Interspecific rice hybrid of Oryza sativa x Oryza nivara reveals a significant increase in seed protein content

Wild species offer a potential reservoir of genetic variation for crop improvement. Besides the valuable genes for disease resistance that the wild species have provided for rice improvement, recent studies have shown that these wild species could also provide favorable alleles for the improvement of yield and yield-related traits. The present study reports yet another potential of wild relatives of rice, which involves the improvement of seed protein content. A significant increase in seed protein content was observed in an interspecific hybrid between Oryza sativa ssp. indica and the wild species Oryza nivara. The hybrid showed a protein content of 12.4%, which was 28 and 18.2% higher than those of the parents O. nivara and IR 64, respectively. The increase in protein content was dependent on the genetic background of the rice variety used in the hybridization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of seed storage proteins demonstrated that a significant increase in prolamins and glutelins was mainly responsible for the elevated protein content of the hybrid. Amino acid analysis of seed proteins revealed that the hybrid had net gains of 19.5% in lysine and 19.4% in threonine over the O. nivara parent on a seed dry weight basis. Molecular analysis indicated that the increase in protein content of the hybrid was not a result of chromosomal rearrangements or transposable element activation, at least in the chromosomal regions containing seed storage protein genes. A preliminary genetic analysis of the F 2 segregating population showed that the inheritance of the increased protein content was polygenic in nature. The development of this interspecific hybrid offers a great potential for selecting new rice cultivars that combine the high yield and superior cooking quality of IR 64 with improved seed protein content.     

Amino acid analysis for meat protein evaluation

The Food Safety and Inspection Service procedure for determination of essential amino acid content of mechanically processed products from red meat animals and poultry is based on hydrolysis of a powder prepared by blending samples in acetone-chloroform. The hydrolysis procedure incorporates thioglycolic acid to prevent loss of tryptophan. Aliquots of prepared hydrolysates are injected into a liquid chromatographic system, using gradient elution on an ion-exchange column for separation. The system also uses post-column hypochlorite oxidation coupled with orthophthalaldehyde reagent and fluorescence detection. Modification of the elution program allows concurrent determination of tryptophan with minimal added cost. Chromatograms from beef, pork, and poultry products show adequate separation and quantitation of beta-alanine, 1-methyl-histidine, and 3-methyl-histidine, indicating that the procedure could be used to estimate muscle content of products. A colorimetric procedure for assay of hydroxyproline was introduced and validated as an adjunct method for protein quality estimation.     

Amino acid rating method for evaluating protein adequacy of infant formulas

Amino acid profiles and/or protein digestibility (by the rat balance method) were determined for various forms (powder, ready-to-use, liquid concentrate, etc.) of cow's milk- and soy-based infant formulas obtained from 4 manufacturers. The essential amino acid data of the formulas were compared with that of human milk for the calculation of amino acid scores (based on the single most limiting amino acid). The product of amino acid score and total protein (g/100 kcal) was then termed "amino acid rating." Amino acid scores for the milk- and soy-based formulas ranged from 59 to 90 and from 59 to 81%, respectively, due to deficiencies in sulfur amino acids and/or tryptophan. Because of significantly higher total protein contents (g/100 kcal) of soy- (2.65-3.68) and milk-based (2.20-2.95) infant formulas compared to human milk (1.5), the relative amino acid ratings (human milk = 100) for all infant formulas except 2 liquid concentrates (having values of 87%) were above 100%. Values for true digestibility of protein in milk- and soy-based formulas ranged from 87 to 97 and from 92 to 95%, respectively. When corrected for protein digestibility, the relative amino acid ratings for all the milk-based liquid concentrates were below 100% (77-98%).     

Amino acid,peptide and protein mineralization dynamics in a taiga forest soil

The availability of inorganic N has been shown to be one of the major factors limiting primary productivity in high latitude ecosystems. The factors regulating the rate of transformation of organic N to nitrate and ammonium, however, remain poorly understood. The aim of this study was to investigate the nature of the soluble N pool in forest soils and to determine the relative rate of inorganic N production from high and low molecular weight (MW) dissolved organic nitrogen (DON) compounds in black spruce forest soils. DON was found to be the dominant N form in soil solution, however, most of this DON was of high MW of which >75% remained unidentified. Free amino acids constituted less than 5% of the total DON pool. The concentration of NO₃⁻ and NH₄⁺ was low in all soils but significantly greater than the concentration of free amino acids. Incubations of low MW DON with soil indicated a rapid processing of amino acids, di- and tri-peptides to NH₄⁺ followed by a slower transformation of the NH₄⁺ pool to NO₃⁻. The rate of protein transformation to NH₄⁺ was slower than for amino acids and peptides suggesting that the block in N mineralization in taiga forest soils is the transformation of high MW DON to low MW DON and not low MW DON to NH₄⁺ or NH₄⁺ to NO₃⁻. Calculated turnover rates of amino acid-derived C and N immobilized in the soil microbial biomass were similar with a half-life of approximately 30 d indicating congruent C and N mineralization.     

Genetic variation in pea seed globulin composition

A quantitative characterization of seeds from 59 pea (Pisum sativum L.) lines and relative taxa with various external characteristics and wide geographical origin was performed to explore the genetic variation of pea concerning its starch and protein contents and globulin composition. Pea lines, which produce round, wrinkled, flat, and round-dimpled seeds, have starch as the major reserve, with an average content of 46%. Protein content varied from 13.7 to 30.7% of the seed dry matter, with an overall average of 22.3%. Densitometric quantification of the individual globulins (legumin, vicilin, convicilin, and globulin-related proteins) based on SDS-PAGE gels showed no lines lacking any particular globulin. Among the lines tested, variation was shown in both their total globulins content and their globulin composition. The total globulin content ranged from 49.2 to 81.8% of the total pea protein extract (TPPE). Legumin content varied between 5.9 and 24.5% of the TPPE. Vicilin was the most abundant protein of pea, and its content varied between 26.3 and 52.0% of the TPPE. Both processed and nonprocessed vicilins occurred. The processed vicilin was the predominant one, with values between 17.8 and 40.8%, whereas the nonprocessed ones constituted between 3.1 and 13.5% of the TPPE. Convicilin was the least abundant globulin, and its content ranged from 3.9 to 8.3%. Finally, the globulin-related proteins were present in amounts ranging from 2.8 to 17.3%. They were less abundant in comparison with legumin and vicilin, but they showed the largest relative variation of the four globulin classes. Correlations between the different external characteristics and globulin composition were determined. Comparison with soybean showed that pea lines show more variety in the abundance of globulin proteins, enabling a wider range of food application.     

Amino acid metabolism in plant leaf

Recent studies have shown that the incorporation of ammonium nitrogen into amino acids in the leaves is strictly dependent on light (1-4). It is speculated that the effect of light on ammonium assimilation may be through the synthesis of the precursors of amino acids, or by the supply of the energy required for amination and amidation with organic acids. In the Vicia faba chloroplasts Givan et al. (1) exhibited that the synthesis of glutamic acid from a-ketoglutarate was linked with the generation of reduced pyridin nucleotide by photosynthetic electron transport. Mitchell and Stocking (2) suggested the direct coupling of glutamine formation with photophosphorylation in the pea chloroplasts. On the other hand. the processes of nitrate assimilation are more indebted to light than those of ammonium assimilation, because the former ones involve the reduction of nitrate to ammonium which is believed to be light-dependent (5). Canvin and Atkins (6). and Atkins and Canvin (7) reported that the incorporation of ¹⁵N-labeled ammonium and nitrate into amino acid fractiom was depressed by the dark treatment and by photosystem inhibitors; 3-(3′,4′-dichlrophenyl)-1-1-dimethylurea (DCMU) and carbonyl-cyanide-m-chlorophenyl-hydrazone(CCCP).     

Amino acid metabolism in plant leaf

¹⁴C-labelled sodium bicarbonate and ¹⁵N-labelled ammonium sulfate were simultaneously vacuum-infiltrated into detached sunflower leaves, and the incorporation of ¹⁴C and ¹⁵N into free amino acids was chased during 60-min period in the light and in the dark.

In the light, the ue specific activity of aspartic acid, alanine, serine and glycine rapidly increased for 5 min and thereafter decreased. On the other hand, that of gultamic acid continued to increase slowly during the entire 60-min period. In the dark, aspartic acid most actively incorporated ¹⁴C. The difference of changes in ¹⁴C specific activity between glutamic acid and other amino acids was also observed in the dark as in the light. These results suggest that the carbon skeleton of glutamic acid is synthesized from aspartic acid, alanine, serine and glycine.

¹⁵N content of glutamine was the highest of all amino acids investigated in the light, and it was followed by glutamic acid. alanine, aspartic acid, serine and glycine, in this order. In the dark, ¹⁵N content of glutamic acid fell remarkably and was lower than that of alanine up to 5 min. From these ¹⁵N tracer experiments, it is suggested that the incorporation of ammonium into glutamic acid is strictly dependent on light and that alanine incorporates ammonium by the direct amination besides the transamination from glutamic acid.     

Abundant class III acidic chitinase homologue in tamarind (Tamarindus indica) seed serves as the major storage protein

The phyla Leguminosae contains protease inhibitors, lectins, chitinases, and glycohydrolases as major defense proteins in their seeds. Electrophoretic analysis of the seed proteins of tamarind ( Tamarindus indica L.), an agri-waste material, indicated the unusual presence of two major proteins comparable to overexpression of recombinant proteins. These proteins were identified by amino-terminal analysis to be (1) Kunitz-type trypsin inhibitor and (2) class III endochitinase (34000 Da). These two proteins were purified to apparent homogeneity by a single-step chitin bead affinity chromatography and characterized. The Kunitz inhibitor was specific toward inhibiting trypsin with a stoichiometry of 1:1. The 33000 +/- 1000 Da protein, accounting for >50% of the total seed protein, is an acidic glycoprotein exhibiting a very low endotype hydrolytic activity toward chitin derivatives. SDS-PAGE followed by densitometry of tamarind seed germination indicates the disappearance of the chitinase with the concomitant appearance of a cysteine endopeptidase. On the basis of its abundance, accumulation without any pathogenesis-related stimulus, temporal regulation, amino acid composition, and very low enzyme activity, this 34000 Da protein designated "tamarinin" physiologically serves as the major storage protein.     

Amino acid cycling in three cold-temperate forests of the northeastern USA

Amino acids play a critical role in soil-N cycling. Much of the current research on amino acid cycling has been conducted in arctic, alpine, boreal, and temperate grassland ecosystems. There are no comparable data for temperate forests. We quantified the concentration and production of amino acid N and inorganic N in three forests varying in parent material and tree species composition in Connecticut, USA. Soil samples were collected on three sample dates in 2001 and 2002. At all three sites, a pool of free amino acids was present in soil on all sample dates. Among-site differences in the production of amino acids were related to variations in the activity of proteolytic enzymes, the sensitivity of proteolytic enzymes to the availability of protein substrate, and the presence or absence of a surface organic horizon. Among-site differences in amino acid turnover appeared to be at least partially related to soil C-to-N ratios and their effect on C vs. N limitation to microbial function. Amino acid concentrations in the top 15 cm of mineral soil in these study sites fell within the range of reported values for ecosystems spanning a wide latitudinal gradient, including ecosystems in which amino acids are thought to contribute substantively to plant-N nutrition. The concentration of amino acid N in the organic horizons of these study sites was considerably higher than those reported in the literature. The implications of the results for N capture by temperate forest trees are discussed.     

Isolation and computer analysis of the 5'-regulatory region of the seed storage protein gene from buckwheat (Fagopyrum esculentum Moench)

Using the modified rapid amplification of cDNA ends (5'-RACE) approach, a fragment containing the 955 bp long 5'-regulatory region of the buckwheat storage globulin gene (FeLEG1) has been amplified from the genomic DNA of buckwheat. The entire fragment was sequenced, and the sequence was analyzed by computer prediction of cis-regulatory elements possibly involved in tissue-specific and developmentally controlled seed storage protein gene expression. The promoter obtained might be interesting not only for fundamental research but also as a useful tool for biotechnological application.     

Comparison of interlaboratory variation in amino acid analysis and rat growth assays for evaluating protein quality

Estimates of inter- and intralaboratory variation of protein efficiency ratio (PER), relative PER (RPER), net protein ratio (NPR), relative NPR (RNPR), and nitrogen utilization (NU) were compared with those of amino acid analysis in the same batches of 7 protein sources (ANRC casein, egg white solids, minced beef, soy assay protein, rapeseed protein concentrate, pea flour, and whole wheat flour). Interlaboratory variation (estimated as between-laboratories coefficients of variation, CV) of NPR and RNPR (up to 6.0%) was lower than that of PER (up to 20.2%) and RPER (up to 18.5%). The interlaboratory determination of NPR and RNPR was also more reproducible than that of most essential amino acids (CV up to 10.0%), especially tryptophan (CV up to 23.7%), cystine (CV up to 17.6%), and methionine (CV up to 16.1%). Intralaboratory variation (estimated as within-laboratories CV) of amino acid analysis (up to 4.7%), however, was comparable to that of protein quality indices in most protein sources (up to 6.0%). The significant (P less than 0.01) positive correlations (r = 0.68-0.74) between amino acid scores and protein quality indices based on rat growth were further improved when amino acid scores were corrected for digestibility of protein (r = 0.73-0.78) or individual amino acids (r = 0.79-0.82).     

Evaluating the quality of protein from hemp seed (Cannabis sativa L.) products through the use of the protein digestibility-corrected amino acid score method

The macronutrient composition and the quality of protein of hemp seed and products derived from hemp seed grown in Western Canada were determined. Thirty samples of hemp products (minimum 500 g), including whole hemp seed, hemp seed meal from cold-press expelling, dehulled, or shelled, hemp seed and hemp seed hulls, were obtained from commercial sources. Proximate analysis, including crude protein (% CP), crude fat (% fat) and fiber, as well as full amino acid profiles, were determined for all samples. Protein digestibility-corrected amino acid score (PDCAAS) measurements, using a rat bioassay for protein digestibility and the FAO/WHO amino acid requirement of children (2-5 years of age) as reference, were conducted on subsets of hemp products. Mean (±SD) percentage CP and fat were 24.0(2.1) and 30.4(2.7) for whole hemp seed, 40.7(8.8) and 10.2(2.1) for hemp seed meal, and 35.9(3.6) and 46.7(5.0) for dehulled hemp seed. The percentage protein digestibility and PDCAAS values were 84.1-86.2 and 49-53% for whole hemp seed, 90.8-97.5 and 46-51% for hemp seed meal, and 83.5-92.1 and 63-66% for dehulled hemp seed. Lysine was the first limiting amino acid in all products. Removal of the hull fraction improved protein digestibility and the resultant PDCAAS value. The current results provide reference data in support of protein claims for hemp seed products and provide evidence that hemp proteins have a PDCAAS equal to or greater than certain grains, nuts, and some pulses.     

Sampling variation in genetic resources of seed crops: a review

Summary The first explorations for the collection of genetic resources of crop plants took place in areas designated as centers of origin. Later, important characters were found to be present in areas other than those indicated by Vavilov. Hence collection missions were extended to cover other areas in addition to the centers of diversity with different ecogeography and agromorphological features. Collecting activities were aimed not only at providing germplasm for immediate use in breeding but also at conserving genetic variation for future contingencies. The analysis of the collected material has revealed the complex nature, amount and distribution of genetic variation present in natural populations as well as in landraces, old varieties and primitive forms. The study of variation for characters from different regions indicated that it is possible to select certain characters for detecting variability in the populations and establishing the minimum number of genotypes to be sampled. A minimum number of 20–130 plants per sample, depending on the crop, is recommended in order to capture 95% of the total variation in the field. However, it has been emphasized that the number of different populations sampled rather than the sample size per population determines the overall efficiency of a collection. The findings reported allow better utilization of the collected material and formulation of optimal and cost-effective sampling strategies for the future.     

A 50 kDa maize gamma-zein has marked cross-reactivity with the almond major protein

Cross-reactivity of antibodies against almond major protein (AMP, a legumin), the major almond allergen, with cereal proteins may cause problems in detecting almond contaminants in cereal products when antibody-based assays are used. Rabbit polyclonal IgG antiserum produced against AMP was used to test cross-reactivity with protein extracts from maize, a cereal commonly found in breakfast and snack foods. Gradient SDS-PAGE followed by Western blotting was performed, and two cross-reactive proteins were detected by chemiluminescence. A fraction of maize proteins purified by elution from an IgG anti-AMP affinity column followed by electrophoreseis and immunoblotting showed a high degree of cross-reactivity with a minor 50 kDa protein of maize, as well as low cross-reactivity with the 27 kDa gamma-zein. The 50 kDa cross-reactive protein was identified as the 50 kDa gamma-zein by immunoreaction with anti-50 kDa gamma-zein antiserum. Notably, the 50 kDa maize gamma-zein also reacted with IgE from pooled human sera from patients with self-reported severe almond allergies. The high immunoreactivity of the 50 kDa gamma-zein should be considered in maize quality improvement programs, and such notable cross-reactivity is of relevance in the design of antibody-based assays for almond allergen detection.     

S02-induced enzymatic changes and ascorbic acid oxidation in Oryza sativa

Field grown rice (Oryza sativa L.) plants exposed separately to 0.25 or 0.5 ppm SO₂ for 1.5 hr daily for 40 days, showed significant decrease of catalase (p < 0.001) and increase of peroxidase (p < 0.001) activities as well as decreases of protein (p < 0.001) and ascorbic acid (p < 0.001) contents associated with leaf lesions, which were proportional to SO₂-dose. Catalase and peroxidase activity levels showed an inverse relationship. It is hypothesized from the molecular structure of both enzymes and from the in vitro relationship between catalase and peroxidase activity that the tetrameric molecules of catalase in vivo might disintegrate into monomeric units with peroxidase activity, which in turn oxidise ascorbic acid and may reduce tolerance of plants to SO₂.     

Identification and variation of glutelin alpha polypeptides in the genus Oryza assessed by two-dimensional electrophoresis and step-by-step immunodetection

To obtain fundamental information for nutritional improvement of rice (Oryza sativa) seed proteins, the alpha polypeptides of the major storage protein glutelin varied over the genus Oryza were qualitatively and quantitatively characterized with unique methods. The polypeptides were maximally separated by two-dimensional electrophoresis (2D-PAGE) composed of nonequilibrium pH gradient gel electrophoresis (NEPHGE) and higher temperature SDS-PAGE. Then the subunit for each polypeptide spot was identified with the sequential immunodetection called a step-by-step detection method, making use of highly subunit-specific antibodies. The comparative analysis showed considerable variation in the accumulation level of A-type and B-type glutelin subunits and found unknown glutelin subunits that were unable to be identified with the antibodies used. Wild species accumulating a high amount of lysine-rich B-type glutelin subunits and unknown unique subunits were identified as they might play a crucial role in nutritional quality improvement of the cultivated rice.     

Assessment of the protein quality of nine northern adapted yellow and brown seed coated soybean cultivars by amino acid analysis

Accurate and detailed amino acid determinations were carried out on nine northern adapted soybean cultivars to ascertain whether their amino acid profiles could be used as potentially useful indices for assessing their protein quality. The cultivars were Maple Amber, Maple Donovan, Maple Glen, Maple Isle, Maple Presto, Maple Ridge, and three brown seed coat near-isogenic lines, Maple Presto Brown, Maple Ridge Brown, and Maple Arrow Brown. Their total protein and amino acid composition were compared with those of an established cultivar, Maple Arrow. Mean protein values for the new cultivars ranged from 30.1 to 33.1% compared to Maple Arrow, which was 33.2%. The total nitrogen content was also variable among these cultivars, ranging from 5.0 to 5.4%. All nine Maple series soybean cultivars were higher in their essential amino acid (EAA) content, that is, EAA(9) = 45.2-46.5%, than the FAO/WHO reference protein pattern value of EAA(9) = 33.9%, for a 2-5-year-old child. Each of the nine new soybean cultivars was limited only in methionine and to a lesser extent in valine and isoleucine and had a protein digestibility corrected amino acid score of 91% for all cultivars, compared to the value of egg protein (97%). These results suggest that the most accurate evaluation of protein quality in soybeans, and possibly other legumes and cereals, is by the protein digestibility-corrected amino acid score.