RESUMO
The present work aimed to study the control of the biosynthesis of the antinutritional factor phytate and its associated Fe-rich protein family, ferritin, in coffee. Phytate has the ability to chelate Fe, making it unavailable to human absorption. The Coffea genome databases were queried for genes associated with phytate metabolism and ferritin genes. The genetic framework for phytate biosynthesis and its reverse pathway was identified in silico analyses and indicate that Coffea phosphatidyl inositol kinase and monophosphatase families play nonredundant roles in phytate metabolism. The transcriptional profiles of phytate biosynthesis key-genes MYO-INOSITOL(3)P1 SYNTHASE, two genes coding for PHOSPHATIDYL INOSITOL KINASE, and three FERRITIN genes were temporally evaluated by qPCR in coffee seeds from two crop locations, Adamantina-SP and Ouro-Fino-MG, the last one traditionally associated with high-quality coffee beverage grain. A targeted metabolome profile of phytic acid contents throughout three fruit maturation stages in association with the transcriptional analysis was also obtained. Taken together, our data indicate that the investigated local conditions did not cause significant alterations in phytate biosynthesis. Futhermore, the temporal transcriptional profiling revealed that candidate gene expression is regulated independently of phytate accumulation. In contrast, the expression profile of ferritin-unit genes is affected by environmental conditions and genetic background. The roles of the investigated genes are discussed concerning the quality of coffee beverage.
Assuntos
Coffea/genética , Ferritinas/genética , Perfilação da Expressão Gênica , Ácido Fítico/biossíntese , Proteínas de Plantas/genética , Coffea/metabolismo , Ferritinas/metabolismo , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Proteínas de Plantas/metabolismoRESUMO
The abundant metabolite myo-inositol hexakisphosphate (InsP6) can form vesicular deposits with cations, a widespread phenomenon in plants also found in the cestode parasite, Echinococcus granulosus. In this organism, the deposits are exocytosed, accumulating in a host-exposed sheath of extracellular matrix termed the laminated layer. The formation and mobilization of InsP6 deposits, which involve precipitation and solubilization reactions, respectively, cannot yet be rationalized in quantitative chemical terms, as the solids involved have not been formally described. We report such a description for the InsP6 deposits from E. granulosus, purified as the solid residue left by mild alkaline digestion of the principal mucin component of the laminated layer. The deposits are largely composed of the compound Ca5H2L.16H2O (L representing fully deprotonated InsP6), and additionally contain Mg2+ (6-9% molar ratio with respect to Ca2+), but not K+. Calculations employing recently available chemical constants show that the precipitation of Ca5H2L.16H2O is predicted by thermodynamics in secretory vesicle-like conditions. The deposits appear to be similar to microcrystalline solids when analysed under the electron microscope; we estimate that each crystal comprises around 200 InsP6 molecules. We calculate that the deposits increase, by three orders of magnitude, the surface area available for adsorption of host proteins, a salient ability of the laminated layer. The major inositol phosphate in the deposits, other than InsP6, is myo-inositol (1,2,4,5,6) pentakisphosphate, or its enantiomer, inositol (2,3,4,5,6) pentakisphosphate. The compound appears to be a subproduct of the intracellular pathways leading to the synthesis and vesicular accumulation of InsP6, rather than arising from extracellular hydrolysis of InsP6.
Assuntos
Echinococcus granulosus/química , Ácido Fítico/análise , Animais , Cálcio/análise , Bovinos , Cromatografia Líquida de Alta Pressão , Echinococcus granulosus/crescimento & desenvolvimento , Exocitose , Matriz Extracelular/química , Matriz Extracelular/ultraestrutura , Hidrólise , Larva/química , Magnésio/análise , Camundongos , Ressonância Magnética Nuclear Biomolecular , Ácido Fítico/biossíntese , Ácido Fítico/isolamento & purificação , Potássio/análise , Sódio/análise , Estereoisomerismo , TermodinâmicaRESUMO
Four cultivars of Phaseolus vulgaris were grown in a greenhouse and each flower was Labeled with date of anthesis. Seeds were collected at six different stages of development and inositol phosphates (InsPs) were analyzed by ion-pair reversed-phase HPLC. Phytate accumulation was similar in all cultivars, and the specific rate of phytate synthesis (Rs) peaked at about 22 days after flowering (DAF). Variations in the concentrations of the InsP3 and InsP4 pools matched changes in Rs in cultivars Una and Aruã. These results suggest mass-action effects. Thus, the rates of conversion of InsP3 to InsP5 appeared to be at least partly dependent on substrate concentration. Proportional increases in size of all InsP pools up to 21 DAF are also consistent with Little regulation in this part of the pathway. However, this did not appear to be the case in cv. Diamante Negro or with the conversion of InsP5 to InsP6 in all cultivars, where concentrations of the InsP precursor pools peaked earlier or even dropped as Rs peaked, suggesting activation of enzyme activity. Therefore, the evidence is consistent with a control point regulating this metabolic route upstream of InsP3 and possibly in the conversion of InsP5 to InsP6.