Update on <Emphasis Type="Italic">transparent testa</Emphasis> mutants from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: characterisation of new alleles from an isogenic collection

Main conclusion

We present a comprehensive overview on flavonoid-related phenotypes of A. thaliana tt and tds mutants, provide tools for their characterisation, increase the number of available alleles and demonstrate that tds3 is allelic to tt12 and tds5 to aha10.Flavonoid biosynthesis is one of the best-studied secondary metabolite pathways in plants. In the model system Arabidopsis thaliana it leads to the synthesis of three phenolic compound classes: flavonol glycosides, anthocyanins and proanthocyanidins (PAs). PAs appear brown in their oxidised polymeric forms, and most A. thaliana mutants impaired in flavonoid accumulation were identified through screens for lack of this seed coat pigmentation. These mutants are referred to as transparent testa (tt) or tannin-deficient seed (tds). More than 20 mutants of these types have been published, probably representing most of the genes relevant for PA accumulation in A. thaliana. However, data about the genes involved in PA deposition or oxidation are still rather scarce. Also, for some of the known mutants it is unclear if they represent additional loci or if they are allelic to known genes. For the present study, we have performed a systematic phenotypic characterisation of almost all available tt and tds mutants and built a collection of mutants in the genetic background of the accession Columbia to minimise effects arising from ecotype variation. We have identified a novel tt6 allele from a forward genetic screen and demonstrated that tds3 is allelic to tt12 and tds5 to aha10.

     

Modified nicotine metabolism in transgenic tobacco plants expressing the human cytochrome P450 2A6 cDNA

In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo.     

ag13 is expressed in Alnus glutinosa nodules in infected cells during endosymbiont degradation and in the nodule pericycle

We isolated and characterized an Alnus glutinosa cDNA clone, pAg13, which corresponds to a gene expressed at higher levels in nodules induced by Frankia than in roots. The deduced polypeptide sequence is rich in glutamic acid and proline and contains a putative signal peptide indicating an extracellular location of Ag13. In situ hybridization showed that ag13 is expressed in the pericycle of the nodule vascular bundle and in infected cells that exhibited degradation of the endosymbiont.     

Conserved function of pex11p and the novel pex25p and pex27p in peroxisome biogenesis

We describe the isolation and characterization of a homologous pair of proteins, Pex25p (YPL112c) and Pex27p (YOR193w), whose C-termini are similar to the entire Pex11p. All three proteins localize to the peroxisomal membrane and are likely to form homo-oligomers. Deletion of any of the three genes resulted in enlarged peroxisomes as revealed by fluorescence and electron microscopy. The partial growth defect on fatty acids of a pex25Δ mutant was not exacerbated by the additional deletion of PEX27; however, when PEX11 was deleted on top of that, growth was abolished on all fatty acids. Moreover, a severe peroxisomal protein import defect was observed in the pex11Δpex25Δpex27Δ triple mutant strain. This import defect was also observed when cells were grown on ethanol-containing medium, where peroxisomes are not required, suggesting that the function of the proteins in peroxisome biogenesis exceeds their role in proliferation. When Pex25p was overexpressed in the triple mutant strain, growth on oleic acid was completely restored and a massive proliferation of laminar membranes and peroxisomes was observed. Our data demonstrate that Pex11p, Pex25p, and Pex27p build a family of proteins whose members are required for peroxisome biogenesis and play a role in the regulation of peroxisome size and number.     

Combined anaerobic ammonium and methane oxidation for nitrogen and methane removal

Anammox (anaerobic ammonium oxidation) is an environment-friendly and cost-efficient nitrogen-removal process currently applied to high-ammonium-loaded wastewaters such as anaerobic digester effluents. In these wastewaters, dissolved methane is also present and should be removed to prevent greenhouse gas emissions into the environment. Potentially, another recently discovered microbial pathway, n-damo (nitrite-dependent anaerobic methane oxidation) could be used for this purpose. In the present paper, we explore the feasibility of simultaneously removing methane and ammonium anaerobically, starting with granules from a full-scale anammox bioreactor. We describe the development of a co-culture of anammox and n-damo bacteria using a medium containing methane, ammonium and nitrite. The results are discussed in the context of other recent studies on the application of anaerobic methane- and ammonia-oxidizing bacteria for wastewater treatment.     

Prevalence and characterization of motile Salmonella in commercial layer poultry farms in Bangladesh

Salmonella is a globally widespread food-borne pathogen having major impact on public health. All motile serovars of Salmonella enterica of poultry origin are zoonotic, and contaminated meat and raw eggs are an important source to human infections. Information on the prevalence of Salmonella at farm/holding level, and the zoonotic serovars circulating in layer poultry in the South and South-East Asian countries including Bangladesh, where small-scale commercial farms are predominant, is limited. To investigate the prevalence of Salmonella at layer farm level, and to identify the prevalent serovars we conducted a cross-sectional survey by randomly selecting 500 commercial layer poultry farms in Bangladesh. Faecal samples from the selected farms were collected following standard procedure, and examined for the presence of Salmonella using conventional bacteriological procedures. Thirty isolates were randomly selected, from the ninety obtained from the survey, for serotyping and characterized further by plasmid profiling and pulsed-field gel electrophoresis (PFGE). Results of the survey showed that the prevalence of motile Salmonella at layer farm level was 18% (95% confidence interval 15-21%), and Salmonella Kentucky was identified to be the only serovar circulating in the study population. Plasmid analysis of the S. Kentucky and non-serotyped isolates revealed two distinct profiles with a variation of two different sizes (2.7 and 4.8 kb). PFGE of the 30 S. Kentucky and 30 non-serotyped isolates showed that all of them were clonally related because only one genotype and three subtypes were determined based on the variation in two or three bands. This is also the first report on the presence of any specific serovar of Salmonella enterica in poultry in Bangladesh.