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

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.

Figure 1

Induction of AtTSPO mRNA by abiotic stresses. (A) Quantitative real-time PCR analyses of AtTspO transcripts upon treatment of different stresses, (a) 150 mM NaCl, (b) 1 μM ABA, (c) 250 mM mannitol and (d) 0.2 μM methyl viologen. Relative expression levels were calculated and ACTIN (At3g18780) and 18S rRNA (At3g41768) here used as reference genes. (B) GUS expression in AtTSPO-437::GUS and LHCB::GUS lines in 15-day-old transgenic Arabidopsis plants either untreated or treated with 150 mM NaCl.

Figure 2

Phenotype of mutants with different levels of AtTSPO expression. (A) Schematic representation of isolated insertional mutant of AtTSPO in Arabidopsis. Two copies of the T-DNA were inserted in tandem 123 bp upstream from the translational initiation codon of AtTSPO. (B) Total RNA was isolated from 5 day-old seedlings, reverse-transcribed and subjected to qRT-PCR. Data shown represent mean values obtained from independent amplification reactions (n = 3) and biological replicates (n = 2). Bars represent the standard error of biological replicates. (C) Root lengths of at least 100 individual 7-day-old seedlings grown in 16 h photoperiods. (D) Chlorophyll concentrations in 14-day-old, in vitro-grown plants of the indicated genotypes were determined spectrophotometrically. Values shown are means derived from three independent samples, each sample containing 100 mg of fresh weight. Units are μg of chlorophyll a + b per g of fresh weight (fw).

Figure 3

Stress-response genes are up-regulated in tspo-1 during salt stress. (A)-(C) Stress-induced gene expression in OxM1TSPO:eGFP and tspo-1 lines compared to wild type plants, by qPCR. 5-day-old seedlings grown under standard conditions and transferred for 3 hours to plates containing 150 mM NaCl. (A) DREB2A, (B) RAB18 and (C) ERD10 mRNA levels were determined by quantitative qRT-PCR. Relative amounts were calculated and normalized relative to Col-0 non-treated (100%). The ACTIN and 18S rRNA were used as reference genes. ACTIN, At3g18780; 18S RNA, At3g41768; RAB18, At5g66400; ERD10, At1g20450; DREB2A, At5g05410. Data shown represent mean values obtained from independent amplification reactions (n = 3) and biological replicates (n = 2). Relative expression levels were calculated. Bars represent the standard error of biological replicates.

Figure 4

Expression of tetrapyrrole biosynthesis genes in tspo-1 mutant. qRT-PCR analyses of tetrapyrrole biosynthesis genes in Col-0, tspo-1 and gun5 5-days-old seedlings grown in constant light. Relative amounts were calculated and normalized relative to Col-0 non-treated (100%). With the exception of HEMA2 and FC1, all the genes have been show to be regulated by light. The data are presented following the enzymes order in the tetrapyrrole biosynthesis. The ACTIN and 18S rRNA genes were used as control. ACTIN, At3g18780; 18S rRNA, At3g41768; (A) HEMA1 (Glutamyl-tRNA reductase 1 - At1g58290) (B) HEMA2 (Glutamyl-tRNA reductase 2 - At1g04490); (C) PPO (Protoporphyrinogen oxidase - At4g01690); (D) FC1 (Ferrochelatase 1 - At5g26030); (E) FC2 (Ferrochelatase 2 - At2g30390); (F) GUN2 (Heme oxygenase 1 - At2g26670); (G) GUN4 (Regulator of Mg-porphyrin synthesis - At3g59400); (H) GUN5 (Mg-chelatase subunit H - At5g13630); (I) CAO (Chlorophyllide A oxygenase - At1g44446); and (J) GUN1 (Pentatricopeptide repeat (PPR) protein - At2g31400). Data shown represent mean values obtained from independent amplification reactions (n = 3) and biological replicates (n = 2). Relative expression levels were calculated. Bars represent the standard error of biological replicates.

Figure 5

Relationship between tetrapyrrole flux and AtTSPO expression. (A) AtTSPO expression in wild-type plants germinated in 50 μM of gabaculine or 500 nM of norflurazon compared to untreated plants. (B) AtTSPO mRNA levels in different mutants of the tetrapyrrole pathway. Relative amounts were calculated and normalized relative to Col-0 non-treated (100%). The ACTIN and 18S rRNA genes were used as control. ACTIN, At3g18780; 18S RNA, At3g41768. Data shown represent mean values obtained from independent amplification reactions (n = 3) and biological replicates (n = 2). Relative expression levels were calculated. Bars represent the standard error of biological replicates.

Figure 6

AtTSPO has different sub-cellular location depending on the translational start site used. Confocal images of OxM1TSPO:eGFP (A-C), OxM21TSPO:eGFP (D-F) and OxM42TSPO:eGFP (G-I) localization. OxM1TSPO:eGFP localizes in the ER and vesicles of unknown function in the root (A), hypocotyl (B) and cotyledon (C). OxM21TSPO:eGFP localizes in the mitochondria of root (D), hypocotyl (E) and cotyledon (F). OxM42TSPO:eGFP show mitochondria localization in root (G), hypocotyls (H) and cotyledons (I). GFP fluorescence is represented by green and chlorophyll auto fluorescence in red. The samples were incubated with Mitotracker to identify mitochondria (see Additional file 3). Homozygous transgenic plants harboring 35S-TSPO:eGFP in wild-type background were used for the analysis. Scale bars = 50 μm.

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.

Figure 8

AtTSPO accumulation and chloroplast localization upon salt stress. (A) Immunoblot analysis of OxAtTSPO:eGFP (OxM1TSPO:eGFP, OxM21TSPO:eGFP and OxM42TSPO:eGFP) fusion proteins detected in plants with an antibody to GFP. Plants were untreated, or treated with 150 mM NaCl. As a control wild-type plants and plants over-expressing GFP (OxeGFP) seedlings were used. (B) Anti-GFP immunoblot of trypsinized chloroplasts from Arabidopsis plants either untreated or treated with 150 mM NaCl. Control immunoblots were probed with antibodies chloroplast proteins RuBisCo and D1; and to cytosolic UGPase. Each lane represents equal amounts of chloroplasts.