Mechanism Of Peel Color Formation In Persimmon(Diospyros Kaki Cv.'heishi')

The skin of mature 'Heishi' is black.The mechanism of the formation of black pigment is still unknown.During development of 'Heishi' fruit,peel that exposed direct to sunlight will eventually develop into orange-red.In this study,we firstly pay attention to pigments that commonly found in persimmon peel,the isolated and identified the black pigment in 'Heishi',and studied the role of PPO in the formation of the black pigment.Finially,metabolism and transcriptome were carried out to study the factors that affect color formation in 'Heishi' peel.The main findings are as follows:1.Pigments in persimmon peel.phenolic compounds and carotenoids are the common pigments in persimmon peel.It was found that phenolic compounds and carotenoids were significantly accumulated in the peel.At mature,?-carotene was the major carotenoids in the 'Heishi' peel,lycopene was the major one in 'Huoguan',?-carotene and lycopene were the major carotenoid compounds in 'Jinping'.The proanthocyanidin B1,rutin,hypericin and epicatechin were the mainly phenolic compounds in these three cultivars.There was no anthocyanin compounds were found in the peel.The black pigment in 'Heishi' peel is still unknown.2.The black pigment in 'Heishi' peel is a kind of melanin.Microstructure analysis showed that the pigment is mainly located in the upper epidermis and subcutaneous tissues.Ultraviolet spectrophotometer(UV-vis)and Fourier infrared spectroscopy(FT-IR)analysis showed that the black pigment in the peel of 'Heishi' was a kind of melanin.The melanin has a typical structure of indole,aliphatic C-H group and amide ? with aromatic ring vibration of C=C or COO-symmetric stretching structure characteristics of the group.The DPPH and ABTS free radical scavenging ability of persimmon increased with time course,and the free radical scavenging ability was relatively stable.The in vitro bacteriostasis analysis showed that persimmon melanin can inhibit the growth of Botrytis cinerea.3.Dk PPO plays a critical role in the formatioin of melanin in 'Heishi' peel.It was found that polyphenol oxidase(PPO)enzyme activity and the Dk PPO expression increased significantly during the color transformation of 'Heishi' peel.It was found that transient overexpression can significantly increase the expression of Dk PPO in the 'Heishi' peel tissue near the injection,and significantly increaseed PPO activity was also found.UV-Vis and FT-IR analysis showed that the black pigment formed in the peel is melanin,which is the same as the naturally formed melanin in 'Heishi' peel.Conversely,silencing the Dk PPO by gene-induced silencing in 'Heishi' fruit can significantly inhibit the formation of melanin in the peel.4.Strong sunlight caused stress inhibits melanin formation in 'Heishi' peel.During fruit development,strong sunlight caused severe oxidative stress,resulting in destruction of chloroplasts and yellow-brown peel.Strong sunlight caused stress changed the primary and secondary metabolism in 'Heishi' peel.The expression of the Dk PPO during the color transformation of the 'Heishi' peel was significantly inhibited by strong sunlight,and PPO activity was also significantly decreaed.The reduced PPO activity inhibits the oxidative polymerization of phenolic compounds,and the production of melanin is strongly inhibited.At the same time,a large amount of lycopene and ?-carotene were synthesized in the stressed peel,so that peel color of stressed 'Heishi' fruit ultimately turned to orange-red.5.Under Srong sunlight stress,differentially expressed genes related to heat stress,light response and free radical scavenging in the skin of 'Heishi' were found by RNA-Seq.Genes c84983.graph_c0(hsf30),c89016.graph_c0(hsf A6),c90096.graph_c0(hsf B2)and c125979.graph_c0(hsp70)were significantly up-regulated in the stressed peel.which indicates that these genes may play important roles in participating in coping with strong sunlight stress.In addition,transcription factors c106044.graph_c0(WRKY70),c109109.graph_c0(Dk WRKY22)and c109740.graph_c0(Dk MYB8)may also play important roles in regulating 'Heishi' against strong sunlight caused stress.In summary,the black pigment in 'Heishi' peel is a kind of melanin.It has good antioxidant effect and certain antibacterial activity.Polyphenol oxidase plays an important role in its synthesis.The changing color pattern of the mature 'Heishi' peel is caused by storng sunlight stress during fruit development.As the reduced PPO activity cannot effectively catalyze the polymerization of oxidized phenolic compounds to produce melanin.At the same time,a large amount of lycopene and ?-carotene are synthesized in the stressed peel.Therefore the peel color of 'Heishi' ultimately turned to orange-red.Transcriptome identified genes c84983.graph_c0(hsf30),c89016.graph_c0(hsf A6),c90096.graph_c0(hsf B2),c125979.graph_c0(hsp70),c106044.graph_c0(WRKY70),c109109.graph_c0(Dk WRK22).graph_c0(Dk MYB8)may play an important role in 'Heishi' in resisting strong sunlight stress.