- Title
- Control of hexose accumulation in developing fruit of tomato (Lycopersicon esculentum mill)
- Creator
- Ruan, Yong-Ling
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 1995
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Hexose is a key component determining quality of commercial tomato fruit. The accumulation of hexose in tomato fruit is realised and regulated during the rapid period of fruit growth. With outer pericarp as the targeted fruit tissue, the aims of this investigation were (1) to determine the cellular pathway of postphloem sugar transport; and thereby (2) attempt to identify the cellular processes controlling hexose accumulation in the storage parenchyma cells. The cellular pathway of postphloem sugar transport was elucidated in fruit at 13 to 14 and 23 to 25 days after anthesis (DAA). These developmental stages are characterised by phloem-imported sugars being accumulated as starch and hexose respectively. The distribution of the membrane impermeant tracer, 5(6)-carboxyfluorescein (CF), and [¹⁴C]glucose introduced into the storage parenchyma cells of pericarp discs, was consistent with their movement through a symplastic postphloem pathway in the younger fruit . In contrast, symplastic movement of these tracers was shown to be absent in fruit at 23 to 25 DAA. An apoplastic continuity for the older fruit was indicated by free movement of the apoplastic tracer, trisodium 3-hydroxy-5,8, 10-pyrenetrisulfonate (PTS) throughout the pericarp tissues. Indeed, the transport capacity of the pericarp apoplast was such that the steady-state rate of in vitro glucose uptake by pericarp discs accounted fully for the estimated rate of in vivo glucose accumulation. Together, these data indicate that the postphloem cellular pathway in the outer fruit pericarp shifts from the symplast during starch accumulation (13 to 14 DAA) to the fruit apoplast for the phase of rapid hexose accumulation (23 to 25 DAA ). The characteristics of plasma membrane hexose transport were investigated further using pericarp discs. For fruit at 23 to 25 DAA, the inhibitory effects of the sulfuydryl group modifier, p-chloromercuribenzenesulfonic acid (PCMBS) on [¹⁴C]glucose and [¹⁴C]fructose uptake by the pericarp discs was most ronounced for pericarp discs enriched in storage parenchyma. Consistent with PCMBS study, pericarp discs exposed to membrane impermeable thiol-binding fluorochrome, onobromotrimethylammoniobimane (qBBr) exhibited strong fluorescent signals by the storage parenchyma cells. The fluorescent weak acid, sulphorhodamine G (SRO), was accumulated preferentially by the storage parenchyma cells and its entry could be halted by the ATPase inhibitor erythrosin B (EB). A linkage between the putative H+-ATPase activity and hexose transport was demonstrated by an EB inhibition of [14C]glucose and [14C]fructose uptake. In contrast, comparable evidence for an energy-coupled hexose porter could not be found in the pericarp of younger fruit at 13 to 14 DAA. The results obtained provide circumstantial evidence that an energy-coupled plasma membrane hexose carrier is expressed specifically in the storage parenchyma cells at the latter stage of fruit development. Based on these data, a proposed cellular pathway of sugar transport in the developing tomato fruit is presented and discussed. Experiments to determine the control step(s) of hexose accumulation in the developing tomato fruit require knowledge of the chemical nature of the apoplastic sap that baths the storage parenchyma cells. In this context, further studies were conducted to develop a valid and convenient experimental procedure for the non-destructive collection of apoplastic fluid from the outer pericarp tissues of intact developing tomato fruit. The most appropriate procedure was found to be pressure dehydration in which sap was expelled under pressure through a cannula inserted into the pericarp tissue of a fruit sealed into a Scholander pressure bomb. Possible symplastic contamination of the exudate was deduced from measurements of the exuded sap including pH, osmolality and concentrations of K⁺, hexoses and infused PTS. These determinations were compared with the respective values obtained from the bulk tissues. When the exuded sap was collected at 4 ° C under applied pressures in the range of 0.6 - 1.0 MPa, the composition of the sap remained constant with time, was independent of the applied pressure and differed markedly from that of the bulk tissues. Moreover, the concentration of pre-infused PTS in the exudate also remained unchanged under these conditions. This procedure permits the collection of a relatively large volume exuded fluid, up to about 500 µl sap per 70 g fruit, at an initial flow rate of 100 -150 µ1 h-1. It is concluded that the pressure dehydration technique, under the described conditions, allows the collection of uncontaminated apoplastic fluid from the pericarp of intact developing tomato fruit. Using the above pressure dehydration technique, apoplastic fluid was collected from developing tomato fruit of both high and low hexose accumulating varieties. Extensive chemical analysis of the apoplastic fluid indicated that there was no significant differences in the concentrations of sugars and other solutes between varieties which differed in bulk tissue hexose levels. The hexose concentration of the apoplastic sap, about 30 mM, was 4 to 4.5 times lower than that of bulk tissue. The main amino acids in the apoplastic sap were glutamine, r- aminobutyric acid, asparagine, aspartic acid and glutamic acid. The concentration of K+ and Ca++ in the apoplastic sap were about 19.5 and 6.0 mM respectively. c1-, the major inorganic anion in the sap, was present at 8.5 to 9.5 and 16 to 19 mM in apoplastic and bulk tissue sap respectively. The identical hexose concentrations in the fruit apoplast between varieties differing in bulk tissue hexose levels suggests that the control step(s) for hexose accumulation in the developing tomato fruit is likely to be located within the storage parenchyma cells themselves. To identify the cellular processes controlling fruit hexose accumulation, the rates of sugar transfer through various transport and metabolic processes within the pericarp tissue need to be quantified. Technically, however, such an endeavour is impeded by the close anatomical relationship between the phloem and storage parenchyma tissues of the tomato pericarp. To avoid the complexity of the in vivo state, a novel in vitro liquid culture system was established based on the chemical composition of the apoplastic sap. Over a five-day culture period, the in vivo behaviour of sugar accumulation could be reproduced using a medium based on the chemical composition of the fruit apoplast. Significantly, the varietal differences in fruit hexose concentrations were maintained under he culture medium containing 30 mM hexose as the sole sugar source. This suggests that varietal differences in hexose levels of the fruit pericarp are not influenced by phloem unloading and cell-wall invertase activity. Rather these are controlled by factors within the storage parenchyma cells. Both in vitro and in vivo quantification of the sugar fluxes into the metabolic and storage pools suggested that membrane transport rather than metabolism plays a major role in the control of the hexose levels within storage parenchyma cells. Significant varietal differences were found in net hexose uptake across the plasma membrane and accumulation into the vacuole. In contrast, starch hydrolysis and metabolic interconversions as well as respiration rates were similar between the varieties examined. The varietal differences in membrane transport of hexoses were further examined using short-term [14C]hexose uptake studies. Both hexose influx across the plasma membranes and the quasie-steady state flux into the vacuoles of the pericarp cells were greater for the high sugar-accumulating variety. More than 73% of the varietal difference in hexose influx could be abolished by PCMBS. This shows that the putative hexose plasma membrane carrier could account for the major proportion of the varietal differences in influx rates. Concentration-dependent uptake studies demonstrated that Km values for hexose influx were similar between the two varieties. The difference in hexose fluxes was fully accounted for by Vmax value suggesting that the varietal difference resided in the amount /activity of the putative plasma membrane hexose carrier. Overall, it is concluded that (1) Postphloem sugar transfer is symplastic in outer pericarp of fruit at 13-14 DAA However, for fruit at 23-25 DAA, a symplastic discontinuity exists and sugar transfer follows an apoplastic route; (2) Hexose uptake by the storage parenchyma cells could be carrier-mediated and energy dependent at the latter stage of fruit development; (3) Steps controlling hexose accumulation in developing tomato fruit are located within the storage parenchyma cells rather than phloem unloading and cell wall sucrose hydrolysis; (4) Membrane transport not metabolism plays a major role in the control of the hexose levels within storage parenchyma cells and the putative hexose carrier appears to be the key control point for hexose influx across the plasma membranes of the storage parenchyma cells.
- Subject
- tomatoes; fruit growth; sugar transport; cellular processes
- Identifier
- http://hdl.handle.net/1959.13/1417691
- Identifier
- uon:37236
- Rights
- Copyright 1995 Yong-Ling Ruan
- Language
- eng
- Full Text
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