Emerging natural hemp seed proteins and their functions for nutraceutical applications

With changing dietary habits and increasing awareness of the nutraceutical role of dietary foods,the demand for natural plant proteins and interest in non-traditional protein sources in the food industry are increasing.Industrial hemp,belonging to the plant family Cannabaceae,is cultivated for its fibre and edible seeds.Due to its nutritional value,it has also been used in the food industry and medicine.In particular,hemp seed proteins have drawn considerable attention in both scientific and industrial fields because of their excellent nutraceutical values,superior digestibility,low allergenicity and diverse techno-functional properties.In this review,we provide a summary of the current research progress on the extraction and purification processes,physiochemical properties,nutraceutical functions,and applications of hemp seed proteins.Perspectives in the application of advanced technologies for hemp seed bioactive peptide mining are also discussed.This review provides up-to-date insights into the nutraceutical values,health benefits,and future applications of this emerging plant source protein.

Analysis of Genetic Variation of Seed Proteins in the Genus Vigna and among Its Relatives Cultivated in China

The genetic variation of seed proteins was assayed by SDSPAGE for 24 cultivars belonging to 5 species in Vigna and 7 species in its 7 relative genera cultivated in China. There were 48 polymorphic subunit bands discriminated from electrophoretic profiles. Two dendrograms were constructed by UPGMA cluster analyses using PHYLIP3.6 respectively. Variation among genera or species was larger than that among lower taxonomic categories level. Little variation among cuhivars of yardlong bean （Vigna sesquipedalis ） and small variation of lablab （ Lablab purpureus）, pea （Pisum sativum）, or sword bean （Canavalia gladiata）, but large variation of soybean or rice bean in their origin of China were all revealed. The seed proteins profiles of traditionally regarded as typical species in Vigna such as yardlong bean, rice bean and small bean were more similar than mungbean （Vigna radiata） and black gram （Vigna mungo） were. Mungbean and black gram had distinct seed proteins pattern, they should be of two species.

Virtual screening and directional preparation of xanthine oxidase inhibitory peptides derived from hemp seed protein

The traditional nutritional and medical hemp(Cannabis sativa L.)seed protein were explored for the discovery and directional preparation of new xanthine oxidase inhibitory(XOI)peptides by structure-based virtual screening,compound synthesis,in vitro bioassay and proteolysis.Six subtypes of hemp seed edestin and albumin were in silico hydrolyzed by 29 proteases,and 192 encrypted bioactive peptides were screened out.Six peptides showed to be XOI peptides,of which four(about 67%)were released by elastase hydrolysis.The peptide DDNPRRFY displayed the highest XOI activity(IC50=(2.10±0.06)mg/mL),acting as a mixed inhibitor.The pancreatic elastase directionally prepared XOI hemp seed protein hydrolysates,from which 6 high-abundance XOI peptides encrypted 3 virtually-screened ones including the DDNPRRFY.The novel outstanding hemp seed protein-derived XOI peptides and their virtual screening and directed preparation methods provide a promising and applicable approach to conveniently and efficiently explore food-derived bioactive peptides.

A Dominant Locus, qBSC-1, Controls β Subunit Content of Seed Storage Protein in Soybean(Glycine max(L.) Merri.)

Soybean seed storage protein is one of the most important plant vegetable proteins, and β subunit is of great significance to enhance soybean protein quality and processing property. F2 segregated population and residual heterozygous lines（RHL） derived from the cross between Yangyandou（low level of β subunit） and Zhonghuang 13（normal level of β subunit） were used for mapping of β subunit content. Our results showed that β subunit content was controlled by a single dominant locus, qBSC-1（β subunit content）, which was mapped to a region of 11.9 cM on chromosome 20 in F2 population of 85 individuals. This region was narrowed down to 2.5 cM between BARCSOYSSR_20_0997 and BARCSOYSSR_20_0910 in RHL with a larger population size of 246 individuals. There were 48 predicted genes within qBSC-1 region based on the reference genome（Glyma 1.0, Williams 82）, including the two copies of β subunit coding gene CG4. An InDel marker developed from a thymine（TT） insertion in one copy of CG4 promoter region in Yangyandou cosegregrated with BARCSOYSSR_20_0975 within qBSC-1 region, suggesting that this InDel marker maybe useful for marker-assisted selection（MAS）.

Characterization of Agro-diversity by Seed Storage Protein Electrophoresis:Focus on Rice Germplasm from Uttarakhand Himalaya,India

The characteristics of 48 rice varieties from Uttarakhand Himalaya, India were detected by morphological and biochemical markers. The grains of the selected rice varieties varied in their morphological （grain length, grain width and grain weight） and biochemical characters （sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE）. Based on the presence of 70, 65, 60, 57, 37-39, 22-23, 13 and 10 kDa protein bands in the 48 rice varieties, seven types of profiles were identified. An unweighted pair group average method with arithmetic mean （UPGMA） dendrogram based on cluster analysis of genetic similarity of the protein bands showed two distinct groups with 1%-78% similarity coefficients. The presence of characteristic bands in selected varieties is a useful parameter for identification of rice germplasm.

Effect of AAPH oxidation on digestion characteristics of seed watermelon(Citrullus lanatus var)kernels protein isolates

Seed watermelon kernel is a typical complex food with high fat and protein contents.During storage and processing,it is often affected by various factors to undergo interactions between components,which lead to its quality change.In this experiment,seed watermelon kernels were used as research objects,and the effects of 2-Azobis(2-amidinopropane)dihydrochloride(AAPH)on seed watermelon kernel protein isolates(WMP)were investigated.The structure and digestion characteristics of WMP after oxidation were studied.The results showed that with the increase of AAPH concentration(0.05−5 mol/L),WMP showed obvious aggregation,and its solubility decreased from 6.76 mg/mL to 9.59 mg/mL.The free sulfhydryl content of WMP was 18.24 mmol/g decreased to 11.25 mmol/g,α-helix decreased andβ-sheet decreased in secondary structure,and its disulfide bond increased by 43.06 mmol/g from 39.57 mmol/g,enthalpy(H)and denaturation temperature increased(Td)(P<0.05).By mass spectrometry results of simulated gastric digestion products,it was found that oxidation adversely affected the digestion characteristics of WMP.It can be seen that the lipid oxidation product APPH of seed watermelon kernel can significantly affect the structure and function of the protein extracted from the seed kernel.

Evaluation of Total Seed Protein Content in Eleven Arkansas Cowpea (Vigna unguiculata (L.) Walp.) Lines

Cowpea is cultivated on more than 11 million hectares with a worldwide production of 5.4 million tons of dried seeds. Cowpea is an affordable source of protein, which is used as an alternative to soybean for people who are allergic to soybean protein. The aim of this research was to assess the variability of the total seed protein content in cowpea. Eleven Arkansas breeding lines were used in this study. Field experiment design was a randomized complete block design (RCBD) with 2 blocks, and conducted in three different locations within Arkansas State (Fayetteville, Alma, and Hope) in 2015. A standard chemical protocol was performed involving an analysis of the total nitrogen by combustion using an Elementar Rapid N III instrument to estimate the protein content. The average protein content was 25.4%, and ranged from 23.7% to 27.4% with a standard deviation of 1.9%. The significant effects of genotype, environment (location), and genotype by environment were observed for the total seed protein content in cowpea. The broad sense heritability (H2) for cowpea seed protein was estimated to be 57.8% based on the eleven cowpea genotypes studied. The cowpea lines, “Early Scarlet” and 09-204 had the highest seed protein content with 27.4% and 26.9% dried seed weight, respectively. This study provides valuable information on cowpea protein content for breeders to select and utilize those breeding lines with high seed protein content to develop new high protein cowpea cultivars.

New combination in Veronica (Scrophulariaceae s.l.) based on morphological characters and the seed storage protein polymorphism

Veronica erciyasdagi （M. A. Fischer） C. Vural comb. ＆ stat. nov., previously regarded as a variety, is proposed as a new combination, based on the morphological characters and seed storage protein polymorphism presented in this study. In addition, information about the ecology and conservation status of Veronica erciyasdagi was reported. This taxon is endemic to central Anatolia, Turkey and is critically endangered.

Orchestrating seed storage protein and starch accumulation toward overcoming yield–quality trade-off in cereal crops

Achieving high yield and good quality in crops is essential for human food security and health.However,there is usually disharmony between yield and quality.Seed storage protein(SSP)and starch,the predominant components in cereal grains,determine yield and quality,and their coupled synthesis causes a yield–quality trade-off.Therefore,dissection of the underlying regulatory mechanism facilitates simultaneous improvement of yield and quality.Here,we summarize current findings about the synergistic molecular machinery underpinning SSP and starch synthesis in the leading staple cereal crops,including maize,rice and wheat.We further evaluate the functional conservation and differentiation of key regulators and specify feasible research approaches to identify additional regulators and expand insights.We also present major strategies to leverage resultant information for simultaneous improvement of yield and quality by molecular breeding.Finally,future perspectives on major challenges are proposed.

Rice seed storage proteins:Biosynthetic pathways and the effects of environmental factors

Rice(Oryza sativa L.)is the most important food crop for at least half of the world’s population.Due to improved living standards,the cultivation of high-quality rice for different purposes and markets has become a major goal.Rice quality is determined by the presence of many nutritional components,including seed storage proteins(SSPs),which are the second most abundant nutrient components of rice grains after starch.Rice SSP biosynthesis requires the participation of multiple organelles and is influenced by the external environment,making it challenging to understand the molecular details of SSP biosynthesis and improve rice protein quality.In this review,we highlight the current knowledge of rice SSP biosynthesis,including a detailed description of the key molecules involved in rice SSP biosynthetic processes and the major environmental factors affecting SSP biosynthesis.The effects of these factors on SSP accumulation and their contribution to rice quality are also discussed based on recent findings.This recent knowledge suggests not only new research directions for exploring rice SSP biosynthesis but also innovative strategies for breeding high-quality rice varieties.

Regulation of seed storage protein synthesis in monocot and dicot plants:A comparative review

Seeds are a major source of nutrients for humans and animal livestock worldwide.With improved living standards,high nutritional quality has become one of the main targets for breeding.Storage protein content in seeds,which is highly variable depending on plant species,serves as a pivotal criterion of seed nutritional quality.In the last few decades,our understanding of the molecular genetics and regulatory mechanisms of storage protein synthesis has greatly advanced.Here,we systematically and comprehensively summarize breakthroughs on the conservation and divergence of storage protein synthesis in dicot and monocot plants.With regard to storage protein accumulation,we discuss evolutionary origins,developmental processes,characteristics of main storage protein fractions,regulatory networks,and genetic modifications.In addition,we discuss potential breeding strategies to improve storage protein accumulation and provide perspectives on some key unanswered problems that need to be addressed.

Genetic Diversity and Classification of Chinese Elite Foxtail Millet [Setaria italica (L.) P. Beauv.] Revealed by Acid-PAGE Prolamin

Arid and semi-arid regions of China account for more than half of the country. Because of drought resistance and high nutritive value, elite foxtail millet (Setaria Italica (L.) P. Beauv.) is one of the most important cereal crops in China. Evaluation of germplasm and genetic diversity of foxtail millet is still in its infancy, but prolamin could play an important role as a protein marker. To investigate the genetic diversity and population structure of foxtail millet from different ecological zones of China, 90 accessions of foxtail millet were collected from three major ecological areas: North, Northwest, and Northeast China. The prolamin contents were examined by acid polyacrylamide gel electrophoresis (acid-PAGE). Five to twenty-two prolamin bands appeared in tested varieties, of which were polymorphic, so prolamin patterns of foxtail millet varieties can be used in variety identification and evaluation. Structure analysis identified six groups, which matches their pedigree information but not their geographic origins. This indicated a high degree (87.78%) of consistency with a phylogenetic classification based on SSR. The results showed prolamin banding patterns were an effective method for analyzing foxtail millet genetic variability.

Disulphide Linkages Occur in Many Polypeptides of Rice Protein Fractions: a Two-dimensional Gel Electrophoretic Study

Most of the work on rice storage proteins focused on the major fraction - glutelin. In contrast to previous reports of the occurrence of single legumin-like polypeptide pair in glutelin, two-dimensional gel electrophoresis revealed many glutelin bands, with some having and some lacking disulphide-linkages. Five legumin-like polypeptide pairs exhibited wide heterogeneity over a range of molecular weight （Mr） 25 to 60 kDa, each having a large subunit （Mr ranged from 18 to 40.5 kDa） disulphide-bonded to a small subunit （Mr ranged from 16.5 to 18.0 kDa）. A band of 49 kDa was homodimeric with two subunits of 29 kDa each; a polypeptide of 51 kDa which altered position to ones corresponding to 53 kDa and 57 kDa on 2-D gels contained intrapolypeptide linkages. Polypeptides of 65 kDa and 60 kDa occurred as aggregates of 110 kDa. The number of polypeptides in other seed protein fractions albumins, globulins and prolamins, varied from four to ten. One of the albumin bands had intrapolypeptide disulphide linkages （20 kDa） and the globulins contained two such bands （13.5 and 20 kDa）. Thus, the present study provides a description of the polypeptide composition of different rice protein fractions that is finely resolved with respect to the occurrence of disulphide linkages.

Effect of Allelic Variation of β-conglycinin α-subunit Locus cgy-2 on Soybean(Glycine max) Quality Ⅰ. Evaluation of Free Amino Acid Content

The effects of the deficiency of the allergenicα-subunit on soybean amino acid(AA)composition were studied using the cultivar Dongnong 47(DN47)as a genetic background.The near-isogenic line(NIL)NIL-DN47-Δα of DN47,with an introgression of theα-null trait allele from the high protein donor parent RiB,was created by marker assisted background selection and used to investigate the AA content and nutritional quality.The contents of crude protein,the total AAs,the total essential amino acids(EAAs)and sulfur-containing(Met and Cys)AAs increased by 4.11%,4.16%,5.20% and 11.96%,respectively in NIL-DN47-Δα compared with DN47.Analyses of the total EAAs(TEAAs)and the EAA index(EAAI)revealed that both parameters in NIL-DN47-Δα were higher than those in DN47.The null-allele of theα-subunit positively affected the AA scores.The quantitative changes in free AAs(FAAs)in the developing seeds of NIL-DN47-Δα and DN47 were compared as of 15 days after flowering(DAF)until maturity.The results showed that the total FAA content in NIL-DN47-Δα was significantly higher than that in the DN47 throughout the late maturation stage(40-60 DAF)of seeds.The high concentration of the FAAs in cgy-2 mutant seeds was a consequence of the high rates of synthesis and/or accumulation of individual FAAs during seed maturation where 25 DAF was an important turning point in the accumulation of the FAAs.The FAA contents of single soybeanα-null,double(α+α′)and triple(α+α′+group I)-null mutant combination lines were investigated.In all of these combinations,introduction of the cgy-2 gene invariably raised the FAA content of mature seeds above that of the DN47.In summary,the enhanced protein quality in cgy-2 mutants resulted from several factors.(1)There was a general increase in the contents of most AAs and FAAs in NIL-DN47-Δα.(2)The induced synthesis of free Arg contributed effectively to the high FAAs of various storage-protein-deficiency mutants.For example,in the S2(null α,group I),the free Arg content was seven times as much as that of DN47,accounting for more than half of the total FAA content in the seed.(3)The increase of sulfur-containing AAs in theα-null type NIL mainly resulted from elevated Met content.These data suggested that the cgy-2 mutation might improve the protein quality of soybean seeds and that lacked of the allergenicα-subunit resulted in increased the FAA content.

Antimicrobial Activity of a Cys-Rich Peptide Derived from a Centrosema virginianum Vicilin

Many antimicrobial peptides (AMPs) have been identified in plants. These peptides are highly divergent at the primary sequence level and vary in their hierarchical structures. Some common biochemical features include the ability to form disulfide bonds, tandemly repeated amino acid sequences and a net charge at pH 7. Unusual Cysteine containing repeats has been identified in several plant seed storage proteins that may act as AMPs. We identified a Cys repeat within a vicilin (seed storage protein) of a wild legume, Centrosema virginianum. Cleavage of the vicilin protein during germination would generate a vicilin derived Cys peptide (VDCP). We investigated the antimicrobial properties of this VDCP and compared its efficacy as an antimicrobial agent to VDCPs from other species. We developed transgenic tobacco plants that expressed cloned sequences encoding the Cysteine repeat unit from C. virginianum, Theobroma cacao and Gossypium hirsutum. Extracts from fully expanded leaves were tested for antimicrobial activity against a fungal pathogen, Botrytis cinerea. The Cys motif from C. virginianum was also expressed in two E. coli cell lines (reducing or oxidizing cytoplasm) and peptide fusion protein fractions were tested for antimicrobial activity against a battery of fungal strains. The unique Cysteine repeat single unit from C. virginianum exhibited antimicrobial properties greater than or equal to the antimicrobial activity associated with expression of the multiple Cys-repeat VDCPs from G. hirsutum or T. cacao in transgenic tobacco. When expressed in bacteria, a C. virginianum VDCP fusion protein exhibited antifungal activity against 3 of the 4 fungi tested. Although the primary role of seed storage proteins is to provide a pool of amino acids and nitrogen for germinating seeds and developing plantlets, it is likely that seed storage protein proteolytic products also provide beneficial antimicrobial properties during germination and young plantlet development.

Defective Kernel 39 encodes a PPR protein required for seed development in maize

RNA editing is a posttranscriptional process that is important in mitochondria and plastids of higher plants. All RNA editing-specific trans-factors reported so far belong to PLS-class of pentatricopeptide repeat(PPR)proteins. Here, we report the map-based cloning and molecular characterization of a defective kernel mutant dek39 in maize. Loss of Dek39 function leads to delayed embryogenesis and endosperm development, reduced kernel size, and seedling lethality. Dek39 encodes an E subclass PPR protein that targets to both mitochondria and chloroplasts, and is involved in RNA editing in mitochondrial NADH dehydrogenase3(nad3) at nad3-247 and nad3-275. C-to-U editing of nad3-275 is not conserved and even lost in Arabidopsis, consistent with the idea that no close DEK39 homologs are present in Arabidopsis. However, the amino acids generated by editing nad3-247 and nad3-275 are highly conserved in many other plant species, and the reductions of editing at these two sites decrease the activity of mitochondria NADH dehydrogenase complex I,indicating that the alteration of amino acid sequence is necessary for Nad3 function. Our results indicate that Dek39 encodes an E sub-class PPR protein that is involved in RNA editing of multiple sites and is necessary for seed development of maize.