The barley MATE gene,HvAACT1, increases citrate efflux and Al3+ tolerance when expressed in wheat and barley

Background and Aims

Aluminium is toxic in acid soils because the soluble Al³⁺ inhibits root growth. A mechanism of Al³⁺ tolerance discovered in many plant species involves the release of organic anions from root apices. The Al³⁺-activated release of citrate from the root apices of Al³⁺-tolerant genotypes of barley is controlled by a MATE gene named HvAACT1 that encodes a citrate transport protein located on the plasma membrane. The aim of this study was to investigate whether expressing HvAACT1 with a constitutive promoter in barley and wheat can increase citrate efflux and Al³⁺ tolerance of these important cereal species.

Methods HvAACT1

was over-expressed in wheat (Triticum aestivum) and barley (Hordeum vulgare) using the maize ubiquitin promoter. Root apices of transgenic and control lines were analysed for HvAACT1 expression and organic acid efflux. The Al³⁺ tolerance of transgenic and control lines was assessed in both hydroponic solution and acid soil.

Key Results and Conclusions

Increased HvAACT1 expression in both cereal species was associated with increased citrate efflux from root apices and enhanced Al³⁺ tolerance, thus demonstrating that biotechnology can complement traditional breeding practices to increase the Al³⁺ tolerance of important crop plants.     

In Vivo Substrate Diversity and Preference of Small Heat Shock Protein IbpB as Revealed by Using a Genetically Incorporated Photo-cross-linker

Small heat shock proteins (sHSPs), as ubiquitous molecular chaperones found in all forms of life, are known to be able to protect cells against stresses and suppress the aggregation of a variety of model substrate proteins under in vitro conditions. Nevertheless, it is poorly understood what natural substrate proteins are protected by sHSPs in living cells. Here, by using a genetically incorporated photo-cross-linker (p-benzoyl-l-phenylalanine), we identified a total of 95 and 54 natural substrate proteins of IbpB (an sHSP from Escherichia coli) in living cells with and without heat shock, respectively. Functional profiling of these proteins (110 in total) suggests that IbpB, although binding to a wide range of cellular proteins, has a remarkable substrate preference for translation-related proteins (e.g. ribosomal proteins and amino-acyl tRNA synthetases) and moderate preference for metabolic enzymes. Furthermore, these two classes of proteins were found to be more prone to aggregation and/or inactivation in cells lacking IbpB under stress conditions (e.g. heat shock). Together, our in vivo data offer novel insights into the chaperone function of IbpB, or sHSPs in general, and suggest that the preferential protection on the protein synthesis machine and metabolic enzymes may dominantly contribute to the well known protective effect of sHSPs on cell survival against stresses.     

Five Conditions Commonly Used to Down-regulate Tor Complex 1 Generate Different Physiological Situations Exhibiting Distinct Requirements and Outcomes

Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc¹³ localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G₁ arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc¹³ localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal.     

Inhibition of the First Step in Synthesis of the Mycobacterial Cell Wall Core,Catalyzed by the GlcNAc-1-phosphate Transferase WecA,by the Novel Caprazamycin Derivative CPZEN-45

Because tuberculosis is one of the most prevalent and serious infections, countermeasures against it are urgently required. We isolated the antitubercular agents caprazamycins from the culture of an actinomycete strain and created CPZEN-45 as the most promising derivative of the caprazamycins. Herein, we describe the mode of action of CPZEN-45 first against Bacillus subtilis. Unlike the caprazamycins, CPZEN-45 strongly inhibited incorporation of radiolabeled glycerol into growing cultures and showed antibacterial activity against caprazamycin-resistant strains, including a strain overexpressing translocase-I (MraY, involved in the biosynthesis of peptidoglycan), the target of the caprazamycins. By contrast, CPZEN-45 was not effective against a strain overexpressing undecaprenyl-phosphate–GlcNAc-1-phosphate transferase (TagO, involved in the biosynthesis of teichoic acid), and a mutation was found in the tagO gene of the spontaneous CPZEN-45-resistant strain. This suggested that the primary target of CPZEN-45 in B. subtilis is TagO, which is a different target from that of the parent caprazamycins. This suggestion was confirmed by evaluation of the activities of these enzymes. Finally, we showed that CPZEN-45 was effective against WecA (Rv1302, also called Rfe) of Mycobacterium tuberculosis, the ortholog of TagO and involved in the biosynthesis of the mycolylarabinogalactan of the cell wall of M. tuberculosis. The outlook for WecA as a promising target for the development of antituberculous drugs as a countermeasure of drug resistant tuberculosis is discussed.     

Solid-state NMR Study Reveals Collagen I Structural Modifications of Amino Acid Side Chains upon Fibrillogenesis

In vivo, collagen I, the major structural protein in human body, is found assembled into fibrils. In the present work, we study a high concentrated collagen sample in its soluble, fibrillar, and denatured states using one and two dimensional {¹H}-¹³C solid-state NMR spectroscopy. We interpret ¹³C chemical shift variations in terms of dihedral angle conformation changes. Our data show that fibrillogenesis increases the side chain and backbone structural complexity. Nevertheless, only three to five rotameric equilibria are found for each amino acid residue, indicating a relatively low structural heterogeneity of collagen upon fibrillogenesis. Using side chain statistical data, we calculate equilibrium constants for a great number of amino acid residues. Moreover, based on a ¹³C quantitative spectrum, we estimate the percentage of residues implicated in each equilibrium. Our data indicate that fibril formation greatly affects hydroxyproline and proline prolyl pucker ring conformation. Finally, we discuss the implication of these structural data and propose a model in which the attractive force of fibrillogenesis comes from a structural reorganization of 10 to 15% of the amino acids. These results allow us to further understand the self-assembling process and fibrillar structure of collagen.     

Heterochromatin Protein 1a (HP1a) Partner Specificity Is Determined by Critical Amino Acids in the Chromo Shadow Domain and C-terminal Extension

Drosophila melanogaster Heterochromatin Protein 1a (HP1a) is an essential protein critical for heterochromatin assembly and regulation. Its chromo shadow domain (CSD) homodimerizes, a requirement for binding protein partners that contain a PXVXL motif. How does HP1a select among its many different PXVXL-containing partners? HP1a binds tightly to Heterochromatin Protein 2 (HP2), but weakly to PIWI. We investigated differences in homodimerization and the impact of the C-terminal extension (CTE) by contrasting HP1a to its paralogue, HP1b. HP1a and HP1b differ in the dimerization interface, with HP1a having an Arg at position 188 rather than Glu. We find that while this substitution reduces the dimerization constant, it does not impact the binding surface as demonstrated by unchanged partner binding affinities. However, the CTE (only 4 residues in HP1a as compared with 87 residues in HP1b) is critical; the charged residues in HP1a are necessary for tight peptide binding. Examining a panel of amino acid substitutions in the HP1a CSD, we find that Leu-165 in HP1a interacts with HP2 but not PIWI, supporting the conclusion that different sites in the binding surface provide discrimination for partner selection. Partner sequence is also critical for affinity, as the remaining difference in binding between HP2 and PIWI polypeptides is eliminated by swapping the PXVXL motifs between the two. Taken together, these studies indicate that the binding surface of the HP1a CSD plus its short CTE provide the needed discrimination among HP1a''s partners, and that the CTE is important for differentiating the interactions of the Drosophila HP1 paralogs.     

Sialylneolacto-N-tetraose c (LSTc)-bearing Liposomal Decoys Capture Influenza A Virus

Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains.     

Dhrs3 Protein Attenuates Retinoic Acid Signaling and Is Required for Early Embryonic Patterning

All-trans-retinoic acid (atRA) is an important morphogen involved in many developmental processes, including neural differentiation, body axis formation, and organogenesis. During early embryonic development, atRA is synthesized from all-trans-retinal (atRAL) in an irreversible reaction mainly catalyzed by retinal dehydrogenase 2 (aldh1a2), whereas atRAL is converted from all-trans-retinol via reversible oxidation by retinol dehydrogenases, members of the short-chain dehydrogenase/reductase family. atRA is degraded by cytochrome P450, family 26 (cyp26). We have previously identified a short-chain dehydrogenase/reductase 3 (dhrs3), which showed differential expression patterns in Xenopus embryos. We show here that the expression of dhrs3 was induced by atRA treatment and overexpression of Xenopus nodal related 1 (xnr1) in animal cap assay. Overexpression of dhrs3 enhanced the phenotype of excessive cyp26a1. In embryos overexpressing aldh1a2 or retinol dehydrogenase 10 (rdh10) in the presence of their respective substrates, Dhrs3 counteracted the action of Aldh1a2 or Rdh10, indicating that retinoic acid signaling is attenuated. Knockdown of Dhrs3 by antisense morpholino oligonucleotides resulted in a phenotype of shortened anteroposterior axis, reduced head structure, and perturbed somitogenesis, which were also found in embryos treated with an excess of atRA. Examination of the expression of brachyury, not, goosecoid, and papc indicated that convergent extension movement was defective in Dhrs3 morphants. Taken together, these studies suggest that dhrs3 participates in atRA metabolism by reducing atRAL levels and is required for proper anteroposterior axis formation, neuroectoderm patterning, and somitogenesis.     

岩溶区不同植被下土壤水溶解无机碳含量及其稳定碳同位素组成特征

自2010年7月至2011年7月对重庆青木关岩溶区典型植被下的土壤水进行了月动态取样,分析了土壤水溶解无机碳含量(DIC浓度)及其稳定碳同位素组成(δ13CDIC值)的时空变化特征,以揭示岩溶土壤系统碳酸盐岩溶蚀作用及其碳汇效应。研究结果表明:草地和针叶林地土壤水的DIC浓度和δ13CDIC值相对较低,分别为59.12 mg/L和-17.22‰,31.47 mg/L和-16.37‰;而旱地、灌丛地、退耕还林地土壤水具有较高的DIC浓度和δ13CDIC值,分别达153.88 mg/L和-12.2‰,221.82 mg/L和-11.9‰,97.30 mg/L和-11.23‰,其中灌丛和退耕还林地的δ13CDIC值与DIC浓度呈正比,且雨季较旱季偏高约4‰—5‰。根据δ13CDIC值,结合各植被类型下土壤水DIC浓度与其相应的土壤碳酸盐含量呈正相关,判断旱地、灌丛地、退耕还林地等岩溶土壤水中的DIC主要来自土壤中碳酸盐岩矿物的碳酸溶蚀,即岩溶土壤中存在着碳酸盐岩碳酸溶蚀作用,从而在一定程度上减少了土壤系统向大气排放的CO2量。