Quality Protein Maize Based on Reducing Sulfur in Leaf Cells.

Low levels of the essential amino acids lysine (Lys) and methionine (Met) in a maize-based diet are a major cost to feed and food. Lys deficiency is due to the abundance of Lys-poor proteins in maize kernels. Although a maize mutant, opaque-2 (o2), has sufficient levels of Lys, its soft kernel renders it unfit for storage and transportation. Breeders overcame this problem by selecting quantitative trait loci (QTL) restoring kernel hardness in the presence of o2, a variety called Quality Protein Maize (QPM). Although at least one QTL acts by enhancing the expression of the γ-zein proteins, we could surprisingly achieve rebalancing of the Lys content and a vitreous kernel phenotype by targeting suppression of γ-zeins without the o2 mutant. Reduced levels of γ-zeins were achieved with RNA interference (RNAi). Another transgenic event, PE5 expresses the Escherichia coli enzyme 3'-phosphoadenosine-5'-phosphosulfate reductase involved in sulfate assimilation, specifically in leaves. The stacked transgenic events produce a vitreous endosperm, which has higher Lys level than the classical opaque W64Ao2 variant. Moreover, due to the increased sulfate reduction in the leaf, Met level is elevated in the seed. Such a combination of transgenes produces hybrid seeds superior to classical QPMs that would neither require a costly feed mix nor synthetic Met supplementation, potentially creating a novel and cost-effective means for improving maize nutritional quality.

Engineering sulfur storage in maize seed proteins without apparent yield loss.

Sulfur assimilation may limit the pool of methionine and cysteine available for incorporation into zeins, the major seed storage proteins in maize. This hypothesis was tested by producing transgenic maize with deregulated sulfate reduction capacity achieved through leaf-specific expression of the Escherichia coli enzyme 3'-phosphoadenosine-5'-phosphosulfate reductase (EcPAPR) that resulted in higher methionine accumulation in seeds. The transgenic kernels have higher expression of the methionine-rich 10-kDa δ-zein and total protein sulfur without reduction of other zeins. This overall increase in the expression of the S-rich zeins describes a facet of regulation of these proteins under enhanced sulfur assimilation. Transgenic line PE5 accumulates 57.6% more kernel methionine than the high-methionine inbred line B101. In feeding trials with chicks, PE5 maize promotes significant weight gain compared with nontransgenic kernels. Therefore, increased source strength can improve the nutritional value of maize without apparent yield loss and may significantly reduce the cost of feed supplementation.

Maize defective kernel mutant generated by insertion of a Ds element in a gene encoding a highly conserved TTI2 cochaperone.

We have used the newly engineered transposable element Dsg to tag a gene that gives rise to a defective kernel (dek) phenotype. Dsg requires the autonomous element Ac for transposition. Upon excision, it leaves a short DNA footprint that can create in-frame and frameshift insertions in coding sequences. Therefore, we could create alleles of the tagged gene that confirmed causation of the dek phenotype by the Dsg insertion. The mutation, designated dek38-Dsg, is embryonic lethal, has a defective basal endosperm transfer (BETL) layer, and results in a smaller seed with highly underdeveloped endosperm. The maize dek38 gene encodes a TTI2 (Tel2-interacting protein 2) molecular cochaperone. In yeast and mammals, TTI2 associates with two other cochaperones, TEL2 (Telomere maintenance 2) and TTI1 (Tel2-interacting protein 1), to form the triple T complex that regulates DNA damage response. Therefore, we cloned the maize Tel2 and Tti1 homologs and showed that TEL2 can interact with both TTI1 and TTI2 in yeast two-hybrid assays. The three proteins regulate the cellular levels of phosphatidylinositol 3-kinase-related kinases (PIKKs) and localize to the cytoplasm and the nucleus, consistent with known subcellular locations of PIKKs. dek38-Dsg displays reduced pollen transmission, indicating TTI2's importance in male reproductive cell development.

Phage M13 for the treatment of Alzheimer and Parkinson disease.

The studies of microbes have been instrumental in combatting infectious diseases, but they have also led to great insights into basic biological mechanism like DNA replication, transcription, and translation of mRNA. In particular, the studies of bacterial viruses, also called bacteriophage, have been quite useful to study specific cellular processes because of the ease to isolate their DNA, mRNA, and proteins. Here, I review the recent discovery of how properties of the filamentous phage M13 emerge as a novel approach to combat neurodegenerative diseases.

Balancing of sulfur storage in maize seed.

BACKGROUND: A balanced composition of amino acids in seed flour is critical because of the demand on essential amino acids for nutrition. However, seed proteins in cereals like maize, the crop with the highest yield, are low in lysine, tryptophan, and methionine. Although supplementation with legumes like soybean can compensate lysine deficiency, both crops are also relatively low in methionine. Therefore, understanding the mechanism of methionine accumulation in the seed could be a basis for breeding cultivars with superior nutritional quality. RESULTS: In maize (Zea mays), the 22- and 19-kDa α-zeins are the most prominent storage proteins, nearly devoid of lysine and methionine. Although silencing synthesis of these proteins through RNA interference (RNAi) raises lysine levels in the seed, it fails to do so for methionine. Computational analysis of annotated gene models suggests that about 57% of all proteins exhibit a lysine content of more than 4%, whereas the percentage of proteins with methionine above 4% is only around 8%. To compensate for this low representation, maize seeds produce specialized storage proteins, the 15-kDa ß-, 18-kDa and 10-kDa δ-zeins, rich in methionine. However, they are expressed at variant levels in different inbred lines. A654, an inbred with null δ-zein alleles, methionine levels are significantly lower than when the two intact δ-zein alleles are introgressed. Further silencing of ß-zein results in dramatic reduction in methionine levels, indicating that ß- and δ-zeins are the main sink of methionine in maize seed. Overexpression of the 10-kDa δ-zein can increase the methionine level, but protein analysis by SDS-PAGE shows that the increased methionine levels occur at least in part at the expense of cysteines present in ß- and γ-zeins. The reverse is true when ß- and γ-zein expression is silenced through RNAi, then 10-kDa δ-zein accumulates to higher levels. CONCLUSIONS: Because methionine receives the sulfur moiety from cysteine, it appears that when seed protein synthesis of cysteine-rich proteins is blocked, the synthesis of methionine-rich seed proteins is induced, probably at the translational level. The same is true, when methionine-rich proteins are overexpressed, synthesis of cysteine-rich proteins is reduced, probably also at the translational level. Although we only hypothesize a translational control of protein synthesis at this time, there are well known paradigms of how amino acid concentration can play a role in differential gene expression. The latter we think is largely controlled by the flux of reduced sulfur during plant growth.

RNA interference can rebalance the nitrogen sink of maize seeds without losing hard endosperm.

BACKGROUND: One of the goals of plant breeding is to create crops to provide better nutrition for humans and livestock. Insufficient intake of protein is one of the most severe factors affecting the growth and development of children in developing countries. More than a century ago, in 1896, Hopkins initiated the well-known Illinois long-term selection for maize seed protein concentration, yielding four protein strains. By continuously accumulating QTLs, Illinois High Protein (IHP) reached a protein level 2.5-fold higher than normal maize, with the most increased fraction being the zein protein, which was shown to contain no lysine soon after the long-term selection program initiated. Therefore, IHP is of little value for feeding humans and monogastric animals. Although high-lysine lines of non-vitreous mutants were based on reduced zeins, the kernel soft texture precluded their practical use. Kernel hardness in opaque 2 (o2) could be restored in quality protein maize (QPM) with quantitative trait loci called o2 modifiers (Mo2s), but those did not increase total protein levels. METHODS: The most predominant zeins are the 22- and 19-kDa α-zeins. To achieve a combination of desired traits, we used RNA interference (RNAi) against both α-zeins in IHP and evaluated the silencing effect by SDS-PAGE. Total protein, amino acid composition and kernel texture were analyzed. CONCLUSIONS: The α-zeins were dramatically reduced, but the high total seed protein level remained unchanged by complementary increase of non-zein proteins. Moreover, the residual zein levels still allowed for a vitreous hard seed. Such dramatic rebalancing of the nitrogen sink could have a major impact in world food supply.