Reduced Tonoplast Fast-Activating and Slow-Activating Channel Activity Is Essential for Conferring Salinity Tolerance in a Facultative Halophyte,Quinoa

Halophyte species implement a “salt-including” strategy, sequestering significant amounts of Na⁺ to cell vacuoles. This requires a reduction of passive Na⁺ leak from the vacuole. In this work, we used quinoa (Chenopodium quinoa) to investigate the ability of halophytes to regulate Na⁺-permeable slow-activating (SV) and fast-activating (FV) tonoplast channels, linking it with Na⁺ accumulation in mesophyll cells and salt bladders as well as leaf photosynthetic efficiency under salt stress. Our data indicate that young leaves rely on Na⁺ exclusion to salt bladders, whereas old ones, possessing far fewer salt bladders, depend almost exclusively on Na⁺ sequestration to mesophyll vacuoles. Moreover, although old leaves accumulate more Na⁺, this does not compromise their leaf photochemistry. FV and SV channels are slightly more permeable for K⁺ than for Na⁺, and vacuoles in young leaves express less FV current and with a density unchanged in plants subjected to high (400 mm NaCl) salinity. In old leaves, with an intrinsically lower density of the FV current, FV channel density decreases about 2-fold in plants grown under high salinity. In contrast, intrinsic activity of SV channels in vacuoles from young leaves is unchanged under salt stress. In vacuoles of old leaves, however, it is 2- and 7-fold lower in older compared with young leaves in control- and salt-grown plants, respectively. We conclude that the negative control of SV and FV tonoplast channel activity in old leaves reduces Na⁺ leak, thus enabling efficient sequestration of Na⁺ to their vacuoles. This enables optimal photosynthetic performance, conferring salinity tolerance in quinoa species.The increasing problem of global land salinization (Flowers, 2004; Rengasamy, 2006) and its associated multibillion dollar losses in agricultural production require a better understanding of the key physiological mechanisms that confer salinity tolerance in crops. One effective way of gaining such knowledge comes from studying halophytes (Glenn et al., 1999; Flowers and Colmer, 2008; Shabala and Mackay, 2011).One of the prominent features of halophytes is their ability to efficiently sequester cytosolically toxic Na⁺ to the cell vacuole. The classic view is that this sequestration is achieved by tonoplast Na⁺/H⁺ antiporters (Barkla et al., 1995; Flowers and Colmer, 2008), a process energized by both vacuolar H⁺ pumps: ATPase (Ayala et al., 1996; Vera-Estrella et al., 1999; Wang et al., 2001) and pyrophosphatase (Parks et al., 2002; Vera-Estrella et al., 2005; Guo et al., 2006; Krebs et al., 2010). However, recent studies have added more complexity to the relationship between Na⁺/H⁺ antiporters and vacuolar Na⁺ sequestration, assigning a role to the transporter in the regulation of K⁺ and H⁺ homeostasis (for review, see Rodríguez-Rosales et al., 2009; Jiang et al., 2010; Bassil et al., 2011). Vacuolar Na⁺/H⁺ antiporters encoded by NHX genes have been shown to also act as K⁺/H⁺ antiporters, with a relatively weak selectivity between Na⁺ and K⁺. The Na⁺/K⁺ selectivity ratio, in turn, is regulated by vacuolar calmodulin in a pH- and Ca²⁺-dependent manner (Yamaguchi et al., 2005). Consequently, other transporters, in addition to and different from NHX, are likely to be involved in vacuolar Na⁺ sequestration. In addition, salt-induced up-regulation of Na⁺/H⁺ antiporter expression levels has been observed in leaves but not in roots (Cosentino et al., 2010), suggesting the importance of Na⁺ exclusion and intracellular sequestration, primarily in photosynthesizing cells. Thus, tissue- and species-specific differences in the respective mechanisms should be considered as well.Whatever the actual mechanisms are for intracellular Na⁺ sequestration, efficient Na⁺ pumping into vacuole is only one side of the coin. To confer salinity tolerance, toxic Na⁺ ions must be prevented from leaking back into the cytosol. Indeed, given the at least 4- to 5-fold concentration gradient between the vacuole and the cytosol (Shabala and Mackay, 2011) and a zero or slightly negative cytosol-to-vacuole voltage difference across the tonoplast, Na⁺ leakage from the vacuole is thermodynamically favorable. Thus, to avoid energy-consuming futile Na⁺ cycling between the cytosol and the vacuole, and to achieve efficient vacuolar sequestration of toxic Na⁺, passive tonoplast Na⁺ conductance has to be kept to an absolute minimum. This implies strict and efficient control over Na⁺-permeable tonoplast channels.Two major types of Na⁺-permeable channels are present in the tonoplast, the slow-activating (SV) and fast-activating (FV) vacuolar channels. The SV channel is permeable to both monovalent and divalent cations and is activated by cytosolic Ca²⁺ and positive vacuolar voltage (Hedrich and Neher, 1987; Ward and Schroeder, 1994; Pottosin et al., 1997, 2001). The FV channel is permeable for monovalent cations only, is activated by large voltages of either sign, and is inhibited by divalent cations from either side of the membrane (Tikhonova et al., 1997; Brüggemann et al., 1999a, 1999b). In Arabidopsis (Arabidopsis thaliana), SV channels are shown to be encoded by a TPC1 (for two-pore channel1) protein (Peiter et al., 2005; Pottosin and Schönknecht, 2007; Hedrich and Marten, 2011). Importantly, recent studies on mammalian two-pore channels have suggested that endolysosomal TPCs are, in fact, Na⁺-selective channels (Wang et al., 2012). In contrast, the molecular identity of FV channels remains elusive. Both SV and FV channels are ubiquitous and abundant (up to several copies per μm²) in plant tissues, including mesophyll cell vacuoles (Pottosin and Muñiz, 2002; Pottosin and Schönknecht, 2007). SV and FV channel activity is strongly controlled at physiologically attainable conditions (physiological tonoplast voltages and vacuolar and cytosolic divalent and polyvalent cation concentrations). Importantly, even with 0.1% to 1% of the total population of channels open at any one time, impressive monovalent cation currents in the range of tens of pA per vacuole can be conducted. This is equivalent to a current mediated by the whole vacuole population of H⁺ pumps (Hedrich et al., 1988). Thus, under saline conditions, SV and FV channel activity probably needs to be further reduced.Early attempts to unravel any dramatic differences between the properties of tonoplast cation channels in salt-tolerant and salt-sensitive plants did not yield a clear outcome. Ivashikina and Hedrich (2005) studied the voltage dependence of the SV channels in vacuoles from Arabidopsis cell culture and found that an increase in luminal Na⁺/K⁺ ratio, mimicking the accumulation of Na⁺ in vacuoles during salt stress, shifted the threshold for SV activation to positive potentials, reducing SV channel open probability under saline conditions. Maathuis and coworkers (1992) found significant SV channel activity in leaf vacuoles isolated from the extreme halophyte Suaeda maritima, even when plants were grown under high (200 mm) NaCl conditions. The estimated activity of the transporter at physiologically relevant cytosolic Ca²⁺ levels and relatively small transmembrane voltage differences was low. Thus, the authors suggested that, rather than possessing some specific salt-induced control over the SV channel, the transporter’s low activity would mean that even under highly saline conditions, it would consume only about 30% of the H⁺-ATPase-generated power. Further studies from this laboratory demonstrated that voltage gating, unitary conductance, and Na⁺/K⁺ selectivity (P_K = P_Na) of SV channels from roots of Plantago media (salt sensitive) and Plantago maritima (salt tolerant) were essentially the same (Maathuis and Prins, 1990). However, when both species were grown under saline conditions, the SV channel activity greatly diminished. Yet, based on the original data of this study, it is not possible to decipher whether the SV channel activity in the two species was the same or different under control conditions and whether it was a statistically significant difference between the salt-induced decrease in the open probability of SV channels between P. media and P. maritima. As for FV channels, we are not aware of a single study on their properties/expression in relation to the salt tolerance.While the total number of halophytic species is relatively small compared with glycophytes, it still amounts to at least several thousand species (Glenn et al., 1999; Flowers et al., 2010). Moreover, halophytes are present in about one-half of higher plant families (Flowers and Colmer, 2008). These species possess a wide range of anatomical and morphological features that may potentially enable their superior performance under saline conditions (Shabala and Mackay, 2011). Nonetheless, the extent to which the above considerations could be extrapolated to all halophytes remains to be assessed. In this work, we used quinoa (Chenopodium quinoa) mesophyll leaf vacuoles to address some of these issues. Quinoa is a facultative halophyte species that originates from the Andean region of South America and was domesticated for human consumption some 3,000 to 4,000 years ago. It can grow under extreme saline conditions with a soil electrical conductivity exceeding 40 dS m⁻¹, approximately 500 mm NaCl (Jacobsen et al., 2003; Razzaghi et al., 2011). Optimal plant growth is usually observed at NaCl concentrations of around 100 mm (Hariadi et al., 2011), but this may be genotype specific (Adolf et al., 2012). Quinoa possesses some degree of leaf succulence as well as epidermal bladder cells (EBC), so it has the potential to employ two different sequestration strategies for cytosolic Na⁺ exclusion: internal (e.g. vacuolar sequestration) and external (sequestration in EBC). This makes quinoa an excellent model species to investigate the role of vacuolar Na⁺ sequestration in the overall salinity tolerance in this crop plant as well as to determine the contribution of SV and FV channels in this process. Here, we report a highly significant difference in SV and FV channel activity between old and young leaves of quinoa plants, a difference that is further enhanced under saline conditions. We conclude that the ability of quinoa plants to control ion leak via SV and FV tonoplast channels is essential for conferring salinity tolerance in this species. The possible implications of these findings for crop breeding for salinity tolerance are discussed.     

Inflammation in disseminated lesions: an analysis of CD4+, CD20+, CD68+, CD31+ and vW+ cells in non-ulcerated lesions of disseminated leishmaniasis

Disseminated leishmaniasis (DL) differs from other clinical forms of the disease due to the presence of many non-ulcerated lesions (papules and nodules) in non-contiguous areas of the body. We describe the histopathology of DL non-ulcerated lesions and the presence of CD4-, CD20-, CD68-, CD31- and von Willebrand factor (vW)-positive cells in the inflamed area. We analysed eighteen biopsies from non-ulcerated lesions and quantified the inflamed areas and the expression of CD4, CD20, CD68, CD31 and vW using Image-Pro software (Media Cybernetics). Diffuse lymphoplasmacytic perivascular infiltrates were found in dermal skin. Inflammation was observed in 3-73% of the total biopsy area and showed a significant linear correlation with the number of vW⁺ vessels. The most common cells were CD68⁺ macrophages, CD20⁺ B-cells and CD4⁺ T-cells. A significant linear correlation between CD4⁺ and CD20⁺ cells and the size of the inflamed area was also found. Our findings show chronic inflammation in all DL non-ulcerated lesions predominantly formed by macrophages, plasmacytes and T and B-cells. As the inflamed area expanded, the number of granulomas and extent of the vascular framework increased. Thus, we demonstrate that vessels may have an important role in the clinical evolution of DL lesions.     

Alginate esters via chemoselective carboxyl group modification

Alginates are (1 → 4) linked linear copolysaccharides composed of β-d-mannuronic acid (M) and its C-5 epimer, α-l-guluronic acid (G). Several strategies for synthesis of carboxyl modified alginate derivatives exist in the literature. Most of these however employ aqueous chemistries, such as carbodiimide coupling reactions. Based on our recently discovered method for homogeneous dissolution of tetrabutylammonium (TBA)-alginate, we now describe use of tetrabutylammonium fluoride (TBAF)-based two component solvent systems as media for synthesis of carboxyl-modified alginate esters. Partially and fully esterified benzyl, butyl, ethyl, and methyl alginates were synthesized via reaction with the corresponding alkyl halides. The newly synthesized derivatives were soluble in polar aprotic solvents without the addition of TBAF. Saponification was performed to demonstrate that alkylation was completely regioselective for carboxylate groups in preference to hydroxyl groups to form esters. We demonstrate the utility of these alginate esters to enhance aqueous solubility of the flavonoid naringenin by formation of solid dispersions.     

New substrates and activity of Phanerochaete chrysosporium Omega glutathione transferases

Omega glutathione transferases (GSTO) constitute a family of proteins with variable distribution throughout living organisms. It is notably expanded in several fungi and particularly in the wood-degrading fungus Phanerochaete chrysosporium, raising questions concerning the function(s) and potential redundancy of these enzymes. Within the fungal families, GSTOs have been poorly studied and their functions remain rather sketchy. In this study, we have used fluorescent compounds as activity reporters to identify putative ligands. Experiments using 5-chloromethylfluorescein diacetate as a tool combined with mass analyses showed that GSTOs are able to cleave ester bonds. Using this property, we developed a specific activity-based profiling method for identifying ligands of PcGSTO3 and PcGSTO4. The results suggest that GSTOs could be involved in the catabolism of toxic compounds like tetralone derivatives. Biochemical investigations demonstrated that these enzymes are able to catalyze deglutathionylation reactions thanks to the presence of a catalytic cysteine residue. To access the physiological function of these enzymes and notably during the wood interaction, recombinant proteins have been immobilized on CNBr Sepharose and challenged with beech wood extracts. Coupled with GC–MS experiments this ligand fishing method allowed to identify terpenes as potential substrates of Omega GST suggesting a physiological role during the wood–fungus interactions.     

Comparison of treatments of peripheral arterial disease with mesenchymal stromal cells and mesenchymal stromal cells modified with granulocyte and macrophage colony-stimulating factor

Background aimsGranulocyte macrophage-colony stimulating factor (GM-CSF) promotes vessel formation through several molecular signaling pathways. Mesenchymal stromal cells (MSCs) have an important role in neovasculogenesis during ischemia because they release pro-angiogenic paracrine factors, pro-survival and immunomodulatory substances and can differentiate into endothelial cells. The objective of this study was to evaluate whether there is synergy between GM-CSF and MSCs in recovering ischemic limbs.MethodsMSCs from mouse bone marrow were transduced with a lentiviral vector expressing GM-CSF and injected into animals with surgically induced limb ischemia, with unmodified MCSs used as control. The evolution of limb necrosis was evaluated for 1 month. Muscle strength was assessed on the 30th day, and the animals were euthanized to determine the muscle mass and to perform histological analyses to determine the degree of cellular infiltration, capillary and microvessel densities, fibrosis, necrosis and tissue regeneration.ResultsBoth treatments were able to ameliorate ischemia, decrease the areas of fibrosis, necrosis, adipocytes and leukocyte infiltrates and increase the number of capillaries. The addition of GM-CSF promoted the formation of larger vessels, but it also resulted in more fibrosis and less muscle mass without affecting muscle force.ConclusionsBoth treatments resulted in a remarkable amelioration of ischemia. More fibrosis and less muscle mass produced by the overexpression of GM-CSF did not affect muscle functionality significantly. Importantly, MSCs overexpressing GM-CSF produced larger vessels, which is an important long-term advantage because larger vessels are more efficient in the reperfusion of ischemic tissues physiologically.     

Interaction between AP-5 and the hereditary spastic paraplegia proteins SPG11 and SPG15

The AP-5 complex is a recently identified but evolutionarily ancient member of the family of heterotetrameric adaptor proteins (AP complexes). It is associated with two proteins that are mutated in patients with hereditary spastic paraplegia, SPG11 and SPG15. Here we show that the four AP-5 subunits can be coimmunoprecipitated with SPG11 and SPG15, both from cytosol and from detergent-extracted membranes, with a stoichiometry of ∼1:1:1:1:1:1. Knockdowns of SPG11 or SPG15 phenocopy knockdowns of AP-5 subunits: all six knockdowns cause the cation-independent mannose 6-phosphate receptor to become trapped in clusters of early endosomes. In addition, AP-5, SPG11, and SPG15 colocalize on a late endosomal/lysosomal compartment. Both SPG11 and SPG15 have predicted secondary structures containing α-solenoids related to those of clathrin heavy chain and COPI subunits. SPG11 also has an N-terminal, β-propeller–like domain, which interacts in vitro with AP-5. We propose that AP-5, SPG15, and SPG11 form a coat-like complex, with AP-5 involved in protein sorting, SPG15 facilitating the docking of the coat onto membranes by interacting with PI3P via its FYVE domain, and SPG11 (possibly together with SPG15) forming a scaffold.     

Habituation of the eyeblink response in humans with stimuli presented in a sequence of incremental intensity

In an experiment we examined whether the repeated presentation of tones of gradually increasing intensities produces greater decrement in the eyeblink reflex response in humans than the repetition of tones of constant intensities. Two groups of participants matched for their initial level of response were exposed to 110 tones of 100-ms duration. For the participants in the incremental group, the tones increased from 60- to 90- dB in 3-dB steps, whereas participants in the constant group received the tones at a fixed 90-dB intensity. The results indicated that the level of response in the last block of 10 trials, in which both groups received 90-dB tones, was significantly lower in the incremental group than in the constant group. These findings support the data presented by Davis and Wagner (7) with the acoustic response in rats, but differ from several reports with autonomic responses in humans, where the advantage of the incremental condition has not been observed unambiguously. The discussion analyzes theoretical approaches to this phenomenon and the possible involvement of separate neural circuits.     

OAT2 catalyses efflux of glutamate and uptake of orotic acid

OAT (organic anion transporter) 2 [human gene symbol SLC22A7 (SLC is solute carrier)] is a member of the SLC22 family of transport proteins. In the rat, the principal site of expression of OAT2 is the sinusoidal membrane domain of hepatocytes. The particular physiological function of OAT2 in liver has been unresolved so far. In the present paper, we have used the strategy of LC (liquid chromatography)-MS difference shading to search for specific and cross-species substrates of OAT2. Heterologous expression of human and rat OAT2 in HEK (human embryonic kidney)-293 cells stimulated accumulation of the zwitterion trigonelline; subsequently, orotic acid was identified as an excellent and specific substrate of OAT2 from the rat (clearance=106 μl·min?1·mg of protein?1) and human (46 μl·min?1·mg of protein?1). The force driving uptake of orotic acid was identified as glutamate antiport. Efficient transport of glutamate by OAT2 was directly demonstrated by uptake of [3H]glutamate. However, because of high intracellular glutamate, OAT2 operates as glutamate efflux transporter. Thus expression of OAT2 markedly increased the release of glutamate (measured by LC-MS) from cells, even without extracellular exchange substrate. Orotic acid strongly trans-stimulated efflux of glutamate. We thus propose that OAT2 physiologically functions as glutamate efflux transporter. OAT2 mRNA was detected, after laser capture microdissection of rat liver slices, equally in periportal and pericentral regions; previous reports of hepatic release of glutamate into blood can now be explained by OAT2 activity. A specific OAT2 inhibitor could, by lowering plasma glutamate and thus promoting brain-to-blood efflux of glutamate, alleviate glutamate exotoxicity in acute brain conditions.     

Liver-Primed Memory T Cells Generated under Noninflammatory Conditions Provide Anti-infectious Immunity

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Highlights? Memory-like CD8⁺ T cells are generated in the liver in the absence of inflammation ? An alternative pathway of T cell priming is facilitated by nonimmune cells ? Liver-primed T cells are rescued from deletion for anti-infectious immunity ? T cell priming in the liver complements conventional memory T cell generation