Phosphorylation of fructose-6-phosphate catalysed by PFK is virtually irreversible while PFP catalyses the reaction in both directions. In citrus juice cells, two PFPs and one PFK were found to be differentially expressed . CitPFP1 and CitPFP2 remained unchanged during the transition from early stage II to stage II. CitPFP1 was down-regulated during the transition from stage II to stage III while CitPFP2 was up-regulated. Gene expression analysis of CitPFP1 and CitPFP2 showed similar expression patterns . In contrast to PFPs, only one PFK was identified and its expression did not change during the transition from early stage II to stage II but was up-regulated during the transition from stage II to stage III. No changes in invertases, an important family of proteins responsible for sucrose degradation to glucose and fructose, were seen in our proteomics analysis. The activity of two citrus acid invertases were detected in juice sac cells with higher activities at the earlier stages of development. Since no differences in invertase proteins were detected in our comparisons, the expression of three citrus invertase genes, the vacuolar/acidic bFruct1 and bFruct2 and the neutral/alkaline invertase CitCINV1, was followed. The expression of these three genes peaked at early stage II and was down-regulated during the later stages of fruit development, suggesting a role of invertases during the early stages of fruit development .
Interestingly, an invertase inhibitor protein was found between early stage II and stage II, plastic pot black and was up-regulated during the transition from stage II to stage III . Invertase inhibitors are responsible for the decrease in invertase activity and function at the later stages of fruit development, highlighting the importance of sucrose synthase activity in sucrose degradation during these developmental stages. Sucrose-phosphate synthase , an enzyme involved in sucrose synthesis, was up-regulated during the transition from early stage II to stage II and remained unchanged during the transition from stage II to stage III, while no changes were seen in sucrose-6-phosphate phosphatase, which mediates the formation of sucrose from sucrose-6-P. Some differences between SPS and SPP protein amounts and their levels of gene expression were noted. The expression of SPS was up-regulated only during the transition from stage II to stage III . Also, although no differences were seen in SPP protein amounts, the gene expression decreased slightly towards stage II and increased towards stage III . SPS and SPP activities correlated well with their protein expression patterns .Citrus fruits accumulate large amount of organic acids, mainly citrate, in juice sac cells. In contrast to sugars, citrate is synthesized in the juice cells and not transported from other organs of the tree. Citrate is produced through the TCA cycle and accumulates in the vacuole during fruit development, reaching a maximum at late stage II and decreasing towards maturation . Citrate is not only an intermediate metabolite in energy production in citrus juice cells, but also accumulates to high concentrations and is stored in the vacuole, contributing more than 90% of citrus fruit juice cells’ organic acids content .
The mechanisms regulating citrate accumulation and degradation during pre- and post-harvest are unknown but play significant roles in determining the quality of many fruit species in general, and citrus fruit in particular. The pyruvate dehydrogenase enzyme complex links the TCA cycle to glycolysis. One of the pyruvate dehydrogenase complex proteins increased during the transition from early stage II to stage II while three others were downregulated . In addition, two other components of the pyruvate dehydrogenase complex, dihydrolipoamide S-acetyltransferase and dihydrolipoamide dehydrogenase were down-regulated during this transition. The pyruvate dehydrogenase complex was up-regulated during the transition from stage II to stage III . Aconitase, isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, succinyl-CoA synthetase, fumarase, and malate dehydrogenase were up-regulated or remained unchanged during the transition from early stage II to stage II, and were up-regulated during the transition from stage II to stage III . Three exceptions to these general trends in the TCA cycle protein expression were noted, pyruvate dehydrogenase, succinate dehydrogenase, and malic enzyme, which were down-regulated during the transition from early stage II to stage II and were upregulated during the transition from stage II to stage III.After determining protein changes in juice cells during fruit development, our focus turned to changes in core metabolite accumulation.
Organic acids of the TCA cycle, citrate, isocitrate, aconitate, and malate, were highest at stage II and decreased towards stage III, while 2-oxoglutarate and fumarate gradually decreased during fruit development and succinate accumulated mainly during stage III . Sucrose, glucose, fructose, maltose, and sedoheptulose accumulated in an essentially linear manner during fruit development . Galactose and trehalose rapidly accumulated in the juice sac cells towards maturation. Mannose gradually decreased during fruit development. Sugar alcohols displayed different accumulation patterns; inositol reached a maximum at stage II and decreased towards stage III, sorbitol decreased towards stage II and accumulated again towards maturation, while myo-inositol reached a maximum at the early stages of development and decreased gradually towards fruit maturation. Mannitol displayed considerable variation during development, increasing at stage II and decreasing towards maturation. Sugar phosphates and gluconate were higher at the earlier stages of fruit development and decreased during fruit maturation.Plants assimilate inorganic nitrogen into four major amino acids, glutamate, glutamine, aspartate, and asparagine. These amino acids are usually transported from source to sink tissues and are used as a nitrogen source for metabolism and growth. The carbon skeletons for amino acids are derived from 3-phosphoglycerate, phosphoenolpyruvate or pyruvate generated during glycolysis or from 2-oxoglutarate and oxaloacetate generated in the citric acid cycle. The amounts of amino acids were highly variable during fruit development . A gradual decline was noted for isoleucine and glutamine while a gradual increase was noted for shikimic acid, histidine, and tyrosine. Aspartate, asparagine, threonine, alanine, valine, leucine, glycine, serine, glutamate, and a-aminobutyrate peaked at stage II and decreased towardsmaturity. Homoserine, proline, arginine, and ornithine decreased towards late stage II and increased again towards maturity. Methionine and tryptophan showed a decrease only towards maturation and phenylalanine, b-alanine, lysine, and GABA did not change during fruit development . Two aspartate aminotransferases were up-regulated during the transition from early stage II to stage II and from stage II to stage III and one was up-regulated during the transition from stage II to stage III . In addition, two glutamine synthetases , catalysing the reaction synthesis of glutamine from glutamate, were up-regulated during the transition from early stage II to stage II, while two other isoforms remained stable. During the transition from stage II to stage III , iCitrus 41697 was up-regulated, iCitrus 2123 and iCitrus 678 remained unchanged, and iCitrus 25117 was down-regulated . Another player in the amino acid core biosynthetic pathway is glutamate dehydrogenase catalysing the reversible conversion of 2-oxoglutarate to glutamate. Two GDH proteins were identified; GDH3 was down-regulated during the transition from early stage II to stage II and was up-regulated during the transition from stage II to stage III and GDH2 was upregulated during the transition from stage II to stage III . The GABA shunt is suggested to be essential for plant growth and to be pivotal in regulating citric acid degradation and fruit acidity during the later stage of citrus fruit development . Although significant changes in GABA were not detected, the three components of the GABA shunt [i.e. glutamate decarboxylase , GABA transaminase, and succinic semialdehyde dehydrogenase ] displayed changes during fruit development. These enzymes, plastic growers pots catalysing the decarboxylation of glutamate to GABA and CO2, were up-regulated during the transition from early stage II to stage II and from stage II to stage III . The level of GABA transaminase, catalysing the conversion of GABA to succinate semialdehyde, increased towards stage III. SSADH, mediating the oxidation of succinate semialdehyde to succinate, accumulated during the transition from early stage II to stage II and remained unchanged during the transition from stage II to stage III . Pyruvate supplies the carbon skeleton for alanine, leucine, and valine. Alanine aminotransferase and alanine:glyoxylate aminotransferase, both mediating alanine synthesis, remained unchanged at the early stages and were up-regulated during the late stage of development . In the aspartatederived amino acid biosynthesis pathway changes were identified in S-adenosyl-L-homocysteine hydrolases, methionine synthase, and ketol-acid reductiosomerase. S-adenosylL-homocysteine hydrolase and methionine synthase, active in the SAM cycle, were both down-regulated during thetransition from early stage II to stage II and were up-regulated during the transition from stage II to stage III .
The use of two-way hierarchical clustering of metabolite amounts allowed the grouping of metabolites according to their accumulation trends . Metabolites were separated into five different clusters: pyruvate, mannose, methionine, tryptophan, leucine, 2-oxoglutarate, isoleucine, fumarate, glutamine, myo-inositol, fructose-6-P, glycerate, and gluconate were clustered together and their amounts declined during development and maturation . In two clusters: lactate, a-aminobutyrate, asparagine, c-aminobutyrate, alanine, serine, valine, threonine, malate, inositol, and glucose-6-P; and citrate, 3-phosphoglycerate, glycine, aconitate, aspartate, and glutamate amounts increased from early stage II to stage II followed by a decline in their amounts in stage III. Two additional clusters of metabolites including b-alanine, tyrosine, phosphoenolpyruvate, xylose, lysine, sorbitol, arginine, and mannose-6-phosphate and phenylalanine, ornithine, trehalose, arabinose, proline, ribose, succinate, shikimate, galactose, sedoheptulose, sucrose, isocitrate, mannitol, histidine, maltose, fructose, and glucose increased during both stage II and stage III. Interestingly, almost all sugars displayed the same trend of accumulation towards fruit maturation.In this study, differential quantitative proteomics and metabolite profiling were used to assess developmental changes of citrus fruits. Most of the organic acids and many of the amino acids branching out from glycolysis and the TCA cycle peaked at stage II and declined during stage III of development. On the other hand, most of the sugars increased during stage III. No correlation was found between citrate accumulation and the expression of enzymes participating in citrate biosynthesis and degradation. Interestingly, citrate synthase protein amounts remained constant while aconitase, mediating the first step of citrate catabolism, isomerizing citrate to isocitrate, was up-regulated during fruit development. The TCA cycle maintains a cyclic flux in order to generate reducing NADH and FADH2 facilitating ATP synthesis by oxidative phosphorylation. Beyond the maintenance of a cyclic flux, the TCA cycle also functions to provide carbon skeletons for biosynthetic pathways as well as to metabolize organic acids generated from other pathways . The reduced expression of some of the proteins involved in the TCA cycle during the transition from early stage II to stage II such as pyruvate dehydrogenase, succinate dehydrogenase, and malic enzyme suggest a non-cyclic flux controlled by the influx of citrate and malate from the vacuole into the cytosol for its use in the TCA cycle. The changes in metabolite amounts throughout fruit development, with little correlation with protein expression levels, would also suggest that a largeproportion of metabolism regulation occurs at the posttranslational level . The increase in glutamate dehydrogenase, glutamate decarboxylase, and c-aminobutyrate transaminase protein expression and the patterns of GABA and succinate accumulation indicates that the GABA shunt is active. In citrus, sucrose is transported into the juice cells through the apoplast and accumulates mainly in the vacuole . Sucrose is then degraded to glucose and fructose by invertases or to fructose and UDP-glucose by sucrose synthase . While invertases did not appear to change at the protein level, the expression of three known invertase genes decreased during fruit development . Early studies have shown that acidic invertase activities in both grapefruit and Satsuma mandarin were initially high and decreased to very low levels at fruit maturation . The role of invertases in plant development is well established and the cleavage of sucrose is of key importance in the generation of hexoses needed for metabolism and signalling . In addition to transcriptional control, invertase activity can be regulated post-translationally by the action of invertase inhibitors . Interestingly, an invertase inhibitor was up-regulated towards maturation, suggesting its potential role in the previously described decrease in invertase activity in citrus fruits . As invertase inhibitor proteins have also been implicated in the regulation of sugar metabolism in grape and peach fruits , their role in the control of fruit quality warrants further investigation. Sucrose synthase proteins decreased during the transition from early stage II to stage II and increased during the transition from stage II to stage III. This pattern correlated well with sucrose synthase enzymatic activity reported by Komatsu et al. .