The Expression of an Abscisic Acid-Responsive Glycine-Rich Protein Coincides with the Level of Seed Dormancy in Fagus sylvatica

By differential screening of a cDNA library constructed frompoly (A⁺) RNA of ABA-treated seeds of Fagus sylvatica L., wehave isolated an ABA-responsive clone that is present in dormantseeds and under conditions that maintain dormancy, but it tendsto disappear under conditions breaking seed dormancy. A searchof the sequence data bases showed that the clone codes for aGlycine-Rich Protein and has sequence similarity to RNA-bindingproteins. The clone, which exibits the characteristics of lea-genes,is up-regulated by ABA and down-regulated by GA3. Paclobutrazolabolishes the effect of GA³, which is restored upon additionof GA3. The possible relationship of this Glycine-Rich Proteinto seed dormancy in F. sylvatica is discussed. (Received May 23, 1997; Accepted September 22, 1997)     

Cloning of cDNA of Prunus serotina (R)-(+)-Mandelonitrile Lyase and Identification of a Putative FAD-Binding Site

A full-length cDNA clone encoding the flavoprotein (R)-(+)-mandelonitrilelyase was isolated from a black cherry (Prunus serotina) cDNAexpression library and sequenced. A putative FAD-binding sitewas identified near the N-terminus of this enzyme by comparingits deduced amino acid sequence with those of other FAD- andNAD-binding proteins. ¹Present address: Department of Pharmacology, University ofMedicine & Dentistry of New Jersey, Piscataway, New Jersey08854, U.S.A.     

Identification of amino-acid substitutions in the proteolipid subunit of the ATP synthase from dicyclohexylcarbodiimide-resistant mutants of Escherichia coli

Polyadenylated mRNA was isolated from chick embryo liver following induction of hepatic porphyria. The RNA was translated in vitro using a wheat germ cell-free system and delta-aminolaevulinate synthase was identified in the translation products by indirect immunoprecipitation. The enzyme was not apparent in the translation products of polyadenylated RNA from non-induced livers. The molecular weight of delta-aminolaevulinate synthase synthesized in vitro was 70000 and the protein was estimated to represent up to 5% of total products synthesised in vitro. These data demonstrate for the first time that induction of chick embryo liver delta-aminolaevulinate synthase activity in hepatic porphyria correlates with a large increase in the translational capacity of isolated polyadenylated RNA for this enzyme and, together with preliminary cDNA . RNA hybridization studies, indicate that an increase in the level of delta-aminolaevulinic synthase mRNA is responsible.     

Studies on Glutamate Synthase from the Leaves of Higher Plants

Ferredoxin-dependent glutamate synthase has been isolated andpurified 170-fold from field bean (Vicia faba cv. Minor) leaves.The enzyme has a molecular weight of 145 000 and does not appearto contain non-haem iron. The K^m values of the enzyme for glutamine,2-oxoglutarate, and ferredoxin are 330, 150, and 2 µMrespectively. The specificity of the enzyme is extremely highand no substitutes for glutamine have been found. Azaserineis a potent inhibitor of glutamate synthase. Chloroplasts from four species of plants have been shown tocarry out a light-dependent glutamate synthesis from glutamine.This reaction is also highly specific for glutamine and purifiedasparagine cannot substitute for glutamine.     

Isolation of cDNA for a Plasma Membrane H+-ATPase from Guard Cells of Vicia faba L.

cDNA encoding the plasma membrane H⁺-ATPase of guard cells ofVicia faba L. was isolated. The clone encoded a 105-kDa polypeptide(956 amino acids) that was 79–85% identical in terms ofamino acid sequence to other plant H⁺-ATPases. High levels ofmRNA explain the high H⁺-ATPase activity of these plasma membranes. (Received December 24, 1994; Accepted April 12, 1995)     

cDNA Cloning and Expression of the Plastidic Copper/Zinc-Superoxide Dismutase from Spinach (Spinacia oleracea L.) Leaves

A cDNA clone for copper/zinc-superoxide dismutase (Cu/Zn-SOD)was isolated from spinach (Spinacia oleracea L.) leaves. Itsnucleotide sequence showed that it codes for a precursor polypeptideof 222 amino acids, including the NH₂-terminal 68-residue extensionwhich corresponds to a plastidic transit peptide. Northern hybridization,using plastidic and cytosolic Cu/Zn-SOD cDNAs as the probes,revealed that these two genes are differentially expressed inthe roots and leaves of spinach. ¹Present address: Department of Biochemistry and Microbiology,Cook College, Rutgers University New Brunswick, NJ 08903-0231,U.S.A.     

The Level of mRNA Transcribed from psaL, Which Encodes a Subunit of Photosystem I, Is Increased by Cytokinin in Darkness in Etiolated Cotyledons of Cucumber

A cDNA clone for an mRNA whose level increased within 2 h ofthe start of treatment with N⁶-benzyladenine in etiolated cotyledonsof cucumber was isolated by differential hybridization. ThecDNA was homologous to psaL, which encodes subunit XI (PSI-L)of photosystem I. The accumulation of psaL mRNA was specificallyinduced by cytokinins or light. (Received April 10, 1996; Accepted July 31, 1996)     

Structure of the Gene for an Auxin-Binding Protein and a Gene for 7SL RNA from Arabidopsis thaliana

A genomic clone encoding an auxin-binding protein (ABP) fromthe endoplasmic reticulum was isolated from Arabidopsis thaliana.The ABP gene consisted of 5 exons and 4 introns and encodeda polypeptide of 198 residues. A gene encoding the 7SL RNA ofthe signal recognition particle was located downstream of theABP gene. ⁴Recipient of a scholarship from the National Education Commission,People's Republic of China.     

Crystal structure determination of aristolochene synthase from the blue cheese mold, Penicillium roqueforti

The 2.5-A resolution crystal structure of recombinant aristolochene synthase from the blue cheese mold, Penicillium roqueforti, is the first of a fungal terpenoid cyclase. The structure of the enzyme reveals active site features that participate in the cyclization of the universal sesquiterpene cyclase substrate, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. Metal-triggered carbocation formation initiates the cyclization cascade, which proceeds through multiple complex intermediates to yield one exclusive structural and stereochemical isomer of aristolochene. Structural homology of this fungal cyclase with plant and bacterial terpenoid cyclases, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of terpene biosynthesis.     

SHORT COMMUNICATION Cyclic Nucleotides inFrankiaand Symbiotic Nodules

Cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine3',5'-monophosphate (cGMP) contents of cultured cells ofFrankiastrainsoriginally isolated from nodules ofAlnus sieboldiana, MyricarubraandElaeagnus macrophyllawere measured by enzyme immunoassays(EIA).Frankiacells, cultured for 59–121 d, had cAMP contentsranging from 2.9 to 76.1 pmol mg^-1protein and cGMP contentsranging from 0.9 to 5.2 pmol mg^-1protein. FollowingFrankiaculture,the media contained extremely large quantities of cAMP and significantlevels of cGMP. The nature of accumulation and secretion ofcyclic nucleotides by slow-growingFrankiacells was comparableto that by a fast-growing actinomyceteStreptomyces lividansTK24,suggesting that secretion of cAMP byFrankiacells may occur throughthe cell membrane but not by cell lysis. cAMP and cGMP contentsin the symbiotic nodules, leaves and roots of actinorrhizalplants and leaves of non-actinorrhizal trees were also measured.The nodules of actinorrhizal woody plants(A. sieboldiana, E.macrophylla, E. umbellata, E. pungensandM. rubra)had cAMP contentsranging from 4 to 258 pmol g^-1f. wt and cGMP contents rangingfrom 1.1 to 5.2 pmol mg^-1protein. Most leaves and some rootsof actinorrhizal plants and all the leaves of non-actinorrhizalwoody plants examined contained small but significant amountsof cAMP and cGMP. This is the first report of significant contentsof cAMP and cGMP in culturedFrankiacells andFrankia-infectednodules. Possible roles of cyclic nucleotides as symbiotic signalsare discussed.Copyright 1998 Annals of Botany Company cAMP, cGMP, actinorrhizal plants, nodules,Frankia,symbiosis.     

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.