Untersuchung von Zusammenhängen zwischen pflanzlichen Emissionen und pflanzeninternen Signalmolekülen und Enzymaktivitäten
- Determination of connections between plant emissions and plant internal signal molecules and enzyme activities
Miebach, Marco; Slusarenko, Alan (Thesis advisor)
Aachen : Publikationsserver der RWTH Aachen University (2008)
Dissertation / PhD Thesis
Aachen, Techn. Hochsch., Diss., 2008
Plants are able to emit flavours over their flowers, leaves and fruits. These flavours serve them as attractants, repellents or signal molecules. Many plants show a constitutive emission of a series of different mono- and sesquiterpenes. Futhermore plants have also inducible emissions. The influence of stressors like e.g. predators, bacteria, fungi or ozone yield to novel synthesis and emission of repellents and signal molecules. They originate mainly form octadecanoid and sikimate pathway. The octadecanoid pathway yields in the formation of alcohols and aldehyds (predominatly C6-compounds). Starting form free fatty acids they were generated and emitted via an enzyme cascade (lipase, lipoxygenase, hydroperoxidelyase). The Shikimat pathway is important for the formation of the important plant hormone salicylic acid (SA). After methylation SA can be emitted as the volatile methylsalicylate MeSA) into the atmosphere. The main task of the work was to approve if it is possible to draw conclusions from stress induced emissions to certain plant internal enzyme activities or substrate concentration in the model plant tomato (cv. Moneymaker). The would allow a non invasive measurement of such endogenous parameters. On the one hand it should be proved if the emission of green leave aldehyds and alcohols (GLAs) can be suitable for the prediction of the enzyme activities lipase, lipoxygenase and hydroperoxidelyase or the substrate concentrations in the octadecanoid pathway. The GLAs were measured by online GC/MS, the intermediates (free fatty acids, hydroperoxy fatty acids)by HPLC and the enzyme activities by UV/VIS spectroscopy. A good correlation between a certain enzyme activity/substrate concentration should then be a hint to the rate-determining step in the cascade. On the other hand it should be proved if emission of MeSA can be suitable for the prediction of endogenous free SA concentration. Also in this case I looked for a correlation between the MeSA emission and the plant internal SA concentration. The measurement of MeSA were measured by online GC/MS as well and the endogenous SA concentrations by HPLC. Ozone exposure and infection by fungi (Botrytis cinerea) were used as abiotic/biotic stressors respectively. The stress response on ozone exposure of the tomato plants started with varying lag times between 1 and 3 hours. The plants reacted with the emission of methanol, GLAs and MeSA as well as increasing of the emissions of mono- and sesquiterpenes. The plants were removed from the chamber, snap frozen and homogenised with pistil and mortar and stored at -80°C until the extraction and measurement of the enzyme activities/SA concentrations respectively. Whereas the GLA emissions showed no good correlations with lipase and hydroperoxidelyase activities (0.19 and 0.41) the correlation with the lipoxygenase activity was quite good (0.82). This points to the lipoxygenase to be the rate-determining step for the GLA emission. Free fatty acids were found in similar concentrations both in control plants and stressed plants. Substrate limitation by low availability of free fatty acids hence seems not to be ratedetermining. The concentration of hydroperoxy fatty acids were always beyond the detection limit, therefore the lack of substrate for the hydroperoxidelyase may be the "bottle neck" for the GLA emission strength. Also this substrate limitation should lead to a good correlation between GLA emission and lipoxygenase activity. Due to this good correlation it is in cases of Botrytis infection and ozone fluxes between 3•10-8 and 8•10-8 mol m-2 s-1 possible to predict internal enzyme activities by measurement of the GLA emissions. However the GLA emission are not in all cases sufficient to predict the lipoxygenase activity without restrictions. In particular high ozone fluxes into the plant yielded in very high GLA emissions and strong variances form the linear correlation between emission and lipoxygenase activity occurred. Also the emission of MeSA and the internal concentration of free SA showed a good correlation (0.91). Though it should possible to estimate the endogenous free SA concentration by measuring the MeSA emission. All in all this work shows that it is possible to conclude different plant internal enzyme activities and amounts of substances using the measurement of volatile compounds. Though these emissions allow the not invasive determination of these parameters in a good demand interval.