Characterisation of promoter and transcription factors of Coffea sp. with special reference to caffeine metabolism

Avinash, Kumar (2015) Characterisation of promoter and transcription factors of Coffea sp. with special reference to caffeine metabolism. PhD thesis, University of Mysore.

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Abstract

With respect to the identification of regulatory elements on the putative promoter (pETSSPI clone) of theobromine synthase and other 22 NMT gene promoters obtained from coffee genome browser involved in caffeine biosynthesis, an initial in silico analysis was carried out revealing presence of several WRKY motifs (based on PLACE software), salinity and drought responsive motif {both ABA dependant (ABRE) and ABA independent (DRE etc)}, light responsive motif (GT1 and GATA) as the major motifs. Promoter deletion analysis indicate the region between -100 to + 57 with respect to start codon as the minimal promoter. In order to have a annotation value to data, phylogenetic analysis of seven NMT sequences (CX8, CX10, CS2A, CS2B, PG-1, PG-4 and PG-5) isolated previously in the Plant Cell Biotechnology Department was performed and these were found to be similar to theobromine synthases. All the NMTs were grouped in accordance to the major substrate specificity into three clades based on the recently assigned catalytic acitivies as first, second and third nitrogen methylation. The third NMT of the pathway diverges early from ancestors of first and second NMTs on the phylogeny. Codon-substitution prediction of C. arabica and C. canephora NMTs indicate positive selection on two sites of importance to substrate binding and on ancestral branches, one subtending to the third NMT and the other to first and second NMT of the pathway. This study proposes that coffee NMTs evolved from a multiple-substrate-specific ancestral enzyme and escape from adaptive conflict model best explains the mechanism of fixation of NMT duplicates. Due to the involvement of light, salicylic acid and methyl jasmonate in the regulation of caffeine, via the theobromine synthase route, it is also necessary to study the influence of light, methyljasmonate and salicylic acid on theobromine synthase along with other related NMTs involved in caffeine biosynthetic pathway. Hence, transcript profiles analysis is desirable to understand the pattern of NMTs in caffeine biosynthesis before actually probe the expression pattern of WRKY and light responsive transcription factors of theobromine synthase (2nd NMT). Apart from this, it is also desirable to find out the influence of various types of stress factors such as salinity and drought on caffeine profiles to make a detailed model system for studying co-expression of NMTs with WRKY transcription factors. Accordingly, experiments were designed and the results revealed that, treatment of leaves of robusta coffee (C. canephora) with SA and MeJ increased the biosynthesis of methyxanthines (theobromine and caffeine) correlating with the augmented expression of the NMT genes as well as over expression of some WRKY transcription factors. Caffeine biosynthesis is subdued in endosperms at the early dry weight accumulation phase until fruit maturity and is related to the repression of both theobromine synthase (2nd NMT) and CcWRKY-69. This factor is overexpressed also during SA and MeJ treatments coinciding with over-expression of NMT genes. 2 Similarly, light exhibited profound influence on transcript profiles of NMTs involved in caffeine biosynthesis. This biochemical response was substantiated by transcript analysis. Influence of both the salinity and drought stress leads to lowered caffeine content. The biochemical profile was supported by transcript expression of the caffeine biosynthetic NMT genes and the analysis of regulatory motifs of the promoters. The contents of upstream methylxanthines (7-methylxanthine and theobromine) and the degradation pathway (theophylline) indicate that salinity and drought might have a negative impact on biosynthesis of caffeine but accelerated the rate of caffeine degradation. Subsequently, preparation of RNAi (RNA interference) constructs for putative theobromine synthase promoter and their use for transcriptional gene silencing of theobromine synthase transcript was accomplished. Promoter invert repeat constructs were constructed by cloning the sense and antisense fragments of promoter flanking on either ends of PDK intron in the pHANNIBAL siRNA construction vector under the constitutively expressing CaMV35S promoter. Three regions of the promoter were utilized for making sense and antisense stands each with or without the region of TATA element. These six siRNA cassettes was subcloned into the binary vector and named pPINC1- pPINC6. Then they were mobilized to A. tumefaciens and then transformed to primary somatic embryos of C. canephora. Fourteen putative pPINC-6 transgenic lines and one putative pPINC-1 transgenic line was obtained after eight months of selection through secondary somatic embryogenesis. Attempts were made to establish efficient somatic embryogenesis and in vitro regeneration protocol for C. canephora, in order to achieve higher rate of genetic transformation. The addition of 60μM silver thiosulphate in place of the earlier optimized 40μM silver nitrate to embryogenic medium gave a better response both in terms of percentage response (~3-3.5 fold higher) as well as number of embryos per explant (~2 folds higher). The silver thiosulphate induced primary embryos exhibited higher Agrobacterium tumefaciens infectivity and showed stronger transient gus expression. Greater than 95% embryos germinated into healthy plantlets compared to the usually 60-70% viability of silver nitrate induced embryos. The reasoning for improved Agrobacterium infectivity was assigned to difference in the cell wall remodelling as observed by differential expression pattern of mannan biosynthesis, mannan degrading and mannan re-branching enzymes in embryos developed on different medium.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	coffee, caffeine metabolism, transcription factor
Subjects:	500 Natural Sciences and Mathematics > 07 Life Sciences > 04 Microbiology > 07 Metabolism 600 Technology > 07 Beverage Technology > 04 Coffee
Divisions:	Plant Cell Biotechnology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	18 May 2016 08:31
Last Modified:	18 May 2016 08:31
URI:	http://ir.cftri.res.in/id/eprint/12161

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