Intracellular pH Regulation in Ricinus communis Grown with Ammonium or Nitrate as N Source: The Role of Long Distance Transport

Allen, S. and Raven, J. A. 1987. Intracellular pH regulationin Ricinus communis grown with ammonium or nitrate as N source:the role of long distance transport.—J. exp. Bot. 38:580–596. Charge balance and intracellular pH regulation were studiedin Ricinus communis grown in water culture with 1–0 molm^–3 or l-0 mol m ^–3 as sole N source. Seedling and 70-d-old plant parts were analysed for total P, C, organic N and S, and ash alkalinity;xylem and phloem saps were analysed for mineral ion content,and amino acids, amides, and dicarboxylates. Excreted H⁺ andbase were also measured. It was shown that in -N plants, H ⁺ produced and excreted directly bythe roots accounted for all net —COOH produced in theplant, but not for cation uptake by net H⁺ exchange. IntracellularpH perturbation in the shoot was regulated partly by reduction and partly by the transportof OH^- -generating dicarboxylates in the xylem. Phloem sap hadthe capacity to transport organic N and carboxylates excessto shoot requirements back to the root. In -N plants, 60% of total reduction occurred in the root, and 70% of all OH^- produced by root and reduction was excreted directly as base. Very little —COO^–from root reduction was stored in the root: most was transported to the shoot as xylem dicarboxylateand stored in the shoot. Of the OH^- produced from shoot and reduction, 40% was stored as shoot carboxylate: the phloem saphad the capacity to transport the rest back to the roots whereit was excreted as base. Key words: Intracellular pH, ammonium nutrition, nitrate nutrition, xylem dicarboxylates, phloem dicarboxylates, charge balance     

Metabolism of Glycollate by Lemna minor L. Grown on Nitrate or Ammonium as Nitrogen Source

Marques, I. A., Oberholzer, M. J. and Erismann, K. H. 1985.Metabolism of glycollate by Lemna minor L. grown on nitrateor ammonium as nitrogen source.—J. exp. Bot. 36: 1685–1697. Duckweed, Lemna minor L., grown on inorganic nutrient solutionscontaining either NH₄⁺ or NO₃^– as nitrogen source wasallowed to assimilate [1-¹⁴C]- or [2-¹⁴C]glycollate during a20 min period in darkness or in light. The incorporation ofradioactivity into water-soluble metabolites, the insolublefraction, and into the CO₂ released was measured. In additionthe extractable activity of phosphoenolpyruvate carboxylasewas determined. During the metabolism of [2-¹⁴C]glycollate in darkness, as wellas in the light, NH₄⁺ grown plants evolved more ¹⁴CO₂ than NO₃^–grown plants. Formate was labelled only from [2-¹⁴C]glycollateand in NH₄⁺ grown plants it was significantly less labelledin light than in darkness. In NO₃^– grown plants formateshowed similar radioactivity after dark and light labelling.The radioactivity in glycine was little influenced by the nitrogensource. Amounts of radioactivity in serine implied that thefurther metabolism of serine was reduced in darkness comparedwith its metabolism in the light under both nitrogen regimes.In illuminated NH₄⁺ plants, serine was labelled through a pathwaystarting from phosphoglycerate. After [1-¹⁴C]glycollate feedingNH₄⁺ grown plants contained markedly more radioactive aspartateand malate than NO₃^– plants indicating a stimulated phosphoenolpyruvatecarboxylation in plants grown on NH₄⁺. Key words: Photorespiration, glycollate, nitrogen, Lemna     

CAM Photosynthesis under Drought Conditions in Kalanchoe blossfeldiana Grown with Nitrate or Ammonium as the Sole Nitrogen Source

K. blossfeldiana Poelln. cv. Hikan was grown in vermiculite,supplied daily with nutrient solution containing 1 mM (or 10mM) nitrate or ammonium as the sole nitrogen source. The nitrate-grownplants had more activity of CAM (Crassulacean acid metabolism)photosynthesis (nocturnal CO₂ uptake in the shoot and nocturnalincreases of titratable acidity and malate content in the leaves)than the ammonium-grown plants. Interruption of the solutionsupply for 5 or more days (drought conditions) increased theactivity of CAM photosynthesis in nitrate- or ammonium-grownplants, and the diurnal CO₂ uptake pattern in the nitrate-grownplants shifted from ‘weak-CAM’ to ‘full-CAM’.The difference in the activity of CAM photosynthesis betweennitrate- and ammonium-grown plants increased under the droughtconditions. When the solution was resupplied, the activity ofCAM photosynthesis rapidly decreased to the levels before theinterruption. The physiological mechanism and ecological significanceof the effect of the nitrogen source on CAM photosynthesis arediscussed (Received January 5, 1988; Accepted April 13, 1988)     

Regulation of Nitrate Transport in Citrus Rootstocks Depending on Nitrogen Availability

Previously, we reported that in Citrus plants, nitrate influx through the plasmalemma of roots cells follows a biphasic pattern, suggesting the existence of at least two different uptake systems, a high and low affinity transport system (HATS and LATS, respectively). Here, we describe a novel inducible high affinity transport system (iHATS). This new nitrate transport system has a high capacity to uptake nitrate in two different Citrus rootstocks (Cleopatra mandarin and Troyer citrange). The iHATS was saturable, showing higher affinity than constitutive high affinity transport system (cHATS) to the substrate NO₃⁻. The V_max for this saturable component iHATS was higher than cHATS, reaching similar values in both rootstocks.Additionally, we studied the regulation of root NO₃⁻ uptake mediated by both HATS (iHATS and cHATS) and LATS. In both rootstocks, cHATS is constitutive and independent of N-status. Concerning the regulation of iHATS, this system is upregulated by NO₃⁻ and down-regulated by the N status and by NO₃⁻ itself when plants are exposed to it for a longer period of time. LATS in Cleopatra mandarin and Troyer citrange rootstocks is repressed by the N-status.The use of various metabolic uncouplers or inhibitors indicated that NO₃⁻ net uptake mediated by iHATS and LATS was an active transport system in both rootstocks.Key Words: Citrus, inducible high affinity transport system (iHATS), constitutive high affinity transport system (cHATS), nitrate uptake, regulation     

Potassium Transport and Regulation of Intracellular pH in Elodea densa Leaves

The effects of extracellular K⁺ concentration ([K⁺]_o) on the pH of cell sap, “bulk cytoplasm” and vacuole have been investigated in Elodea densa leaves under conditions of either low or high activity of the plasmalemma electrogenic H⁺ pump. Cell sap pH was evaluated directly in the cell sap expressed after freezing and thawing. Cytoplasmic and vacuolar pH were calculated by the weak base and weak acid distribution method, DMO and benzylamine appearing to be a suitable acid and base, respectively, for this purpose in this material. When added to the basal medium (no rapidly permeating ions present), 5 mM K⁺ induced an increase in intracellular pH, larger for the cell sap and the vacuole (about 0.2 units), and smaller but still significant for the cytoplasm (0.07 units). This alkalinizing effect of K⁺ was thus associated with a significant decrease in the pH difference across the tonoplast. The alkalinizing effect of K⁺ was markedly and synergistically enhanced by the presence of fusicoccin, a condition inducing a marked activation of H⁺ extrusion and of K⁺ uptake. The correlation between these effects of [K⁺]_o on intracellular pH and those on H⁺ extrusion indicates that changes in extracellular K⁺ concentration, and thus in K⁺ influx, can influence cytoplasmic and vacuolar pH by modulating the rate of H⁺ extrusion by the plasmalemma H⁺ pump.     

Nitrogen Fixation, Assimilation and Transport During the Initial Growth Stage of Phaseolus vulgaris L

In nodulated common bean (Phaseolus vulgaris L.), there is typicallya period of N stress between 15 and 20 d after emergence (DAE),due to a lack of synchronization between the depletion of Nin the cotyledons and the beginning of N² fixation and transport.Screening trials identified some Rhizobium leguminosarum bv.phaseoli strains with which symptoms of N deficiency were notvisible (‘precocious’ strains). Cultivar Negro Argelwas then inoculated with two ‘traditional’ strains(C-05 and CIAT 727) and two ‘precocious’ strains(CNPAF 146 and CNPAF 512), and plants were harvested from 8to 30 DAE. There were no differences between the two groupsof strains in nodule dry weight or in the acetylene reductionrates between 8 and 16 DAE. However, nodules induced by the‘precocious’ strains showed earlier onset of glutaminesynthetase (GS) (EC 6.3.1.2 [EC] ) and glutamate synthase (GOGAT)(EC 1.4.1.14 [EC] ) activities, and ureide synthesis. The N concentrationin the nodules formed by ‘precocious’ strains variedfrom 4.2 to 4.5%, whereas with the ‘traditional’strains, it increased from 3.2% at 8 DAE to 65% at 18 DAE, atwhich time plants exhibited N-deficiency symptoms. By 21 DAE,GS and GOGAT activities in ‘traditional’ noduleswere increased, as well as the ureide-N-concentration in thexylem sap, nodule N content declined to 4.5% and the leavesbecame green. These results suggest that the N stress with ‘traditional’strains is not a limitation in early N² fixation activity butrather in the rates of expression of the processes of N assimilationand transport. Key words: Glutamate synthase, glutamine synthetase, nitrogen fixation, Phaseolus vulgaris, Rhizobium     

Nitrogen Source Regulation of Growth and Photosynthesis in Beta vulgaris L

A chimeric gene containing the patatin promoter and the transit-peptide region of the small-subunit carboxylase gene was utilized to direct expression of Escherichia coli glycogen synthase (glgA) to potato (Solanum tuberosum) tuber amyloplasts. Expression of the glgA gene product in tuber amyloplasts was between 0.007 and 0.028% of total protein in independent potato lines as determined by immunoblot analysis. Tubers from four transgenic potato lines were found to have a lowered specific gravity, a 30 to 50% reduction in the percentage of starch, and a decreased amylose/amylopectin ratio. Total soluble sugar content in these selected lines was increased by approximately 80%. Analysis of the starch from these potato lines also indicated a reduced phosphorous content. A very high degree of branching of the amylopectin fraction was detected by comparison of high and low molecular weight carbohydrate chains after debranching with isoamylase and corresponding high-performance liquid chromatography analysis of the products. Brabender viscoamylograph analysis and differential scanning calorimetry of the starches obtained from these transgenic potato lines also indicate a composition and structure much different from typical potato starch. Brabender analysis yielded very low stable paste viscosity values (about 30% of control values), whereas differential scanning calorimetry values indicated reduced enthalpy and gelatinization properties. The above parameters indicate a novel potato starch based on expression of the glgA E. coli gene product in transgenic potato.     

Feedback Regulation of Nitrate Influx in Barley Roots by Nitrate,Nitrite, and Ammonium

The short-lived radiotracer 13N was used to study feedback regulation of nitrate influx through the inducible high-affinity transport system of barley (Hordeum vulgare L. cv Steptoe) roots. Both wild-type plants and the mutant line Az12:Az70 (genotype nar1a;nar7w), which is deficient in the NADH-specific and NAD(P)H-bispecific nitrate reductases (R.L. Warner, R.C. Huffaker [1989] Plant Physiol 91: 947-953) showed strong feedback inhibition of nitrate influx within approximately 5 d of exposure to 100 fmu]M nitrate. The result with the mutant, in which the flux of nitrogen into reduced products is greatly reduced, indicated that nitrate itself was capable of exercising feedback regulation upon its own influx. This conclusion was supported by the observation that feedback in wild-type plants occurred in both the presence and absence of L-methionine sulfoximine, an inhibitor of ammonium assimilation. Nitrite and ammonium were also found to be capable of exerting feedback inhibition upon nitrate influx, although it was not determined whether these ions themselves or subsequent metabolites were responsible for the effect. It is suggested that feed-back regulation of nitrate influx is potentially mediated through several nitrogen pools, including that of nitrate itself.     

Nodule Nitrate Reductase as a Source of Reduced Nitrogen in Soybean, Glycine max

Hydroponically grown soybeans were fed ¹⁵N-enriched NaNO₃ at nine reproductive stages of development. The stem exudates contained excess ¹⁵N in the fully reduced nitrogen fraction. The soybean nodules had high nitrate reductase activity, whereas the roots had no detectable nitrate reductase activity. Based on these results, we concluded that the nodule nitrate reductase system has the potential of contributing significantly to the nitrogen economy of the plant.     

Intracellular pH Regulation in an Acidophilic Unicellular Alga, Cyanidium caldarium: 31P-NMR Determination of Intracellular pH

The intracellular pH of an acidophilic unicellular alga, Cyanidiumcaldarium, was determined as a function of external pH by ³¹Pnuclear magnetic resonance. The algal cells incubated underaerobic conditions or under anaerobic and illuminated conditionsmaintained the intracellular pH in the range from 6.8 to 7.0even when the external pH was changed from 1.2 to 8.4. Underanaerobic and dark conditions, however, the intracellular pHacidified at the acidic pH region of the external medium. Theacidified intracellular pH reversibly returned to neutral eitheron aeration or illumination. The results indicate that, in Cyanidiumcells growing in extremely acidic environments, an active H⁺efflux (H⁺ pump) which depends on metabolic activity (respirationor photosynthesis) is essential to maintain the intracellularpH at a constant physiological level against the passive H⁺leakage due to the steep pH gradient across the cell membrane. (Received March 19, 1986; Accepted July 17, 1986)     

Role of Intracellular pH in Proliferation, Transformation, and Apoptosis

Both cellular proliferation and apoptosis (programmed cell death) have been claimed to be modulated, perhaps even triggered by, changes in intracellular pH. In this review, we summarize the evidence that gave rise to these hypotheses. To facilitate a critical appraisal of the existing data, we briefly review the main pathways involved in cytosolic pH homeostasis and their regulation by mitogens and by apoptosis-inducing agents. The information available at present suggests that cytosolic pH plays a permissive role in cellular growth and proliferation, but is neither a trigger nor an essential step in the mitogenic signal transduction cascade. Concerning apoptosis, it is clear that lowering the pH in vitro can activate DNase II. However, the evidence linking cytosolic acidification with DNA degradation in vivois presently not convincing. We conclude that the cytosolic pH, an essential physiological parameter that is tightly controlled by multiple, complementary, or redundant systems, is unlikely to play a role in signalling either cell growth or death.     

Rates of Hydrogen Ion Efflux by Nodulated Legumes Grown in Flowing Solution Culture with Continuous pH Monitoring and Adjustment

The net efflux of H⁺ from lucerne (Medicago saliva L.), redclover (Trifolium pratense L.) and white clover (Trifolium repensL.) growing in flowing solution culture and dependent upon symbioticfixation of atmospheric N, was measured over a 75 d experimentalperiod. Considerable and rapid increases in acidity of the nutrientsolution of up to 1.45 pH units were recorded when the pH wasriot held constant over a 30 h period. There was little differencein H⁺ efflux when solution pH was held constant at 4.75, 5.75or 6.75, but there was an immediate cessation when it was adjustedto 3.75. Differences in the daily net efflux of H⁺ closely followedthe pattern of daily differences in incoming radiation, andthere was also evidence of a diurnal pattern of H⁺ efflux. Althoughthere were initially distinct differences between the speciesin the calculated rate of net H⁺ efflux (µg H⁺ g^–1dry shoot d^–), by day 75 these had diminished. In allspecies, however, the maximum rate of efflux per unit of shootsoccurred during the earlier rapid phases of growth. The measuredefflux of H⁺ was well equated with the plant content of excesscations (as measured by ash alkalinity) and, on average, theratio of acidity produced to N assimilated (expressed as anequivalent) was 0-24. Medicago sativa L., Trifolium pratense L., Trifolium repens L., lucerne, red clover, white clover, acidification, cation/anion balance, flowing solution culture, H⁺ efflux, nitrogen fixation     

Depression of Nitrate and Ammonium Transport in Barley Plants with Diminished Sulphate Status. Evidence of Co-regulation of Nitrogen and Sulphate Intake

When barley plants were grown in a solution with nitrate asthe sole N-source but deprived of sulphate (–Splants)for 1 to 5 d, the capacity for sulphate transport by the rootsincreased very markedly; subsequent measurement of influx using³⁵S-labelled showed increases of > 10-fold compared to plants continuously supplied with sulphate (+S plants).There were only small effects on plant growth over a 5 d periodand yet the influx of , labelled with the short-lived tracer ¹³N, was diminished by approximately 30%.By contrast, the influx of phosphate was little affected bysulphate-deprivation. When a sulphate supply was restored to– S plants, the sulphate influx was quickly repressedover the subsequent 24 h and the nitrate influx was restoredto >90% of the value in +S plants. When plants were grown in a solution with a mixed nitrate andammonium supply and deprived of sulphate for 1 d or 5 d thedepression of nitrate influx was more strongly marked (up to55% depression). The influx of ammonium was also depressed after5 d of sulphate-deprivation, but not at 1 d, nor where the concentrationof ammonium in the uptake solution was lowered to 20 mmol m^–3or less. Additional measurements with ¹⁵N-labelled nitrate and ammoniumover longer periods were used to determine net uptake. Net uptakeof nitrate was depressed to a similar extent to efflux, butnet ammonium uptake was depressed only in unbuffered uptakesolution where the pH decreased to pH 4.9 during the uptakeperiod. The ¹⁵N-tracer experiments showed that the translocationof label to the shoot, from both nitrate and ammonium, was depressedto a greater extent than net uptake in –S plants. Thedepression of nitrate influx, caused by 5 d of sulphate deprivation,could be relieved almost completely by providing plants with1.0 mol m^–3 L-methionine during the day prior to influxmeasurement. This treatment substantially decreased sulphateand potassium (⁸⁶Rb-labelled) influx in both +S and –Splants, but greatly increased total S-status of the plants.This methionine treatment had no effect on ammonium influx ornet uptake in – S plants but increased influx significantlyin +S ones. When plants were grown with sulphate but deprived of nitratefor 4 d there was a marked depression of the sulphate influx(by 48–65%) but a smaller effect on phosphate influx (21–37%of +N). The results are discussed in relation to the effects of sulphate-deprivationon growth rate and the root: shoot weight ratio. It is concludedthat the effects on influx and net uptake of nitrogen are moresevere than could be accounted for by these factors. The decreasedtranslocation of either nitrate, or the products of nitrateand ammonium assimilation from the roots, is suggested as areason for the depression of influx. The restoration of nitrateinflux and net uptake by methionine suggests that, for thision at least, a shortage of S-amino acids within the plant maylead to the accumulation of inhibitory concentrations of non-Samino acids in the transport pool. Key words: ¹³N, sulphate, nitrate, ammonium, ion-uptake, barley     

Uptake and Distribution of Nitrogen in Young Apple Trees after Application of Nitrate or Ammonium, with Special Reference to Asparagine and Arginine

Apple rootstocks M.7 were given either nitrate or ammonium at the end of September. Until the following June, total and protein nitrogen and the composition of the soluble nitrogen fraction were followed in the roots, stem-bark, and new growth. Nitrogen from both sources was readily absorbed in roughly the same amounts. Absorption occurred in the autumn and especially in the following spring. Incorporation of the absorbed nitrogen took place exclusively in the young roots. Arginine and, to a lower degree, asparagine were by far the most abundant of the soluble amino compounds and reached levels far above those in the unfertilized trees. The asparagine/arginine ratio was rather high during the first few weeks after fertilization but then dropped steadily to a low winter level. During May and June there was a gradual decrease to very low levels, especially in the new growth. The data suggest that the absorbed nitrogen is incorporated into asparagine and that arginine is formed from asparagine if the supply of nitrogen exceeds the immediate needs. Throughout the experimental period, ammonium fertilization led to higher values of the asparagine/arginine ratio than did nitrate nutrition. This is explained in terms of an enhanced production of asparagine and a retarded conversion into arginine. It is argued that asparagine is the main translocation compound for nitrogen. The possibility is discussed that, in addition, arginine moves upwards by a process of exchange along the negatively charged walls of the xylem vessels.     

Regulation of photosynthetic electron-transport in Phaseolus vulgaris L., as determined by room-temperature chlorophyll a fluorescence

The regulation of photosystem II (PSII) by light-, CO₂-, and O₂-dependent changes in the capacity for carbon metabolism was studied. Estimates of the rate of electron transport through PSII were made from gas-exchange data and from measurements of chlorophyll fluorescence. At subsaturating photon-flux density (PFD), the rate of electron transport was independent of O₂ and CO₂. Feedback on electron transport was observed under two conditions. At saturating PFD and low partial pressure of CO₂, p(CO₂), the rate of electron transport increased with p(CO₂). However, at high p(CO₂), switching from normal to low p(O₂) did not affect the net rate of photosynthetic CO₂ assimilation but the rate of electron-transport decreased by an amount related to the change in the rate of photorespiration. We interpret these effects as 1) regulation of ribulose-1,5-bisphosphatecarboxylase (RuBPCase, EC 4.1.1.39) activity to match the rate of electron transport at limiting PFD, 2) regulation of electron-transport rate to match the rate of RuBPCase at low p(CO₂), and 3) regulation of the electron-transport rate to match the capacity for starch and sucrose synthesis at high p(CO₂) and PFD. These studies provide evidence that PSII is regulated so that the capacity for electron transport is matched to the capacity for other processes required by photosynthesis, such as ribulose-bisphosphate carboxylation and starch and sucrose synthesis. We show that at least two mechanisms contribute to the regulation of PSII activity and that the relative engagement of these mechanisms varies with time following a step change in the capacity for ribulose-bisphosphate carboxylation and starch and sucrose synthesis. Finally, we take advantage of the relatively slow activation of deactivated RuBPCase in vivo to show that the activation level of this enzyme can limit the rate of electron transport as evidenced by increased feedback on PSII following a step change in p(CO₂). As RuBPCase as activated, the feedback on PSII declined.Abbreviations and symbols J_C electron-transport rate calculated from CO₂-assimilation measurements - J_F electron-transport rate calculated from fluorescence parameters - PFD photon-flux density - q_E energy-dependent quenching - PSII photosystem II - q_Q Q-dependent quenching - QY quantum yield - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) C.I.W. publication No. 1015     

Nitrogen Enhancement of Phosphate Transport in Roots of Zea mays L. : I. Effects of Ammonium and Nitrate Pretreatment

The effect of nitrogen status on phosphorous uptake and translocation was examined in 6-day-old dark-grown decapitated maize seedlings exposed to 25 micromolar phosphorous. Transfer to complete solutions containing 1 millimolar ammonium resulted in an increase in phosphorous uptake rate after 6 to 8 hours. The stimulus remained effective for at least 5.5 hours upon subsequent transfer to nitrogen-free solutions. Pretreatments for 16 hours with either nitrate or ammonium resulted in enhanced rates of subsequent phosphorous uptake and in enhanced translocation to the xylem of the exogenously supplied phosphorous. Both processes reached a plateau following pretreatment with 0.1 to 1.0 millimolar concentrations of either nitrogen ion. Further enhancement occurred with 10 millimolar nitrate, but not with 10 millimolar ammonium pretreatment. Although nitrogen pretreatments slightly increased the quantity of exogenous phosphorous retained in the root tissue, most of the extra phosphorous taken up by the nitrogen-pretreated seedlings was translocated to the xylem. The enhanced translocation, however, did not totally account for the increase in uptake implying a specific stimulation of the uptake process.     

Influence of Ni Supply on Growth and Nitrogen Metabolism ofBrassica napusL. Grown with NH4NO3or Urea as N Source

The significance of nickel (Ni), which is essential for ureaseactivity, for growth and nitrogen (N) metabolism ofBrassicanapusgrown in nutrient solution with either NH₄NO₃or urea assole N source was investigated. Although Ni contents were below25 µg kg^-1d. wt, growth of plants relying on NH₄NO₃wasnot affected by the Ni status. However, supplementing the growthmedium with 0.04 µMNi enhanced dry matter production ofurea-grown plants significantly. Urease activity was significantlyreduced in leaves and roots of plants grown without supplementaryNi irrespective of N source. Plants grown with urea withoutadditional Ni accumulated large amounts of urea and had loweramino acid contents indicating impaired usage of the N supplied,while those grown with NH₄NO₃under Ni-deprived conditions accumulatedendogenous urea in their older leaves. It is suggested thatNi may not be strictly essential for plants receiving mineralN, or that the critical level is well below 25 µg kg^-1d.wt. These results confirm that Ni is required for urease activityand thus for growth of plants on urea-based media, as well asfor recycling endogenous urea.Copyright 1999 Annals of BotanyCompany. Brassica napusvar.annua, amino acids, N nutrition, nickel, spring rape, urea, urease activity.     

Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands

Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pH_i) was measured and the pH_i recovery rate from cell acidification induced by an NH₄Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pH_i recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pH_i recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pH_i recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH₄Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pH_i regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1.     

Effects of Nitrogen, Sucrose, IAA and Kinetin on Explants of Beta vulgaris Grown in vitro

Hypocotyl explants of Beta vulgaris L. were grown on defined agar media with different combinations of IAA and kinetin at varying concentrations of nitrogen or sucrose. The cultures were kept in light (18 h a day) at 27°C for 5 weeks. Root initiation and callus growth were recorded and the callus tissue was analysed for N and K. Root formation was found to increase with increasing nitrogen concentration (from 5 mM to 23.3 mM) in the medium at 10.0 mg/1 of IAA, whereas no stimulation was found at 0.1 mg/1 of IAA. When raising the sucrose level from 20 g/1 to 100 mg/1 at 10.0 mg/1 of IAA and 1.0 mg/1 of kinetin, root initiation was also stimulated. At a lower kinetin and auxin level, however, no increase was recorded. Callus growth was affected by changes in the nitrogen or sucrose concentration of the culture media. The nitrogen content of the callus tissue increased with rising nitrogen concentration of the media. When raising the sucrose level instead of the nitrogen level, the nitrogen content of the tissue decreased.