ANTI-SILENCING FUNCTION1 proteins are involved in ultraviolet-induced DNA damage repair and are cell cycle regulated by E2F transcription factors in Arabidopsis.

ANTI-SILENCING FUNCTION1 (ASF1) is a key histone H3/H4 chaperone that participates in a variety of DNA- and chromatin-related processes, including DNA repair, where chromatin assembly and disassembly are of primary relevance. Information concerning the role of ASF1 proteins in the post-ultraviolet (UV) response in higher plants is currently limited. In Arabidopsis (Arabidopsis thaliana), an initial analysis of in vivo localization of ASF1A and ASF1B indicates that both proteins are mainly expressed in proliferative tissues. In silico promoter analysis identified ASF1A and ASF1B as potential targets of E2F corresponds to Adenovirus E2 Binding Factor. [corrected]. These observations were experimentally validated, both in vitro, by electrophoretic mobility shift assays, and in vivo, by chromatin immunoprecipitation assays and expression analysis using transgenic plants with altered levels of different E2F transcription factors. These data suggest that ASF1A and ASF1B are regulated during cell cycle progression through E2F transcription factors. In addition, we found that ASF1A and ASF1B are associated with the UV-B-induced DNA damage response in Arabidopsis. Transcript levels of ASF1A and ASF1B were increased following UV-B treatment. Consistent with a potential role in UV-B response, RNA interference-silenced plants of both genes showed increased sensitivity to UV-B compared with wild-type plants. Finally, by coimmunoprecipitation analysis, we found that ASF1 physically interacts with amino-terminal acetylated histones H3 and H4 and with acetyltransferases of the Histone Acetyl Transferase subfamily, which are known to be involved in cell cycle control and DNA repair, among other functions. Together, we provide evidence that ASF1A and ASF1B are regulated by cell cycle progression and are involved in DNA repair after UV-B irradiation.

Potential of Salvinia biloba Raddi for removing atrazine and carbendazim from aquatic environments.

In this exploratory study, naturally occurring Salvinia biloba Raddi specimens were assessed for atrazine and carbendazim polluted water remediation. Experiments were carried out over 21 days in glass vessels containing deionized water artificially contaminated with 0, 5, 10, and 20 mg L-1 of atrazine or carbendazim. Atrazine had a pronounced detrimental impact on S. biloba, as no biomass development was observed in all macrophytes exposed to this herbicide in the entire concentration range. However, carbendazim-treated plants were able to grow and survive in the polluted medium even when subjected to the highest concentration of this fungicide (i.e., 20 mg L-1). In addition, increased chlorosis and necrosis were also detected in plants subjected to carbendazim as a result of the high phytotoxicity caused by atrazine. A maximal removal efficiency of ~ 30% was observed for both pesticides at 5 mg L-1 and decreased with increasing concentrations of the pollutants. The spectrum of the FTIR-ATR analysis revealed the existence of various functional groups (e.g., amide, carboxyl, hydroxyl, phosphate, sulfate) on the plants, which could be related to pesticide biosorption. In addition, at the end of the 21-day assay, seven carbendazim-resistant bacteria could be isolated from the roots of fungicide-treated plants. Therefore, the use of autochthonous free-floating S. biloba macrophytes for phytoremediation of aquatic environments contaminated with carbendazim shows great promise. Still, additional research is required to further elucidate the plant-mediated carbendazim elimination process and the role of the herbicide-resistant bacteria, and seek alternative species capable of mitigating atrazine contamination.

Regulation of plant MSH2 and MSH6 genes in the UV-B-induced DNA damage response.

Deleterious effects of UV-B radiation on DNA include the formation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). These lesions must be repaired to maintain the integrity of DNA and provide genetic stability. Of the several repair systems involved in the recognition and removal of UV-B-induced lesions in DNA, the focus in the present study was on the mismatch repair system (MMR). The contribution of MutSα (MSH2-MSH6) to UV-induced DNA lesion repair and cell cycle regulation was investigated. MSH2 and MSH6 genes in Arabidopsis and maize are up-regulated by UV-B, indicating that MMR may have a role in UV-B-induced DNA damage responses. Analysis of promoter sequences identified MSH6 as a target of the E2F transcription factors. Using electrophoretic mobility shift assays, MSH6 was experimentally validated as an E2F target gene, suggesting an interaction between MMR genes and the cell cycle control. Mutations in MSH2 or MSH6 caused an increased accumulation of CPDs relative to wild-type plants. In addition, msh2 mutant plants showed a different expression pattern of cell cycle marker genes after the UV-B treatment when compared with wild-type plants. Taken together, these data provide evidence that plant MutSα is involved in a UV-B-induced DNA damage response pathway.

From bacteria to plants: a compendium of mismatch repair assays.

Mismatch repair (MMR) system maintains genome integrity by correcting mispaired or unpaired bases which have escaped the proofreading activity of DNA polymerases. The basic features of the pathway have been highly conserved throughout evolution, although the nature and number of the proteins involved in the mechanism vary from prokaryotes to eukaryotes and even between humans and plants. Cells deficient in MMR genes have been observed to display a mutator phenotype characterized by an increased rate in spontaneous mutation, instability of microsatellite sequences and illegitimate recombination between diverged DNA sequences. Studies of the mutator phenotype have demonstrated a critical role for the MMR system in mutation avoidance and genetic stability. Here, we briefly review our current knowledge of the MMR mechanism and then focus on the in vivo biochemical and genetic assays used to investigate the function of the MMR proteins in processing DNA mismatches generated during replication and mitotic recombination in Escherichia coli, Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana. An overview of the biochemical assays developed to study mismatch correction in vitro is also provided.

Primary metabolism changes triggered in soybean leaves by Fusarium tucumaniae infection.

Sudden death syndrome (SDS) of soybean can be caused by at least four distinct Fusarium species, with F. tucumaniae being the main causal agent in Argentina. The fungus is a soil-borne pathogen that is largely confined to the roots, but damage also reaches aerial part of the plant and interveinal chlorosis and necrosis, followed by premature defoliation can be observed. In this study, two genetically diverse soybean cultivars, one susceptible (NA 4613) and one partially resistant (DM 4670) to SDS infection, were inoculated with F. tucumaniae or kept uninoculated. Leaf samples at 7, 10, 14 and 25 days post-inoculation (dpi) were chosen for analysis. With the aim of detecting early markers that could potentially discriminate the cultivar response to SDS, gas chromatography-mass spectrometry (GC-MS) analyses and biochemical studies were performed. Metabolic analyses show higher levels of several amino acids in the inoculated than in the uninoculated susceptible cultivar starting at 10 dpi. Biochemical studies indicate that pigment contents and Rubisco level were reduced while class III peroxidase activity was increased in the inoculated susceptible plant at 10 dpi. Taken together, our results indicate that the pathogen induced an accumulation of amino acids, a decrease of the photosynthetic activity, and an increase of plant-specific peroxidase activity in the susceptible cultivar before differences of visible foliar symptoms between genotypes could be observed, thus suggesting that metabolic and biochemical approaches may contribute to a rapid characterization of the cultivar response to SDS.

Improvement in extracellular protease production by the marine antarctic yeast Rhodotorula mucilaginosa L7.

Microorganisms from extreme and restrictive eco systems, such as the Antarctic continent, are of great interest due to their ability to synthesize products of commercial value. Among these, enzymes from psychrotolerant and psychrophilic microorganisms offer potential economical benefits due to their high activity at low and moderate temperatures. The cold adapted yeast Rhodotorula mucilaginosa L7 was selected out of 97 yeasts isolated from Antarctica as having the highest extracellular proteolytic activity in preliminary tests. The present study was aimed at evaluating the effects of nutrient composition (peptone, rice bran extract, ammonium sulfate, sodium chloride) and physicochemical parameters (temperature and pH) on its proteolytic activity. A 26-2 fractional factorial design experiment followed by a central composite design (CCD 23) was performed to optimize the culture conditions and improve the extracellular proteolytic activity. The results indicated that the presence of peptone in the medium was the most influential factor in protease production. Enzymatic activity was enhanced by the interaction between low glucose and peptone concentrations. The optimization of culture conditions with the aid of mathematical modeling enabled a c. 45% increase in proteolytic activity and at the same time reduced the amount of glucose and peptone required for the culture. Thus culture conditions established in this work may be employed in the biotechnological production of this protease.