Label-free shotgun proteomics and metabolite analysis reveal a significant metabolic shift during citrus fruit development

Label-free LC-MS/MS-based shot-gun proteomics was used to quantify the differential protein synthesis and metabolite profiling in order to assess metabolic changes during the development of citrus fruits. Our results suggested the occurrence of a metabolic change during citrus fruit maturation, where the organic acid and amino acid accumulation seen during the early stages of development shifted into sugar synthesis during the later stage of citrus fruit development. The expression of invertases remained unchanged, while an invertase inhibitor was up-regulated towards maturation. The increased expression of sucrose-phosphate synthase and sucrose-6-phosphate phosphatase and the rapid sugar accumulation suggest that sucrose is also being synthesized in citrus juice sac cells during the later stage of fruit development.     

The role of neutral lipid nanospheres in Plasmodium falciparum haem crystallization

The intraerythrocytic malaria parasite constructs an intracellular haem crystal, called haemozoin, within an acidic digestive vacuole where haemoglobin is degraded. Haem crystallization is the target of the widely used antimalarial quinoline drugs. The intracellular mechanism of molecular initiation of haem crystallization, whether by proteins, polar membrane lipids or by neutral lipids, has not been fully substantiated. In the present study, we show neutral lipid predominant nanospheres, which envelop haemozoin inside Plasmodium falciparum digestive vacuoles. Subcellular fractionation of parasite-derived haemozoin through a dense 1.7 M sucrose cushion identifies monoacylglycerol and diacylglycerol neutral lipids as well as some polar lipids in close association with the purified haemozoin. Global MS lipidomics detects monopalmitic glycerol and monostearic glycerol, but not mono-oleic glycerol, closely associated with haemozoin. The complex neutral lipid mixture rapidly initiates haem crystallization, with reversible pH-dependent quinoline inhibition associated with quinoline entry into the neutral lipid microenvironment. Neutral lipid nanospheres both enable haem crystallization in the presence of high globin concentrations and protect haem from H2O2 degradation. Conceptually, the present study shifts the intracellular microenvironment of haem crystallization and quinoline inhibition from a polar aqueous location to a non-polar neutral lipid nanosphere able to exclude water for efficient haem crystallization.     

Glucose Induces Sensitivity to Oxygen Deprivation and Modulates Insulin/IGF-1 Signaling and Lipid Biosynthesis in Caenorhabditis elegans

Diet is a central environmental factor that contributes to the phenotype and physiology of individuals. At the root of many human health issues is the excess of calorie intake relative to calorie expenditure. For example, the increasing amount of dietary sugars in the human diet is contributing to the rise of obesity and type 2 diabetes. Individuals with obesity and type 2 diabetes have compromised oxygen delivery, and thus it is of interest to investigate the impact a high-sugar diet has on oxygen deprivation responses. By utilizing the Caenorhabditis elegans genetic model system, which is anoxia tolerant, we determined that a glucose-supplemented diet negatively impacts responses to anoxia and that the insulin-like signaling pathway, through fatty acid and ceramide synthesis, modulates anoxia survival. Additionally, a glucose-supplemented diet alters lipid localization and initiates a positive chemotaxis response. Use of RNA-sequencing analysis to compare gene expression responses in animals fed either a standard or glucose-supplemented diet revealed that glucose impacts the expression of genes involved with multiple cellular processes including lipid and carbohydrate metabolism, stress responses, cell division, and extracellular functions. Several of the genes we identified show homology to human genes that are differentially regulated in response to obesity or type 2 diabetes, suggesting that there may be conserved gene expression responses between C. elegans fed a glucose-supplemented diet and a diabetic and/or obesity state observed in humans. These findings support the utility of the C. elegans model for understanding the molecular mechanisms regulating dietary-induced metabolic diseases.     

Ascorbate peroxidase 1 plays a key role in the response of Arabidopsis thaliana to stress combination

Within their natural habitat plants are subjected to a combination of different abiotic stresses, each with the potential to exacerbate the damage caused by the others. One of the most devastating stress combinations for crop productivity, which frequently occurs in the field, is drought and heat stress. In this study we conducted proteomic and metabolic analysis of Arabidopsis thaliana plants subjected to a combination of drought and heat stress. We identified 45 different proteins that specifically accumulated in Arabidopsis in response to the stress combination. These included enzymes involved in reactive oxygen detoxification, malate metabolism, and the Calvin cycle. The accumulation of malic enzyme during the combined stress corresponded with enhanced malic enzyme activity, a decrease in malic acid, and lower amounts of oxaloacetate, suggesting that malate metabolism plays an important role in the response of Arabidopsis to the stress combination. Cytosolic ascorbate peroxidase 1 (APX1) protein and mRNA accumulated during the stress combination. When exposed to heat stress combined with drought, an APX1-deficient mutant (apx1) accumulated more hydrogen peroxide and was significantly more sensitive to the stress combination than wild type. In contrast, mutants deficient in thylakoid or stromal/mitochondrial APXs were not more sensitive to the stress combination than apx1 or wild type. Our findings suggest that cytosolic APX1 plays a key role in the acclimation of plants to a combination of drought and heat stress.     

Vacuolar citrate/H+ symporter of citrus juice cells

We have isolated a cDNA, designated Citrus sinensis citrate transporter 1 CsCit1 encoding a novel vacuolar citrate/symporter. Immunoblots using antibodies raised against CsCit1 showed that the protein is localized to the juice sac cell vacuoles. The highest expression of CsCit1 and the amount of protein in the juice sac cell vacuoles coincided with the developmental stage at which the vacuolar citrate content began declining with the concomitant increase in vacuolar pH. Vacuoles from Sacharomyces cereviseae expressing CsCit1 displayed a citrate-dependent H⁺ efflux, and our results clearly demonstrate that CsCit1 is able to mediate the electroneutral co-transport of H⁺ and citrate ions, since the citrate-dependent H⁺ fluxes are not affected by changing the electrical potential difference across the tonoplast. The roles of CsCit1 in mediating citrate efflux from the vacuole and on citric acid homoestasis in Citrus juice sac cells are discussed. T. Shimada and R. Nakano contributed equally to this work.