Regulation Of Light And Exogenous Ethylene On Phenolics Synthesis In Grape Berry Skins

Ethylene as a gaseous plant hormones,plays an important role in improving the metabolism of phenolic compounds anthocyanins in grape berry skin.However,there are complex signal networks between light and ethylene,and the light signal could regulate the ethylene signal pathway in Arabidopsis thaliana.Therefore,it has a great theoretical significance to study the mediation effect of light on the effect of ethylene to promote phenolic accumulation.In this study,four treatments(light exposure with(+L+E)and without(+L-E)ethephon,and box-shading with(-L+E)andwithout(-L-E)ethephon)were carried out on Vitis vinifera cv.Cabernet Sauvignonberriesat the onset of veraisonin(coloring of 5～10 % of the berries)to study the effects of light and ethylene treatments and their interactions on the synthesis of phenolic compounds in grape,and also to study the molecular mechanism of the effect of light on the promotion of anthocyanin synthesis by ethylene.It provides a theoretical basis for the next step to verify the key pathway and site of interaction between light and ethylene signaling pathways,and provide reference for the microbiological function of ethylene on non-climacteric fruits.The main results are as follows:(1)light treatment increased fruit weight,berry size,maturity and skin weight,ethylene had no effect on fruit weight and fruit,but increased total soluble solid content,reducing sugar content,and decreased titratable acidity.Both light and ethylene treatments promoted the decrease of L *,C *,and h * values in fruit ripening process,and accelerated the process of changing from green to red and then to purple.Both light and ethylene treatments under light could significantly increase the skin weight and the contents of total phenolics,total flavonoids,total anthocyanins and tannins,but the effect of ethylene treatments under dark was not significant.There were interaction effects between light and ethylene treatments on maturity,total phenolics,total flavonoids,total anthocyanins and tannin indicators.(2)Light mediated the regulation of ethylene on anthocyanin synthesis.Twenty kinds of anthocyanin individuals were detected in the grape.Light significantly promoted the accumulation of anthocyanins and increase the ratio of Peonidin-glucosides and Petunidin-glucosides,and then increase the proportion of 3‘5‘/3‘-substituted,and reduced the ratio of methoxylated/non-methoxylated,acylated /non-acylated anthocyanins.Ethylene treatment also significantly increased the content of all anthocyanins individuals,and.The increased ratios were higher in ethylene treatment under light than ethylene treatment under dark.While,the effects of ethylene treatment on the composition of anthocyanin individuals were different under light and dark conditions.The treatment of ethylene under the light improved the contents of most of the anthocyanin individuals other than Malvidin-3-O-(6-O-acetyl)-glucoside and Malvidin-3-O-glucoside,while ethylene treatment under dark had a greater effect on the above-methioned two kinds of anthocyanins.The ethylene treatment under light reduced the ratio of methoxylated/non-methoxylated,3‘5‘/3‘-substituted and acylated/non-acylated,while the ethylene treatment under dark increase these modified anthocyanin ratios.(3)The light also mediated the regulation of ethylene on synthesis of non-anthocyanin phenolics.Sixteen kinds of non-anthocyanin individuals were detected in grape skin.Light significantly increased the contents of all non-anthocyanin individuals and the proportion of flavonols,also reduced the proportion of flavanols.Ethylene treatment also significantly improved the accumulations of most non-anthocyanin individuals,and the increased ratio were higher in ethylene treatment under light than ethylene treatment under dark.While,the effects of ethylene treatment on the composition of non-anthocyanin individuals were different under light and dark.Ethylene treatment under light reduced the contents of Quercetin-3-O-glucuronide,Syringetin-3-O-glucoside and Procyanin B1,as well as the proportion of flavanols,while ethylene treatment under dark increased the contents of Quercetin-3-O-glucuronide,Syringetin-3-O-glucoside and Procyanin B1,and increased the proportion of flavanols.(4)Light affected the regulation of ethylene on phenolic synthesis-related genes and signal transduction related genes.Light had no effect on ethylene production,endogenous ABA content and endogenous IAA content,but down-regulated ethylene receptors Vv ETR1,Vv ETR2 and transcription factors Vv EIN2,Vv EIL1,Vv EIN3 and Vv ERF1 genes.Ethylene treatment increased ethylene production,endogenous ABA content and reduced endogenous IAA content.The expression patterns of ethylene biosythesic and signal transduction related genes(Vv ACO1,Vv ACO2,Vv ETR1,Vv ERS1,Vv EIN4 and Vv EIN2)regulated by ethylene treatment under light were opposite to that of ethylene treatment under dark.Ethylene treatment down-regulated Vv EIL1 and Vv EIN3 genes under both light and dark conditions,but up-regulated downstream-transcription factor Vv ERF1 gene.The expression profiles of Vv ACO2 and Vv ERF1 genes were consistent to those of phenolic synthesis-related genes,but the expression patterns of Vv EIN3 and Vv EIL1 were not consistent with those of phenolic synthesis-related genes.(5)Tissue culture experiments showed that light inhibited the expressions of Vv ACO2,Vv EIL1 and Vv ERF1 genes,but increased the expressios of Vv UFGT and Vv MYBA1 genes.Ethylene promoted the expressions of Vv ACO2,Vv ERF1,Vv UFGT and Vv MYBA1 genes,but inhibited the expressions of Vv EIL1 gene.Light affected the regulations of ethylene on the expressions of Vv ACO2,Vv ERF1,Vv UFFT,Vv UFGT and Vv MYBA1 genes,and the expressions of Vv ACO2,Vv ERF1 and Vv MYBA1 genes were higher in ethylene treatment under light than those in ethylene treatment under dark.The alternation of light and dark experiments further showed that Vv ACO2,Vv ERF1,Vv UFGT and Vv MYBA1 genes were down-regulated by ethylene traetment under dark,but after turned into light conditions,Vv ACO2,Vv ERF1,Vv UFGT and Vv MYBA1 genes were showed up-regulated by ethylene.The ethylene-regulated expression of Vv EIL1 was not affected by light at the transcriptional level.(6)Two EIN3 gene family members Vv EIL1 and Vv EIN3 were cloned from grape skin,which were 1851 bp and 1833 bp,encoding 616 and 610 amino acids respectively.Phylogenetic tree analysis showed that the amino acids encoded by Vv EIL1 and Vv EIN3 were similar to EIN3 protein in other plants like peony and apple.The results of q PCR showed that Vv EIL1 and Vv EIN3 had higher expression levels in berries,leaves,stemand buds,and the expressions of Vv EIL1 and Vv EIN3 were down-regulated by light and ethylene.p ET32a(+)-Vv EIL1 and p ET32a(+)-Vv EIN3 recombinant plasmid were constructed and transferred into E.coli Rossete(DE3),which successfully expressed the target protein.Construction of fusion expression vectors p BI221-Vv EIL1-GFP and p BI221-Vv EIN3-GFP,and further transformation of Arabidopsis thaliana protoplasts by PEG-mediated further confirmed that two transcription factors Vv EIL1 and Vv EIN3 were localized in the nucleus.Vv EIL1/Col-0,Vv EIN3/Col-0,Vv EIL1/ein3eil1,Vv EIN3/ein3eil1 transgenic lines were obtained by constructing plant expression vector p CG1301-Vv EIL1,p CG1301-Vv EIN3 and transfering into wild-type Arabidopsis thaliana(Col-0)and double mutant ein3eil1.The transgenic plants significantly enhanced or restored the elongated hypocotyls of Col-0 and ein3eil1 mutants by ethylene treatement under light.The regulation of ethylene under light on elongation of hypocotyls was dependent on Vv EIL1 and Vv EIN3.Transcription factors Vv EIL1 and Vv EIN3 were the key factor of light-mediated the regulation of ethylene on transgenic lines phenotypic differences.