Study On The Mechanism Of Anthocyanin Biosynthesis During Autumn Leaf Coloration In Pistacia Chinensis

Pistacia chinensis Bunge is the only native species of Pistacia in China.The leaves of P.chinensis change from green to yellow,orange or red in autumn,making it become important resource for excellent native colored-leaf species cultivation.The leaf coloring period of P.chinensis is unstable under natural conditions.Anthocyanins are the main compounds in plant coloration,however,the mechanism of anthocyanin biosynthesis during leaf senescence of perennial woody plants is still unclear,and the molecular regulatory network of anthocyanin biosynthesis in autumn has not been established.Therefore,the study on the leaf coloring mechanism of P.chinensis will lay a theoretical foundation for the cultivation of colored-leaf P.chinensis.In this study,the leaves of P.chinensis which turned red in autumn were taken as the research object.The anthocyanins,flavones and proanthocyanidins in autumn leaves were analyzed qualitatively and quantitatively.Based on the analysis of transcriptome and metabolome,the regulatory network of anthocyanin biosynthesis in P.chinensis leaves in autumn under natural conditions was established.Meanwhile,the key genes regulating anthocyanin biosynthesis were screened and their functions were verified.The main results are as follows:? The main type of anthocyanin in P.chinensis is cyanidin,accounting for 98.32% of the total content of anthocyanins;the main type of anthocyanin C ring 3-position binding glycosides was galactoside,accounting for 97.17% of the total content of anthocyanins;Cyanidin 3-O-galactoside is the main anthocyanin in P.chinensis leaves,and its content is 121.10ng/g,accounting for 95.88% of the total content of anthocyanins.In addition,the anthocyanins in the leaves of P.chinensis are not prone to be methylated and acylated.? Light and low temperature can affect the accumulation of anthocyanin in leaves of P.chinensis.The absence of light signal or low temperature signal would make the leaves unable to color.A total of6379 DEGs and 33 differential metabolites were obtained during leaf coloration.The transcriptomic data suggested that 31 structural genes in the anthocyanin biosynthesis pathway were upregulated during leaf coloration.Several genes involved in light and low temperature responses were significantly induced or reduced during autumn leaf coloration.HY5 is considered as an integrator of light and low temperature signals,which was significantly upregulated both in low temperature and light conditions.Interestingly,jasmonate content was significantly enriched during leaf coloration,thereby indicating a consistent change with anthocyanin content.Furthermore,under shading treatment,the expression of the structural genes was maintained at a low level,less consistent with anthocyanin accumulation.Notably,the expression of MYB113 was consistent with anthocyanin content under both natural and shading conditions.Combining our data with previous reports,we proposed an anthocyanin biosynthesis model of P.chinensis during autumn leaf coloration.? A total of 158 members of R2R3-MYB gene family were identified in Pistacia genome.R2R3-MYB gene family members EVM0017061,EVM0007925 and EVM0016534 were induced by the combined signal of light and low temperature.The expression levels of EVM0017061 and EVM0016534 were positively correlated with anthocyanin content,suggesting that they were involved in the positive regulation of anthocyanin biosynthesis.The homologous gene PcMYB113 of EVM0016534 was isolated from P.chinensis,and the nucleotide sequence identity of EVM0016534 and PcMYB113 was 97.98%.Subcellular localization showed that PcMYB113 protein was localized in the nucleus.Overexpression of PcMYB113 in Arabidopsis thaliana promoted anthocyanin accumulation in seed coat,cotyledon and leaf.Under normal conditions,the anthocyanin content in leaves of transgenic plants was more than 1000 times that of wild-type plants.PcTT8 and PcTTG1 were isolated from P.chinensis.Protein interaction experiments showed that PcMYB113,PcTT8 and PcTTG1 interacted with each other.? PcHY5 was isolated from P.chinensis.Subcellular localization showed that PcHY5 protein was localized in the nucleus.Overexpression of PcHY5 in Arabidopsis enhanced low temperature-induced anthocyanin accumulation.However,there was no significant difference in anthocyanin content between transgenic and wild-type Arabidopsis under shading or non-low temperature temperature.Also,we isolated the potential interacting factors PcBBX1 and PcBBX2 of PcHY5 protein from P.chinensis and verified their interaction in yeast.In conclusion,the results showed that cyanidin 3-o-galactoside was the main type of anthocyanin in P.chinensis leaves.Light and low temperature are the necessary conditions for leaf coloring of P.chinensis in autumn.The combined signal induced the expression of PcMYB113 during leaf coloration.The MBW complex formed by PcMYB113 and potential interacting proteins PcTT8 and PcTTG1 activated the anthocyanin biosynthesis,resulting in the accumulation of anthocyanin.Light and low temperature can also activate the transcription of PcHY5,which may be involved in the regulation of anthocyanin biosynthesis by binding with interacting factors PcBBX1 or PcBBX2.