Calcium-mediated signaling during sandalwood somatic embryogenesis. Role for exogenous calcium as second messenger

The possible involvement of Ca2+-mediated signaling in the induction/regulation of somatic emioryogenesis from proembryogenic cells of sandalwood (Santalum album) has been investigated. Ca-45(2+)-uptake studies and fura-2 fluorescence ratio photometry were used to measure changes in [Ca2+](cyt) of pro-embryogenic cells in response to culture conditions conducive for embryo development. Sandalwood pro-embryogenic cell masses (PEMs) are obtained in the callus proliferation medium that contains the auxin 2,2-dichlorophenoxyacetic acid. Subculture of PEMs into the embryo differentiation medium, which lacks 2,4-dichlorophenoxyacetic acid and has higher osmoticum, results in a 4-fold higher Ca-45(2+) incorporation into the symplast. Fura-2 ratiometric analysis corroboratively shows a 10- to 16-fold increase in the [Ca2+](cyt) of PEMs, increasing from a resting concentration of 30 to 50 nM to 650 to 800 nM. Chelation of exogenous Ca2+ with ethyleneglycol-bis(aminoethyl ether)-N,N'-tetraacetic acid arrests such an elevation in [Ca2+](cyt). Exogenous Ca2+ when chelated or deprived also arrests embryo development and inhibits the accumulation of a sandalwood Ca2+-dependent protein kinase. However, such culture conditions do not cause cell death as the PEMs continue to proliferate to form larger cell dumps. Culture treatment with N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide reduced embryogenic frequency by 85%, indicating that blockage of Ca2+-mediated signaling pathway(s) involving sandalwood Ca2+-dependent protein kinase and/or calmodulin causes the inhibition of embryogenesis. The observations presented are evidence to suggest a second messenger role for exogenous Ca2+ during sandalwood somatic embryogenesis.