Acid mine drainage (AMD) endangers pomegranate trees nearby a copper mine.

Acid mine drainage (AMD) is known as an important source of environmental pollution with potentially toxic elements. High concentrations of minerals in soil were observed in a pomegranate garden nearby a copper mine, Chaharmahal and Bakhtiari, Iran. In the vicinity of this mine, AMD locally caused distinct chlorosis in pomegranate trees. As expected, potentially toxic concentrations of Cu, Fe, and Zn were accumulated in the leaves of the chlorotic pomegranate trees (YLP), i.e., increased by 69 %, 67 % and 56 %, respectively, compared to the non-chlorotic trees (GLP). Remarkably, also some other elements, including Al (82 %), Na (39 %), Si (87 %), and Sr (69 %) were considerably enhanced in YLP, compared to GLP. On the other hand, the foliar Mn concentration in YLP was strongly decreased, about 62 % lower than that in GLP. The most plausible reasons for chlorosis in YLP are either toxicity of Al, Cu, Fe, Na, and Zn, or a deficiency of Mn. In addition, AMD led to oxidative stress, shown by a high accumulation of H2O2 in YLP, and a strong upregulation of enzymatic and non-enzymatic antioxidants. AMD apparently caused chlorosis, reduced the size of individual leaves, and caused lipid peroxidation. A further analysis of the adverse effect of the responsible AMD component(s) could be helpful to reduce the risk of food chain contamination.

Zinc- and nickel-induced changes in fatty acid profiles in the zinc hyperaccumulator Arabidopsis halleri and non-accumulator Arabidopsis lyrata.

This pilot study aimed at comparing zinc (Zn) and nickel (Ni) effects on the fatty acid (FA) profiles, oxidative stress and desaturase activity in the Zn hyperaccumulator Arabidopsis halleri and the excluder Arabidopsis lyrata to allow a better picture of the physiological mechanisms which may contribute to metal tolerance or acclimation. The most significant changes in the FA composition were observed in the shoots of the hyperaccumulator and in the roots of the excluder, and were not only metal-dependent, but also species-specific, since the most significant changes in the shoots of A. halleri were observed under Ni treatment, though Ni, in contrast to Zn, was accumulated mainly in its roots. Several FAs appeared in the roots and shoots of A. lyrata only upon metal exposure, whereas they were already found in control A. halleri. In both species, there was an increase in oleic acid under Ni treatment in both organs, whereas in Zn-treated plants the increase was shown only for the shoots. A rare conjugated α-parinaric acid was identified only in the shoots of metal-treated A. halleri. In the shoots of the hyperaccumulator, there was an increase in the content of saturated FAs and a decrease in the content of unsaturated FAs, while in the roots of the excluder, the opposite pattern was observed. These metal-induced changes in FA composition in the shoots of A. halleri can lead to a decrease in the fluidity of membranes, which could diminish the penetration of ROS into the membrane and thus maintain its stability.

Transcriptome Profiling of Cu Stressed Petunia Petals Reveals Candidate Genes Involved in Fe and Cu Crosstalk.

Copper (Cu) is an essential element for most living plants, but it is toxic for plants when present in excess. To better understand the response mechanism under excess Cu in plants, especially in flowers, transcriptome sequencing on petunia buds and opened flowers under excess Cu was performed. Interestingly, the transcript level of FIT-independent Fe deficiency response genes was significantly affected in Cu stressed petals, probably regulated by basic-helix-loop-helix 121 (bHLH121), while no difference was found in Fe content. Notably, the expression level of bHLH121 was significantly down-regulated in petals under excess Cu. In addition, the expression level of genes related to photosystem II (PSII), photosystem I (PSI), cytochrome b6/f complex, the light-harvesting chlorophyll II complex and electron carriers showed disordered expression profiles in petals under excess Cu, thus photosynthesis parameters, including the maximum PSII efficiency (FV/FM), nonphotochemical quenching (NPQ), quantum yield of the PSII (ΦPS(II)) and photochemical quenching coefficient (qP), were reduced in Cu stressed petals. Moreover, the chlorophyll a content was significantly reduced, while the chlorophyll b content was not affected, probably caused by the increased expression of chlorophyllide a oxygenase (CAO). Together, we provide new insight into excess Cu response and the Cu-Fe crosstalk in flowers.

Independent variation in copper tolerance and copper accumulation among crop species and varieties.

Copper (Cu) locally contaminates soils and might negatively affect growth and yield of crops. A better understanding of plant copper tolerance and accumulation is needed in order to breed more Cu-tolerant or Cu-efficient crops. Cu tolerance was evaluated in different varieties of seven species (Brassica carinata, B. juncea, B. napus, Cynara cardunculus, Helianthus annuus, Nicotiana tabacum and Raphanus sativus) exposed to a series of CuSO4 concentrations (0.1-8 µM CuSO4) in the nutrient solution. Plants were further exposed to 0.1 µM CuSO4 and to their variety-specific concentrations that reduced root growth to 50% of the maximum rate (EC50). Among all the varieties of all the species the EC50 varied from 0.7 up to 3.1 µM Cu. B. carinata was significantly more Cu-sensitive than the other species, which were not significantly different among each other, and B. carinata and H. annuus accommodated significant intra-specific, inter-varietal variation. There were significant differences between species in Cu uptake efficiency and nutrient status. When under EC50 exposure, all the Brassicaceae, except B. carinata, maintained low Cu concentrations in shoots, whereas the other species and B. carinata exhibited significantly increased shoot Cu concentrations, compared to the control. There was no apparent relationship between Cu tolerance and Cu accumulation in roots and shoots, suggesting that the observed variation in tolerance, both between and within species, is not explained by differential exclusion capacity. Discriminant analysis and treatment comparisons suggest possible contribution of lignin, saturated fatty acids, manganese (Mn) and zinc (Zn) in tolerance to high Cu concentrations in shoot.

Effects of antimony on enzymatic and non-enzymatic antioxidants in a metallicolous and a non-metallicolous population of Salvia spinosa L.

The present study aimed at elucidating the role of antioxidants and stress metabolites in antimony (Sb) tolerance in a metallicolous (M), Sb[V]-hypertolerant population, and a non-metallicolous (NM) population of Salvia spinosa, particularly with regard to the question of whether they could be involved in constitutive Sb tolerance or, specifically, in Sb[V] hypertolerance in the M population. Plants were exposed in hydroponics to 0, 8, 24, 74, 221 µM Sb (Ш or V). Superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities, and the concentrations of phenolics, flavonoids, and proline in leaves were measured after 20 d. As potential stress/tolerance markers, the concentrations of chlorophyll a and b, anthocyanins, and those of total soluble and reducing sugars were also measured. Chlorophyll a concentration reflected the difference, both in Sb[III] and Sb[V] tolerance, between N and NM, and the higher toxicity of Sb[III], compared to Sb[V]. APX and proline accumulation were more induced in M than in NM, and more by Sb[V] than by Sb[III], which is theoretically compatible with a role in Sb[V] hypertolerance. CAT was more induced in M than in NM, but more by Sb[III] than Sb[V], suggesting that is not functional in Sb[V] hypertolerance. The other enzymes and compounds did not exhibit significant Sb redox status*population interactions, suggesting that they don't play a role in, specifically, Sb[V] hypertolerance in M, but at most in the constitutive Sb[III] or Sb[V] tolerance of the species.

Analysis of Arabidopsis thaliana HKT1 and Eutrema salsugineum/botschantzevii HKT1;2 Promoters in Response to Salt Stress in Athkt1:1 Mutant.

Soil salinity imposes a serious threat to the productivity of agricultural crops. Among several other transporters, high-affinity K+ transporter (HKT)'s play an important role in reducing the phytotoxicity of Na+. Expression of Eutrema salsugineum (a halophyte) HKT1;2 is induced upon salt exposure. To elucidate the role of its promoter, we compared the sequences of HKT1;2 promoters from E. salsugineum (1822 bp) and E. botschantzevii (1811 bp) with Arabidopsis thaliana HKT1;1 (846 bp) promoter. In silico analysis predicted several cis-acting regulatory elements (GT-1 elements, core motifs of DRE/CRT, MYC/MYB-recognition sites and ACGT elements). Activities of the three promoters were analyzed by measuring HKT1;1 and/or HKT1;2 transcript level in the Athkt1;1 mutant plants. NaCl tolerance of the transgenics was also assessed. Our results depicted that expressing either AtHKT1;1 or EsHKT1;2 coding regions under the control of AtHKT1;1 promoter, almost reversed the hypersensitivity of the mutant for salt, on contrarily, when AtHKT1;1 coding sequence expressed under either Es or EbHKT1;2 promoters did not. Changes in shoot Na+/K+ concentrations under salt exposure is significantly consistent with the complementation ability of the mutant. The transcript concentration for genes under the control of either of Eutrema promoters, at control level was very less. This may suggest that either an important upstream response motif is missed or that A. thaliana misses a transcriptional regulator that is essential for salt-inducible HKT1 expression in Eutrema.

Transcriptional effects of cadmium on iron homeostasis differ in calamine accessions of Noccaea caerulescens.

Calamine accessions of the zinc/cadmium/nickel hyperaccumulator, Noccaea caerulescens, exhibit striking variation in foliar cadmium accumulation in nature. The Ganges accession (GA) from Southern France displays foliar cadmium hyperaccumulation (>1000 µg g-1 DW), whereas the accession La Calamine (LC) from Belgium, with similar local soil metal composition, does not (<100 µg g-1 DW). All calamine accessions are cadmium hypertolerant. To find out the differences between LC and GA in their basic adaptation mechanisms, we bypassed the cadmium excluding phenotype of LC by exposing the plants to 50 µm cadmium in hydroponics, achieving equal cadmium accumulation in the shoots. The iron content increased in the roots of both accessions. GA exhibited significant decreases in manganese and zinc contents in the roots and shoots, approaching those in LC. Altogether 702 genes responded differently to cadmium exposure between the accessions, 157 and 545 in the roots and shoots, respectively. Cadmium-exposed LC showed a stress response and had decreased levels of a wide range of photosynthesis-related transcripts. GA showed less changes, mainly exhibiting an iron deficiency-like response. This included increased expression of genes encoding five iron deficiency-regulated bHLH transcription factors, ferric reduction oxidase FRO2, iron transporters IRT1 and OPT3, and nicotianamine synthase NAS1, and decreased expression of genes encoding ferritins and NEET (a NEET family iron-sulfur protein), which is possibly involved in iron transfer, distribution and/or management. The function of the IRT1 gene in the accessions was compared. We conclude that the major difference between the two accessions is in the way they cope with iron under cadmium exposure.

Evolutionary dynamics of quantitative variation in an adaptive trait at the regional scale: The case of zinc hyperaccumulation in Arabidopsis halleri.

Metal hyperaccumulation in plants is an ecological trait whose biological significance remains debated, in particular because the selective pressures that govern its evolutionary dynamics are complex. One of the possible causes of quantitative variation in hyperaccumulation may be local adaptation to metalliferous soils. Here, we explored the population genetic structure of Arabidopsis halleri at fourteen metalliferous and nonmetalliferous sampling sites in southern Poland. The results were integrated with a quantitative assessment of variation in zinc hyperaccumulation to trace local adaptation. We identified a clear hierarchical structure with two distinct genetic groups at the upper level of clustering. Interestingly, these groups corresponded to different geographic subregions, rather than to ecological types (i.e., metallicolous vs. nonmetallicolous). Also, approximate Bayesian computation analyses suggested that the current distribution of A. halleri in southern Poland could be relictual as a result of habitat fragmentation caused by climatic shifts during the Holocene, rather than due to recent colonization of industrially polluted sites. In addition, we find evidence that some nonmetallicolous lowland populations may have actually derived from metallicolous populations. Meanwhile, the distribution of quantitative variation in zinc hyperaccumulation did separate metallicolous and nonmetallicolous accessions, indicating more recent adaptive evolution and diversifying selection between metalliferous and nonmetalliferous habitats. This suggests that zinc hyperaccumulation evolves both ways-towards higher levels at nonmetalliferous sites and lower levels at metalliferous sites. Our results open a new perspective on possible evolutionary relationships between A. halleri edaphic types that may inspire future genetic studies of quantitative variation in metal hyperaccumulation.

Two Silene vulgaris copper transporters residing in different cellular compartments confer copper hypertolerance by distinct mechanisms when expressed in Arabidopsis thaliana.

Silene vulgaris is a metallophyte of calamine, cupriferous and serpentine soils all over Europe. Its metallicolous populations are hypertolerant to zinc (Zn), cadmium (Cd), copper (Cu) or nickel (Ni), compared with conspecific nonmetallicolous populations. These hypertolerances are metal-specific, but the underlying mechanisms are poorly understood. We investigated the role of HMA5 copper transporters in Cu-hypertolerance of a S. vulgaris copper mine population. Cu-hypertolerance in Silene is correlated and genetically linked with enhanced expression of two HMA5 paralogs, SvHMA5I and SvHMA5II, each of which increases Cu tolerance when expressed in Arabidopsis thaliana. Most Spermatophytes, except Brassicaceae, possess homologs of SvHMA5I and SvHMA5II, which originate from an ancient duplication predating the appearance of spermatophytes. SvHMA5II and the A. thaliana homolog AtHMA5 localize in the endoplasmic reticulum and upon Cu exposure move to the plasma membrane, from where they are internalized and degraded in the vacuole. This resembles trafficking of mammalian homologs and is apparently an extremely ancient mechanism. SvHMA5I, instead, neofunctionalized and always resides on the tonoplast, likely sequestering Cu in the vacuole. Adaption of Silene to a Cu-polluted soil is at least in part due to upregulation of two distinct HMA5 transporters, which contribute to Cu hypertolerance by distinct mechanisms.

Cerium negatively impacts the nutritional status in rapeseed.

Cerium (Ce) has been reported to be both beneficial and harmful to plants. This contradiction deserves explanation in the light of increased anthropogenic release of Ce in the environment. Ce tolerance and accumulation were evaluated in hydroponically cultivated Brassica napus L. (rapeseed). Ce and other nutrient concentrations were measured with increasing Ce concentration in the nutrient solution. Moreover, Ce and calcium (Ca) accumulation were evaluated at different Ca and Ce concentrations in nutrient solution and a Michaelis-Menten type inhibition model considering Ce and Ca competition was tested. Plants were also sprayed with Ce solution in Ca-deficient media. Ce decreased the growth and root function, which affected shoot nutritional status. Calcium was the most severely inhibited nutrient in both roots and shoots. High Ca concentrations in the nutrient solution inhibited Ce accumulation in a non-competitive way. Moreover, phosphorus (P) precipitated Ce inside root cells. Ce spraying did not alleviate Ca deficiency symptoms and the results were critically compared to the available literature.

Salt and heavy metal tolerance and expression levels of candidate tolerance genes among four extremophile Cochlearia species with contrasting habitat preferences.

To test the concept of a general "mineral stress tolerance", we compared four extremophile Cochlearia species for salt (NaCl), zinc (Zn) and cadmium (Cd) tolerance and accumulation, and for expression of candidate tolerance genes for salt and Zn tolerance. Salt tolerance decreased in the order C. anglica>C. x hollandica>C. danica>C. pyrenaica, corresponding with the average salinity levels in the species' natural environments. The glycophytic metallophyte, C. pyrenaica, showed a relatively high level of salt tolerance, compared to other glycophytic Brassicaceae. Salt tolerance was positively correlated with HKT1 expression and the K+ concentration in roots under salt exposure, but uncorrelated with the Na+ concentrations in roots and shoots. All the species accumulated Na+ primarily in their leaves, and exhibited a high NHX1 expression in leaves, in comparison with other glycophytic Brassicaceae, suggesting that salt tolerance in Cochlearia is based on an efficient vacuolar sequestration of Na+ in leaves. The metallicolous C. pyrenaica population was hypertolerant to Zn, but not to Cd, in comparison with the other Cochlearia species. All the Cochlearia species accumulated Zn and Cd primarily in roots, and showed high levels of Cd and Zn tolerance, with unusually low rates of metal accumulation, in comparison with non-metallophytes, or non-metallicolous metallophyte populations, of species belonging to other genera or families. Although Cochlearia, as a genus, shows relatively high levels of tolerance to both salt and heavy metals, this is most probably not due to a common 'mineral stress tolerance' mechanism.

Genome interrogation for novel salinity tolerant Arabidopsis mutants.

Soil salinity is becoming an increasingly large problem in agriculture. In this study, we have investigated whether a capacity to withstand salinity can be induced in the salinity sensitive plant species Arabidopsis thaliana, and whether it can be maintained in subsequent generations. To this end, we have used zinc finger artificial transcription factor (ZF-ATFs) mediated genome interrogation. Already within a relatively small collection Arabidopsis lines expressing ZF-ATFs, we found 41 lines that were tolerant to 100 mM NaCl. Furthermore, ZF-ATF encoding gene constructs rescued from the most strongly salinity tolerant lines were indeed found to act as dominant and heritable agents for salinity tolerance. Altogether, our data provide evidence that a silent capacity to withstand normally lethal levels of salinity exists in Arabidopsis and can be evoked relatively easily by in trans acting transcription factors like ZF-ATFs.

Expression of the ZNT1 Zinc Transporter from the Metal Hyperaccumulator Noccaea caerulescens Confers Enhanced Zinc and Cadmium Tolerance and Accumulation to Arabidopsis thaliana.

Prompt regulation of transition metal transporters is crucial for plant zinc homeostasis. NcZNT1 is one of such transporters, found in the metal hyperaccumulator Brassicaceae species Noccaea caerulescens. It is orthologous to AtZIP4 from Arabidopsis thaliana, an important actor in Zn homeostasis. We examined if the NcZNT1 function contributes to the metal hyperaccumulation of N. caerulescens. NcZNT1 was found to be a plasma-membrane located metal transporter. Constitutive overexpression of NcZNT1 in A. thaliana conferred enhanced tolerance to exposure to excess Zn and Cd supply, as well as increased accumulation of Zn and Cd and induction of the Fe deficiency response, when compared to non-transformed wild-type plants. Promoters of both genes were induced by Zn deficiency in roots and shoots of A. thaliana. In A. thaliana, the AtZIP4 and NcZNT1 promoters were mainly active in cortex, endodermis and pericycle cells under Zn deficient conditions. In N. caerulescens, the promoters were active in the same tissues, though the activity of the NcZNT1 promoter was higher and not limited to Zn deficient conditions. Common cis elements were identified in both promoters by 5' deletion analysis. These correspond to the previously determined Zinc Deficiency Responsive Elements found in A. thaliana to interact with two redundantly acting transcription factors, bZIP19 and bZIP23, controlling the Zn deficiency response. In conclusion, these results suggest that NcZNT1 is an important factor in contributing to Zn and Cd hyperaccumulation in N. caerulescens. Differences in cis- and trans-regulators are likely to account for the differences in expression between A. thaliana and N. caerulescens. The high, constitutive NcZNT1 expression in the stele of N. caerulescens roots implicates its involvement in long distance root-to-shoot metal transport by maintaining a Zn/Cd influx into cells responsible for xylem loading.

Both the concentration and redox state of glutathione and ascorbate influence the sensitivity of arabidopsis to cadmium.

BACKGROUND AND AIMS: Cadmium (Cd) is a non-essential trace element that elicits oxidative stress. Plants respond to Cd toxicity via increasing their Cd-chelating and antioxidative capacities. They predominantly chelate Cd via glutathione (GSH) and phytochelatins (PCs), while antioxidative defence is mainly based on the use and recycling of both GSH and ascorbate (AsA), complemented by superoxide dismutase (SOD) and catalase (CAT). In addition, both metabolites act as a substrate for the regeneration of other essential antioxidants, which neutralize and regulate reactive oxygen species (ROS). Together, these functions influence the concentration and cellular redox state of GSH and AsA. In this study, these two parameters were examined in plants of Arabidopsis thaliana exposed to sub-lethal Cd concentrations. METHODS: Wild-type plants and mutant arabidopsis plants containing 30-45 % of wild-type levels of GSH (cad2-1) or 40-50 % of AsA (vtc1-1), together with the double-mutant (cad2-1 vtc1-1) were cultivated in a hydroponic system and exposed to sub-lethal Cd concentrations. Cadmium detoxification was investigated at different levels including gene expression and metabolite concentrations. KEY RESULTS: In comparison with wild-type plants, elevated basal thiol levels and enhanced PC synthesis upon exposure to Cd efficiently compensated AsA deficiency in vtc1-1 plants and contributed to decreased sensitivity towards Cd. Glutathione-deficient (cad2-1 and cad2-1 vtc1-1) mutants, however, showed a more oxidized GSH redox state, resulting in initial oxidative stress and a higher sensitivity to Cd. In order to cope with the Cd stress to which they were exposed, GSH-deficient mutants activated multiple alternative pathways. CONCLUSIONS: Our observations indicate that GSH and AsA deficiency differentially alter plant GSH homeostasis, resulting in opposite Cd sensitivities relative to wild-type plants. Upon Cd exposure, GSH-deficient mutants were hampered in chelation. They experienced phenotypic disturbances and even more oxidative stress, and therefore activated multiple alternative pathways such as SOD, CAT and ascorbate peroxidase, indicating a higher Cd sensitivity. Ascorbate deficiency, however, was associated with enhanced PC synthesis in comparison with wild-type plants after Cd exposure, which contributed to decreased sensitivity towards Cd.

A comparative study of antimony accumulation in plants growing in two mining areas in Iran, Moghanlo, and Patyar.

Antimony occurs locally at high concentrations in some mineralized soils. Very little is known about behavior of antimony in plants. In this study, we analyzed the soil and vegetation of two mining areas in Iran, Patyar, and Moghanlo. Total Sb concentrations in soil were 358-3482 mg/kg in Moghanlo and 284-886 mg/kg in Patyar. Corresponding Sb concentrations in plant shoots were 0.8-287 and 1.3-49 mg/kg, respectively. In both areas, foliar Sb concentrations increased with acid-extractable soil Sb, although the slope was about 2-fold steeper for Patyar than for Moghanlo. Regressing the foliar concentrations on water-soluble Sb yielded identical slopes for both areas, suggesting that the soluble fraction of Sb rather than total Sb is the direct determinant of foliar Sb accumulation. Both in Patyar and Moghanlo, only a minor part of the total variance of shoot Sb was explained by soluble Sb. The major part was explained by plant species, demonstrating that plant taxonomic identity is the most important determinant of foliar Sb accumulation capacity in both areas. The translocation factor (TF) was highly variable too, with species as the only significant variance component. Only four species were able to accumulate more than 100 mg/kg Sb in their leaves. Among these species, Achillea wilhelmsii and Matthiola farinosa were by far the best Sb accumulators, with, on average, 141 and 132 mg/kg Sb in their leaves. Of these two, only Matthiola farinosa consistently maintained TF values far above unity across the whole range of soluble Sb in Moghanlo.

Variation in Heavy Metal Accumulation and Genetic Diversity at a Regional Scale Among Metallicolous and Non-Metallicolous Populations of the Facultative Metallophyte Biscutella laevigata subsp. laevigata.

Biscutella laevigata is a facultative metallophyte, with populations on non-metalliferous and metalliferous soils. Some of its metallicolous populations have been shown to hyperaccumulate thallium or lead in nature. Only Tl hyperaccumulation has been experimentally confirmed. We aimed to compare the patterns of metal (hyper)accumulation and genetic diversity among populations of B. laevigata subsp. laevigata in NE Italy. None of the populations exhibited foliar hyperaccumulation of Cu, Zn, or Pb. The root-to-shoot accumulation rates for these metals were unchanged or decreased rather than enhanced in the metallicolous populations, in comparison with the non-metallicolous ones. Hyperaccumulation of Tl was confined to the population of the Cave del Predil mine. This population was genetically very distinct from the others, as demonstrated by AFLP-based cluster analysis. The two other mine populations did not surpass the threshold for Tl hyperaccumulation, but showed enhanced foliar Tl concentrations and root-to-shoot translocation rates, in comparison with the non-metallicolous populations. Genetic analysis suggested that adaptation to metalliferous soil must have been independently evolved in the metallicolous populations.

Differential response of Arabidopsis leaves and roots to cadmium: glutathione-related chelating capacity vs antioxidant capacity.

This study aims to uncover the spatiotemporal involvement of glutathione (GSH) in two major mechanisms of cadmium (Cd)-induced detoxification (i.e. chelation and antioxidative defence). A kinetic study was conducted on hydroponically grown Arabidopsis thaliana (L. Heyhn) to gain insight into the early events after exposure to Cd. Cadmium detoxification was investigated at different levels, including gene transcripts, enzyme activities and metabolite content. Data indicate a time-dependent response both within roots and between plant organs. Early on in roots, GSH was preferentially allocated to phytochelatin (PC) synthesis destined for Cd chelation. This led to decreased GSH levels, without alternative pathways activated to complement GSH's antioxidative functions. After one day however, multiple antioxidative pathways increased including superoxide dismutase (SOD), ascorbate (AsA) and catalase (CAT) to ensure efficient neutralization of Cd-induced reactive oxygen species (ROS). As a consequence of Cd retention and detoxification in roots, a delayed response occurred in leaves. Together with high leaf thiol contents and possibly signalling responses from the roots, the leaves were protected, allowing them sufficient time to activate their defence mechanisms.

Differences in proton pumping and Na/H exchange at the leaf cell tonoplast between a halophyte and a glycophyte.

The tonoplast Na(+)/H(+) antiporter and tonoplast H(+) pumps are essential components of salt tolerance in plants. The objective of this study was to investigate the transport activity of the tonoplast Na(+)/H(+) antiporter and the tonoplast V-H(+)-ATPase and V-H(+)-PPase in a highly tolerant salt-accumulating halophyte, Salicornia dolichostachya, and to compare these transport activities with activities in the related glycophyte Spinacia oleracea. Vacuolar membrane vesicles were isolated by density gradient centrifugation, and the proton transport and hydrolytic activity of both H(+) pumps were studied. Furthermore, the Na(+)/H(+)-exchange capacity of the vesicles was investigated by 9-amino-6-chloro-2-methoxyacridine fluorescence. Salt treatment induced V-H(+)-ATPase and V-H(+)-PPase activity in vesicles derived from S. oleracea, whereas V-H(+)-ATPase and V-H(+)-PPase activity in S. dolichostachya was not affected by salt treatment. Na(+)/H(+)-exchange capacity followed the same pattern, i.e. induced in response to salt treatment (0 and 200 mM NaCl) in S. oleracea and not influenced by salt treatment (10 and 200 mM NaCl) in S. dolichostachya. Our results suggest that S. dolichostachya already generates a high tonoplast H(+) gradient at low external salinities, which is likely to contribute to the high cellular salt accumulation of this species at low external salinities. At high external salinities, S. dolichostachya showed improved growth compared with S. oleracea, but V-H(+)-ATPase, V-H(+)-PPase and Na(+)/H(+)-exchange activities were comparable between the species, which might imply that S. dolichostachya more efficiently retains Na(+) in the vacuole.