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Amyloids are protein aggregates with a highly ordered spatial structure giving them unique physicochemical properties. Different amyloids not only participate in the development of numerous incurable diseases but control vital functions in archaea, bacteria and eukarya. Plants are a poorly studied systematic group in the field of amyloid biology. Amyloid properties have not yet been demonstrated for plant proteins under native conditions in vivo. Here we show that seeds of garden pea Pisum sativum L. contain amyloid-like aggregates of storage proteins, the most abundant one, 7S globulin Vicilin, forms bona fide amyloids in vivo and in vitro. Full-length Vicilin contains 2 evolutionary conserved β-barrel domains, Cupin-1.1 and Cupin-1.2, that self-assemble in vitro into amyloid fibrils with similar physicochemical properties. However, Cupin-1.2 fibrils unlike Cupin-1.1 can seed Vicilin fibrillation. In vivo, Vicilin forms amyloids in the cotyledon cells that bind amyloid-specific dyes and possess resistance to detergents and proteases. The Vicilin amyloid accumulation increases during seed maturation and wanes at germination. Amyloids of Vicilin resist digestion by gastrointestinal enzymes, persist in canned peas, and exhibit toxicity for yeast and mammalian cells. Our finding for the first time reveals involvement of amyloid formation in the accumulation of storage proteins in plant seeds.

In Arabidopsis, vacuolar sorting receptor isoform 1 (VSR1) sorts 12S globulins to the protein storage vacuoles during seed development. Vacuolar sorting is mediated by specific protein–protein interactions between VSR1 and the vacuolar sorting determinant located at the C terminus (ctVSD) on the cargo proteins. Here, we determined the crystal structure of the protease-associated domain of VSR1 (VSR1-PA) in complex with the C-terminal pentapeptide (468RVAAA472) of cruciferin 1, an isoform of 12S globulins. The 468RVA470 motif forms a parallel β-sheet with the switch III residues (127TMD129) of VSR1-PA, and the 471AA472 motif docks to a cradle formed by the cargo-binding loop (95RGDCYF100), making a hydrophobic interaction with Tyr99. The C-terminal carboxyl group of the ctVSD is recognized by forming salt bridges with Arg95. The C-terminal sequences of cruciferin 1 and vicilin-like storage protein 22 were sufficient to redirect the secretory red fluorescent protein (spRFP) to the vacuoles in Arabidopsis protoplasts. Adding a proline residue to the C terminus of the ctVSD and R95M substitution of VSR1 disrupted receptor–cargo interactions in vitro and led to increased secretion of spRFP in Arabidopsis protoplasts. How VSR1-PA recognizes ctVSDs of other storage proteins was modeled. The last three residues of ctVSD prefer hydrophobic residues because they form a hydrophobic cluster with Tyr99 of VSR1- PA. Due to charge–charge interactions, conserved acidic residues, Asp129 and Glu132, around the cargo-binding site should prefer basic residues over acidic ones in the ctVSD. The structural insights gained may be useful in targeting recombinant proteins to the protein storage vacuoles in seeds.

Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein-protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.

Rice (Oryza sativa) seed storage proteins (SSPs) are synthesized and deposited in storage organelles in the endosperm during seed maturation as a nitrogen source for germinating seedlings. We have generated glutelin, globulin, and prolamin knockdown lines and have examined their effects on seed quality. A reduction of one or a few SSP(s) was compensated for by increases in other SSPs at both the mRNA and protein levels. Especially, reduction of glutelins or sulfur-rich 10-kD prolamin levels was preferentially compensated by sulfur-poor or other sulfur-rich prolamins, respectively, indicating that sulfur-containing amino acids are involved in regulating SSP composition. Furthermore, a reduction in the levels of 13-kD prolamin resulted in enhancement of the total lysine content by 56% when compared with the wild type. This observation can be mainly accounted for by the increase in lysine-rich proteins. Although reducing the level of glutelins slightly decreased protein storage vacuoles (PSVs), the simultaneous reduction of glutelin and globulin levels altered the inner structure of PSVs, implicating globulin in framing PSV formation. Knock down of 13-kD prolamins not only reduced the size of endoplasmic reticulum-derived protein bodies (PBs) but also altered the rugged peripheral structure. In contrast, PBs became slightly smaller or unchanged by severe suppression of 10- or 16-kD prolamins, respectively, indicating that individual prolamins have distinct functions in the formation of PBs. Extreme increases or decreases in sulfur-poor prolamins resulted in the production of small PBs, suggesting that the ratio of individual prolamins is crucial for proper aggregation and folding of prolamins.

Proteomics approaches have been a useful tool for determining the biological roles and functions of individual proteins and identifying the molecular mechanisms that govern seed germination, vigour and viability in response to ageing. In this work the dry seed proteome of four Arabidopsis thaliana genotypes, that carry introgression fragments at the position of seed longevity quantitative trait loci and as a result display different levels of seed longevity, was investigated. Seeds at two physiological states, after-ripened seeds that had the full germination ability and aged (stored) seeds of which the germination ability was severely reduced, were compared. Aged dry seed proteomes were markedly different from the after-ripened and reflected the seed longevity level of the four genotypes, despite the fact that dry seeds are metabolically quiescent. Results confirmed the role of antioxidant systems, notably vitamin E, and indicated that protection and maintenance of the translation machinery and energy pathways are essential for seed longevity. Moreover, a new role for seed storage proteins (SSPs) was identified in dry seeds during ageing. Cruciferins (CRUs) are the most abundant SSPs in Arabidopsis and seeds of a triple mutant for three CRU isoforms (crua crub cruc) were more sensitive to artificial ageing and their seed proteins were highly oxidized compared with wild-type seeds. These results confirm that oxidation is involved in seed deterioration and that SSPs buffer the seed from oxidative stress, thus protecting important proteins required for seed germination and seedling formation.

Summary Increasing grain yield and improving grain quality are two important goals for rice breeding. A better understanding of the factors that contribute to the overall grain quantity and nutritional quality of rice will lay the foundation for developing new breeding strategies. RAG2 is a member of 14‐to‐16‐kDa α‐amylase/trypsin inhibitors in rice, which belong to the albumin of seed storage proteins. We found that RAG2 was specifically expressed in ripening seed and its transcription peak was between 14 and 21 days after flowering. Grain size and 1000‐grain weight were obviously increased in RAG2‐overexpressed lines compared with wild type, and grain size was reduced in RAG2‐suppressed lines. In addition, the major storage substances of the seeds differed significantly in RAG2‐overexpressed and RAG2‐suppressed lines compared to wild type. The protein content and amount of total lipids were increased and decreased, respectively, in the seeds of RAG2‐overexpressed and RAG2‐suppressed lines. Overexpression of RAG2 significantly increased grain size and improved grain quality and yield simultaneously. These results imply that RAG2 might play an important role in regulating grain weight and seed quality of rice. The functional characterization of rice RAG2 facilitates a further understanding of the mechanisms involved in grain size and seed quality and may be helpful in improving grain yield and quantity in cereal crops.

Dense vesicles (DVs) are vesicular carriers, unique to plants, that mediate post-Golgi trafficking of storage proteins to protein storage vacuoles (PSVs) in seeds. However, the molecular mechanisms regulating the directional targeting of DVs to PSVs remain elusive. Here, we show that the rice (Oryza sativa) glutelin precursor accumulation5 (gpa5) mutant is defective in directional targeting of DVs to PSVs, resulting in discharge of its cargo proteins into the extracellular space. Molecular cloning revealed that GPA5 encodes a plant-unique phox-homology domain-containing protein homologous to Arabidopsis (Arabidopsis thaliana) ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN. We show that GPA5 is a membrane-associated protein capable of forming homodimers and that it is specifically localized to DVs in developing endosperm. Colocalization, biochemical, and genetic evidence demonstrates that GPA5 acts in concert with Rab5a and VPS9a to regulate DV-mediated post-Golgi trafficking to PSVs. Furthermore, we demonstrated that GPA5 physically interacts with a class C core vacuole/endosome tethering complex and a seed plant–specific VAMP727-containing R-soluble N-ethylmaleimide sensitive factor attachment protein receptor complex. Collectively, our results suggest that GPA5 functions as a plant-specific effector of Rab5a required for mediating tethering and membrane fusion of DVs with PSVs in rice endosperm.

Protein rich meal is a valuable co-product of canola/rapeseed oil extraction. Seed storage proteins that include cruciferin (11S) and napin (2S) dominate the protein complement of canola while oleosins, lipid transfer proteins and other minor proteins of non-storage nature are also found. Although oil-free canola meal contains 36–40% protein on a dry weight basis, non-protein components including fibre, polymeric phenolics, phytates and sinapine, etc. of the seed coat and cellular components make protein less suitable for food use. Separation of canola protein from non-protein components is a technical challenge but necessary to obtain full nutritional and functional potential of protein. Process conditions of raw material and protein preparation are critical of nutritional and functional value of the final protein product. The storage proteins of canola can satisfy many nutritional and functional requirements for food applications. Protein macromolecules of canola also provide functionalities required in applications beyond edible uses; there exists substantial potential as a source of plant protein and a renewable biopolymer. Available information at present is mostly based on the protein products that can be obtained as mixtures of storage protein types and other chemical constituents of the seed; therefore, full potential of canola storage proteins is yet to be revealed.

Key messageNanobody-heavy chain (VHH-Fc) antibody formats have the potential to immunomodulate even highly accumulating proteins and provide a valuable tool to experimentally modulate the subcellular distribution of seed storage proteins.Recombinant antibodies often obtain high accumulation levels in plants, and thus, besides being the actual end-product, antibodies targeting endogenous host proteins can be used to interfere with the localization and functioning of their corresponding antigens. Here, we compared the effect of a seed-expressed nanobody-heavy chain (VHH-Fc) antibody against the highly abundant Arabidopsis thaliana globulin seed storage protein cruciferin with that of a VHH-Fc antibody without endogenous target. Both antibodies reached high accumulation levels of around 10% of total soluble protein, but strikingly, another significant part was present in the insoluble protein fraction and was recovered only after extraction under denaturing conditions. In seeds containing the anti-cruciferin antibodies but not the antibody without endogenous target, the amount of soluble, processed globulin subunits was severely reduced and a major part of the cruciferin molecules was found as precursor in the insoluble fraction. Moreover, in these seeds, aberrant vacuolar phenotypes were observed that were different from the effects caused by the depletion of globulins in knock-out seeds. Remarkably, the seeds with strongly reduced globulin amounts are fully viable and germinate with frequencies similar to wild type, illustrating how flexible seeds can retrieve amino acids from the stored proteins to start germination.

β-Conglycinin has been identified as one of the major feed allergens. However, studies of β-conglycinin on fish are scarce. This study investigated the effects of β-conglycinin on the growth, digestive and absorptive ability, inflammatory response, oxidative status and gene expression of juvenile Jian carp (Cyprinus carpio var. Jian) in vivo and their enterocytes in vitro. The results indicated that the specific growth rate (SGR), feed intake, and feed efficiency were reduced by β-conglycinin. In addition, activities of trypsin, chymotrypsin, lipase, creatine kinase, Na(+),K(+)-ATPase and alkaline phosphatase in the intestine showed similar tendencies. The protein content of the hepatopancreas and intestines, and the weight and length of the intestines were all reduced by β-conglycinin. β-Conglycinin increased lipid and protein oxidation in the detected tissues and cells. However, β-conglycinin decreased superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) activities and glutathione (GSH) content in the intestine and enterocytes. Similar antioxidant activity in the hepatopancreas was observed, except for GST. The expression of target of rapamycin (TOR) gene was reduced by β-conglycinin. Furthermore, mRNA levels of interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β) genes were increased by β-conglycinin. However, β-conglycinin increased CuZnSOD, MnSOD, CAT, and GPx1b gene expression. In conclusion, this study indicates that β-conglycinin induces inflammation and oxidation, and causes dysfunction of intestinal digestion and absorption in fish, and finally reduces fish growth. The results of this study provide some information to the mechanism of β-conglycinin-induced negative effects.