomatal conductance), DMADP content and Isoprene emission of the leaves followed the diurnal course of light and temperature. In vitro ISPS activity showed no significant diurnal changes. Strongest influence on the Isoprene emission was shown by leaf temperature. Stomatal conductance had no influence on the Isoprene emission. 3. Quantitative RT-PCR elucidated the gene expression pattern of PcDXR and PcISPS in parallel to diurnal gas exchange measurements. Gene expression of PcISPS showed distinct diurnal courses with maximum values on the late morning, whereas PcDXR transcript levels stayed consistent over the day. No short-term influence of PPFD and leaf temperature has been observed on the expression rates of PcDXR or PcISPS. 4. Daytime variation of physiological and biochemical parameters of the monoterpene emission of Holm Oak was measured the same way as for Grey Poplar. Main components of the monoterpene emission were α-pinene, β-pinene/sabinene, myrcene, camphene, limonene, p-cymol and A-3-carene and also isoprene was detected. The photosynthetic gas exchange of the plants (netto-assimilation, C i , transpiration and stomatal conductance) as well as monoterpene emission followed the diurnal course of light and temperature. For leaf DMADP content and for the sum of Mono-TPS activities no diurnal changes have been detected. The leaf temperature showed strongest influence on monoterpene emission. In contrast to the measurements on Grey Poplar, a regulating influence of the stomatal conductance on the monoterpene emission could have been observed. A weak negative correlation has been detected for DMADP contents and the rate of monoterpene emission, although DMADP-content of the leaves showed no correlations to leaf temperatures and light intensity. DMADP content and in vitro Mono-TPS-activity of the Holm Oak leaves were correlated negatively to each other. 6. In order to test the influence of isoprene within or in the nearest surrounding of poplar leaves on the concentrations of superoxideanionradicals (O 2 . - ) Nitrobluetetrazoliumchloride (NBT) was used to make them visible. Isoprene concentration was raised in the near surrounding of the leaf by isoprene fumigation. Distinct influence of isoprene on the 02 .- concentrations compared to non fumigated leaves has been demonstrated under the chosen conditions. Specific inhibition of the isoprene synthesis and emission by Fosmidomycin showed no further confirmation of these results and also isoprene fumigation of the inhibited leaves showed no distinct effect. Considering the influence of the endogenous isoprene content, again no distinct effect of increased endogenous isoprene concentrations on the 02 .- -concentration could have been determined. The results concerning a possible protection effect against oxidative stress are inconsistent and can not clarify this question.