Regulation Role Of PcPS And PcPKS1from Polygonum Cuspidatum In The Resveratrol Biosynthesis

Resveratrol is a phytoalexin accumulated in some plant species in response to fungalinfection or UV irradiation. Resveratrol have been tested to have significant pharmaceuticaleffects in prevention of cancers, heart diseases and neurodegenerative diseases. Resveratrolsynthase (RS), competing with chalcone synthase(CHS) for the same substrates, is the keyenzyme to synthesize resveratrol. Although RS and CHS genes have been well characterizedin many plant species, there are only a few descriptions about RS and CHS genes fromPolygonum cuspidatum Sieb.Recently, a resveratrol synthase gene (PcRS) and a chalcone synthase gene (PcPKS1)were isolated in P. cuspidatum, but there are only a few descriptions about the metabolicfunction of PcRS and PcPKS1in planta. In this study, to understand the regulation of PcRSand PcPKS1in the resveratrol biosynthesis, the PcRS gene was over-expressed in Arabidopsisand P. cuspidatum. On the other hand, we use RNAi technology to inhibite the geneexpression of PcPKS1in P. cuspidatum. The main results were as follows:(1)Northern blot analysis revealed that the accumulation of PcRS transcripts was mostabundant in rhizomes. This distribution corresponds well with the accumulation of theresveratrol, suggesting that PcRS might play an important role in resveratrol biosynthesis.(2)To understand the metabolic function of PcRS in planta, it was placed under thecontrol of the cauliflower mosaic virus (CaMV)35S promoter and overexpressed intransgenic Arabidopsis thaliana via Agrohacterium-mediated transfer methods. Transgenicplants accumulated a new compound, which was identified as piceid by high-pressure liquidchromatography (HPLC) and electrospray mass spectrometry (HPLC–ESI–MS). Nosignificant difference in PcRS transcript levels was observed in leaves, stems and roots, whilepiceid concentration was highest in the leaves of4-week-old Arabidopsis plants, up to183.73μg/g FW. We showed that overexpression of PcRS in transgenic Arabidopsis resultedin accumulation of piceid. But the successful accumulation of resveratrol is likely to bedependent upon a combination of sufficient PcRS levels and the availability of commonsubstrates. (3)Piceid was purified by preparative HPLC from transgenic lines and tested inagar-plate bioassays in order to assess its effectiveness against fungal. At the concentration of50μg/mL, hyphal growth was approximately reduced (50%). Results showed that themycelial growth of Colletotrichum higginsianum was greatly inhibited. The detached leavesof plants were used for spot inoculations.The lesion diameter of the transgenic Arabidopsisplants was56-65%smaller than that of the wild-type plants. Spore production of thetransgenic plants was75-80%less than the wild-type plants. Overexpression of PcRS intransgenic Arabidopsis caused restriction of C. higginsianum colonization by inhibition ofspore production, resulting in enhanced resistance against C. higginsianum.(4)We investigated whether the accumulation of piceid in transgenic Arabidopsis plantswould affect other secondary metabolite pools. The mature seed coats of wild-type plantswere brown, whereas the seeds of transgenic Arabidopsis appeared pale fawn. The transgeniclines had a remarkable decrease of46-50%in anthocyanin accumulation. These resultsindicate that both tannin and anthocyanin accumulation are decreased due to ectopicexpression of PcRS. Northern analysis showed that expression of endogenous Arabidopsischalcone synthase gene (AtCHS) in the transgenic lines was not decreased. These resultssuggest that the significant reduction in flavonoid levels in transgenic plants may be due tocompetition for the substrates, instead of suppression of endogenous AtCHS expression.(5)To understand the metabolic function of PcRS in P. cuspidatum, PcRS wasoverexpressed in transgenic P. cuspidatum. Prior to the transformation experiments, thefactors influencing A.tumefaciens-mediated transformation of P. cuspidatum were explored tooptimize the transformation system, which included sensitivity of shoot tips to hygromycin,preculture period, Agrobacterium concentration, infection time, co-cultivation period andpre-culturing time. Finally, a system of high efficiency of genetic transformation andregeneration of P. cuspidatum was established. The plant expression vectorspCAMBIA1380-35S-PcRS was introduced into P. cuspidatum. No significant phenotypicdifference was observed in transgenic plants and the second asexual multiplicationgeneration. Nouthern blot exhibited a increase expression in all positive lines and expressionof PcRS were1.4to1.9times as control. Transgenic P. cuspidatum produced1.2to1.9-foldhigher piceid than control plant. These results suggest that overexpression of PcRS activated resveratrol biosynthesis pathway in P. cuspidatum.(6)To understand the metabolic function of PcPKS1, the plant expression vectorpRNAi-PcPKS1were introduced into Polygonum cuspidatum. The transgenic lines hadremarkable decrease of39-87%in PcPKS1transcript levels,14-42%in flavonoidaccumulation,22-62%in quercetin accumulation. But transgenic P. cuspidatum produced2.5to4.1-fold higher piceid than control plant. The transgenic line B5had a remarkable decreaseof52%in anthocyanin accumulation, with altered stem color. These results suggest that theflavonoid content can be reduced by restrained PcPKS1gene expression using RNAi method,at the same time, resveratrol biosynthesis pathway was activated.In this study, the effectiveness of PcRS in improving stilbene accumulation in foreignspecies has been proved. Our results suggest that PcRS overexpression and PcPKS1geneexpression using RNAi activated resveratrol biosynthesis pathway.