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.     

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.     

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.     

The reactivation of nitrate reductase from spinach (Spinacea oleracea L.) inactivated by NADH and cyanide: effects of peroxidase and associated systems

Nitrate reductase of spinach (Spinacea oleracea L.) leaves which had been inactivated in vitro by treatment with NADH and cyanide, was reactivated by incubation with oxidant systems and measured as FMNH₂-dependent activity. Ferricyanide, a purely chemical oxidant, produced rapid maximal reactivation (100%) which was 90% complete in less than 3 min. Reactivation occurred slowly and less completely (30–75% in 30 or 60 min) when the enzyme was incubated with pure horseradish peroxidase alone, depending on using one or 20 units and time. Addition of glucose and glucose oxidase to generate hydrogen peroxide increased reactivation slightly (10–15%) with 20 units of peroxidase but more (30–50%) with one unit and to 75–90% of ferricyanide values. Adding catalase decreased reactivation by more than half either with or without glucose oxidase. Glucose and glucose oxidase alone did not cause reactivation. Addition of superoxide dismutase increased reactivation from 50–75% of ferricyanide values with one unit of peroxidase alone but had no effect on greater reactivation obtained in the presence of glucose oxidase. The addition of p-cresol and manganese together increased reactivation with one unit of peroxidase and in the presence of glucose oxidase by about double, but omission of manganese had no effect. However, as shown previously, although trivalent manganese was formed, the residual presence of manganous ions inhibited reactivation. Nevertheless, peroxidase systems either alone or with additionally generated hydrogen peroxide can induce substantial reactivation of nitrate reductase in physiologically relevant conditions.Abbreviations EDTA ethylenediaminetetraacetic acid - FMN flavine mononucleotide     

Multiplicity of soluble glucan-synthase activity in spinach leaves: Enzyme pattern and intracellular location

Buffer-extractable proteins from leaves of Spinacia oleracea L. were separated by non-denaturing polyacrylamide gel electrophoresis. Gels were stained for adenosine diphosphoglucose (ADPglucose)-dependent glucan-synthase (GS) activity (EC 2.4.1.21). Three major forms of activity were observed. No staining was detectable when ADPglucose was replaced by an equimolar concentration of either uridine, guanosine or cytosine diphosphoglucose. Two of the three GS forms exhibited both primed and citrate-stimulated unprimed activity whereas one enzyme form was strictly dependent upon the presence of an exogenous glucan. For intracellular localization, mesophyll protoplasts and intact chloroplasts were isolated and their enzyme pattern was compared with that of the leaf extract. Intactness and purity of the chloroplast preparations were ascertained by polarographic measurement of the ferricyanide- or CO₂-dependent oxygen evolution, by determination of marker-enzyme activities, and by electrophoretic evaluation of the content of chloroplast- and cytosol-specific glucanphosphorylase forms (EC 2.4.1.1). The three GS forms were present in mesophyll protoplasts. Intact chloroplasts possessed both primer-independent enzyme forms but lacked the primer-dependent one. The latter form was enriched in supernatant fractions of leaf homogenates when the intact chloroplasts had been pelleted by centrifugation. Thus, in spinach-leaf mesophyll cells soluble ADPglucose-dependent GS is located both inside and outside the chloroplast.Abbreviations GS glucan synthase - PAGE polyacrylamide gel electrophoresis This work has been made possible by grants from the Deutsche Forschungsgemeinschaft and from the Minister für Wissenschaft und Forschung des Landes Nordrhein-Westfalen. The authors gratefully acknowledge the generous permission to use the laser densitometer of Professor Dr. W. Barz (Biochemie der Pflanzen, Universität Münster, FRG). They are indebted to Dr. H.-J. Witt (Pflanzenphysiologie, Universität Kassel, FRG) for helpful discussions and to Mr. W. Lamkemeyer for skilfull technical assistance.     

The reactivation of nitrate reductase from spinach (Spinacia oleracea L.) inactivated by NADH and cyanide,using trivalent manganese either generated by illuminated chloroplasts or as manganipyrophosphate

Nitrate reductase of spinach (Spinacia oleracea L.) leaves which had been inactivated in vitro by treatment with NADH and cyanide, was reactivated by incubation with oxidant systems and measured as FMNH₂-dependent activity. Reactivation was produced with trivalent manganese compounds represented either by manganipyrophosphate or produced by oxidation of Mn²⁺ ions in the presence of illuminated chloroplasts and compared with reactivation obtained with ferricyanide. Reactivation in the chloroplast system was equivalent to that with ferricyanide when orthophosphate was used but was variable and weak in the presence of pyrophosphate, although manganipyrophosphate was formed, freely. Reactivation by manganipyrophosphate in dark reaction conditions was less effective than with ferricyanide but was not inhibited by the addition of pyrophosphate. Reactivation with illuminated unheated chloroplasts was dependent on added manganese and oxidation of manganese in the presence of pyrophosphate was abolished by boiling the chloroplasts. In the presence of orthophosphate however, boiled, illuminated chloroplasts reactivated the enzyme in the absence of added manganese. Reactivation occurred spontaneously in air, more slowly than with the other oxidants, but to a similar extent to that produced by manganipyrophosphate. The results provide a possible model for physiological reactivation mechanisms.     

Elicitor-induced metabolic changes in cell cultures of chickpea (Cicer arietinum L.) cultivars resistant and susceptible to Ascochyta rabiei

Cell-suspension cultures of two chickpea (Cicer arietinum L.) cultivars, resistant (ILC 3279) and susceptible (ILC 1929) to the fungus Ascochyta rabiei (Pass.) Lab., showed differential accumulation of the phytoalexins medicarpin and maackiain, and transient induction of related enzyme activities after application of an A. rabiei-derived elicitor. The chalcone-synthase (CHS) activity (EC 2.3.1.74) which is involved in the first part of phytoalexin biosynthesis exhibited a maximum 8–12 h after elicitation in the cells of both cultivars. Concomitant with the fivefold-higher phytoalexin accumulation, CHS activity increased twofold in the cells of the resistant cultivar. The maximum of the elicitor-induced CHS-mRNA activity was determined 4 h after onset of induction in the cultures of both cultivars, although in cells of cultivar ILC 3279 this mRNA activity was induced at a level twofold higher than that in cells of the susceptible race ILC 1929. Investigations of CHS isoenzymes by two-dimensional gel electrophoresis of immunoprecipitated in-vitro-translated protein indicated the presence of five proteins. In the cells of both cultivars only two of the isoenzymes were induced after elicitor treatment. Analysis of the total in-vitro-translated proteins by two-dimensional gel electrophoresis showed that the constitutively expressed patterns of mRNA activities in the cell cultures of the two cultivars were identical. After elicitation, considerably more translatable mRNAs were induced in the cells of cultivar ILC 3279. The few induced proteins, and their respective mRNA activities, which could be detected in the cells of the susceptible cultivar, all existed in the cells of the resistant cultivar, too. One highly induced protein (M_r 18 kDa) found in the cells of cultivar ILC 3279 reached its maximum mRNA activity 6 h after elicitor application. The amount of this protein was hardly increased in the cells of the susceptible cultivar. This protein appears to be excreted from the cells into the growth medium.Abbreviations CHS chalcone synthase - IEF isoelectric focussing - ILC international legume chickpea - PR-protein pathogenesis-related protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. The authors thank Dr. K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for provision of antisera and the International Centre for Agricultural Research in the Dry Areas (Aleppo, Syria) for plant material.     

Resolution of two molecular forms of sucrose-phosphate synthase from maize,soybean and spinach leaves

Two forms of sucrose-phosphate synthase (EC 2.4.1.14) were resolved from leaves of three species, maize (Zea mays L. cv. Pioneer 3184), soybean (Glycine max (L.) Merr., cv. Ransom) and spinach (Spinacia oleracea L. cv. Resistoflay) by hydroxyapatite Ultrogel chromatography, using a 75-mM (designated peak 1) and 250-mM (peak 2) K-phosphate discontinuous-gradient elution. Rechromatography of the two forms showed that they were not readily interconvertible. The distribution of activity between the two forms differed among species and changed during purification of the enzyme. Recovery of peak-1 activity was specifically lowered when maize leaf extracts were prepared in the absence of magnesium, indicating that the two forms may differ in stability. In addition, the forms of the enzyme from maize differed in the extent of glucose-6-phosphate activation. These results provide evidence for the existence of multiple forms of sucrose-phosphate synthase in leaves of different species and that the forms differ in regulatory properties.Abbreviations Fru6P fructose 6-phosphate - Glc6P glucose 6-phosphate - HAU hydroxyapatite Ultrogel - Pi inorganic phosphate - SPS sucrose-phosphate synthase - UDP uridine 5-diphosphate - UDPG uridinediphosphate glucose Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh. Paper No. 10511 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh. Supported in part by USDA Competitive Research Grant No. 85-CRCR-1-1568     

Chalcone synthase in rice (Oryza sativa L.): Detection of the CHS protein in seedlings and molecular mapping of the chs locus

The chalcone synthase is a key enzyme that catalyses the first dedicated reaction of the flavonoid pathway in higher plants. The chs gene and its protein product in rice has been investigated. The presence of a chalcone synthase (CHS) protein in rice seedlings and its developmental stage-specific expression has been demonstrated by western analysis. The chalcone synthase of rice was found to be immunologically similar to that of maize. A rice cDNA clone, Os-chs cDNA, encoding chalcone synthase, isolated from a leaf cDNA library of an indica rice variety Purpleputtu has been mapped to the centromeric region of chromosome 11 of rice. It was mapped between RFLP markers RG2 and RG103. RG2 is the nearest RFLP marker located at a genetic distance of 3.3 cM. Some segments of chromosome 11 of rice including chs locus are conserved on chromosome 4 of maize. The markers, including chs locus on chromosome 11 of rice are located, though not in the same order, on chromosome 4 of maize. Genetic analysis of purple pigmentation in two rice lines, Abhaya and Shyamala, used in the present mapping studies, indicated the involvement of three genes, one of which has been identified as a dominant inhibitor of leaf pigmentation. The Os-chs cDNA shows extensive sequence homology, both for DNA and protein (deduced), to that of maize, barley and also to different monocots and dicots.     

Effect of phosphon-D on photosynthetic light reactions and on reactions of the oxidative and reductive pentose phosphate cycle in a reconstituted spinach (Spinacia oleracea L.) chloroplast system

Phosphon-D (tributyl-2, 4-dichlorobenzylphosphonium chloride), known as an inhibitor of gibberellin biosynthesis, enhances photosynthetic electron transport by up to 200%, with Fe(CN) ₆ ^3- and NADP⁺ being the electron acceptors. Maximum stimulation is reached at phosphon-D concentrations around 2–5 M. At the same time photosynthetic ATP formation is gradually inhibited. Phosphon-D concentrations over 0.1 mM inhibit electron transport. The uncoupling activity of phosphon-D is manifested by inhibition of noncyclic ATP synthesis and by stimulation of light-induced electron flow. The inhibition of ATP synthesis drastically decreases photosynthetic carbon assimilation in a reconstituted spinach chloroplast system. The two ATP-dependent kinase reactions of the reductive pentose phosphate cycle become the rate-limiting steps. On the other hand a stimulated photoelectron transport increases the NADPH/NADP⁺ ratio, resulting in a drastic inhibition of chloroplast glucose-6-phosphate dehydrogenase (EC 1.1.1.49), the key enzyme of the oxidative pentose phosphate cycle. When light-induced electron flow is inhibited by high phosphon-D concentrations and the NADPH/NADP⁺ ratio is low, the light-dependent inhibition of glucose-6-phosphate dehydrogenase is gradually abolished.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride - B-Nine N-dimethylaminosuccinamic acid - CCC (2-chloroethyl)-trimethylammonium chloride - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DCPIP dichlorophenolindophenol - G-6-PDH glucose-6-phosphate dehydrogenase - FBP fructose bisphosphate - F-6-P fructose-6-phosphate - 3-PGA 3-phosphoglyceric acid - Posphon-D tributyl-2,4-dichlorobenzylphosphonium chloride - PMP pentose monophosphates - PPC pentose phosphate cycle - RuBP ribulose bisphosphate - Ru-5-P ribulose-5-phosphate Dedicated to Prof. Dr. Drs.h.c. Adolf Butenandt on the occasion of his 75. birthday     

The uptake of gibberellin A1 by suspension-cultured Spinacia oleracea cells has a carrier-mediated component

The kinetics of the uptake of [³H]gibberellin A₁ (GA₁) by light- and dark-grown suspension-cultured cells of Spinacia oleracea (spinach) have been studied. Use of nonradioactive GA₁ and gibberellic acid (GA₃) show that the uptake has a saturable and a nonsaturable component. The nonsaturable component increases as the pH is lowered at a fixed concentration of [³H]GA₁ and is probably caused by non-mediated diffusion of the uncharged protonated species of GA₁. The saturable component is not the result of metabolic transformation or to GA₁ binding to the cell wall and is suggested to represent the operation of a transport carrier for which GA₁ and GA₃ are substrates. Auxin, abscisic acid and a cytokinin did not alter the GA₁ uptake. The K_m is approx. 0.3 mol dm^-3 at pH 4.4 in light- and dark-grown cells. The V_max of the carrier is higher in the light-grown cells. The optimum pH for the carrier at a physiological GA₁ concentration (3 nmol dm^-3) was pH 4.0, with no activity detectable at pH 7.0. Both saturable and nonsaturable components were decreased by protonophores indicating that the pH gradient between the cells and the medium may be a component of the driving forces for both types of transport. Both the permeability coefficient for the undissociated GA₁ and the ratio V _max/K _m for the carrier are lower than the corresponding values for the indole-3-acetic acid and abscisic acid carriers studied in other species.Abbreviations and symbols ABA abscisic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - GA gibberellin - GA₃ gibberellic acid - IAA indole-3-acetic acid - P permeability coefficient     

Enzymatic Reactions by Five Chalcone Synthase Homologs from Hop (Humulus lupulus L.)

The enzyme activities encoded in five cDNAs for chalcone synthase (CHS) homologs from hop were investigated. Only valerophenone synthase (VPS) and CHS_H1 showed both naringenin-chalcone and phlorisovalerophenone forming activity. Narigenin-chalcone production by VPS was much lower than by CHS_H1. Therefore, it is highly possible that flavonoid depends mainly on CHS_H1, while bitter acid biosynthesis depends mainly on VPS and CHS_H1.     

The role of the gas phase in the greening and growth of illuminated cell suspension cultures of spinach (Spinacia oleracea,L.)

Summary The gas phase developed above spinach suspension cultures critically affected their growth and greening. Ethylene accumulation inhibited greening; this effect of ethylene was antagonised when the culture gas phase was enriched with carbon dioxide. Greening was enhanced by reducing the partial pressure of oxygen below the air level; this effect was observed when oxygen supply did not restrict growth. One of the authors (C.C.D.) was supported by an S.R.C. studentship grant during this work.     

矮牵牛中查尔酮合成酶基因A (chsA) 2个启动子的克隆和分析

查尔酮合成酶(chalcone synthase,CHS)是类黄酮类物质生物合成途径中的第一个关键酶,其控制基因chs为超家族基因.根据前人报道的矮牵牛查尔酮合成酶基因A(chsA)启动子的保守序列设计1对特异性引物,从矮牵牛基因组DNA中通过1次PCR同时扩增出长为550 bp和354 bp的启动子(分别命名为PchsA-L和PchsA-S,GenBank登录号:EF199747和EF199748),其中PchsA-L与PchsA-S相比,除个别碱基有差异外,在88～269 bp多出一段182 bp的序列,其中103～201 bp含有典型的内含子特征.应用DNAStar软件分析表明2条序列均含有普通启动子的保守序列TATA box、CCAAT box、capsite(CCATAA),并含有花中特异表达启动子的特征序列TACPyAT box、anther box(TAGAAGTGACAGAAAT)、G-box(CACGTG)、box1元件(ATGTCACGTGCCATC)和box2元件(TGTGTTGAAGGTTTGCTA).对克隆启动子所用的矮牵牛后代群体进行分析,130个单株中只含有PchsA-S的有13株,只含有PchsA-L的有20株,同时含有2个启动子的有97株.2个启动子在后代中发生了分离,但其分离比并不符合1∶2∶1.克隆启动子所用的矮牵牛有14条染色体,为二倍体.DNA印迹表明2个启动子在基因组中均是多拷贝.qRT-PCR分析显示:未经过紫外光处理的花中以及经过紫外光处理的花中,PchsA-L启动子驱动的chsA基因与PchsA-S启动子驱动的chsA基因表达都未见明显差异;在紫外光处理的幼苗叶片中表达量相应地比紫外光处理的花中的表达量增高;在紫外光处理的幼苗叶片中,PchsA-L启动子驱动的chsA基因比PchsA-S启动子驱动的chsA基因表达量显著增高;而未经过紫外光处理的幼苗叶片中,PchsA-L启动子驱动的chsA基因、PchsA-S启动子驱动的chsA基因都没有检测到明显的表达信号.结果表明:在矮牵牛中chsA基因存在2个独立的启动子PchsA-L和PchsA-S;启动子PchsA-L中182 bp类内含子特征的序列具有显著提高chsA基因在紫外光处理的幼苗叶片中表达量的功能.     

Regulation of adenylate levels in intact spinach chloroplasts

Starch phosphorylase activity in extracts of spinach or pea leaves and of isolated chloroplasts was determined and separated by electrophoresis in polyacrylamide gels. In spinach leaf extracts, a specific activity of 16 nmol glucose 1-phosphate formed per min per mg protein was found, whereas a lower value (6 nmol per min per mg protein) was observed in preparations of isolated chloroplasts which were about 75% intact. In the spinach leaf extracts two forms of phosphorylase were found; chloroplast preparations almost exclusively contained one of these. In pea leaf extracts the specific activity was 10 nmol glucose 1-phosphate formed per min per mg protein. Three forms of phosphorylase contributed to this activity. Preparations of isolated chloroplasts with an intactness of about 85% exhibited a lower specific activity (5nmol per min per mg protein) and contained two of these three phosphorylase forms.Abbreviations G1P Glucose 1-phosphate - P_i orthophosphate - Tris Tris (hydroxymethyl)aminomethane - MES 2(N-morpholino)ethane sulphonic acid - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Regulation of sucrose-phosphate-synthase activity in spinach leaves by protein level and covalent modification

A dot-blot technique was developed using monoclonal antibodies to measure, rapidly and accurately, the amount of sucrose-phosphate synthase (SPS; EC 2.4.1.14) protein present in a crude extract from spinach (Spinacia oleracea L. cv. Dark Green Bloomsdale) leaves; this was compared with SPS activity in this material. During leaf development, increased SPS activity followed closely the increase in enzyme-protein level, indicating denovo synthesis or altered turn-over rates for SPS. In contrast, activation of SPS by illumination of leaves or by mannose treatment of leaf discs in the dark (M. Stitt et al. Planta 174, 217–230) occurred without a significant change in the level of enzyme protein. Since conditions which altered SPS activity did not affect immunoprecipitation or mobility of the 120-kilodalton (kDa) subunit of the enzyme during denaturing gel electrophoresis, some form of protein modification other than proteolysis must be involved. Overall, the results indicate that regulation of SPS activity can involve changes in the level of enzyme protein and-or covalent modification.Abbreviations kDa kilodalton - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - SPS sucrosephosphate synthase Cooperative investigations of the U.S. Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Reseach Service, Raleigh. Paper No. 11789 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643, USA