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Autophagy contributes to regulation of the hypoxia response during submergence in Arabidopsis thaliana.

Chen L, Liao B, Qi H, Xie LJ, Huang L, Tan WJ, Zhai N, Yuan LB, Zhou Y, Yu LJ, Chen QF, Shu W, Xiao S - Autophagy (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: a State Key Laboratory of Biocontrol; Guangdong Provincial Key Laboratory of Plant Resources; Collaborative Innovation Center of Genetics and Development; School of Life Sciences; Sun Yat-sen University ; Guangzhou , China.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Induction of autophagy by submergence. (A) qRT-PCR analyses showing the relative expression of autophagy-related genes (ATG2, ATG5, ATG7, ATG8a, ATG10, and ATG18a) upon submergence. Total RNA was isolated from rosettes of 4-wk-old soil-grown plants upon dark submergence (DS), and from control plants under constant darkness (Dark) and normal light/dark cycle (Light). The samples were harvested at 0, 3, 6, 12, and 24 h after treatment. Transcript levels relative to 0 h for each time point were normalized to the levels of ACT2 (Actin2). The experiments have been repeated 3 times (biological replicates) with similar results and the representative data from one replicate are shown. Data are average values ± SD (n = 3) of 3 technical replicates. Asterisks indicate significant differences from the untreated control (**, P < 0.01 by the Student t test). (B) GFP fluorescence in the root cells of GFP-ATG8e transformants under Light and light submergence (LS) conditions. One-wk-old Arabidopsis seedlings expressing GFP-ATG8e were not treated or LS-treated for 6 h with (+ConA) or without concanamycin A (−ConA) and visualized by confocal microscopy. Scale bar: 50 μm. (C) Immunoblot analysis showing the processing of GFP-ATG8e under Light, and upon LS, DS, and Dark treatments. Leaves from 4-wk-old plants were collected at 0, 6, 12, 24, and 48 h after treatment (hpt) and anti-GFP antibodies were used for immunoblotting. The GFP-ATG8e fusion and free GFP form are indicated on the right. Coomassie blue–stained total proteins are shown below the blots to indicate the amount of protein loaded per lane.
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f0001: Induction of autophagy by submergence. (A) qRT-PCR analyses showing the relative expression of autophagy-related genes (ATG2, ATG5, ATG7, ATG8a, ATG10, and ATG18a) upon submergence. Total RNA was isolated from rosettes of 4-wk-old soil-grown plants upon dark submergence (DS), and from control plants under constant darkness (Dark) and normal light/dark cycle (Light). The samples were harvested at 0, 3, 6, 12, and 24 h after treatment. Transcript levels relative to 0 h for each time point were normalized to the levels of ACT2 (Actin2). The experiments have been repeated 3 times (biological replicates) with similar results and the representative data from one replicate are shown. Data are average values ± SD (n = 3) of 3 technical replicates. Asterisks indicate significant differences from the untreated control (**, P < 0.01 by the Student t test). (B) GFP fluorescence in the root cells of GFP-ATG8e transformants under Light and light submergence (LS) conditions. One-wk-old Arabidopsis seedlings expressing GFP-ATG8e were not treated or LS-treated for 6 h with (+ConA) or without concanamycin A (−ConA) and visualized by confocal microscopy. Scale bar: 50 μm. (C) Immunoblot analysis showing the processing of GFP-ATG8e under Light, and upon LS, DS, and Dark treatments. Leaves from 4-wk-old plants were collected at 0, 6, 12, 24, and 48 h after treatment (hpt) and anti-GFP antibodies were used for immunoblotting. The GFP-ATG8e fusion and free GFP form are indicated on the right. Coomassie blue–stained total proteins are shown below the blots to indicate the amount of protein loaded per lane.

Mentions: To investigate the potential involvement of autophagy in the submergence response, we examined the abundance of ATG transcripts in Arabidopsis rosettes subjected to submergence treatment under constant darkness (DS), with plants subjected to constant darkness (Dark) and the normal light/dark cycle (Light) as controls. Reverse transcription quantitative PCR (qRT-PCR) analyses showed that ATG transcript levels were unchanged under light conditions, and increased under dark conditions (Fig. 1A), consistent with previous observations.34 After DS treatment, the transcript levels of all 6 ATG genes increased significantly more than in the dark controls (Fig. 1A). We also confirmed the induction of ATG expression by examining transgenic Arabidopsis expressing ATG promoter fusions with the β-glucuronidase (GUS) reporter: ATG7pro-GUS, ATG8epro-GUS, ATG8hpro-GUS, and ATG8ipro-GUS. GUS staining revealed that the expression of all 4 fusions increased after DS or LS (submergence under light conditions) from 6 to 24 h after treatment (Fig. S1A), suggesting that submergence activated autophagy under both DS and LS treatments, 2 submergence conditions leading to hypoxia around the underwater leaves at various times after treatment (Fig. S1B).Figure 1.


Autophagy contributes to regulation of the hypoxia response during submergence in Arabidopsis thaliana.

Chen L, Liao B, Qi H, Xie LJ, Huang L, Tan WJ, Zhai N, Yuan LB, Zhou Y, Yu LJ, Chen QF, Shu W, Xiao S - Autophagy (2015)

Induction of autophagy by submergence. (A) qRT-PCR analyses showing the relative expression of autophagy-related genes (ATG2, ATG5, ATG7, ATG8a, ATG10, and ATG18a) upon submergence. Total RNA was isolated from rosettes of 4-wk-old soil-grown plants upon dark submergence (DS), and from control plants under constant darkness (Dark) and normal light/dark cycle (Light). The samples were harvested at 0, 3, 6, 12, and 24 h after treatment. Transcript levels relative to 0 h for each time point were normalized to the levels of ACT2 (Actin2). The experiments have been repeated 3 times (biological replicates) with similar results and the representative data from one replicate are shown. Data are average values ± SD (n = 3) of 3 technical replicates. Asterisks indicate significant differences from the untreated control (**, P < 0.01 by the Student t test). (B) GFP fluorescence in the root cells of GFP-ATG8e transformants under Light and light submergence (LS) conditions. One-wk-old Arabidopsis seedlings expressing GFP-ATG8e were not treated or LS-treated for 6 h with (+ConA) or without concanamycin A (−ConA) and visualized by confocal microscopy. Scale bar: 50 μm. (C) Immunoblot analysis showing the processing of GFP-ATG8e under Light, and upon LS, DS, and Dark treatments. Leaves from 4-wk-old plants were collected at 0, 6, 12, 24, and 48 h after treatment (hpt) and anti-GFP antibodies were used for immunoblotting. The GFP-ATG8e fusion and free GFP form are indicated on the right. Coomassie blue–stained total proteins are shown below the blots to indicate the amount of protein loaded per lane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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f0001: Induction of autophagy by submergence. (A) qRT-PCR analyses showing the relative expression of autophagy-related genes (ATG2, ATG5, ATG7, ATG8a, ATG10, and ATG18a) upon submergence. Total RNA was isolated from rosettes of 4-wk-old soil-grown plants upon dark submergence (DS), and from control plants under constant darkness (Dark) and normal light/dark cycle (Light). The samples were harvested at 0, 3, 6, 12, and 24 h after treatment. Transcript levels relative to 0 h for each time point were normalized to the levels of ACT2 (Actin2). The experiments have been repeated 3 times (biological replicates) with similar results and the representative data from one replicate are shown. Data are average values ± SD (n = 3) of 3 technical replicates. Asterisks indicate significant differences from the untreated control (**, P < 0.01 by the Student t test). (B) GFP fluorescence in the root cells of GFP-ATG8e transformants under Light and light submergence (LS) conditions. One-wk-old Arabidopsis seedlings expressing GFP-ATG8e were not treated or LS-treated for 6 h with (+ConA) or without concanamycin A (−ConA) and visualized by confocal microscopy. Scale bar: 50 μm. (C) Immunoblot analysis showing the processing of GFP-ATG8e under Light, and upon LS, DS, and Dark treatments. Leaves from 4-wk-old plants were collected at 0, 6, 12, 24, and 48 h after treatment (hpt) and anti-GFP antibodies were used for immunoblotting. The GFP-ATG8e fusion and free GFP form are indicated on the right. Coomassie blue–stained total proteins are shown below the blots to indicate the amount of protein loaded per lane.
Mentions: To investigate the potential involvement of autophagy in the submergence response, we examined the abundance of ATG transcripts in Arabidopsis rosettes subjected to submergence treatment under constant darkness (DS), with plants subjected to constant darkness (Dark) and the normal light/dark cycle (Light) as controls. Reverse transcription quantitative PCR (qRT-PCR) analyses showed that ATG transcript levels were unchanged under light conditions, and increased under dark conditions (Fig. 1A), consistent with previous observations.34 After DS treatment, the transcript levels of all 6 ATG genes increased significantly more than in the dark controls (Fig. 1A). We also confirmed the induction of ATG expression by examining transgenic Arabidopsis expressing ATG promoter fusions with the β-glucuronidase (GUS) reporter: ATG7pro-GUS, ATG8epro-GUS, ATG8hpro-GUS, and ATG8ipro-GUS. GUS staining revealed that the expression of all 4 fusions increased after DS or LS (submergence under light conditions) from 6 to 24 h after treatment (Fig. S1A), suggesting that submergence activated autophagy under both DS and LS treatments, 2 submergence conditions leading to hypoxia around the underwater leaves at various times after treatment (Fig. S1B).Figure 1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: a State Key Laboratory of Biocontrol; Guangdong Provincial Key Laboratory of Plant Resources; Collaborative Innovation Center of Genetics and Development; School of Life Sciences; Sun Yat-sen University ; Guangzhou , China.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus