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

摘要

Calcium (Ca2+) 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 Ca2+ immobilized metal affinity chromatography (Ca2+ IMAC), revealed the presence of Ca2+ binding proteins (CaBPs), while Ca2+-dependent amylase activity peaked 18 h PI, implying Ca2+ presence before its release from Ca-phytate, indicating an alternative source of Ca2+. 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. Ca2+-binding capacity as well as cell-wall bound Ca2+, 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 Ca2+ binding capacity, as well as cell-wall bound Ca2+. Imbibition in the presence of chelators, Ca2+-channel blockers, and H+-pump inhibitors, interfered with germination and radical extension. Further, EDTA-treated cotyledon protein extracts separated on Ca2+ IMAC showed a larger CaBP peak than control cotyledon extracts. Pooled fractions clearly showed Ca2+-induced extrinsic fluorescence with anilino-napthalene sulfonate. The results strongly implicate the apoplast may be a major source of Ca2+ in the initial phase of germination and seedling establishment in Vigna seeds.