Rapid changes in gene expression were studied during incubation of cucumber (Cucumis sativus L.) cotyledons with cytokinins in darkness. Complementary-DNA clones for mRNAs whose levels decreased within 4 h of treatment with N6-benzyladenine (BA) were isolated by differential hybridization. One of them (CR9) was sequenced. It is 588 bp long, and would encode a protein consisting of 137 amino-acid residues and having a molecular mass of 15 kDa. The sequence shows a high homology with a light-induced gene from rice. Northern blot analysis of the CR9 transcript showed the level of the mRNA (0.7 kb) to decrease tenfold within 4 h of BA treatment, i.e. well before BA-induced cotyledon expansion was observed. The repression became greater with increasing concentration of BA (10(-8)-10(-5) M). The expression of the CR9 gene was repressed specifically by cytokinins (BA, isopentenyladenine and t-zeatin), but not by adenine or 2,4-dichlorophenoxyacetic acid (auxin). The results are discussed in relation to the primary action of cytokinin.
H. 1993. Benzyladenine-induced changes in the translatable mRNA population in excised cucumber cotyledons. -Physio! Plant. 87: 584-591.Benzyladenine-induced changes in the translatable mRNA population in excised cucumber cotyledons were studied. Poly (A)* RNA was prepared from etiolated cotyledons incubated with or without benzyladenine (BA) for various periods in the dark. Using nonequilibrium pH gradient eiectrophoresis-SDS poiyaeryiamide gel electrophoresis and isoelectric focusing-SDS polyacrylamide gel electrophoresis. both basic and neutral proteins translated in vitro were separated. About 240 spots were detected and 16 of them changed within 6 h after BA application. Some spots changed quickly (within 1-2 hj. Among them, three were repressed markedly.
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.
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