Abstract
BackgroundEnvironmentally inflicted stresses such as salinity and drought limit the plant productivity both in natural and agricultural system. Increasing emphasis has been directed to molecular breeding strategies to enhance the intrinsic ability of plant to survive stress conditions. Functional screens in microorganisms with heterologous genes are a rapid, effective and powerful tool to identify stress tolerant genes in plants. Jatropha curcas (Physic nut) has been identified as a potential source of biodiesel plant. In order to improve its productivity under stress conditions to benefit commercial plantations, we initiated prospecting of novel genes expressed during stress in J. curcas that can be utilized to enhance stress tolerance ability of plant.ResultsTo identify genes expressed during salt tolerance, cDNA expression libraries were constructed from salt-stressed roots of J. curcas, regulated under the control of the yeast GAL1 system. Using a replica based screening, twenty thousand yeast transformants were screened to identify transformants expressing heterologous gene sequences from J. curcas with enhanced ability to tolerate stress. From the screen we obtained 32 full length genes from J. curcas [GenBank accession numbers FJ489601-FJ489611, FJ619041-FJ619057 and FJ623457-FJ623460] that can confer abiotic stress tolerance. As a part of this screen, we optimized conditions for salt stress in J. curcas, defined parameters for salt stress in yeast, as well as isolated three salt hypersensitive yeast strains shs-2, shs-6 and shs-8 generated through a process of random mutagenesis, and exhibited growth retardation beyond 750 mM NaCl. Further, we demonstrated complementation of the salt sensitive phenotypes in the shs mutants, and analyzed the expression patterns for selected J. curcas genes obtained from the screen in both leaf and root tissues after salt stress treatments.ConclusionsThe approach described in this report provides a rapid and universal assay system for large scale screening of genes for varied abiotic stress tolerance within a short span of time. Using this screening strategy we could isolate both genes with previously known function in stress tolerance as well as novel sequences with yet unknown function in salt stress tolerance from J. curcas. The isolated genes could be over-expressed using plant expression system to generate and evaluate transgenic plants for stress tolerance as well as be used as markers for breeding salt stress tolerance in plants.
Highlights
Inflicted stresses such as salinity and drought limit the plant productivity both in natural and agricultural system
Functional screening of microorganisms that express heterologous cDNA libraries is a powerful tool for identifying genes with specific functions, independent of the regulation of their expression [3]
These results demonstrate that microbial functional screening is an effective tool to quickly identify stress tolerant gene candidates in plants
Summary
Inflicted stresses such as salinity and drought limit the plant productivity both in natural and agricultural system. In order to improve its productivity under stress conditions to benefit commercial plantations, we initiated prospecting of novel genes expressed during stress in J. curcas that can be utilized to enhance stress tolerance ability of plant. Inflicted stresses such as extreme temperatures, low water availability, high salt levels, mineral deficiency and toxicity are frequently encountered by plants both in natural and agricultural system that affect difficulty of breeding tolerance associated traits from diverse plant backgrounds. These results demonstrate that microbial functional screening is an effective tool to quickly identify stress tolerant gene candidates in plants
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