Strains of Escherichia coli that express two different cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, were used to study the relative sensitivity of these receptors to various cytokinins. Both receptors were most sensitive to the bases of the isoprenoid-type cytokinins trans-zeatin and isopentenyladenine but differed significantly in the recognition of other cytokinin compounds. In particular, CRE1/AHK4 recognized at 1 microm concentration only trans-zeatin while AHK3 recognized cis-zeatin and dihydrozeatin as well, although with a lower sensitivity. Similarly, CRE1/AHK4 was not activated by cytokinin ribosides and ribotides, but AHK3 was. Comparisons using the ARR5::GUS fusion gene as a cytokinin reporter in Arabidopsis showed similar relative degrees of responses in planta, except that cytokinins with aromatic side chains showed much higher activities than in the bacterial assay. These results indicate that the diverse cytokinin compounds might have specific functions in the numerous cytokinin-regulated processes, which may depend in turn on different receptors and their associated signalling pathways. The importance of precise control of local concentrations of defined cytokinin metabolites to regulate the respective downstream event is corroborated.

We tested the hypothesis that nitric oxide (NO) plays an important role in cytokinin signaling. Inhibitors of NO-synthase (NOS), L-NMMA and L-NAME, inhibited the expression of the GUS gene controlled by the cytokinin-responsive ARR5 promoter. However, the inactive analogues D-NMMA and D-NAME had a similar inhibitory activity. NO donors alone did not induce GUS activity and the NO scavenger cPTIO did not prevent the induction of the ARR5 promoter by cytokinin. Northern blot analysis of the P ARR5 ::GUS transgene and the host ARR5 gene revealed that cytokinin-induced transcript accumulation was not altered by NMMA-treatment, indicating that NMMA acts post-transcriptionally. Together the data show that NO has no direct role in eliciting the primary cytokinin response in plants.

Using Amaranthus and transgenic Arabidopsis P ARR5 : GUS seedlings in bioassays, we studied the effects of exogenous polyamines on the development of cytokinin-induced effects based on the expression of the cytokinin primary response genes. Transgenic Arabidopsis contained a reporter GUS gene under the control of Arabidopsis ARR5 promoter. In both bioassays, we found that all polyamines tested suppressed the effects induced by 5 µ M benzyladenine (BA). Spermine and putrescine were more efficient as BA antagonists than cadaverine and spermidine. Polyamines affected posttranscriptional stages of BA action but did not interfere with the accumulation of cytokinin-dependent mRNA. This fact contradicts the hypotheses about the existence of a common receptor for polyamines and BA or about polyamines as second messengers in the cytokinin signaling pathway. In Arabidopsis seedlings, exogenous polyamines suppressed BA effects at low (micromolar) concentrations comparable with BA concentrations. The incubation of Arabidopsis seedlings with aminoguanidine (an inhibitor of putrescine and cadaverine catabolism) for 24 h resulted in a significant reduction in the activity of the cytokinin primary response gene. These data indicate that endogenous polyamines can modulate considerably the amplitude of the cytokinin primary response gene expression in vivo.Abbreviations and designations : AG-aminoguanidine; BAbenzyladenine; MU-4-methylunbelliferone; MUG-4-methylumbelliferyl-β -D-glucuronide; P ARR5 : GUS -the reporter GUS gene under the control of the promoter of the cytokinin-dependent ARR5 gene.

No abstract

No abstract