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.

Sulfur assimilation in developing lupin cotyledons could contribute significantly to the accumulation of organic sulfur reserves in the seed.

It is currently assumed that the assimilation of sulfur into reduced forms occurs predominantly in the leaves of plants. However, developing seeds have a strong requirement for sulfur amino acids for storage protein synthesis. We have assessed the capacity of developing seeds of narrow-leaf lupin (Lupinus angustifolius) for sulfur assimilation. Cotyledons of developing lupin seeds were able to transfer the sulfur atom from 35S-labeled sulfate into seed proteins in vitro, demonstrating the ability of the developing cotyledons to perform all the steps of sulfur reduction and sulfur amino acid biosynthesis. Oxidized sulfur constituted approximately 30% of the sulfur in mature seeds of lupins grown in the field and almost all of the sulfur detected in phloem exuded from developing pods. The activities of three enzymes of the sulfur amino acid biosynthetic pathway were found in developing cotyledons in quantities theoretically sufficient to account for all of the sulfur amino acids that accumulate in the protein of mature lupin seeds. We conclude that sulfur assimilation by developing cotyledons is likely to be an important source of sulfur amino acids for the synthesis of storage proteins during lupin seed maturation.

Expression of the insecticidal bean alpha-amylase inhibitor transgene has minimal detrimental effect on the nutritional value of peas fed to rats at 30% of the diet.

The effect of expression of bean alpha-amylase inhibitor (alpha-AI) transgene on the nutritional value of peas has been evaluated by pair-feeding rats diets containing transgenic or parent peas at 300 and 650 g/kg, respectively, and at 150 g protein/kg diet, supplemented with essential amino acids to target requirements. The results were also compared with the effects of diets containing lactalbumin with or without 0.9 or 2.0 mg bean alpha-AI, levels equivalent to those in transgenic pea diets. When 300 and 650 g peas/kg diet were fed, the daily intake of alpha-AI was 11.5 or 26.3 mg alpha-AI, respectively. At the 300 g/kg level, the nutritional value of the transgenic and parent line peas was not significantly different. The weight gain and tissue weights of rats fed either of the two pea diets were not significantly different from each other or from those of rats given the lactalbumin diet even when this was supplemented with 0.9 g alpha-AI/kg. The digestibilities of protein and dry matter of the pea diets were slightly but significantly lower than those of the lactalbumin diet, probably due to the presence of naturally occurring antinutrients in peas. The nutritional value of diets containing peas at the higher (650 g) inclusion level was less than that of the lactalbumin diet. However, the differences between transgenic and parent pea lines were small, possibly because neither the purified recombinant alpha-AI nor that in transgenic peas inhibited starch digestion in the rat small intestine in vivo to the same extent as did bean alpha-AI. This was the case even though both forms of alpha-AI equally inhibited alpha-amylase in vitro. Thus, this short-term study indicated that transgenic peas expressing bean alpha-AI gene could be used in rat diets at 300 g/kg level without major harmful effects on their growth, metabolism and health, raising the possibility that transgenic peas may also be used at this level in the diet of farm animals.

The influences of two plant nuclear matrix attachment regions (MARs) on gene expression in transgenic plants.

Nuclear matrix attachment regions (MARs) are thought to influence gene expression by anchoring active chromatin to the proteinaceous nuclear matrix. In this study, two plant DNA fragments with strong MAR activity were selected and tested for their effects on expression of a linked reporter gene in transgenic tobacco. One MAR was isolated from the 5' flanking region of a pea vicilin gene previously reported to be expressed in a copy number-dependent manner in transgenic tobacco. A second MAR was isolated from the genome of Arabidopsis thaliana by preselection for autonomously replicating sequence (ARS) activity in yeast. Flanking copies of the A. thaliana MAR stimulated median reporter gene expression in transgenic plants by five to ten fold. Neither MAR significantly reduced the variation in transgene expression between individual transformants, or conferred copy number-dependence in gene expression.

Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a sunflower seed albumin gene.

With the aim of improving the nutritive value of an important grain legume crop, a chimeric gene specifying seed-specific expression of a sulfur-rich, sunflower seed albumin was stably transformed into narrow-leafed lupin (Lupinus angustifolius L.). Sunflower seed albumin accounted for 5% of extractable seed protein in a line containing a single tandem insertion of the transferred DNA. The transgenic seeds contained less sulfate and more total amino acid sulfur than the nontransgenic parent line. This was associated with a 94% increase in methionine content and a 12% reduction in cysteine content. There was no statistically significant change in other amino acids or in total nitrogen or total sulfur contents of the seeds. In feeding trials with rats, the transgenic seeds gave statistically significant increases in live weight gain, true protein digestibility, biological value, and net protein utilization, compared with wild-type seeds. These findings demonstrate the feasibility of using genetic engineering to improve the nutritive value of grain crops.

A biotechnological approach to improving the nutritive value of alfalfa.

The postruminal supply of the sulfur-containing amino acids, methionine and cysteine, has been reported to be a major limitation to wool growth in sheep. We aim to improve the protein quality of forage for ruminants by introducing into alfalfa chimeric genes encoding a ruminally stable, sulfur amino acid-rich protein from sunflower seeds. Four gene constructs were transferred to Australian commercial cultivars of alfalfa using Agrobacterium tumefaciens-mediated transformation and selection with phosphinothricin (PPT). Modification of the sunflower seed albumin protein-coding region by addition of the coding information for an endoplasmic reticulum (ER) retention signal was found to greatly increase the level to which the sulfur amino acid-rich protein accumulated in the leaves of transgenic alfalfa plants. The Cauliflower Mosaic Virus (CaMV) 35S promoter and two light-regulated plant gene promoter regions were compared for their ability to direct high-level expression of the introduced genes in alfalfa leaves. The highest expression of sunflower seed albumin was found in transformants bearing a gene incorporating the promoter from the Arabidopsis thaliana ats1A gene, which encodes the ribulose bisphosphate carboxylase small subunit. The highest level of sunflower seed albumin found in transgenic alfalfa leaves was estimated to constitute .1% of soluble leaf protein. This level of accumulation of the foreign protein would be predicted to supply an extra 40 mg of sulfur amino acids daily to sheep fed the modified forage. Published studies in which wool growth rates were significantly increased employed supplementation of approximately 1 to 2 g of sulfur amino acids daily.

Bean [alpha]-Amylase Inhibitor Confers Resistance to the Pea Weevil (Bruchus pisorum) in Transgenic Peas (Pisum sativum L.).

Bruchid larvae cause major losses of grain legume crops through-out the world. Some bruchid species, such as the cowpea weevil and the azuki bean weevil, are pests that damage stored seeds. Others, such as the pea weevil (Bruchus pisorum), attack the crop growing in the field. We transferred the cDNA encoding the [alpha]-amylase inhibitor ([alpha]-AI) found in the seeds of the common bean (Phaseolus vulgaris) into pea (Pisum sativum) using Agrobacterium-mediated transformation. Expression was driven by the promoter of phytohemagglutinin, another bean seed protein. The [alpha]-amylase inhibitor gene was stably expressed in the transgenic pea seeds at least to the T5 seed generation, and [alpha]-AI accumulated in the seeds up to 3% of soluble protein. This level is somewhat higher than that normally found in beans, which contain 1 to 2% [alpha]-AI. In the T5 seed generation the development of pea weevil larvae was blocked at an early stage. Seed damage was minimal and seed yield was not significantly reduced in the transgenic plants. These results confirm the feasibility of protecting other grain legumes such as lentils, mungbean, groundnuts, and chickpeas against a variety of bruchids using the same approach. Although [alpha]-AI also inhibits human [alpha]-amylase, cooked peas should not have a negative impact on human energy metabolism.

Agrobacterium-Mediated Transformation of Subterranean Clover (Trifolium subterraneum L.).

We have developed a rapid and reproducible transformation system for subterranean clover (Trifolium subterraneum L.) using Agrobacterium tumefaciens-mediated gene delivery. Hypocotyl segments from seeds that had been allowed to imbibe were used as explants, and regeneration was achieved via organogenesis. Glucose and acetosyringone were required in the co-cultivation medium for efficient gene transfer. DNA constructs containing four genes encoding the enzymes phosphinothricin acetyl transferase, [beta]-glucuronidase (GUS), neomycin phosphotransferase, and an [alpha]-amylase inhibitor were used to transform subterranean clover. Transgenic shoots were selected on a medium containing 50 mg/L of phosphinothricin. Four commercial cultivars of subterranean clover (representing all three subspecies) have been successfully transformed. Southern analysis revealed the integration of T-DNA into the subterranean clover genome. The expression of the introduced genes has been confirmed by enzyme assays and northern blot analyses. Transformed plants grown in the glasshouse showed resistance to the herbicide Basta at applications equal to or higher than rates recommended for killing subterranean clover in field conditions. In plants grown from the selfed seeds of the primary transformants, the newly acquired gene encoding GUS segregated as a dominant Mendelian trait.

Genetic engineering of grain and pasture legumes for improved nutritive value.

This review describes work aimed at the improvement of the nutritive value of grain and forage legumes using gene transfer techniques. Two traits which are amenable to manipulation by genetic engineering have been identified. These are plant protein quality and lignin content. In order to increase the quality of protein provided by the legume grains peas and lupins, we are attempting to introduce into these species chimeric genes encoding a sunflower seed protein rich in the sulphur-containing amino acids methionine and cysteine. These genes are designed to be expressed only in developing seeds of transgenic host plants. Chimeric genes incorporating a similar protein-coding region, but different transcriptional controls, are being introduced into the forage legumes lucerne and subterranean clover. In this case the genes are highly expressed in the leaves of transformed plants, and modifications have been made to the sunflower seed protein-coding sequences in order to increase the stability of the resultant protein in leaf tissue. Another approach to increasing plant nutritive value is represented by attempts to reduce the content of indigestible lignin in lucerne.