Autophagy-mediated regulation of phytohormone metabolism during rice anther development

<p>Autophagy has recently been shown to be required for postmeiotic anther development including anther dehiscence, programmed cell death-mediated degradation of the tapetum and pollen maturation in rice. Several phytohormones are known to play essential roles during male reproductive development including pollen maturation. However, the relationship between phytohormone metabolism and autophagy in plant reproductive development is unknown. We here comprehensively analyzed the effect of autophagy disruption on phytohormone contents in rice anthers at the flowering stage, and found that endogenous levels of active-forms of gibberellins (GAs) and cytokinin, trans-zeatin, were significantly lower in the autophagy-defective mutant, Os<i>atg7–1</i>, than in the wild type. Treatment with GA₄ partially recovered maturation of the mutant pollens, but did not recover the limited anther dehiscence as well as sterility phenotype. These results suggest that autophagy affects metabolism and endogenous levels of GAs and cytokinin in rice anthers. Reduction in bioactive GAs in the autophagy-deficient mutant may partially explain the defects in pollen maturation of the autophagy-deficient mutant, but tapetal autophagy also plays other specific roles in fertilization.</p

Effect of <i>SLAC1</i>-overexpression on cryptogein-induced hypersensitive cell death.

<p>Cryptogein-induced cell death in a dose-dependent manner in <i>SLAC1</i>-overexpressing cells. BY-2 cells 24 h after the addition of the cryptogein elicitor at various concentrations. Evans blue staining was applied to quantify the level of cryptogein-induced cell death. Data are the mean ± SE of five independent experiments. **** <i>p</i><0.001, significantly different from the control line.</p

Effect of <i>SLAC1</i>-overexpression on cryptogein-induced ion fluxes.

<p>(A) Time course of cryptogein-induced [Cl^–]_ext changes in BY-2 cells. BY-2 cells were treated with cryptogein (0.25 µM). (B) Effect of DIDS on cryptogein-induced [Cl^–]_ext changes. DIDS (100 µM) was added to BY-2 cells 15 min prior to the elicitor (0.25 µM) treatment. DMSO was used as a control. (C) Time course of cryptogein-induced [pH]_ext changes. BY-2 cells were treated with cryptogein (1 µM). ** <i>p</i><0.005, *** <i>p</i><0.001, significantly different from the control line. (D) Effect of DIDS on cryptogein-induced [pH]_ext changes. DIDS (50 µM) was added to BY-2 cells 15 min prior to the elicitor treatment. DMSO was used as a control. Data are the mean ± SE of three independent experiments. * <i>p</i><0.05, significantly different from the control line.</p

Intracellular localization of the SLAC1 protein in tobacco BY-2 cells.

<p>Confocal fluorescence images (<b>a</b>-<b>f</b>) of BY-2 protoplast expressing SLAC1-GFP (<b>a</b>–<b>c</b>) or GFP (<b>d–f</b>) stained with the fluorescent styryl membrane probe FM4-64. Fluorescence of GFP (<b>a</b> and <b>d</b>) and FM4-64 (<b>b</b> and <b>e</b>). Scale bar: 10 µm. FM4-64 was kept as a 17 mM stock solution in sterile water, and used at a final concentration of 4.25 µM. Tobacco BY-2 protoplasts were treated with FM4-64 for 10 min and washed twice with the wash buffer at room temperature to label the PM.</p

An S-Type Anion Channel SLAC1 Is Involved in Cryptogein-Induced Ion Fluxes and Modulates Hypersensitive Responses in Tobacco BY-2 Cells

<div><p>Pharmacological evidence suggests that anion channel-mediated plasma membrane anion effluxes are crucial in early defense signaling to induce immune responses and hypersensitive cell death in plants. However, their molecular bases and regulation remain largely unknown. We overexpressed Arabidopsis <i>SLAC1</i>, an S-type anion channel involved in stomatal closure, in cultured tobacco BY-2 cells and analyzed the effect on cryptogein-induced defense responses including fluxes of Cl⁻ and other ions, production of reactive oxygen species (ROS), gene expression and hypersensitive responses. The SLAC1-GFP fusion protein was localized at the plasma membrane in BY-2 cells. Overexpression of <i>SLAC1</i> enhanced cryptogein-induced Cl⁻ efflux and extracellular alkalinization as well as rapid/transient and slow/prolonged phases of NADPH oxidase-mediated ROS production, which was suppressed by an anion channel inhibitor, DIDS. The overexpressor also showed enhanced sensitivity to cryptogein to induce downstream immune responses, including the induction of defense marker genes and the hypersensitive cell death. These results suggest that SLAC1 expressed in BY-2 cells mediates cryptogein-induced plasma membrane Cl⁻ efflux to positively modulate the elicitor-triggered activation of other ion fluxes, ROS as well as a wide range of defense signaling pathways. These findings shed light on the possible involvement of the SLAC/SLAH family anion channels in cryptogein signaling to trigger the plasma membrane ion channel cascade in the plant defense signal transduction network.</p></div

Effect of <i>SLAC1</i>-overexpression on cryptogein-induced defense gene expressions.

<p>(A and B) Cryptogein-induced the expression of <i>HIN1</i> in a dose-dependent manner (A) and <i>Hsr203j</i> (B) in <i>SLAC1</i>-overexpressing cells. The amount of each mRNA was calculated from the threshold point located in the log-linear range of the RT-PCR. The relative level of each gene in the control cells at time 0. Total RNA was isolated from BY-2 cells harvested 5 h after the addition of cryptogein at various concentrations. Data are the mean ± SE of three independent experiments. * <i>p</i><0.05, ** <i>p</i><0.005, significantly different from the control line.</p

Involvement of SLAC1 in the regulation of cryptogein-induced biphasic ROS production mediated by NADPH oxidases.

<p>(A) Cryptogein (0.25 µM)-induced rapid transient ROS production within 15 min in BY-2 cells. Data is representative of three experiments (B) The effects of inhibitors on cryptogein-induced ROS production within 15 min. To quantify the effects of inhibitors on ROS production, the peak intensity of luminol chemiluminescence was compared with the control. DIDS or an NADPH oxidase inhibitor, DPI, was added to BY-2 cells 15 min prior to the elicitor treatment. DMSO was used as a control. Data are the mean ± SE of three independent experiments. ** <i>p</i><0.005, significantly different from the control line. (C) Effect of <i>SLAC1</i>-overexpression on cryptogein-induced prolonged ROS production. BY-2 cells were treated with cryptogein (0.25 µM). Data are the mean ± SE of three independent experiments. * <i>p</i><0.05, ** <i>p</i><0.005, *** <i>p</i><0.001, significantly different from the control line. (D) Quantitative expression levels of <i>NtRbohD</i> mRNAs in <i>SLAC1</i>-overexpressing cells by real-time quantitative PCR. Total RNA was isolated from BY-2 cells harvested 5 h after the addition of cryptogein at various concentrations. The amount of each mRNA was calculated from the threshold point located in the log-linear range of the RT-PCR. The relative level of each gene in the control cells at time 0. Data are the mean ± SE of four independent experiments. * <i>p</i><0.005, ** <i>p</i><0.005, significantly different from the control line.</p

Effect of DIDS on cryptogein-induced mitochondrial dysfunction and expression of <i>HIN1</i> in BY-2 cells.

<p>(A) Cryptogein (1 µM)-induced reductions in MTT reductase activity. MTT reductase activity was used as a putative marker for mitochondrial dysfunction. Black circle: cryptogein treatment, white circle: water treatment as a control. *** <i>p</i><0.001, significantly different from the control. (B) Inhibitory effect of DIDS on cryptogein-induced reductions in MTT reductase activity. BY-2 cells 3 h after the addition of the cryptogein elicitor (1 µM). DIDS or DMSO was added to BY-2 cells 15 min prior to the elicitor treatment. Data are the mean ± SE of three independent experiments. * <i>p</i><0.05, ** <i>p</i><0.005, significantly different from the control. DMSO was used as a control. (C) Effect of DIDS on cryptogein-induced <i>HIN1</i> expression. Total RNA was isolated from BY-2 cells harvested 2 h after the addition of cryptogein. DIDS (white bars) or DMSO (black bars) was added to BY-2 cells 15 min prior to the elicitor treatment. The amount of each mRNA was calculated from the threshold point located in the log-linear range of RT-PCR. The relative level of each gene in control cells at time 0 was standardized as 1. Data are the mean ± SE of three independent experiments. ** <i>p</i><0.005, *** <i>p</i><0.001, significantly different from the control.</p

OsATG7 is required for autophagy-dependent lipid metabolism in rice postmeiotic anther development

<div><p>In flowering plants, the tapetum, the innermost layer of the anther, provides both nutrient and lipid components to developing microspores, pollen grains, and the pollen coat. Though the programmed cell death of the tapetum is one of the most critical and sensitive steps for fertility and is affected by various environmental stresses, its regulatory mechanisms remain mostly unknown. Here we show that autophagy is required for the metabolic regulation and nutrient supply in anthers and that autophagic degradation within tapetum cells is essential for postmeiotic anther development in rice. Autophagosome-like structures and several vacuole-enclosed lipid bodies were observed in postmeiotic tapetum cells specifically at the uninucleate stage during pollen development, which were completely abolished in a retrotransposon-insertional <i>OsATG7</i> (autophagy-related 7)-knockout mutant defective in autophagy, suggesting that autophagy is induced in tapetum cells. Surprisingly, the mutant showed complete sporophytic male sterility, failed to accumulate lipidic and starch components in pollen grains at the flowering stage, showed reduced pollen germination activity, and had limited anther dehiscence. Lipidomic analyses suggested impairment of editing of phosphatidylcholines and lipid desaturation in the mutant during pollen maturation. These results indicate a critical involvement of autophagy in a reproductive developmental process of rice, and shed light on the novel autophagy-mediated regulation of lipid metabolism in eukaryotic cells.</p></div