Role of calcium–calmodulin in auxin-induced somatic embryogenesis in leaf base cultures of wheat (Triticum aestivum var. HD 2329)

Abstract

Wheat leaf bases cultured for 1 day on 2,4-D (10 lM) display the induction of somatic embryogenesis. The induction of somatic embryogenesis by 2,4-D appears to be calcium-mediated as treatment of leaf bases with the calcium chelator, EGTA, prior to 2,4-D treatment, inhibited the induction of somatic embryogenesis. This sensitivity of auxin to reduced calcium levels can be reversed by calcium ions alone and not any other divalent cation like magnesium or zinc. Additionally, the expression of the three calciumregulated genes, Triticum aestivum calmodulin binding protein kinase, calcium-dependent protein kinase, and putative calcium binding protein was analyzed in wheat leaf bases which suggest a specific role for Ca 2+ in somatic embryogenesis. Application of the calcium ionophore, A23187, either alone or along with 2,4-D, induced somatic embryogenesis. This specificity for calcium was verified both by treatment with the calcium antagonist TMB8, and the elimination of calcium from the medium, resulting in reduction of somatic embryogenesis by 80%. Treatment with calcium channel blockers like verapamil and nifedipine, calcium antagonist, lanthanum, and calmodulin inhibitors chlorpromazine and fluphenazine, prior to the 2,4-D treatment, inhibited induction of somatic embryogenesis. The present study thus provides evidence for the involvement of calcium-calmodulin in the stimulus-response coupling of auxin-induced somatic embryogenesis in wheat leaf base system.

Analysis of expression profile of selected genes expressed during auxin-induced somatic embryogenesis in leaf base system of wheat (Triticum aestivum) and their possible interactions

“…The transcript levels of BHLH-like protein could be enhanced by heat, cold, salt and mannitol, but ABA did not have much effect. Calcium ions, known to transduce auxin signal for somatic embryogenesis (Li et al 2003;Du and Poovaiah 2005;Quint and Gray 2006;Mahalakshmi et al 2007), also induced the expression of genes encoding LTP, spermidine synthase, BHLH-like protein and putative malate dehydrogenase and its effect could be reversed partially or completely by the calcium chelator, EGTA, for the four genes studied (Fig. 7B).…”

“…The enhancement of somatic embryogenesis by calcium (Jansen et al 1990;Mahalakshmi et al 2007) and the presence of vacuolar Ca 2+ as the first signal that allows the recognition of embryogenic cells (Verdus et al 1993) is well documented. Calmodulin-mediated developmental regulation during various stages of somatic embryogenesis has also been demonstrated (Timmers et al 1989;Overvoorde and Grimes 1994;.…”

“…1; Mahalakshmi et al 2003Mahalakshmi et al , 2007. This response is auxin specific and provides a unique opportunity to monitor the molecular events associated with auxin-mediated induction of somatic embryogenesis.…”

“…Once embryogenesis has been induced, the role of auxin changes in that the embryos begin to synthesize their own auxin (Michalczuk et al 1992a, b). In the present investigation, a cDNA library was constructed from 13-day-old wheat leaf bases after short-term 2,4-D application, which has been previously demonstrated to undergo somatic embryogenesis in culture (Mahalakshmi et al 2003(Mahalakshmi et al , 2007, and screened for the isolation of genes that are expressed differentially on auxin application during somatic embryogenesis.…”

Isolation and characterization of a novel ammonium overly sensitive mutant, amos2, in Arabidopsis thaliana

“…supply have been documented in a variety of experimental systems (Wilcox et al 1973;Gerendas et al 1995Gerendas et al , 1997Cao and Tibbitts 1998;Roosta and Schjoerring 2007), and these are seen both in WT and amos2, reflecting no difference in this target of ammonium toxicity. Calcium is widely known to regulate growth and development of plants (Hepler 1988;Mahalakshmi et al 2007), including the control of flowering and vegetative organogenic pathways (Capitani and Altamura 2004;Iwano et al 2009), and adventitious root formation (Bellamine et al 1998;Lanteri et al 2006;Li et al 2008). In addition to effects on Ca 2?…”