Análise quantitativa de rna mensageiros, proteínas e atividades enzimáticas no estudo da rede de regulação controlada pelo gene Opaco-2

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 alpha-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.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 (alfa-zeínas de 22 kDa, beta-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. Análises de correlação foram realizadas entre os polimorfismos de RFLP e (i) quantidade de RNAm do O2, cyPpdk, Lor/Sdh e Ahas (ii) quantidade de isoformas de zeínas e (iii) atividade da enzima SDH. Sítios polimórficos foram correlacionados com a quantidade de RNAm do próprio O2, do gene Lor/Sdh e com a quantidade de duas isoformas de a-zeinas de 19 kDa. Nossos resultados indicam a presença de relações entre o polimorfismo do locus O2 e o nível de expressão de genes sob o seu controle. A utilização de um maior número de linhagens e o uso de dados de seqüência do O2 permitirá uma análise precisa da conseqüência do polimorfismo deste fator de transcrição sobre o controle do seu próprio nível de expressão e dos genes por ele controlados

LKR/SDH Plays Important Roles throughout the Tick Life Cycle Including a Long Starvation Period

BACKGROUND:Lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) is a bifunctional enzyme catalyzing the first two steps of lysine catabolism in plants and mammals. However, to date, the properties of the lysine degradation pathway and biological functions of LKR/SDH have been very little described in arthropods such as ticks. METHODOLOGY/PRINCIPAL FINDINGS:We isolated and characterized the gene encoding lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) from a tick, Haemaphysalis longicornis, cDNA library that encodes a bifunctional polypeptide bearing domains similar to the plant and mammalian LKR/SDH enzymes. Expression of LKR/SDH was detected in all developmental stages, indicating an important role throughout the tick life cycle, including a long period of starvation after detachment from the host. The LKR/SDH mRNA transcripts were more abundant in unfed and starved ticks than in fed and engorged ticks, suggesting that tick LKR/SDH are important for the starved tick. Gene silencing of LKR/SDH by RNAi indicated that the tick LKR/SDH plays an integral role in the osmotic regulation of water balance and development of eggs in ovary of engorged females. CONCLUSIONS/SIGNIFICANCE:Transcription analysis and gene silencing of LKR/SDH indicated that tick LKR/SDH enzyme plays not only important roles in egg production, reproduction and development of the tick, but also in carbon, nitrogen and water balance, crucial physiological processes for the survival of ticks. This is the first report on the role of LKR/SDH in osmotic regulation in animals including vertebrate and arthropods

Soluble amino acid profile, mineral nutrient and carbohydrate content of maize kernels harvested from plants submitted to ascorbic acid seed priming

ABSTRACT Both the scientific community and society have shown interest in improving the content of amino acids, carbohydrates and mineral nutrients in maize because it represents an important staple food in many developing countries. Earlier studies demonstrated that the treatment of seeds using ascorbic acid (AsA-seed priming) enhanced soluble carbohydrates, proteins and soluble amino acids for other species. AsA seed priming in maize showed the potential for reducing abiotic stresses. The effects on grain quality have not been previously demonstrated. This study investigated the impacts of AsA seed priming on maize kernel quality of seeds produced by the plants generated from the primed seeds, based on the amino acid profile and carbohydrate and mineral nutrient contents. AsA seed priming improved the maize kernel quality with respect to the ascorbate content, boron allocation, total carbohydrate content and increased soluble amino acid levels, including serine, tyrosine, alanine, valine, glutamate, arginine, proline, aspartate, lysine and isoleucine, whereas soluble methionine was decreased. Therefore, AsA seed priming can represent a potential technique for improving maize grain quality

Degradation of lysine in rice seeds : effects of calcium, ionic strength, S-adenosylmethionine and S-2-aminoethyl-L-cysteine on lysine 2-oxoglutarate reductase-saccharopine dehydrogenase bifunctional enzyme.

Lysine biosynthesis has been extensively studied and the regulatory enzymes characterized in some of the most important crop plants, however, much less is known about the lysine degradation pathway. Lysine 2-oxoglutarate reductase (LOR) and saccharopine dehydrogenase (SDH) have recently been partially purified and characterized from plants, and have been shown to exist as a single bifunctional polypeptide. We have further characterized these enzymes from rice endosperm in relation to Ca2+ and ionic strength modulation. Optimum pH values of 7.0 and 8.0 were obtained for LOR and SDH, respectively. The LOR domain of the polypeptide was modulated by Ca2+ and ionic strength, whereas the SDH domain was not. It would appear that the modulation by Ca2+ and ionic strength of LOR is a common feature among plant LOR enzymes. S-adenosylmethionine (SAM) did not produce any significant effect on either enzyme activity, indicating that it only plays a role in the regulation of lysine biosynthesis. The effect of S-2-aminoethyl- l-cysteine (AEC) as both a substrate and an inhibitor of LOR activity was also tested. AEC was shown to partially substitute for lysine as a substrate for LOR, but was also able to inhibit LOR activity, possibly competing with lysine at the active site. The higher Km for AEC compared to lysine may reflect a lower binding affinity for AEC

Isolation And Characterization Of Enzymes Involved In Lysine Catabolism From Sorghum Seeds.

Lysine is an essential amino acid synthesized in plants via the aspartic acid pathway. The catabolism of lysine is performed by the action of two consecutive enzymes, lysine 2-oxoglutarate reductase (LOR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9). The final soluble lysine concentration in cereal seeds is controlled by both synthesis and catabolism rates. The production and characterization of high-lysine plants species depends on knowledge of the regulatory aspects of lysine metabolism and manipulation of the key enzymes. We have for the first time isolated, partially purified, and characterized LOR and SDH from developing sorghum seeds, which exhibited low levels of activity. LOR and SDH were only located in the endosperm and were very unstable during the isolation and purification procedures. LOR and SDH exhibited some distinct properties when compared to the enzymes isolated from other plant species, including a low salt concentration required to elute the enzymes during anion-exchange chromatography and the presence of multimeric forms with distinct molecular masses.531791-

Manipulating cereal crops for high lysine accumulation in seeds.

A lisina é um aminoácido essencial cuja via de biossíntese faz parte da via metabólica do ácido aspártico, pela qual são também sintetizados os aminoácidos treonina, metionina e isoleucina. Além disso, a lisina é o principal aminoácido limitante em todos os cereais e por cerca de 30 anos a via do ácido aspártico tem sido estudada em plantas, com o intuito de desvendar e caracterizar os principais pontos chave na regulação das vias de biossíntese desses aminoácidos. Duas etapas distintas, uma primeira originada a partir do desenvolvimento da cultura de tecidos (anos 70-80) e a segunda a partir do desenvolvimento de técnicas para a transformação de plantas (anos 90), permitiram que mutantes bioquímicos e plantas trangênicas fossem produzidos com alterações específicas em passos metabólicos chave, levando à superprodução e acúmulo de treonina em vários tecidos das plantas. Entretanto, a acumulação de lisina em sementes não foi obtida. Tal fato, associado a estudos bioquímicos da via de degradação da lisina em cereais e em leguminosas, indicou que a manipulação da degradação seria tão ou mais importante que a manipulação da biossíntese de lisina para o acúmulo deste aminoácido em sementes dos cereais . Em milho, o uso e estudo de outros mutantes tais como o opaco-2 e variedades QPM (Quality Protein Maize) contribuíram significativamente para a compreensão dos eventos regulatórios. As estratégias para a obtenção de materiais ricos em lisina e sua relevância à manipulação de outros aminoácidos são revisados.The nutrition value of a protein is directly related to its amino acid composition. Some of these amino acids, termed essential amino acids, cannot be synthesized by humans and therefore must be supplied in the diet for adults and in particular for infants and children. Lysine is an essential amino acid synthesized via the aspartic acid metabolic pathway, in which threonine, methionine and isoleucine are also endproducts. Moreover, lysine is the first limiting amino acid in all cereal grains. For over 30 years, the aspartic acid metabolic pathway has been studied in higher plants with the aim of identifying and characterizing the key regulatory points controlling the biosynthetic pathway. Two clear distinct time periods, one begining with the development of tissue culture techniques (1970-80's) and the second with the development of plant transformation techniques (90's), has encouraged the production of biochemical mutants and transgenic plants with specific alterations in key enzymes of the pathway, leading to the overproduction and accumulation of threonine in all plant tissues. However, the accumulation of lysine in seeds has been particularly difficult to achieve. Such an observation, associated with the recent biochemical studies on lysine degradation in cereal and legume plant species, has indicated that the manipulation of lysine degradation is as important as the manipulation of lysine synthesis, if the goal of accumulating this amino acid in cereal seeds is to be achieved. In maize, the study and use of other mutants such as the opaque-2 and QPM (Quality Protein Maize) varieties, has contributed significantly to our understanding of the regulatory aspects of the aspartate pathway. The strategies of obtaining cereals rich in lysine and their relevance to the manipulation of other amino acids have been discussed

Isolation of the biunctional enzyme lysine 2-oxoglutarate reductase-saccharopine dehydrogenase from Phaseolus vulgaris.

Lysine is catabolyzed by the bifunctional enzyme lysine 2-oxoglutarate reductase-saccharopine dehydrogenase (LOR-SDH) in both animals and plants. LOR condenses lysine and 2-oxoglutarate into saccharopine, using NADPH as cofactor and SDH converts saccharopine into α-aminoadipate δ-semialdehyde and glutamic acid, using NAD as cofactor. The distribution pattern of LOR and SDH among different tissues of Phaseolus vulgaris was determined. The hypocotyl contained the highest specific activity, whereas in seeds the activities of LOR and SDH were below the limit of detection. Precipitation of hypocotyl proteins with increasing concentrations of PEG 8000 revealed one broad peak of SDH activity, indicating that two isoforms may be present, a bifunctional LOR-SDH and possibly a monofunctional SDH. During the purification of the hypocotyl enzyme, the LOR activity proved to be very unstable, following ion-exchange chromatography. Depending on the purification procedure, the protein eluted as a monomer of 91–94 kDa containing only SDH activity, or as a dimer of 190 kDa with both, LOR and SDH activities, eluting together