Type-B Arabidopsis thaliana response regulators (ARRs) are transcription factors that function in the final step of twocomponent signaling systems. To characterize their role in plant growth and development, we isolated T-DNA insertions within six of the genes (ARR1, ARR2, ARR10, ARR11, ARR12, and ARR18) from the largest subfamily of type-B ARRs and also constructed various double and triple combinations of these mutations. Higher order mutants revealed progressively decreased sensitivity to cytokinin, including effects on root elongation, lateral root formation, callus induction and greening, and induction of cytokinin primary response genes. The triple mutant arr1,10,12 showed almost complete insensitivity to cytokinin under many of the assay conditions used. By contrast, no significant change in the sensitivity to ethylene was found among the mutants examined. These results indicate that there is functional overlap among the type-B ARRs and that they act as positive regulators of cytokinin signal transduction.

The type B Arabidopsis Response Regulators (ARRs) of Arabidopsis thaliana are transcription factors that act as positive regulators in the two-component cytokinin signaling pathway. We employed a mutant-based approach to perform a detailed characterization of the roles of ARR1, ARR10, and ARR12 in plant growth and development. The most pronounced phenotype was found in the arr1-3 arr10-5 arr12-1 triple loss-of-function mutant, which showed almost complete insensitivity to high levels of exogenously applied cytokinins. The triple mutant exhibited reduced stature due to decreased cell division in the shoot, enhanced seed size, increased sensitivity to light, altered chlorophyll and anthocyanin concentrations, and an aborted primary root with protoxylem but no metaxylem. Microarray analysis revealed that expression of the majority of cytokinin-regulated genes requires the function of ARR1, ARR10, and ARR12. Characterization of double mutants revealed differing contributions of the type B ARRs to mutant phenotypes. Our results support a model in which cytokinin regulates a wide array of downstream responses through the action of a multistep phosphorelay that culminates in transcriptional regulation by ARR1, ARR10, and ARR12.

Two-component signaling systems, involving His kinases, His-containing phosphotransfer proteins, and response regulators, have been implicated in plant responses to hormones and environmental factors. Genomic analysis of Arabidopsis supports the existence of 22 response regulators (ARRs) that can be divided into at least two distinct groups designated type-A and type-B. Phylogenetic analysis indicates that the type-B family is composed of one major and two minor subfamilies. The expression of the type-B ARRs was examined by using both reverse transcription-PCR and b-glucuronidase fusion constructs. The major subfamily of type-B ARRs showed particularly high expression in regions where cytokinins play a significant role, including cells in the apical meristem region and in young leaves that would be undergoing cell division. Multiple members within this same subfamily of type-B ARRs were expressed near the root tip with highest expression in the root elongation zone. b-Glucuronidase-fusions to full-length ARR2, ARR12, and ARR19 were nuclear localized, consistent with a role in transcriptional regulation. These data suggest that differing expression levels of the type-B ARRs may play a role in modulating the cellular responses to cytokinin.Plant two-component signaling systems have been implicated in vital cellular processes such as the responses to cytokinins, ethylene, red light, and osmosensing (Schaller, 2000;Hutchison and Kieber, 2002;Hwang et al., 2002;Schaller et al., 2002). Twocomponent systems were originally identified in bacteria, and in their simplest form involve a receptor kinase that autophosphorylates itself on a conserved His residue in response to an environmental stimulus (Mizuno, 1997;Stock et al., 2000). This phosphate is then transferred to a conserved Asp residue within the receiver domain of a response regulator. Phosphorylation of the response regulator modulates its ability to mediate downstream signaling in the pathway. Of particular relevance to plants is a permutation on the twocomponent system known as the multi-step phosphorelay (Swanson et al., 1994;Schaller, 2000). The multi-step phosphorelay makes use of three components: a hybrid receptor kinase that contains both His-kinase and receiver domains in one protein, a His-containing phosphotransfer (HPt) protein, and a separate response regulator. In these multi-step phosphorelays the phosphate is transferred from amino acid to amino acid in sequence His to Asp to His to Asp.In Arabidopsis, proteins with homology to all elements of the two-component system have been identified, including His kinases, response regulators, and HPt proteins (Schaller, 2000(Schaller, , 2002Hutchison and Kieber, 2002;Hwang et al., 2002). Phosphorylation activity has been confirmed in each case. Thus, all elements needed to establish a histidyl-aspartyl phosphorelay are represented in plants. Analysis of the Arabidopsis genome reveals the existence of 8 His kinases, 22 response regulators, and 5 HPt proteins that contain all the conserved residues required ...

The ethylene receptor family of Arabidopsis consists of five members, one of these being ETR1. The effect of ethylene pathway mutations upon expression of ETR1 was examined. For this purpose, ETR1 levels were quantified in mutant backgrounds containing receptor loss-of-function mutations, ethylene-insensitive mutations, and constitutive ethylene response mutations. Ethylene-insensitive mutations of ETR1 resulted in a posttranscriptional increase in levels of the mutant receptor. Treatment of seedlings with silver, which leads to ethylene insensitivity, also resulted in an increase in levels of ETR1. Loss-of-function mutations of ETR1 resulted in both transcriptional and posttranscriptional changes in levels of the receptor. Most other ethylene pathway mutations, including a newly isolated T-DNA insertion mutation in the gene encoding the ethylene receptor ERS1, had relatively minor effects upon the expression of ETR1. Our results indicate that mutations in ETR1 can affect expression at the posttranscriptional level, and suggest that these posttranscriptional changes may contribute to the phenotypes observed in the mutants. Our results also refine the model on how mutations in ethylene receptors are able to confer dominant ethylene insensitivity upon plants.Ethylene (C 2 H 4 ) is a simple gaseous hydrocarbon that has profound effects upon plant growth and development. Ethylene regulates seed germination, seedling growth, leaf and petal abscission, organ senescence, ripening, stress responses, and pathogen responses (Mattoo and Suttle, 1991; Abeles et al., 1992). An important contribution to our understanding of ethylene signal transduction has come from the identification of mutants in Arabidopsis with altered ethylene sensitivity (Chang and Shockey, 1999;Stepanova and Ecker, 2000). These mutations fall into two main classes: (a) mutations that render a plant insensitive to ethylene, and (b) mutations that result in a constitutive ethylene response. Characterization of Arabidopsis mutants has led to the identification of ethylene receptors and additional components in the ethylene signal transduction pathway.The ethylene receptor family of Arabidopsis contains five members (ETR1, ETR2, ERS1, ERS2, and EIN4; Schaller, 2000; Chang and Stadler, 2001), with ethylene binding confirmed for ETR1 and ERS1 Rodriguez et al., 1999; Hall et al., 2000). The receptors contain three Nterminal transmembrane domains that encompass the ethylene-binding site Rodriguez et al., 1999). The binding site contains a copper cofactor that is required for the high-affinity ethylene binding that receptors display (Rodriguez et al., 1999). In the C-terminal half, the receptors contain regions with similarity to His kinases and, in some cases, the receiver domains of response regulators (Schaller, 2000; Chang and Stadler, 2001), signaling elements originally identified as parts of bacterial twocomponent systems (Parkinson, 1993;Schaller, 2000). His kinase activity has been confirmed in vitro for ETR1 (Gamble et al., 1998), but the role of th...

Cytokinins play critical roles in plant growth and development, with the transcriptional response to cytokinin being mediated by the type-B response regulators. In Arabidopsis (Arabidopsis thaliana), type-B response regulators (ARABIDOPSIS RESPONSE REGULATORS [ARRs]) form three subfamilies based on phylogenic analysis, with subfamily 1 having seven members and subfamilies 2 and 3 each having two members. Cytokinin responses are predominantly mediated by subfamily 1 members, with cytokinin-mediated effects on root growth and root meristem size correlating with type-B ARR expression levels. To determine which type-B ARRs can functionally substitute for the subfamily 1 members ARR1 or ARR12, we expressed different type-B ARRs from the ARR1 promoter and assayed their ability to rescue arr1 arr12 double mutant phenotypes. ARR1, as well as a subset of other subfamily 1 type-B ARRs, restore the cytokinin sensitivity to arr1 arr12. Expression of ARR10 from the ARR1 promoter results in cytokinin hypersensitivity and enhances shoot regeneration from callus tissue, correlating with enhanced stability of the ARR10 protein compared with the ARR1 protein. Examination of transfer DNA insertion mutants in subfamilies 2 and 3 revealed little effect on several well-characterized cytokinin responses. However, a member of subfamily 2, ARR21, restores cytokinin sensitivity to arr1 arr12 roots when expressed from the ARR1 promoter, indicating functional conservation of this divergent family member. Our results indicate that the type-B ARRs have diverged in function, such that some, but not all, can complement the arr1 arr12 mutant. In addition, our results indicate that type-B ARR expression profiles in the plant, along with posttranscriptional regulation, play significant roles in modulating their contribution to cytokinin signaling.

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SUMMARYThe life cycle of higher plants alternates between the diploid sporophytic and the haploid gametophytic phases. In angiosperms, male and female gametophytes develop within the sporophyte. During female gametophyte (FG) development, a single archesporial cell enlarges and differentiates into a megaspore mother cell, which then undergoes meiosis to give rise to four megaspores. In most species of higher plants, including Arabidopsis thaliana, the megaspore closest to the chalaza develops into the functional megaspore (FM), and the remaining three megaspores degenerate. Here, we examined the role of cytokinin signaling in FG development. We characterized the FG phenotype in three triple mutants harboring non-overlapping T-DNA insertions in cytokinin AHK receptors. We demonstrate that even the strongest mutant is not a complete null for the cytokinin receptors. Only the strongest mutant displayed a near fully penetrant disruption of FG development, and the weakest triple ahk mutant had only a modest FG phenotype. This suggests that cytokinin signaling is essential for FG development, but that only a low threshold of signaling activity is required for this function. Furthermore, we demonstrate that there is elevated cytokinin signaling localized in the chalaza of the ovule, which is enhanced by the asymmetric localization of cytokinin biosynthetic machinery and receptors. We show that an FM-specific marker is absent in the multiple ahk ovules, suggesting that disruption of cytokinin signaling elements in Arabidopsis blocks the FM specification. Together, this study reveals a chalazal-localized sporophytic cytokinin signal that plays an important role in FM specification in FG development.

Hormonal interactions are crucial for plant development. In Arabidopsis, cytokinins inhibit root growth through effects on cell proliferation and cell elongation. Here, we define key mechanistic elements in a regulatory network by which cytokinin inhibits root cell elongation in concert with the hormones auxin and ethylene. The auxin importer AUX1 functions as a positive regulator of cytokinin responses in the root; mutation of AUX1 specifically affects the ability of cytokinin to inhibit cell elongation but not cell proliferation. AUX1 is required for cytokinin-dependent changes of auxin activity in the lateral root cap associated with the control of cell elongation. Cytokinin regulates root cell elongation through ethylene-dependent and -independent mechanisms, both hormonal signals converging on AUX1 as a regulatory hub. An autoregulatory circuit is identified involving the control of ARR10 and AUX1 expression by cytokinin and auxin, this circuit potentially functioning as an oscillator to integrate the effects of these two hormones. Taken together, our results uncover several regulatory circuits controlling interactions of cytokinin with auxin and ethylene, and support a model in which cytokinin regulates shootward auxin transport to control cell elongation and root growth.