Eukaryotic and prokaryotic phytochelatin synthases differ less in functional terms than previously thought: a comparative analysis of Marchantia polymorpha and Geitlerinema sp. PCC 7407

This paper reports functional studies on the enzyme phytochelatin synthase in the liverwortMarchantia polymorphaand the cyanobacteriumGeitlerinemasp. strain PCC 7407. In vitro activity assays in control samples (cadmium-untreated) showed that phytochelatin synthase was constitutively expressed in both organisms. In the presence of 100 mu M cadmium, in both the liverwort and the cyanobacterium, the enzyme was promptly activated in vitro, and produced phytochelatins up to the oligomer PC4. Likewise,in vivoexposure to 10-36 mu M cadmium for 6-120 h induced in both organisms phytochelatin synthesis up to PC4. Furthermore, the glutathione (GSH) levels inM. polymorphawere constitutively low (compared with the average content in higher plants), but increased considerably under cadmium stress. Conversely, the GSH levels inGeitlerinemasp. PCC 7407 were constitutively high, but were halved under metal treatments. At odds with former papers, our results demonstrate that, as inM. polymorphaand other plants, the cyanobacterial phytochelatin synthase exposed to cadmium possesses manifest transpeptidasic activity, being able to synthesize phytochelatins with a degree of oligomerization higher than PC2. Therefore, prokaryotic and eukaryotic phytochelatin synthases differ less in functional terms than previously thought

Ancestral function of the phytochelatin synthase C-terminal domain in inhibition of heavy metal-mediated enzyme overactivation

Phytochelatin synthases (PCSs) play essential roles in detoxification of a broad range of heavy metals in plants and other organisms. Until now, however, no PCS gene from liverworts, the earliest branch of land plants and possibly the first one to acquire a PCS with a C-terminal domain, has been characterized. In this study, we isolated and functionally characterized the first PCS gene from a liverwort, Marchantia polymorpha (MpPCS). MpPCS is constitutively expressed in all organs examined, with stronger expression in thallus midrib. The gene expression is repressed by Cd2+ and Zn2+. The ability of MpPCS to increase heavy metal resistance in yeast and to complement cad1-3 (the null mutant of the Arabidopsis ortholog AtPCS1) proves its function as the only PCS from M. polymorpha. Site-directed mutagenesis of the most conserved cysteines of the C-terminus of the enzyme further uncovered that two twin-cysteine motifs repress, to different extents, enzyme activation by heavy metal exposure. These results highlight an ancestral function of the PCS elusive C-terminus as a regulatory domain inhibiting enzyme overactivation by essential and non-essential heavy metals. The latter finding may be relevant for obtaining crops with decreased root to shoot mobility of cadmium, thus preventing its accumulation in the food chain

Retaining unlogged patches in Mediterranean oak forests may preserve threatened forest macrolichens

Forest management practices may heavily impact epiphytic (tree inhabiting) organisms. Retaining tree patches and buffer strips in logged stands may contribute to preserve ecosystem functioning and the vitality of epiphytic organisms in managed forests. To test these statements, the threatened forest macrolichen Lobaria pulmonaria (L.) Hoffm. was used as a model species, since it is a “flag” indicator species of forest ecosystems with long ecological continuity. To this purpose, photosynthetic performances, thallus anatomy and water holding capacity (WHC) of samples of L. pulmonaria were investigated in a logged mixed oak forest (Tuscany, Italy), confronting lichen thalli from retained- forest patches and retained-isolated trees, 18 months after logging. Compared with those of retained-forest patches, thalli on the trunks of retained- isolated trees were thinner and showed lower vitality (as indicated by the potential quantum yield of primary photochemistry – FV/FM and the index of overall photosynthetic performance – PIABS), as well as lower water holding capacity. In contrast, thalli from forest patches had performances comparable to those of healthy samples from unlogged forests

Cadmium tolerance and phytochelatin content of Arabidopsis seedlings over-expressing the phytochelatin synthase gene AtPCS1

Previous studies demonstrated that expression of the Arabidopsis phytochelatin (PC) biosynthetic gene AtPCS1 in Nicotiana tabacum plants increases the Cd tolerance in the presence of exogenous glutathione (GSH). In this paper, the Cd tolerance of Arabidopsis plants over-expressing AtPCS1 (AtPCSox lines) has been analysed and the differences between Arabidopsis and tobacco are shown. Based on the analysis of seedling fresh weight, primary root length, and alterations in root anatomy, evidence is provided that, at relatively low Cd concentrations, the Cd tolerance of AtPCSox lines is lower than the wild type, while AtPCS1 over-expressing tobacco is more tolerant to Cd than the wild type. At higher Cd concentrations, Arabidopsis AtPCSox seedlings are more tolerant to Cd than the wild type, while tobacco AtPCS1 seedlings are as sensitive as the wild type. Exogenous GSH, in contrast to what was observed in tobacco, did not increase the Cd tolerance of AtPCSox lines. The PC content in wild-type Arabidopsis at low Cd concentrations is more than three times higher than in tobacco and substantial differences were also found in the PC chain lengths. These data indicate that the differences in Cd tolerance and in its dependence on exogenous GSH between Arabidopsis and tobacco are due to species-specific differences in the endogenous content of PCs and GSH and may be in the relative abundance of PCs of different length

Correlation between hormonal homeostasis and morphogenic responses in Arabidopsis thaliana seedlings growing in a Cd/Cu/Zn multi-pollution context

To date, almost no information is available in roots and shoots of the model plant Arabidopsis thaliana about the hierarchic relationship between metal accumulation, phytohormone levels, and glutathione/phytochelatin content, and how this relation affects root development. For this purpose, specific concentrations of cadmium, copper and zinc, alone or in triple combination, were supplied for 12 days to in vitro growing seedlings. The accumulation of these metals was measured in roots and shoots, and a significant competition in metal uptake was observed. Microscopic analyses revealed that root morphology was affected by metal exposure, and that the levels of trans-zeatin riboside, dihydrozeatin riboside, indole-3-acetic acid, and the auxin/cytokinin ratio varied accordingly. By contrast, under metal treatments, minor modifications in gibberellic acid and abscisic acid levels occurred. RT-PCR analysis of some genes involved in auxin and cytokinin synthesis (e.g., AtNIT in roots and AtIPT in shoots) showed on average a metal up-regulated transcription. The production of thiol-peptides was induced by all the metals, alone or in combination, and the expression of the genes involved in thiol-peptide synthesis (AtGSH1, AtGSH2, AtPCS1 and AtPCS2) was not stimulated by the metals, suggesting a full post-transcriptional control. Results show that the Cd/Cu/Zn-induced changes in root morphology are caused by a hormonal unbalance, mainly governed by the auxin/cytokinin ratio

The Arabidopsis thaliana knockout mutant for phytochelatin synthase1 (cad1-3) Is defective in callose deposition, bacterial pathogen defense and auxin content, but shows an increased stem lignification

The enzyme phytochelatin synthase (PCS) has long been studied with regard to its role in metal(loid) detoxification in several organisms, i.e., plants, yeasts, and nematodes. It is in fact widely recognized that PCS detoxifies a number of heavy metals by catalyzing the formation of thiol-rich oligomers, namely phytochelatins, from glutathione and related peptides. However, recent investigations have highlighted other possible roles played by the PCS enzyme in the plant cell, e.g., the control of pathogen-triggered callose deposition. In order to examine novel aspects of Arabidopsis thaliana PCS1 (AtPCS1) functions and to elucidate its possible roles in the secondary metabolism, metabolomic data of A. thaliana wild-type and cad1-3 mutant were compared, the latter lacking AtPCS1. HPLC-ESI-MS analysis showed differences in the relative levels of metabolites from the glucosinolate and phenylpropanoid pathways between cad1-3 and wild-type plants. Specifically, in control (Cd-untreated) plants, higher levels of 4-methoxy-indol 3-ylmethylglucosinolate were found in cad1-3 plants vs. wild-type. Moreover, the cad1-3 mutant showed to be impaired in the deposit of callose after Cd exposure, suggesting that AtPCS1 protects the plant against the toxicity of heavy metals not only by synthesizing PCs, but also by contributing to callose deposition. In line with the contribution of callose in counteracting Cd toxicity, we found that another callose-defective mutant, pen2-1, was more sensitive to high concentrations of Cd than wild-type plants. Moreover, cad1-3 plants were more susceptible than wild-type to the hemibiotrophic bacterial pathogen Pseudomonas syringae. The metabolome also revealed differences in the relative levels of hydroxycinnamic acids and flavonols,with consequences on cell wall properties and auxin content, respectively. First, increased lignification in the cad1-3 stems was found, probably aimed at counteracting the entry of Cd into the inner tissues. Second, in cad1-3 shoots, increased relative levels of kaempferol 3,7 dirhamnoside and quercetin hexoside rhamnoside were detected. These flavonols are endogenous inhibitors of auxin transport in planta; auxin levels in both roots and shoots of the cad1-3 mutant were in fact lower than those of the wild-type. Overall, our data highlight novel aspects of AtPCS1 functions in A. thaliana

Does air pollution influence the success of species translocation? Trace elements, ultrastructure and photosynthetic performances in transplants of a threatened forest macrolichen

Species translocation can be considered as a primary conservation strategy with reference to in situ conservation. In the case of lichens, translocations often risk to fail due stress factors associated with unsuitable receptor sites. Considering the bioecological characteristics of lichens, air pollution is among the most limiting stress factors. In this study, the forest macrolichen Lobaria pulmonaria was used as a model to test the hypothesis that the translocation of sensitive lichens is effective only in unpolluted environments. At purpose, 500 fragments or whole thalli were translocated in selected beech forests of Central Europe (the Western Carpathians, Slovakia) where the species disappeared in the past and in oak forests of Southern Europe (Tuscany, Central Italy) where native populations are present. Prior to the translocation (May 2016) and after one year, morphological and ultrastructural features, trace elements as well as chlorophyll a fluorescence emission were analysed. Four years later, the effectiveness of lichen translocation was further evaluated as presence of the transplants and of newly formed individuals. After one year, the translocation ensured an effective survival of the thalli in remote oak and beech forests characterized by a negligible or low contamination by heavy metals. The transplants were considered successful and developed new lobules and rhizines, attaching by themselves to the bark of the host trees, looking overall healthy, without evident signs of alteration also at ultrastructural level. Moreover, in a few cases newly formed individuals were observed after four years. On the other hand, the results highlighted the link between the unsuccess of the translocation and air pollution in other areas of the Western Carpathians and suggested that current air quality still limits the possibility of recolonization in areas where the model species disappeared

Interaction and accumulation of manganese and cadmium in the manganese accumulator Lupinus albus

NOTICE: this is the author’s version of a work that was accepted for publication in Journal of Plant Physiology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Plant Physiology, 167, 13 (2010) DOI http://dx.doi.org/10.1016/j.jplph.2010.02.011The effects of the interaction between Mn and Cd on the growth of the white lupin (Lupinus albus), uptake of these metals, their accumulation, and effects on heavy metal stress indicators were studied under glasshouse conditions. Plants were grown with and without Mn and/or Cd for 4 weeks. The absence of Mn and Cd led to lipid peroxidation-induced loss of flavonoids and anthocyanins in the roots, reduced the size of the plant canopy, and led to the appearance of proteoid roots. Sensitivity to Cd in white lupin was enhanced by a low Mn supply, despite lower Cd uptake and accumulation (leaf Mn:Cd concentration ratio <3), as evidenced by increased lipid peroxidation in the leaves and strong inhibition of growth. However, when the Mn supply was adequate, the plants showed few symptoms of Cd toxicity, even though Cd uptake and accumulation increased. A Mn:Cd ratio of up to 20 was enough to minimize Cd stress in the leaf, reflecting the plants' relative tolerance to Cd under such conditions. Irrespective of the Mn supply, the increase in antioxidant compounds observed in the roots of Cd-treated plants might act as a protective mechanism by minimizing the oxidative stress caused by Cd exposure. In summary, high leaf Mn concentrations seem to render white lupins more tolerant to Cd stressThis work was funded by the Spanish Ministry of Education and Science (project CTM2005-04809/TECNO