Potassium transport in suspension culture cells and protoplasts of carrot

The properties of potassium transport in carrot (Daucus carota L.) suspension culture cells and their isolated protoplasts were examined. Cells cultured in Murashige and Skoog (MS) medium (Plant Physiol 15: 473-497) were potassium saturated and, consequently, they exhibited little net potassium accumulation. Cells that transport and accumulate potassium were derived from the MS-grown cells by culturing them in a potassium-free modified medium. The transport properties of the modified medium cells included: (a) smooth nonsaturating kinetics with 80% of the maximum rates occurring at 0.1 millimolar KCl, (b) linear transport for at least 75 min, (c) alkaline pH optimum, (d) little accompanying anion uptake with increased malate concentrations balancing net increases in positive charge, and (3) little effect on transport by plasmolysis. Potassium transport activity appeared to be 50% lower in protoplasts isolated from the modified medium cells. Nevertheless, the protoplasts exhibited essentially the same kinetics, time course, pH response, and malate adjustment as the intact cells. We concluded from these results that the low potassium cells and their isolated protoplasts are ideally suited to investigating potassium transport at the cell level without the complications associated with multilayered and highly differentiated tissues.     

Dicarboxylate transport in maize mesophyll chloroplasts

Evidence is presented for high rates of carrier-mediated dicarboxylate anion transport in maize mesophyll chloroplasts. Radioactively labeled malate is transported across the chloroplast envelope leading to accumulation in the stroma. Malate in the stroma will exchange for external malate, oxaloacetate, glutamate, aspartate, and oxoglutarate. At 4 °C the V of malate uptake is 50 μmol·h^?1·mg Chl^?1 and the K_m for malate is 0.5 mm. Oxaloacetate competitively inhibits malate uptake with a K_i estimated to be 0.3 mm. The temperature dependence of malate uptake indicates an activation energy of 12 kcal/mol, and extrapolation using this value gives a rate of transport at 30 °C of approximately 300 μmol·h^?1·mg Chl^?1. This rate approximates the rates of photosynthetic malate production by these chloroplasts.     

Citrate transport in corn mitochondria

Citrate uptake by corn mitochondria (Zea mays L. B73 × Mol9) was investigated by osmotic swelling and [¹⁴C]citrate accumulation. Uptake driven by passive influx, ammonium gradients, and respiration was followed. There was no requirement for phosphate and/or malate to secure citrate uptake, although under some conditions these additives were promotive. Inhibition of the phosphate and dicarboxylate carriers did not eliminate citrate uptake. Citrate_in/malate_out exchange occurs, but at a rate too slow to account for observed citrate uptake, and depletion of endogenous malate only reduced citrate uptake by 38%. It was concluded that citrate can be rapidly accumulated by a mechanism other than by exchange for dicarboxylates. The effect of uncoupler on respiration-driven [¹⁴C]citrate accumulation, and studies of passive swelling using ionophores and uncouplers indicated that the major avenue of citrate uptake is by H⁺/citrate co-transport with a pH optimum near 4.5. The in vivo role of this mechanism is not yet understood.     

Transport of dicarboxylic acids in castor bean mitochondria

Mitochondria from castor bean (Ricinus communis cv Hale) endosperm, purified on sucrose gradients, were used to investigate transport of dicarboxylic acids. The isolated mitochondria oxidized malate and succinate with respiratory control ratios greater than 2 and ADP/O ratios of 2.6 and 1.7, respectively. Net accumulation of ¹⁴C from [¹⁴C]malate or [¹⁴C]succinate into the mitochondrial matrix during substrate oxidation was examined by the silicone oil centrifugation technique. In the presence of ATP, there was an appreciable increase in the accumulation of ¹⁴C from [¹⁴C]malate or [¹⁴C]succinate accompanied by an increased oxidation rate of the respective dicarboxylate. The net accumulation of dicarboxylate in the presence of ATP was saturable with apparent K_m values of 2 to 2.5 millimolar. The ATP-stimulated accumulation of dicarboxylate was unaffected by oligomycin but inhibited by uncouplers (2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone) and inhibitors of the electron transport chain (antimycin A, KCN). Dicarboxylate accumulation was also inhibited by butylmalonate, benzylmalonate, phenylsuccinate, mersalyl and N-ethylmaleimide. The optimal ATP concentration for stimulation of dicarboxylate accumulation was 1 millimolar. CTP was as effective as ATP in stimulating dicarboxylate accumulation, and other nucleotide triphosphates showed intermediate or no effect on dicarboxylate accumulation. Dicarboxylate accumulation was phosphate dependent but, inasmuch as ATP did not increase phosphate uptake, the ATP stimulation of dicarboxylate accumulation was apparently not due to increased availability of exchangeable phosphate.

The maximum rate of succinate accumulation (14.5 nanomoles per minute per milligram protein) was only a fraction of the measured rate of oxidation (100-200 nanomoles per minute per milligram protein). Efflux of malate from the mitochondria was shown to occur at high rates (150 nanomoles per minute per milligram protein) when succinate was provided, suggesting dicarboxylate exchange. The uptake of [¹⁴C]succinate into malate or malonate preloaded mitochondria was therefore determined. In the absence of phosphate, uptake of [¹⁴C]succinate into mitochondria preloaded with malate was rapid (27 nanomoles per 15 seconds per milligram protein at 4°C) and inhibited by butylmalonate, benzylmalonate, and phenylsuccinate. Uptake of [¹⁴C]succinate into mitochondria preloaded with malonate showed saturation kinetics with an apparent K_m of 2.5 millimolar and V_max of 250 nanomoles per minute per milligram protein at 4°C. The measured rates of dicarboxylate-dicarboxylate exchange in castor bean mitochondria are sufficient to account for the observed rates of substrate oxidation.

     

Malate oxidation, rotenone-resistance, and alternative path activity in plant mitochondria

The effect of cyanide and rotenone on malate (pH 6.8), malate plus glutamate (pH 7.8), citrate, α-ketoglutarate, and succinate oxidation by cauliflower (Brassica oleracea L.) bud, sweet potato (Ipomoea batatis L.) tuber, and spinach (Spinacia oleracea and Kalanchoë daigremontiana leaf mitochondria was investigated. Cyanide inhibited all substrates equally with the exception of malate plus glutamate; in this case, inhibition of O₂ uptake was more severe due to an effect of cyanide on aspartate aminotransferase. Azide and antimycin A gave similar inhibitions with all substrates. Subsequent addition of NAD had no effect with any substrate. Providing that oxalacetate accumulation was prevented, rotenone inhibited all NAD-linked substrates equally and caused ADP:O ratios to decrease by one-third. Addition of succinate to mitochondria oxidizing malate stimulated oxygen uptake, but adding citrate and α-ketoglutarate did not. These results indicate that there is no direct link between malic enzyme and the rotenone- and cyanide-resistant respiratory pathways, and that there is no need to postulate separate compartmentation of malic enzyme and the other NAD-linked enzymes in the matrix.     

Regulation of hydrogen utilisation in Rhizobium japonicum by cyclic AMP

Utilisation (uptake) of hydrogen gas by whole cells of Rhizobium japonicum was found to be influenced by the carbon source(s) present in the growth medium, with activity being highest in a medium containing sugars. Tricarboxylic acid cycle intermediates, such as malate, significantly reduced H₂ utilisation. No reduction in the hydrogenase activity is observed when the enzyme is assayed directly by the tritium exchange method, indicating that the decrease in hydrogen uptake activity is not due to repression of hydrogenase biosynthesis. Cyclic AMP was found to alleviate the inhibition of H₂ uptake by malate, and this requires new protein synthesis. Addition of chloramphenicol or rifampicin simultaneously with cyclic AMP eliminated the stimulation of H₂ uptake in the malate medium. These results show that in R. japonicum cyclic AMP plays a major role in the regulation of H₂ metabolism.     

Rapid auxin- and fusicoccin-enhanced Rb+ uptake and malate synthesis in Avena coleoptile sections

The short-term effects of auxin (indole-3-acetic acid) and fusicoccin (FC) on Rb⁺ uptake and malate accumulation in Avena sativa L. coleoptile sections have been investigated. FC stimulates ⁸⁶Rb⁺ uptake within 1 min while auxin-enhanced uptake begins after a 15–20-min lag period. Auxin has little or no effect on ⁸⁶Rb⁺ uptake at external pHs of 6.0 or less, but substantial auxin effects can be observed in the range of pH 6.5 to 7.5. Competition studies indicate that the uptake mechanism is specific for Rb⁺ and K⁺. After 3 h of auxin treatment the total amount of malate in the coleoptile sections is doubled compared to control sections. FC causes a doubling of malate levels within 60 min of treatment. Auxin-induced malate accumulation exhibits a sensitivity to inhibitors and pH which is similar to that observed for the H⁺-extrusion and Rb⁺-uptake responses. Both auxin- and FC-enhanced malate accumulation are stimulated by monovalent cations but this effect is not specific for K⁺.Abbreviations FC fusicoccin - IAA indole-3-acetic acid     

Effect of carbon dioxide on nitrate accumulation and nitrate reductase induction in corn seedlings

Exposure of the leaf canopy of corn seedlings (Zea mays L.) to atmospheric CO₂ levels ranging from 100 to 800 μl/l decreased nitrate accumulation and nitrate reductase activity. Plants pretreated with CO₂ in the dark and maintained in an atmosphere containing 100 μl/l CO₂ accumulated 7-fold more nitrate and had 2-fold more nitrate reductase activity than plants exposed to 600 μl/l CO₂, after 5 hours of illumination. Induction of nitrate reductase activity in leaves of intact corn seedlings was related to nitrate content. Changes in soluble protein were related to in vitro nitrate reductase activity suggesting that in vitro nitrate reductase activity was a measure of in situ nitrate reduction. In longer experiments, levels of nitrate reductase and accumulation of reduced N supported the concept that less nitrate was being absorbed, translocated, and assimilated when CO₂ was high. Plants exposed to increasing CO₂ levels for 3 to 4 hours in the light had increased concentrations of malate and decreased concentrations of nitrate in the leaf tissue. Malate and nitrate concentrations in the leaf tissue of seven of eight corn genotypes grown under comparable and normal (300 μl/l CO₂) environments, were negatively correlated. Exposure of roots to increasing concentrations of potassium carbonate with or without potassium sulfate caused a progressive increase in malate concentrations in the roots. When these roots were subsequently transferred to a nitrate medium, the accumulation of nitrate was inversely related to the initial malate concentrations. These data suggest that the concentration of malate in the tissue seem to be related to the accumulation of nitrate.     

Regulation of succinate oxidation by NAD+ in mitochondria purified from potato tubers

NAD⁺ supplied to purified Solanum tuberosum mitochondria caused progressive inhibition of succinate oxidation in State 3. This inhibition was especially pronounced at alkaline pH and at low succinate concentrations. Glutamate counteracted the inhibition. NAD⁺ promoted oxaloacetate accumulation in State 3; supplied oxaloacetate inhibited O₂ uptake in the presence of succinate much more severely in State 3 than in State 4. NAD reduction linked to succinate oxidation by ATP-dependent reverse electron transport was likewise inhibited by oxaloacetate. We conclude that NAD⁺-induced inhibition of succinate oxidation is due to an inhibition of succinate dehydrogenase resulting from increased accumulation of oxaloacetate generated from malate oxidation via malate dehydrogenase. The results are discussed in the context of the known regulatory characteristics of plant succinate dehydrogenase.     

Diurnal Ion Fluctuations in the Mesophyll Tissue of the Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Both laboratory- and field-grown Mesembryanthemum crystallinum plants exhibited large scale diurnal ion fluctuations. In mesophyll tissue, potassium and sodium levels varied in conjunction with acid levels while chloride levels varied in opposition. Thus, dark CO₂ fixation in this Crassulacean acid metabolism species seems analogous to the common plant process of malate synthesis to balance cation surplus. Sodium levels in the epidermis appeared to fluctuate in opposition to those in the mesophyll. It is proposed that inorganic cations cycle between mesophyll and epidermal tissue to balance malate accumulation and to produce stomatal opening in the dark.     

Vacuolar efflux of malate and its influence of nitrate accumulation in Catharanthus roseus cells

Catharanthus roseus cell suspension cultures may accumulate large quantities of malate in the vacuolar space. Upon transfer into a fresh medium malate moves out of the vacuole. This compound is then oxidized and its assimilatory products (CO₂ + HCO₃^?) are excreted into the medium. The malate concentration decreases concurrently with an intracellular accumulation of nitrate. The opposite time course changes in malate and nitrate concentrations can be slowed down by treatment with synthetic auxins and fusicoccin which increase the HCO₃^? concentration in the cytoplasm. A line of evidence is presented which shows that malate consumption is causally related with the uptake of nitrate. The involvement of a HCO₃^?/NO₃^? antiport is proposed.     

Alternate pathways of D-fructose transport and metabolism in Arthrobacter pyridinolis

Phosphoenolpyruvate: fructose phosphotransferase negative mutants of $\text{Arthrobacter}\text{pyridinolis}$ used fructose in the presence of malate. In isolated membrane vesicles from fructose-grown wild type cells, fructose uptake was stimulated by malate, but not by a variety of other compounds, to almost the same extent as by phosphoenolpyruvate. The stimulation by malate but not that by phosphoenolpyruvate was prevented by 2,4-dinitrophenol or potassium cyanide. D-Fructokinase activity was elevated in cells grown in malate plus fructose or sucrose.     

Changes in the electron transport chain of pea leaf mitochondria metabolizing malate

Pea leaf mitochondria showed complex kinetics for malate metabolism. O₂ uptake increased as malate concentration increased from 0 to 10 mm, reached a plateau between 10 and 20 mm malate, and then increased again up to 40 mm malate. Analysis of the products of malate oxidation by high-performance liquid chromatography revealed that the first phase of O₂ uptake coincided with the synthesis of both pyruvate and oxalacetate (OAA) while the second phase of O₂ uptake at higher malate levels usually occurred with a large increase in OAA formation. The biphasic response in O₂ uptake and the changing ratios of pyruvate and OAA synthesis did not appear to be the direct result of the differing K_m values of malate dehydrogenase and malic enzyme. Rather, they resulted from thermodynamic properties of these two malate oxidases and the kinetics of the two NADH dehydrogenases found in plant mitochondria. At low malate concentrations the rotenone-sensitive NADH dehydrogenase was active and could accept electrons from both malate oxidases. This NADH dehydrogenase became saturated at about 10 mm malate. At higher malate concentrations the rotenone-insensitive NADH dehydrogenase was increasingly important and its increased electron transport capacity was best exploited by malate dehydrogenase. At the higher malate concentrations an increasing portion of the electrons from malate reduce O₂ through the alternative oxidase. Although this coincided with the second phase of malate-dependent O₂ uptake it was not required for this phase to be seen.     

Osmoregulation in Cotton Fiber: Accumulation of Potassium and Malate during Growth

Kinetics and osmoregulation of cotton (Gossypium hirsutum L.) fiber growth (primarily extension) have been studied. Growth is dependent on turgor pressure in the fiber. It is inhibited when a decrease in the water potential of the culture medium due to an addition of Carbowax 6000, equals the turgor pressure of the fiber. Potassium and malate accumulate in the fiber and reach peak levels when the growth rate is highest. Maximum concentrations of potassium and malate reached in the fiber can account for over 50% of the osmotic potential of the fiber. As growth slows down, levels of potassium and malate decrease and turgor pressure declines. Cotton ovules are capable of fixing H¹⁴CO₃⁻ in the dark, predominantly into malate. Fiber growth is inhibited by the absence of potassium and/or atmospheric CO₂. We suggest that potassium and malate act as osmoregulatory solutes and that malate, at least in part, arises from dark CO₂ fixation reactions.     

Effect of phosphate and uncouplers on substrate transport and oxidation by isolated corn mitochondria

A study was made to determine conditions under which malate oxidation rates in corn (Zea mays L.) mitochondria are limited by transport processes. In the absence of added ADP, inorganic phosphate increased malate oxidation rates by processes inhibited by mersalyl and oligomycin, but phosphate did not stimulate uncoupled respiration. However, the uncoupled oxidation rates were inhibited by butylmalonate and mersalyl. When uncoupler was added prior to substrate, subsequent O₂ uptake rates were reduced when malate and succinate, but not exogenous NADH, were used. Uncoupler and butylmalonate also inhibited swelling in malate solutions and malate accumulation by these mitochondria, which were found to have a high endogenous phosphate content. Addition of uncoupler after malate or succinate produced an initial rapid oxidation which declined as the mitochondria lost solute and contracted. This decline was not affected by addition of ADP or AMP, and was not observed when exogenous NADH was substrate. Increasing K⁺ permeability with valinomycin increased the P-trifluoromethoxy (carboxylcyanide)phenyl hydrazone inhibition. Kinetic studies showed the slow rate of malate oxidation in the presence of uncoupler to be characterized by a high Km and a low V_max, probably reflecting a diffusion-limited process.     

Inhibitory effects of 2,3,5-triiodobenzoic Acid on ion absorption, respiration, and carbon metabolism in excised barley roots

The ability of 2,3,5-triiodobenzoic acid (TIBA) to alter ion absorption, respiration, carbon metabolism, and the permeability of the cell membranes of excised barley roots has been examined. Roots pretreated in either H₂O, KCl, or TIBA followed by treatment in KCl, TIBA, or KCl and TIBA demonstrated that inhibition of ion uptake due to TIBA was reversible. These studies also suggest that ions already accumulated within the vacuole remain sequestered after the addition of TIBA, whereas cytoplasmic ions leak out into the external medium. A 20-minute lag period was present prior to the onset of inhibition of O₂ consumption by TIBA. A b-type cytochrome from corn that is apparently associated with the plasmalemma and possibly involved in respiration or ion uptake, or both, was unaffected by TIBA. The addition of TIBA to treatment solutions resulted in the synthesis and accumulation of ethanol. Analysis of organic acids showed that only the malate concentration was affected by treatment with TIBA. A reduction of 26% was noted for malate in the presence of 2 micromolar TIBA. These combined results suggest that the inhibitory action of TIBA in barley roots involves an alteration of mitochondrial respiration and not a direct depolarization of the plasmalemma.     

Role of potassium and malate in nitrate uptake and translocation by wheat seedlings

Wheat seedlings (Triticum 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, translocation, accumulation and reduction; cation uptake and accumulation; and malate accumulation. Seedlings treated with KNO₃ absorbed 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 seedlings 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 seedlings had only slightly greater levels of phosphoenolpyruvate carboxylase than leaves of NaNO₃-treated seedlings. NADP-malic enzyme levels increased only slightly in leaves and roots of both KNO₃- and NaNO₃-treated seedlings. 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 phosphoenolpyruvate carboxylase activity increased about 2-fold during 36 hours of KNO₃ treatment, and increased only slightly in the KCl control.     

Nitrate Uptake by Roots as Regulated by Nitrate Reduction Products of the Shoot

A mechanism is proposed by which secondary products of nitrate reduction in the shoot control the uptake of nitrate by the roots. KNO₃ enters the roots and is translocated to the shoot where nitrate is reduced and, at the same time, malate is produced. The reduction of nitrate is stoichiometric to the synthesis of malate (1). Part of the K-malate moves down to the root system in which malate is oxidized, yielding KHCO₃ which exchanges for KNO₃. Nitrate reduction in the shoot promotes the synthesis of malate which, after its translocation to the root, allows the preferential uptake of nitrate. Thus, plants reducing large amounts of nitrate may take up the anion without a superfluous accumulation of the cation. Furthermore, the utilization of nitrate by the shoot regulates its uptake by the root.     

Protein phosphorylation stimulates the rate of malate uptake across the peribacteroid membrane of soybean nodules

Incubation of intact isolated symbiosomes with [gamma-32P]ATP, followed by isolation of the peribacteroid membrane and polypeptide analysis, showed that a single major polypeptide at 26 kDa was labelled. Antibodies raised against nodulin 26 reacted with a similar sized polypeptide. Incubation of the symbiosomes with alkaline phosphatase removed the label from this polypeptide. Pre-incubation with ATP stimulated malate accumulation by isolated symbiosomes, but only slightly (10-30%). Pre-treatment of symbiosomes with alkaline phosphatase inhibited malate uptake substantially and this inhibition was completely relieved by addition of ATP. The ATP stimulation of malate uptake was not affected by ATPase inhibitors. It is suggested that the rate of malate uptake across the peribacteroid membrane is controlled by phosphorylation of nodulin 26.