The possible role of metallothioneins in copper tolerance of Silene cucubalus

Growth and copper-binding of a copper-tolerant and a copper-sensitive population of Silene cucubalus (L.) Wib. have been studied. The copper-tolerant plants showed a much lower uptake and a proportionally higher transport of copper from root to shoot. A copper-binding protein with an apparent M_r of 8500 resembling metallothionein has been isolated from the roots of copper-treated plants of the tolerant population. After 20 d, the protein was observed to be inducible upon copper supply in the copper-tolerant plants, but not yet in the sensitive ones. This could be an indication of a difference in metalregulated synthesis of the protein. Ion-exchange chromatography of the 8500 protein yielded a major copper-containing fraction eluting at high ionic strength. Other characteristics such as UV absorption and amino-acid composition resembled strongly those of metallothioneins. The involvement of metallothioneins in the detoxification of copper within Cu-tolerant plants is discussed in relation to other mechanisms.Abbreviation DEAE diethyloaminoethyl     

Contribution of plastocyanin isoforms to photosynthesis and copper homeostasis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> grown at different copper regimes

In land plants plastocyanin is indispensable and therefore copper (Cu) availability is a prerequisite for growth. When Cu supply is limited, higher plants prioritize the Cu delivery to plastocyanin by down-regulation of other Cu proteins. Arabidopsis has two plastocyanin genes (PETE1 and PETE2). PETE2 is the predominant isoform in soil-grown plants and in hydroponic cultures it is accumulated in response to Cu addition. It functions as a Cu sink when more Cu is available, in addition to its role as an electron carrier. PETE1 is not affected by Cu feeding and it is the isoform that drives electron transport under Cu-deficiency. Cu feeding rescued the defect in photosystem II electron flux (Φ_PSII) in the pete1 mutant whereas Φ_PSII was not changed in the pete2 mutant as Cu was added. Plants with mutations in the plastocyanin genes had altered Cu homeostasis. The pete2 mutant accumulated more Cu/Zn superoxide dismutase (CSD2 and CSD1) and Cu chaperone (CCS) whereas the pete1 mutant accumulated less. On the other hand, less iron superoxide dismutase (FeSOD) and microRNA398b were observed in the pete2 mutant, whereas more were accumulated in the pete1 mutant. Our data suggest that plastocyanin isoforms are different in their response to Cu and the absence of either one changes the Cu homeostasis. Also a small amount of plastocyanin is enough to support efficient electron transport and more PETE2 is accumulated as more Cu is added, presumably, to buffer the excess Cu. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Photosynthesis in Two Wheat Cultivars Differing in Salt Susceptibility

Salinised (150 mM NaCl for 15 d) roots excised from salt sensitive wheat cultivar Giza 163 showed about 15-fold increase in the ratio of Na/K while salt tolerant Sakha 92 exhibited only 7.5-fold increase compared to their control ratios. Root ratio of saturated/unsaturated fatty acids was stimulated twice in the sensitive cultivar versus 1.7-fold increase in the tolerant ones. Salinity enhanced greatly the accumulation of spermine (Spm) and spermidine (Spd) contents associated with a decrease in putrescine (Put) content in both wheat cultivars. Higher ratios of Spm+Spd/Put associated with lower content of proline and low ethylene evolution were detected in shoots and roots of salt tolerant cultivar. Chlorophyll a/b ratio showed an increase from 1.3 in control of both cultivars to 1.6 and 1.4 in stressed Giza 163 and Sakha 92, respectively. A reduced Hill reaction activity (19 %) was observed in stressed chloroplasts isolated from leaves of the tolerant cultivar versus 40 % inhibition in the sensitive ones. Moreover, chloroplasts isolated from stressed leaves of the sensitive cultivar showed about 25 % reduction in fluorescence emission at 685 nm as well as shifts in the peaks in the visible region.     

Excess copper effect on growth, chloroplast ultrastructure, oxygen-evolution activity and chlorophyll fluorescence in Glycine max cell suspensions

The influence of excess copper on soybean photosynthetic cell suspensions was investigated. The cell suspensions grew well in the presence of 5–20 µ M CuSO₄ and developed tolerance to even higher levels of CuSO₄ (i.e. up to 50 µ M ), indicating that copper was not toxic to the cells at that high concentrations. Cu-adapted cell suspensions grew faster than the control in limiting light conditions and had higher content of chlorophyll per dry weight of cells. Copper was accumulated within the cells, and this event was accompanied by (1) increased oxygen evolution activity; (2) increased number of chloroplasts per cell, smaller chloroplasts, increased thylakoid stacking and grana size; (3) higher fluorescence emission of photosystem II antenna complexes and (4) stimulation of plastocyanin protein synthesis compared with untreated cells. Microanalysis of cross-sections revealed an increase of copper content in chloroplasts as well as vacuole, cytoplasm and cell wall in Cu-adapted cells. No antagonist interaction between copper and iron uptake took place in these cell suspensions. On the other hand, copper at subtoxic concentrations stimulated oxygen evolution activity in thylakoids from control cells, but this event did not take place in those from Cu-adapted ones. Furthermore, the loss of activity by copper inhibitory action at toxic concentrations was two-fold slower in thylakoids from Cu-adapted cells compared with the control ones. The data strongly indicate that copper plays a specific positive role on photosynthesis and stimulates the growth and the oxygen evolution activity in soybean cell suspensions.     

Aluminum tolerance in maize is correlated with increased levels of mineral nutrients, carbohydrates and proline, and decreased levels of lipid peroxidation and Al accumulation

We investigated the uptake of aluminum (Al) and transport to shoots in two inbred maize lines (Zea mays L., VA-22 and A(4/67)) differing in Al tolerance. Seedlings were grown for 7 days in hydroponic culture with nutrient solution that contained 0, 240, 360, and 480muM Al at pH 4.2. After 7 days of exposure to Al, roots of sensitive maize line (A(4/67)) plants accumulated 2-2.5 times more Al than roots of tolerant line (VA-22) plants. Inductively coupled plasma atomic emission spectrometry (ICP-AES) showed that the tolerant line retained higher concentrations of Ca(2+), Mg(2+), and K(+) compared with the sensitive line. In response to Al treatment, proline (Pro) concentration increased three-fold in roots of tolerant plants, while a slight increase was observed in roots of sensitive-line plants. A substantial carbon surplus (two-fold increase) was observed in roots of the Al-tolerant maize line. Carbohydrate concentration remained almost unchanged in roots of Al-sensitive line plants. Al treatment triggered the enhancement of lipid peroxidation in the sensitive line, while no change in lipid peroxidation level was observed in the tolerant maize line. These data provide further support to the hypothesis that a mechanism exists that excludes Al from the roots of the tolerant maize line, as well as an internal mechanism of tolerance that minimizes accumulation of lipid peroxides through a higher Pro and carbohydrate content related to osmoregulation and membrane stabilization.     

Selection for tolerance to copper during pollen formation in Mimulus guttatus Fischer ex DC

In Mimulus guttatus, copper tolerance is determined largely by a single gene and is expressed in both the sporophyte and microgametophyte. This study explores the extent to which selection during pollen formation affects copper tolerance in the sporophytic generation. Two sets of plants heterozygous for copper tolerance, produced by reciprocal crosses between different copper-tolerant or sensitive families, and the plant on which the original observations were based, were cloned and grown in control or copper-supplemented solutions. Pollen viability and the number of tolerant progeny produced in backcrosses to sensitive plants were compared. In addition, the effect of copper treatment on pollen viability in vitro was compared for plants tolerant, sensitive and heterozygous for copper tolerance. The extent to which in vitro pollen viability decreased in response to copper treatment corresponded to the copper tolerance of the pollen source. When grown with added copper, four of the five plants showed significant reductions in pollen viability, ranging from 18% to 48% of control values. The reductions in pollen viability were correlated with an increase in tolerant progeny (r= 0.679, p=0.004). Increases in tolerant progeny could be large, ranging from 119% to 170% of that of controls, but were usually smaller than was predicted from the reductions in viable pollen. In addition, plants derived from reciprocal crosses differed significantly in the extent to which pollen viability was decreased and sporophytic tolerance increased. Thus, while selection during pollen formation could increase sporophytic tolerance, sporophytic factors, perhaps including cytoplasmic or epigenetic ones, moderated the effectiveness of pollen selection for copper tolerance.     

Glutathione Depletion Due to Copper-Induced Phytochelatin Synthesis Causes Oxidative Stress in Silene cucubalus

The relation between loss of glutathione due to metal-induced phytochelatin synthesis and oxidative stress was studied in the roots of copper-sensitive and tolerant Silene cucubalus (L.) Wib., resistant to 1 and 40 micromolar Cu, respectively. The amount of nonprotein sulfhydryl compounds other than glutathione was taken as a measure of phytochelatins. At a supply of 20 micromolar Cu, which is toxic for sensitive plants only, phytochelatin synthesis and loss of total glutathione were observed only in sensitive plants within 6 h of exposure. When the plants were exposed to a range of copper concentrations for 3 d, a marked production of phytochelatins in sensitive plants was already observed at 0.5 micromolar Cu, whereas the production in tolerant plants was negligible at 40 micromolar or lower. The highest production in tolerant plants was only 40% of that in sensitive plants. In both varieties, the synthesis of phytochelatins was coupled to a loss of glutathione. Copper at toxic concentrations caused oxidative stress, as was evidenced by both the accumulation of lipid peroxidation products and a shift in the glutathione redox couple to a more oxidized state. Depletion of glutathione by pretreatment with buthionine sulfoximine significantly increased the oxidative damage by copper. At a comparably low glutathione level, cadmium had no effect on either lipid peroxidation or the glutathione redox couple in buthionine sulfoximine-treated plants. These results indicate that copper may specifically cause oxidative stress by depletion of the antioxidant glutathione due to phytochelatin synthesis. We conclude that copper tolerance in S. cucubalus does not depend on the production of phytochelatins but is related to the plant's ability to prevent glutathione depletion resulting from copper-induced phytochelatin production, e.g. by restricting its copper uptake.     

Zonal responses of sensitive vs. tolerant wheat roots during Al exposure and recovery

Aluminium (Al) irreversibly inhibits root growth in sensitive, but not in some tolerant genotypes. To better understand tolerance mechanisms, seedlings from tolerant ('Barbela 7/72' line) and sensitive ('Anahuac') Triticum aestivum L. genotypes were exposed to AlCl(3) 185 μM for: (a) 24 h followed by 48 h without Al (recovery); (b) 72 h of continuous exposure. Three root zones were analyzed (meristematic (MZ), elongation (EZ) and hairy (HZ)) for callose deposition, reserves (starch and lipids) accumulation, endodermis differentiation and tissue architecture. Putative Al-induced genotoxic or cytostatic/mytogenic effects were assessed by flow cytometry in root apices. Tolerant plants accumulated less Al, presented less root damage and a less generalized callose distribution than sensitive ones. Starch and lipid reserves remained constant in tolerant roots but drastically decreased in sensitive ones. Al induced different profiles of endodermis differentiation: differentiation was promoted in EZ and HZ, respectively, in sensitive and tolerant genotypes. No ploidy changes or clastogenicity were observed. However, differences in cell cycle blockage profiles were detected, being less severe in tolerant roots. After Al removal, only the 'Barbela 7/72' line reversed Al-induced effects to values closer to the control, mostly with respect to callose deposition and cell cycle progression. We demonstrate for the first time that: (a) cell cycle progression is differently regulated by Al-tolerant and Al-sensitive genotypes; (b) Al induces callose deposition >3 cm above root apex (in HZ); (c) callose deposition is a transient Al-induced effect in tolerant plants; and (d) in HZ, endodermis differentiation is also stimulated only in tolerant plants, probably functioning in tolerant genotypes as a protective mechanism in addition to callose.     

Interaction between a copper-tolerant and a copper-sensitive population of Silene cucubalus

Interactions between copper-tolerant and copper-sensitive plants of Silene cucubalus (L.) Wib. were absent when grown in mixed culture in a nutrient solution with a normal Cu²⁺ concentration (0.5 μ M ). When grown in mixed culture in a nutrient solution with 40.5 μ M CuSO₄, however, the biomass production of the sensitive plants was less affected than when grown in monoculture. At 40.5 μ M Cu²⁺, in the presence of tolerant plants, the concentration of copper in both roots and shoots of sensitive plants was significantly diminished in comparison to a monoculture without tolerant plants. At the same time the copper concentration in the roots of the tolerant plants was higher in the presence of sensitive plants. The possibility of external detoxification of the copper by tolerant plants as a mechanism of heavy metal resistance is discussed.     

The influence of cold acclimation on antioxidative enzymes and antioxidants in sensitive and tolerant barley cultivars

In order to better understand the role of cold acclimation in alleviating freezing injury, two barley cultivars with different cold tolerance, i.e. a sensitive cv. Chumai 1 and a tolerant cv. Mo 103, were used. The freezing treatment increased leaf soluble protein content more in the tolerant cultivar than in the sensitive one. Cold acclimation increased H₂O₂ content of the two cultivars during freezing treatment, especially in Mo 103. Glutathione and ascorbate contents during freezing and recovery were significantly higher in cold-acclimated plants than in non-acclimated ones. Activities of peroxidase, ascorbate peroxidase and glutathione reductase were also higher in cold-acclimated plants than non-acclimated plants during freezing treatment. However, there was no significant difference between cold-acclimated plants and the control plants in catalase activity. It may be assumed that cold acclimation induced H₂O₂ production, which in turn enhanced activities of antioxidative enzymes and synthesis of antioxidants, resulting in alleviation of oxidative stress caused by freezing.     

Chloroplast development and the synthesis of chlorophyll and protochlorophyllide in Zostera transferred to darkness

Intact plants and isolated leaves of Zostera capricornii Martens ex Aschers were transferred from daylight to darkness. Substantial amounts of chloropyll a and b continued to accumulate in immature and mature tissue in the same ratio as in the light and were incorporated into chlorophyll-protein complexes in the thylakoids. A small amount of protochlorophyllide also accumulated in immature tissue in the dark. Proplastids and immature chloroplasts continued to develop into mature chloroplasts in the dark in the normal manner but prolamellar bodies, which are a conspicuous feature of immature chloroplasts, took longer to disperse than in the light. Protochlorophyllide accumulation and prolamellar-body formation were not correlated. The results indicate that Zostera has a genetic capacity for dark chlorophyll synthesis which is expressed in immature and mature leaf tissue and enables this plant to continue synthesising chlorophyll and assembling chloroplasts at night.Abbreviations Chl chlorophyll - T _o time of transfer to darkness     

Phytochelatin is not a primary factor in determining copper tolerance

Phytochelatin (PC) is involved in the detoxification of harmful, non-essential heavy metals and the homeostasis of essential heavy metals in plants. Its synthesis can be induced by either cadmium (Cd) or copper (Cu), and can form stable complexes with either element. This might suggest that PC has an important role in determining plant tolerance to both. However, this is not clearly apparent, as evidenced by a PC-deficient and Cd-sensitiveArabidopsis mutant (cad1-3) that shows no significant increase in its sensitivity to copper. Therefore, we investigated whether the mechanism for Cu tolerance differed from that for Cd by analyzing copper sensitivity in Cd-tolerant transgenics and Cd-sensitive mutants ofArabidopsis. Cadmium-tolerant transgenic plants that over-expressedA. thaliana phytochelatin synthase 1 (AtPCS1) were not tolerant of copper stress, thereby supporting the hypothesis that PC is not primarily involved in this tolerance mechanism. We also investigated Cu tolerance incad2-1, a Cd-sensitive and glutathione (GSH)-deficientArabidopsis mutant. Paradoxically,cad2-1 was more resistant to copper stress than were wild-type plants. This was likely due to the high level of cysteine present in that mutant. However, when the growth medium was supplemented with cysteine, the wild types also exhibited copper tolerance. Moreover,Saccharomyces cerevisiae that expressedAtPCS1 showed tolerance to Cd but hypersensitivity to Cu. All these results indicate that PC is not a major factor in determining copper tolerance in plants.     

The effect of NaCl on proline accumulation in potato seedlings and calli

The effects of salt stress were studied on the accumulation and metabolism of proline and its correlation with Na⁺ and K⁺ content in shoots and callus tissue of four potato cultivars, viz., Agria, Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive). Na⁺ and proline contents increased in all cultivars under salt stress. However, K⁺ and protein contents decreased in response to NaCl treatments. The activities of enzymes involved in proline metabolism, Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) increased and decreased, respectively, in response to elevated NaCl concentrations. The changes of P5CS and ProDH activities in more salt sensitive cultivars (Diamant, Ajax) were more than those in the tolerant ones. Then the stimulation of synthesis in combination with a partially increase of protein proteolysis, a decrease in proline utilization and inhibition of oxidation resulted in high proline contents in seedlings and calli under salt stress. In callus tissue, reduced growth and cell size may be partially responsible for high proline accumulation in response to high NaCl levels. However, although the basic proline contents in the seedlings of more salt tolerant cultivars were higher than the sensitive ones, a clear relationship was not generally observed between accumulation of proline and salt tolerance in potato.     

Increased Zinc Tolerance in Silene vulgaris (Moench) Garcke Is Not Due to Increased Production of Phytochelatins

The concentration of acid-soluble thiols other than reduced glutathione (SH - GSH) increases in the roots of zinc-sensitive and zinc-tolerant Silene vulgaris (Moench) Garcke after exposure to zinc for 1 to 3 d. The concentration of SH - GSH in the roots is higher in the sensitive plants than in the tolerant ones, both at equal external zinc concentrations and at zinc concentrations causing the same level of root-length growth inhibition. High performance liquid chromatography analyses show that the increase in the concentration of SH - GSH is not only due to the production of phytochelatins, but is also due to an increase in the concentration of cysteine and the production of nonidentified thiols. The cysteine concentration increases equally in the roots of sensitive and tolerant plants. The accumulation of phytochelatins is higher in the roots of the sensitive plants, whereas the chain length distribution of phytochelatins is the same in sensitive and tolerant plants. It is concluded that increased zinc tolerance in S. vulgaris is not due to increased production of phytochelatins.     

Transgenic tobacco plants overexpressing the Met25 gene of Saccharomyces cerevisiae exhibit enhanced levels of cysteine and glutathione and increased tolerance to oxidative stress

Summary. The cysteine biosynthesis pathway differs between plants and the yeast Saccharomyces cerevisiae. The yeast MET25 gene encoded to O-acetylhomoserine sulfhydrylase (AHS) catalyzed the reaction that form homocysteine, which later can be converted into cystiene. In vitro studies show that this enzyme possesses also the activity of O-acetyl(thiol)lyase (OASTL) that catalyzes synthesis of cysteine in plants. In this study, we generated transgenic tobacco plants expressing the yeast MET25 gene under the control of a constitutive promoter and targeted the yeast protein to the cytosol or to the chloroplasts. Both sets of transgenic plants were taller and greener than wild-type plants. Addition of SO₂, the substrate of the yeast enzyme caused a significant elevation of the glutathione content in representative plants from each of the two sets of transgenic plants expressing the yeast gene. Determination of non-protein thiol content indicated up to four-folds higher cysteine and 2.5-fold glutathione levels in these plants. In addition, the leaf discs of the transgenic plants were more tolerant to toxic levels of sulphite, and to paraquat, an herbicide generating active oxygen species.     

Detection and characterization of a lectin from non-seed tissue ofPhaseolus vulgaris

The distribution of lectin in various tissues ofPhaseolus vulgaris L. (cv. red) has been investigated using a sensitive solid-phase enzyme immunoassay. Roots, leaves and stems from 3- to 4-week-old plants were screened for their lectin content; low levels could be detected in all organs, with a relative distribution of 37% in roots, 20% in leaves and 43% in stems. The lectin from stemsleaves and roots was then isolated from 5- to 6-week-old plants using extraction, salt fractionation and affinity chromatography on immobilized porcine thyroglobulin. A comparative study of the seed lectin and the lectin isolated from 5- to 6-week-old plants was made using hemagglutination, inhibition of hemagglutination, immunodiffusion, polyacrylamide and agarose electrophoresis. The results showed that lectin isolated from the different tissues was immunologically identical and exhibited the same subunit structure and similar isolectin composition as the seed lectin.Abbreviations EDTA ethylenediaminetetraacetic acid - PHA phytohemagglutinin - SDS sodium dodecyl sulfate     

Constitutive and aluminium-induced patterns of phenolic compounds in two maize varieties differing in aluminium tolerance

Aluminium tolerance in maize is mainly due to more efficient Al exclusion. Nonetheless, even in tolerant varieties Al can gain access into the cells. Detoxification by binding to strong organic ligands should therefore play a role also in plants with high Al exclusion capacity. To test this hypothesis in this study the concentrations of soluble, free and bound, phenolics were analyzed in roots of two maize varieties differing in Al tolerance. Exposure for 24 h to 50 μM Al in nutrient solution strongly inhibited root elongation in the sensitive variety 16 × 36, but not in the Al-tolerant variety Cateto. Cateto accumulated about half the concentration of Al in roots than 16 × 36 (analysis performed after root desorption with citrate). Roots of Al-tolerant Cateto contained higher concentrations of caffeic acid, catechol and catechin than roots of the sensitive variety. Exposure to Al induced the accumulation of taxifolin in roots of both varieties. However, Al-tolerant Cateto accumulated about twice the concentration than Al-sensitive 16 × 36 of this pentahydroxyfavonol. The molar ratio for phenolics with catecholate groups to Al was about unity in roots of Cateto, while in those of 16 × 36 the ratio was ten times lower. Both the fact that these phenolics are strong ligands for Al and their high antioxidant and antiradical activity suggest that these compounds may provide protection against the Al fraction that is able to surpass the exclusion mechanisms operating in the tolerant maize variety.     

Gene flow inAgrostis tenuis (Poaceae)

The copper tolerance of both adult plants and their seedlings, ofAgrostis tenuis from sites within and outside, but neighbouring the Parys Mountain mine (North Wales) were studied. Intensive sampling was carried out along a line starting from the mine boundaries to a distance of five miles away in the direction of the prevailing wind, and of two miles in an upwind direction. The test have shown that mine populations are very tolerant. In outside populations on soils with reduced of very low copper content the mean tolerance level of the seedlings is considerably higher than that of corresponding adult plants, while both show a gradual decrease in tolerance with distance from the mine. The gene flow moves in the direction of the prevailing wind transfering tolerant characters from the mine to the normal populations. The selective pressures on the toxic soil of the mine are quite strong and permit ± tolerant plants only, while on normal soils these pressures are not so severe, so that populations may be rather heterogenous regarding their tolerance towards copper.     

Localization of the reaction side of plastocyanin from immunological and kinetic studies with inside-out thylakoid vesicles

(1) The effect of four active antisera against plastocyanin on Photosystem I-driven electron transport and phosphorylation was investigated in spinach chloroplasts. Partial inhibition of electron transport and stimulation of plastocyanin-dependent phosphorylation were sometimes observed after adding amounts of antibodies which were in large excess and not related to the plastocyanin content of the chloroplasts. This indicates effects of the antibodies on the membrane. (2) The antibodies against plastocyanin neither directly nor indirectly agglutinated unbroken chloroplast membranes. (3) The plastocyanin content of right-side-out and inside-out thylakoid vesicles isolated by aqueous polymer two-phase partition from chloroplasts disrupted by Yeda press treatment was determined by quantitative rocket electroimmunodiffusion. Right-side-out vesicles retained about 25%, inside-out vesicles none of the original amount of plastocyanin. (4) The effect of externally added plastocyanin on the reduction of P-700 was studied by monitoring the absorbance changes at 703 nm after a long flash. In inside-out vesicles P-700 was reduced by the added plastocyanin but not in right-side-out vesicles and class II chloroplasts. These results provide strong evidence for a function of plastocyanin at the internal side of the thylakoid membrane.