Occurrence of phosphorylated proteins and kinase activity in coconut tissues cultured in vitro in a medium that induces somatic embryogenesis

The presence of tyrosine kinase and tyrosine-phosphorylated proteins was in vestigated in coconut tissues cultured in vitro. In order to study this phe nomenon, plumular explants were taken from mature zygotic embryos and cultu red in a medium that induces somatic embryogenesis. Immunoblot analyses of soluble proteins of coconut cultured tissues with a recombinant monoclonal antibody against phosphotyrosine detected protein bands with molecular mass es ranging from 170 to 27 kDa. The highest response was exhibited by plumul e-forming callus, which decreased both in number and intensity of bands wit h a longer time of in vitro culture. The specific immunodetection was corro borated by incubating the membranes with anti-phosphotyrosine antibody in t he presence of 1 mM phosphotyrosine. Tyrosine phosphorylated proteins was a lso suggested by the presence of phosphoproteins resistant to alkaline trea tment. In plumule, plumular callus and callus with globular embryos and sho ots, a 41-kDa protein remained phosphorylated after alkaline treatment. In plumule, most [P-32]-proteins remained phosphorylated after alkaline treatm ent. Phosphoaminoacid analysis in protein hydrolysates from [P-32]-labelled 41-kDa protein showed the presence of [P-32]-tyrosine and [P-32]-threonine . Evaluation of tyrosine kinase activity in these tissues by the use of RR- SRC, a synthetic peptide substrate (derived from the amino acid sequence su rrounding the phosphorylation site), showed that the activity was highest i n plumule forming callus and initial explant, whereas in other tissues, tyr osine kinase activity decreased to values close to zero. Genistein, a speci fic tyrosine kinase inhibitor, diminished the ability of soluble extracts f rom coconut tissues cultured in vitro to incorporate P-32 into RR-SRC. Thes e results suggest the presence of tyrosine phosphorylated proteins and tyro sine kinase activity in coconut tissues that have been cultured in vitro. ( C) 2000 Editions scientifiques et medicales Elsevier SAS.