Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine

We have increased the methionine content of the seed proteins of a commercial winter variety of canola by expressing a chimeric gene encoding a methionine-rich seed protein from Brazil nut in the seeds of transgenic plants. Transgenic canola seeds accumulate the heterologous methionine-rich protein at levels which range from 1.7% to 4.0% of the total seed protein and contain up to 33% more methionine. The precursor of the methionine-rich protein is processed correctly in the seeds, resulting in the appearance of the mature protein in the 2S protein fraction. The 2S methionine-rich protein accumulates in the transgenic seeds at the same time in development as the canola 11S seed proteins and disappears rapidly upon germination of the seed. The increase in methionine in the canola seed proteins should increase the value of canola meal which is used in animal feed formulations.     

Enhanced levels of methionine and cysteine in transgenic alfalfa (Medicago sativa L.) plants over-expressing the Arabidopsis cystathionine gamma-synthase gene

With the aim of increasing the methionine level in alfalfa (Medicago sativa L.) and thus improving its nutritional quality, we produced transgenic alfalfa plants that expressed the Arabidopsis cystathionine gamma-synthase (AtCGS), the enzyme that controls the synthesis of the first intermediate metabolite in the methionine pathway. The AtCGS cDNA was driven by the Arabidopsis rubisco small subunit promoter to obtain expression in leaves. Thirty transgenic plants were examined for the transgene protein expression, and four lines with a high expression level were selected for further work. In these lines, the contents of methionine, S-methylmethionine (SMM), and methionine incorporated into the water-soluble protein fraction increased up to 32-fold, 19-fold, and 2.2-fold, respectively, compared with that in wild-type plants. Notably, in these four transgenic lines, the levels of free cysteine (the sulphur donor for methionine synthesis), glutathione (the cysteine storage and transport form), and protein-bound cysteine increased up to 2.6-fold, 5.5-fold, and 2.3-fold, respectively, relative to that in wild-type plants. As the transgenic alfalfa plants over-expressing AtCGS had significantly higher levels of both soluble and protein-bound methionine and cysteine, they may represent a model and target system for improving the nutritional quality of forage crops.     

High accumulation of bioactive peptide in transgenic rice seeds by expression of introduced multiple genes

We have developed a simple binary vector construction system for the simultaneous expression of multiple genes in plants. Up to three independent gene cassettes can be easily integrated into one binary vector using the MultiSite Gateway System. Using this system, we produced transgenic rice plants that accumulated high levels of the hypocholesterolaemic peptide lactostatin (IIAEK) in endosperm. Binary vectors were constructed that could accommodate up to three independent modified glutelin gene cassettes encoding multimer lactostatin in the variable regions. Eight construct permutations were used for rice transformation. We measured the accumulation of lactostatin expressed as a glutelin fusion protein in the mature seeds of 105 independent transgenic rice lines. A general correlation was observed between accumulation level and gene number in the vector constructs, indicating that a higher accumulation of lactostatin was obtained from transgenic rice plants containing the maximum number of gene inserts. These results indicate that this strategy is applicable for the selection of transgenic lines containing large amounts of bioactive peptides in rice seeds.     

Enhanced available methionine concentration associated with higher phaseolin levels in common bean seeds

Summary The relationship between available methionine concentration and the levels of phaseolin — the major seed storage proteins of the common bean — was studied using three groups of genetic materials: First, the F₂ progenies of interspecific crosses between P. vulgaris cultivars and aP. coccineus subsp. coccineus line (cv. Mexican Red Runner) having no detectable phaseolin; second, the F₂ progenies and segregating F₃ families of crosses between cultivated P. vulgaris lines and a Mexican wild bean accession (PI 325690-3) carrying a gene producing a reduction in phaseolin content; third, two inbred backcross populations: SanilacxBush Blue Lake 240 (population 2) and Sanilacx15R 148 (population 6). Total seed N levels were determined by micro-Kjeldahl, phaseolin levels by rocket immunoelectrophoresis and available methionine levels by the Streptococcus zymogenes bioassay. Our results indicate that in all the genetic materials studied, with the exception of population 6, higher phaseolin levels lead to increased available methionine concentration. Although phaseolin has a low methionine concentration, it is actually a major source of available methionine in common bean seeds, because it represents a large part of total seed nitrogen and because limited differences exist between the methionine concentrations of the different protein fractions. This contrasts with the situation in cereals such as maize, barley and sorghum, where increased levels of the major limiting amino acid (lysine) can be achieved through a decrease in the amounts of the main seed storage protein fraction (prolamines). In population 6, no relationship was observed between available methionine and phaseolin content. Other factors, such as additional methionine-rich polypeptides or the presence of tannins, might obscure the positive relationship between phaseolin and available methionine content in population 6.     

Iron accumulation does not parallel the high expression level of ferritin in transgenic rice seeds

To answer the question whether iron accumulation in transgenic rice seeds depends on the expression level of exogenous soybean ferritin, we generated two kinds of ferritin hyper-expressing rice lines by introducing soybean ferritin SoyferH-1 gene under the control of the rice seed storage glutelin gene promoter, GluB-1 and the rice seed storage globulin gene promoter, Glb-1, (GluB-1/SoyferH-1 and Glb-1/SoyferH-1, DF lines), and by introducing the SoyferH-1 gene under the control of Glb-1 promoter alone (Glb-1/SoyferH-1, OF lines). Ferritin expression was restricted to the endosperm in both lines and protein levels determined by western blot analysis were up to 13-fold higher than in a construct previously reported FK22 (GluB-1/SoyferH-1, in genetically Kitaake bachground); however, the maximum iron concentrations in seeds of both of the new lines were only about 30% higher than FK22. The maximum iron concentration in the OF and DF lines was about threefold higher than in the non-transformant. The mean Fe concentration in leaves of ferritin over-expressing lines decreased to less than half of the non-transformant while that the plant biomasses and seed yields of the ferritin-transformed lines were not significantly different from those of the non-transformant, suggesting that accumulation of Fe in seeds of hyper-expression ferritin rice did not always depend on the expression level of exogenous ferritin but may have been limited by Fe uptake and transport. No obvious differences were observed for other divalent-metal concentrations (Ca, Cd, Cu, Mg, Mn and Zn) in the seeds among all experimental lines and non-transformant.     

Differential effects of cysteine and methionine residues in the antioxidant activity of human serum albumin

Antioxidant properties of human serum albumin (HSA) may explain part of its beneficial role in various diseases related to free radical attack. In the present study, the antioxidant role of Cys and Met was studied by copper-mediated oxidation of human low density lipoproteins and by free radical-induced blood hemolysis which essentially assessed metal-chelating and free radical scavenging activities, respectively. Mild conditions were set up to specifically modify Cys and Met residues by N-ethylmaleimide (NEM) and chloramine T treatments, respectively. We found that Met and Cys accounted for 40–80% of total antioxidant activity of HSA. Copper binding to HSA was decreased by about 50% with chloramine T treatment of Met whereas no change was observed after NEM treatment of Cys. Although other amino acid residues are likely to be involved in anti-/prooxidant properties of HSA, from our data, we propose that Cys chiefly works as a free radical scavenger whereas Met mainly acts as a metal chelator.     

Differential effects of cysteine and methionine residues in the antioxidant activity of human serum albumin

Antioxidant properties of human serum albumin (HSA) may explain part of its beneficial role in various diseases related to free radical attack. In the present study, the antioxidant role of Cys and Met was studied by copper-mediated oxidation of human low density lipoproteins and by free radical-induced blood hemolysis which essentially assessed metal-chelating and free radical scavenging activities, respectively. Mild conditions were set up to specifically modify Cys and Met residues by N-ethylmaleimide (NEM) and chloramine T treatments, respectively. We found that Met and Cys accounted for 40-80% of total antioxidant activity of HSA. Copper binding to HSA was decreased by about 50% with chloramine T treatment of Met whereas no change was observed after NEM treatment of Cys. Although other amino acid residues are likely to be involved in anti-/prooxidant properties of HSA, from our data, we propose that Cys chiefly works as a free radical scavenger whereas Met mainly acts as a metal chelator.     

Relationship between sulphur content of leaf with methionine,cysteine and cysteine contents in the seeds ofPhaseolus aureus L. As affected by S,P, and N application

Summary The sulphur content in leaves increased with increasing levels of sulphur and with the advancement of age and was maximum at 50 days stage and declined towards maturity. There was a significant positive correlation between sulphur content of leaf at various stages of growth and sulphur-containing amino acids viz. methionine, cystine, and cysteine in seed. The sulphur containing amino acids in seeds also increased with increasing levels of sulphur upto 90 ppm and with further increase in the dose it decreased.     

Expression of a fungal glucoamylase in transgenic rice seeds

Glucoamylase, which catalyses the hydrolysis of the α-1,4 glycosidic bonds of starch, is an important industrial enzyme used in starch enzymatic saccharification. In this study, a glucoamylase gene from Aspergillus awamori, under the control of the promoter of seed storage protein Gt1, was introduced into rice by Agrobacterium-mediated transformation. Significant glucoamylase activity was detected specifically in the seeds but not other tissues of the transgenic rice lines. The highest enzymatic activity was found in the transgenic line Bg17-2, which was estimated to have about 500 units per gram of seeds (one unit is defined as the amount of enzyme that produces 1 μmol of reducing sugar in 1 min at 60 °C using soluble starch as substrate). The optimum pH for the activity of the rice produced enzyme is 5.0–5.5, and the optimum temperature is around 60 °C. One part of this transgenic glucoamylase rice seed flour fully converted 25 parts of corn starch pre-liquefied by an α-amylase also produced by a transgenic rice into glucose in 16 h incubation. This study suggests that this hydrolysis enzyme may substitute commercial fermentation enzymes for industrial starch conversion.     

High-level tryptophan accumulation in seeds of transgenic rice and its limited effects on agronomic traits and seed metabolite profile

Metabolic manipulation of plants to improve their nutritional quality is an important goal of plant biotechnology. Expression in rice (Oryza sativa L.) of a transgene (OASA1D) encoding a feedback-insensitive alpha subunit of rice anthranilate synthase results in the accumulation of tryptophan (Trp) in calli and leaves. It is shown here that the amount of free Trp in the seeds of such plants is increased by about two orders of magnitude compared with that in the seeds of wild-type plants. The total Trp content in the seeds of the transgenic plants was also increased. Two homozygous lines, HW1 and HW5, of OASA1D transgenic rice were generated for characterization of agronomic traits and aromatic metabolite profiling of seeds. The marked overproduction of Trp was stable in these lines under field conditions, although spikelet fertility and yield, as well as seed germination ability, were reduced compared with the wild type. These differences in agronomic traits were small, however, in HW5. In spite of the high Trp content in the seeds of the HW lines, metabolic profiling revealed no substantial changes in the amounts of other phenolic compounds. The amount of indole acetic acid was increased about 2-fold in the seeds of the transgenic lines. The establishment and characterization of these OASA1D transgenic lines have thus demonstrated the feasibility of increasing the Trp content in the seeds of rice (or of other crops) as a means of improving its nutritional value for human consumption or animal feed.     

Seed-specific increased expression of 2S albumin promoter of sesame qualifies it as a useful genetic tool for fatty acid metabolic engineering and related transgenic intervention in sesame and other oil seed crops

The sesame 2S albumin (2Salb) promoter was evaluated for its capacity to express the reporter gusA gene encoding β-glucuronidase in transgenic tobacco seeds relative to the soybean fad3C gene promoter element. Results revealed increased expression of gusA gene in tobacco seed tissue when driven by sesame 2S albumin promoter. Prediction based deletion analysis of both the promoter elements confirmed the necessary cis-acting regulatory elements as well as the minimal promoter element for optimal expression in each case. The results also revealed that cis-regulatory elements might have been responsible for high level expression as well as spatio-temporal regulation of the sesame 2S albumin promoter. Transgenic over-expression of a fatty acid desaturase (fad3C) gene of soybean driven by 2S albumin promoter resulted in seed-specific enhanced level of α-linolenic acid in sesame. The present study, for the first time helped to identify that the sesame 2S albumin promoter is a promising endogenous genetic element in genetic engineering approaches requiring spatio-temporal regulation of gene(s) of interest in sesame and can also be useful as a heterologous genetic element in other important oil seed crop plants in general for which seed oil is the harvested product. The study also established the feasibility of fatty acid metabolic engineering strategy undertaken to improve quality of edible seed oil in sesame using the 2S albumin promoter as regulatory element.     

Expression of an Acidothermus cellulolyticus endoglucanase in transgenic rice seeds

The thermostable endo-1,4-β-glucanase (E1) from Acidothermus cellulolyticus, is a useful enzyme for commercial hydrolysis of cellulose into glucose. A codon-optimized synthetic gene encoding this enzyme was transformed into rice (Oryza sativa L. ssp. japonica) under the control of the rice seed storage protein Gt1 promoter. The transgenic line C19 was identified as the one with the highest endoglucanase activity among the total of 36 independent transgenic lines obtained. The cellulase activity in the C19 seeds was estimated at about 830U/g of dried seeds using CMC as substrate. The enzymes produced in the seeds had an optimum pH of 5.0 and optimum temperature of 80°C, which is similar to the enzymes produced by the native bacterium host. This study demonstrates that the transgenic rice seeds could be used as a bioreactor for production of enzymes for cellulosic biomass conversion.     

Biologically active human GM-CSF produced in the seeds of transgenic rice plants

Rice flour is a well-known and characterized source of pharmaceutical ingredients, which are gluten-free and incorporated in many drug delivery applications such as excipient starch. To further exploit this uniqueness, the synthetic capacity of rice endosperm tissue, the basis of rice flour, was extended by genetic transformation. Recombinant human GM-CSF, a cytokine used in treating neutropenia and with other potential clinical applications, has been expressed in transgenic rice seeds using a rice glutelin promoter. Rice seeds accumulated human GM-CSF to a level of 1.3% of total soluble protein. The rice seed-produced human GM-CSF was found to be biologically active when tested using a human cell line TF-1. Use of rice as a host plant offers not only attractive features of safe production in seeds but also self-containment of foreign genes, as rice is primarily a self-pollinated crop plant. Ravinder Sardana and Anil K. Dudani contributed equally to this work.     

Vacuole accumulation of storage protein and lectin expressed in transgenic tobacco seeds

The expression of proteins in transgenic plants offers an elegant means to examine targeting signals used for transport to intracellular sites of accumulation. We have used electron microscopic immunogold procedures to localize several different storage proteins and lectins expressed in transgenic tobacco seeds. The objective of these studies is to characterize targeting signals which permit translocation and accumulation in protein storage vacuoles (protein bodies). Vacuolar proteins such as phaseolin and phytohemagglutinin (PHA) are correctly transported to the protein storage vacuoles of transgenic tobacco seeds. Site-directed mutagenesis was used to change Asn-linked glycosylation sites of PHA. Minus glycan PHA was accumulated in the protein storage vacuoles indicating that glycans do not confer targeting information. Zein the non-vacuolar storage protein of maize accumulates in the protein storage vacuoles indicating that deposition occurs in some proteins which may lack vacuolar targeting signals.     

Quantitative trait loci controlling sulfur containing amino acids, methionine and cysteine, in soybean seeds

Soybean [Glycine max (L.) Merr.] is the single largest source of protein in animal feed. However, a major limitation of soy proteins is their deficiency in sulfur-containing amino acids, methionine (Met) and cysteine (Cys). The objective of this study was to identify quantitative trait loci (QTL) associated with Met and Cys concentration in soybean seed. To achieve this objective, 101 F₆-derived recombinant inbred lines (RIL) from a population developed from a cross of N87-984-16 × TN93-99 were used. Ground soybean seed samples were analyzed for Met and Cys concentration using a near infrared spectroscopy instrument. Data were analyzed using SAS software and QTL Cartographer. RIL differed (P<0.01) in Met and Cys concentrations, with a range of 5.1–7.3 (g kg⁻¹ seed dry weight) for Cys and 4.4–8.8 (g kg⁻¹ seed dry weight) for Met. Heritability estimates on an entry mean basis were 0.14 and 0.57 for Cys and Met, respectively. A total of 94 polymorphic simple sequence repeat molecular genetic markers were screened in the RIL. Single factor ANOVA was used to identify candidate QTL, which were confirmed by composite interval mapping using QTL Cartographer. Four QTL linked to molecular markers Satt235, Satt252, Satt427 and Satt436 distributed on three molecular linkage groups (MLG) D1a, F and G were associated with Cys and three QTL linked to molecular markers Satt252, Satt564 and Satt590 distributed on MLG F, G and M were associated with Met concentration in soybean seed. QTL associated with Met and Cys in soybean seed will provide important information to breeders targeting improvements in the nutritional quality of soybean.     

Oxidation of cysteine and homocysteine by bovine albumin

The autooxidation of cysteine and homocysteine to their disulfide forms was determined by measuring the time course of thiol groups disappearance. We found the oxidative chemistry of cysteine and homocysteine to be quite different. In the absence of added Cu(II), cysteine autooxidized at a slower rate than homocysteine, though in its presence cysteine oxidation was much faster, homocysteine being found to be a poor responder to copper catalysis. Albumin speeded up the spontaneous oxidation of both aminothiols, the reaction being faster with cysteine than with homocysteine. The copper content of different albumins was found to be highly variable, ranging from 12.75 to 0.64 microg Cu(II)/g albumin. We propose that copper bound to albumin possesses redox cycling activity to perform cysteine oxidation since: (i) copper elimination by copper chelators markedly reduces oxidation; and (ii) a positive correlation exists between the albumin copper content and the oxidation reaction rate.     

Additive effects of the feed-back insensitive bacterial aspartate kinase and the Brazil nut 2S albumin on the methionine content of transgenic narbon bean (Vicia narbonensis L.)

So far two different strategies for engineering high methionine (Met) grain legumes were followed separately in several laboratories: a) The transfer of foreign genes encoding Met-rich proteins, and b) the engineering of Met biosynthesis pathways. In some cases a down regulation of the formation of endogenous sulfur-containing compounds was observed due to the expression of Met-rich foreign proteins. Since this might result from competition of the foreign protein with endogenous compounds for limited Met supply both strategies were combined in the present work. Double transformants of narbon bean (Vicia narbonensis L.) were generated which express seed-specifically the Met-rich Brazil nut 2S albumin (BNA) as well as a feed-back insensitive bacterial aspartate kinase (AK) known to stimulate Met biosynthesis in transgenic tobacco seeds. In order to produce double transformants a homozygous transgenic BNA line of narbon bean was either retransformed with the AK gene or crossed with an AK line. For the first time the influence of a deregulated AK on amino acids of the aspartate pathway was studied in seeds of a transgenic legume. Effects of expressing the foreign genes on inorganic sulphate, free and protein-bound Met and other amino acids of the aspartate pathway as well as on free sulphhydryl compounds of mature seeds were analysed. AK lines had 10 to 12 percent and the BNA line 80 percent increased Met in mature seeds. Double transformants showed additive but not synergistic effects of the expression of AK and BNA gene on seed Met. In their mature seeds protein-bound Met reached levels 2.0 to 2.4 times higher than in the wildtype. The Met level of best line corresponds approximately to the FAO standard for Met in a nutritionally balanced protein for human food or for feeding monogastric animals.     

Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by constitutive expression of rice chitinase

Rice blast is the most devastating plant disease in Japan. Our goal is to create new rice varieties which show enhanced resistance against blast, regardless of the race of blast. By an Agrobacterium-mediated transformation method, we reintroduced a rice class-I chitinase gene, Cht-2 or Cht-3, under the control of the enhanced CaMV 35S promoter and a hygromycin phosphotransferase gene, as a selection marker into the Japonica rice varieties Nipponbare and Koshihikari, which have retained the best popularity over a long period in Japan. In regenerated plants (R₀), the Cht-2 product was found to accumulate intracellularly whereas the Cht-3 product was found to be targeted extracellularly. The transgenic rice plants which constitutively expressed either chitinase gene showed significantly higher resistance against the rice blast pathogen Magnaporthe grisea races 007.0 and 333. Both high-level expression of the chitinase and blast-resistance were stably inherited by the next generation in several lines. Received: 16 November 1998 / Accepted: 30 January 1999