Plant Material and Preparation
Celery (Apium graveolens L.) and courgette (Curbita pepo L.) were purchased locally. Other plants were grown in unheated glasshouses under conditions of natural light in a standard mixture of peat, sand, limestone, vermiculite and celcote containing sincrostart and sincrocel 6 fertilisers. Plants were hand watered daily. Celery vascular bundles and parenchyma discs were prepared as described . Vascular exudates were collected from courgettes and other Cucurbitaceae by positive displacement pipette from mature cut fruit and directly transferred to the appropriate ice-cold buffer for either enzyme activity or AsA determinations (see below). Phloem exudates from other plants were collected directly from severed stylets of Myzus persicae feeding on source leaf petioles. Prior to HPLC injection, phloem exudate was diluted into ice-cold 5% metaphosphoric acid with or without 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP).
Chemicals and labelled substrates
D-[U-14C]glucose (S.A. 11.47 MBq mmol-1), D-[U-14C]mannose (S.A. 11.36 MBq mmol-1), [U-14C]sucrose (S.A. 17.09 MBq mmol-1) and L-[1-14C]AsA (S.A. 0.52 MBq mmol-1) were obtained from Amersham Pharmacia Biotech, Little Chalfont, UK. L-[1-14C]galactose (S.A. 2.04 MBq mmol-1) was obtained from American Radiolabelled Chemicals, St. Louis, USA. [14C]DHA was obtained by incubating 0.037 MBq L-[1-14C]AsA with 10 μg ascorbate oxidase (E.C. 126.96.36.199; Roche, Lewes, UK) in 50 mM sodium phosphate buffer pH 5.6 (final volume 100 μl). L-[1-14C]galactonic acid was synthesised by incubation of 0.037 MBq L-[1-14C]galactose with 1U fucose dehydrogenase (E.C. 188.8.131.52; Sigma, Dorset, UK) and 0.5 mM NADP in 50 mM glycine-KOH buffer pH 9.5 (final volume 100 μl). L-[1-14C]galactonolactone was obtained by incubation of 0.037 MBq L-[1-14C]galactose with 1U fucose dehydrogenase and 0.5 mM NADP in 50 mM MOPS buffer pH 7.8 (100 μl). GDP-L-[1-14C]galactose was a gift from Dr. Ken Lawrie, GaxoSmithKline Pharmaceuticals, Marlow, UK and was synthesised according to the method of Baisch and Ohrlein  from L-[1-14C]galactose [ARC, St. Louis, USA]. The final product had a specific activity 0.37 MBq mmol-1, radiochemical purity of >95% as estimated by anion exchange HPLC and overall purity >90% as estimated by 1H and 13C NMR.
With the exception of L-fucose dehydrogenase and phosphomannose isomerase, which were purchased from Sigma, Dorset, UK, all enzymes were purchased from Roche, East Sussex, UK.
In Situ Staining of AsA using Acidic Silver Nitrate
Transverse sections (approximately 1 mm) of either celery petiole or courgette fruit were cut by hand and washed briefly in distilled water then stained and fixed as described by Chinoy . Control reactions were undertaken in which AsA in the tissue was first oxidised by exposure to aqueous 5% CuSO4 as described .
Tissue Incubation, Metabolite Extraction and HPLC Analysis
Celery tissues were incubated in buffer A consisting of 20 mM MES pH 5.5, 300 mM mannitol, 5 mM MgCl2, 2 mM KCl, 1 mM CaCl2 and 1 mM CaSO4  in petri dishes with rotary shaking of 100 rpm. For experiments with unlabelled precursors, the appropriate compound was added to a final concentration of 25 mM and the incubation continued for 18 h. For labelling experiments, 400 mg tissue were incubated in 650 μl buffer containing 111 kBq of either D-[U-14C]glucose, D-[U-14C]mannose or L-[1-14C]galactose in sealed vessels with 100 rpm rotary shaking for 4 h. For experiments with unlabelled precursors, AsA was extracted by grinding the celery tissue in liquid N2 followed by the addition of 5 volumes of 5% (w/v) metaphosphoric acid and further homogenisation. Cell debris was removed by centrifugation (16000 g, 1°C, 5 min). The supernatant was used directly for analysis of reduced AsA or incubated with 5 mM TCEP (1 h, 4°C) prior to analysis for measurement of total AsA by HPLC . Recoveries of authentic AsA added to tissue samples immediately prior to extraction exceeded 90%. In radiolabelling experiments the tissue was removed from the incubation medium and washed three times for 5 min in 5 ml of buffer A. The tissue was then blotted, ground in liquid N2 and homogenised in 5 volumes of 5% perchloric acid (PCA) containing 5 mM TCEP. After standing in ice for 30 min, cell debris was removed by centrifugation (16000 g, 5 min, 1°C) and a pH indicator (BDH 4080 indicator, 20 μl ml-1) was added to the supernatant. The pH range of the sample was adjusted to 6–7 by the drop-wise addition of a solution of 5 M K2CO3. Insoluble KClO3 salts were removed by centrifugation (16000 g, 5 min, 1°C), the supernatant was divided into 500 μl aliquots and applied to SAX SPE cartridges (100 mg, acetate counter ion, Alltech, Carnforth, UK). After washing the column with 4 ml H2O, L-[14C]AsA was released with 4 ml 300 mM formic acid. After lyophilisation and resuspension in 200 μl H2O, L-[14C]AsA was further purified and quantified by HPLC as previously described  with the exception that radioactivity was detected and quantified using a Packard 150TR radiodetector in the homogenous mode (500 μl flow cell) with postcolumn addition of Ultima Flo M scintillant at 3 ml min-1. Under these conditions, recovery of authentic L-[1-14C]AsA added to the tissue samples immediately prior to extraction exceeded 85%. Total carbohydrate determination in Cucurbitaceae fruit exudates was carried out using the phenol-sulphuric acid method .
Extraction and Determination of Enzyme Activities
Hexose kinase (HK), phosphoglucose isomerase (PGI), phosphomannose isomerase (PMI), phosphomannose mutase (PMM), GDP-L-galactose pyrophosphatase (GDP-L-gal PPPase) and L-galactose dehydrogenase activities (L-GalDH) were extracted from courgette fruit tissue by grinding in a mortar and pestle in ice cold 50 mM HEPES pH 8.0, 5 mM MgCl2, 5 mM DTT, 1 mM EDTA, 1 mM EGTA, 1 mM benzamidine hydrochloride and 0.5 mM PMSF (1:3 w/v). Cell debris was removed by centrifugation (10000 g, 10 min, 1°C) and sample supernatants were desalted on Sephadex G25 PD10 columns (Amersham Biosciences, UK) equilibrated with the same buffer prior to use. GDP-D-mannose pyrophosphorylase activity was extracted and desalted as described above with the exception that the buffer used throughout consisted of 100 mM tris pH 7.5, 15 mM 2-mercaptoethanol and 0.5 mM PMSF as described by Keller et al. . GDP-D-mannose-3,5-epimerase activity was extracted and desalted using 100 mM tris pH 7.6, 5 mM DTT and 1 mM EDTA .
L-Galactono-1,4-lactone dehydrogenase (L-GalLDH) activity was extracted as described  with the exception that mitochondria were collected by centrifugation at 30000 g. In all cases, vascular exudates were treated exactly as whole tissue with the exception that exudates were diluted directly into extraction buffer and were not ground in a mortar and pestle.
HK activity was determined in a reaction mixture consisting of enzyme extract, 50 mM HEPES pH 8.0, 5 mM MgCl2, 2 mM ATP, 0.5 mM NAD and 1 U NAD-dependent glucose-6-phosphate dehydrogenase (from Leuconostoc mesnteroides) in a final volume of 1 ml. The reaction was undertaken at 30°C and started by the addition of 2 mM D-glucose. Reaction kinetics were followed by measurement of NAD reduction at 340 nm in a Hitachi U-3010 spectrophotometer. Control reactions were undertaken using boiled extract. HK activity was also measured using fructose as substrate in which case the reaction mixture additionally contained 1 U PGI or mannose as substrate where the mixture contained 1 U PMI in addition to PGI.
PGI activity was determined in a reaction mixture consisting of enzyme extract, 50 mM HEPES pH 8.0, 5 mM MgCl2, 0.5 mM NAD, 5 μM ZnSO4 and 1 U glucose-6-phosphate dehydrogenase in a final volume of 1 ml. The reaction was undertaken at 30°C and started by the addition of 2 mM D-fructose-6-phopshate. Reaction kinetics were measured as described for hexokinase. PMI activity was measured as for PGI but the reaction mixture additionally contained 1 U PGI and was started by the addition of D-mannose-6-phosphate. PMM activity was assayed by including 1 U PMI in addition to PGI and starting the reaction with D-mannose-1-phosphate. In all cases control reactions were undertaken using boiled enzyme preparations.
GDP-D-mannose pyrophosphorylase activity was measured as described  with the exception that 2 mM NaF was included in the reaction mixture and control reactions lacked inorganic pyrophosphate. The reaction was linear with time for up to 2 h. GDP-D-mannose-3,5-epimerase activity was determined as described . GDP-L-galactose pyrophosphatase activity was determined in a reaction mixture consisting of enzyme extract, 50 mM HEPES pH 8.0 and 5 mM MgCl2in a volume of 0.5 ml. The reaction was started by the addition of 1.11 kBq GDP-L-[1-14C]galactose. The reaction was stopped by the addition of 500 μl 10% (w/v) activated charcoal followed by brief vortexing. The charcoal was removed by centrifugation (16000 g; 10 min; 1°C) and radioactivity in the supernatant estimated by scintillation counting (Packard Tri-Carb 2000CA).
L-GalDH activity was measured in a reaction mixture consisting of enzyme extract, 50 mM HEPES pH 8.0, 5 mM MgCl2 and 0.5 mM NAD. The reaction was started by the addition of 2 mM L-galactose. Reaction conditions and kinetic measurements were as described for hexokinase. L-Galactose dehydrogenase activity was also analysed in crude fruit exudate by addition of L-[1-14C]galactose. Samples were incubated at 30°C and the reaction stopped by addition of exudate to an equal volume of 5% (w/v) H3PO3.
Precipitated material was removed by centrifugation and formation of L-[1-14C]galactonic acid monitored by HPLC on a Metacarb 87C 300 × 7.8 mm column (MetaChem Technologies Inc., Torrance, CA) maintained at 70°C with a mobile phase of ultrapure water flowing at 1 ml min-1. HPLC equipment used was as described in Hancock et al.  with the exception that radioactivity was detected and quantified using a Packard 150TR radiodetector in the homogenous mode (500 μl flow cell) with postcolumn addition of Ultima Flo M scintillant at 3 ml min-1.
L-GalLDH activity was assayed as described by Oba et al.  with the exception that the buffer additionally contained 0.03% triton X-100 and 0.1 mM KCN to prevent reoxidation of cytochrome C by cytochrome C oxidase . In another deviation from the published protocol, the reaction was started by addition of L-galactono-1,4-lactone to 5 mM. The value for molar extinction coefficient of cytochrome c at 550 nm used was 25300.
Source Leaf Loading and Autoradiography
Loading of source leaves with 14C-assimilates was undertaken as described by Turgeon and Gowan . Leaves were removed from turgid plants and moistened with buffer B (25 mM MES pH 5.5, 20 mM CaCl2). The abaxial surface was gently abraded with carbarundum powder and 16 mm discs cut using a sharp cork borer. Leaf discs were floated on buffer B under light (200 μmol m-2 sec-1) for 15 h at 25°C with gentle rotary shaking. Finally, discs were transferred to 10 ml fresh buffer B containing 1 mM [U-14C]sucrose, D-[U-14C]mannose, L-[1-14C]galactose, L-[1-14C]AsA or [1-14C]DHA at a final S.A. of 18.5 kBq mmol-1. Incubation was continued under light for a further 3 h and subsequently, leaf discs were removed and washed five times (20 ml, 5 min) in buffer B. Small samples of incubation buffer were taken for HPLC analysis to ensure no metabolism had occurred at the beginning and end of the incubation period. Discs were surface dried and lyophilised between filter paper held flat between metal plates secured with G-clamps. Dried leaves were exposed to Kodak Biomax single sided X-ray film for 1 week at -80°C prior to development.