Effects of low molecular weight organic acids on Cu accumulation by castor bean and soil enzyme activities.

Chelating agents have been considered as an important phytoremediation strategy to enhance heavy metal extraction from contaminated soil. A pot experiment was conducted to explore the effects of low molecular weight organic acids (LMWOAs) on the phytoremediation efficiency of copper (Cu) by castor bean, and soil enzyme activities. Results indicated that the addition of all the three kinds of LMWOAs (citric, tartaric, oxalic acids) did not decrease the biomass of castor bean, despite the fact they reduced the concentration of chlorophyll-a in leaves compared to the control. The Cu concentrations in the roots and shoots significantly increased by 6-106% and 5-148%, respectively, in the LMWOAs treatments so that the total accumulation of Cu by whole plants in all the LMWOAs treatments increased by 21-189% in comparison with the control. The values of the translocation factor (TF) and bio-concentration factor (BCF) of Cu in castor bean also rose following the addition of LMWOAs, indicating that the LMWOAs enhanced the uptake and transportation of Cu. Moreover, the application of LMWOAs did not significantly change the soil pH but significantly increased the activity of soil enzymes (urease, catalase, and alkaline phosphatase). The addition of exogenous LMWOAs increased the available Cu significantly in the soil, thus promoted the phytoextraction efficiency of Cu by castor bean. These results will provide some new insights into the practical use of LMWOAs for the phytoremediation of heavy-metal-contaminated soil employing castor bean.

Comparison of chelates for enhancing Ricinus communis L. phytoremediation of Cd and Pb contaminated soil.

We studied chelate effects on castor bean (Ricinus communis L.) growth. These effects included Cd and Pb accumulation in plant tissues and the chemical behavior of Cd and Pb in the plant rhizosphere and non-rhizosphere. Tests were conducted in a glasshouse using the rhizobag method. Two castor bean cultivars (Zibo-3 and Zibo-9) were grown in soil contaminated with 3.53mg/kg Cd and 274mg/kg Pb. The soil was treated with citric acid (CA), ethylenediamine disuccinic acid (EDDS) or ethylenediamine tetraacetic acid (EDTA) (5mmol/kg). EDDS-treated soil produced 28.8% and 59.4% greater biomass for Zibo-3 and Zibo-9 respectively. In contrast, CA and EDTA inhibited the growth of the two cultivars. Zibo-9 had greater tolerance than Zibo-3 to chelate toxicity. Based on Cd and Pb plant uptake, EDDS could substitute for EDTA for phytoremediation of Cd in soil. EDTA was the most effective of the three chelates for Pb phytoremediation but it is less suitable for field use due to toxicology environmental persistence. Acid extractable Cd and Pb in the rhizosphere or reducible Cd and Pb in the non-rhizosphere of soil were the main influences on Cd and Pb accumulation in castor bean.

Phloem flow and sugar transport in Ricinus communis†L. is inhibited under anoxic conditions of shoot or roots.

Anoxic conditions should hamper the transport of sugar in the phloem, as this is an active process. The canopy is a carbohydrate source and the roots are carbohydrate sinks. By fumigating the shoot with N2 or flooding the rhizosphere, anoxic conditions in the source or sink, respectively, were induced. Volume flow, velocity, conducting area and stationary water of the phloem were assessed by non-invasive magnetic resonance imaging (MRI) flowmetry. Carbohydrates and δ(13) C in leaves, roots and phloem saps were determined. Following flooding, volume flow and conducting area of the phloem declined and sugar concentrations in leaves and in phloem saps slightly increased. Oligosaccharides appeared in phloem saps and after 3 d, carbon transport was reduced to 77%. Additionally, the xylem flow declined and showed finally no daily rhythm. Anoxia of the shoot resulted within minutes in a reduction of volume flow, conductive area and sucrose in the phloem sap decreased. Sugar transport dropped to below 40% by the end of the N2 treatment. However, volume flow and phloem sap sugar tended to recover during the N2 treatment. Both anoxia treatments hampered sugar transport. The flow velocity remained about constant, although phloem sap sugar concentration changed during treatments. Apparently, stored starch was remobilized under anoxia.

Concentrations of metals and potential metal-binding compounds and speciation of Cd, Zn and Cu in phloem and xylem saps from castor bean plants (Ricinus communis) treated with four levels of cadmium.

We examined the concentrations of metals (Cd, Zn, Cu, Fe and Mn) and potential metal-binding compounds [nicotianamine (NA), thiol compounds and citrate] in xylem and phloem saps from 4-week-old castor bean plants (Ricinus communis) treated with 0 (control), 0.1, 1.0, and 10 µM Cd for 3 weeks. Treatment with 0.1 and 1 µM Cd produced no visible damage, while 10 µM Cd retarded growth. Cadmium concentrations in both saps were higher than those in the culture solution at 0.1 µM, similar at 1.0 µM and lower at 10 µM. Cd at 10 µM reduced Cu and Fe concentrations in both saps. NA concentrations measured by capillary electrophoresis-mass spectrometry (MS) in xylem sap (20 µM) were higher than the Cu concentrations, and those in phloem sap (150 µM) were higher than those of Zn, Fe and Cu combined. Reduced glutathione concentrations differed in xylem and phloem saps (1-2 and 30-150 µM, respectively), but oxidized glutathione concentrations were similar. Phloem sap phytochelatin 2 concentration increased from 0.8 µM in controls to 8 µM in 10 µM Cd. Free citrate was 2-4 µM in xylem sap and 70-100 µM in phloem sap. Total bound forms of Cd in phloem and xylem saps from 1 µM Cd-treated plants were 54 and 8%, respectively. Treatment of phloem sap with proteinaseK reduced high-molecular compounds while increasing fractions of low-molecular Cd-thiol complexes. Zinc-NA, Fe-NA and Cu-NA were identified in the phloem sap fraction of control plants by electrospray ionization time-of-flight MS, and the xylem sap contained Cu-NA.

Understanding the effects of lithium exposure on castor bean (Ricinus communis) plants, a potential bioindicator of lithium-contaminated areas.

As lithium (Li) stands out as a crucial component of batteries, the inappropriate disposal of electronic gadgets might drive Li pollution in environmentally sensitive environments, such as dumps, where castor bean (Ricinus communis) plant communities are usually found. The exposure to high Li concentration is potentially harmful to the environment and humans. Therefore, it is opportune to evaluate the potential of bioindicator species to monitor Li contamination. In this scenario, the present study assessed the effects of Li exposure on the development of castor bean plants exposed to lithium chloride at five Li dosages (0, 5, 10, 20, and 30 mg dm-3). Significant symptoms of phytotoxicity were observed at all doses. Li dosage exhibited increasing impairment effects on plant biometrics, such as stem diameter and the number of leaves, as well as on the SPAD index, nutritional balance, and biomass production. Our findings suggest castor bean as a potential model species for biomonitoring Li-contaminated areas.

Growth, tolerance efficiency and phytoremediation potential of Ricinus communis (L.) and Brassica juncea (L.) in salinity and drought affected cadmium contaminated soil.

We have previously reported that Ricinus communis (castor) is more tolerant to soil cadmium (Cd) and more efficient for Cd phytoremediation than Brassica juncea (Indian mustard) (Bauddh and Singh, 2012). In the present study, R. communis was found more tolerant to salinity and drought in presence of Cd and removed more Cd in a given time than Indian mustard. R. communis produced 23 and twelve folds higher biomass in terms of fresh weight and dry weight, respectively than that in B. juncea during three months when grown in Cd contaminated soil in presence of 100mM NaCl salinity and ten day water withdrawal based drought at 90 day after sowing (DAS). Castor plants showed stronger self-protection ability in form of proline bioaccumulation (r(2)=0.949) than Indian mustard (r(2)=0.932), whereas a lower r(2) for malondialdehyde (MDA) and total soluble protein in R. communis (r(2)=0.914 and r(2)=0.915, respectively) than that of B. juncea (r(2)=0.947 and r(2)=0.927, respectively) indicated a greater damage to cell membrane in Indian mustard during the multiple stress conditions. Though, the amount of Cd accumulated in the roots and shoots of Indian mustard was higher as per unit biomass than that in castor, total removal of the metal from soil was much higher in castor on per plant basis in the same period in presence of the stresses. R. communis accumulated about seventeen and 1.5 fold higher Cd in their roots and shoots, respectively than that of B. juncea in 90 DAS under the multiple stresses. Salinity alone enhanced Cd uptake, whereas drought stress reduced its uptake in both the plants.

Hydrogen peroxide induced oxidation of peroxisomal malate synthase and catalase.

Peroxisomes contain oxidases that produce H(2)O(2), which can result in protein oxidation. To test the vulnerability of peroxisomal proteins to oxidation in vivo the organelles were isolated from castor bean endosperm incubated with H(2)O(2). When peroxisomes were exposed to H(2)O(2)in vivo, the peroxisomal proteins exhibited an increase in carbonylation as detected in avidin blots of biotin hydrazide derivatized samples. Biotin-tagged peptides from trypsin digests of the proteins were analyzed by mass spectroscopy and compared to the masses of peptides from the same protein that had not been biotin-tagged and from proteins not exposed to excess H(2)O(2). H(2)O(2) exposure was found to increase the activity of catalase (CAT), and to increase the number of oxidized peptides found in CAT and malate synthase (MS). CAT had 10 peptides that were affected by in vivo exposure to H(2)O(2) and MS had 8. These sites of oxidation have definable locations within the proteins' structures.

Respiration-department uncoupler-stimulated ATPase activity in castor bean endosperm mitochondria and submitochondrial particles.

1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitchondrial particles has been studied. The rate of ATP hydrolysis catalyzed by intact mitochondria was slow and little enhanced by addition of uncouplers at the concentration required for uncoupling the oxidative phosphorylation. ATP-ase activity was stimulated at higher concentrations of uncouplers. 2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response. 3. The addition of succinate, NADH or ascorbate/N,N,N'-N'-tetramethyl-p-phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin. 4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide-p-trifluoromethoxyphenylhydrazone at 10-8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin. 5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity. 6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation.

Methyl jasmonate promotes the transient reduction of the levels of 2-Cys peroxiredoxin in Ricinus communis plants.

Jasmonates are signaling molecules that play a key role in the regulation of metabolic processes, reproduction and defense against insects and pathogens. This study investigated the effects of methyl jasmonate on the protein pattern of Ricinus communis plants and the activity of guaiacol peroxidase, an antioxidant enzyme. Methyl jasmonate treatment caused a transient reduction in guaiacol peroxidase activity. A similar response was observed for the levels of 2-Cys peroxiredoxin protein. Moreover, the levels of the small and large chains of Rubisco were also reduced. The transient reduction of the levels and activity of antioxidant enzymes could account for the increase in the levels of H2O2, an important signaling molecule in plant defense.

Glucose positions affect the phloem mobility of glucose-fipronil conjugates.

In our previous work, a glucose-fipronil (GTF) conjugate at the C-1 position was synthesized via click chemistry and a glucose moiety converted a non-phloem-mobile insecticide fipronil into a moderately phloem-mobile insecticide. In the present paper, fipronil was introduced into the C-2, C-3, C-4, and C-6 positions of glucose via click chemistry to obtain four new conjugates and to evaluate the effects of the different glucose isomers on phloem mobility. The phloem mobility of the four new synthetic conjugates and GTF was tested using the Ricinus seedling system. The results confirmed that conjugation of glucose at different positions has a significant influence on the phloem mobility of GTF conjugates.

Seeds' physicochemical traits and mucilage protection against aluminum effect during germination and root elongation as important factors in a biofuel seed crop (Ricinus communis).

We determined the length, volume, dry biomass, and density in seeds of five castor bean cultivars and verified notable physicochemical trait differences. Seeds were then subjected to different toxic aluminum (Al) concentrations to evaluate germination, relative root elongation, and the role of root apices' rhizosphere mucilage layer. Seeds' physicochemical traits were associated with Al toxicity responses, and the absence of Al in cotyledons near to the embryo was revealed by Al-hematoxylin staining, indicating that Al did not induce significant germination reduction rates between cultivars. However, in the more sensitive cultivar, Al was found around the embryo, contributing to subsequent growth inhibition. After this, to investigate the role of mucilage in Al tolerance, an assay was conducted using NH4Cl to remove root mucilage before or after exposure to different Al concentrations. Sequentially, the roots were stained with hematoxylin and a quantitative analysis of staining intensity was obtained. These results revealed the significant contribution of the mucilage layer to Al toxicity responses in castor bean seedlings. Root growth elongation under Al toxicity confirmed the role of the mucilage layer, which jointly indicated the differential Al tolerance between cultivars and an efficient Al-exclusion mechanism in the tolerant cultivar.

Environmental effects of nanosilver: impact on castor seed germination, seedling growth, and plant physiology.

Increasing use of nanoparticles in daily products is of great concern today, especially when their positive and negative impact on environment is not known. Hence, in current research, we have studied the impact of silver nanoparticle (AgNPs) and silver nitrate (AgNO3) application on seed germination, root, and shoot length of castor bean, Ricinus communis L. plant. Silver nanoparticles had no significant effects on seedling growth even at higher concentration of 4,000 mg L(-1), while the silver in bulk form as AgNO3 applied on the castor bean seeds inhibited the seed germination. Silver uptake in seedlings of the castor seeds on treatment with both the forms of silver was confirmed through atomic absorption spectroscopy studies. The silver nanoparticle and silver nitrate application to castor seeds also caused an enhanced enzymatic activity of ROS enzymes and phenolic content in castor seedlings. High-performance liquid chromatography analysis of individual phenols indicated enhanced content of parahydroxy benzoic acid. These kinds of studies are of great interest in order to unveil the movement and accumulation of nanoparticles in plant tissues for assessing future applications in the field or laboratory.

Simultaneous allergen inactivation and detoxification of castor bean cake by treatment with calcium compounds.

Ricinus communis L. is of great economic importance due to the oil extracted from its seeds. Castor oil has been used for pharmaceutical and industrial applications, as a lubricant or coating agent, as a component of plastic products, as a fungicide or in the synthesis of biodiesel fuels. After oil extraction, a castor cake with a large amount of protein is obtained. However, this by-product cannot be used as animal feed due to the presence of toxic (ricin) and allergenic (2S albumin) proteins. Here, we propose two processes for detoxification and allergen inactivation of the castor cake. In addition, we establish a biological test to detect ricin and validate these detoxification processes. In this test, Vero cells were treated with ricin, and cell death was assessed by cell counting and measurement of lactate dehydrogenase activity. The limit of detection of the Vero cell assay was 10 ng/mL using a concentration of 1.6 x 10(5) cells/well. Solid-state fermentation (SSF) and treatment with calcium compounds were used as cake detoxification processes. For SSF, Aspergillus niger was grown using a castor cake as a substrate, and this cake was analyzed after 24, 48, 72, and 96 h of SSF. Ricin was eliminated after 24 h of SSF treatment. The cake was treated with 4 or 8% Ca(OH)2 or CaO, and both the toxicity and the allergenic properties were entirely abolished. A by-product free of toxicity and allergens was obtained.

Assessing the tolerance of castor bean to Cd and Pb for phytoremediation purposes.

This study evaluated Cd and Pb accumulation by castor bean (Ricinus communis cv. Guarany) plants grown in nutrient solution, aiming to assess the plant's ability and tolerance to grow in Cd- and Pb-contaminated solutions for phytoremediation purposes. The plants were grown in individual pots containing Hoagland and Arnon's nutrient solution with increasing concentrations of Cd and Pb. The production of root and shoot dry matter and their contents of Cd, Pb, Ca, Mg, Cu, Fe, Mn, and Zn were evaluated in order to calculate the translocation and bioaccumulation factors, as well as toxicity of Cd and Pb. Cadmium caused severe symptoms of phytotoxicity in the plant's root and shoot, but no adverse effect was observed for Pb. Castor bean is an appropriate plant to be used as indicator plant for Cd and tolerante for Pb in contaminated solution and it can be potentially used for phytoremediation of contaminated areas.

Simultaneous allergen inactivation and detoxification of castor bean cake by treatment with calcium compounds

Ricinus communis L. is of great economic importance due to the oil extracted from its seeds. Castor oil has been used for pharmaceutical and industrial applications, as a lubricant or coating agent, as a component of plastic products, as a fungicide or in the synthesis of biodiesel fuels. After oil extraction, a castor cake with a large amount of protein is obtained. However, this by-product cannot be used as animal feed due to the presence of toxic (ricin) and allergenic (2S albumin) proteins. Here, we propose two processes for detoxification and allergen inactivation of the castor cake. In addition, we establish a biological test to detect ricin and validate these detoxification processes. In this test, Vero cells were treated with ricin, and cell death was assessed by cell counting and measurement of lactate dehydrogenase activity. The limit of detection of the Vero cell assay was 10 ng/mL using a concentration of 1.6 x 10(5) cells/well. Solid-state fermentation (SSF) and treatment with calcium compounds were used as cake detoxification processes. For SSF, Aspergillus niger was grown using a castor cake as a substrate, and this cake was analyzed after 24, 48, 72, and 96 h of SSF. Ricin was eliminated after 24 h of SSF treatment. The cake was treated with 4 or 8% Ca(OH)2 or CaO, and both the toxicity and the allergenic properties were entirely abolished. A by-product free of toxicity and allergens was obtained.

Adenosine stimulates anabolic metabolism in developing castor bean (Ricinus communis L.) cotyledons.

In previous experiments it was shown that Castor-bean (Ricinus communis) endosperm releases carbohydrates, amino acids and nucleoside derivatives, which are subsequently imported into the developing cotyledons (Kombrink and Beevers in Plant Physiol 73:370-376, 1983). To investigate the importance of the most prominent nucleoside adenosine for the metabolism of growing Ricinus seedlings, we supplied adenosine to cotyledons of 5-days-old seedlings after removal of the endosperm. This treatment led to a 16% increase in freshweight of intact seedlings within 16 h, compared to controls. Using detached cotyledons, we followed uptake of radiolabelled adenosine and identified 40% of label in solubles (mostly ATP and ADP), 46% incorporation in RNA and 2.5% in DNA, indicating a highly active salvage pathway. About 7% of freshly imported adenosine entered the phloem, which indicates a major function of adenosine for cotyledon metabolism. Import and conversion of adenosine improved the energy content of cotyledons as revealed by a substantially increased ATP/ADP ratio. This effect was accompanied by slight increases in respiratory activity, decreased levels of hexose phosphates and increased levels of fructose-1,6-bisphosphate and triose phosphates. These alterations indicate a stimulation of glycolytic flux by activation of phosphofructokinase, and accordingly we determined a higher activity of this enzyme. Furthermore the rate of [(14)C]-sucrose driven starch biosynthesis in developing castor-bean is significantly increased by feeding of adenosine. In conclusion, our data indicate that adenosine imported from mobilizing endosperm into developing castor-bean cotyledons fulfils an important function as it promotes anabolic reactions in this rapidly developing tissue.

The breakdown of lipid reserves in the endosperm of germinating castor beans.

1. Lipid extracts were obtained from castor-bean endosperm tissue at various times during germination and, after purification, the total lipid content was determined. Quantitative measurements of the triglyceride and phospholipid content together with the fatty acid composition were made. 2. The total lipid content of the endosperm rapidly decreased during germination; after 10 days less than 20% of the original weight of lipid remained. In contrast, the phospholipid content (initially less than 0.5% of the total lipid) increased slightly during this time. The fatty acid composition and the relative proportions of the triglyceride species of the total lipid extract remained constant during 10 days of germination. 3. Gibberellic acid (0.3 mM) markedly stimulated the rate of lipid breakdown but did not alter either the fatty acid composition or the relative proportion of triglyceride species. 4. The embryo had little effect on lipid metabolism in the endosperm tissue; only after 6 days of germination were differences observed in the rate of fat utilization in the presence and absence of the embryo.

Pyruvate metabolism in castor-bean mitochondria.

We report the isolation of mitochondria from the endosperm of castor beans (Ricinus communis). These mitochondria oxidized succinate, external NADH, malate and pyruvate with respiratory-control and ADP/O ratios consistent with those found previously with mitochondria from other plant sources. The mitochondria exhibited considerable sensitivity to the electron-transport-chain inhibitors antimycin A and cyanide when oxidizing succinate and external NADH. Pyruvate-dependent O2 uptake was relatively insensitive to these inhibitors, although the residual O2 uptake could be inhibited by salicylhydroxamic acid. We conclude that a cyanide-insensitive alternative terminal oxidase is functional in these mitochondria. However, electrons from the succinate dehydrogenase or external NADH dehydrogenase seem to have no access to this pathway. There is little interconnection between the salicylhydroxamic acid-sensitive and cyanide-sensitive pathways of electron transport. alpha-Cyanocinnamate and its analogues, compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and alpha-cyano-4-hydroxycinnamate, were all found to be potent non-competitive inhibitors of pyruvate oxidation in castor-bean mitochondria. The accumulation of pyruvate by castor-bean mitochondria was determined by using a silicone-oil-centrifugation technique. The accumulation was shown to observe Michaelis-Menten kinetics, with a Km for pyruvate of 0.10 mM and a Vmax. of 0.95 nmol/min per mg of mitochondrial protein. However, the observed rates of pyruvate accumulation were insufficient to account for the pyruvate oxidation rates found in the oxygen-electrode studies. We were able to demonstrate that this is due to the immediate export of the accumulated radiolabel in the form of malate and citrate. Compound UK5099 inhibited the accumulation of [2-14C]pyruvate by castor-bean mitochondria at concentrations similar to those required to inhibit pyruvate oxidation.

Kinetic characterization, stereoselectivity, and species selectivity of the inhibition of plant acetyl-CoA carboxylase by the aryloxyphenoxypropionic acid grass herbicides.

The selective grass herbicides diclofop, haloxyfop, and trifop were found to be potent reversible inhibitors of acetyl-CoA carboxylase from the susceptible species barley, corn, and wheat. Kis values with variable concentrations of acetyl-CoA ranged from 0.01 to 0.06 microM at pH 8.5 depending on the species of grass. Inhibition of the wheat enzyme by diclofop was noncompetitive versus acetyl-CoA with Kis less than Kii and noncompetitive versus MgATP and bicarbonate, but with Kis approximately equal to Kii. Since the apparent inhibition constant was most sensitive to the level of acetyl-CoA, these compounds probably interact with the transcarboxylase site rather than the biotin carboxylation site. With the wheat enzyme the Kis value for the R-(+)-enantiomer of trifop was 1.98 +/- 0.22 times lower than that of the racemic mixture. This confirms the stereoselectivity observed in the whole plant. The enzyme from tolerant broadleaf species (spinach and mung bean) was much less sensitive to these herbicides (Kis values varied from 16 to 515 microM). These data confirm that acetyl-CoA carboxylase is the site of action for the aryloxyphenoxypropionic acid herbicides and may explain their selectivity for monocotyledenous species.