Lipid Droplets Study In Camellia Sinensis Leaves

Tea is a kind of beverage that people love deeply.Aroma is one of the important factors that determine the quality of tea.Fatty substances are one of the components of the aroma.Fat substances are stored in lipid droplets mainly in the form of neutral lipids in the body of cells.In recent years,LDs have also been found to participate in the life activities such as lipid metabolism,biological signal transduction,and stress responses.LDs in Camellia sinensis have not been studied,its composition and metabolism remains unclear.In this study,we stained Tieguanyin leaves,extracted LDs from its leaves,and analyzed its composition.We cloned CsPSAT1 gene which is the key enzyme for sterol ester(SEs)biosynthesis,and analyzed its expression pattern by QPCR.The major findings are as following:The staining of Tieguanyin leaves by Nile red confirmed the presence of LDs.We stained tea tree leaves with two different specific dyes for neutral lipid(Nile red and topfluor cholesterol),we found that the LDs from tea leaves can be divided into two types:one type LDs can be stained by both Nile red and topfluor cholesterol;another type only can be stained by Nile red.The number of LDs that can be stained by both dyes accounted for more than 80%of the total LDs.The analysis of sterol ester lipid droplets of green tea varieties in Fujian Province showed that there was no significant difference in the diameter and distribution of lipid droplets among different varieties.The difference of sterol ester lipid droplets was mainly caused by the number of lipid droplets.LDs were isolated from Tieguanyin leaves and analyzed by thin-layer chromatography,we found that sterol ester(SEs)is the major component of lipid droplets in Tieguanyin leaves.The size and number of LDs were quantified,we found that the average diameter of LDs increased with leaf maturation,and the proportion of small size LDs gradually decreased and the proportion of large size LDs gradually increased.The SEs was purified from Tieguanyin leaves,then analyzed by gas chromatography-mass spectrometer,we found that the sterol head group was constituted by stigmasterol,beta-sitosterol,cycloartenol and campesterol.The composition of sterol ester in leaves of different green tea varieties was the same,the highest total sterol ester content was Fuyun 595,and the lowest was Zhenghedabai.Different leaf position in Tieguanyin showed similar composition of SEs.Total SEs content was increased with leaf maturation.The SEs content in the fifth leaf were significant higher compared with that of the first leaf and the second leaf.Four different sterol esters varied with leaf maturation.The contents of stigmasterol in the first four leaves were similar,and became higher in the fifth leaf.The content of β-sitosterol showed an increasing trend with leaf maturation.The content of cycloartenol and campesterol in the first four leaves showed a trend of increase,then decreased in the fifth leaf.The diurnal changes of SEs from Tieguanyin leaves were analyzed,we found that the total SEs content decreased in the morning,followed by increase in the afternoon.During night,first decrease then increase.This suggested that the total SEs exhibited a cycle of consumptionaccumulation-consumption-accumulation.The four different SEs components exhibited similar trend.Quantitative expression analysis of CsPSAT1which is a key gene for SEs synthesis.The CsPSAT1 homolog was cloned from Tieguanyin,and its cDNA sequence was obtained.Gene-specific primers were designed for QPCR to monitor CsPSAT1 expression during leaf maturation.We found that CsPSAT1 showed upward trend with leaf maturation.There was significant differences in CsPSAT1 expression level between different leaves except the first leaf and second leaf(Tukey HSD).