Molecular characterization of transformants of Coffea canephora Pierre. for lowered caffeine.

Shibin, Mohanan (2013) Molecular characterization of transformants of Coffea canephora Pierre. for lowered caffeine. PhD thesis, University of Mysore.

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Abstract

Coffee is one of the largest consumed natural beverages produced from beans of coffee plants grown in semi arid tropics. Caffeine is an important constituent of coffee. A growing belief that intake of excessive caffeine has adverse effect on human health have resulted in the increased demand of decaffeinated coffee. At present there are numerous solvent based methods to decaffeinate coffee. Cloning of the genes involved in caffeine biosynthesis has been an important advancement towards the production of transgenic caffeine deficient coffee and tea using post transcriptional gene silencing (PGTS). Different silencing constructs have been used at C .F.T.R.I. to reduce caffeine levels. Preliminary analysis has indicated that caffeine is extremely low in the transformed somatic embryos bearing the inverse repeat constructs. Further regeneration of the transformed somatic embryos and analysis of individual plants is needed especially with regard to site of integration and metabolic profiling of metabolites involved in caffeine biosynthesis. A class of endogenously produced small RNA namely microRNAs (miRNA) are also known to post transcriptionally regulate mRNA. Since miRNA and RNAi share a common biogenesis pathway it was postulated that RNAi (invert repeat) transcribed by a strong viral promoter may affect miRNA biosynthesis and this in turn affect gene regulation indirectly. Hence it would be significant to characterize the miRNA of the transgenic plants, since they play an important role in gene regulation. It appeared that a comparison of the miRNA profiling in plants with and without the RNAi for the theobromine synthase would reveal something about the competition between the syntheses of the two forms of RNA, if such exists. Caffeine biosynthesis involves sequential methylation of the purine ring by the transfer of methyl group from the methyl donor. Genes that code for the individual methyl transferase have a high degree of sequence homology. In order to achieve decaffeination by genetic engineering of coffee, the effect and efficacy of different post-transcriptional gene silencing (PTGS) constructs were evaluated. Sense, antisense and invert repeat constructs were developed using a 360 bp fragment from the conserved region of the coffee N-methyltransferase genes involved in caffeine biosynthesis. The silencing constructs were mobilized into Agrobacterium tumefaciens (EHA101). Sonication assisted Agrobacterium-mediated transformation of the somatic embryos of Coffea canephora was performed, followed by ix regeneration of the transformants. Molecular analysis and identification of flanking regions confirmed stable integration of the transgenes in the transformants and their integration in non coding regions of the genome, respectively. Effect and effectiveness of the individual silencing constructs was evaluated by transcript analysis of the targeted genes by RT-PCR and northern blotting. The silencing efficiency was further evaluated by estimation of the purine alkaloid content in the transgenic lines using HPLC. The three constructs differed in their silencing efficiencies and specificity. Though the constructs were not specific to a single Nmethyltransferase, transformants obtained were mainly affected in one of the Nmethyltransferases. Results show that the use of homologous coding sequence in the PTGS constructs results in a much higher efficiency in silencing the caffeine biosynthetic pathway when compared to previous attempts. Based on the results of the present study it is postulated that the lowering of caffeine content should be the preferred over complete decaffeination due to extremely low survival rate of transgenic plants with extremely low levels of caffeine. The successful transformation of C.canephora using Agrobacterium tumefaciens brought forward few significant observations; increase in accumulation of caffeine and theophylline was observed in regenerated transformed plants when compared to that found in transformed somatic embryos. Even though the NMT genes involved in caffeine biosynthesis show high degree of similarity, RT-PCR results suggested that the CcMTL, a closely related NMT gene, was not silenced. At the same time an increase in transcript accumulation indicated a negative relationship between CcMTL and the genes silenced. In order to substantiate this hypothesis all previously reported cDNA and genomic clones of genes involved in caffeine biosynthesis was analyzed computationally to predict the origin of guide strand from the inverse repeat and its target sequence using siRNA design rules. The sequences were also checked for a possible origin or target of microRNA and other regulatory factors in the coding and non coding regions. A probable region for the origin of the RNAi guide strand was identified. The predicted region showed mismatches on the cDNA sequence of CcMTL. Further investigations into the sequences of caffeine biosynthetic genes showed the presence of an intronic microRNA originating from the second intron of theobromine synthase gene, regulating CcMTL. Northern blot analysis and cloning of the cleavage product by RLM-RACE confirmed the presence of this miRNA and their x activity in the C. canephora. The absence of the newly identified miRNA in the knockdown plants explains the altered accumulation of CcMTL mRNA in transgenic plants. The observation in the present study throws a new insight into regulation of gene expression and the role of introns in it. A possible role of miRNAs in regulation of secondary metabolite production especially those which involved multigene families was evidenced. At the same time the results also show the possible effects of RNAi on the endogenous miRNA population. The identification of intronic miRNA originating from the second intron of theobromine synthase gene revealed the importance of understanding the role of small RNAs especially miRNAs in C. canephora. As a prelude towards identifying the role of small non coding RNAs in C.canephora, identification of conserved miRNA from C. canephora using in silico methods of miRNA was carried out. Comparative genomic tools have been successfully used to predict new miRNAs in different plant and animal species using expressed sequence tag (EST) and genome survey sequence (GSS) analysis. With the availability of a comparatively large EST database conserved miRNAs in C. canephora was identified by EST analysis using a modified comparative genomics method. The modification made in the existing methods helped in eliminating false positives to a greater extent. 18 miRNAs belonging to 12 miRNA families was identified. All identified miRNA were used to predict their potential target genes from the SGN (Solanaceace Genome Network) EST database. A total of 42 potential targets were identified for miRNA families from C. canephora. Most of the target genes identified for the predicted miRNAs were transcription factors involved in plant growth, development and stress responses. Detailed studies on the role of identified miRNA can help in developing a better understanding of growth and development Coffea. Two commercially important species C. arabica and C. canephora differ from each other morphologically, physiologically and genetically. Studies have shown that most of these factors are regulated or linked to auxin homeostasis. Phytohormone auxin is potent regulator of plant growth and development. In auxin signaling pathways, the tiny RNAs i.e., miRNAs show their strong potential in modulating the auxin signaling transduction. MiRNAs like 160 and 164 target ARFs, a class of transcription factors that play important role in auxin signaling. Till date auxin signaling data have been published for Arabidopsis and Oryza but no reports are there xi for Coffea. Based on the conserved miRNAs identified during the present study, an attempt is made to initiate an investigation at molecular level into the role of miRNAs in the differences between C. arabica and C. canephora. Three conserved miRNAs known to regulate directly or indirectly genes involved in lateral root formation, flower development and disease resistance were selected. Results indicate that the levels of miRNA targeting mRNA coding for proteins involved in auxin signaling pathways were differentially expressed in the leaf, endosperm and suspension culture of C. arabica and C. canephora. Further studies into the genes regulated by the miRNA were correlated with the difference in morphological and physiological differences between the two species. Based on the results, it is hypothesized that, though an identical auxin signaling pathway exists in both the species a major difference exists in auxin signaling between them and miRNAs play an important role in modulating it. Expression of these miRNA was checked in auxin free media to check the control of auxin on miRNA. Results showed that exogenous auxin showed positively affected the miRNA expression. Efficient silencing of caffeine biosynthesis was carried out using homologus coding sequence. Results show that in transgenic plants RNAi does affect the miRNA in transgenic. Effect of RNAi on other endogenous RNA should also be explored in detail. The cross regulation of identical genes by an intronic miRNA will help us decipher the evolution of multigene families. Further studies into the role of miRNA in Coffea will help us understand the growth, development and adaptations within the genus that can be used to develop or increase desirable traits for agricultural applications.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	caffeine biosynthesis, Cloning, coffee, C.canephora, Agrobacterium tumefaciens
Subjects:	500 Natural Sciences and Mathematics > 07 Life Sciences > 03 Biochemistry & Molecular Biology > 04 Biosynthesis 600 Technology > 07 Beverage Technology > 04 Coffee
Divisions:	Plant Cell Biotechnology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	19 Mar 2015 10:42
Last Modified:	19 Mar 2015 10:42
URI:	http://ir.cftri.res.in/id/eprint/11760

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