RESUMO
Fructans are the main storage carbohydrates of temperate grasses, sustaining regrowth immediately after defoliation, as well as contributing to the nutritive value of feed. Fructan metabolism is based on the substrate sucrose and involves fructosyltransferases (FTs) for biosynthesis and fructan exohydrolases (FEHs) for degradation. Sucrose is also utilized by invertases (INVs), which hydrolyse it into its constituent monosaccharides for use in metabolism. The isolation, molecular characterization, functional analysis, and phylogenetic relationships of genes encoding FTs, FEHs, and INVs from temperate grasses are reviewed, with an emphasis on perennial ryegrass (Lolium perenne L.). The roles these enzymes play in fructan accumulation and remobilization, and future biotechnological applications in molecular plant breeding are discussed.
RESUMO
A sucrose: sucrose 1-fructosyltransferase (1-SST) gene and cDNA (Lp 1-SST) from perennial ryegrass (Lolium perenne) were isolated. The Lp 1-SST gene was fully sequenced and shown to contain three exons and two introns. Nucleotide sequence analysis of the 4824 bp Lp 1-SST genomic sequence revealed 1618 bp of 5' UTR and an open reading frame of 1962 bp encoding a protein of 653 amino acids. Lp 1-SST is 95% identical to the tall fescue 1-SST and contains plant fructosyltransferase functional domains. Lp 1-SST corresponds to a single copy gene in perennial ryegrass, and is expressed in young leaf bases and mature leaf sheaths. The recombinant Lp 1-SST protein from corresponding cDNA expression in Pichia pastoris showed 1-SST activity.
Assuntos
Hexosiltransferases/genética , Lolium/enzimologia , Proteínas de Plantas/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hexosiltransferases/isolamento & purificação , Hexosiltransferases/metabolismo , Lolium/genética , Dados de Sequência Molecular , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNARESUMO
An invertase (LpFT2) cDNA from perennial ryegrass was isolated and sequenced. Nucleotide sequence analysis revealed an ORF of 2016 bp encoding a protein of 671 amino acids. LpFT2 is 76% identical to sugarcane soluble acid invertase, and contains invertase and fructosyltransferase functional domains. LpFT2 is present as a single copy gene and maps to the distal region of LG6 in perennial ryegrass. The expression pattern analysis of LpFT2 revealed transcript accumulation in seedlings and in mature leaf sheaths. The LpFT2 recombinant protein expressed in yeast showed invertase and fructan exohydrolase-like activities with complete breakdown of sucrose, 1-kestose (DP3), 1,1-kestotetraose (DP4) and 1,1,1-kestopentaose (DP5) into glucose and fructose.
Assuntos
DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Lolium/genética , beta-Frutofuranosidase/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA de Plantas/isolamento & purificação , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Lolium/classificação , Lolium/enzimologia , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , beta-Frutofuranosidase/químicaRESUMO
Synthesis of the wound-inducible alkaloid, nicotine, in roots of the allotetraploid species Nicotiana tabacum L. is strongly influenced by the presence of two non-allelic genes, A and B. Together, these loci affect baseline transcript levels of genes dedicated to secondary metabolism (e.g. PMT and A622) as well as genes with roles in separate areas of primary metabolism (e.g. ODC, ADC, SAMS - polyamines; QPT - pyridine nucleotide cycle). Experiments comparing high alkaloid variety NC 95 (AABB genotype) and near-isogenic low alkaloid N. tabacum variety LAFC 53 (aabb genotype) indicate that together, mutations in the A and B loci diminish, but do not ablate, the propensity of roots to increase transcript levels of genes involved in alkaloid metabolism after damage to aerial tissues or direct treatment with the wound hormone, methyl jasmonate. Accordingly, roots of aabb genotype can increase their nicotine content somewhat in response to these treatments.
RESUMO
Invasive forms of apicomplexan parasites contain secretory organelles called rhoptries that are essential for entry into host cells. We present a detailed characterization of an unusual rhoptry protein of the human malaria parasite Plasmodium falciparum, the rhoptry-associated membrane antigen (RAMA) that appears to have roles in both rhoptry biogenesis and host cell invasion. RAMA is synthesized as a 170-kDa protein in early trophozoites, several hours before rhoptry formation and is transiently localized within the endoplasmic reticulum and Golgi within lipid-rich microdomains. Regions of the Golgi membrane containing RAMA bud to form vesicles that later mature into rhoptries in a process that is inhibitable by brefeldin A. Other rhoptry proteins such as RhopH3 and RAP1 are found in close apposition with RAMA suggesting direct protein-protein interactions. We suggest that RAMA is involved in trafficking of these proteins into rhoptries. In rhoptries, RAMA is proteolytically processed to give a 60-kDa form that is anchored in the inner face of the rhoptry membrane by means of the glycosylphosphatidylinositol anchor. The p60 RAMA form is discharged from the rhoptries of free merozoites and binds to the red blood cell membrane by its most C-terminal region. In early ring stages RAMA is found in association with the parasitophorous vacuole.