Stimulation of proton extrusion by K+ and divalent cations (Ni2+, Co2+ Zn2+) in maize root segments

Entry of the divalent cations Ni²⁺, Co²⁺ and Zn²⁺ into cells of maize ( Zea mays L. cv. Dekalb XL 85) root tissue is accompanied by an acidification of the incubation medium, a decrease in both the pH of the cell sap and the level of malate in the cells, and by an inhibition of dark fixation of CO₂. K⁺, on the contrary, induces only a very low acidification of the incubation medium, does not change either the pH of the cell sap or the malate level in the cells, and induces an increase in CO₂ dark fixation. Different mechanisms are postulated for the stimulation of proton extrusion by divalent cations and K⁺.     

Effects of Ca2+ and polyamines on Na+ and Rb+ influx in excised maize roots

When 1 m M spermidine or spermine was included in an absorption solution which contained 20 m M Na⁺ and 1 m M Rb⁺, Na⁺ influx into excised maize roots ( Zea mays L. cv. Golden Cross Bantam) was reduced. Rb⁺ influx was reduced in the presence of spermidine and uneffected in the presence of spermine when compared with control solutions. When 1 m M Ca²⁺ replaced the polyamines, Na⁺ influx was strongly reduced and Rb⁺ influx was promoted. Rb⁺ influx from 1 m M Rb⁺ solutions which did not contain Na⁺ was also promoted by 1 m M Ca²⁺, but was inhibited by 1 m M spermidine. This Ca²⁺ promotion of Rb⁺ influx could be reversed by 10 times greater concentration of spermidine in the absorption solution. H⁺ efflux from excised roots was inhibited by spermidine when compared with Ca²⁺ or control solutions, however, the plasma membrane ATPase was not inhibited by spermidine. It is concluded that external Ca²⁺ plays two separate roles in membrane function, only one of which can be substituted for by polyamines. The first role, maintenance of membrane integrity, can be substituted for by spermidine or spermine. The second function, maintenance of the Rb⁺ transport mechanism, is Ca²⁺ specific and cannot be substituted for by spermidine or spermine. The results of this study are discussed in terms of electrostatic interactions between the plasma membrane and the Ca²⁺ or polyamines.     

Effects of 2-iodobenzanilide on potassium uptake, H+ extrusion and K+-stimulated ATPase of corn roots

The carboxanilide systemic fungicide 2-iodobenzanilide (2-IB) after 2 h pretreatment at 0.25 m M inhibited K⁺ and SO₄^2- uptake by excised corn roots ( Zea mays L., cv. Dekalb 342) up to ca 70 and 40%, respectively. Proton extrusion from corn roots was also reduced by ca 50% after 1 h contact, and the microsomal K⁺-stimulated ATPase activity from corn roots and pea stems ( Pisum sativum L., cv. Alaska) inhibited by 50 and 72%, respectively. In contrast, the Mg²⁺-ATPase activities of microsomes and mitochondria at pH 6.0 and 8.7, respectively, were unaffected. After 2 h of preincubation with 0.25 m M 2-IB, O₂ consumption by corn roots and pea stems was inhibited by 12 and 18%, respectively. ATP content of corn roots was not altered by 2-IB treatment. Therefore, energy availability "in vivo" was unaffected and the primary effect on corn roots is suggested to be at the plasmalemma ATPase which forms the proton gradient.
With isolated pea stem mitochondria, 0.25 m M 2-IB inhibited O₂ consumption by ca 60% when NADH or malate plus pyruvate were added as substrates; when succinate was used O₂ consumption was unaffected. The mode of action on isolated mitochondria was different from that shown for carboxin and also formerly attributed to the whole class of carboxanilide fungicides.     

A comparison of NH4+ and NO3– net fluxes along roots of rice and maize

Net fluxes of NH₄⁺ and NO₃^– along adventitious roots of rice ( Oryza sativa L.) and the primary seminal root of maize ( Zea mays L.) were investigated under nonperturbing conditions using ion-selective microelectrodes. The roots of rice contained a layer of sclerenchymatous fibres on the external side of the cortex, whereas this structure was absent in maize. Net uptake of NH₄⁺ was faster than that of NO₃^– at 1 mm behind the apex of both rice and maize roots when these ions were supplied together, each at 0·1 mol m^–3. In rice, NH₄⁺ net uptake declined in the more basal regions, whereas NO₃^– net uptake increased to a maximum at 21 mm behind the apex and then it also declined. Similar patterns of net uptake were observed when NH₄⁺ or NO₃^– was the sole nitrogen source, although the rates of NO₃^– net uptake were faster in the absence of NH₄⁺. In contrast to rice, rates of NH₄⁺ and NO₃^– net uptake in the more basal regions of maize roots were similar to those near the root apex. Hence, the layer of sclerenchymatous fibres may have limited ion absorption in the older regions of rice roots.     

Effects of amiodarone on K+, internal pH and Ca2+ homeostasis in Saccharomyces cerevisiae

In this study, amiodarone, at very low concentrations, produced a clear efflux of K⁺. Increasing concentrations also produced an influx of protons, resulting in an increase of the external pH and a decrease of the internal pH. The K⁺ efflux resulted in an increased plasma membrane potential difference, responsible for the entrance of Ca²⁺ and H⁺, the efflux of anions and the subsequent changes resulting from the increased cytoplasmic Ca²⁺ concentration, as well as the decreased internal pH. The Δ tok1 and Δ nha1 mutations resulted in a smaller effect of amiodarone, and Δ trk1 and Δ trk2 showed a higher increase of the plasma membrane potential. Higher concentrations of amiodarone also produced full inhibition of respiration, insensitive to uncouplers and a partial inhibition of fermentation. This phenomenon appears to be common to a large series of cationic molecules that can produce the efflux of K⁺, through the reduction of the negative surface charge of the cell membrane, and the concentration of this cation directly available to the monovalent cation carriers, and/or producing a disorganization of the membrane and altering the functioning of the carriers, probably not only in yeast.     

CO2 and intracellular pH

Abstract The experimental determination of cytoplasmic and vacuolar pH values is discussed. Despite variation in these values evidence indicates that intracellular pH values are normally regulated within narrow limits. The regulatory mechanisms proposed involve the metabolic consumption of OH^& and the active efflux of H ⁺. The evidence for intracellular pH modification in response to CO₂ hydration and the production of HCO^?₃ and H⁺ is examined. Theoretical calculations and experimental data indicate that CO₂ concentrations as high as 5% will lower intracellular pH. Conversely, variation in CO₂ levels around atmospheric concentrations is unlikely to perturb intracellular pH. High CO₂ levels are found in bulky tissues, and flooded root systems. Evidence is presented that the slow diffusion of dissolved CO₂ compared to gaseous CO₂ results in its accumulation. It is proposed that the accumulation of respiratory CO₂ may reduce intracellular pH values when plant tissues, cells or protoplasts are maintained in a liquid culture medium. Finally, the possible role of dark CO₂ fixation and organic acid synthesis in the regulation of intracellular pH is examined.     

Effects of ATP analogs on the proton pumping by the vacuolar H+-ATPase from maize roots

Understanding the regulatory properties of the activities of the V-type adenosine triphosphatase (ATPase) on tonoplast membranes is important in determining the mechanisms by which this enzyme controls cytoplasmic and vacuolar pH. The possible existence of a regulatory site for adenine nucleotides was examined by comparing the effects of ADP, adenylylimidodiphosphate (AMP-PNP) and 3'- o -(4-benzoyl) benzoyladenine 5'-triphosphate (BzATP) to those of the 2',3'-dialdehyde derivative of AMP (oAMP) and ATP by using highly purified tonoplast vesicles from maize ( Zea mays L. cv. FRB 73) roots. The addition of either AMP-PNP or BzATP reversibly inhibited the initial rate of proton transport catalyzed by the H⁺-ATPase in a concentration-dependent manner. Less than 20 μ M AMP-PNP or 50 μ M BzATP was sufficient to inhibit half the initial rate of proton transport in the presence of 2 m M ATP and an excess of Mg. Both analogs increased the K_m for ATP and reduced the maximum enzyme velocity. The presence of ADP also inhibited proton transport. The characteristics of ADP-induced inhibition were similar to those of BzATP and AMP-PNP. The addition of the periodated derivative of AMP (oAMP) irreversibly inhibited the ATPase in a concentration and time-dependent manner similar to that reported previously (Chow et al. 1992, Plant Physiology 98: 44–52). Irreversible inhibition by oAMP reduced the maximum velocity of the tonoplast ATPase and was prevented by the addition of ATP. The presence of ADP, AMP-PNP or BzATP had no effect on irreversible inhibition by oAMP. The effects of ADP, AMP-PNP and BzATP on the kinetics of ATP utilization and the lack of protection against inhibition by oAMP argue in favor of at least two types of nucleotide binding sites on the V-type ATPase from maize root tonoplast membranes.     

Influence of elevated CO2 and nitrogen nutrition on photosynthesis and nitrate photo-assimilation in maize (Zea mays L.)

Measurements of CO₂ and O₂ gas exchange and chlorophyll a fluorescence were used to test the hypothesis that elevated atmospheric CO₂ inhibits nitrate (NO₃ ^– ) photo‐assimilation in the C₄ plant, maize (Zea mays L.). The assimilatory quotient (AQ), the ratio of net CO₂ assimilation to net O₂ evolution, decreases as NO₃ ^– photo‐assimilation increases so that the difference in AQ between the ammonium‐ and nitrate‐fed plants (ΔAQ) provided an in planta estimate of NO₃ ^– photo‐assimilation. In fully expanded maize leaves, NO₃ ^– photo‐assimilation was detectable only under high light and was not affected by CO₂ treatments. Furthermore, CO₂ assimilation and O₂ evolution were higher under NO₃ ^– than ammonia (NH₄⁺) regardless of CO₂ levels. In conclusion, NO₃ ^– photo‐assimilation in maize primarily occurred at high light when reducing equivalents were presumably not limiting. Nitrate photo‐assimilation enhanced C₄ photosynthesis, and in contrast to C₃ plants, elevated CO₂ did not inhibit foliar NO₃^– photo‐assimilation.     

Products of photosynthetic 14CO2 fixation and related enzyme activities in fruiting structures of chickpea

Fruiting structures of a number of legumes including chickpea are known to carry out photosynthetic CO₂ assimilation, but the pathway of CO₂ fixation and particularly the role of phosphoenolpyruvate carboxylase (EC 4.1.1.31) in these tissues is not clear. Activities of some key enzymes of the Calvin cycle and C₄ metabolism, rates of ¹⁴CO₂ fixation in light and dark, and initial products of photosynthetic ¹⁴CO₂ fixation were determined in podwall and seedcoat (fruiting structures) and their subtending leaf in chickpea (Cicer arietinum L.). Compared to activities of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and other Calvin cycle enzyme, viz. NADP⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13), NAD⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and ribulose-5-phosphate kinase (EC 2.7.1.19), the levels of phosphoenolpyruvate carboxylase and other enzymes of C₄ metabolism viz. NADP⁺-malate dehydrogenase (EC 1.1.1.82), NAD⁺-malate dehydrogenase (EC 1.1.1.37), NADP⁺ malic enzyme (EC 1.1.1.40), NAD⁺-malic enzyme (EC 1.1.1.39), glutamate oxaloacetate transaminase (EC 2.6.1.1) and glutamate pyruvate transaminase (EC 2.6.1.2), were generally much higher in podwall and seedcoat than in the leaf. Podwall and seedcoat fixed ¹⁴CO₂ in light and dark at much higher rates than the leaf. Short-term assimilation of ¹⁴CO₂ by illuminated fruiting structures produced malate as the major labelled product with less labelling in 3-phosphoglycerate, whereas the leaf showed a major incorporation into 3-phosphoglycerate. It seems likely that the fruiting structures of chickpea utilize phosphoenolpyruvate carboxylase for recapturing the respired carbon dioxide.     

Effect of gibberellic acid on K+ (Rb+) uptake and transport in sunflower roots

Four-week-old sunflower plants ( Helianthus annuus L. cv. Halcón), grown in different nutrient solutions, were used to study the effects of gibberellic acid (GA₃) on K⁺ (Rb⁺) uptake by roots or transport to the shoot. Gibberellic acid application to the nutrient solution did not affect the exudation process of excised roots. When GA₃ was sprayed on leaves 2 to 6 days before excising the roots, the rate of exudation and the K⁺ flux increased. When the exudation study was done keeping the roots in a nutrient solution in which Rb⁺ replaced K⁺, the GA₃ effects were evident also on Rb⁺ uptake and transport. In intact plants, GA₃ increased the Rb⁺ transported to the shoot but did not affect Rb⁺ accumulation in the root. It is suggested that these GA₃ effects can be explained if it is assumed that GA₃ acts on the transport of ions to the xylem vessels.     

Short term 22Na+ and 42K+ uptake in intact, mid-vegetative plants

Spergularia marina (L.) Griseb. is. a rapidly growing, annual, coastal halophyte. Because of its small size, it is suitable for isotope studies of ion transport well beyond the seedling stage. The purpose of this report is to establish the similarities and differences between ²²Na⁺ and ⁴²K⁺ uptake in S. marina and in more commonly used mesophytic crop species. Vegetative plants were used 18 days after transfer to solution culture. Plants were grown either on Na⁺-free medium or on 0.2 × sea water. ²²Na⁺ uptake was linear with time for several hours. The rate was relatively insensitive to external concentration between 1 and 180 mol Na⁺ m?³, particularly in Na⁺-free plants. Transport to the shoot accounted for 40 to 70% of the total uptake, dependent on salinity but largely independent of time. ⁴²K⁺ uptake decreased with increasing salinity in Na⁺-free plants and increased in 0.2 × sea water plants. Both uptake and transport to the shoot were non-linear with time, upward concavity suggesting recovery from a manipulative and/or osmotic injury. Steady state root contents were compared with predicted contents based on cortical cell electrical potentials using the Nernst equation. Reasonable agreement was found in all cases except Na⁺ content of 0.2 × sea water plants, in which active efflux was indicated. Uptake studies conducted in the presence of chemical modifiers (dicyclohexylcarbodiimide, dinitrophenol and fusicoccin) showed responses of ⁴²K⁺ uptake as expected from studies on agronomic species, and implied the presence of a similar active uptake here despite the appearance of equilibrium. Active Na⁺ uptake was suggested at low Na⁺ levels. We conclude that S. marina is a promising experimental system combining the rapid nutrient acquisition strategy of agionomically important annuals with a high degree of salt tolerance.     

Binding of Cd2+, Ni2+, Cu2+ and Zn2+ by isolated envelopes of Pseudomonas fluorescens

Abstract Cell envelopes of Pseudomonas fluorescens , cytoplasmic membrane, peptidoglycan and outer membrane were obtained from a fractionation procedure and tested for their metal binding capacity. Isolated envelopes (cytoplasmic membrane, peptidoglycan and outer membrane) were chemically modified and functional carboxyl groups transformed to electropositive amine groups, using carbodiimide ethylenediamine. Transformation of carboxyl groups was evaluated by measuring total amine groups in all fractions (modified or not). Using equilibrium dialysis and Scatchard plots for the data, we have established that isolated unmodified cell envelopes (cytoplasmic membrane, peptidoglycan and outer membrane) possess at least two types of metal binding sites with different association constants ( K _a and K '_a). Introduction of positive charges into the bacterial envelopes resulted in the disappearance of one type of metal binding site which had the highest association constant value for Ni²⁺, Cu²⁺ and Zn²⁺. All fractions, modified or not, always presented at least two types of binding sites with different association constants for Cd²⁺.     

On the presence of inside-out plasma membrane vesicles and vanadate-inhibited K+,Mg2+-ATPase in microsomal fractions from wheat and maize roots

We have estimated the amount of inside-out plasma membrane (PM) vesicles in microsomal fractions from wheat (Triticum aestivum L. cv. Drabant) and maize (Zea mays L.) roots; non-latent activities of the PM markers vanadate-inhibited K⁺, Mg²⁺-ATPase (ΔVO₄-ATPase) and glucan synthase II (GS II, EC 2.4.1.34) were used as markers for inside-out PM vesicles, latent activities as markers for right-side-out PM vesicles, and specific staining with silicotungstic acid (STA) as a general marker for the PM. Separation of presumptive inside-out PM vesicles from right-side-out ones was achieved by counter-current-distribution (CCD) in an aqueous polymer two-phase system. Most of the GS II activity was latent and was found in material partitioning into the upper phase; a distribution which correlated well with that of STA-stained vesicles. Thus, most of the PM vesicles had a right-side-out orientation. ΔVO₄-ATPase, on the other hand, had a dual distribution (particularly pronounced in wheat) and was recovered both in material partitioning into the lower phase and into the upper phase. This indicates that ΔVO₄-ATPase activity was present also in membranes other than the PM. Additional evidence for this interpretation came from sucrose gradient centrifugation of wheat root material. This produced two peaks of ΔVO₄-ATPase activity with the membranes partitioning into the lower phase, none of which coincided with the peak obtained with right-side-out PM vesicles. Taken together, these results indicate that only very few inside-out PM vesicles are present in the microsomal fraction, and that ΔVO₄-ATPase as a marker for the PM, in contrast to GS II, may give quite misleading results with some plant materials. This stresses the need to use well-defined preparations of scaled, inside-out PM vesicles in solute uptake studies. The distribution of Ca²⁺-inhibited ATPase, on the other hand, agreed well with those of GS II and STA-stained vesicles both after CCD and sucrose gradient centrifugation, which suggests that Ca²⁺ inhibition may be a more specific property of the PM H⁺-ATPase than vanadate inhibition.     

Mycorrhiza formation on Norway spruce (Picea abies) roots affects the pathway of anaplerotic CO2 fixation

Activities of carboxylation enzymes were analyzed in the mycelium of the mycorrhizal fungus Amanita muscaria (L. ex Fr.) Hooker, in non-mycorrhizal short roots of Norway spruce ( Picea abies [L.] Karst.) and in myconhizas of these two partners. While pyruvale carboxylase (PC, EC 6.4.1.1) and phosphoenolpyruvate carboxykinase activities (PEPCK.EC 4.1.1.49) could be detected in the mycelium of A. muscaria , phosphoenolpyruvate carboxyknase (PEPC, EC 4.1.1.31) was only active in root tissue. In A. muscaria , PC activity was generally low (around 10 nmol mg^−tprotein min⁻) but PEPCK activity was above 250 nmol mg⁻¹ protein min⁻¹. Mycorrhizal development on short roots decreased PEPC activity by more than 75%, although dilution by the fungal biomass in mycorrhizas was only 35%. This reduction in activity was paralleled by a decreased content of PEPC protein. By means of micro-analytical methods it was shown that PEPC activity was lowest in the central zones of the mycorrhizas, Whereas PEPC activity was highest in the corresponding central sections in non-mycorrhizal short roots. ¹⁴CO₂ labelling, on the other hand, revealed that in vivo CO₂ fixation was higher in mycorrhizas compared to non-mycorrhizal short roots. It is concluded that fungal carboxylases (probably PEPCK) are important for anaplerotic CO₂ fixation during nitrogen assimilation in mycorrhizas of Norway spruce.     

Effect of potassium status on K+ (Rb)+ uptake and transport in sunflower roots

Young sunflower plants ( Helianthus annuus L. cv. Halcón), grown in nutrient solution at two K⁺ levels (0.25 and 2.5 m M ) were used to study the effect of K⁺ content in the root on uptake and transport of K⁺ to the exuding stream of decapitated plants. Roots of plants grown in low K⁺ gave higher exudation flux, higher K⁺ concentration in exudate and higher K⁺ flux than high K⁺ roots. After 6 h of uptake the K⁺ flux in low K⁺ roots was about three times that in high K⁺ roots. When the roots were kept in a nutrient solution in which Rb⁺ replaced K⁺, low K⁺ roots exuded much more Rb⁺ than K⁺ after the first 2 h, whereas high K⁺ roots exuded about similar amounts of K⁺ and Rb⁺. In intact plants grown at three different K⁺ levels (0.1, 1.0 and 10.0 m M ), there was an inverse relationship between the K⁺ level in the nutrient solution and the Rb⁺ accumulated in the roots or transported to the shoot. The results suggest that the transport of ions from xylem parenchyma to stele apoplast may be controlled by ions coming down from the shoot in sieve tubes.     

Free space uptake and influx of Ni2+ in excised barley roots

The effects of switches between high and low nutrient supplies on growth and mineral nutrition of winter wheat ( Triticum aestivum L. cv. Martonvásári-8) were followed in four main developmental phases: tillering, shooting, heading and grain filling. Growth of the shoots was significantly affected by switches. Under low nutrient supply the life cycle was shortened. Root growth was only slightly affected by switches, but an early high nutrient supply followed by low nutrient supply gave an impetus for root development. In general, the growth data indicate that the nutrient status of the plants is determined by the nutrient level supplied during shooting. A high level of nutrients during shooting leads also to high vegetative growth, whereas the best grain yield was obtained by a high dose of nutrients during tillering followed by low nutrient conditions during the shooting stage and later. K⁺(⁸⁶Rb) influx in the roots decreased with age. The potential for K⁺ (⁸⁶Rb) influx was low in plants of high-salt status, but it became high in response to switching to low supply at shooting, whereas later switches had no influence on this function in high-salt plants. The highest K⁺(⁸⁶Rb) influx was found in plants starting with high nutrient supply followed by low-salt conditions; this plant group was outstanding also with respect to its high grain yield.     

Localisation of translocated 14C in roots and root exudates of field-grown maize

The roots of a mature, field-grown maize plant are dimorphic: the primary root and those from the oldest nodes are bare with a heavily lignified cortex arid sloughed epidermis; those from younger nodes, except for a bare elongation zone, have an intact epidermis surrounded by a persistent soil sheath. Sheathed roots consistently have more layers of cortical cells, but the ratio of volumes of cortex to stele (ca 4) and the cross-sectional area of phloem (ca³× 10⁻² mm²) are similar in each type. Assimilated carbon (from ¹⁴ C applied to a small area of one leaf) was translocated to all roots and actively metabolized in cortex and stele of both types. After 1 to 2 days the proportion of ¹⁴C exuded from a given length of mature root into its soil sheath, or into the adjacent unattached soil in the case of bare roots, was the same (5%) in both root types when compared with the ethanol-soluble ¹⁴C in the tissues of this length. Up to 75% of the ethanol-soluble label in the roots was in a cationic fraction (amino acids and unidentified compounds), ca 1% was in an anionic fraction and the remainder was in a neutral fraction (sugars). Approximately equal amounts of soluble ¹⁴C were found in the stele, cortex and laterals.