Alterations in the accumulation patterns of seed storage proteins enhance the nutritive value of maize kernels
Creators: name: Planta, Jose Ramon
Publisher: name: No Publisher Supplied
Year: 2017

description: The phenotypic plasticity of the zeins, the major seed storage proteins in maize, allows for manipulation of the quality of amino acids to enhance the nutritive value of maize kernels. As the primary sink of nitrogen (N) and sulfur (S) for the germinating seedling and not as a repository of specific amino acids, it can tolerate significant alterations in its amino acid composition. Using transgenic approaches to improve the essential amino acid (EAA) content of maize, my dissertation is formatted into two chapters and focuses on disparate mechanisms that we have utilized to increase methionine and/or lysine in the kernels.   The first chapter relates to the generation and characterization of a high-methionine maize obtained by metabolic engineering of the cysteine biosynthetic pathway.  Under limiting S availability, transgenic maize kernels overexpressing its own high-methionine 10-kDa δ-zein displays redistribution of protein S in the seeds, a sink tissue, whereby the increased accumulation of the δ-zein is accompanied by a concomitant decrease in the cysteine-rich β- and γ-zeins. This reflects the limitation on S availability in the maize kernels. Increasing S availability or supply from the leaf, a source tissue, by overexpression of the assimilatory reductase EcPAPR, 3’-phosphoadenosine-5’-phosphosulfate reductase enzyme from Escherichia coli, overcame the rebalancing of S storage in the zeins. Transgenic high-methionine kernels have high levels of the δ-zein without the accompanying loss of protein S from other zeins. In addition, cysteine-rich non-zein proteins in the transgenic kernels were also upregulated. This overall increase in the accumulation of the S-rich zeins and cysteine-rich non-zein proteins enhanced the kernel methionine and cysteine contents. One transgenic event, PE5, promoted weight gains in chicks when formulated as an animal feed without synthetic methionine supplementation. Increased expression of the S-rich zeins under enhanced sulfur assimilation in source tissues constitutes a newly-described aspect of regulation of S amino acid accumulation in maize kernels.  The second chapter involves the combination of two different mechanisms – deregulation of the cysteine biosynthetic pathway, as mentioned, and reduction of zeins by RNA interference – to produce maize kernels with enhanced lysine and methionine contents. Reduction in zeins are generally associated with increased nutritional quality of maize kernels, and this is exemplified by the classical opaque-2 (o2) mutant of maize that conditions a severe reduction in the abundant α-zeins. o2 has high nutritional value as determined by feeding trials with rats and infants and a biological value that approaches that of milk. However, O2 as a transcription factor recognizes several genes, including the 22-kDa a-zeins, and its loss results to pleiotropic changes in gene expression in the o2 seeds, leading to the opaque, or soft endosperm, phenotype. The opaque phenotype renders the kernel susceptible to insect and fungal damage and the soft texture makes the kernels prone to breakage during handling and storage. In combination with genetic modifiers that can restore the vitreous endosperm in o2 kernels, quality protein maize (QPM) was developed that has the high-lysine trait of o2 but with a hard endosperm. However, the complexity of introducing multiple, unlinked loci of these modifiers into an o2 background makes the process of generating QPMs laborious and complicated.  To explore QPMs with elevated levels of methionine, PE5 maize was crossed with different transgenic zein reduction lines (RNAi lines that target zeins of classes: α, β, γ, α + γ, or γ + β). Although it was already known that loss of the abundant α-zeins redistributed N to the non-zein protein fraction, thereby increasing the synthesis of some lysine-rich non-zein proteins. Addition of the PE5 event to α-RNAi further increased the lysine content. Although an additional reduction in γ-zeins in PE5;α-/γ- resulted in a reduction of lysine, the methionine level were nearly as good as with PE5 alone. Despite this favorable outcome, PE5;α-/γ- shares the same problems as α-RNAi alone in that it had an opaque kernel phenotype. Surprisingly, leaving off α-RNAi still gave elevated levels of lysine and methionine over classical mutants without the opaque phenotype.    Aside from downregulation of zein proteins, upregulation of the S-rich zeins as displayed in the PE5 kernels show that altering zein synthesis in the maize endosperm can indeed significantly improve the nutritional quality of maize kernels. Combining PE5 with the loss of γ-zeins produced kernels with superior Met and Lys contents and hard endosperm compared to o2 seeds. From our observations, a common mechanism in the regulation of EAA accumulation in maize kernels appears to be the redistribution of macronutrients, particularly N and S, under limiting nutrient availability. We show here that this redistribution occurs due to the altered synthesis of zeins and can improve the amino acid quality of the seeds. This redistribution of nutrients leads to rebalancing of nutrient storage by altering the synthesis of other proteins. Increasing nutrient availability from the source tissues by deregulation of the corresponding metabolic pathway can overcome this restriction on EAA accumulation in the sink.