Identification and Functional Characterization of P2- type Calcium ATPases in Selected Species of Gramineae

Abstract

Calcium and manganese perform various important biological functions within the plant body, including signal transduction, proper cell wall formation, photosynthesis, respiration and nitrogen assimilation etc. Calcium and manganese exist in the form of ions within cells. P2- type calcium ATPases are responsible for maintaining homeostasis of these ions in both domains of life. P2- type ATPases are further divided into P2A (ECAs) and P2B- types (ACAs). In present research work, BLAST searches in different databases were conducted for the retrieval of calcium ATPase sequences of selected monocots and a phylogenetic analysis was performed. Furthermore, Triticum aestivum plants were grown under calcium and manganese stress using hydroponic culture. The tissue generated was used to find out the expression of selected P2A and P2B- type calcium ATPases under manganese and calcium stress. Moreover, an ECA3 construct was generated and it was transformed into yeast model (Pmr1) to find out the possible role of this gene in manganese nutrition. Overall, phylogenetic analysis revealed a high degree of evolutionary relatedness among P2- type calcium ATPases. High expression of selected ACAs was observed in T. aestivum plants grown under calcium stress, which gave clues to the role of ACAs in signal transduction in T. aestivum. High expression of selected T. aestivum ECAs under calcium and manganese stress was also observed which may indicate the potential roles of these ATPases in calcium and manganese nutrition in T. aestivum. Additionally, ECA3 gene (which is a P2A- type ATPase) was amplified using T. aestivum cDNA and sequenced subsequently. The data indicated that it retained introns in some of the transcripts. The qRT-PCR analysis revealed that events of intron retention in ECA3 transcripts were much enhanced when plants were exposed to calcium stress. ECA3 construct was further transformed into yeast mutant Pmr1, which is a null mutant for xi its own manganese pump and cannot grow on high manganese concentration in the medium. Interestingly, ECA3 rescued the phenotype and helped the mutant to grow successfully on manganese toxic media. This further indicated, the importance of ECA3 in manganese translocation in T. aestivum. Hence, ECA3 gene is reported here as first P2A- type pump identified in T. aestivum and is required for manganese management in this species. its own manganese pump and cannot grow on high manganese concentration in the medium. Interestingly, ECA3 rescued the phenotype and helped the mutant to grow successfully on manganese toxic media. This further indicated, the importance of ECA3 in manganese translocation in T. aestivum. Hence, ECA3 gene is reported here as first P2A- type pump identified in T. aestivum and is required for manganese management in this species.