Changes in the Arabidopsis RNA-binding proteome reveal novel stress response mechanisms.

BACKGROUND: RNA-binding proteins (RBPs) are increasingly recognized as regulatory component of post-transcriptional gene expression. RBPs interact with mRNAs via RNA-binding domains and these interactions affect RNA availability for translation, RNA stability and turn-over thus affecting both RNA and protein expression essential for developmental and stimulus specific responses. Here we investigate the effect of severe drought stress on the RNA-binding proteome to gain insights into the mechanisms that govern drought stress responses at the systems level. RESULTS: Label-free mass spectrometry enabled the identification 567 proteins of which 150 significantly responded to the drought-induced treatment. A gene ontology analysis revealed enrichment in the "RNA binding" and "RNA processing" categories as well as biological processes such as "response to abscisic acid" and "response to water deprivation". Importantly, a large number of the stress responsive proteins have not previously been identified as RBPs and include proteins in carbohydrate metabolism and in the glycolytic and citric acid pathways in particular. This suggests that RBPs have hitherto unknown roles in processes that govern metabolic changes during stress responses. Furthermore, a comparative analysis of RBP domain architectures shows both, plant specific and common domain architectures between plants and animals. The latter could be an indication that RBPs are part of an ancient stress response. CONCLUSION: This study establishes mRNA interactome capture technique as an approach to study stress signal responses implicated in environmental changes. Our findings denote RBP changes in the proteome as critical components in plant adaptation to changing environments and in particular drought stress protein-dependent changes in RNA metabolism.

The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling.

The brassinosteroid receptor brassinosteroid insensitive 1 (BRI1) is a member of the leucine-rich repeat receptor-like kinase family. The intracellular kinase domain of BRI1 is an active kinase and also encapsulates a guanylate cyclase catalytic centre. Using liquid chromatography tandem mass spectrometry, we confirmed that the recombinant cytoplasmic domain of BRI1 generates pmol amounts of cGMP per µg protein with a preference for magnesium over manganese as a co-factor. Importantly, a functional BRI1 kinase is essential for optimal cGMP generation. Therefore, the guanylate cyclase activity of BRI1 is modulated by the kinase while cGMP, the product of the guanylate cyclase, in turn inhibits BRI1 kinase activity. Furthermore, we show using Arabidopsis root cell cultures that cGMP rapidly potentiates phosphorylation of the downstream substrate brassinosteroid signaling kinase 1 (BSK1). Taken together, our results suggest that cGMP acts as a modulator that enhances downstream signaling while dampening signal generation from the receptor.

Multi-omics analysis of thermal stress response in a zooxanthellate cnidarian reveals the importance of associating with thermotolerant symbionts.

Corals and their endosymbiotic dinoflagellates of the genus Symbiodinium have a fragile relationship that breaks down under heat stress, an event known as bleaching. However, many coral species have adapted to high temperature environments such as the Red Sea (RS). To investigate mechanisms underlying temperature adaptation in zooxanthellate cnidarians we compared transcriptome- and proteome-wide heat stress response (24 h at 32°C) of three strains of the model organism Aiptasia pallida from regions with differing temperature profiles; North Carolina (CC7), Hawaii (H2) and the RS. Correlations between transcript and protein levels were generally low but inter-strain comparisons highlighted a common core cnidarian response to heat stress, including protein folding and oxidative stress pathways. RS anemones showed the strongest increase in antioxidant gene expression and exhibited significantly lower reactive oxygen species (ROS) levels in hospite However, comparisons of antioxidant gene and protein expression between strains did not show strong differences, indicating similar antioxidant capacity across the strains. Subsequent analysis of ROS production in isolated symbionts confirmed that the observed differences of ROS levels in hospite were symbiont-driven. Our findings indicate that RS anemones do not show increased antioxidant capacity but may have adapted to higher temperatures through association with more thermally tolerant symbionts.

Phosphorylation of the dimeric cytoplasmic domain of the phytosulfokine receptor, PSKR1.

Phytosulfokines (PSKs) are plant peptide hormones that co-regulate plant growth, differentiation and defense responses. PSKs signal through a plasma membrane localized leucine-rich repeat receptor-like kinase (phytosulfokine receptor 1, PSKR1) that also contains a functional cytosolic guanylate cyclase with its cyclase catalytic center embedded within the kinase domain. To functionally characterize this novel type of overlapping dual catalytic function, we investigated the phosphorylation of PSKR1 in vitro Tandem mass spectrometry of the cytoplasmic domain of PSKR1 (PSKR1cd) revealed at least 11 phosphorylation sites (8 serines, 2 threonines and 1 tyrosine) within the PSKR1cd. Phosphomimetic mutations of three serine residues (Ser686, Ser696 and Ser698) in tandem at the juxta-membrane position resulted in enhanced kinase activity in the on-mutant that was suppressed in the off-mutant, but both mutations reduced guanylate cyclase activity. Both the on and off phosphomimetic mutations of the phosphotyrosine (Tyr888) residue in the activation loop suppressed kinase activity, while neither mutation affected guanylate cyclase activity. Size exclusion and analytical ultracentrifugation analysis of the PSKR1cd suggest that it is reversibly dimeric in solution, which was further confirmed by biflourescence complementation. Taken together, these data suggest that in this novel type of receptor domain architecture, specific phosphorylation and dimerization are possibly essential mechanisms for ligand-mediated catalysis and signaling.

Cyclic Nucleotide Monophosphates in Plants and Plant Signaling.

Cyclic nucleotide monophosphates (cNMPs) and the enzymes that can generate them are of increasing interest in the plant sciences. Arguably, the major recent advance came with the release of the complete Arabidopsis thaliana genome that has enabled the systematic search for adenylate (ACs) or guanylate cyclases (GCs) and did eventually lead to the discovery of a number of GCs in higher plants. Many of these proteins have complex domain architectures with AC or GC centers moonlighting within cytosolic kinase domains. Recent reports indicated the presence of not just the canonical cNMPs (i.e., cAMP and cGMP), but also the noncanonical cCMP, cUMP, cIMP, and cdTMP in plant tissues, and this raises several questions. Firstly, what are the functions of these cNMPs, and, secondly, which enzymes can convert the substrate triphosphates into the respective noncanonical cNMPs? The first question is addressed here by comparing the reactive oxygen species (ROS) response of cAMP and cGMP to that elicited by the noncanonical cCMP or cIMP. The results show that particularly cIMP can induce significant ROS production. To answer, at least in part, the second question, we have evaluated homology models of experimentally confirmed plant GCs probing the substrate specificity by molecular docking simulations to determine if they can conceivably catalytically convert substrates other than ATP or GTP. In summary, molecular modeling and substrate docking simulations can contribute to the evaluation of cyclases for noncanonical cyclic mononucleotides and thereby further our understanding of the molecular mechanism that underlie cNMP-dependent signaling in planta.

Changes in the Arabidopsis thaliana Proteome Implicate cAMP in Biotic and Abiotic Stress Responses and Changes in Energy Metabolism.

The second messenger 3',5'-cyclic adenosine monophosphate (cAMP) is increasingly recognized as having many different roles in plant responses to environmental stimuli. To gain further insights into these roles, Arabidopsis thaliana cell suspension culture was treated with 100 nM of cell permeant 8-bromo-cAMP for 5 or 10 min. Here, applying mass spectrometry and comparative proteomics, 20 proteins were identified as differentially expressed and we noted a specific bias in proteins with a role in abiotic stress, particularly cold and salinity, biotic stress as well as proteins with a role in glycolysis. These findings suggest that cAMP is sufficient to elicit specific stress responses that may in turn induce complex changes to cellular energy homeostasis.

The dual nature of trehalose in citrus canker disease: a virulence factor for Xanthomonas citri subsp. citri and a trigger for plant defence responses.

Xanthomonas citri subsp. citri (Xcc) is a bacterial pathogen that causes citrus canker in susceptible Citrus spp. The Xcc genome contains genes encoding enzymes from three separate pathways of trehalose biosynthesis. Expression of genes encoding trehalose-6-phosphate synthase (otsA) and trehalose phosphatase (otsB) was highly induced during canker development, suggesting that the two-step pathway of trehalose biosynthesis via trehalose-6-phosphate has a function in pathogenesis. This pathway was eliminated from the bacterium by deletion of the otsA gene. The resulting XccΔotsA mutant produced less trehalose than the wild-type strain, was less resistant to salt and oxidative stresses, and was less able to colonize plant tissues. Gene expression and proteomic analyses of infected leaves showed that infection with XccΔotsA triggered only weak defence responses in the plant compared with infection with Xcc, and had less impact on the host plant's metabolism than the wild-type strain. These results suggested that trehalose of bacterial origin, synthesized via the otsA-otsB pathway, in Xcc, plays a role in modifying the host plant's metabolism to its own advantage but is also perceived by the plant as a sign of pathogen attack. Thus, trehalose biosynthesis has both positive and negative consequences for Xcc. On the one hand, it enables this bacterial pathogen to survive in the inhospitable environment of the leaf surface before infection and exploit the host plant's resources after infection, but on the other hand, it is a tell-tale sign of the pathogen's presence that triggers the plant to defend itself against infection.

The type III protein secretion system contributes to Xanthomonas citri subsp. citri biofilm formation.

BACKGROUND: Several bacterial plant pathogens colonize their hosts through the secretion of effector proteins by a Type III protein secretion system (T3SS). The role of T3SS in bacterial pathogenesis is well established but whether this system is involved in multicellular processes, such as bacterial biofilm formation has not been elucidated. Here, the phytopathogen Xanthomonas citri subsp. citri (X. citri) was used as a model to gain further insights about the role of the T3SS in biofilm formation. RESULTS: The capacity of biofilm formation of different X. citri T3SS mutants was compared to the wild type strain and it was observed that this secretion system was necessary for this process. Moreover, the T3SS mutants adhered proficiently to leaf surfaces but were impaired in leaf-associated growth. A proteomic study of biofilm cells showed that the lack of the T3SS causes changes in the expression of proteins involved in metabolic processes, energy generation, exopolysaccharide (EPS) production and bacterial motility as well as outer membrane proteins. Furthermore, EPS production and bacterial motility were also altered in the T3SS mutants. CONCLUSIONS: Our results indicate a novel role for T3SS in X. citri in the modulation of biofilm formation. Since this process increases X. citri virulence, this study reveals new functions of T3SS in pathogenesis.

Exploring the Arabidopsis proteome: influence of protein solubilization buffers on proteome coverage.

The study of proteomes provides new insights into stimulus-specific responses of protein synthesis and turnover, and the role of post-translational modifications at the systems level. Due to the diverse chemical nature of proteins and shortcomings in the analytical techniques used in their study, only a partial display of the proteome is achieved in any study, and this holds particularly true for plant proteomes. Here we show that different solubilization and separation methods have profound effects on the resulting proteome. In particular, we observed that the type of detergents employed in the solubilization buffer preferentially enriches proteins in different functional categories. These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism. This data may offer a functional bias on comparative analysis studies. In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins.

Insights into xanthomonas axonopodis pv. citri biofilm through proteomics.

BACKGROUND: Xanthomonas axonopodis pv. citri (X. a. pv. citri) causes citrus canker that can result in defoliation and premature fruit drop with significant production losses worldwide. Biofilm formation is an important process in bacterial pathogens and several lines of evidence suggest that in X. a. pv. citri this process is a requirement to achieve maximal virulence since it has a major role in host interactions. In this study, proteomics was used to gain further insights into the functions of biofilms. RESULTS: In order to identify differentially expressed proteins, a comparative proteomic study using 2D difference gel electrophoresis was carried out on X. a. pv. citri mature biofilm and planktonic cells. The biofilm proteome showed major variations in the composition of outer membrane proteins and receptor or transport proteins. Among them, several porins and TonB-dependent receptor were differentially regulated in the biofilm compared to the planktonic cells, indicating that these proteins may serve in maintaining specific membrane-associated functions including signaling and cellular homeostasis. In biofilms, UDP-glucose dehydrogenase with a major role in exopolysaccharide production and the non-fimbrial adhesin YapH involved in adherence were over-expressed, while a polynucleotide phosphorylase that was demonstrated to negatively control biofilm formation in E. coli was down-regulated. In addition, several proteins involved in protein synthesis, folding and stabilization were up-regulated in biofilms. Interestingly, some proteins related to energy production, such as ATP-synthase were down-regulated in biofilms. Moreover, a number of enzymes of the tricarboxylic acid cycle were differentially expressed. In addition, X. a. pv. citri biofilms also showed down-regulation of several antioxidant enzymes. The respective gene expression patterns of several identified proteins in both X. a. pv. citri mature biofilm and planktonic cells were evaluated by quantitative real-time PCR and shown to consistently correlate with those deduced from the proteomic study. CONCLUSIONS: Differentially expressed proteins are enriched in functional categories. Firstly, proteins that are down-regulated in X. a. pv. citri biofilms are enriched for the gene ontology (GO) terms 'generation of precursor metabolites and energy' and secondly, the biofilm proteome mainly changes in 'outer membrane and receptor or transport'. We argue that the differentially expressed proteins have a critical role in maintaining a functional external structure as well as enabling appropriate flow of nutrients and signals specific to the biofilm lifestyle.

Structural and functional characteristics of cGMP-dependent methionine oxidation in Arabidopsis thaliana proteins.

BACKGROUND: Increasing structural and biochemical evidence suggests that post-translational methionine oxidation of proteins is not just a result of cellular damage but may provide the cell with information on the cellular oxidative status. In addition, oxidation of methionine residues in key regulatory proteins, such as calmodulin, does influence cellular homeostasis. Previous findings also indicate that oxidation of methionine residues in signaling molecules may have a role in stress responses since these specific structural modifications can in turn change biological activities of proteins. FINDINGS: Here we use tandem mass spectrometry-based proteomics to show that treatment of Arabidopsis thaliana cells with a non-oxidative signaling molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by cGMP-dependent methionine oxidation is functionally enriched for stress response proteins. Furthermore, we also noted distinct signatures in the frequency of amino acids flanking oxidised and un-oxidised methionine residues on both the C- and N-terminus. CONCLUSIONS: Given both a structural and functional bias in methionine oxidation events in response to a signaling molecule, we propose that these are indicative of a specific role of such post-translational modifications in the direct or indirect regulation of cellular responses. The mechanisms that determine the specificity of the modifications remain to be elucidated.

Flower Development in Arabidopsis.

Like in other angiosperms, the development of flowers in Arabidopsis starts right after the floral transition, when the shoot apical meristem (SAM) stops producing leaves and makes flowers instead. On the flanks of the SAM emerge the flower meristems (FM) that will soon differentiate into the four main floral organs, sepals, petals, stamens, and pistil, stereotypically arranged in concentric whorls. Each phase of flower development-floral transition, floral bud initiation, and floral organ development-is under the control of specific gene networks. In this chapter, we describe these different phases and the gene regulatory networks involved, from the floral transition to the floral termination.

(De)Activation (Ir)Reversibly or Degradation: Dynamics of Post-Translational Protein Modifications in Plants.

The increasing dynamic functions of post-translational modifications (PTMs) within protein molecules present outstanding challenges for plant biology even at this present day. Protein PTMs are among the first and fastest plant responses to changes in the environment, indicating that the mechanisms and dynamics of PTMs are an essential area of plant biology. Besides being key players in signaling, PTMs play vital roles in gene expression, gene, and protein localization, protein stability and interactions, as well as enzyme kinetics. In this review, we take a broader but concise approach to capture the current state of events in the field of plant PTMs. We discuss protein modifications including citrullination, glycosylation, phosphorylation, oxidation and disulfide bridges, N-terminal, SUMOylation, and ubiquitination. Further, we outline the complexity of studying PTMs in relation to compartmentalization and function. We conclude by challenging the proteomics community to engage in holistic approaches towards identification and characterizing multiple PTMs on the same protein, their interaction, and mechanism of regulation to bring a deeper understanding of protein function and regulation in plants.

Citrullination of Proteins as a Specific Response Mechanism in Plants.

Arginine deimination, also referred to as citrullination of proteins by L-arginine deiminases, is a post-translational modification affecting histone modifications, epigenetic transcriptional regulation, and proteolysis in animals but has not been reported in higher plants. Here we report, firstly, that Arabidopsis thaliana proteome contains proteins with a specific citrullination signature and that many of the citrullinated proteins have nucleotide-binding regulatory functions. Secondly, we show that changes in the citrullinome occur in response to cold stress, and thirdly, we identify an A. thaliana protein with peptidyl arginine deiminase activity that was shown to be calcium-dependent for many peptide substrates. Taken together, these findings establish this post-translational modification as a hitherto neglected component of cellular reprogramming during stress responses.

Functional Roles of RNA-Binding Proteins in Plant Signaling.

RNA-binding proteins (RBPs) are typical proteins that bind RNA through single or multiple RNA-binding domains (RBDs). These proteins have a functional role in determining the fate or function of the bound RNAs. A few hundred RBPs were known through in silico prediction based on computational assignment informed by structural similarity and the presence of classical RBDs. However, RBPs lacking such conventional RBDs were omitted. Owing to the recent mRNA interactome capture technology based on UV-crosslinking and fixing proteins to their mRNA targets followed by affinity capture purification and identification of RBPs by tandem mass spectrometry, several hundreds of RBPs have recently been discovered. These proteome-wide studies have colossally increased the number of proteins implicated in RNA binding and unearthed hundreds of novel RBPs lacking classical RBDs, such as proteins involved in intermediary metabolism. These discoveries provide wide insights into the post-transcriptional gene regulation players and their role in plant signaling, such as environmental stress conditions. In this review, novel discoveries of RBPs are explored, particularly on the evolving knowledge of their role in stress responses. The molecular functions of these RBPs, particularly focusing on those that do not have classical RBDs, are also elucidated at the systems level.

Drought Stress Causes Specific Changes to the Spliceosome and Stress Granule Components.

The spliceosome processes RNAs from a pre-RNA state to a mature mRNA thereby influencing RNA availability for translation, localization, and turnover. It consists of complex structures containing RNA-binding proteins (RBPs) essential for post-transcriptional gene expression control. Here we investigate the dynamic modifications of spliceosomal RBPs under stress and in particular drought stress. We do so by mRNA interactome capture in Arabidopsis thaliana using label free quantitation. This approach identified 44 proteins associated with the spliceosome and further 32 proteins associated with stress granules. We noted a high enrichment in the motifs RDRR and RSRSRS that are characteristic of RNA interacting proteins. Identification of splicing factors reflect direct and/or indirect stress induced splicing events that have a direct effect on transcriptome and proteome changes under stress. Furthermore, detection of stress granule components is consistent with transcriptional arrest. Identification of drought induced stress granule components is critical in determining common abiotic stress-induced foci that can have biotechnological applications. This study may therefore open ways to modify plant stress responses at a systems level through the modification of key spliceosome components.

Proteome changes and physiological adaptations of the phytopathogen Xanthomonas citri subsp. citri under salt stress and their implications for virulence.

The bacterium Xanthomonas citri subsp. citri (Xcc) is responsible for the widely distributed disease citrus canker. In the last years, Xcc has become a model for the study of plant pathogens, and here we used this bacterium to examine stress on the pathogen during adaptions required for leaf colonization. In the first stages of citrus canker cycle, bacteria encounter low water availability and osmotic stress that can affect their maintenance on plant surfaces. To examine such conditions, we conducted a proteome analysis of Xcc grown in culture medium supplemented with 0.25 M sodium chloride and compared it to control conditions. We found that salt stress induced changes in known stress-induced proteins and also revealed novel stress response proteins. Moreover, some of the bacterial processes associated with bacterial fitness and virulence were modified under salt stress conditions. In particular, swimming, twitching and surface motilities were decreased, while biofilm formation was increased under salt stress. Other adaptations to high salt included reduced bacterial size and increased survival of bacteria exposed to oxidative stress. Furthermore, expression of type III protein secretion system related genes were augmented under salt stress condition. Our results offer new insight into molecular mechanisms that govern phytopathogen adaptation to harsh environments. These adaptations affect life cycle progression which in turn influences virulence.

Discovery of Novel Functional Centers With Rationally Designed Amino Acid Motifs.

Plants are constantly exposed to environmental stresses and in part due to their sessile nature, they have evolved signal perception and adaptive strategies that are distinct from those of other eukaryotes. This is reflected at the cellular level where receptors and signalling molecules cannot be identified using standard homology-based searches querying with proteins from prokaryotes and other eukaryotes. One of the reasons for this is the complex domain architecture of receptor molecules. In order to discover hidden plant signalling molecules, we have developed a motif-based approach designed specifically for the identification of functional centers in plant molecules. This has made possible the discovery of novel components involved in signalling and stimulus-response pathways; the molecules include cyclic nucleotide cyclases, a nitric oxide sensor and a novel target for the hormone abscisic acid. Here, we describe the major steps of the method and illustrate it with recent and experimentally confirmed molecules as examples. We foresee that carefully curated search motifs supported by structural and bioinformatic assessments will uncover many more structural and functional aspects, particularly of signalling molecules.

Intramolecular crosstalk between catalytic activities of receptor kinases.

Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

Towards a tailored indoor horticulture: a functional genomics guided phenotypic approach.

As indoor horticulture gathers momentum, electric (also termed artificial) lighting systems with the ability to generate specific and tunable wavelengths have been developed and applied. While the effects of light quality on plant growth and development have been studied, authoritative and reliable sets of light formulae tailored for the cultivation of economically important plants and plant traits are lacking as light qualities employed across laboratories are inconsistent. This is due, at least in part, to the lack of molecular data for plants examined under electric lights in indoor environments. It has hampered progress in the field of indoor horticulture, in particular, the transition from small-scale indoor farming to commercial plant factories. Here, we review the effects of light quality on model and crop plants studied from a physiological, physical and biochemical perspective, and explain how functional genomics can be employed in tandem to generate a wealth of molecular data specific for plants cultivated under indoor lighting. We also review the current state of lighting technologies in indoor horticulture specifically discussing how recent narrow-bandwidth lighting technologies can be tailored to cultivate economically valuable plant species and traits. Knowledge gained from a complementary phenotypic and functional genomics approach can be harvested not only for economical gains but also for sustainable food production. We believe that this review serves as a platform that guides future light-related plant research.