In the phosphorelay-mediated cytokinin signal transduction of Arabidopsis thaliana, certain members of the type-B authentic response regulator (ARR) family are implicated in the regulatory networks that are primarily propagated by the cytokinin-receptors [authentic histidine kinases (AHKs)]. Clarification of the involvement of each type-B ARR transcription factor in cytokinin-responsive phenomena is still at a very early stage. Here we analyzed the redundant function of two type-B ARR genes, ARR10 and ARR12, by constructing an arr10/arr12 double mutant. The resulting mutant plants showed stronger phenotypes with special reference to the cytokinin action in roots (e.g. inhibition of root elongation, green callus formation from root explants) than those for each single mutant, suggesting that ARR10 and ARR12 redundantly play an important role in the cytokinin signaling in roots. This idea was further supported by results from root-specific microarray analyses with the double mutant plant. We also showed that ARR10 and ARR12 are involved in the AHK-dependent signaling pathway that negatively regulates protoxylem specification in root vascular tissues. When the double mutant is combined with an arr1 allele, the resultant arr1/arr10/arr12 triple mutant showed phenotypes displaying a very poor growth, quite similar to those of the wooden leg (wol) mutant that virtually lacks cytokinin receptor activities in plants. In this triple arr mutant, the specification of root vascular tissues is also affected as severely as in wol. Taken together, we propose that ARR10 and ARR12, together with ARR1, redundantly play pivotal roles in the AHK-dependent phosphorelay signaling in response to cytokinin in roots.

Increased-branching mutants of garden pea (Pisum sativum; ramosus [rms]) and Arabidopsis (Arabidopsis thaliana; more axillary branches) were used to investigate control of cytokinin export from roots in relation to shoot branching. In particular, we tested the hypothesis that regulation of xylem sap cytokinin is dependent on a long-distance feedback signal moving from shoot to root. With the exception of rms2, branching mutants from both species had greatly reduced amounts of the major cytokinins zeatin riboside, zeatin, and isopentenyl adenosine in xylem sap compared with wild-type plants. Reciprocally grafted mutant and wild-type Arabidopsis plants gave similar results to those observed previously in pea, with xylem sap cytokinin down-regulated in all graft combinations possessing branched shoots, regardless of root genotype. This long-distance feedback mechanism thus appears to be conserved between pea and Arabidopsis. Experiments with grafted pea plants bearing two shoots of the same or different genotype revealed that regulation of root cytokinin export is probably mediated by an inhibitory signal. Moreover, the signaling mechanism appears independent of the number of growing axillary shoots because a suppressed axillary meristem mutation that prevents axillary meristem development at most nodes did not abolish long-distance regulation of root cytokinin export in rms4 plants. Based on double mutant and grafting experiments, we conclude that RMS2 is essential for long-distance feedback regulation of cytokinin export from roots. Finally, the startling disconnection between cytokinin content of xylem sap and shoot tissues of various rms mutants indicates that shoots possess powerful homeostatic mechanisms for regulation of cytokinin levels.