C1 metabolism inParacoccus denitrificans: Genetics ofParacoccus denitrificans

Paracoccus denitrificans is able to grow on the C₁ compounds methanol and methylamine. These compounds are oxidized to formaldehyde which is subsequently oxidized via formate to carbon dioxide. Biomass is produced by carbon dioxide fixation via the ribulose biphosphate pathway. The first oxidation reaction is catalyzed by the enzymes methanol dehydrogenase and methylamine dehydrogenase, respectively. Both enzymes contain two different subunits in an ₂₂ configuration. The genes encoding the subunits of methanol dehydrogenase (moxF andmoxI) have been isolated and sequenced. They are located in one operon together with two other genes (moxJ andmoxG) in the gene ordermoxFJGI. The function of themoxJ gene product is not yet known.MoxG codes for a cytochromec _551i , which functions as the electron acceptor of methanol dehydrogenase. Both methanol dehydrogenase and methylamine dehydrogenase contain PQQ as a cofactor. These so-called quinoproteins are able to catalyze redox reactions by one-electron steps. The reaction mechanism of this oxidation will be described. Electrons from the oxidation reaction are donated to the electron transport chain at the level of cytochromec. P. denitrificans is able to synthesize at least 10 differentc-type cytochromes. Five could be detected in the periplasm and five have been found in the cytoplasmic membrane. The membrane-bound cytochromec ₁ and cytochromec ₅₅₂ and the periplasmic-located cytochromec ₅₅₀ are present under all tested growth conditions. The cytochromesc _551i andc _553i , present in the periplasm, are only induced in cells grown on methanol, methylamine, or choline. The otherc-type cytochromes are mainly detected either under oxygen limited conditions or under anaerobic conditions with nitrate as electron acceptor or under both conditions. An overview including the induction pattern of allP. denitrificans c-type cytochromes will be given. The genes encoding cytochromec ₁, cytochromec ₅₅₀, cytochromec _551i , and cytochromec _553i have been isolated and sequenced. By using site-directed mutagenesis these genes were mutated in the genome. The mutants thus obtained were used to study electron transport during growth on C₁ compounds. This electron transport has also been studied by determining electron transfer rates inin vitro experiments. The exact pathways, however, are not yet fully understood. Electrons from methanol dehydrogenase are donated to cytochromec _551i . Further electron transport is either via cytochromec ₅₅₀ or cytochromec _553i to cytochromeaa ₃. However, direct electron transport from cytochromec _551i to the terminal oxidase might be possible as well. Electrons from methylamine dehydrogenase are donated to amicyanin and then via cytochromec ₅₅₀ to cytochromeaa ₃, but other routes are used also.P. denitrificans is studied by several groups by using a genetic approach. Several genes have already been cloned and sequenced and a lot of mutants have been isolated. The development of a host/vector system and several techniques for mutation induction that are used inP. denitrificans genetics will be described.     

Identification of novel genes specifically expressed in Chlamydomonas reinhardtii zygotes

The maturation of zygotes formed by the fusion of two gametes is the essential part of the diploid phase of the Chlamydomonas reinhardtii sexual life cycle and results in mature zygotes competent to germinate. To understand the molecular mechanisms underlying zygote maturation and the attainment of competence for germination we isolated genomic clones representing three different genes that are specifically expressed in Chlamydomonas reinhardtii zygotes. Accumulation of the RNAs started more than 24 h after mating, setting these genes apart from genes expressed in young zygotes [9]. Upon light-induced germination of zygotes, the mRNAs disappeared. The patterns of RNA accumulation and disappearance were gene-specific and suggested a function of these genes in maturation and/or in initial steps of germination.     

Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana

We have examined the cold-induced enhancement of freezing tolerance and expression of cold-regulated (cor) genes in Arabidopsis thaliana (L.) Heynh (Landsberg erecta) and abscisic acid (ABA)-deficient (aba) and ABA-insensitive (abi) mutants derived from it. The results indicate that the abi mutations had no apparent effect on freezing tolerance, while the aba mutations did: cold-acclimated aba mutants were markedly impaired in freezing tolerance compared to wild-type plants. In addition, it was observed that non-frozen leaves from both control and cold-treated aba mutant plants were more ion-leaky than those from corresponding wild-type plants. These data are consistent with previous observations indicating that ABA levels can affect freezing tolerance. Whether ABA has a direct role in the enhancement of freezing tolerance that occurs during cold acclimation, however, is uncertain. Several studies have suggested that ABA might mediate certain changes in gene expression that occur during cold acclimation. Our data indicate that the ABA-induced expression of three ABA-regulated Arabidopsis cor genes was unaffected in the abi2, abi3, and aba-1 mutants, but was dramatically impaired in the abi1 mutant. Cold-regulated expression of all three cor genes, however, was nearly the same in wild-type and abi1 mutant plants. These data suggest that the cold-regulated and ABA-regulated expression of the three cor genes may be mediated through independent control mechanisms.     

Promoters of auxin-induced genes from tobacco can lead to auxin-inducible and root tip-specific expression

In previous studies we have identified several mRNAs which accumulate after addition of 2,4-dichlorophenoxyacetic-acid (2,4-D) to auxin-starved tobacco cells [45, 46]. The mRNAs corresponding to cDNA clone pCNT103 were found to accumulate transiently prior to the cell division response due to auxin treatment. In this study we determined the sequences of three 103-like cDNAs and two 103-like genes, GNT1 and GNT35. To further study the regulation of the expression of these genes their 5 regions were translationally fused with the -D-glucuronidase reporter gene (GUS). The GNT1 5 region led to GUS expression only in the root tips of transgenic plants. By using transgenic hairy-root cultures and transformed cell suspension cultures it was shown that the 5 regions of both GNT1 and GNT35 lead to 2,4-D-inducible expression of GUS activity. The homology of the 103-like genes with other auxin-regulated genes is evaluated.Department of Plant Molecular Biology, Leiden University     

High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection

Endochitinases (E.C. 3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.     

Sex determination in cucumber (Cucumis sativus) as a model system for molecular biology

Floral biology and sex determination are reviewed in cucumber, one of the best studied monoecious plant systems. Sexual differentiation is controlled by genotypic and environmental factors. Sex conversion has been achieved by a variety of chemical treatments, some of which being extensively used for commercial purposes. Sex expression can be shifted in either direction: femaleness is promoted by ethylene, auxines and ethylene releasing compounds, while maleness is induced by gibberellins and chemicals counteracting ethylene action. Agrobacterium transformation affects, albeit rather nonspecifically, sex expression. An important collection of sex and floral mutants has been developed. The expression of sex genes has been shown to be under the control of modifier genes or the environment. Cloning strategies can take profit of the fact that sex conversion can be modulated alone or in combination by genetical, chemical and/or environmental parameters.