Role of apoplastic calcium during germination and initial stages of seedling establishment in Vigna radiata seeds.

Calcium (Ca) is implicated in the initial phase of seed germination and seedling establishment. It is stored complexed with phytic acid during seed development and released by phytase action during germination. We observed phytase activity 18 h post-imbibition (PI) in Vigna seeds, while radicle protrusion occurred approximately 12 h PI. Cotyledon protein extracts prepared 4, 8, 16 and 24 h PI, subjected to Ca immobilized metal affinity chromatography (Ca IMAC), revealed the presence of Ca binding proteins (CaBPs), while Ca-dependent amylase activity peaked 18 h PI, implying Ca presence before its release from Ca-phytate, indicating an alternative source of Ca. Vigna cotyledon cell-wall preparations 4 h and 24 h PI, titrated against alkali, revealed high cation-binding capacity, and seeds 4 h PI demonstrated high rates of H extrusion. Ca-binding capacity as well as cell-wall bound Ca, measured in cotyledon cell-wall preparations from unimbibed seeds as well as seeds 24 h PI, using a novel competitive chelation technique, showed a marked decline in Ca binding capacity, as well as cell-wall bound Ca. Imbibition in the presence of chelators, Ca-channel blockers, and H-pump inhibitors, interfered with germination and radical extension. Further, EDTA-treated cotyledon protein extracts separated on CaIMAC showed a larger CaBP peak than control cotyledon extracts. Pooled fractions clearly showed Ca-induced extrinsic fluorescence with anilino -napthalene sulfonate. The results strongly implicate the apoplast may be a major source of Ca in the initial phase of germination and seedling establishment in Vigna seeds.