Cytokinin and Auxin-Induced Regulation of Protein Synthesis and Poly (A) + RNA Accumulation in Catharanthus roseus Cell Cultures

Abstract

2,4-dichlorophenoxyacetic acid (2,4-D)-dependent C. roseus cells were groven for three days in a 2,4-D-free medium, then treated with 4.5 μM 2,4-D, 5 μM zeatin or 4.5 μM 2,4-D + 5 μM zeatin. Hormonetreated cells were labelled in vivo with [35S]-methionine and polypeptides were analyzed by two-dimensional polyacrylamide gel electrophoresis. Translation products of poly (A)+ RNAs isolated from cells treated with hormones were also taken as indicators of differential gene expression. Hormone treatments did not achieve dramatic changes in polypeptide patterns and changes in in vitro polypeptide synthesis were fewer than those encountered in vivo. However, accumulation of specific RNAs coding for 18 and 28 kDa polypeptides was demonstrated under conditions of alkaloid production in the cells (i.e., when cells veere grown in 2,4-D-free, zeatin-containing medium). Their molecular masses and isoelectric points are identical to those of two polypeptides whose in vivo synthesis are similarly regulated by 2,4-D and for zeatin. These polypeptides are candidates for a direct or indirect regulatory role in alkaloid synthesis in C. roseus cells, and particularly the polypeptide of 28 kDa whose in vivo and in vitro synthesis are repressed by 2,4D, yet enhanced by zeatin. Another polypeptide of 16 kDa might be derived from the 18 kDa polypeptide in a post-translational process.