Abstract
M.Sc.
A common feature of multi gene families is that their members are expressed in different ways in response to environmental and developmental signals. In the present study the expression of a CHS multi gene family in bean (Phase°lus vulgari.), was studied. using a RT-PCR technique that focuses on the 3' divergent regions of the isogenes. Tissue-specific expression in roots, stems, leaves and the flowers of Phaseolus vulgaris, as well as in callus tissue, were investigated. Patterns and levels of gene expression were investigated after treatment with different elicitors as well as light. In most cases time and concentration studies were performed. The four CHS transcripts (CHS4. CHS1. CHS17 and CHS14) showed tissue-specific expression. The four CHS transcripts were differently expressed in the seven organs investigated: and different levels of activity were observed. The highest level of transcript expression for CHS14 and CHS4 could be observed in the roots, whereas relatively low levels were obtained in the leaves. stems and flowers of the green as well as etiolated seedlings. Higher levels of CHS were found in flower buds. High levels of all four transcripts were also found in callus. Elicitor treatment with structurally diverse abiotic agents showed induction of all four CHS mRNA transcripts. Concentration studies revealed high levels of CHS transcript levels. Elicitation with different concentrations of the elicitors: glutathione. mercuric chloride and sodium salicylate showed high levels of the CHS transcripts after exposure of 6 h to the different elicitation agents. The transcript levels increased significantly to levels above those observed in untreated (control) plants. The CHS transcripts showed higher levels of induction after elicitation with mercuric chloride (1 mM) relative to treatment with sodium salicvlate (10 rnM), suggesting differential regulation at the transcriptional level. The expression patterns observed with glutathione were very similarly to those induced by mercuric chloride. The kinetics of induction of all CHS transcripts. except for CHS1 were low at 2 and at 8 h postelicitation and maximal levels of transcript. although transiently induced, could be observed at 4 - 6 h. The use of 4 mM mercuric chloride did not give any induction. most probably because it was a lethal concentration. Etiolated and green bean seedlings, exposed to UV light. showed expression of all four CHS transcripts. In green leaves no significant differences in the induction kinetics between the different chs genes were observed. Three of the transcripts (CHS4. CHSI7. CHS14) accumulated rapidly (within ca. 3h). reaching a maximum after 6 h of irradiation. followed by a decline. CHS4 revealed a 18.2 fold induction. CHSI7 showed a 4.8 fold increase and CHS14 a 4 fold increase after 6 h of illumination in green leaves. In contrast. CHS1 showed a delay ed response which was still observable after 15 h. It was also demonstrated that CHS transcripts accumulated rapidly but transiently. Following illumination of etiolated leaves with white light. except for CHS I. CHS17 and CHS4 showed similar expression levels and patterns. with maximal induction at 1,5 h after white light exposure, whereas maximal induction for CHS14 was at 2 h. At 2.5 Ii the levels for all three transcripts dropped to preinduction levels. It is therefore evident that CHS is a key metabolic control point in the phenylpropanoid pathway leading specifically to isollavonoid biosynthesis. The results strongly suggest that the activation of plant defence genes are regulated in a tissue-specific manner and that induction by different elicitor-active agents. may be regulated by different. But convergent signal transduction regulatory networks.