Enhancement of the methionine content of seed proteins by the expression of a chimeric gene encoding a methionine-rich protein in transgenic plants

We have constructed a chimeric gene encoding a Brazil nut methionine-rich seed protein which contains 18% methionine. This gene has been transferred to tobacco and expressed in the developing seeds. Tobacco seeds are able to process the methionine-rich protein efficiently from a larger precursor polypeptide of 17 kDa to the 9kDa and 3 kDa subunits of the mature protein, a procedure which involves three proteolytic cleavage steps in the Brazil nut seed. The accumulation of the methionine-rich protein in the seeds of tobacco results in a significant increase (30%) in the levels of the methionine in the seed proteins of the transgenic plants. Our data indicate that the introduction of a chimeric gene encoding a methionine-rich seed protein into crop plants, particularly legumes whose seeds are deficient in the essential sulfur-containing amino acids, represents a feasible method for improving the nutritional quality of seed proteins.     

Amino acid and cDNA sequences of a methionine-rich 2S protein from sunflower seed (Helianthus annuus L.)

The amino acid sequence of a methionine-rich 2S seed protein from sunflower (Helianthus annuus L.) and the sequence of a cDNA clone which codes for the entire primary translation product have been determined. The mature protein consists of a single polypeptide chain of 103 amino acids (molecular mass 12133 Da) which contains 16 residues of methionine and 8 residues of cysteine. The cDNA sequence established that the protein is synthesized as a precursor of 141 residues with a typical hydrophobic signal sequence of 25 residues followed by a further 13-residue hydrophobic pro-sequence which is presumably removed by post-translational cleavage. The sequence of the mature protein and that deduced from the cDNA were identical with no evidence of processing at the C-terminus. Comparison of the sunflower methionine-rich protein sequence with sequences of other seed 2S proteins from dicotyledons and monocotyledons showed limited but distinct sequence similarities; in particular the arrangement of the cysteine residues was conserved. The sunflower protein shows 34% identity with the methionine-rich Brazil nut 2S protein and the prepro regions of the precursors of these two proteins show about 50% identity. This similarity indicates that these methionine-rich 2S proteins have diverged as a subclass of the 2S superfamily of proteins which contain only 2-3% methionine. While the related 2S proteins from other dicotyledons are processed to a small and large subunit, the sunflower protein is not cleaved in this way.     

Stable Accumulation of Modified 2S Albumin Seed Storage Proteins with Higher Methionine Contents in Transgenic Plants

We present the results of two sets of experiments designed to express high methionine proteins in transgenic seeds in three different plant species. In the first approach, two chimeric genes were constructed in which parts of the Arabidopsis 2S albumin gene 1 (AT2S1) were fused at different positions to a Brazil nut 2S albumin cDNA clone. Brazil nut 2S albumin was found to accumulate stably in transgenic Arabidopsis, Brassica napus, and tobacco seeds. In the second approach, methionine-enriched AT2S1 genes were constructed by deleting sequences encoding a region of the protein which is not highly conserved among 2S albumins of different species and replacing them with methioninerich sequences. Introduction of the modified AT2S1 genes into three different plant species resulted in the accumulation of the methionine-enriched 2S albumins in all three species at levels reaching 1 to 2% of the total high salt-extractable seed protein.     

Role of source-to-sink transport of methionine in establishing seed protein quantity and quality in legumes

Grain legumes such as pea (Pisum sativum L.) are highly valued as a staple source of protein for human and animal nutrition. However, their seeds often contain limited amounts of high-quality, sulfur (S) rich proteins, caused by a shortage of the S-amino acids cysteine and methionine. It was hypothesized that legume seed quality is directly linked to the amount of organic S transported from leaves to seeds, and imported into the growing embryo. We expressed a high-affinity yeast (Saccharomyces cerevisiae) methionine/cysteine transporter (Methionine UPtake 1) in both the pea leaf phloem and seed cotyledons and found source-to-sink transport of methionine but not cysteine increased. Changes in methionine phloem loading triggered improvements in S uptake and assimilation and long-distance transport of the S compounds, S-methylmethionine and glutathione. In addition, nitrogen and carbon assimilation and source-to-sink allocation were upregulated, together resulting in increased plant biomass and seed yield. Further, methionine and amino acid delivery to individual seeds and uptake by the cotyledons improved, leading to increased accumulation of storage proteins by up to 23%, due to both higher levels of S-poor and, most importantly, S-rich proteins. Sulfate delivery to the embryo and S assimilation in the cotyledons were also upregulated, further contributing to the improved S-rich storage protein pools and seed quality. Overall, this work demonstrates that methionine transporter function in source and sink tissues presents a bottleneck in S allocation to seeds and that its targeted manipulation is essential for overcoming limitations in the accumulation of high-quality seed storage proteins.

Methionine transporter function in pea phloem and embryo affects sulfur, nitrogen, and carbon acquisition, metabolism, and partitioning, resulting in increased seed yield, protein levels, and quality.     

Differential expression of a gene for a methionine-rich storage protein in maize

Summary A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A fulllength cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.     

Enhanced methionine and cysteine levels in transgenic rice seeds by the accumulation of sesame 2S albumin

A chimeric gene encoding a precursor polypeptide of sesame 2S albumin, a sulfur-rich seed storage protein, was expressed in transgenic rice plants under the control of the glutelin promoter with the aim of improving the nutritive value of rice. Rice grains harvested from the first generation of ten different transformed lines inherited the transgene, and the accumulated sesame 2S albumin was presumably processed correctly as its mature form in sesame seed. This transgene was specifically expressed in maturing rice seeds with its encoded sesame 2S albumin exclusively accumulated in the seeds. The crude protein content in rice grains from five putative homozygous lines was increased by 0.64-3.54%, and the methionine and cysteine contents of these transgenic rice grains were respectively elevated by 29-76% and 31-75% compared with those of wild-type rice grains.     

Sulphur and nitrogen nutrition influence the response of chickpea seeds to an added, transgenic sink for organic sulphur

In order to increase the concentration of the nutritionally essential sulphur amino acids in seed protein, a transgene encoding a methionine- and cysteine-rich protein, sunflower seed albumin (SSA), was transferred to chickpeas (Cicer arietinum L). Transgenic seeds that accumulated SSA contained more methionine and less oxidized sulphur than the controls, suggesting that additional demand for sulphur amino acids from the expression of the transgene stimulated sulphur assimilation. In addition, the activity of trypsin inhibitors, a known family of endogenous, sulphur-rich chickpea seed proteins, was diminished in transgenic, SSA-containing seeds compared with the non-transgenic controls. Together, these results indicate that the reduced sulphur sequestered into SSA was supplied partly by additional sulphur assimilation in the developing transgenic seeds, and partly by some diversion of sulphur amino acids from endogenous seed proteins. Growth of chickpeas on nutrient with a high sulphur-to-nitrogen ratio increased the total seed sulphur content and the accumulation of sulphur amino acids in the seeds, and partly mitigated the effect of SSA accumulation on the trypsin inhibitor amount. The results suggest that free methionine and O-acetylserine (OAS) acted as signals that modulated chickpea seed protein composition in response to the variation in sulphur demand, as well as in response to variation in the nitrogen and sulphur status of the plant.     

Characterization of a Small Sulphur-Rich Storage Albumin in Seeds of Alfalfa (Medicago sativa L.)

A 2S albumin fraction was characterized in seeds of alfalfa{^{Medicago sativa} L.). This low molecular weight (LMW) familyof disulphide-bonded proteins represents a major nitrogen andsulphur storage reserve for the alfalfa seed Characteristicof seed storage proteins, the 2S albumins are abundant in nitrogen-richglutarrune/glutamate/asparagine/aspartate (32%) In addition,this LMW fraction is high in cysteine (9%) and methionine (4%),amino acids which are under-represented in legume seed globulins.These 2S proteins start to accumulate during the early cotyledonstage of development, and are mobilized following germinationPulse-chase labelling experiments show that the 2S proteinsare synthesized as 'preproproteins', similar to 2S proteinsin other seeds. However, alfalfa 2S albumins are immunologicallyunrelated to these proteins. Key words: Seed development, sulphur-containing 2S storage protein, alfalfa (Medicago sativa)     

Increasing lysine levels in pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millsp) seeds through genetic engineering

In higher plants the essential amino acids lysine, threonine, methionine and isoleucine are synthesised through a branched pathway starting from aspartate. The key enzyme of lysine biosynthesis in this pathway—dihydrodipicolinate synthase (DHDPS)—is feedback-inhibited by lysine. The dhdps-r1 gene from a mutant Nicotiana sylvestris, which encodes a DHDPS enzyme insensitive to feedback inhibition, was used to improve the lysine content in pigeonpea seeds. The dhdps-r1 coding region driven by a phaseolin or an Arabidopsis 2S2 promoter was successfully overexpressed in the seeds of pigeonpea by using Agrobacterium transformation and particle bombardment. In 11 lines analysed, a 2- to 6-fold enhanced DHDPS activity in immature seeds at a late stage of maturation was found in comparison to wild type. The overexpression of dhdps-r1 led to an enhanced content of free lysine in the seeds of pigeonpea from 1.6 to 8.5 times compared with wild type. However, this was not reflected in an increase in total seed lysine content. This might be explained by a temporal discrepancy between maximal expression of dhdps-r1 and the rate of amino acid incorporation into storage proteins. Assays of the lysine degradative enzyme lysine-ketoglutarate reductase in these seeds showed no co-ordinated regulation of lysine biosynthesis and catabolism during seed maturation. All transgenic plants were fertile and produced morphologically normal seeds.     

Effect of processing on the recovery of recombinant beta-glucuronidase (rGUS) from transgenic canola

This study addresses the processing of transgenic canola seed for production of recombinant proteins by using beta-glucuronidase (rGUS) as a model protein. The major processing steps that were investigated included dry and wet grinding of the seed, solvent extraction of canola oil, and protein extraction. rGUS in canola seed was stable for at least 2 weeks of incubation at 38 degrees C and for more than 5 months at 10 degrees C. At 70 degrees C, the residual activity changed inversely to the initial moisture content of the seed. The comparison of wet and dry processing revealed no significant differences in protein recovery. rGUS was stable during the defatting of transgenic canola flakes with hexane at 66 degrees C, whereas 2-propanol extraction at the same temperature reduced the extractable enzyme activity by almost 50%. The particle size of the ground seed was important for the extraction efficiency. A faster extraction and greater protein yield was achieved by extracting particles with an average diameter equal to or smaller than 255 microm. More than 80% rGUS was extracted in one stage with sodium phosphate buffer of pH 7.5.     

Bioengineering approaches to improve the nutritional values of seeds by increasing their methionine content

Methionine is a nutritionally essential, sulfur-containing amino acid found at low levels in plants and in their seeds. Methionine levels often limit the plant’s value as a source of dietary protein for humans and animals. Despite recent accumulated knowledge of methionine metabolism in vegetative tissues, there is still little knowledge of methionine metabolism in seeds. In this review, we summarize the efforts made to increase the levels of methionine in seeds using genetic engineering methods. Two main approaches were tested: the first was the expression of methionine-rich storage proteins in a seed-specific manner, with the goal of trapping the soluble methionine into protein form and competing with the catabolism of methionine to its essential metabolites. However, in many cases this approach does not lead to a significant increase in total methionine content. The second approach aimed to increase the soluble content of methionine in seeds. Despite the nutritional significance of methionine, the factors regulating soluble methionine content in seeds are not fully known. Evidence shows that two biosynthetic pathways, the aspartate family pathway and the S-methylmethionine pathway, contribute to soluble methionine content in seeds. However, their roles in soluble methionine synthesis and accumulation are not fully understood. In recent years, combinations of these two approaches have been tested; however, they have not yet succeeded in elevating total methionine content in seeds. More emphasis should be applied to gaining knowledge of the biosynthesis pathways that could contribute to an increase in methionine content in seeds.     

Expression of an 11 kDa methionine-rich delta-zein in transgenic soybean results in the formation of two types of novel protein bodies in transitional cells situated between the vascular tissue and storage parenchyma cells

Soybean (Glycine max (L.) Merr.) is an important protein source in human diets and animal feeds. The sulphur content of soybean seed proteins, however, is not optimal for ration formulations. Thus, increasing the methionine and cysteine content of soybean seed proteins would enhance the nutritional quality of this widely utilized legume. We have earlier reported the isolation of an 11 kDa delta-zein protein rich in methionine from the endosperm of the maize (Zea mays L.) inbred line W23a1 [Kim, W.-S. and Krishnan, H.B. (2003) Allelic variation and differential expression of methionine-rich-delta-zeins in maize inbred lines B73 and W23a1. Planta, 217, 66-74]. Using Agrobacterium-mediated transformation, a construct consisting of the coding region of the cloned delta-zein gene under regulation of the beta-conglycinin alpha'-promoter was introduced into the soybean genome. The 11 kDa delta-zein gene was expressed in the seeds of transgenic soybeans, although low-level expression was also detected in the leaves. In situ hybridization indicated that the 11 kDa delta-zein mRNA was expressed predominantly in transitional cells located between the vascular tissue and storage parenchyma cells. Immunohistochemistry of developing transgenic soybeans revealed that the accumulation of the 11 kDa delta-zein occurred primarily in these transitional cells. Expression of the 11 kDa delta-zein gene in transgenic soybean resulted in the formation of two endoplasmic reticulum-derived protein bodies that were designated as either spherical or complex. Immunocytochemical localization demonstrated that both the spherical and complex protein bodies accumulated the 11 kDa delta-zein. Although expression of the 11 kDa delta-zein gene elevated the methionine content of the alcohol-soluble protein fraction 1.5-1.7-fold above that of the non-transgenic line, the overall methionine content of seed flour was not increased. Our results suggest that the confined expression of the 11 kDa delta-zein gene in transitional cells could be limiting the increase in methionine content in transgenic soybean seeds.     

Importance of methionine biosynthesis for Arabidopsis seed germination and seedling growth

Proteomics of Arabidopsis seeds revealed the differential accumulation during germination of two housekeeping enzymes. The first corresponded to methionine synthase that catalyses the last step in the plant methionine biosynthetic pathway. This protein was present at low level in dry mature seeds, and its level was increased strongly at 1-day imbibition, prior to radicle emergence. Its level was not increased further at 2-day imbibition, coincident with radicle emergence. However, its level in 1-day imbibed seeds strongly decreased upon subsequent drying of the imbibed seeds back to the original water content of the dry mature seeds. The second enzyme corresponded to S -adenosylmethionine synthetase that catalyses the synthesis of S -adenosylmethionine from methionine and ATP. In this case, this enzyme was detected in the form of two isozymes with different p I and M r. Both proteins were absent in dry mature seeds and in 1-day imbibed seeds, but specifically accumulated at the moment of radicle protrusion. Arabidopsis seed germination was strongly delayed in the presence of dl -propargylglycine, a specific inhibitor of methionine synthesis. Furthermore, this compound totally inhibited seedling growth. These phenotypic effects were largely alleviated upon methionine supplementation in the germination medium. The results indicated that methionine synthase and S -adenosylmethionine synthetase are fundamental components controlling metabolism in the transition from a quiescent to a highly active state during seed germination. Moreover, the observed temporal patterns of accumulation of these proteins are consistent with an essential role of endogenous ethylene in Arabidopsis only after radicle protrusion.     

Enhanced seed oil production in canola by conditional expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in developing seeds

The seed oil content in oilseed crops is a major selection trait to breeders. In Arabidopsis (Arabidopsis thaliana), LEAFY COTYLEDON1 (LEC1) and LEC1-LIKE (L1L) are key regulators of fatty acid biosynthesis. Overexpression of AtLEC1 and its orthologs in canola (Brassica napus), BnLEC1 and BnL1L, causes an increased fatty acid level in transgenic Arabidopsis plants, which, however, also show severe developmental abnormalities. Here, we use truncated napin A promoters, which retain the seed-specific expression pattern but with a reduced expression level, to drive the expression of BnLEC1 and BnL1L in transgenic canola. Conditional expression of BnLEC1 and BnL1L increases the seed oil content by 2% to 20% and has no detrimental effects on major agronomic traits. In the transgenic canola, expression of a subset of genes involved in fatty acid biosynthesis and glycolysis is up-regulated in developing seeds. Moreover, the BnLEC1 transgene enhances the expression of several genes involved in Suc synthesis and transport in developing seeds and the silique wall. Consistently, the accumulation of Suc and Fru is increased in developing seeds of the transgenic rapeseed, suggesting the increased carbon flux to fatty acid biosynthesis. These results demonstrate that BnLEC1 and BnL1L are reliable targets for genetic improvement of rapeseed in seed oil production.     

Seed-specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects

A bacterial phytoene synthase (crtB) gene was overexpressed in a seed-specific manner and the protein product targeted to the plastid in Brassica napus (canola). The resultant embryos from these transgenic plants were visibly orange and the mature seed contained up to a 50-fold increase in carotenoids. The predominant carotenoids accumulating in the seeds of the transgenic plants were alpha and beta-carotene. Other precursors such as phytoene were also detected. Lutein, the predominant carotenoid in control seeds, was not substantially increased in the transgenics. The total amount of carotenoids in these seeds is now equivalent to or greater than those seen in the mesocarp of oil palm. Other metabolites in the isoprenoid pathway were examined in these seeds. Sterol levels remained essentially the same, while tocopherol levels decreased significantly as compared to non-transgenic controls. Chlorophyll levels were also reduced in developing transgenic seed. Additionally, the fatty acyl composition was altered with the transgenic seeds having a relatively higher percentage of the 18 : 1 (oleic acid) component and a decreased percentage of the 18 : 2 (linoleic acid) and 18 : 3 (linolenic acid) components. This dramatic increase in flux through the carotenoid pathway and the other metabolic effects are discussed.     

Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola

Transgenic plants offer advantages for biomolecule production because plants can be grown on a large scale and the recombinant macromolecules can be easily harvested and extracted. We introduced an Aspergillus phytase gene into canola (Brassica napus) (line 9412 with low erucic acid and low glucosinolates) by Agrobacterium-mediated transformation. Phytase expression in transgenic plant was enhanced with a synthetic phytase gene according to the Brassica codon usage and an endoplasmic reticulum (ER) retention signal KDEL that confers an ER accumulation of the recombinant phytase. Secretion of the phytase to the extracellular fluid was also established by the use of the tobacco PR-S signal peptide. Phytase accumulation in mature seed accounted for 2.6% of the total soluble proteins. The enzyme can be glycosylated in the seeds of transgenic plants and retain a high stability during storage. These results suggest a commercial feasibility of producing a stable recombinant phytase in canola at a high level for animal feed supplement and for reducing phosphorus eutrophication problems.