Subcellular localization of H+-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation

A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H⁺-ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H⁺ pump was directly demonstrated. The H⁺ pump was substrate-specific for Mg·ATP and 0.1 mM diethylstilbestrol completely prevented the development of a pH. The presence of unsupecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg·ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K⁺-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because -mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. Nigericinstimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DES dethyltilbestrol     

Auxin binding to subcellular fractions from Cucurbita hypocotyls: In vitro evidence for an auxin transport carrier

An auxin binding sive, with characteristics different from the previously described auxin binding sites I and II in maize coleoptiles, is reported in homogenates of zucchini (Cucurbita pepo L. cv. Black Beauty) hypocotyls. Evidence from differential centrifugation and sucrose and metrizamide density gradients indicates that the site is localized on the plasma membrane. The site has a K_D of 1–2×10^–6 M for indole acetic acid and has a pH optimum of 5.0. Binding specificity measured with several auxins, weak auxins, and anti-auxins generally parallels the activities of the same compounds as inhibitors of auxin transport. 1-N-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid (2,3,5-TIBA), both auxin transport inhibitors in vivo, increase specific auxin binding to this site. 3,4,5-TIBA, which can partially reverse 2,3,5-TIBA's transport inhibition when the two substances are added together in vivo, partially reverses 2,3,5-TIBA's increase in specific auxin binding to the plasma membrane site when added with 2,3,5-TIBA in vitro. Preliminary investigations indicate that a similar plasma membrane site exists in maize (Zea mays L.) coleoptiles. It is suggested that different conformations of this site may function during active auxin transport.Abbreviations IAA indole-3-acetic acid - NPA 1-N-naphthylphthalamie acid - 2,3,5-TIBA 2,3,5-triiodobenzoic acid - 3,4,5-TIBA 3,4,5-triiodobenzoic acid - 1-NAA 1-naphthaleneacetic acid - 2-NAA 2-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - DTE dithioerythritol - MOPS N-morpholino-3-propansulfonic acid - CCO cytochrome c oxidase - CCR NADH: cytochrome c reductase - glu I glucan synthetase I - ER endoplasmic reticulum     

Phytochrome and phosphotungstate-chromate-positive vesicles from Cucurbita pepo L.

The phosphotungstic acid-chromic acid (PTA-CrO₃) stain, putatively specific for the plasma membrane of plants, has been used in an attempt to monitor the distribution of this membrane in a 20,000 x g particulate fraction from Cucurbita hypocotyl hooks. On discontinuous sucrose gradients, the relative distributions of the phytochrome and PTA-CrO₃-positive vesicles present in this fraction appear to be correlated. When intact tissue is stained, however, other components, in addition to the plasma membrane, react positively to the stain. These components include prolamellar-body membranes, lipid droplets, and ribosomes. This lack of specificity calls into question the reliability of the technique for the unequivocal identification and accurate quantitation of plasma-membrane fragments in isolated particulate fractions. The present data do not, therefore, provide unambiguous evidence that phytochrome is associated with plasma membrane in tissue homogenates from Cucurbita.Abbreviations PTA-CrO₃ phosphotungstate-chromate - RNP ribonucleoprotein     

Host-tissue differences in transformation of pumpkin (Cucurbita pepo L.) by Agrobacterium rhizogenes

Agrobacterium rhizogenes (wild-type strains 8196 and 15834) transformation of pumpkin (Cucurbita pepo L.) intact seedlings grown in vivo, and 6–8-day-old excised cotyledons cultured in axenic conditions was investigated. Transformed (hairy) roots were successfully induced only on the excised cotyledons with the strain 8196, while intact seedlings failed to form hairy roots with either of the two different bacterial strains. Axenic hairy-root cultures established on MS medium without hormones grew vigorously. Mannopine was detected in all transgenic root clones examined. The peroxidase activity in transformed roots was higher compared with normal roots. Electrophoretic analyses of soluble proteins and isoperoxidases showed substantial differences between transformed and normal pumpkin roots.     

Heterogeneity of auxin-accumulating membrane vesicles from Cucurbita and Zea: a possible reflection of cell polarity

When microsomes from hypocotyls of Cucurbita pepo L. or coleoptiles of Zea mays L. were centrifuged on dextran-sucrose gradients a heterogeneity of auxin-accumulating vesicles was observed. Vesicles from the top part of the gradient showed saturable, specific accumulation of indole-3-acetic acid with only a small stimulation by phytotropins, and with very few binding sites for 1-N-naphthylphthalamic acid. In the vesicles from the lower part of the gradient, net accumulation of indole-3-acetic acid could be strongly increased by addition of phytotropins; binding of 1-N-naphthylphthalamic acid was high in this region. After two-phase partitioning, both kinds of vesicles were found in the upper-phase membrane fraction considered to be purified plasma membrane. The hypothesis is discussed that vesicles can be separated from the apical and basal parts of the cell's plasmalemma.Abbreviations CCO cytochrome-c oxidase - CCR KCN-insensitive NADH-dependent cytochrome-c reductase - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IDPase inosine 5-diphosphatase - ION3 ionophore mixture of carbonylcyanide-3-chlorophenylhydrazone, nigericin and valinomycin - 1-NAA 1-naphthaleneacetic acid - NPA 1-N-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - UDPG uridine diphosphoglucose     

Auxin carriers in Cucurbita vesicles

Carrier-mediated uptake of indole-3-acetic acid (IAA) by microsomal vesicles from Cucurbita pepo L. hypocotyls was strongly inhibited by 2,4-dichlorophenoxyacetic acid (2,4-D; i ₅₀= 0.3 M) but only weakly by 1-naphthylacetic acid (NAA). The fully ionised auxin indol-3-yl methanesulphonic acid also inhibited (i ₅₀=3 M). The same affinity ranking of these auxins for the uptake carrier, an electroimpelled auxin anion-H⁺ symport, is demonstrable in hypocotyl segments. The specificity of the auxin-anion eflux carrier was tested by the ability of different nonradioactive auxins to compete with [³H]IAA and reduce the stimulation of net radioactive uptake by N-1-naphthylphthalamic acid (NPA), a noncompetitive inhibitor of this carrier. By this criterion, NAA and IAA had comparable affinities, with 2,4-D interaction more weakly. Stimulation of [³H]IAA uptake by NAA, as a result of competition for the efflux carrier, could also be demonstrated when a suitable concentration of 2,4-D was used selectively to inhibit the uptake carrier. However, when [³H]NAA was used, no stimulation of its association with vesicles by NPA, 2,3,5-triiodobenzoic acid, or nonradioactive NAA was found. In hypocotyl segments, [³H]NAA net uptake was much less sensitive to NPA stimulation than was [¹⁴C]IAA uptake. The apparent contradictions concerning NAA could be explained by carrier-mediated auxin efflux making a smaller relative contribution to the overall transport of NAA than of IAA. The relationship between carrier specificity as manifested in vitro and the specificity of polar auxin transport is discussed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - ION3 mixture of 4 M carbonylcyanide m-chlorophenylhydrazone, nigericin and valinomycin - IMS indol-3-yl methanesulphonic acid - NAA 1-naphthylacetic aci - NPA N-1-naphthylphthalamic acid     

Stimulation of ATPase activity by auxin is dependent on ATP concentration

The effect of auxin on membrane-bound ATPase activity was studied in a plasma membrane-enriched fraction from zucchini hypocotyls. The apparent K_M of ATPase activity for ATP was decreased in the presence of 10^-6 M auxin so that at very low ATP concentrations the stimulation of ATPase activity was most obvious. The weak auxin analogue, 2,3-dichlorophenoxyacetic acid, stimulated much less than the active auxin 2,4-dichlorophenoxyacetic acid.     

Phytochrome modulation of ATPase activity in a membrane fraction from Phaseolus

Membrane-bound phytochrome and ATPase (ATP phosphohydrolase EC 3.6.1.3.) activity extracted from hypocotyl hooks of etiolated Phaseolus aureus Roxb. were both separated from solute proteins by gel filtration on Sepharose C1-2B. The amount of phytochrome detected in the membrane fraction was very small and was not significantly increased by red irradiation (in vivo or in vitro). Membrane-bound ATPase activity was modulated in vitro by the phytochrome in the membrane fraction, being lower after red light than after far-red light. This effect was potentiated by a preliminary light reaction which occurred only in vivo and, in continuous red light, required 60 to 90 s at 25°C. Thus a two minute, in vivo, red irradiation reduced membrane-bound ATPase activity to about half that of the etiolated state. Subsequently bound-ATPase activity was determined by the form of phytochrome (P_r or P_fr) irrespective of whether established in vivo or in vitro. These results indicate that binding or release (of enzyme, cofactors or inhibitors) is not involved in phytochrome modulation of enzyme activity in the membrane fraction.Abbreviations R red light - F far red light - P_r inactive form of phytochrome (_max=660 nm) - P_fr active form of phytochrome (_max=730 nm) - MOPS N-morpholino-3-propansulphonic acid     

In vitro binding of riboflavin to subcellular particles from maize coleoptiles and Cucurbita hypocotyls

Saturable and reversible in vitro binding of [¹⁴C]riboflavin was found to occur on subcellular, sedimentable particles from maize coleoptiles and Cucurbita hypocotyls. The K_D was ca. 6 M, the pH optimum was near 6.0, and the number of binding sites amounted to 0.1–0.5 M on a fresh-weight basis. When the reducing agent dithionite was present, riboflavin binding increased-the K_D was 2.5 M, and the pH optimum above 8.0. The binding was specific: flavin mononucleotide (FMN) and flavin adenosine-dinucleotide (FAD) bound less tightly to these sites than riboflavin and another major soluble flavin, the previously described riboflavin-analog FX, occurring in grass coleoptiles. These flavin-binding sites were localized on vesicles derived from plasmalemma and endoplasmic reticulum by analyzing sucrose and metrizamide density gradients and marker enzymes.Abbreviations CCO cytochrome-c oxidase - CCR NADH-cytochrome-c oxidoreductase - ER endoplasmic reticulum - FAD flavin-adenosinedinucleotide - FMN flavin mononucleotide - MOPS N-morpholino-3-propansulfonic acid - NADH reduced -nicotinamide dinucleotide - nKP n thousand times g pellet - NPA l-naphthylphthalamic acid - PM plasma membrane, plasmalemma - RBF riboflavin - IAA indoleacetic acid - BA benzoic acid     

Purification and partial amino-acid sequence of gibberellin 20-oxidase from Cucurbita maxima L. endosperm

Gibberellin (GA) 20-oxidase was purified to apparent homogeneity from Cucurbita maxima endosperm by fractionated ammonium-sulphate precipitation, gel-filtration chromatography and anion-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC). Average purification after the last step was 55-fold with 3.9% of the activity recovered. The purest single fraction was enriched 101-fold with 0.2% overall recovery. Apparent relative molecular mass of the enzyme was 45 kDa, as determined by gel-filtration HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, indicating that GA 20-oxidase is probably a monomeric enzyme. The purified enzyme degraded on two-dimensional gel electrophoresis, giving two protein spots: a major one corresponding to a molecular mass of 30 kDa and a minor one at 45 kDa. The isoelectric point for both was 5.4. The amino-acid sequences of the amino-terminus of the purified enzyme and of two peptides from a tryptic digest were determined. The purified enzyme catalysed the sequential conversion of [¹⁴C]GA₁₂ to [¹⁴C]GA₁₅, [¹⁴C]GA₂₄ and [¹⁴C]GA₂₅, showing that carbon atom 20 was oxidised to the corresponding alcohol, aldehyde and carboxylic acid in three consecutive reactions. [¹⁴C]Gibberellin A₅₃ was similarly converted to [¹⁴C]GA₄₄, [¹⁴C]GA₁₉, [¹⁴C]GA₁₇ and small amounts of a fourth product, which was preliminarily identified as [¹⁴C]GA₂₀, a C₁₉-gibberellin. All GAs except [¹⁴C]GA₂₀ were identified by combined gas chromatography-mass spectrometry. The cofactor requirements in the absence of dithiothreitol were essentially as in its presence (Lange et. al, Planta 195, 98–107, 1994), except that ascorbate was essential for enzyme activity and the optimal concentration of catalase was lower.     

Phytotropin-binding sites and auxin transport in Cucurbita pepo: evidence for two recognition sites

Two properties of phytotropins, their ability to bind to 1-N-naphthylphthalamic acid (NPA) receptors located on microsomal vesicles isolated from Cucurbita pepo L. hypocotyls, and to stimulate auxin (indol-3-yl acetic acid, IAA) accumulation into such vesicles by blocking its efflux from them, were assessed in double labelling experiments using [2,3,4,5-³H]1-N-naphthylphthalamic acid and 3-indolyl-[2-¹⁴C]acetic acid. Two sites of differing affinities and activities on IAA accumulation were found. 1-N-Naphthylphthalamic acid was found to have high affinity (K_D at 10^-8mol·l^-1) for one site and low affinity (K_D at 10^-6 mol·l^-1) for the other, whereas 2-(1-pyrenoyl)benzoic acid displaced NPA with high efficiency (K_D below 10^-8 mol·l^-1) from both sites. Other phytotropins had intermediate affinities for either site. Occupation of the site with low affinity for NPA stimulated auxin accumulation, while occupation of the high-affinity site with a phytotropin did not interfere with auxin accumulation into vesicles.Abbreviations IAA Indol-3-yl acetic acid - NPA 1-N-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - TIBA 2,3,5-triiodobenzoic acid W.M. was supported in part by an allowance from CSIRO and in part by a fellowship of the Deutsche Forschungsgemeinschaft; he acknowledges the friendly hospitality of the CSIRO Division of Plant Industry. The authors thank R. Hertel (Freiburg) for valuable discussion.     

Immunocytochemical localisation of phloem lectin from Cucurbita maxima using peroxidase and colloidal-gold labels

Antibodies were raised against lectin purified from the sieve-tube exudate of Cucurbita maxima. Immunocytochemistry, using peroxidase-labelled antibodies and Protein A-colloidal gold, was employed to determine the location of the lectin within the tissues and cells of C. maxima and other cucurbit species. The anti-lectin antibodies bound to P-protein aggregates in sieve elements and companion cells, predominantly in the extrafascicular phloem of C. maxima. This may reflect the low rate of translocation in these cells. Under the electron microscope, the lectin was shown to be a component of P-protein filaments and was also found in association with the sieve-tube reticulum which lines the plasmalemma. The anti-lectin antibodies reacted with sieve-tube proteins from other species of the genus Cucurbita but showed only limited reaction with other genera. We suggest that the lectin serves to anchor P-protein filaments and associated proteins to the parietal layer of sieve elements.Abbreviation SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Proton-transport activity,sidedness, and morphometry of tonoplast and plasma-membrane vesicles purified by free-flow electrophoresis from roots of Lepidium sativum L. and hypocotyls of Cucurbita pepo L.

Large-scale preparations of highly purified tonoplast and plasma-membrane vesicles were obtained from roots (garden cress, Lepidium sativum L.) and shoots (etiolated zucchini hypocotyl, Cucurbita pepo L.) of representative dicotyledonous seedlings. When tonoplast-enriched fractions of cress roots were prepared by centrifugation and then subjected to free-flow electrophoresis a highly purified tonoplast fraction was obtained. This fraction from cress roots was characterized by morphometry of filipin-treated freeze-fractured preparations and by enzymology to be about 90% homogeneous. Using latency of nitrate-inhibited ATPase and H⁺-pumping as criteria we found that the majority of the tonoplast vesicles from both sources were oriented right(cytoplasmic)-side-out. Plasma-membrane vesicles were first purified by two-phase partitioning and then subjected to free-flow electrophoresis for further purification. From cress roots, the fraction of highest purity contained 89% plasma-membrane vesicles as judged by morphometry of filipin-treated, freeze-fractured preparations and by enzymology. From both sources, the major plasma-membrane subfraction in the upper phase after two-phase partitioning was shown to have the least electrophoretic mobility in free-flow electrophoresis and to be oriented right(extracytoplasmic)-side-out a slightly more mobile plasma-membrane subfraction was oriented inside-out and originated after freezing thawing from outside-out plasma-membrane vesicles.Part of the doctoral thesis (D5) of B. vom DorpWe thank the Bundesministerium für Forschung und Technologie for financial support.     

Somatic embryogenesis in pumpkin (Cucurbita pepo L.): control of somatic embryo development by nitrogen compounds

Embryogenic cultures of pumpkin (Cucurbita pepo L.) were initiated from mechanically wounded mature zygotic embryos on 2,4-D-containing MS medium, and on hormone-free, semisolid modified MS medium containing NH4Cl as the sole source of nitrogen. The habituated line was derived from the embryogenic tissue induced with 2,4-D and maintained on medium without growth regulators. Sustained subculturing of the three embryogenic lines on a medium with NH4Cl as the sole source of nitrogen enabled the establishment of highly uniform cultures in which no further development into mature embryo stages occurred. The tissue consisting of proembryogenic globules or globular stage embryos was maintained, without decline, for over six years. Globular embryos proceeded to maturity when a combination of reduced (NH4) and unreduced (NO3) forms of nitrogen was provided in the medium. Different nitrogen sources in the medium caused changes of medium pH during subculture in the pH range of 4.0-6.5. The tissue growth and embryo development were blocked on medium with pH adjusted and stabilized at 4.0 or at 3.2.     

Genetics of resistance to red pumpkin beetle (A ulacophora foveicollis) in summer squash (Cucurbita pepo L.)

Summary Resistance to red pumpkin beetle in summer squash was found to be controlled by polygenes. Diallel and Triple test cross analysis revealed the preponderance of non-additive and additive gene effects for resistance respectively. Absence of epistasis for resistance was indicated by both tests.     

The effects of calcium deficiency on Cucurbita pepo L. hypocotyl cells: A 31P nuclear-magnetic-resonance study

Calcium deficiency in zucchini (Cucurbita pepo L.) is associated with reduced growth and a reduced ability to transport auxin (Allan and Rubery, 1991, Planta 183, 604–612). An investigation of the effects of calcium-deficiency on zucchini hypocotyl cells was made using weak-acid uptake and ³¹P-nuclear-magneticresonance (³¹P-NMR) spectroscopy in vivo and in tissue extracts. Calcium-deficient tissue had the same cytoplasmic and vacuolar pHs as normal tissue when extracellular pH was near neutral. At acidic external pH the vacuolar pH was lower in deficient tissue. Adenine nucleotides were present predominantly as ATP in both control and calcium-deficient tissues. Addition of calcium to calcium-deficient tissue, under conditions which cause recovery of auxin transport induced no changes in the ³¹P-NMR spectra of deficient tissue. The content of mobile, phosphorylated metabolites was reduced in calcium-deficient tissue in comparison to control tissue. However, a substantial increase in the content of phosphorylcholine occurs in calcium-deficient tissues compared with controls; this may reflect changes in lipid turnover in calcium-stressed cells. We wish to thank Drs. Terry Moore and Jamie Vandenberg for technical assistance and Professor Peter Morris for providing the gated oxygen device. A.C.A. thanks the Cambridge Commonwealth Trust for a Prince of Wales Scholarship and the O.R.S. Awards Scheme for an award.     

Characterisation of a salt-stimulated ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

Ion stimulation and some other properties of an ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.) have been determined. The ATPase had a specific requirement for Mg²⁺ and in the presence of Mg²⁺ it was stimulated by salts of monovalent cations. The degree of stimulation by monovalent salts was influenced mainly by the anion and the order of effectiveness of the anions tested was Cl^->HCO ₃ ^- >Br^->malate>acetate>SO ₄ ^2- . For any given series of anions the magnitude of the stimulation obtained was influenced by the accompanying cation (NH ₄ ⁺ Na⁺>K⁺). This cation effect was abolished by 0.01% (v/v) Triton X-100 and it is suggested that it is the result of different permeabilities of membrane vesicles to the cations. There was no evidence of synergistic stimulation of the ATPase by mixtures of Na⁺ and K⁺. KCl- and NaCl-stimulation was maximal with salt concentrations in the range 60–150 mM. The true substrate of the enzyme was shown to be MgATP. It was shown that KCl stimulation was the result of an increase in V_max rather than a change in the affinity of the enzyme for MgATP. The ATPase was inhibited by N,N-dicyclohexylcarbodiimide, diethylstilbestrol, mersalyl and KNO₃ but other inhibitors tested (azide, oligomycin, orthovanadate, K₃[Cr(oxalate)₆] and ethyl-3-[3-dimethylaminopropyl]carbodiimide) were without effect or caused only partial inhibition at the highest concentration tested. The ATPase activity was equally distributed between pellet and supernatant fractions obtained after the subfractionation of vacuoles but the properties of the ATPase in each fraction were the same. It is suggested that beet vacuoles possess only one ATPase. The properties of the ATPase are compared with those of ATPases associated with other plant membranes and organelles and its possible role in transport at the tonoplast is discussed.Abbreviations ATP_F free ATP - ATP_T total ATP - BSA bovine serum albumen - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - DNP 2,4-dinitrophenol - EDAC ethyl-3-(3-dimethylaminopropyl)carbodiimide - K_m apparent Michaelis constant - MgATP complex of Mg²⁺ and ATP - Mg _F ²⁺ free Mg²⁺ - Mg _T ² total Mg²⁺ - MES 2-(N-Morpholino)ethanesulphonic acid - Na₂EDTA disodium ethylenediaminetetraacetic acid - NEM N-ethylmaleimide - P_i inorganic phosphate - TCA trichloroacetic acid - Tris tris(hydroxymethyl)methylamine - V_max maximum velocity     

Ammonium-related metabolic changes affect somatic embryogenesis in pumpkin (Cucurbita pepo L.)

Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1 mM ammonium (NH₄⁺) as the sole source of nitrogen. Growth of NH₄⁺-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH₄⁺ medium with 25 mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH₄⁺ induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH₄⁺ as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20 mM) or Gln (10 mM) in combination with NH₄⁺ (1 mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin.     

In vivo and in vitro effects of cadmium on H+ ATPase activity of plasma membrane vesicles from oat (Avena sativa L.) roots

The effect of an in vivo and in vitro treatment with cadmium on transport activities of root plasma membrane enriched vesicles was studied in oat (Avena sativa L. cv. Argentina) plants. Addition of 100 mumol/L CdSO4 to nutrient solution decreases both proton transport activity and ATPase activity to the same level. In vitro experiments show that cadmium seems to have a differential inhibiting effect on proton transport activity and ATPase activity, the most pronounced one on ATP-dependent H(+)-accumulation, suggesting that cadmium would interfere with membrane permeability properties. This is indeed the case. The results demonstrate that cadmium decreases passive permeability to protons.