Relationships between puroindoline A-prolamin interactions and wheat grain hardness.

Grain hardness is an important quality trait of cereal crops. In wheat, it is mainly determined by the Hardness locus that harbors genes encoding puroindoline A (PINA) and puroindoline B (PINB). Any deletion or mutation of these genes leading to the absence of PINA or to single amino acid changes in PINB leads to hard endosperms. Although it is generally acknowledged that hardness is controlled by adhesion strength between the protein matrix and starch granules, the physicochemical mechanisms connecting puroindolines and the starch-protein interactions are unknown as of this time. To explore these mechanisms, we focused on PINA. The overexpression in a hard wheat cultivar (cv. Courtot with the Pina-D1a and Pinb-D1d alleles) decreased grain hardness in a dose-related effect, suggesting an interactive process. When PINA was added to gliadins in solution, large aggregates of up to 13 µm in diameter were formed. Turbidimetry measurements showed that the PINA-gliadin interaction displayed a high cooperativity that increased with a decrease in pH from neutral to acid (pH 4) media, mimicking the pH change during endosperm development. No turbidity was observed in the presence of isolated α- and γ-gliadins, but non-cooperative interactions of PINA with these proteins could be confirmed by surface plasmon resonance. A significant higher interaction of PINA with γ-gliadins than with α-gliadins was observed. Similar binding behavior was observed with a recombinant repeated polypeptide that mimics the repeat domain of gliadins, i.e., (Pro-Gln-Gln-Pro-Tyr)8. Taken together, these results suggest that the interaction of PINA with a monomeric gliadin creates a nucleation point leading to the aggregation of other gliadins, a phenomenon that could prevent further interaction of the storage prolamins with starch granules. Consequently, the role of puroindoline-prolamin interactions on grain hardness should be addressed on the basis of previous observations that highlight the similar subcellular routing of storage prolamins and puroindolines.

The Spatiotemporal Deposition of Lysophosphatidylcholine Within Starch Granules of Maize Endosperm and its Relationships to the Expression of Genes Involved in Endoplasmic Reticulum-Amyloplast Lipid Trafficking and Galactolipid Synthesis.

The presence of lipids within starch granules is specific to cereal endosperm starches. These starch lipids are composed of lysophospholipids, especially lysophosphatidylcholine (LysoPC) and free fatty acids that strongly impact the assembly and properties of cereal starches. However, the molecular mechanisms associated with this specific lipid routing have never been investigated. In this study, matrix-assisted laser desorption ionization mass spectrometry imaging revealed decreasing gradients in starch LysoPC concentrations from the periphery to the center of developing maize endosperms. This spatiotemporal deposition of starch LysoPC was similar to that previously observed for endoplasmic reticulum (ER)-synthesized storage proteins, i.e. zeins, suggesting that LysoPC might originate in the ER, as already reported for chloroplasts. Furthermore, a decrease of the palmitate concentration of amyloplast galactolipids was observed during endosperm development, correlated with the preferential trapping of palmitoyl-LysoPC by starch carbohydrates, suggesting a link between LysoPC and galactolipid synthesis. Using microarray, the homologous genes of the Arabidopsis ER-chloroplast lipid trafficking and galactolipid synthesis pathways were also expressed in maize endosperm. These strong similarities suggest that the encoded enzymes and transporters are adapted to managing the differences between chloroplast and amyloplast lipid homeostasis. Altogether, our results led us to propose a model where ER-amyloplast lipid trafficking directs the LysoPC towards one of two routes, the first towards the stroma and starch granules and the other towards galactolipid synthesis.

Responses to Hypoxia and Endoplasmic Reticulum Stress Discriminate the Development of Vitreous and Floury Endosperms of Conventional Maize (Zea mays) Inbred Lines.

Major nutritional and agronomical issues relating to maize (Zea mays) grains depend on the vitreousness/hardness of its endosperm. To identify the corresponding molecular and cellular mechanisms, most studies have been conducted on opaque/floury mutants, and recently on Quality Protein Maize, a reversion of an opaque2 mutation by modifier genes. These mutant lines are far from conventional maize crops. Therefore, a dent and a flint inbred line were chosen for analysis of the transcriptome, amino acid, and sugar metabolites of developing central and peripheral endosperm that is, the forthcoming floury and vitreous regions of mature seeds, respectively. The results suggested that the formation of endosperm vitreousness is clearly associated with significant differences in the responses of the endosperm to hypoxia and endoplasmic reticulum stress. This occurs through a coordinated regulation of energy metabolism and storage protein (i.e., zein) biosynthesis during the grain-filling period. Indeed, genes involved in the glycolysis and tricarboxylic acid cycle are up-regulated in the periphery, while genes involved in alanine, sorbitol, and fermentative metabolisms are up-regulated in the endosperm center. This spatial metabolic regulation allows the production of ATP needed for the significant zein synthesis that occurs at the endosperm periphery; this finding agrees with the zein-decreasing gradient previously observed from the sub-aleurone layer to the endosperm center. The massive synthesis of proteins transiting through endoplasmic reticulum elicits the unfolded protein responses, as indicated by the splicing of bZip60 transcription factor. This splicing is relatively higher at the center of the endosperm than at its periphery. The biological responses associated with this developmental stress, which control the starch/protein balance, leading ultimately to the formation of the vitreous and floury regions of mature endosperm, are discussed.

Lipid partitioning in maize (Zea mays L.) endosperm highlights relationships among starch lipids, amylose, and vitreousness.

Content and composition of maize endosperm lipids and their partition in the floury and vitreous regions were determined for a set of inbred lines. Neutral lipids, i.e., triglycerides and free fatty acids, accounted for more than 80% of endosperm lipids and are almost 2 times higher in the floury than in the vitreous regions. The composition of endosperm lipids, including their fatty acid unsaturation levels, as well as their distribution may be related to metabolic specificities of the floury and vitreous regions in carbon and nitrogen storage and to the management of stress responses during endosperm cell development. Remarkably, the highest contents of starch lipids were observed systematically within the vitreous endosperm. These high amounts of starch lipids were mainly due to lysophosphatidylcholine and were tightly linked to the highest amylose content. Consequently, the formation of amylose-lysophosphatidylcholine complexes has to be considered as an outstanding mechanism affecting endosperm vitreousness.

Tomato GDSL1 is required for cutin deposition in the fruit cuticle.

The plant cuticle consists of cutin, a polyester of glycerol, hydroxyl, and epoxy fatty acids, covered and filled by waxes. While the biosynthesis of cutin building blocks is well documented, the mechanisms underlining their extracellular deposition remain unknown. Among the proteins extracted from dewaxed tomato (Solanum lycopersicum) peels, we identified GDSL1, a member of the GDSL esterase/acylhydrolase family of plant proteins. GDSL1 is strongly expressed in the epidermis of growing fruit. In GDSL1-silenced tomato lines, we observed a significant reduction in fruit cuticle thickness and a decrease in cutin monomer content proportional to the level of GDSL1 silencing. A significant decrease of wax load was observed only for cuticles of the severely silenced transgenic line. Fourier transform infrared (FTIR) analysis of isolated cutins revealed a reduction in cutin density in silenced lines. Indeed, FTIR-attenuated total reflectance spectroscopy and atomic force microscopy imaging showed that drastic GDSL1 silencing leads to a reduction in ester bond cross-links and to the appearance of nanopores in tomato cutins. Furthermore, immunolabeling experiments attested that GDSL1 is essentially entrapped in the cuticle proper and cuticle layer. These results suggest that GDSL1 is specifically involved in the extracellular deposition of the cutin polyester in the tomato fruit cuticle.

Integrating genes and phenotype: a wheat-Arabidopsis-rice glycosyltransferase database for candidate gene analyses.

Glycosyltransferases (GTs) constitute a very large multi-gene superfamily, containing several thousand members identified in sequenced organisms especially in plants. GTs are key enzymes involved in various biological processes such as cell wall formation, storage polysaccharides biosynthesis, and glycosylation of various metabolites. GTs have been identified in rice (Oryza sativa) and Arabidopsis thaliana, but their precise function has been demonstrated biochemically for only a few. In this work we have established a repertoire of virtually all the wheat (Triticum aestivum) GT sequences, using the large publicly available banks of expressed sequences. Based on sequence similarity with Arabidopsis and rice GTs compiled in the carbohydrate active enzyme database (CAZY), we have identified and classified these wheat sequences. The results were used to feed a searchable database available on the web ( http://wwwappli.nantes.inra.fr:8180/GTIDB ) that can be used for initiating an exhaustive candidate gene survey in wheat applied to a particular biological process. This is illustrated through the identification of GT families which are expressed during cell wall formation in wheat grain maturation.

Ha-DEF1, a sunflower defensin, induces cell death in Orobanche parasitic plants.

Plant defensins are small basic peptides of 5-10 kDa and most of them exhibit antifungal activity. In a sunflower resistant to broomrape, among the three defensin encoding cDNA identified, SF18, SD2 and HaDef1, only HaDef1 presented a preferential root expression pattern and was induced upon infection by the root parasitic plant Orobanche cumana. The amino acid sequence deduced from HaDef1 coding sequence was composed of an endoplasmic reticulum signal sequence of 28 amino acids, a standard defensin domain of 50 amino-acid residues and an unusual C-terminal domain of 30 amino acids with a net positive charge. A 5.8 kDa recombinant mature Ha-DEF1 corresponding to the defensin domain was produced in Escherichia coli and was purified by means of a two-step chromatography procedure, Immobilized Metal Affinity Chromatography (IMAC) and Ion Exchange Chromatography. Investigation of in vitro antifungal activity of Ha-DEF1 showed a strong inhibition on Saccharomyces cerevisiae growth linked to a membrane permeabilization, and a morphogenetic activity on Alternaria brassicicola germ tube development, as already reported for some other plant defensins. Bioassays also revealed that Ha-DEF1 rapidly induced browning symptoms at the radicle apex of Orobanche seedlings but not of another parasitic plant, Striga hermonthica, nor of Arabidopsis thaliana. FDA vital staining showed that these browning areas corresponded to dead cells. These results demonstrate for the first time a lethal effect of defensins on plant cells. The potent mode of action of defensin in Orobanche cell death and the possible involvement in sunflower resistance are discussed.

Plant lipid binding proteins: properties and applications.

Lipid transfer proteins (LTP) and puroindolines are abundant lipid binding proteins of plant seeds. While LTP are ubiquitous plant proteins, puroindolines are only found in the seeds of plants from the Triticae and Avenae tribes. These proteins display a similar overall folding pattern but different lipid binding properties. The unique and diverse biological and technological functions of LTPs and puroindolines are closely related to their structural and lipid binding properties. These proteins are attractive to improve the agronomic performances and food quality of crops. Heterologous expression and genetic engineering should allow industrial production and enlarge applications of these lipid binding proteins.

Puroindoline-a and alpha1-purothionin form ion channels in giant liposomes but exert different toxic actions on murine cells.

Puroindoline-a (PIN-a) and alpha1-purothionin (alpha1-PTH), isolated from wheat endosperm of Triticum aestivum sp., have been suggested to play a role in plant defence mechanisms against phytopathogenic organisms. We investigated their ability to form pores when incorporated into giant liposomes using the patch-clamp technique. PIN-a formed cationic channels (approximately 15 pS) with the following selectivity K(+) > Na(+) >> Cl(-). Also, alpha1-PTH formed channels of approximately 46 pS and 125 pS at +100 mV, the selectivity of which was Ca(2+) > Na(+) approximately K(+) >> Cl(-) and Cl(-) >> Na(+), respectively. In isolated mouse neuromuscular preparations, alpha1-PTH induced muscle membrane depolarization, leading to blockade of synaptic transmission and directly elicited muscle twitches. Also, alpha1-PTH caused swelling of differentiated neuroblastoma NG108-15 cells, membrane bleb formation, and disorganization of F-actin. In contrast, similar concentrations of PIN-a had no detectable effects. The cytotoxic actions of alpha1-PTH on mammalian cells may be explained by its ability to induce cationic-selective channels.

Heterologous expression and purification of active divercin V41, a class IIa bacteriocin encoded by a synthetic gene in Escherichia coli.

Divercin V41, a class IIa bacteriocin with strong antilisterial activity, is produced by Carnobacterium divergens V41. To express a recombinant version of divercin V41, we constructed a synthetic gene that encodes the mature divercin V41 peptide and then overexpressed the gene in pET-32b by using the T7 RNA polymerase promoter in the Escherichia coli Origami (DE3)(pLysS) strain. The DvnRV41 peptide was expressed as a translational fusion protein with thioredoxin and accumulated in the cell cytoplasm in a soluble anti-Listeria active form. The fusion protein was then purified and cleaved to obtain pure, soluble, folded DvnRV41 (462 microg per 20 ml of culture). This paper describes the first design of a synthetic bacteriocin gene and the first bacteriocin expressed in the E. coli cytoplasm.

A bacterial expression system revisited for the recombinant production of cystine-rich plant lipid transfer proteins.

Non-specific lipid transfer proteins (nsLTPs) are abundant and ubiquitous cystine-rich proteins that are capable, in vitro, of binding lipids and hydrophobic molecules. In view to probe the lipid binding properties of the wheat nsLTP1, mutant variants may represent a powerful tool. To this end, a synthetic gene, encoding a mature wheat nsLTP1 polypeptide, was designed to ensure high level expression in Escherichia coli. The bacterial expression host strain, a translational fusion strategy, and convenient cleavage and purification procedures were optimized to produce in standard fermentation conditions, a significant amount (15 mg/L final yield), of a soluble and correctly folded recombinant nsLTP1. This highly amenable expression system is helpful in order to investigate structure-activity relationships of plant nsLTP.

Neuronal and muscular alterations caused by two wheat endosperm proteins, puroindoline-a and alpha1-purothionin, are due to ion pore formation.

Using the patch-clamp technique it was found that the toxicity of the two wheat endosperm proteins puroindoline-a and alpha1-purothionin probably results from the dissipation of ion concentration gradients essential for the maintenance of cellular homeostasis.

High microbial production and characterization of strictly periodic polymers modelled on the repetitive domain of wheat gliadins.

Primary structures of wheat prolamins contain repetitive domains involved in the mechanical properties of gluten. In order to experience the ability of recombinant strictly periodic polypeptides, modelled on a consensus sequence of wheat gliadins (PQQPY)(8) and (PQQPY)(17) (SPR8 and SPR17 polypeptides, respectively), to be formulated in film solutions, their heterologous expression conditions, in batch culture and low cell densities, were optimized to match the high requirements of this process. A convenient and general purification procedure was also devised. Moreover, FTIR-ATR characterizations indicated that these periodic polypeptides prepared as hydrated doughy state and dried have the tendency to form a protein network through intermolecular beta-sheets, strongly maintained by hydrogen bonds. Accordingly, these recombinant polypeptides are assumed to be a suitable candidate for potential application.