Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel alpha-amylase inhibitory properties

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1). Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein`s biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1), showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of alpha-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian alpha-amylase activity in vitro.

Storage proteins and cell wall mobilisation in seeds of Sesbania virgata (Cav.) Pers. (Leguminosae)

The endosperm of seeds of Sesbania virgata (Cav.) Pers. accumulates galactomannan as a cell wall storage polysaccharide. It is hydrolysed by three enzymes, one of them being alpha-galactosidase. A great amount of protein bodies is found in the cytoplasm of endospermic cells, which are thought to play the major role as a nitrogen reserve in this seed. The present work aimed at understanding how the production of enzymes that degrade storage compounds is controlled. We performed experiments with addition of inhibitors of transcription (actinomycin-d and alpha-amanitin) and translation (cycloheximide) during and after germination. In order to follow the performance of storage mobilisation, we measured fresh mass, protein contents and alpha-galactosidase activity. All the inhibitors tested had little effect on seed germination and seedling development. Actinomycin-d and cycloheximide provoked a slight inhibition of the storage protein degradation and concomitantly lead to an elevation of the alpha-galactosidase activity. Although alpha-amanitin showed some effect on seedling development at latter stages, it presented the former effect and did not change galactomannan degradation performance. Our data suggest that some of the proteases may be synthesised de novo, whereas alpha-galactosidase seems to be present in the endosperm cells probably as an inactive polypeptide in the protein bodies, being probably activated by proteolysis when the latter organelle is disassembled. These evidences suggest the existence of a connection between storage proteins and carbohydrates mobilisation in seeds of S. virgata, which would play a role by assuring a balanced afflux of the carbon and nitrogen to the seedling development.

GRASSIUS: A Platform for Comparative Regulatory Genomics across the Grasses

Transcription factors (TFs) are major players in gene regulatory networks and interactions between TFs and their target genes furnish spatiotemporal patterns of gene expression. Establishing the architecture of regulatory networks requires gathering information on TFs, their targets in the genome, and the corresponding binding sites. We have developed GRASSIUS (Grass Regulatory Information Services) as a knowledge-based Web resource that integrates information on TFs and gene promoters across the grasses. In its initial implementation, GRASSIUS consists of two separate, yet linked, databases. GrassTFDB holds information on TFs from maize (Zea mays), sorghum (Sorghum bicolor), sugarcane (Saccharum spp.), and rice (Oryza sativa). TFs are classified into families and phylogenetic relationships begin to uncover orthologous relationships among the participating species. This database also provides a centralized clearinghouse for TF synonyms in the grasses. GrassTFDB is linked to the grass TFome collection, which provides clones in recombination-based vectors corresponding to full-length open reading frames for a growing number of grass TFs. GrassPROMDB contains promoter and cis-regulatory element information for those grass species and genes for which enough data are available. The integration of GrassTFDB and GrassPROMDB will be accomplished through GrassRegNet as a first step in representing the architecture of grass regulatory networks. GRASSIUS can be accessed from www.grassius.org.