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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 enhanced submergence and ethanol sensitivity in autophagy-defective mutants requires salicylic acid signaling. (A) Measurement of endogenous SA contents in the wild type (WT) and atg mutants (atg5-1 and atg7-3). WT, atg5-1, and atg7-3 mutants were treated with submergence (LS) and leaves were collected at 0, 1, 3, 6, and 12 h after treatment for SA extraction and then analyzed by LC/MS. D6-SA was added as an internal quantitative standard. The experiments were repeated (biological replicates) twice, with similar results, and the representative data from one replicate are shown. Data are means ± SD calculated from 8 technical replicates (200 mg leaves harvested from 3 independent plants were pooled for each technical replicate). Asterisks with “a” indicate significant differences from that of the wild type at each time point and with “b” indicate significant differences from untreated wild-type control (*, P < 0.05; **, P < 0.01 by the Student t test). (B) Relative expression levels of SA biosynthesis genes in WT and atg mutants (atg5–1 and atg7–3) upon LS treatment. Total RNA was isolated from 4-wk-old WT and atg mutants under LS treatment for 0, 3, 6, and 24 h. The relative transcript levels were normalized to that of ACT2. *, P < 0.05; **, P < 0.01 by Student t test. (C) Images of WT, atg5–1, atg5 sid2, sid2, atg5 npr1, and npr1-5 seeds germinated on MS medium containing no ethanol or 50 mM ethanol, for 2 wk. (D) Images of WT, atg5-1, atg5 sid2, sid2, atg5 npr1, and npr1-5 plants before treatment (Day 0) and after a 6-d recovery after a 5-d LS treatment (Day 5). (E) DAB staining showing ROS levels in the leaves of 4-wk-old WT, atg5-1, atg5 sid2, sid2, atg5 npr1, and npr1-5 mutants under normal growth conditions (Light) and after 3-d LS treatment. Scale bar: 500 μm. (F) MDC staining showing autophagosome formation in the root cells of WT, atg5-1, atg5 sid2, sid2, atg5 npr1, and npr1-5 seedlings under normal light/dark (Light) and LS conditions. One-wk-old seedlings were not treated or LS-treated for 24 h following by staining with MDC. The labeled autophagosomes (arrows) were visualized by epifluorescence microscopy. Scale bar: 50 μm.
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f0007: The enhanced submergence and ethanol sensitivity in autophagy-defective mutants requires salicylic acid signaling. (A) Measurement of endogenous SA contents in the wild type (WT) and atg mutants (atg5-1 and atg7-3). WT, atg5-1, and atg7-3 mutants were treated with submergence (LS) and leaves were collected at 0, 1, 3, 6, and 12 h after treatment for SA extraction and then analyzed by LC/MS. D6-SA was added as an internal quantitative standard. The experiments were repeated (biological replicates) twice, with similar results, and the representative data from one replicate are shown. Data are means ± SD calculated from 8 technical replicates (200 mg leaves harvested from 3 independent plants were pooled for each technical replicate). Asterisks with “a” indicate significant differences from that of the wild type at each time point and with “b” indicate significant differences from untreated wild-type control (*, P < 0.05; **, P < 0.01 by the Student t test). (B) Relative expression levels of SA biosynthesis genes in WT and atg mutants (atg5–1 and atg7–3) upon LS treatment. Total RNA was isolated from 4-wk-old WT and atg mutants under LS treatment for 0, 3, 6, and 24 h. The relative transcript levels were normalized to that of ACT2. *, P < 0.05; **, P < 0.01 by Student t test. (C) Images of WT, atg5–1, atg5 sid2, sid2, atg5 npr1, and npr1-5 seeds germinated on MS medium containing no ethanol or 50 mM ethanol, for 2 wk. (D) Images of WT, atg5-1, atg5 sid2, sid2, atg5 npr1, and npr1-5 plants before treatment (Day 0) and after a 6-d recovery after a 5-d LS treatment (Day 5). (E) DAB staining showing ROS levels in the leaves of 4-wk-old WT, atg5-1, atg5 sid2, sid2, atg5 npr1, and npr1-5 mutants under normal growth conditions (Light) and after 3-d LS treatment. Scale bar: 500 μm. (F) MDC staining showing autophagosome formation in the root cells of WT, atg5-1, atg5 sid2, sid2, atg5 npr1, and npr1-5 seedlings under normal light/dark (Light) and LS conditions. One-wk-old seedlings were not treated or LS-treated for 24 h following by staining with MDC. The labeled autophagosomes (arrows) were visualized by epifluorescence microscopy. Scale bar: 50 μm.

Mentions: The senescence- and immunity-related programmed cell death phenotypes in the autophagy-defective mutants require the salicylic acid (SA) signal transducer NPR1 (NON-EXPRESSOR OF PR 1).45,46 To determine whether the enhanced submergence sensitivity in the atg mutants relies on an complete SA pathway, we first measured the endogenous SA levels in the wild type and atg mutants at various time points (0, 1, 3, 6, and 12 h) upon submergence treatment. In wild-type plants, SA contents decreased at 1 and 3 h upon submergence, followed by a slight increase at 6 and 12 h after treatment (Fig. 7A). In the atg mutants, however, the SA contents remained higher at all time points, compared with the wild type (Fig. 7A). These results were confirmed by analyzing mRNA abundance of the SA biosynthesis genes ICS1 (isochorismate synthase 1), EDS1 (enhanced disease susceptibility 1), and PAD4 (phytoalexin deficient 4); these were all upregulated at 0, 3, and 24 h upon submergence treatment in the atg mutants in comparison to the wild type (Fig. 7B).Figure 7.


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 enhanced submergence and ethanol sensitivity in autophagy-defective mutants requires salicylic acid signaling. (A) Measurement of endogenous SA contents in the wild type (WT) and atg mutants (atg5-1 and atg7-3). WT, atg5-1, and atg7-3 mutants were treated with submergence (LS) and leaves were collected at 0, 1, 3, 6, and 12 h after treatment for SA extraction and then analyzed by LC/MS. D6-SA was added as an internal quantitative standard. The experiments were repeated (biological replicates) twice, with similar results, and the representative data from one replicate are shown. Data are means ± SD calculated from 8 technical replicates (200 mg leaves harvested from 3 independent plants were pooled for each technical replicate). Asterisks with “a” indicate significant differences from that of the wild type at each time point and with “b” indicate significant differences from untreated wild-type control (*, P < 0.05; **, P < 0.01 by the Student t test). (B) Relative expression levels of SA biosynthesis genes in WT and atg mutants (atg5–1 and atg7–3) upon LS treatment. Total RNA was isolated from 4-wk-old WT and atg mutants under LS treatment for 0, 3, 6, and 24 h. The relative transcript levels were normalized to that of ACT2. *, P < 0.05; **, P < 0.01 by Student t test. (C) Images of WT, atg5–1, atg5 sid2, sid2, atg5 npr1, and npr1-5 seeds germinated on MS medium containing no ethanol or 50 mM ethanol, for 2 wk. (D) Images of WT, atg5-1, atg5 sid2, sid2, atg5 npr1, and npr1-5 plants before treatment (Day 0) and after a 6-d recovery after a 5-d LS treatment (Day 5). (E) DAB staining showing ROS levels in the leaves of 4-wk-old WT, atg5-1, atg5 sid2, sid2, atg5 npr1, and npr1-5 mutants under normal growth conditions (Light) and after 3-d LS treatment. Scale bar: 500 μm. (F) MDC staining showing autophagosome formation in the root cells of WT, atg5-1, atg5 sid2, sid2, atg5 npr1, and npr1-5 seedlings under normal light/dark (Light) and LS conditions. One-wk-old seedlings were not treated or LS-treated for 24 h following by staining with MDC. The labeled autophagosomes (arrows) were visualized by epifluorescence microscopy. Scale bar: 50 μm.
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f0007: The enhanced submergence and ethanol sensitivity in autophagy-defective mutants requires salicylic acid signaling. (A) Measurement of endogenous SA contents in the wild type (WT) and atg mutants (atg5-1 and atg7-3). WT, atg5-1, and atg7-3 mutants were treated with submergence (LS) and leaves were collected at 0, 1, 3, 6, and 12 h after treatment for SA extraction and then analyzed by LC/MS. D6-SA was added as an internal quantitative standard. The experiments were repeated (biological replicates) twice, with similar results, and the representative data from one replicate are shown. Data are means ± SD calculated from 8 technical replicates (200 mg leaves harvested from 3 independent plants were pooled for each technical replicate). Asterisks with “a” indicate significant differences from that of the wild type at each time point and with “b” indicate significant differences from untreated wild-type control (*, P < 0.05; **, P < 0.01 by the Student t test). (B) Relative expression levels of SA biosynthesis genes in WT and atg mutants (atg5–1 and atg7–3) upon LS treatment. Total RNA was isolated from 4-wk-old WT and atg mutants under LS treatment for 0, 3, 6, and 24 h. The relative transcript levels were normalized to that of ACT2. *, P < 0.05; **, P < 0.01 by Student t test. (C) Images of WT, atg5–1, atg5 sid2, sid2, atg5 npr1, and npr1-5 seeds germinated on MS medium containing no ethanol or 50 mM ethanol, for 2 wk. (D) Images of WT, atg5-1, atg5 sid2, sid2, atg5 npr1, and npr1-5 plants before treatment (Day 0) and after a 6-d recovery after a 5-d LS treatment (Day 5). (E) DAB staining showing ROS levels in the leaves of 4-wk-old WT, atg5-1, atg5 sid2, sid2, atg5 npr1, and npr1-5 mutants under normal growth conditions (Light) and after 3-d LS treatment. Scale bar: 500 μm. (F) MDC staining showing autophagosome formation in the root cells of WT, atg5-1, atg5 sid2, sid2, atg5 npr1, and npr1-5 seedlings under normal light/dark (Light) and LS conditions. One-wk-old seedlings were not treated or LS-treated for 24 h following by staining with MDC. The labeled autophagosomes (arrows) were visualized by epifluorescence microscopy. Scale bar: 50 μm.
Mentions: The senescence- and immunity-related programmed cell death phenotypes in the autophagy-defective mutants require the salicylic acid (SA) signal transducer NPR1 (NON-EXPRESSOR OF PR 1).45,46 To determine whether the enhanced submergence sensitivity in the atg mutants relies on an complete SA pathway, we first measured the endogenous SA levels in the wild type and atg mutants at various time points (0, 1, 3, 6, and 12 h) upon submergence treatment. In wild-type plants, SA contents decreased at 1 and 3 h upon submergence, followed by a slight increase at 6 and 12 h after treatment (Fig. 7A). In the atg mutants, however, the SA contents remained higher at all time points, compared with the wild type (Fig. 7A). These results were confirmed by analyzing mRNA abundance of the SA biosynthesis genes ICS1 (isochorismate synthase 1), EDS1 (enhanced disease susceptibility 1), and PAD4 (phytoalexin deficient 4); these were all upregulated at 0, 3, and 24 h upon submergence treatment in the atg mutants in comparison to the wild type (Fig. 7B).Figure 7.

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