Modifying lysine biosynthesis and catabolism in corn with a single bifunctional expression/silencing transgene cassette.

Although it is one of the major crops in the world, corn has poor nutritional quality for human and animal consumption due to its low lysine content. Here, we report a method of simultaneous expression of a deregulated lysine biosynthetic enzyme, CordapA, and reduction of a bifunctional lysine degradation enzyme, lysine-ketoglutarate reductase/saccharophine dehydrogenase (LKR/SDH), in transgenic corn plants by a single transgene cassette. This is accomplished by inserting an inverted-repeat sequence targeting the maize LKR/SDH gene into an intron of a transgene cassette that expresses CordapA. This combination of LKR/SDH silencing and CordapA expression led to the accumulation of free lysine to over 4000 p.p.m. in transgenic corn grain, compared to less than 100 p.p.m. in wild-type controls. This intron-embedded silencing cassette design reduces the number of transgene cassettes needed in transgenic approaches for manipulating metabolic pathways that sometimes require expression of one gene and silencing of another.

High-lysine corn generated by endosperm-specific suppression of lysine catabolism using RNAi.

Because of the limited lysine content in corn grain, synthetic lysine supplements are added to corn meal-based rations for animal feed. The development of biotechnology, combined with the understanding of plant lysine metabolism, provides an alternative solution for increasing corn lysine content through genetic engineering. Here, we report that by suppressing lysine catabolism, transgenic maize kernels accumulated a significant amount of lysine. This was achieved by RNA interference (RNAi) through the endosperm-specific expression of an inverted-repeat (IR) sequence targeting the maize bifunctional lysine degradation enzyme, lysine-ketoglutarate reductase/saccharopine dehydrogenase (ZLKR/SDH). Although plant-short interfering RNA (siRNA) were reported to lack tissue specificity due to systemic spreading, we confirmed that the suppression of ZLKR/SDH in developing transgenic kernels was restricted to endosperm tissue. Furthermore, results from our cloning and sequencing of siRNA suggested the absence of transitive RNAi. These results support the practical use of RNAi for plant genetic engineering to specifically target gene suppression in desired tissues without eliciting systemic spreading and the transitive nature of plant RNAi silencing.

High-lysine corn produced by the combination of enhanced lysine biosynthesis and reduced zein accumulation.

Corn is one of the major crops in the world, but its low lysine content is often problematic for animal consumption. While exogenous lysine supplementation is still the most common solution for today's feed corn, high-lysine corn has been developed through genetic research and biotechnology. Reducing the lysine-poor seed storage proteins, zeins, or expressing a deregulated lysine biosynthetic enzyme, CordapA, has shown increased total lysine or free lysine content in the grains of modified corn plants, respectively. Here, by combining these two approaches through genetic crosses, the total lysine content has more than doubled in F1 progeny. We also observe a synergy between the transgenic zein reduction and the enhanced lysine biosynthesis by CordapA expression. The zein reduction plants are found to accumulate higher levels of aspartate, asparagine and glutamate, and therefore, provide excess precursors for the enhanced lysine biosynthesis.

Developmental expression of the mitochondrial pyruvate dehydrogenase complex in pea (Pisum sativum) seedlings.

In order to better understand control of the mitochondrial pyruvate dehydrogenase complex (PDC), total catalytic activity was determined during development of the primary leaves of pea (Pisum sativum L.) seedlings, as well as in each leaf pair of 21-day-old plants. Activity of the PDC in clarified homogenates was highest in the youngest organs and then dropped dramatically as the leaves matured and became photosynthetically competent. As leaves began to senesce, total PDC activity dropped to zero. Steady-state mRNA levels were determined using E1 and E3 cDNA probes. The overall pattern of transcript abundance matched the pattern observed for total PDC activity; transcript levels for E1alpha and E1beta approached zero during senescence. Levels of the E1alpha, E1beta, E2 and E3 subunits of the PDC were analyzed in the same samples, using specific antibodies. Quantitation of the immunoblotting results throughout this developmental series showed a pattern in parallel with that of catalytic activity and mRNA levels, although the relative changes in subunit protein levels were not as extreme as the changes in activity. The exception to the global pattern was that of the E3 subunit: lipoamide dehydrogenase. Expression of this enzyme was highest in mature, fully expanded leaves, which were active in photosynthesis and photorespiration, reflecting the additional role of E3 as a component of glycine decarboxylase.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of zeins in mature maize kernels.

A high-throughput method has been developed to allow rapid analysis of maize seed storage proteins by matrix-assisted laser desorption time-of-flight mass spectrometry. The extraction solution containing an organic solvent, a reducing agent, and a volatile base has been optimized to enable extraction of all classes of zein proteins (alpha-, beta-, gamma-, and delta-). A near-saturating concentration of matrix, 2-(4-hydroxyphenylazo)benzoic acid, was necessary to obtain strong peaks for the most lipophilic zeins, the alpha-zeins. Zein proteins with small mass differences, difficult to separate by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were resolved through this analysis. Mass signals corresponding to the 10-kDa delta-, 15-kDa beta-, 16-kDa gamma-, 27-kDa gamma-, and several 19 and 22-kDa alpha-zeins were detected. The zein identities were further confirmed by the association of the number of cysteine residues in each zein MS peak, as determined by iodoacetamide derivatization, with the number predicted from its coding sequence. The relative zein abundance in the zein MS peaks was also correlated with the relative zein EST abundance among endosperm EST libraries. This method was utilized to examine the zein composition of a number of corn inbred lines and opaque mutants.

Improving nutritional quality of maize proteins by expressing sense and antisense zein genes.

The predominant proteins in maize grain are a family of alcohol-soluble prolamin storage proteins called zeins. They account for >50% of total seed proteins but are deficient in several essential amino acids. As a result, the corn grain is considered to be nutritionally poor for monogastric animals with respect to key essential amino acids, most notably lysine, tryptophan, and methionine. Thus, corn mutants with reduced levels of zeins, such as opaque-2 (o2), have been demonstrated to possess grain with improved nutritional quality characteristics. The o2 mutant has a superior amino acid composition and has been used through conventional breeding to develop Quality Protein Maize (QPM) for human and animal consumption in developing countries. With the understanding of molecular genetics of zeins and progress in biotechnology, an alternative approach to zein reduction is explored here. Through the targeted reduction of the 19-kDa alpha-zeins, increased levels of lysine, tryptophan, and methionine have been engineered in grain of transgenic hybrids. Currently, the agronomic properties and nutritional values of these transgenic lines are being evaluated.

High lysine and high tryptophan transgenic maize resulting from the reduction of both 19- and 22-kD alpha-zeins.

The major maize seed storage proteins, zeins, are deficient in lysine and tryptophan content, which contribute to the poor nutritional quality of corn. Whether through the identification of mutations or genetic engineering, kernels with reduced levels of zein proteins have been shown to have increased levels of lysine and tryptophan. It has been hypothesized that these increases are due to the reduction of lysine-poor zeins and a pleiotropic increase in the lysine-rich non-zein proteins. By transforming maize with constructs expressing chimeric double-stranded RNA, kernels derived from stable transgenic plants displayed significant declines in the accumulation of both 19- and 22-kD alpha-zeins, which resulted in higher lysine and tryptophan content than previously reported for kernels with reduced zein levels. The observation that lysine and tryptophan content is correlated with the protein levels measured in transgenic maize kernels is consistent with the hypothesis that a pleiotropic increase in non-zein proteins is contributing to an improved amino acid balance. In addition, a large increase in accumulation of free amino acids, consisting predominantly of asparagine, aspartate and glutamate, was observed in the zein reduction kernels.

Generation of marker-free transgenic maize by regular two-border Agrobacterium transformation vectors.

By introducing additional T-DNA borders into a binary plasmid used in Agrobacterium-mediated plant transformation, previous studies have demonstrated that the marker gene and the gene of interest (GOI) can be carried by independent T-strands, which sometimes integrate in unlinked loci in the plant genome. This allows the recovery of marker-free transgenic plants through genetic segregation in the next generation. In this study, we have found that by repositioning the selectable marker gene in the backbone and leaving only the GOI in the T-DNA region, a regular two-border binary plasmid was able to generate marker-free transgenic maize plants more efficiently than a conventional single binary plasmid with multiple T-DNA borders. These results also provide evidence that both the right and left borders can initiate and terminate T-strands. Such non-canonical initiation and termination of T-strands may be the basis for the elevated frequencies of cotransformation and unlinked insertions.