8th INTERNATIONAL WHEAT CONFERENCE

IdeNTIfICATIoN ANd ChARACTeRIzATIoN of hIgh
TemPeRATuRe STReSS ReSPoNSIVe geNeS IN BReAd
WheAT (TRITICum AeSTIVum L.) ANd TheIR ReguLATIoN
AT VARIouS deVeLoPmeNTAL STAgeS
Paramjit Khurana and Harsh Chauhan
Department of Plant Molecular Biology, University of Delhi South Campus, Dhaula Kuan,
New Delhi-110021, India.
E-mail Address of presenting author: param@genomeindia.org
Climate change and abiotic stress affects agriculture and crop production adversely. Wheat
is one of the most important crop of our country and India is the second largest producer
globally. Wheat is a temperate crop and prone to heat stress during the grain filling stages
(seed maturation/embryogenesis) in the northern regions of India, while heat stress during
seedling stages is more prevalent in the mid-western regions of India. High temperatures
reduce both yield and quality of wheat by decreasing the duration of developmental
phases, leading to fewer and smaller organs, and changes in the assimilation processes.
Despite its importance, genomic information in wheat is inadequate for functional analysis.
Hence, a program has been initiated to understand the gene expression profile of the
sensitive and tolerant cultivars of wheat under heat stress by substractive hybridization,
both at the seedling, flowering and the embryogenic stages of grain filling. To elucidate
the effect of high temperature, wheat plants (Triticum aestivum cv. CPAN 1676) were given
heat shock at 37 o C and 42 o C for two hrs, and responsive genes were identified through
PCR-Select Subtraction technology. Four subtractive cDNA libraries, including three forward
and one reverse subtraction, were constructed from three different developmental
stages. A total of 5500 ESTs were generated and 3516 high quality ESTs were submitted
to Genbank. More than one third of the ESTs generated fall in unknown/no hit category
upon homology search through BLAST analysis. A large number of high temperature
responsive genes were identified and characterized. Reverse subtraction analysis in developing
grains showed extensive transcriptional changes upon heat stress as revealed by
comparative analysis with forward subtraction. Differential expression was confirmed
by cDNA macroarray and by northern/RT-PCR analysis. Expression analysis of wheat
plants subjected to high temperature stress, after one and four days of recovery, showed
fast recovery in seedling tissue. However, recovery was little in the developing seed tissue
after two hours of heat stress. Ten selected genes were analyzed in further detail including
one unknown protein and a new heat shock factor, by quantitative real-time PCR in
an array of 35 different wheat tissues representing major developmental stages as well as
different abiotic stresses. Tissue specificity was examined along with cross talk with other
abiotic stresses and putative signalling molecules. Results obtained contribute towards
understanding the regulation of genes at different developmental stages in wheat crucial
to withstanding and recovery from heat stress. Some of these genes are being functionally
validated in transgenics in wheat is inadequate for functional analysis. One such gene, a
chloroplastic small heat shock protein (sHSP26) was found essential for tolerance to heat
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