A., SWAROOPA RANI

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ContentsCaffeine (1,3,7-trimethyl xanthine) is found in seeds and leaves of coffee, cola, mate and tea. Along with other methylxanthines, including theobromine, paraxanthine and methyluric acids, caffeine is a member of a group of compounds known collectively as purine alkaloids. The biosynthetic pathway of theobromine and caffeine has been the subject of much study over the years. Caffeine is synthesized through three sequential steps involving methylation of xanthine derivatives at positions 7-N, 3-N and 1-N (xanthosine  7-Methylxanthosine  7-Methylxanthine  Theobromine  Caffeine), catalyzed by N -Methyltransferases that use S-Adenosyl-L-Methionine as methyl donor.    A survey of literature suggests that the N-methyltransferases (NMTs) that are involved in the caffeine biosynthetic pathway in coffee comprise a multigene family. Several cDNA clones for NMTs from coffee have been reported. However, there are no reports on the genomic sequences for the NMT genes. The studies on NMT gene structure and organisation are lacking. Therefore, it was envisaged that PCR based genomic cloning of the NMT genes could provide valuable information regarding the number and size of introns and exons present in NMT genes. The study of introns and exons could possibly shed light on the mechanisms for origin of NMT multigene family in coffee and possible functional divergence among different members of the gene.   The survey of literature and patents indicated a distinct lacuna in the studies related to NMT gene regulation. The isolation of promoter for the NMT genes has not been hitherto reported and hence it became one of the major objectives of this investigation. The promoter isolation could help in understanding the NMT gene regulation and also open up the possibilities of regulating caffeine content in coffee plants.    There are wide variations in the caffeine contents of different coffee species, the reasons for which are unknown. Very little is known about the NMT genes in Coffea species other than C. arabica. It may be speculated that differential transcription of various NMT genes could be one of the reasons for such variations in the caffeine content. Study of possible polymorphisms for the NMT genes and their corresponding promoters in different Coffea species could be vital in understanding differential transcription of various NMT genes.    The production of transgenic caffeine deficient coffee plants by RNAi using specific region of the theobromine synthase gene has been reported earlier, wherein spreading of silencing to other NMT genes was observed. The use of a conserved region common to all the NMT genes should theoretically result in silencing of all the NMT genes. Moreover, other posttranscriptional gene silencing strategies were not explored for caffeine down regulation. The use of conserved region of NMT genes for different posttranscriptional gene silencing strategies could be a viable option for obtaining phenotypes with desirable purine alkaloid profiles.    With this background knowledge, the present research work was undertaken. The research work carried out to meet the proposed objectives is presented in five chapters in the thesis. The references are cited at the end in alphabetical order. The title and summary of the work carried out under each chapter are presented as follows: Chapter IIntroduction and review of literature   This chapter provides an overview of the current literature regarding the purine alkaloid biosynthesis in coffee. The taxonomy of coffee, distribution of various purine alkaloids and their biosynthesis in different Coffea species are highlighted. The properties of N-methyltransferases that catalyze the conversion of xanthosine to caffeine and the genes encoding them have been described in detail. The comparisons of the NMT sequences in tea and coffee with related methyltransferases are also presented. The pathway for caffeine catabolism in plants is discussed. The uses of biotechnological approaches for regulating the caffeine content in plants are discussed at the end of the chapter. This chapter also highlights the unexplored areas in coffee NMT research, which forms the basis for the present investigation.Chapter IIMolecular Cloning of the N - Methyltransferase Gene from Coffea canephora   Primers for PCR amplification of N-methyltransferase (NMT) genes from Coffea canephora were designed based on the available cDNA sequences for NMT genes of Coffea arabica. Different sets of primers were used to obtain amplicons for partial and full-length NMT gene. Various PCR products were cloned and sequenced. Variations were observed with respect to restriction patterns among the clones for partial NMT gene. Sequence analysis of these clones revealed the presence of introns in the gene. High degree of similarity in the nucleotide sequences of exon regions and differences in the length and sequence of introns were observed, suggesting that these clones may belong to different members of the NMT multigene family. Using primers based on 5΄ and 3΄ untranslated regions specific for theobromine synthase, involved in the second methylation step, amplicons for full-length theobromine synthase gene were obtained. Sequencing of the clones for full-length gene revealed the presence of four exons interrupted by three introns. Based on the insertions and deletions present in the cDNA sequences of various NMT genes reported so far and the sizes of their open reading frames, the genomic clone corresponded to the theobromine synthase gene encoding for 378 amino acids. Variations with respect to the size and sequence of introns in two of the full-length clones for theobromine synthase were observed, though one of them contained one base nucleotide deletion in fourth exon. From this study, it appears that the coffee plants possess multiple alleles and/or multiple copies of the same gene; some of which may have undergone pseudogenisation.Chapter IIICloning of Promoter for an N-Methyltransferase Gene   A PCR based genome walking method was adopted for isolation and cloning of the promoter for one of the coffee NMT genes. Inspection of the promoter sequence revealed the presence of several motifs important for the regulation of the gene expression. The isolated promoter fragment was fused to the β-glucuronidase (GUS) reporter gene and used in Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum and also for electroporation of coffee endosperms. GUS assays proved that the isolated promoter was able to direct the expression of the reporter gene in transgenic tobacco. The cellular localization of GUS activity in transgenic tobacco confirms that coffee NMT gene is a cytosolic enzyme. Transient assays demonstrated the ability of the isolated promoter fragment to drive gene expression in endosperms. The cloning of promoter for a gene involved in caffeine biosynthetic pathway opens up the possibility of studying the molecular mechanisms that regulate the production of caffeine. Based on the promoter sequence, primer was designed and the genomic fragment comprising the promoter and its corresponding gene was amplified and cloned. Sequencing of one of the genomic clones revealed the presence of four exons and three introns in NMT gene. Sequence comparisons were made between coffee NMT gene and Arabidopsis Jasmonic acid carboxyl methyltransferase, which belongs to coffee N-methyltransferase related family. Though the gene structure was highly conserved in terms of number and position of introns, AtJMT revealed great variation for coding regions, introns, and the promoter region sequences, suggesting that the two genes have diverged greatly after their origin from common ancestor. Chapter IVPolymorphism for Coffee NMT Genes   Based on the promoter sequence for theobromine synthase gene-1, primers were designed and the genomic fragment comprising the promoter and its corresponding gene was amplified and cloned. Differences in the restriction pattern among the genomic clones of theobromine synthase gene-1 were studied by performing PCR-RFLP. Though differences were observed in restriction pattern for the clones, Southern blotting of the restricted PCR products from these clones with probes for promoter as well as the gene suggested that genomic clones are highly homologous and possibly belonged to different alleles/ members of NMT gene family. Sequencing of some of these clones from Coffea canephora revealed differences in intron length and sequences, but the promoter regions and the coding regions were highly conserved. It may be concluded that C. canephora possesses several alleles/ copies of theobromine synthase gene-1. NMT gene fragment from Indian wild species Psilanthus bengalensis was also cloned and partially sequenced. The conservation of NMT gene structure was studied by comparing the introns from various genomic clones. To assess relationships between coffee NMT family members within and across species, their exon-intron organization patterns (e.g. number, positions, and phases of introns) were determined. Intron phase is strictly conserved in all genomic NMT clones available. All the three introns were located at conserved positions, being inserted between two codons (phase 0). Intron positions are absolutely conserved in the NMT genes from various species. Intron sequences have the consensus dinucleotide GT at the 5΄-end and AG at the 3΄-end. Although the splicing sites are conserved, the sizes of introns varied for different NMT genes. Two of the genomic clones obtained in this study are probably pseudogenes, due to a single nucleotide deletion in fourth and third exon respectively. Gene duplication followed by sequence divergence has been proposed as a model to explain how novel biochemical functions may have arisen for different duplicated genes of Coffee NMTs. The promoter regions were PCR amplified from different Coffea species. The sequence analyses of promoter regions from different species revealed a very high degree of conservation. The recent origin of coffee could be the reason for this high level of conservation of promoter sequences within the species and across the species.  Chapter VPosttranscriptional Gene Silencing of Coffee N-Methyl transferases   A highly conserved region among coffee N-Methyltransferase genes involved in caffeine biosynthetic pathway was used in making sense, antisense and RNA interference constructs. Sonication Assisted Agrobacterium tumefaciens (SAAT) mediated transformation was used to mobilize these constructs into Coffea canephora. Somatic embryos were used for the transformation experiments. The transformed coffee plantlets, selected on hygromycin containing selection media, were used for estimating the purine alkaloids content by High Pressure Liquid Chromatography (HPLC). The purine alkaloids viz: 7-methylxanthine, theobromine, caffeine and theophylline were estimated from the transgenic plantlets. The three constructs differed in their efficiencies and specificity of silencing. RNAi was found to be more efficient in reducing caffeine content in transformed coffee plantlets, when compared to sense and antisense constructs. Though the constructs were not specific for a single gene, sense and RNAi transformants were obtained that were mainly affected in one of the N-Methyltransferases. The results suggest that the use of highly homologous coding region is highly effective in down regulating caffeine biosynthesis, encoded by N-methyltransferase multigene family. Increased transcription of CaMTL1 gene, substrate specificity of which is not known, was observed indicating possible activation of parallel or bypass pathways. Chapter VI Conclusions and Future perspectives    The major findings of this investigation and the future prospects are briefly highlighted in the chapter. PAGE  vi

STUDIES ON HIGH PERFORMANCE BIOREACTORS FOR PENICILLIN PRODUCTION

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STUDIES ON HIGH PERFORMANCE BIOREACTORS FOR PENICILLIN PRODUCTION

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