Peptide signaling for drought-induced tomato flower drop.

The premature abscission of flowers and fruits limits crop yield under environmental stress. Drought-induced flower drop in tomato plants was found to be regulated by phytosulfokine (PSK), a peptide hormone previously known for its growth-promoting and immune-modulating activities. PSK formation in response to drought stress depends on phytaspase 2, a subtilisin-like protease of the phytaspase subtype that generates the peptide hormone by aspartate-specific processing of the PSK precursor in the tomato flower pedicel. The mature peptide acts in the abscission zone where it induces expression of cell wall hydrolases that execute the abscission process. Our results provide insight into the molecular control of abscission as regulated by proteolytic processing to generate a small plant peptide hormone.

A two-way molecular dialogue between embryo and endosperm is required for seed development.

The plant embryonic cuticle is a hydrophobic barrier deposited de novo by the embryo during seed development. At germination, it protects the seedling from water loss and is, thus, critical for survival. Embryonic cuticle formation is controlled by a signaling pathway involving the ABNORMAL LEAF SHAPE1 subtilase and the two GASSHO receptor-like kinases. We show that a sulfated peptide, TWISTED SEED1 (TWS1), acts as a GASSHO ligand. Cuticle surveillance depends on the action of the subtilase, which, unlike the TWS1 precursor and the GASSHO receptors, is not produced in the embryo but in the neighboring endosperm. Subtilase-mediated processing of the embryo-derived TWS1 precursor releases the active peptide, triggering GASSHO-dependent cuticle reinforcement in the embryo. Thus, a bidirectional molecular dialogue between embryo and endosperm safeguards cuticle integrity before germination.

Nitrifying moving bed biofilm reactor (MBBR) biofilm and biomass response to long term exposure to 1 °C.

This study aims to investigate moving bed biofilm reactor (MBBR) nitrification rates, nitrifying biofilm morphology, biomass viability as well as bacterial community shifts during long-term exposure to 1 °C. Long-term exposure to 1 °C is the key operational condition for potential ammonia removal upgrade units to numerous northern region treatment systems. The average laboratory MBBR ammonia removal rate after long-term exposure to 1 °C was measured to be 18 ± 5.1% as compared to the average removal rate at 20 °C. Biofilm morphology and specifically the thickness along with biomass viability at various depths in the biofilm were investigated using variable pressure electron scanning microscope (VPSEM) imaging and confocal laser scanning microscope (CLSM) imaging in combination with viability live/dead staining. The biofilm thickness along with the number of viable cells showed significant increases after long-term exposure to 1 °C. Hence, this study observed nitrifying bacteria with higher activities at warm temperatures and a slightly greater quantity of nitrifying bacteria with lower activities at cold temperatures in nitrifying MBBR biofilms. Using DNA sequencing analysis, Nitrosomonas and Nitrosospira (ammonia oxidizers) as well as Nitrospira (nitrite oxidizer) were identified and no population shift was observed between 20 °C and after long-term exposure to 1 °C.

Plant defense in the absence of jasmonic acid: the role of cyclopentenones.

The Arabidopsis opr3 mutant is defective in the isoform of 12-oxo-phytodienoate (OPDA) reductase required for jasmonic acid (JA) biosynthesis. Oxylipin signatures of wounded opr3 leaves revealed the absence of detectable 3R,7S-JA as well as altered levels of its cyclopentenone precursors OPDA and dinor OPDA. In contrast to JA-insensitive coi1 plants and to the fad3 fad7 fad8 mutant lacking the fatty acid precursors of JA synthesis, opr3 plants exhibited strong resistance to the dipteran Bradysia impatiens and the fungus Alternaria brassicicola. Analysis of transcript profiles in opr3 showed the wound induction of genes previously known to be JA-dependent, suggesting that cyclopentenones could fulfill some JA roles in vivo. Treating opr3 plants with exogenous OPDA powerfully up-regulated several genes and disclosed two distinct downstream signal pathways, one through COI1, the other via an electrophile effect of the cyclopentenones. We conclude that the jasmonate family cyclopentenone OPDA (most likely together with dinor OPDA) regulates gene expression in concert with JA to fine-tune the expression of defense genes. More generally, resistance to insect and fungal attack can be observed in the absence of JA.

Fast biological iron chelators: kinetics of iron removal from human diferric transferrin by multidentate hydroxypyridonates.

For decades, desferrioxamine B (Desferal) has been the therapeutic iron chelator of choice for iron-overload treatment, despite numerous problems associated with its use. Consequently, there is a continuous search for new iron chelating agents with improved properties, particularly oral activity. We have studied new potential therapeutic iron sequestering agents: multidentate ligands containing the hydroxypyridonate (HOPO) moiety. The ligands TRENCAM-3,2-HOPO, TRPN-3,2-HOPO, TREN-Me-3,2-HOPO, TREN-1,2,3-HOPO, 5LIO-3,2-HOPO, and BU-O-3,4-HOPO have been examined for their ability to remove iron from human diferric transferrin. The iron removal ability of the HOPO ligands is compared with that of the hydroxamate desferrioxamine B, the catecholates TRENCAM and enterobactin, as well as the bidentate hydroxypyridonate deferiprone, a proposed therapeutic substitute for Desferal. All the tested HOPO ligands efficiently remove iron from diferric transferrin at millimolar concentrations, with a hyperbolic dependence on ligand concentration. At high ligand concentrations, the fastest rates are found with the tetra- and bidentate hydroxypyridonates 5LIO-3,2-HOPO and deferiprone, and the slowest rates with the catecholate ligands. At low concentrations, closer to therapeutic dosage, hexadentate ligands which possess high pM values have the fastest rates of iron removal. TRENCAM-3,2-HOPO and TREN-Me-3,2-HOPO are the most efficient at lower doses and are regarded as having high potential as therapeutic agents. The kinetics of removal of Ga(III) from transferrin [in place of the redox active Fe(III)] were performed with TRENCAM and TREN-Me-3,2-HOPO to determine that there is no catalytic reduction step involved in iron removal.

Microbial iron transport via a siderophore shuttle: a membrane ion transport paradigm.

A mechanism of ion transport across membranes is reported. Microbial transport of Fe(3+) generally delivers iron, a growth-limiting nutrient, to cells via highly specific siderophore-mediated transport systems. In contrast, iron transport in the fresh water bacterium Aeromonas hydrophila is found to occur by means of an indiscriminant siderophore transport system composed of a single multifunctional receptor. It is shown that (i) the siderophore and Fe(3+) enter the bacterium together, (ii) a ligand exchange step occurs in the course of the transport, and (iii) a redox process is not involved in iron exchange. To the best of our knowledge, there have been no other reports of a ligand exchange mechanism in bacterial iron transport. The ligand exchange step occurs at the cell surface and involves the exchange of iron from a ferric siderophore to an iron-free siderophore already bound to the receptor. This ligand exchange mechanism is also found in Escherichia coli and seems likely to be widely distributed among microorganisms.

The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis.

Jasmonic acid (JA) and its precursor 12-oxophytodienoic acid (OPDA) act as plant growth regulators and mediate responses to environmental cues. To investigate the role of these oxylipins in anther and pollen development, we characterized a T-DNA-tagged, male-sterile mutant of Arabidopsis, opr3. The opr3 mutant plants are sterile but can be rendered fertile by exogenous JA but not by OPDA. Cloning of the mutant locus indicates that it encodes an isozyme of 12-oxophytodienoate reductase, designated OPR3. All of the defects in opr3 are alleviated by transformation of the mutant with an OPR3 cDNA. Our results indicate that JA and not OPDA is the signaling molecule that induces and coordinates the elongation of the anther filament, the opening of the stomium at anthesis, and the production of viable pollen. Just as importantly, our data demonstrate that OPR3 is the only isoform of OPR capable of reducing the correct stereoisomer of OPDA to produce JA required for male gametophyte development.

Amonabactin-mediated iron acquisition from transferrin and lactoferrin by Aeromonas hydrophila: direct measurement of individual microscopic rate constants.

The effectiveness and mechanism of iron acquisition from transferrin or lactoferrin by Aeromonas hydrophila has been analyzed with regard to the pathogenesis of this microbe. The ability of A. hydrophila's siderophore, amonabactin, to remove iron from transferrin was evaluated with in vitro competition experiments. The kinetics of iron removal from the three molecular forms of ferric transferrin (diferric, N- and C-terminal monoferric) were investigated by separating each form by urea gel electrophoresis. The first direct determination of individual microscopic rates of iron removal from diferric transferrin is a result. A. hydrophila 495A2 was cultured in an iron-starved defined medium and the growth monitored. Addition of transferrin or lactoferrin promoted bacterial growth. Growth promotion was independent of the level of transferrin or lactoferrin iron saturation (between 30 and 100%), even when the protein was sequestered inside dialysis tubing. Siderophore production was also increased when transferrin or lactoferrin was enclosed in a dialysis tube. Cell yield and growth rate were identical in experiments where transferrin was present inside or outside the dialysis tube, indicating that binding of transferrin was not essential and that the siderophore plays a major role in iron uptake from transferrin. The rate of iron removal from diferric transferrin shows a hyperbolic dependence on amonabactin concentration. Surprisingly, amonabactin cannot remove iron from the more weakly binding N-terminal site of monoferric transferrin, while it is able to remove iron from the more strongly binding C-terminal site of monoferric transferrin. Iron from both sites is removed from diferric transferrin and it is the N-terminal site (which does not release iron in the monoferric protein) that releases iron more rapidly! It is apparent that there is a significant interaction of the two lobes of the protein with regard to the chelator access. Taken together, these results support an amonabactin-dependent mechanism for iron removal by A. hydrophila from transferrin and lactoferrin. The implications of these findings for an amonabactin-dependent mechanism for iron removal by A. hydrophila from transferrin and lactoferrin are discussed.

The pvc gene cluster of Pseudomonas aeruginosa: role in synthesis of the pyoverdine chromophore and regulation by PtxR and PvdS.

A putative operon of four genes implicated in the synthesis of the chromophore moiety of the Pseudomonas aeruginosa siderophore pyoverdine, dubbed pvcABCD (where pvc stands for pyoverdine chromophore), was cloned and sequenced. Mutational inactivation of the pvc genes abrogated pyoverdine biosynthesis, consistent with their involvement in the biosynthesis of this siderophore. pvcABCD expression was negatively regulated by iron and positively regulated by both PvdS, the alternate sigma factor required for pyoverdine biosynthesis, and PtxR, a LysR family activator previously implicated in exotoxin A regulation.

The ferripyoverdine receptor FpvA of Pseudomonas aeruginosa PAO1 recognizes the ferripyoverdines of P. aeruginosa PAO1 and P. fluorescens ATCC 13525.

FpvA, the ferripyoverdine outer membrane receptor of Pseudomonas aeruginosa ATCC 15692 (PAO1 strain), is not specific to the pyoverdine produced by PAO1, but is also able to recognize the structurally different (ferri)pyoverdine of P. fluorescens ATCC 13525. The specificity of FpvA was assessed by iron uptake competitions using the wild-type strains P. aeruginosa ATCC 15692 and P. fluorescens ATCC 13525 and their respective ferripyoverdines, and by fpvA gene complementation of a FpvA-deficient mutant of P. aeruginosa ATCC 15692. The receptor mutant was able to utilize none of the two pyoverdines, while the same but fpvA-complemented mutant recovered simultaneously the ability to incorporate iron thanks to each of the two siderophores. The broad specificity of recognition of FpvA is viewed as an advantage for the strain in iron competition. Moreover, it allows an interesting approach for the understanding of the recognition mechanism between a (ferri)pyoverdine and its cognate outer membrane receptor.

Quorum-sensing and siderophore biosynthesis in Pseudomonas aeruginosa: lasR/lasI mutants exhibit reduced pyoverdine biosynthesis.

Cell density-dependent gene expression in Pseudomonas aeruginosa is controlled, in part, by the quorum-sensing regulator LasR. lasR null mutants exhibited a reproducible 2-fold decrease in production of the catecholate-hydroxamate siderophore pyoverdine during grown under iron-limiting conditions. Similarly, lasI mutants defective in the biosynthesis of the autoinducer PAI-1 also exhibited a 2-fold decrease in pyoverdine production which could be largely restored upon addition of exogenous PAI-1. lasR mutants were not altered with respect to expression of the pvdD gene involved in the synthesis of the peptide portion of pyoverdine, indicating that some other pyoverdine biosynthetic gene(s) were affected by the LasRI status of the cell. This represents the first report of quorum-sensing regulation of siderophore production in bacteria and highlights the fact that cell density, while not an essential signal for pyoverdine expression, does enhance production of this siderophore.

Substrate specificities of tobacco chitinases.

Ten tobacco chitinases (1,4-N-acetyl-beta-D-glucosaminide glycanhydrolase, EC 3.2.1.14) were purified from tobacco leaves hypersensitively reacting to tobacco mosaic virus. The 10 enzymes, which belong to five distinct structural classes of plant chitinases, were incubated with several potential substrates such as chitin, a beta-1,4 N-acetyl-D-glucosamine (GlcNAc) polymer, chitosan (partially deacetylated chitin), chitin oligomers of variable length and bacterial cell wall. Tobacco chitinases are all endotype enzymes that liberate oligomers from chitin and are capable of processing the chito-oligomers further at differential rates. Chitin reaction products were separated and quantified by HPLC and differential kinetics of oligomer accumulation and degradation were observed with the distinct classes of chitinases. Depending on the substrate to be hydrolysed, each isoform displayed a different spectrum of activity. For example, class I isoforms were the most active on chitin and (GlcNAc)4-6 whereas class III basic isoforms were the most efficient in inducing bacterial lysis. Class V and class VI chitinases were shown to more readily hydrolyse chitin oligomers than the chitin polymer itself. Together, these data indicate that the 10 tobacco chitinases represent complementary enzymes which may have synergistic effects on their substrates. This paper discusses their implication in plant defense by attacking pathogen's structural components and in plant development by maturing signal molecules.

Pyoverdin is essential for virulence of Pseudomonas aeruginosa.

The role of pyoverdin, the main siderophore in iron-gathering capacity produced by Pseudomonas aeruginosa, in bacterial growth in vivo is controversial, although iron is important for virulence. To determine the ability of pyoverdin to compete for iron with the human iron-binding protein transferrin, wild-type P. aeruginosa ATCC 15692 (PAO1 strain) and PAO pyoverdin-deficient mutants were grown at 37 degrees C in bicarbonate-containing succinate medium to which apotransferrin had been added. Growth of the pyoverdin-deficient mutants was fully inhibited compared with that of the wild type but was restored when pyoverdin was added to the medium. Moreover, when growth took place at a temperature at which no pyoverdin production occurred (43 degrees C), the wild-type PAO1 strain behaved the same as the pyoverdin-deficient mutants, with growth inhibited by apotransferrin in the presence of bicarbonate and restored by pyoverdin supplementation. Growth inhibition was never observed in bicarbonate-free succinate medium, whatever the strain and the temperature for growth. In vivo, in contrast to results obtained with the wild-type strain, pyoverdin-deficient mutants demonstrated no virulence when injected at 10(2) CFU into burned mice. However, virulence was restored when purified pyoverdin originating from the wild-type strain was supplemented during the infection. These results strongly suggest that pyoverdin competes directly with transferrin for iron and that it is an essential element for in vivo iron gathering and virulence expression in P. aeruginosa. Rapid removal of iron from [59Fe]ferritransferrin by pyoverdin in vitro supports this view.

Ornibactin production and transport properties in strains of Burkholderia vietnamiensis and Burkholderia cepacia (formerly Pseudomonas cepacia).

Several strains of Burkholderia vietnamiensis, isolated from the rhizosphere of rice plants, and four strains formerly known as Pseudomonas cepacia including two collection strains and two clinical isolates were compared for siderophore production and iron uptake. The B. vietnamiensis (TVV strains) as well as the B. cepacia strains (ATCC 25416 and ATCC 17759) and the clinical isolates K132 and LMG 6999 were all found to produce ornibactins under iron starvation. The two ATCC strains of B. cepacia additionally produced the previously described siderophores, pyochelin and cepabactin. Analysis of the ratio of isolated ornibactins (C4, C6 and C8) by HPLC revealed nearly identical profiles. Supplementation of the production medium with ornithine (20 mM) resulted in a 2.5-fold increase in ornibactin synthesis. Ornibactin-mediated iron uptake was independent of the length of the acyl side chain and was observed with all strains of B. vietnamiensis and B. cepacia, but was absent with strains of Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas stutzeri, known to produce pyoverdines or desferriferrioxamines as siderophores. These results suggest that ornibactin production is a common feature of all Burkholderia strains and that these strains develop an ornibactin-specific iron transport system which is distinct from the pyoverdine-specific transport in Pseudomonas strains.

Pathogenesis-related PR-1 proteins are antifungal. Isolation and characterization of three 14-kilodalton proteins of tomato and of a basic PR-1 of tobacco with inhibitory activity against Phytophthora infestans.

Three distinct basic 14-kD proteins, P14a, P14b, and P14c, were isolated from tomato (Lycopersicon esculentum Mill. cv Baby) leaves infected with Phytophthora infestans. They exhibited antifungal activity against P. infestans both in vitro (inhibition of zoospore germination) and in vivo with a tomato leaf disc assay (decrease in infected leaf surface). Serological cross-reactions and amino acid sequence comparisons showed that the three proteins are members of the PR-1 group of pathogenesis-related (PR) proteins. P14a and P14b showed high similarity to a previously characterized P14, whereas P14c was found to be very similar to a putative basic-type PR-1 from tobacco predicted from isolated DNA clones. This protein, named PR-1 g, was purified from virus-infected tobacco (Nicotiana tabacum Samsun NN) leaves and characterized by amino acid microsequencing, along with the well-known acidic tobacco PR-1a, PR-1b, and PR-1c. The various tomato and tobacco PR-1 proteins were compared for their biological activity and found to display differential fungicidal activity against P. infestans in both the in vitro and in vivo assays, the most efficient being the newly characterized tomato P14c and tobacco PR-1g.

cDNA cloning and gene expression analysis of the microbial proteinase inhibitor of tobacco.

Tobacco mosaic virus-infected tobacco (Nicotiana tabacum var. Samsun NN) leaves produce a serine proteinase inhibitor that has evolved a specificity for microbial proteinases. We have isolated two closely related cDNAs that were shown to encode two active inhibitors. Southern analysis of genomic DNA, comparison of deduced amino acid sequences, and characterization of the two separated proteins suggest that the two genes of tobacco are homologous originating from each parent. Amino acid sequences deduced from the cDNAs exhibit a glutamic residue at the P1 position of the active site, known to determine the specificity of this type of inhibitors. Nevertheless, the V8 proteinase from Staphylococcus aureus, an enzyme that cleaves polypeptides after glutamic acid residues, was found to be unaffected by the tobacco inhibitor. We demonstrate strong accumulation of the two mRNAs and proteins during the hypersensitive reaction of tobacco to tobacco mosaic virus. Messengers and products of the two genes are present in a 3:2 ratio, in infected leaves as well as in upper uninfected leaves, the induction being markedly lower at distance from the infection site. The transcripts were also found in sepals and petals of healthy plants, indicating that these genes are also developmentally regulated. Unlike the tomato and potato I inhibitors, the tobacco inhibitor was only weakly induced by wounding, but was expressed upon salicylic acid or ethephon treatment, as many pathogenesis-related proteins.

Plant 'pathogenesis-related' proteins and their role in defense against pathogens.

The hypersensitive reaction to a pathogen is one of the most efficient defense mechanisms in nature and leads to the induction of numerous plant genes encoding defense proteins. These proteins include: 1) structural proteins that are incorporated into the extracellular matrix and participate in the confinement of the pathogen; 2) enzymes of secondary metabolism, for instance those of the biosynthesis of plant antibiotics; 3) pathogenesis-related (PR) proteins which represent major quantitative changes in soluble protein during the defense response. The PRs have typical physicochemical properties that enable them to resist to acidic pH and proteolytic cleavage and thus survive in the harsh environments where they occur: vacuolar compartment or cell wall or intercellular spaces. Since the discovery of the first PRs in tobacco many other similar proteins have been isolated from tobacco but also from other plant species, including dicots and monocots, the widest range being characterized from hypersensitively reacting tobacco. Based first on serological properties and later on sequence data, the tobacco PRs have been classified in five major groups. Group PR-1 contains the first discovered PRs of 15-17 kDa molecular mass, whose biological activity is still unknown, but some members have been shown recently to have antifungal activity. Group PR-2 contains three structurally distinct classes of 1,3-beta-glucanases, with acidic and basic counterparts, with dramatically different specific activity towards linear 1,3-beta-glucans and with different substrate specificity. Group PR-3 consists of various chitinases-lysozymes that belong to three distinct classes, are vacuolar or extracellular, and exhibit differential chitinase and lysozyme activities. Some of them, either alone or in combination with 1,3-beta-glucanases, have been shown to be antifungal in vitro and in vivo (transgenic plants), probably by hydrolysing their substrates as structural components in the fungal cell wall. Group PR-4 is the less studied, and in tobacco contains four members of 13-14.5 kDa of unknown activity and function. Group PR-5 contains acidic-neutral and very basic members with extracellular and vacuolar localization, respectively, and all members show sequence similarity to the sweet-tasting protein thaumatin. Several members of the PR-5 group from tobacco and other plant species were shown to display significant in vitro activity of inhibiting hyphal growth or spore germination of various fungi probably by a membrane permeabilizing mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)