Phosphatidylinositol 5-Phosphate Links Dehydration Stress to the Activity of ARABIDOPSIS TRITHORAX-LIKE Factor ATX1

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.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.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

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

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 \u3ci\u3eArabidopsis\u3c/i\u3e 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) trimethylates 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

PtdIns5<i>P</i> in <i>Arabidopsis</i> tissues determined by the PIP4Kα assay.

<p>Thin layer chromatographic separation of the products of the <i>in vitro</i> phosphorylation reaction between PIP4Kάandendogenous phosphoinositides isolated from leaves (lane 2), stems (lane 3), flowers (lane 4) and siliques (lane 5). A PtdIns(4,5)<i>P</i>₂ standard is shown (lane 1) (panel a). HPLC analysis of deacylated products of the <i>in vitro</i> phosphorylation reaction between endogenous phosphoinositides (from leaves) and PIP4Kα. Deacylated PtdIns(3,4)<i>P</i>₂ and PtdIns(4,5)<i>P</i>₂ used as standards (panel b). The products of the <i>in vitro</i> phosphorylation reaction between endogenous phosphoinositides isolated from rosette leaves and PIP4Kα were incubated in the absence (lane 1) or presence (lane 2) of yeast YNK-5 phosphatase. The reaction products were analyzed by thin layer chromatography. The position of a PtdIns4<i>P</i> standard is shown (lane 3) (panel c).</p

<i>FLC</i> and <i>AG</i> transcripts are not regulated by AtMTM activity.

<p>Relative <i>FLC</i> transcripts determined in leaves of 12 day old seedlings in non-stressed and in stressed wild-type, <i>atx1</i>, <i>OX-AtMTM</i>, and <i>mtm</i> mutants, quantitated by real-time PCR and normalized against actin (panel a). Relative <i>AG</i> transcripts in inflorescences of wild-type, <i>atx1</i>, <i>OX-AtMTM</i>, and <i>mtm</i> mutants, under non-stressed and stressed conditions (panel b). Bars are s.d.</p

Time-course of water loss from detached leaves.

<p>Water-loss in detached rosette leaves after exposure to ambient air and a temperature of 20°C determined as percentage of residual tissue mass taken as 100%. Data are from four independent experiments; bars are s.d. (panel a). Lipids extracted from leaves after 30 minutes or 120 minutes of air-exposure were processed for PtdIns5<i>P</i> content and expressed as the percentage of the fresh sample (indicated as 100%). Data are from six independent experiments; bars are s.d. (panel b). Detached (column 1), air-exposed (column 2) and water-submerged leaves (column 3) were treated in parallel for 120 minutes. Cellular PtdIns5<i>P</i> is indicated as pmoles PtdIns5<i>P</i>/mg initial fresh tissue mass. Bars are s.d. (panel c).</p

Cells showing nuclear GFP-signal associated with ATX1 protein or its derivatives.

<p>The number of cells showing nuclear localization of transiently expressed ATX1 alone is set at 100%. ATX+M represents the population of cells displaying nuclear signal when co-expressing the entire ATX1 and AtMTM; ATX-N+M represents cells co-expressing the entire N-terminal portion of ATX1 and AtMTM; ATX-C+M are cells co-expressing the ATX1-C-terminal portion and AtMTM, respectively; bars are s.d. from four independent experiments.</p