Development of a cell-free translation system from Cucumis melo: preparation, optimization and evaluation of sensitivity to some translational inhibitors

A very active cell-free translation system was prepared from 4–5-day-old embryonic axes of melon (Cucumis melo L.), a species whose dry seeds contain a powerful translational inhibitor. The system was optimized for Mg²⁺, K⁺, NH₄⁺, high speed supernatant, total wheat germ tRNA, time and temperature. Using a 30 000 × g supernatant, the system translates endogenous messengers and polyuridylic acid very efficiently. Melon ribosomes were inhibited in vitro by several well-known eukaryotic inhibitors including melonin, the protein inhibitor present in the dry seeds of C. melo. Our results suggest that the protein inhibitor does not affect the activity of melon ribosomes neither in vivo nor during their isolation.     

Uptake and translocation of calcium in cucumber

Uptake and translocation of Ca²⁺(⁴⁵Ca) were compared with water translocation in 12-day old intact plants and excised roots of cucumber ( Cucumis sativus L. var. Cilla), which had been cultivated in nutrient solution. No immediate reduction of Ca²⁺ uptake was found when water translocation was reduced by excision of the shoot. In the presence of 2,4-dinitrophenol Ca²⁺ translocation was reduced in the intact plants while water translocation was unchanged. It is suggested that regulation of Ca²⁺ uptake is primarily achieved in the root. The DNP-sensitive mechanism of Ca²⁺ uptake was associated with the root and probably represented transport through the endodermis into the stele.     

Uptake of calcium in wheat and cucumber roots

Uptake of Ca²⁺(⁴⁵Ca) was investigated in plants of wheat ( Triticum aestivum L. var. Svenno) and cucumber ( Cucumis sativus L. var. Cilla) cultivated in a nutrient solution with various Ca²⁺ concentrations. The adsorption of Ca²⁺ was higher in cucumber roots than in wheat roots especially at lower Ca²⁺ levels in the external medium. The intracellular fraction of Ca²⁺ was less than 20% of the total Ca²⁺ in wheat roots and less than 10% of the total Ca²⁺ in cucumber roots. The uptake of Ca²⁺ in cucumber was about 40 times higher than in wheat. Transport of Ca²⁺ in the roots towards the endodermis is suggested to take place mainly in the apoplastic pathway regulated by the availability of negatively charged binding sites along the cell wall continuum. Further transport of Ca²⁺ towards the stele may involve diffusion of Ca²⁺ into the symplasm in the vicinity of the endodermis. An active extrusion of Ca²⁺ towards the stele or towards the external medium is suggested to play a role in the regulation of Ca²⁺ uptake.     

Uptake and distribution of calcium, magnesium and potassium in cucumber of different age

Uptake and distribution of Ca⁺, Mg²⁺ and K²⁺ were investigated in plants of cucumber ( Cucumis sativus L. var. Cila) which had been cultivated for 12, 19, 32, or 53 days in complete nutrient solution with 1.0 m M Ca²⁺, 2.0 m M Mg²⁺ and 2.0 m M K⁺. The ⁺ concentration was about the same in roots and shoots, while the Ca²⁺ and Mg²⁺ concentrations were low in roots compared to shoots. The K⁺ concentration decreased with increasing leaf age, while the Ca²⁺ and Mg²⁺ concentrations increased, except in older plants with flowers and fruits, where an increased concentration was found in the youngest leaves. This is discussed in connection with increased indoleacetic acid (IAA) synthesis in the shoot. Excision of leaves at different levels from 21-day-old plants, followed by uptake for 24 h from the nutrient solution on days 22 and 23, resulted in no immediate reduction in Ca²⁺ (⁴⁵Ca) uptake. Transport of Ca²⁺ increased to leaves above and below the excision point and total Ca²⁺ uptake remained at the same level as for the intact plant. It is suggested that regulation of Ca²⁺ uptake is primarily achieved in the root while the distribution in the shoot is regulated by the accessability of negative binding sites.     

Effects of sulfate and nitrate on K+ uptake and growth of wheat and cucumber

The uptake of K⁺ ion was studied in the roots of wheat ( Triuicum aestivum L. cv. GK Szeged) and cucumber ( Cucumis sativus L. cv. Budai csemege) seedlings grown in nutrient solution under nitrogen and sulfate stress conditions. Seedlings pretreated with 1 or 10 m M NaNO₃, absorbed more K⁺ than those treated with 0.1 m M NaNO₃. However, the posteffect of NaNO₃ was considerably influenced by the Na₂SO₄, treatment. The results suggest that, at least partly, a feed-back regulation of K⁺ uptake may occur. However, due to the high Na⁺ contents of the roots, a Na⁺ effect in this process cannot be excluded. The growth and dry matter yields of the roots and shoots were strongly influenced by the SO²⁻/₄ and NO⁻/₃ supply of the plants. Appreciable differences were experienced between wheat and cucumber seedlings. The optimum SO²⁻/₄ concentration of the growth solution for maximal growth varied considerably between the species, and was also different for the roots and the shoots in a given species.     

Plasma membrane-bound H+-ATPase and reductase activities in Fe-deficient cucumber roots

Physiological and biochemical modifications induced by Fe-deficiency have been studied in cucumber ( Cucumis sativus L. cv. Marketer) roots, a Strategy I plant that initiates a rapid acidification of the medium and an increase in the electric potential difference when grown under Fe-deficiency. Using the aqueous two-phase partitioning method, a membrane fraction which has the plasmalemma characteristics was purified from roots of plants grown in the absence and in the presence of iron. The plasma membrane vesicles prepared from Fe-deficient plants showed an H⁺-ATPase activity (EC 3.6.1.35) that is twice that of the non-deficient control. Furthermore, membranes from Fe-deficient plants showed a higher capacity to reduce Fe³⁺-chelates. The difference observed in the reductase activity was small with ferricyanide (only 30%) but was much greater with Fe³-EDTA and Fe³-citrate (210 and 250%, respectively). NADH was the preferred electron donor for the reduction of Fe³⁺ compounds. Fe³⁺ reduction in plasma membrane from cucumber roots seems to occur with utilisation of superoxide anion, since addition of superoxide dismutase (SOD; EC 1.15.1.1) "in vitro" decreased Fe³⁺ reduction by 60%.
The response and the difference induced by iron starvation on these two plasma membrane activities together with a possible involvement of O₂ in controlling the Fe³⁺/Fe²⁺ ratio in the rhizosphere are discussed.     

NADP+-isocitrate dehydrogenase in germinating cucumber cotyledons: Purification and characterization of a cytosolic isoenzyme

The activity of NADP⁺-dependent isocitrate dehydrogenase (ICDH, EC 1.1.1.42) was investigated during the post-germinative growth of cucumber ( Cucumis sativus L. cv. Marketmore) seedlings. Isoelectric focusing showed the presence of several isoenzymes, two of which represented 70–80% of the total NADP⁺-ICDH activity in cotyledons of seedlings grown in the dark. They had pI values between 4.8 and 5.8. The isoenzyme with higher pI was purified to homogeneity by hydrophobic interaction, affinity, hydroxylapatite and anion exchange chromatography. The purified isoenzyme is a dimeric protein, consisting of two apparently identical 43-kDa subunits. It is specific for NADP⁺, inhibited by ATP and by 2-oxoglutarate, whereas it is not inhibited by citrate, succinate, and glyoxylate. The data indicate that NADP⁺-ICDH from cucumber is structurally similar to ICDHs from other plants, but it shows some peculiar biochemical characteristics.     

Partial purification and some biochemical properties of acid phosphatase in germinating chickpea (Cicer arietinum) seeds

An acid phosphatase (EC 3.1.3.2.) from the embryonic axes of chickpea seeds ( Cicer arietinum L. cv. Castellana) was purified by ammonium sulphate precipitation, chromatography on Sephacryl S-200 and polyacrylamide gel electrophoresis. The preparation has an apparent molecular weight of 39 kDa, pH optimum for p -nitrophenylphosphate hydrolysis of 5.25, and K _m of 0.57 m M . The enzyme hydrolyzed all the mono- and di-phosphorylated sugars tested, but had no effect on ATP, ADP, AMP and phosphoenolpyruvate. Phosphate was a competitive inhibitor. Mg²⁺. Ca²⁺, Hg²⁺, Fe³⁺, arsenate, K⁺ and Zn²⁺ were inhibitory. Mn²⁺, dithiothreitol and EDTA had no effect, and polyamines were activators.     

Plasmalemma from the roots of cucumber: Isolation by two-phase partitioning and characterization

Plasmalemma was isolated from the roots of 2-week-old cucumber plants ( Cucumis sativus L. cv. Rhensk druv) by utilizing an aqueous polymer two-phase system with 6.5%:6.5% (w/w) Dextran T500 and polyethylene glycol (PEG) 3350 at pH 7.8. The plasmalemma fraction comprised ca 6% of the membrane proteins contained in the microsomal fraction. The specific activity of the plasma membrane marker enzyme (K⁺, Mg²⁺-ATPase) was 14- to 17-times higher in the upper (PEG-rich) than in the lower (Dextran-rich) phase, and the reverse was true for marker enzymes (cytochrome c oxidase, EC 1.9.3.1, and antimycin A-resistant NADPH cytochrome c reductase) of intracellular membranes. The ATPase was highly stimulated by the addition of detergent (Triton X-100), so that the isolated plasmalemma vesicles appear tightly sealed and in a right-side-out orientation. Further characterization of the ATPase activities showed a pH optimum at 6.0 in the presence of Mg²⁺. This optimum was shifted to pH 5.8 after addition of K⁺. K⁺ stimulated the ATPase activity below pH 6 and inhibited above pH 6. The ATPase activity was specific for ATP and sensitive to N,N-dicyclohexylcarbodiimide and sodium vanadate, with K⁺ enhancing the vanadate inhibition. The enzyme was insensitive to sodium molybdate, NO⁻₃, azide and oligomycin. No Ca²⁺-ATPase was detected, and even as little as 0.05 m M Ca²⁺ inhibited the Mg²⁺-ATPase activity.     

Effect of nonanoic acid and other short chain fatty acids on exchange properties in embryonic axes of Cicer arietinum during germination

Nonanoic acid, which inhibits germination in several seeds, enhanced ion efflux from embryonic axes of Cicer arietinum L., especially at temperatures above 25°C. Other short chain fatty acids had little effect on germination and ion leakage. Nonanoic acid also decreased uptake of ⁸⁶Rb⁺ and ²²Na⁺ and increased efflux of both isotopes from the embryonic axes into the incubation solution. Fusicoccin, which stimulates early germination in C. arietinum , counteracted the effects of nonanoic acid at both 25 and 30°C. These results suggest that nonanoic acid affects the integrity of plasmalemma and other membrane systems. Nonanoic acid thus inhibits cell elongation during early germination by disturbing ion exchange and inhibiting water uptake.     

Vicia sativa L. 'Run-off' and Purified Ribosomes: Polyphenylalanine Synthesis and Molecular Action of Ribosome-inactivating Proteins

A ‘run-off’ cell-free translation system (a 30000x g supernatant; S 30) has been prepared from 4–5-d-oldembryonic axes of Vicia sativa L., a plant lacking ribosome-inactivatingprotein activities which is very sensitive to certain RIPs butnot to others. The system was able to generate a high rate ofpolyphenylalanine synthesis upon addition of polyuridylic acid.From this supernatant, purified ribosomes and a 100000 x g supernatantwere prepared which were able to perform polyphenylalanine synthesiswhen mixed together (reconstituted system). The most importanttranslation parameters were optimized in each case. Both theS 30 and the reconstituted system displayed differential sensitivitiesto certain RIPs. The purified RIP-inactivated ribosomes wereable to release a 370 nucleotide rRNA fragment diagnostic forRIPs upon treatment of the isolated rRNA with acid aniline. Key words: Translation, polyphenylalanine synthesis, ribosomes, ribosome-inactivating proteins, Vicia sativa     

Does calcium ameliorate the negative effect of NaCl on melon root water transport by regulating aquaporin activity?

The hydraulic conductance ( L ₀) of detached, exuding root systems from melon ( Cucumis melo cv. Amarillo oro) was measured. All plants received a half-strength Hoagland nutrient solution, and plants stressed either solely with NaCl (50 mM) or with NaCl (50 mM) following treatment (2 d) with CaCl₂ (10 mM) were compared with controls and CaCl₂-treated (10 mM) plants. The L ₀ of NaCl-treated plants was markedly decreased when compared to control and CaCl₂-treated plants, but the decrease was smaller when NaCl was added to plants previously treated with CaCl₂. A similar effect was observed when the flux of Ca²⁺ into the xylem and the Ca²⁺ concentration in the plasma membrane of the root cells were determined. In control, CaCl₂- and NaCl + CaCl₂-treated plants, HgCl₂ treatment (50 μM) caused a sharp decline in L ₀ to values similar to those of NaCl-stressed roots, but L ₀ was restored by treatment with 5 mM DTT. However, in NaCl roots only a slight effect of Hg²⁺ and DTT were observed. The effect of all treatments on L ₀ was similar to that on osmotic water permeability ( P _f) of individual protoplasts isolated from roots. The results suggest that NaCl decreased the passage of water through the membrane and roots by reducing the activity of Hg-sensitive water channels. The ameliorative effect of Ca²⁺ on NaCl stress could be related to water-channel function.     

Cell-Free Protein Synthesis by Rumen Protozoa

SYNOPSIS. The characteristics of protein synthesis by cell-free extracts of mixed rumen protozoa have been investigated. ATP,¹ GTP, and an energy supply system were necessary for amino acid incorporation which was partially inhibited by cycloheximide but not by chloramphenicol (100 μg/ml). The system was particularly sensitive to the cation concentration of the incubation mixture, maximal incorporation requiring 5 mM Mg⁺⁺ and 50 mM K⁺ Incorporation was further stimulated by the addition of 0.25 mM spermidine or 0.25 mM MnCl₂. Sucrose gradient centrifugation of the cell sap after amino add incorporation showed that most of the incorporated radioactivity was associated with free polysomes. These polysomes contained 82 S ribosomes which dissociated in high Tris concentrations to yield 40 S and 55 S ribosomes.     

Role of the testa epidermis in the leakage of intracellular substances from imbibing soybean seeds and its implications for seedling survival

The generation of ethylene from 1-aminocyclopropane-1-carboxylic acid (ACC) added to a cell-free preparation from etiolated pea ( Pisum sativum L. cv. Alaska) epicotyls was found not to be due to a specific ACC oxidase or to oxygen radicals. Rather, endogenously produced H₂O and manganese ions are coupled in a reaction sequence which produces ethylene from ACC. In a model system, H₂O and Mn²⁺ converted ACC to ethylene under conditions similar to those in the pea preparation. Ultrafiltration of the pea preparation inhibited ethylene production, but it could be reconstituted either by adding an H₂O₂-generating system to the ultrafiltrate or Mn²⁺ to the retentate. H₂O₂-generating systems could reconstitute ethylene formation in a heat-inactivated cell-free sample while the loss of ability to produce ethylene upon dialysis of the pea preparation correlated with the loss of Mn²⁺ from the sample. Studies using cell-free preparations to investigate ethylene synthesis should take care to exclude the possible involvement of H₂O₂ and Mn^2+.     

Light modulation and in vitro effects of adenine nucleotides on leaf nitrate reductase activity in cucumber (Cucumis sativus)

Nitrate reductase (NR, EC 1.6.6.1) activity in attached cucumber ( Cucumis sativus L. cv. Ashley) leaves changed rapidly and reversibly during light/dark transitions, especially when assayed in the presence of free Mg²⁺. Light decreased and darkness increased the sensitivity of the enzyme to inhibition by Mg²⁺. The NR activation state, i.e. activity in the presence of Mg²⁺ relative to activity in the absence of Mg²⁺, increased with light intensity up to 400 μmol m⁻² s⁻¹ PAR (photosynthetically active radiation). When a desalted crude extract from illuminated leaves was preincubated with ATP, NR was gradually inactivated. Inactivation was only observed when activity was assayed in the presence of Mg²⁺. The ATP-inactivated NR remained inactive after removing the excess of ATP by gel filtration and it did not occur in partially purified NR preparations. NR extracted from darkened attached leaves was markedly activated when preincubated with 5'-AMP. These results support the view that inactivation/activation of cucumber-leaf NR in response to light/dark signals most likely involves phosphorylation/dephosphorylation of the enzyme catalysed by endogenous proteins. A substantial activation of NR by preincubation with 5'-AMP was also observed when activity was assayed in the absence of Mg²⁺, thus indicating that 5'-AMP can directly activate NR. Irradiation of an extract from darkened leaves containing FAD promoted a partial activation of NR. This effect was observed both in the +Mg²⁺ and in the −Mg²⁺ assay, indicating that activation was caused by photoexcited flavin and did not involve dephosphorylation of the enzyme.     

Partial purification and some biochemical properties of nonspecific alkaline phosphatase in germinating chick-pea (Cicer arietinum) seeds

A procedure for the partial purification of a non-specific alkaline phosphatase (EC 3.1.3.1.) from the embryonic axes of chick-pea seeds is described. Ammonium sulphate precipitation, DEAE-cellulase chromatography, Sephacryl S-200 chroma-tography and polyacrylamide gel electrophoresis are the most important steps. The molecular weight of this non-specific enzyme, as determined by Sephacryl S–200 gel filtration and SDS–polyacrylamide gel electrophoresis, was estimated as being 68 and 78 kDa respectively; the optimum pH for p-nitrophenylphosphate hydrolysis was 7.5, and the K_m for this artificial substrate was 0.5 mM. The enzyme catalyzes the hydrolysis of a variety of organic phosphate esters. The best substrates are: phos-phoenolpymvate (K_m= 2.4 m M ), NADP⁺ (K_m= 4.0 m M ), 5'-AMP (K_m= 4.5 m M ), 5'-ADP (K_m= 6.1 m M ) and ribose-5P (K_m= 5.8 m M ); but it is unable to hydrolyze 5'-ATP, phosphocreatine and tripolyphosptiate. Phospate was a competitive inhibitor. Zn²⁺, K⁺, Hg²⁺ and Mo⁶⁺ were strong inhibitors, whereas F⁻ and Ca²⁺ inhibited weakly; Co²⁺ and Ni²⁺ were activators.     

Ca2+ release from intracellular stores by thapsigargin in sea urchin eggs: Relationship to larval development and relevance in egg activation

Thapsigargin (Tg), an inhibitor of microsomal Ca²⁺ ATPase, is used as a tool to study the changes in Ca²⁺ sequestration in sea urchin eggs and their relationship to embryonic development. Micromolar amounts of Tg inhibit ATP-dependent Ca²⁺ sequestration in a dose-dependent and non-reversible manner, depending on the bulk of biological material used. IC_5O values are 1 nmol/L and 1–10μmol/L, respectively, in the cortical Ca²⁺ stores (isolated cortices preparation) and in digitonin-permeabilized eggs, a preparation giving access to the deeper reticulum compartment. Micromolar Tg does not induce Ca²⁺ release from ⁴⁵Ca pre-loaded cortices but leads to a loss of 25% of the total Ca²⁺ content from the cortical area. Using microspectrofluorimetry of fura-2-loaded eggs, we found that 10 μmol/L Tg induced a moderate rise in cytosolic Ca²⁺ activity as compared with the fertilization-induced Ca²⁺ transient whether eggs were fertilized or not. Early events related to fertilization as, for example, elevation of the fertilization envelope, proton excretion and sustained increase of amino acid uptake, are triggered by 10μmol/L Tg but with a delayed onset relative to sperm-induced effects. The present findings indicate that although it triggers most fertilization-related events, Tg cannot be considered as a true mitotic agent in sea urchin eggs. When added after fertilization, Tg affects cleavage and the further embryonic development giving rise to abnormalities comparable to the animalized larvae obtained with other compounds responsible for the inhibition of reticular Ca²⁺ sequestration.     

5'-Methylthioadenosine nucleosidase and 5-methylthioribose kinase activities in relation to stress-induced ethylene biosynthesis

The activities of 5'-methylthioadenosine (MTA) nucleosidase (EC 2.2.2.28) and 5-methylthioribose (MTR) kinase (EC 2.7.1.100) were related to changes in ethylene biosynthesis in tomato ( Lycopersicon esculentum Mill. cv. Rutgers) and cucumber ( Cucumis sativus Mill. cv. Poinsett 76) fruit following wounding and chemically induced stresses. Stress ethylene formation in wounded tomato and cucumber tissue continued to increase after wounding, reached its peak by 3h, and then declined. The activities of MTA nucleosidase and MTR kinase increased parallel to stress ethylene in both tissues. At peak ethylene formation, MTA and MTR kinase activities were 2- to 4-fold higher in wounded than in intact tissue. Wounded, mature-green tomato tissue treated with specific inhibitors of MTA nucleosidase and MTR kinase showed a significant reduction in the activities of these enzymes, which was concomitant with a decline in stress ethylene biosynthesis. When mature-green tomato discs were infiltrated with [¹⁴CH₃] MTA and wounded, radioactive MTR and methionine were formed. Incubation of mature-green tomato discs with Cu²⁺ and Li⁺ in the presence of kinetin increased ethylene biosynthesis. MTA nucleosidase activity was higher than that of the control in the presence of Cu²⁺ but not in the presence of Li⁺, while MTR kinase activity was lower than that of the control in both Cu²⁺ and Li⁺ treatments. Data indicate that MTA nucleosidase and MTR kinase are required for wound-induced ethylene biosynthesis but not for chemical stress-induced ethylene by Cu²⁺ or Li⁺ treatments.     

Patterns of ammonium absorption and acetylene reduction during soybean developmental growth

Nodulated and unnodulated soybean plants ( Glycine max (L.) Merr. cv. Amsoy 71) were grown in nutrient solution either lacking or containing N. Nodulated plants, dependent on N₂ fixation, exhibited a generalized N-stress and were less vigorous than unnodulated plants dependent on inorganic N assimilation.
Starting at preflowering throughout mid pod-filling, NH₄⁺ absorption, expressed on the basis of root dry weight, was determined for intact nodulated and unnodulated plants in short-term kinetic experiments. Depletion of NH₄⁺ was measured from the liquid phase of a mist chamber. Maximum NH₄⁺ absorption occurred for both nodulated and unnodulated plants during vegetative growth. A pattern of progressive decrease in NH₄⁺ absorption was similar in nodulated and unnodulated plants, however. NH₄⁺ absorption was consistently greater in unnodulated plants. Simultaneous measurements of C₂H₂ reduction from the gas phase of the mist chamber revealed and 41-day-old plants, corresponding to late flowering and early pod-filling.     

Effect of thiourea on the ionic content and dark fixation of CO2 in embryonic axes of Cicer arietinum seeds

During the first 12 h of germination of Cicer arietinum L. (cv. Castellans) seeds, K⁺ is first lost into the surrounding medium and is later reabsorbed. Thiourea accelerates this reabsorption. Since there is an increase in the mobilization of K⁺ in response to thiourea, a greater accumulation of malate due to the carboxylation of phosphoenolpyruvic acid takes place as compared with that occurring in water. The subapical zone of the radicle accumulates the greatest amounts of water, K⁺ and malate. The variation in the "in vitro" activity of phosphoenolpyruvate carboxylase does not explain the difference in malate in response to the different treatments, consequently there must be chemical changes in the cytoplasm which favour this carboxylation "in vivo". These results show that thiourea accelerates the mobilization of K⁺ and stimulates the dark fixation of CO₂ in embryonic axes of Cicer arietinum.