Overexpression of Arabidopsis response regulators,ARR4/ATRR1/IBC7 and ARR8/ATRR3, alters cytokinin responses differentially in the shoot and in callus formation

Arabidopsis ARR4/ATRR1/IBC7 and ARR8/ATRR3 are homologous genes of prokaryotic response regulators that are involved in the His-Asp phosphorelay signal transduction. We analyzed the function of these genes as response regulators using transgenic plants. Overexpression of ARR4 in cultured stems of the transgenics markedly promoted shoot formation in the presence of cytokinin, while overexpression of ARR8 repressed shoot formation and greening of calli. The expression level of cytokinin-inducible genes, cycD3 and cab increased in the ARR4 overexpressor but decreased in the ARR8 overexpressor. By contrast, two drought stress-inducible genes, rd29A and erd1, were expressed in both overexpressors as that in control plants. These results suggest that ARR4 and ARR8 are involved in cytokinin signal transduction, and that ARR4 functions as a positive-regulator, whereas ARR8 functions as a negative-regulator. Furthermore, microarray analysis showed that several genes were up-regulated in the ARR4 overexpressor. Consistent with these results, ARR4 and ARR8 might play important roles in the sensoring system of cytokinin signal transduction pathway in various developmental and environmental conditions and the regulation of gene expression.     

Biochemical characterization of a putative cytokinin-responsive His-kinase, CKI1, from Arabidopsis thaliana

His-Asp phosphorelays are evolutionary-conserved powerful biological tactics for intracellular signal transduction. Such a phosphorelay is generally made up of "sensor histidine (His)-kinases", "response regulators", and "histidine-containing (HPt) phosphotransmitters". Results from recent intensive studies suggested that in the higher plant Arabidopsis thaliana, His-Asp phosphorelays may be widely used for propagating environmental stimuli, such as phytohormones (e.g., ethylene and cytokinin). In this study, we characterized, in vitro, the putative cytokinin-responsive CKI1 His-kinase, in terms of His-Asp phosphorelays. It was demonstrated for the first time that the receiver domain in this sensor exhibits a strong phosphohistidine phosphatase activity toward some Arabidopsis HPt phosphotransmitters (AHP1 and AHP2), suggesting the functional importance of the receiver domain for a resumed interaction of the sensor His-kinase with other His-Asp phosphorelay components.     

Compilation and characterization of Arabidopsis thaliana response regulators implicated in His-Asp phosphorelay signal transduction.

His-Asp phosphorelays are evolutionary-conserved powerful biological tactics for intracellular signal transduction. Such a phosphorelay is generally made up of "sensor histidine (His)-kinases", "response regulators", and "histidine-containing (HPt) phosphotransmitters". In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that His-Asp phosphorelays may be widely used for propagating environmental stimuli, such as phytohormones (e.g., ethylene and cytokinin). In this study, we first inspected extensively the occurrence of Arabidopsis response regulators in order to compile and characterize them. The results showed that this higher plant has, at least, 14 members of the family of response regulators that can be classified into two distinct subtypes (type-A and type-B), as judged from their structural designs, biochemical properties, and expression profiles. Comparative studies were conducted for each representative (ARR3 and ARR4 for type-A, and ARR10 for type-B). It was suggested that expression of the type-A response regulator is cytokinin-inducible, while that of the type-B response regulator appears to be not. Results from yeast two-hybrid analyses suggested that the type-B response regulator may have an ability to stably interact with a set of HPt phosphotransmitters (AHPs). These and other results will be discussed with special reference to the His-Asp phosphorelay signaling network in Arabidopsis thaliana.     

His-Asp phosphorelay signaling: a communication avenue between plants and their environment

His-Asp phosphorelay systems have been recently discovered in plants and have emerged as some of the most important signaling systems. The phosphorelay systems in plants include components with sensor (His-protein kinase) domains, His-containing phosphotransfer (HPt) domains, and receiver (response regulator) domains. Recent studies implicate phosphorelay systems in sensing and propagating signals from a wide variety of external and/or internal stimuli such as ethylene, cytokinin, and osmolarity. In maize and Arabidopsis, some response regulators are up-regulated by both cytokinins and nitrate. These findings imply that the His-Asp phosphorelay may operate in an inorganic nitrogen-signaling pathway mediated by cytokinin in plants.     

The altered responses of a new mutant long life span 1 to cytokinin in Arabidopsis thaliana

Cytokinins are a class of essential plant hormones regulating plant growth and development.Although the two-component phosphorelay pathway of cytokinin has been well characterized,the intact cytokinin responses regulation picture still needs to be fully depicted.Here we report a new mutant,long life span 1(lls1),which displays dwarf stature,curled leaves,numerous axillary branches and nearly 5-month life span.Exogenous cytokinin could not recover the phenotypes of the mutant.Moreover,mutation in lls1 suppressed the cytokinin-responsive phenotypes,including root and hypocotyl growth inhibition,anthocyanin accumulation,metaxylem promotion in primary root development.The induction of cytokinin-responsive genes,ARR5,AHP5,and CKX3,was also suppressed in lls1.According to quantitative RT-PCR(qRT-PCR) and microarray results,the basal expression of positive factors AHP5,ARR1,and ARR10 were down-regulated,while the negative factors ARR4 and ARR5 were up-regulated.Our results suggested that LLS1 gene might be involved in the regulation of cytokinin signaling.It was mapped to chromosome 4 where no other cytokinin relevant gene has been reported.