Endosperm Proteins Research Articles

Breadmaking stability of wheat flours: Relation between mixing properties and molecular weight distribution of polymeric glutenins

The environmental stability of breadmaking quality of wheat flours i.e. the absence of fluctuation across environments and genotype×environment interactions is as important as their performance in milling and baking. Many statistical approaches have been used to assess cultivar stability parameters across environments. Quality stability parameters were studied using a set of eleven bread wheat cultivars and five hybrid wheats grown in France at eight locations in two consecutive years. Three different statistical variances: ecovalence Wi, environmental variance S X i 2 and genotypic stability were used to assess the stability of quality parameters measured on wheat kernels at harvest, on the technological properties of their flours and on the molecular weight distribution of total endosperm proteins extracted using 2% SDS in phosphate buffer with sonication. Asymmetrical flow field-flow fractionation coupled with a multi-angle laser light scattering photometer allowed the molar masses of polymeric glutenins to be measured without size limits. Marked variation in the quality parameters was observed due to genetic and environmental effects. The three stability values calculated for protein content were not significantly correlated to the stability of the polymeric fraction, the bread loaf volume (LV) or the bread quality score (BQS). The three stability values of LV and BQS were significantly correlated to the stability of the weight-average (〈 M〉w) molecular weight of the polymeric fraction. Multiple regressions showed that 60–84% of the stability values of LV and BQS were explained by the stability of only few quality parameters among which the 〈 M〉w of polymers played the major role.

Fine mapping and marker-assisted selection (MAS) of a low glutelin content gene in rice

Rice with low glutelin content is suitable as functional food for patients affected with diabetes and kidney failure. The fine mapping of the gene(s) responsible for low glutelin content will provide information regarding the distribution of glutelin related genes in rice genome and will generate markers for the selection of low glutelin rice varieties. Following an SDS-PAGE screen of rice germplasm from Taihu Valley of China, Japonica selection W3660 is identified to be a novel mutant characterized with low glutelin content. For fine mapping the mutant gene for low glutelin content, F2 and F3 populations were derived from a cross between W3660 and Jingrennuo. SDS-PAGE analysis of the total endosperm protein showed that the low glutelin content trait was controlled by a single dominant nuclear gene. Genetic mapping, using SSRs, located this gene to chromosome 2, in the region between SSR2-001/SSR2-004 and RM1358. The distances of the two markers to the target gene were 1.1 cM and 3.8 cM respectively. By semi-quantitative RT-PCR analysis, the transcripts of GluB4/GluB5 genes located within the region do not change. However, GluB5 gene located proximal to SSR2-001/SSR2-004 was specifically reduced. SSR profiles of seven Japonica varieties were compared with that of W3660 for loci in the relevant genetic region. The markers SSR2-004 and RM1358 were used for marker-assisted selection. The selection efficiencies of SSR2-004 and RM1358 were 96.8% and 92.7% respectively. This provides a standard starting point for the breeding of low glutelin content rice varieties in China.

Characteristics of Thiol:Protein Disulfide Oxidoreductase from Wheat (Triticum aestivum L.) Grain

Biochemical properties of a homogenous preparation of thiol:protein disulfide oxidoreductase (TPDO, EC 1.8.4.2) isolated for the first time from mature wheat (Triticum aestivum L.) grain were studied. According to polyacrylamide gel electrophoresis data, the molecular weight of TPDO is around 167 kD, the enzyme consisting of two subunits of 77 and 73 kD, which differentiates TPDO from known enzymes of SH/SS-metabolism of wheat caryopses. In substrate specificity and enzymatic characteristics (pH and temperature optima) TPDO is similar to analogous enzymes of animal tissues. Inhibition of disulfide reductase activity by alkylating agents and heavy metal ions suggests the participation of active center SH-groups in the catalytic act and classes the enzyme as a member of the thioredoxin superfamily. The SS-reductase reduces aggregating capacity of acetic acid-soluble fraction of wheat storage proteins. The proposed physiological role of TPDO is participation in creation and regulation of SH/SS-status of wheat endosperm proteins and formation of the rheological properties of gluten.

Two-generation marker-aided backcrossing for rapid conversion of normal maize lines to quality protein maize (QPM)

The low nutritive value of maize endosperm protein is genetically corrected in quality protein maize (QPM), which contains the opaque 2 gene along with numerous modifiers for kernel hardness. We report here a two generation marker-based backcross breeding program for incorporation of the opaque 2 gene along with phenotypic selection for kernel modification in the background of an early maturing normal maize inbred line, V25. Using the flanking marker distances from opaque 2 gene in the cross V 25 xCML 176, optimum population size for the BC(2) generation was computed in such a way that at least one double recombinant could be obtained. Whole genome background selection in the BC(2) generation identified three plants with 93 to 96% recurrent parent genome content. The three BC(2)F(2) families derived from marker identified BC(2) individuals were subjected to foreground selection and phenotypic selection for kernel modification. The tryptophan concentration in endosperm protein was significantly enhanced in all the three classes of kernel modification viz., less than 25%, 25--50% and more than 50% opaqueness. BC(2)F(3) lines developed from the hard endosperm kernels were evaluated for desirable agronomic and biochemical traits in replicated trials and the best line was chosen to represent the QPM version of V25, with tryptophan concentration of 0.85% in protein. The integrated breeding strategy reported here can be applied to reduce genetic drag as well as the time involved in a conventional line conversion program, and would prove valuable in rapid development of specialty corn germ plasm.

Effect of the opaque and floury mutations on the accumulation of dry matter and protein fractions in maize endosperm

Grains of nine opaque ( o) and floury ( fl) mutants of maize (Oh43 o1, Oh43 o2, B79 o5, B37 o7, W22 o10, W22 o11, W22 o13, Oh43 fl1 and Oh43 fl2) were examined for the weight proportions of their component tissues and the content of eight nitrogen fractions in their endosperms. A linear regression was found connecting the amounts (mg per endosperm) of zeins and true proteins (crude proteins minus non-protein nitrogen) for the non- opaque2 mutants. The data points connecting zeins to true proteins present in the mature endosperms of six wild-type (+) inbred lines and their o2 versions were located outside (+) or within ( o2) the 95% confidence range of the regression line. The data obtained from the developing and mature endosperms of the W22 o7 inbred line (Di Fonzo et al., Plant Sci. Lett., 1979, 77) and the floury portion of mature endosperms of three other wild-type inbred lines fell practically on the regression line. The effects of genotype and environmental factors upon the relative accumulation rate of zeins were assessed from the present results and the data taken from the literature concerning the quantitative interdependence between zeins and true proteins in immature and mature endosperms.

Molecular genetic mapping of a high-lysine mutant gene (opaque-16) and the double recessive effect with opaque-2 in maize

The lysin content in maize endosperm protein is considered to be one of the most important traits for determining the nutritional quality of food and feed. Improving the protein quality of the maize kernel depends principally on finding a mutant with a higher lysine content. Two high-lysine mutant lines with opaque endosperm, QCL3024 and QCL3021, were isolated from a self-cross population derived from Robertson’s Mutator stocks. The gene controlling this mutation is temporarily termed opaque-16 (o16). In order to illuminate the genetic locus and effect of the o16 gene, two F2:3 populations, one developed from a cross between QCL3024 and QCL3010 (a wild type line) and another from a cross between Qi205 (opaque-2 line) and QCL3021, were created, and F3 seeds from the F2 plants in the two populations were evaluated for lysine content. The distributions of lysine content and tests for their normality indicate that the lysine content in the two populations is regulated by the major gene of o16 and genes of o2 and o16, respectively. Based on two data sets of the linkage maps of the F2 plant marker genotypes and the lysine content of F3 seeds originating from the two F2:3 populations, the o16 gene was located within 5 cM, at either 3 or 2.2 cM from umc1141 in the interval between umc1121 and umc1141 on the long arm of chromosome 8, depending on the recombination rate in the two populations as determined by composite interval mapping. According to the data of the F2:3 population constructed from the o2 and o16 lines, the double recessive mutant effect was analyzed. The average lysine content of the F3 ‘o2o2o16o16’ families identified by the umc1066 and umc1141 markers was approximately 30% higher than that of the F3 o2o2 and ‘‘o16o16’ families, respectively. The lysine content of seven F3 families among nine F3 double recessive mutant families showed different increments, with an average increase of some 6% compared with that of the maternal o2 line. The potential application of the o16 mutant for maize high-lysine breeding may be to combine it with the o2 mutant bearing modifier genes, thus obtaining a mutant with much higher lysine content. For the purpose of pyramiding the o16 with o2 genes, the availability of closely linked markers of the o16 and o2 loci will facilitate marker-assisted selection and greatly reduce breeding time and effort.

Developmental changes in the metabolic protein profiles of wheat endosperm

A combined two-dimensional gel electrophoresis-mass spectrometry approach was utilized to identify over 250 proteins of wheat (Triticum aestivum L., cv. Butte 86) starchy endosperm that participate in 13 biochemical processes: ATP interconversion reactions, carbohydrate metabolism, cell division, cytoskeleton, lipid metabolism, nitrogen metabolism, protein synthesis/assembly, protein turnover, signal transduction, protein storage, stress/defense, transcription/translation, and transport. Endosperm protein populations were compared at early (10 days post-anthesis, dpa) and late (36 dpa) stages of grain development. Analysis of protein number and spot volume revealed that carbohydrate metabolism, transcription/translation, and protein synthesis/assembly were the principal endosperm functions at 10 dpa followed by nitrogen metabolism, protein turnover, cytoskeleton, cell division, signal transduction, and lipid metabolism. Carbohydrate metabolism and protein synthesis/assembly were also major functions at 36 dpa, but stress/defense and storage were predominant. The results provide insight into biochemical events taking place during wheat grain development and highlight the value of proteomics in characterizing complex biochemical processes. Further, the proteome maps will facilitate future studies addressing the effects of genetic and environmental factors on the development and quality of wheat grain.

Relationship between common wheat (Triticum aestivum L.) gluten proteins and dough rheological properties

This paper reports the correlation between the rheological properties of bread wheat dough and the types and quantities of endosperm proteins in 28 common wheat cultivars. Different methods were used to analyse the allelic composition of these cultivars and the relative quantities of the different proteins contributing to the gluten structure. Neither dough strength (W) nor tenacity/extensibility (P/L) correlated with allelic composition. Different wheats with the same allelic composition (i.e., with respect to glutenins) showed different rheological properties. The glutenins were the most influential components of W and P/L, especially the high molecular weight (HMW) glutenin subunits and in particular the type x form. These proteins seem to increase W and are the main constituents of the gluten network. The gliadins and low molecular weight (LMW) glutenin subunits appear to act as a “solvent”, and thus modify the rheological properties of the dough by either interfering with the polymerisation of the HMW glutenin subunits, or by altering the relative amounts of the different types of glutenin available. Thus, the protein subunits coded for by the alleles Glu-B1x7 and Glu-D1x5 stabilised the gluten network, whereas those coded for by Glu-B1x17 and Glu-D1x2 had the opposite effect. Dough properties therefore appear to depend on the glutenin/gliadins balance, and on the ratio of the type x and type y HMW proteins. The influence of external factors seems to depend on the allelic composition of each cultivar.

Mechanical and Physicochemical Characterization of Vitreous and Mealy Durum Wheat Endosperm

ABSTRACTThe mechanical, physical, and biochemical characteristics of mealy and vitreous endosperm were investigated. Endosperm were obtained from four durum wheat cultivars grown under different nitrogen fertilization designs. The textural properties and the density of the endosperm were measured on hand‐shaped parallelepiped endosperm samples. Endosperm protein content and composition and also gliadin composition were investigated by HPLC. Mechanical tests showed that mealy and vitreous endosperm differed in hardness and vitreousness. Vitreousness increased with nitrogen fertilization supply whereas there was no variation among the different cultivars. Hardness seemed to be linked to genotype and insensitive to nitrogen supply. From this result, we concluded that hardness and vitreousness are not related. Endosperm protein content and gliadin‐to‐glutenin ratio were related to nitrogen supply and increased especially when nitrogen supply was applied at flowering. At the same time, endosperm vitreousness increased. Further biochemical analyses were performed on 270 kernels, mealy or vitreous, hand‐picked from 148 different crops. Results showed that protein content of vitreous endosperm exceeded 9.7% in >90% of the cases. The glia/glu ratio was a less accurate predictor of kernel vitreousness, indicating that, by itself, it cannot account for the change in kernel vitreousness. Endosperm vitreous texture would rise above a threshold content of 9.7% protein within the endosperm.

Microwave‐Assisted Rapid Modification of Zein by Octenyl Succinic Anhydride

ABSTRACTCorn is one of the most important food and industrial crops in the United States. Zein constitutes about half of the endosperm proteins in corn. Potential applications of zein include use in fibers, adhesives, inks, cosmetics, textiles, and chewing gum. Recently, attempts have been made to utilize zein for food coatings and biodegradable materials. The new applications of zein require it to be resistant to water. Thus, we are interested in new routes for chemical modification of zein (Biswas et al, unpublished). 2‐Octenyl succinic anhydride (OSA) is extensively used in modifying food starches. Our objective was to take advantage of OSA hydophobicity, flexibility, and compatibility with nonpolar groups and incorporate OSA onto zein. This OSA‐modified zein would possess the best of both OSA and zein. A zein solution in dimethyl formamide (DMF) was reacted with various levels of OSA to give modified zein. In a microwave reactor, the reactions proceeded tremendously fast and they were over in 5 min. The anhydride group of OSA reacted with the hydroxyl/amine group of zein to form ester/amide of zein. The reaction of OSA‐modified zein was confirmed by proton nuclear magnetic resonance (NMR) and IR spectroscopy. The preliminary evaluation showed that zein and OSA‐modified zein had comparable mechanical properties.

Identification of new puroindoline genotypes and their relationship to flour texture among wheat cultivars

Puroindoline genotypes (Pina and Pinb) and their encoded proteins related to grain hardness were studied in various common wheat cultivars from Australia, China, Japan, Korea and North America. Most of the hard wheats had the Pinb-D1b genotype with a glycine to serine mutation at position 46. In addition to the known Pina and Pinb genotypes, cultivars were found with Pina and Pinb double-null mutations (Pina-D1b/Pinb-D1h (t)) and a new Pinb frameshift mutation (designated Pinb-D1i (t)) within the region encoding a tryptophan-rich domain. This new Pinb frameshift mutation was found only in Chinese cultivars. Endosperm proteins encoded by Pina and Pinb in these cultivars were analysed by 2D-gel electrophoresis (IPG×SDS-PAGE). Cultivars with Pina and Pinb double-null mutations showed no PIN-a or PIN-b protein, and cultivars with Pinb-D1i (t) had no PIN-b protein. Surprisingly, cultivars with Pinb-D1b had severely reduced amounts of PIN-b and cultivars with Pinb-D1c showed no PIN-b proteins. Grain hardness among cultivars having mutated Pinb may be explained by the amount of PIN-b protein and not by the type of amino acid substitutions.

Isolation and characterisation of friabilin genes in rye

The Hardness locus on chromosome 5D is the main determinant of grain texture in hexaploid wheat. Puroindoline-a (pin-a), puroindoline-b (pin-b) and Grain Softness Protein (GSP) genes are tightly linked at this locus and their products are the predominant components of friabilin, a 15 kDa endosperm protein complex. Differences in grain texture are mainly caused by specific puroindoline genes mutations and are known to play a large impact on the end-product quality of wheat, contributing to the distinction of well-suited market classes for specific end-uses. We investigated friabilin genes in rye (Secale cereale L.), aiming at an increase information on the friabilin molecular system in cereals and to further investigate their potential use for the genetic improvement of this crop. Using a PCR approach, puroindoline-b and GSP-like sequences were identified for the first time in several rye and triticale cultivars, sequenced and located on specific chromosomal arms. The primary structures of deduced proteins were determined and compared with those from other cereal species. In contrast with data from previous studies, secaloindoline-a was not found. Our results introduce new evidence for a discrete allelic variation at the secaloindoline loci in rye, indicating that future larger screenings may facilitate preliminary selection of germplasm for rye breeding purposes and triticale production.

Improved methods for separation of wheat endosperm proteins and analysis by two-dimensional gel electrophoresis

Fractionation of seed proteins has been a significant area of interest and activity, since Osborne (1924) introduced his solubility fractionation methods. In particular, the fractionation of wheat endosperm proteins has presented numerous challenges, because of their unique solubilities. Recently, we combined solubility fractionation and 2-DE protein analysis methods that taken separately may not seem unusual, but together resulted in an approach enabling us to identify a number of thioredoxin target proteins in wheat endosperm (Wong et al., 2003, 2004). Proteins are recovered from the various fractions by precipitation with methanol or acetone. A finding of interest to cereal scientists is that SDS can be used to solubilize the gluten proteins and, following acetone precipitation, the recovered proteins can be further characterized by IEF, HPLC, and mass spectroscopy. Wheat (Triticum aestivum L. ‘Butte 86’) was grown in a climate-controlled greenhouse as described previously (Wong et al., 2003). Mature grain was tempered and milled to flour with a Brabender Quadrumat Junior (South Hakensack, NJ) using standard procedures at the Western

Intraspecific Structure and Reproductive Relationships between Elymus mutabilis and E. transbaicalensis (Poaceae) in Southern Siberia from the Viewpoint of Taxonomical Genetics

The relationships between StH-genomic species Elymus mutabilis (Drob.) Tzvel. and E. transbaicalensis (Nevski) Tzvel. were examined using biosystematic and biochemical (SDS electrophoresis of endosperm proteins) methods. The results showed the following. (1) There is interspecific morphological discreteness that permits assigning most individuals from the wild to a definite species. However, self-fertile intermediate individuals of introgressive origin were found. (2) Both taxa exhibited marked specificity of electrophoretic characteristics of endosperm proteins. (3) The species form two separate recombination gene pools (RGPs). Thus, E. mutabilis and E. transbaicalensis should be regarded as two distinct species. Nevertheless, there is some genetic exchange and unidirectional gene flow between the RGPs, especially in secondary (successive) habitats where mixed populations are observed. Two RGPs form the single introgressive gene pool (IGP), which includes other StH-genomic species of Northern Eurasia.

Proteomic analysis of aneuploid lines in the homeologous group 1 of the hexaploid wheat cultivar Courtot.

Three monosomic lines (MSLs) and three nullisomic lines (NSLs) of the homeologous group 1 and one euploid line of the bread wheat Triticum aestivum cultivar Courtot were used in a proteomic approach to investigate the effects of zero, one or two doses of chromosomes 1A, 1B and 1D on the amount of endosperm proteins. Polypeptides whose amounts changed significantly between each aneuploid line and the euploid line were identified using image analyses of two-dimensional gel electrophoresis patterns resulting from specific endosperm protein extractions. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry were also used for protein identification. Removing one chromosome or a chromosome pair allowed varying responses to be observed for the remaining endosperm protein genes. Compensation phenomena for the high molecular weight glutenin subunits (HMW-GS) were detected only in the MSLs. Subunits Bx7, By8 and Dy12 were the only HMW-GS overexpressed (from 152-737%) when chromosomes 1A or 1B or 1D were at hemizygous state. Thirteen new protein spots were detected only in the NSL1D, and seven were identified as HMW-GS analogs. These seven new spots may result from the expression of inactive genes. The HMW-GS were of significantly higher volume in MSLs, whereas the low molecular weight glutenin subunits and the gamma-gliadins were of lower volume in aneuploid lines. Most of the down-regulated proteins in the MSLs were storage proteins encoded at loci located on another chromosome pair. Complex regulations between chromosomes and loci of the homeologous groups 1 and 6 in bread wheat are discussed.

Regulation of lysine metabolism and endosperm protein synthesis by the opaque-5 and opaque-7 maize mutations.

Two high lysine maize endosperm mutations, opaque-5 (o5) and opaque-7 (o7), were biochemically characterized for endosperm protein synthesis and lysine metabolism in immature seeds. Albumins, globulins, and glutelins, which have a high content of lysine, were shown to be increased in the mutants, whereas zeins, which contain trace concentrations of lysine, were reduced in relation to the wild-type lines B77xB79+ and B37+. These alterations in the storage protein fraction distribution possibly explain the increased concentration of lysine in the two mutants. Using two-dimensional polyacrylamide gel electrophoresis of proteins of mature grains, variable amounts of zein polypeptides were detected and considerable differences were noted between the four lines studied. The analysis of the enzymes involved in lysine metabolism indicated that both mutants have reduced lysine catabolism when compared to their respective wild types, thus allowing more lysine to be available for storage protein synthesis.

Endosperm protein polymorphism of common and tartary buckwheat

The endosperm protein polymorphism of seven accessions of common buckwheat (Fagopyrum esculentum Moench) and two accessions of tartary buckwheat (Fagopyrum tataricum Gaertn.) were studied by SDS-PAGE electrophoresis. The position and the intensity of the main four electrophoretic protein bands (marked as “a”, “b”, “c” and “d”) of cultivar Siva (common buckwheat) were estimated and the polymorphism was studied using seeds from open pollinated heterozygous plants. Two high significant correlations (correlation coefficient 0.559 for “a” and “c” bands, and 0.264 for “a” and “d” bands) were established among the intensities of electrophoretic protein bands of common buckwheat, while other four possible relations were not significant. The results indicate significantly higher protein polymorphism of endosperm within individual accessions than among accessions of common buckwheat. In contrary to common buckwheat, there was in material studied found no variability in endosperm protein bands between or within two accessions of tartary buckwheat.

The genetics and properties of cereal ribosome-inactivating proteins.

Plants contain proteins that are capable of inactivating ribosomes, commonly referred to as Ribosome Inactivating Proteins (RIPs). These particular plant proteins have received attention in biological and biomedical research because of their unique biological activities towards animals and human cells as cell-killing agents. Some of the best-characterised RIPs have been isolated from exotic plants, but they have also been found in cereals and other food crops. Cereals contain, in general, RIPs in the endosperm protein pool: they share a high similarity with all the other RIPs retaining, however, characteristic features forming a distinct class which diversified significantly during evolution. They appear to be involved in quite different physiological roles, such as defence against pathogens and/or involved in regulatory and developmental processes. This review aims to provide a critical assessment to work related to cereal RIP with particular emphasis to the maize RIPs.

Differentiation in the Elymus dahuricus complex (Poaceae): evidence from grain proteins, DNA, and crossability.

The status of the taxa Elymus dahuricus Turcz. ex Griseb., E. excelsus Turcz. ex Griseb., E. woroschilowii Probat. and E. tangutorum (Nevski) Hand.-Mazz., all of which are sometimes treated as members of E. dahuricus s.l. or as constituting the E. dahuricus complex, has been investigated using morphological, biochemical (SDS-PAGE of endosperm proteins) and molecular (DNA-AFLP) variation characteristics. Populations include accessions from Siberia, Far East (Russia), Tien-Shan (Kirghizstan), and Tibet and Sichuan Provinces (China). Variation in the morphological characteristics and endosperm proteins patterns have been found in all populations of the complex. DNA variation was very low within populations but was different among populations; this was attributed to genetic drift. Seed fertility of artificial hybrids and F2-F3 progenies in 38 combinations was analyzed. All biotypes studied form a common recombination genepool (RGP). The reproductive compatibility of hybrid combinations decreases with increased geographical separation of the parents. Differences in protein patterns and in genetic variation drawn from DNA-AFLPs and phenotypical segregation in F2 on series of diagnostic characters does not support existing taxonomic treatments of the E. dahuricus complex in Southern Russia, and Central-Asian area. The E. dahuricus complex seems to be a polymorphic species having a wide geographical range and wide genetic variation. The different species need probably be relegated to infraspecific rank. We have refrained from making the necessary combinations pending examination of additional specimens, including specimens of taxa not included in this study and the type specimens of all taxa involved.

Ozone‐Aided Corn Steeping Process

ABSTRACTThe present research evaluated the feasibility of using ozone (O3) to replace sulfur dioxide (SO2) in corn steeping. Traditionally, steep water contains 0.1–0.2% sulfur dioxide to promote starch‐protein separation and high starch yields, and to control microbial growth. However, residual SO2 in starch products affects product quality and jeopardizes the “organic products” claims. Also, SO2 discharged to the environment pollutes water and air. Ozone is a strong oxidant and disinfectant with a capability to control the growth of putrefactive microorganisms in steeping systems, and to break down the endosperm protein matrix and, hence, improve starch release. This study demonstrates that an ozone‐aided steeping (OAS) process had starch yields as high as conventional SO2 steeping. OAS processes can be conducted at a lower temperature (20°C vs. 50°C) and for shorter times (36 hr vs. 48 hr) than the conventional SO2 processes, suggesting significant energy savings and increased productivity. We have found that the timing of ozone application is of great importance to the performance of the OAS process.