Rice Storage Proteins:Focus on Composition,Distribution,Genetic Improvement and Effects on Rice Quality

Rice storage proteins(RSPs)are plant proteins with high nutritional quality.As the second largest type of storage substance in rice,it is the main source of protein intake for people who consume rice as a staple food.The content and type of RSPs affect the appearance,processing quality and eating quality of rice.These effects involve the distribution of RSPs in rice grains as well as the interactions of RSPs with other components such as starch in rice grains.In the past two decades,some progress has been made in the genetic improvement of RSPs.However,the determination mechanism of protein content and composition in rice is still unclear,and the mechanism of the effect of RSPs on rice quality has not been elucidated.In this review,the composition,biosynthesis and distribution of RSPs,and quantitative trait loci mapping and cloning of RSP genes are summarized,the research progress of the influence of RSPs and their components on rice quality are reviewed,and the research directions in the future are proposed.

Effects of water deficit and high N fertilization on wheat storage protein synthesis,gluten secondary structure,and breadmaking quality

The content and composition of wheat storage proteins are the major determinants of dough rheological properties and breadmaking quality and are influenced by cultivation conditions.This study aimed to investigate the effects of water deficit and high N-fertilizer application on wheat storage protein synthesis,gluten secondary structure,and breadmaking quality.Reverse-phase ultrahigh-performance liquid chromatography analysis showed that storage protein and gluten macropolymer accumulation was promoted under both independent applications and a combination of water-deficit and high N-fertilizer treatments.Fourier-transform infrared spectroscopy showed that water deficit and high N-fertilizer treatments generally improved protein secondary structure formation and lipid accumulation,and reduced flour moisture.In particular,high N-fertilizer application increasedβ-sheet content by 10.4%and the combination of water-deficit and high N-fertilizer treatments increased random coil content by 7.6%.These changes in gluten content and secondary structure led to improved dough rheological properties and breadmaking quality,including superior loaf internal structure,volume,and score.Our results demonstrate that moderately high N-fertilizer application under drought conditions can improve gluten accumulation,gluten secondary structure formation,and baking quality.

Genetic Diversity of Seed Storage Proteins in Different Ecotype Varieties of japonica Rice and Its Application

One hundred and fifteen varieties （including cultivars and lines） with different ecotypes in japonica rice （Oryza sativa L.） were analyzed for endosperm storage proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） to estimate their genetic diversity for the purpose of genetic improving and variety identification. Nineteen types of profile were identified according to 1） presence/absence of 65 kDa bands, 2） staining intensity of 70, 60, 57, 37-39, 22-23, 13 and 10 kDa bands, 3） migration velocity of 35 kDa （α-4） and 4） band number at 57 kDa location. An unweighted-pair group average method with arithmetic mean （UPGMA） dendregram based on the cluster analysis of genetic similarity of the protein bands showed a small genetic variation among the tested materials, with the similarity coefficients varying between 0.75 and 1.00. Three distinct groups were identified from the cluster analysis of the rice varieties studied at the similarity coefficient level of 0.894. The first group included eight varieties with high amylose content, the second group contained fifteen varieties with high protein content, and the third group had the remaining ninety-two varieties, which accounted for 80% of the total materials. Clear relationship between ecotypes distinguished by maturity and groups revealed by cluster analysis was not found in this study. Only the group of high amylose linked with medium-maturity medium japonica ecotype. The bands of 70 kDa and 65 kDa can be used as protein markers to identify F1 seed purity of japonica hybrid rice Liuyanyou 422.

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.

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）.

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.

Isolation and Characterization of Storage Protein Subunit-null-dwarf Mutants in Soybean(Glycine max(L.) Merr.)

Dwarfing is useful to reduce plant height,when breeding high-yielding and non-lodging crops.In this study,a set of natural storage protein subunit-null dwarf mutants of soybean was reported that showed strongly reduced plant stature and deficiency in various 7S and 11S subunits,designated as snd1 mutants.Under normal growth conditions,the snd1 mutants showed a severe dwarf phenotype,with plant height of about 25 cm.Compared with wild-type DN47,the mutant snd1 exhibited no obvious morphological differences at the early stage of development.All the snd1 mutants examined had fewer nodes and shorter than normal internodes;the leaves were similar in shape to normal parents,but were dark-green at the mature stage.The flower size was similar to DN47;however,the flowering period was shorter than in the wild-type.Significant variation was noted for protein content,oil content of the seeds and size of seeds(weight of 100 seeds)among 17 snd1 dwarf lines.Genetic analysis indicated that the dwarfism of snd1 was controlled by a single recessive gene.The snd1 dwarf mutant had markedly different dynamic levels of the endogenous hormones gibberellin(GA),brassinosteroid,indole-3-acetic acid and abscisic acid,at the seedling stage.Exogenous GA3 treatment led to recovery of the plant height phenotype of the snd1 mutant;GA3 at 0.1 mm had the largest effect on enhancing plant height.Using molecular markers,snd1 gene was approximately mapped in an interval of 603 kb between markers Satt166 and Satt561 on chromosome 19.Snd1 mutant provided valuable material for hypoallergenic soybean breeding and the snd1 gene might be a novel gene related to plant height in soybean.

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.

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.

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.

Poplar Trees （Populus canadensis Moench） Initiate Vegetative Storage Protein Accumulation During New Shoot Development in Spring

It was generally assumed that the accumulation of vegetative storage protein (VSP) in poplar trees and/or temperate hardwoods did not occur in spring. To test this assumption, the accumulation of the 32-kDa VSP and the differential expression of a gene encoding for the protein in poplars were investigated using light and electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reverse transcription-polymerase chain reaction (RT-PCR). We report, for the first time, that poplar trees initiated VSP accumulation in new shoots during the development of new shoots in spring under conditions of high temperature and long days. The amount of 32-kDa VSP increased gradually in the stem of new shoots and in two-year-old branches, but there were no detectable changes in its abundance in the bark tissues of trunks during new shoot development. Based on the presence of a 286-bp DNA fragment that is identical to the VSP gene bspA, encoding for the 32-kDa VSP in Populus deltoids Bartr. ex Marsh., the differential expression of the 32-kDa VSP gene in P. canadensis Moench was revealed by RT-PCR. The results indicated that the 32-kDa VSP gene was expressed strongly in new shoots, relative weakly in two-year-old branches and was not expressed in the trunk during new shoot development. This pattern of VSP accumulation and VSP gene space-time differential expression may be an important mechanism by which stored nitrogen compounds are used preferentially to exogenously available nitrogen and, in addition, the dynamic pattern may also have a role in the regulation of nitrogen metabolism, especially nitrogen uptake by the roots.

Characterization of Three Novel Wheat LThinopyrum intermedium Addition Lines with Novel Storage Protein Subunits and Resistance to Both Powdery Mildew and Stripe Rust

Wide hybridization is an effective approach for enhancing the resistance of bread wheat （Triticum aestivum L.） to biotic and abiotic stresses by introducing favorable alien genes （Sepsi et al., 2008）. Wheatgrass, Thinopyrum intermedium （Host） Barkworth ＆ D.R. Dewey or Agropyron intermedium （Host） Beauvoir （2n = 42; genome formula JJjSjSstst）, is a perennial species in the tribe Triticeae and an important source of wheat improvement for biotic and abiotic stress resistance and quality-related traits, such as high grain protein concentration （Chen et al., 1998; 2001; 2003; Han et al., 2004; Li and Wang, 2009）. In addition, the ready crossing ability of wheatgrass with various Triticum species has made it popular in germ- plasm development.

cDNA of an arylphorin-type storage protein from Pieris rapae with parasitism inducible expression by the endoparasitoid wasp Pteromalus puparum

This report presents the cDNA cloning of a storage protein, PraAry, from Pieris rapae and investigates its expression regulated by parasitism of an endoparasitoid wasp Pteromalus puparum. The full-length eDNA of PraAry is 2 270 nucleotides and contains a 2 121 nucleotide open reading frame encoding 707 amino acids with calculated molecular weights of approximately 83 kDa. Analysis of the primary protein sequence revealed that it possesses a signal peptide of 16 amino acids at the N-terminus and contains two highly conserved storage protein signature motifs. According to both phylogenetic analysis and the criteria for amino acid composition, PraAry belongs to the subfamily of arylphorin-type storage protein （1.42% methionine and 18.82% aromatic amino acids）. Reverse transcription- polymerase chain reaction analysis indicated that the transcriptional level of PraAry mRNA in P. rapae pupae fat body is inducible in response to parasitism by P. puparum.

Fluctuation of Vegetative Storage Proteins in the Seedlings of Swietenia macrophylla, Analogous to the Seasonal Changes of Those in the Shoot of the Adult Tree

In order to Identify appropriate plant materials for studying the gene expression and biological function of vegetative storage proteins （VSPs） in woody plants, the VSPs in the seedlings of Swietenla rnacrophylla King were investigated by using light microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western.blotting. The seed of S. macrophylla was rich in storage proteins that accumulated In the vacuoles of cotyledon parenchyma cells in appearance of compact spherical grains. The growth and development of S. macrophylla seedlings were characterized by an obvious growth rhythm. The storage proteins In seeds disappeared during seedling growth while VSPs appeared in the stem 2 weeks after seedling leaves matured. Thereafter, the VSPs In the seedling stem almost exhausted during new shoot growth, and when the leaves of new shoot Just matured, both the stem beneath the new shoot of seedlings and the stem of new shoot started to accumulate VSPs. Nitrogen application dramatically Increased the level of VSPs, but had little influence on the dynamics of VSP consumption and accumulation in seedling stem. Together with these data, the fluctuation of VSPs in seedlings was very similar to that in the branches of the adult trees. In addition, seedlings are easy to be treated due to their small size. Our results suggested that S. rnacrophylla seedlings were suitable for Investigating the biological roles of VSPs and the mechanism of nitrogen storage in trees.

Identification in Lupin Seed of a Serine- Endopeptidase Activity Cleaving between Twin Arginine Pairs and Causing Limited Proteolysis of Seed Storage Proteins

The occurrence of twin-arginine motifs （-R-R-） in the amino acid sequences of animal pro-proteins frequently defines the cleavage site（s） for their structural/functional maturation. No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins. In this work, a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds. The endopeptidase was tested with a number of endogenous and exogenous protein substrates, which were selected according to the pres- ence of one or more twin-arginine residue motifs in their amino acid sequences. The observed hydrolysis patterns were limited and highly specific. Partial proteolysis led to stable polypeptide fragments that were characterized by 1- and 2-D electrophoresis. Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry anal- yses, These approaches, supported by bioinformatic analysis of the available sequences, allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates. The endopeptidase activity was inhibited by 4-（2-AminoEthyl）Benzene-Sulphonyl Fluoride hy- drochloride （AEBSF）, leupeptin, and serine proteinase protein inhibitors, while it was not affected by pepstatin, trans- EpoxysuccinyI-L-leucylamido（4-guanidino）butane （E64）, and ethylenediaminetetraacetic acid （EDTA）, thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase. The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving -R-R- bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.

Effects of nitrogen fertilizer on protein synthesis, accumulation, and physicochemical properties in common buckwheat

Nitrogen(N)fertilization affects grain quality in common buckwheat(Fagopyrum esculentum Moench).But the effects of N fertilizer on various buckwheat protein parameters are not fully understood.This study aimed to investigate the synthesis,accumulation,and quality of buckwheat protein under four N application rates in the Loess Plateau,China.Optimal N application(180 kg N ha-1)improved yield,agronomic traits,and N transport and increased protein yield and protein component accumulation.Prolamin and glutelin accumulation first increased and then decreased with increasing N application.The relationships between the contents of protein components and the amount of applied N generally followed quadratic functions.Nitrate reductase and glutamine synthetase activities first increased and then decreased with increasing N levels.Optimal N fertilizer increased the waterholding capacity and thermal stability of buckwheat protein and reduced its emulsification capacity,but negligibly changed its oil-absorption capacity.Hydrophobic amino acids and glutelin content were the main factors affecting protein quality.

Influence of protein characteristics and the proportion of gluten on end-use quality in Korean wheat cultivars

The effects of protein characteristics and the proportion of gluten on end-use quality in 13 Korean wheat cultivars for three years were verified in this study. Year, cultivar, and the interaction between the year and the cultivar influenced protein characteristics, the proportion of gluten except for γ-and ω-gliadin using RP-HPLC(reversed-phase high-performance liquid chromatography), and end-use quality. Protein characteristics and the proportion of gluten in Korean wheat cultivars were between those of Australian standard white(ASW) and hard wheat(AH). Korean wheat cultivars exhibited a higher average α+β gliadin proportion than imported wheat, a γ-gliadin proportion similar to that of dark northern spring wheat, and the same ω-gliadin proportion as AH. They showed a bread-loaf volume intermediate between those of ASW and AH and a texture of cooked noodles similar to that of soft white wheat, but less springiness than imported wheat. The cookie diameter of Korean wheat cultivars was similar to that of hard red winter wheat. There was a correlation between bread-loaf volume and protein characteristics, except for the protein content in Korean wheat cultivars. Springiness and cohesiveness of cooked noodles were not correlated with protein characteristics, while hardness was correlated with the protein content and water absorption of a mixograph. Cookie diameter was negatively correlated with the sodium dodecyl sulfate(SDS) sedimentation volume and water absorption of a mixograph. The end-use quality was not correlated with any proportion of gluten composition. Principal component analysis(PCA) showed that the proportion of gluten was not related to the quality of the bread(both PCs, 81.3%), noodle(77.7%), and cookie(82.4%). PCA explained that Keumkang is suitable for superior bread, while Uri is good for cooked noodles and cookies.

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.

Molecular identification of ω-secalin gene expression activity in a wheat 1B/1R translocation cultivar

ω-Secalin was an important factor influencing processing quality of wheat 1BL/1RS translocations. On the basis of four ω-secalin gene sequences cloned from Lankao 906 （a wheat cultivar with 1BL/1RS translocation） with putative transcription activity, a pair of primers with suitable restriction endonucleases added at their 5＂ ends were designed to amplify the mature protein-coding regions of the four genes. After digestion with restriction endonucleases, the amplified products were ligated into the prokaryotic expression vector pET30a（＋）. The prokaryotically expressed recombinant proteins and gliadin isolated from the Lankao 906 seed were analyzed on the same acid polyacrylamide gel electrophoresis. All four prokaryotically expressed secalin bands had corresponding seed-expressed gliadin bands. The four corresponding gliadin bands were confirmed to be the expression products of the four ω-secalin genes by liquid chromatography tandem mass spectrometry （LC-MS/MS）. This conclusion was further confirmed because the four ω-secalin bands could be detected in all 14 wheat 1BL/1 RS translocation cultivars used in the study, although there was some interference for the detection of one ω-secalin band from nearby wheat gliadin bands. The sequence information of ω-secalin genes with expression activity will be helpful for improving the processing quality of wheat with 1BL/1RS translocations by using RNA interference method to silence the expression of the ω-secalin genes.

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.