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

Abstract

The possible involvement of Ca²⁺-mediated signaling in the induction/regulation of somatic embryogenesis from pro-embryogenic cells of sandalwood (Santalum album) has been investigated. ⁴⁵Ca²⁺-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 2,4-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 higher⁴⁵Ca²⁺ 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 clumps. Culture treatment withN-(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.