Effect Of STAY-GREEN And Light-harvesting Complex Ⅱ Chlorophyll A/b Binding Protein On The Albinism Of ’Huabai 1’

In recent years,many albino tea cultivars with white or yellow leaf color have received increasing attention from tea researchers and manufacturers due to their unique phenotypes,great flavor and good economic value.Based on the environmental responsive factor,albino tea cultivars are divided into two main types namely light-sensitive and temperature-sensitive,of these two varieties,temperature-sensitive albino tea plants are the most common.The widely cultivated and most studied temperature sensitive albino tea cultivars is the ‘Baiye 1’.However,the small number of varieties and short production period significantly reduce the economic benefits of albino tea cultivation.Therefore,it is important to breed for new types of albino tea with more stable albino phenotype and longer albino period.The‘Huabai 1’ is a new natural albino mutant tea germplasm.It has white jade tender shoots under low temperature,and the albino period is longer,with higher degree of albino is higher than other albino tea cultivars.The tender shoots are white at the beginning of germination,and the mesophyll,veins,calyx and pericarp are all albino.Dried tea made from tender shoots of ‘Huabai 1’ showed excellent quality,which makes it a good new variety breeding value.Therefore,it is of great significance to study the albinism mechanism of ‘Huabai 1’.Despite research advances to uncover the molecular mechanisms of leaf albinism in albino tea cultivars,the phenomenon still remains unclear.Previous studies using transcriptome analysis reported that the expression levels of STAY-GREEN(CsSGR,CSA024979)and light-harvesting complex Ⅱ chlorophyll a/b binding protein(CsLHCⅡ,CSA035910)varied in the albino and regreened leaves of ‘Huabai 1’.Therefore,it is speculated that they may play important roles in albinism of tea plants.In the present study,transgenic function verification was perform using the albino-related genes CsSGR and CsLHCⅡ in order to explore their roles in the regulation of albino.Yeast two-hybrid library was constructed using the albino and regreened shoots of ‘Huabai 1’,and the corresponding interaction proteins were screened using CsSGR and CsLHCⅡ as ‘bait’ proteins to reveal the mechanism of albinism in ‘Huabai 1’ tender shoots at the protein level.Widely targeted metabolomics analysis was performed to uncover the metabolic differences of albino shoots between ‘Huabai 1’ and ‘Baiye 1’.The main research results are as follows:1.The chlorophyll content of the albino and regreened leaves of ‘Huabai 1’ was measured,and the ultrastructure of chloroplasts was observed by transmission electron microscope(TEM).The results showed that the ultrastructure of chloroplasts in both albino and regreened shoots of ‘Huabai 1’ were abnormal,and the albino degree of white leaves of ‘Huabai 1’ was higher than that of ‘Baiye 1’.2.Transgenic experiments were performed to investigate the function of CsSGR in chlorophyll degradation.The results showed that the constitutive overexpression of CsSGR rescued the stay-green phenotype of nye1-1 mutant in Arabidopsis and resulted in albino or variegated transgenic seedlings;transient overexpression of CsSGR in N.benthamiana leaves caused a rapid etiolated phenotype.This results indicate that CsSGR accelerates chlorophyll degradation.qRT-PCR analysis and chlorophyll detection showed that the transcription level of CsSGR was positively correlated with chlorophyll degradation ability,and CsSGR may induce the expression of chlorophyll catabolic enzymes(CCEs)gene to promote chlorophyll degradation.3.Transgenic experiments were performed to investigate the function of CsLHCⅡ in albinism of tea plants.The results showed that overexpression of CsLHCⅡ rescues the light green leaves of lhcb and deepen the green of wild-type(WT)Arabidopsis leaves.This results indicate that CsLHCⅡ promotes the accumulation of chlorophyll.qRT-PCR analysis and chlorophyll detection showed that key enzymes in chlorophyll biosynthesis were up-regulated and chlorophyll content increased.This indicates that up-regulation of CsLHCⅡ transcription levels affects regulation of the chlorophyll biosynthetic pathway.4.In order to explore the relationship between protein interactions and the albinism in tender shoots of ‘Huabai 1’,a yeast two-hybrid library was constructed using albino and regreened shoots of ‘Huabai 1’,and the corresponding interaction proteins were screened using CsSGR and CsLHCⅡ as ‘bait’ proteins,respectively.The results showed that a chloroplast small heat shock protein(s HSP,XM＿028206028.1)was screened out.The specific interactions of this protein with CsSGR and CsLHCⅡ on the chloroplast were confirmed by yeast two-hybrid(Y2H)assay,bimolecular fluorescence complementary(BIFC)and in vitro protein binding(Pull-down)analysis.Moreover,the results of qRT-PCR analysis showed that the expression levels of CssHSP in ‘Huabai 1’ were significantly higher than that of controls,and CssHSP was significantly down-regulated in albino shoots of ‘Huabai 1’ compared with regreened shoots.It shows that CssHSP play a role in the albino process of ‘Huabai 1’.5.The widely the widely targeted metabolomics analysis was performed to uncover the metabolic differences of albino shoots between ‘Huabai 1’ and ‘Baiye 1’.KEGG enrichment analysis and differential metabolite analysis revealed that the phenylpropanoid and flavonoids biosynthesis are the key metabolic pathways,contributing to the color change in ‘Huabai 1’,while the purine metabolism and lipid-related metabolites may play important roles in regulating albinism in ‘Baiye 1’,which suggests that there are different metabolic mechanisms associated with albinism in ‘Huabai 1’ and ‘Baiye 1’.In addition,albino shoots of ‘Huabai 1’ possess highly Antioxidant components,such as apigenin and genistein,suggesting that‘Huabai 1’ have high application value.