Isolation And Functional Characterization Of Genes Regulating Anthocyanin Biosynthesis In The Pericarp Of Litchi Chinensis

Litchi(Litchi chinensis Sonn.), an important subtropical fruit in South China, has plentiful germplasm resources which varied in fruit color phenotype. However, studies on their color development and the coloration mechanism in litchi are poorly documented. Recent studies showed that anthocyanin biosynthesis was regulated at transcriptional level. In generally, transcriptional regulation of the structural genes is controlled by MYB-basic helix-loop-helix(b HLH)-WD40 complex. In this study, candidate MYB and b HLH transcription factors may involved in anthocyanin regulation were identified based on transcriptiome database from litchi pericarp and litchi genome database. The mechanisms of these transcription factors regulating anthocyanin biosynthesis in litchi pericarp were discussed in this study. The main results are as follows:1. Litchi red coloration derives from anthocyanin accumulation in the pericarp. Previous study showed that the expression of Lc MYB1 is highly correlated with anthocyanin contents. In this paper, we overexpressed Lc MYB1 in tobacco to verify the function of this gene. Overexpression of Lc MYB1 enhanced anthocyanin accumulation in all tissues in tobacco, confirming the function of Lc MYB1 in regulating the biosynthesis of anthocyanin. The upregulation of Nt An1 b in response to Lc MYB1 overexpression seems to be essential for anthocyanin accumulation in leaf and pedicel. In the reproductive tissues of transgenic tobacco, however, increased anthocyanin accumulation is independent of endogenous MYB and b HLH transcriptional factors in tobacco, but associated with the upregulation of late structural genes, Nt DFR, Nt ANS and Nt UFGT.2. Analysis of the transcriptome of litchi pericarp was performed to obtain information regarding the molecular mechanisms underlying the physiological changes in the pericarp, including those leading to fruit coloration. Coincident with the rapid break down of chlorophyll, but substantial increase of anthocyanins in litchi pericarp as fruit developed, two major physiological changes, degreening and pigmentation were visually apparent. A c DNA library of litchi pericarp with three different coloration stages was constructed. A total of 4.6 Gb of raw RNA-Seq data was generated and this was then de novo assembled into 51,089 unigenes with a mean length of 737 bp. Approximately 70% of the unigenes(34,705) could be annotated based on public protein databases and, of these, 3,649 genes were significantly differentially expressed between any two coloration stages, while 156 genes were differentially expressed among all three stages. Genes encoding enzymes involved in chlorophyll degradation and flavonoid biosynthesis were identified in the transcriptome dataset. The transcript expression patterns of the Stay Green(SGR) protein suggested a key role in chlorophyll degradation in the litchi pericarp, and this conclusion was supported by the result of an assay over-expressing the SGR protein in tobacco leaves. We also found that the expression levels of most genes especially late anthocyanin biosynthesis genes were co-ordinated up-regulated coincident with the accumulation of anthocyanins, and that candidate MYB transcription factors that likely regulate flavonoid biosynthesis were identified. This study provides a large collection of transcripts and expression profiles associated with litchi fruit maturation processes, including coloration. Since most of the unigenes were annotated, they provide a platform for litchi functional genomic research within this species.3. Anthocyanins biosynthesis was regulated by MYB-b HLH-WD40 protein complex at transcriptional level. The present study describes the functional characterization of three litchi b HLH candidate anthocyanin regulators, Lcb HLH1, Lcb HLH2 and Lcb HLH3. Although these three litchi b HLHs phylogenetically clustered with b HLH proteins related to anthcoyanin biosynthesis in other plant, only Lcb HLH1 and Lcb HLH3 were found to localize in the nucleus and to physically interact with Lc MYB1. None of these b HLHs transcription level coordinated with anthocyanin accumulation in different tissues and during development. However, when co-infiltrated with Lc MYB1, both Lcb HLH1 and Lcb HLH3 enhanced anthocyanin accumulation in tobacco leaves with Lcb HLH3 being the best inducer. Significant accumulation of anthocyanin in leaves transformed with the combination of Lc MYB1 and Lcb HLH3 involves the up-regulation of two tobacco endogenous b HLH regulators, Nt An1 a and Nt An1 b and late structural genes, like Nt DFR and Nt ANS. Significant activity of the ANS promoter was observed in transient expression assays either with Lc MYB1-Lcb HLH1 or Lc MYB1-Lcb HLH3, while only minute activity was detected after transformation with only Lc MYB1. Instead no activity was measured after induction with the combination of Lcb HLH2 and Lc MYB1. Lcb HLH1 and Lcb HLH3 are essential partner of Lc MYB1 in regulating the anthocynin production in tobacco and probably also in litchi. The Lc MYB1-Lcb HLH complex enhanced anthocyanin accumulation may associate with activating the transcription of DFR and ANS.4. Two candidate MYB transcription factors named Lc MYB5 and Lc MYB2 were identified in litchi trascriptome. Lc MYB5 displays high similarity with Vv MYB5 b and p H4, two other another MYB factors shown to regulate flavonoid synthesis and p H value in grapevine and petunia. Overexpression of Lc MYB5 in tobacco(Nicotiana tabacum) leads to an up-regulation of genes encoding enzymes of the flavonoid pathway i.e. Nt CHI, Nt F3 H and Nt DFR, and b HLH endogenous regulators, Nt AN1aå’ŒNt AN1 b, which paralleled with significate accumulation of anthocyanins. Lc MYB5 was found to interact with Lcb HLH1 which was previously reported to be involved in anthocyanin regulation. That Lc MYB5 activate the promoters of Lc F3 H and Lc DFR confirmed its role in regulating anthocyanin biosynthesis. More interesting, the p H value in flower and leaf of transgenic plants was significant lower than wild type, indicating Lc MYB5 also involved in p H regulation. In addition, the flowers of transgenic plants were obviously larger than that of wild type. Taken together, our finding indicated that the transcriptional mechanisms associated with the regulation of flavonoid pathway in litchi, and also provided insights in p H and organ size regulation.5. Lc MYB2 transcription factor clustered with Vv MYB5 subgroup. It was constitutive expressed in litchi tiusses. It was highly expressed in the pericarp of young fruit, but decreased sharply as fruit development. Its expression pattern agreed with the expression of two key proanthocyanidin biosynthesis pathway genes, Lc LAR and Lc ANR. Lc MYB2 located in nuclei of tobacco cell and could interact with Lcb HLH3, an anthocyanin biosynthesis regulator. Lc MYB2 interacted with Lcb HLH3 activated the promoters of Lc LAR and Lc ANR. These results suggested that Lc MYB2 might involve in regulation of proanthocyanidin biosynthetic pathway. Overexpression Lc MYB2 in tobacco resulted in pigmented anther without pollen, indicating that Lc MYB2 also could regulate anthocyanin accumulation in specific tissue and might involve in other physiological processes.