Functional genomics analyses of a DREB-related gene in durum wheat
Author(s)
Latini, Arianna
Date Issued
April 4, 2008
Type
Doctoral Thesis
Abstract
Adverse environmental stresses, such as drought, cold and high salinity, have a strong influence on
agricultural production. The study of the mechanisms involved in plant response and tolerance to
these stresses represents a major challenge for plant scientists especially in light of foreseen global
climate changes. Plant genomics research is beginning to provide detailed information related to the
molecular mechanisms and crosstalks of the signalling pathways involved in the different kinds of
abiotic stress response.
Wheat, together with maize and rice, is the most important crop grown worldwide. Durum wheat is
a minor crop, grown on only 8 to 10 % of all the wheat cultivated area, but it is better adapted to
semiarid climates than bread wheat. Significant advances in understanding the molecular biology of
this tetraploid species must be achieved to improve its tolerance to drought and other environmental
insults and, actually, this PhD research work focuses on dehydration tolerance in durum wheat by
means of functional genomics techniques.
A number of genes have been described that respond to water stress at the transcriptional level and
several cis- and trans-acting factors involved in the expression of dehydration-responsive genes,
such as the DRE element (Dehydration Responsive Element) and the DREB factors (DRE-Binding
factors), have been extensively dissected. In a previous study, my collaborators at ENEA Research
Center (Rome, Italy) and I have isolated a DREB2-related gene in durum wheat, designated as
TdDRF1 (Triticum durum Dehydration Responsive Factor 1), and established that it is expressed in
response to dehydration. This gene produces three transcript variants, namely, TdDRF1.1,
TdDRF1.2 and TdDRF1.3, through alternative splicing. The transcript isoforms TdDRF1.1 and
TdDRF1.3 encode AP2 transcriptional activators and TdDRF1.2 encodes a putatively truncated
protein, lacking the AP2 DNA-binding domain.
The current work is composed of three experimental sections. In the fist section, I report the
analysis of the expression profiles of the three TdDRF1 alternatively spliced transcripts upon
dehydration, in four durum wheat and one triticale cultivars grown in greenhouse, and reveal that,
even though these cultivars exhibit a very similar water retention in their leaves, their tolerance to
dehydration may depend on a genotype-specific TdDRF1 expression pattern together with many
other additional genotype-specific traits. Furthermore, the genetic variability of TdDRF1 sequences
was explored among the five cultivars, in search of genotype-specific polymorphisms.
In the second section, I describe a notable experimental work, that is still in progress, for assessing
the expression profile of the TdDRF1 gene and its relation to the tolerance of durum wheat
cultivars, grown under water stressed (reduced irrigation) and non-stressed (full irrigation)
conditions at CIMMYT s experimental fields (Obregón, Mexico). Two groups of durum cultivars
were chosen for this scope: one group exhibiting good drought tolerance and the other one
exhibiting strong drought susceptibility. The TdDRF1 transcripts expression levels were monitored
by real-time RT-PCR and results of one tolerant (Duilio) and one susceptible (Creso) cultivars are
reported.
While studying the TdDRF1 transcripts expression profile, dependent on the particular plant water
status and on the genotype, many efforts have been concentrated to attempt clarify the function of
the three gene products, for which no direct information is available in literature. In the third
section, I describe an approach towards the investigation of the function of all the three gene
products, using a heterologous overexpression system PVX (Potato Virus X)-mediated. Initially, the
complete codifying sequences (CDSs) of the three TdDRF1 transcripts were isolated and cloned
and, after that, the PVX-derived constructs for the transient overexpression in Nicotiana
benthamiana and Nicotiana tabacum were engineered. An increased mRNA expression level, in
comparison to wild type plants, was detected in all inoculated plants. The immediate next step will
be the protein analysis with the double aim of isolating the target proteins produced in plant and
investigating their molecular partners in the networks involved in the dehydration stress signalling
and response.
All the information and experimental data reported in this PhD work, as basic research, will be
useful to enrich the actual knowledge about the molecular basis of drought stress and, possibly, they
will draw together to a future application in molecular assisted breeding.
Additional information
Dottorato di ricerca in Genetica e biologia cellulare
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