Cytokinins regulate spatially specific ethylene production to control root growth in Arabidopsis.

Two principal growth regulators, cytokinins and ethylene, are known to interact in the regulation of plant growth. However, information about the underlying molecular mechanism and positional specificity of cytokinin/ethylene crosstalk in the control of root growth is scarce. We have identified the spatial specificity of cytokinin-regulated root elongation and root apical meristem (RAM) size, both of which we demonstrate to be dependent on ethylene biosynthesis. Upregulation of the cytokinin biosynthetic gene ISOPENTENYLTRANSFERASE (IPT) in proximal and peripheral tissues leads to both root and RAM shortening. By contrast, IPT activation in distal and inner tissues reduces RAM size while leaving the root length comparable to that of mock-treated controls. We show that cytokinins regulate two steps specific to ethylene biosynthesis: production of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) by ACC SYNTHASEs (ACSs) and its conversion to ethylene by ACC OXIDASEs (ACOs). We describe cytokinin- and ethylene-specific regulation controlling the activity of ACSs and ACOs that are spatially discrete along both proximo/distal and radial root axes. Using direct ethylene measurements, we identify ACO2, ACO3, and ACO4 as being responsible for ethylene biosynthesis and ethylene-regulated root and RAM shortening in cytokinin-treated Arabidopsis. Direct interaction between ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), a member of the multistep phosphorelay cascade, and the C-terminal portion of ETHYLENE INSENSITIVE 2 (EIN2-C), a key regulator of canonical ethylene signaling, is involved in the cytokinin-induced, ethylene-mediated control of ACO4. We propose tight cooperation between cytokinin and ethylene signaling in the spatially specific regulation of ethylene biosynthesis as a key aspect of the hormonal control of root growth.

The Arabidopsis B-type response regulator 18 homomerizes and positively regulates cytokinin responses.

In higher plants, the two-component system (TCS) is a signaling mechanism based on a His-to-Asp phosphorelay. The Arabidopsis TCS involves three different types of proteins, namely the histidine kinases (AHKs), the histidine phosphotransfer proteins (AHPs) and the response regulators (ARRs). The ARRs comprise three different families, namely A, B and C types, according to their protein structure. While some members of the B-type family of ARRs have been studied extensively and reported to act as DNA-binding transcriptional regulators, very limited information is available for other B-type ARRs such as ARR18. In this study, we characterize in detail the molecular and functional properties of ARR18. ARR18 acts as a transcriptional regulator in plant cells and forms homodimers in planta as shown by FRET-FLIM studies. As demonstrated by mutational analysis, the aspartate at position 70 (D70) in the receiver domain of ARR18 acts as crucial phosphorylation site. The modification of D70 affects the response regulator's ability to homodimerize and to activate its target genes. Furthermore, physiological investigations of Arabidopsis lines ectopically expressing ARR18 introduce ARR18 as a new member within the cytokinin-regulated response pathway regulating root elongation.

Exploring the Binding Affinity of the ARR2 GARP DNA Binding Domain via Comparative Methods.

Plants have evolved signaling mechanisms such as the multi-step phosphorelay (MSP) to respond to different internal and external stimuli. MSP responses often result in gene transcription regulation that is modulated through transcription factors such as B-type Arabidopsis response regulator (ARR) proteins. Among these proteins, ARR2 is a key component that is expressed ubiquitously and is involved in many aspects of plant development. Although it has been noted that B-type ARRs bind to their cognate genes through a DNA-binding domain termed the GARP domain, little is known about the structure and function of this type of DNA-binding domain; thus, how ARRs bind to DNA at a structural level is still poorly understood. In order to understand how the MSP functions in planta, it is crucial to unravel both the kinetics as well as the structural identity of the components involved in such interactions. For this reason, this work focusses on resolving how the GARP domain of ARR2 (GARP2) binds to the promoter region of ARR5, one of its native target genes in cytokinin signaling. We have established that GARP2 specifically binds to the ARR5 promoter with three different bi-molecular interaction systems-qDPI-ELISA, FCS, and MST-and we also determined the KD of this interaction. In addition, structural modeling of the GARP2 domain confirms that GARP2 entails a HTH motif, and that protein-DNA interaction most likely occurs via the α3-helix and the N-terminal arm of this domain since mutations in this region hinder ARR2's ability to activate transcription.

New Insights into Multistep-Phosphorelay (MSP)/ Two-Component System (TCS) Regulation: Are Plants and Bacteria that Different?

The Arabidopsis multistep-phosphorelay (MSP) is a signaling mechanism based on a phosphorelay that involves three different types of proteins: Histidine kinases, phosphotransfer proteins, and response regulators. Its bacterial equivalent, the two-component system (TCS), is the most predominant device for signal transduction in prokaryotes. The TCS has been extensively studied and is thus generally well-understood. In contrast, the MSP in plants was first described in 1993. Although great advances have been made, MSP is far from being completely comprehended. Focusing on the model organism Arabidopsis thaliana, this review summarized recent studies that have revealed many similarities with bacterial TCSs regarding how TCS/MSP signaling is regulated by protein phosphorylation and dephosphorylation, protein degradation, and dimerization. Thus, comparison with better-understood bacterial systems might be relevant for an improved study of the Arabidopsis MSP.

ARR22 overexpression can suppress plant Two-Component Regulatory Systems.

In plants, several developmental processes are co-coordinated by cytokinins via phosphorylation dependent processes of the Two-Component System (TCS). An outstanding challenge is to track phosphorelay flow from cytokinin perception to its molecular outputs, of which gene activation plays a major role. To address this issue, a kinetic-based reporter system was expounded to track TCS phosphorelay activity in vivo that can distinguish between basal and cytokinin dependent effects of overexpressed TCS members. The TCS phosphorelay can be positively activated by cytokinin and inhibited by pharmaceuticals or naturally interfering components. In this case we took advantage of the phosphohistidine-phosphatase Arabidopsis Response Regulator (ARR) 22 and investigated its phosphocompetition with other TCS members in regulating promoters of ARR5 and WUS in Arabidopsis thaliana cell culture protoplasts. In congruency with the proposed function of ARR22, overexpression of ARR22 blocked the activation of all B-type ARRs in this study in a TCS dependent manner. Furthermore, this effect could not be mimicked by A-type response regulator overexpression or compensated by AHP overexpression. Compared to other reporter assays, ours mimicked effects previously observed only in transgenic plants for all of the TCS proteins studied, suggesting that it is possible to expose phosphocompetition. Thus, our approach can be used to investigate gene signaling networks involving the TCS by leveraging ARR22 as a TCS inhibitor along with B-type ARR overexpression.

Arabidopsis response regulator 22 inhibits cytokinin-regulated gene transcription in vivo.

Cytokinin signaling in Arabidopsis is carried out by a two-component system (TCS) multi-step phosphorelay mechanism that involves three different protein families: histidine kinases (AHKs), phosphotransfer proteins (AHPs), and response regulators (ARRs) that are in turn, subdivided into A-, B- and C-type ARRs depending on their function and structure. Upon cytokinin perception, AHK proteins autophosphorylate; this phosphate is then transferred from the AHKs to the AHPs to finally reach the ARRs. When B-type ARRs are activated by phosphorylation, they function as transcription factors that regulate the expression of cytokinin-dependent genes such as the A-type ARRs, among many others. In cytokinin signaling, while A- and B-type ARR function is well understood, it is still unclear if C-type ARRs (ARR22 and ARR24) play a role in this mechanism. Here, we describe a novel method suitable to study TCS activity natively as an in vivo system. We also show that ARR22 inhibits gene transcription of an A-type ARR upon cytokinin treatment in vivo. Consequently, we propose that ARR22, by acting as a phosphatase on specific AHPs, disrupts the TCS phosphorelay and prevents B-type ARR phosphorylation, and thus their activation as transcription factors, explaining the observed deactivation of cytokinin-responsive genes.

The interaction of the Arabidopsis response regulator ARR18 with bZIP63 mediates the regulation of PROLINE DEHYDROGENASE expression.

As the first and rate-limiting enzyme of proline degradation, PROLINE DEHYDROGENASE1 (PDH1) is tightly regulated during plant stress responses, including induction under hypoosmolarity and repression under water deficit. The plant receptor histidine kinases AHKs, elements of the two-component system (TCS) in Arabidopsis thaliana, are proposed to function in water stress responses by regulating different stress-responsive genes. However, little information is available concerning AHK phosphorelay-mediated downstream signaling. Here we show that the Arabidopsis type-B response regulator 18 (ARR18) functions as a positive osmotic stress response regulator in Arabidopsis seeds and affects the activity of the PDH1 promoter, known to be controlled by C-group bZIP transcription factors. Moreover, direct physical interaction of ARR18 with bZIP63 was identified and shown to be dependent on phosphorylation of the conserved aspartate residue in the ARR18 receiver domain. We further show that bZIP63 itself functions as a negative regulator of seed germination upon osmotic stress. Using reporter gene assays in protoplasts, we demonstrated that ARR18 interaction negatively interferes with the transcriptional activity of bZIP63 on the PDH1 promoter. Our findings provide new insight into the function of ARR18 and bZIP63 as antagonistic regulators of gene expression in Arabidopsis.

Identification of two-component system elements downstream of AHK5 in the stomatal closure response of Arabidopsis thaliana.

To optimize water use efficiency, plants regulate stomatal closure through a complex signaling process. Hydrogen peroxide (H2O2) is produced in response to several environmental stimuli, and has been identified as a key second messenger involved in the regulation of stomatal aperture. The Arabidopsis histidine kinase 5 (AHK5) has been shown to regulate stomatal closure in response to H2O2 and other stimuli that depend on H2O2. AHK5 is a member of the two-component system (TCS) in Arabidopsis. The plant TCS comprises three different protein types: the hybrid histidine kinases (HKs), the phosphotransfer proteins (HPs) and the response regulators (RRs). Here we determined TCS elements involved in H2O2- and ethylene-dependent stomatal closure downstream of AHK5. By yeast and in planta interaction assays and functional studies, AHP1, 2 and 5 as well as the response regulators ARR4 and ARR7 were identified acting downstream of AHK5 in the ethylene and H2O2 response pathways of guard cells. Furthermore, we demonstrate that aspartate phosphorylation of ARR4 is only required for the H2O2- but not for the ethylene-induced stomatal closure response. Our data suggest the presence of a complex TCS signaling network comprising of at least AHK5, several AHPs and response regulators, which modulate stomatal closure in response to H2O2 and ethylene.

The histidine kinase AHK5 integrates endogenous and environmental signals in Arabidopsis guard cells.

BACKGROUND: Stomatal guard cells monitor and respond to environmental and endogenous signals such that the stomatal aperture is continually optimised for water use efficiency. A key signalling molecule produced in guard cells in response to plant hormones, light, carbon dioxide and pathogen-derived signals is hydrogen peroxide (H(2)O(2)). The mechanisms by which H(2)O(2) integrates multiple signals via specific signalling pathways leading to stomatal closure is not known. PRINCIPAL FINDINGS: Here, we identify a pathway by which H(2)O(2), derived from endogenous and environmental stimuli, is sensed and transduced to effect stomatal closure. Histidine kinases (HK) are part of two-component signal transduction systems that act to integrate environmental stimuli into a cellular response via a phosphotransfer relay mechanism. There is little known about the function of the HK AHK5 in Arabidopsis thaliana. Here we report that in addition to the predicted cytoplasmic localisation of this protein, AHK5 also appears to co-localise to the plasma membrane. Although AHK5 is expressed at low levels in guard cells, we identify a unique role for AHK5 in stomatal signalling. Arabidopsis mutants lacking AHK5 show reduced stomatal closure in response to H(2)O(2), which is reversed by complementation with the wild type gene. Over-expression of AHK5 results in constitutively less stomatal closure. Abiotic stimuli that generate endogenous H(2)O(2), such as darkness, nitric oxide and the phytohormone ethylene, also show reduced stomatal closure in the ahk5 mutants. However, ABA caused closure, dark adaptation induced H(2)O(2) production and H(2)O(2) induced NO synthesis in mutants. Treatment with the bacterial pathogen associated molecular pattern (PAMP) flagellin, but not elf peptide, also exhibited reduced stomatal closure and H(2)O(2) generation in ahk5 mutants. SIGNIFICANCE: Our findings identify an integral signalling function for AHK5 that acts to integrate multiple signals via H(2)O(2) homeostasis and is independent of ABA signalling in guard cells.

Functional cross-talk between two-component and phytochrome B signal transduction in Arabidopsis.

The A-type response regulator ARR4 is an element in the two-component signalling network of Arabidopsis. ARR4 interacts with the N-terminus of the red/far-red light photoreceptor phytochrome B (phyB) and functions as a modulator of photomorphogenesis. In concert with other A-type response regulators, ARR4 also participates in the modulation of the cytokinin response pathway. Here evidence is presented that ARR4 directly modulates the activity state of phyB in planta, not only under inductive but also under extended irradiation with red light. Mutation of the phosphorylatable aspartate to asparagine within the receiver domain creates a version of ARR4 that negatively affects photomorphogenesis. Additional evidence suggests that ARR4 activity is regulated by a phosphorelay mechanism that depends on the AHK family of cytokinin receptors. Accordingly, the ability of ARR4 to function on phyB is modified by exogenous application of cytokinin. These results implicate a cross-talk between cytokinin and light signalling mediated by ARR4. This cross-talk enables the plant to adjust light reponsiveness to endogenous requirements in growth and development.