9 research outputs found
Latest News on Arabidopsis Brassinosteroid Perception and Signaling
Brassinosteroids (BRs) are plant hormones regulating growth and development. In interaction with other hormones, they are involved in environmental cue responses. The present model of the BR response pathway in Arabidopsis includes the perception of the hormone by the plasma membrane (PM) receptor brassinosteroid insensitive 1 (BRI1) and its hetero-oligomerization with the co-receptor BRI1-associated receptor kinase 1 (BAK1), followed by the activation of a signaling-cascade finally resulting in the expression of BR-responsive genes. New findings have shed light on the receptor density in the PM and on the molecular mechanism of BR perception, which includes the hormone-induced formation of a platform in the BRI1 extracellular domain for interaction with BAK1. Furthermore, new knowledge on early, BRI1-initiated signaling events at the PMâcytoplasm interface has recently been gained. In addition, a fast BR response pathway that modifies the membrane potential and the expansion of the cell wall â both crucial processes preceding cell elongation growth â have been identified. In this review, these latest findings are summarized and discussed against the background of the present model of BRI1 signaling
Evidence for the localization of the Arabidopsis cytokinin receptors AHK3 and AHK4 in the endoplasmic reticulum
Cytokinins are hormones that are involved in various processes of plant growth and development. The model of cytokinin signalling starts with hormone perception through membrane-localized histidine kinase receptors. Although the biochemical properties and functions of these receptors have been extensively studied, there is no solid proof of their subcellular localization. Here, cell biological and biochemical evidence for the localization of functional fluorophor-tagged fusions of Arabidopsis histidine kinase 3 (AHK3) and 4 (AHK4), members of the cytokinin receptor family, in the endoplasmic reticulum (ER) is provided. Furthermore, membrane-bound AHK3 interacts with AHK4 in vivo. The ER localization and putative function of cytokinin receptors from the ER have major impacts on the concept of cytokinin perception and signalling, and hormonal cross-talk in plants
Analysis of plant protein kinases and their downstream components
Zum tieferen VerstĂ€ndnis pflanzlicher Signaltransduktionswege ist es essentiell, die molekulare Wirkungsweise ihrer einzelnen Komponenten im Detail zu untersuchen. AuĂerdem stellt sich die Frage, ob molekulare Eigenschaften wie deren subzellulĂ€re Lokalisation oder ihr Phosphorylierungszustand fĂŒr ihre Funktion ausschlaggebend sind.
DafĂŒr wurde exemplarisch die subzellulĂ€re Lokalisation der Cytokininrezeptoren AHK3 und AHK4 untersucht. Diese sind Vertreter der Familie der Histidinkinasen des Zweikomponentensystems (TCS), einem bedeutenden Signaltransduktionsmechanismus in Pflanzen, basierend auf einem Histidin zu Aspartat Phosphorelay. Bisher wurde angenommen, dass die Cytokininrezeptoren an der Plasmamembran lokalisiert sind (Kim et al. 2006). In dieser Arbeit wurde jedoch gezeigt, dass GFP- beziehungsweise RFP-Fusionsproteine von AHK3 und AHK4 in transient transformierten TabakblĂ€ttern sowie Arabidopsis Keimlingen am ER lokalisiert sind. AuĂerdem sind die GFP-Fusionen von AHK3 funktional, da sie den cytokinininsensitiven PhĂ€notyp der ahk2ahk3 Doppelmutante komplementieren. AHK3-GFP wurde auch in stabil transformierten Arabidopsis-Linien am ER detektiert. Die gezeigte SensitivitĂ€t von AHK3-GFP und GFP-AHK3 auf die Endoglykosidase H untermauert deren ER-Lokalisation und lĂ€sst ferner vermuten, dass AHK3 so orientiert ist, dass die cytokininbindende CHASE-DomĂ€ne ins ER-Lumen reicht. Aufgrund der hier erzielten Ergebnisse muss der Cytokininsignalweg neu ĂŒberdacht werden.
Abgesehen von der subzellulĂ€ren Lokalisation der Cytokininrezeptoren, war die der A-Typ Responseregulatoren (ARRs) von Interesse. Diese gehören zu den Output-Elementen des TCS und sind, obwohl sie kein offensichtliches Kernlokalisationssignal (NLS) besitzen, gröĂtenteils im Zellkern lokalisiert (Grefen & Harter 2004). Durch gezielte EinzelaminosĂ€ureaustausche (Lysin zu Alanin) konnte gezeigt werden, dass ARR3, 4, 7 und 15, typische Vertreter der A-Typ ARRs, kurze Sequenzmotive besitzen, deren Mutation zur einer ĂŒberwiegend zytoplasmatischen Lokalisation der entsprechenden GFP-Fusionen fĂŒhrt und die daher als NLSs dienen. Die NLS-Sequenzen und ihre intramolekulare Lokalisation sind interessanterweise auch zwischen eng verwandten A-Typ ARRs nicht konserviert und befinden sich ausschlieĂlich im C-Terminus der Proteine. Es ist daher zu vermuten, dass der bisher wenig untersuchte C-terminale Bereich der A-Typ ARRs fĂŒr deren subzellulĂ€re Lokalisation verantwortlich ist und ihm daher eine gröĂere Bedeutung zukommt als bislang angenommen.
Des Weiteren wurde die bis dahin am wenigsten charakterisierte Histidinkinase des TCS, AHK5, hinsichtlich ihrer Funktion beim SchlieĂen der Stomata untersucht. Bisher war AHK5 nur als negativer Regulator der ABA- und ethyleninduzierten Hemmung des Wurzelwachstums bekannt (Iwama et al. 2007). In der vorliegenden Arbeit konnte jedoch gezeigt werden, dass das SchlieĂen der Stomata als Reaktion auf H2O2, welches aufgrund exo- oder endogener Auslöser gebildet wird, von AHK5 abhĂ€ngt. ahk5-Mutanten reagierten weder mit SchlieĂen der Stomata auf exogen appliziertes H2O2 noch auf Faktoren wie Dunkelheit, NO oder Ethylen, die die endogene H2O2-Produktion stimulieren. Durch Interaktionsstudien mit weiteren Elementen des TCS â Phosphotransferproteine (AHPs) und ARRs â wurden auĂerdem Komponenten des Signalwegs downstream von AHK5 identifiziert und zwar AHP1, AHP2 und ARR4. Durch die Analyse verschiedener ahp- und einer arr4-Mutante konnte bestĂ€tigt werden, dass AHP1, AHP2 und ARR4 fĂŒr die Reaktion der Stomata auf verschiedene exo- oder endogene Faktoren essentiell sind. DarĂŒber hinaus konnte gezeigt werden, dass das SchlieĂen der Stomata in Reaktion auf H2O2 vom Phosphorylierungszustand von ARR4 abhĂ€ngt. Die KomplexitĂ€t dieses Signalwegs unterstreicht die Bedeutung einer prĂ€zisen Feinregulierung des Stomataschlusses fĂŒr die Pflanze.
UnabhĂ€ngig vom TCS, werden auch andere Phytohormone wie die Brassinosteroide (BR) durch membrangebundene Rezeptoren wie in diesem Fall BRI1, perzipiert. In dieser Arbeit konnte ein frĂŒher BR-induzierter und BRI1-abhĂ€ngiger Signaltransduktionsvorgang in der Plasmamembran (PM) charakterisiert werden. BR fĂŒhrt zur Hyperpolarisation der PM und zu einer damit verbundenen Verbreiterung der Zellwand, was durch die Aufzeichnung der Fluoreszenzlebensdauer von BRI1-GFP gezeigt werden konnte, die auf Ănderungen der physikochemischen Umgebung eines Fluoreszenzproteins reagiert. Die beobachteten Prozesse stehen mit der AktivitĂ€t der PM-gebundenen H+-ATPase in Zusammenhang, die jedoch offensichtlich in diesem Fall nicht durch Phosphorylierung eines konservierten ThreoninÂŹrestes in ihrer C-terminalen autoinhibitorischen DomĂ€ne reguliert wird. Die BR-induzierte Hyperpolarisation der PM und die Verbreiterung der Zellwand sind daher frĂŒhe Ereignisse bei der BR-regulierten Zellstreckung, welche hier zum ersten Mal mit hoher rĂ€umlich-zeitlicher Auflösung in planta beschrieben werden konnten.In order to elucidate the mechanisms of plant signal transduction pathways, it is essential to analyze in detail the molecular function of the different signalling components and to determine whether aspects such as their subcellular localization or phosphorylation state play an important role in regulating their function.
Here, the subcellular localization of the cytokinin receptors AHK3 and AHK4 was analyzed. They are representatives of the histidine kinase family of the two-component system (TCS) which is an important signalling mechanism in Arabidopsis, based on a histidine to aspartate phosphorelay. Until now, it has been assumed that the cytokinin receptors reside at the plasma membrane (Kim et al. 2006). However, we showed that GFP- and RFP-fusions of AHK3 and AHK4, respectively, are localized at the endoplasmic reticulum (ER) of transiently transformed tobacco epidermal leaf cells and Arabidopsis cotyledon cells. Furthermore, the N- and C-terminal GFP fusions of AHK3 represent functional cytokinin receptors since they complement the cytokinin insensitive phenotype of the ahk2ahk3 double mutant. The observed ER-localization of AHK3-GFP in transiently transformed leaves could be confirmed in these Arabidopsis lines. The sensitivity of AHK3-GFP and GFP-AHK3 to the endoglycosidase H substantiates their ER localization and leads to the assumption that the cytokinin-binding CHASE domain of AHK3 is exposed to the ER lumen. Apparently, the current model of cytokinin signal perception at the plasma membrane needs to be reconsidered.
Apart from the cytokinin receptors, the subcellular localization of the A-type response regulators (ARRs), representing output elements of the TCS, was examined. Although they do not have an obvious nuclear localization signal (NLS), they are mainly localized in the nucleus (Grefen & Harter 2004). It could be shown by single amino acid exchanges (lysine to alanine) that ARR3, 4, 7 and 15, which are representatives of the A-type ARRs, have short sequence motifs, whose mutation led to a mainly cytoplasmic localization of the corresponding GFP-fusions and, therefore serve as NLSs. Interestingly, the determined NLS positions are not conserved even between phylogenetically tightly related A-type ARRs and are located in the C-terminus of the proteins. Therefore, it can be assumed that this less examined C-terminal part of the A-type ARRs is relevant for their subcellular distribution, in contrast to the receiver domain which confers signalling.
Furthermore, AHK5, the least characterized histidine kinase of the TCS, was analyzed regarding its functional relevance in the stomatal closure response. Until now, AHK5 has only been identified as a negative regulator of ABA- and ethylene-induced root growth inhibition (Iwama et al. 2007). Here, it could be shown that stomatal closure, mediated by H2O2, which is produced in response to exogenous or endogenous stimuli, depends on AHK5. Stomata of different ahk5 mutants did neither respond to exogenously applied H2O2 nor to factors like darkness, NO or ethylene that are known to enhance the endogenous H2O2 production. By protein-protein interaction studies of AHK5 with downstream elements of the TCS â phosphotransfer proteins (AHPs) and ARRs â it was possible to identify further compoÂŹnents of the pathway, namely AHP1, AHP2 and ARR4. Using different ahp mutants as well as an arr4 mutant, it could be confirmed that AHP1, AHP2 and ARR4 are crucial for stomatal closure in response to different stimuli. Furthermore, it could be shown that the stomatal closure in response to H2O2 is depending on the phosphorylation state of ARR4. The complexity of this pathway leads to the conclusion that fine-tuning of the stomatal movement has a high impact on the plantâs response to environmental stimuli.
Independently of the TCS, other phytohormones like brassinosteroids (BR) are also perceived by memÂŹbrane bound receptors, namely BRI1 in the case of BR. Here, a fast BR-induced and BRI1-depending process in the plasma membrane (PM) of Arabidopsis could be described. BR leads to the hyperpolarisation of the PM accompanied by a cell wall expansion. This was shown by recording the fluorescence lifetime of BRI1-GFP which responds to changes in the physico-chemical environment of fluorescent proteins. The observed processes depend on the activity of the PM-bound H+-ATPase (P-ATPase), which is in this case not regulated by the phosphorylation of a conserved threonine residue (T948) in its C-terminal autoÂŹinhibitory domain. The BR-induced hyperpolarisation of the PM and the cell wall expansion are therefore early events in the BR-regulated cell elongation that are described for the first time in planta with high spatio-temporal resolution
Concerted expression of a cell cycle regulator and a metabolic enzyme from a bicistronic transcript in plants
Identification of two-component system elements downstream of AHK5 in the stomatal closure response of Arabidopsis thaliana
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 (H2O2). The mechanisms by which H2O2 integrates multiple signals via specific signalling pathways leading to stomatal closure is not known. Principal Findings: Here, we identify a pathway by which H2O2, 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 H2O2, 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 H2O2, 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 H2O2 production and H2O2 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 H2O2 generation in ahk5 mutants. Significance: Our findings identify an integral signalling function for AHK5 that acts to integrate multiple signals via H2O2 homeostasis and is independent of ABA signalling in guard cells. © 2008 Desikan et al