ISOLATION AND CHARACTERIZATION OF CDNAS FOR CYTOKININ-REPRESSED GENES

As an approach to the primary action of cytokinins, we studied the rep ression of gene expression which occurs shortly after the application of this hormone. First we studied the changes in the translatable mRNA population during dark incubation of etiolated cucumber cotyledons wi th benzyladenine (BA). Two dimensional gel electrophoresis of basic an d neutral proteins showed that several spots changed 1 or 2 h after BA application. Among them, three were markedly repressed. Next we isola ted cDNA clones for the cytokinin-repressed genes CR9 and CR20 by diff erential screening. The CR9 cDNA is 588 bp long, and would encode a pr otein consisting of 137 amino-acid residues, having a molecular mass o f 15 kDa. The composition of amino-acid residues indicates that the pr otein is either neutral or weakly acidic. The hydropathy plot showed t hat it is probably soluble rather than associated with membranes. The deduced amino-acid sequence shows that it contains two similar sequenc es of 18 amino-acid residues, each containing two conserved cysteines at an interval of 7 residues. It shows 48% identity with lirl, a light -induced gene from rice [24]. The CR9 transcript began to decrease as early as 1 h after BA application, reaching an extremely low level at 4 h, preceding the initiation of BA-induced cotyledon expansion, altho ugh, it began to recover after 8 h. The repression is BA-dose dependen t, and highly specific for cytokinins. The CR9 transcript was abundant in mature and senescent leaves, but was not found in roots or young l eaves. Wounding and illumination also caused a transient decrease in t he CR9 transcript level. When seedlings were grown under a light/dark cycle, expression of CR9 exhibited diurnal fluctuation with an increas e in the light period. Expression of another cytokinin-repressed gene, CR20, showed the same pattern of changes as that of CR9 in terms of c ytokinin-repression, BA-dose response, cytokinin-specificity, wounding and light effects, although it showed a broad organ specificity. It a lso exhibited diurnal changes, but opposite to those observed with CR9 , showing an increase in the dark period. The nucleotide sequence of C R20 cDNA is quite different from that of CR9 and shows no significant homology with any sequences in the databases. There are many stop codo ns, hence no long open reading frame to encode a polypeptide. Possible roles of CR9 and CR20 in cytokinin-induced physiological changes are discussed.