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Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H(+)-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8.

ABSTRACT: Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H(+)-ATPase inhibitors concanamycin A and bafilomycin A1, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H(+)-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.

SUBMITTER: Yano K

PROVIDER: S-EPMC4883836 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H(+)-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8.

Yano Kanako K Yanagisawa Takahiro T Mukae Kyosuke K Niwa Yasuo Y Inoue Yuko Y Moriyasu Yuji Y

Plant signaling & behavior 20150101 11

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H(+)-ATPase inhibitors concanamycin A and bafilomycin A1, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolyso ...[more]

PMID: 26368310

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Project description:Sugar is an important resource for energy generation and developmental regulation in plants, and sucrose starvation causes enormous changes in cellular morphology, enzyme activities and gene expression. Genome-wide gene expression profiling provides a comprehensive knowledge into gene expression under nutrients depletion and senescence, however, that of monocot model plant rice under sucrose depletion is still under investigation. Here, the time-course monitoring of gene expression profiles in sucrose-starved rice (Oryza sativa cv Tainung67) suspension cells was investigated by 21495 probes-containing Agilent rice chip. In sucrose-starved cells, the induced vacuolar biogenesis was coincided with the significantly upregulated expression of genes encoding H+-pyrophosphatase, delta-TIP, one putative alpha-TIP, several vacuolar proteases and proteinase inhibitors, and one OsATG3. To survey the overall metabolic adaptations under sucrose depletion the genes significantly alternating expression level were incorporated into multiple metabolic pathways. The majority of genes encoding enzymes involved in biosynthesis and degradation pathways of various macromolecules were comprehensively down- and upregulated, respectively, by sucrose starvation. Transcriptional regulation of gene expression is important for the physiological adaptations to environmental stress and many transcription factors, including bZIPs, NACs, and WRKY showed significant increase in transcript levels under sucrose starvation. Concurrently, statistical analysis reveals that their corresponding consensus cis-elements, such as ABA-responsive element, CACG, ACI, ACII and CTTATCC, are frequently found in the promoter regions of many Suc starvation-upregulated genes. Particle bombardment-mediated transient promoter activity assays further showed that the CTTATCC, derived form TATCCA, and the AC motifs, are the promising sucrose starvation responsive activators in sucrose-starved rice suspension cells. Keywords: stress response

Dataset Information

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H(+)-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8.

Publications

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H(+)-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8.

Similar Datasets

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