Fig. ? Fig.1 1 shows HPLC chromatograms of potato source leaf phloem exudates collected from severed petioles in buffer containing either EDTA or CaCl2. Total AsA (AsAt; L -ascorbic acid + dehydroascorbic acid) appeared as the largest peak of absorbance at 245 nm retained by the column in both cases. 2 instead of EDTA, all exudate derived peaks were strongly reduced due to the reduction in exudation caused by callose coffee meets bagel desktop gelation . In experiments to test the stability of authentic AsA (final concentration 0.1 ?M) in the presence of EDTA or CaCl2 exudation buffers less than 5% oxidation was observed in either case over 90 min (data not shown). AsA localisation to the vascular tissue was confirmed histochemically in sections of potato stems and tubers incubated with ethanolic AgNO3 at 3°C (Fig. ? (Fig.2). 2 ). Intense deposition of metallic silver was observed in the vasculature of stems. In tubers metallic silver deposits appeared as short strands or well defined spots, sometimes in the perimedullary zone and also in the cortex. Although AgNO3 staining and CFDA treatments could not be carried out on the same section due to interference between the treatments, a clear similarity was observed in the pattern of metallic silver deposits and the distribution of fluorescence in stem and tuber sections. No AgNO3 staining was observed in control sections pre-incubated with 1% CuSO4 for 18 h in order to oxidise AsA (data not shown) .
HPLC traces of potato leaf exudates. The terminal leaflets of potato leaves were excised from glasshouse plants, petioles were re-cut under water and the freshly cut end transferred to 200 ?l 5 mM CaCl2 (trace A) or 200 ?l 15 mM EDTA (trace B). Samples were transferred to a saturated atmosphere to exude in the dark for 90 min. The exudation medium was brought to 5% MPA, 5 mM TCEP, centrifuged and injected onto a Coregel 64H column. Traces were recorded at 245 nm using a diode array detector and insets show the absorbance spectra of the peaks at 12.2 min. Trace C was authentic AsA (2.27 nmol) which gave a peak height of 42 mAU. The unbound peak at 6.3 min contained MPA.
Comparison of CF and AgNO3 staining in potato tissues. Plants were labelled with aqueous CFDA and left to translocate for 5 h prior to harvesting. Tubers or stems were hand sectioned and sections were either incubated in mineral oil and examined under a confocal microscope for CF fluorescence or stained for AsA using ethanolic AgNO3. Confocal images are shown on the left and silver stained sections on the right of each panel. A, stem (bar = 2 mm); B, developing tuber (bar = 3 mm); C, large developing tuber (bar = 5 mm).
AsA distribution throughout tuber advancement
Fig. ? Fig.3 3 shows AsAt distribution along the axis of stolons and developing tubers. In non-swelling stolons the AsAt content was maximal in the apical section with a sharp basipetal decline such that the more basal sections contained approximately 10% of the AsAt content found in the apex. Tuberising stolons showed a 50% reduction in the AsAt content of the apical section. This was accompanied by increases in the subapical 3–10 mm sections. In developing tubers there was a very substantial increase in the AsAt content in the sub-apical region, corresponding to the swelling area.
Distribution of AsAt along the axes of stolons and developing tubers. Non-swelling stolons, tuberising stolons or developing tubers were excised from glasshouse grown plants and sliced into 1 mm transverse sections which were quickly weighed and immediately frozen in liquid nitrogen. Samples were ground to a powder, extracted in 5% MPA, 5 mM TCEP (9:1 v/w) and AsAt in the extract supernatant estimated by HPLC. Values are represented as mean ± SE, n = 3.