Comparative Proteomic Analysis of Wild and Cultivated Amaranth Species Seeds by 2-DE and ESI-MS/MS.

Amaranth is a promising staple food that produces seeds with excellent nutritional quality. Although cultivated species intended for grain production have interesting agronomic traits, relatively little is known about wild species, which can prosper in diverse environments and could be a rich genetic source for crop improvement. This work focuses on the proteomic comparison between the seeds of wild and cultivated amaranth species using polarity-based protein extraction and two-dimensional gel electrophoresis. Differentially accumulated proteins (DAPs) showed changes in granule-bound starch synthases and a wide range of 11S globulin isoforms. The electrophoretic profile of these proteins suggests that they may contain significant phosphorylation as post-translational modifications (PTMs), which were confirmed via immunodetection. These PTMs may impact the physicochemical functionality of storage proteins, with potential implications for seed agronomic traits and food system applications. Low-abundant DAPs with highly variable accumulation patterns are also discussed; these were involved in diverse molecular processes, such as genic regulation, lipid storage, and stress response.

Mo-CBP3, a 2S albumin from Moringa oleifera, is a complex mixture of isoforms that arise from different post-translational modifications.

Mo-CBP3 is a chitin-binding 2S albumin from Moringa oleifera. This seed storage protein is resistant to thermal denaturation and shows biological activities that might be of practical use, such as antifungal properties against Candida sp., a pathogen that causes candidiasis, and against Fusarium solani, a soil fungus that can cause diseases in plants and humans. Previous work has demonstrated that Mo-CBP3 is a mixture of isoforms encoded by members of a small multigene family. Mature Mo-CBP3 is a small protein (∼14 kDa), constituted by a small chain of approximately 4 kDa and a large chain of 8 kDa, which are held together by disulfide bridges. However, a more comprehensive picture on the spectrum of Mo-CBP3 isoforms which are found in mature seeds, is still lacking. In this work, genomic DNA fragments were obtained from M. oleifera leaves, cloned and completely sequenced, thus revealing new genes encoding Mo-CBP3. Moreover, mass spectrometry analysis showed that the mature protein is a complex mixture of isoforms with a remarkable number of molecular mass variants. Using computational predictions and calculations, most (∼86%) of the experimentally determined masses were assigned to amino acid sequences deduced from DNA fragments. The results suggested that the complex mixture of Mo-CBP3 isoforms originates from proteins encoded by closely related genes, whose products undergo different combinations of distinct post-translational modifications, including cleavage at the N- and C-terminal ends of both subunits, cyclization of N-terminal Gln, as well as Pro hydroxylation, Ser/Thr phosphorylation, and Met oxidation.

Protein analysis reveals differential accumulation of late embryogenesis abundant and storage proteins in seeds of wild and cultivated amaranth species.

BACKGROUND: Amaranth is a plant naturally resistant to various types of stresses that produces seeds of excellent nutritional quality, so amaranth is a promising system for food production. Amaranth wild relatives have survived climate changes and grow under harsh conditions, however no studies about morphological and molecular characteristics of their seeds are known. Therefore, we carried out a detailed morphological and molecular characterization of wild species A. powellii and A. hybridus, and compared them with the cultivated amaranth species A. hypochondriacus (waxy and non-waxy seeds) and A. cruentus. RESULTS: Seed proteins were fractionated according to their polarity properties and were analysed in one-dimensional gel electrophoresis (1-DE) followed by nano-liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS). A total of 34 differentially accumulated protein bands were detected and 105 proteins were successfully identified. Late embryogenesis abundant proteins were detected as species-specific. Oleosins and oil bodies associated proteins were observed preferentially in A. cruentus. Different isoforms of the granule-bound starch synthase I, and several paralogs of 7S and 11S globulins were also identified. The in silico structural analysis from different isoforms of 11S globulins was carried out, including new types of 11S globulin not reported so far. CONCLUSIONS: The results provide novel information about 11S globulins and proteins related in seed protection, which could play important roles in the nutritional value and adaptive tolerance to stress in amaranth species.

Cloning of cDNA sequences encoding cowpea (Vigna unguiculata) vicilins: Computational simulations suggest a binding mode of cowpea vicilins to chitin oligomers.

Vicilins are 7S globulins which constitute the major seed storage proteins in leguminous species. Variant vicilins showing differential binding affinities for chitin have been implicated in the resistance and susceptibility of cowpea to the bruchid Callosobruchus maculatus. These proteins are members of the cupin superfamily, which includes a wide variety of enzymes and non-catalytic seed storage proteins. The cupin fold does not share similarity with any known chitin-biding domain. Therefore, it is poorly understood how these storage proteins bind to chitin. In this work, partial cDNA sequences encoding ß-vignin, the major component of cowpea vicilins, were obtained from developing seeds. Three-dimensional molecular models of ß-vignin showed the characteristic cupin fold and computational simulations revealed that each vicilin trimer contained 3 chitin-binding sites. Interaction models showed that chito-oligosaccharides bound to ß-vignin were stabilized mainly by hydrogen bonds, a common structural feature of typical carbohydrate-binding proteins. Furthermore, many of the residues involved in the chitin-binding sites of ß-vignin are conserved in other 7S globulins. These results support previous experimental evidences on the ability of vicilin-like proteins from cowpea and other leguminous species to bind in vitro to chitin as well as in vivo to chitinous structures of larval C. maculatus midgut.