Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.)

GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5′ flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, ¹⁵NH₄⁺ was incorporated into [5−¹⁵N]glutamine and [2−¹⁵N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2−¹⁵N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2−¹⁵N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15–20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO₂ contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides amino acids for nitrogen translocation. The nucleotide sequence data of the GLU1 gene reported in the present study is available from GenBank with the following accession number: AY189525     

Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163–168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.     

Action of light,nitrate and ammonium on the levels of NADH- and ferredoxin-dependent glutamate synthases in the cotyledons of mustard seedlings

In mustard (Sinapis alba L.) cotyledons, NADH-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14) is only detectable during early seedling development with a peak of enzyme activity occurring between 2 and 2.5 d after sowing. With the beginning of plastidogenesis at approximately 2 d after sowing, ferredoxindependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) appears while NADH-GOGAT drops to a very low level. The enzymes were separated by anion exchange chromatography. Both enzymes are stimulated by light operating through phytochrome. However, the extent of induction is much higher in the case of Fd-GOGAT than in the case of NADH-GOGAT. Moreover, NADH-GOGAT is inducible predominantly by red light pulses, while the light induction of Fd-GOGAT operates predominantly via the high irradiance response of phytochrome. The NADH-GOGAT level is strongly increased if mustard seedlings are grown in the presence of nitrate (15 mM KNO₃,15 mM NH₄NO₃) while the Fd-GOGAT level is only slightly affected by these treatments. No effect on NADH-GOGAT level was observed by growing the seedlings in the presence of ammonium (15 mM NH₄Cl) instead of water, whereas the level of Fd-GOGAT was considerably reduced when seedlings were grown in the presence of NH₄Cl. Inducibility of NADH-GOGAT by treatment with red light pulses or by transferring water-grown seedlings to NO ₃ ^- -containing medium follows a temporal pattern of competence. The very low Fd-GOGAT level in mustard seedlings grown under red light in the presence of the herbicide Norflurazon, which leads to photooxidative destruction of the plastids, indicates that the enzyme is located in the plastids. The NADH-GOGAT level is, in contrast, completely independent of plastid integrity which indicates that its location is cytosolic. It is concluded that NADH-GOGAT in the early seedling development is mainly concerned with metabolizing stored glutamine whereas Fd-GOGAT is involved in ammonium assimilation.Abbreviations and symbols c continuous - D darkness - Fd-GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - FR far-red light (3.5 W·m^-2) - NADH-GOGAT NADH-dependent glutamate synthase (EC 1.4.1.14) - Pfr far-red absorbing form of phytochrome - Ptot total phytochrome - R red light (6.8 W· m^-2) - RG9-light long wavelength FR (10 W·m^-2, _RG9<0.01) - () Pfr/Ptot=wavelength-dependent photoequilibrium of the phytochrome system     

Cloning and sequence analysis of a cDNA for barley ferredoxin-dependent glutamate synthase and molecular analysis of photorespiratory mutants deficient in the enzyme

The NH₂-terminal sequences of ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) purified from barley (Hordeum vulgare L.) and Chlamydomonas reinhardtii (Dangeard), and of a barley peptide, were determined and the barley sequences were used to design oligonucleotide primers for the polymerase chain reaction. A specific 1.3-kilobase (kb) cDNA fragment specifying the NH₂-terminal one-third of the mature barley polypeptide, was amplified, cloned and sequenced. The NH₂-terminus of plant Fd-GOGAT is highly conserved and homologous to the NH₂-terminus of the heavy subunit of Escherichia coli NADPH-GOGAT. Based on sequence homologies, we tentatively identified the NH₂-terminal region of Fd-GOGAT as the glutamine-amidotransferase domain, which is related to the corresponding domain of the purF-type amidotransferases. The Fd-GOGAT cDNA clone, and polyclonal antibodies raised against the barley enzyme, were used to analyse four Fd-GOGAT-deficient photorespiratory mutants. Three mutants (RPr 82/1, RPr 82/9 and RPr 84/82) had no detectable Fd-GOGAT protein in leaves, while the fourth (RPr 84/42) had a small amount of cross-reacting material. Hybridization to Northern blots of total leaf RNA revealed that both RPr 82/9 and RPr 84/82 were indistinguishable from the parental line (Maris Mink), having normal amounts of a 5.7-kb mRNA species. On the other hand, RPr 82/2 and RPr 84/42 each contained two distinct hybridizing RNA species, one of which was larger than 5.7 kb, the other smaller. Using a set of wheat-barley telosomic addition lines we have assigned the Fd-GOGAT structural locus to the short arm of chromosome 2.Abbreviations bp kbase pairs - cDNA copy DNA - Fd ferredoxin - GOGAT glutamate synthase - GAT glutamine amidotransferase - kb kilobase - PCR polymerase chain reaction C.A. was the holder of a Fleming award from the British Council and the Spanish Ministry of Education and Science. A.J.M. was for part of the work the recipient of a European Molecular Biology Organization postdoctoral fellowship. The research was also partly supported by contract no. BAP/O354/E of the Biotechnology Action Programme of the E.C., by an Acciones Integradas award (no. 40/125) from the British Council and the Spanish Ministry of Education and Science, by the Junta de Andalucia (to Group 3263) and by project PB91-0613 from DGICYT (Spain). We thank Daryl Pappin (Department of Biochemistry, University of Leeds) for amino-acid sequencing, and Martin Cornelius (Rothamsted Experimental Station, Harpenden, Herts., UK) for synthesis of oligonuleotides.     

Modulation of amino acid metabolism in transformed tobacco plants deficient in Fd-GOGAT

Tobacco (Nicotiana tabacum) plants expressing a partial ferredoxin-dependent glutamine-2-oxoglutarate aminotransferase (Fd-GOGAT) cDNA in the antisense orientation under the control of the 35S promoter, were used to study the metabolism of amino acids, 2-oxoglutarate and ammonium following the transition from CO₂ enrichment (where photorespiration is inhibited) to air (where photorespiration is a major process of ammonium production in leaves). The leaves of the lowest Fd-GOGAT expressors accumulated more foliar glutamine (Gln) and α-ketoglutarate (α-KG) than the untransformed controls in both growth conditions. Photorespiration-dependent increases in foliar ammonium, glutamine, α-KG and total amino acids were proportional to the decreases in foliar Fd-GOGAT activity. No change in endoprotease activity was observed following transfer to air in the Fd-GOGAT transformants or the untransformed controls which has similar activities over a broad range of pH values. We conclude that several pathways of amino acid biosynthesis are modified when NH₃ ⁺ and Gln accumulate in leaves. This revised version was published online in June 2006 with corrections to the Cover Date.     

Structure and expression of a nuclear gene for the PSI-D subunit of photosystem I inNicotiana sylvestris

The PSI-D subunit is the ferredoxin-binding site of photosystem I, and is encoded by the nuclear genepsaD. We isolated apsaD genomic clone fromNicotiana sylvestris, by screening a genomic library with apsaD cDNA which we previously cloned fromN. sylvestris (Yamamotoet al., Plant Mol Biol 17: 1251, 1991). Nucleotide sequence analysis revealed that this genomic clone contains apsaD gene, which does not correspond to thepsaD cDNA, so we designated these genespsaDb andpsaDa, respectively. ThepsaDb clone encodes a protein of 214 amino acids uninterrupted by introns. The N-terminal sequence determined for theN. sylvestris PSI-D protein encoded bypsaDb begins at the 49th residue. The products ofpsaDa andpsaDb share 82.7% and 79.5% identity at the amino acid and nucleotide levels, respectively. Genomic Southern analysis showed that two copies ofpsaD are present in theN. sylvestris genome. Ribonuclease protection assays and immunoblot analysis inN. sylvestris indicate that both genes are expressed in leaves, stems and flower buds, but neither is expressed in roots. During leaf development, the ratio ofpsaDb topsaDa mRNA increases from 0.12 in leaf buds to 0.36 in mature leaves. The relative abundance of the corresponding proteins decreased over the same developmental period. These results indicate that differential regulation mechanisms controlpsaDa andpsaDb expression at both the mRNA and protein levels during leaf development.     

Expression of soybean-embryo lipoxygenase 2 in transgenic tobacco tissue

To assess the role of lipoxygenase (LOX; EC 1.13.11.12) in plants, we increased the expression of LOX in the tissues of Nicotiana tabacum L. cv. KY 14 by over-expression of the LOX2 gene from the soybean (Glycine max (L.) Merrill) embryo. The LOX2 cDNA was manipulated by replacing its 5-untranslated sequence with the translational enhancer of the alfalfa mosaic virus (AMV), and subcloned into a plant expression vector, 3 to a duplicated cauliflower mosaic virus 35S promoter. The AMV-LOX2 construct was transferred into tobacco using Agrobacterium tumefaciens strain A281. The LOX2 was expressed in transgenic tobacco calli, leaves of transgenic plants, and their seed progeny at levels up to 0.1–0.2% of the total extracted protein. The introduced LOX2 affected fatty-acid oxidative metabolism as evidenced by a 50–529% increase in C₆-aldehyde production. The impact on C₆-aldehyde formation was greater than the effect on production of fatty-acid hydroperoxides. This is consistent with other studies indicating the greater propensity of soybean embryo LOX2 in generating C₆-aldehydes than that of other well-characterized LOX isozymes.Abbreviations AMV alfalfa mosaic virus - CaMV cauliflower mosaic virus - IEF isoelectric focusing - kDa kilodalton - LOX lipoxygenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We thank Bernard Axelrod (Purdue University) for supplying the lipoxygenase 2 cDNA, and Arthur G. Hunt (University of Kentucky) for supplying the pKYLX71² and pBS/AMV. The advice of Arthur G. Hunt, Chris L. Schardl, Sadik Tuzun and Dwight Tomes is greatly appreciated, as is the technical assistance of Udaya Chand and Robert Versluys.     

Photoregulation of germination in seed of transgenic lines of tobacco and Arabidopsis which express an introduced cDNA encoding phytochrome A or phytochrome B

Photoinduction and photoinhibition of germination in seed from a homozygous tobacco (Nicotiana tabacum L.) line containing an introduced oat phyA cDNA (encoding phytochrome A) is compared with that of isogenic wild-type (WT) tobacco. Under continuous irradiation by a light source with a low redfar-red (RFR) ratio the transgenic tobacco seed appeared to be less susceptible to photoinhibition of germination compared with WT seed. However, induction of germination following a short pulse by R (666 nm) was not enhanced in the genotype transformed by oat phyA cDNA compared with the WT; neither did germination of the transgenic tobacco seed show an increased sensitivity to saturating pulses of light of longer wavelengths (666–730 nm). In seeds of transgenic Arabidopsis thaliana (L.) Heynh. which contained an introduced phytochrome-B-encoding cDNA, levels of dark germination were enhanced, consistent with mediation of response by phytochrome B-P_fr. The germination behaviour of Arabidopsis genotypes wich contained an introduced cDNA encoding phytochrome A, however, did not significantly differ from that of the WT.Abbreviations ABO seed transformed with Arabidopsis phyB - cDNA; CaMV cauliflower mosaic virus - FR far-red light - P_fr far-red-absorbing form of phytochrome - P_tot total phytochrome - P_fr/P_tot phytochrome photoequilibrium - R red light - RBO seed transformed with rice phyB cDNA - RFR quantum ratio of red and far-red light - WL white light - WL + FR whitelight supplemented with far-red light - WT wild type The authors wish to thank R.D. Vierstra (Department of Horticulture, University of Wisconsin-Madison, USA) for providing the transgenic tobacco line, and M.T. Boylan, D. Wagner and P.H. Quail (U.C. Berkeley/USDA Plant Gene Expression Center, Albany, Calif. USA) for providing the transgenic Arabidopsis lines. The work presented in this paper was funded by grants from the Agricultural and Food Research Council (H.S., A.C.M., G.C.W.).     

Coaction of blue/ultraviolet-A light and light absorbed by phytochrome in controlling the appearance of ferredoxin-dependent glutamate synthase in the Scots pine (Pinus sylvestris L.) seedling

The appearance of NADH- and ferredoxin (Fd)-dependent glutamate synthases (GOGATs) was investigated in the major organs (roots, hypocotyl and cotyledonary whorl) of the Scots pine seedling. It was found that cytosolic NADH-GOGAT (EC 1.4.1.14) dropped to a low level during the experimental period (from 4 to 12 d after sowing) and was not significantly affected by light. On the other hand, plastidic Fd-GOGAT (EC 1.4.7.1) increased strongly in response to light. Whereas similar amounts of NADH-GOGAT were found in the different organs, Fd-GOGAT was mainly found in the cotyledons even in the presence of nitrate. Protein chromatography revealed only a single Fd-GOGAT peak. No isoforms were detected. Experiments to investigate regulation of the appearance of Fd-GOGAT in the cotyledonary whorl yielded the following results: (i) In darkness, neither nitrate (15 mM KNO₃) nor ammonium (15 mM NH₄Cl) had an effect on the appearance of Fd-GOGAT. In the light, nitrate stimulated Fd-GOGAT activity by 30% whereas ammonium had no effect. The major controlling factor is light. (ii) The action of long-term white light (100 W · m^–2) could be replaced quantitatively by blue light (B, 10 W · m^–2). Since the action of long-term far-red light was very weak, operation of the High Irradiance Reaction of phytochrome is excluded. On the other hand, light-pulse experiments with dark-grown seedlings showed the involvement of phytochrome. (iii) Red light, operating via phytochrome, could fully replace B, but only up to 10 d after sowing. Thereafter, there was an absolute requirement for B for a further increase in the enzyme level. It appears that the operation of phytochrome was replaced by the operation of cryptochrome (B/UV-A photoreceptor). (iv) However, dichromatic experiments (simultaneous treatment of the seedlings with two light beams to vary the level of the far-red-absorbing form of phytochrome (Pfr) in blue light) showed that B does not affect enzyme appearance if the Pfr level is low. It is concluded that B is required to maintain responsiveness of Fd-GOGAT synthesis to phytochrome (Pfr) beyond 10 d after sowing.Abbreviations and Symbols B blue light - c continuous - D darkness - Fd-GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - FR far-red light - HIR high-irradiance reaction of phytochrome - NADH-GOGAT nicotinamide-dinucleotide-dependent glutamate synthase (EC 1.4.1.14) - R red light - RG9 long-wavelength far-red light defined by the properties of the Schott glass filter (_RG9<0.01) - Pfr/Ptot far-red-absorbing form of phytochrome/total phytochrome, wavelength-dependent photoequilibrium of the phytochrome system Research supported by Deutsche Forschungsgemeinschaft (SFB 46 und Schwerpunkt Physiologie der Bäume). We thank E. Fernbach for his help with the dichromatic experiments.     

Monocotyledonous C4 NADP+ -malate dehydrogenase is efficiently synthesized,targeted to chloroplasts and processed to an active form in transgenic plants of the C3 dicotyledon tobacco

Chloroplastic NADP⁺-malate dehydrogenase (cpMDH, EC 1.1.1.82) is a key enzyme in the carbonfixation pathway of some C₄ plants such as the monocotyledons maize or Sorghum. We have expressed cpMDH from Sorghum vulgare Pers. in transgenic tobacco (Nicotiana tabacum L.) (a dicotyledonous C₃ plant) by using a gene composed of the Sorghum cpMDH cDNA under the control of cauliflower mosaic virus 35S promoter. High steady-state levels of cpMDH mRNA were observed in isogenic dihaploid transgenic tobacco lines. Sorghum cpMDH protein was detected in transgenic leaf extracts, where a threefold higher cpMDH activity could be measured, compared with control tobacco leaves. The recombinant protein was identical in molecular mass and in N-terminal sequence to Sorghum cpMDH. The tobacco cpMDH protein which has a distinct N-terminal sequence, could not be detected in transgenic plants. Immunocytochemical analyses showed that Sorghum cpMDH was specifically localized in transgenic tobacco chloroplasts. These data indicate that Sorghum cpMDH preprotein was efficiently synthesized, transported into and processed in tobacco chloroplasts. Thus, C₃-C₄ photosynthesis specialization or monocotyledon-dicotyledon evolution did not affect the chloroplastic proteinimport machinery. The higher levels of cpMDH in transgenic leaves resulted in an increase of l-malate content, suggesting that carbon metabolism was altered by the expression of the Sorghum enzyme.     

Cloning of a cDNA encoding 1-aminocyclopropane-1-carboxylate synthase and expression of its mRNA in ripening apple fruit

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) purified from apple (Malus sylvestris Mill.) fruit was subjected to trypsin digestion. Following separation by reversed-phase high-pressure liquid chromatography, ten tryptic peptides were sequenced. Based on the sequences of three tryptic peptides, three sets of mixed oligonucleotide probes were synthesized and used to screen a plasmid cDNA library prepared from poly(A)⁺ RNA of ripe apple fruit. A 1.5-kb (kilobase) cDNA clone which hybridized to all three probes were isolated. The clone contained an open reading frame of 1214 base pairs (bp) encoding a sequence of 404 amino acids. While the polyadenine tail at the 3-end was intact, it lacked a portion of sequence at the 5-end. Using the RNA-based polymerase chain reaction, an additional sequence of 148 bp was obtained at the 5-end. Thus, 1362 bp were sequenced and they encode 454 amino acids. The deduced amino-acid sequence contained peptide sequences corresponding to all ten tryptic fragments, confirming the identity of the cDNA clone. Comparison of the deduced amino-acid sequence between ACC synthase from apple fruit and those from tomato (Lycopersicon esculentum Mill.) and winter squash (Cucurbita maxima Duch.) fruits demonstrated the presence of seven highly conserved regions, including the previously identified region for the active site. The size of the translation product of ACC-synthase mRNA was similar to that of the mature protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that apple ACC-synthase undergoes only minor, if any, post-translational proteolytic processing. Analysis of ACC-synthase mRNA by in-vitro translation-immunoprecipitation, and by Northern blotting indicates that the ACC-synthase mRNA was undetectable in unripe fruit, but was accumulated massively during the ripening proccess. These data demonstrate that the expression of the ACC-synthase gene is developmentally regulated.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AdoMet S-adenosyl-l-methionine - HPLC high-pressure liquid chromatography - kDa kilodalton - kb kilobase - mAb monoclonal antibody - Met methionine - PCR polymerase chain reaction - poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by grants DCB-9004129 and INT-8915155 from the National Science Foundation.     

Organization,inheritance and expression of acetohydroxyacid synthase genes in the cotton allotetraploid Gossypium hirsutum

The acetohydroxyacid synthase (AHAS) gene family of the cotton AD allotetraploid Gossypium hirsutum has been cloned and characterized. We have identified six different AHAS genes from an analysis of genomic clones and Southern blots of genomic DNA. Four of the six genes are organized as tandem pairs, in which the genes are separated by only 2–3 kb. Conservation of restriction fragment length polymorphisms between G. hirsutum and A-genome and D-genome-containing diploid cottons was sufficient to assign the single genes in clones A5 and A19 to the A and D subgenomes, respectively. Each diploid genome has one tandem pair, but in these cases we could not make specific subgenomic assignments. DNA and deduced amino acid sequences were determined for the A5 and A19 genes, and an AHAS cDNA clone isolated from a leaflibrary. The sequence of the A19 gene matches that of the cDNA clone, while the A5 gene is 97.8% similar. The four genes comprising the tandem pairs are much less similar to the cDNA clone. The deduced amino acid sequences of the mature polypeptides encoded by the A5 and A19 genes are collinear with the housekeeping forms of AHAS from Arabidopsis thaliana, Nicotiana tabacum and Brassica napus. The constitutive expression of A5 and A19 was confirmed with RNase protection assays and northern blots. We conclude that these genes encode the main house-keeping froms of AHAS in G. hirsutum. Among the four AHAS genes comprising the two tandem pairs, at least two are functional. These genes exhibit either low-level constitutive expression (one or both of the downstream genes of each pair), or highly specific expression in reproductive tissue (one or both of the upstream genes of each pair). The AHAS gene family of G. hirsutum is more complex than that of other plants so far examined.     

Developmental,hormonal, and pathogenesis-related regulation of the tobacco class I β-1,3-glucanase B promoter

The class I -1,3-glucanases are antifungal vacuolar proteins implicated in plant defense that show developmental, hormonal, and pathogenesis-related regulation. The tobacco enzymes are encoded by a small gene family with members derived from ancestors related to the present-day species Nicotiana sylvestris and N. tomentosiformis. We studied the expression in transgenic tobacco plants of a chimeric -glucuronidase (GUS) reporter gene fused to 1.6 kb of upstream sequence of the tobacco class I -1,3-glucanase B (GLB) gene, which is of N. tomentosiformis origin. Expression of the GUS reporter gene and the accumulation of class I -1,3-glucanase and its mRNA showed very similar patterns of regulation. In young seedlings the reporter gene was expressed in the roots. In mature tobacco plants it was preferentially expressed in lower leaves and roots and was induced in leaves by ethylene treatment and by infection with tobacco mosaic virus (TMV). Furthermore, it was down-regulated in cultured leaf discs by combinations of the hormones auxin and cytokinin. Histological studies of GUS activity showed that the GLB promoter shows highly localized expression in roots of seedlings. It is also expressed in a ring of cells around necrotic lesions induced by TMV infection, but not in cells immediately adjacent to the lesions or in the lesions themselves. The results of deletion analyses suggest that multiple positive and negative elements in the GLB promoter regulate its activity. The region from –1452 to –1193 containing two copies of the heptanucleotide AGCCGCC, which is highly conserved in plant-stress and defense-related genes, is necessary for high level expression in leaves. Additional regions important for organ-specific and regulated expression were: –568 to –402 for ethylene induction of leaves; –402 to –211 for expression in lower leaves and cultured leaf discs and for TMV induction of leaves; and –211 to –60 for expression in roots.     

RFLP mapping of three new loci for resistance genes to powdery mildew (Erysiphe graminis f. sp. hordei) in barley

Three new major, race-specific, resistance genes to powdery mildew (Erysiphe graminis f. sp. hordei) were identified in three barley lines, RS42-6*O, RS137-28*E, and HSY-78*A, derived from crosses with wild barley (Hordeum vulgare ssp. spontaneum). The resistance gene origining from wild barley in line RS42-6*O, showed a recessive mode of inheritance, whereas the other wild barley genes were (semi)-dominant. RFLP mapping of these three genes was performed in segregating F₂ populations. The recessive gene in line RS42-6*O, was localized on barley chromosome 1S (7HS), while the (semi)-dominant genes in lines RS137-28*E, and HSY-78*A, were localized on chromosomes 1L (7HL) and 7L (5HL), respectively. Closely linked RFLP clones mapped at distances between 2.6cM and 5.3 cM. Hitherto, specific loci for powdery mildew resistance in barley had not been located on these chromosomes. Furthermore, tests for linkage to the unlocalized resistance gene Mlp revealed free segregation. Therefore, these genes represent new loci and new designations are suggested: mlt (RS42-6*O), Mlf (RS137-28*E), and Mlj (HSY-78*A). Comparisons with mapped QTLs for mildew resistance were made and are discussed in the context of homoeology among the genomes of barley (H-vulgare), wheat (Triticum aestivum), and rye (Secale cereale). Duplications of RFLP bands detected in the neighbourhood of Mlf and mlt might indicate an evolutionary interrelationship to the Mla locus for mildew resistance.     

Isolation of a novel RNA-binding protein and its association with a large ribonucleoprotein particle present in the nucleoplasm of tobacco cells

A cDNA encoding a protein with a consensus sequence-type RNA-binding domain (CS-RBD) has been isolated from a Nicotiana sylvestris cDNA library. The deduced protein (designated RZ-1) contains CS-RBD in its N-terminal half, arginine/aspartic acid repeats in its center and a glycine-rich C-terminal region in which a zinc finger motif of the CCHC type is present. The corresponding gene appears to be expressed constitutively in all tobacco organs. Immunocytochemical assays revealed that RZ-1 is localized in the nucleoplasm of tobacco cultured cells. Glycerol gradient fractionation of tobacco nuclear lysates showed that RZ-1 is associated with a large ribonucleoprotein particle of around 60 S in size. Nucleic acid-binding assays indicated that RZ-1 binds preferentially to poly (G) and both the CS-RBD and glycine-rich region are necessary for its binding activity. A possible role of RZ-1 is discussed.     

An S-RNase promoter from Nicotiana alata functions in transgenic N. alata plants but not Nicotiana tabacum

Nicotiana tabacum and Nicotiana alata plants were transformed with genomic clones of two S-RNase alleles from N. alata. Neither the S ₂ clone, with 1.6 kb of 5 sequence, nor the S ₆ clone, with 2.8 kb of 5 sequence, were expressed at detectable levels in transgenic N. tabacum plants. In N. alata, expression of the S ₂ clone was not detected, however the S ₆ clone was expressed (at low levels) in three out of four transgenic plants. An S ₆-promoter-GUS fusion gene was also expressed in transgenic N. alata but not N. tabacum. Although endogenous S-RNase genes are expressed exclusively in floral pistils, the GUS fusion was expressed in both styles and leaves.     

Detection of PR-b proteins in tobacco roots infected with Chalara elegans

All previous studies on pathogenesis-related (b) protein (PR-b) induction in tobacco have been carried out on leaves or callus tissue. This paper reports the production of PR-b proteins also in roots of tobacco plants (Nicotiana tabacum cv. Xanthi NC) infected with Chalara elegans. Antiserum against PR-b₁ reacted with PR-b₁, PR-b₂ and PR-b₃ and gave the same pattern of reaction as for leaves. Antiserum against PR-b₅ revealed the presence of PR-b₄, PR-b₅ and, very weakly, PR-b₆ which have been shown to be -1,3 glucanases. Antiserum against PR-b₇ reacted with both PR-b₇ and PR-b₈ which are chitinases.     

Purification of chloroplast elongation factor Tu and cDNA analysis in tobacco: the existence of two chloroplast elongation factor Tu species

We have purified a chloroplast elongation factor Tu (EF-Tu) from tobacco (Nicotiana tabacum) and determined its N-terminal amino acid sequence. Two distinct cDNAs encoding EF-Tu were isolated from a leaf cDNA library of N. sylvestris (the female progenitor of N. tabacum) using an oligonucleotide probe based on the EF-Tu protein sequence. The cDNA sequence and genomic Southern analyses revealed that tobacco chloroplast EF-Tu is encoded by two distinct genes in the nuclear genome of N. sylvestris. We designated the corresponding gene products EF-Tu A and B. The mature polypeptides of EF-Tu A and B are 408 amino acids long and share 95.3% amino acid identity. They show 75–78% amino acid identity with cyanobacterial and chloroplast-encoded EF-Tu species.