This research was conducted to extend the knowledge on the differential regulation of laccase genes in response to dyes. In order to accomplish this, we analyzed both, the expression of five laccase genes by real time RT-qPCR, and also the laccase activity and isoforms patterns during the time-course of a Pleurotus ostreatus submerged fermentation supplemented with either acetyl yellow G (AYG) or remazol brilliant blue R (RBBR) dyes. For the purpose of obtaining a stable reference gene for optimal normalization of RT-quantitative PCR gene expression assays, we tested four candidate reference genes. As a result of this analysis, gpd was selected as reference index for data normalization. The addition of dyes had an induction effect on the enzymatic activity and also modified the zymogram profile. Fermentation with RBBR showed the highest laccase activity and number of isoforms along the course of the fermentation. Laccase gene expression profiles displayed up/down regulation along the fermentation time in four laccase genes (pox4, pox3, poxa1b and pox2), while pox1 was not expressed in either of the fermentation conditions. AYG addition caused the highest induction and repression levels for genes pox3 and poxa1b respectively. The expression level for all genes in the presence of RBBR were lower than in AYG, being in both conditions this response growth time dependent. These results show the influence of the nature of dyes on the induction level of laccase activity and on the differential regulation of the laccase genes expression in P. ostreatus.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0263-3) contains supplementary material, which is available to authorized users.

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Studies of gene expression are very important for the identification of genes that participate in different biological processes. Currently, reverse transcription quantitative real-time PCR (RT-qPCR) is a high-throughput, sensitive and widely used method for gene expression analysis. Nevertheless, RT-qPCR requires precise normalisation of data to avoid the misinterpretation of experimental data. In this sense, the selection of reference genes is critical for gene expression analysis. At this time, several studies focus on the selection of reference genes in several species. However, the identification and validation of reference genes for the normalisation of RT-qPCR have not been described in amaranth. A set of seven housekeeping genes were analysed using RT-qPCR, to determine the most stable reference genes in amaranth for normalisation of gene expression analysis. Transcript stability and gene expression level of candidate reference genes were analysed in different tissues, at different developmental stages and under different types of stress. The data were compared using the geNorm, NormFinder and Bestkeeper statistical methods. The reference genes optimum for normalisation of data varied with respect to treatment. The results indicate that AhyMDH, AhyGAPDH, AhyEF-1α and AhyACT would be optimum for accurate normalisation of experimental data, when all treatment are analysed in the same experiment. This study presents the most stable reference genes for normalisation of gene expression analysis in amaranth, which will contribute significantly to future gene studies of this species.

The degradation of 2,6-dimethoxyphenol (DMP) and decolorization of Remazol brilliant blue R dye (RBB), added to culture media of Pleurotus ostreatus developed in submerged fermentation, and the laccase, manganese peroxidase and veratryl alcohol oxidase activities produced in these systems were evaluated. Both compounds were removed from the culture medium mainly by enzymatic action. These compounds decreased the specific growth rate and the effect on the maximal biomass values was not important. The enzymatic activities were increased by DMP and/or RBB; however, the DMP showed a higher inducer effect on all enzymes than RBB. On the other hand, the RBB showed a larger inducer effect on manganese peroxidase activity than on the laccases and veratryl alcohol oxidase activities. These results show that DMP was a better inducer of ligninolytic enzymes than dye, and the process of dye decolorization and degradation of DMP requires the action of all enzymes of the ligninolytic complex.

Background: Enzymatic activity and laccase isoenzymes number of Pleurotus ostreatus grown in different pH values of the growing medium in submerged fermentation and incubated in buffer solutions of different initial pH values were determined. The expression profiles of five laccase genes (Lacc1, Lacc4, Lacc6, Lacc9 and Lacc10) in these cultures were also studied. Results: The highest laccases activity was obtained in cultures grown at initial pH of 4.5 and the lowest in cultures grown at initial pH of 8.5. Isoenzyme profiles were different in all the cases. Lacc1, Lacc4, Lacc6 and Lacc10 were expressed in all the cultures. Conclusions: The initial pH of the growing medium is an important factor for regulating the expression of laccase genes, having an effect on the activity and on the laccase isoenzymes number produced by P. ostreatus in SmF. This is the first report on the influence of different initial pH values of the growing medium on the laccases activity, laccase isoenzymes number and laccases expression profiles of P. ostreatus grown in submerged fermentation.