Carotenoids Accumulation And Expression Pattern Of Carotenogenic Genes In Tea [Camellia Sinensis (L.) O.Kuntze.]

Tea [Camellia sinensis (L.) O. Kuntze] is a perennial crop and widely cultivated for the production of a popular non-alcoholic beverage. Known as the traditional 'Asian beverage', tea plays a significant role in the economy of the producer countries. With the increased participation of producer countries in the global tea market, research efforts in recent years, diverted from quantity to quality. Quality of tea is a complex character and the volatile flavor compounds in the finished product are strong determinant of the quality. Many of the flavor compounds reported from tea were of carotenoids origin, however, literature on this aspect in tea is sporadic. Understanding of the carotenoids content in different tea cultivars used for production of black, green and oolong tea, their fluctuation in different harvesting seasons and relation to different kinds of tea are required for understanding the relation of carotenoids content to tea quality.Carotenoids, along with their extraordinary variety of functions in plants, are important component of animal diets, including human. In the past few years, genes and cDNAs encoding nearly all the enzymes required for carotenoids biosynthesis in green plants have been identified and sequenced and their products characterized. Identification and sequencing of the carotenogenic genes are required to gather information how the expression of carotenogenic genes regulates the accumulation of the carotenoid and to manipulate the caretenoids pathway for enhancing the carotenoids accumulation.In the present investigation we studied the carotenoids accumulation pattern in twelve tea cultivars representing elite tea cultivars of black, green and oolong tea in different harvesting seasons. Phytoene synthase (PSY) and phytoene desaturase (PDS) are the two regulatory enzymes in the carotenoids pathway catalyzing the first two steps of the pathway. Violaxanthin deepoxidase (VDE) is another key enzyme in the xanthophylls cycle within the carotenoids pathways. We identified these three genes in tea (Accession no: PSY, EF545005; PDS, EU275984 and VDE, EU091350) and studied the expression pattern of these genes in different harvesting season. The expressions of these genes were also studied in the white and green tissues of a thermo-sensitive chlorophyll deficient albino mutant of tea.A significant variation was found for the carotenoids contents in all the studied cultivars and in the plucking seasons. The variation was more pronounced between seasons than between cultivars. Longjing-43, a popular green tea cultivar in China, accumulated the highest carotenoids. In every season, high accumulation of the carotenoids was detected in the later period of the season than the early period. Since flavoury oolong tea is produced from shoot plucked in the late of the season, it implies the role of carotenoids in the flavour formation in tea.To establish a relation of the flavour compounds with that of the carotenoids content, we estimated the volatile flavour compounds (VFC) in the steam fixed material of the twelve cultivars plucked in the early spring season. However, our investigation could not establish a stable relation of VFC to the carotenoids content. More systematic study, particularly accurate identification of VFC of carotenoids origin and their modification, determination of the quantity and activity of carotenoids cleavage enzymes will provide more insights into the role of carotenoids on flavour formation.Polyphenol content was reported to hinder the formation of VFC. We determined the polyphenol content in the material used to estimate the VFC content. However, our investigation could not establish such a relation.The expression of the PSY, PDS and VDE were studied in two cultivars: Longjing-43, a green tea cultivar with small leaf and Yulan, a black tea cultivar with large leaf. The expression of these genes in the early and late period of the three plucking seasons were measured by a semi-quantitative RT-PCR method and the carotenoids content were estimated in the respective seasons. The study revealed a strong relation of PSY and PDS expression to that of carotenoids accumulation. However, the transcript abundance of PDS is much more low than that of PSY and this gene even failed to amplify in some seasons. VDE catalyzes the conversion of Violaxanthin to zeaxanthin, a compound having photo-protective function and high expression of this gene was reported in stress conditions. As expected, in our investigation, we observed a higher expression of this gene in sunny summer periods. However, expression of this gene did not show a high relation to the Violaxanthin content.The expression analysis of the PSY, PDS and VDE in the white tissue of the albino mutant revealed a high expression in comparison with that in its green counterpart. The white tissue was found to accumulate low carotenoids and have damaged chloroplast. The carotenogenic genes are nuclear encoded but participate in the biosynthetic pathway in the plastid. According to the plastid signal hypotheses, the expression of the nuclear encoded plastid enzymes is regulated by the signal from the plastid. Since high expression of these genes were observed in the tissue with damaged plastids, expression of these genes may be regulated by negative plastidic signal.The expression of the PSY gene was found to positively correlated with the carotenoids accumulation in tea and the full length PSY gene was obtained by performing 3'- and 5'- RACE. The evolutionary relationship of the deduced amino acid sequence of the obtained PSY from tea with that reported from other crop species was inferred by constructing a phylogenetic tree. Result showed that PSY in tea was closely related with that of Adonis palaestina (90%) and of Citrus sinensis (90%).It can be concluded from this investigation that carotenoids content, as reported in other plant system, is also an important chemical component in tea and plays a significant role in determining tea quality. Since the PSY gene can be easily amplified and its expression showed a positive correlation with the carotenoids accumulation, this gene can be a suitable candidate for designing a molecular marker for carotenoids content.