NADPH oxidases differentially regulate ROS metabolism and nutrient uptake under cadmium toxicity.

The role of NADPH oxidases under cadmium (Cd) toxicity was studied using Arabidopsis thaliana mutants AtrbohC, AtrbohD and AtrbohF, which were grown under hydroponic conditions with 25 and 100 µM Cd for 1 and 5 days. Cadmium reduced the growth of leaves in WT, AtrbohC and D, but not in AtrbohF. A time-dependent increase in H2 O2 and lipid peroxidation was observed in all genotypes, with AtrbohC showing the smallest increase. An opposite behaviour was observed with NO accumulation. Cadmium increased catalase activity in WT plants and decreased it in Atrboh mutants, while glutathione reductase and glycolate oxidase activities increased in Atrboh mutants, and superoxide dismutases were down-regulated in AtrbohC. The GSH/GSSG and ASA/DHA couples were also affected by the treatment, principally in AtrbohC and AtrbohF, respectively. Cadmium translocation to the leaves was severely reduced in Atrboh mutants after 1 day of treatment and even after 5 days in AtrbohF. Similar results were observed for S, P, Ca, Zn and Fe accumulation, while an opposite trend was observed for K accumulation, except in AtrbohF. Thus, under Cd stress, RBOHs differentially regulate ROS metabolism, redox homeostasis and nutrient balance and could be of potential interest in biotechnology for the phytoremediation of polluted soils.

Effect of Hg, As and Pb on biomass production, photosynthetic rate, nutrients uptake and phytochelatin induction in Pfaffia glomerata.

Plantlets of Pfaffia glomerata (Spreng.) were exposed for 28 days to three different metal/metalloid (Hg, Pb and As) with different levels (Hg 1; As 25, 50, 100 and Pb 100 and 400 µM) to analyze the possible phytochelatin initiation and affects on growth and photosynthetic pigments vis-à-vis metal accumulation potential of plants. The plantlets showed significant Hg, As and Pb accumulation in roots (150, 1267.67 and 2129 µg g(-1) DW respectively); however, a low root to shoot metal translocation was observed. It was interesting to note that all tested macronutrient (Mg, K, Ca) was higher in shoots and just opposite in case of micronutrients (Cu, Fe, Zn), was recorded highest in roots. The growth of plantlets (analyzed in terms of length and dry weight) was negatively affected by various metal treatments. In addition, the level of photosynthetic pigments alters significantly in response to all metal/metalloid treatment. In response to all tested metal/metalloids in plants only As induced phytochelatins (PC2, PC3 and PC4) in roots, and in shoots, GSH was observed in all tested metal/metalloids. In conclusion, P. glomerata plantlets could not cooperatively induce phytochelatins under any of Hg and Pb levels.

Oxidative stress and arsenic toxicity: role of NADPH oxidases.

The effect of arsenic (25 and 50 µM As for 1 and 5d) was analysed in wild type (WT) and Arabidopsis thaliana (L.) Heynh plants deficient in NADPH oxidase C (AtrbohC). The content of H(2)O(2) and malondialdehyde (MDA) increased with the As concentration, while the opposite effect was found for NO in WT and AtrbohC plants. The As treatment reduced catalase and increased glutathione reductase activities to the same extent in WT and AtrbohC plants, although the induction of all SOD isoforms (mainly CuZn-SODs) was observed in WT plants, the opposite effects being found in AtrbohC plants. Glycolate oxidase (H(2)O(2) producers) considerably increased with the concentration and time of treatment with As in WT and AtrbohC mutants. Arsenic induced the uptake and translocation of P, S, Cu, Zn, and Fe in WT plants, while in AtrbohC plants the opposite trend was noted and the uptake of As became considerably lower than in WT plants. These results suggest that As causes oxidative stress by inducing glycolate oxidase, while NADPH oxidase does not appear to participate in ROS overproduction but could be critical in regulating antioxidant defences as well as the transport and translocation of As and macro/micronutrients.

The detoxification of lead in Sedum alfredii H. is not related to phytochelatins but the glutathione.

Two ecotypes of S. alfredii [Pb accumulating (AE) and Pb non-accumulating (NAE)] differing in their ability in accumulating Pb were exposed to different Pb levels to evaluate the effects on plant length, photosynthetic pigments, antioxidant enzymes (SOD and APX), cysteine, non-protein thiols (NP-SH), phytochelatins (PCs) and glutathione (GSH) vis-à-vis Pb accumulation. Both ecotypes showed significant Pb accumulation in roots, however only the AE showed significant Pb accumulation in shoots. We found that both AE and NAE of S. alfredii-induced biosynthesis of GSH rather than phytochelatins in their tissue upon addition of even high Pb levels (200 microM). Root and shoot length were mostly affected in both ecotypes after addition of higher Pb concentrations and on longer durations, however photosynthetic pigments did not alter upon addition of any Pb treatment. Both superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities of AE were higher than NAE. The levels of cysteine and NP-SH were also higher in AE than in NAE. Hence, the characteristic Pb accumulation of ecotypes differed presumably in relation to their capacity for detoxification of Pb. These results suggest that enzymatic and non-enzymatic antioxidants play a key role in the detoxification of Pb-induced toxic effects in Sedum alfredii. This plant can be used as an indicator species for Pb contamination.

Arsenic accumulation in root and shoot vis-a-vis its effects on growth and level of phytochelatins in seedlings of Cicer arietinum L.

Arsenic (As) contamination of water and soil has become a subject of prime interest due to its direct effect on human health through drinking water and food. In present study two varieties (CSG-8962 and C-235) of chickpea, Cicer arietinum L., which is a major supplementary food in many parts of India and a valuable source of protein, has been selected to estimate the level of arsenate in root and shoot of five day old seedlings vis-à-vis effect of arsenate on seedling growth and induction of thiols including glutathione (GSH) and phytochelatins (PCs) and their homologues. Both varieties accumulated arsenate to similar levels and most of the metalloid was confined to roots, only about 2.5% was translocated to shoot. Plant growth was also not affected significantly in both the varieties. Arsenate exposure significantly induced the levels of thiols including PCs and homophytochelatins (hPCs). The induction of thiols was much higher in roots than shoots and was greater in var C-235 between the two tested ones. Thus, both varieties tolerated and detoxified arsenic through chelation with GSH, PCs and hPCs, primarily in roots, however var C-235 performed better

Changes in amino acid profile and metal content in seeds of Cicer arietinum L. (chickpea) grown under various fly-ash amendments.

Seeds of Cicer arietinum L. plants are edible and a valuable source of protein. Accumulation of toxic metals in the edible part of the plant, grown in fields close to fly-ash (FA) landfills, may pose a threat to human health. In the present study, the effects of FA and its amendments with different ameliorants viz., garden soil (GS), press mud (PM) and saw dust (SD), on total soluble protein contents, amino acid composition and metal accumulation in seeds were investigated in var. CSG-8962 and var. C-235 of C. arietinum. Plants accumulated adequate amounts of essential metals viz. Fe, Cu, Zn in seeds, while the toxic metals such as Cd and Cr were taken up in smaller quantities. The accumulation of Cr and Cd was less in var. C-235 than var. CSG-8962. Amendment of FA with PM enhanced the amount of soluble protein and amino acids in both varieties and was found to be superior among all tested ameliorants. Both quantitative and qualitative analysis of amino acids showed better response in var. C-235 as compared to var. CSG-8962. Thus var. C-235 seems to be suitable for cultivation in FA contaminated areas due to more accumulation of essential metals and less accumulation of toxic metals in seeds. Application of PM may further improve the growth of plants and nutritional quality of seeds.

Fly-ash induced synthesis of phytochelatins in chickpea (Cicer arietinum L.) plants.

Phytochelatins and related metabolites (cysteine and GSH) were found to be induced in the shoots of two varieties of Cicer arietinum viz., CSG-8962 and C-235 grown under different amendments of fly-ash with garden soil and press mud. Cysteine, GSH, PCs and its speciation were found in higher concentrations in amended fly-ash than in the control 100% soil. Two species of metal binding peptides i.e., PC2 and PC4 were found in both varieties and in amendments, however, their concentration varied depending upon the fly-ash concentrations in both amendments. Further, var. CSG-8962 was found more tolerant than var. C-235 because of higher concentrations of PCs and related metabolites.

Induction for the expression of yeast metallothionein gene, CUP1, by cobalt.

Induction for the expression of the metallothionein gene, CUP1, in the yeast Saccharomyces cerevisiae by cobalt was examined using a reporter gene with the promoter of this gene fused to the coding region of lacZ. The expression of the gene was induced by cobalt as well as by copper and silver ions. The activity of beta-galactosidase showed high levels after treatment with 1.0 mM cobalt chloride. It has been reported that the induction for the transcription of CUP1 by copper and silver is mediated by the Ace1 transcription factor. However, the expression of the gene by cobalt occurred in yeast cells lacking the Ace1 factor. These results suggest the presence of a novel cobalt-specific transcription factor for the CUP1 gene.

Azuki bean cells are hypersensitive to cadmium and do not synthesize phytochelatins.

Suspension-cultured cells of azuki bean (Vigna angularis) as well as the original root tissues were hypersensitive to Cd (<10 microM). Repeated subculturings with a sublethal level of Cd (1-10 microM) did not affect the subsequent response of cells to inhibitory levels of Cd (10-100 microM). The azuki bean cells challenged to Cd did not contain phytochelatin (PC) peptides, unlike tomato (Lycopersicon esculentum) cells that have a substantial tolerance to Cd (>100 microM). Both of the cell suspensions contained a similar level of reduced glutathione (GSH) when grown in the absence of Cd. Externally applied GSH to azuki bean cells recovered neither Cd tolerance nor PC synthesis of the cells. Furthermore, enzyme assays in vitro revealed that the protein extracts of azuki bean cells had no activity converting GSH to PCs, unlike tomato. These results suggest that azuki bean cells are lacking in the PC synthase activity per se, hence being Cd hypersensitive. We concluded that the PC synthase has an important role in Cd tolerance of suspension-cultured cells.

Endo-1,3;1,4-beta-glucanase from coleoptiles of rice and maize: role in the regulation of plant growth.

The Matrix Polymer Hydrolysis Model for regulation of growth in plants is based on the simultaneous hydrolysis and incorporation of new glucans into the cell wall observed in growing plant tissues. The inhibition of growth in rice coleoptile tissues treated with glucanase antibodies confirms similar results observed previously in maize coleoptiles and provides direct evidence for a role of glucanase in control of plant growth. Analysis of two-maize coleoptile endo-glucanase ESTs shows that these sequences are not related to any other previously known family of glycosyl hydrolase. Thus, the coleoptile endo-glucanase enzyme should be classified as a new enzyme group (E.C. 3.2.1.xx). These discoveries enable new initiatives for further investigation of the glucanase role in control of plant growth.

Cell wall autolytic activities and distribution of cell wall glucanases in Zea mays L. seedlings.

Exo- and endo-glucanases mediate specific degradation of cell wall (1,3)(1,4)-beta-D-glucans and these enzymes have been related to auxin-mediated growth and development of cereal coleoptiles. However, their distribution and functions have not been well established in other tissues. In this study the glucanase activities and cell wall autolytic activities of different maize organs were determined. Autolysis assays serve to evaluate the hydrolysis of cell wall polymers in situ by measuring the sugars released from the insoluble cell wall matrix resulting from the action of bound enzymes. Autolytic activities were observed in the cell walls of elongating young leaves, mesocotyl and roots of maize. Wall proteins extracted from all of these structures are enriched in several types of glucanases and other wall polysaccharide hydrolases. These enzymes therefore appear to have a widespread and fundamental role in wall metabolism in growing tissues.

The cadmium-resistant gene, CAD2, which is a mutated putative copper-transporter gene (PCA1), controls the intracellular cadmium-level in the yeast S. cerevisiae.

Yeast cells carrying the CAD2 gene exhibit a resistance to cadmium. We cloned this gene and demonstrated that it was a mutated form derived from the gene of a putative copper-transporting ATPase (PCA1). By site-directed mutagenesis, it appeared that the mutation conferring cadmium resistance was a R970G-substitution in the C-terminal region of Pca1 protein. The intracellular cadmium level of cells carrying CAD2 was lower than that of cells carrying either PAC1 or delta cad2. Furthermore, cells with overexpression of CAD2 showed a much lower intracellular cadmium level than that of cells with a single-copy CAD2. From these results, we conclude that the Cad2 protein controls the intracellular cadmium level through an enhanced cadmium efflux system.

Exo- and endoglucanases of maize coleoptile cell walls: their interaction and possible regulation.

Glucanase-mediated degradation of beta-(1,3)(1,4)-glucans has been attributed to auxin-induced cell wall loosening and thus tissue growth in cereal plants, but the regulatory mechanisms for the auxin-enhancement of glucanase activities in situ are not fully understood. Here, we report evidence for possible mechanisms which might account for auxin-induced changes in glucanase activities. A likely cause for acceleration of wall glucan degradation is the change in the ratio of exo- and endoglucanases. The combined enzymes synergistically promote beta-(1,3)(1,4)-glucan hydrolysis. In addition, these enzyme activities are enhanced by other enzymic and non-enzymic proteins and are also partially stimulated by divalent cations such as Ca(2+) and Mg(2+) at certain pH values. The acceleration of glucan degradation mediated by auxin may be mediated by changes and/or interactions of any of these factors in situ.

Effect of some heavy metal ions on copper-induced metallothionein synthesis in the yeast Saccharomyces cerevisiae.

Copper-induced metallothionein (MT) synthesis in Saccharomyces cerevisiae was investigated in order to associate this exclusively with Cu2+ in vivo, when cultured in nutrient medium containing other heavy metal ions. Expression of the CUP1 promoter/lacZ fusion gene was inhibited by all heavy metal ions tested, especially Cd2+ and Mn2+. By adding Cd2+ and Mn2+ at 10 microM concentration, the beta-galactosidase activity decreased by about 80% and 50% of the maximum induction observed with 1 mM CuSO4, respectively. Furthermore, cell growth was markedly inhibited by combinations of 1 mM-Cu2+ and 1 microM-Cd2+. Therefore, the yeast S. cerevisiae could not rely on MT synthesis as one of the copper-resistance mechanisms, when grown in a Cd2+ environment. In contrast, the presence of Mn2+ in the nutrient medium showed alleviation rather than growth inhibition by high concentrations of Cu2+. The recovery from growth inhibition by Mn2+ was due to decreased Cu2+ accumulation. Inhibitory concentrations of Co2+, Ni2+ and Zn2+ on expression of the CUP1p/lacZ fusion gene were at least one order of magnitude higher than that of Cd2+ and Mn2+. These results are discussed in relation to Cu2+ transport and Cu-induced MT synthesis in the copper-resistance mechanism of the yeast S. cerevisiae.

Cell wall metabolism and autolytic activities of the yeast Saccharomyces exiguus.

Autolytic activities were measured in cell walls prepared from the yeast Saccharomyces exiguus. Walls of yeast cells exhibited higher autolytic activities directed toward glucans at the exponential phase of growth when compared to cells at the stationary phase, while glucanase activities in the soluble extract fraction were higher at the stationary phase when compared to exponential phase, suggesting an important role of cell wall glucanases in growth of the yeast cells. Yeast cell walls also exhibited a substantially high autolytic activity of glycoproteins containing mannose throughout growth. These results illustrate the diverse metabolism related to functions of yeast cell walls.

Regulation of cell wall glucanase activities by non-enzymic proteins in maize coleoptiles.

Auxin stimulates cell wall glucanase activities in isolated segments of maize coleoptiles with no detectable change in the amounts of the enzymic peptides. To disclose the regulatory mechanism for this enhanced growth, we examined the possibility that protein interactions account for stimulating cell wall enzyme activities. One putative regulatory peptide, an acidic wall protein (AW), was separated from the wall protein fractionation profile, and this non enzymic protein was able to stimulate both exo- and endoglucanase activities in vitro. The results are consistent with the hypothesis that a pertinent glucanase/glucan interaction might be facilitated by AW mobilization within the wall in response to auxin-treatment.

Developmental regulation of polysaccharide metabolism and growth in the primary cell walls of maize.

During development of the intact maize (Zea mays L.) coleoptile a correspondence exists between the rate of change in length and fresh weight but these parameters, that reflect growth, are all preceded by the accumulation of dry weight by the tissue. Coleoptile dry weight increases rapidly for the first 3-4 days commensurate with tissue formation with evidence for specific biosynthesis including the net incorporation into the cell wall of protein and substantial deposition of beta-(1,3)(1,4) glucan. After day 4 the extractable wall protein and after day 5 the beta-(1,3)(1,4) glucan begin to decline while the deposition of glucuronoarabinoxylan continues. Despite the continued incorporation of specific polymers into the wall the accumulated mass does not augment the overall dry weight. The shifts in polysaccharide distribution with development are consistent with what would be expected in terms of changes in the autolytic degradation of the wall. Glucan autolysis in isolated cell walls is relatively inactive initially then increases to a maximum by day 4 while arabinoxylan autolysis undergoes only relatively minor changes. Changes in the extent of wall glucan autolysis is proportional to the levels of glucanase activities extracted from the cell wall during the developmental sequence. Glucan in walls of the first three leaves of the maize seedlings also reflects dynamic turnover in correspondence with growth much like that of the coleoptile, suggesting that glucan metabolism coincides with growth in other maize tissues. The acceleration of glucan turnover mediated by auxin in situ may result from combinations and/or interactions of proteins that promote synergistic polysaccharide hydrolysis.

Resistance to cadmium ions and formation of a cadmium-binding complex in various wild-type yeasts.

The resistance to cadmium ions (Cd-resistance) and possible formation of cadmium-binding complexes were examined in eight different wild-type yeasts. Saccharomyces exiguus, Pichia farinosa, Torulaspora delbrueckii and Schizosaccharomyces octosporus exhibited partial Cd-resistance, as compared to the Cd-resistant strain 301N and the Cu-resistant but Cd-sensitive strain X2180-1B of Saccharomyces cerevisiae. Saccharomyces carlsbergensis, Pichia mogii, Zygosaccharomyces rouxii and Kluyveromyces lactis were all Cd-sensitive. The partially Cd-sensitive species, with the exception of S. exiguus, accumulated Cd2+ ions in the cytoplasmic fraction to varying extents. This fraction from S. octosporus included a Cd-binding complex that contained (gamma EC)nG peptides known as cadystins or phytochelatins, while P. farinosa and T. delbrueckii synthesized Cd-binding proteins that were similar to the Cd-metallothionein produced by S. cerevisiae 301N in terms of molecular weight and amino acid composition. These results suggest that such cytoplasmic molecules play a role in the Cd-tolerance of the above three species of yeast. S. exiguus retained most cadmium in the cell wall fraction and no Cd-binding complex was found in the cytoplasm, an indication of the important role of the cell wall in its Cd-tolerance. Different modes of binding of Cd2+ ions appear to be involved in the Cd-resistance of wild-type yeasts and fungi.