3-Deoxy-D-arabino heptulosonate 7-phosphate synthase from Zea mays: General properties and regulation by tryptophan

In extracts from Zea mays shoots, the presence of thiol compoundsin the extraction buffer was necessary to get an active 3 deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP) synthase. Its pH optimumfor activity was about 7.5. Of the different cations tested,only Mn⁺⁺ was an activator. Enzyme stability was optimal inTris-HCl buffer, pH 7.5, that contained a reducing agent, Mn⁺⁺and a polyol. Contrary to other reports, phosphoenolpyruvate(PEP) did not stabilize the preparation significantly. The synthaseexhibited high affinities for both erythrose-4-phosphate (Km:0.24 mM) and PEP (Km: 0.31 mM). Its specific activity was highestin young shoots. Corn DAHP synthase was inhibited in vitro by tryptophan. Moreover,the enzyme was retarded on a tryptophan agarose affinity column,but it was removed with the bulk of protein from the same supportwhen eluted with buffer containing tryptophan. Inhibition whichwas easily lost during storage at 4°C was pH dependent andincreased during development. Maximal inhibition, about 60%with 1 mM tryptophan, was observed in extracts from 8 day-oldshoots. Phenylalanine and tyrosine were not inhibitory, andno synergistic effects were observed when the aromatic aminoacids were tested in combination. Isoenzymes could not be demonstrated. (Received April 23, 1980; )     

Purification and Some Properties of Plant Serine Acetyltransferase

Serine acetyltransferase (SATase) that catalyzes the conversionof L-serine to O-acetyl-L-serine (OAS) in the presence of acetyl-CoAwas highly purified from rape leaf extract, using a coupledassay system in which OAS is converted to cysteine by enzymaticaction of exogenous cysteine synthase. Through purificationprocedures including heat treatment, ammonium sulfate fractionationand successive column chromatographies on DEAE-Toypearl, Blue-Toyopearland Toyopearl HW-55, the specific activity was raised to about4,800-fold over that in the crude extract. The molecular weightof rape enzyme was estimated to be about 350,000 by gel filtrationcolumn chromatography. The cysteine-forming activity of thefinal preparation was completely dependent on L-serine, acetyl-CoAand sulfide. However, this preparation had low activity forL-cysteine synthesis from L-serine even in the absence of exogenouscysteine synthase, suggesting that plant SATase exists as ahigh-molecular weight enzyme complexed with cysteine synthase. (Received November 6, 1987; Accepted March 25, 1988)     

Wound-Induced Ethylene Production and 1-Aminocyclopropane-1-carboxylic Acid Synthase in Mesocarp Tissue of Winter Squash Fruit

Discs (9 mm in diameter and 2 mm in thickness) sliced from mesocarpof winter squash fruit (Cucurbita maxima Duch.) upon incubationat 24°C produced ethylene at an increasing rate after alag period of 3 h. 1-Aminocydopropane-l-carboxylic acid (ACC)synthase activity also increased at a rapid rate after lag periodof less than 3 h, reaching a peak 14 h after incubation andthen declining sharply. The rise in ACC synthase activity precededa rapid increase in ACC formation and ethylene production. Inductionof ACC synthase by wounding in sliced discs was strongly suppressedby the application of cycloheximide, actinomycin D and cordycepin,suggesting that the rise in ACC synthase activity may resultfrom de novo synthesis of protein. ACC synthase extracted from wounded tissue of winter squashmesocarp required pyridoxal phosphate for its maximum activity.The optimum pH of the reaction was 8.5. Km value for S-adenosylmethioninewas 120 µM. The reaction was markedly inhibited by aminoethoxyvinylglycinewith Ki value being 2.7 µM. (Received March 23, 1983; Accepted May 23, 1983)     

Transformation of Brassica napus L. with a 'Disarmed' Octopine Plasmid of Agrobacterium tumefaciens: Molecular Analysis and Inheritance of the Transformed Phenotype

Phenotypically normal and fertile transgenic Brassica napuscv. Westar plants were obtained following co-cultivation ofstem epidermal explants with an Agrobacterium tumefaciens straincontaining a disarmed octopine tumour-inducing plasmid pTiB6S3-SE.The A. tumefaciens cells also contained pMON316, a cointegratevector carrying genes for kanamycin resistance and a scorablemarker nopaline synthase. Transformants were selected by theirability to grow in the presence of 100 µg cm^-3 kanamycin.Transformation was confirmed by the activities of neomycin phosphotransferaseII and nopaline synthase enzymes and by Southern blots. Thekanamycin resistance trait was transferred to the progeny ofthe self-fertilized plants. Key words: Transformation, octopine Ti-plasmid, oilseed rape     

氟虫脲对东亚飞蝗和中华稻蝗几丁质合成酶基因表达的影响

为了探讨氟虫脲可能的作用靶标及毒性机制, 本研究以重要农业害虫东亚飞蝗Locusta migratoria manilensis (Meyen)和中华稻蝗Oxya chinensis (Thunberg)为材料, 采用简并引物扩增中华稻蝗几丁质合成酶1基因（OcCHS1）的部分cDNA序列; 以氟虫脲浸渍法处理2龄中期中华稻蝗及1, 2和3龄东亚飞蝗若虫为处理组, 丙酮处理为对照组, 使用RT-PCR和实时荧光定量PCR方法分析氟虫脲对蝗虫几丁质合成酶基因mRNA表达的影响。结果获得的OcCHS1部分cDNA序列, 其长度为312 bp, 编码104个氨基酸, GenBank登录号为HM214491, 与东亚飞蝗几丁质合成酶1基因（LmCHS1）在氨基酸水平上相似度达95％。RT-PCR结果显示, 处理组几丁质合成酶1扩增带均强于对照组。实时荧光定量PCR结果表明: 与对照组相比, 处理组中华稻蝗2龄中期若虫OcCHS1 mRNA表达提高了1.02倍, 东亚飞蝗1, 2, 3龄若虫LmCHS1 mRNA表达分别提高了34%, 82%和89%, 差异显著（P<0.05）。分析基因表达提高的原因是几丁质合成受阻后基因表达水平的一种代偿性增加, 由此推测几丁质合成酶可能是氟虫脲作用的靶标之一。     

Molecular Cloning of Plant Spermidine Synthases

Four cDNAs for spermidine synthase (SPDS), which converts thediamine putrescine to the higher polyamine spermidine usingdecarboxylated S-adenosylmethionine as the co-factor, were isolatedfrom Nicotiana sylvestris, Hyoscyamus niger, and Arabidopsisthaliana. When the N. sylvestris SPDS cDNA was expressed ina SPDS-deficient E. coli mutant, the recombinant protein showedhigh SPDS activity, but did not have any spermine synthase activity.The plant SPDSs have molecular masses of about 34 kDa, possessthe co-factor binding motifs which have been proposed for S-adenosylmethionine,and are more homologous in amino acid sequence to tobacco putrescineN-methyltransferase (PMT) than to SPDSs from mammals and E.coli. The SPDS gene is expressed in root, stem, and leaf inN. sylvestris, whereas the PMT gene is expressed only in root.The potential evolution of plant SPDS and PMT, and their evolutionaryrelationships with animal SPDS are discussed. (Received September 3, 1997; Accepted November 5, 1997)     

Cloning and Functional Expression of a Novel Geranylgeranyl Pyrophosphate Synthase Gene from Arabidopsis thaliana in Escherichia coli

A gene encoding a novel geranylgeranyl pyrophosphate (GGPP)synthase from Arabidopsis thaliana has been identified and termedGGPS5. The gene has been sequenced and expressed in Escherichiacoli. The deduced amino acid sequence showed 64.5% and 57.5%identity with a putative GGPP synthase from Arabidopsis andCapsicum annuum, respectively. GGPP enzymatic activity was detectedin E. coli cells expressing the GGPS5 gene in two differentways. One was the direct measurement of GGPP synthase activityin cell extracts and the other was the yellow color productionof cells when the GGPS5 gene was co-expressed with crtB, crtI,crtX, crtY and crtZ genes derived from Erwinia uredovora. (Received May 20, 1996; Accepted December 14, 1996)     

Distinctive Enzymes of Aromatic Amino Acid Biosynthesis That Are Highly Conserved in Land Plants Are Also Present in the Chlorophyte Alga Chlorella sorokiniana

Considerable enzymological diversity underlies the capacityfor biosynthesis of aromatic amino acids in nature. For thisbiochemical pathway, higher plants as a group exhibit a uniformpattern of pathway steps, compartmentation, and catalytic, physicaland allosteric properties of enzymes. This biochemical patternof higher plants contains a collection of features which arecompletely different from photosynthetic prokaryotes such asthe cyanobacteria. A unicellular representative of the chlorophytealgae, Chlorella sorokiniana, was found to be strikingly similarto higher-plant plastids in possessing the following distinctiveenzymes: a Mn²⁺-stimulated, dithiothreitol-activated isoenzymeof 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase,a probable bifunctional protein competent as both dehydroquinaseand shikimate dehydrogenase, an allosterically controlled isoenzymeof chorismate mutase, a highly thermotolerant species of prephenateaminotransferase, an NADP⁺-dependent, tyrosine-inhibited arogenatedehydrogenase, and an arogenate dehydratase. In addition anisoenzyme of DAHP synthase shown in higher plants to be cytosolic,absolutely dependent upon the presence of divalent metals, andable to substitute other sugars for erythrose-4-phosphate, wasalso demonstrated in this alga. A broad-specificity 3-deoxy-D-manno-octulosonate8-phosphate synthase, recently discovered in higher plants,is also present in this Chlorella species. (Received March 25, 1995; Accepted June 14, 1995)     

Induction of 1-Aminocyclopropane-1-carboxylic Acid Synthase in Wounded Mesocarp Tissue of Winter Squash Fruit and the Effects of Ethylene

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase activityincreased rapidly after wounding of mesocarp tissue of wintersquash fruit (Cucurbita maxima Duch.) and reached a peak at16 h after excision and then declined sharply. The rise in ACCsynthase activity was followed by increases in the endogenousACC content and the rate of ethylene production. The activityof ethylene forming enzyme (EFE) also increased rapidly in theexcised discs of mesocarp of winter squash fruit. ACC synthase activity was strongly inhibited by aminoethoxyvinylglycinewith a Ki value of 2.1 µM. Michaelis-Menten constant ofACC synthase for S-adenosylmethionine was 13.3 µM. Ethylene suppressed the induction of ACC synthase in the woundedmesocarp tissue. The suppression by ethylene increased withthe increasing concentrations of applied ethylene and the maximumeffect was obtained at about 100 µl 1^–1 ethylene,at which point the induction was suppressed by 54%. Ethylenedid not inhibit ACC synthase activity, nor did it suppress theinduction of EFE, but rather it slightly enhanced the latter. (Received August 24, 1984; Accepted October 29, 1984)     

Structure and Expression of Mitochondrial Citrate Synthases from Higher Plants

Mitochondrial citrate synthase (EC 4.1.3.7 [EC] ) represents the firstenzyme of the tricarboxylic acid cycle, catalyzing the condensationof acetyl-CoA and oxaloacetate, finally yielding citrate andCoA. We report here the isolation of cDNA clones encoding citratesynthase from Nicotiana tabacum, Beta vulgaris and Populus.Nucleotide and deduced amino acid sequences were compared withpreviously published sequences of mitochondrial citrate synthasesfrom Arabidopsis thaliana and potato, as well as with the sequenceof glyoxysomal citrate synthase from pumpkin. Homologies betweenthe various plant mitochondrial enzymes were in the range from77.2% (potato vs. Arabidopsis) to 94.2% (potato vs. tobacco)on the nucleotide level (coding regions only), and in the rangefrom 70.1% to 90.4% (potato vs. Arabidopsis, and potato vs.tobacco, respectively) on the amino acid level. Identities ofthe mitochondrial isozymes to the pumpkin glyoxysomal enzymewere below 30% on the nucleotide and amino acid level. In Northernblot experiments citrate synthase mRNA was detected in all tissuesanalyzed. However, levels of expression showed tissue dependencydespite the fact that citrate synthase is usually considereda house-keeping enzyme. Whether these different levels of expressionreflect tissuespecifc variations with respect to basic metabolismawaits further analysis. (Received May 20, 1996; Accepted August 20, 1996)     

Chalcone Synthase Localization in Shoot Apices of Fagopyrum, Brassica and Pisum

Chalcone synthase (CHS), a key enzyme of flavonoid synthesis,was localized in shoot apices of Fagopyrum, Brassica and Pisum.The enzyme was detected in initial cells of the shoot apex,which gives rise to the whole plant body. In Fagopyrum and BrassicaCHS was located in the rib and flank meristems, whereas in theArgenteum mutant of Pisum this enzyme was localized at an earlierstage in the ontogenesis of the shoot. It occurs in a few cellsof the tunica, which gives rise to the protoderm, and then tothe epidermis which contains anthocyanins in these plants. Chalcone synthase, immunogold labelling, promeristem, shoot apex, Brassica, Fagopyrum, Pisum     

Investigation of sucrose synthase from rice for the synthesis of various nucleotide sugars and saccharides

The unique character of the plant glucosyltransferase sucrosesynthase, to catalyse in vitro the synthesis and cleavage ofsucrose under appropriate conditions, can be exploited for theenzymatic synthesis of carbohydrates. The present paper describesthe potential utilization of sucrose synthase from rice forthe enzymatic synthesis of activated sugars and saccharides.In the cleavage reaction of sucrose, the nucleoside diphosphatescan be used in the order UDP > TDP > ADP > CDP >GDP to obtain the corresponding activated glucoses. In batchreactions, >90% conversion of UDP and TDP could be achieved.Substituting different di- and trisaccharides for sucrose inthe cleavage reaction with UDP 2-deoxysucrose was the most promisingsubstrate. Sucrose synthase was combined with UDP-galactose4'-epimerase and ß1–4 galactosyltransferaseto synthesize N-acetyllactosamine with in situ regenerationof UDP-glucose. In the synthesis reaction of sucrose synthase,different donor (UDP-sugars) and acceptor substrates were investigated.UDP-N-acetylglucosamine and UDP-xylose could be used in combinationwith fructose as acceptor. D-Xylulose, D-tagatose, D-lyxose,D-psicose, L-sorbose, D-mannose, L-arabinose, 1, 6 anhydroglucose,lactulose, raffinose and isomaltulose can serve as acceptorsfor UDP-glucose. N-acetyllactosamine nucleotide sugars saccharides sucrose synthase     

Molecular Size of Wound-Induced 1-Aminocyclopropane-1-carboxylate Synthase from Cucurbita maxima Duch. and Change of Translatable mRNA of the Enzyme after Wounding

Wound-induced 1-aminocyclopropane-1-carboxylate (ACC) synthasewas purified by an immunoaffinity column from wounded mesocarpof winter squash (Cucurbita maxima Duch. cv. Ebisu) fruit, anda specific antibody was raised in rabbit. Translatable mRNAcoding for ACC synthase was barely detectable in fresh tissuebut clearly increased after wounding. The apparent molecularsize of the purified enzyme as estimated by SDS-polyacrylamidegel electrophoresis (PAGE) was about 50 kDa. However, SDS-PAGEfluorograms of in vitro translation product of ACC synthasemRNA and the in vivo labeled enzyme as well as Western blotanalysis showed that the subunit size of the enzyme was 58 kDa.The enzyme was partially degraded or processed to a 50 kDa peptideboth in vivo and in vitro. (Received December 19, 1987; Accepted June 13, 1988)     

Influence of abscisic acid on nitrogen partitioning, sucrose metabolism and nitrate reductase activity of chicory suspension cells

Batch suspension cultures of chicory cells (Cichorium intybusL. var. Witloof) possess a NADH-specific nitrate reductase activitythat peaks on day 3 of a 10 d growth cycle. When both nitrateand ammonium are used as nitrogen sources, chicory cells absorbnitrate irst. Ammonium uptake becomes predominant at day 3,even though NO₃^– was still present in the medium. Althoughabscisic acid impairs growth as well as ¹⁵NO₃^– uptakeand reduction, it promotes nitrate reductase activity as measuredboth in vivo and in vitro. Specific activity is 50% higher inABA-treated cells than in controls. These conflicting data maybe explained either in erms of nitrate reductase levels or bythe availability of reducing power and energy. Since NRA isgenerally controlled by the availability of the reducing power,the energy status of the cell, the adenylate nucleotide pools,were measured simultaneously with the carbohydrate levels withinthe cell and the growth medium. The energy charge was not modifiedduring the growth cycle, regardless of the rowth conditions.Yet ABA modified the intracellular carbohydrate metabolism andinhibited the acidic invertase, the sucrose synthase and thesucrose phosphate synthase activities. Modified assimilationrates of nitrate in chicory cells grown in the presence of ABA,were probably correlated to modified carbohydrate metabolismpathways leading to increased availability of reducing power,energy and C-skeletons. Key words: Abscisic acid, Cichorium intybus L, nitrate reductase, reductase, invertase, sucrose synthase, sucrose phosphate synthase     

The Synthesis of Ethylene in Melon Fruit during the Early Stage of Ripening

The levels of mRNA and polypeptide for a 1-aminocyclopropane-1-carboxylate(ACC) oxidase were studied to identify the tissues in whichthe synthesis of ethylene first occurs during the initial stageof ripening. RNA and immunoblot analysis showed that the levelsof the mRNA and polypeptide for ACC oxidase were very low inunripe fruit. They first became detectable in the placentaltissue at the pre-climacteric stage, and then their levels increasedin the mesocarp tissue during the climacteric increase in theproduction of ethylene. Two mRNAs for ACC synthase (transcribedfrom ME-ACS1 and ME-ACS2) were detected in the placental tissueand seeds at the pre-climacteric stage, but only the level ofME-ACS1 mRNA, which has been characterized as the mRNA for awound-inducible ACC synthase, increased in mesocarp, placentaltissues and seeds during ripening. The level of ME-ACS2 mRNAthat was isolated from etiolated seedlings of melon, did notchange markedly during ripening. These results suggest thatthe central region of melon fruit (placental tissue and seeds)plays a major role in the production of ethylene during theearly stage of ripening. ³These three authors made equal contribution to this study.     

Preparation of Chlorophyll a, Chlorophyll b and Bacteriochlorophyll a by Column Chromatography with DEAE-Sepharose CL-6B and Sepharose CL-6B

Chlorophylls extracted from spinach leaves were made free fromcarotenoids, pheophytins, chlorophyllides and plastoquinonesby column chromatography with DEAE-Sepharose CL-6B. Chlorophylla and b were separated by column chromatography with SepharoseCL-6B. By a combined use of DEAE-Sepharose CL-6B and SepharoseCL-6B, pure chlorophyll a and b were prepared from the leavesin a short time. Bacteriochlorophyll a_p extracted from a photosyntheticbacterium Chromatium vinosum was made free from carotenoids,bacteriopheophytin, ubiquinone and lipids by column chromatographywith Sepharose CL-6B. (Received April 19, 1983; Accepted June 16, 1983)     

The Effects of 2,5-Norbornadiene on the Induction of the Activity of 1-Aminocyclopropane-l-carboxylate Synthase and of Phenylalanine Ammonia-lyase in Wounded Mesocarp Tissue of Cucurbita maxima

Activities of both 1-aminocyclopropane-l-carboxylate (ACC) synthaseand phenylalanine ammonia-lyase (PAL) were rapidly induced inexcised mesocarp discs of Cucurbita maxima Duch. The increasein activity of ACC synthase preceded that of PAL. 2,5-Norbornadiene(NBD), an inhibitor of the action of ethylene [Sisler and Yang(1984) Phytochemistry 12: 2765-2768.], enhanced the level ofactivity of ACC synthase in excised mesocarp disc and overcamethe suppression by exogenous ethylene. NBD, by contrast, suppressedthe level of PAL activity induced in the wounded tissue. Theseresults suggest that endogenous ethylene produced in the woundedmesocarp tissue suppresses the induction of ACC synthase butpromotes the induction of PAL. (Received March 9, 1989; Accepted June 14, 1989)