Summary: Arsenic (As) contamination of rice (Oryza sativa) is a worldwide concern and elucidating the molecular mechanisms of As accumulation in rice may provide promising solutions to the problem. Previous studies using microarray techniques to investigate transcriptional regulation of plant responses to As stress have identified numerous differentially expressed genes. However, little is known about the metabolic and regulatory network remodelings, or their interactions with microRNA (miRNA) in plants upon As(III) exposure. We used Illumina sequencing to acquire global transcriptome alterations and miRNA regulation in rice under As(III) treatments of varying lengths of time and dosages. We found that the response of roots was more distinct when the dosage was varied, whereas that of shoots was more distinct when the treatment time was varied. In particular, the genes involved in heavy metal transportation, jasmonate (JA) biosynthesis and signaling, and lipid metabolism were closely related to responses of rice under As(III) stress. Furthermore, we discovered 36 new As(III)‐responsive miRNAs, 14 of which were likely involved in regulating gene expression in transportation, signaling, and metabolism. Our findings highlight the significance of JA signaling and lipid metabolism in response to As(III) stress and their regulation by miRNA, which provides a foundation for subsequent functional research.

Autophagy involves massive degradation of intracellular components and functions as a conserved system that helps cells to adapt to adverse conditions. In mammals, hypoxia rapidly stimulates autophagy as a cell survival response. Here, we examine the function of autophagy in the regulation of the plant response to submergence, an abiotic stress that leads to hypoxia and anaerobic respiration in plant cells. In Arabidopsis thaliana, submergence induces the transcription of autophagy-related (ATG) genes and the formation of autophagosomes. Consistent with this, the autophagy-defective (atg) mutants are hypersensitive to submergence stress and treatment with ethanol, the end product of anaerobic respiration. Upon submergence, the atg mutants have increased levels of transcripts of anaerobic respiration genes (alcohol dehydrogenase 1, ADH1 and pyruvate decarboxylase 1, PDC1), but reduced levels of transcripts of other hypoxia- and ethylene-responsive genes. Both submergence and ethanol treatments induce the accumulation of reactive oxygen species (ROS) in the rosettes of atg mutants more than in the wild type. Moreover, the production of ROS by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases is necessary for plant tolerance to submergence and ethanol, submergence-induced expression of ADH1 and PDC1, and activation of autophagy. The submergence- and ethanol-sensitive phenotypes in the atg mutants depend on a complete salicylic acid (SA) signaling pathway. Together, our findings demonstrate that submergence-induced autophagy functions in the hypoxia response in Arabidopsis by modulating SA-mediated cellular homeostasis.

Autophagy is a highly conserved system in eukaryotes for the bulk degradation and recycling of intracellular components. Autophagy is involved in many physiological processes including development, senescence, and responses to abiotic and biotic stress. The adenosine 5'-monophosphate (AMP)-activated protein kinase AMPK positively regulates autophagy in mammals; however, the potential function of AMPK in plant autophagy remains largely unknown. Here, we identified KIN10, a plant orthologue of the mammalian AMPK, as a positive regulator of plant autophagy and showed that it acts by affecting the phosphorylation of ATG1 (AUTOPHAGY-RELATED GENE 1) proteins in Arabidopsis. Transgenic Arabidopsis lines overexpressing KIN10 (KIN10-OE) showed delays in leaf senescence, and increased tolerance to nutrient starvation, these phenotypes required a functional autophagy pathway. Consistent with KIN10 having a potential role in autophagy, the nutrient starvation-induced formation of autophagosomes and cleavage of GFP-ATG8e were accelerated in the KIN10-OE lines compared to the wild type. Moreover, the KIN10-OE lines were less sensitive to drought and hypoxia treatments, compared with wild type. Carbon starvation enhanced the level of phosphorylated YFP-ATG1a in the KIN10-OE lines compared to that of wild type. Together, these findings suggest that KIN10 is involved in positive regulation of autophagy, possibly by affecting the phosphorylation of ATG1s in Arabidopsis.

Lipid remodeling is crucial for hypoxic tolerance in animals, whilst little is known about the hypoxia-induced lipid dynamics in plants. Here we performed a mass spectrometry-based analysis to survey the lipid profiles of Arabidopsis rosettes under various hypoxic conditions. We observed that hypoxia caused a significant increase in total amounts of phosphatidylserine, phosphatidic acid and oxidized lipids, but a decrease in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Particularly, significant gains in the polyunsaturated species of PC, PE and phosphatidylinositol, and losses in their saturated and mono-unsaturated species were evident during hypoxia. Moreover, hypoxia led to a remarkable elevation of ceramides and hydroxyceramides. Disruption of ceramide synthases LOH1, LOH2 and LOH3 enhanced plant sensitivity to dark submergence, but displayed more resistance to submergence under light than wild type. Consistently, levels of unsaturated very-long-chain (VLC) ceramide species (22:1, 24:1 and 26:1) predominantly declined in the loh1, loh2 and loh3 mutants under dark submergence. In contrast, significant reduction of VLC ceramides in the loh1-1 loh3-1 knockdown double mutant and lacking of VLC unsaturated ceramides in the ads2 mutants impaired plant tolerance to both dark and light submergences. Evidence that C24:1-ceramide interacted with recombinant CTR1 protein and inhibited its kinase activity in vitro, enhanced ER-to-nucleus translocation of EIN2-GFP and stabilization of EIN3-GFP in vivo, suggests a role of ceramides in modulating CTR1-mediated ethylene signaling. The dark submergence-sensitive phenotypes of loh mutants were rescued by a ctr1-1 mutation. Thus, our findings demonstrate that unsaturation of VLC ceramides is a protective strategy for hypoxic tolerance in Arabidopsis. Hypoxia is one of the most important abiotic stresses of worldwide concern that determines the crop productivity and the natural distribution of plant species. Flooding events such as root waterlogging and submergence strongly affect diffusion of gasses into plant cells, which eventually leads to hypoxia and carbohydrate shortages in terrestrial plants. The gaseous hormone ethylene is considered as the primary determinant of hypoxia response in plants. In particular, the Group VII ethylene-responsive factors (ERFs) have been demonstrated to be master regulators for oxygen sensing through an N-end rule protein degradation mechanism. Recent investigations have suggested that lipid molecules such VLC ceramides may play crucial roles in hypoxia signaling in both animal and plant cells. Here, we identified the unsaturated VLC ceramide species including C22:1-, C24:1- and C26:1-Cers which were essential for hypoxia response by interacting with CTR1 protein, inhibiting its kinase activity, and modulating subsequent ethylene signaling in Arabidopsis. The dynamic unsaturation of VLC ceramides is likely to serve as a novel protective strategy for enhancing plant tolerance to the frequent environmental stresses, including flooding.

Autophagy is a major degradation process of cytoplasmic components in eukaryotes, and executes both bulk and selective degradation of targeted cargos. A set of autophagy-related (ATG) proteins participate in various stages of the autophagic process. Among ATGs, ubiquitin-like protein ATG8 plays a central role in autophagy. The ATG8 protein is conjugated to the membrane lipid phosphatidylethanolamine in a ubiquitin-like conjugation reaction that is essential for autophagosome formation. In addition, ATG8 interacts with various adaptor/receptor proteins to recruit specific cargos for degradation by selective autophagy. The ATG8-interacting proteins usually contain the ATG8-interacting motif (AIM) or the ubiquitin-interacting motif (UIM) for ATG8 binding. Unlike a single ATG8 gene in yeast, multiple ATG8 orthologs have been identified in the plant kingdom. The large diversity within the ATG8 family may explain the various functions of selective autophagy in plants. Here, we discuss and summarize the current view of the structure and function of ATG8 proteins in plants.