Sulfur Amino Acid Content Research Articles

Folate‐independent remethylation of homocysteine is increased by dietary egg white protein

Proper maintenance and regulation of methyl group and homocysteine (hcy) metabolism is essential for optimization of health. Methyl deficiency and/or elevations in plasma hcy concentrations have been associated with numerous pathologies, such as vascular disease. It is important to identify dietary components that positively impact these metabolic pathways; thus, we examined dietary protein sources with varying sulfur amino acid content. Rats were fed either a casein or egg white protein (20%) diet for 2 wk after which half the rats in each group were fed folate‐deficient diets for 4 wk. Compared to casein, egg white protein resulted in: (1) elevated hepatic betaine‐hcy S‐methyltransferase activity, responsible for folate‐independent hcy remethylation; (2) increased hepatic activity of glycine N‐methyltransferase, a key methyl group regulatory protein; and (3) reduced plasma glutathione concentrations. Moreover, folate deficiency was without affect on plasma hcy levels in the egg white group. In a parallel study, soy protein did not differ from casein with respect to altering methyl group and hcy metabolism. In conclusion, dietary egg white protein appears to promote hcy remethylation at the expense of catabolism via transsulfuration. These findings may be attributed to the greater met‐cys content of egg white protein compared to casein and soy (Support: Amer. Egg Board, Egg Nutr. Center, IA Egg Council).

3T3-L1 Adipocytes and Rat Adipose Tissue Have a High Capacity for Taurine Synthesis by the Cysteine Dioxygenase/Cysteinesulfinate Decarboxylase and Cysteamine Dioxygenase Pathways

Taurine is the most abundant free amino acid in the body and is synthesized in mammals by 2 pathways. Taurine is synthesized either from the oxidation of cysteine via cysteine dioxygenase (CDO), which generates cysteinesulfinate that is decarboxylated by cysteinesulfinic acid decarboxylase (CSAD), or from the oxidation of cysteamine by cysteamine (2-aminoethanethiol) dioxygenase (ADO). Both pathways generate hypotaurine, which is oxidized to taurine. To determine whether these pathways for taurine synthesis are present in the adipocyte, we studied 3T3-L1 cells during their adipogenic conversion and fat from rats fed diets with varied sulfur-amino acid content. CDO, CSAD, and ADO protein levels increased during adipogenic differentiation of 3T3-L1 cells and all of these enzymes were significantly increased when cells achieved a mature adipocyte phenotype. Furthermore, these changes were accompanied by an increased hypotaurine and taurine production, particularly when cells were treated with cysteine or cysteamine. CDO mRNA levels also responded robustly to cysteine or cysteamine treatment in adipocytes but not in undifferentiated 3T3-L1 cells. Furthermore, CDO protein and activity were greater in adipose tissue from rats fed a high protein or cystine-supplemented low protein (LP) diet than in adipose tissue from rats fed a LP diet. Overall, our results demonstrate that CDO is regulated at both the level of enzyme abundance and the level of mRNA in mature adipocytes.

Feeding layers of different genotypes in organic feed environments

1. Two experiments were carried out to evaluate the suitability of an experimental laying hen genotype (SH) in organic egg production. SH had been selected over 25 generations on a low protein diet (130 g/kg) based on home grown feedstuffs. SH hens were compared with LSL (Experiment 1) and Hyline (Experiment 2) hens. The birds were kept in 12 aviary pens with access to outdoor runs. 2. Four diets, which were different in each experiment, were used. A control diet was compared with diets based on feedstuffs supposed to be able to be grown and produced according to organic standards. The diets differed in the content of sulphur amino acids. 3. There were no significant differences in mortality caused by diets or genotypes but an incidence of cannibalism occurred in one Hyline group fed on the diet with the lowest methionine content. 4. Severe feather pecking of LSL birds occurred and was worst on the low protein diet. In Experiment 2, signs of feather pecking of birds on the low protein diet occurred at the end of the experiment, but there was no difference between the genotypes. There was no significant difference in number of eggs produced between diets. Egg weight was influenced by diets in both experiments and by genotype in Experiment 2. Number of misplaced eggs was higher in SH hens in both experiments. There were few significant diet × genotype interactions. 5. The use of the outdoor area was influenced by diets and genotype in both experiments. 6. In conclusion, diets low in methionine content influenced predominantly plumage condition and egg weight, although production of eggs by number was unaffected. The SH hen adapted well to organic conditions, as did the Hyline genotype.

Some physicochemical and antinutritional properties of raw flours and protein isolates from Mucuna pruriens (velvet bean) and Canavalia ensiformis (jack bean)

SummaryThe legumes Canavalia ensiformis and Mucuna pruriens are underexploited in tropical Mexico. Their seeds have good nutritional potential, but contain antinutritional factors. Physicochemical and antinutritional properties were determined for raw flours (RF) and protein isolates (PI) produced from these legumes. Protein content in the PI was 737 g kg–1 for C. ensiformis and 666 g kg–1 for M. pruriens. Protein isolation improved in vitro digestibility, while maintaining high lysine levels and adequate sulphur amino acids content. Antinutritional factors such as cyanogenic glucosides, cyanide precursors from hydrolysis, tannins and trypsin inhibitors were lower in the PIs than in the RFs. The reduction in canavanine levels, a structural analogue of arginine, in the C. ensiformis PI was noteworthy. These PIs thus have potential applications in the development of new food ingredients in tropical regions using processes that improve nutritional value.

Dl-Methionine Is as Efficacious as l-Methionine, but Modest l-Cystine Excesses Are Anorexigenic in Sulfur Amino Acid-Deficient Purified and Practical-Type Diets Fed to Chicks

Three bioassays were conducted to elucidate the effects of dietary cystine on the efficacy of L-Met and DL-Met fed to chicks. In assay 1, a purified diet markedly deficient in Met (0.12%) and cyst(e)ine (0.05%) was used to compare the relative effectiveness of L-Met and DL-Met in the presence of graded levels of dietary cystine. Addition of 0.05% Met improved (P < 0.01) weight gain when added to diets with 0 or 0.07% added L-cystine, but weight gain decreased linearly (P < 0.01) with greater cystine supplementation up to 0.35%, regardless of Met supplementation. There were no differences in growth performance due to supplementation of L-Met vs. DL-Met. In assay 2, a corn-soybean meal-peanut meal diet (0.25% Met and 0.25% cyst(e)ine) was supplemented with 0, 0.025, 0.05, or 0.075% L-Met plus 0 or 0.2% added L-cystine. Supplemental Met improved (P < 0.01) growth performance, but weight gain and feed intake were depressed (P < 0.01) by cystine supplementation. Whereas 0.2% added L-cystine depressed feed intake 6.9%, weight gain was reduced only 3.6%. Thus, cystine supplementation actually improved (P < 0.01) gain:feed. In assay 3, the corn-soybean meal-peanut meal diet was supplemented with 0 or 0.03% L-Met or DL-Met in the absence or presence of 0.2% added L-cystine. Again, Met supplementation improved (P < 0.01) growth performance, whereas supplemental cystine reduced (P < 0.05) weight gain and feed intake, but increased (P < 0.01) gain:feed. From these bioassays, it may be concluded that there is no evidence to suggest differences in effectiveness between L-Met and DL-Met in purified or practical-type low-protein diets of varying sulfur amino acid (SAA) content fed to chicks from 8 to 20 d of age. However, supplemental cystine has a negative impact on voluntary feed intake when supplemented in diets markedly deficient in SAA. This effect is evident with minimal SAA intake and when greater than 50% of SAA intake is provided by cyst(e)ine, rather than Met.

Analyzing Sulfur Amino Acids in Selected Feedstuffs Using Least-Squares Nonlinear Regression

Five feedstuffs were oxidized using performic acid, and these, along with their unoxidized counterparts, were acid hydrolyzed for multiple times (0-144 h) in degassed and vacuum-sealed glass tubes. The methionine sulfone, cysteic acid, methionine, and cysteine contents were determined for each hydrolysis time. Least-squares nonlinear regression of the sulfur amino acid contents and hydrolysis time was used to predict the actual sulfur amino acid content as well as the hydrolysis and loss rates. Least-squares nonlinear regression estimates for methionine content compared well with those of methionine sulfone for most of the feedstuffs tested. In contrast, the estimates for cysteine agreed poorly with cysteic acid. The loss rates during acid hydrolysis for methionine, methionine sulfone, and cysteic acid were low. Overall, acid hydrolysis in an evacuated sealed tube for 24 h without prior oxidation is suitable for methionine, but not cysteine, quantitation in some complex feedstuffs.

Replacement of Fish Meal with Soybean Meal in the Production Diets of Juvenile Red Snapper, Lutjanus campechanus

The replacement of fish meal with soybean meal in fish diets has met with varying degrees of success. Quite often, poor responses to high soybean meal diets are either due to shifts in the nutrient profile or a reduced palatability of the diet when fish meal is removed. The present research was designed to evaluate the replacement of menhaden fish meal with solvent-extracted soybean meal in practical diets containing 10% poultry by-product meal and formulated to contain 40% protein, 8% lipid, and a total sulfur amino acid content of > 3.0% of the protein. The response of red snapper (mean initial weight 10.9 g) to diets containing graded levels of fish meal (30,20, 10, 0%) as well as the response to a low fish meal diet (10%) without poultry by-product meal were evaluated over a 6-wk growth period. Significant (P± 0.05) differences in final mean weight, percent weight gain, and feed conversion were observed. Final weights (percent gain) ranged from 30.9 g (185.5%) for fish offered diets with 30% fish meal to 12.6 g (16.3%) for fish offered diets with 0% fish meal. Corresponding feed conversion efficiencies ranged from 60.1% to 7.7%. No significant differences were observed for survival between treatment means. Although there was a clear reduction in performance as the fish meal was replaced with soybean meal, the use of 10% poultry by-product meal or 10% fish meal resulted in similar performance of the fish. This is a good indication that poultry by-product meal does not have palatability problems and could be used as a substitute. The present findings suggest that replacing fish meal with high levels of soybean meal appears to reduce the palatability of the diet. While the cost reducing benefit, with respect to the replacement of fish meal, has been shown with other species, before high levels of inclusion can be efficiently utilized further research is needed to address the palatability problems observed with red snapper.

A Chemically Defined Diet With Insufficient Sulfur Amino Acids Induces Oxidation of Plasma Cysteine/Cystine and Glutathione/Glutathione Disulfide Redox State in Humans

Sulfur amino acid (SAA) availability is critical for glutathione (GSH) synthesis. GSH has an important role in detoxification and maintenance of intracellular glutathione/glutathione disulfide (GSH/GSSG) redox as well as extracellular cysteine/cystine (Cys/CySS) redox in vitro. Intermittent periods of SAA insufficiency are common due to food selection, dieting, and fasting. To examine the effects of dietary SAA insufficiency on redox status in vivo, young, healthy volunteers were studied on 2 separate occasions for 60 hours in the General Clinical Research Center (GCRC) at Emory University Hospital. During each study period, volunteers consumed 1 of 2 chemically defined diets modified for SAA content (100% or 0% of the Recommended Dietary Allowance for SAA intake) for 3 days. Blood was drawn hourly for 12 hours on day 3. Results from 4 individuals show that consumption of the 0% SAA diet resulted in a significant oxidation (~ 9 mV) of plasma Cys/CySS redox status as compared to consumption of the 100% SAA diet (−73± 0.8 mV vs. −82± 0.8 mV respectively). In contrast, consumption of the 0% SAA diet did not affect plasma GSH/GSSG redox status as compared to control diet (−123± 2 mV vs. −124.3± 1 mV respectively). We conclude that 3-day consumption of a diet insufficient in SAA induces significant oxidation of the plasma Cys/CySS redox state while having no affect on plasma GSH/GSSG redox status in humans. Support: NIEHS RO1 09047.

Nutrient retention capabilities of Nile tilapia ( Oreochromis niloticus) fed bio-regenerative life support system (BLSS) waste residues

Nile tilapia were evaluated as a bio-regenerative sub-process for reducing solid waste potentially encountered in bio-regenerative life support systems. Ten juvenile Nile tilapia (mean weight = 2.05 g) were stocked into triplicate aquaria and fed one of seven experimental diets consisting of vegetable, bacterial, or food waste for a period of seven weeks. Weight gain (g), specific growth rate (mg/d), and daily consumption (g) was significantly higher ( p < 0.05) in the control group (13.80, 281.60, and 47.49, respectively) followed by the wheat bran/wheat germ group (4.25, 86.87, and 24.24). Carbon and crude lipid retention was significantly higher ( p < 0.001) in fish fed the control diet (37.99 and 68.54, respectively) followed by fish fed the wheat bran/wheat germ diet (23.19 and 63.67, respectively). Nitrogen, sulfur, and crude protein retention was significantly higher ( p < 0.001) in fish fed the wheat bran/wheat germ group (40.73, 98.65, and 40.75, respectively) followed by fish fed the control diet (23.68, 21.89, and 23.68, respectively). A general loss of minerals was observed among all groups. Strong associations were observed between crude lipid retention and sulfur retention ( r 2 = 0.94), crude lipid retention and carbon retention ( r 2 = 0.92), WG and fiber content of dietary treatments ( r 2 = 0.92), WG and carbon retention and ( r 2 = 0.88), WG and lysine content of waste residues ( r 2 = 0.86), crude protein retention and carbon retention ( r 2 = 0.84), sulfur retention and crude protein retention ( r 2 = 0.84), and total sulfur amino acid (TSAA) content of residues and WG ( r 2 = 0.81). Weaker associations existed between WG and crude lipid retention ( r 2 = 0.77), crude fiber content and carbon retention ( r 2 = 0.76), and WG and methionine content of waste residues ( r 2 = 0.75). Additional research is needed to improve the nutritional quality of fibrous residues as a means to improve tilapia’s ability to utilize these residues as a food source in bio-regenerative support systems.

Critical appraisal of the management of severe malnutrition: 2. Dietary management

In the dietary management of severe acute malnutrition in children, there is evidence to support the WHO Manual's protocol of cautious feeding of a low energy and protein formula with small frequent feeds in the initial phase of treatment, particularly in kwashiorkor. However, this initial milk diet (WHO F-75) might benefit from increasing the sulphur amino acid, phosphorus and potassium content and reducing the lactose content, but further studies are needed. Careful tube-feeding results in faster initial recovery and weight gain, but has a significant risk of aspiration in poorly supervised settings. Ready-to-use therapeutic food is an important recent advance in the dietary management of malnutrition in ambulatory settings, allowing more effective prevention programmes and earlier discharge from hospital where community follow-up is available. It should be included in future protocols. There is very good evidence on the use of micronutrients such as zinc, and preliminary evidence suggests that smaller doses of daily vitamin A are preferable to a single large dose on admission for severe malnutrition.

Preparation of a rice bran enzymatic extract with potential use as functional food

The production, stabilization, by enzymatic treatment, physicochemical composition, and biological properties (including the anti-proliferative activity), of a water-soluble rice bran enzymatic extract (RBEE) are described. The main component of RBEE is proteins (38.1%) – in the form of peptide and free amino acids – having a 6% content of sulfur amino acids. The second component is fat (30.0%), with oleic and linoleic acids as the major components, and 1.2 mg/g of γ-oryzanol. Carbohydrates (14.2%) are comprised mainly of slowly absorbed carbohydrates. Preliminary studies on the anti-proliferative effect of RBEE on leukemia tumor cell growth in vitro are also reported. This property makes RBEE potentially useful as a functional food for the treatment and prevention of chronic pathological states associated with abnormal proliferation of cells, as is the case with cancer.

Nitrogen Lowers the Sulfur Amino Acid Content of Soybean (Glycine max [L.] Merr.) by Regulating the Accumulation of Bowman−Birk Protease Inhibitor

Soybeans in general contain 35-40% protein. Efforts are underway to increase further this protein content, thus enhancing their nutritive value. Even though higher protein is a desirable characteristic, whether such an increase will be accompanied by enhanced protein quality is not known. Soybean protein quality could be significantly improved by increasing the concentration of the sulfur-containing amino acids, cysteine and methionine. To ascertain if a correlation existed between protein quantity and quality, a comparison of the amino acids of soybeans differing in protein content was made. Soybeans with higher protein content had a significantly lower percentage of sulfur amino acids, while those with lower protein exhibited a higher content of cysteine and methionine. Nitrogen application elevated the protein content but lowered that of the sulfur amino acids. Transmission electron microscopy examination of thin sections of low protein soybean seeds revealed several protein storage vacuoles that were partially filled with storage proteins. Fluorescence two-dimensional difference gel electrophoresis of soybean seed proteins revealed that nitrogen application favored the accumulation of the beta-subunit of beta-conglycinin while decreasing the accumulation of Bowman-Birk protease inhibitor (BBI), a protein rich in cysteine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 60% 2-propanol-extracted proteins showed a drastic reduction in the accumulation of BBI with increasing protein content. Northern blot analysis indicated that nitrogen had a negative influence on the expression of the BBI gene. Our results indicate that the negative correlation between total protein and sulfur amino acid content is mostly mediated by the differential accumulation of BBI.

Combined Proteome and Metabolite-profiling Analyses Reveal Surprising Insights into Yeast Sulfur Metabolism

Metabolomics is considered as an emerging new tool for functional proteomics in the identification of new protein function or in projects aiming at modeling whole cell metabolism. When combined with proteome studies, metabolite-profiling analyses revealed unanticipated insights into the yeast sulfur pathway. In response to cadmium, the observed overproduction of glutathione, essential for the detoxification of the metal, can be entirely accounted for by a marked drop in sulfur-containing protein synthesis and a redirection of sulfur metabolite fluxes to the glutathione pathway. A kinetic analysis showed sequential and dramatic changes in intermediate sulfur metabolite pools within the first hours of the treatment. Strikingly, whereas proteome and metabolic data were positively correlated under cadmium conditions, proteome and metabolic data were negatively correlated during other growth conditions, i.e. methionine supplementation or sulfate starvation. These differences can be explained by alternative mechanisms in the regulation of Met4, the activator of the sulfur pathway. Whereas Met4 activity is controlled by the cellular cysteine content in response to sulfur source and availability, the present study suggests that Met4 activation under cadmium conditions is cysteine-independent. The results clearly indicate that the metabolic state of a cell cannot be safely predicted based solely on proteomic and/or gene expression data. Combined metabolome and proteome studies are necessary to draw a comprehensive and integrated view of cell metabolism.

Nutritional and protein quality evaluation of thermally treated seeds of Canavalia maritima in the rat

Roasted and pressure-cooked seeds of Canavalia maritima of coastal sand dunes of the southwest coast of India were assessed for proximate and mineral composition, amino acid and fatty acid profiles and antinutritional features. Growth and nitrogen balance studies were carried out to assess the nutritive value of thermally treated seeds in the rat. Proteins and energy values of Canavalia seeds surpassed common pulse crops. The carbohydrates and ratio of polyunsaturated-saturated fatty acids of the test seed were superior to soybean. The essential amino acids of treated seeds were higher than the Food and Agriculture Organization (FAO)/ World Health Organization (WHO)/ United Nations University (UNU) pattern recommended for adults. The sulfur amino acid content exceeded that for rice, soybean, and FAO/WHO reference pattern. Phenylalanine (roasted and pressure-cooked seeds) and lysine (roasted seeds) was also higher than the FAO/WHO pattern. The food efficiency ratio, net protein retention, protein retention efficiency, and biological value were significantly different between thermal treatments ( P < .05). Pressure-cooked seeds exhibited better biological indices than roasted seeds. Partial detoxification of lectins by thermal treatments in the current study resulted in low protein quality, suggesting that a more appropriate method of processing is needed to eliminate the antinutritional factors. This is the first study providing data on the biochemical and protein quality of thermally treated seeds of C maritima.

Engineering Soybean for Enhanced Sulfur Amino Acid Content

Soybean [Glycine max (L.) Merr.] is an important protein source in human diets and animal rations. Additionally, human consumption of soybean protein is reported to provide specific health benefits. Since methionine and cysteine contents limit the nutritional value of soybean, efforts involving both traditional breeding and genetic engineering have been employed in attempts to increase the presence of these essential amino acids. Traditional breeding has been primarily utilized to increase the total protein content but not to enhance the sulfur amino acid content of soybean. Although mutagenesis in conjunction with traditional breeding is a viable approach for enhancing the sulfur amino acid content of soybean, genetic engineering appears to be a more realistic approach. Introduction of methionine‐rich heterologous proteins has resulted in a modest increase of this amino acid in soybean. Either elevating the expression of endogenous methionine‐rich proteins or introducing synthetic proteins containing a high percentage of essential amino acids are other possible approaches that may increase the nutritional quality of the seed. Even though considerable progress has been made in enhancing the methionine content of soybean, several obstacles remain. A thorough understanding the sulfur assimilatory pathway in soybean is a prerequisite for improving the sulfur amino acid content. Expression of feedback‐insensitive forms of serine acetyl transferase and O‐acetylserine (thiol) lyase, two key enzymes in sulfur assimilatory pathway, could lead to an increase in the availability of sulfur amino acids. An adequate supply of sulfur amino acids in developing seeds may facilitate accumulation of sulfur‐rich proteins to a level sufficient to meet the nutritional requirement of livestock and poultry.

Neurolathyrism risk depends on type of grass pea preparation and on mixing with cereals and antioxidants

To study an array of household individual and dietary risk factors for neurolathyrism. Case-control study using recent cases in a district highly affected by the recent neurolathyrism epidemic in Ethiopia: 108 households with cases and 104 households with no cases; 170 neurolathyrism cases, 370 intra-family controls and 170 community controls frequency matched for age and sex. A history of acute illness was associated with a two- and threefold increased risk of paralysis in community and intra-family controls, respectively. Soaking grass pea in water before cooking roughly halved the risk of neurolathyrism but cooking in clay utensils more than quadrupled it. Consumption of grass pea in the green unripe and boiled forms increased the risk 10 times or more. Mixing the food with gravy that contains condiments with antioxidant activity reduced it by a factor of 4. The consumption of grass pea mixed with cereals rich in sulphur amino acids was also highly protective, but the magnitude of the effect depended on the grass pea preparation consumed. Consumption of pure grass pea, especially in the green unripe and boiled forms, should be avoided. Communities at risk of neurolathyrism during famine crises should be encouraged to combine and use grass pea with cereals before the household cereal stock is fully depleted. Breeding programmes, alongside traditional attempts to reduce the toxin content, should enhance the content of sulphur amino acids and antioxidants in grass pea.

The effect of graded levels of dietary casein, with or without methionine supplementation, on glutathione concentration in unstressed and endotoxin-treated rats.

Glutathione (GSH) concentration was measured in rats fed either graded levels of dietary casein (experiment 1; 180 g, 120 g, 80 g, or 60 g protein/kg diet) or graded levels of dietary casein, supplemented with methionine to equalize dietary sulfur amino acid content to that seen in an 180 g/kg casein diet supplemented with 0.3 g L-methionine/kg diet (experiment 2; 180 g protein +0.3 g L-methionine, 80 g protein +6.70 g L-methionine, or 60 g protein +7.45 g L-methionine/kg diet). Rats were given an inflammatory challenge by intraperitoneal injection of endotoxin (lipopolysaccharide from Escherichia coli), and were compared with ad libitum and pair-fed controls. Glutathione concentration in various organs (liver, lung, spleen, and thymus) decreased in animals fed the low-protein diets (80 g or 60 g/kg diet). Addition of the sulfur amino acid, methionine, to the low-protein diets restored glutathione concentrations in animals fed ad libitum and prevented the fall in GSH concentration, which occurred in lung, spleen, and thymus in response to the endotoxin. Despite the similarity in the amount of sulfur amino acid consumed between the groups fed the 180 g protein +0.3 g L-methionine and the 60 g protein +7.45 g L-methionine/kg diet, in experiment 2, hepatic GSH concentration significantly increased in the latter group, in animals fed ad libitum and in the endotoxin-treated animals, but not in the pair-fed controls.

Sulfur Assimilation in Soybean

Soybean [Glycine max (L.) Merr.] is a good protein source for both humans and livestock. However, soybean seed proteins are deficient in the sulfur‐containing amino acids cysteine and methionine. This deficiency has stimulated efforts to improve the amino acid composition of soybean seed proteins. Our overall goal is to improve the sulfur amino acid content of soybean seed proteins by genetic manipulation. The objective of this study was to isolate and characterize O‐acetylserine (thiol) lyase (OAS‐TL), a key enzyme that catalyzes the last step in the production of cysteine. A full‐length cDNA clone encoding a cytosolic isoform of OAS‐TL was isolated by screening a soybean seed cDNA library with a 32P‐labeled expressed sequence tag (EST). Nucleotide sequence analysis of the cDNA revealed a single open‐reading frame of 978 base pairs (bp) encoding a 34‐kDa protein. The authenticity of the isolated cDNA was confirmed by the functional complementation of an Escherichia coli cysteine auxotrophic mutant. Reverse transcriptase polymerase chain reaction (RT‐PCR) analysis revealed that OAS‐TL mRNA was abundant at early stages of seed development. Western blot analysis using antibodies generated against the recombinant soybean OAS‐TL demonstrated that the abundance of this protein gradually declined during later stages of seed development. The OAS‐TL activity peaked in young developing seeds and declined steadily during the time period when the bulk of seed storage protein accumulation occurred. Thus, elevating the specific activity of OAS‐TL during later stages of seed development could lead to an increase in cysteine synthesis in soybean seeds.

The redistribution of protein sulfur in transgenic rice expressing a gene for a foreign, sulfur-rich protein.

Sulfur amino acid composition is an important determinant of seed protein quality. A chimeric gene encoding sunflower seed albumin (SSA), one of the most sulfur-rich seed storage proteins identified so far, was introduced into rice (Oryza sativa) in order to modify cysteine and methionine content of the seed. Analysis of a transgenic line expressing SSA at approximately 7% of total seed protein revealed that the mature grain showed little change in the total sulfur amino acid content compared to the parental genotype. This result indicated that the transgenic rice grain was unable to respond to the added demand for cysteine and methionine imposed by the production of SSA. Analysis of the protein composition of the transgenic grain showed changes in the relative levels of the major seed storage proteins, as well as some non-storage proteins, compared to non-transgenic controls. Changes observed at the protein level were concomitant with differences in mRNA accumulation but not always with the level of transcription. The limited sulfur reserves appeared to be re-allocated from endogenous proteins to the new sulfur sink in the transgenic grain. We hypothesize that this response is mediated by a signal transduction pathway that normally modulates seed storage protein composition in response to environmental fluctuations in sulfur availability, via both transcriptional and post-transcriptional control of gene expression.

Limits to Sulfur Accumulation in Transgenic Lupin Seeds Expressing a Foreign Sulfur-Rich Protein

The low sulfur amino acid content of legume seeds restricts their nutritive value for animals. We have investigated the limitations to the accumulation of sulfur amino acids in the storage proteins of narrow leaf lupin (Lupinus angustifolius) seeds. Variation in sulfur supply to lupin plants affected the sulfur amino acid accumulation in the mature seed. However, when sulfur was in abundant supply, it accumulated to a large extent in oxidized form, rather than reduced form, in the seeds. At all but severely limiting sulfur supply, addition of a transgenic (Tg) sink for organic sulfur resulted in an increase in seed sulfur amino acid content. We hypothesize that demand, or sink strength for organic sulfur, which is itself responsive to environmental sulfur supply, was the first limit to the methionine (Met) and cysteine (Cys) content of wild-type lupin seed protein under most growing conditions. In Tg, soil-grown seeds expressing a foreign Met- and Cys-rich protein, decreased pools of free Met, free Cys, and glutathione indicated that the rate of synthesis of sulfur amino acids in the cotyledon had become limiting. Homeostatic mechanisms similar to those mediating the responses of plants to environmental sulfur stress resulted in an adjustment of endogenous protein composition in Tg seeds, even when grown at adequate sulfur supply. Uptake of sulfur by lupin cotyledons, as indicated by total seed sulfur at maturity, responded positively to increased sulfur supply, but not to increased demand in the Tg seeds.