Atsushi Deji

Molecular characterization of His-Asp phosphorelay signaling factors in maize leaves: Implications of the signal divergence by cytokinin-inducible response regulators in the cytosol and the nuclei

Genes for histidyl-aspartyl (His-Asp) phosphorelay components (His-containing phosphotransfer proteins, HP, and response regulators, RR) were isolated from Zea mays L. to characterize their function in cytokinin signaling. Six type-A RRs (ZmRR1, ZmRR2, ZmRR4–ZmRR7), 3 type-B RRs (ZmRR8–ZmRR10), and 3 HPs (ZmHP1–ZmHP3) were found in leaves. All type-A RR genes expressed in leaves were up-regulated by exogenous cytokinin. Transient expression of fusion products of the signaling modules with green fluorescent protein in epidermal leaf cells suggested cytosolic and nuclear localizations of ZmHPs, whereas type-B ZmRR8 was restricted to the nucleus. Type-A RRs were localized partly to the cytosol (ZmRR1, ZmRR2, and ZmRR3) and partly to the nucleus (ZmRR4, ZmRR5, and ZmRR6). In the yeast two-hybrid assay, ZmHP1 and ZmHP3 interacted with both cytosolic ZmRR1 and nuclear type-B ZmRRs. In vitro experiments demonstrated that ZmHPs function as a phospho-donor for ZmRRs; turnover rates of the phosphorylated state were tenfold lower in ZmRR8 and ZmRR9 than in ZmRR1 and ZmRR4. These results suggest that the His-Asp phosphorelay signaling pathway might diverge into a cytosolic and a nuclear branch in leaves of maize, and that the biochemical nature of ZmRRs is different in terms of stability of the phosphorylated status.

Hormone-mediated nitrogen signaling in plants: implication of participation of abscissic acid in negative regulation of cytokinin-inducible expression of maize response regulator

Abstract In addition to nitrate ion itself, plant growth regulators such as cytokinin function to communicate nitrogen availability between organs. The cytokinin-mediated nitrogen signal is transmitted by a His-Asp phosphorelay system in the target cells. Although mutual crosstalk among the growth regulators in various aspects has been reported, the effect of other hormones on the cytokinin-mediated nitrogen signaling has not been characterized well. In maize (Zea mays L.), abscissic acid (ABA) concentration in tissue is affected by nitrogen availability: it is remarkably higher in the aged leaves than younger leaves under a nitrogen-limited condition, whereas the tendency is reversed in a sufficient condition. The delivery rate of ABA from root to shoot via xylem is also increased under a nitrogen-limited cultivation. These profiles of ABA are reciprocal to those of cytokinin. Cytokinin-dependent induction of ZmRR 1 and ZmRR 2, response regulators, is repressed by co-treatment with ABA in a dose-dependent manner. These results imply that ABA participates in cytokinin-mediated nitrogen signaling by means of negative regulation of ZmRRs.

Accumulation of Maize Response Regulator Proteins in Mesophyll Cells after Cytokinin Treatment

The maize response regulator genes ZmRR1 and ZmRR2 respond to cytokinin, and the translated products seem to be involved in nitrogen signal transduction mediated by cytokinin through the His-Asp phosphorelay. To elucidate the physiological function of the proteins, we examined the temporal and spatial distribution in maize leaves by immunochemical analysis and use of transgenic plants. ZmRR1 and ZmRR2 polypeptides could be distinctively detected by western blotting. The polypeptides accumulated in leaves within 5 h of the supply of nitrate to nitrogen-depleted maize, and the accumulation was transient. The extent of induction was larger in the leaf tip, which is rich in photosynthetically matured cells, than elsewhere. In leaves, the polypeptides accumulated mostly in mesophyll cells. Histochemical analyses of transgenic maize harboring a ZmRR1 promoter-β-glucuronidase fusion gene also showed most of the expression to be in these cells. These results suggest that ZmRR1 and ZmRR2 are induced in mesophyll cells and function in nitrogen signal transduction mediated by cytokinin.