Identification and regulation of the stilbene biosynthetic pathway genes from grapevine
Plant Physiology
Final Report Abstract
This project had the ambitious overarching goal to identify and characterize genes in the stilbene biosynthesis and modification pathway. Using VvMYB15 overexpression tissues and subsequent microarray experiments by Janine Höll, it was possible to identify several promising candidates for modification and transport of stilbenes in Vitis vinifera. Experiments on transient promoter activation and expression profiling for the developmental series (berry ripening) and infection series (challenge with P. viticola) aimed to provide additional support for promising candidate genes and shed some light on their potential functions. Further characterization of candidate genes included the yeast expression system, the enzymatic characterization of the corresponding recombinant proteins (performed in Colmar), and the ectopic expression in tobacco. Together, these different experimental approaches were expected to provide evidence on whether the four most interesting candidates are directly linked to the production of their suspected metabolites. The glycosyltransferases VvGT2, VvGT4 and VvGT5 showed promoter induction by VvMYB15 in grapevine. Expression profiles in correlation with piceid content indicated that VvGT2 and VvGT5 were active in grape berry development, but not during infection with P. viticola. Taken together, these observations suggest that they most likely were involved in the stilbene modification pathway, however not involved in an active defense response but rather in storage or detoxification of resveratrol. Although other researchers had already described the role of VvGT2 (or closely related enzymes form other cultivars) in previous publications, it was decided to still analyze the gene further as a “positive” control but also with the perspective to find additional functions of this enzyme. It could be shown that VvGT2 in fact was able to use resveratrol as substrate, although with low efficiency and not as the preferred substrate. We were not able to show whether VvGT5 accepts resveratrol as substrate. For both enzymes there are other potential substrates that could be investigated in the future. Unfortunately, VvGT4 could not be cloned in time for experimental analysis (due to consequences of the Covid19 pandemic). In summary, it appears that the glycosyltransferases are not specific for the stilbene pathway. Since stilbenes evolved from the flavonoid pathway, it might be that they play a role and have a low affinity to substrates from both pathways. Here, further elucidation of their relative substrate preferences by more quantitative approaches is required. The laccase gene under study was considered to be a very promising candidate due to its promoter induction by VvMYB15 as well as its correlation with viniferin in infected Regent leaf discs. This was shown very clearly in the 2017 series and also with a little more background in the 2019 series. Since viniferin is an active compound in defense, this encouraged us to further investigate this gene. Although successfully cloned, the bacterial expression systems did unfortunately not allow to produce recombinant laccase protein. It was therefore attempted to use tobacco as an expression system. While the time to develop a complete assay including protein purification was not available, a faster assay by co-infiltrating the gene with VvSTS29 yielded first results, i.e. production of a substance that eluted within fractions of a second of viniferin that has yet to be identified, although in small amount. In summary, the particular laccase under study appears to be involved in plant defense. This result may be of interest for future studies. VvABC12 and VvABC13, the two ABC transporters found in the microarray analysis are candidate genes for the transport of stilbenes. Unfortunately, several attempts to clone these genes failed, presumably because of their large size. Nevertheless, expression of the two genes were analyzed in the developmental series, where their expression profiles correspond to stilbene accumulation. Also, their promoters were activated by VvMYB15, though not exclusively. They therefore remain interesting candidate genes. Additional candidate genes (e.g. VvbGluc 1 + 2, VvStoxy; data not shown) have been identified by the transcriptome analysis, however due to time constraints further analysis was not possible. Finally, VvROMT, an already characterized and published gene was also included in our analysis to see how an already established gene would behave in our experimental systems. Similar to laccase gene and viniferin, VvROMT displayed co-expression with pterostilbene accumulation in the infection experiment. First attempts were made to discover the regulatory pathway upstream of VvMYB15. However, our approach to use the µ-MACS FactorFinder method unfortunately yielded as yet no promising results.
Publications
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(2016). The photomorphogenic factors UV-B RECEPTOR 1, ELONGATED HYPOCOTYL 5, and HY5 HOMOLOGUE are part of the UV-B signalling pathway in grapevine and mediate flavonol accumulation in response to the environment. Journal of Experimental Botany, 67(18), 5429-5445 (IF 5.9)
Loyola, R., Herrera, D., Mas, A., Wong, D. C. J., Höll, J., Cavallini, E., Castellarin, S. D.
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(2018). Combinatorial regulation of stilbene synthase genes by WRKY and MYB transcription factors in grapevine (Vitis vinifera L.). Plant and Cell Physiology, 59(5), 1043-1059
Vannozzi, A., Wong, D. C. J., Höll, J., Hmmam, I., Matus, J. T., Bogs, J., Lucchin, M.
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(2019). Impact of pulsed UV‐B stress exposure on plant performance: How recovery periods stimulate secondary metabolism while reducing adaptive growth attenuation. Plant, Cell & Environment, 42(3), 801-814 (IF 6.2)
Höll, J., Lindner, S., Walter, H., Joshi, D., Poschet, G., Pfleger, S., Rausch, T.
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(2019). Rpv3–1 mediated resistance to grapevine downy mildew is associated with specific host transcriptional responses and the accumulation of stilbenes. BMC Plant Biology, 19(1), 343 (IF 3.5)
Eisenmann, B., Czemmel, S., Ziegler, T., Buchholz, G., Kortekamp, A., Trapp, O., Bogs, J.
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(2019). The in vivo impact of MsLAC1, a Miscanthus laccase isoform, on lignification and lignin composition contrasts with its in vitro substrate preference. BMC Plant Biology, 19(1), 552
He, F., Machemer-Noonan, K., Golfier, P., Unda, F., Dechert, J., Zhang, W., Wolf, S.
