ABSTRACT
ß-1,3-glucanases are found in organisms as diverse as plants, animals, bacteria and fungi. In plants, such enzymes are not only associated with defense mechanisms against pathogens, but also play critical roles in physiological and developmental processes. Here we identified a new ß-1,3-glucanase in maize seeds, and named it ZmGlucA. Sequence analysis revealed that ZmGlucA belongs to the class A of ß-1,3-glucanase, a class related to defense and physiological processes in plants. mRNA and protein assays showed that zmGlucA is expressed exclusively in seeds, and it is differentially regulated during seed development. Additionally, zmGlucA expression is strongly induced in seeds of the mutant dek 827Kpro1, which is defective for embryo and endosperm development. Our data support the idea that ZmGlucA protein is relevant to seed development.
Subject(s)
Glycoside Hydrolases/metabolism , Plants, Genetically Modified/growth & development , Seeds/enzymology , Seeds/growth & development , Zea mays/enzymology , Zea mays/growth & development , Blotting, Northern , Gene Expression Regulation, Enzymologic/genetics , Gene Expression Regulation, Enzymologic/physiology , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Glycoside Hydrolases/genetics , Plants, Genetically Modified/genetics , Seeds/genetics , Zea mays/geneticsABSTRACT
BACKGROUND: Hematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: Hz formation activity of an alpha-glucosidase was investigated. Hz formation was inhibited by specific alpha-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect alpha-glucosidase was able to inhibit Hz formation. The alpha-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that alpha-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of alpha-glucosidase gene, was injected into R. prolixus females' hemocoel. Gene silencing was accomplished by reduction of both alpha-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of alpha-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of alpha-glucosidase shows a high similarity to the insect alpha-glucosidases, with critical histidine and aspartic residues conserved among the enzymes. CONCLUSIONS/SIGNIFICANCE: Herein the Hz formation is shown to be associated to an alpha-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that alpha-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.
Subject(s)
Hemeproteins/chemistry , Intestinal Mucosa/metabolism , alpha-Glucosidases/chemistry , Animals , Binding Sites , Catalysis , Evolution, Molecular , Female , Gene Expression Regulation , Heme/chemistry , Hemoglobins/chemistry , Hydrolysis , Insecta , Microvilli/metabolism , RNA, Double-Stranded/chemistry , Rhodnius/metabolismABSTRACT
The SALT protein is a 14.5 kDa mannose-binding lectin, originally described as preferentially expressed in rice plant roots in response to NaCl stress. Recombinant SALT lectin was produced in Escherichia coli from a cDNA clone encoding protein. After isopropyl-beta-d-thiogalactopyranoside induction, the expression level achieved was 23% of the soluble protein. The recombinant agglutinin was purified by a single-step process by dialyses against a high concentrated salt solution. After purification, hemagglutination assays of rabbit erythrocytes revealed that the recombinant SALT protein is a potent agglutinin (0.078 microg ml(-1) minimal concentration). The purified recombinant lectin was also used for comparative estimation of native protein amounts in protein extracts from rice plants by Western blot assay.
Subject(s)
Oryza/metabolism , Plant Proteins/biosynthesis , Plant Proteins/isolation & purification , Animals , Electrophoresis, Polyacrylamide Gel/methods , Erythrocytes/drug effects , Escherichia coli/genetics , Escherichia coli/metabolism , Hemagglutination Tests/methods , Oryza/genetics , Plant Lectins/biosynthesis , Plant Lectins/genetics , Plant Lectins/immunology , Plant Lectins/isolation & purification , Plant Proteins/genetics , Plant Proteins/immunology , Rabbits , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purificationABSTRACT
A practical and low cost system for isoelectric protein focusing (IEF) was developed. The system uses a multi-cell glass plate compatible with a common vertical one-dimensional electrophoresis chamber, dispensing specific IEF apparatus. After focusing, the IEF gels are easily recovered. The resulting two-dimensional (2-D) gels have provided efficient protein separation for different concentrations and extracts.
