Influence of short- and long-chain AHLs on phytohormone levels

3.3.2.1 Determination of salicylic acid

The SA content was determined in leaves during a time course of 24 h after 10-day-old barley plants were treated with 10 µM C8- and C12-HSL (fig. 3.15). The determination of the phytohormone content was kindly performed by the Department of Animal Biology, Plant Biology and Ecology of the UAB in Spain. To better illustrate the data structure a Bayesian analysis was performed. The treatments with short- and long-chain AHLs caused 13.5- and 14-fold credible increases compared to the control after 4 h, leading to phytohormone levels of 22.7 and 24.2 pmol per gram plant fresh weight, respectively. This shows that both AHL derivatives were able to induce an SA response in barley leaves. The incubation time of 8 h with C8-HSL caused only a slight credible SA increase, whereas a decrease of SA occurred after 12 h with C8-HSL treatment and remained at this level until 24 h. The application of the long-chain AHL induced a slight decline in SA compared to the previous time point, but the SA level still remains credibly elevated compared to the control, while further treatment led to credible 2-fold increase after 12 h compared to controls. Subsequently the SA concentration shows a decline after additional 12 h of C12-HSL. After 24 h the controls reached 9.4-fold up-regulated SA contents compared to both AHL treatments. Taken together, the highest AHL-mediated SA level was achieved 4 h after C8 and C12-HSL treatment, whereas the C12-HSL induced an additional small SA peak after 12 h, but showed generally a credible difference within the whole measurement series to the control.

RESULTS

58 Figure 3.16 Contents of JA and JA-Ile in barley leaves after 4, 10, and 22 h of control, 10 µM C8- or C12-HSL application. Controls received the same amount of solvent (DMSO) as used for AHL treatments. Application of each substance was carried out for axenically grown, 10-day-old barley plants. Values indicated are most probable means of n=5 (with 2 technical replicates per biological experiment) according to Bayesian analysis with error bars representing the 95 % highest density interval.

3.3.2.2 Determination of jasmonic acid and its derivative jasmonic acid-isoleucine

The content of JA and its amino acid conjugate JA-Ile were determined in leaves of 10-day-old barley plants during a time course of 22 h and are displayed in figure 3.16. For this, samples were taken 4, 10, and 22 h after C8- and C12-HSL treatments (for experimental procedure chapter 2.2.16). The determination of the jasmonates was kindly performed by the Department of Cell and Metabolic Biology of the Leibnitz Institute of Plant Biochemistry. For the present data a Bayesian analysis was applied to better illustrate the data structure. Both levels of JA and JA-Ile equal the control values with small deviations till the time point 22 h. It seems that at 4 h after C12-HSL and 10 h after C8-HSL a slight increase of JA is induced, but the standard deviations are too high in their variance that a reliable statement can be done. Additionally, JA-Ile levels seems to be repressed after 4 and 22 h of C12-HSL treatment, but also here high standard deviations prevent a clear statement.

RESULTS

59 Figure 3.17 ABA content of barley leaves after 4, 10, and 22 h of control, 10 µM C8-, or C12-HSL application. Controls received the same amount of solvent (DMSO) as used for AHL treatments.

Application of each substance was carried out for axenically grown, 10-day-old barley plants. Values indicated are most probable means of n=5 (with 2 technical replicates per biological experiment) according to Bayesian analysis with error bars representing the 95 % highest density interval. * shows a credible difference to the control measurement within each time point.

3.3.2.3 Determination of abscisic acid

The AHL mediated influence on the ABA-level in barley leaves was determined during a time course of 22 h (for experimental procedure chapter 2.2.16) and the results are presented in the line graph 3.17. For the present data a Bayesian analysis was applied to better illustrate the data structure. The analysis of the phytohormone content was kindly performed by the Department of Cell and Metabolic Biology of the Leibnitz Institute of Plant Biochemistry. The short-term incubation of 4 h caused a credible 2-fold up-regulation of the ABA level in C12-HSL treated plants. Further, the ABA content of C12-C12-HSL treated plants approximated to the control-values at the last sample time point at 22 h after AHL application, whereby the values declined below control values at the 22 h time point, but always displayed a credible difference to the control treatment. Accordingly, the treatments with the short-chain AHL displayed kinetics similar to control plants throughout the entire treatment period, and showed additionally a credible difference to controls after 4 and 22 h of application. At this time point, the values were even below controls. In summary, C12-HSL mediated an enhanced ABA concentration after 4 h compared to controls and lowered the phytohormone level after 10 h to control levels.

RESULTS

60 Figure 3.18 Content of the flavonoids lutonarin and saponarin in barley leaves after control, 72 h, and 96 h of 10 µM AHL treatment. 0 h shows the flavonoid level before starting the experiment. Controls received the same amount of solvent (DMSO) as used for AHL treatments.

Application of each substance was carried out for axenically grown, 10-day-old barley plants. Values indicated are most probable means of n=2 (with 2 technical replicates per biological experiment) according to Bayesian analysis with error bars representing the 95 % highest density interval. No credible difference to the control measurement could be determined.