An artificial increase of AsAt content in the totality of source foliage of whole plants was obtained through the supply of the direct AsA precursor L -GalL via the flap technique for dos4 h to all terminal leaflets of the four lowermost nodes. t content of source leaves was 2.2-fold higher than in control plants (Fig. ? (Fig.6). 6 ). AsAt content also significantly increased in sink organs such as flowers (33%) and, more substantially in developing tubers (80%) compared with the control. No changes in AsAt levels were observed in petioles, stems or non-tuberising stolons.
Effect of precursor supply to source leaves on AsAt content in sink tissues. Terminal leaflets of all leaves of the lower four nodes of each stem of four potato plants were incubated with 500 ?l 20 mM MES pH 5.5, 2 mM CaCl2 alone (control) or containing 25 mM L -GalL for 24 h. At the end of incubation the indicated tissues were removed from plants, snap frozen in liquid nitrogen and lyophilised. After lyophilisation, tissues were powdered and extracted in 5% MPA, 5 mM TCEP (19:1 v/w) prior to estimation of the AsA content in each tissue by HPLC. Values are presented as means ± SE, n = 4.
AsA regarding the potato phloem
We have previously shown that AsA represented the largest peak of absorption at 245 nm retained by the column when phloem sap of potato source leaves obtained by aphid stylectomy was analysed by HPLC . In the present study we obtained very similar results from phloem samples collected from different organs of the potato plant by a different approach i.e. the EDTA exudation technique . Localisation of AsA to the vascular tissue of stems and developing tubers was further demonstrated histochemically exploiting the specific interaction between AsA and AgNO3 at low temperature with ensuing formation of metallic silver deposits . In particular, the distribution of metallic silver in developing tubers generated a pattern reflecting the intense phloem anastomosis typical of these storage organs and resembling the phloem network involved in the unloading of labelled assimilates in plants supplied with 14 CO2 . This was further evidenced by the close similarity between the silver nitrate staining and distribution of fluorescence in tubers following supply of CFDA to source leaves. We have previously demonstrated that the distribution of CF in developing potato tubers can be used to identify symplastic phloem unloading in these organs beautifulpeople, thus the similar distribution of CF and metallic silver deposits in developing tubers implies that transfer of phloem AsA to storage parenchyma cells occurs with the mass flow of assimilates. This hypothesis is further supported by the changes in AsAt distribution along the stolon axis following tuber induction which closely resemble the changes in sucrose content and radiolabelled assimilate distribution observed in plants labelled with 14 CO2 . The decline of AsAt in the apices of tuberising stolons accompanied by its accumulation in the subapical region may thus reflect the induction of symplastic phloem unloading in this region resulting in a distal migration of sink activity from the apex. However, this pattern also correlates with changes in the mitotic index along the axis as a result of tuber induction with cessation of cell division in the apical region of the stolon and its activation in secondary meristems within the swelling region . This may be relevant in view of the purported role of AsA in cell division .
Long-range AsA transportation during the potato
In preliminary experiments, we observed diurnal changes in the AsAt content of mature source leaves of potato. Circadian or diurnal oscillations of AsA content in photosynthetic tissues are known [e.g. ] as well as light-induction of AsA accumulation in leaves [e.g. ] and sink organs. Light-induced expression of specific AsA biosynthetic enzymes has been observed [e.g. ] as well as a general increase in the AsA biosynthetic flux . By growing plants in cabinets with out-of-phase light/dark regimes we were able to simultaneously sample plants in the dark or light phase when maximal differences in foliage AsAt content occurred. Leaf AsAt content showed diurnal changes with the maximal levels (observed during the light-phase) 2-fold higher than the lowest levels observed in the dark. Phloem exudates obtained from source and sink organs of light phase plants always showed significantly higher AsAt levels than exudates obtained from dark phase plants. Indeed, the AsAt content of exudates from tuberising stolons of light phase plants was over 4-fold higher that of dark-phase plants. Our findings indicate that changes in source leaf AsA biosynthesis rapidly impact on the phloem AsAt content resulting in transport of de novo synthesised AsA directly to developing sinks. This was also confirmed in experiments where exogenous AsA precursors such as L -GalL or L -Gal were supplied to source leaves via the flap technique, a treatment which resulted in substantial AsAt enrichment in phloem exudates. We were also able to more than double the AsAt content of the source foliage of whole plants by “bulk” supply of exogenous L -GalL to the majority of source leaves for 24 h. This resulted in significant AsAt increases in sink organs such as flowers and developing tubers.