Isolation and characterisation of cDNAs for cytokinin-repressed genes

Abstract

As an approach to the primary action of cytokinins, we studied the repression 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 with benzyladenine (BA). Two dimensional gel electrophoresis of basic and neutral proteins showed that several spots changed 1 or 2 h after BA application. Among them, three were markedly repressed. Next we isolated cDNA clones for the cytokinin-repressed genes CR9 and CR20 by differential screening. The CR9 cDNA is 588 bp long, and would encode a protein consisting of 137 amino-acid residues, having a molecular mass of 15 kDa. The composition of amino-acid residues indicates that the protein is either neutral or weakly acidic. The hydropathy plot showed that it is probably soluble rather than associated with membranes. The deduced amino-acid sequence shows that it contains two similar sequences 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, although, it began to recover after 8 h. The repression is BA-dose dependent, and highly specific for cytokinins. The CR9 transcript was abundant in mature and senescent leaves, but was not found in roots or young leaves. Wounding and illumination also caused a transient decrease in the CR9 transcript level. When seedlings were grown under a light/dark cycle, expression of CR9 exhibited diurnal fluctuation with an increase in the light period. Expression of another cytokinin-repressed gene, CR20, showed the same pattern of changes as that of CR9 in terms of cytokininrepression, BA-dose response, cytokinin-specificity, wounding and light effects, although it showed a broad organ specificity. It also exhibited diurnal changes, but opposite to those observed with CR9, showing an increase in the dark period. The nucleotide sequence of CR20 cDNA is quite different from that of CR9 and shows no significant homology with any sequences in the databases. There are many stop codons, hence no long open reading frame to encode a polypeptide. Possible roles of CR9 and CR20 in cytokinin-induced physiological changes are discussed.