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.

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.

Cadmium-binding protein in a cadmium-resistant strain of Saccharomyces cerevisiae.

A Cd-binding protein in the Cd2+-resistant strain 301N of Saccharomyces cerevisiae was induced by administration to 0.5 mM CdSO4. The protein was purified by a gel-permeation and subsequent ion-exchange column chromatographies. The purified Cd-binding protein had the characteristics of metallothioneins: (1) low molecular weight (9.0 kDa), (2) high Cd content (63 micrograms/mg protein), (3) amino-acid composition rich in cysteine (18%), basic and acidic amino acids and free from aromatic amino acids, and (4) an absorption shoulder at near 250 nm. Acid pH or EDTA treatments abolished 250 nm absorption of the Cd-binding protein, and the formed apoprotein was capable of binding Cd2+, Cu2+ and Zn2+, respectively. Heat treatment (75 degrees C) little affected the ultraviolet absorption or sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of Cd-binding protein. These results suggest that metallothionein generally found in animals also occurs in Cd-adapted yeast cells and thus has a role in its Cd-resistance.

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.

A possible role of histidine in a nickel resistant mechanism of Saccharomyces cerevisiae.

When a nickel resistant strain N08 of S. cerevisiae was grown in a Ni-supplemented medium, approximately 70% of the nickel is distributed in the soluble fraction. The soluble fraction was chromatographed on Sephadex G-10 and the fraction contained both nickel and large amounts of histidine. When cells were grown in medium containing various combinations of nickel and magnesium and which exhibited approximately 50% growth inhibition, a molar ratio of intracellular histidine and nickel contents remained constant at 1.2-1.4, indicating that the increase in histidine content is correlated with nickel accumulation. The wild type strain 0605-S6, however, exhibits no increase in histidine content when grown in a Ni-supplemented medium, and, therefore, a nickel-resistant mechanism of yeast appears to be the formation of histidine-nickel complexes.

Constitutive transcription of the gene for metallothionein in a cadmium-resistant yeast.

A cadmium-resistant strain of Saccharomyces cerevisiae produces a cadmium metallothionein encoded by the CUP1 gene as does a copper-resistant strain. The mechanism of expression of the gene is inducible by copper ions in the copper-resistant strain. However, assays of CUP1-specific mRNA revealed that the transcription of the CUP1 gene in the cadmium-resistant strain is constitutive and the rate of transcription is further increased by exposure to cadmium or copper ions. This result was confirmed by the appearance of constitutive-expression segregants from diploid crosses between the cadmium-resistant strain and a strain with a reporter gene having the promoter of CUP1.

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.

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.

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.

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.

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.

Galactose inhibition of auxin-induced growth of mono- and dicotyledonous plants.

Galactose inhibited auxin-induced cell elongation of oat coleoptiles but not that of azuki bean stems. Galactose decreased the level of UDP-glucose in oat coleoptiles but not in azuki bean hypocotyls. Glucose-1-phosphate uridyltransferase activity (EC 2.7.7.9), in a crude extract from oat coleoptiles, was competitively inhibited by galactose-1-phosphate, but that enzyme from azuki bean was not. A correlation was found between inhibition of growth by galactose and inhibition of glucose-1-phosphate uridyltransferase activity by galactose-1-phosphate using oat, wheat, maize, barley, azuki bean, pea, mung bean, and cucumber plants. Thus, it is concluded that galactose is converted into galactose-1-phosphate, which interferes with UDP-glucose formation as an analog of glucose-1-phosphate.

Auxin-enhanced glucan autohydrolysis in maize coleoptile cell walls.

Cell walls isolated from auxin-pretreated maize (Zea mays L.) coleoptile segments were assayed to disclose evidence for the existence of enhanced autolysis. To improve the sensitivity of the measurements and to facilitate kinetic analysis, isolated cell walls were consolidated within a small column, and the autolysis rate was directly determined from the sugar content of the effluent. This protocol revealed that the maximum rate of autohydrolysis of walls prepared from segments occurs within the first 2 hours and a steady decline commences almost immediately. Walls from indoleacetic acid pretreated segments (0.5-4 hours) released sugar at a higher rate initially (110-125% of controls) and the enhanced rate of autolysis continued for 6 to 8 hours, but then it became equivalent to that of the controls. Pretreatment of the segments at acidic pH had no effect on the measurable rates of autolysis. The (1-->3), (1-->4)-beta-d-glucan content of the walls and the extractable glucanase activities support the hypothesis that temporal enhancement of autohydrolysis is a function of auxin on enzyme activity. The progressive decline in autolysis during prolonged incubations is consistent with the decrease in the quantity of the beta-d-glucan in the wall. The relationship between glucan content and autolysis rate is supported by the observation that while glucose pretreatment of segments had only a small effect on initial autolysis rates, the presence of the sugar during pretreatment served to extend the interval over which higher rates of autolysis could be sustained. The results demonstrate that autolysis is related to auxin-induced wall metabolism in maize coleoptiles.

Inhibition of auxin-induced cell elongation of maize coleoptiles by antibodies specific for cell wall glucanases.

Polyclonal antibodies were raised in rabbits in response to the administration of purified exo- and endoglucanases extracted from cell walls of maize (Zea mays L. B37 x Mo17) coleoptiles. Since the antibodies formed specific conjugates when challenged with the glucanase antigens in immunoblot assays they were employed to evaluate the participation of glucanases in tissue growth. Indole-3-acetic acid induced cell elongation of abraded coleoptile segments was inhibited when the antibodies were supplied as a short term pretreatment (25-200 microgram/milliliter of serum protein). The extent of inhibition of IAA induced cell elongation was additive when endo- and exoglucanase antibodies were applied together. The results suggest that both enzymes have a role in mediating IAA-induced cell elongation. Pretreatment with exo- and endoglucanases antibodies also inhibited IAA induced degradation of noncellulosic beta-d-glucans and the increased level of cellulosic polymers in maize coleoptiles. Antibodies also inhibited the expression of the autohydrolytic degradation of glucans in isolated cell walls. The extent of inhibition was dependent on the antibody concentration applied. The results support the contention that enzymatic processes mediated by exo- and endoglucanases are responsible for cell wall autolytic reactions and that these reactions are linked to the mechanism for expressing auxin induced cell elongation in maize coleoptiles.