The cDNA Sequence of NS3-Glycoprotein from Brassica campestris and its Homology to Related Proteins

A cDNA clone encoding NS₃-glycoprotein was isolated from stigmasof Brassica campestris. NS-glycoproteins correspond to the SLRI-glycoproteinsof B. oleracea and are highly conserved within the species.These data suggest that the NS-glycoproteins may play a rolein discrimination between species in the fertilization systemof Brassica. (Received September 4, 1992; Accepted November 9, 1992)     

The Mode of Expression and Promoter Analysis of the arcA Gene, an Auxin-Regulated Gene in Tobacco BY-2 Cells

The arcA, a member of the G protein rß-subunit family,was isolated from tobacco BY-2 cells as an auxin-responsivegene. Characterization of arcA, which should help to elucidatethe function of the gene product in the plant cells, was performedwith emphasis on the mode of expression and the analysis ofits promoter. Accumulation of the arcA message was detectedonly after treatments with auxins and not after treatments withother phytohormones or CdCl₂, implying that responsiveness ofarcA was exclusive to auxin. The putative arcA promoter regionwas fused to a reporter gene for rß-glucuronidase(GUS), and transient expression was analyzed in tobacco BY-2cells. Two series of arcA promoter/GUS chimeric genes were constructed.One consisted of a set of 5' nested deletions of the arcA promoterconnected to the gene for GUS and the other consisted of a varietyof the arcA promoter fragments fused to a minimal promoter-GUSconstruct. The results indicated that the promoter sequencecovering four sets of direct repeats (– 562 to –167)was necessary for the sufficient response of arcA promoter toauxin in BY-2 cells. Moreover, irrespective of auxin treatment,elevated activity of GUS driven by this promoter fragment wasdetected, a result that implies that this region behaves anenhancer in BY-2 cells. (Received September 30, 1995; Accepted March 1, 1996)     

Isolation of the Photoactive Reaction Center Complex that Contains Three Types of Fe-S Centers and a Cytochrome c Subunit from the Green Sulfur Bacterium Chlorobium limicola f. thiosulfatophilum, Strain Larsen

The photoactive reaction center (RC) complex from the greensulfur bacterium Chlorobium limicola f. thiosulfatophilum, strainLarsen, was isolated after solubilization and ammonium sulfatefractionation followed by ion-exchange chromatography. The spectrumof the complex was almost identical with that of the similarRC complex isolated by Feiler et al. [(1992) Biochemistry 31:2608–2614] except for the presence of cytochrome c₅₅₁instead of c₅₅₃ in the latter study. A molecular ratio of BChla to P840 of the isolated RC complex was assayed to be 25–35.SDSPAGE analysis revealed that the isolated complex containedthree major polypeptides with apparent molecular masses of 68,41 and 21 kDa, respectively. The 21-kDa polypeptide was identifiedto be a heme-binding protein by staining the gel for peroxidaseactivity. The cytochrome c₅₅₁ was oxidized by flash light ina biphasic manner with half times of 90 and 390 µs, respectively,that coincided with the reduction half times of P840⁺. Threedistinct iron-sulfur centers assigned to F_A, F_B and F_x, respectively,from their g-values were detected by EPR spectroscopy at cryogenictemperature. These results suggest that the present preparationcontains a minimal functional unit of the RC of this bacterium,and that this complex appears to lie on a evolutionary linebetween RC's of purple bacteria and photosystem I. (Received August 18, 1992; Accepted October 28, 1992)     

The GUS reporter-aided analysis of the promoter activities of Arabidopsis ACC synthase genes AtACS4, AtACS5, and AtACS7 induced by hormones and stresses

Ethylene biosynthesis in higher plants is regulated developmentallyand environmentally. To investigate the regulation of ACC synthasegene expression, the promoters of Arabidopsis ACS genes, AtACS4,AtACS5, and AtACS7, were fused to a GUS reporter gene, and therecombinant transgenes were introduced into Arabidopsis to producethree groups of AtACS::GUS transgenic plants. Histochemic andfluorometric study of these transgenic plants revealed thatpromoters of AtACS4, AtACS, and AtACS7 are all active in dark-germinatedseedlings. AtACS5 has the highest promoter activity in leavesof 2-week-old light-grown seedlings among the three AtACS genesstudied. In the mature leaves, AtACS4 and AtACS7 genes are expressedin both veins and areoles, whereas AtACS5 is expressed at ahigher level in the areoles and epidermal cells surroundingtrichomes. The promoter activities of all these AtACS genesare found in the reproductive organs. AtACS5 and AtACS7 arehighly expressed in petals, sepals, carpels, stamens, caulineleaves, inflorescence stems, and siliques, while AtACS4 expressionis undetectable in the petals of open flowers. All three AtACSgenes are expressed in root tissue. In the 2-week-old light-grownArabidopsis, the AtACS4 promoter is responsive to the planthormones IAA, ethylene, and ABA, and to darkness and wounding;the AtACS5 promoter to IAA, ABA, salt, high temperature, andwounding; and the AtACS7 promoter to GA₃, ethylene, and ABA,and to darkness and salt. Low-temperature treatment abolishesthe darkness-induced AtACS7 gene expression, but not that ofAtACS4. Each AtACS gene has a unique expression profile duringgrowth and development. It appears that at any developmentalstage or any growth period of Arabidopsis, there is always amember of AtACS multigene family that is actively expressed. Key words: ACC synthase, Arabidopsis, ethylene, gene expression, GUS histochemical staining, reporter, stress treatments     

Isolation and Characterization of a Cytokinin Up-Regulated Gene from Tobacco Mesophyll Protoplasts

We have isolated a cytokinin up-regulated cDNA clone, H13, froman early stage of cultured tobacco mesophyll protoplasts bya differential display method. The expression of this gene wasspecifically induced by natural and synthetic cytokinins includingN-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30), a diphenylurea-typecytokinin, although the simultaneous presence of auxin was alsorequired. It seems that the preceding treatment of the tobaccomesophyll protoplasts by auxin is necessary for the gene torespond to cytokinin. The addition of a cytokinin antagonist,compound 182, which suppressed the induction of cell divisionin tobacco mesophyll protoplasts, completely abolished the expressionof this gene. Though the predicted gene product of H13 did notsuggest us any sequences of defined functions, two domains ofthe predicted sequence had significant homology to several reportedsequences in the data base. The gene product of H13 is proposedto have a role in regenerating cell wall in cultured protoplasts,since a cDNA clone E6, from cotton fiber cells, which has themost closely related structure to H13, has been isolated fromcells which showed active cellulose synthesis. This suppositionis supported by the evidence that in the absence of cytokinin,cell wall regeneration was significantly suppressed, resultingin failure of the induction of cell division. Thus, the geneproduct of H13 is supposed to have a role in regenerating cellwalls and facilitating the progression of the cell cycle, resultingin the sustained cell division of tobacco mesophyll protoplasts. ¹These authors are equally contributed to this work.     

Nucleotide Sequence and Genetic Analysis of the Region Essential for Functional Expression of the Gene for Ferredoxin I, fdxN, in Rhodobacter capsulatus: Sharing of One Upstream Activator Sequence in Opposite Directions by Two Operons Related to Nitrogen Fixation

Nucleotide sequencing of the region upstream of two ferredoxingenes, fdxC and fdxN, of Rhodobacter capsulatus revealed theexistence of one open reading frame (ORF), ORFU1,in the sameorientation as these genes and two other ORFs, ORFU2 and ORFU3,in the opposite orientation. Two potential –24/–12promoters were found in front of ORFU1 and ORFU2, respectively,and there was a putative upstream activator sequence (UAS) orNifA-binding site between them. The ORFs corresponded to noknown nif genes. However, analysis of their putative productsshowed that the product of ORFU1 (M_r 47,912) and that of ORFU3(M_r 19,090) flavodoxin-like domain and a 2[4Fe-4S] ferredoxin-likedomain, respectively, and that the product of ORFU2 (M_r 20,424)was a hydrophobic protein with six potential membrane-spanningportions. Results of interposon mutagenesis and complementationexperiments indicated ORFU2 but not ORFU1 is essential for nitrogenfixation and that additional gene(s) essential nitrogen fixationmust be present in the unsequenced region adjacent to ORFU3.Translational fusion analysis involving lacZYA and fdxN or ORFU3provided evidence that the putative UAS responsible for regulationof both ORFUl-fdxC-fdxNand ORFU2-ORFU3 operons in opposite orientations,and that the control of the latter is stricter than that ofthe former. (Received August 19, 1992; Accepted November 16, 1992)     

Transgenic Rice: Characterization of Protoplastderived Plants and their Seed Progeny

Thirty eight green and 2 albino plants were regenerated from400 kanamycin-resistant colonies derived from protoplasts isolatedfrom cell suspensions of Oryza sativa variety Taipei 309 andelectroporated with pCaMVNEO carrying the neomycin phosphotransferaseII (nptII) gene. Twenty of the green transgenic R_o plants weretransferred to the glasshouse, where 3 flowered after 7 months.Of 15 plants analysed by DNA hybridization, all carried thenptll gene, but only 2 of 11 plants assayed for NPTII activityexpressed the nptll gene. One transgenic R_o plant produced 59seeds following self-pollination. The seeds, when germinatedon medium containing kanamycin sulphate, gave 16 green transgenicR, plants. Five transgenic R₁ plants flowered and set seed,7 flowered but failed to produce seeds, while 4 did not producepanicles. Transgenic R_o and R₁ plants were shorter, requiredlonger to flower, and had reduced pollen viability comparedto non-transformed R₁ protoplast-derived plants. The nptII genewas present in all 16 transgenic R₁ plants, but NPTII activitywas detected in only 8 of these plants. Key words: Oryza sativa variety Taipei 309, rice, protoplasts, direct DNA uptake, kanamycin-resistant tissues, transgenic plants, DNA hybridization, neomycin phosphotransferase II (NPTII), gene expression and inheritance     

Impact of chloroplastic- and extracellular-sourced ROS on high light-responsive gene expression in Arabidopsis

The expression of 28 high light (HL)-responsive genes of Arabidopsiswas analysed in response to environmental and physiologicalfactors known to influence the expression of the HL-responsivegene, ASCORBATE PEROXIDASE2 (APX2). Most (81%) of the HL-responsivegenes, including APX2, required photosynthetic electron transportfor their expression, and were responsive to abscisic acid (ABA;68%), strengthening the impression that these two signals arecrucial in the expression of HL-responsive genes. Further, fromthe use of mutants altered in reactive oxygen species (ROS)metabolism, it was shown that 61% of these genes, includingAPX2, may be responsive to chloroplast-sourced ROS. In contrast,apoplastic/plasma membrane-sourced H₂O₂, in part directed bythe respiratory burst NADPH oxidases AtrbohD and AtrbohF, wasshown to be important only for APX2 expression. APX2 expressionin leaves is limited to bundle sheath parenchyma; however, forthe other genes in this study, information on their tissue specificityof expression is sparse. An analysis of expression in petioles,enriched for bundle sheath tissue compared with distal leafblade, in HL and control leaves showed that 25% of them had>10-fold higher expression in the petiole than in the leafblade. However, this did not mean that these petiole expressiongenes followed a pattern of regulation observed for APX2. Key words: Arabidopsis, chloroplast, excess light, gene expression, plasma membrane, reactive oxygen species, signalling     

Effects of a Plant-Growth Regulator, Prohexadione, on the Biosynthesis of Gibberellins in Cell-Free Systems Derived from Immature Seeds

Inhibition of the biosynthesis of gibberellins by prohexadione,3,5-dioxo-4-propionylcyclo-hexanecarboxylic acid, was studiedwith cell-free systems derived from immature seeds of Cucur-bitamaxima, Phaseolus vulgaris and Pisum sativum. Prohexadione,at a concentration of 10–⁴ M, inhibited C-7 oxidationof GA₁₂-aldehyde, C-20 oxidation of GA₁₅, conversion of C₂₀-gib-berellinsto C₁₉-gibberellins, 3ß-hydroxylation, 2,3-dehydrogenationof GA²⁰, 2,3-epoxidation of GA₅ and 2ß-hydroxylationof GA₉ and GA₂₀. The 3ß-hydroxylase activity appearedto be more sensitive to prohexadione than were the C-20 oxygenaseand the 2ß-hydroxylase activities. The conversionof mevalonic acid to GA₁₂-aldehyde and the 13-hydroxylationof GA₁₂ were not affected by prohexadione at a concentrationof 3 ? 10^–4 M. All of the steps inhibited by prohexadioneare oxidation steps catalyzed by soluble enzymes that require2-oxoglutarate, Fe²⁺ and oxygen, and all of them occur distalto the synthesis of GA₁₂-aldehyde in the biosynthesis of gibberellins. (Received April 4, 1990; Accepted September 14, 1990)     

Differentiation of Photorespiratory Activity between Mesophyll and Bundle Sheath Cells of C4 Plants I. Glycine Oxidation by Mitochondria

Mitochondria were isolated from mesophyll protoplasts and bundlesheath protoplasts or strands which were obtained by enzymaticdigestion of six C₄ species: Zea mays, Sorghum bicolor, Panicummiliaceum, Panicum capillare, Panicum maximum and Chloris gayana,representative of three C₄ types. Photorespiratory glycine oxidationand related enzyme activities of mesophyll and bundle sheathmitochondria were compared. Mesophyll mitochondria showed good P/O ratios with malate andsuccinate as substrate but lacked the ability to oxidize glycine.On the other hand, mitochondria isolated from bundle sheathprotoplasts of P. miliaceum and bundle sheath strands of Z.mays possessed glycine oxidation activity similar to that ofmitochondria from C₃ plant leaves. The two enzymes involvedin glycine metabolism in mitochondria, serine hydroxymethyltransferaseand glycine decarboxylase, were also assayed in the mitochondriaof the two cell types. The activities of the two enzymes inbundle sheath mitochondria were in the range found in C₃ mitochondria.In contrast, the activities in mesophyll mitochondria were eithernot detectable or far lower than those in bundle sheath mitochondriaand ascribed to contaminating bundle sheath mitochondria. The present results indicate the deficiency of a complete glycineoxidation system in mesophyll mitochondria and also a differentiationbetween mesophyll and bundle sheath cells of C₄ plants withrespect to the photorespiratory activities of the mitochondria. (Received June 8, 1983; Accepted August 29, 1983)     

Hydrogen Peroxide-Dependent Oxidation of 3,4-Dihydroxyphenylalanine in Vacuoles of Mesophyll Cells of Vicia faba L. Participation of Peroxidase in the Oxidation

Peroxidase activity and 3,4-dihydroxyphenylalanine (DOPA) werefound in vacuoles isolated from mesophyll protoplasts of Viciafaba L. A peroxidase isozyme localized in vacuoles migratedto the cathode during electrophoresis at pH 8.7, indicatingthat the vacuole peroxidase was a basic isozyme. When isolatedvacuoles were treated with 2 mM H₂O₂, dopachrome, a productof oxidation of DOPA, was formed in a reaction that was inhibitedby KCN and NaN₃. These results suggest that DOPA can serve asa donor of electrons to the peroxidase in vacuoles. (Received December 25, 1989; Accepted March 22, 1990)     

The Gene for Pyruvate,Orthophosphate Dikinase in C4 Plants: Structure, Regulation and Evolution

Pyruvate, orthophosphate dikinase (PPDK; EC 2.7.9.1 [EC] ) is a keyenzyme in photosynthesis in plants that exploit the C4 photosyntheticpathway for the fixation of CO₂. This review focuses on thestructure, regulation and evolution of the C4-type ppdk genein the maize genome. The C4-ppdk gene in maize consists of 19exons spanning about 12 kbp. The gene is transcribed from twodifferent initiation sites under the control of two promotersto produce two mRNAs of different sizes. The larger one containsthe exon 1 sequence that encodes the chloroplast transit peptideand its product acts as C4-PPDK in chloroplasts, while the smallerone does not contain the sequence and its product may functionas a C3-enzyme in the cytosol. This unusual dual promoter systemis not unique to the maize C4-type ppdk gene since the sameorganization is also observed in the rice (C3 plant) ppdk geneand in Flaveria. Thus, the two-promoter system is common toplant ppdk genes from C3 and C4, monocot and dicot plants. Adiscussion is also presented of the generation of a system forregulation of the expression of the C4-type ppdk gene. A chimericgene consisting of a reporter gene under the control of thepromoter of maize CA-ppdk is exclusively expressed in photosynthetictissues and not in roots or stems of transgenic rice. The expressionof the introduced gene is also regulated by light: it is lowin etiolated leaves and is enhanced by illumination. These resultsindicate that the regulatory system that controls ppdk expressionin maize is not unique to C4 plants. ¹Recipient of the JSPP Young Investigator Award, 1995.     

A Novel Gene, ITM, Located between p57KIP2 and IPL, Is Imprinted in Mice

We searched for new imprinted genes using a positional cloningmethod in a region of human chromosome 11p15.5, which containsseveral imprinted genes including p57^KIP2 and IPL, and founda novel ITM gene located between p57^KIP2 and IPL. We also obtainedthe mouse homologue Itm in itss yntenic region of mouse chromosome7. In humans, its location is 17 kb centromeric to p57^KIP2 and3 kb telomeric to IPL, and in mice, 15 kb and 2.5 kb, respectively.They are expressed in most tissue, but especially in the kidneyand liver, and moderately in the heart, lung and testis. Miceexhibit a functional imprinting resulting in higher expressionof maternal alleles in fetal, newborn and most adult tissues,but it is biallelically expressed in the adult kidney and liverwhere expression is the highest. In addition to the discrepancybetween the level of expression and the strength of the imprint,Itm has several unusual features for an imprinted gene, includinglarge introns, moderate GC content and the absence of directrepeats. Our results will be helpful in understanding the intricateregulatory mechanism of imprinted genes.     

The Conductance of the Plasmalemma for CO2

Permeability coefficients (P_S values) for CO₂ of the plasmamembrane (PM) of the unicellular green algae Eremosphaera viridis,Dunaliella parva, and Dunaliella acidophila, and of mesophyllprotoplasts isolated from Valerianella locusta were determinedfrom ¹⁴CO₂ uptake experiments using the rapid separation ofcells by the silicone oil layer centrifugation technique. Theexperimental P_S values were compared with calculated numbersobtained by interpolation of Collander plots, which are basedon lipid solubility and molecular size, for D. parva cells,mesophyll protoplasts isolated from Spinacia oleracea, mesophyllcells and guard cells of Valerianella, and guard cell protoplastsisolated from Vicia faba. The conductivity of algal plasma membranes for CO₂ varies between0.1 and 9 ? 10^–6 m s^–1, whereas for the plasmalemmaof cells and protoplasts isolated from leaves of higher plantsvalues between 0.3 and 11 ? 10^–6 m s^–1 were measured.By assuming that these measurements are representative for plantsand algae in general, it is concluded that the CO₂ conductivityof algal PM is of the same order of magnitude as that of thehigher plant cell PM. P_s values of plasma membranes for CO₂are lower than those for SO₂, but are in the same order of magnitudeas those measured for H₂O. On the basis of these results itis concluded that theoretical values of about 3000 ? 10^–6m s^–1 believed to be representative for higher plant cells(Nobel, 1983) and which are frequently used for computer-basedmodels of photosynthesis, lack experimental confirmation andrepresent considerable overestimations. However, with severalsystems, including higher plant cells, the conductance of thePM for CO₂ was significantly higher in light than in darkness.This suggests that in light, additional mechanisms for CO₂ uptakesuch as facilitated diffusion or active uptake may operate inparallel with diffusional uptake. Key words: Conductivity, CO₂, permeability coefficient, photosynthesis, plasmalemma