Plant Two-Component Signaling Systems and the Role of Response Regulators

Two-component signaling systems, in their most simple form, involve a histidine kinase and a response regulator that initiate cellular responses to environmental stimuli. Two-component signaling systems were originally thought to be unique to prokaryotes. However, in the early 1990s researchers identified three plant genes that bear similarity to bacterial two-component system members, showing that these systems also exist in some eukaryotic species.In a review published in the February 2002 issue of Plant Physiology, (vol 128:363-9), Jens Lohrmann and Klaus Harter at the Institute of Biology, Freiburg University, Germany, describe the latest developments on two-component signaling systems in higher plants. Unlike the prokaryotic systems, multi-step two-component signaling systems are prevalent in plants. These systems involve additional proteins, aside from the initial kinase, involved in phosphotransfer to the ultimate response regulator protein target. Sequencing of the Arabidopsis genome has made it possible to identify 17 histidine and hybrid kinase proteins (AHKs), five histidine phosphotransfer kinases (HPts) and 23 response regulars (ARRs). Preliminary studies have shown that, with the exception of the phytochromes, the majority of AHKs, HPts and ARRs are involved in the phosphotransfer process.Histidine kinase and hybrid kinase receptors are the first members of two-component signaling systems. Plant receptors such as CRE1, a cytokinin receptor, and ETR1, an ethylene receptor, have been identified at the molecular level. Both ETR1 and CRE1 are examples of hybrid kinases. The proteins encoded by these genes have a conserved histidine residue in the transmitter domain and a conserved aspartate residue in the receiver domain. Upon binding to the hormone ligand, a phosphate group derived from ATP is transferred first to the conserved His, then to the conserved Asp. The phosphate group is then shifted to a Hpt protein and then to one or more ARR proteins.

The authors describe the structures of two-component signaling system elements in Arabidopsis. CRE1 and ETR1 have characteristic amino-terminal input domains and hormone-binding domains, followed by a transmitter domain and receiver- or receiver-like domains. Hpt proteins, designated as AHPs in Arabidopsis, are short proteins with a conserved His residue. ARR proteins may be of two types, type A or type B. Type A ARRs are shorter, having a receiver domain and only a minor output domain. The expression of type A ARRs is induced by cytokinin. Type B ARRs are longer, having a receiver domain and complex output domain that includes a nuclear localization sequence, Pro- and Gln-rich regions that serve as transactivation domains, and a GARP motif (named for proteins – Golden2, ARR and Psr1 – that contain this domain) that is responsible for DNA binding. Recent research has suggested that type B ARRs function as transcription factors. The expression of type B ARRs is not induced by cytokinin.

The authors describe a model phosphorelay multistep two-component system in more detail, focusing on cytokinin signal transduction as an example. The input domain the membrane-bound receptor CRE1 binds to cytokinin, resulting in autophosphorylation of the transmitter domain and then transfer of phosphate to the receiver domain of CRE1. The phosphate is relayed to a His residue of AHP2, which then enters the nucleus and transfers the phosphate to an Asp residue of the type B response regulators ARR1 and ARR2. After phosphotransfer, AHP2 ferries back to the cytosol to repeat the process. The output domains of ARR1 and ARR2 undergo conformational changes as a result of phosphorylation of the receiver domain, inducing binding to target promoter sequences (in this case, the GAT box). Transcription of downstream target genes, such as nCland type-A ARR genes, is facilitated by the phosphorylated type B ARR proteins.

The authors also describe a model, arising from recent research on ARRs, that provides a mechanism for cross-talk between cytokinin and red light signal transduction pathways. Upon exposure to red light, phytochrome B (phyB) is converted from an inactive form to an active form. Conversion back to the inactive form can occur either by exposure to far-red light (a process called photoconversion), or in a light-independent process (termed dark reversion). Evidence indicates that the type A ARR4 can stabilize the active (Pfr) form of phyB, thus slowing dark reversion. Consistent with this model, plants over-expressing ARR4show hypersensitivity to red light, while plants expressing a version of ARR4 in which the conserved Asp residue has been mutated show hyposensitivity to red light. The cross-talk enabled by this system may allow interplay between cytokinin and red light signals.

As multi-step two-component systems are studied in more detail, a network of interactions between members of such systems is beginning to emerge. The authors describe the conclusions of research on these networks. For example, the hybrid kinase receptors ETR1 and CRE1 can both interact with AHP1 and AHP2, but the hybrid kinase CKII can only interact with AHP2. The type B response regulator ARR2 can interact with both AHP1 and AHP2, whereas the type A response regulator ARR4 can only interact with AHP1. The authors state that the functional implication of such interactions is that two-component systems may allow plants to sense and integrate a broad range of signals. Complex interconnections between these systems may help explain the overlapping physiological responses that have been observed, for example, in studies on responses to ethylene and cytokinin.

The authors emphasize two possible explanations for the existence of multi-step two-component systems in plants. The membrane compartmentation occuring in eukaryotic cell results in hybrid kinases that may be localized to the plasma membrane and response regulator proteins that may be localized within the nucleus. In Arabidopsis, it seems that AHPs function to transfer phosphate groups between kinase factors and response regulators. In addition, the presence of multi-step systems, in principle, enables a network of interactions between various two-component system members. This may allow a more finely tuned set of physiological responses to arise from the sum total of interactions occurring in the cell at any given time. Although there has been a considerable progress in recent years elucidating the mechanisms of multi-step two-component signaling systems in plants, the authors conclude that there is a long way to go before the complexity of these pathways is fully understood. However, the authors advocate the use of molecular, cell biological, genetic and biochemical approaches to shed light on the mechanisms of these systems.

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