Changes in sugar beet leaf plasma membrane Fe(III)-chelate reductase activities mediated by Fe-deficiency, assay buffer composition, anaerobiosis and the presence of flavins

Summary Different assay conditions induce changes in the ferric chelate reductase activities of leaf plasma membrane preparations from Fe-deficient and Fe-sufficient sugar beet. With an apoplasttype assay medium the ferric chelate reductase activities did not change significantly when Fe(III)-EDTA was the substrate. However, with ferric citrate as substrate, the effect depended on the citrateto-Fe ratio. When the citrate-to-Fe ratio was 20 1, the effects were practically unappreciable. However, with a lower citrate-to-Fe ratio of 5 1 the activities were significantly lower with the apoplast-type medium than with the standard assay medium. Our data also indicate that anaerobiosis during the assay facilitates the reduction of ferric malate and Fe(III)-EDTA by plasma membrane preparations. Anaerobiosis increased by approximately 50% the plasma membrane ferric chelate reductase activities when Fe(III)-EDTA was the substrate. With ferric malate anaerobiosis increased activities by 70–90% over the values obtained in aerobic conditions. However, with ferric citrate the increase in activity by anaerobiosis was not significant. We have also tested the effect of riboflavin, flavin adenine dinucleotide, and flavin mononucleotide on the plasma membrane ferric chelate reductase activities. The presence of flavins generally increased activities in plasma membrane preparations from control and Fe-deficient plants. Increases in activity were generally moderate (lower than twofold). These increases occurred with Fe(III)-EDTA and Fe(III)-citrate as substrates.Abbreviations BPDS bathophenantroline disulfonate - FC ferric chelate - FC-R ferric chelate reductase - PM plasma membrane     

Iron deficiency decreases the Fe(III)-chelate reducing activity of leaf protoplasts

The ferric-chelate reductase (FC-R) activity of mesophyll protoplasts isolated from Fe-sufficient (control) and Fe-deficient sugar beet (Beta vulgaris L.) leaves has been characterized. Measurements were made in an ionic environment similar to that in the apoplastic space of the sugar beet mesophyll cells. The FC-R activity of Fe-sufficient and Fe-deficient protoplasts was dependent on light. Fe deficiency decreased markedly the FC-R activity per protoplast surface unit. The optimal pH for the activity of the FC-R in mesophyll protoplasts was in the range 5.5 to 6.0, typical of the apoplastic space. Beyond pH 6.0, the activity of the FC-R in mesophyll protoplasts decreased markedly in both Fe-sufficient and Fe-deficient protoplasts. These data suggest that both the intrinsic decrease in FC-R activity per protoplast surface and a possible shift in the pH of the apoplastic space could lead to the accumulation of physiologically inactive Fe pools in chlorotic leaves.     

Effects of heavy metals on both induction and function of root Fe(lll) reductase in Fe-deficient cucumber (Cucumis sativus L.) plants

Heavy metals are known to induce Fe chlorosis in different plantspecies. Heavy-metal-induced chlorosis is generally correlatedwith low plant Fe contents, suggesting effects of heavy metalson Fe mobilization and uptake. Under Fe-deficient conditions,dicotyledonous plants enhance root Fe(lll) reductase activity,thus increasing the capacity to reduce Fe(MI) to Fe(ll), theform in which roots absorb Fe. We studied the effect of severalheavy metals (Mn, Pb, Zn, Mo, Ni, Cu, and Cd) on the inductionof enhanced root Fe(lll) reductase by 11-d-old Fe-deficientcucumber [Cucumis sativus L. cv. Ashley). The effect of theseheavy metals on the function of the induced Fe(lll) reductasewas also investigated. Results showed that some heavy metalscan inhibit both the induction and function of root Fe(lll)reductase. Ni, at 20//M, and Cu and Cd, at 5 fiM concentrationor higher, severely inhibited the induction of root Fe(lll)reductase while Mn, Pb, Zn, and Mo had little effect, even atconcentrations higher than 20 //M. Function of the induced rootFe(lll) reductase only was negatively affected by Cu and Ni. Key words: Cucumis sativus, iron deficiency, iron reduction, heavy metals     

Effects of Cd or/and Pb on soil enzyme activities and microbial community structure

Heavy metals could have long-term hazardous impacts on the health of soil ecosystems and adverse influences on soil biological processes. A study of Cd or/and Pb effects on soil enzyme activities and microbial community structure was undertaken with brown soil in a greenhouse for a period of 10 weeks. The experiment results showed that urease, acid phosphatase and dehydrogenase activities were significantly lower (p < 0.05) in Cd or/and Pb treatments than in control. Three enzyme activities decreased with the increasing metal concentrations. The effects of Cd and Pb combined on enzyme activities were higher than Cd or Pb alone. The soil microbial populations were far lower in heavy metal treatments than in control, and soil microbial populations under different heavy metals levels showed a significant difference (p < 0.05). The PCR-DGGE banding patterns confirmed that the addition of metals had a significant impact on microbial community structure.     

Effects of Municipal Sewage Sludge Doses on the Chlorophyll Contents and Heavy Metal Concentration of Sugar Beet (Beta vulgaris var. saccharifera)

The present study was conducted to assess the suitability of sewage sludge amendment (SSA) in soil for Beta vulgaris var. saccharifera (sugar beet) by evaluating the heavy metal accumulation and physiological responses of plants grown at a 10%, 25%, and 50% sewage sludge amendment rate. The sewage sludge amendment was modified by the physicochemical properties of soil, thus increasing the availability of heavy metals in the soil and consequently increasing accumulation in plant parts. Cd, Pb, Ni, and Cu concentrations in roots were significantly higher in plants grown at 25% as compared to 50% SSA; however, Cr and Zn concentration was higher at 50% than 25% SSA. The concentrations of heavy metal showed a trend of Zn > Ni > Cu > Cr > Pb > Cd in roots and Zn > Cu > Ni > Cr > Pb > Cd in leaves. The only instance in which the chlorophyll content did not increase after the sewage sludge treatments was 50%. There were approximately 1.12-fold differences between the control and 50% sewage sludge application for chlorophyll content. The sewage sludge amendment led to a significant increase in Pb, Cr, Cd, Cu, Zn, and Ni concentrations of the soil. The heavy metal accumulation in the soil after the treatments did not exceed the limits for the land application of sewage sludge recommended by the US Environmental Protection Agency (US EPA). The increased concentration of heavy metals in the soil due to the sewage sludge amendment led to increases in heavy metal uptake and the leaf and root concentrations of Ni, Zn, Cd, Cu, Cr, Pb, and Zn in plants as compared to those grown on unamended soil. More accumulation occurred in roots and leaves than in shoots for most of the heavy metals. The concentrations of Cd, Cr, and Pb were more than the permissible limits of national standards in the edible portion of sugar beet grown on different sewage sludge amendment ratios. The study concludes that the sewage sludge amendment in the soil for growing sugar beet may not be a good option due to risk of contamination of Cr, Pb, and Cd.     

Iron deficiency enhances the levels of ascorbate,glutathione, and related enzymes in sugar beet roots

Summary.  The effects of Fe deficiency stress on the levels of ascorbate and glutathione, and on the activities of the enzymes ferric chelate reductase, glutathione reductase (EC 1.6.4.2), ascorbate free-radical reductase (EC 1.6.5.4) and ascorbate peroxidase (EC 1.11.1.11), have been investigated in sugar beet (Beta vulgaris L.) roots. Plasma membrane vesicles and cytosolic fractions were isolated from the roots of the plants grown in nutrient solutions in the absence or presence of Fe for two weeks. Plants responded to Fe deficiency not only with a 20-fold increase in root ferric chelate reductase activity, but also with moderately increased levels of the general reductants ascorbate (2-fold) and glutathione (1.6-fold). The enzymes of the ascorbate-glutathione cycle in roots were also affected by Fe deficiency. Glutathione reductase activity was enhanced 1.4-fold with Fe deficiency, associated to an increased ratio of reduced to oxidized glutathione, from 3.1 to 5.2. The plasma membrane fraction from iron-deficient roots showed 1.7-fold higher ascorbate free-radical reductase activity, whereas in the cytosolic fraction the enzyme activity was not affected by Fe deficiency. The activity of the cytosolic hemoprotein ascorbate peroxidase decreased approximately by 50% with Fe deprivation. These results show that sugar beet responds to Fe deficiency with metabolic changes affecting components of the ascorbate-glutathione cycle in root cells. This suggests that the ascorbate-glutathione cycle would play certain roles in the general Fe deficiency stress responses in strategy I plants. Received November 19, 2001; accepted September 30, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, CSIC, Apartado 202, 50080 Zaragoza, Spain.     

Physiological responses and differential gene expression in Prunus rootstocks under iron deficiency conditions

Two Prunus rootstocks, the Myrobalan plum P 2175 and the interspecific peach-almond hybrid, Felinem, were studied to characterize their biochemical and molecular responses induced under iron-Deficient conditions. Plants of both genotypes were submitted to different treatments using a hydroponic system that permitted removal of Fe from the nutrient solution. Control plants were grown in 90 μM Fe (III)-EDTA, Deficient plants were grown in an iron free solution, and plants submitted to an Inductor treatment were resupplied with 180 μM Fe (III)-EDTA over 1 and 2 days after a period of 4 or 15 days of growth on an iron-free solution. Felinem increased the activity of the iron chelate reductase (FC-R) in the Inductor treatment after 4 days of iron deprivation. In contrast, P 2175 did not show any response after at least 15 days without iron. The induction of the FC-R activity in this genotype was coincident in time with the medium acidification. These results suggest two different mechanisms of iron chlorosis tolerance in both Strategy I genotypes. Felinem would use the iron reduction as the main mechanism to capture the iron from the soil, and in P 2175, the mechanism of response would be slower and start with the acidification of the medium synchronized with the gradual loss of chlorophyll in leaves. To better understand the control of these responses at the molecular level, the differential expression of PFRO2, PIRT1 and PAHA2 genes involved in the reductase activity, the iron transport in roots, and the proton release, respectively, were analyzed. The expression of these genes, estimated by quantitative real-time PCR, was different between genotypes and among treatments. The results were in agreement with the physiological responses observed.     

The pH Requirement for in Vivo Activity of the Iron-Deficiency-Induced "Turbo" Ferric Chelate Reductase (A Comparison of the Iron-Deficiency-Induced Iron Reductase Activities of Intact Plants and Isolated Plasma Membrane Fractions in Sugar Beet)

The characteristics of the Fe reduction mechanisms induced by Fe deficiency have been studied in intact plants of Beta vulgaris and in purified plasma membrane vesicles from the same plants. In Fe-deficient plants the in vivo Fe(III)-ethylenediaminetetraacetic complex [Fe(III)-EDTA] reductase activity increased over the control values 10 to 20 times when assayed at a pH of 6.0 or below ("turbo" reductase) but increased only 2 to 4 times when assayed at a pH of 6.5 or above. The Fe(III)-EDTA reductase activity of root plasma membrane preparations increased 2 and 3.5 times over the controls, irrespective of the assay pH. The Km for Fe(III)-EDTA of the in vivo ferric chelate reductase in Fe-deficient plants was approximately 510 and 240 [mu]M in the pH ranges 4.5 to 6.0 and 6.5 to 8.0, respectively. The Km for Fe(III)-EDTA of the ferric chelate reductase in intact control plants and in plasma membrane preparations isolated from Fe-deficient and control plants was approximately 200 to 240 [mu]M. Therefore, the turbo ferric chelate reductase activity of Fe-deficient plants at low pH appears to be different from the constitutive ferric chelate reductase.     

Accumulation and distribution of iron, cadmium, lead and nickel in cucumber plants grown in hydroponics containing two different chelated iron supplies

Cucumber plants grown in hydroponics containing 10 μM Cd(II), Ni(II) and Pb(II), and iron supplied as Fe(III) EDTA or Fe(III) citrate in identical concentrations, were investigated by total-reflection X-ray fluorescence spectrometry with special emphasis on the determination of iron accumulation and distribution within the different plant compartments (root, stem, cotyledon and leaves). The extent of Cd, Ni and Pb accumulation and distribution were also determined. Generally, iron and heavy-metal contaminant accumulation was higher when Fe(III) citrate was used. The accumulation of nickel and lead was higher by about 20% and 100%, respectively, if the iron supply was Fe(III) citrate. The accumulation of Cd was similar. In the case of Fe(III) citrate, the total amounts of Fe taken up were similar in the control and heavy-metal-treated plants (27-31 μmol/plant). Further, the amounts of iron transported from the root towards the shoot of the control, lead- and nickel-contaminated plants were independent of the iron(III) form. Although Fe mobility could be characterized as being low, its distribution within the shoot was not significantly affected by the heavy metals investigated.     

Investigation of the Effects of Phosphate Fertilizer Application on the Heavy Metal Content in Agricultural Soils with Different Cultivation Patterns

The use of phosphate fertilizers is essential in agriculture, because they supply farmland with nutrients for growing plants. However, heavy metals might be included as impurities in natural materials and minerals, so heavy metals can also be present in phosphate fertilizers or other chemical fertilizers. The aim of this work was to assess the heavy metal content and contamination status of agricultural soils in the Hamadan province of Iran used for the cultivation of different crops, including cucumber, potatoes, and sugar beet. Surface soil samples were collected and analyzed to determine the total concentration of specific elements (As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn), before the pollution index was calculated for each element. Soils used for the cultivation of the three types of crop were not contaminated with As, Cr, Cu, Pb, or Zn. However, the pollution indices for Cd were 1.1, 4.4, and 3.8 in cucumber, potato, and sugar beet fields, respectively, which indicated moderate, high, and high levels of contamination, respectively. Soils from potato and sugar beet fields were heavily contaminated with Cd, which may have resulted from long-term overuse of phosphate fertilizers.     

Bioaccumulation and rhizofiltration potential of Pistia stratiotes L. for mitigating water pollution in the Egyptian wetlands

The bioaccumulation and rhizofiltration potential of P. stratiotes for heavy metals were investigated to mitigate water pollution in the Egyptian wetlands. Plant and water samples were collected monthly through nine quadrats equally distributed along three sites at Al-Sero drain in Giza Province. The annual mean of the shoot biomass was 10 times that of the root. The concentrations of shoot heavy metals fell in the order: Fe < Mn < Cr < Pb < Cu < Zn < Ni < Co < Cd, while that of the roots were: Fe < Mn < Cr < Pb < Zn < Ni < Co < Cu < Cd. The bio-concentration factor (BCF) of most investigated heavy metals, except Cr and Pb, was greater than 1000, while the translocation factor (TF) of most investigated metals, except Pb and Cu, did not exceed one. The rhizofiltration potential (RP) of heavy metals was higher than 1000 for Fe, and 100 for Cr, Pb and Cu. Significant positive correlations between Fe and Cu in water with those in plant roots and leaves, respectively were recorded, which, in addition to the high BCF and RP, indicate the potential use of P. stratiotes in mitigating these toxic metals.     

Nitric oxide stimulates seed germination and counteracts the inhibitory effect of heavy metals and salinity on root growth of Lupinus luteus

Nitric oxide (NO) is a bioactive molecule, which in plants was found to function as prooxidant as well as antioxidant. In the present study, we found that NO donor sodium nitroprusside (SNP) stimulates seed germination and root growth of lupin (Lupinus luteus L. cv. Ventus). Seed germination is promoted at concentrations between 0.1 and 800 μM SNP in a dose-dependent manner. The stimulation was most pronounced after 18 and 24 h and ceased after 48 h of imbibition. The promoting effect of NO on seed germination persisted even in the presence of heavy metals (Pb, Cd) and sodium chloride. Pretreatment of lupin seedlings for 24 h with 10 μM SNP resulted in efficient reduction of the detrimental effect of the abiotic stressors on root growth and morphology. The inhibitory effect of heavy metals on root growth was accompanied by increased activity of superoxide dismutase (SOD, EC 1.15.1.1.), which in roots preincubated with SNP was significantly higher. Some changes in the activity of other antioxidant enzymes, peroxidase (POX, EC 1.11.1.7) and catalase (CAT, EC 1.11.1.6) were also detected. Using the superoxide anion (O₂^•–)-specific indicator dihydroethidium (DHE), we found intense DHE-derived fluorescence in heavy metal-stressed roots, whereas in those pretreated with SNP the fluorescence was very low, comparable to the level in unstressed roots. On the basis of the above data, we conclude that the protective effect of NO in stressed lupin roots may be at least partly due to the stimulation of SOD activity and/or direct scavenging of the superoxide anion.     

Influence of acid rain and ozone on soil heavy metals under loblolly pine trees: A field study

A field growth chamber study was conducted to determine the effects of ozone and simulated acid rain (SAR) on soil heavy metals. Loblolly pine (Pinus taeda L.), grown in open-top chambers, was exposed to three concentrations of ozone (charcoal filtered air with 0.026 µL O₃ L^-1, and two non-filtered treatments in which ozone concentrations were 0.074 µL L^-1 and 0.147 µL L^-1, respectively) and two levels of SAR (pH 3.5 and 5.2). Ozone was applied for 12 h d^-1 for 9 months and acid rain deposition was 125 mm event^-1. After 9 months exposure, soil pH, organic matter and DTPA-extractable heavy metals (Cd, Pb, Zn, Mn, Fe, Cu) were determined on soil samples collected from exposed chambers at two depths (0–15 cm and 15–30 cm). Simulated acid rain decreased the original soil pH. The concentrations of Cd, Pb and Mn at SAR pH 3.5 were significantly higher than at SAR pH 5.2. Ozone did not affect Zn, Fe and Cu, but a significant interaction between pH and O₃ on Mn, Pb and Cd was observed. Due to the poor drainage capacity of this soil, leaching of heavy metals was not observed.     

Comparison of cadmium-induced iron-deficiency responses and genuine iron-deficiency responses in Malus xiaojinensis

The effects of the heavy metal Cd in Malus xiaojinensis were investigated using hydroponic cultures. Chlorophyll and Fe concentrations in young leaves were markedly decreased by Cd treatment, although Fe concentration was significantly enhanced in the roots. A comparative examination of the Fe-deficiency responses due to Fe deficiency and Cd treatment was also performed. Both Fe deficiency and Cd treatment induced responses similar to those of Fe-deficiency in M. xiaojinensis, including acidification of the rhizosphere, enhanced Fe(III) chelate reductase activity, and upregulation of the Fe-deficiency-responsive genes MxIRT1 and MxFRO2-Like. However, the Fe-deficiency responses induced by Cd treatment were different in intensity and timing from those induced by Fe deficiency.     

Heavy Metal Phytotoxicity to Radish (Raphanus sativus L.) in a Digested Sludge-amended Gangetic Alluvium

A limiting factor in land application of sewage sludge is the resultant heavy metal accumulation in soils followed by biomagnification in the food chain, posing a potential hazard to animal and human health. In view of this fact, pot experiments were conducted to evaluate the effect of digested sludge application to soil on phytotoxicity of heavy metals such as Cd, Cr, Ni, and Pb to radish (Raphanus sativus L.) plants. Increasing sludge levels resulted in increased levels of DTPA-extractable heavy metals in the soil. Cadmium was the dominant metal extracted by DTPA followed by Ni, Pb, and Cr. The extractability of metals by DTPA tended to decrease from the first to the second crop. Dry matter yield of radish increased significantly as a function of increasing sludge treatments. Soil application of sludge raised the concentration of one or more heavy metals in plants. Shoots contained higher concentrations of Cd, Cr, and Ni than the roots of radish plants. Shoot concentrations of Cd, Cr, Ni, and Pb were within the tolerance levels of this crop at all rates of sludge application. Shoot as well as root concentration of Cd was above 0.5 mg kg^?1, considered toxic for human and animal consumption. The levels of DTPA-extractable Cd and Ni were less correlated while those of Cr and Pb were more correlated with their respective shoot and root contents. The results emphasize that accumulation of potentially toxic heavy metals in soil and their build-up in vegetable crops should not be ignored when sludge is applied as an amendment to land.     

The induction of the “turbo reductase” is inhibited by cycloheximide, cordycepin and ethylene inhibitors in Fe-deficient cucumber (Cucumis sativus L.) plants

Summary Dicotyledonous plants respond to Fe deficiency by enhancing the capacity of their roots to reduce Fe(III) to Fe(II). It has been suggested that there are two different ferric redox systems in the roots: the standard reductase, active with ferricyanide and not inducible by Fe deficiency, and the turbo reductase, active with both ferricyanide and ferric chelates and inducible by Fe deficiency. We have used different experimental approaches to test whether or not the Fe(III)-reducing capacity of cucumber (Cucumis sativus L. cv. Ashley) roots can be explained by considering the standard and the turbo reductase as the same enzyme. For this, we used both Fe-sufficient and Fe-deficient plants, which were treated with ethylene inhibitors (cobalt or silver thiosulfate; found to inhibit the turbo reductase in a previous work), a protein synthesis inhibitor (cycloheximide), or an mRNA polyadenylation inhibitor (cordycepin). At different times after application of these inhibitors, reduction of both ferricyanide and Fe(III)-EDTA were determined. In addition, we studied the effects of pH and temperature on the reduction of ferricyanide and Fe(III)-EDTA by both Fe-sufficient and Fe-deficient plants. Results suggest that there are, at least, two different ferric redox systems in the roots. Enhancement of Fe(III)-reducing capacity (turbo reductase) by Fe-deficient plants probably requires the de novo synthesis of a (or several) protein(s), which has a high turnover rate and whose expression is presumably regulated by ethylene.Abbreviations Ch-R ferric chelate reductase - CHM cycloheximide - CN-R ferricyanide reductase - EDDHA N,N-ethylene bis[2-(2-hydroxyphenyl)-glycine] - EDTA ethylenediamine-tetraacetic acid - Ferrozine 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine - HEDTA N-hydroxyethylethylene-diaminetriacetic acid - STS silver thiosulfate     

Impact of two iron(III) chelators on the iron, cadmium, lead and nickel accumulation in poplar grown under heavy metal stress in hydroponics

Poplar (Populus jacquemontiana var. glauca cv. Kopeczkii) was grown in hydroponics containing 10 μM Cd(II), Ni(II) or Pb(II), and Fe as Fe(III) EDTA or Fe(III) citrate in identical concentrations. The present study was designed to compare the accumulation and distribution of Fe, Cd, Ni and Pb within the different plant compartments. Generally, Fe and heavy-metal accumulation were higher by factor 2-7 and 1.6-3.3, respectively, when Fe(III) citrate was used. Iron transport towards the shoot depended on the Fe(III) chelate and, generally, on the heavy metal used. Lead was accumulated only in the root. The amounts of Fe and heavy metals accumulated by poplar were very similar to those of cucumber grown in an identical way, indicating strong Fe uptake regulation of these two Strategy I plants: a cultivar and a woody plant. The Strategy I Fe uptake mechanism (i.e. reducing Fe(III) followed by Fe(II) uptake), together with the Fe(III) chelate form in the nutrient solution had significant effects on Fe and heavy metal uptake. Poplar appears to show phytoremediation potential for Cd and Ni, as their transport towards the shoot was characterized by 51-54% and 26-48% depending on the Fe(III) supply in the nutrient solution.     

Studies on the role of iron in the reversal of cadmium toxicity in chicks

Studies were conducted to determine the effect of dietary iron (Fe) levels ranging from a deficiency to an excess on the toxicity of cadmium (Cd) in chicks. In Fe-deficient animals, cadmium was found to be more toxic than in Fe supplemented animals as measured by growth. The liver Cd burdens were increased significantly in the presence of dietary Fe supplementation, and there was a significant Cd−Fe interaction in the Cd concentration of the kidney, indicating that iron deficiency increased the concentration of Cd in the kidneys of those chicks receiving this element. Cd tended to reduce the Fe concentration in both the liver and kidney. The absorption of Cd as measured by the amount of¹⁰⁹Cd that disappeared from an isolated duodenal segment in one h was not affected by the Fe content of the diet, but the amount of isotope appearing in the liver compared to the amount present in the blood was increased in the Fe supplemented chicks. Separation of the Cd binding ligands by column chromatography revealed that more of the Cd in the liver, but not the kidney, was associated with ligands which eluted in a column volume that contained metallothionein in those chicks receiving Fe than in the livers from Fe deficient animals. The inverse relationship between the amount of Cd bound to the metallothionein containing fraction and toxicity may be related causally. Paper No. 10538 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. The use of trade names in this publication does not imply endorsement by the NC Agricultural Research Service of the products named nor criticism of similar ones not mentioned.     

Relationships among iron deficit-induced potato callus growth inhibition,Fe distribution,chlorosis, and oxidative stress amplified by reduced antioxidative enzyme activities

Callus cultures were used to investigate and delineate responses of potato to iron (Fe) deficiency conditions over different culture durations. The morphological responses included chlorotic symptoms, reduced fresh weight and area of callus growth on Fe-deficient medium compared to calli grown under Fe sufficient conditions. Biochemically, potato calli under Fe deficit exhibited decreases in chlorophyll and carotenoid contents, reduction in activities of antioxidant enzymes (peroxidase, catalase and ascorbate peroxidase), as well as an increase in ferric chelate reductase (FCR) activity, lipid peroxidation, phenolic production and hydrogen peroxide (H₂O₂) level. Perls staining revealed sparse Fe distribution in Fe-deficient callus cells whereas Fe was widely distributed and intensely stained among numerous actively dividing cells in Fe-sufficient calli. These responses of calli to Fe deficiency were more pronounced with prolonged exposure to such stress leading to severe chlorosis and/or death of cells in chlorosis-susceptible calli but potential chlorosis-tolerant callus cells maintained their greenness and viability. Over a prolonged period in culture, significantly positive correlations were found among callus fresh weight, chlorophyll and carotenoid contents, antioxidant enzyme activities and lipid peroxidation as Fe supplies to the medium was increased. FCR activity was strongly correlated in a negative manner with Fe deficiency, chlorophyll content and peroxidase activity. The responses of calli to Fe supply can serve as reliable indicators for detecting chlorosis tolerance and/or nutrient deficiency stress.     

Geochemical cycling of heavy metals in a marine coastal area: benthic flux determination from pore water profiles and in situ measurements using benthic chambers

Concentrations of the heavy metals Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn were measured in sea water, suspended matter, sediments and pore water samples collected in a coastal area of the middle Tyrrhenian Sea. Concentration factors between pore water (extracted from the first centimeter of the sediments) and the overlying sea water (taken 30 cm above the sea bed) were less than 1 for Cr, Cu and Pb, 1–10 for Cd and Ni, 10–100 for Fe and Co, 100–1000 for Mn, and 1–100 for Zn.The benthic fluxes of heavy metals at the sediment-water interface were measured directly using in situ benthic chambers and calculated using Fick's first law during two experimental periods, one in 1986 and the other in 1988. The fluxes of Cu, Ni, Pb and Zn varied significantly over time; this appeared to be related to their relatively low ( 10) concentration factors. From the benthic chamber experiments, metals with positive fluxes were in the order: Mn > Fe > Co > Cd, while those with negative fluxes were: Zn > Pb > Ni Cu. Fluxes calculated using Fick's Law were: positive – Mn > Fe > Zn (or Zn > Fe) > Ni > Co > Cd, negative fluxes Pb > Cu > Cr.Measured (benthic chamber) and calculated (Fick's first law) fluxes for Co, Cd, Mn, Pb and Fe were comparable within an order of magnitude, although less agreement was found for Cu, Ni and Zn. Removal of Ni and Zn at the sediment-water interface has been proposed to explain the fact that the measured and calculated fluxes have opposite directions for these metals.