Dissecting the Opaque-2 regulatory network using transcriptome and proteome approaches along with enzyme activity measurements

Abstract

The Opaque-2 (O2) gene encodes a transcriptional activator specifically expressed for grain development of maize. o2 mutants have an opaque and chalky kernel, with a decrease in zein storage protein content, and an increase in the proportions of lysine and tryptophan. In this review, we present recent results investigating genetic properties of the O2 network, using transcriptome and proteome approaches, associated with measurements of activities of enzymes of the aspartate pathway and lysine degradation. The structural polymorphism at the O2 locus was investigated by RFLP in a collection of 51 maize inbred lines. Most polymorphic sites were found outside the coding regions. We then searched for relationships between RFLP polymorphism and (i) mRNA abundance of O2 and of known or suspected target genes, (ii) activity of SDH and (iii) amount of zein isoforms. Polymorphic restriction sites in the 5' upstream regions of the O2 gene were found associated with O2 mRNA abundance (three sites) and the amount of two 19 kDa α-zein isoforms (two sites). One restriction site on the 3' side of the O2 gene was found associated with Lor/Sdh mRNA abundance. Our results indicate relationships between polymorphism at the O2 locus and the expression of some of its target genes. Evidence of these associations has to be confirmed on larger samples, and the analysis of the O2 gene sequence should allow more precise testing of the actual involvement of O2 polymorphism in its own transcriptional expression, and in the expression of its target genes. Key words: zein, aspartate kinase, lysine oxoglutarate reductase/saccharopine dehydrogenase, polymorphism ANÁLISE QUANTITATIVA DE RNA MENSAGEIROS, PROTEÍNAS E ATIVIDADES ENZIMÁTICAS NO ESTUDO DA REDE DE REGULAÇÃO CONTROLADA PELO GENE OPACO-2RESUMO: O gene Opaco-2 (O2), expresso especificamente no grão de milho, transcreve para um fator de transcrição da família "leucine-zipper". Mutantes o2 apresentam grãos opacos, redução na quantidade de zeínas e aumento na proporção de lisina e triptofano. Genes cuja expressão é controlada diretamente pelo O2 são conhecidos (α-zeínas de 22 kDa, β-zeínas de 14 kDa, b-32 e cyPpdk1). Nesta revisão, nós apresentamos resultados da caracterização genética de genes relacionados com o O2, através de abordagens de transcritoma, proteoma e de atividades enzimáticas da via metabólica do aspartato e da degradação da lisina. O polimorfismo do locus O2 foi avaliado utilisando-se a técnica de RFLP em 51 linhagens de milho. A maioria dos polimorfismos foi observada nas regiões não codificadoras da proteína.

Lysine catabolism: flow, metabolic role and regulation

“…Through transcriptome and proteome approaches, the regulatory role of the opaque-2 gene has been confirmed, since a 3' restriction site was shown to be associated with LOR-SDH mRNA abundance (Lefèvre et al, 2002). The use of such techniques certainly will contribute significantly in the future.…”

“…This mutation is characterized by an opaque phenotype with a farinaceous endosperm. The high lysine concentration observed in the endosperm is related to an increase in the concentration of soluble lysine and storage proteins with the simultaneous reduction of the prolamin fraction, which has only trace amounts of lysine (Lefèvre et al, 2002). The introduction of the opaque phenotype modifier genes allowed the production of opaque-2 maize lines with good grain productivity, that also exhibit characteristics of high lysine and tryptophan contents, but with a translucent phenotype, which have been denominated as quality protein maize -(QPM) (Vasal, 1994;Gaziola et al, 1999).…”

Determination of aspartate kinase activity in maize tissues

“…Lysine and threonine, which are in low concentrations in cereal seeds are derived from aspartate (Lefèvre et al, 2002;Toro et al, 2003). The in vitro manipulation of AK has proved to be difficult and in order to isolate and characterize the enzyme from different plant tissues, the assay used to determine enzyme activity has to be efficient.…”

“…Amino acids are the major nitrogen containing compounds of plants and are the building-blocks of proteins (Helm et al, 2004). The essential amino acids lysine, threonine, methionine and isoleucine are derived from aspartic acid in a strongly regulated metabolic pathway (Lefèvre et al, 2002;Toro et al, 2003).…”