Record Details

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

IR@CSIR-CFTRI

View Archive Info
 
 
Field Value
 
Relation http://ir.cftri.com/12161/
 
Title Characterisation of promoter and
transcription factors of Coffea sp. with special reference to caffeine metabolism
 
Creator Avinash, Kumar
 
Subject 07 Metabolism
04 Coffee
 
Description 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.
 
Contributor Giridhar, P.
 
Date 2015
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Language en
 
Rights —
 
Identifier http://ir.cftri.com/12161/1/AVINASH%20KUMAR%20%20Ph.D%20Thesis.pdf
Avinash, Kumar (2015) Characterisation of promoter and transcription factors of Coffea sp. with special reference to caffeine metabolism. PhD thesis, University of Mysore.