Genetics of the peroxidase isoenzymes inPetunia

Summary The structural geneprxD inPetunia codes for a slow moving anodic peroxidase whose activity is sensitive to high concentrations of hydrogen peroxide. The PRXd enzyme could be found in mature and old leaf and stem tissue of full-grown flowering plants. PRXd was found to be absent in tissues from flower corolla and root. The geneprxD is the fourth gene that codes for peroxidases in leaf and stem. Two mobility variants of the PRXd enzyme have been found among our inbred lines using starch gel system II electrophoresis. The geneprxD could be located on chromosome III by a four-point-cross involving the genesprxA, prxD, Mf1 andHt1. The order of the genes established is:Ht1 — Mf1 — prxD — prxA.     

On the origin of anthocyanin methyltransferase isozymes of Petunia hybrida and their role in regulation of anthocyanin methylation

Summary Four genes controlling anthocyanin methylation in flowers of Petunia hybrida have been described. Three of them, Mt2, Mf1 and Mf2, caused a dosage effect on anthocyanin methyltransferase activity and degree of methylation of anthocyanins. Antiserum raised against partially purified Mf2-enzyme precipitated three of the four anthocyanin methyltransferases. In two subspecies of one of the ancestral species of P. hybrida: Petunia integrifolia, different anthocyanin methyltransferases were found as determined by immunoprecipitation. The methyltransferase isozymes showed no differences in subcellular or tissue location, and had no physiologically important difference in time course of activity during bud development. The methylation-system in Petunia is discussed with regard to anthocyanin methylation in other plant species.     

Genetics of the peroxidase isoenzymes in Petunia

Summary Two alleles of the structural gene prxA from Petunia, prxA6 and prxA7, could be identified by their differential temporal expression. The alleles prxA6 and prxA7 code for peroxidases with a similar electrophoretic mobility as the products of the previously described alleles prxA1 and prxA5, respectively. The former two alleles differ in that they have a different temporal expression with regard to the temporal expression of the allele prxA2. Crossing experiments indicated that the mutations involved are (cisacting) internal site mutations. In the case of the allele prxA6, the experiments indicated a difference with respect to the allele prxA1 in responsiveness to the action of a trans-acting factor.     

Antigenic relationships between petunia peroxidase a and specific peroxidase isoenzymes in other Solanaceae

Summary A highly specific rabbit antiserum raised against peroxidase (PRXa) from petunia (Petunia hybrida) was used to investigate the antigenic relatedness of peroxidases in the Solanaceae. After SDS-PAGE of crude leaf extracts from a large number of species of this family, immunoblotting revealed that cross-reacting protein bands were present in all species tested. In order to determine whether these protein bands represent peroxidases, the peroxidase isoenzymes in thorn apple (Datura stramonium L.), tobacco (Nicotiana tabacum L.), sweet pepper (Capsicum annuum L.), potato (Solanum tuberosum L.), and tomato (Lycopersicon esculentum Mill.) were further analyzed. Immunoblots obtained after native PAGE revealed that the antiserum only recognized fast-moving peroxidase isoenzymes that are localized in the apoplast. Despite their serological relatedness, these peroxidases differed with respect to heat stability and apparent molecular weight. Differences in avidity for the petunia PRXa antiserum were suggested by immunoprecipitation with antibodies bound to protein A-Sepharose. The antiserum did not react with peroxidases from horseradish (Armoracea rusticana Gaertn., Mey and Scherb), turnip (Brassica napus L.), African marigold (Tagetes cresta L.), maize (Zea mays L.), and oats (Avena sativa L.). Apparently, the Solanaceae contain orthologous genes encoding the fast-moving anionic peroxidases homologous to petunia PRXa.     

Molecular cloning of two tandemly arranged peroxidase genes from Populus kitakamiensis and their differential regulation in the stem

A genomic library was prepared from Populus kitakamiensis and screened with the cDNA for an anionic peroxidase from P. kitakamiensis. One genomic clone was isolated that contained two tandemly oriented genes for anionic peroxidases, prxA3a and prxA4a. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, namely, GT and AG, at their 5 and 3 ends, respectively. The prxA3a and prxA4a genes encoded 347 and 343 amino acid residues, respectively, including putative signal sequences at the amino-termini. Putative promoters and polyadenylation signals were found in the flanking regions of both genes. The sequence of the coding region of prxA3a was completely identical to that of the cDNA clone pA3, whereas the sequence of the coding region of prxA4a was only 73% identical to that of the cDNA clone pA3. Northern blot analysis showed that the patterns of expression of the mRNAs that corresponded to prxA3a and prxA4a differed in stems of P. kitakamiensis.     

Down-regulation of an anionic peroxidase in transgenic aspen and its effect on lignin characteristics

It is generally accepted that peroxidases catalyze the final step in the biosynthesis of lignin. In this study, to examine how expression of prxA3a, a gene for an anionic peroxidase, might be related to lignification in plant tissues, we produced transgenic tobacco plants that harbored a gene for β-glucuronidase (GUS) fused to the prxA3a promoter. Histochemical staining for GUS activity indicated that the prxA3a promoter was active mainly in the lignifying cells of stem tissues. Further, to examine the effects of suppressing the expression of prxA3a, we transferred an antisense prxA3a gene construct into the original host, hybrid aspen (Populus sieboldii ×P. gradidentata), under the control of the original promoter of the prxA3a gene. Eleven transformed aspens were obtained and characterized, and the stable integration of the antisense construct was confirmed by PCR and Southern blotting analysis in all these lines. Assays of enzymatic activity showed that both total peroxidase activity and acidic peroxidase activity were lower in most transgenic lines than in the control plants. In addition, the reduction of peroxidase activity was associated with lower lignin content and modified lignin composition. Transgenic lines with the highest reduction of peroxidase activity displayed a higher syringyl/vanillin (S/V) ratio and a lower S+V yield, mainly because of a decreased amount of V units. Thus, our results indicate that prxA3a is involved in the lignification of xylem tissue and that the down-regulation of anionic peroxidase alters both lignin content and composition in hybrid aspen.     

Genetics of the peroxidase isoenzymes in Petunia

Summary Antibodies were raised against the peroxidases encoded by the allele prxA1 to determine the specific activities of the peroxidases encoded by the alleles prxA1, prxA2, prxA3, and prxA5. The results from double diffusion experiments indicated that all peroxidases encoded by the four alleles are antigenically identical. By rocket immuno electrophoresis it was shown that the peroxidases encoded by the alleles prxA1, prxA2, prxA3, and prxA5 have different specific activities. The results presented are discussed in relation to differential expression of the alleles involved.     

A mitochondrial cytochrome b mutation but no mutations of nuclearly encoded subunits in ubiquinol cytochrome c reductase (complex III) deficiency

Ubiquinol cytochrome c reductase (complex III) deficiency represents a clinically heterogeneous group of mitochondrial respiratory chain disorders that can theoretically be subject to either a nuclear or a mitochondrial mode of inheritance. In an attempt to elucidate the molecular bases of the disease, we first determined the nucleotide sequence of three unknown subunits (9.5 kDa, 7.2 kDa, 6.4 kDa) by cyberscreening of human expressed sequence tag data bases and sequenced the 11 cDNA subunits encoding complex III in five patients with isolated complex III deficiency. No mutation in the nuclearly encoded complex III subunits was observed, but a mutation in the cd2 helix of the mitochondrial (mt) cytochrome b gene was found to alter the conformation of the bc ₁ complex in one patient with severe hypertrophic cardiomyopathy. The present study is highly relevant to genetic counseling as the absence of mtDNA mutations in all but one patient in our series strongly supports autosomal rather than maternal inheritance in the majority of patients with complex III deficiency. Received: 15 January 1999 / Accepted: 31 March 1999     

Petunia peroxidase a: isolation, purification and characteristics

The fast-moving anionic peroxidase isoenzyme variant PRXa was purified from leaves of petunia (Petunia hybrida). Over 1300-fold purification was achieved by subjecting extracellular extracts to two sequential acetone precipitations and resuspending the pellets at pH 5.0 and pH 8.0, respectively, followed by gel filtration and chromatofocusing. The purified enzyme had an absorbance ratio (A405 nm/A280 nm) of 3.6, a molecular mass of about 37 kDa and a pI of 3.8. Three molecular forms with slightly different molecular masses were separated by concanavalin-A--Sepharose affinity chromatography, indicating that these three forms differ in their carbohydrate moieties. The absorption spectrum of PRXa had maxima at 496 and 636 nm and a Soret band at 405 nm. Spectra of compounds I and IV were obtained by titrating a batch of PRXa stored for several months at -20 degrees C with H2O2. The addition of 1 mol H2O2/mol freshly purified PRXa caused the formation of compound II, indicating that freshly isolated PRXa contains a bound hydrogen donor which is lost upon storage. Compound III was obtained from both preparations in the presence of excess H2O2. The pH optimum of PRXa for the reaction with H2O2 and guaiacol was 5.0 and its specific activity 61 mkat/g protein. Among various aromatic compounds, coniferyl alcohol was polymerized by PRXa to presumed lignin-like material. The extracellular localization and high affinity of PRXa for the cinnamic acid derivatives suggest that this isoenzyme functions in the polymerization or cross-linking of lignin in the plant cell wall.     

Cloning and analysis of two genes for chalcone synthase from Petunia hybrida

Summary With the help of a cDNA probe for a chalcone synthase gene of Petroselinum a cDNA clone for a chalcone synthase gene of Petunia hybrida could be identified. The homologous cDNA allowed the cloning of two genomic EcoRI fragments from Petunia hybrida containing complete chalcone synthase genes. It could be demonstrated that the genes on the two fragments are different and are not allelic but members of a gene family. The two genes are found in a variety of different Petunia lines including in the two conditional mutants affected in chalcone synthase expression in floral buds, White Joy and Red Star. The structure of the two chs genes from Petunia is compared to the chs gene from Antirrhinum majus.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Regulation of synthesis of nitrite reductase in pea leaves: in-vivo and in-vitro studies

The cellular location of three peroxidase isoenzymes (PRX) in mature leaf tissue of Petunia and their affinity for Concanavalin A-Sepharose were investigated. The isoenzymes PRXa, PRXb and PRXc were identified by their positions in starch-gel zymograms. The fast-moving anodic and cathodic peroxidase bands, the isoenzymes PRXa and PRXc respectively, were the most active peroxidases in extracellular extracts. The molecular forms of PRXa showed a tissue-specific distribution between midrib and remaining leaf tissue. An intermediate-moving anodic peroxidase band, the isoenzyme PRXb, was the most active peroxidase released after extraction of isolated mesophyll protoplasts. Small amounts of the peroxidase isoenzymes were present in cell-wall-bound fractions. Incubation of a crude protein fraction with Concanavalin A-Sepharose showed that the isoenzyme PRXb bound more firmly to Concanavalin A-Sepharose than the isoenzymes PRXa and PRXc, of which only one molecular form bound partly. The results are discussed with respect to a possible function of one of the peroxidase isoenzymes, and a possible role of oligosaccharide chains in determining the cellular location of plant peroxidases is suggested.Abbreviations Con A Concanavalin A - PRX peroxidase (isoenzyme)     

Genetics of the peroxidase isoenzymes in Petunia

Summary The structural gene prxE, coding for a slow cathodic peroxidase in Petunia, has been located to chromosome II, linked to F1. The presence of two mobility alleles in Petunia hybrida can be ascribed to its hybrid descent. Some properties of peroxidase e are mentioned. A gene prxJ is postulated for a still slower cathodic band. The gene Rp1, regulating the onset of expression of the allele prxB2, has been located on chromosome VII (gene order Rp1-prxF-An4). A synopsis of the isoperoxidases and the corresponding genes is given.     

Chromosomal localization of foreign genes in Petunia hybrida

Summary A F1 hybrid of Petunia hybrida, heterozygous for at least one marker on each of the seven chromosomes, was transformed with a modified strain of Agrobacterium tumefaciens in which the phytohormone biosynthetic genes in the transferred DNA (T-DNA) were replaced with a NOS/NPTII/NOS chimeric gene and a wildtype nopaline synthase (NOS) gene. The chimeric gene, which confers kanamycin resistance, was used as selectable marker during the transformation process and the NOS gene was used as a scorable marker in the genetic studies. After plants had been regenerated from the transformed tissues, the transgenic plants that expressed both of these markers were backcrossed to the parental lines. The offspring were examined for the segregation of the NOS gene and the Petunia markers. Genetic mapping was thus accomplished in a single generation.By Southern hybridization analysis we confirmed the presence of the expected T-DNA fragments in the transformed plants. Four out of the six plants presented here, had just one monomeric T-DNA insertion. The sizes of the plant/T-DNA junction fragments suggest that the integration occurred in different sites of the Petunia genome. One transformant gave a more complicated hybridization pattern and possibly has two T-DNA inserts. Another transgenic plant was earlier reported (Fraley et al. 1985) to have two, possibly tandemly repeated T-DNAs.Data is presented on the genetic localization of the T-DNA inserts in six independently obtained transgenic plants. The T-DNA inserts in three plants were mapped to chromosome I. However, the distances between the NOS gene and the marker gene on this chromosome were significantly different. In another transgenic plant the NOS gene was coinherited with the marker on chromosome IV. Two other transgenic plants have the T-DNA insert on chromosome III. A three point cross enabled us to determine that both plants have the NOS gene distally located from the peroxidaseA (prxA) marker and both plants showed about 18% recombination. However, Southern hybridization analysis shows that the sizes of the plant/T-DNA junction fragments in these transgenic plants are different, thus suggesting that the integrations occurred in different sites.     

Two modes of mitochondrial dysfunction lead independently to lifespan extension in Caenorhabditis elegans

In Caenorhabditis elegans, longevity is increased by a partial loss‐of‐function mutation in the mitochondrial complex III subunit gene isp‐1. Longevity is also increased by RNAi against the expression of a variety of mitochondrial respiratory chain genes, including isp‐1, but it is unknown whether the isp‐1(qm150) mutation and the RNAi treatments trigger the same underlying mechanisms of longevity. We have identified nuo‐6(qm200), a mutation in a conserved subunit of mitochondrial complex I (NUDFB4). The mutation reduces the function of complex I and, like isp‐1(qm150), results in low oxygen consumption, slow growth, slow behavior, and increased lifespan. We have compared the phenotypes of nuo‐6(qm200) to those of nuo‐6(RNAi) and found them to be distinct in crucial ways, including patterns of growth and fertility, behavioral rates, oxygen consumption, ATP levels, autophagy, and resistance to paraquat, as well as expression of superoxide dismutases, mitochondrial heat‐shock proteins, and other gene expression markers. RNAi treatments appear to generate a stress and autophagy response, while the genomic mutation alters electron transport and reactive oxygen species metabolism. For many phenotypes, we also compared isp‐1(qm150) to isp‐1(RNAi) and found the same pattern of differences. Most importantly, we found that, while the lifespan of nuo‐6, isp‐1 double mutants is not greater than that of the single mutants, the lifespan increase induced by nuo‐6(RNAi) is fully additive to that induced by isp‐1(qm150), and the increase induced by isp‐1(RNAi) is fully additive to that induced by nuo‐6(qm200). Our results demonstrate that distinct and separable aspects of mitochondrial biology affect lifespan independently.     

Acetohydroxy acid synthase isoenzymes of Escherichia coli K-12: A trans-acting regulatory locus for ilvHI gene expression

Summary We isolated an Escherichia coli K-12 regulatory mutation affecting the acetohydroxy acid synthase III isoenzyme. This mutation was found to lie outside the structural genes ilvHI for this isoenzyme and was designated lrs-1. A strain carrying this mutation was found to be altered in the leucine-mediated control of ilvHI mRNA and acetohydroxy acid synthase III synthesis observed in the isogenic lrs ⁺ strain. These alterations appeared to be a consequence of a reduced intracellular concentration of a single one of five tRNA^Leu isoaccepting species.     

Structure,expression, chromosomal location and product of the gene encoding ADH1 inPetunia

A genomic clone for an alcohol dehydrogenase (Adh) gene has been isolated fromPetunia hybrida cv. V30 by screening aPetunia genomic library with a maizeAdh1 probe. A combination of RFLP and allozyme segregation data failed to demonstrate which of twoAdh loci, both of which map to chromosome 4, was the source of the cloned gene. The product of the cloned genes has been identified unequivocally by a transient expression assay inPetunia protoplasts. We have designated this genePetunia Adh1. The expression of this gene is tightly regulated in the developing anther, where its gene product is the predominant ADH isozyme. It is anaerobically inducible in roots, stems and leaves of seedlings. The induction of enzyme activity is correlated with induction ofAdh1 mRNA.     

Search for Frequent Mutations in Genes Predisposing to Breast Cancer

DNA of oncological patients, including Ashkenazi Jews and Slavs, living in St. Petersburg was collected, and the resultant collection was screened for three common mutations of genes BRCA1andBRCA2by means of heteroduplex analysis. The mutation 5382insC in exon 20 of the BRCA1gene was found in four unrelated patients, including three Slavs and one Ashkenazi Jew, with a positive family history of breast cancer. The mutations 185delAG and 6174delT in the BRCA1and BRCA2genes, respectively, which are typical of Ashkenazi Jewish patients with breast cancer, were not found in the patients of either ethnicity living in St. Petersburg, although the 6174delT mutation was found in the control group of Ashkenazi Jews. A new 12-nucleotide duplication g.71741ins12nt found in intron 20 of the BRCA1gene was described. The high frequency of the 5382insC mutation in the BRCA1gene in patients with familial breast cancer in both St. Petersburg and Moscow indicates that Russian families with the history of breast cancer should be primarily tested for this mutation.     

Mutations in the UQCC1-Interacting Protein,UQCC2, Cause Human Complex III Deficiency Associated with Perturbed Cytochrome b Protein Expression

Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis.