Toward an interaction map of the two-component signaling pathway of Arabidopsis thaliana

Among the signal transduction pathways in higher eukaryotes, the two-component system (TCS) is unique to plants. In the model plant Arabidopsis thaliana, it consists of more than 30 proteins, including eight receptors, five phosphotransmitters and 23 response regulators. One of its important functions is to perceive and transduce the signal of the plant hormone cytokinin. The basic signal flow within the TCS is well-understood, but it is unclear how this pathway is integrated with the remainder of the proteome. Thus, knowledge about the interactions of TCS proteins should contribute to the understanding of their mode of action. Therefore, we conducted medium-scale yeast two-hybrid screens focusing on those members of the TCS, which are thought to be involved in cytokinin signaling. In total, more than 6.3 x 10 (7) transformants were screened resulting in the identification of 160 different interactions, of which 136 were novel. Most of the interacting proteins belong to the functional categories of signal transduction and protein metabolism. TCS proteins and their interactors localized to the same subcellular compartment in many cases, a prerequisite to being of biological relevance. The resulting interaction network map revealed large differences in the connectivity. Cytokinin receptors (AHK2, CRE1/AHK4) showed the highest numbers of different interaction partners. This study is the first systematic protein-protein interaction experiment for a plant signal system and provides numerous starting points for further analysis of the molecular mechanisms used to convert the signal carried by the TCS into biological processes.     

Analysis of protein interactions within the cytokinin-signaling pathway of Arabidopsis thaliana

The signal of the plant hormone cytokinin is perceived by membrane-located sensor histidine kinases and transduced by other members of the plant two-component system. In Arabidopsis thaliana, 28 two-component system proteins (phosphotransmitters and response regulators) act downstream of three receptors, transmitting the signal from the membrane to the nucleus and modulating the cellular response. Although the principal signaling mechanism has been elucidated, redundancy in the system has made it difficult to understand which of the many components interact to control the downstream biological processes. Here, we present a large-scale interaction study comprising most members of the Arabidopsis cytokinin signaling pathway. Using the yeast two-hybrid system, we detected 42 new interactions, of which more than 90% were confirmed by in vitro coaffinity purification. There are distinct patterns of interaction between protein families, but only a few interactions between proteins of the same family. An interaction map of this signaling pathway shows the Arabidopsis histidine phosphotransfer proteins as hubs, which interact with members from all other protein families, mostly in a redundant fashion. Domain-mapping experiments revealed the interaction domains of the proteins of this pathway. Analyses of Arabidopsis histidine phosphotransfer protein 5 mutant proteins showed that the presence of the canonical phospho-accepting histidine residue is not required for the interactions. Interaction of A-type response regulators with Arabidopsis histidine phosphotransfer proteins but not with B-type response regulators suggests that their known activity in feedback regulation may be realized by interfering at the level of Arabidopsis histidine phosphotransfer protein-mediated signaling. This study contributes to our understanding of the protein interactions of the cytokinin-signaling system and provides a framework for further functional studies in planta.     

Pathological hormone imbalances

Plant hormones play important roles in regulating developmental processes and signalling networks involved in plant responses to a wide range of biotic and abiotic stresses. Salicylic acid (SA), jasmonates (JA) and ethylene (ET) are well known to play crucial roles in plant disease and pest resistance. However, the roles of other hormones such as abscisic acid (ABA), auxin, gibberellin (GA), cytokinin (CK) and brassinosteroid (BL) in plant defence are less well known. Much progress has been made in understanding plant hormone signalling and plant disease resistance. However, these studies have mostly proceeded independently of each other, and there is limited knowledge regarding interactions between plant hormone-mediated signalling and responses to various pathogens. Here, we review the roles of hormones other than SA, JA and ET in plant defence and the interactions between hormone-mediated signalling, plant defence and pathogen virulence. We propose that these hormones may influence disease outcomes through their effect on SA or JA signalling.     

Cytokinin receptor-dependent and receptor-independent pathways in the dehydration response of Arabidopsis thaliana

Cytokinin signaling in Arabidopsis thaliana utilizes a multi-step two-component signaling (TCS) system comprised of sensor histidine kinases (AHKs), histidine phosphotransfer proteins (AHPs), and response regulators (ARRs). Recent studies have suggested that the cytokinin TCS system is involved in a variety of other signaling and metabolic pathways. To further explore a potential function of the cytokinin TCS in the Arabidopsis dehydration stress response, we investigated the expression of all type-A ARR genes and a type-C ARR, ARR22, in both wild type and ahk single, double, and triple mutants in response to dehydration compared to cytokinin as well as dehydration tolerance of ahk mutants. We found that drought significantly induced the expression of a subset of ARR genes, ARR5, ARR7, ARR15, and ARR22. The results of expression analyses in ahk single, double, and triple mutants demonstrated that the cytokinin receptors AHK2 and AHK3 are redundantly involved in dehydration-inducible expression of ARR7, but not that of ARR5, ARR15, or ARR22. Dehydration tolerance assays showed that ahk2 and ahk3 single mutants exhibited enhanced dehydration tolerance compared with that of wild-type plants and ahk4 mutants, and that ahk2 ahk3 double mutants exhibited stronger drought tolerance than that of ahk3 ahk4, which exhibited more enhanced drought tolerance than that of wild-type plants and ahk single mutants. Taken together, these results demonstrate that while the cytokinin receptors AHK2 and AHK3 are critically involved in the dehydration tolerance response, both cytokinin receptor-dependent pathway and receptor-independent pathway occur in the dehydration response regulating ARR gene expression. In addition, preincubating ahk2, ahk3, ahk4, and the wild-type plants with cytokinin induced enhanced dehydration stress tolerance in these plants, demonstrating that cytokinins are involved in regulating plant response to dehydration stress.     

Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling

Cytokinins are plant hormones involved in regulation of diverse developmental and physiological processes in plants whose molecular mechanisms of action are being intensely researched. However, most rapid responses to cytokinin signals at the proteomic and phosphoproteomic levels are unknown. Early cytokinin responses were investigated through proteome-wide expression profiling based on image and mass spectrometric analysis of two-dimensionally separated proteins and phosphoproteins. The effects of 15 min treatments of 7-day-old Arabidopsis thaliana seedlings with four main cytokinins representing hydroxyisopentenyl, isopentenyl, aromatic, and urea-derived type cytokinins were compared to help elucidate their common and specific function(s) in regulating plant development. In proteome and phosphoproteome maps, significant differences were reproducibly observed for 53 and 31 protein spots, respectively. In these spots, 96 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), providing a snapshot of early links in cytokinin-regulated signalling circuits and cellular processes, including light signalling and photosynthesis, nitrogen metabolism, the CLAVATA pathway, and protein and gene expression regulation, in accordance with previously described cytokinin functions. Furthermore, they indicate novel links between temperature and cytokinin signalling, and an involvement of calcium ions in cytokinin signalling. Most of the differentially regulated proteins and phosphoproteins are located in chloroplasts, suggesting an as yet uncharacterized direct signalling chain responsible for cytokinin action in chloroplasts. Finally, first insights into the degree of specificity of cytokinin receptors on phosphoproteomic effects were obtained from analyses of cytokinin action in a set of cytokinin receptor double mutants.     

The STIR-domain superfamily in signal transduction,development and immunity

We have identified a conserved sequence segment in transmembrane receptors (including SEFs, IL17Rs) and soluble factors (including CIKS/ACT1) in eukaryotes and bacteria - the SEFIR domain. This sequence domain is part of the new STIR domain superfamily comprising also the TIR domain known to mediate TIR-TIR homotypic interactions. In TOLL/IL1R-like pathways, the cytoplasmically localized TIR domain of a receptor and the TIR domain of a soluble adaptor interact physically and activate signalling. The similarity between the SEFIR and TIR domains involves the conserved boxes 1 and 2 of the TIR domain that are implicated in homotypic dimerization, but there is no sequence similarity between SEFIR domains and the TIR sequence box 3. By analogy, we suggest that SEFIR-domain proteins function as signalling components of Toll/IL-1R-similar pathways and that their SEFIR domain mediates physical protein-protein interactions between pathway components.     

Plant hormone perception and action: a role for G-protein signal transduction?

Plants perceive and respond to a profusion of environmental and endogenous signals that influence their growth and development. The G-protein signalling pathway is a mechanism for transducing extracellular signals that is highly conserved in a range of eukaryotes and prokaryotes. Evidence for the existence of G-protein signalling pathways in higher plants is reviewed, and their potential involvement in plant hormone signal transduction evaluated. A range of biochemical and molecular studies have identified potential components of G-protein signalling in plants, most notably a homologue of the G-protein coupled receptor superfamily (GCR1) and the G alpha and G beta subunits of heterotrimeric G-proteins. G-protein agonists and antagonists are known to influence a variety of signalling events in plants and have been used to implicate heterotrimeric G-proteins in gibberellin and possibly auxin signalling. Antisense suppression of GCR1 in Arabidopsis leads to a phenotype which supports a role for this receptor in cytokinin signalling. These observations suggest that higher plants have at least some of the components of G-protein signalling pathways and that these might be involved in the action of certain plant hormones.     

植物TCP转录因子的作用机理及其应用研究进展

TCP转录因子是一类植物特有蛋白,含有保守的TCP domain,其中由60个氨基酸组成的b HLH结构是结合DNA和蛋白互作所必需的。TCP转录因子由于其广泛参与调控植物的生长发育过程(如分枝、株高、叶型、花型等)而备受关注。最近有报道显示,TCP转录因子在植物逆境胁迫应答中(如低温和高盐)同样发挥重要作用。TCP蛋白参与多种信号转导途径(如油菜素内酯、茉莉酸、赤霉素、细胞分裂素等),可能是连接生长发育和介导胁迫响应的一个交叉点。本文从分子生物学角度,系统综述了植物TCP转录因子的作用机理及其在激素应答、发育调控及环境胁迫响应等过程中的功能,以期为基因工程方法改良作物生长模式和抗性提供参考。     

Cyanide is an adequate agonist of the plant hormone ethylene for studying signalling of sensor kinase ETR1 at the molecular level

The plant hormone ethylene is involved in many developmental processes and responses to environmental stresses in plants. Although the elements of the signalling cascade and the receptors operating the ethylene pathway have been identified, a detailed understanding of the molecular processes related to signal perception and transfer is still lacking. Analysis of these processes using purified proteins in physical, structural and functional studies is complicated by the gaseous character of the plant hormone. In the present study, we show that cyanide, a π-acceptor compound and structural analogue of ethylene, is a suitable substitute for the plant hormone for in vitro studies with purified proteins. Recombinant ethylene receptor protein ETR1 (ethylene-resistant 1) showed high level and selective binding of [(14)C]cyanide in the presence of copper, a known cofactor in ethylene binding. Replacement of Cys(65) in the ethylene-binding domain by serine dramatically reduced binding of radiolabelled cyanide. In contrast with wild-type ETR1, autokinase activity of the receptor is not reduced in the ETR1-C65S mutant upon addition of cyanide. Additionally, protein-protein interaction with the ethylene signalling protein EIN2 (ethylene-insensitive 2) is considerably sustained by cyanide in wild-type ETR1, but is not affected in the mutant. Further evidence for the structural and functional equivalence of ethylene and cyanide is given by the fact that the ethylene-responsive antagonist silver, which is known to allow ligand binding but prevent intrinsic signal transduction, also allows specific binding of cyanide, but shows no effect on autokinase activity and ETR1-EIN2 interaction.     

Histidine kinases in plants: Cross talk between hormone and stress responses

Two-component signaling pathways involve sensory histidine kinases (HK), histidine phosphotransfer proteins (HpT) and response regulators (RR). Recent advancements in genome sequencing projects for a number of plant species have established the TCS family to be multigenic one. In plants, HKs operate through the His–Asp phosphorelay and control many physiological and developmental processes throughout the lifecycle of plants. Despite the huge diversity reported for the structural features of the HKs, their functional redundancy has also been reported via mutant approach. Several sensory HKs having a CHASE domain, transmembrane domain(s), transmitter domain and receiver domain have been reported to be involved in cytokinin and ethylene signaling. On the other hand, there are also increasing evidences for some of the sensory HKs to be performing their role as osmosensor, clearly indicating toward a possible cross-talk between hormone and stress responsive cascades. In this review, we bring out the latest knowledge about the structure and functions of histidine kinases in cytokinin and ethylene signaling and their role(s) in development and the regulation of environmental stress responses.     

Structure prediction, evolution and ligand interaction of CHASE domain

Cytokinins are plant hormones involved in the essential processes of plant growth and development. They bind with receptors known as CRE1/WOL/AHK4, AHK2, and AHK3, which possess histidine kinase activity. Recently, the sensor domain cyclases/histidine kinases associated sensory extracellular (CHASE) was identified in those proteins but little is known about its structure and interaction with ligands. Distant homology detection methods developed in our laboratory and molecular phylogeny enabled the prediction of the structure of the CHASE domain as similar to the photoactive yellow protein-like sensor domain. We have identified the active site pocket and amino acids that are involved in receptor-ligand interactions. We also show that fold evolution of cytokinin receptors is very important for a full understanding of the signal transduction mechanism in plants.     

Trichosanthin interacts with acidic ribosomal proteins P0 and P1 and mitotic checkpoint protein MAD2B.

Trichosanthin is a ribosome-inactivating protein with multiple pharmacological properties. By a yeast two-hybrid system, ribosomal phosphoproteins P0 and P1 and a putative mitotic checkpoint protein, MAD2B, were found to interact with an active-site mutated trichosanthin (TCS). The interactions were verified by an in vitro binding assay of recombinant wild-type TCS and target proteins. The interaction domain of P0 was mapped to amino acids 220-273, which had been previously reported to be involved in the interaction with P1 and P2 in yeast. Consistent with our previous finding that the last seven residues of TCS are not essential for an active conformation, the same deletion did not affect the interaction with P0. Our present study suggests that TCS may disrupt the binding of elongation factors to the P-complex, in addition to the well-known N-glycosidase activity for ribosome inactivation.     

Environmental perception avenues: the interaction of cytokinin and environmental response pathways

Cytokinins were discovered in the 1950s by their ability to promote cell division in cultured plant cells. Recently, there have been significant breakthroughs in our understanding of the biosynthesis, metabolism, perception and signal transduction of this phytohormone. These advances, coupled with physiological and other approaches, have enabled remarkable progress to be made in our understanding of the interactions between cytokinin function and environmental inputs. In this review, we first highlight the most recent advances in our understanding of cytokinin biosynthesis, metabolism and signalling. We then discuss how various environmental signals interact with these pathways to modulate plant growth, development and physiology.