Purification and properties of tobacco ferredoxin-dependent glutamate synthase,and isolation of corresponding cDNA clones

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco (Nicotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C₁₆) from a tobacco gt11 expression library. A longer Fd-GOGAT cDNA clone (C₃₅) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco gt10 cDNA library using C₁₆ as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C₃₅ was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression.Abbreviations bp base pairs - Fd-GOGAT ferredoxin-dependent glutamate synthase - GS glutamine synthetase - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate The authors wish to thank Juan Luis Gómez Pinchetti (Marine Plant Biotechnology Laboratory) for his assistance during the experiments. This study was supported by grants received from SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Tryggers Fund for Scientific Research (K. Haglund), SJFR (Swedish Council for Forestry and Agricultural Research) (M. Björk, M. Pedersén), CITYT Spain (SAB 89-0091 and MAR 91-1237, M. Pedersén) and CICYT Spain (Z. Ramazanov, invited professor of Ministerio de Educatión y Ciencia, Spain). The planning of this cooperation was facilitated by COST-48.     

Two different modes of early development and nitrogen assimilation in gymnosperm seedlings†

Light-independent chloroplast development and expression of genes encoding chloroplast proteins occur in many but not all species of gymnosperms. Early development in maritime pine (Pinus pinaster) seedlings was strongly light-independent, whereas Ginkgo biloba seedlings exhibited a typical angiosperm-like morphogenesis with differentiated patterns in light and dark. In pine, chloroplast polypeptides were undetectable in the seed embryo and accumulated in cotyledons of both light- and dark-grown plants in good correlation with light-independent chlorophyll synthesis. In contrast, chlorophyll and chloroplast proteins were only detected in light-grown ginkgo. Pine cytosolic glutamine synthetase (GS) and ferredoxin glutamate synthase (Fd-GOGAT) were present at low levels in the seeds and accumulated at comparable amounts in light- and dark-grown seedlings. Fd-GOGAT was also barely detectable in the seeds of ginkgo and only accumulated in green plants with mature chloroplasts. In G. biloba seeds and etiolated plants only cytosolic GS was identified, while in light-grown seedlings this molecular form was present at low abundance and choroplastic GS was the predominant isoenzyme. The above results have been confirmed by immunolocalization of GS protein in pine and ginkgo plantlets. In pine, GS was present in the peripheral cytoplasm of mesophyll cells and also in the phloem region of the vascular bundle. Immunocytochemical analysis showed that the labelling of mesophyll and phloem cells was only cytoplasmic. In developing ginkgo, GS antigens were present in the chloroplasts of mesophyll parenchyma cells of leaflets and green cotyledons. In contrast, a weak labelling of GS was observed in the parenchyma and phloem cells of non-green cotyledons enclosed in the seed coat. Taking all this into account, our data indicate the existence of two different modes of GS and GOGAT regulation in gymnosperms in close correlation with the differential response of plants to light. Furthermore, the results suggest that glutamine and glutamate biosynthesis is confined to the chloroplast of mesophyll cells in species with light-dependent chloroplast, development whereas compartmentation would be required in species with light-independent plastid development.     

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     

Molecular characterization of a cDNA clone encoding glutamine synthetase from a gymnosperm,Pinus sylvestris

A full-length cDNA clone (pGSP114) encoding glutamine synthetase was isolated from a gt11 library of the gymnosperm Pinus sylvestris. Nucleotide sequence analysis showed that pGSP114 contains an open reading frame encoding a protein of 357 amino acid residues with a calculated molecular mass of 39.5 kDa. The derived amino acid sequence was more homologous to cytosolic (GS1) (78–82%) than to chloroplastic (GS2) (71–75%) glutamine synthetase in angiosperms. The lack of N-terminal presequence and C-terminal extension which define the primary structure of GS2, also supports that the isolated cDNA encodes cytosolic GS. Southern blot analysis of genomic DNA from P. sylvestris and P. pinaster suggests that GS may be encoded by a small gene family in pine. GS mRNA was more abundant in cotyledons and stems than in roots of both Scots and maritime pines. Western blot analysis in P. sylvestris seedlings showed that only one GS polypeptide, similar in size to GS1 in P. pinaster, could be detected in several different tissues. Our results suggest that cytosolic GS is mainly responsible for glutamine biosynthesis in pine seedlings.This paper is dedicated to the memory of Dr. Jesús S. Olavarría.     

Location of two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L.

The two isoenzymes of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), previously identified in root nodules of Phaseolus vulgaris L., have both been shown to be located in root-nodule plastids. The nodule specific NADH-GOGAT II accounts for the majority of the activity in root nodules, and is present almost exclusively in the central tissue of the nodule. However about 20% of NADH-GOGAT I activity is present in the nodule cortex, at about the same specific activity as this isoenzyme is found in the central tissue. Glutamine synthetase (GS; EC 6.3.1.2) occurs predominantly as the polypeptide in the central tissue, whereas in the cortex, the enzyme is represented mainly by the polypeptide. Over 90% of both GS and NADH-GOGAT activities are located in the central tissue of the nodule and GS activity exceeds NADH-GOGAT activity by about twofold in this region. Using the above information, a model for the subcellular location and stoichiometry of nitrogen metabolism in the central tissue of P. vulgaris root nodules is presented.Abbreviations Fd-GOGAT ferredoxin-dependent glutamate synthase - GOGAT glutamate synthase - GS glutamine synthetase - NADH-GOGAT NADH-dependent glutamate synthase - IEX-HPLC ion-exchange high-performance liquid chromatography     

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.     

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     

Glutamine synthetase in Scots pine seedlings and its control by blue light and light absorbed by phytochrome

The appearance of glutamine synthetase (GS. EC 6.3.1.2) in response to light and nitrogen (NO ₃ ^- , NH ₄ ⁺ ) was studied in the organs (roots, hypocotyl, cotyledonary whorl) of the Scots pine (Pinus sylvestris L.) seedling. Although GS activity was found to be mainly (> 80%) located in the whorl where it increased strongly in response to light, a significant GS synthesis was also detected in dark-grown seedlings. Anion-exchange chromatography was used to resolve two GS isoforms which appeared to be regulated differentially in the cotyledonary whorls. The isoform (presumably plastidic GS2) which eluted from the column at 90 mM KCl increased drastically in response to light. The other isoform (presumably cytosolic GS1), which eluted at 200 mM KCl, was not stimulated by light but tended to disappear during the experimental period (4 to 12 d after sowing). Immunoblotting of pine extract yielded a prominent band with a molecular weight of 43 kDa. The linear correlation between GS activity and immunodetectable GS protein could be extrapolated through zero, showing that any increase of GS2 activity is to be attributed to the de-novo synthesis of GS protein. Gelfiltration chromatography yielded a molecular mass for the GS holoenzyme of 340 kDa, a value which supports an octameric quarternary structure as previously suggested for angiosperms. While supplying seedlings with 10 mM NO ₃ ^- stimulated GS synthesis in the whorl by 12%, 10 mM NH ₄ ⁺ caused an incipient ammonium toxicity. Experiments using dischromatic light (simultaneous treatment with two light beams to vary the level of the physiologically active form of phytochrome, Pfr, in blue light) revealed that synthesis of GS2 was controlled by light in the same way as previously shown for ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1). Up to 10 d after sowing the strong light effect could be attributed to phytochrome action whereas between 10 and 12 d after sowing phytochrome control of GS-synthesis failed if no blue/ultraviolet-A light was provided. The data show that blue light is required to maintain responsiveness of GS2 synthesis to phytochrome. Both enzymes, GS2 as well as Fd-GOGAT, appear to be regulated coordinately to meet the demands of ammonium assimilation.Abbreviations and Symbols B blue light - D darkness - Fd-GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1); - GS glutamine synthetase (EC 6.3.1.2) - R red light - RG9 long-wavelength far-red light defined by the properties of Schott glass filter RG9 - =Pfr/Ptot far-red absorbing form of phytochrome/total phytochrome, wavelength-dependent photoequilibrium of the phytochrome system Research supported by Deutsche Forschungsgemeinschaft (SFB 46 and Schwerpunkt Physiologie der Bäume). We thank J.M. Penther, (Institut für Biologie II, Freiburg, FRG) for his advice on the chromatographic techniques.     

The rate of nuclear gene transcription in barley endosperm syncytia increases sixfold before cell-wall formation

In seedlings of the Scots pine (Pinus sylvestris L.), alanine aminotransferase (AlAT EC 2.6.1.2.) is present in the shoot and in the primary root but most activity is found in the cotyledons. During the experimental period (from 6 to 12 d after sowing), AlAT activity increased steadily. Anion exchange chromatography and native polyacrylamide gel electrophoresis were used to show that AlAT activity in extracts from cotyledons is associated with two isoforms of the enzyme. One isoform (AlAT 1) dominated in the cotyledons of lightgrown seedlings, but was absent from primary roots. Its accumulation was strongly increased by light, and both phytochrome and cryptochrome were shown to be involved in this effect. Results of experiments using dichromatic irradiation indicate that cryptochrome acts indirectly by establishing responsiveness towards phytochrome. When plastids were damaged by photooxidation, the accumulation of AlAT 1 decreased; however, AlAT 1 which had accumulated before the onset of photooxidative treatment seemed to remain undamaged. Therefore, and because of the absence of AlAT 1 from primary roots, it is suggested that this isoform is localized in leaf peroxisomes. The isoform AlAT 2 is the only one found in primary roots, and the predominant one in the cotyledons of dark-grown seedlings. It is unaffected by light. Upon photodestruction of plastids, a pronounced increase of its activity was found. This is taken as evidence that AlAT 2 is a cytosolic enzyme. Total AlAT activity in cotyledons was unaffected by feeding nitrate to the seedlings; supplying exogenous ammonium led to a considerably slower accumulation of AlAT compared with water controls. In contrast, AlAT accumulation in the primary roots was augmented by up to 45% if nitrogenous ions were supplied, ammonium being more effective than nitrate.Abbreviations and Symbols AlAT alanine aminotransferase (EC 2.6.1.2.) - B blue light - c continuous - D darkness - Fd-GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1.) - FR far-red light - HPR hydroxypyruvate reductase (EC 1.1.1.81.) - FPLC fast protein liquid chromatography - PAGE polyacrylamide gel electrophoresis - R red light - RG9 long-wavelength far-red light defined by the properties of the Schott glass filter RG9 (_RG9 < 0.01) - =Pfr/Ptot far-red-absorbing form of phytochrome/total phtochrome, wavelength-dependent photoequilibrium of the phytochrome system This work was supported by Heidelberger Akademie der Wissenschaften (Forschungsstelle Nitratassimilation). We are very grateful to Ms. B. Seith for measuring the DNA contents of the seedlings.     

Regulation of loblolly pine (Pinus taeda L.) arginase in developing seedling tissue during germination and post-germinative growth

After seed germination, hydrolysis of storage proteins provides a nitrogen source for the developing seedling. In conifers the majority of these reserves are located in the living haploid megagametophyte tissue. In the developing loblolly pine (Pinus taeda L.) seedling an influx of free amino acids from the megagametophyte accompanies germination and early seedling growth. The major component of this amino acid pool is arginine, which is transported rapidly and efficiently to the seedling without prior conversion. This arginine accounts for nearly half of the total nitrogen entering the cotyledons and is likely a defining factor in early seedling nitrogen metabolism. In the seedling, the enzyme arginase is responsible for liberating nitrogen, in the form of ornithine and urea, from free arginine supplied by the megagametophyte. In this report we investigate how the seedling uses arginase to cope with the large arginine influx. As part of this work we have cloned an arginase cDNA from a loblolly pine expression library. Analysis of enzyme activity data, accumulation of arginase protein and mRNA abundance indicates that increased arginase activity after seed germination is due to de novo synthesis of the enzyme. Our results suggest that arginase is primarily regulated at the RNA level during loblolly pine seed germination and post-germinative growth.     

Chloroplast chlB gene is required for light-independent chlorophyll accumulation in Chlamydomonas reinhardtii

Light-independent chlorophyll synthesis occurs in some algae, lower plants, and gymnosperms, but not in angiosperms. We have identified a new chloroplast gene, chlB, that is required for the light-independent accumulation of chlorophyll in the green alga Chlamydomonas reinhardtii. The chlB gene was cloned, sequenced, and then disrupted by performing particle gun-mediated chloroplast transformation. The resulting homoplasmic mutant was unable to accumulate chlorophyll in the dark and thus exhibited a yellow-in-the-dark phenotype. The chlB gene encodes a polypeptide of 688 amino acid residues, and is distinct from two previously characterized chloroplast genes (chlN and chlL) also required for light-independent chlorophyll accumulation in C. reinhardtii. Three unidentified open reading frames in chloroplast genomes of liverwort, black pine, and Chlamydomonas moewusii were also identified as chlB genes, based on their striking sequence similarities to the C. reinhardtii chlB gene. A chlB-like gene is absent in chloroplast genomes of tobacco and rice, consistent with the lack of light-independent chlorophyll synthesis in these plants. Polypeptides encoded by the chloroplast chlB genes also show significant sequence similarities with the bchB gene product of Rhodobacter capsulatus. Comparisons among the chloroplast chlB and the bacterial bchB gene products revealed five highly conserved sequence areas that are interspersed by four stretches of highly variable and probably insertional sequences.     

Characterization of cDNAs encoding CuZn-superoxide dismutases in Scots pine

A Scots pine (Pinus sylvestris L.) cDNA library was screened with two heterologous cDNA probes (P31 and T10) encoding cytosolic and chloroplastic superoxide dismutases (SOD) from tomato. Several positive clones for cytosolic and chloroplastic superoxide dismutases were isolated, subcloned, mapped and sequenced. One of the cDNA clones (PS3) had a full-length open reading frame of 465 bp corresponding to 154 amino acid residues and showed approximately 85% homology with the amino acid sequences of angiosperm cytosolic SOD counterparts. Another cDNA clone (PST13) was incomplete, but encoded a putative protein with 93% homology to pea and tomato chloroplastic superoxide dismutase. The derived amino acid sequence from both cDNA clones matched the corresponding N-terminal amino acid sequence of the purified mature SOD isozymes. Northern blot hybridizations showed that, cytosolic and chloroplastic CuZn-SOD are expressed at different levels in Scots pine organs. Sequence data and Southern blot hybridization confirm that CuZn-SODs in Scots pine belong to a multigene family. The results are discussed in relation to earlier observations of CuZn-SODs in plants.     

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.     

Chloroplast DNA of black pine retains a residual inverted repeat lacking rRNA genes: nucleotide sequences of trnQ, trnK, psbA, trnI and trnH and the absence of rps16

Summary A physical map of black pine (Pinus thunbergii) chloroplast DNA (120 kb) was constructed and two separate portions of its nucleotide sequence were determined. One portion contains trnQ-UUG, ORF510, ORF83, trnK-UUU (ORF515 in the trnK intron), ORF22, psbA, trnI-CAU (on the opposing strand) and trnH-GUG, in that order. Sequence analysis of another portion revealed the presence of a 495 by inverted repeat containing trnI-CAU and the 3 end of psbA but lacking rRNA genes. The position of trnI-CAU is unique because most chloroplast DNAs have no gene between psbA and trnH (trnI-CAU is usually located further downstream). Black pine chloroplast DNA lacks rps16, which has been found between trnQ and trnK in angiosperm chloroplast DNAs, but possesses ORF510 instead. This ORF is highly homologous to ORF513 found in the corresponding region of liverwort chloroplast DNA and ORF563 located downstream from trnT in Chlamydomonas moewusii chloroplast DNA. A possible pathway for the evolution of black pine chloroplast DNA is discussed.     

Cloning of an Arabidopsis thaliana cDNA encoding cystathionine β-lyase by functional complementation in Escherichia coli

Cystathionine -lyase, the second enzyme involved in the methionine biosynthetic pathway in plants, catalyses the synthesis of homocysteine from cystathionine. A cDNA encoding cystathionine -lyase was cloned from an Arabidopsis thaliana expression library by complementation of an Escherichia coli mutant deficient in this enzyme. As deduced from the full-length nucleotide sequence (1.7 kb), the polypeptide contains 464 amino acids and presents a predicted M _r of 50372. A. thaliana cystathionine -lyase exhibits 22% sequence identity with the E. coli corresponding enzyme and contains a 70 amino acid N-terminal additional sequence compared with the bacterial protein. Since the general features of chloroplast transit peptides could be observed in this amino-terminal extension, we propose a chloroplast localization for the cDNA-encoded enzyme. Southern blot analysis suggested that cystathionine -lyase is encoded by a single copy gene in A. thaliana.     

Immunocharacterization of Vitis vinifera L. ferredoxin-dependent glutamate synthase,and its spatial and temporal changes during leaf development

The grapevine (Vitis vinifera L.) partial fragment of cDNA clone pGOGAT1 [Loulakakis and Roubelakis-Angelakis (1997) Physiol Plant 101:220-228], encoding the ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1), was overexpressed in Escherichia coli cells. A hybrid between the Fd-GOGAT fragment and maltose-binding protein was purified and used to raise a polyclonal antibody in a rabbit. The prepared antibody appeared to be specific towards Fd-GOGAT; it recognized a protein band of approximately 160 kDa on nitrocellulose blots after SDS-PAGE of total proteins from leaves, internodes, roots and calluses, and precipitated most of the enzyme activity present in grapevine protein extracts. The quantity of Fd-GOGAT protein was substantially higher in leaves than in other grapevine tissues tested, coincident with a similar distribution of the enzyme specific activity. Intracellular localization studies revealed that both the enzyme activity and the 160-kDa immunoreactive protein were associated with the chloroplastic fraction. Furthermore, the accumulation of Fd-GOGAT, glutamine synthetase (GS) and glutamate dehydrogenase (GDH), at the activity and protein levels, was monitored during leaf development of field-grown plants, from the stage of the newly expanding leaf to the senescing old leaf. Both the specific activity and quantity of the 160-kDa polypeptide of Fd-GOGAT were higher in the mature, full sized leaves and substantially lower in young and senescing leaves. GS specific activity and immunoreactive protein followed the same trend as Fd-GOGAT, while GDH showed opposite developmental patterns of accumulation. The biological significance of the presence of Fd-GOGAT in the various grapevine tissues and its physiological role during early development and natural senescence of the leaves are discussed.     

Homologues of the green algal gidA gene and the liverwort frxC gene are present on the chloroplast genomes of conifers

Strong hybridization signals were obtained from total DNA of two conifers, lodgepole pine (Pinus contorta) and Norway spruce (Picea abies), in a Southern blot analysis using a probe derived from the chloroplast gidA gene of the green alga Chlamydomonas reinhardtii. The pine fragments detected by the probe were found to originate from the chloroplast genome and, as judged by the signal intensity, this was also true for the spruce fragments. Sequence analysis of the hybridizing pine chloroplast DNA region revealed an open reading frame potentially encoding a 459 amino acid polypeptide, highly homologous to that deduced from the algal gene and to ORF465 of liverwort chloroplast DNA. Upstream of the gidA sequence, we found a trnN(GUU) gene and an open reading frame of 291 codons which was 78% identical to the frxC gene of liverwort. Since ORF465 is located immediately downstream of trnN and frxC in liverwort, the genetic organization of this region is very similar in the two plants. In contrast, neither the gidA nor the frxC gene is present in the chloroplast DNA of tobacco or rice. It was recently reported that deletions in the gidA region of the chloroplast genome of Chlamydomonas reinhardtii abolish the light-independent pathway of chlorophyll synthesis which exists in many algae and lower plants. The presence of the gidA gene on the chloroplast genomes of conifers may therefore be of significance with respect to the ability of these plants to synthesize chlorophyll in the dark.     

Are NADP-dependent isocitrate dehydrogenases and ferredoxin-dependent glutamate synthase co-regulated by the same photoreceptors?

Activities of NADP-dependent isocitrate dehydrogenases (cytosolic and plastidic isoforms, ICDH1 and ICDH2; EC 1.1.1.42) and ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) in turions of Spirodela polyrhiza were all stimulated by light. Single or repeated red light (R) pulses induced the activity of the enzymes and this effect was reverted by subsequent far-red light (FR) pulses. The enzymes are, therefore, co-regulated by the low-fluence response of phytochrome. For ICDH, this is reported here for the first time. Neither an effect of the very low-fluence response nor of the FR-mediated high-irradiance response was detectable. Irradiance with continuous R resulted in enhanced enzyme activities and protein levels (Western analysis using polyclonal antibodies against ICDH1 and Fd-GOGAT). These additional effects of continuous R (called a non-induction effect) could be inhibited for ICDH1 and ICDH2 by the inhibitor of photosynthetic electron transport, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and are therefore related to the effect of photosynthesis. In contrast, the non-induction effect of Fd-GOGAT was resistant against this inhibitor. Moreover, hourly R pulses did not replace the effect of continuous R. The non-induction effect of light on the activity and protein level of Fd-GOGAT was therefore tentatively classified as an R-mediated high-irradiance response. The activity of Fd-GOGAT but not that of ICDHs was additionally regulated by a specific blue-light receptor. It can be concluded that the levels of ICDHs and Fd-GOGAT were coordinated by light but were not co-regulated by the same photoreceptors. Nitrate is necessary for the light regulation of both enzymes, contributing to the coordinated expression of the relevant genes.Abbreviations DCMU 3-(3,4-Dichlorophenyl)-1,1-dimethylurea - Fd-GOGAT Ferredoxin-dependent glutamate synthase - FR Far-red light - HIR High-irradiance response - ICDH NADP-dependent isocitrate dehydrogenase - ICDH1 Cytosolic ICDH - ICDH2 Chloroplastic ICDH - LFR Low-fluence response - R Red light - SDS–PAGE Denaturing polyacrylamide gel electrophoresis - VLFR Very low-fluence response     

Type I and Type II genes for the chlorophyll a/b-binding protein in the gymnosperm Pinus sylvestris (Scots pine): cDNA cloning and sequence analysis

A cDNA library was constructed from mRNA prepared from light-treated seedlings of Scots pine (Pinus sylvestris L.) and cDNAs for the chlorophyll a/b-binding protein LHC-II were identified using a pea gene as the heterologous probe. Three cDNA clones were sequenced. The deduced amino acid sequences of two of the genes corresponded to Type I and one to Type II LHC-II proteins which were ca. 90% homologous to their angiosperm counterparts. The transit peptides of the Scots pine preLHC-II showed features common to angiosperm transit peptides. The three cDNAs had a 70 to 75% preference for G+C in the third base position. CpG and GpC profiles and degenerate codon position bias suggested that two of the corresponding genes lie within CpG islands.