Root Plasmalemma Research Articles

9-22 Root plasmalemma H^+-ATPase involvement in the BNI release in sorghum

9-22 Root plasmalemma H^+-ATPase involvement in the BNI release in sorghum

Is Boron involved solely in structural roles in vascular plants?

It is very well proved that boron (B) plays a primary structural role in the plant cell wall. In addition, this micronutrient has been involved in a great variety of physiological processes in vascular plants. It has been reported that B deficiency induces stress-responsive genes and, in tobacco plants, it seems to decrease net nitrate uptake by repressing expression of root plasmalemma H+-ATPase gene. Moreover, root asparagine concentration is clearly increased under B deficiency, as also observed for other abiotic stresses. Accumulation of asparagine in response to abiotic stresses could be an ammonium detoxification mechanism when high amounts of ammonium are internally generated by deamination of soluble amino acids released from enhanced proteolysis under stress conditions. Nevertheless, the mechanisms underlying the several effects caused by B deficiency are unknown. Although a mechanism has been reported to explain B effects based on signals via the cell wall-plasma membrane-cytoskeleton continuum, we propose and discuss the possible role of B as a cellular signal through transcriptional factors. This hypothetical mechanism could explain not only its diverse effects on so many physiological processes, but also that a negligible amount of boron into the protoplast can be decisive for the normal development of such events.

Changes in plasmalemma K +Mg 2+-ATPase dephosphorylating activity and H + transport in relation to seasonal growth and freezing tolerance of Festuca pratensis Huds

Changes in plasmalemma K(+)Mg(2+)-ATPase dephosphorylating activity and H(+) transport were examined in freezing-tolerant and non-tolerant genotypes of the perennial grass species Festuca pratensis Huds. Enzyme activity and DeltamuH(+) were measured in plasmalemma fractions isolated from basal nodes and roots. Three types of experiments were undertaken: (i) a field experiment, utilizing the seasonal growth and cessation cycle of a perennial plant; (ii) a cold acclimation experiment in hydroponics; and (iii) an instant freezing test. A specific fluctuation in K(+)Mg(2+)-ATPase activity was found throughout the seasonal growth of the plants (i). The K(+)Mg(2+)-ATPase activity peaks for both the basal node and the root plasmalemma were determined early in the spring before the renewal of growth. The lowest activity values in roots occurred at the time approaching flowering, and in basal nodes at the transition into the growth cessation. The K(+)Mg(2+)-ATPase activity was approximately 50% lower in the basal node plasmalemma of freezing-tolerant plants than of non-tolerant ones, when assessed at the optimal growth stage in hydroponics. In hydroponics (ii) and in the freezing test (iii), temperature stress was followed by a more pronounced change in the level of K(+)Mg(2+)-ATPase activity than in that of H(+) transport, and this change was more clearly differentiated in the basal node plasmalemma of contrasting genotypes than in the roots. Stress response was manifested differently in freezing-tolerant and non-tolerant plants at cold acclimation (4-2 degrees C) and at freezing (-8 degrees C) temperatures. Proton transport regulation via coupled changes in the hydrolysed ATP/transported proton ratio, as an attribute of freezing-tolerant plants, is discussed.

Redox modulation of osmotic water permeability in plasma membranes isolated from roots and shoots of pea seedlings

Partitioning in a biphasic polymer system was used to isolate plasmalemma (PM) from roots and shoots of etiolated pea seedlings. The membrane preparations were used to assess the osmotic water permeability (Pos) with the stopped-flow method. The Western-blot technique was employed to determine the membrane content of the PIP-family of aquaporins, and their activity was estimated by measuring the rate of osmotic vesicle shrinking in the presence of inhibitors, HgCl2 and AgNO3. Monobromobimane fluorescent dye was used to determine the quantity of sulfhydryl groups in cell membranes and follow the effect of SH-oxidizing (diamide) and SH-reducing (dithiothreitol and tributylphosphine) agents on Pos of the root PM and oligomerization of aquaporins. The shoot PM was shown to combine high Pos with low aquaporin content. In the root PM, Pos was lower and the aquaporin content greatly exceeded that in the shoots. HgCl2 and AgNO3 did not decrease the rate of osmotic shrinking in root membrane vesicles, whereas considerably (by 40–50%) inhibited this index in the shoot membranes. Root and shoot PM preparations dramatically differed in their SH-group contents: the former exceeded the latter sixfold. When added to the homogenization medium, diamide and tributylphosphine affected the content of SH-groups and Pos in the root PM. In the roots, diamide decreased the quantity of SH-groups almost twofold and increased Pos fourfold, and the introduction of tributylphosphine produced a twofold increase in the quantity of SH-groups with only slight decrease in the Pos. Immunological analysis of membranes isolated in the presence of diamide showed that the ratio between the monomer and dimer forms of aquaporins in two membrane preparations depended on the presence of dithiothreitol in the denaturing buffer apparently because dithiothreitol exposed and reduced disulfide bonds essential for monomer interactions and inaccessible for interaction with redox modifiers of SH-groups in the membrane. Because of their inaccessibility, these modifiers could not cause the changes of Pos in the root PM produced by oxidation and reduction of SH-groups. This phenomenon is probably related to the change in the status of SH-groups in two cysteine residues at the N-end of the aquaporin loop C oriented outward into the apoplast.

Effect of Fertilization on Plant Growth and Nutrient Uptake in Oilseed Rape Under Varying Boron Supply

Both pot experiment and solution culture were conducted with two contrasting oilseed rape cultivars to investigate the effects of NPK fertilization with varied boron (B) status on plant growth, nutrient uptake, root activity (as TTC reducing capacity by fresh roots) and root cell plasmalemma H+-ATPase activity. The tolerant and sensitive cultivars used were ZY 821 and WY 324, respectively. The soil used for pot experiment was an alluvial soil. The pot experiment consisted of two nitrogen–phosphorus–potassium (NPK) levels (mg kg−1 air-dried soil): high NPK with N 335; P2O5 150 and K2O 150; low NPK with N 285; P2O5 100 and K2O 100 and two B levels (as borax, mg kg−1 air-dried soil): 16 (+B) and 0 (−B, no B supply). The solution culture consisted of two B levels: 0 (−B, no B added) and 100 μmol/L (+B, added as H3BO3). Compared to the treatment with no B added, the incorporation of B into the B-deficient soil significantly increased shoot and root dry weight (DW), ratio of root to shoot (RRS), seed yield (SY), and uptake of nutrients [N, P, K, calcium (Ca), magnesium (Mg), and B], irrespective of NPK supply levels. Increasing rate of NPK could significantly decrease the parameters described above, regardless of B status. In contrast to −B treatment, addition of B to the solution obviously enhanced root activity and plasmalemma H+-ATPase activity. Under the same conditions, cultivar ZY 821 produced more DW, larger RRS, higher SY, more nutrient uptake, higher root activity and H+-ATPase activity than cultivar WY 324. It is suggested that RRS, root activity and plasmalemma H+-ATPase activity be used as useful parameters to screen oilseed rape cultivars, which are genotypically different in response to B deficiency.

Flux of protons released by wild type and ferritin over-expressor tobacco plants : effect of phosphorus and iron nutrition

Tobacco ( Nicotiana tabacum) plants over-expressing the iron storage protein ferritin, either in the cytoplasm or in the plastids, were grown under various P and Fe conditions. The crossed effects of both the genotypes and the environmental conditions on iron and chlorophyll concentrations in leaves, ferric reductase (EC 1.6.99.13) and plasmalemma H +-ATPase (EC 3.6.3.6) activities in roots, and fluxes of H + released by roots were determined. The increase in leaf Fe concentration observed in plants over-expressing ferritin was accompanied by an increase in root ferric reductase and H +-ATPase activities. Iron deficient conditions induced a decrease in Fe and chlorophyll concentrations in leaves, an increase in ferric reductase and H +-ATPase activities in roots and an increase in H +-flux released by roots in all genotypes. Phosphorus abundant conditions induced also an increase in ferric reductase and H +-ATPase activities in roots in all genotypes, and an increase in H +-flux released by roots in the genotype over-expressing ferritin in the cytoplasm. These results suggest that P could regulate the root Fe uptake system, at the gene expression level, as already reported for Zn in barley. Moreover, they show that H +-flux was not always consistent with H +-ATPase activity, revealing that the global measurement of root plasmalemma H +-ATPase activity has some limits compared to the localized determination of the H +-flux. These limits could be due to the fact that part of the H +-flux is not directly linked to H +-ATPase activity in roots.

Effects of bicarbonate, nitrate and other environmental factors on iron deficiency chlorosis. A review

The influence of several environmental factors on the occurrence of iron deficiency chlorosis on calcareous soils, also called bicarbonate induced chlorosis, is described. Bicarbonate in the soil solution is a strong pH buffer, mainly in the presence of calcium carbonate. Since bicarbonate is quite mobile, and CO2 diffusion is a slow process, the pH decrease in such soils after proton release by plants is small. Also, the ferric reductase activity of plant roots declines sharply at high pHs. The chemical Fe(III) reduction depends on the pe+pH; so, the lower the pH, the more favored is the formation of Fe(II) from the Fe(III) in the rizosphere. Nitrate can be acquired by the roots along with a proton cotransport that increase the pH outside the root plasmalemma, but a redox effect may be also important. In the rizosphere, the conditions for the reduction of nitrate (N(V)) to nitrite (N(III)) and ammonia (N(‐III)) are attained before than the conditions for the Fe(III) reduction from a solid phase. So, from the thermodynamic point of view, the electrons released by the plant can be taken directly by nitrate instead of Fe(III), or the Fe(II) formed can be reoxidized by the nitrate. Furthermore, if nitrate and nitrite reductions occurs in the rizosphere, microsite pH increases at the root surface. Since controversial data have been reported on the pH variations in the xylem sap in the presence of an excess of bicarbonate in the medium, a slower transport of iron does not appear to be the main reason for bicarbonate induced chlorosis. The “iron paradox” is well known (Römheld, 1987): sometimes, in soil culture, chlorotic leaves can have higher iron concentration than green ones. Some authors suggest that bicarbonate may affect the Fe distribution within the leaves. Apoplastic pH would control the entry of iron to the cytosol in leaves in a similar way than occurs in the root. Bicarbonate in the nutrient solution could act as a buffer in the leaf apoplast. Nitrate uptake in the meristem also produces a pH increase, with the same effect than the bicarbonate. Also, nitrate is an oxidant that could reduce the availability of the electrons for the Fe reduction for the membrane transport.

The influence of elevated rhizosphere dissolved inorganic carbon concentrations on respiratory O2 and CO2 flux in tomato roots

Respiratory CO 2 and O 2 flux were measured in hydroponically grown Lycopersicon esculentum (L.) Mill, cv. F144 plants at either low (0 μmol mol -1 ) or elevated CO 2 concentrations (>2000 μmol mol -1 ) supplied to the roots. In NO 3 - -fed plants the consumption of O 2 and the engagement of the alternative pathway were increased by elevated dissolved inorganic carbon (DIC = CO 2 + HCO 3 - ) concentrations. This was ascribed to the influence of organic acids on the TCA cycle and electron transport pathways. Inhibition of O 2 consumption by elevated DIC in NH 4 + -fed plants may be due to the reduction requirements of anaplerotic carbon entering the TCA cycle or the removal of carbon from the TCA cycle for amino acid synthesis. In both NO 3 - and NH 4 + -fed plants elevated DIC inhibited root CO 2 release due to high rates of DIC incorporation by phosphoenolpyruvate carboxylase (PEPc) activity. Transient net CO 2 consumption due to the inhibition of respiration by salicylhydroxamic acid and KCN, together with high respiratory quotients after the addition of inhibitors of carbonic anhydrase (CA) activity, were also ascribed to high rates of DIC incorporation at elevated DIC concentrations. Ethoxyzolamide, an inhibitor of CA activity, inhibited both DI 14 C incorporation into organic products and NO 3 - uptake by 81% and 40%, respectively. This, together with a 32% increase in DI 14 C accumulation and inhibition of NO 3 - uptake upon inhibition of anion transport by diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) may indicate the exchange of HCO 3 - for NO 3 - across the root plasmalemma. It was concluded that dark incorporation of HCO 3 - by PEPc increased at elevated rhizosphere DIC concentrations and that the products of DIC incorporation may stimulate respiratory electron transport. Additional reducing energy and carbon skeletons from the tricarboxylic acid (TCA) cycle would therefore be available for respiration and the reduction and incorporation of NO 3 - into amino acids.

Comparison of Mg 2+-dependent ATP hydrolase activities of pea nodule symbiosomes and of pea root plasmalemma, obtained by an aqueous polymer two-phase system

The characteristics of the Mg 2+-dependent ATPase activity from the peribacteroid membrane of pea symbiosomes was compared with that from pea root plasma membranes. Enzyme inhibitors, optimum reaction pH, substrate specificity and antibody recognition were the main parameters examined. Both the symbiosomes and the root plasma membrane were purified with an aqueous polymer two-phase system (APS). The final concentration of the APS for the purification of symbiosomes were: 6.3% w/w dextran T500, 6.3% w/w poly(ethylene glycol) 3350, 5 m M KH 2PO 4–K 2HPO 4, 5 m M KCl, 0.33 M sucrose, (pH 7.85); for the root plasma membrane was: 6.2% (w/w) dextran T500, 6.2% poly(ethylene glycol) 3350, 330 m M sucrose, 5 m M K 2HPO 4 and 4 m M KCl (pH 7.8). The lack of contamination of pea symbiosomes with endoplasmic reticulum, mitochondria, broken bacteroids and/or tonoplast vesicles was established. Similarly, the aqueous two-phase system used here provided a fairly enriched root plasma membrane with low cross-contamination from other sources. Both symbiosomal and root plasma membrane ATPase activities were highly specific to ATP. The symbiosome ATPase apparently corresponds to an E 1E 2–ATPase mechanism, similar to that found at the plasma membrane. The similarity between these two ATPases was further supported by immuno-analysis. Mg 2+-ATPase of pea symbiosome and root plasma membranes were very similar, by all parameters tested.

Kinetics of NH4+ Influx in Spruce.

Influxes of 13NH4+ across the root plasmalemma were measured in intact seedlings of Picea glauca (Moench) Voss. Two kinetically distinct uptake systems for NH4+ were identified. In N-deprived plants, a Michaelis-Menten-type high-affinity transport system (HATS) operated in a 2.5 to 350 [mu]M range of external NH4+ concentration ([NH4 +]o). The Vmax of this HATS was 1.9 to 2.4 [mu]mol g-1 h-1, and the Km was 20 to40 [mu]M. At [NH4+]o from 500 [mu]M to 50 mM, a linear low-affinity system (LATS) was apparent. Both HATS and LATS were constitutive. A time-dependence study of NH4+ influx in previously N-deprived seedlings revealed a small transient increase of NH4+ influx after 24 h of exposure to 100 [mu]M [NH4+]o. This was followed by a decline of influx to a steady-state value after 4 d. In seedlings exposed to 100 [mu]M external NO3- concentration for 3 d, the Vmax for NH4+ uptake by HATS was increased approximately 30% compared to that found in N-deprived seedlings, whereas LATS was down-regulated. The present study defines the much higher uptake capacity for NH4+ than for N03- in seedlings of this species.

Kinetics of NO3- Influx in Spruce.

Influxes of 13NO3- across the root plasmalemma were measured in intact seedlings of Picea glauca (Moench) Voss. Three kinetically distinct uptake systems for NO3- were identified. In seedlings not previously exposed to external NO3-, a single Michaelis-Menten-type constitutive high-affinity transport system (CHATS) operated in a 2.5 to 500 [mu]M range of external NO3- [NO3-]o. The Vmax of this system was 0.1 [mu]mol g-1 h-1, and the Km was approximately 15 [mu]M. Following exposure to NO3- for 3 d, this CHATS activity was increased approximately 3-fold, with no change of Km. In addition, a NO3--inducible high-affinity system became apparent with a Km of approximately 100[mu]M. The combined Vmax for these discrete saturable components was 0.7 [mu]mol g-1 h-1. In both uninduced and induced plants a linear low-affinity system, additive to CHATS and an NO3--inducible high-affinity system, operated at [NO3-]o [greater than or equal to] 1 mM. The time taken to achieve maximal rates of uptake (full induction) was 2 d from 1.5 mM [NO3-]o and 3 d from 200 [mu]M [NO3-]o.

FATTY ACID COMPOSITION OF PLASMALEMMA LIPIDS ASSOCIATED WITH GENETIC VARIABILITY IN COLD TOLERANCE OF SWEETPOTATO IPOMOEA BATATAS. (L). LAM

Storage roots of `Beauregard' and `Centennial' were used to identify varietal differences in fatty acid composition in plasmalemma lipids during storage conditions. Total plasmalemma fatty acid composition of glycolipids and phospholipids in storage roots of `Beauregard' and `Centennial' did not differ. The fatty acid composition of MGDG and DGDG in storage root plasmalemma was >50% unsaturated fatty acids in `Beauregard'. The high percentage of 18:2 (65.44%) fatty acid compared to `Centennial' (19.70%) and 79.35% total unsaturated fatty acid content in MGDG may contribute to low temperature tolerance in `Beauregard'. The higher percentages of 16:1 and 22:1 fatty acids in `Centennial' compared to `Beauregard' contributed to MGDG fatty acid unsaturation. However, these fatty acids have not been related to chilling tolerance.

Aluminium and calcium transport interactions in intact roots and root plasmalemma vesicles from aluminium-sensitive and tolerant wheat cultivars

Recent research from our laboratory indicates that aluminium (Al) and calcium (Ca) transport interactions may play an important role in the mechanisms of Al phytotoxicity. In this study, we investigated the effects of Al on Ca2+ transport in intact roots of winter wheat (Triticum aestivum L.) cultivars (Al-tolerant Atlas 66 and Al-sensitive Scout 66). We used both a vibrating Ca2+-microelectrode technique and 45Ca2+ to monitor Ca2+ influx in intact roots. Root apical Ca2+ uptake was immediately inhibited, when roots were exposed to Al levels that ultimately decreased root growth in Al-sensitive Scout 66. The Al-tolerant cultivar was able to resist this Al inhibition of Ca2+ uptake, and to resist Al inhibition of 45Ca2+ translocation from roots to shoots. We also studied Ca2+ transport in right-side out plasmalemma vesicles isolated from roots of Al-sensitive and tolerant wheat cultivars. Calcium influx into the vesicles was mediated by a voltage-gated Ca2+ channel. Aluminium blocks the Ca2+ channel equally well in the plasmalemma vesicles isolated from Al-sensitive and Al-tolerant wheat roots. The results indicate that the differential response observed in intact roots is not due to differences in Ca2+ channels. The Al-tolerant wheat cultivar may have an ability to reduce Al3+ activity in the rhizosphere, thus reducing the Al-inhibition of Ca2+ influx.

32P uptake and transport to shoots in Pinuus serotina seedlings under aerobic and hypoxic growth conditions

We examined the effects o long‐term hypoxic growth conditions on net uptake and transport of P to shoots of pond Pine (Pinus serotina Michx.), a moderately flood‐tolerant southern pine. Seedlings were grown under aerobic orhypoxic solution conditions for 4–5 weeks in continuously flowing solution culture containing 100 μM P. Short – and long‐term 32P. experiments were then concluded with intact seedlings to determine rates of 32P influx, efflux and net transport to the shoot. Shoot fresh weight/root fresh weight ratios were significantly higher under hypoxic gorwth conditions, reflecting the larger reduction in root growth than shoot growth, despite extensive aerechyma formation in roots. Estimates for the unidirectional influx of 32P in aerobic and hypoxic seedlings were 1.43 and 3.20 μmol P (gFW root)−1 h−1, respectively. However, 32P accumulation between the two treatments became similar within 8 h, suggesting that efflux was also higer in seedlings from the hypoxic treatment. Indeed in a separate experiment, hypoxic growth conditions increased efflux by over 60%. Transport of 32P to shoots was significantly reduced under hypoxic growth conditions, despite higher root P concentrations and lower shoot P concentrations. After 48 h, 32P accumulation in roots was similar between the two treatments. Yet total accumulation of seedling 32P decrcased by 31% under the hypoxic treatment, largely because of reduced transport of 32p to the shoot. The lower accumulation of 32 by shoots of seedlings in the hypoxic treatment may be the result of a direct inhibition on the transport process in O2‐defident tissues, but could also reflect a slower turnover or labeling of the ool available for transport. Indeed, the percentage of total 32P in. roots present in the soluble P. (or transportable form of P) was about 33% lower in seedlings from the hypoxic treatment, probably reflecting increased assimilation into organic compounds as well as chelation with iron. Our results suggest that P transport to the shoots of acclimated seedlings may be more sensitive to hypoxic solution conditions than influx at the root Plasmalemma.

Aluminum Effects on Calcium Fluxes at the Root Apex of Aluminum-Tolerant and Aluminum-Sensitive Wheat Cultivars

The role of Ca(2+) transport in the mechanism of Al toxicity was investigated, using a Ca(2+)-selective microelectrode system to study Al effects on root apical Ca(2+) fluxes in two wheat (Triticum aestivum L.) cultivars: Al-tolerant Atlas 66 and Al-sensitive Scout 66. Intact 3-day-old low-salt-grown (100 micromolar CaCl(2), pH 4.5) wheat seedlings were used, and it was found that both cultivars maintained similar rates of net Ca(2+) uptake in the absence of Al. Addition of Al concentrations that were toxic to Scout (5-20 micromolar AlCl(3)) immediately and dramatically inhibited Ca(2+) uptake in Scout, whereas Ca(2+) transport in Atlas was relatively unaffected. The Al-induced inhibition of Ca(2+) uptake in Scout 66 was rapidly reversed following removal of Al from the solution bathing the roots. Similar studies with morphologically intact root cell wall preparations indicated that the Al effects did not involve Al-Ca interactions in the cell wall. These results suggest that Al inhibits Ca(2+) influx across the root plasmalemma, possibly via blockage of calcium channels. The differential effect of Al on Ca(2+) transport in Al-sensitive Scout and Al-tolerant Atlas suggests that Al blockage of Ca(2+) channels could play a role in the cellular mechanism of Al toxicity in higher plants.

In vivo and in vitro effects of nickel and cadmium on the plasmalemma ATPase from rice ( Oryza sativa L.) shoots and roots

Plasmalemma vesicles isolated from rice ( Oryza sativa L.) shoots and roots were used to study the effect of different nickel and cadmium concentrations on the Mg 2+-ATPase activity. In vivo application of the metals for 5 or 10 days resulted in a stimulation of the enzyme activity in shoots, while either increases or decreases were observed in roots depending on the concentration and the type of ion tested. In contrast, an inhibitory effect was always observed on shoot and root plasmalemma ATPase activity when these ions were applied in vitro. At the organ level, the content of Cd in plants treated with 0.5 mM metal was higher than that of Ni, especially in roots. No correlation between the metal ion levels in the plasmalemma vesicles and ATPase activties could be established. The possible mechanisms of heavy metal induced changes on ATPase activities are discussed.

Characterization of ATPase activities in rice root plasmalemma vesicles isolated by two‐phase partitioning

Plasma membrane vesicles were isolated from the roots of 7‐day‐old rice plants (Oryza sativa L. cv. Bahía) by utilizing an aqueous polymer two‐phase system with 6.2%:6.2% (w/w) Dextran T500 and polyethylene glycol 3350 (PEG) at pH 7.6. Plasmalemma vesicles of high purity were obtained as indicated by the vanadate‐sensitive K+, Mg2+‐ATPase activity that was 18 times higher in the upper (PEG‐rich) phase than in the lower (Dextran‐rich) phase and by specific staining with sodium silicotungstate. Two peaks of ATPase activity were found. One showed a pH optimum at 6.0 in the presence of 150 mM KCl and 3 mM ATP with apparent Km (ATP) and Vmax of 0.75 mM and 79 μmol (mg protein)−1 h−1, respectively. With 50 mM KCl and 7 mM ATP a pH optimum of 6.5, an apparent Km (ATP) of 6.3 mM and Vmax of 159 μmol (mg protein)−1 h−1 were determined. Both activities were specific for ATP, unspecific for monovalent cations, sensitive to sodium vanadate and Ca2+ but insensitive to azide and nitrate.

Characterization of ATPase activities in rice root plasmalemma vesicles isolated by two-phase partitioning

Characterization of ATPase activities in rice root plasmalemma vesicles isolated by two-phase partitioning

Some properties of the H-ATPase activity present in root plasmalemma of Avena sativa L. Two different enzymes or one enzyme with two ATP sites?

The effects of Mg 2+, K + and ATP on a H-ATPase activity from a native plasmalemma fraction of oat roots were explored at 20°C and pH 6.5. In the presence of 3 mM ATP and no K +, H-ATPase activity vs. [Mg 2+] approached a monotonic activation but it became biphasic, with a decline above 3 mM Mg 2+, in the presence of 20 mM K +. Mg 2+ inhibition occurred also in K-free solutions when [ATP] was lowered to 0.05 mM. Also, an apparent monotonic H-ATPase activation by [K +] at 3.0 mM ATP was transformed in biphasic (inhibition by high [K +]) when [ATP] was reduced to 0.05 mM. The best fits of the ATP stimulation curves of hydrolysis satisfied the sum of two Michaelian functions where that with higher affinity had lower V max. Taking into consideration all conditions of activity assay, the high-affinity component (1) had a K m about 11–16 μM and a V max around 0.14–0.28 μmol P i/mg per min whereas that with lower affinity (2) had a K m of 220–540 μM and a V max of 0.5–1.0 μmol P i/mg per min. K m2 was markedly affected by the [K +] and [Mg 2+]; at optimal concentrations of these cations (1 mM Mg 2+ and 10 mM K +) it had a value of 235±24 μM which was increased to 540±35 μM at 20 mM [Mg 2+] and 60 mM [K +]. In addition, V mx1 was reduced to about a half when the concentrations of Mg 2+ and K + were increased to inhibitory levels. These results could be explained by the existence of two different enzymes or one enzyme with two ATP sites. In the second case, we could not tell at this stage if both are catalytic or one is regulatory.

The effects of mineral nutrition and benzyladenine on the plasmalemma ATPase activity from roots of wheat and Plantago major ssp. pleiosperma

There is an increasing awareness of the possibilities of mineral nutrition as regulator of growth substance action and vice versa. The present paper focuses on the effects of mineral nutrition and benzyladenine at the level of the plasma membrane. Seedlings of wheat (Triticum aestivum L. cv. Drabant) and juvenile plants of Plantago major L. ssp. pleiosperma (Pilger) were grown hydroponically at different mineral levels with or without benzyladenine. Purified plasmalemma preparations from roots of wheat and P. major ssp. pleiosperma were obtained by the two phase partitioning method, using 6.5% (w/w) of each of Dextran T‐500 and polyethylene glycol 3350. The Mg2+ and (Mg2++ K+) dependent ATPase activities of the root plasmalemma in both species and the (Mg2++ Cl−) one in P. major ssp. pleiosperma increased with increasing mineral levels, but the ionic strength did not influence the substrate specificity, the sensitivity to inhibitors or the pH optima.The addition of 10−8M benzyladenine to a nutrient solution increased the ATPase activities. The pH optima and the sensitivity to several inhibitors were not affected by benzyladenine, but the substrate specificity for ATP decreased, except for the K+ stimulation. In conclusion, benzyladenine mimics the effects of a higher mineral level than actually applied. Data from this and previous experiments indicate that benzyladenine exerts its effects by increasing the endogenous cytokinin concentrations and by modulating membrane components.