Abstract
Low temperature injury is one of the most significant causes of crop damage worldwide. Cold acclimatization processes improve the freezing tolerance of plants. To identify genes of potential importance for acclimatzation to the cold and to elucidate the pathways that regulate this process, global transcriptome expression of the chickpea (Cicer arietinum L), a species of legume, was analyzed using the cDNA-AFLP technique. In total, we generated 4800 transcript-derived fragments (TDFs) using cDNA-AFLP in conjunction with 256 primer combinations. We only considered those cDNA fragments that seemed to be up-regulated during cold acclimatization. Of these, 102 TDFs with differential expression patterns were excised from gels and re-amplified by PCR. Fifty-four fragments were then cloned and sequenced. BLAST search of the GenBank non-redundant (nr) sequence database demonstrated that 77 percent of the TDFs belonged to known sequences with putative functions related to metabolism (31), transport (10), signal transduction pathways (15) and transcription factors (21). The last group of expressed transcripts showed homology to genes of unknown function (22). To further analyze and validate our cDNA-AFLP experiments, the expression of 9 TDFs during cold acclimatzatiion was confirmed using real time RT-PCR. The results of this research show that cDNA-AFLP is a powerful technique for investigating the expression pattern of chickpea genes under low-temperature stress. Moreover, our findings will help both to elucidate the molecular basis of low-temperature effects on the chickpea genome and to identify those genes that could increase the cold tolerance of the chickpea plant.
Highlights
In worldwide agriculture, abiotic stress, such as extreme temperature, drought and salinity, is the primary cause of crop damage and reduces average yields for most crop plants by more than 50% [1]
Homology search analysis using the BLASTn and BLASTx programs revealed 22% of transcript-derived fragments (TDFs) with high homology to ESTs or unknown proteins; 77% of these belonged to known sequences with putative functionality during biotic and abiotic stresses in different plant species
Among those cloned TDFs that had homology with genes of known functions, 31% were involved in metabolism, while 10%, 15% and 21% were involved in transport, signal transduction pathway and transcription factors, respectively One sequence showed no significant homology to any known sequences in public databases
Summary
Abiotic stress, such as extreme temperature, drought and salinity, is the primary cause of crop damage and reduces average yields for most crop plants by more than 50% [1]. Cold acclimatization can lead to changes in lipid composition, protein, and nucleic acid conformation, as well as the accumulation of carbohydrates. These modifications assist plants in withstanding and repressing the intensive dehydration produced by freezing stress [5,8]. The key roles of cold acclimatization are to protect and stabilize the integrity of cell membrane rigidification and to prevent disruption by freezing. This ability is vital for plants because cellular membranes have a fundamental role in metabolism
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