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The Arabidopsis translocator protein (AtTSPO) is regulated at multiple levels in response to salt stress and perturbations in tetrapyrrole metabolism.

Balsemão-Pires E, Jaillais Y, Olson BJ, Andrade LR, Umen JG, Chory J, Sachetto-Martins G - BMC Plant Biol. (2011)

Bottom Line: The location of the AtTSPO:eGFP fusion protein was found to depend on the translational start position and the conditions under which plants were grown.Salt-responsive genes are increased in a tspo-1 knock-down mutant compared to wild type under conditions of salt stress, while they are decreased when AtTSPO is overexpressed.In addition, our results show that AtTSPO is regulated at the transcriptional level in tetrapyrrole biosynthetic mutants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratório de Genômica Funcional e Transdução de Sinal, Departamento de Genética, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT

Background: The translocator protein 18 kDa (TSPO), previously known as the peripheral-type benzodiazepine receptor (PBR), is important for many cellular functions in mammals and bacteria, such as steroid biosynthesis, cellular respiration, cell proliferation, apoptosis, immunomodulation, transport of porphyrins and anions. Arabidopsis thaliana contains a single TSPO/PBR-related gene with a 40 amino acid N-terminal extension compared to its homologs in bacteria or mammals suggesting it might be chloroplast or mitochondrial localized.

Results: To test if the TSPO N-terminal extension targets it to organelles, we fused three potential translational start sites in the TSPO cDNA to the N-terminus of GFP (AtTSPO:eGFP). The location of the AtTSPO:eGFP fusion protein was found to depend on the translational start position and the conditions under which plants were grown. Full-length AtTSPO:eGFP fusion protein was found in the endoplasmic reticulum and in vesicles of unknown identity when plants were grown in standard conditions. However, full length AtTSPO:eGFP localized to chloroplasts when grown in the presence of 150 mM NaCl, conditions of salt stress. In contrast, when AtTSPO:eGFP was truncated to the second or third start codon at amino acid position 21 or 42, the fusion protein co-localized with a mitochondrial marker in standard conditions. Using promoter GUS fusions, qRT-PCR, fluorescent protein tagging, and chloroplast fractionation approaches, we demonstrate that AtTSPO levels are regulated at the transcriptional, post-transcriptional and post-translational levels in response to abiotic stress conditions. Salt-responsive genes are increased in a tspo-1 knock-down mutant compared to wild type under conditions of salt stress, while they are decreased when AtTSPO is overexpressed. Mutations in tetrapyrrole biosynthesis genes and the application of chlorophyll or carotenoid biosynthesis inhibitors also affect AtTSPO expression.

Conclusion: Our data suggest that AtTSPO plays a role in the response of Arabidopsis to high salt stress. Salt stress leads to re-localization of the AtTSPO from the ER to chloroplasts through its N-terminal extension. In addition, our results show that AtTSPO is regulated at the transcriptional level in tetrapyrrole biosynthetic mutants. Thus, we propose that AtTSPO may play a role in transporting tetrapyrrole intermediates during salt stress and other conditions in which tetrapyrrole metabolism is compromised.

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OxM1TSPOeGFP localizes in chloroplasts upon salt stress. (A-F) Confocal analyses show OxM1TSPO:eGFP localization in the ER and vesicles of unknown function in hypocotyls of 5-day-old seedlings grown in the standard conditions. (G-L) Confocal analyses show OxM1TSPO:eGFP chloroplast localization in hypocotyls of 5-day-old seedlings grown in the presence of 150 mM NaCl. GFP fluorescence channel is represented in green and chlorophyll auto fluorescence channel is represented in red. Homozygous transgenic plants harboring 35S-TSPO:eGFP in wild-type background were used for the analysis. Scale bars = 50 μm.
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Figure 7: OxM1TSPOeGFP localizes in chloroplasts upon salt stress. (A-F) Confocal analyses show OxM1TSPO:eGFP localization in the ER and vesicles of unknown function in hypocotyls of 5-day-old seedlings grown in the standard conditions. (G-L) Confocal analyses show OxM1TSPO:eGFP chloroplast localization in hypocotyls of 5-day-old seedlings grown in the presence of 150 mM NaCl. GFP fluorescence channel is represented in green and chlorophyll auto fluorescence channel is represented in red. Homozygous transgenic plants harboring 35S-TSPO:eGFP in wild-type background were used for the analysis. Scale bars = 50 μm.

Mentions: Having established a key role for AtTSPO in response to abiotic stress, we next examined the localization of AtTSPO:eGFP fusion proteins in plants subjected to various stress conditions. 5 day-old seedlings were treated with 250 mM mannitol, 1 μM ABA, 0.2 μM MV and 150 mM NaCl. After 18 hours of treatment, OxM1TSPO:eGFP became localized to the plastid (Figure 7G, H, I, J, K and 7L), while neither OxM21TSPO:eGFP nor OxM42TSPO:eGFP had altered localization even with 5 day extended NaCl treatment (data not shown). AtTSPO:GFP localization did not change when plants were treated with mannitol, ABA or MV (data not shown).


The Arabidopsis translocator protein (AtTSPO) is regulated at multiple levels in response to salt stress and perturbations in tetrapyrrole metabolism.

Balsemão-Pires E, Jaillais Y, Olson BJ, Andrade LR, Umen JG, Chory J, Sachetto-Martins G - BMC Plant Biol. (2011)

OxM1TSPOeGFP localizes in chloroplasts upon salt stress. (A-F) Confocal analyses show OxM1TSPO:eGFP localization in the ER and vesicles of unknown function in hypocotyls of 5-day-old seedlings grown in the standard conditions. (G-L) Confocal analyses show OxM1TSPO:eGFP chloroplast localization in hypocotyls of 5-day-old seedlings grown in the presence of 150 mM NaCl. GFP fluorescence channel is represented in green and chlorophyll auto fluorescence channel is represented in red. Homozygous transgenic plants harboring 35S-TSPO:eGFP in wild-type background were used for the analysis. Scale bars = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141639&req=5

Figure 7: OxM1TSPOeGFP localizes in chloroplasts upon salt stress. (A-F) Confocal analyses show OxM1TSPO:eGFP localization in the ER and vesicles of unknown function in hypocotyls of 5-day-old seedlings grown in the standard conditions. (G-L) Confocal analyses show OxM1TSPO:eGFP chloroplast localization in hypocotyls of 5-day-old seedlings grown in the presence of 150 mM NaCl. GFP fluorescence channel is represented in green and chlorophyll auto fluorescence channel is represented in red. Homozygous transgenic plants harboring 35S-TSPO:eGFP in wild-type background were used for the analysis. Scale bars = 50 μm.
Mentions: Having established a key role for AtTSPO in response to abiotic stress, we next examined the localization of AtTSPO:eGFP fusion proteins in plants subjected to various stress conditions. 5 day-old seedlings were treated with 250 mM mannitol, 1 μM ABA, 0.2 μM MV and 150 mM NaCl. After 18 hours of treatment, OxM1TSPO:eGFP became localized to the plastid (Figure 7G, H, I, J, K and 7L), while neither OxM21TSPO:eGFP nor OxM42TSPO:eGFP had altered localization even with 5 day extended NaCl treatment (data not shown). AtTSPO:GFP localization did not change when plants were treated with mannitol, ABA or MV (data not shown).

Bottom Line: The location of the AtTSPO:eGFP fusion protein was found to depend on the translational start position and the conditions under which plants were grown.Salt-responsive genes are increased in a tspo-1 knock-down mutant compared to wild type under conditions of salt stress, while they are decreased when AtTSPO is overexpressed.In addition, our results show that AtTSPO is regulated at the transcriptional level in tetrapyrrole biosynthetic mutants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratório de Genômica Funcional e Transdução de Sinal, Departamento de Genética, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT

Background: The translocator protein 18 kDa (TSPO), previously known as the peripheral-type benzodiazepine receptor (PBR), is important for many cellular functions in mammals and bacteria, such as steroid biosynthesis, cellular respiration, cell proliferation, apoptosis, immunomodulation, transport of porphyrins and anions. Arabidopsis thaliana contains a single TSPO/PBR-related gene with a 40 amino acid N-terminal extension compared to its homologs in bacteria or mammals suggesting it might be chloroplast or mitochondrial localized.

Results: To test if the TSPO N-terminal extension targets it to organelles, we fused three potential translational start sites in the TSPO cDNA to the N-terminus of GFP (AtTSPO:eGFP). The location of the AtTSPO:eGFP fusion protein was found to depend on the translational start position and the conditions under which plants were grown. Full-length AtTSPO:eGFP fusion protein was found in the endoplasmic reticulum and in vesicles of unknown identity when plants were grown in standard conditions. However, full length AtTSPO:eGFP localized to chloroplasts when grown in the presence of 150 mM NaCl, conditions of salt stress. In contrast, when AtTSPO:eGFP was truncated to the second or third start codon at amino acid position 21 or 42, the fusion protein co-localized with a mitochondrial marker in standard conditions. Using promoter GUS fusions, qRT-PCR, fluorescent protein tagging, and chloroplast fractionation approaches, we demonstrate that AtTSPO levels are regulated at the transcriptional, post-transcriptional and post-translational levels in response to abiotic stress conditions. Salt-responsive genes are increased in a tspo-1 knock-down mutant compared to wild type under conditions of salt stress, while they are decreased when AtTSPO is overexpressed. Mutations in tetrapyrrole biosynthesis genes and the application of chlorophyll or carotenoid biosynthesis inhibitors also affect AtTSPO expression.

Conclusion: Our data suggest that AtTSPO plays a role in the response of Arabidopsis to high salt stress. Salt stress leads to re-localization of the AtTSPO from the ER to chloroplasts through its N-terminal extension. In addition, our results show that AtTSPO is regulated at the transcriptional level in tetrapyrrole biosynthetic mutants. Thus, we propose that AtTSPO may play a role in transporting tetrapyrrole intermediates during salt stress and other conditions in which tetrapyrrole metabolism is compromised.

Show MeSH
Related in: MedlinePlus