Coproporphyrinogen III oxidase from barley and tobacco — sequence analysis and initial expression studies

Coproporphyrinogen III oxidase (coprogen oxidase; EC 1.3.3.3) is part of the pathway from 5-aminolevulinate to protoporphyrin IX which is common in all organisms and catalyses oxidative decarboxylation at two tetrapyrrole side chains. We cloned and sequenced fulllength cDNAs encoding coprogen oxidase from barley (Hordeum vulgare L.) and tobacco (Nicotiana tabacum L.). They code for precursor peptides of 43.6 kDa and 44.9 kDa, respectively. Import into pea plastids resulted in a processed tobacco protein of approx. 39 kDa, which accumulated in the stroma fraction. Induction of synthesis of recombinant putative tobacco mature coprogen oxidase consisting of 338 amino-acid residues in Escherichia coli at 20°C result in a catalytically active protein of approx. 39 kDa, while induction of its formation at 37°C immediately terminated bacterial growth, possibly due to toxic effects on the metabolic balance of tetrapyrrole biosynthesis. The plant coprogen oxidase gene was expressed to different extents in all tissues investigated. This is most likely due to the differing requirements for tetrapyrroles in different organs. The steady-state level of mRNA did not significantly differ in etiolated and greening barley leaves. The content of coprogen oxidase RNA reached its maximum in developing cells and decreased drastically when cells were completely differentiated. Functioning of the two photosystems apparatus requires the synthesis of all pigment and protein components during plant development. It is speculated that the enzymes involved in tetrapyrrole synthesis are developmentally rather than light-dependently regulated. Regulation of these enzymes also guarantees a constant flux of metabolic intermediates and avoids photodynamic damage by accumulating porphyrins. Accession number: The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X82830 (barley coprogen oxidase) and X82831 (tobacco coprogen oxidase).     

Reduction of Uroporphyrinogen Decarboxylase by Antisense RNA Expression Affects Activities of Other Enzymes Involved in Tetrapyrrole Biosynthesis and Leads to Light-Dependent Necrosis

We introduced a full-length cDNA sequence encoding tobacco (Nicotiana tabacum) uroporphyrinogen III decarboxylase (UROD; EC 4.1.1.37) in reverse orientation under the control of a cauliflower mosaic virus 35S promoter derivative into the tobacco genome to study the effects of deregulated UROD expression on tetrapyrrole biosynthesis. Transformants with reduced UROD activity were characterized by stunted plant growth and necrotic leaf lesions. Antisense RNA expression caused reduced UROD protein levels and reduced activity to 45% of wild type, which was correlated with the accumulation of uroporphyrin(ogen) and with the intensity of necrotic damage. Chlorophyll levels were only slightly reduced (up to 15%), indicating that the plants sustained cellular damage from accumulating photosensitive porphyrins rather than from chlorophyll deficiency. A 16-h light/8-h dark regime at high-light intensity stimulates the formation of leaf necrosis compared with a low-light or a 6-h high-light treatment. Transgenic plants grown at high light also showed inactivation of 5-aminolevulinate dehydratase and porphobilinogen deaminase, whereas the activity of coproporphyrinogen oxidase and the 5-aminolevulinate synthesizing capacity were not altered. We conclude that photooxidation of accumulating uroporphyrin(ogen) leads to the generation of oxygen species, which destabilizes other enzymes in the porphyrin metabolic pathway. This porphyrin-induced necrosis resembles the induction of cell death observed during pathogenesis and air pollution.     

A barley cDNA clone encoding a type III chlorophyll a/b-binding polypeptide of the light-harvesting complex II

The nucleotide sequence of a leaf cDNA clone encoding a Type III chlorophyll a/b-binding (CAB) protein of light-harvesting complex II (LHCII) in barley is reported. Sequence comparisons and results from in vitro import into chloroplasts demonstrate that the cDNA clone encodes a functional transit peptide of 45 amino acid residues and a mature polypeptide of 223 residues with a predicted molecular mass of 24.3 kDa. After insertion into thylakoids, the mature protein is resistant to protease attack. Hybridization analysis using a gene-specific probe shows that the gene is expressed in dark-grown seedlings and that the amount of mRNA increases during illumination.     

Comparison of two assay methods for activities of uroporphyrinogen decarboxylase and coproporphyrinogen oxidase

Uroporphyrinogen decarboxylase (UROD) and coproporphyrinogen oxidase (copro'gen oxidase) are two of the least well understood enzymes in the heme biosynthetic pathway. In the fifth step of the pathway, UROD converts uroporphyrinogen III to coproporphyrinogen III by the decarboxylation of the four acetic acid side chains. Copro'gen oxidase then converts coproporphyrinogen III to protoporphyrinogen IX via two sequential oxidative decarboxylations. Studies of these two enzymes are important to increase our understanding of their mechanisms. Assay comparisons of UROD and copro'gen oxidase from chicken blood hemolysates (CBH), using a newly developed micro-assay, showed that the specific activity of both enzymes is increased in the micro-assay relative to the large-scale assay. The micro-assay has distinct advantages in terms of cost, labor intensity, amount of enzyme required, and sensitivity.     

Cloning and characterization of cDNAs coding for Vicia faba polyphenol oxidase

Three cDNA clones were isolated which code for the ubiquitous chloroplast enzyme, polyphenol oxidase (PPO), from Vicia faba. Analysis of the cloned DNA reveals that PPO is synthesized with an N-terminal extension of 92 amino acid residues, presumed to be a transit peptide. The mature protein is predicted to have a molecular mass of 58 kDa which is in close agreement to the molecular mass estimated for the in vivo protein upon SDS-PAGE. Differences in the DNA sequence of two full-length and one partial cDNA clones indicate that PPO is encoded by a gene family. Analysis of the deduced amino acid sequence shows that the chloroplast PPO shares homology with the 59 kDa PPOs in glandular trichomes of solanaceous species. A high degree of sequence conservation was found with the copper-binding domains of the 59 kDa tomato PPO as well as hemocyanins and tyrosinases from a wide diversity of taxa.     

Mg-chelatase of tobacco: identification of a Chl D cDNA sequence encoding a third subunit, analysis of the interaction of the three subunits with the yeast two-hybrid system, and reconstitution of the enzyme activity by co-expression of recombinant CHL D, CHL H and CHL I

Mg-protoporphyrin IX chelatase catalyzes insertion of the magnesium ion into protoporphyrin IX, the last common intermediate precursor in chlorophyll and heme biosynthesis, to form Mg-protoporphyrin IX. In Rhodobacter sphaeroides, and Synechocystis, the three open reading frames bchD/chlD, bchH/chlH and bchl/chll encode proteins which are required for in vitro Mg-chelatase activity. In higher plants also, three proteins are necessary for the Mg chelation, and genes homologous to bchH and bchl have been isolated previously. In this study, a novel tobacco cDNA sequence homologous to bchD is isolated and initially characterized. Together with the tobacco clones encoding the other two subunits, full-length cDNAs are now available for the first time for all three subunits of one plant species. The CHL D polypeptide deduced from the open reading frame encodes a protein of 758 aa (82.9 kDa) with an amino terminal extension that resembles a plastid transit peptide. Sequence comparison of tobacco CHL D revealed similarities to the D subunit of Rhodobacter and Synechocystis of 44% and 75%. The amino terminal half of CHL D shows significant similarity (46%) to the entire CHL I peptide sequence, indicating a gene duplication from an ancestral gene. The carboxy terminal half seemed to be unique. Both parts of CHL D are linked with a glutamine/asparagine/proline-rich region flanked by a highly acid-rich segment. Protein-protein interaction among the three subunits CHL D, H and I was studied using the yeast two-hybrid system. Physical interaction was demonstrated between CHL D and CHL I indicating that CHL D is part of the Mg-chelatase. Heterodimer formation of CHL H with CHL I or CHL D could not be demonstrated by transactivation of the lacZ reporter gene. Homodimerization of the CHL D subunit was indicated in the more sensitive assay on X-Gal-containing agar plates. In vitro Mg²⁺ insertion into protoporphyrin IX was demonstrated in protein extracts of yeast strains expressing the three subunits of tobacco Mg-chelatase. The reconstitution of the recombinant enzyme activity required additional ATP.     

Identification of a mutation in the ovine uroporphyrinogen decarboxylase (UROD) gene associated with a type of porphyria

Porphyria is a group of at least eight diseases caused by abnormalities in the chemical steps that lead to haeme production. The different types of porphyria show different signs and symptoms and can be strongly influenced by environmental factors. Mutations of the uroporphyrinogen decarboxylase (UROD) gene have been shown to be causative for porphyria cutanea tarda (PCT) in humans. Porphyria is a rare disorder in livestock. Although disorders of haeme biosynthesis have been described in cattle, pigs, sheep and cats, PCT has only been reported in pigs. We observed typical signs of porphyria (photosensitivity and porphyrinuria) in a flock of German Blackface sheep and postulated that the porphyria could be caused by a mutation in the UROD gene. To investigate this, we cloned and sequenced the ovine UROD gene. We identified a single point mutation (C --> T) in UROD which leads to an amino acid substitution at Leu 131 Pro, which is located within the active cleft site of the UROD protein.     

Influence of cesium on tetrapyrrole biosynthesis in etiolated and greening barley leaves

Cesium chloride (CsCl) treatment of greening primary leaves of barley for 8 h inhibited chlorophyl] accumulation in a concentration-dependent manner and led to the accumulation of excessive amounts of uroporphyrin(ogen) III (URO[gen]) and to a minor extent of heptacarboxylporphyrin(ogen). When dark-grown leaves were incubated with CsCl, accumulation of URO(gen) was observed only after feeding of the tetrapyrrole precursor 5-aminolevulinic acid. Western blot analysis showed no apparent difference in content of uroporphyrinogen decarboxylase (EC 4.1.1.37, UROD) or selected proteins involved in tetrapyrrole biosynthesis in extracts of CsCl-incubated (15 m M ) versus control leaves. UROD activity was drastically decreased upon CsCl treatment in leaves incubated in the dark or in the light (44 and 86%, respectively). Selected preceding enzymes of the tetrapyrrole biosynthetic pathway, 5-aminolevulinic acid dehydratase (EC 4.2.1.24, ALAD) and porphobilinogen deaminase (EC 4.3.1.8, PBGD), were influenced only to a minor extent under standard incubation conditions (15 m M CsCl). Furthermore, the ALA synthesizing capacity did not differ in leaves incubated with and without Cs⁻ cations. UROD activity of crude homogenates from control plants and after partial purification was reduced to 56 and 80%, respectively, upon addition of 10 m M CsCl. Equal concentrations of KCl were not inhibitory. Enzyme assays of the same barley extract in the presence of CsCl yielded no effect on ALAD and a minor loss of PBGD activity. The initial visible cytotoxic effect of CsCl appeared to be a selective inhibition of UROD resulting in accumulation of photosensitizing URO (gen). Consequences of the diminished UROD activity on early steps of the tetrapyrrole biosynthesis and its functional and regulatory significance for the porphyrin synthesis are discussed.     

Partial Purification and N-Terminal Amino Acid Sequencing of a β-1,3-Glucanase from Sorghum Leaves

A protein with an apparent molecular mass of 30 kDa that cross-reacts with barley glucanase antiserum was detected in healthy leaves of sorghum (Sorghum bicolor (L.) Moench). When sorghum leaves were infected with Exserohilum turcicum, the causal agent of leaf blight, the 30-kDa glucanase was substantially induced. The 30-kDa glucanase was partially purified from sorghum leaves using ammonium sulfate fractionation and anion exchange chromatography on DEAE-sephacel. The N-terminal amino acid sequence of the 30-kDa glucanase shows homology to glucanases of maize, barley, bean, soybean, tobacco and pea. The purified 30-kDa glucanase showed antifungal activity against Trichoderma viride.     

Expression of the ribosome-inactivating protein JIP60 from barley in transgenic tobacco leads to an abnormal phenotype and alterations on the level of translation

In this paper we report the in-planta activity of the ribosome-inactivating protein JIP60, a 60-kDa jasmonate-induced protein from barley (Hordeum vulgare L.), in transgenic tobacco (Nicotiana tabacum L.) plants. All plants expressing the complete JIP60 cDNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited conspicuous and similar phenotypic alterations, such as slower growth, shorter internodes, lanceolate leaves, reduced root development, and premature senescence of leaves. Microscopic inspection of developing leaves showed a loss of residual meristems and higher degree of vacuolation of mesophyll cells as compared to the wild type. When probed with an antiserum which was immunoreactive against both the N- and the C-terminal half of JIP60, a polypeptide with a molecular mass of about 30 kDa, most probably a processed JIP60 product, could be detected. Phenotypic alterations could be correlated with the differences in the detectable amount of the JIP60 mRNA and processed JIP60 protein. The protein biosynthesis of the transformants was characterized by an increased polysome/monosome ratio but a decreased in-vivo translation activity. These findings suggest that JIP60 perturbs the translation machinery in planta. An immunohistological analysis using the JIP60 antiserum indicated that the immunoreactive polypeptide(s) are located mainly in the nucleus of transgenic tobacco leaf cells and to a minor extent in the cytoplasm. Received: 31 July 1996 / Accepted: 18 February 1997     

Characterisation and functional analysis of two barley caleosins expressed during barley caryopsis development

Two full-length cDNA sequences homologous to caleosin, a seed-storage oil-body protein from sesame, were identified from a series of barley grain development cDNA libraries and further characterised. The cDNAs, subsequently termed HvClo1 and HvClo2, encode proteins of 34 kDa and 28 kDa, respectively. Real-time RT-PCR indicated that HvClo1 is expressed abundantly during the later stages of embryogenesis and is seed-specific, accumulating in the scutellum of mature embryos. HvClo2 is expressed mainly in the endosperm tissues of the developing grain. We show that HvClo1 and HvClo2 are paralogs that co-segregate on barley chromosome 2HL. Transient expression of HvClo1 in lipid storage and non-storage cells of barley using biolistic particle bombardment indicates that caleosins have different subcellular locations from the structural oil-body protein oleosin, and by inference participate in different sorting pathways. We observe that caleosin sorts via small vesicles, suggesting a likely association with lipid trafficking, membrane expansion and oil-body biogenesis.     

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.     

Expression of enzymatically active and correctly targeted strictosidine synthase in transgenic tobacco plants

Strictosidine, a precursor to over 1000 indole alkaloids including the anti-tumor drugs vinblastine, vincristine, and camptothecin, is produced by the condensation of tryptamine and secologanin. Strictosidine synthase, the enzyme responsible for this condensation, is the first committed step in the indole-alkaloid pathway. We have introduced a modified cDNA encoding Strictosidine synthase from Catharanthus roseus (L.) Don. (McKnight et al. 1990, Nucl. Acids Res. 18, 4939) driven by the CaMV 35S promoter into tobacco (Nicotiana tabacum L.). Transgenic tobacco plants expressing this construct had from 3 to 22 times greater strictosidinesynthase activity than C. roseus plants. Ultrastructural immunolocalization demonstrated that strictosidine synthase is a vacuolar protein in C. roseus and is correctly targeted to the vacuole in transgenic tobacco. Immunoblot analysis of strictosidine synthase showed that two distinct forms of the enzyme were produced in transgenic tobacco plants but that only a single form was made in C. roseus. This observation indicates that the second form of the protein is not simply a result of overexpression in tobacco, but may reflect differences in protein processing between tobacco and C. roseus.Abbreviations cDNA complementary DNA - TLC thin-layer chromatography We thank Dr. C.A. Roessner for providing the E. coli strain expressing strictosidine synthase, Dr. J. Balsevich for providing alkaloid standards, and Dr. L. Cloney for assisting with antibody preparation. This work was supported by a National Institutes of Health Biomedical Research Support Grant to T.D.M and by a grant from the US Department of Agriculture, Competitive Research Grants Office (90-37262-5375) to C.L.N.     

Analysis of a cDNA encoding arginine decarboxylase from oat reveals similarity to the Escherichia coli arginine decarboxylase and evidence of protein processing

Summary Arginine decarboxylase is the first enzyme in one of the two pathways of putrescine synthesis in plants. We purified arginine decarboxylase from oat leaves, obtained N-terminal amino acid sequence, and then used this information to isolate a cDNA encoding oat arginine decarboxylase. Comparison of the derived amino acid sequence with that of the arginine decarboxylase gene from Escherichia coli reveals several regions of sequence similarity which may play a role in enzyme function. The open reading frame (ORF) in the oat cDNA encodes a 66 kDa protein, but the arginine decarboxylase polypeptide that we purified has an apparent molecular weight of 24 kDa and is encoded in the carboxyl-terminal region of the ORF. A portion of the cDNA encoding this region was expressed in E. coli, and a polyclonal antibody was developed against the expressed polypeptide. The antibody detects 34 kDa and 24 kDa polypeptides on Western blots of oat leaf samples. Maturation of arginine decarboxylase in oats appears to include processing of a precursor protein.     

A possible strategy against head and neck cancer: in silico investigation of three-in-one inhibitors

Overexpression of epidermal growth factor receptor (EGFR), Her2, and uroporphyrinogen decarboxylase (UROD) occurs in a variety of malignant tumor tissues. UROD has potential to modulate tumor response of radiotherapy for head and neck cancer, and EGFR and Her2 are common drug targets for the treatment of head and neck cancer. This study attempts to find a possible lead compound backbone from TCM Database@Taiwan (http://tcm.cmu.edu.tw/) for EGFR, Her2, and UROD proteins against head and neck cancer using computational techniques. Possible traditional Chinese medicine (TCM) lead compounds had potential binding affinities with EGFR, Her2, and UROD proteins. The candidates formed stable interactions with residues Arg803, Thr854 in EGFR, residues Thr862, Asp863 in Her2 protein, and residues Arg37, Arg41 in UROD protein, which are key residues in the binding or catalytic domain of EGFR, Her2, and UROD proteins. Thus, the TCM candidates indicated a possible molecule backbone for evolving potential inhibitors for three drug target proteins against head and neck cancer.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:35     

Cloning and expression of transaldolase from potato

We have isolated a cDNA encoding transaldolase, an enzyme of the pentose-phosphate pathway, from potato (Solanum tuberosum). The 1.5 kb cDNA encodes a protein of 438 amino acid residues with a molecular mass of 47.8 kDa. When the potato cDNA was expressed in Escherichia coli a 45 kDa protein with transaldolase activity was produced. The first 62 amino acids of the deduced amino acid sequence represent an apparent plastid transit sequence. While the potato transaldolase has considerable similarity to the enzyme from cyanobacteria and Mycobacterium leprae, similarity to the conserved transaldolase enzymes from humans, E. coli and Saccharomyces cerevisiae is more limited. Northern analysis indicated that the transaldolase mRNA accumulated in tubers in response to wounding. Probing the RNA from various potato tissues indicated that the transaldolase mRNA accumulation to higher levels in the stem of mature potato plants than in either leaves or tubers. These data are consistent with a role for this enzyme in lignin biosynthesis.     

Cloning and sequence analysis of lily and tobacco guanylate kinases

Guanylate kinase is an essential enzyme in the nucleotide biosynthetic pathway, catalyzing the reversible transfer of the terminal phospharyl group of ATP to GMP or dGMP. This enzyme has been well studied from several organisms and many structural and functional details have been characterized. Animal GMP kinases have also been implicated in signal transduction pathways. However, the corresponding role by plant derived GMP kinases remains to be elucidated. Full-length cDNA clones encoding enzymatically active guanylate kinases were isolated from cDNA libraries of lily and tobacco. Lily cDNA is predicted to encode a 392-amino acid protein with a molecular mass of 43.1 kDa and carries amino- and carboxy- terminal extensions of the guanylate kinase (GK)-like domain. But tobacco cDNA is predicted to encode a smaller protein of 297-amino acids with a molecular mass of 32.7 kDa. The amino acid residues known to participate in the catalytic activity of functionally characterized GMP kinases, are also conserved in GK domains of LGK-1 and NGK-1. The GK domains of NGK-1, LGK-1 and previously characterized AGK-1 from Arabidopsis exhibit 74–84% identity, whereas their N- and C-terminal domains are more divergent with amino acid conservation in the order of 48-55%. Phylogenetic analysis on the deduced amino acid sequences reveals that NGK-1 and LGK-1 form one distinct subgroup along with AGK-1 and AGK-2 homologues from Arabidopsis. Isolation of GMP kinases from diverse plant species like lily and tobacco adds a new dimension in understanding their role in cell signaling pathways that are associated with plant growth and development.     

A cDNA clone from barley encoding the precursor from the photosystem I polypeptide PSI-G: Sequence similarity to PSI-K

A cDNA clone encoding the photosystem I subunit, PSI-G was isolated from barley using an oligonucleotide specifying a partial amino acid sequence from a 9 kDa polypeptide of barley photosystem I. The 724 bp sequence contains an open reading frame encoding a precursor polypeptide of 15 107 kDa. Import studies using the in vitro expressed barley PsaG cDNA clone demonstrate that PSI-G migrates with an apparent molecular mass of 9 kDa on SDS-polyacrylamide gels together with PSI-C (subunit-VII). The previous assignment of the gene product of PsaG from spinach as subunit V (Steppuhn J, Hermans J, Nechushtai R, Ljungberg U, Thümmler F, Lottspeich F, Herrmann RG, FEBS Lett 237: 218–224, 1988) needs to be re-examined. The expression of the psaG gene is light-induced similar to other barley photosystem I genes. A significant sequence similarity to PSI-K from Chlamydomonas reinhardtii was discovered when a gene database was searched with the barley PSI-G amino acid sequence. Extensive sequence similarity between the nuclear-encoded photosystem I subunits has not previously been found. The observed sequence similarity between PSI-G and PSI-K suggests a symmetric location of these subunits in the photosystem I complex. The hydropathy plot of the barley PSI-G polypeptide indicates two membrane-spanning regions which are also found at the corresponding locations in the PSI-K polypeptide. PSI-G and PSI-K probably have evolved from a gene duplication of an ancestral gene.     

A reporter gene under the control of tms or aux promoters is differentially expressed in tobacco and barley protoplasts

Summary Agrobacterium tumefaciens and some Agrobacterium rhizogenes strains possess auxin biosynthesis genes (tms and aux genes respectively), responsible for a de novo auxin biosynthetic pathway in transformed plant cells. A comparison is presented of the potential expression of these genes in a monocotyledonous (barley) and a dicotyledonous plant (tobacco). The promoters of the genes were translationally fused to the -glucuronidase reporter gene and analysed in transient expression experiments. The tms and aux fusions were highly expressed in tobacco, but not in barley. However, the aux enhancer active in tobacco, conferred low -glucuronidase expression in barley when fused to a truncated cauliflower mosaic virus 35S promoter. The results are discussed in relation to the differential responses to Agrobacterium infection in monocots and dicots.Abbreviations CaMV cauliflower mosaic virus - PCR polymerase chain reaction - PEG polyethylene glycol - Mu 4-methyl umbelliferone