Preparation of Corn Root Plasmalemma with Low Mg-ATPase Latency and High Electrogenic H Pumping Activity after Phase Partitioning

Crude plasma membranes of corn (Zea mays L.) roots were obtained according to MI De Michelis and RM Spanswick (1986 Plant Physiol 81: 542-547). This preparation, which contained tightly sealed vesicles displaying Mg-ATP dependent H⁺-transport, was purified by phase partitioning. The percentage of inside-out vesicles (10%) was determined from the Mg-ATPase latency, revealed with lysophosphatidylcholine. A Triton X-100 treatment described previously (JP Grouzis, R Gibrat, J Rigaud, C Grignon 1987 Biochim Biophys Acta 903: 449-464) was applied to phase-partitioned plasma membranes. The percentage of catalytic sites freely accessible to Mg-ATP increased to 50% after Triton X-100 treatment. Treated vesicles remained capable of electrogenic H⁺-pumping, as demonstrated by Mg:ATP-dependent quinacrine fluorescence quenching and oxonol absorbance shift. As expected from the large increase of the catalytic sites accessibility, increases of the dye responses were observed. Concanavalin A binding was estimated from microelectrophoretic measurements of individual vesicles. Statistical analysis of concanavalin A binding and Mg-ATPase latency suggest that treated membranes have lost their asymmetric structure.     

Effect of Low Root Medium pH on Net Proton Release, Root Respiration, and Root Growth of Corn (Zea mays L.) and Broad Bean (Vicia faba L.)

The effect of low pH on net H⁺ release and root growth of corn (Zea mays L.) and broad bean (Vicia faba L.) seedlings was investigated in short-term experiments at constant pH. Broad bean was more sensitive to low pH than corn: the critical values (pH values below which net H⁺ release and root growth ceased) were pH 4.00 (broad bean) and pH 3.50 (corn) at 1 millimolar Ca²⁺. Both proton release and root growth were progressively inhibited as the medium pH declined. Additional Ca²⁺ in the root medium helped to overcome the limitations of low pH for net H⁺ release and root growth. Potassium (for corn) and abscisic acid (for broad bean) increased both net H⁺ release and root growth rate at the critical pH value. It is concluded that poor root growth at low pH is caused by a lack of net H⁺ release that may decrease cytoplasmic pH values. Inhibited net H⁺ release at high external H⁺ activity is not due to a shortage of energy supply to the H⁺ ATPase. Instead, a displacement of Ca²⁺ by H⁺ at the external side of the plasmalemma may enhance reentry of H⁺ into root cells.     

H Cotransports in Corn Roots as Related to the Surface pH Shift Induced by Active H Excretion

The surface pH shift induced by active H⁺ excretion in corn (Zea mays L.) roots was estimated using acetic acid influx as a pH probe (H Sentenac, C Grignon 1987 Plant Physiol 84: 1367-1372). At constant bulk pH, buffering the medium strongly reduced the magnitude of the surface pH shift. This was used to study the effect of surface pH shift on H⁺ cotransports. In the absence of buffers, the surface pH shift increased with the bulk pH. Buffers decreased ³²Pi influx and this effect was stronger at pH 7.2 than at pH 5.8, and stronger in the absence than in the presence of an inhibitor of the proton pump (vanadate). Buffers exerted a similar depressive and pH-dependent effect on net NO₃⁻ uptake. They hyperpolarized the cell membrane, and stimulated ⁸⁶Rb⁺ influx, K⁺:H⁺ net exchange, and malate accumulation. These results are consistent with the hypothesis that H⁺ accumulation at the cell surface is effective in driving H⁺ reentry. We concluded that the surface pH shift due to proton pump activity is involved in the energetic coupling of H⁺ cotransports.     

Adaptation of Denitrifying Populations to Low Soil pH

Natural denitrification rates and activities of denitrifying enzymes were measured in an agricultural soil which had a 20-year past history of low pH (pH ca. 4) due to fertilization with acid-generating ammonium salts. The soil adjacent to this site had been limed and had a pH of ca. 6.0. Natural denitrification rates of these areas were of similar magnitude: 158 ng of N g⁻¹ of soil day⁻¹ for the acid soil and 390 ng of N g⁻¹ of soil day⁻¹ at the neutral site. Estimates of in situ denitrifying enzyme activity were higher in the neutral soil, but substantial enzyme activity was also detected in the acid soil. Rates of nitrous oxide reduction were very low, even when NO₃⁻ and NO₂⁻ were undetectable, and were ca. 400 times lower than the rates of N₂O production from NO₃⁻. Denitrification rates measured in slurries of the acid and neutral soil showed distinctly different pH optima (pH 3.9 and pH 6.3) which were near the pH values of the two soils. This suggests that an acid-tolerant denitrifying population had been selected during the 20-year period of low pH.     

Proton Fluxes and the Activity of a Stelar Proton Pump in Onion Roots

The xylem vessels of excised adventitious roots of onion, Alliumcepa, were perfused with unbuffered nutrient solution adjustedinitially to either pH 9·3 or 3·9; the pH of thesolution after passage through the xylem, at rates not lessthan 2 xylem volume changes min^–1, was close to pH 6·5in both instances. The flux of H⁺ across the xylem/symplastboundary into mildly alkaline, phosphate-buffered solutionsperfusing the vessels could be increased greatly with increasingbuffer strength, up to a maximum value between 0·5–1·0pmol H⁺ mm^–2 s^–1. The apparent neutralization ofacidic malic acid buffers had a slightly lower maximum capacity,equivalent to –0·3 to –0·5 pmol H⁺mm^–2 s^–1. The addition of 5·0 pmol m^–3fusicoccin (FC) to the xylem perfusion solution stimulated theentry of H⁺ into the xylem; in unbuffered perfusion solutionsthe pH fell to pH 3·6 after a lag of 25–35 min.FC additions to phosphate-buffered solutions also stimulatedthe H⁺ flux to an extent similar to that in unbuffered solution,viz. 0·2–0·4 pmol mm^–2 s^–1. The release of K⁺ (³⁶Rb-labelled) into xylem sap transientlyincreased as the [K⁺] in weakly buffered perfusion solutionswas raised stepwise; a very marked increase being seen whenthe concentration was raised to 100 mol m^–3 from 40 molm^–3. The addition of 5·0 mmol m^–3 FC to theperfusing solution containing 100 mol m^–3 K⁺ rapidly decreasedthe K⁺ flux to the xylem as the H⁺ flux increased. Fusicoccinalso inhibited the flux of K⁺ into unbuffered perfusion solutionsbut the effect appeared reversible. Addition of 10 mmol m^–3abscisic acid (ABA) to the perfusion solution quickly producedtransient increases in both K⁺ and H⁺ fluxes into the xylem.In this and other experiments using weakly phosphate-bufferedperfusing solutions, H⁺ fluxes were comparable in size to thoseof K⁺ The results are consistent with the idea that the stele of onionroots contains a proton trarislocating ATPase whose activityresponds to the pH of the xylem sap. It is evident that theactivity of the proton secreting and proton neutralizing mechanismsin the xylem parenchyma control the movement of other ions acrossthe xylem/symplast boundary. Key words: Xylem perfusion, fusicoccin, abscisic acid, pH gradient     

Reduced Root Elongation of Lupinus angustifolius L. by High pH is not Due to Decreased Membrane Integrity of Cortical Cells or Low Proton Production by the Roots

Root elongation and cell expansion were decreased markedly bypH     

A Novel Interaction between Plant-Beneficial Rhizobacteria and Roots: Colonization Induces Corn Resistance against the Root Herbivore Diabrotica speciosa

A number of soil-borne microorganisms, such as mycorrhizal fungi and rhizobacteria, establish mutualistic interactions with plants, which can indirectly affect other organisms. Knowledge of the plant-mediated effects of mutualistic microorganisms is limited to aboveground insects, whereas there is little understanding of what role beneficial soil bacteria may play in plant defense against root herbivory. Here, we establish that colonization by the beneficial rhizobacterium Azospirillum brasilense affects the host selection and performance of the insect Diabrotica speciosa. Root larvae preferentially orient toward the roots of non-inoculated plants versus inoculated roots and gain less weight when feeding on inoculated plants. As inoculation by A. brasilense induces higher emissions of (E)-β-caryophyllene compared with non-inoculated plants, it is plausible that the non-preference of D. speciosa for inoculated plants is related to this sesquiterpene, which is well known to mediate belowground insect-plant interactions. To the best of our knowledge, this is the first study showing that a beneficial rhizobacterium inoculant indirectly alters belowground plant-insect interactions. The role of A. brasilense as part of an integrative pest management (IPM) program for the protection of corn against the South American corn rootworm, D. speciosa, is considered.     

Ethylene Signaling Causing Tolerance of Arabidopsis thaliana Roots to Low pH Stress is Linked to Class III Peroxidase Activity

Journal of Plant Growth Regulation - One irreversible consequence of acidic pH for roots is cell death. Growing evidence suggests the role of hormones and cell wall-related enzymes in response to...     

Cell-Specific Production and Antimicrobial Activity of Naphthoquinones in Roots of Lithospermum erythrorhizon

Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots. Normal pigment development is limited to root hairs and root border cells in hairy roots grown on “noninducing” medium, whereas induction of additional pigment production by abiotic (CuSO₄) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and β-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed “hairy-root” cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested. Hyphae of the pathogenic fungi Rhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots. Challenge by R. solani crude elicitor increased shikonin derivative production 30-fold. We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere.     

Nematode Control Related to Fusarium Wilt in Soybean and Root Rot and Zinc Deficiency in Corn

Nematode and disease problems of irrigated, double-cropped soybean and corn, and zinc deficiency of corn were investigated. Ethylene dibromide, phenamiphos, and aldicarb were equally effective for controlling nematodes and increasing yields of corn planted minimum-till and soybean planted in a moldboard plow prepared seedbed. The residual effects on yields of nematicides applied to the preceeding crop occurred during 3 years for soybean and 1 year for corn. Fusarium wilt symptoms of soybean that developed during 2 years of the study were less severe in all nematicide-treated plots than in control plots. Typical zinc deficiency symptoms on 30-day-old corn plants were observed during 1 year of the study in certain plots. Symptoms were not evident on plants grown on plots treated with ethylene dibromide, and only occasional plants had symptoms on plots treated with phenamiphos and aldicarb. The amount of yield response directly related to nematode control could not be determined because of the apparent interaction of nematodes on the expression of Fusarium wilt of soybean. Our study strongly indicates that the expression of Fusarium wilt of soybean and zinc deficiency in corn are influenced by nematodes and that nematicides will reduce their severity.     

Nitrate-Sensitive ATPase Activity and Proton Pumping in Guard Cell Protoplasts of Commelina

ATPase activity was measured in crude homogenates of guard cellprotoplasts of Commelina communis L. using a linked enzyme assay.A low level of azide-sensitive ATPase activity was detectedwith a pH optimum of 6.8. This activity was stimulated by 0.01%(v/v) Triton X-100, and the pH optimum shifted to pH 7.4. Nitrate-sensitiveATPase activity was measured in the presence of azide and showeda pH optimum around pH 8.0. Proton pumping activity in a mixedpopulation of vesicles from GCP was monitored using fluorescencequenching of quinacrine. Mg-ATP dependent proton pumping wasobserved at pH 8.0, but not at pH 6.6. The activity at pH 8.0was inhibited by nitrate and DCCD but not vanadate. These dataindicate that activity of the tonoplast proton pump was beingmeasured. There was, however, no evidence for a tonoplast cation(K⁺)/proton antiporter under these assay conditions as potassiumdid not reduce the initial rate of pH gradient formation orincrease the rate of collapse of a pre-formed gradient afterinhibition of the pump. Key words: Tonoplast ATPase, proton pump, guard cell protoplasts, Commelina     

Relationship of Transplasmalemma Redox Activity to Proton and Solute Transport by Roots of Zea mays

Transplasmalemma redox activity, monitored in the presence of exogenous ferricyanide stimulates net H⁺ excretion and inhibits the uptake of K⁺ and α-aminoisobutyric acid by freshly cut or washed, apical and subapical root segments of corn (Zea mays L. cv “Seneca Chief”). H⁺ excretion is seen only following a lag of about 5 minutes after ferricyanide addition, even though the reduction of ferricyanide occurs before 5 minutes and continues linearly. Once detected, the enhanced rate of H⁺ excretion is retarded by the ATPase inhibitors N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and vanadate. A model is presented in which plasmalemma redox activity in the presence of ferricyanide involves the transport only of electrons across the plasmalemma, resulting in a depolarization of the membrane potential and activation of an H⁺-ATPase. Such a model implies that this class of redox activity does not provide an additional and independent pathway for H⁺ transport, but that the activity may be an important regulator of H⁺ excretion. The 90% inhibition of K⁺ (⁸⁶Rb⁺) uptake within 2 minutes after ferricyanide addition can be contrasted with the 5 to 15% inhibition of uptake of α-aminoisobutyric acid. The possibility exists that a portion of the K⁺ and most of the α-aminoisobutyric acid uptake inhibitions are related to the ferricyanide-induced depolarization of the membrane potential, but that the redox state of some component of the K⁺ uptake system may also regulate K⁺ fluxes.     

Distribution of Field Corn Roots and Parasitic Nematodes in Subsoiled and Nonsubsoiled Soil

A field trial was conducted for 2 years in an Arredondo fine sand containing a tillage pan at 15-20 cm deep to determine the influence of subsoiling on the distribution of corn roots and plant-parasitic nematodes. Soil samples were taken at various depths and row positions at 30, 60, and 90 days after planting in field corn subsoiled under the row with two chisels and in non-subsoiled corn. At 30 and 60 days, in-row nematode population densities to 60 cm deep were not affected by subsoiling compared with population densities in nonsubsoiled plots. After 90 days, subsoiling had not affected total root length or root weight at the 20 depth-row position sampling combinations, but population densities of Meloidogyne incognita and Criconemella spp. had increased in subsoiled corn. Numbers of Pratylenchus zeae were not affected. Subsoiling generally resulted in a change in distribution of corn roots and nematodes in the soil profile but caused little total increase in either roots or numbers of nematodes. Corn yield was increased by subsoiling.     

Characterization of Anion Effects on the Nitrate-Sensitive ATP-Dependent Proton Pumping Activity of Soybean (Glycine max L.) Seedling Root Microsomes

The ATP-dependent proton-pumping activity of soybean (Glycine max L.) root microsomes is predominantly nitrate sensitive and presumably derived from the tonoplast. We used microsomes to characterize anion effects on proton pumping of the tonoplast vesicles using two distinctly different techniques.

Preincubation of the vesicles with nitrate caused inhibition of proton pumping and ATPase activity, with similar concentration dependence. Fluoride, which preferentially inhibits the plasma membrane ATPase, inhibited ATPase activity strongly at concentrations which did not affect proton pumping activity.

Addition of potassium salts, after a steady-state pH gradient is established in the absence of such salts, caused an increased pH gradient which was due to alleviation of Δ Ψ and subsequent increased influx of H⁺ into these vesicles. This anion-induced increase in the pH gradient could be used as a measure of the relative anion permeabilities, which were of the order Br⁻ = NO₃⁻ > Cl⁻ SO₄²⁻. Phosphate and fluoride caused no increase in the pH gradient. Since the concentration dependence of KCl- and KNO₃-induced quenching exhibited a saturable component, and since H⁺ uptake was increased by only certain anions, the data suggest that there may be a relatively specific anion channel associated with tonoplast-derived vesicles.

     

Penetration and Development of Meloidogyne incognita in Roots of Resistant and Susceptible Corn Genotypes

Rates of penetration and development ofMeloidogyne incognita race 4 in roots of resistant (inbred Mp307, and S4 lines derived from the open-pollinated varieties Tebeau and Old Raccoon) and susceptible (Pioneer 3110) corn genotypes were determined. Seedlings grown in styrofoam containers were inoculated with 5,000 eggs of M. incognita. Roots were harvested at 3-day intervals starting at 3 days after inoculation (DAI) to 27 DAI and stained with acid fuchsin. Penetration of roots by second-stage juveniles (J2) at 3 DAI was similar for the four corn genotypes. Meloidogyne incognita numbers in Tebeau, Old Raccoon, Mp307, and Pioneer 3110 peaked at 12, 12, 15, and 27 DAI, respectively. Nematode development in the resistant genotypes was greatly suppressed compared to Pioneer 3110. Resistance to M. incognita in these genotypes appears to be expressed primarily as slower nematode development rather than differences in J2 penetration.     

Potassium Stimulation of Corn Root Plasmalemma ATPase : II. H-Pumping in Native and Reconstituted Vesicles with Purified ATPase

The stimulation by K⁺ of the initial rate of H⁺-pumping by ATPase was studied in native plasmalemma (Zea mays L. var Mona) vesicles and in reconstituted vesicles with enzyme purified on a glycerol gradient. In reconstituted vesicles, a very high H⁺-pumping rate (200,000% quenching per minute per milligram protein) was obtained with 9-amino-6-chloro-2-methoxyacridine provided that the pump was short-circuited by K⁺-valinomycin. A constant ionic strength was used to prevent indirect stimulation by the electrostatic effects of K⁺ salts. Indirect stimulation of H⁺-pumping by the short-circuiting effect of internal K⁺, could be abolished by using the permeant anions NO₃⁻ and Br⁻ in native, but not in reconstituted vesicles. In both materials, half-stimulation of the H⁺-pumping by K⁺ was observed at about 5 millimolar. The same stimulation was obtained when K⁺ was present only in the external solution or when it was present both outside and inside the vesicles. It was concluded that the stimulating effect of K⁺ on the H⁺-pumping evidenced in these experiments on both native and reconstituted vesicles was due to a direct effect of the cation on the cytoplasmic face of the ATPase. These results are discussed within the context of the hypothesis of an active K⁺ transport driven by the ATPase through a direct H⁺/K⁺ exchange mechanism.