The activities of vacuolar and cell-wall invertases were not reported, and it might therefore be assumed that most of the cell-to-cell movement is through the symplastic pathway.Photo assimilates were detectable in the stalk of the juice sacs as early as 6 h after 14CO2 feeding, as found by pulse-chase experiment. However, with continuous exposure, the kinetics of radioactivity accumulation were higher between 24 and 48 h of exposure . Sugar-metabolizing enzymes were not monitored in the stalk separately from the juice sac, but the same mechanisms are likely to be operating in both parts of the juice sacs.As the edible part of the fruit, sugar metabolism and transport in the juice sac have received more attention than in other fruit parts. Photo assimilate transport proceeds to the inner part of the juice sac . Following 1 h of 14CO2 feeding to a source leaf next to grapefruit fruit, and 1 week of translocation, about 60% of the label was found in the juice sacs, with similar results in Satsuma mandarin . A maximal rate of radiolabel accumulation in pulse-chase experiments was reached between 24 and 48 h of labeling . Movement from the stalk to the distal part of the juice vesicle is relatively slow, and may take up to 96 h in the case of pomelo juice vesicles, which can reach 3 cm in length . Interestingly, drainage planter pot whereas in grapefruit juice sacs, most of the labeled assimilates were recovered as sucrose, in Satsuma mandarin, fructose was predominant .
The accumulation of sucrose per fresh weight peaked in the juice sacs during stage II of fruit development . Sucrose hydrolysis seemed to be mediated by all enzymes, as the activity of SuSy and that of the three forms of invertase were detected in the juice sacs . However, most studies showed that the activity of vacuolar invertase was relatively high, followed by SuSy activity. The activity of cell-wall invertase was also detected, but at a lower level, and soluble invertase activity was lowest. The presence of plasmodesmata has so far not been demonstrated, and cell-to cell movement might also follow a symplasmic pathway. Considering the relatively high activity of the vacuolar invertase, temporal storage and compartmentalization of sugars should occur during transport. Moreover, as the activity of cell wall invertase was also demonstrated, apoplasmic movement cannot be ruled out, and it might also play a role in temporal storage. Lowell et al. indicated that young fruit might behave differently than mature ones, as the former displayed uphill transport in terms of sugar concentration whereas fully grown fruit displayed downhill transport .
Interestingly, out of the six SuSy genes in the citrus genome, two were induced in juice sacs during development, with one of them induced in the segment epidermis as well, suggesting that SuSy acts in sucrose mobilization within the juice sacs . As expected, invertase activity in all cellular compartments was reduced toward fruit maturation, in good correlation with the reduction in the invertase transcripts . The activity and transcript levels of sucrose phosphate synthase genes were induced in Satsuma fruit juice sacs toward maturation, in accordance with an increase in sucrose level; however, in grapefruit, enzyme activity was induced from stage I to stage II of fruit development, and decreased toward maturation . This might explain the difference in sucrose levels between the two cultivars, as grapefruit accumulates less sucrose than Satsuma mandarin. Sucrose phosphate phosphatase was also induced during later stages of fruit development, suggesting that sucrose accumulation did not result only from translocation from the leaves but also from active synthesis within the juice sac cells . Nonutilized sucrose is stored in the vacuole and therefore, sucrose transport across the tonoplast might well play a role in regulating its levels within the cell and even its unloading rate. Sucrose and hexose uptake into tonoplast vesicles of sweet lime was not induced by ATP, suggesting facilitated diffusion .
Inclusion of acid invertase protein in the vesicles induced sucrose uptake, suggesting that sucrose hydrolysis by invertase or chemical acid hydrolysis within the vacuole provided the driving force for its uptake . An endocytic mechanism for sucrose transport across the tonoplast was also suggested .While being transported into the fruit, sucrose can undergo metabolism in a few directions. Hexose phosphate synthesis is an important metabolic step, with the reversible conversion of fructose-1-phosphate and fructose-1,6-biphosphate providing a link between sugar and organic metabolism via glycolysis/gluconeogenesis pathways . The reaction is catalyzed by two independent mechanisms . One involves two enzymes, an ATP-dependent phosphofructokinase catalyzing the glycolytic conversion of Fru-6-P to Fru-1,6-P2, and fructose-1,6-bisphosphatase , catalyzing the reverse, gluconeogenic reaction. The other mechanism is composed of one bidirectional enzyme, pyrophosphate-dependent PFK composed of two subunits, PFPα and PFPβ . Whereas PFK is generally considered ubiquitous, PFP has been described in prokaryotes and lower eukaryotes, including some bacteria, and some protozoan parasites . In addition, it is found in higher plants, where it is expressed in various tissues . While plants contain both PFP and PFK, bacteria and protozoa appear to have either one or the other, and yeast and animals contain only the latter . PFK is considered the more abundant enzyme, but its activity in plants is less characterized than that of PFP, due to its instability upon purification. PFK is found in both the cytosol and the plastids, whereas PFP is a cytosolic enzyme. Several hypotheses have been raised to explain the role of PFP in plants, including activation during stress . Transgenic up/downregulation of PFP in tobacco, potato, and sugarcane resulted in only minor alternations in plant growth and metabolism . However, reduced expression of PFP in Arabidopsis resulted in delayed development, while higher expression resulted in induced development . Moreover, knockout mutants suggested that PFP is required for adaptation to salt and osmotic stress during germination and seedling growth . While Fru-2,6-P2 is the major PFK activator in microorganisms and animals, in plants it does not activate PFK but rather PFP . Citrate was found to be an inhibitor of PFP activity, especially in the glycolytic direction , and was suggested to affect the affinity of Fru-2,6-P2 binding . PFP was detected in the juice sac cells of Valencia orange and grapefruit along with PFK and FBPase . While grapefruit PFP was strongly induced by Fru-2,6-P2 in the forward reaction, it was barely affected by the activator in the reverse reaction , as also demonstrated for potato, pineapple and tomato fruit . It was also shown that citrate, and to some extent other intermediates of the tricarboxylic acid cycle, inhibit the glycolytic reaction of PFP in grapefruit, whereas the gluconeogenic reaction was barely affected . Reduction in PFP activity in the ovaries of open versus closed flowers paralleled the reduction in protein levels of the two subunits, suggesting that the enzyme activity was regulated by its protein levels in the ovary . However, more complex relationships were detected in the fruit, demonstrating the involvement of other mechanisms in regulating PFP activity. Recently, the two subunits of citrus PFP were coexpressed and expressed separately in bacteria . Monomeric forms of both subunits were able to catalyze phosphorylation of Fru-1-P, but when coexpressed, plant pot with drainage the heteromeric form generated activity that was two orders of magnitude larger. While the activity of the heteromeric form was induced by Fru-2,6-P2, that of the β-monomer was repressed and the activity of the α-monomer was barely affected.Pulp acidity in citrus fruit is determined by two separate processes, citrate content in the vacuole of the juice sac celland vacuolar acidification, which can reach 0.3 M and pH 2.0, respectively in lemon and other acidic cultivars . Although separate, these two processes are bioenergetically coregulated . During the first half of fruit development, citrate accumulation is accompanied by proton influx which reduces the vacuolar pH. Citrate has three dissociation constants — 6.39, 4.77 and 3.14 — and in the vacuole it acts as a buffer by binding protons as they accumulate and reducing the pH, thus providing a driving force for additional proton influx . On the other hand, proton influx provides a driving force for citrate uptake, and probably also for its synthesis.
When the vacuolar pH of Navel orange juice sacs was below 3.5, two forms of citrate were detected, citrateH3 and citrateH2 – . CitrateH2− and citrateH3− could be detected in pH ≥ 3.5 and pH ≥ 5.0, respectively. During the second half of fruit development, when the acid level declines, citrate removal is accompanied by proton efflux and increasing pH. There is a good correlation among different citrus cultivars between the level of juice pH and citric acid concentration , and there are no reported cases in which pulp pH and citrate level are both low; therefore, altering citrate concentration will change pH homeostasis, and vice versa. However, early in fruit development, the two processes can be distinguished . Citrate accumulation in Minneola tangelo starts in early June and continues for approximately 3 weeks; during this time, pH is slightly increased, probably due to the dilution effect associated with cell division. Significant pH reduction is only detected after 4 weeks, suggesting that the buildup of some citrate accumulation is required to induce proton influx into the vacuole. This also suggests that citrate accumulation precedes proton accumulation. In other fruit of low and moderate acidity levels, such as melons, i.e., pH 4.5–6.5, some inbred lines with higher pH and higher citrate + malate content than their parents were reported . Although citrate is the major organic acid accumulated in citrus fruit, accounting for 90% of the total acids, the synthesis and accumulation of other organic acids have also been reported . For instance, in orange, there is a transient increase in quinic and oxalic acids early in fruit development. Malic acid also accumulates to some extent during the maturation of lemon, lime and orange fruit.So far, three mechanisms associated with proton movement across the tonoplast have been identified and characterized in citrus juice sac cells : V-type H+-ATPase, the major enzyme driving proton influx; H+-pyrophosphatase; and citrate/H+ symporter, most likely acting to remove citrate−2 out of the vacuole along with 2H+ . Other transport mechanisms, associated with citrate transport across the mitochondrial membrane and citrate movement into the vacuole, have been predicted for other plant species, but not for citrus fruit . A P-type ATPase, homologous to the petunia PH5 and PH8, was suggested to play a role in vacuolar hyperacidification . PH5 and PH5 were recently shown to be highly expressed in acid cultivars and down regulated in acidless cultivars, due to mutations in the MYB, HLH and/or WRKY transcription factors . While PH5 and PH8 were shown to localize to the vacuole in petunia, their membrane localization and biochemical properties in citrus require further research . The identification and characterization of vacuolar transport mechanisms require isolating purified tonoplast vesicles or intact vacuoles . An array of experimental tools can then be used to study transport across the membranes, such as radiolabeled molecules , pH-dependent fluorescent dyes such as acridine orange or quinacrine . An acidic-inside can be generated in isolated tonoplast vesicles or intact vacuoles through the activation of the V-type H+-ATPase or the H+-pyrophosphate with Mg–ATP or Mg– PPi and the use of inhibitors or protonophores to alter the pH gradient. For example, the addition of bafilomycin A inhibits the V-ATPase activity while gramicidin permeabilize the membrane to protons, thus abolishing the DpH across the membrane without affecting the pump hydrolytic activity. Tonoplast vesicles of juice sacs were isolated and purified from acidic cultivars and their acidless counterparts. 14C-citrate uptake of acidless pomelo vesicles was about 20% higher than that of acid pomelo, eliminating the possibility that the difference in fruit acidity between these two cultivars was due to citrate transport into the vacuole . The uptake was enhanced by ATP . Generation of a pH gradient was investigated in tonoplast vesicles of acid and acidless lime. As expected, it was induced by Mg–ATP, while bafilomycin and nitrate inhibited ATP hydrolysis and abolished the pH-gradient formation . Sweet lime tonoplast vesicles appeared to generate a DpH four times faster than those of acid lime, but they had higher H+ leakage following H+-ATPase inhibition by EDTA than the acid lime, possibly representing their limited in-vivo capacity for H+ retention.