[show abstract][hide abstract] ABSTRACT: Bryophytes, a paraphyletic group which includes liverworts, mosses, and hornworts, have been stated as land plants that under metal stress (particularly cadmium) do not synthesize metal-binding peptides such as phytochelatins. Moreover, very little information is available to date regarding phytochelatin synthesis in charophytes, postulated to be the direct ancestors of land plants, or in lycophytes, namely very basal tracheophytes. In this study, it was hypothesized that basal land plants and charophytes have the capability to produce phytochelatins and possess constitutive and functional phytochelatin synthases. To verify this hypothesis, twelve bryophyte species (six liverworts, four mosses, and two hornworts), three charophytes, and two lycophyte species were exposed to 0-36 μM cadmium for 72h, and then assayed for: (i) glutathione and phytochelatin quali-quantitative content by HPLC and mass spectrometry; (ii) the presence of putative phytochelatin synthases by western blotting; and (iii) in vitro activity of phytochelatin synthases. Of all the species tested, ten produced phytochelatins in vivo, while the other seven did not. The presence of a constitutively expressed and functional phytochelatin synthase was demonstrated in all the bryophyte lineages and in the lycophyte Selaginella denticulata, but not in the charophytes. Hence, current knowledge according to phytochelatins have been stated as being absent in bryophytes was therefore confuted by this work. It is argued that the capability to synthesize phytochelatins, as well as the presence of active phytochelatin synthases, are ancestral (plesiomorphic) characters for basal land plants.
Journal of Experimental Botany 01/2014; · 5.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: Very few studies have provided information about the effects of cadmium (Cd) at histoanatomical and ultrastructural levels, along with potential localization of the metal in planta. In particular, from this standpoint, almost nothing is known in Daucus carota L. (carrot), a particularly important species for in vitro and in vivo functional investigations. In this work we hypothesized that 36 μM Cd, supplied for 1, 2, 3, 4, 7 and 14 days to 30-day-old in vitro-cultured plants, might induce an early acclimation, but a final collapse of roots and leaves. In fact, as a general feature, a biphasic root response to Cd stress actually took place: in the first phase (1-4 days of Cd exposure), the cytological and functional events observed - by light microscopy, TEM, epifluorescence, as well as by the time-course of thiol-peptide compounds - can be interpreted as acclimatory responses aimed at diminishing the movement of Cd across the root. The second phase (from 4 to 14 days of Cd exposure) was instead characterized by cell hypertrophy, cell-to-cell separation events, increase in α-β-γ-tocopherol levels and, not least, endocytogenic processes, coupled with a dramatic drop in the amount of thiol-peptide compounds. These events led to a progressive root collapse, even if they did not ingenerate macro/microscopic injury symptoms in leaf blades and petioles.
Plant Physiology and Biochemistry 07/2012; 58:269-79. · 2.78 Impact Factor
[show abstract][hide abstract] ABSTRACT: Indian mustard (Brassica juncea L. Czern.) tolerates high concentrations of heavy metals and is a promising species for the purpose of phytoextraction of cadmium (Cd) from metal-contaminated soils. This work investigates the extent to which antioxidant and metal sequestering mechanisms are responsible for this tolerance. To this end, seedlings of Indian mustard were grown for 7 days in 0, 50 or 200 μM Cd. Increasing Cd concentrations led to a progressive Cd accumulation in roots and shoots, accompanied by an organ-dependent alteration in mineral uptake, and a decrease in root/shoot length and fresh/dry weight. Cd negatively affected chlorophyll and carotenoid contents and activated the xanthophyll cycle, suggesting the need to protect the photosynthetic apparatus from photoinhibition. Shoots seemed to be less efficient than roots in ROS scavenging, as indicated by the different response to Cd stress shown by peroxidase and catalase activities and, solely with regard to the highest Cd concentration, by ascorbate level. Such a different antioxidant capacity might at least partly explain differences in the trend of lipid peroxidation observed in the two organs. Moreover, in both roots and shoots, glutathione and phytochelatin content markedly increased under Cd stress, regardless of the metal concentration involved.
Plant Physiology and Biochemistry 05/2012; 57:15-22. · 2.78 Impact Factor
[show abstract][hide abstract] ABSTRACT: Both Fe deficiency and Cd exposure induce rapid changes in the S nutritional requirement of plants. The aim of this work was to characterize the strategies adopted by plants to cope with both Fe deficiency (release of phytosiderophores) and Cd contamination [production of glutathione (GSH) and phytochelatins] when grown under conditions of limited S supply. Experiments were performed in hydroponics, using barley plants grown under S sufficiency (1.2 mM sulphate) and S deficiency (0 mM sulphate), with or without Fe(III)-EDTA at 0.08 mM for 11 d and subsequently exposed to 0.05 mM Cd for 24 h or 72 h. In S-sufficient plants, Fe deficiency enhanced both root and shoot Cd concentrations and increased GSH and phytochelatin levels. In S-deficient plants, Fe starvation caused a slight increase in Cd concentration, but this change was accompanied neither by an increase in GSH nor by an accumulation of phytochelatins. Release of phytosiderophores, only detectable in Fe-deficient plants, was strongly decreased by S deficiency and further reduced after Cd treatment. In roots Cd exposure increased the expression of the high affinity sulphate transporter gene (HvST1) regardless of the S supply, and the expression of the Fe deficiency-responsive genes, HvYS1 and HvIDS2, irrespective of Fe supply. In conclusion, adequate S availability is necessary to cope with Fe deficiency and Cd toxicity in barley plants. Moreover, it appears that in Fe-deficient plants grown in the presence of Cd with limited S supply, sulphur may be preferentially employed in the pathway for biosynthesis of phytosiderophores, rather than for phytochelatin production.
Journal of Experimental Botany 11/2011; 63(3):1241-50. · 5.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: The hypothesis that a daily water supply allows a lichen to endure the negative effects of environmental concentrations of NO(x) and O(3) was tested with a transplant experiment. Five groups (0, A-D) of Flavoparmelia caperata samples derived from the same thalli were used for destructive, pre-exposure measurements (0), or exposed for 5 weeks in the rural collection site (A), and in a urban site with high levels of NO(x) and O(3) (B-D). Two groups (C, D) were daily watered half an hour before the daily peak of NO(x) (C), and O(3) (D). The comparison between pre- and post-exposure measurements of stress biomarkers revealed that the different thallus hydration regime modified the pollution tolerance as well as the physiology of the exposed samples. The non-watered group B suffered an evident decrease in F(v)/F(m) and reduced glutathione, but increased ion leakage, whereas the watered groups C and D showed only decreased non-photosynthetic-quenching, possibly derived from NO(x) exposure. Ozone, which was higher in the rural than in the urban site, did not significantly affect the lichen metabolism. Our results re-open the discussion on the so-called "drought hypothesis", which suggests that the lichen desert observed in urban areas of central and eastern Europe is more a matter of dry microclimate than of air pollution.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Experimental Botany 08/2011; 62(15):5509-19. · 5.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cuscuta sp., known with the common name of “dodder”, is an obligate parasite capable of invading stems and leaves of a wide range of host plants. Dodder stem usually coils counterclockwise around the host and, within a few days, develops haustorial structures at each point of contact. As soon as dodder haustoria reach host vascular bundles, they start tapping water, photosynthates and minerals. Metal ions such as zinc (Zn) and copper (Cu) are essential for dodder growth and metabolism, although an exceedingly high (over-homeostatic) supply of these micronutrients can result in growth inhibition and cellular toxicity. Even more so, non-essential metals such as cadmium (Cd), if transferred from the host to the parasite, need to be neutralized by timely detoxification mechanisms. In this work, we showed that Cuscuta campestris Yuncker establishes effective haustorial connections with leaf petioles and blades of Daucus carota L. (carrot), with the consequent transfer of Cd and essential metals (such as Zn and Cu) from the host vascular bundles to the parasite. Following up to this point, we detected the presence in the parasite of significant amounts of glutathione and phytochelatins, even in the absence of Cd exposure. This suggests that thiol peptides in dodder might be particularly important for Zn and Cu homeostasis as well as for Cd detoxification. Finally, we demonstrated that dodder is capable of synthesizing phytochelatins on its own, rather than massively importing them from the host, and also provided evidence for the existence of an endogenous, constitutively expressed, dodder's phytochelatin synthase.
Environmental and Experimental Botany 08/2011; 72(1):26-33. · 2.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pteris vittata can tolerate very high soil arsenic concentration and rapidly accumulates the metalloid in its fronds. However, its tolerance to arsenic has not been completely explored. Arbuscular mycorrhizal (AM) fungi colonize the root of most terrestrial plants, including ferns. Mycorrhizae are known to affect plant responses in many ways: improving plant nutrition, promoting plant tolerance or resistance to pathogens, drought, salinity and heavy metal stresses. It has been observed that plants growing on arsenic polluted soils are usually mycorrhizal and that AM fungi enhance arsenic tolerance in a number of plant species. The aim of the present work was to study the effects of the AM fungus Glomus mosseae on P. vittata plants treated with arsenic using a proteomic approach. Image analysis showed that 37 spots were differently affected (21 identified). Arsenic treatment affected the expression of 14 spots (12 up-regulated and 2 down-regulated), while in presence of G. mosseae modulated 3 spots (1 up-regulated and 2 down-regulated). G. mosseae, in absence of arsenic, modulated 17 spots (13 up-regulated and 4 down-regulated). Arsenic stress was observed even in an arsenic tolerant plant as P. vittata and a protective effect of AM symbiosis toward arsenic stress was observed.
Journal of proteomics 03/2011; 74(8):1338-50. · 5.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ectomycorrhizal fungi are thought to enhance mineral nutrition of their host plants and to confer increased tolerance toward toxic metals. However, a global view of metal homeostasis-related genes and pathways in these organisms is still lacking. Building upon the genome sequence of Tuber melanosporum and on transcriptome analyses, we set out to systematically identify metal homeostasis-related genes in this plant-symbiotic ascomycete. Candidate gene products (101) were subdivided into three major functional classes: (i) metal transport (58); (ii) oxidative stress defence (32); (iii) metal detoxification (11). The latter class includes a small-size metallothionein (TmelMT) that was functionally validated in yeast, and phytochelatin synthase (TmelPCS), the first enzyme of this kind to be described in filamentous ascomycetes. Recombinant TmelPCS was shown to support GSH-dependent, metal-activated phytochelatin synthesis in vitro and to afford increased Cd/Cu tolerance to metal hypersensitive yeast strains. Metal transporters, especially those related to Cu and Zn trafficking, displayed the highest expression levels in mycorrhizae, suggesting extensive translocation of both metals to root cells as well as to fungal metalloenzymes (e.g., laccase) that are strongly upregulated in symbiotic hyphae.
Fungal Genetics and Biology 11/2010; 48(6):573-84. · 3.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: Reversed-phase liquid chromatography (RPLC) and electrospray (ESI)-linear ion trap (LIT) mass spectrometry was applied to the direct characterization of in vivo S-nitrosylated (SNO) phytochelatins (PCs) expressed in cadmium-stressed Arabidopsis thaliana cells. Cys-nitrosylation is under discussion as in vivo redox-based post-translational modification of proteins and peptides in plants in which the -NO group is involved as signal molecule in different biological functions. The gas-phase ion chemistry of in vivo and in vitro generated SNO-PC(s) was compared with the aim of evaluating NO binding stability and improving MS knowledge about peptide nitrosation. Using RPLC separation and ESI-LIT-MS, mono-nitrosylated PCs were identified in in vivo cadmium treated A. thaliana cells without derivatization. The in vivo binding of the NO group to PC(2), PC(3) and PC(4) resulted to occur selectively on only one cystein residue. The fragmentation pathway energies of the in vitro GSNO-generated NO-PCs with respect to the in vivo NO-PCs were investigated, suggesting the presence of a different internal stability for these molecules. By carrying out MS(2) experiments on these quasi-symmetric peptides, the different stability degree of the NO group was demonstrated to be correlated with the PC chain length. In addition, the data obtained highlight a putative role of the adjacent Glu/Cys motif in the gas-phase stability of the NO-containing molecule.
Journal of chromatography. A 02/2010; 1217(25):4120-6. · 4.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Thalli of epiphytic lichen Hypogymnia physodes (L.) Nyl. and terricolous Cladonia furcata (Huds.) Schrad., collected from an area with background arsenic concentrations, were exposed to 0, 0.1, 1 and 10 microg mL(-1) arsenate (As(V)) solutions for 24 h. After exposure they were kept in the metabolically active state for 0, 24 and 48 h in a growth chamber. In the freeze dried samples glutathione (GSH), glutathione disulphide (GSSG), cysteine (Cys) and cystine were analysed and induction of phytochelatin (PC) synthesis measured by reversed-phase high-performance liquid chromatography in combination with fluorescence detection or UV spectrometry. Total arsenic content in thalli was measured by instrumental neutron activation analysis (INAA). In H. physodes, which contained higher amounts of arsenic compared to C. furcata, total glutathione content significantly decreased in samples exposed to 10 microg mL(-1) As(V), whereas in C. furcata a significant increase was observed. In both species PC synthesis was induced in thalli exposed to 10 microg mL(-1).
Biology of Metals 11/2009; 23(2):207-19. · 3.17 Impact Factor
[show abstract][hide abstract] ABSTRACT: Exposure to cadmium (Cd(2+)) can result in cell death, but the molecular mechanisms of Cd(2+) cytotoxicity in plants are not fully understood. Here, we show that Arabidopsis (Arabidopsis thaliana) cell suspension cultures underwent a process of programmed cell death when exposed to 100 and 150 microm CdCl(2) and that this process resembled an accelerated senescence, as suggested by the expression of the marker senescence-associated gene12 (SAG12). CdCl(2) treatment was accompanied by a rapid increase in nitric oxide (NO) and phytochelatin synthesis, which continued to be high as long as cells remained viable. Hydrogen peroxide production was a later event and preceded the rise of cell death by about 24 h. Inhibition of NO synthesis by N(G)-monomethyl-arginine monoacetate resulted in partial prevention of hydrogen peroxide increase, SAG12 expression, and mortality, indicating that NO is actually required for Cd(2+)-induced cell death. NO also modulated the extent of phytochelatin content, and possibly their function, by S-nitrosylation. These results shed light on the signaling events controlling Cd(2+) cytotoxicity in plants.
[show abstract][hide abstract] ABSTRACT: Two bread wheat (Triticum aestivum L.) cultivars (Albimonte, traditional cultivar very important in Italy since long time; and Manital, more recent, evincing better productive performances) were grown for 10 d in presence of 0.7 (control), 70 or 350 μM ZnSO4, to verify whether Zn excess was differently managed at inter-varietal and at inter-organ level. Roots were found to be the main site of Zn accumulation, although a moderate metal translocation to leaves occurred in both cultivars. Despite only slight differences in internal Zn concentrations between cultivars, Albimonte seemed to be more sensitive to Zn excess in terms of growth reduction and H2O2 accumulation, suggesting that the diversities in responses to Zn stress should be ascribed here to inter-varietal metabolic differences. In both cultivars, increased NAD(P)H oxidation rate by pH-dependent peroxidases, and reduced detoxification activity by catalase and peroxidases, were responsible for Zn-induced H2O2 accumulation, while total superoxide dismutase content and activity seemed in general to not change or even depress. Moreover, differences in the content of thiol-peptide compounds (glutathione and phytochelatins) were detected, suggesting indeed the setting up of differential response mechanisms to Zn excess at an inter-varietal and inter-organ level.
[show abstract][hide abstract] ABSTRACT: Potted one-year-old plants of Thymus vulgaris L. (thyme, Lamiaceae, C3 metabolism), were grown for three months (10 June–10 September, 2004) in a “mini-free-air-CO2-enrichment” (“mini-FACE”) system, under 500 μmol mol−1 and ambient concentrations of atmospheric carbon dioxide (CO2). Compared to ambient CO2, elevated CO2 stimulated leaf superoxide dismutase (SOD, EC 22.214.171.124) activity only at the first sampling-time (July), followed by no variation or even a trend of decreased activity on the other two sampling-times (August and September). Under high CO2, guaiacol peroxidase (GPX, EC 126.96.36.199) and catalase (CAT, EC 188.8.131.52) leaf activities showed no variation or drop throughout the duration of the experiment. By contrast, under elevated CO2, leaf glutathione reductase (GR, EC 184.108.40.206) activity increased on all the sampling-times, and also a duration-dependent upward trend of glutathione (GSH) content was recorded, with this increase becoming significant – compared with ambient CO2 – at the third sampling-time (September). Simultaneously, leaves from plants grown under high CO2 showed a marked increase in essential oil yield, with slight increments in phenolic component and decrements in mono- and sesquiterpene components. Also, a drop in thiobarbituric acid reactive substances (TBARS) content under elevated CO2 was displayed. Thus, in general, the results reported here point to a downregulation of leaf antioxidant enzymes under elevated CO2, supporting the notion of reduced reactive oxygen species (ROS) formation under these circumstances. Relying instead on antioxidant-regenerating enzymes, namely GR, fairly high GSH content and essential oils, might be a ‘low cost’ life strategy for growth under elevated CO2, not requiring synthesis/activation of energy-intensive and expensive metabolic processes.
Environmental and Experimental Botany 01/2009; 65(1):99-107. · 2.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: This work was undertaken to explore the potential of proteomics to dissect parallel and consecutive events of cadmium stress response in the lichen Physcia adscendens (Fr.) H. Olivier. Thalli were exposed to 0 (control) and 36 microM Cd for 6, 18, 24 and 48 h. Two-dimensional electrophoresis and mass spectrometry analyses showed an 80-85% spot identity between 6 and 18 h vs. 24 and 48 h of Cd exposure. Putative heat-shock proteins and glutathione S-transferase generally increased their expression all over the Cd treatments. By contrast, ABC transporters were underexpressed after 6-18 h, but in some cases induced after 24-48 h of Cd exposure. The cytochrome P450 appeared to have a variable expression pattern over time. Overall these data suggest that a considerable importance in the response of P. adscendens thalli to Cd stress can be assumed by differential expression of various protein families.
[show abstract][hide abstract] ABSTRACT: Durum wheat plants (Triticum durum cv Creso) were grown in the presence of cadmium (0-40 microM) and analysed after 3 and 7 d for their growth, oxidative stress markers, phytochelatins, and enzymes and metabolites of the ascorbate (ASC)-glutathione (GSH) cycle. Cd exposure produced a dose-dependent inhibition of growth in both roots and leaves. Lipid peroxidation, protein oxidation and the decrease in the ascorbate redox state indicate the presence of oxidative stress in the roots, where H2O2 overproduction and phytochelatin synthesis also occurred. The activity of the ASC-GSH cycle enzymes significantly increased in roots. Consistently, a dose-dependent accumulation of Cd was evident in these organs. On the other hand, no oxidative stress symptoms or phytochelatin synthesis occurred in the leaves; where, at least during the time of our analysis, the levels of Cd remained irrelevant. In spite of this, enzymes of the ASC-GSH cycle significantly increased their activity in the leaves. When ASC biosynthesis was enhanced, by feeding plants with its last precursor, L-galactono-gamma-lactone (GL), Cd uptake was not affected. On the other hand, the oxidative stress induced in the roots by the heavy metal was alleviated. GL treatment also inhibited the Cd-dependent phytochelatin biosynthesis. These results suggest that different strategies can successfully cope with heavy metal toxicity. The changes that occurred in the ASC-GSH cycle enzymes of the leaves also suggest that the whole plant improved its antioxidant defense, even in those parts which had not yet been reached by Cd. This precocious increase in the enzymes of the ASC-GSH cycle further highlight the tight regulation and the relevance of this cycle in the defense against heavy metals.
Plant and Cell Physiology 04/2008; 49(3):362-74. · 4.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: "First line" defence mechanisms, such as phytochelatin biosynthesis, and "second line" mechanisms, such as stress protein induction, were investigated in cadmium-exposed cells of Trebouxia impressa Ahmadjian, a green microalgal species that is a common photobiont of the lichen Physcia adscendens (Fr.) H. Olivier. When T. impressa cells were exposed to 0, 9 and 18 microM Cd for 6, 18 and 48 h, glutathione and phytochelatins efficiently protected the cells against Cd damage. By contrast, the highest Cd concentration (36 microM) at the longest exposure-time (48 h) caused marked drops in glutathione and phytochelatin content, several types of ultrastructural damage, and decreases in cell density and total chlorophyll concentration. In this case, induction of stress proteins was observed, but only long after the induction of phytochelatins. Thus, stress proteins could represent a "second line" mechanism to counteract Cd stress, activated when there is a decline in the "first line" mechanism of Cd detoxification given by phytochelatins.
[show abstract][hide abstract] ABSTRACT: Mechanisms underlying cadmium (Cd) detoxification were compared in two aquatic macrophytes commonly used in phytoremediation, namely Pistia stratiotes L. and Eichhornia crassipes (Mart.) Solms. To simulate Cd pollution in the open environment, plants growing in outdoor artificial lakes were exposed for 21d to either 25 or 100microM Cd, in two consecutive years. Toxicity symptoms were absent or mild in both species. Metal accumulation was much higher in the roots of P. stratiotes, whereas in E. crassipes a comparatively higher fraction was translocated to the leaves. In both species, Cd was neither included in phenolic polymers or Ca-oxalate crystals, nor altered the levels of Cd-complexing organic acids. Glutathione levels were constitutively remarkably higher and much more responsive to Cd exposure in P. stratiotes than in E. crassipes. Abundant phytochelatin synthesis occurred only in P. stratiotes, both in roots and in leaves. In E. crassipes, on the other side, the constitutive levels of some antioxidant enzymes and ascorbate were higher and more responsive to Cd than in P. stratiotes. Thus, in these two aquatic plants grown in the open, different detoxification mechanisms might come into play to counterbalance Cd acute stress.
[show abstract][hide abstract] ABSTRACT: Three dipeptides, belonging to the family of diketopiperazines (DKPs), were extracted from broth culture of the grapevine endophyte Alternaria alternata, and were tested against Plasmopara viticola on leaves of grapevine plants grown in greenhouse. DKPs, used at different concentrations (10(-3), 10(-4), 10(-5) and 10(-6)M) both singularly and in mixtures, demonstrated real effectiveness in inhibiting P. viticola sporulation when applied 2 or 24h after pathogen inoculation. Moreover, no necrotic lesions or other phytotoxicity symptoms were observed on DKP-treated grapevine leaf tissues. Ultrastructural analysis performed on grapevine leaf tissues revealed that the DKPs used singularly and in mixture, at above reported concentrations, did not cause leaf tissue damages. By contrast, hyphae of P. viticola exhibited marked structural changes, similar to those induced by the endophyte A. alternata. This demonstrates the involvement of these metabolites in the relationship of P. viticola and the endophyte. Further experimental trials will be carried out in the next future in order to test the effectiveness of these molecules also under field conditions, and to better understand the mechanism of action involved in the pathogen inhibition.