Study On The Regulation Of Stilbene Synthase From Chinese Wild Grapevine

As one of the most ancient crops,grapevine?Vitis vinifera L.?is of economic and cultural significance.However,this crop is challenged by several diseases.China is a major biodiversity centre for Vitis.Chinese wild grapevines are valuable gene pools with a number of resistance factors that are generally understood to be fundamental to immunity.In our study,Chinese wild V.pseudoreticulata accession'Baihe-35-1'and V.quinquangularis accession'Danfeng-2'were used for various biotic and abiotic treatments.We dissect the upstream signals conferring the response to SA as well as the regulation of MAPK cascades in stilbene synthase transcription and stilbene accumulation.The main results described as followings.1.The two full-length genomic stilbene synthase genes were cloned from Chinese wild V.pseudoreticulata accession'Baihe-35-1'?VpSTS?and V.vinifera cvs.Carigane?VvSTS?.Comparative analysis revealed that the nucleotide homology was 98.63%among two stilbene synthases,both of them contained two exons and one intron.In contrast,the promoter sequences,despite some degree of congruence,displayed specific differences.In particular,the promoter of the wild allele?VpSTS?harboured two stretches?of 13 bp and 244 bp,respectively?that were absent in the VvSTS allele,whereas the VpSTS allele lacked a stretch of 18 bp found in VvSTS.2.The two promoters sequences of stilbene synthases were fused to GUS reporter,then transformed in a stable manner into Arabidopsis as the heterologous system.We found that VpSTS could be actived in all tissues,whereas the activity of the VvSTS promoter could only be measured in leaves and even here its activity was consider ably lower than that of the VpSTS promoter.Both the VpSTS and VvSTS promoters can be induced by powdery mildew but the expression profiles were significantly different.Furthermore,the two stilbene synthase promoters were respectively fused with its stilbene synthases and also transformed into Arabidopsis as the heterologous system.We found that the phenotype of the VpSTS::STS transgenic Arabidopsis lines showed stronger resistance to powdery mildew and contained more stilbenes.3.To dissect the upstream signalling driving the activation of this promoter,we used a dual-luciferase reporter system in a grapevine cell culture.We show elevated responsiveness of the promoter from the wild grape to the pathogen-associated molecular pattern?PAMP?flg22,and a lack of response to the UV-C and cell death-inducing elicitor Harpin.We show elevated responsiveness of the VpSTS promoter from the wild grape to salicylic acid?SA?and equal induction of both alleles by jasmonic acid?JA?.The elevated SA response of the VpSTS promoter depends on calcium influx,oxidative burst by RboH,mitogen-activated protein kinase?MAPK?signalling,and JA synthesis.4.VqMAPKKK38 was obtained from the leaves of V.quinquangularis accession'Danfeng-2'.The PCR amplified fragment was 1419 bp in length,encoding a 472-amino acid protein which contained a kinase catalytic domain from residue Gln²⁰³ to Leu⁴⁵⁰ and a Ser/Thr kinase active site?VIHCDLKPKNILL?.Expression analysis indicated that VqMAPKKK38 transcripts were elevated by powdery mildew infection,salinity and chilling stresses,as well as various hormonal signals?SA,MeJA,Eth and ABA?.5.As concluded from transient overexpression and RNAi experiments in grapevine leaves,VqMAPKKK38 positively regulated stilbene biosynthesis by modulating the transcripts of STS.Moreover,the calcium and ROS signalling events function upstream of VqMAPKKK38,both of them contributed to STS induction and stilbene accumulation.In summary,the expression of stilbene synthase was regulated by SA in Chinese wild V.pseudoreticulata accession'Baihe-35-1',while the activation of VqMAPKKK38 might be positively regulate stilbene biosynthesis in V.quinquangularis accession'Danfeng-2'.