Role of Potassium and Malate in Nitrate Uptake and Translocation by Wheat Seedlings

Abstract

Wheat seedlings (Tddcum vulgare) treated with 1 mM KNO3 or NaNO3, in the presence of 0.2 mM CaSO4, were compared during a 48-hour period with respect to nitrate uptake, translocatlon, accumulation and reduction; cation uptake and accumulation; and malate accumulation. Seedlngs treated with KNO3 absord and accumulated more nitrate, had higher nitrate reductase levels in leaves but less in roots, accumulated 17 times more malate in leaves, and accumulated more of the accompanying cation than seedlings treated with NaNO3. Within seedlns of each treatment, changes in nitrate reductase activity and malate accumulation were parallel in leaves and in roots. Despite the great difference in malate accumulation, leaves of the KNO3-treated seedlgs had only slightly greater levels of phosphoenolpyruvate carboxylase than leaves of NaNO3-treated seedlings. NADP-malic enzyme levels increased only slightiy in leaves and roots of both KNO3-and NaNO3-treated seedlns. The effects of K+ and Na+ on all of these parameters can best be explained by their effects on nitrate translocation, which in turn affects the other parameters. In a separate experiment, we confirmed that phospboenolpyruvate carboxylase activity increased about 2-fold during 36 hours of KNO3 treatment, and increased only slightly in the KCI control. (2), in which K+ is an integral part of a NO3-malate shuttle between roots and shoots. Jackson and Coleman (10) first showed the presence of PEP carboxylase in roots, and proposed the PEP carboxylase-malic dehydrogenase pathway for malate accumulation in roots. This pathway, together with malic enzyme for decarboxylation of malate, have been proposed as a mechanism for pH regulation during nitrate assimilation (18, 21), but the hypothesis has not been explicitly tested. We report here the effects of NO3 and its salts on changes in activity of carboxylating and decarboxylating enzymes. Dijkshoorn et al. (6) have shown that internal HCO3, from the decarboxylation of organic acids, was exchanged for external N03. Also, Ben-Zioni et al. (2) have shown that tobacco roots can secrete more H"CO3-into the medium when N03 is supplied than when Cl-is supplied. The source of H14CO3 was most likely malate, previously synthesized from 14C02 supplied to the leaves. These data also implied an exchange of HCO3 for external NO3. Since there is no a priori reason why this exchange should depend on the presence of K+, we predicted that activity of the reputed decarboxylase, NADP malic enzyme (5), should not be affected by the cation accompanying N03 in the medium. This prediction was fulfilled by experiment. Both the effects of N03-on cation uptake (3, 11) and the effects of different cations on N03-uptake have been studied (4, 15). In some experiments N03 uptake from KNO3 and NaNO3 were nearly the same (16). Ca21 was not routinely included in the treatment solutions. Minotti et al. (15) showed that wheat seedlings absorbed no more N03 from KNO3 solution in the absence of Ca2" than from Ca(NO3)2 solution. In the presence of CaSO4, N03 u?take and assimilation were much greater with K+ than with Ca + as the countercation (4). We confirm here the effect of K+ on increasing N03 uptake and assimilation in the presence of CaSO4, and report on the effects of K+ and Na+ on other parameters associated with N03 uptake and assimilation. We predicted that KNO3 treatment, as compared with NaNO3 treatment, should result in greater NO3 uptake, translocation, and accumulation; greater nitrate reductase activity (NRA3); and greater malate accumulation. Since malate accumulation generally depends on the activity of PEP carboxylase, we expected shoot PEP carbox-ylase activity to be higher in shoots of plants treated with KNO3 rather than with NaNO3. Measuring the above parameters should also help test portions of the Dijkshoorn (6)-Ben-Zioni hypothesis 1 'Abbreviations used: NRA: nitrate reductase activity; PEP: phospho-enolpyruvate. MATERIALS AND METHODS Seeds of wheat (Triticum vulgare L. cv. Arthur) were cultured according to the methods of Blevins et al. (3). The seeds were soaked for 24 hr in continuously aerated deionized H20, which was changed three times during the 24-hr period. Fifteen seeds per culture were then placed on cheesecloth suspended by concentric plastic rings over 600-ml beakers containing 450 ml of 0.2 mm CaSO4. All cultures were continuously aerated with filtered air throughout the experiments. After 2 days in the dark, the cultures were culled to 10 plants each and placed in a growth chamber. They were given 16 hr of light (37,000 lux, 23 C) and 8 hr of darkness (20 C) alternately throughout the experiment. The CaSO4 solutions were changed every other day during the growing period. Cultures were grown 7 days in 0.2 mm CaSO4, then transferred to beakers containing 450 ml of treatment solution consisting of 0.2 mM CaSO4 plus 1.0 mm KNO3, 1.0 mm NaNO3 or 1.0 mm KC1. The treatment solutions were changed every 12 hr. Seedlings to be used for the PEP carboxylase, malic enzyme, and xylem exudate studies were grown similarly, except 1-literjars were used with 870 ml of solution as described by Frost et al. (8). These solutions were changed every 24 hr. The seedlings were harvested after 0, 12, 24, 36, and 48 hr of treatment. The 48-hr harvest was eliminated in the PEP carboxylase-malic enzyme study. The volume and pH of the remaining solution were taken at each change and at harvest. A sample of each culture solution was frozen at-80 C for later analyses. At harvest, the roots were excised from the shoots, rinsed thoroughly with deionized H20, blotted dry, weighed, and im-784