Tobacco plants over-expressing the sweet orange tau glutathione transferases (CsGSTUs) acquire tolerance to the diphenyl ether herbicide fluorodifen and to salt and drought stresses.

The glutathione transferases (GSTs) are members of a superfamily of enzymes with pivotal role in the detoxification of both xenobiotic and endogenous compounds. In this work, the generation and characterization of transgenic tobacco plants over-expressing tau glutathione transferases from Citrus sinensis (CsGSTU1 and CsGSTU2) and several cross-mutate forms of these genes are reported. Putative transformed plants were verified for the presence of the transgenes and the relative quantification of transgene copy number was evaluated by Taqman real time PCR. The analysis of gene expression revealed that transformed plants exhibit high levels of CsGSTU transcription suggesting that the insertion of the transgenes occurred in transcriptional active regions of the tobacco genome. In planta studies demonstrate that transformed tobacco plants gain tolerance against fluorodifen. Simultaneously, the wild type CsGSTU genes were in vitro expressed and their kinetic properties were determined using fluorodifen as substrate. The results show that CsGSTU2 follows a Michaelis-Menten hyperbolic kinetic, whereas CsGSTU1 generates a sigmoid plot typical of the regulatory enzymes, thus suggesting that when working at sub-lethal fluorodifen concentrations CsGSTU2 can counteract the herbicide injury more efficiently than the CsGSTU1. Moreover, the transgenic tobacco plant over-expressing CsGSTs exhibited both drought and salinity stress tolerance. However, as we show that CsGSTUs do not function as glutathione peroxidase in vitro, the protective effect against salt and drought stress is not due to a direct scavenging activity of the oxidative stress byproducts. The transgenic tobacco plants, which are described in the present study, can be helpful for phytoremediation of residual xenobiotics in the environment and overall the over-expression of CsGSTUs can be helpful to develop genetically modified crops with high resistance to abiotic stresses.

The glutathione S-transferase gene superfamily: an in silico approach to study the post translational regulation.

The use of plants to reclaim contaminated soils and groundwater, known as phytoremediation, is a promising biotechnological strategy which has gained a lot of attention in the last few years. Plants have evolved sophisticated detoxification systems against the toxin chemicals: following the uptake, the compounds are activated so that certain functional groups can conjugate hydrophilic molecules, such as thiols. The resulting conjugates are recognized by the tonoplast transporters and sequestered into the vacuoles. The xenobiotic conjugation with glutathione is mediated by enzymes which belong to the superfamily of glutathione S-transferases (GSTs) catalyzing the nucleophylic attack of the sulphur of glutathione on the electrophilic groups of the cytotoxic substrates therefore playing a crucial role in their degradation. This study was designed to identify the putative correlation between structural and functional characteristics of plant GST classes belonging to different plant species. Consequently, the protein sequences of the expressed GSTs have been retrieved from UniGene, classified and then analyzed in order to assess the evolutionary trend and to predict secondary structure. Moreover, the fingerprint analysis was performed with SCAN Prosite in the attempt to correlate meaningful signature profile and biological information. The results evidenced that all the soluble GSTs have a tendency to assume the α-helix secondary structure followed by random coil and ß-sheet. The fingerprint analysis revealed that specific signature profiles related mainly to protein phosphorylation are in the GST classes of all considered species thus suggesting that they might be subjected to reversible activation by phosphorylation-mediated regulation. This approach provides the knowledge of the relationship between presence of conserved signature profile and biological function in the view of future selection of GSTs which might be employed in either mutagenesis or genetic engineering studies.

Short cold storage enhances the anthocyanin contents and level of transcripts related to their biosynthesis in blood oranges.

The health benefits associated with the consumption of anthocyanin-containing foods are extensively documented. Mature fruits of blood oranges and their hybrids are characterized by the presence of these bioactive pigments, the abundance of which can be enhanced by storing fruit at cooling nonfreezing temperature. In this work the effects of short low-temperature exposure (4 °C × 15 days) upon orange anthocyanin content and the expression of structural genes belonging to the pigment biosynthesis pathway were investigated. The results highlight that anthocyanin levels of fruit exposed to cold sharply increase, reaching, after 6 days of storage, a value 8 times higher than that observed in the time zero samples, thus suggesting that fruit with enhanced health-related attributes might be obtained at this storage stage. The analysis of gene expression shows that the amount of transcripts of all considered genes (CM1, PAL, CHS, DFR, ANS, UFGT, and GST) sharply increased after 3-6 days of cold storage, confirming previous data showing that the biosynthesis of anthocyanins is a cold-regulated pathway. By comparing the expression of selected genes (PAL, DFR, and UFGT) between blood and common oranges, it turns out that those genes strictly involved in anthocyanin biosynthesis are not cold responsive in common oranges. Moreover, the data highlight that the EST encoding the transcription factor NAC domain protein is selectively induced by cold in blood oranges but not in common oranges, thus proposing it as a candidate gene specifically involved in blood orange response to cold exposure.