Chalcone synthases from spinach (Spinacia oleracea L.)

The two chalcone-synthase forms from leaves ofSpinacia oleracea L. were purified to apparent homogeneity. Antibodies were raised against both proteins in rabbits. The specificity of the antibodies was tested using immunotitration, immunoblotting, and immunoelectrophoresis techniques. The antibodies exhibited exclusive specificity for chalcone synthase and did not discriminate between the two antigens. The homodimeric chalcone synthases had the same subunit molecular weight but differed in their apparent native molecular weights. The peptide maps indicated extensive homology between the proteins. Chalcone-synthase activity was not detected in isolated spinach chloroplasts. Both enzyme forms were present in spinach cell-suspension cultures in which they were induced by light.Abbreviations DEAE diethylaminoethyl - DTE 1,4-dithioerythritol - EDTA ethylenediaminetetraacetic acid - HPLC high-performance liquid chromatography - IgG immunoglobulin G - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Parts of the results were presented at the 14^th International Botanical Congress at Berlin in July 1987     

Fractionation analysis and bioavailability of manganese in spinach (Spinacia oleracea L.) leaves

Abstract

This paper introduces a fractionation scheme using water, acetone, chloroform, diethyl ether, ethanol, n-hexane, and methanol as extractants for the determination of manganese in spinach samples by inductively coupled plasma-mass spectrometry (ICP-MS). Simulated gastric and intestinal digestions as well as n-octanol extraction and activated carbon adsorption were performed for the bioavailability assessments. Comparative studies of the various extraction treatments were evaluated for confirmation analysis. The total elemental concentrations were determined after digesting the samples in a microwave digestion system. The method validation parameters were defined in terms of the detection limits, accuracy, and precision. Additional validation was performed by comparing the ICP-MS method with atomic absorption spectrometry. The limits of detection and quantification were 0.046 and 0.154 mg kg^-1, respectively. Additionally, the repeatability and reproducibility, calculated from the relative standard deviation (%RSD), were 2.4% and 3.7%, respectively.     

Embryogenesis and plant regeneration of spinach (Spinacia oleracea L.) from hypocotyl segments

A system for somatic embryogenesis and plant regeneration of spinach from hypocotyl segments has been established. Callus was induced on solid media supplemented with 8.5–15.0 mg.l⁻¹ of indole-3-acetic acid and 3.46–34.64 mg.l⁻¹ gibberellic acid. Callus was then subcultured on different media (solid or liquid) with or without IAA, or continuously maintained on the initiating media. Somatic embryos were obtained in subcultures on IAA-containing media as well as in long-term cultures on initiating media. The best results were achieved in liquid subcultures. About 60% of plantlets survived after transplanting in pots.     

Diurnal variations in growth rate and growth substrate levels of spinach (Spinacia oleracea L.) under nitrogen-limiting conditions

Spinach plants were grown in hydroponic culture provided with variable limiting amounts of N. During a complete diurnal cycle, growth of the root and shoot parts, as well as levels of soluble and insoluble sugars and of free amino acids, were monitored. No clear relationship could be detected between the level of N feeding and the levels of free sugars and amino acids. Analysis of variance revealed that the variances in the relative growth rates of plant root and shoot could be correlated with the levels of sugars and amino acids. Root amino acid concentration could be correlated with shoot amino acid concentration and root sugar concentration. No relationship was found between the variances in root and shoot free sugar concentrations.     

Ecdysteroids in Spinacia oleracea and Chenopodium bonus-henricus

The roots of Chenopodium bonus-henricus and the seeds of Spinacia oleracea contain 20-hydroxyecdysone and polypodine B. The seeds of S. oleracea also contain a compound with properties similar to those of 24(28)-dehydromakisterone-A and may contain small amounts of ecdysone.     

Purification of UDP-galactose: diacylglycerol galactosyltransferase from chloroplast envelopes of spinach (Spinacia oleracea L.)

Uridine 5-diphosphate(UDP)-galactose: 1,2-diacylglycerol 3-O--d-galactopyranosyltransferase (EC 2.4.1.46) is an integral protein of chloroplast envelope membranes from which it has been partially purified (Covès et al., 1986, FEBS Lett. 208, 401–406). We have worked out a purification procedure which after removal of peripheral membrane proteins, solubilization and two chromotographic steps allowed us to identify a 22-kDa protein as the galactosyltransferase. Enrichment of enzymatic activity was paralleled by an enrichment of this protein and its radioactive derivative obtained by photoaffinity labelling with [-^–32P]UDP which is a potent inhibitor of the enzyme. The purification factor of about 350 is substantially higher than achieved previously and indicates that the enzyme represents less than 0.3% of the envelope proteins. The purified enzyme has a Km of 87 M for UDP-galactose with dioleoylglycerol as acceptor and could not be activated by addition of other lipids.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonate - DTE dithioerythritol - MGD monogalactosyl diacylglycerol - PMSF phenylmethanesulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by the Deutsche Forschungsgemeinschaft.     

Production and diurnal utilization of assimilates in leaves of spinach (Spinacia oleracea L.) and barley (Hordeum vulgare L.)

Rates of CO₂ fixation during the light period and the rates of CO₂ release during the night period were measured using mature leaves from 39- to 49-d-old spinach (Spinacia oleracea L., US Hybrid 424; grown in 9 h light, 15 h darkness, daily) and mature leaves from 21-d-old barley (Hordeum vulgare L., cv. Apex; grown in 14 h light, 10 h darkness, daily). At certain times during the light and dark periods leaves were harvested for assay of their contents of soluble carbohydrates, starch, malate and the various amino acids. Evaluation of the results of these measurements shows that in spinach and barley leaves 46% and 26%, respectively, of the carbon assimilated during the light period is deposited in the leaves for export during the night period. Taking into account the carbon consumption in the source leaves by dark respiration, it is evaluated that rates of assimilate export during the light period from spinach and barley leaves [38 and 42 atom C · (mg Chl)^–1 · h^–1] are reduced in the dark period to 16 atom C · (mg Chl)^–1 · h^–1 in both species. The calculated C/N ratios of the photoassimilates exported during the dark period were 0.029 and 0.015 for spinach and barley leaves, respectively.This work was supported by the Deutsche Forschungsgemeinschaft. We thank Dr. Dieter Heineke for stimulating discussions and Mrs. Petra Hoferichter and Mrs. Marita Feldkämper for their technical assistance.     

Interactions between selenium and iodine uptake by spinach (Spinacia oleracea L.) in solution culture

Interactions between Se (as selenate) and I (as iodate) uptake by spinach plants (Spinacia oleracea L.) were studied under controlled conditions using solution culture. Spinach readily accumulated both Se and I in the edible parts, the leaves, with solution-to-leaf transfer factors ranging from 3.5 to 13.4. The distribution coefficients between leaves and roots ranged from 4.07 to 5.66 for I and 4.51 to 8.59 for Se. Selenium concentrations in plant tissues were unaffected by addition of I to the nutrient solution. Similarly, plant I concentrations were unaffected by addition of Se to the nutrient solution, except in nutrient solution with I at a concentration of 50 μM, in which addition of Se lowered shoot I concentrations significantly, but the effect was of low magnitude. These results indicate the possible feasibility of dual supplementation of plant growth substrates with Se and I to improve human nutrition where these two elements are deficient in the diet. The data also indicate the involvement of a positive feedback mechanism in the uptake of Se by spinach plants, since Se concentrations in leaves increased disproportionately with increasing Se concentration in the nutrient solution.     

Nitrate reductases from leaves of Ricinus (Ricinus communis L.) and spinach (Spinacia oleracea L.) have different regulatory properties

The activity of nitrate reductase (+Mg(2+), NR(act)) in illuminated leaves from spinach, barley and pea was 50-80% of the maximum activity (+EDTA, NR(max)). However, NR from leaves of Ricinus communis L. had a 10-fold lower NR(act), while NR(max) was similar to that in spinach leaves. The low NR(act) of Ricinus was independent of day-time and nitrate nutrition, and varied only slightly with leaf age. Possible factors in Ricinus extracts inhibiting NR were not found. NR(act) from Ricinus, unlike the spinach enzyme, was very low at pH 7.6, but much higher at more acidic pH with a distinct maximum at pH 6.5. NR(max) had a broad pH response profile that was similar for the spinach and the Ricinus enzyme. Accordingly, the Mg(2+)-sensitivity of NR from Ricinus was strongly pH-dependent (increasing sensitivity with increasing pH), and as a result, the apparent activation state of NR from a Ricinus extract varied dramatically with pH and Mg(2+)concentration. Following a light-dark transition, NR(act) from Ricinus decreased within 1 h by 40%, but this decrease was paralleled by NR(max). In contrast to the spinach enzyme, Ricinus-NR was hardly inactivated by incubating leaf extracts with ATP plus okadaic acid. A competition analysis with antibodies against the potential 14-3-3 binding site around ser 543 of the spinach enzyme revealed that Ricinus-NR contains the same site. Removal of 14-3-3 proteins from Ricinus-NR by anion exchange chromatography, activated spinach-NR but caused little if any activation of Ricinus-NR. It is suggested that Mg(2+)-inhibition of Ricinus-NR does not require 14-3-3 proteins. The rather slow changes in Ricinus-NR activity upon a light/dark transient may be mainly due to NR synthesis or degradation.     

Sexual modification of female spinach seeds (Spinacia oleracea L.) by irradiation with ion particles

The female seeds of a spinach plant (Spinacia orelacea L.) were exposed to He (12.5 MeV/n) and C (18.3 MeV/n) ions in order to investigate the effects of ion particles on sex expression. He ions did not affect germination rates or flowering at doses up to 50 Gy. C ions did not affect germination rates or flowering at doses up to 15 Gy, but a dose of 25 Gy resulted in many plants with morphological aberrations. When unexposed female plants were grown without cross-fertilization for 10 weeks after sowing, 5.6-14.3% of the plants produced anthers from female flowers. These sex-modified plants could self-pollinate and form seeds, which expressed only female organs. Conversely, gynomonoecious plants were induced from these female seeds by exposure to He ions (5-50 Gy) and C ions (5-25 Gy) without any difference in the rates of flowered progeny. Moreover, andromonoecious plants were induced from female seeds by exposure to He ions at 50 Gy. These results suggest that the sex of a spinach plant is expressed as a flexible phenotype, converging from female to gyno- and andromonoecy after exposure to ion particles.     

An efficient Agrobacterium tumefaciens-mediated transformation and regeneration system for cotyledons of spinach (Spinacia oleracea L.)

An efficient transformation and regeneration system was established for the production of transgenic spinach (Spinacia oleracea L.) plants. Cotyledon explants were infected with Agrobacterium tumefaciens strain LBA4404 carrying the selectable marker gene, neomycin phosphotransferase II (nptII), and the reporter gene smgfp, encoding soluble-modified green-fluorescent protein, driven by the cauliflower mosaic virus 35S promoter. The infected explants were cultured on Murashige and Skoog medium, containing 1 mg/l benzyladenine and 0.4 mg/l naphthaleneacetic acid. Shoots were regenerated on selection medium containing 50 mg/l kanamycin. Regenerated kanamycin-resistant shoots were rooted on medium containing 1 mg/l indolebutyric acid and subsequently grown in soil in the greenhouse. Southern blot analysis indicated that the smgfp gene had been integrated into the spinach genome. Northern and Western blots showed that the smgfp gene was expressed in progeny plants. Received: 31 March 1998 / Revision received: 27 September 1998 / Accepted: 10 Ocotber 1998     

Nitrogen-molybdenum-manganese co-fertilization reduces nitrate accumulation and enhances spinach (Spinacia oleracea L.) yield and its quality

Spinach (Spinacia oleracea L.) is considered a nitrogen (N) intensive plant with high nitrate (NO₃^?) accumulation in its leaves. The current study via a two-year field trial introduced an approach by combining N fertilization from different sources (e.g., ammonium nitrate; 33.5 % N, and urea; 48 % N) at different rates (180, and 360 kg N ha^?1) with the foliar spraying of molybdenum (Mo) as sodium molybdate, and/or manganese (Mn) as manganese sulphate at rates of 50 and 100 mgL^?1 of each or with a mixture of Mo and Mn at rates of 50 and 50 mg L^?1, respectively on growth, chemical constituents, and NO₃^? accumulation in spinach leaves. Our findings revealed that the highest rate of N fertilization (360 kg N ha^?1) significantly increased most of the measured parameters e.g., plant length, fresh and dry weight plant^?1, number of leaves plant^?1, leaf area plant^?1, leaf pigments (chlorophyll a, b and carotenoids), nutrients (N, P, K, Fe, Mn, Zn), total soluble carbohydrates, protein content, net assimilation rate, and NO₃^? accumulation, but decreased leaf area ratio and relative growth rate. Moreover, plants received urea-N fertilizer gave the highest values of all previous attributes when compared with ammonium nitrate –N fertilizers, and the lowest values of NO₃^? accumulation. The co-fertilization of N-Mo-Mn gave the highest values in all studied attributes and the lowest NO₃^? accumulation. The best treatment was recorded under the treatment of 360 kg N-urea ha^?1 in parallel with the combined foliar application of Mo and Mn (50 + 50 mg L^?1). Our findings proposed that the co-fertilization of N-Mo-Mn could enhance spinach yield and its quality, while reducing NO₃^? accumulation in leaves, resulting agronomical, environmental and economic benefits.     

Nodularia spumigena extract induces upregulation of mitochondrial respiratory chain complexes in spinach (Spinacia oleracea L.)

Nodularin, a cyclic hepatotoxic pentapeptide produced by the nitrogen-fixing cyanobacterium Nodularia spumigena, induces oxidative stress in various organisms including higher plants and algae. We have monitored the physiological consequences of N. spumigena AV1 extract exposure on terrestrial plants, specifically focusing on the mitochondrial function of Spinacia oleracea L. Our results show that exposure of the plants to the nodularin-containing extract leads to significantly increased activity of respiratory complex I and citrate synthase, as well as increased accumulation of various subunits of respiratory enzyme complexes. Moreover, upregulation of the stress-induced alternative oxidase as well as the NAD⁺-specific isocitrate dehydrogenase and mitochondrial ascorbate peroxidase was detected in the mitochondria of plants exposed to N. spumigena AV1 extract, while no difference in the carbonylation level of the mitochondrial proteins could be detected between the control and the exposed plants.     

Influence of nitrate concentration at the root surface on yield and nitrate uptake of kohlrabi (Brassica oleracea gongyloides L.) and spinach (Spinacia oleracea L.)

The objective of this study was to determine if plant roots have to take up nitrate at their maximum rate for achieving maximum yield. This was investigated in a flowing-solution system which kept nutrient concentrations at constant levels. Nitrate concentrations were maintained in the range 20 to 1000 μM. Maximum uptake rate for both species was obtained at 100 μM. Concentrations below 100 μM resulted in decreases in uptake rate per cm root (inflow) for both spinach and kohlrabi by 1/3 and 2/3, respectively. However, only with kohlrabi this caused a reduction in N uptake and yield. Thus indicating that this crop has to take up nitrate at the maximum inflow. Spinach, however, compensated for lower inflows by enhancing its root absorbing surface with more and longer roots hairs. Both species increased their root length by 1/3 at low nitrate concentrations.     

Calmodulin-dependent and independent NAD kinase activities from cytoplasmic and chloroplastic fractions of spinach (Spinacia oleracea L.)

NAD kinase activity has been found in a soluble, cytoplasmic fraction and in the chloroplasts prepared from green spinach leaves. A small amount of both the cytoplasmic and the chloroplastic NAD kinase activities was retained on a calmodulin-Sepharose affinity column. The cytoplasmic NAD kinase eluted from the affinity column was found to be enhanced by calmodulin in a Ca²⁺-dependent manner. The chloroplastic enzyme which is located exclusively in the stroma and not in the envelope and thylakoid fractions was not affected by Ca²⁺ and calmodulin. The stromal fraction of purified chloroplasts contained only a negligible amount of calmodulin, most probably due to cytoplasmic contamination. Based on these data, two different mechanisms for the light-dependent modulation of spinach NAD kinase activity are suggested.     

Shoot regeneration from cultured root explants of spinach (Spinacia oleracea L.): a system for Agrobacterium transformation

A reliable plant regeneration system is described for the production of adventitious shoots from root explants of spinach. Explants from roots of axenic shoots and roots induced on cultured hypocotyl explants were used for adventitious shoot induction. Explants from apical, middle and basal root regions were incubated on Nitsch and Nitsch medium supplemented with α-naphthaleneacetic acid, gibberellic acid and kinetin. Optimum shoot regeneration was from explants of apical and middle root regions on medium with 20 μm α-naphthaleneacetic acid and 5.0 μm gibberellic acid. Shoots originated directly from root tissues without an intervening callus phase. Adventitious shoots were rooted and were grown to maturity in the glasshouse. This plant regeneration procedure has been exploited in preliminary studies of Agrobacterium-mediated transformation. Received: 27 February 1996 / Revision received: 22 August 1996 / Accepted: 30 September 1996     

Effects of different sewage sludge applications on heavy metal accumulation,growth and yield of spinach (Spinacia oleracea L.)

In this study, we present the response of spinach to different amendment rates of sewage sludge (0, 10, 20, 30, 40 and 50 g kg^?1) in a greenhouse pot experiment, where plant growth, biomass and heavy metal uptake were measured. The results showed that sewage sludge application increased soil electric conductivity (EC), organic matter, chromium and zinc concentrations and decreased soil pH. All heavy metal concentrations of the sewage sludge were below the permissible limits for land application of sewage sludge recommended by the Council of the European Communities. Biomass and all growth parameters (except the shoot/root ratio) of spinach showed a positive response to sewage sludge applications up to 40 g kg^?1 compared to the control soil. Increasing the sewage sludge amendment rate caused an increase in all heavy metal concentrations (except lead) in spinach root and shoot. However, all heavy metal concentrations (except chromium and iron) were in the normal range and did not reach the phytotoxic levels. The spinach was characterized by a bioaccumulation factor <1.0 for all heavy metals. The translocation factor (TF) varied among the heavy metals as well as among the sewage sludge amendment rates. Spinach translocation mechanisms clearly restricted heavy metal transport to the edible parts (shoot) because the TFs for all heavy metals (except zinc) were <1.0. In conclusion, sewage sludge used in the present study can be considered for use as a fertilizer in spinach production systems in Saudi Arabia, and the results can serve as a management method for sewage sludge.     

Availability of iodide and iodate to spinach (Spinacia oleracea L.) in relation to total iodine in soil solution

A greenhouse pot experiment was carried out to investigate the availability of iodide and iodate to soil-grown spinach (Spinacia oleracea L.) in relation to total iodine concentration in soil solution. Four iodine concentrations (0, 0.5, 1, 2 mg kg⁻¹) for iodide (I⁻) and iodate (IO₃⁻) were used. Results showed that the biomass productions of spinach were not significantly affected by the addition of iodate and iodide to the soil, and that iodine concentrations in spinach plants on the basis of fresh weights increased with increasing addition of iodine. Iodine concentrations in tissues were much greater for plants grown with iodate than with iodide. In contrast to the iodide treatments, in iodate treatment leaves accounted for a larger fraction of the total plant iodine. The soil-to-leaf transfer factors (TF_leaf) for plants grown with iodate were about tenfold higher than those grown with iodide. Iodine concentrations in soil solution increased with increasing iodine additions to the soil irrespective of iodine species. However, total iodine in soil solution was generally higher for iodate treatments than iodide both in pots with and without spinach. According to these results, iodate can be considered as potential iodine fertilizer to increase iodine content in vegetables.     

Rapid sex identification method of spinach (Spinacia oleracea L.) in the vegetative stage using loop-mediated isothermal amplification

Planta - A LAMP-mediated, simple and rapid method for sex identification in spinach was developed. Nutrient compositional analysis showed a higher iron content in male than female plants. Spinach...