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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

The rboh mutants show enhanced sensitivity to ethanol and submergence stresses. (A) Images of the wild type (WT) and rboh mutants (rbohd, rbohf, and rbohd rbohf) germinated on MS medium containing no ethanol or 50 mM ethanol, for 2 wk. (B) Images of WT and rboh mutants (rbohd, rbohf, and rbohd rbohf) before treatment (Light) and at 6 d after light submergence (LS) treatment followed by a 9-d recovery. (C) Relative expression levels of hypoxia-responsive marker genes in WT and rboh (rbohd, rbohf, and rbohd rbohf) mutants upon LS treatment. Four-wk-old plants were LS-treated for 0 and 3 h, and leaves were collected for RNA extraction. Transcript levels relative to WT at 0 h were normalized to that of ACT2. The experiments have been repeated 3 times, with biological replicates, and similar results were obtained. Data are average values ± SD (n = 3) of 3 technical replicates. Asterisks indicate significant differences from WT (*, P < 0.05; **, P < 0.01 by the Student t test). (D) MDC staining of root cells from WT, rbohd, rbohf, and rbohd rbohf seedlings. One-wk-old seedlings were untreated and LS-treated for 24 h followed by staining with MDC. The labeled autophagosomes (arrows) were visualized by epifluorescence microscopy. Scale bar: 50 μm.
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f0006: The rboh mutants show enhanced sensitivity to ethanol and submergence stresses. (A) Images of the wild type (WT) and rboh mutants (rbohd, rbohf, and rbohd rbohf) germinated on MS medium containing no ethanol or 50 mM ethanol, for 2 wk. (B) Images of WT and rboh mutants (rbohd, rbohf, and rbohd rbohf) before treatment (Light) and at 6 d after light submergence (LS) treatment followed by a 9-d recovery. (C) Relative expression levels of hypoxia-responsive marker genes in WT and rboh (rbohd, rbohf, and rbohd rbohf) mutants upon LS treatment. Four-wk-old plants were LS-treated for 0 and 3 h, and leaves were collected for RNA extraction. Transcript levels relative to WT at 0 h were normalized to that of ACT2. The experiments have been repeated 3 times, with biological replicates, and similar results were obtained. Data are average values ± SD (n = 3) of 3 technical replicates. Asterisks indicate significant differences from WT (*, P < 0.05; **, P < 0.01 by the Student t test). (D) MDC staining of root cells from WT, rbohd, rbohf, and rbohd rbohf seedlings. One-wk-old seedlings were untreated and LS-treated for 24 h followed by staining with MDC. The labeled autophagosomes (arrows) were visualized by epifluorescence microscopy. Scale bar: 50 μm.

Mentions: Previous work showed that the RBOHD transcript was significantly upregulated by anoxia and the rbohd mutants displayed reduced survival of anoxic stress.44 To gain genetic evidence on the involvement of ROS in the autophagy-mediated submergence response, we obtained the rbohd and rbohf transposon insertion mutants and the rbohd rbohf double mutant,42 and characterized their responses to ethanol and submergence stresses. When their seeds were germinated on MS medium, or MS containing 50 mM ethanol, the rbohd, rbohf, and rbohd rbohf mutants showed less resistance to ethanol stress in comparison with the wild type (Fig. 6A). Moreover, upon submergence exposure for 6 d followed by a 9-d recovery, all of the wild-type plants survived submergence, but most of the rbohd and rbohf mutants displayed enhanced damages in their rosette leaves and all of rbohd rbohf double mutants died (Fig. 6B). Upon a 3-h submergence exposure, the transcripts of the hypoxia-responsive genes ADH1, PDC1, and HB1 were downregulated in the rbohd, rbohf, and rbohd rbohf mutants compared to the wild type (Fig. 6C). Further, MDC staining showed that the submergence-induced accumulation of autophagic structures was reduced in the rbohd, rbohf, and rbohd rbohf mutants compared to the wild type (Fig. 6D). Moreover, DAB staining results showed that the ROS accumulation in the leaves of rbohd, rbohf, and rbohd rbohf mutants were much lower than that of the wild type after submergence (Fig. S7A). Consistently, the submerged rbohd, rbohf, and rbohd rbohf mutants displayed more severe cell death staining by trypan blue than that of wild-type leaves (Fig. S7B).Figure 6.


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)

The rboh mutants show enhanced sensitivity to ethanol and submergence stresses. (A) Images of the wild type (WT) and rboh mutants (rbohd, rbohf, and rbohd rbohf) germinated on MS medium containing no ethanol or 50 mM ethanol, for 2 wk. (B) Images of WT and rboh mutants (rbohd, rbohf, and rbohd rbohf) before treatment (Light) and at 6 d after light submergence (LS) treatment followed by a 9-d recovery. (C) Relative expression levels of hypoxia-responsive marker genes in WT and rboh (rbohd, rbohf, and rbohd rbohf) mutants upon LS treatment. Four-wk-old plants were LS-treated for 0 and 3 h, and leaves were collected for RNA extraction. Transcript levels relative to WT at 0 h were normalized to that of ACT2. The experiments have been repeated 3 times, with biological replicates, and similar results were obtained. Data are average values ± SD (n = 3) of 3 technical replicates. Asterisks indicate significant differences from WT (*, P < 0.05; **, P < 0.01 by the Student t test). (D) MDC staining of root cells from WT, rbohd, rbohf, and rbohd rbohf seedlings. One-wk-old seedlings were untreated and LS-treated for 24 h followed by staining with MDC. The labeled autophagosomes (arrows) were visualized by epifluorescence microscopy. Scale bar: 50 μm.
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f0006: The rboh mutants show enhanced sensitivity to ethanol and submergence stresses. (A) Images of the wild type (WT) and rboh mutants (rbohd, rbohf, and rbohd rbohf) germinated on MS medium containing no ethanol or 50 mM ethanol, for 2 wk. (B) Images of WT and rboh mutants (rbohd, rbohf, and rbohd rbohf) before treatment (Light) and at 6 d after light submergence (LS) treatment followed by a 9-d recovery. (C) Relative expression levels of hypoxia-responsive marker genes in WT and rboh (rbohd, rbohf, and rbohd rbohf) mutants upon LS treatment. Four-wk-old plants were LS-treated for 0 and 3 h, and leaves were collected for RNA extraction. Transcript levels relative to WT at 0 h were normalized to that of ACT2. The experiments have been repeated 3 times, with biological replicates, and similar results were obtained. Data are average values ± SD (n = 3) of 3 technical replicates. Asterisks indicate significant differences from WT (*, P < 0.05; **, P < 0.01 by the Student t test). (D) MDC staining of root cells from WT, rbohd, rbohf, and rbohd rbohf seedlings. One-wk-old seedlings were untreated and LS-treated for 24 h followed by staining with MDC. The labeled autophagosomes (arrows) were visualized by epifluorescence microscopy. Scale bar: 50 μm.
Mentions: Previous work showed that the RBOHD transcript was significantly upregulated by anoxia and the rbohd mutants displayed reduced survival of anoxic stress.44 To gain genetic evidence on the involvement of ROS in the autophagy-mediated submergence response, we obtained the rbohd and rbohf transposon insertion mutants and the rbohd rbohf double mutant,42 and characterized their responses to ethanol and submergence stresses. When their seeds were germinated on MS medium, or MS containing 50 mM ethanol, the rbohd, rbohf, and rbohd rbohf mutants showed less resistance to ethanol stress in comparison with the wild type (Fig. 6A). Moreover, upon submergence exposure for 6 d followed by a 9-d recovery, all of the wild-type plants survived submergence, but most of the rbohd and rbohf mutants displayed enhanced damages in their rosette leaves and all of rbohd rbohf double mutants died (Fig. 6B). Upon a 3-h submergence exposure, the transcripts of the hypoxia-responsive genes ADH1, PDC1, and HB1 were downregulated in the rbohd, rbohf, and rbohd rbohf mutants compared to the wild type (Fig. 6C). Further, MDC staining showed that the submergence-induced accumulation of autophagic structures was reduced in the rbohd, rbohf, and rbohd rbohf mutants compared to the wild type (Fig. 6D). Moreover, DAB staining results showed that the ROS accumulation in the leaves of rbohd, rbohf, and rbohd rbohf mutants were much lower than that of the wild type after submergence (Fig. S7A). Consistently, the submerged rbohd, rbohf, and rbohd rbohf mutants displayed more severe cell death staining by trypan blue than that of wild-type leaves (Fig. S7B).Figure 6.

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