Regulation of dye-decolorizing peroxidase gene expression in Pleurotus ostreatus grown on glycerol as the carbon source.

Dye-decolorizing peroxidases (DyPs) (E.C. 1.11.1.19) are heme peroxidases that catalyze oxygen transfer reactions similarly to oxygenases. DyPs utilize hydrogen peroxide (H2O2) both as an electron acceptor co-substrate and as an electron donor when oxidized to their respective radicals. The production of both DyPs and lignin-modifying enzymes (LMEs) is regulated by the carbon source, although less readily metabolizable carbon sources do improve LME production. The present study analyzed the effect of glycerol on Pleurotus ostreatus growth, total DyP activity, and the expression of three Pleos-dyp genes (Pleos-dyp1, Pleos-dyp2 and Pleos-dyp4), via real-time RT-qPCR, monitoring the time course of P. ostreatus cultures supplemented with either glycerol or glucose and Acetyl Yellow G (AYG) dye. The results obtained indicate that glycerol negatively affects P. ostreatus growth, giving a biomass production of 5.31 and 5.62 g/L with respective growth rates (micra; m) of 0.027 and 0.023 h-1 for fermentations in the absence and presence of AYG dye. In contrast, respective biomass production levels of 7.09 and 7.20 g/L and growth rates (µ) of 0.033 and 0.047 h-1 were observed in equivalent control fermentations conducted with glucose in the absence and presence of AYG dye. Higher DyP activity levels, 4,043 and 4,902 IU/L, were obtained for fermentations conducted on glycerol, equivalent to 2.6-fold and 3.16-fold higher than the activity observed when glucose is used as the carbon source. The differential regulation of the DyP-encoding genes in P. ostreatus were explored, evaluating the carbon source, the growth phase, and the influence of the dye. The global analysis of the expression patterns throughout the fermentation showed the up- and down- regulation of the three Pleos-dyp genes evaluated. The highest induction observed for the control media was that found for the Pleos-dyp1 gene, which is equivalent to an 11.1-fold increase in relative expression (log2) during the stationary phase of the culture (360 h), and for the glucose/AYG media was Pleos-dyp-4 with 8.28-fold increase after 168 h. In addition, glycerol preferentially induced the Pleos-dyp1 and Pleos-dyp2 genes, leading to respective 11.61 and 4.28-fold increases after 144 h. After 360 and 504 h of culture, 12.86 and 4.02-fold increases were observed in the induction levels presented by Pleos-dyp1 and Pleos-dyp2, respectively, in the presence of AYG. When transcription levels were referred to those found in the control media, adding AYG led to up-regulation of the three dyp genes throughout the fermentation. Contrary to the fermentation with glycerol, where up- and down-regulation was observed. The present study is the first report describing the effect of a less-metabolizable carbon source, such as glycerol, on the differential expression of DyP-encoding genes and their corresponding activity.

Biotechnological Advances to Improve Abiotic Stress Tolerance in Crops.

The major challenges that agriculture is facing in the twenty-first century are increasing droughts, water scarcity, flooding, poorer soils, and extreme temperatures due to climate change. However, most crops are not tolerant to extreme climatic environments. The aim in the near future, in a world with hunger and an increasing population, is to breed and/or engineer crops to tolerate abiotic stress with a higher yield. Some crop varieties display a certain degree of tolerance, which has been exploited by plant breeders to develop varieties that thrive under stress conditions. Moreover, a long list of genes involved in abiotic stress tolerance have been identified and characterized by molecular techniques and overexpressed individually in plant transformation experiments. Nevertheless, stress tolerance phenotypes are polygenetic traits, which current genomic tools are dissecting to exploit their use by accelerating genetic introgression using molecular markers or site-directed mutagenesis such as CRISPR-Cas9. In this review, we describe plant mechanisms to sense and tolerate adverse climate conditions and examine and discuss classic and new molecular tools to select and improve abiotic stress tolerance in major crops.

The decolorization and degradation of azo dyes by two Stenotrophomonas strains isolated from textile effluent (Tepetitla, Mexico).

Stenotrophomonas' metabolic versatility plays important roles in the remediation of contaminated environment and plant growth promotion. We investigated two Stenotrophomonas strains isolated from textile polluted sewage for their ability to decolorize and degrade azo dyes. Two Stenotrophomonas strains (TepeL and TepeS) were isolated from textile effluents (Tepetitla, Mexico) using the selective agar Stenotrophomonas vancomycin, imipenem, amphotericin B agar (SVIA). Isolates' identity was determined by the sequencing of their partial 16S rRNA fragments. Their abilities to decolorize dyes were tested in a Luria broth supplemented with varying concentrations (50 mg/L-1 g/L) of textile dyes (acidic red, methyl orange, reactive green, acidic yellow, and reactive black). Fourier-transform infrared (FTIR) spectroscopy and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) metabolite analyses were used to determine the effect of the isolates' growth on the dyes (acidic red, methyl orange). We also identified the enzymes that may be involved in the degradation process. Phylogenetic analysis based on the 16S rDNA sequences showed that the isolates belong to the genus Stenotrophomonas. Stenotrophomonas sp. TepeL and TepeS respectively decolorize all the azo dyes at the tested concentration except at 1 g/L and degraded the azo dyes. The degradation resulted in the formation of N, N-dimethyl p-phenylenediamine, and sodium 4-amino-1-naphthalenesulfonate from methyl orange and acid red. TepeL and TepeS rapidly decolorized and degraded the azo dyes tested. This result showed that the two isolates have a good potential for the decontamination of textile effluents.

Pseudocrossidium replicatum (Taylor) R.H. Zander is a fully desiccation-tolerant moss that expresses an inducible molecular mechanism in response to severe abiotic stress.

KEY MESSAGE: The moss Pseudocrossidium replicatum is a desiccation-tolerant species that uses an inducible system to withstand severe abiotic stress in both protonemal and gametophore tissues. Desiccation tolerance (DT) is the ability of cells to recover from an air-dried state. Here, the moss Pseudocrossidium replicatum was identified as a fully desiccation-tolerant (FDT) species. Its gametophores rapidly lost more than 90% of their water content when exposed to a low-humidity atmosphere [23% relative humidity (RH)], but abscisic acid (ABA) pretreatment diminished the final water loss after equilibrium was reached. P. replicatum gametophores maintained good maximum photosystem II (PSII) efficiency (Fv/Fm) for up to two hours during slow dehydration; however, ABA pretreatment induced a faster decrease in the Fv/Fm. ABA also induced a faster recovery of the Fv/Fm after rehydration. Protein synthesis inhibitor treatment before dehydration hampered the recovery of the Fv/Fm when the gametophores were rehydrated after desiccation, suggesting the presence of an inducible protective mechanism that is activated in response to abiotic stress. This observation was also supported by accumulation of soluble sugars in gametophores exposed to ABA or NaCl. Exogenous ABA treatment delayed the germination of P. replicatum spores and induced morphological changes in protonemal cells that resembled brachycytes. Transcriptome analyses revealed the presence of an inducible molecular mechanism in P. replicatum protonemata that was activated in response to dehydration. This study is the first RNA-Seq study of the protonemal tissues of an FDT moss. Our results suggest that P. replicatum is an FDT moss equipped with an inducible molecular response that prepares this species for severe abiotic stress and that ABA plays an important role in this response.

High levels of glucose alter Physcomitrella patens metabolism and trigger a differential proteomic response.

Sugars act not only as substrates for plant metabolism, but also have a pivotal role in signaling pathways. Glucose signaling has been widely studied in the vascular plant Arabidopsis thaliana, but it has remained unexplored in non-vascular species such as Physcomitrella patens. To investigate P. patens response to high glucose treatment, we explored the dynamic changes in metabolism and protein population by applying a metabolomic fingerprint analysis (DIESI-MS), carbohydrate and chlorophyll quantification, Fv/Fm determination and label-free untargeted proteomics. Glucose feeding causes specific changes in P. patens metabolomic fingerprint, carbohydrate contents and protein accumulation, which is clearly different from those of osmotically induced responses. The maximal rate of PSII was not affected although chlorophyll decreased in both treatments. The biological process, cellular component, and molecular function gene ontology (GO) classifications of the differentially expressed proteins indicate the translation process is the most represented category in response to glucose, followed by photosynthesis, cellular response to oxidative stress and protein refolding. Importantly, although several proteins have high fold changes, these proteins have no predicted identity. The most significant discovery of our study at the proteome level is that high glucose increase abundance of proteins related to the translation process, which was not previously evidenced in non-vascular plants, indicating that regulation by glucose at the translational level is a partially conserved response in both plant lineages. To our knowledge, this is the first time that metabolome fingerprint and proteomic analyses are performed after a high sugar treatment in non-vascular plants. These findings unravel evolutionarily shared and differential responses between vascular and non-vascular plants.

Genome-wide transcriptional changes triggered by water deficit on a drought-tolerant common bean cultivar.

BACKGROUND: Common bean (Phaseolus vulgaris L.) is a relevant crop cultivated over the world, largely in water insufficiency vulnerable areas. Since drought is the main environmental factor restraining worldwide crop production, efforts have been invested to amend drought tolerance in commercial common bean varieties. However, scarce molecular data are available for those cultivars of P. vulgaris with drought tolerance attributes. RESULTS: As a first approach, Pinto Saltillo (PS), Azufrado Higuera (AH), and Negro Jamapa Plus (NP) were assessed phenotypically and physiologically to determine the outcome in response to drought on these common bean cultivars. Based on this, a Next-generation sequencing approach was applied to PS, which was the most drought-tolerant cultivar to determine the molecular changes at the transcriptional level. The RNA-Seq analysis revealed that numerous PS genes are dynamically modulated by drought. In brief, 1005 differentially expressed genes (DEGs) were identified, from which 645 genes were up-regulated by drought stress, whereas 360 genes were down-regulated. Further analysis showed that the enriched categories of the up-regulated genes in response to drought fit to processes related to carbohydrate metabolism (polysaccharide metabolic processes), particularly genes encoding proteins located within the cell periphery (cell wall dynamics). In the case of down-regulated genes, heat shock-responsive genes, mainly associated with protein folding, chloroplast, and oxidation-reduction processes were identified. CONCLUSIONS: Our findings suggest that secondary cell wall (SCW) properties contribute to P. vulgaris L. drought tolerance through alleviation or mitigation of drought-induced osmotic disturbances, making cultivars more adaptable to such stress. Altogether, the knowledge derived from this study is significant for a forthcoming understanding of the molecular mechanisms involved in drought tolerance on common bean, especially for drought-tolerant cultivars such as PS.

The Polycyclic Aromatic Hydrocarbon (PAH) degradation activities and genome analysis of a novel strain Stenotrophomonas sp. Pemsol isolated from Mexico.

BACKGROUND: Stenotrophomonas are ubiquitous gram-negative bacteria, which can survive in a wide range of environments. They can use many substances for their growth and are known to be intrinsically resistant to many antimicrobial agents. They have been tested for biotechnological applications, bioremediation, and production of antimicrobial agents. METHOD: Stenotrophomonas sp. Pemsol was isolated from a crude oil contaminated soil. The capability of this isolate to tolerate and degrade polycyclic aromatic hydrocarbons (PAH) such as anthraquinone, biphenyl, naphthalene, phenanthrene, phenanthridine, and xylene was evaluated in Bushnell Hass medium containing PAHs as the sole carbon sources. The metabolites formed after 30-day degradation of naphthalene by Pemsol were analyzed using Fourier Transform Infra-red Spectroscopic (FTIR), Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS). The genome of Pemsol was also sequenced and analyzed. RESULTS: Anthraquinone, biphenyl, naphthalene, phenanthrene, and phenanthridine except xylene can be used as sole carbon sources for Pemsol's growth in Bushnell Hass medium. The degradation of naphthalene at a concentration of 1 mg/mL within 30 days was tested. A newly formed catechol peak and the disappearance of naphthalene peak detected on the UPLC-MS, and GC-MS analyses spectra respectively confirmed the complete degradation of naphthalene. Pemsol does not produce biosurfactant and neither bio-emulsify PAHs. The whole genome was sequenced and assembled into one scaffold with a length of 4,373,402 bp. A total of 145 genes involved in the degradation of PAHs were found in its genome, some of which are Pemsol-specific as compared with other 11 Stenotrophomonas genomes. Most specific genes are located on the genomic islands. Stenotrophomonas sp. Pemsol's possession of few genes that are associated with bio-emulsification gives the genetic basis for its inability to bio-emulsify PAH. A possible degradation pathway for naphthalene in Pemsol was proposed following the analysis of Pemsol's genome. ANI and GGDH analysis indicated that Pemsol is likely a new species of Stenotrophomonas. It is the first report on a complete genome sequence analysis of a PAH-degrading Stenotrophomonas. Stenotrophomonas sp. Pemsol possesses features that make it a good bacterium for genetic engineering and will be an excellent tool for the remediation of crude oil or PAH-contaminated soil.

Desiccation-induced viable but nonculturable state in Pseudomonas putida KT2440, a survival strategy.

The potential of Pseudomonas putida KT2440 to act as a plant-growth promoter or as a bioremediator of toxic compounds can be affected by desiccation. In the present work, the bacterial survival ratio (BSR) in response to air desiccation was evaluated for P. putida KT2440 in the presence of different protectors. The BSR in the presence of nonreducing disaccharides, such as trehalose, was high after 15 days of desiccation stress (occurring at 30°C and 50% relative humidity), whereas in the absence of a protector the bacterial counts diminished to nondetectable numbers (ca 2.8 log CFU/mL). The LIVE/DEAD staining method showed that bacteria protected with trehalose maintained increased numbers of green cells after desiccation while cells without protection were all observed to be red. This indicated that nonprotected bacteria had compromised membrane integrity. However, when nonprotected bacteria subjected to 18 days of desiccation stress were rehydrated for a short time with maize root exudates or for 48 h with water (prolonged rehydration), the bacterial counts were as high as that observed for those not subjected to desiccation stress, suggesting that the cells entered the viable but nonculturable (VBNC) state under desiccation and that they returned to a culturable state after those means of rehydration. Interestingly an increase in the green color intensity of cells that returned to a culturable state was observed using LIVE/DEAD staining method, indicating an improvement in their membrane integrity. Cellular activity in the VBNC state was determined. A GFP-tagged P. putida strain expressing GFP constitutively was subjected to desiccation. After 12 days of desiccation, the GFP-tagged strain lost culturability, but it exhibited active GFP expression, which in turn made the cells green. Furthermore, the expression of 16S rRNA, rpoN (housekeeping), mutL, mutS (encoding proteins from the mismatch repair complex), and oprH (encoding an outer membrane protein) were examined by RT-PCR. All evaluated genes were expressed by both types of cells, culturable and nonculturable, indicating active molecular processes during the VBNC state.

Complete Genome Sequence of Stenotrophomonas maltophilia Strain SVIA2, Isolated from Crude Oil-Contaminated Soil in Tabasco, Mexico.

Stenotrophomonas maltophilia strain SVIA2 was isolated from crude oil-contaminated soil from Tabasco, Mexico, and displayed a good potential for the degradation of polycyclic aromatic hydrocarbons (PAHs), using naphthalene, anthracene, phenanthridine, or biphenyl as the unique source of carbon. The SVIA2 genome contains essential genes involved in the degradation of PAHs.

Effect of textile dyes on activity and differential regulation of laccase genes from Pleurotus ostreatus grown in submerged fermentation.

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.

The Actions of Lyophilized Apple Peel on the Electrical Activity and Organization of the Ventricular Syncytium of the Hearts of Diabetic Rats.

This study was designed to examine the effects of lyophilized red delicious apple peel (RDP) on the action potentials (APs) and the input resistance-threshold current relationship. The experiments were performed on isolated papillary heart muscles from healthy male rats, healthy male rats treated with RDP, diabetic male rats, and diabetic male rats treated with RDP. The preparation was superfused with oxygenated Tyrode's solution at 37°C. The stimulation and the recording of the APs, the input resistance, and the threshold current were made using conventional electrophysiological methods. The RDP presented no significant effect in normal rats. Equivalent doses in diabetic rats reduced the APD and ARP. The relationship between input resistance and threshold current established an inverse correlation. The results indicate the following: (1) The functional structure of the cardiac ventricular syncytium in healthy rats is heterogeneous, in terms of input resistance and threshold current. Diabetes further accentuates the heterogeneity. (2) As a consequence, conduction block occurs and increases the possibility of reentrant arrhythmias. (3) These modifications in the ventricular syncytium, coupled with the increase in the ARP, are the adequate substrate so that, with diabetes, the heart becomes more arrhythmogenic. (4) RDP decreases the APD, the ARP, and most syncytium irregularity caused by diabetes.