Ion localisation in floating leaves of Nymphoides Indica

Abstract

Examination of the lower epidermis of floating leaves of Nymphoides indica showed the presence of 7-8 hydropotes mm -2 of the type described originally by Perrott (1898). These hydropotes consist of circular groups of transfer cells which are formed just before the young leaf beg:l.ns to unroll by delayed anticlinal divisions of epidermal cells. The wall protuberances are confined to the internal surface of walls adjacent to the external medium and the cells contain a relatively large amount of cytoplasm with prominent mitochondria. Ultrastructural features of secretory cells such as Golgi bodies and vesicles were not common and there was no evidence suggesting a function in secretion. The transfer cell walls are unlike those of surrounding epidermal cells where the outermost layer shows some staining capability suggestlng restricted passage of ions and water. A function for hydropotes in ion uptake was shown by autoradiography using 32P, 36C1, and 86Rb. A 'rim' of cells apparently not involved in uptake was revealed, corresponding with a region which failed to stain with cationic dyes. Autoradiography also showed that hydropotes first become functional in uptake of ions as the leaf begins to unroll near the water surface, which coincided with the earliest uptake of these stains. Although short term uptake experiments using tracers suggested that the leaf apoplast is partially dominated by negative charges, efflux analysis showed little difference between Rb+ and C1- contents of this compartment and exchange constants representing DFS and WFS for cations were not resolved. The electron opaque tracers, lanthanum hydroxide and uranyl acetate, entered the hydropotes passing across the wall protuberances or along anticlinal walls showing a possible pathway for ions. Movement of C1- in the transpiration stream (WFS) was confirmed by increased uptake under conditions favouring high transpiration rates, and some accumulation at sites of evaporation adjacent to internal spaces was found. The entry of ions into the symplast of the leaf is supported by evidence including enhanced uptake in the light, reduced uptake at low temperature, and by the localisation of ATP-ase activity in association with the enlarged plasmalemma of transfer cells and the sub-epidermal cells immediately above. Efflux analysis showed a predominance of cation exchangesites in the cytoplasm. Inhibitor experiments suggested the utilisation of both respiratory and photosynthetic energy for C1- uptake but perhaps a dependence on respiratory process only for Rb+ uptake. Attempts to study further the intracellular location of ions and sites of uptake by precipitation methods were successful only in the case of C1- precipitation with Ag+. Using X-ray microanalysis and a C1--free embedding medium this method was extensively examined with respect to losses during processing, formation of non-specific deposits, their removal with HNO3, the effect of CaSO4 rinses and the effect of external pH. Br- was also used, since C1- cannot be entirely removed from the cells. Results indicated that C1- enters the leaf mainly via the outer transfer cells and peripheral cells of hydropotes and moves either apoplastically in anticlinal cell walls or across transfer cell wall protuberances to the plasmalemma where it is taken up in 'packets' and transported in the cytoplasm in these same aggregates. Symplastic movement was confirmed by the presence of deposjts in plasmodesmata. Precipitation with cobaltinitrite is unsuitable for high resolution localisation of K+ because of poor cytoplasmic preservation, the low mobility of the precipitant ion, and solubility of the product which results in few, large deposits. The addition of Ag+ failed to overcome these problems. The use of NaTPB overcame problems of penetration and cytoplasmic preservation, but smaller deposits are probably lost during GU embedding and those remaining are difficult to locate. X-ray microanalysis is essential for identification of deposits containing K. The antimonate precipitation reaction for Na+ was shown to be non-specific, the ions precipitated possibly depending on the reagent used. Preliminary experiments with methods based on initial freezing of the specimen showed that ice crystal formation is a problem in this tissue, but low resolution analysis of frozen hydrated bulk specimens could yield useful information. At the present time several different procedures should be applied to check the validity of ion localisation methods.