Stuart Meier

The Arabidopsis Wall Associated Kinase-Like 10 Gene Encodes a Functional Guanylyl Cyclase and Is Co-Expressed with Pathogen Defense Related Genes

Background Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3′,5′-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. Principal Findings We have identified an Arabidopsis receptor type wall associated kinase–like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10431–700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently co-expressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors. Conclusions We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP.

Defence Responses of Arabidopsis thaliana to Infection by Pseudomonas syringae Are Regulated by the Circadian Clock

The circadian clock allows plants to anticipate predictable daily changes in abiotic stimuli, such as light; however, whether the clock similarly allows plants to anticipate interactions with other organisms is unknown. Here we show that Arabidopsis thaliana (Arabidopsis) has circadian clock-mediated variation in resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), with plants being least susceptible to infection in the subjective morning. We suggest that the increased resistance to Pst DC3000 observed in the morning in Col-0 plants results from clock-mediated modulation of pathogen associated molecular pattern (PAMP)-triggered immunity. Analysis of publicly available microarray data revealed that a large number of Arabidopsis defence-related genes showed both diurnal- and circadian-regulation, including genes involved in the perception of the PAMP flagellin which exhibit a peak in expression in the morning. Accordingly, we observed that PAMP-triggered callose deposition was significantly higher in wild-type plants inoculated with Pst DC3000 hrpA in the subjective morning than in the evening, while no such temporal difference was evident in arrhythmic plants. Our results suggest that PAMP-triggered immune responses are modulated by the circadian clock and that temporal regulation allows plants to anticipate and respond more effectively to pathogen challenges in the daytime.

A transcriptional analysis of carotenoid, chlorophyll and plastidial isoprenoid biosynthesis genes during development and osmotic stress responses in Arabidopsis thaliana

BackgroundThe carotenoids are pure isoprenoids that are essential components of the photosynthetic apparatus and are coordinately synthesized with chlorophylls in chloroplasts. However, little is known about the mechanisms that regulate carotenoid biosynthesis or the mechanisms that coordinate this synthesis with that of chlorophylls and other plastidial synthesized isoprenoid-derived compounds, including quinones, gibberellic acid and abscisic acid. Here, a comprehensive transcriptional analysis of individual carotenoid and isoprenoid-related biosynthesis pathway genes was performed in order to elucidate the role of transcriptional regulation in the coordinated synthesis of these compounds and to identify regulatory components that may mediate this process in Arabidopsis thaliana.ResultsA global microarray expression correlation analysis revealed that the phytoene synthase gene, which encodes the first dedicated and rate-limiting enzyme of carotenogenesis, is highly co-expressed with many photosynthesis-related genes including many isoprenoid-related biosynthesis pathway genes. Chemical and mutant analysis revealed that induction of the co-expressed genes following germination was dependent on gibberellic acid and brassinosteroids (BR) but was inhibited by abscisic acid (ABA). Mutant analyses further revealed that expression of many of the genes is suppressed in dark grown plants by Phytochrome Interacting transcription Factors (PIFs) and activated by photoactivated phytochromes, which in turn degrade PIFs and mediate a coordinated induction of the genes. The promoters of PSY and the co-expressed genes were found to contain an enrichment in putative BR-auxin response elements and G-boxes, which bind PIFs, further supporting a role for BRs and PIFs in regulating expression of the genes. In osmotically stressed root tissue, transcription of Calvin cycle, methylerythritol 4-phosphate pathway and carotenoid biosynthesis genes is induced and uncoupled from that of chlorophyll biosynthesis genes in a manner that is consistent with the increased synthesis of carotenoid precursors for ABA biosynthesis. In all tissues examined, induction of β-carotene hydroxylase transcript levels are linked to an increased demand for ABA.ConclusionsThis analysis provides compelling evidence to suggest that coordinated transcriptional regulation of isoprenoid-related biosynthesis pathway genes plays a major role in coordinating the synthesis of functionally related chloroplast localized isoprenoid-derived compounds.

Ozone and nitric oxide induce cGMP‐dependent and ‐independent transcription of defence genes in tobacco

Here, we analyse the temporal signatures of ozone (O3)-induced hydrogen peroxide(H2O2) and nitric oxide (NO) and the role of the second messenger guanosine3′,5′-cyclic monophosphate (cGMP) in transcriptional changes of genes diagnostic for biotic and abiotic stress responses. Within 90 min O3 induced H2O2 and NO peaks and we demonstrate that NO donors cause rapid H2O2 accumulation in tobacco (Nicotiana tabacum) leaf. Ozone also causes highly significant, late (> 2 h) and sustained cGMP increases, suggesting that the second messenger may not be required in all early (< 2 h) responses to O3,but is essential and sufficient for the induction of some O3-dependent pathways.This hypothesis was tested resolving the time course of O3-induced transcript accumulation of alternative oxidase (AOX1a), glutathione peroxidase (GPX),aminocyclopropancarboxylic acid synthase (ACS2) that is critical for the synthesis of ethylene, phenylalanine ammonia lyase (PALa) and the pathogenesis-related protein PR1a.The data show that early O3 and NO caused transcriptional activation of the scavenger encoding proteins AOX1a, GPX and the induction of ethylene production through ACS2 are cGMP independent. By contrast, the early response of PALa and the late response of PR1a show critical dependence on cGMP.

Co-expression and promoter content analyses assign a role in biotic and abiotic stress responses to plant natriuretic peptides

BackgroundPlant natriuretic peptides (PNPs) are a class of systemically mobile molecules distantly related to expansins. While several physiological responses to PNPs have been reported, their biological role has remained elusive. Here we use a combination of expression correlation analysis, meta-analysis of gene expression profiles in response to specific stimuli and in selected mutants, and promoter content analysis to infer the biological role of the Arabidopsis thaliana PNP, AtPNP-A.ResultsA gene ontology analysis of AtPNP-A and the 25 most expression correlated genes revealed a significant over representation of genes annotated as part of the systemic acquired resistance (SAR) pathway. Transcription of these genes is strongly induced in response to salicylic acid (SA) and its functional synthetic analogue benzothiadiazole S-methylester (BTH), a number of biotic and abiotic stresses including many SA-mediated SAR-inducing conditions, as well as in the constitutive SAR expressing mutants cpr5 and mpk4 which have elevated SA levels. Furthermore, the expression of AtPNP-A was determined to be significantly correlated with the SAR annotated transcription factor, WRKY 70, and the promoters of AtPNP-A and the correlated genes contain an enrichment in the core WRKY binding W-box cis-elements. In constitutively expressing WRKY 70 lines the expression of AtPNP-A and the correlated genes, including the SAR marker genes, PR-2 and PR-5, were determined to be strongly induced.ConclusionThe co-expression analyses, both in wild type and mutants, provides compelling evidence that suggests AtPNP-A may function as a component of plant defence responses and SAR in particular. The presented evidence also suggests that the expression of AtPNP-A is controlled by WRKY transcription factors and WRKY 70 in particular. AtPNP-A shares many characteristics with PR proteins in that its transcription is strongly induced in response to pathogen challenges, it contains an N-terminal signalling peptide and is secreted into the extracellular space and along with PR-1, PR-2 and PR-5 proteins it has been isolated from the Arabidopsis apoplast. Based on these findings we suggest that AtPNP-A could be classified as a newly identified PR protein.

Deciphering cGMP signatures and cGMP-dependent pathways in plant defence.

The second messenger, 3’, 5’-cyclic monophosphate (cGMP), is a critical component of many different processes in plants while guanylyl cyclases that catalyse the formation of cGMP from GTP have remained somewhat elusive in higher plants. Consequently, two major aims are the discovery of novel GCs and the identification of cGMP mediated processes. Recently, we have reported temporal signatures of ozone (O3)-induced hydrogen peroxide (H2O2) and nitric oxide (NO) generation, their effect on cGMP generation, and consequent transcriptional changes of genes diagnostic for stress responses in tobacco. We demonstrated that O3 and NO induced early transcriptional activation of the scavenger encoding proteins, alternative oxidase (AOX1a), glutathione peroxidase (GPX) and the induction of ethylene production through aminocyclopropancarboxylic acid synthase (ACS2) are cGMP-independent. By contrast, the early response of the phenylalanine ammonia lyase gene (PALa) and the late response of the gene encoding the pathogenesis-related protein (PR1a) show critical dependence on cGMP. Here we show differential cGMP responses to virulent and avirulent Pseudomonas syringae strains and propose that host-pathogen recognition and/or down-stream processes are transduced by complex cGMP signatures. This is in accordance with the identification of a growing number of multi-domain molecules in Arabidopsis that are reported to contain putative functional GC catalytic centres.

Gibberellic acid and cGMP-dependent transcriptional regulation in Arabidopsis thaliana.

An ever increasing amount of transcriptomic data and analysis tools provide novel insight into complex responses of biological systems. Given these resources we have undertaken to review aspects of transcriptional regulation in response to the plant hormone gibberellic acid (GA) and its second messenger guanosine 3’,5’-cyclic monophosphate (cGMP) in Arabidopsis thaliana, both wild type and selected mutants. Evidence suggests enrichment of GA-responsive (GARE) elements in promoters of genes that are transcriptionally up-regulated in response to cGMP but down-regulated in a GA insensitive mutant (ga1-3). In contrast, in the genes up-regulated in the mutant, no enrichment in the GARE is observed suggesting that GARE elements are diagnostic for GA-induced and cGMP-dependent transcriptional up-regulation. Further, we review how expression studies of GA-dependent transcription factors and transcriptional networks based on common promoter signatures derived from ab initio analyses can contribute to our understanding of plant responses at the systems level.

A guide to the integrated application of on‐line data mining tools for the inference of gene functions at the systems level

Genes function in networks to achieve a common biological response. Thus, inferences into the biological role of individual genes can be gained by analyzing their association with other genes with more precisely defined functions. Here, we present a guide, using the well‐characterized Arabidopsis thaliana pathogenesis‐related protein 2 gene (PR‐2) as an example, to document how the sequential use of web‐based tools can be applied to integrate information from different databases and associate the function of an individual gene with a network of genes and additionally identify specific biological processes in which they collectively function. The analysis begins by performing a global expression correlation analysis to build a functionally associated gene network. The network is subsequently analyzed for Gene Ontology enrichment, stimuli and mutant‐specific transcriptional responses and enriched putative promoter regulatory elements that may be responsible for their correlated relationships. The results for the PR‐2 gene are entirely consistent with the published literature documenting the accuracy of this type of analysis. Furthermore, this type of analysis can also be performed on other organisms with the appropriate data available and will greatly assist in understanding individual gene functions in a systems context.

Plant nucleotide cyclases: an increasingly complex and growing family.

Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, cGMP, has long been known to be critical to many different processes in higher plants while guanylyl cyclases (GCs), enzymes that catalyse the formation of cGMP from GTP have largely remained elusive. This is somewhat surprising considering that the unicellular green alga Chlamydomonas reinhardtii contains > 90 annotated GCs. We have recently shown (PLoS ONE 2(5): e449) that a recombinant cytoplasmic domain of the Arabidopsis brassinosteroid receptor AtBRI has GC activity in vitro. This finding may suggest that other leucine-rich receptor kinases such as the phystosulfokine receptor may also confer GC activity as it has a high degree of similarity in the domain that has been delineated as essential for catalysis. In addition, the discovery of increasing complexities in the molecular architecture of higher plant nucleotide cyclases (NCs) is entirely compatible with findings in Chlamydomonas where such domains appear in > 20 different combinations suggesting a role in highly diverse and complex signaling events.

cGMP in ozone and NO dependent responses

We have recently reported (Plant Physiology 2006; 142:595-608) that ozone (O3) can inhibit mitochondrial respiration and induce activation of the alternative oxidase (AOX) pathway and in particular AOX1a in tobacco. While O3 causes mitochondrial H2O2, early leaf nitric oxide (NO) as well as transient ethylene (ET) accumulation, the levels of jasmonic acid and 12-oxo-phytodienoic acid remained unchanged. It was shown that both, NO and ET dependent pathways can induce AOX1a transcription by O3. AOX plays a role in reducing reactive oxygen species (ROS) which in turn are linked to biotic and abiotic plant stresses, much like the second messengers guanosine 3’, 5’-cyclic monophosphate (cGMP). The goal is to unravel specific cGMP signatures and induction pathways downstream from O3 and NO, including transcription of AOX1a. Here we propose that some late (> 3 hours) responses to NO, e.g. the accumulation of phenylalanine lyase (PAL) transcripts, are critically cGMP dependent, while the early (< 2 hours) responses, including AOX1a induction are not.