Synergistic interactions of assorted ameliorating agents to enhance the potential of heavy metal phytoremediation.

Pollution by toxic heavy metals creates a significant impact on the biotic community of the ecosystem. Nowadays, a solution to this problem is an eco-friendly approach like phytoremediation, in which plants are used to ameliorate heavy metals. In addition, various amendments are used to enhance the potential of heavy metal phytoremediation. Symbiotic microorganisms such as phosphate-solubilizing bacteria (PSB), endophytes, mycorrhiza and plant growth-promoting rhizobacteria (PGPR) play a significant role in the improvement of heavy metal phytoremediation potential along with promoting the growth of plants that are grown in contaminated environments. Various chemical chelators (Indole 3-acetic acid, ethylene diamine tetra acetic acid, ethylene glycol tetra acetic acid, ethylenediamine-N, N-disuccinic acid and nitrilotri-acetic acid) and their combined action with other agents also contribute to heavy metal phytoremediation enhancement. With modern techniques, transgenic plants and microorganisms are developed to open up an alternative strategy for phytoremediation. Genomics, proteomics, transcriptomics and metabolomics are widely used novel approaches to develop competent phytoremediators. This review accounts for the synergistic interactions of the ameliorating agent's role in enhancing heavy metal phytoremediation, intending to highlight the importance of these various approaches in reducing heavy metal pollution.

Gene expression profiling of tolerant barley in response to Diuraphis noxia (Hemiptera: Aphididae) feeding.

Aphids are, arguably, the single most damaging group of agricultural insect pests throughout the world. Plant tolerance, which is a plant response to an insect pest, is viewed as an excellent management strategy. Developing testable hypotheses based on genome-wide and more focused methods will help in understanding the molecular underpinnings of plant tolerance to aphid herbivory. As a first step in this process, we undertook transcript profiling with Affymetrix GeneChip Barley Genome arrays using RNA extracted from tissues of tolerant and susceptible genotypes collected at three hours, three days and six days after Diuraphis noxia introduction. Acquired data were compared to identify changes unique to the tolerant barley at each harvest date. Transcript abundance of 4086 genes was differentially changed over the three harvest dates in tolerant and susceptible barley in response to D. noxia feeding. Across the three harvest dates, the greatest number of genes was differentially expressed in both barleys at three days after aphid introduction. A total of 909 genes showed significant levels of change in the tolerant barley in response to D. noxia feeding as compared to susceptible plants infested with aphids. Many of these genes could be assigned to specific metabolic categories, including several associated with plant defense and scavenging of reactive oxygen species (ROS). Interestingly, two peroxidase genes, designated HvPRXA1 and HvPRXA2, were up-regulated to a greater degree in response to D. noxia feeding on tolerant barley plants, indicating that specific peroxidases could be important for the tolerance process. These findings suggest that the ability to elevate and sustain levels of ROS-scavenging enzymes could play an important role in the tolerant response.

Western immunoblotting in avian shell gland sample immunoblotting methods.

Avian shell gland tissue was subjected to Western blot analysis using anti-human estrogen receptor-alpha antibody H222. Initial attempts to obtain consistent, high-quality blots were unsuccessful because, as it turned out, excessive lipid in tissue preparations interfered with protein separation. Incremental additions of acetone eventually proved to be the critical step in solubilizing lipids and allowing consistent separation of bands on gels. A detailed description of the methodology is presented.

Estrogen receptor-alpha populations change with age in commercial laying hens.

Older hens in production lay larger but fewer eggs than younger birds, and the incidence of soft and broken shells is greater in older hens than younger. These changes are attributable at least in part to changing hormone profiles and diminished ability of the hen to transport calcium at the duodenum. In further exploration of this relationship, a study was conducted with three ages of Hy-Line W-36 birds: prelay pullets (PL; 19 wk, 0% production), peak-production hens (PP; 29 wk, approximately 93% production), and late-stage hens (LS; 71 wk, approximately 80% production). Hens from the PP and LS groups were palpated for presence of an egg in the shell gland; hens were then euthanized and tissues (kidney, shell gland, hypothalamus) were removed for quantification of estrogen receptor-alpha (ERalpha) populations via immunocytochemical and Western blot analyses. Localization of ERalpha by immunostaining in the shell gland showed differences among age groups; however, no differences were noted in localization of ERalpha between age groups in the kidney and hypothalamus. In both the kidney and the shell gland there was a decrease in the amount of ERalpha, as detected by immunoblotting, in the LS hens compared to PL and PP birds (P < 0.05). The results suggest that failure of calcium regulating mechanisms with age may be mediated at least in part through the reduced populations of estrogen receptors in certain critical tissues.

Identification of IgE-binding proteins in soy lecithin.

BACKGROUND: Soy lecithin is widely used as an emulsifier in processed foods, pharmaceuticals and cosmetics. Soy lecithin is composed principally of phospholipids; however, it has also been shown to contain IgE-binding proteins, albeit at a low level. A few clinical cases involving allergic reactions to soy lecithin have been reported. The purpose of this investigation is to better characterize the IgE-binding proteins typically found in lecithin. METHODS: Soy lecithin proteins were isolated following solvent extraction of lipid components and then separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated lecithin proteins were immunoblotted with sera from soy-sensitive individuals to determine the pattern of IgE-binding proteins. The identity of IgE-reactive bands was determined from their N-terminal sequence. RESULTS: The level of protein in six lecithin samples obtained from commercial suppliers ranged from 100 to 1,400 ppm. Lecithin samples showed similar protein patterns when examined by SDS-PAGE. Immunoblotting with sera from soy-sensitive individuals showed IgE binding to bands corresponding to 7, 12, 20, 39 and 57 kD. N-terminal analysis of these IgE-binding bands resulted in sequences for 3 components. The 12-kD band was identified as a methionine-rich protein (MRP) and a member of the 2S albumin class of soy proteins. The 20-kD band was found to be soybean Kunitz trypsin inhibitor. The 39-kD band was matched to a soy protein with unknown function. CONCLUSIONS: Soy lecithin contains a number of IgE-binding proteins; thus, it might represent a source of hidden allergens. These allergens are a more significant concern for soy-allergic individuals consuming lecithin products as a health supplement. In addition, the MRP and the 39-kD protein identified in this study represent newly identified IgE-binding proteins.

Cloning and expression analysis of hemoglobin genes from maize (Zea mays ssp. mays) and teosinte (Zea mays ssp. parviglumis).

With the exception of barley and rice, little is known about the existence of hemoglobins (Hbs) in cereals. This work reports the cloning and analysis of hb genes from maize (Zea mays ssp. mays) and teosinte (Zea mays ssp. parviglumis). Coding sequences of maize and teosinte hb genes (hbm and hbt, respectively) are highly similar to each other and are interrupted by three introns located at identical positions as other plant hb genes. Sequences of predicted Hbm and Hbt proteins are identical. The hydropathic profile of Hbm and Hbt is highly similar to that of rice Hb1, suggesting that Hbm, Hbt and Hb1 have the same tertiary structure and biochemical properties. Expression analysis showed that low levels of Hb transcripts, but considerable levels of Hb proteins exist in maize embryonic organs. No Hb transcripts and proteins were detected in teosinte embryonic organs. Low levels of Hb proteins, but no Hb transcripts, were detected in maize and teosinte vegetative organs. These observations suggest that the regulation of hb genes is different in maize and teosinte embryonic organs, and that the expression of hb genes is down- or up-regulated in maize and teosinte, respectively, from germination to vegetative growing.

Synthesis of hemoglobins in rice (Oryza sativa var. Jackson) plants growing in normal and stress conditions.

In rice (Oryza sativa var. Jackson) at least three copies of hemoglobin (hb) gene exist. Rice hb1 and hb2 genes are differentially expressed in roots and leaves from mature plants. We used polyclonal antibodies raised to recombinant rice Hb1 and Western blotting to analyze the synthesis of Hbs in rice plants growing under normal or stress conditions. Results showed that rice Hbs are synthesized in coleoptiles, seminal roots and embryos from seeds germinated for 6 days, and also in leaves and roots from plants 2-14 weeks after germination. Analysis of Hb synthesis in stressed rice showed that: (i) level of Hbs was higher in etiolated than control plants, (ii) level of Hbs increased in roots from flooded rice, and (iii) level of Hbs did not change under oxidative (H(2)O(2)), nitrosative (SNP) and hormonal (2,4-D) stresses. These results suggest that the effect of light withdrawal in etiolated leaves and O(2)-limiting conditions in flooded roots, but not oxidative, nitrosative and hormonal stresses, modulate the synthesis of rice Hbs.

Oxidative responses of resistant and susceptible cereal leaves to symptomatic and nonsymptomatic cereal aphid (Hemiptera: Aphididae) feeding.

The impact of the leaf-chlorosis-eliciting Russian wheat aphid, Diuraphis noxia (Mordvilko), and the nonchlorosis-eliciting bird cherry-oat aphid, Rhopalosiphum padi (L.), feeding on D. noxia-susceptible and -resistant cereals was examined during the period (i.e., 3, 6, and 9 d after aphid infestation) that leaf chlorosis developed. After aphid number, leaf rolling and chlorosis ratings, and fresh leaf weight were recorded on each sampling date, total protein content, peroxidase, catalase, and polyphenol oxidase activities of each plant sample were determined spectrophotometrically. Although R. padi and D. noxia feeding caused significant increase of total protein content in comparison with the control cereal leaves, the difference in total protein content between R. padi and D. noxia-infested leaves was not significant. Although R. padi-feeding did not elicit any changes of peroxidase specific activity in any of the four cereals in comparison with the control leaves, D. noxia feeding elicited greater increases of peroxidase specific activity only on resistant 'Halt' wheat (Triticum aestivum L.) and susceptible 'Morex' barley (Hordeum vulgare L.), but not on susceptible 'Arapahoe' and resistant 'Border' oat (Avena sativa L.). D. noxia-feeding elicited a ninefold increase in peroxidase specific activity on Morex barley and a threefold on Halt wheat 9 d after the initial infestation in comparison with control leaves. Furthermore, D. noxia feeding did not elicit any differential changes of catalase and polyphenol oxidase activities in comparison with either R. padi feeding or control leaves. The findings suggest that D. noxia feeding probably results in oxidative stress in plants. Moderate increase of peroxidase activity (approximately threefold) in resistant Halt compared with susceptible Arapahoe wheat might have contributed to its resistance to D. noxia, whereas the ninefold peroxidase activity increase may have possibly contributed to barley's susceptibility. Different enzymatic responses in wheat, barley, and oat to D. noxia and R. padi feeding indicate the cereals have different mechanisms of aphid resistance.

Chitinases from the Plant Disease Biocontrol Agent, Stenotrophomonas maltophilia C3.

ABSTRACT Stenotrophomonas maltophilia strain C3, a biocontrol agent of Bipolaris sorokiniana in turfgrass, produced chitinases in broth media containing chitin. Chitinases were partially purified from culture fluid by ammonium sulfate precipitation and chitin affinity chromatography. The chromatography fraction with the highest specific chitinase activity was inhibitory to conidial germination and germ-tube elongation of B. sorokiniana, but it was less inhibitory than the protein fraction or the raw culture filtrate. The fraction exhibited strong exochitinase and weak endo-chitinase activity. Optimum temperature and pH for chitinase activity were 45 to 50 degrees C and 4.5 to 5.0, respectively. Chitinase activity was inhibited by Hg(2+) and Fe(3+), but not by other metal ions or enzyme inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the chromatography fraction revealed the presence of five protein bands of 25, 32, 48, 65, and 75 kDa. Partial amino acid sequences of the 32-, 65-, and 75-kDa proteins indicated that they are homologous to known bacterial chitinases. There was no homology found in the partial amino acid sequences of the 25- and 48-kDa proteins to any known chitinases. Five chitinase-active proteins were detected in the protein and chromatography fractions by activity gels, but when each protein was extracted and re-electrophoresed separately under denaturing conditions, only 32- or 48-kDa proteins were revealed. It was concluded that strain C3 produces at least two chitinases that are antifungal.

Nonsymbiotic hemoglobins in rice are synthesized during germination and in differentiating cell types.

Nonsymbiotic hemoglobins (ns-Hbs) previously have been found in monocots and dicots; however, very little is known about the tissue and cell type localization as well as the physiological function(s) of these oxygen-binding proteins. We report the immunodetection and immunolocalization of ns-Hbs in rice (Oryza sativa L.) by Western blotting and in situ confocal laser scanning techniques. Ns-Hbs were detected in soluble extracts of different tissues from the developing rice seedling by immunoblotting. Levels of ns-Hbs increased in the germinating seed for the first six days following imbibition and remained relatively constant thereafter. In contrast, ns-Hb levels decreased during leaf maturation. Roots and mesocotyls contained detectable, but low levels of ns-Hbs. Split-seed experiments revealed that ns-Hbs are synthesized de novo during seed germination and are expressed in the absence of any signal originating from the embryo. Immunolocalization of ns-Hbs by confocal microscopy indicated the presence of ns-Hbs primarily in differentiated and differentiating cell types of the developing seedling, such as the aleurone, scutellum, root cap cells, sclerenchyma, and tracheary elements. To our knowledge, this is the first report of the specific cellular localization of these proteins during seedling development.

Rapid spectrophotometric determination of 2,4,6-trinitrotoluene in a Pseudomonas enzyme assay.

Although TNT (2,4,6-trinitrotoluene) and its degradation products can be quantified by HPLC, this method is not suitable for simultaneous analyses of the numerous samples typically encountered in enzyme studies. To solve this problem, we developed a simple and rapid spectrophotometric assay for TNT and tested the procedure using partially purified nitroreductase(s) from a Pseudomonas aeruginosa isolate, which transformed TNT in the culture medium. In highly alkaline solution, TNT (pK(a)=11.99) exhibits significant absorbance at 447 nm, while major metabolites, 2-amino-4, 6-dinitrotoluene (2ADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and 2,6-diamino-4-nitrotoluene (2,6DANT) display no absorbance at this wavelength. Assay mixtures of TNT, Tris-HCl buffer, a reductant, and the enzyme(s) were analyzed by measuring absorbance 4 min after adjusting the pH to 12.2. TNT transformation to colorless metabolites was linear with respect to protein and substrate concentrations. Using the assay, we determined that TNT nitroreductase(s) from the isolate required an electron donor and preferred NADH to NADPH. TNT transformation increased when NAD was recycled to NADH using glucose-6-phosphate (GP) and glucose-6-phosphate dehydrogenase (GPDH). Enzymatic transformation of TNT was completely inhibited by Cu(2+) (5 mM) and was partially inhibited by other divalent metallic cations. Because the assay is sensitive to ammonium sulfate, dithiothreitol, ascorbic acid, and sodium phosphate, extracts should be assayed in the absence of these components.

Crystal structure of a nonsymbiotic plant hemoglobin.

BACKGROUND: Nonsymbiotic hemoglobins (nsHbs) form a new class of plant proteins that is distinct genetically and structurally from leghemoglobins. They are found ubiquitously in plants and are expressed in low concentrations in a variety of tissues including roots and leaves. Their function involves a biochemical response to growth under limited O(2) conditions. RESULTS: The first X-ray crystal structure of a member of this class of proteins, riceHb1, has been determined to 2.4 A resolution using a combination of phasing techniques. The active site of ferric riceHb1 differs significantly from those of traditional hemoglobins and myoglobins. The proximal and distal histidine sidechains coordinate directly to the heme iron, forming a hemichrome with spectral properties similar to those of cytochrome b(5). The crystal structure also shows that riceHb1 is a dimer with a novel interface formed by close contacts between the G helix and the region between the B and C helices of the partner subunit. CONCLUSIONS: The bis-histidyl heme coordination found in riceHb1 is unusual for a protein that binds O(2) reversibly. However, the distal His73 is rapidly displaced by ferrous ligands, and the overall O(2) affinity is ultra-high (K(D) approximately 1 nM). Our crystallographic model suggests that ligand binding occurs by an upward and outward movement of the E helix, concomitant dissociation of the distal histidine, possible repacking of the CD corner and folding of the D helix. Although the functional relevance of quaternary structure in nsHbs is unclear, the role of two conserved residues in stabilizing the dimer interface has been identified.

Targeting of the soybean leghemoglobin to tobacco chloroplasts: effects on aerobic metabolism in transgenic plants.

Several attempts have been made to alter the aerobic metabolism of plants, especially those related to the oxygenation or carboxylation of Rubisco. However, designing a more efficient Rubisco protein is rather problematic since its structural manipulation leads frequently to an enhancement of oxygenase activity, which is responsible for photorespiratory losses. In order to reduce oxygen availability inside the chloroplast, a chimeric gene consisting of a soybean leghemoglobin cDNA (lba) ligated to the chloroplast targeting signal sequence of the Rubisco small subunit gene, was introduced and expressed in Nicotiana tabacum. Lb was efficiently imported and correctly processed inside the chloroplasts of transgenic tobacco plants. Furthermore, the level of Lb expression in leaf tissue ranged from 0.01 to 0.1%. Analysis of photosynthesis, starch, sucrose and enzymes involved in aerobic metabolism, revealed that despite the high affinity of Lb for oxygen, no significant difference was observed in relation to the control plants. These results suggest that higher Lb concentrations would be required inside the chloroplasts in order to interfere on aerobic metabolism.

Analysis of a ferric leghemoglobin reductase from cowpea (Vigna unguiculata) root nodules.

Ferric leghemoglobin reductase (FLbR), an enzyme reducing ferric leghemoglobin (Lb) to ferrous Lb, was purified from cowpea (Vigna unguiculata) root nodules by sequential chromatography on hydroxylapatite followed by Mono-Q HR5/5 FPLC and Sephacryl S-200 gel filtration. The purified cowpea FLbR had a specific activity of 216 nmol Lb(2+)O(2) formed min(-1) mg(-1) of enzyme for cowpea Lb(3+) and a specific activity of 184 nmol Lb(2+)O(2) formed min(-1) mg(-1) of enzyme for soybean Lb(3+). A cDNA clone of cowpea FLbR was obtained by screening a cowpea root nodule cDNA library. The nucleotide sequence of cowpea FLbR cDNA exhibited about 88% similarity with soybean (Glycine max) FLbR and 85% with pea (Pisum sativum) dihydrolipoamide dehydrogenase (DLDH, EC 1.8.1.4) cDNAs. Conserved regions for the FAD-binding site, NAD(P)H-binding site, and disulfide active site were identified among the deduced amino acid sequences of cowpea FLbR, soybean FLbR, pea DLDH and other enzymes in the family of the pyridine nucleotide-disulfide oxido-reductases.

Further analysis of maize C(4) pyruvate,orthophosphate dikinase phosphorylation by its bifunctional regulatory protein using selective substitutions of the regulatory Thr-456 and catalytic His-458 residues.

In C(4) plants such as maize, pyruvate,orthophosphate dikinase (PPDK) catalyzes the regeneration of the initial carboxylation substrate during C(4) photosynthesis. The primary catalytic residue, His-458 (maize C(4) PPDK), is involved in the ultimate transfer of the beta-phosphate from ATP to pyruvate. C(4) PPDK activity undergoes light-dark regulation in vivo by reversible phosphorylation of a nearby active-site residue (Thr-456) by a single bifunctional regulatory protein (RP). Using site-directed mutagenesis of maize recombinant C(4) dikinase, we made substitutions at the catalytic His residue (H458N) and at this regulatory target Thr (T456E, T456Y, T456F). Each of these affinity-purified mutant enzymes was assayed for changes in dikinase activity. As expected, substituting His-458 with Asn results in a catalytically incompetent enzyme. Substitutions of the Thr-456 residue with Tyr and Phe reduced activity by about 94 and 99%, respectively. Insertion of Glu at this position completely abolished activity, presumably by the introduction of negative charge proximal to the catalytic His. Furthermore, neither the T456Y nor inactive H458N mutant enzyme was phosphorylated in vitro by RP. The inability of the former to serve as a phosphorylation substrate indicates that RP is functionally a member of the Ser/Thr family of protein kinases rather than a "dual-specificity" Ser-Thr/Tyr kinase, since our previous work showed that RP effectively phosphorylated Ser inserted at position 456. The inability of RP to phosphorylate its native target Thr residue when Asn is substituted for His-458 documents that RP requires the His-P catalytic intermediate form of PPDK as its protein substrate. For these latter studies, synthetic phosphopeptide-directed antibodies specific for the Thr(456)-P form of maize C(4) PPDK were developed and characterized.

Soybean glycinin G1 acidic chain shares IgE epitopes with peanut allergen Ara h 3.

BACKGROUND: The identification of IgE epitopes for proteins is the first step in understanding the interaction of allergens with the immune system. Proteins from the legume family have shown in vitro cross-reactivity in IgE-binding assays, but this cross-reactivity is rarely clinically significant. Resolution of this discrepancy requires IgE epitope mapping of legume family protein allergens. METHODS: We constructed six fusion proteins representing overlapping regions of soybean glycinin G1 acidic chain. These fusion proteins were used in immunoblotting and a novel sandwich ELISA with pooled sera from soy-allergic individuals to reveal a common IgE-binding region. This region was the focus for IgE epitope mapping using overlapping synthetic peptides. RESULTS: Data from the fusion protein experiments revealed an IgE-binding region consisting of residues F192-I265. Analysis of the overlapping synthetic peptides to this region indicated that IgE epitopes to glycinin G1 acidic chain consist of residues G217-V235 and G253-I265. The epitopes identified for glycinin G1 acidic chain are homologous to IgE epitopes previously identified for the peanut allergen Ara h 3 [1]. However, residues identified by alanine scanning in the peanut epitopes as being important for IgE binding are different in the natural soybean epitopes. CONCLUSIONS: The IgE epitopes identified for glycinin G1 acidic chain apparently represent an allergenic region of several legume family seed storage proteins. Our findings indicate that the identification of IgE epitopes and structural analysis of legume family proteins will provide valuable information to the study of food allergies.

Soybean nodule sucrose synthase (nodulin-100): further analysis of its phosphorylation using recombinant and authentic root-nodule enzymes.

Sucrose synthase (SS) is a known phosphoserine-containing enzyme in legume root nodules and various other plant "sink" tissues. In order to begin to investigate the possible physiological significance of this posttranslational modification, we have cloned a full-length soybean nodule SS (nodulin-100) cDNA and overexpressed it in Escherichia coli. Authentic nodule SS and recombinant wild-type and mutant forms of the enzyme were purified and characterized. We document that a conserved serine near the N-terminus (Ser(11)) is the primary phosphorylation site for a nodule Ca(2+)-dependent protein kinase (CDPK) in vitro. Related tryptic digestion and mass spectral analyses indicated that this target residue was also phosphorylated in planta in authentic nodulin-100. In addition, a secondary phosphorylation site(s) in recombinant nodule SS was implicated given that all active mutant enzyme forms (S11A, S11D, S11C, and N-terminal truncation between Ala(2) and Arg(13)) were phosphorylated, albeit weakly, by the CDPK. This secondary site(s) likely resides between Glu(14) and Met(193) as evidenced by CNBr cleavage and phosphopeptide mapping. Phosphorylation of the recombinant and authentic nodule Ser(11) enzymes in vitro by the nodule CDPK had no major effect on the sucrose-cleavage activity and/or kinetic properties. However, phosphorylation decreased the apparent surface hydrophobicity of the recombinant wild-type enzyme, suggesting that this covalent modification could potentially play some role in the documented partitioning of nodulin-100 between the nodule symbiosome/plasma membranes and cytosol in planta.