Molecular characterization of a new arcelin-5 gene.

Arcelins are insecticidal proteins found in some wild accessions of the common bean, Phaseolus vulgaris. They are grouped in six allelic variants and arcelin-5 is the variant with the highest inhibitory effect on the development of Zabrotes subfasciatus larvae. Characterization of the protein and its genes resulted in the identification of three polypeptides and the isolation of two genes that encode the Arc5a and Arc5b polypeptides. Here we describe a new gene, Arc5-III. The protein it encodes has 81% amino acid identity with the derived amino acid sequences of Arc5-I and Arc5-II. The Arc5-III gene is highly expressed in developing seeds and at a much lower level in roots. Data obtained by a combination of two-dimensional gel electrophoresis, protein sequencing and MALDI-TOF mass spectrometry analysis support the conclusion that Arc5-III encodes a polypeptide present in Arc5c band. Using ion-exchange chromatography, three fractions containing arcelin-5 polypeptides were eluted by increasing the salt concentration. The three fractions contain various amounts of the three arc-5 polypeptides and inhibit the growth of Zabrotes subfasciatus larvae differentially, suggesting differences in insecticidal activity among the arcelin-5 isoforms.

Analysis of structural and physico-chemical parameters involved in the specificity of binding between alpha-amylases and their inhibitors.

Enzyme-inhibitor specificity was studied for alpha-amylases and their inhibitors. We purified and cloned the cDNAs of two different alpha-amylase inhibitors from the common bean (Phaseolus vulgaris) and have recently cloned the cDNA of an alpha-amylase of the Mexican bean weevil (Zabrotes subfasciatus), which is inhibited by alpha-amylase inhibitor 2 but not by alpha-amylase inhibitor 1. The crystal structure of AI-1 complexed with pancreatic porcine alpha-amylase allowed us to model the structure of AI-2. The structure of Zabrotes subfasciatus alpha-amylase was modeled based on the crystal structure of Tenebrio molitor alpha-amylase. Pairwise AI-1 and AI-2 with PPA and ZSA complexes were modeled. For these complexes we first identified the interface forming residues. In addition, we identified the hydrogen bonds, ionic interactions and loss of hydrophobic surface area resulting from complex formation. The parameters we studied provide insight into the general scheme of binding, but fall short of explaining the specificity of the inhibition. We also introduce three new tools-software packages STING, HORNET and STINGPaint-which efficiently determine the interface forming residues and the ionic interaction data, the hydrogen bond net as well as aid in interpretation of multiple sequence alignment, respectively.

Alpha-amylases of the coffee berry borer (Hypothenemus hampei) and their inhibition by two plant amylase inhibitors.

The adult coffee berry borer (Hypothenemus hampei Ferrari [Coleoptera: Scolytidae]), a major insect pest of coffee, has two major digestive alpha-amylases that can be separated by isoelectric focusing. The alpha-amylase activity has a broad pH optimum between 4.0 and 7.0. Using pH indicators, the pH of the midgut was determined to be between 4.5 and 5.2. At pH 5.0, the coffee berry borer alpha-amylase activity is inhibited substantially (80%) by relatively low levels of the amylase inhibitor (alphaAI-1) from the common bean, Phaseolus vulgaris L., and much less so by the amylase inhibitor from Amaranthus. We used an in-gel zymogram assay to demonstrate that seed extracts can be screened to find suitable inhibitors of amylases. The prospect of using the genes that encode these inhibitors to make coffee resistant to the coffee berry borer via genetic engineering is discussed.

The effect of arcelin-1 on the structure of the midgut of bruchid larvae and immunolocalization of the arcelin protein.

Some wild accessions of the common bean (Phaseolus vulgaris) contain a family of proteins called arcelins, that are toxic to the larvae of certain bruchid species. Among the six allelic variants of arcelin tested so far, arcelin-5 and arcelin-1 confer the highest level of resistance against the Mexican bean weevil, Zabrotes subfasciatus. The same proteins are not toxic to the bean weevil, Acanthoscelides obtectus, which is also a serious pest of cultivated beans. Arcelins belong to the bean lectin family that includes phytohemaggutinins and alpha-amylase inhibitors. Although homologous to lectins, arcelins are themselves only very weak lectins, and their binding properties have not been clearly established. The toxic properties of arcelins may be related to their recognition of and interaction with the glycoproteins and other constituents of the membranes along the digestive tract of insects. Since arcelin-1 was shown to have growth inhibitory effects for the larvae of Z. subfasciatus but not of A. obtectus, we examined the effect of an arcelin-1 containing diet on the structure of the cells that line the intestinal tract of the larvae of these two bruchid species, and used antibodies against arcelin to examine the distribution of arcelin within the cells and tissues. Here we show that dietary arcelin-1 caused an alteration of the gut structure and the penetration of arcelin into the haemolymph in Z. subfasciatus but not in A. obtectus. These results lead us to suggest that arcelins exert their toxic effect by severely damaging the epithelial cells.

Signal transduction networks and the biology of plant cells.

The development of plant transformation in the mid-1980s and of many new tools for cell biology, molecular genetics, and biochemistry has resulted in enormous progress in plant biology in the past decade. With the completion of the genome sequence of Arabidopsis thaliana just around the corner, we can expect even faster progress in the next decade. The interface between cell biology and signal transduction is emerging as a new and important field of research. In the past we thought of cell biology strictly in terms of organelles and their biogenesis and function, and researchers focused on questions such as, how do proteins enter chloroplasts? or, what is the structure of the macromolecules of the cell wall and how are these molecules secreted? Signal transduction dealt primarily with the perception of light (photomorphogenesis) or hormones and with the effect such signals have on enhancing the activity of specific genes. Now we see that the fields of cell biology and signal transduction are merging because signals pass between organelles and a single signal transduction pathway usually involves multiple organelles or cellular structures. Here are some examples to illustrate this new paradigm. How does abscisic acid (ABA) regulate stomatal closure? This pathway involves not only ABA receptors whose location is not yet known, but cation and anion channels in the plasma membrane, changes in the cytoskeleton, movement of water through water channels in the tonoplast and the plasma membrane, proteins with a farnesyl tail that can be located either in the cytosol or attached to a membrane, and probably unidentified ion channels in the tonoplast. In addition there are highly localized calcium oscillations in the cytoplasm resulting from the release of calcium stored in various compartments. The activities of all these cellular structures need to be coordinated during ABA-induced stomatal closure. For another example of the interplay between the proteins of signal transduction pathways and cytoplasmic structures, consider how plants mount defense responses against pathogens. Elicitors produced by pathogens bind to receptors on the plant plasma membrane or in the cytosol and eventually activate a large number of genes. This results in the coordination of activities at the plasma membrane (production of reactive oxygen species), in the cytoskeleton, localized calcium oscillations, and the modulation of protein kinases and protein phosphatases whose locations remain to be determined. The movement of transcription factors into the nucleus to activate the defense genes requires their release from cytosolic anchors and passage through the nuclear pore complexes of the nuclear envelope. This review does not cover all the recent progress in plant signal transduction and cell biology; it is confined to the topics that were discussed at a recent (November 1998) workshop held in Santiago at which lecturers from Chile, the USA and the UK presented recent results from their laboratories.

Molecular characterization of a bean alpha-amylase inhibitor that inhibits the alpha-amylase of the mexican bean weevil Zabrotes subfasciatus.

Cultivated varieties of the common bean (Phaseolus vulgaris L.) contain an alpha-amylase inhibitor (alpha AI-1) that inhibits porcine pancreatic alpha-amylase (PPA; EC 3.2.1.1) and the amylases of certain seed weevils, but not that of the Mexican bean weevil, Zabrotes subfasciatus. A variant of alpha AI-1, called alpha AI-2, is found in certain arcelin-containing wild accessions of the common bean. The variant alpha AI-2 inhibits Z. subfasciatus alpha-amylase (ZSA), but not PPA. We purified alpha AI-2 and studied its interaction with ZSA. The formation of the alpha AI-2-ZSA complex is time-dependent and occurs maximally at pH 5.0 or below. When a previously isolated cDNA assumed to encode alpha AI-2 was expressed in transgenic tobacco seeds, the seeds contained inhibitory activity toward ZSA but not toward PPA, confirming that the cDNA encodes alpha AI-2. The inhibitors alpha AI-1 and alpha AI-2 share 78% sequence identity at the amino acid level and they differ in an important region that is part of the site where the enzyme binds the inhibitor. The swap of a tripeptide in this region was not sufficient to change the specificity of the two inhibitors towards their respective enzymes. The three-dimensional structure of the alpha AI-1/PPA complex has just been solved and we recently obtained the derived amino acid sequence of ZSA. This additional information allows us to discuss the results described here in the framework of the amino acid residues of both proteins involved in the formation of the enzyme-inhibitor complex and to pinpoint the amino acids responsible for the specificity of the interaction.

Protective mechanism of the Mexican bean weevil against high levels of alpha-amylase inhibitor in the common bean.

Alpha-amylase inhibitor (alpha AI) protects seeds of the common bean (Phaseolus vulgaris) against predation by certain species of bruchids such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (Callosobruchus chinensis), but not against predation by the bean weevil (Acanthoscelides obtectus) or the Mexican bean weevil (Zabrotes subfasciatus), insects that are common in the Americas. We characterized the interaction of alpha AI-1 present in seeds of the common bean, of a different isoform, alpha AI-2, present in seeds of wild common bean accessions, and of two homologs, alpha AI-Pa present in seeds of the tepary bean (Phaseolus acutifolius) and alpha AI-Pc in seeds of the scarlet runner bean (Phaseolus coccineus), with the midgut extracts of several bruchids. The extract of the Z. subfasciatus larvae rapidly digests and inactivates alpha AI-1 and alpha AI-Pc, but not alpha AI-2 or alpha AI-Pa. The digestion is caused by a serine protease. A single proteolytic cleavage in the beta subunit of alpha AI-1 occurs at the active site of the protein. When degradation is prevented, alpha AI-1 and alpha AI-Pc do not inhibit the alpha-amylase of Z. subfasciatus, although they are effective against the alpha-amylase of C. chinensis. Alpha AI-2 and alpha AI-Pa, on the other hand, do inhibit the alpha-amylase of Z. subfasciatus, suggesting that they are good candidates for genetic engineering to achieve resistance to Z. subfasciatus.