| Leucoanthocyanidin reductase(VvLAR)converts leucoanthocyanidins to 2R,3S-flavan-3-ols,especially(+)-catechins,which is one of the main units of proanthocyanidins.Proanthocyanidins in Vitis vinifera grape berries not only determine the intensity and the quality of bitterness and astringence in wines,but also affect the color and the shelf-life of wines.However,studies to date on the regulation of proanthocyanidin biosynthesis in grape berries remain at transcriptional level.Little is known about its regulation at protein level.The interacting relationship between VvLARl and a small heat shock protein(VvsHSP1)was observed by Yeast-Two-Hybrid(Y2H)technique in this study.Their interaction region was firstly defined at position 182-346 of VvLAR1 and position 251-367 of VvsHSP1 by Y2H.The analysis of subcellular localization of Arabidopsis thaliana protoplasts revealed that VvLAR1 and VvsHSP1 were co-located in the cytoplasm.Besides,the existence of the interaction between these two proteins were further proven by four experiments,including co-immunoprecipitation(CoIP)of yeast proteins,luciferase complementation imaging(LCI)experiment by tobacco leaf transient expression system,in vitro CoIP experiment of recombinant proteins and in vivo CoIP experiment of the proteins from F1 hybrid of two homozygous single gene transgenic plants.To further explore the biological function of the interaction between VvLAR1 and VvsHSP1 in developing grape berries,a method of high perfonnance liquid chromatography-mass spectrometry(HPLC-MS)was established to detect the activity of VvLAR1,followed by the study of the effect of VvsHSP1 recombinant protein addition on its activity.The result showed that the addition of VvsHSP1 in the reaction system could promote the activity of VvLARl and this activation would be strengthened by high temperature(50℃ or 40℃)or low temperature(10℃).Arabidopsis thaliana F1 hybrids of the two transgenic plants with homozygous single gene were subjected to high temperature(40℃)and low temperature(10°C),respectively.Afterwards,the transcript abundance of gene expression and the protein amount as well as the catechin level were assessed by Real-time PCR,Western bolt and HPLC-MS.The results clearly confirmed that VvsHSP1 could effectively protect VvLAR1 from damage of temperature stress,and stabilize the function of VvLAR1 under environmental stress.Additionally,the effects of different temperatures,ultraviolet(UV)treatments and environmental factors on the expression of VvLARl and VvsHSP1 were studied by using Vitis vinifera L.cv.Cabernet Sauvignon berries of different developmental stages as materials.The expression of VvLAR1 and VvsHSP1 in the pre-veraison berries could be reduced and enhanced synchronously by 40℃ and 10℃ treatments,respectively,but in terms of the fold difference of the expression level of these two gene,the Pearson Correlation coefficients were very low between the treated and the control groups in grape berries at veraison and post-veraison.Reductions of 89%and 99%UV radiation could respectively reduce and enhance the expression of VvLAR1 and VvsHSP1 in grape berries with low Pearson Correlation coefficients.It was observed that 1.8 kJ/m2 UV-C radiation enhanced the expression of VvsHSP1 but reduced that of VvLAR1 in grape berries at pre-veraison,while 0.9 kJ/m2 UV-C radiation could enhance VvLAR1 and VvsHSP1 expression in grape berries at veraison synchronously.The expression of VvsHSP1 and VvLAR1 in grape berries at v6raison was higher in samples of Gaotai region than those of Changli region.These findings elucidate for the first time the regulatory mechanism of proanthocyanidin biosynthesis in grape berries at protein-level,therefore providing certain guiding significance for the production of high-quality wine grape berries. |
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