Screening of anthocyanin-related key light-response transcription factor AaMYB1 in Actinidia arguta

Abstract：【Objective】Fourteen transcription factor genes related to flesh coloration were screened from previous transcriptome data of‘Tianyuanhong’flesh during different fruit developmental stages. Expression level of these genes were analyzed to identify the key transcription factor in response to light, which provided molecular basis for understanding the influencing mechanism of light on anthocyanin biosynthesis and accumulation.【Methods】All-red-fleshed kiwifruit‘Tianyuanhong’(Actinidia arguta) was selected as experimental materials and the fruits at 30th day after full bloom were bagged. A total of eight stages including 30th, 50th, 70th, 80th, 90th, 100th, 110th, and 120th day after full bloom were set as sampling times. The same stage of non-bagging fruits were also sampled as the control. The CR- 400 colorimeter was used for identification of phenotype. Five anthocyanin components, including cyanidin, delphinidin, cyanidin-3-O-galactoside, delphinidin-3-O-galactoside and cyanidin-3-O-xylo-galactoside, were analyzed qualitatively and quantitatively by ultra- performance liquid chromatography cou-pled with tandem mass spectrometry (UPLC-MS/MS). Extraction and identification of total anthocyanin were carried out with the Micro Plant Anthocyanin Assay Kit according to manufacturer’s instructions. Based on previous RNA- seq data, a total of 14 transcription factor genes, including MYB1, MYB5, MYB110, MYB123, MYB14, MYC1, MYC2, MYC3, bHLH, HD-ZIP1, HD-ZIP2, HD-ZIP3, HDZIP4 and HD-ZIP5, were served as candidate genes used for RT-qPCR (real-time fluorescent quantitative polymerase chain reaction) analysis. In addition, the expression profiles of fourteen transcription factors were conducted by Roche LightCycler 480 system. All analysis was combined to screen and identify key candidate transcription factor genes that responded to light.【Results】Phenotypic identification of non- bagging and bagging fruits at eight stages revealed that the flesh color changed from green to red during fruit development, and the flesh was the reddest at 120th day after full bloom, when the color of non-bagging flesh was significantly redder than that of bagging flesh, which indicated bagging treatment could suppress flesh coloring, and 120 day after full bloom was the stage with significantly different flesh color between non-bagging and bagging treatments. The results of measurement of color indexes, including color ratio and hue angle, showed the stage with significant difference between non-bagging and bagging treatments occurred at 120th day after full bloom, which was consistent with the result of phenotipic identification. The contents of five anthocyanin components showed that cyanidin and delphinidin contents were higher in early stage and lower in later stage during fruit development. Delphinidin-3-O-galactoside content presented no obvious change during eight stages. Cyanidin-3-Ogalactoside and cyanidin-3-O-xylo-galactoside contents showed lower and higher level in early and later stages, respectively, reaching the highest level at 120th day after full bloom. The changing trend of total anthocyanin content was similar with cyanidin-3-O-galactoside and cyanidin-3-O-xylo-galactoside during fruit development, suggesting the appearance of A. arguta red flesh was due to the synthesis and accumulation of anthocyanin. The correlation analysis between total anthocyanin and five specific anthocyanin components presented that cyanidin-3-O-galactoside and cyanidin-3-O-xylo-galactoside were significantly correlated to total anthocyanin, which indicated the main specific components that contributed to the redness of flesh were cyanidin-3-O- galactoside and cyanidin-3-O- xylo- galactoside. In addition, cyanidin-3-O- galactoside content in non-bagging flesh was significantly higher than that in bagging flesh at 120th day after full bloom, which indicated bagging could inhibit anthocyanin biosynthesis and accumulation mainly by suppressing synthesis of cyanidin-3-O-galactoside. Expression profiles of 14 transcription factor genes were conducted by RT-qPCR and the results revealed that different transcription factor presented different expression patterns and the expression level of MYB1 in non-bagging flesh was significantly higher than that in bagging flesh at 120 th day after full bloom when the non-bagging flesh color was obviously red. The correlation analysis between expression level of MYB1 and contents of cyanidin-3-O-galactoside and cyanidin-3- O-xylo-galactoside showed MYB1 and MYB110 expressions were extremely significantly correlated at 0.01 level with non-bagging treatment, while only MYB1 was not significantly correlated to cyanidin-3-O-galactoside and cyanidin-3-O-xylo-galactoside in bagging flesh, which not only indicated bagging treatment inhibited anthocyanin biosynthesis probably by suppressing MYB1 expression, but also suggested MYB1 might be a regulatory gene that could respond to light. Finally, based on all the results presented in this study, a possible regulating mode that the AaMYB1 transcription factor participated in was established to show the role of AaMYB1 that responded to light and regulated the anthcyanin biosynthesis in A. arguta flesh.【Conclusion】The candidate key light-response transcription factor AaMYB1 related to anthocyanin biosynthesis was screened out for further studies. Bagging treatment probably inhibited anthocyanin (main component is cyanidin-3-O-galactoside) biosynthesis and accumulation by suppressing the ex-pression of AaMYB1, thus hindering the normal flesh coloration in Actinidia arguta.