Myotubularins, PtdIns5P, and ROS in ABA-mediated stomatal movements in dehydrated Arabidopsis seedlings.

Myotubularins (MTMs) are lipid phosphoinositide 3-phosphate phosphatases and the product of their enzyme activity - phosphoinositide 5-phosphate (PtdIns5P) - functions as a signalling molecule in pathways involved in membrane dynamics and cell signalling. Two Arabidopsis genes, AtMTM1 and AtMTM2, encode enzymatically active phosphatases but although AtMTM1 deficiency results in increased tolerance to dehydration stress and a decrease in cellular PtdIns5P, the role of AtMTM2 is less clear, as it does not contribute to the PtdIns5P pool upon dehydration stress. Here we analysed the involvement of AtMTM1, AtMTM2 and PtdIns5P in the response of Arabidopsis seedlings to dehydration stress/ABA, and found that both AtMTM1 and AtMTM2 were involved but affected oppositely stomata movement and the accumulation of reactive oxygen species (ROS, e.g. H2O2). Acting as a secondary messenger in the ABA-induced ROS production in guard cells, PtdIns5P emerges as an evolutionarily conserved signalling molecule that calibrates cellular ROS under stress. We propose the biological relevance of the counteracting AtMTM1 and AtMTM2 activities is to balance the ABA-induced ROS accumulation and cellular homeostasis under dehydration stress.

Subcellular localizations of Arabidopsis myotubularins MTM1 and MTM2 suggest possible functions in vesicular trafficking between ER and cis-Golgi.

The two Arabidopsis genes AtMTM1 and AtMTM2 encode highly similar phosphoinositide 3-phosphatases from the myotubularin family. Despite the high-level conservation of structure and biochemical activities, their physiological roles have significantly diverged. The nature of a membrane and the concentrations of their membrane-anchored substrates (PtdIns3P or PtdIns3,5P2) and/or products (PtdIns5P and PtdIns) are considered critical for determining the functional specificity of myotubularins. We have performed comprehensive analyses of the subcellular localization of AtMTM1 and AtMTM2 using a variety of specific constructs transiently expressed in Nicotiana benthamiana leaf epidermal cells under the control of 35S promoter. AtMTM1 co-localized preferentially with cis-Golgi membranes, while AtMTM2 associated predominantly with ER membranes. In a stark contrast with animal/human MTMs, neither AtMTM1 nor AtMTM2 co-localizes with early or late endosomes or with TGN/EE compartments, making them unlikely participants in the endosomal trafficking system. Localization of the AtMTM2 is sensitive to cold and osmotic stress challenges. In contrast to animal myotubularins, Arabidopsis myotubularins do not associate with endosomes. Our results suggest that Arabidopsis myotubularins play a role in the vesicular trafficking between ER exit sites and cis-Golgi elements. The significance of these results is discussed also in the context of stress biology and plant autophagy.

The extracytoplasmic function sigma factor EcfO protects Bacteroides fragilis against oxidative stress.

The anaerobe Bacteroides fragilis is a highly aerotolerant, opportunistic pathogen that is an important component of the human intestinal microbiota. Aerotolerance has been linked to a robust oxidative stress response, which in turn is necessary for maximal virulence in a mouse intra-abdominal abscess model. During oxidative stress, there is a dynamic change in gene expression that encompasses a third of the genome, but there is a paucity of information on factors that control this response. A large number of transcription regulators, including about 14 extracytoplasmic function (ECF) sigma factors, are affected by oxidative stress, and one of these, EcfO, was used as a model of ECF sigma factor activity during stress. Genetic and biochemical experiments showed that EcfO was located in an operon with a structurally unique anti-sigma factor, Reo. Cells deleted for EcfO were impaired during exposure to oxygen or other forms of oxidative stress, whereas reo mutants were more resistant to stress. Protein-protein interaction experiments demonstrated that Reo directly interacts with and regulates the activity of EcfO. Expression microarray and chromatin affinity precipitation assays were used to identify target genes regulated by EcfO, and an EcfO recognition sequence was identified. The results revealed that EcfO controls a regulon of novel lipoproteins whose distribution in nature is restricted to members of the Bacteroidetes phylum.

Divergent functions of the myotubularin (MTM) homologs AtMTM1 and AtMTM2 in Arabidopsis thaliana: evolution of the plant MTM family.

Myotubularin and myotubularin-related proteins are evolutionarily conserved in eukaryotes. Defects in their function result in muscular dystrophy, neuronal diseases and leukemia in humans. In contrast to the animal lineage, where genes encoding both active and inactive myotubularins (phosphoinositide 3-phosphatases) have appeared and proliferated in the basal metazoan group, myotubularin genes are not found in the unicellular relatives of green plants. However, they are present in land plants encoding proteins highly similar to the active metazoan enzymes. Despite their remarkable structural conservation, plant and animal myotubularins have significantly diverged in their functions. While loss of myotubularin function causes severe disease phenotypes in humans it is not essential for the cellular homeostasis under normal conditions in Arabidopsis thaliana. Instead, myotubularin deficiency is associated with altered tolerance to dehydration stress. The two Arabidopsis genes AtMTM1 and AtMTM2 have originated from a segmental chromosomal duplication and encode catalytically active enzymes. However, only AtMTM1 is involved in elevating the cellular level of phosphatidylinositol 5-phosphate in response to dehydration stress, and the two myotubularins differentially affect the Arabidopsis dehydration stress-responding transcriptome. AtMTM1 and AtMTM2 display different localization patterns in the cell, consistent with the idea that they associate with different membranes to perform specific functions. A single amino acid mutation in AtMTM2 (L250W) results in a dramatic loss of subcellular localization. Mutations in this region are linked to disease conditions in humans.

ATX1-generated H3K4me3 is required for efficient elongation of transcription, not initiation, at ATX1-regulated genes.

Tri-methylated H3 lysine 4 (H3K4me3) is associated with transcriptionally active genes, but its function in the transcription process is still unclear. Point mutations in the catalytic domain of ATX1 (ARABIDOPSIS TRITHORAX1), a H3K4 methyltransferase, and RNAi knockdowns of subunits of the AtCOMPASS-like (Arabidopsis Complex Proteins Associated with Set) were used to address this question. We demonstrate that both ATX1 and AtCOMPASS-like are required for high level accumulation of TBP (TATA-binding protein) and Pol II at promoters and that this requirement is independent of the catalytic histone modifying activity. However, the catalytic function is critically required for transcription as H3K4me3 levels determine the efficiency of transcription elongation. The roles of H3K4me3, ATX1, and AtCOMPASS-like may be of a general relevance for transcription of Trithorax-activated eukaryotic genes.

A cytoplasm-specific activity encoded by the Trithorax-like ATX1 gene.

Eukaryotes produce multiple products from a single gene locus by alternative splicing, translation or promoter usage as mechanisms expanding the complexity of their proteome. Trithorax proteins, including the Arabidopsis Trithorax-like protein ATX1, are histone modifiers regulating gene activity. Here, we report that a novel member of the Trithorax family has a role unrelated to chromatin. It is encoded from an internal promoter in the ATX1 locus as an isoform containing only the SET domain (soloSET). It is located exclusively in the cytoplasm and its substrate is the elongation factor 1A (EF1A). Loss of SET, but not of the histone modifying ATX1-SET activity, affects cytoskeletal actin bundling illustrating that the two isoforms have distinct functions in Arabidopsis cells.

Phosphatidylinositol 5-phosphate links dehydration stress to the activity of ARABIDOPSIS TRITHORAX-LIKE factor ATX1.

BACKGROUND: Changes in gene expression enable organisms to respond to environmental stress. Levels of cellular lipid second messengers, such as the phosphoinositide PtdIns5P, change in response to a variety of stresses and can modulate the localization, conformation and activity of a number of intracellular proteins. The plant trithorax factor (ATX1) tri-methylates the lysine 4 residue of histone H3 (H3K4me3) at gene coding sequences, which positively correlates with gene transcription. Microarray analysis has identified a target gene (WRKY70) that is regulated by both ATX1 and by the exogenous addition of PtdIns5P in Arabidopsis. Interestingly, ATX1 contains a PtdIns5P interaction domain (PHD finger) and thus, phosphoinositide signaling, may link environmental stress to changes in gene transcription. PRINCIPAL FINDINGS: Using the plant Arabidopsis as a model system, we demonstrate a link between PtdIns5P and the activity of the chromatin modifier ATX1 in response to dehydration stress. We show for the first time that dehydration leads to an increase in cellular PtdIns5P in Arabidopsis. The Arabidopsis homolog of myotubularin (AtMTM1) is capable of generating PtdIns5P and here, we show that AtMTM1 is essential for the induced increase in PtdIns5P upon dehydration. Furthermore, we demonstrate that the ATX1-dependent gene, WRKY70, is downregulated during dehydration and that lowered transcript levels are accompanied by a drastic reduction in H3K4me3 of its nucleosomes. We follow changes in WRKY70 nucleosomal K4 methylation as a model to study ATX1 activity at chromatin during dehydration stress. We found that during dehydration stress, the physical presence of ATX1 at the WRKY70 locus was diminished and that ATX1 depletion resulted from it being retained in the cytoplasm when PtdIns5P was elevated. The PHD of ATX1 and catalytically active AtMTM1 are required for the cytoplasmic localization of ATX1. CONCLUSIONS/SIGNIFICANCE: The novelty of the manuscript is in the discovery of a mechanistic link between a chromatin modifying activity (ATX1) and a lipid (PtdIns5P) synthesis in a signaling pathway that ultimately results in altered expression of ATX1 dependent genes downregulated in response to dehydration stress.

The Arabidopsis chromatin modifier ATX1, the myotubularin-like AtMTM and the response to drought.

Plants respond to environmental stresses by altering transcription of genes involved in the response. The chromatin modifier ATX1 regulates expression of a large number of genes; consequently, factors that affect ATX1 activity would also influence expression from ATX1-regulated genes. Here, we demonstrate that dehydration is such a factor implicating ATX1 in the plant's response to drought. In addition, we report that a hitherto unknown Arabidopsis gene, At3g10550, encodes a phosphoinositide 3'-phosphatase related to the animal myotubularins (AtMTM1). Myotubularin activities in plants have not been described and herein, we identify an overlapping set of genes co-regulated by ATX1 and AtMTM under drought conditions. We propose that these shared genes represent the ultimate targets of partially overlapping branches of the pathways of the nuclear ATX1 and the cytoplasmic AtMTM1. Our analyses offer first genome-wide insights into the relationship of an epigenetic factor and a lipid phosphatase from the other end of a shared drought responding pathway in Arabidopsis.

Wall-modifying genes regulated by the Arabidopsis homolog of trithorax, ATX1: repression of the XTH33 gene as a test case.

The plant cell wall is a dynamic structure playing important roles in the control of plant cell growth and differentiation. These processes involve global reprogramming of the genome driven by dynamic changes in chromatin structure. The chromatin modifier ARABIDOPSIS HOMOLOG OF TRITHORAX (ATX1) methylates lysine residue 4 on histone H3 (H3K4me), acting as an epigenetic mark on associated genes. The remarkable overrepresentation in the ATX1-regulated gene fraction of genes encoding plasma membrane and cell wall-remodeling activities suggested a link between two separate factors affecting growth, development and adaptation in Arabidopsis: the wall-modifying activities regulating cell extension, growth and fate, and the epigenetic mechanisms regulating chromatin structure and gene expression. A co-regulated fraction of specific wall-modifying proteins suggests that they may function together. Here, we study the ATX1-dependent expression of the gene encoding the wall-loosening factor XTH33 as a test case for development- and tissue-specific effects displayed by the chromatin modifier. In addition, we show that XTH33 is, most likely, an integral plasma membrane protein. A putative transmembrane domain is conserved in some, but not all, XTH family members, suggesting that they may be differently positioned when functioning as wall modifiers.

The Arabidopsis homologs of trithorax (ATX1) and enhancer of zeste (CLF) establish 'bivalent chromatin marks' at the silent AGAMOUS locus.

Tightly balanced antagonism between the Polycomb group (PcG) and the Trithorax group (TrxG) complexes maintain Hox expression patterns in Drosophila and murine model systems. Factors belonging to the PcG/TrxG complexes control various processes in plants as well but whether they participate in mechanisms that antagonize, balance or maintain each other's effects at a particular gene locus is unknown. CURLY LEAF (CLF), an Arabidopsis homolog of enhancer of zeste (EZ) and the ARABIDOPSIS HOMOLOG OF TRITHORAX (ATX1) control the expression of the flower homeotic gene AGAMOUS (AG). Disrupted ATX1 or CLF function results in misexpression of AG, recognizable phenotypes and loss of H3K4me3 or H3K27me3 histone H3-tail marks, respectively. A novel idea suggested by our results here, is that PcG and TrxG complexes function as a specific pair generating bivalent chromatin marks at the silent AG locus. Simultaneous loss of ATX1 and CLF restored AG repression and normalized leaf phenotypes. At the molecular level, disrupted ATX1 and CLF functions did not lead to erasure of the CLF- and ATX1-generated epigenetic marks, as expected: instead, in the double mutants, H3K27me3 and H3K4me3 tags were partially restored. We demonstrate that ATX1 and CLF physically interact linking mechanistically the observed effects.

SA-inducible Arabidopsis glutaredoxin interacts with TGA factors and suppresses JA-responsive PDF1.2 transcription.

Salicylic acid (SA) is a plant signaling molecule that mediates the induction of defense responses upon attack by a variety of pathogens. Moreover, it antagonizes gene induction by the stress signaling molecule jasmonic acid (JA). Several SA-responsive genes are regulated by basic/leucine zipper-type transcription factors of the TGA family. TGA factors interact with NPR1, a central regulator of many SA-induced defense responses including SA/JA antagonism. In order to identify further regulatory proteins of SA-dependent signaling pathways, a yeast protein interaction screen with tobacco TGA2.2 as bait and an Arabidopsis thaliana cDNA prey library was performed and led to the identification of a member of the glutaredoxin family (GRX480, encoded by At1g28480). Glutaredoxins are candidates for mediating redox regulation of proteins because of their capacity to catalyze disulfide transitions. This agrees with previous findings that the redox state of both TGA1 and NPR1 changes under inducing conditions. Transgenic Arabidopsis plants ectopically expressing GRX480 show near wild-type expression of standard marker genes for SA- and xenobiotic-inducible responses. In contrast, transcription of the JA-dependent defensin gene PDF1.2 was antagonized by transgenic GRX480. This, together with the observation that GRX480 transcription is SA-inducible and requires NPR1, suggests a role of GRX480 in SA/JA cross-talk. Suppression of PDF1.2 by GRX480 depends on the presence of TGA factors, indicating that the GRX480/TGA interaction is effective in planta.