Making light of it: the role of plant haem oxygenases in phytochrome chromophore synthesis.

The haem oxygenase (HO) enzyme catalyses the oxidation of haem to biliverdin IX alpha, CO and Fe(2+), and performs a wide variety of roles in Nature, including degradation of haem from haemoglobin, iron acquisition and phycobilin biosynthesis. In plants, HOs are required for the synthesis of the chromophore of the phytochrome family of photoreceptors. There are four HO genes in the Arabidopsis genome. Analysis of a mutant deficient in HO1 (the hy1 mutant) has demonstrated that this plastid-localized protein is the major HO in the phytochrome chromophore synthesis pathway. HO2 may also have a minor role in this pathway, but our understanding of the divergent roles of this small gene family is still far from complete.

Functional genomic analysis of the HY2 family of ferredoxin-dependent bilin reductases from oxygenic photosynthetic organisms.

Phytobilins are linear tetrapyrrole precursors of the light-harvesting prosthetic groups of the phytochrome photoreceptors of plants and the phycobiliprotein photosynthetic antennae of cyanobacteria, red algae, and cryptomonads. Previous biochemical studies have established that phytobilins are synthesized from heme via the intermediacy of biliverdin IX alpha (BV), which is reduced subsequently by ferredoxin-dependent bilin reductases with different double-bond specificities. By exploiting the sequence of phytochromobilin synthase (HY2) of Arabidopsis, an enzyme that catalyzes the ferredoxin-dependent conversion of BV to the phytochrome chromophore precursor phytochromobilin, genes encoding putative bilin reductases were identified in the genomes of various cyanobacteria, oxyphotobacteria, and plants. Phylogenetic analyses resolved four classes of HY2-related genes, one of which encodes red chlorophyll catabolite reductases, which are bilin reductases involved in chlorophyll catabolism in plants. To test the catalytic activities of these putative enzymes, representative HY2-related genes from each class were amplified by the polymerase chain reaction and expressed in Escherichia coli. Using a coupled apophytochrome assembly assay and HPLC analysis, we examined the ability of the recombinant proteins to catalyze the ferredoxin-dependent reduction of BV to phytobilins. These investigations defined three new classes of bilin reductases with distinct substrate/product specificities that are involved in the biosynthesis of the phycobiliprotein chromophore precursors phycoerythrobilin and phycocyanobilin. Implications of these results are discussed with regard to the pathways of phytobilin biosynthesis and their evolution.

The Arabidopsis HY2 gene encodes phytochromobilin synthase, a ferredoxin-dependent biliverdin reductase.

Light perception by the plant photoreceptor phytochrome requires the tetrapyrrole chromophore phytochromobilin (P Phi B), which is covalently attached to a large apoprotein. Arabidopsis mutants hy1 and hy2, which are defective in P Phi B biosynthesis, display altered responses to light due to a deficiency in photoactive phytochrome. Here, we describe the isolation of the HY2 gene by map-based cloning. hy2 mutant alleles possess alterations within this locus, some of which affect the expression of the HY2 transcript. HY2 encodes a soluble protein precursor of 38 kD with a putative N-terminal plastid transit peptide. The HY2 transit peptide is sufficient to localize the reporter green fluorescent protein to plastids. Purified mature recombinant HY2 protein exhibits P Phi B synthase activity (i.e., ferredoxin-dependent reduction of biliverdin IX alpha to P Phi B), as confirmed by HPLC and by the ability of the bilin reaction products to combine with apophytochrome to yield photoactive holophytochrome. Database searches and hybridization studies suggest that HY2 is a unique gene in the Arabidopsis genome that is related to a family of proteins found in oxygenic photosynthetic bacteria.

Responses of wild watermelon to drought stress: accumulation of an ArgE homologue and citrulline in leaves during water deficits.

Wild watermelon from the Botswana desert had an ability to survive under severe drought conditions by maintaining its water status (water content and water potential). In the analysis by two-dimensional electrophoresis of leaf proteins, seven spots were newly induced after watering stopped. One with the molecular mass of 40 kilodaltons of the spots was accumulated abundantly. The cDNA encoding for the protein was cloned based on its amino-terminal sequence and the amino acid sequence deduced from the determined nucleotide sequences of the cDNA exhibited homology to the enzymes belong to the ArgE/DapE/Acy1/Cpg2/YscS protein family (including acetylornithine deacetylase, carboxypeptidase and aminoacylase-1). This suggests that the protein is involved in the release of free amino acid by hydrolyzing a peptidic bond. As the drought stress progressed, citrulline became one of the major components in the total free amino acids. Eight days after withholding watering, although the lower leaves wilted significantly, the upper leaves still maintained their water status and the content of citrulline reached about 50% in the total free amino acids. The accumulation of citrulline during the drought stress in wild watermelon is an unique phenomenon in C3-plants. These results suggest that the drought tolerance of wild watermelon is related to (1) the maintenance of the water status and (2) a metabolic change to accumulate citrulline.

Characterization of a novel gene encoding a putative single zinc-finger protein, ZIM, expressed during the reproductive phase in Arabidopsis thaliana.

By differential screening of an arrayed normalized cDNA library from the inflorescence apex in Arabidopsis, a cDNA clone having a deduced amino acid sequence with a motif for a zinc finger was isolated as one of the genes expressed specifically in the reproductive phase. The deduced protein has a modular structure with a putative single C2-C2 zinc-finger motif distantly related to a GATA-1-type finger, a basic region with a sequence resembling a nuclear localization signal, and an acidic region. The gene seemed to have been formed by the exon-shuffling during its molecular evolution, since individual domains are encoded by discrete exons. RNA gel blot analysis showed its expression in shoot apex and flowers in the reproductive phase. The gene was named ZIM for Zinc-finger protein expressed in Inflorescence Meristem. The nuclear localization of ZIM was detected using GFP as a reporter. These results suggest that ZIM is a putative transcription factor involved in inflorescence and flower development.

Systematic isolation of highly transcribed genes in inflorescence apices in Arabidopsis thaliana from an equalized cDNA library.

To analyze plant meristem functions, we systematically isolated thirteen cDNA clones for transcripts that are highly expressed in the inflorescence apices of Arabidopsis by differential screening, using an equalized cDNA library with the clones arrayed on membranes. Their deduced amino acid sequences indicate important functions for proteins in determining the cell wall architecture, translation capacity and mitotic activity in the shoot apex during reproductive growth.

Microinjection of heme oxygenase genes rescues phytochrome-chromophore-deficient mutants of the moss Ceratodon purpureus.

In protonemal tip cells of the moss Ceratodon purpureus (Hedw.) Brid., phototropism and chlorophyll accumulation are regulated by the photoreceptor phytochrome. The mutant ptr116 lacks both responses as a result of a defect in the biosynthesis of phytochromobilin, the chromophore of phytochrome, at the point of biliverdin formation. The rescue of the phototropic response and of chlorophyll synthesis were tested by injecting different substances into tip cells of ptr116. Microinjection was first optimised with the use of fluorescent dyes and an expression plasmid containing a green fluorescent protein (GFP) gene. Injected phycocyanobilin, which substitutes for phytochromobilin, rescued both the phototropic response and light-induced chlorophyll accumulation in ptr116. The same results were obtained when expression plasmids with heme oxygenase genes of rat (HO-1) and Arabidopsis thaliana (L.) Heynh. (HY1) were injected. Heme oxygenase catalyses the conversion of heme into biliverdin. Whereas HY1 has a plastid target sequence and is presumably transferred to plastids, HO-1 is proposed to be cytosolic. The data show that ptr116 lacks heme oxygenase enzyme activity and indicate that heme oxygenases of various origin are active in Ceratodon bilin synthesis. In addition, it can be inferred from the data that the intracellular localisation of the expressed heme oxygenase is not important since the plastid enzyme can be replaced by a cytosolic one.

Systematic isolation of genes expressed at low levels in inflorescence apices of Arabidopsis thaliana.

We constructed an equalized cDNA library from Arabidopsis inflorescence shoot apices including inflorescence meristem, floral meristem and flower tissue collected before stage 5 of flower development. The cDNA clones were arrayed on membranes and were differentially screened using cDNA pools from vegetative and inflorescence tissues as probes. Each clone was classified by expression specificity and expression level. By removing the clones that displayed hybridization signals, 384 out of 3264 clones in this library remained as candidates for inflorescence-specific mRNAs expressed at low levels. Sequence analysis of all selected clones indicated that 53 were identical and 120 were homologous to genes in public protein databases. The remaining 211 selected clones had no significant amino acid sequence similarities with those deduced from any reported genes, though 62 of them appeared in Arabidopsis expressed sequenced tags (ESTs). About 40% of the selected clones were novel, validating the present approach for gene discovery. Northern blot analysis of 22 randomly selected clones confirmed that most were expressed preferentially in inflorescence tissues. In addition, many clones were transcribed at relatively low levels. We demonstrate that the screening method of the present study is useful for systematic classification of cDNA species based on expression specificity.

The Arabidopsis photomorphogenic mutant hy1 is deficient in phytochrome chromophore biosynthesis as a result of a mutation in a plastid heme oxygenase.

The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light. We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation. The product of the HY1 gene shows significant similarity to animal heme oxygenases and contains a possible transit peptide for transport to plastids. Heme oxygenase activity was detected in the HY1 protein expressed in Escherichia coli. Heme oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis. The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts. The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum. These results indicate that the Arabidopsis HY1 gene encodes a plastid heme oxygenase necessary for phytochrome chromophore biosynthesis.

Construction of an equalized cDNA library from Arabidopsis thaliana.

Using a kinetic approach, a cDNA library composed of almost equal representations of all genes expressed in the aerial parts of 2-week-old Arabidopsis was constructed. A cDNA was synthesized with an oligo dT primer containing a Notl site. A linker containing the nucleotide sequence of Sse8387I which recognizes octanucleotides was added at the ends of the synthesized cDNA. The cDNA was amplified by the polymerase chain reaction (PCR), denatured, and reassociated under modified conditions. Thereafter, the remaining single-stranded DNA was converted to double-stranded DNA and amplified by PCR. These equalization steps were repeated three times and the products were cloned unidirectionally into a plasmid vector. Equalization was evaluated by colony hybridization and DNA sequencing. This approach will be applicable to construct a cDNA library suitable for subtraction, differential screening, and expression screening, especially for mRNA species present at very low concentrations in a few cells of a specific tissue.

Mitochondrial DNA of Marchantia polymorpha as a single circular form with no incorporation of foreign DNA.

A cosmid library and physical maps of mitochondrial DNA (mtDNA) from a liverwort, Marchantia polymorpha, were constructed using the cosmid clones. Electrophoresis profile and the physical maps indicated that the liverwort mtDNA was approximately 183 kb long, the smallest among plant mtDNAs, and that it consisted of a single circular molecule. Southern hybridization analysis showed that genes typical to the mitochondrial genome existed in a single copy, and also that there was no incorporation of chloroplast DNA fragments into the mitochondrial genome.

Gene organization deduced from the complete sequence of liverwort Marchantia polymorpha mitochondrial DNA. A primitive form of plant mitochondrial genome.

Analysis of the mitochondrial DNA of a liverwort Marchantia polymorpha by electron microscopy and restriction endonuclease mapping indicated that the liverwort mitochondrial genome was a single circular molecule of about 184,400 base-pairs. We have determined the complete sequence of the liverwort mitochondrial DNA and detected 94 possible genes in the sequence of 186,608 base-pairs. These included genes for three species of ribosomal RNA, 29 genes for 27 species of transfer RNA and 30 open reading frames (ORFs) for functionally known proteins (16 ribosomal proteins, 3 subunits of H(+)-ATPase, 3 subunits of cytochrome c oxidase, apocytochrome b protein and 7 subunits of NADH ubiquinone oxidoreductase). Three ORFs showed similarity to ORFs of unknown function in the mitochondrial genomes of other organisms. Furthermore, 29 ORFs were predicted as possible genes by using the index of G + C content in first, second and third letters of codons (42.0 +/- 10.9%, 37.0 +/- 13.2% and 26.4 +/- 9.4%, respectively) obtained from the codon usages of identified liverwort genes. To date, 32 introns belonging to either group I or group II intron have been found in the coding regions of 17 genes including ribosomal RNA genes (rrn18 and rrn26), a transfer RNA gene (trnS) and a pseudogene (psi nad7). RNA editing was apparently lacking in liverwort mitochondria since the nucleotide sequences of the liverwort mitochondrial DNA were well-conserved at the DNA level.

Identification and map position of YAC clones comprising one-third of the Arabidopsis genome.

YAC clones corresponding to 125 Arabidopsis thaliana RFLP markers have been identified. At least one YAC clone has been isolated for each of the RFLP markers tested. Based on CHEF gel analysis of 196 clones, the mean insert size of the available Arabidopsis YAC libraries is approximately 160 kb. The YACs of known genetic map location encompass about 30% of the Arabidopsis genome. The results presented here represent a first step towards assembly of an overlapping YAC library of the A. thaliana genome.

Respiratory inductance plethysmography and pulse oximetry in the assessment of upper airway patency in a child with Goldenhar's syndrome.

The anaesthetic management of a child with Goldenhar's syndrome and upper airway dysmorphology is presented. She had a history of severe dyspnoea due to deterioration of cor pulmonale caused by upper airway obstruction. The patency of the upper airway and oxygenation were evaluated during the perioperative period with respiratory inductive plethysmography (RIP) and pulse oximetry, which did not show severe upper airway obstruction or oxygen saturation below 80 per cent. Tracheal intubation was performed under inhalational anaesthesia with spontaneous breathing. This case suggests that RIP and pulse oximetry may be useful monitoring devices in the anaesthetic management of patients with upper airway problems as in Goldenhar's syndrome.

[Colorimetric studies on hiding effect of Cosmotech porcelain for laminate restoration].

The hiding effect on color tone of discolored teeth restored with ceramic laminate veneer crown was examined. Translucent (T1, T2), Enamel (E1, E2, E3), Dentin (DA2, DA3, DB2, DB3), Masking dentin (MDA2, MDA3, MDB2, MDB3) in 13 colors and Modifire (M61, M62, M65, M66, M67, M68, M69, M70, M79) in 7 colors of the ceramic materials (Cosmotech porcelain G C Co., Ltd) were examined in this experiments. The colorimetric examination of the color tone backing with the white or black color was carried out by testing for color difference (delta E*ab), value difference (delta L*), chrom and hue of the samples. The result obtained were as follows; 1. Color difference was higher in order of T2 greater than T1 in Translucent greater than E1 greater than E3 greater than E2 in Enamel greater than DB3 greater than DA2 greater than DA3 greater than DB2 in Dentin greater than MDB3 greater than MDA2 greater than MDB2 greater than MDA3 in Masking dentin, and M69 greater than M79 greater than M70 greater than M68 greater than M66 greater than M61 greater than M62 greater than M67 greater than M65 in Modifire. 2. Value difference was higher in order of T2 greater than T1 in Translucent greater than E1 greater than E3 greater than E2 in Enamel greater than DB3 greater than DA2 greater than DB2 greater than DA3 in Dentin greater than MDA2 greater than MDB3 greater than MDB2 greater than MDA3 in Masking dentin, and M69 greater than M70 greater than M79 greater than M68 greater than M66 greater than M61 greater than M67 greater than M65 greater than M62 in Modifire.(ABSTRACT TRUNCATED AT 250 WORDS)

[Studies on the influence of composite resin filling material (Command Ultra fine) on the dental pulp].

Studies on the influence of composite resin filling material on the dental pulp of human 1st premolar are reported in this thesis. An other experiment was carried out on rabbit with the left 1st molar in their lower jaw as subject and the right 1st molar in the lower jaw as a control. In both experiments with human and rabbit, routine operations were performed to prepare the cavities and filled with composite resin material. The human teeth were extracted 21 days afterwards and the rabbit teeth were extracted 7 days afterwards. Edema was found in the dental pulp of both human and rabbit teeth by pathologic examination and was accompanied by hemangiectasis and lymphaniectasis. There was also vacuolar degeneration in the odontoblasts. Some of these cells were reticulated and the of number of odontoblasts was fewer than in the control. Hemangiectasis was even more serious in the rabbit's teeth than those of human.

Study of estimation of color recognition on the dentist. On the ability of subjects to discriminate color in terms of hue, value and chroma.

The tooth crown color space model was manufactured and the improvement of the color representation and communication on the dental clinic have been trying. The study of color discrimination by dentist and student viewing with the use of tooth crown color chips reported here in basic knowledge on the color sensitivity of human eye. The test card were 55 x 90 mm in size and the color chip, 5 x 10 mm. The paired color chips were placed on the test card to have a 2 mm distance between them. Color difference of two tooth crown color chips on the test cards was 3.5 (delta E*ab), the combination of hue, value and chroma was constant of all cards. The subjects were 22 people, 6 dentists and 16 dental school students with normal color sensibility. In proportion of method of comparison for surface color (JIS Z 8723), the subject observed the test card which were different of hue, value and chroma on the their naked-eye, were investigated to evaluate their judgement of the test cards. The following results were obtained. The standard of judgment for the color discrimination was made firstly in terms of hue, secondly in terms of value, and thirdly in terms of chroma.

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha.

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572-574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31,000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31,945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67,000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.