The multistress-induced Translocator protein (TSPO) differentially modulates storage lipids metabolism in seeds and seedlings.

Translocator proteins (TSPO) are conserved membrane proteins extensively studied in mammals, but their function is still unclear. Angiosperm TSPO are transiently induced by abiotic stresses in vegetative tissues. We showed previously that constitutive expression of the Arabidopsis TSPO (AtTSPO) could be detrimental to the cell. Degradation of AtTSPO requires an active autophagy pathway. We show here that genetic modifications of TSPO expression in plant and yeast cells reduce the levels of cytoplasmic lipid droplets (LD). Transgenic Arabidopsis seedlings overexpressing AtTSPO contain less LD as compared with wild type (WT). LD levels were increased in Arabidopsis AtTSPO knockout (KO) seedlings. Deletion of the Schizosaccharomyces pombe TSPO resulted in an increase in LD level in the cell. As compared with the WT, the mutant strain was more sensitive to cerulenin, an inhibitor of fatty acids and sterol biosynthesis. We found that in contrast with seedlings, overexpression of AtTSPO (OE) resulted in an up to 50% increase in seeds fatty acids as compared with WT. A time course experiment revealed that after 4 days of seed imbibition, the levels of triacylglycerol (TAG) was still higher in the OE seeds as compared with WT or KO seeds. However, the de novo synthesis of phospholipids and TAG after 24 h of imbibition was substantially reduced in OE seeds as compared with WT or KO seeds. Our findings support a plant TSPO role in energy homeostasis in a tissue-specific manner, enhancing fatty acids and LD accumulation in mature seeds and limiting LD levels in seedlings.

Enigmatic Translocator protein (TSPO) and cellular stress regulation.

Translocator proteins (TSPOs) are conserved, ubiquitous membrane proteins identified initially as benzodiazepine-binding proteins in mammalian cells. Recent genetic and biochemical studies have challenged the accepted model that TSPOs are essential and required for steroidogenesis in animal cells. Instead, evidence from different kingdoms of life suggests that TSPOs are encoded by nonessential genes that are temporally upregulated in cells encountering conditions of oxidative stress, including inflammation and tissue injury. Here we discuss how TSPOs may be involved in complex homeostasis signaling mechanisms. We suggest that the main physiological role of TSPOs may be to modulate oxidative stress, irrespective of the cell type or subcellular localization, in part through the subtle regulation of tetrapyrrole metabolism.

Translocator proteins, porphyrins and abiotic stress: new light?

After four decades of extensive studies, the role of membrane-bound Translocator proteins (TSPOs) remains unclear and even controversial. In light of recent insights into the structure and activity of TSPOs, showing that they cannot only bind, but also enzymatically photodegrade protoporphyrin IX, we discuss their emerging physiological roles and regulation.

The Arabidopsis abiotic stress-induced TSPO-related protein reduces cell-surface expression of the aquaporin PIP2;7 through protein-protein interactions and autophagic degradation.

The Arabidopsis thaliana multi-stress regulator TSPO is transiently induced by abiotic stresses. The final destination of this polytopic membrane protein is the Golgi apparatus, where its accumulation is strictly regulated, and TSPO is downregulated through a selective autophagic pathway. TSPO-related proteins regulate the physiology of the cell by generating functional protein complexes. A split-ubiquitin screen for potential TSPO interacting partners uncovered a plasma membrane aquaporin, PIP2;7. Pull-down assays and fluorescence imaging approaches revealed that TSPO physically interacts with PIP2;7 at the endoplasmic reticulum and Golgi membranes in planta. Intriguingly, constitutive expression of fluorescently tagged PIP2;7 in TSPO-overexpressing transgenic lines resulted in patchy distribution of the fluorescence, reminiscent of the pattern of constitutively expressed yellow fluorescent protein-TSPO in Arabidopsis. Mutational stabilization of TSPO or pharmacological inhibition of the autophagic pathway affected concomitantly the detected levels of PIP2;7, suggesting that the complex containing both proteins is degraded through the autophagic pathway. Coexpression of TSPO and PIP2;7 resulted in decreased levels of PIP2;7 in the plasma membrane and abolished the membrane water permeability mediated by transgenic PIP2;7. Taken together, these data support a physiological role for TSPO in regulating the cell-surface expression of PIP2;7 during abiotic stress conditions through protein-protein interaction and demonstrate an aquaporin regulatory mechanism involving TSPO.

Selective autophagy of non-ubiquitylated targets in plants: looking for cognate receptor/adaptor proteins.

Cellular homeostasis is essential for the physiology of eukaryotic cells. Eukaryotic cells, including plant cells, utilize two main pathways to adjust the level of cytoplasmic components, namely the proteasomal and the lysosomal/vacuolar pathways. Macroautophagy is a lysosomal/vacuolar pathway which, until recently, was thought to be non-specific and a bulk degradation process. However, selective autophagy which can be activated in the cell under various physiological conditions, involves the specific degradation of defined macromolecules or organelles by a conserved molecular mechanism. For this process to be efficient, the mechanisms underlying the recognition and selection of the cargo to be engulfed by the double membrane autophagosome are critical, and not yet well understood. Ubiquitin (poly-ubiquitin) conjugation to the target appears to be a conserved ligand mechanism in many types of selective autophagy, and defined receptors/adaptors recognizing and regulating the autophagosomal capture of the ubiquitylated target have been characterized. However, non-proteinaceous and non-ubiquitylated cargoes are also selectively degraded by this pathway. This ubiquitin-independent selective autophagic pathway also involves receptor and/or adaptor proteins linking the cargo to the autophagic machinery. Some of these receptor/adaptor proteins including accessory autophagy-related (Atg) and non-Atg proteins have been described in yeast and animal cells but not yet in plants. In this review we discuss the ubiquitin-independent cargo selection mechanisms in selective autophagy degradation of organelles and macromolecules and speculate on potential plant receptor/adaptor proteins.

Molecular cloning and biochemical characterization of two UDP-glycosyltransferases from poplar.

Two pathogen-induced uridine diphosphate glycosyltransferases (UGTs) identified previously via co-expression with induced proanthocyanidin (PA) synthesis in poplar were cloned and characterized. Phylogenetic analysis grouped both genes with other known flavonoid UGTs that act on flavonols and anthocyanins. Recombinant enzymes were produced in order to test if they could glycoslate flavonoids. PtUGT78L1 accepted the flavonols quercetin and kaempferol as well as cyanidin, and used UDP-galactose as a sugar donor. PtUGT78M1 did not accept any of the flavonoids tested as a substrate, but did transfer glucose from UDP-glucose to the universal substrate 2,4,6-trichlorophenol. However, neither enzyme acted on the flavan-3-ols catechin or epicatechin, intermediates in the PA biosynthetic pathway.

Induction of acid phosphatase transcripts, protein and enzymatic activity by simulated herbivory of hybrid poplar.

Herbivory and wounding upregulate a large suite of defense genes in hybrid poplar leaves. A strongly wound- and herbivore-induced gene with high similarity to Arabidopsis vegetative storage proteins (VSPs) and acid phosphatase (AP) was identified among genes strongly expressed during the poplar herbivore defense response. Phylogenetic analysis showed that the putative poplar acid phosphatase (PtdAP1) gene is part of an eight-member AP gene family in poplar, and is most closely related to a functionally characterized soybean nodule AP. Unlike the other poplar APs, PtdAP1 is expressed in variety of tissues, as observed in an analysis of EST data. Following wounding, the gene shows an expression profile similar to other known poplar defense genes such as protease inhibitors, chitinase, and polyphenol oxidase. Significantly, we show for the first time that subsequent to the wound-induction of PtdAP1 transcripts, AP protein and activity increase in extracts of leaves and other tissues. Although its mechanism of action is as yet unknown, these results suggest in hybrid poplar PtdAP1 is likely a component of the defense response against leaf-eating herbivores.

Biochemical and molecular characterization of AtPAP26, a vacuolar purple acid phosphatase up-regulated in phosphate-deprived Arabidopsis suspension cells and seedlings.

A vacuolar acid phosphatase (APase) that accumulates during phosphate (Pi) starvation of Arabidopsis (Arabidopsis thaliana) suspension cells was purified to homogeneity. The final preparation is a purple APase (PAP), as it exhibited a pink color in solution (A(max) = 520 nm). It exists as a 100-kD homodimer composed of 55-kD glycosylated subunits that cross-reacted with an anti-(tomato intracellular PAP)-IgG. BLAST analysis of its 23-amino acid N-terminal sequence revealed that this PAP is encoded by At5g34850 (AtPAP26; one of 29 PAP genes in Arabidopsis) and that a 30-amino acid signal peptide is cleaved from the AtPAP26 preprotein during its translocation into the vacuole. AtPAP26 displays much stronger sequence similarity to orthologs from other plants than to other Arabidopsis PAPs. AtPAP26 exhibited optimal activity at pH 5.6 and broad substrate selectivity. The 5-fold increase in APase activity that occurred in Pi-deprived cells was paralleled by a similar increase in the amount of a 55-kD anti-(tomato PAP or AtPAP26)-IgG immunoreactive polypeptide and a >30-fold reduction in intracellular free Pi concentration. Semiquantitative reverse transcription-PCR indicated that Pi-sufficient, Pi-starved, and Pi-resupplied cells contain similar amounts of AtPAP26 transcripts. Thus, transcriptional controls appear to exert little influence on AtPAP26 levels, relative to translational and/or proteolytic controls. APase activity and AtPAP26 protein levels were also up-regulated in shoots and roots of Pi-deprived Arabidopsis seedlings. We hypothesize that AtPAP26 recycles Pi from intracellular P metabolites in Pi-starved Arabidopsis. As AtPAP26 also exhibited alkaline peroxidase activity, a potential additional role in the metabolism of reactive oxygen species is discussed.