Record Details

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

IR@CSIR-CFTRI

View Archive Info
 
 
Field Value
 
Relation http://ir.cftri.com/11760/
 
Title Molecular
characterization of transformants of Coffea canephora Pierre. for lowered
caffeine.
 
Creator Shibin, Mohanan
 
Subject 04 Biosynthesis
04 Coffee
 
Description 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.
 
Contributor Arun, Chandrashekar
 
Date 2013
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Language en
 
Rights —
 
Identifier http://ir.cftri.com/11760/1/N.Shibin%20Mohanan%20thesis.pdf
Shibin, Mohanan (2013) Molecular characterization of transformants of Coffea canephora Pierre. for lowered caffeine. PhD thesis, University of Mysore.