Osmotically induced proton extrusion from carrot cells in suspension culture

Addition of 200 mm of a polyol to anthocyanin containing carrot (Daucus carota L.) cells in suspension culture decreased turgor pressure to zero and induced hyperpolarization of the membrane potential and acidification of the medium due to H⁺ extrusion. These changes were shown to be slightly affected by vanadate. In parallel, a decrease in intracellular ATP and total adenylate concentrations were observed. However, when the osmoticum was NaCl acidification of the medium occurred in the absence of considerable changes in intracellular ATP concentration. These results are interpreted as indicating that a drop of turgor, by addition of a polyol, triggers a proton extrusion activity which is only slightly inhibited by vanadate but apparently ATP utilizing. The observed decrease in ATP level occurs without a change in respiration rate and is accompanied by a drop in total adenylate pool. However when NaCl is the osmoticum it is assumed that Δ_μH+ is enhanced through a Na⁺/H⁺ antiporter. The difference between the two types of osmotica as related to their ability to penetrate through the cellular membrane is discussed.     

Characterization of the Stimulation of Ethylene Production by Galactose in Tomato (Lycopersicon esculentum Mill.) Fruit

We have characterized the stimulation of ethylene production by galactose in tomatoes (Lycopersicon esculentum Mill.). The effect of concentration was studied by infiltrating 0, 4, 40, 100, 200, 400, or 800 micrograms galactose for each gram of fresh fruit weight into mature green `Rutgers' fruit. Both 400 and 800 micrograms per gram fresh weight consistently stimulated a transient increase in ethylene approximately 25 hours after infiltration; the lower concentrations did not. Carbon dioxide evolution of fruit infiltrated with 400 to 800 micrograms per gram fresh weight was greater than that of lower concentrations. The ripening mutants, rin and nor, also showed the transient increase in ethylene and elevated CO₂ evolution by 400 micrograms per gram fresh weight galactose. 1-Aminocyclopropane-1-carboxylic acid (ACC) content and ACC-synthase activity increased concurrently with ethylene production. However, galactose did not stimulate ACC-synthase activity in vitro. The infiltrated galactose in pericarp tissue was rapidly metabolized, decreasing to endogenous levels within 50 hours. Infiltrated galacturonic acid, dulcitol, and mannose stimulated transient increases in ethylene production similar to that of galactose. The following sugars produced no response: sucrose, fructose, glucose, rhamnose, arabinose, xylose, raffinose, lactose, and sorbitol.     

Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes

An NAD(P)H dehydrogenase stimulated by quinone (P Pupillo, V Valenti, L de Luca, R Hertel 1986 Plant Physiol 80: 384-389) was solubilized from washed microsomes of zucchini squash hypocotyls (Cucurbita pepo L.) by use of 1% Triton X-100. The solubilized enzyme remained in solution in aqueous buffer and could be purified by a combination of Sepharose 6B chromatography and Blue Ultrogel chromatography. Of the three peaks of activity eluted from the latter column with a salt gradient, peak 3 had 50% or more of the activity and was almost pure enzyme. The preparation examined in SDS-gel electrophoresis consisted of two types of subunits, a (molecular weight 39,500) and b (37,000) in equal amounts. Peak 2 was less pure but had a similar polypeptide pattern. The active protein is proposed to be a heterotetramer (a₂b₂) having a molecular weight of about 150,000, as found by gel exclusion chromatography. The purified enzyme can reduce several quinones, DCPIP, cytochrome c, and with best efficiency ferricyanide, and is therefore a diaphorase. The kinetics for the substrates are negatively cooperative with Hill coefficients n_H = 0.55 ± 0.05 for NADPH and 0.22 ± 0.04 for duroquinone. A weak inhibition by p-hydroxymercuric benzoate and mersalyl (stronger with microsomal preparations) suggests the presence of essential sulfhydryl group(s). The possibility is discussed that the dehydrogenase is an NAD(P)H-P450 reductase or similar flavoprotein, and that it is responsible for the NADPH-cytochrome c reductase activity of plant microsomes.     

Control of wild carrot somatic embryo development by antioxidants : a probable mode of action of 2,4-dichlorophenoxyacetic Acid

As we previously reported for glutathione (GSH), both ascorbic acid (AA) and vitamin E were observed to suppress wild carrot (Daucus carota L.) somatic embryogenesis with little concomitant effect on biomass. Endogenous concentrations of AA were lower during embryo development than during cell proliferation, exhibiting a temporal pattern nearly identical to that of GSH. GSSG (oxidized GSH) reductase was found to be considerably more active in proliferating than in developing cultures, whereas no difference was evident in the case of dehydroascorbate (DHA) reductase. Both GSH and AA concentrations in these cells are governed by 2,4-D. These results show that redox status is a strong determinant of proliferative versus developmental growth and indicate that the mode of action of 2,4-D in this system may be explained at least in part by its influence on endogenous antioxidant levels.     

Polysomes from winter rye seedlings grown at low temperature : I. Size class distribution, composition, and stability

We have studied the influence of growth at low temperature on size class distribution, stability and composition of leaf cytoplasmic polysomes from rye seedlings (Secale cereale, cv Puma) grown at 5°C and at 20°C. Leaves of seedlings grown at 5°C contain 2.7 times more cytoplasmic polysomes (expressed on a DNA basis) and the polysome size class distribution is skewed toward larger polysomes. These changes were more pronounced in the free polysome fraction than in the membrane-bound fraction. The melting point of the total ribosome fraction from cold-grown leaves was decreased by 3.7°C. Electrophoresis did not reveal any difference in the rRNA or in core-ribosomal proteins (KCl nondissociable) following growth at low temperature. Some differences were noted in peripheral ribosomal proteins. This study is the first to examine the effect of growth at low and high temperatures on polysome metabolism using plants of similar developmental stage. Polysome quantity, polymerization, melting point and peripheral ribosomal proteins in rye seedlings are modified during growth at low temperature.     

Nonphotosynthetic CO(2) Fixation by Alfalfa (Medicago sativa L.) Roots and Nodules

The dependence of alfalfa (Medicago sativa L.) root and nodule nonphotosynthetic CO₂ fixation on the supply of currently produced photosynthate and nodule nitrogenase activity was examined at various times after phloem-girdling and exposure of nodules to Ar:O₂. Phloemgirdling was effected 20 hours and exposure to Ar:O₂ was effected 2 to 3 hours before initiation of experiments. Nodule and root CO₂ fixation rates of phloem-girdled plants were reduced to 38 and 50%, respectively, of those of control plants. Exposure to Ar:O₂ decreased nodule CO₂ fixation rates to 45%, respiration rates to 55%, and nitrogenase activities to 51% of those of the controls. The products of nodule CO₂ fixation were exported through the xylem to the shoot mainly as amino acids within 30 to 60 minutes after exposure to ¹⁴CO₂. In contrast to nodules, roots exported very little radioactivity, and most of the ¹⁴C was exported as organic acids. The nonphotosynthetic CO₂ fixation rate of roots and nodules averaged 26% of the gross respiration rate, i.e. the sum of net respiration and nonphotosynthetic CO₂ assimilation. Nodules fixed CO₂ at a rate 5.6 times that of roots, but since nodules comprised a small portion of root system mass, roots accounted for 76% of the nodulated root system CO₂ fixation. The results of this study showed that exposure of nodules to Ar:O₂ reduced nodule-specific respiration and nitrogenase activity by similar amounts, and that phloem-girdling significantly reduced nodule CO₂ fixation, nitrogenase activity, nodule-specific respiration, and transport of ¹⁴C photoassimilate to nodules. These results indicate that nodule CO₂ fixation in alfalfa is associated with N assimilation.     

Tabtoxinine-beta-Lactam Transport into Cultured Corn Cells : Uptake via an Amino Acid Transport System

Tabtoxinine-β-lactam (T-β-L), a unique amino acid, is a toxin produced by several closely related pathovars of Pseudomonas syringae. These chlorosis-inducing pathogens establish themselves in the apoplastic space of their hosts where they release the toxin. We have examined the transport of T-β-L into cultured corn (Zea mays cv Black Mexican) cells using [¹⁴C]T-β-L. The pH optimum of the uptake of the toxin was between 4.0 and 5.5 pH units. Toxin uptake was inhibited by the protonophore, carbonyl cyanide m-chlorophenyl hydrazone, and by the sulfhydryl re-agent, N-ethylmaleimide. Tabtoxinine-β-lactam transport exhibited saturation kinetics that were described by the Michaelis-Menton equation for toxin concentrations of 1 millimolar and less. However, the transport of toxin in concentrations greater than 1 millimolar was not described by Michaelis-Menten kinetics. Glutamate and alanine exhibited similar transport kinetics with a transition to non-Michaelis-Menten kinetics when the amino acid concentration exceeded 1 millimolar. Hill numbers for glutamate, alanine, and T-β-L ranged from 0.6 to 0.8. Methionine, alanine, tyrosine, glutamine, glutamate, and arginine were inhibitors of toxin transport. Alanine was a competitive inhibitor of the transport of T-β-L and of glutamate. The data are consistent with T-β-L being transported into the plant cell through an amino acid transport system.     

Promotion of Stomatal Opening by Indoleacetic Acid and Ethrel in Epidermal Strips of Vicia faba L

Indole-3-acetic acid (IAA), at concentrations of 0.01 to 1.0 millimolar, and ethephon (0.3% v/v Ethrel) promote stomatal opening when applied to epidermal peels of Vicia faba L. in light or dark. The effect of ethylene is seen by 30 minutes and maximal opening (over two times that of untreated controls) occurs after only 60 to 90 minutes in the light. Stomatal opening by IAA and Ethrel in both light and dark is prevented by 0.14 millimolar AgCl. It is suggested that the effect of added IAA, but not that of light, is linked to ethylene production. The possible role of ethylene in stomatal opening during fungal infection is discussed. The stomates of Vicia faba provide a new system to study the effects of ethylene on certain membrane-regulated processes.     

Active Glucose Transport and Proton Pumping in Tonoplast Membrane of Zea mays L. Coleoptiles Are Inhibited by Anti-H-ATPase Antibodies

A tonoplast enriched fraction was obtained from Zea mays L. coleoptiles by isopycnic centrifugation of microsomal membranes in a sucrose step gradient. At the 18/26% interface chloride-stimulated and nitrate-inhibited proton pumping activity coincided with a Mg²⁺-ATP dependent accumulation of 3-O-methyl-d-glucose (OMG) as determined by a membrane filtration technique using ¹⁴C-labeled substrate. OMG transport showed an apparently saturable component with a K_m of 110 micromolar, and was completely inhibited by 10 micromolar carbonyl cyanide m-chlorophenylhydrazone. Polyclonal antibodies against solubilized native tonoplast H⁺-ATPase and its 62 and 72 kilodalton subunits were assayed for their ability to inhibit proton pumping and OMG accumulation. Antibodies against both the native enzyme and the putative catalytic subunit (72 kilodalton) strongly inhibited proton pumping and OMG transport whereas antibodies against the 62 kilodalton subunit had only a slight effect on both processes.     

Photoinduced Seed Germination of Oenothera biennis L: II. Analysis of the Photoinduction Period

The photoinduction period of Oenothera biennis L. seed germination was analyzed by varying the photoinduction temperature and by substituting red light pulses for continuous red light. At 24°C, seeds require 36 hours of continuous red light for maximal percent germination. The optimal photoinduction temperature is 32°C, with higher and lower temperatures being strongly inhibitory. A 30 minute exposure to far-red light, given immediately after a red light period of 1 to 36 hours, reduces germination by about 25%. Seeds escape from far-red inhibition with a half-time of 5 to 10 hours, depending on the length of the red exposure that precedes the far-red light. Periodic 15 minute pulses of red light can substitute for continuous red light in stimulating germination. Ted red light pulses, with 6 hours of darkness between successive pulses, cause maximal germination. The response to periodic red light is fully reversible by far-red light. Probit analysis of the periodic light response shows that as the length of the dark periods between successive pulses increases, less incident light is needed to induce germination but the population variance in light sensitivity remains constant. Probit analysis of the temperature response shows that as the photoinduction temperature increases from 16 to 32°C, less incident light is needed to induce germination and the population variance in light sensitivity also increases.