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Systems scale characterization of circadian rhythm pathway in Camellia sinensis

ABSTRACT: Graphical abstract Tea (Camellia sinensis) is among the most valuable commercial crops being a non-alcoholic beverage having antioxidant properties. Like in other plants, circadian oscillator in tea modulates several biological processes according to earth’s revolution dependent variations in environmental cues like light and temperature. In the present study, we report genome wide identification and characterization of circadian oscillator (CO) proteins in tea. We first mined the genes (24, in total) involved in circadian rhythm pathway in the 56 plant species having available genomic information and then built their hidden Markov models (HMMs). Using these HMMs, 24 proteins were identified in tea and were further assessed for their functional annotation. Expression analysis of all these 24 CO proteins was then performed in 3 abiotic (A) and 3 biotic conditions (B) stress conditions and co-expressed as well as differentially expressed genes in the selected 6 stress conditions were elaborated. A methodology to identify the differentially expressed genes in specific types of stresses (A or B) is proposed and novel markers among CO proteins are presented. By mapping the identified CO proteins against the recently reported genome wide interologous protein–protein interaction network of tea (TeaGPIN), an interaction sub-network of tea CO proteins (TeaCO-PIN) is developed and analysed. Out of 24 CO proteins, structures of 4 proteins could be successfully predicted and validated using consensus of three structure prediction algorithms and their stability was further assessed using molecular dynamic simulations at 100 ns. Phylogenetic analysis of these proteins is performed to examine their molecular evolution.

SUBMITTER: Singh G

PROVIDER: S-EPMC8790616 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Green tea (Camellia sinensis, Cs) abundantly produces a diverse array of phenylpropanoid compounds benefiting human health. To date, the regulation of the phenylpropanoid biosynthesis in tea remains to be investigated. Here, we report a cDNA isolated from leaf tissues, which encodes a R2R3-MYB transcription factor. Amino acid sequence alignment and phylogenetic analysis indicate that it is a member of the MYB4-subgroup and named as CsMYB4a. Transcriptional and metabolic analyses show that the expression profile of CsMYB4a is negatively correlated to the accumulation of six flavan-3-ols and other phenolic acids. GFP fusion analysis shows CsMYB4a's localization in the nucleus. Promoters of five tea phenylpropanoid pathway genes are isolated and characterized to contain four types of AC-elements, which are targets of MYB4 members. Interaction of CsMYB4a and five promoters shows that CsMYB4a decreases all five promoters' activity. To further characterize its function, CsMYB4a is overexpressed in tobacco plants. The resulting transgenic plants show dwarf, shrinking and yellowish leaf, and early senescence phenotypes. A further genome-wide transcriptomic analysis reveals that the expression levels of 20 tobacco genes involved in the shikimate and the phenylpropanoid pathways are significantly downregulated in transgenic tobacco plants. UPLC-MS and HPLC based metabolic profiling reveals significant reduction of total lignin content, rutin, chlorogenic acid, and phenylalanine in CsMYB4a transgenic tobacco plants. Promoter sequence analysis of the 20 tobacco genes characterizes four types of AC-elements. Further CsMYB4a-AC element and CsMYB4a-promoter interaction analyses indicate that the negative regulation of CsMYB4a on the shikimate and phenylpropanoid pathways in tobacco is via reducing promoter activity. Taken together, all data indicate that CsMYB4a negatively regulates the phenylpropanoid and shikimate pathways. Highlight: A tea (Camellia sinensis) MYB4a is characterized to encode a R2R3-MYB transcription factor. It is shown to repressively control the phenylpropanoid and shikimate pathway.

Project description:BACKGROUND: Despite great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in tea (Camellia sinensis L.). The development of microsatellite markers will have a major impact on genetic analysis, gene mapping and marker assisted breeding. Unigene derived microsatellite (UGMS) markers identified from publicly available sequence database have the advantage of assaying variation in the expressed component of the genome with unique identity and position. Therefore, they can serve as efficient and cost effective alternative markers in such species. RESULTS: Considering the multiple advantages of UGMS markers, 1,223 unigenes were predicted from 2,181 expressed sequence tags (ESTs) of tea (Camellia sinensis L.). A total of 109 (8.9%) unigenes containing 120 SSRs were identified. SSR abundance was one in every 3.55 kb of EST sequences. The microsatellites mainly comprised of di (50.8%), tri (30.8%), tetra (6.6%), penta (7.5%) and few hexa (4.1%) nucleotide repeats. Among the dinucleotide repeats, (GA)n.(TC)n were most abundant (83.6%). Ninety six primer pairs could be designed form 83.5% of SSR containing unigenes. Of these, 61 (63.5%) primer pairs were experimentally validated and used to investigate the genetic diversity among the 34 accessions of different Camellia spp. Fifty one primer pairs (83.6%) were successfully cross transferred to the related species at various levels. Functional annotation of the unigenes containing SSRs was done through gene ontology (GO) characterization. Thirty six (60%) of them revealed significant sequence similarity with the known/putative proteins of Arabidopsis thaliana. Polymorphism information content (PIC) ranged from 0.018 to 0.972 with a mean value of 0.497. The average heterozygosity expected (HE) and observed (Ho) obtained was 0.654 and 0.413 respectively, thereby suggesting highly heterogeneous nature of tea. Further, test for IAM and SMM models for the UGMS loci showed excess heterozygosity and did not show any bottleneck operating in the tea population. CONCLUSION: UGMS markers identified and characterized in this study provided insight about the abundance and distribution of SSR in the expressed genome of C. sinensis. The identification and validation of 61 new UGMS markers will not only help in intra and inter specific genetic diversity assessment but also be enriching limited microsatellite markers resource in tea. Further, the use of these markers would reduce the cost and facilitate the gene mapping and marker-aided selection in tea. Since, 36 of these UGMS markers correspond to the Arabidopsis protein sequence data with known functions will offer the opportunity to investigate the consequences of SSR polymorphism on gene functions.

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Systems scale characterization of circadian rhythm pathway in Camellia sinensis

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