| dc.description.abstracteng | Lipid-derived hydrocarbons have many important tasks in land plants. They prevent uncontrolled water loss as constituents of the hydrophobic properties of the cuticula. Another important task of lipid-derived hydrocarbons is the storage of energy. A common, lipid-derived plant storage compound is triacylglycerol (TAG). In woody plants, TAGs are localized in the stem in ray cells and utilized to power cambial reactivation in spring. The storage pools in the stem of woody plants are used not only to cope with seasonal changes but also to encounter sudden stress events such as wounding, extreme cold and drought periods. Regarding climate change, especially drought events are expected more frequently and may require enhanced protection against water loss. In woody perennial plants, TAG and wax synthesis and their physiological consequences for wood properties and stress resistance are poorly understood.
The main goal of this thesis was to generate transgenic P. x canescens trees with enhanced amounts of wax esters and TAGs in vegetative tissues and to investigate the impact of these modifications on growth, biomass production, wood properties and physiological performance. Two main hypotheses were tested: (a) enhanced amounts of wax esters or TAGs increase the hydrophobicity of wood evident from reduced wood swelling, thereby, affecting an important technological feature of wood, and (b) the accumulation of wax esters in the cuticula enhances the drought resistance of transgenic trees. Therefore, two key genes involved in wax ester and TAG synthesis were expressed in poplar: (i) a wax ester synthase derived from the desert shrub Simmondsia chinensis (ScWS) and (ii) the Arabidopsis diacylglycerol O-acyltransferase 1 (AtDGAT1), the key enzyme in TAG production via the Kennedy pathway. Efforts were undertaken to co-express two further genes to increase the yield of lipids: the Marinobacter aquaolei fatty alcohol reductase (MaFAR) in combination with the ScWS and the transcription factor WRINKLED1 of Arabidopsis (AtWRI1) in combination with AtDGAT. The genes were expressed in poplar under the 35S promoter or the DX15 promoter, the latter being cloned and characterized in this study. While the commonly used 35S promoter was expected to lead to constitutive overexpression of the target gene, the DX15 promoter was demonstrated to express the target gene strongly in wood. Although a co-expression of two genes could not be achieved, presumably due to the size of the resulting constructs, several viable transgenic lines of Populus x canescens were produced overexpressing ScWS or AtDGAT1. The constitutive overexpression of the ScWS under the 35S promoter led to significant physiological changes of the transgenic plants. Compared to the wildtype, transgenic plants showed a lower stomatal conductance caused by smaller stomata, but similar photosynthesis rates. In long term experiments, the 35S::ScWS expressing plants showed slightly decreased water consumption, and enhanced water use efficiency. The biomass production was significantly influenced negatively by overexpression of ScWS in young plants under only during long term growth under greenhouse conditions. Gas chromatography–mass spectrometry analyses revealed a significant decrease in wax ester precursor molecules on the surface of leaves and – to a lower extent – also on the surface of stems. To test the drought responses of the ScWS-overexpressing poplar lines, the plants were exposed to mild and severe drought stress conditions. Under sudden, severe drought conditions, the transgenic plants showed an enhanced vitality compared to WT plants. Under mild drought conditions, the pre-dawn leaf water potential of the transgenic plants was slightly lower than that of the wildtype plants. Anatomical studies of wood that had been produced during long term mild drought revealed no differences between the transgenic lines and the wildtype. Taken together, we speculate that overexpression of the wax ester synthase might deplete the precursor molecule pool due to higher wax biosynthesis. The synthesis of other compounds being part in the cuticular wax layer (e.g. alkanes) might thus be limited. Since the cuticular composition has been demonstrated to be controlled by drought stress in many annual plants, feed-back mechanisms of an altered cuticular composition might have induced a mild drought stress in the transgenic polar plants. This might have resulted in “pre-acclimation” and subsequently in plants with higher resistance under severe stress.
Transformation of poplars with AtDGAT1 yielded only viable plants under the tissue-specific DX15 promoter but not under the 35S promoter. Previous studies in annual plants suggested that overexpression of DGAT1 resulted only in an enhanced accumulation of TAGs when the production of the precursor molecules for TAGs were enhanced by co-expression of a second gene, such as the transcription factor WRI1 or the enzyme FAR. Therefore, we speculate that a constitutive overexpression of AtDGAT1 without enhancing the precursor pool might unbalance the lipid metabolism too harshly, thereby, precluding the production of viable lines of 35S::AtDGAT1. However, when expressed tissue-specific with the DX15 promoter, the impact might be sufficiently limited to prevent effects on the poplar’s physiology. This idea was supported by the observation that the presence of lipid droplets in ray cells and other xylem regions of the DX15::AtDGAT1 transgenic poplar was not different from that in the wildtype. No significant differences were found in growth rates, biomass production and wood formation, but the water uptake of dry wood was significantly decreased.
In conclusion, the overexpression of ScWS led to improved drought performance in poplar and the overexpression of AtDGAT1 in developing xylem improved a technological wood feature since the swelling of dry wood was diminished compared to the wildtype. In future studies, the impact of these transgenic modifications on the composition of the lipid profiles and the wax load on the plant´s surfaces have to be further characterized. It will also be important to clarify the links between drought signals, stomatal size and wax production. This thesis, thereby, opens new perspectives not only for the development of trees with improved wood properties but also for the selection of trees for future, drier climatic conditions. | de |