The Role of Aquaporin Overexpression in the Modulation of Transcription of Heavy Metal Transporters under Cadmium Treatment in Poplar.

Populus alba 'Villafranca' clone is well-known for its tolerance to cadmium (Cd). To determine the mechanisms of Cd tolerance of this species, wild-type (wt) plants were compared with transgenic plants over-expressing an aquaporin (aqua1, GenBank GQ918138). Plants were maintained in hydroponic conditions with Hoagland's solution and treated with 10 µM of Cd, renewed every 5 d. The transcription levels of heavy metal transporter genes (PaHMA2, PaNRAMP1.3, PaNRAMP2, PaNRAMP3.1, PaNRAMP3.2, PaABCC9, and PaABCC13) were analyzed at 1, 7, and 60 d of treatment. Cd application did not induce visible toxicity symptoms in wt and aqua1 plants even after 2 months of treatment confirming the high tolerance of this poplar species to Cd. Most of the analyzed genes showed in wt plants a quick response in transcription at 1 d of treatment and an adaptation at 60 d. On the contrary, a lower transcriptional response was observed in aqua1 plants in concomitance with a higher Cd concentration in medial leaves. Moreover, PaHMA2 showed at 1 d an opposite trend within organs since it was up-regulated in root and stem of wt plants and in leaves of aqua1 plants. In summary, aqua1 overexpression in poplar improved Cd translocation suggesting a lower Cd sensitivity of aqua1 plants. This different response might be due to a different transcription of PaNRAMP3 genes that were more transcribed in wt line because of the importance of this gene in Cd compartmentalization.

Characterization and quantification of thiol-peptides in Arabidopsis thaliana using combined dilution and high sensitivity HPLC-ESI-MS-MS.

Although thiol-peptide compounds, such as reduced glutathione (GSH), γ-glutamylcysteine (γ-EC), and phytochelatins, play fundamental roles in plants, their analytical determination and characterization is still somewhat problematic, mainly due to their high polarity and oxidation propensity. Thus, in this work a reliable and sensitive HPLC-ESI-MS-MS method was developed, in order to simultaneously assay, within 14-min instrumental runs, γ-EC, GSH, and phytochelatins up to phytochelatin 4. This analytical method was validated in shoot and root extracts of the model plant Arabidopsis thaliana (Brassicaceae) and guaranteed accurate quantification by using specific isotope labelled-internal standards for both GSH and phytochelatins, as well as standards for external calibration. Good linearities in the method performance were observed (R > 0.99), with a dynamic range over three orders of magnitude in thiol-peptide concentrations. In MRM mode, the detection sensitivity of the thiol-peptides was equal to approximately 16, 6, 7, 13, 10 fmol for γ-EC, GSH, phytochelatin 2, phytochelatin 3, and phytochelatin 4, respectively (20 µl injection each). The reproducibility of the method was confirmed by high intra- and inter-day accuracy and precision values. The recovery rates were estimated approximately in the range of 73.8-91.0% and the matrix effect evaluation revealed that all analytes exhibited ionization suppression. The use of stable isotope-labelled analogs of the thiol-peptides as internal standards was particularly worthy of note: it offered the considerable advantage of overcoming the consequences of matrix effect and thiol-peptide loss through sample preparation, by normalizing the analyte signal during the quantification process. Thus, by validating the method's sensitivity, accuracy, precision, reproducibility, stability, recovery, and matrix effect, data reliability and robustness were ensured.

The phytochelatin synthase from Nitella mucronata (Charophyta) plays a role in the homeostatic control of iron(II)/(III).

Although some charophytes (sister group to land plants) have been shown to synthesize phytochelatins (PCs) in response to cadmium (Cd), the functional characterization of their phytochelatin synthase (PCS) is still completely lacking. To investigate the metal response and the presence of PCS in charophytes, we focused on the species Nitella mucronata. A 40 kDa immunoreactive PCS band was revealed in mono-dimensional western blot by using a polyclonal antibody against Arabidopsis thaliana PCS1. In two-dimensional western blot, the putative PCS showed various spots with acidic isoelectric points, presumably originated by post-translational modifications. Given the PCS constitutive expression in N. mucronata, we tested its possible involvement in the homeostasis of metallic micronutrients, using physiological concentrations of iron (Fe) and zinc (Zn), and verified its role in the detoxification of a non-essential metal, such as Cd. Neither in vivo nor in vitro exposure to Zn resulted in PCS activation and PC significant biosynthesis, while Fe(II)/(III) and Cd were able to activate the PCS in vitro, as well as to induce PC accumulation in vivo. While Cd toxicity was evident from electron microscopy observations, the normal morphology of cells and organelles following Fe treatments was preserved. The overall results support a function of PCS and PCs in managing Fe homeostasis in the carophyte N. mucronata.

Transcriptome analyses of Populus x euramericana clone I-214 leaves exposed to excess zinc.

Zinc (Zn) is an essential element for plant growth and development, but at high levels this metal can become toxic. Hyperaccumulator species are often not suitable for phytoremediation technologies because they need to be fast growing and have high biomass production, such as those of the Populus genus. Comparative genomics studies of poplars subjected to stress conditions such as heavy metal contamination have generated resources useful for improving the annotation of genes and have provided novel insights in the defense/tolerance mechanisms governing adaptation in non-hyperaccumulator plants. Using a microarray-based comparative analysis, we identified functional gene sets that are differentially regulated in the leaves of Populus × euramericana clone I-214 subjected to an excess but sub-lethal dose of Zn (1 mM). Eco-physiological and chemical analyses confirmed the results obtained in previous similar experiments. A total of 3861 expressed sequence tags (ESTs) were differentially expressed and grouped into two distinct libraries of up-regulated (40%) and down-regulated (60%) putative genes. The annotation of genes and gene products according to the Gene Ontology vocabularies was performed using Blast2GO software. The two transcriptome data sets were used to query all known Kyoto Encyclopedia of Genes and Genomes (KEGG) biosynthetic pathways of the genes identified in this study. The most represented molecular functions and biological processes were nucleotide binding and transcription, transport and response to stress and abiotic and biotic stimuli. The chloroplast, mitochondrion and their membrane systems were the cellular components most affected by excess Zn, as well as the photosynthetic, defense, sulfur and glutathione (GSH) metabolic pathways. The most up-regulated genes encoded electron carriers associated with ferrodoxin, the small subunit of ribulose-bisphosphate carboxylase oxygenase, and enzymes involved in GSH metabolism. This study is the most in-depth transcriptome and gene-annotation analysis of a hybrid poplar to date. The results are presented and critically discussed in terms of poplar response/tolerance to Zn stress for the characterization of non-hyperaccumulator phenotypes and the identification of candidate genes in perennial plants. These genetic findings provide useful information on tree species' adaptation to metal stress and provide powerful tools for the selection and/or genetic manipulation of stress-tolerant poplar clones.