t content of source leaves of potato plants acclimatised for 14 days in cabinets with artificial diurnal dark/light cycles. The cabinets were off-phased so that plants at different stages in their lighting regime were available at any given time. The AsAt content of source leaves was monitored for dos4 h in plants sampled simultaneously from the two cabinets. At the end of the dark phase, leaf AsAt content was approximately 12 mg/100 gFW and it progressively increased following artificial sunrise. After 10 h of light, the leaf AsAt content increased to approximately 30 mg/100 gFW after which it leveled off. Following artificial sunset the AsAt level gradually fell back to approximately 12 mg/100 gFW within 6–10 h.
Changes in AsAt content of potato leaves as a function of light. Environment chambers were on a 10 h dark – 14 h light cycle as indicated by the brown and yellow panels respectively. At the times shown, source leaves (leaves on the lower four nodes of each stem) were removed from each of three plants, ground in liquid nitrogen and extracted in 5% MPA, 5 mM TCEP (9:1 v/w) prior to quantification by HPLC. Values are represented as mean ± SE, n = 3.
Glasshouse adult plants were relocated to of-phased regulated ecosystem spaces (boards A and B) 2 weeks prior to the start of the test
Table ? Table1 1 shows the AsAt content of source leaves and tuberising stolons collected at h from the light-phase or dark-phase plants (after collection of phloem exudate for 90 min in a prehumidified atmosphere in leaves). In the table are also reported the chromatographic AsAt peak areas from phloem exudates collected from source leaves and tuberising stolons of the same plants. The exudate data are reported as peak areas as the exact volume of exudate collected could not be established. Whilst the leaves from light-phase plants contained over twice the AsAt level of leaves from dark-phase plants, no significant difference was found in the AsAt content of tuberising stolons from the two sets of plants. The AsAt peak area in chromatograms of light-phase leaf exudates was 1.8-fold larger than that obtained from dark-phase leaves. In tuberising stolons the increase in exudate peak area was more pronounced (4.6-fold).
Table step 1
Glasshouse grown plants were transferred to off-phased controlled environment chambers 14 days prior to the start of the experiment. Environment chambers were on a 10 h dark – 14 h light cycle (see Fig. 4). At h source leaves and tuberising stolons were removed from 3 plants and a sub-sample used for tissue AsAt quantification. For the determination of AsAt in leaf phloem exudates, petioles were re-cut under water and placed into EDTA or CaCl2 exudation buffer for 90 min in a prehumidified chamber in the dark. For determination of stolon phloem exudates the cut end of the stolon attached to the plant was re-cut under water and placed in the appropriate exudation solution. Values are represented as mean ± SE, n = 6. mAUt = milli absorbance units (?245 nm) ? time.
The correlation between leaf AsAt content and the AsAt levels of phloem exudates was also investigated in glasshouse-grown plants following the supply of a range of AsA biosynthetic precursors using the flap technique . Figure ? Figure5 5 shows the chromatographic AsAt peak area of exudates from leaves pre-treated for 24 h with 20 mM MES pH 5.5, 2 mM CaCl2 alone (control) or containing precursors at a final concentration of 25 mM. Incubation with D -glucose ( jak wysÅ‚aÄ‡ komuÅ› wiadomoÅ›Ä‡ na beetalk D -Glc) resulted in a slight (10%) reduction in leaf AsAt content compared with the control but no significant change in AsAt was detected in the exudates. By contrast, supply of L -galactose ( L -Gal) or L -galactono-1,4-lactone ( L -GalL) increased the AsAt content of leaves (4.9 and 6.2-fold respectively) and, more substantially, of exudates (10.8 and 11.2-fold respectively). With all treatments, the replacement of EDTA with CaCl2 in collection wells resulted in a significant reduction of AsAt peak area in the exudate chromatograms.