Functional validation of the potential EcCAM, EcCAX1 and EcCAX 3 genes in grain calcium accumulation through over-expression studies in Arabidopsis thaliana: Development of an efficient plant regeneration protocol towards calcium biofortification in finger millet
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Date
2019-02
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
Mineral malnutrition is one of the major problems crippling by one-half world population mostly women’s and pre-schooled children. Calcium deficiency is also considered as major problem which may lead to defects in bones, teeth, and osteoporosis. Finger millet (Eleusine coracana) is an orphan crop, rich in calcium with high nutritional significance and antioxidant properties to grow under harsh drought conditions. Multi-pronged molecular approaches were used in our Lab to identify the candidate genes of calcium sensor and transporter families associated with differential grain calcium in different genotypes due to induction of differential calcium signaling and transport machinery. The identified genes were functionally validated in the present investigation through over-expression studies in a model plant of Arabidopsis thaliana. Complete CDS of EcCAM, EcCAX1 and EcCAX3 genes were retrieved from transcriptomic data of pooled spikes of high calcium containing genotype; GP-45. In-silico molecular characterization of these genes based on protein sequences showed the presence of conserved domains as characteristic features. The phylogenetic analysis showed the genes are closely related to Siteria italic and Oryza sativa while distinctly related with Arabidopsis. To decipher the function of EcCaM, EcCAX1 and EcCAX3 genes, the gain-offunction approach was used to generate the transformed lines in Arabidopsis thaliana and expression analysis by semi-quantitative RT-PCR of such genes carried out in T3 homozygous transgenic lines showed higher expression as compared to no expression in wild type. Phenotypic assays were performed under various abiotic conditions such as EcCAM under PEG induced drought stress, IAA induced homeostasis, NaCl induced salt stress and calcium induced ionic stress; EcCAX1 and EcCAX3 under calcium & magnesium
ionic stress, and IAA induced homeostasis. The observations recorded in 7 days old seedlings suggested that over-expressed transgenic lines were more tolerant as compared to wild type as evident from root elongation with lateral growth and better physiology. The anti-oxidative potential of over-expressed transgenic lines was higher in transgenic lines showing less ROS accumulation while more ROS accumulation in wild type plants as indicated by NBT and DAB staining method. Thus, more oxidative damage was observed in wild type as compared to over-expressed transgenic lines. Finger millet is highly adaptive in harsh conditions and nutritionally superior necessitate its further improvement using transgenic technology. In order to develop superior genetically modified plants, an efficient plant tissue culture protocol is essentially required taking innate nutritional, biochemical and hormonal attributes of finger millet genotypes as indices of plant tissue culture responsiveness. In order to determine the influence of stress tolerant behavior and inherent composition on plant regeneration, four genotypes of finger millet (GP-45, GP-1 GE-1437 and GE-3385) were taken in the present study. The results indicate that GP-45 was found to show maximum stress tolerance whereas GP-1 was the least tolerant. Further estimation of endogenous total calcium, carbohydrates, protein, total phenols, total flavonoids and phytohormones (ABA and GA3) showed genotype dependent variations and high calcium is related with stress tolerance and in turn plant tissue culture responsiveness. The results of the present study clearly elucidate the importance of selection of genotypes based on biochemical indices such as innate phytonutrients, phytochemicals and phytohormones for the development of an efficient regeneration protocol in finger millet to introgres the potential genes whose functions validated through genetic transformation studies in Arabidopsis could further be harnessed for crop improvement especially for improving calcium
nutrition and stress tolerance.