Plants continuously maintain pools of totipotent stem cells in their apical meristems from which elaborate root and shoot systems are produced. In Arabidopsis thaliana, stem cell fate in the shoot apical meristem is controlled by a regulatory network that includes the CLAVATA (CLV) ligand-receptor system and the homeodomain protein WUSCHEL (WUS). Phytohormones such as auxin and cytokinin are also important for meristem regulation. Here we show a mechanistic link between the CLV/WUS network and hormonal control. WUS, a positive regulator of stem cells, directly represses the transcription of several two-component ARABIDOPSIS RESPONSE REGULATOR genes (ARR5, ARR6, ARR7 and ARR15), which act in the negative-feedback loop of cytokinin signalling. These data indicate that ARR genes might negatively influence meristem size and that their repression by WUS might be necessary for proper meristem function. Consistent with this hypothesis is our observation that a mutant ARR7 allele, which mimics the active, phosphorylated form, causes the formation of aberrant shoot apical meristems. Conversely, a loss-of-function mutation in a maize ARR homologue was recently shown to cause enlarged meristems.
Type-A Arabidopsis (Arabidopsis thaliana) response regulators (ARRs) are a family of 10 genes that are rapidly induced by cytokinin and are highly similar to bacterial two-component response regulators. We have isolated T-DNA insertions in six of the type-A ARRs and constructed multiple insertional mutants, including the arr3,4,5,6,8,9 hextuple mutant. Single arr mutants were indistinguishable from the wild type in various cytokinin assays; double and higher order arr mutants showed progressively increasing sensitivity to cytokinin, indicating functional overlap among type-A ARRs and that these genes act as negative regulators of cytokinin responses. The induction of cytokinin primary response genes was amplified in arr mutants, indicating that the primary response to cytokinin is affected. Spatial patterns of ARR gene expression were consistent with partially redundant function of these genes in cytokinin signaling. The arr mutants show altered red light sensitivity, suggesting a general involvement of type-A ARRs in light signal transduction. Further, morphological phenotypes of some arr mutants suggest complex regulatory interactions and gene-specific functions among family members.
The phytohormone cytokinin is an important regulator of plant growth and development; however, relatively few genes that mediate cytokinin responses have been identified. Genome-wide analyses of Arabidopsis seedlings using the approximately 8,300-element Affymetrix Arabidopsis GeneChips (Affymetrix, Santa Clara, CA) to examine cytokinin-responsive genes were conducted, revealing at least 30 genes whose steady-state level of mRNA was elevated and at least 40 that were down-regulated at multiple time points after application of cytokinin. The cytokinin up-regulated genes include the type-A Arabidopsis response regulators (ARRs), which had been shown previously to be cytokinin primary response genes, cytokinin oxidase, which encodes an enzyme that degrades cytokinins, and several transcription factors. Cytokinin down-regulated genes include several peroxidases and kinases and an E3 ubiquitin ligase. We identified a common sequence motif enriched in the upstream regions of the most consistently cytokinin up-regulated genes. This motif is highly similar to the optimal DNA-binding sites for ARR1/ARR2, type-B ARRs that have been implicated in the transcriptional elevation of the type-A ARRs. Additionally, genome-wide analyses of cytokinin receptor mutants (wol/cre1) revealed large-scale changes in gene expression, including down-regulation of the type-A ARRs and several meristem and cell cycle genes, such as CycD3. Mutations in CRE1 reduced but did not eliminate the effect of cytokinin on gene expression for a subset of cytokinin-responsive genes and had little or no effect on others, suggesting functional redundancy among the cytokinin receptors.Cytokinins are a group of adenine derivatives that affect multiple aspects of plant growth and development, including cell division, vascular development, sink/source relationships, apical dominance, and leaf senescence (Binns, 1994; Mok, 1994, 2001). A pathway for cytokinin biosynthesis and metabolism is emerging from molecular and biochemical studies. This has been highlighted by the recent cloning of several genes encoding enzymes involved in cytokinin biosynthesis or metabolism, including ipt (Kakimoto, 2001; Takei et al., 2001), which catalyzes the first committed step in cytokinin biosynthesis; cytokinin oxidase (Houba-Hérin et al., 1999;Morris et al., 1999), which cleaves the N 6 side chain from cytokinins; and several enzymes that catalyze the conjugation of sugar moieties to cytokinins (Martin et al., 1999a(Martin et al., , 1999b(Martin et al., , 2001. A model for cytokinin perception and signal transduction has emerged that is similar to prokaryotic two-component response pathways (Haberer and Kieber, 2001; Hutchison and Kieber, 2002;Lohrmann and Harter, 2002). A family of genes that are similar to bacterial two-component response regulators, the type-A Arabidopsis response regulators (ARRs), was identified as cytokinin primary response genes (Brandstatter and Kieber, 1998;Sakakibara et al., 1998;Taniguchi et al., 1998; D'Agostino et al., 2000). The cytokinin re...
The plant hormone cytokinin regulates many aspects of growth and development. Cytokinin signaling involves His kinase receptors that perceive cytokinin and transmit the signal via a multistep phosphorelay similar to bacterial two-component signaling systems. The final targets of this phosphorelay are a set of Arabidopsis thaliana Response Regulator (ARR) proteins containing a receiver domain with a conserved Asp phosphorylation site. One class of these, the type-A ARRs, are negative regulators of cytokinin signaling that are rapidly transcriptionally upregulated in response to cytokinin. In this study, we tested the role of phosphorylation in type-A ARR function. Our results indicate that phosphorylation of the receiver domain is required for type-A ARR function and suggest that negative regulation of cytokinin signaling by the type-A ARRs most likely involves phosphorylation-dependent interactions. Furthermore, we show that a subset of the type-A ARR proteins are stabilized in response to cytokinin in part via phosphorylation. These studies shed light on the mechanism by which type-A ARRs act to negatively regulate cytokinin signaling and reveal a novel mechanism by which cytokinin controls type-A ARR function.
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