Abstract
Wheat leaf bases cultured for 1 day on 2,4-d (10 μM) 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 calcium-regulated 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 Ca2+ 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.
Similar content being viewed by others
References
Abel S, Nguyen MD, Theologis A (1995) The PS-IAA4/5-like family of early auxin-inducible mRNAs in Arabidopsis thaliana. J Mol Biol 251:533–549
Anil VS, Rao KS (2000) Calcium-mediated signaling during sandalwood somatic embryogenesis. Role for exogenous calcium as second messenger. Plant Physiol 123:1301–1311
Bhatla SC, Haschke HP, Hartmann E (2003) Distribution of activated calmodulin in the chloronema tip cells of the moss Funaria hygrometrica. J Plant Physiol 160:469–474
Braybrook SA, Stone SL, Park S, Bui AQ, Brandon HL, Fischer R, Goldberg RB, Harada JJ (2006) Genes directly regulated by LEAFY COTYLEDON2 provide insight into the control of embryo maturation and somatic embryogenesis. Proc Natl Acad Sci 103:3468–3473
Brummell DA, Maclachlan GA (1989) Calcium antagonist TMB-8 inhibits cell wall formation and growth in pea. J Exp Bot 40:559–565
Chugh A, Khurana P (2002) Gene expression during somatic embryogenesis—recent advances. Curr Sci 83:715–730
Cohen JD, Nadler KD (1976) Calcium requirements for indoleacetic acid-induced acidification by Avena coleoptiles. Plant Physiol 57:347–350
Cunninghame ME, Hall JL (1986) The effect of calcium antagonists and inhibitors of secretory processes on auxin-induced elongation and fine structure of Pisum sativum stem segments. Protoplasma 133:149–159
Davletava S, Meszaros T, Miskolczi P, Obersschall A, Torok K, Magyar Z, Dudits D, Deak M (2001) Auxin and heat shock activation of a novel member of the calmodulin like domain protein kinase gene family in cultured alfalfa cells. J Exp Bot 52:215–221
Du L, Poovaiah BW (2005) Ca2+/calmodulin is critical for brassinosteroid biosynthesis and plant growth. Nature 437:741–745
Feher A, Pasternak TP, Dudits D (2003) Transition of somatic cells to an embryogenic state. Plant Cell Tissue Organ Cult 74:201–228
Gaj MD, Zhang S, Harada JJ, Lemaux G (2005) Leafy cotyledon genes are essential for induction of somatic embryogenesis of Arabidopsis. Planta [Epub]
Harada JJ (2001) Role of Arabidopsis LEAFY COTYLEDON genes in seed development. J Plant Physiol 158:405–409
Hepler PK (1988) Calcium and development. In: Koeltz, Gruelter W, Zimmer B (eds) Proceedings of XIV international botanical congress, Konigstein/Taunus, pp 225–240
Hepler PK, Wayne RO (1985) Calcium and plant development. Annu Rev Plant Physiol 36:397–439
Huang ML, Cangelosi GA, Halperin W, Nester EW (1990) A chromosomal Agrobacterium tumefaciens gene required for effective plant signal transduction. J Bacteriol 172:1814–1822
Hush JM, Overall RI, Newman IA (1991) A calcium influx precedes organogenesis in Graptopetalum. Plant Cell Environ 14:657–665
Jackson CL, Cassanova JE (2000) Turning on ARF: the Sec7 family of guanine-nucleotide-exchange factors. Trends Cell Biol 10:60–67
Jansen MAK, Booij H, Schell JHN, De Vries SC (1990) Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures. Plant Cell Rep 9:221–223
Li S, Xing GM, Cui KR, Yu CH, Zhang X, Xu HX, Wang YF (2003) Ultracytochemical localization of calcium and ATPase activity on the 2,4-d induced somatic embryogenesis of Lycium barbarum L. Shi Yan Sheng Wu Xue Bao 36:414–420 (in Chinese)
Mahalakshmi A, Khurana JP, Khurana P (2003) Rapid induction of somatic embryogenesis by 2,4-d in leaf base cultures of wheat (Triticum aestivum L). Plant Biotechnol 20:267–273
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Muto S (1982) Distribution of calmodulin within wheat leaf cells. FEBS Lett 147:161–164
Nagy F, Schafer E (2000) Nuclear and cytosolic events of light-induced, phytochrome-regulated signaling in higher plants. EMBO J 19:157–163
Nomura K (1987) Mechanisms of somatic embryogenesis in carrot suspension cultures. Ph.D. thesis, University of Tokyo, Japan
Overvoorde PI, Grimes HD (1994) The role of calcium and calmodulin in carrot somatic embryo. Plant Cell Physiol 35:135–144
Patnaik D, Khurana P (2005) Identification of a phosphoprotein expressed during somatic embryogenesis in wheat leaf base cultures. J Plant Biochem Biotechnol 14:149–154
Pfluger J, Zambryski P (2004) The role of SUESS in auxin response and floral organ pattering. Development 131:4697–4707
Poovaiah BW, Reddy ASN (1987) Calcium messenger system in plants. CRC Crit Rev Plant Sci 6:47–103
Poovaiah BW, Reddy ASN (1993) Calcium and signal transduction in plants. CRC Crit Rev Plant Sci 12:185–211
Quint M, Gary WM (2006) Auxin signaling. Curr Opin Plant Biol. DOI 10.1016/j.pbi.2006.07.006
Raghothama KG, Mizrah IY, Poovaiah BW (1985) Effect of calmodulin antagonists on auxin-dependent elongation. Plant Physiol 79:28–33
Reddy VS, Ali GS, Reddy ASN (2002) Molecular and biochemical characterization of a calcium/calmodulin-binding protein kinase from rice. Biochem J 368:145–157
Sanders D, Pelloux J, Brouwnlee C, Harper JF (2002) Calcium at the crossroads of signaling. Plant Cell (Suppl.)14:S401–S417
Singla B, Khurana JP, Khurana P (2006) An early auxin-responsive Aux/IAA gene from wheat (Triticum aestivum) is induced by epibrassinolide and differentially regulated by light and calcium. J Exp Bot. DOI 10.1093/jxb/erl182
Snedden WA, Fromm H (2001) Calmodulin as a versatile signal transducer in plants. New Phytol 151:35–66
Steenhoudt O, Vanderleyden J (2000) Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. FEMS Microbiol Rev 24:487–506
Stone SL, Kwong LW, Yee KM, Pelletier J, Lepiniec L, Fischer RL, Goldberg RB, Harada JJ (2001) LEAFY COTYLEDON2 encodes B3 domain transcription factor that induces embryo development. Proc Natl Acad Sci USA 98:11806–11811
Tanimoto S, Harada H (1986) Involvement of calcium in adventitious bud initiation in Torenia stem segments. Plant Cell Physiol 27:1–10
Timmers ACJ, Schel JHN (1990) Immunocytochemical localization of calmodulin during carrot somatic embryogenesis. In: Nijkamp HJJ, Van der Plas LHW, Van Aartrijk J (eds) Progress in plant cellular and molecular biology. Kluwer Academic Publishers, Dordrecht, pp 443–448
Timmers ACJ, De Vries SC, Schel JHN (1989) Distribution of membrane-bound calcium and activated calmodulin during somatic embryogenesis of carrot (Daucus carota L.). Protoplasma 153:24–29
Trewavas AJ, Malho R (1998) Ca2+ signaling in plant cells: the big network! Curr Opin Plant Biol 1:428–433
Xie QG, Wang W, Liang S, Lu Y (2002) Characterization of a calmodulin-binding protein kinase from Arabidopsis thaliana. Chin Sci Bull 47:1650–1655
Xing GM, Jing RF, Li S, Zhang X, Xu HX, Cui KR, Yu CH, Wang YF (2004) Role of exogenous calcium in the somatic embryogenesis of Lycium barbarum L. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 30:261–268
Zhang L, Lu Y-T (2003) Calmodulin-binding protein kinases in plants. Trends Plant Sci 8:123–127
Acknowledgments
AM and BS acknowledges the award of Senior Research Fellowship from the University of Grants Commission (UGC), New Delhi. This work was financially supported by the Department of Biotechnology, Government of India and the UGC, New Delhi.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mahalakshmi, A., Singla, B., Khurana, J.P. et al. Role of calcium–calmodulin in auxin-induced somatic embryogenesis in leaf base cultures of wheat (Triticum aestivum var. HD 2329). Plant Cell Tiss Organ Cult 88, 167–174 (2007). https://doi.org/10.1007/s11240-006-9186-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-006-9186-z