Citrus fruit quality: insights into citrus fruit metabolism by using proteomics.
We are interested in citrus fruit quality and focus mainly on sugar accumulation and homeostasis during fruit development. We are taking few approaches to understand processes affecting fruit quality traits and to understand sugar accumulation and homeostasis in citrus fruit identification and characterization of vacuolar sugar transporters and proteomics analysis of fruit development.
1) Sugar transport into vacuoles — Sucrose is the main sugar transported from source to sink tissues in citrus, as in many plant species. Since the vascular system surrounding the citrus fruit flesh is not connected symplasically to the juice cells sucrose arrived to the fruit is transported to the apoplast and then is must be taken into juice cell through sugar transporters. Sucrose can be transported as it is or hydrolyzed to glucose and fructose by the cell-wall invertases then taken up via plasma membrane monosaccharide transporters. Most of the sugars accumulated in juice sac cells are transported into the vacuole and stored there. It was assumed that several members of the diverse sugar transporters family serve to transport sugars into the vacuole but only recently the first tonoplast monosaccharide transporters were identified and partially characterized in Arabidopsis. In the citrus ESTs database there are 114 ESTs and 48 unigenes corresponding to the definition of sugar transporter. We cloned two genes from juice sacs tissues, determine their full sequence and expressed them in yeast. We confirmed their localization to the tonoplast by western blot analysis of isolated vacuoles of transformed yeast expressing these genes and by fusing these genes to GFP. The expression of both genes is induced during citrus fruit development accompanying sugar accumulation. We are applying an array of techniques in order to determine these genes functions; among them 1) Fluorescence quenching, 2) sugar nanosensors and 3) Radioisotope techniques.
We are currently developing glucose and sucrose nanosensors based on FRET technology consist of YFP, CFP and a sugar binding protein. Upon sugar binding, the fused protein is going under conformational change which induces fluorescence energy transfer between the two GFPs and to a change in fluorescence emission. In this way we can isolate yeast vacuoles expressing our genes and detect fluorescence changes in the vacuole after applying different concentrations of a given sugar. This tool will enable us to detect and measure sugar transport across membranes and intracellular sugar concentrations.
2) Proteomics analysis of citrus juice cells during development — For the last few years a citrus genome-wide ESTs project has been initiated and consists of 229,570 clones corresponding to 23,577 contigs and 22,586 singletons. Our goal is to identify changes in proteins profile during fruit development, to classify and reveal their function, in particular those which are involved in sugar metabolism and sugar build-up, citric acid metabolism and secondary metabolites using proteomics tools. We are using LTQ-FT LC-MS/MS analyses of ER/Golgi, plasma membrane, tonoplast, mitochondria and soluble enriched fractions from mature citrus juice sac cells to identify changes in the juice sac cell. So far we have identified ca. 1,400 proteins by searching NCBI-nr (green plants) and citrus ESTs databases. Using the extended citrus ESTs database we have identified proteins involved in sugar metabolism, citrate cycle, signaling, transport, processing, vesicular trafficking and more. This data, that has already been published, is the first extensive analysis of protein expression in citrus
We have recently developed LC-MS/MS label-free technique to identify quantitative differences in high throughput protein expression. We also developed methods for fractionation of membranes and protein purification for LC-MS/MS analysis. These techniques allow for the quantification in protein expression differences between different developmental stages, thus facilitating the identification of metabolic pathways. We identified many biochemical changes during fruit development among them changes in pathways affecting sugar homeostasis, amino acid metabolism, plant hormones biosynthesis, perception, signaling and response. We also identified changes in many transporters responsible for protons, amino acids, sugars and ions transport across membranes.