STTM165/166 plants exhibit drought stress resistance phenotype.

<p>(A) Drought resistance phenotype. 3-week-old plants (upper panel) were grown under the same conditions but without irrigation for 14 days (middle panel), and then rewatered for 2 days (lower panel). (B) Quantification of the survival rate. Forty plants of wild type and STTM165/166 were used in each experiment, and the survival rate was calculated from the results of four independent experiments. (C) Water loss assay. Aerial parts of 3-week-old plants were detached and weighed at the indicated time points. Water content at any time point was calculated as percentage of the fresh weight at time zero. Data were derived from four independent experiments (±SD).</p

The miR165/166 Mediated Regulatory Module Plays Critical Roles in ABA Homeostasis and Response in <i>Arabidopsis thaliana</i>

<div><p>The function of miR165/166 in plant growth and development has been extensively studied, however, its roles in abiotic stress responses remain largely unknown. Here, we report that reduction in the expression of miR165/166 conferred a drought and cold resistance phenotype and hypersensitivity to ABA during seed germination and post-germination seedling development. We further show that the ABA hypersensitive phenotype is associated with a changed transcript abundance of ABA-responsive genes and a higher expression level of <i>ABI4</i>, which can be directly regulated by a miR165/166 target. Additionally, we found that reduction in miR165/166 expression leads to elevated ABA levels, which occurs at least partially through the increased expression of <i>BG1</i>, a gene that is directly regulated by a miR165/166 target. Taken together, our results uncover a novel role for miR165/166 in the regulation of ABA and abiotic stress responses and control of ABA homeostasis.</p></div

STTM165/166 plants are hypersensitive to ABA during seed germination and early seedling development.

<p>(A) The phenotype of wild type and STTM165/166 plants grown on MS medium supplemented with 1.0 μM ABA. Seedlings were photographed 9 days after stratification. (B, C) Germination and cotyledon greening analysis of the STTM165/166 and wild type plants in response to different concentrations of ABA (0, 1.0, 1.5 and 2.0 μM). Germination was scored at 4 days and greening was scored at 9 days after stratification. Three independent experiments were performed, and >100 seeds for each treatment were used for each experiment. Values are means ± standard deviation. (D, E) Quantitative RT-PCR analysis of the expression of both mature miR165/166 and its targets at seedling stage. Three independent experiments were performed, and values are means ± standard deviation.</p

ABA content is altered in STTM165/166 plants.

<p>(A) Quantitative RT-PCR analysis of the expression of genes involved in ABA conjugation and de-conjugation. (B) Quantitative RT-PCR analysis of <i>BG1</i> expression in various tissues of wild type and STTM165/166 plants. (C) Comparison of the ABA content between wild type and STTM165/166 plants.</p

Summary of AdipoR-mediated signaling pathways in mammalian cells and IZH2-mediated signaling pathways in <i>S.</i><i>cerevisiae</i> cells.

<p>(A) The components of the AdipoR1 signaling pathway of mammalian cells are shown in black font and their <i>S. cerevisiae</i> homologs, if known are shown in red font. In mammalian cells, adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1 (APPL1) interacts directly with AdipoR1. Interaction of adiponectin (ADPN) with AdipoR1 stimulates AdipoR1-APPL1 interaction. This results in release of Ca²⁺ from the ER to the cytosol and also increases export of LKB1 kinase from the nucleus. Influx of extracellular Ca²⁺ to the cytosol is also stimulated by adiponectin, although the mechanism by which this occurs remains to be clarified. Increase in cytosolic Ca²⁺ concentration activates Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKK) which in turn activates AMP activated protein kinase (AMPK) by phosphorylating its α subunit. However, phosphorylation of AMPK α subunit by the cytosol-localized kinase LKB1 is the major pathway for activation of AMPK. Adiponectin-AdipoR1 interaction also increases cellular ceramidase activity which in turn leads to phosphorylation of AMPKα subunit. The details of this pathway are not clear yet. Activation of AMPK is required for many of the anti-diabetic and anti-atherosclerotic effects of adiponectin. In vascular endothelial cells, interaction of AdipoR1 with adiponectin activates protein kinase A (PKA) which has the effect of lowering accumulation of reactive oxygen species (ROS) and thereby reducing inflammation. (B) Subunit structure of <i>S. cerevisiae</i> AMPK (ScAMPK). Like mammalian AMPK, ScAMPK is a trimer composed of an α, β, and γ subunit. The genes encoding the β subunit isoforms as well as the sole α and γ subunits are indicated. (C) Components of the <i>IZH2</i>-mediated signaling pathways in <i>S. cerevisiae</i> are shown in red font. Interaction of IZH2 (homolog of AdipoRs) with osmotin (OSM) activates PKA <i>via</i> a RAS2-cAMP pathway. Overexpression of <i>IZH2</i>, overexpression of <i>AdipoR1</i>, treatment of <i>S. cerevisiae</i> cells expressing <i>AdipoR1</i> with adiponectin and treatment of <i>S. cerevisiae</i> cells expressing various levels of <i>IZH2</i> with thaumatin (THN, a homolog of osmotin) has been shown to activate PKA by increasing cellular ceramidase activity. Activation of PKA leads to decreased transcription from a stress responsive promoter element (<i>STRE</i>) and increased cellular ROS content. Activated PKA promotes export of ScAMPK from the nucleus which leads to decreased transcription from the ferroxidase <i>(FET3</i>) promoter. Activated PKA also represses <i>FET3</i> transcription <i>via</i> the stress-responsive transcription factors MSN2/4. Mutational analyses show that genes encoding the APPL1-lke protein Sip3, the LKB1-like protein Sak1, the ScAMPK β subunit Sip1 and the ScAMPK γ subunit Snf4 are components of the pathway leading from <i>IZH2</i> (or <i>AdipoR1</i>) to <i>FET3</i> repression in <i>S. cerevisiae</i>.</p

AdipoRs are expressed on the plasma membrane in <i>S. cerevisiae</i>.

<p>(A) Subcellular localization of AdipoRs by confocal microscopy. <i>S. cerevisiae</i> strain BY4741 carrying plasmid pYES-EGFP (GFP), pYES-EGFP-AdipoR1 (GFP-AdipoR1) and pYES-EGFP-AdipoR2 (GFP-AdipoR2) were cultured in selective minimal medium containing 2% galactose. Shown are images of cells that were in the early log phase of growth. (B) Western blot analysis of total membrane protein extracts that were fractionated by 10% SDS-PAGE. The predicted molecular weights of GFP-AdipoR1 and GFP-AdipoR2 are around 65 kDa.</p

Final phenotypes of the female gametophyte in the wild type and <i>siz1-2</i> mutant.

<p>(A) LSCM images of an ovule derived from a wild-type flower; the pistil was harvested 2 day after emasculation. (B)–(D) LSCM images for ovules derived from <i>siz-1-2</i> flowers; the pistils were harvested 2 days after emasculation. Percentages of abnormal female gametophytes among the examined ovules are indicated below. Cn, central cell nucleus; En, egg cell nucleus; Sn, synergid cell nucleus. Bar = 40 µm.</p

PHB promotes <i>ABI4</i> expression by directly binding to its promoter.

<p>(A) <i>ABI4</i> expression was analyzed in wild type and STTM165/166 2-day-old seedlings using qRT-PCR. (B) <i>ABI4</i> expression was analyzed in <i>PHB</i>:<i>PHB G202G-YFP</i> lines using qRT-PCR. (C) Analysis of <i>ABI4</i> promoter. A 3.0kb fragment upstream of ATG was chosen for the promoter analysis. (D) EMSA assay showed that PHB binds to an <i>ABI4</i> promoter region. Labeled probe of the <i>ABI4</i> promoter region was incubated with GST-PHB fusion protein. For the competition test, a non-labeled probe was added at 10-fold and 100-fold concentrations.</p

The AdipoR1 ligands, adiponectin and osmotin, induce increase in Luc reporter activity.

<p>Cells of strain BY4741carrying pESC-URA-CLuc-AdipoR1-APPL1-NLuc were grown for 16 h at 30°C in selective minimal medium at the indicated galactose concentrations, treated for 4 h at 30°C with the indicated test compounds and then assayed for Luc activity. (A) Imaging of Luc activity. (B) Quantitative measurement of Luc activity as a function of osmotin concentration. A representative image of relative Luc activity at the different osmotin concentrations is shown for each galactose concentration. Data represent the means ± SD from three experiments with triplicate samples. For each galactose concentration, significant differences by a Student’s t-test between osmotin treated samples and untreated control are indicated by asterisks. Symbols: PBS, 1/8 X PBS; f-ADPN, bacterially expressed full length adiponectin; BSA, bovine serum albumin, OSM, osmotin; g-ADPN, bacterially expressed globular adiponectin; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

Expression patterns of selected genes from <i>siz1-2</i> and wild-type siliques.

<p>The tubulin α-2 gene (AT1G04820) was used as the internal control, and its expression level was set arbitrarily as 1.</p