Mechanisms Of CsClpP3 On Pale And Re-greening Of Albino Tea(Camellia Sinensis)

China has kinds of tea varieties,which gives a great chance for tea breeding.Albino tea,which possess mellow taste,low bitter and bright color due to the higher ratio of amino acids and catechins,and is more and more popular in China nowadays.In early spring,the newly produced young leaves of albino tea appear yellowish or whitish,then gradually return to green along with the development and growth of leaves.Many previous studies emphasized on the characteristics and mechanisms of albino tea through transcriptome,proteome and metabolome,however,the mechanisms of pale and re-greening of albino teas are still unclear.In the present study,we analyzed the transcriptome data of the albino tea’Yujinxiang’ and the normal leaf tea ’Shuchazao’,results showed that the transcriptional level of genes encoding the chloroplast protease complex subunits(Clp)had significant differences between the two varieties.Therefore,qRT-PCR were used to confirm the difference of transcriptional levels,results revealed that the expression levels of P-ring subunits of Clp had remarkably differences.Subsequently,genes encoding the P-Ring subunits of Clp were cloned.Interestingly,a gene encoding CsClpP3 were proved that undergo alternative splicing(AS),which had a intron retention of 16 bp,which resulted in a premature stop codon,and shortened the amino acid of the CsClpP3 from 344aa to 166aa.This gene,losing the predicted amino acid active site Ser-His-Asp,was named CsClpP3m.CsClpP3 and its AS variants CsClpP3m simultaneously present in both albino tea and normal tea,but with different transcript levels,suggesting that the ratio of transcript levels between CsClpP3 and CsClpP3m in tea plants is responsible for pale and re-greening of albino tea.The Agrobacterium-mediated genetic transformation system was used in this study,and the CsClpP3 and its mutant gene were heterologously overexpressed in the model plant tobacco under the promoter 35S.The results showed that transgenic plants compared with wild type tobacco were no significant difference in chlorophyll content and phenotype.According to these results,NtClpP3 gene in tobacco plant were knockout,and NtClpP3RNAi transgenic plants were obtained.These NtClpP3-RNAi transgenic plants appear albino leaves,and the concentration of chlorophyll a and b in transgenic plants were also investigated,showed a lower level compared to that of wild type.For the sake of investigating the structures of chloroplast,transmission electron microscope were used.The results showed that the structure of chloroplast was incomplete,and thylakoid membranes were premature,indicating that the function of chloroplast in NtClpP3-RNAi transgenic plants presumably were weaken.Furthermore,qRT-PCR were used to analyze the expression level of gene encoding the Clp subunits in NtClpP3-RNAi transgenic.The results revealed that NtClpP3 gene was significantly down-regulated,while genes encoding other subunits were dramatically up-regulated.These results indicated that konck-down of a single Clp subunit results in the change of expression level of genes encoding other Clp subunit,which imply the compensatory response of these characteristic subunits to the lack of specific subunits in each knock-down lines.Therefore,there is a synergistic effect between the subunits of the Clp complex,which is involved in the regulation of chloroplast protease.To verify the influence of CsClpP3 and CsClpP3m genes on the pale mechanism of albino tea CsClpP3 and CsClpP3m were heterologously expressed in NtClpP3-RNAi transgenic tobacco for the sake of recovering the structure and function of Clp complex.The results indicated that overexpression of CsClpP3 gene could change the pale leaves of NtClpP3-RNAi plants to green,proved that the function of CsClpP3 is complete.Furthermore,we predicted that CsClpP3m failed to recover the pale leaves of NtClpP3-RNAi plants due to loss of function.Thus,these results further proved that the alternative splicing of CsClpP3 gene in tea plants is responsible for the mutant mechanism of albino tea.In present study,we provide an insight into the mutant mechanism of albino tea,according to the analysis of gene ecoding P-Ring subunits of Clp and its mutant gene.