Cloning and Functional Verification of Endogenous U6 Promoters for the Establishment of Efficient CRISPR/Cas9-Based Genome Editing in Castor (Ricinus communis).

Castor (Ricinus communis) seeds are rich in a type of hydroxy fatty acid called ricinoleic acid, which is in high demand for the production of plant-based plastics, lubricants, and hydraulic oils. However, the high content of ricin, a toxic protein, in these seeds has restricted further expansion in the area of castor cultivation. Therefore, the development of ricin-free castor is needed. Genome editing technology, although successfully applied in several plant species, is still in the developing stages in castor and awaits the identification of an endogenous U6 promoter with robust function. Here, we searched for U6 small nuclear RNA (snRNA) genes in the castor genome. This led to the identification of six U6 snRNA genes. The promoters of these U6 snRNA genes were cloned, and their function was examined in castor cells using the particle delivery method. The results showed that a U6 promoter length of approximately 300 bp from the transcription start site was sufficient to activate gene expression. This study provides insights into the endogenous castor U6 promoter sequences and outlines a method for verifying the function of U6 promoters in plants using the particle delivery system.

Genome-wide identification of core components of ABA signaling and transcriptome analysis reveals gene circuits involved in castor bean (Ricinus communis L.) response to drought.

Castor bean (Ricinus communis L.) can withstand long periods of water deficit and high temperatures, and therefore has been recognized as a drought-resistant plant species, allowing the study of gene networks involved in drought response and tolerance. The identification of genes networks related to drought response in this plant may yield important information in the characterization of molecular mechanisms correlating changes in the gene expression with the physiological adaptation processes. In this context, gene families related to abscisic acid (ABA) signaling play a crucial role in developmental and environmental adaptation processes of plants to drought stress. However, the families that function as the core components of ABA signaling, as well as genes networks related to drought response, are not well understood in castor bean. In this study 7 RcPYL, 63 RcPP2C, and 6 RcSnRK2 genes were identified in castor bean genome, which was further supported by chromosomal distribution, gene structure, evolutionary relationships, and conserved motif analyses. The castor bean general expression profile was investigated by RNAseq in root and leaf tissues in response to drought stress. These analyses allowed the identification of genes differentially expressed, including genes from the ABA signaling core, genes related to photosynthesis, cell wall, energy transduction, antioxidant response, and transcription factors. These analyses provide new insights into the core components of ABA signaling in castor bean, allow the identification of several molecular responses associated with the high physiological adaptation of castor bean to drought stress, and contribute to the identification of candidate genes for genetic improvement.

Regulation of capsule spine formation in castor.

Castor (Ricinus communis L.) is a dicotyledonous oilseed crop that can have either spineless or spiny capsules. Spines are protuberant structures that differ from thorns or prickles. The developmental regulatory mechanisms governing spine formation in castor or other plants have remained largely unknown. Herein, using map-based cloning in 2 independent F2 populations, F2-LYY5/DL01 and F2-LYY9/DL01, we identified the RcMYB106 (myb domain protein 106) transcription factor as a key regulator of capsule spine development in castor. Haplotype analyses demonstrated that either a 4,353-bp deletion in the promoter or a single nucleotide polymorphism leading to a premature stop codon in the RcMYB106 gene could cause the spineless capsule phenotype in castor. Results of our experiments indicated that RcMYB106 might target the downstream gene RcWIN1 (WAX INDUCER1), which encodes an ethylene response factor known to be involved in trichome formation in Arabidopsis (Arabidopsis thaliana) to control capsule spine development in castor. This hypothesis, however, remains to be further tested. Nevertheless, our study reveals a potential molecular regulatory mechanism underlying the spine capsule trait in a nonmodel plant species.

Substrate profiling of the Arabidopsis Ca2+-dependent protein kinase AtCPK4 and its Ricinus communis ortholog RcCDPK1.

AtCPK4 and AtCPK11 are Arabidopsis thaliana Ca2+-dependent protein kinase (CDPK) paralogs that have been reported to positively regulate abscisic acid (ABA) signal transduction by phosphorylating ABA-responsive transcription factor-4 (AtABF4). By contrast, RcCDPK1, their closest Ricinus communis ortholog, participates in the control of anaplerotic carbon flux in developing castor oil seeds by catalyzing inhibitory phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser451. LC-MS/MS revealed that AtCPK4 and RcCDPK1 transphosphorylated several common, conserved residues of AtABF4 and its castor ortholog, TRANSCRIPTION FACTOR RESPONSIBLE FOR ABA REGULATON. Arabidopsis atcpk4/atcpk11 mutants displayed an ABA-insensitive phenotype that corroborated the involvement of AtCPK4/11 in ABA signaling. A kinase-client assay was employed to identify additional AtCPK4/RcCDPK1 targets. Both CDPKs were separately incubated with a library of 2095 peptides representative of Arabidopsis protein phosphosites; five overlapping targets were identified including PLANT INTRACELLULAR RAS-GROUP-RELATED LEUCINE-RICH REPEAT PROTEIN-9 (AtPIRL9) and the E3-ubiquitin ligase ARABIDOPSIS TOXICOS EN LEVADURA 6 (AtATL6). AtPIRL9 and AtATL6 residues phosphorylated by AtCPK4/RcCDPK1 conformed to a CDPK recognition motif that was conserved amongst their respective orthologs. Collectively, this study provides evidence for novel AtCPK4/RcCDPK1 substrates, which may help to expand regulatory networks linked to Ca2+- and ABA-signaling, immune responses, and central carbon metabolism.

Response of sugar metabolism in the cotyledons and roots of Ricinus communis subjected to salt stress.

Ricinus communis is an important oilseed crop worldwide and is also considered one of the best potential plants for salt-affected soil improvement in northeast China. However, little is known about photosynthesis and carbohydrate metabolism in this plant, nor the distribution of carbohydrates in cotyledons and roots under salinity stress. In the present study, seedling growth, gas exchange parameters (PN , E, gs and Ci ), carbohydrate (fructose, sucrose, glucose, soluble sugar and starch) metabolism and related enzymes and genes were measured in Ricinus plants. Under salt stress, PN of cotyledons decreased significantly (P < 0.05), resulting in weak photosynthetic capacity. Furthermore, salt stress increased sucrose and glucose content in cotyledons, but decreased soluble sugar and starch content. However, sucrose increased and starch decreased in roots. This may be correlated with the increasing sugar metabolism under salinity, including notable changes in sugar-related enzyme activities (SPS, SuSy, α-amylase and ß-amylase) and gene expression of RcINV, RcSUS, RcAmY, RcBAM and RcGBE1. The results suggest that salinity reduces photosynthesis of cotyledons, alters carbohydrate allocation between cotyledons and roots and also promotes starch utilization in cotyledons and starch biosynthesis in roots, leading to a functional imbalance between cotyledons and roots. Together, these findings provide insights into the crucial role of sugar metabolism in improving salt-tolerance of Ricinus during the early seedling growth stage.

An Exploration of the Effect of the Kleier Model and Carrier-Mediated Theory to Design Phloem-Mobile Pesticides Based on Researching the N-Alkylated Derivatives of Phenazine-1-Carboxylic Acid-Glycine.

The Kleier model and Carrier-mediated theory are effective for molecularly designing pesticides with phloem mobility. However, the single Kleier model or Carrier-mediated theory cannot achieve a reliable explanation of the phloem mobility of all exogenous substances. A detailed investigation of the two models and the scope of their applications can provide a more accurate and highly efficient basis for the guidance of the design and development of phloem-mobile pesticides. In the present paper, a strategy using active ingredient-amino acid conjugates as mode compounds is developed based on Carrier-mediated theory. An N-alkylated amino acid is used to improve the pesticide's physicochemical properties following the Kleier model, thus allowing the conjugates to fall on the predicted and more accessible transportation region of phloem. Moreover, the influence of this movement on phloem is inspected by the Kleier model and Carrier-mediated theory. To verify this strategy, a series of N-alkylated phenazine-1-carboxylic acid-glycine compounds (PCA-Gly) were designed and synthesized. The results related to the castor bean seeds (R. communis L.) indicated that all the target compounds (4a−4f) had phloem mobility. The capacity for phloem mobility shows that N-alkylated glycine containing small substituents can significantly improve PCA phloem mobility, such as 4c(i-C3H7-N) > 4a(CH3-N) ≈ 4b(C2H5-N) > 4d (t-C4H9-N) > PCA-Gly > 4e(C6H5-N) > 4f(CH2COOH-N), with an oil−water partition coefficient between 1.2~2.5. In particular, compounds 4a(CH3-N), 4b(C2H5-N), and 4c(i-C3H7-N) present better phloem mobility, with the average concentrations in phloem sap of 14.62 µΜ, 13.98 µΜ, and 17.63 µΜ in the first 5 h, which are 8 to 10 times higher than PCA-Gly (1.71 µΜ). The results reveal that the Kleier model and Carrier-mediated theory play a guiding role in the design of phloem-mobile pesticides. However, the single Kleier model or Carrier-mediated theory are not entirely accurate. Still, there is a synergism between Carrier-mediated theory and the Kleier model for promoting the phloem transport of exogenous compounds. Therefore, we suggest the introduction of endogenous plant compounds as a promoiety to improve the phloem mobility of pesticides through Carrier-mediated theory. It is necessary to consider the improvement of physicochemical properties according to the Kleier model, which can contribute to a scientific theory for developing phloem-mobile pesticides.

Different acyl-CoA:diacylglycerol acyltransferases vary widely in function, and a targeted amino acid substitution enhances oil accumulation.

Triacylglycerols (TAGs) are the major component of plant storage lipids such as oils. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step of the Kennedy pathway, and is mainly responsible for plant oil accumulation. We previously found that the activity of Vernonia DGAT1 was distinctively higher than that of Arabidopsis and soybean DGAT1 in a yeast microsome assay. In this study, the DGAT1 cDNAs of Arabidopsis, Vernonia, soybean, and castor bean were introduced into Arabidopsis. All Vernonia DGAT1-expressing lines showed a significantly higher oil content (49% mean increase compared with the wild-type) followed by soybean and castor bean. Most Arabidopsis DGAT1-overexpressing lines did not show a significant increase. In addition to these four DGAT1 genes, sunflower, Jatropha, and sesame DGAT1 genes were introduced into a TAG biosynthesis-defective yeast mutant. In the yeast expression culture, DGAT1s from Arabidopsis, castor bean, and soybean only slightly increased the TAG content; however, DGAT1s from Vernonia, sunflower, Jatropha, and sesame increased TAG content >10-fold more than the former three DGAT1s. Three amino acid residues were characteristically common in the latter four DGAT1s. Using soybean DGAT1, these amino acid substitutions were created by site-directed mutagenesis and substantially increased the TAG content.

Free amino acids, carbon and nitrogen isotopic compositions responses to cadmium stress in two castor (Ricinus communis L.) species.

Cadmium (Cd) toxicity induce various disturbances in metabolic processes and impair plant establishment. The composition of carbon and nitrogen stable isotopes (δ13C and δ15N) and free amino acids (FAAs) can reflect the response of plants to environmental stress. In the present study, a solution culture experiment was carried out, and the secretion characteristics of FAAs as well as δ13C and δ15N were evaluated as indicative of the functional performance of two castor species (Zibo-3 and Zibo-9) under various Cd concentrations stress (0, 1, 2, and 5 mg L-1). The results indicated that: 1) The treatment of the plants with 5 mg L-1 of a Cd solution resulted in a significant decline of biomasses by 22.4% and 11.6% in Zibo-3 and Zibo-9, respectively, relative to controls; additionally, the accumulation levels for Cd in Zibo-9 were higher than those in Zibo-3, thus Zibo-9 showed higher tolerance and enrichment ability to Cd. 2) The exposure of castor to Cd treatments results in significant modifications in individual FAAs, suggesting a differential sensitivity of each biosynthetic pathway to this stress; however, a positive correlation was found between the accumulation of total FAAs and Cd treatment dosages; higher proportion of asparagine and glutamate in total amino acids for Zibo-9, and abundant secretion of arginine in Cd treated Zibo-9 may be associated with the higher Cd-tolerance and Cd-accumulation in Zibo-9. 3) Cd stress increased leaf δ13C and δ15N values regardless of the castor species; δ13C and δ15N could be used as monitoring tools for heavy metal stress in plants.

Autophosphorylation Inhibits RcCDPK1, a Dual-Specificity Kinase that Phosphorylates Bacterial-Type Phosphoenolpyruvate Carboxylase in Castor Oil Seeds.

Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated enzyme that plays a crucial anaplerotic role in central plant metabolism. Bacterial-type PEPC (BTPC) of developing castor oil seeds (COS) is highly expressed as a catalytic and regulatory subunit of a novel Class-2 PEPC heteromeric complex. Ricinus communis Ca2+-dependent protein kinase-1 (RcCDPK1) catalyzes in vivo inhibitory phosphorylation of COS BTPC at Ser451. Autokinase activity of recombinant RcCDPK1 was detected and 42 autophosphorylated Ser, Thr or Tyr residues were mapped via liquid chromatography-tandem mass spectrometry. Prior autophosphorylation markedly attenuated the ability of RcCDPK1 to transphosphorylate its BTPC substrate at Ser451. However, fully dephosphorylated RcCDPK1 rapidly autophosphorylated during the initial stages of a BTPC transphosphorylation assay. This suggests that Ca2+-dependent binding of dephospho-RcCDPK1 to BTPC may trigger a structural change that leads to rapid autophosphorylation and subsequent substrate transphosphorylation. Tyr30 was identified as an autophosphorylation site via LC-MS/MS and immunoblotting with a phosphosite-specific antibody. Tyr30 occurs at the junction of RcCDPK1's N-terminal variable (NTVD) and catalytic domains and is widely conserved in plant and protist CDPKs. Interestingly, a reduced rate and extent of BTPC transphosphorylation occurred with a RcCDPK1Y30F mutant. Prior research demonstrated that RcCDPK1's NTVD is essential for its Ca2+-dependent autophosphorylation or BTPC transphosphorylation activities but plays no role in target recognition. We propose that Tyr30 autophosphorylation facilitates a Ca2+-dependent interaction between the NTVD and Ca2+-activation domain that primes RcCDPK1 for transphosphorylating BTPC at Ser451. Our results provide insights into links between the post-translational control of COS anaplerosis, Ca2+-dependent signaling and the biological significance of RcCDPK1 autophosphorylation.

Characteristics of cadmium translocation and isotope fractionation in Ricinus communis seedlings: Effects from split/cut-root and limited nutrients.

Studying cadmium (Cd) transport in plants will improve the current understanding of Cd tolerance mechanisms. Due to the influence of analytical techniques, the application of Cd isotopes in plants is still in its early stages. Therefore, the relationships between Cd isotope fractionation and Cd translocation in plants remain unclear. In this study, we cultured Ricinus communis in hydroponic solutions during split/cut-root experiments and limited and infinite nutrient experiments. To understand the Cd transport process, the Cd2+ and other ion concentrations in different tissues (i.e., roots, stems, and leaves) and nutrient solutions, Cd isotope composition and the soluble protein in tissues were measured. The results showed that although significant effects were evident in the top leaves, the principal roots had less pronounced effects on Cd2+ translocation in the stems. Moreover, Cd underwent homolateral transport before it was translocated from the principal roots to the leaves on the side without Cd. It was apparent that the stems were responsible for translocating Cd2+ in plants. In addition, the continuous supply of high Cd2+ concentrations inhibited the growth of the top leaves, while in low Cd2+ concentrations, it was gradually transferred to the top leaves. Moreover, the tissues of R. communis were enriched with lighter Cd isotopes compared with the solutions. The clear differences between the Cd isotope fractionation of leaves under infinite and limited nutrient experiments may be attributed to plant growth and Cd uptake rates. This study provides important information for understanding Cd2+ translocation in R. communis and furthers our understanding of its tolerance and hyperaccumulation.

6-Benzylaminopurine Alleviates the Impact of Cu2+ Toxicity on Photosynthetic Performance of Ricinus communis L. Seedlings.

Copper (Cu) is an essential element involved in various metabolic processes in plants, but at concentrations above the threshold level, it becomes a potential stress factor. The effects of two different cytokinins, kinetin (KIN) and 6-benzylaminopurine (BAP), on chlorophyll a fluorescence parameters, stomatal responses and antioxidation mechanisms in castor (Ricinus communis L.) under Cu2+ toxicity was investigated. Ricinus communis plants were exposed to 80 and 160 µM CuSO4 added to the growth medium. Foliar spraying of 15 µM KIN and BAP was carried out on these seedlings. The application of these cytokinins enhanced the tissue water status, chlorophyll contents, stomatal opening and photosynthetic efficiency in the castor plants subjected to Cu2+ stress. The fluorescence parameters, such as Fm, Fv/Fo, Sm, photochemical and non-photochemical quantum yields, energy absorbed, energy trapped and electron transport per cross-sections, were more efficiently modulated by BAP application than KIN under Cu2+ toxicity. There was also effective alleviation of reactive oxygen species by enzymatic and non-enzymatic antioxidation systems, reducing the membrane lipid peroxidation, which brought about a relative enhancement in the membrane stability index. Of the various treatments, 80 µM CuSO4 + BAP recorded the highest increase in photosynthetic efficiency compared to other cytokinin treatments. Therefore, it can be concluded that BAP could effectively alleviate the detrimental effects of Cu2+toxicity in cotyledonary leaves of R. communis by effectively modulating stomatal responses and antioxidation mechanisms, thereby enhancing the photosynthetic apparatus' functioning.

Lupeol Accumulation Correlates with Auxin in the Epidermis of Castor.

Lupeol, a natural lupane-type pentacyclic triterpene, possesses various pharmacological properties, and its production attracts attention. Significant quantities of lupeol are deposited on the castor aerial organ surface and are easily extractable as a predominant wax constituent. Thus, castor might be considered as a potential bioreactor for the production of lupeol. The lupeol biosynthesis pathway is well known, but how it is regulated remains largely unknown. Among large numbers of castor cultivars, we targeted one accession line (337) with high levels of lupeol on its stem surface and low levels thereof on its hypocotyl surface, implicating that lupeol synthesis is differentially regulated in the two organs. To explore the underlying mechanisms, we did comparative transcriptome analysis of the first internode of 337 stem and the upper hypocotyl. Our results show that large amounts of auxin-related genes are differentially expressed in both parts, implying some possible interactions between auxin and lupeol production. We also found that several auxin-responsive cis-elements are present in promoter regions of HMGR and LUS genes encoding two key enzymes involved in lupeol production. Furthermore, auxin treatments apparently induced the expression levels of RcHMGR and RcLUS. Furthermore, we observed that auxin treatment significantly increased lupeol contents, whereas inhibiting auxin transport led to an opposite phenotype. Our study reveals some relationships between hormone activity and lupeol synthesis and might provide a promising way for improving lupeol yields in castor.

Green Synthesis, Characterization, Enzyme Inhibition, Antimicrobial Potential, and Cytotoxic Activity of Plant Mediated Silver Nanoparticle Using Ricinus communis Leaf and Root Extracts.

The need of non-toxic synthesis protocols for nanoparticles arises developing interest in biogenic approaches. The present project was focused on cost effective, environment congenial synthesis of Ag nanoparticles and their biological applications. Leaf and root extracts of Ricinus communis were used as a reducing and stabilizing agent in synthesis process. A Proposed mechanism in published literature suggested that Indole-3-acetic acid, l-valine, triethyl citrate, and quercetin-3-0-p-d-glucopyranoside phytoconstituents of Ricinus communis act as reducing and capping agents. The synthesized Ag NPs were characterized with a help X-ray diffractometer, Transmission electron microscopy, UV-Vis spectrophotometry and Fourier Transform Infrared Spectroscopy (FTIR). The XRD results inveterate the synthesis of pure nano size crystalline silver particles. The FTIR data revealed the possible functional groups of biomolecules involved in bio reduction and capping for efficient stabilization of silver nanoparticles. TEM analysis confirmed the almost spherical morphology of synthesized particles with mean size 29 and 38 nm for R-Ag-NPs (root) and L-Ag-NPs (leaf), respectively. The stability of synthesized nanoparticles was examined against heat and pH. It was observed that synthesized nanoparticles were stable up to 100 °C temperature and also showed stability in neutral, basic and slightly acidic medium (pH 05-06) for several months while below pH 5 were unstable. The synthesized silver nanoparticles had promising inhibition efficiency in multiple applications, including as bactericidal/fungicidal agents and Urease/Xanthine oxidase enzymes inhibitors. The cytotoxicity of synthesized nanoparticles shows that the concentration under 20 µg/mL were biologically compatible.

Ricin Antibodies' Neutralizing Capacity against Different Ricin Isoforms and Cultivars.

Ricin, a highly toxic protein from Ricinus communis, is considered a potential biowarfare agent. Despite the many data available, no specific treatment has yet been approved. Due to their ability to provide immediate protection, antibodies (Abs) are an approach of choice. However, their high specificity might compromise their capacity to protect against the different ricin isoforms (D and E) found in the different cultivars. In previous work, we have shown the neutralizing potential of different Abs (43RCA-G1 (anti ricin A-chain) and RB34 and RB37 (anti ricin B-chain)) against ricin D. In this study, we evaluated their protective capacity against both ricin isoforms. We show that: (i) RB34 and RB37 recognize exclusively ricin D, whereas 43RCA-G1 recognizes both isoforms, (ii) their neutralizing capacity in vitro varies depending on the cultivar, and (iii) there is a synergistic effect when combining RB34 and 43RCA-G1. This effect is also demonstrated in vivo in a mouse model of intranasal intoxication with ricin D/E (1:1), where approximately 60% and 40% of mice treated 0 and 6 h after intoxication, respectively, are protected. Our results highlight the importance of evaluating the effectiveness of the Abs against different ricin isoforms to identify the treatment with the broadest spectrum neutralizing effect.

Assisted phytostabilization of Pb-spiked soils amended with charcoal and banana compost and vegetated with Ricinus communis L. (Castor bean).

A greenhouse experiment was performed to elucidate the potency of Prosopis juliflora charcoal (PJC) and banana waste compost (BWC) to improve soil fertility and enhance plant growth rate. Plantlets of Ricinus communis were grown in 0, 400, and 800 mg kg-1 Pb-spiked soil ameliorated with P. juliflora charcoal and banana waste compost at 0, 5%, and 10% (w/w) for 60 days. PJC and BWC significantly (p < 0.05) increased plant growth parameters, that is, number of leaves, node number, plant height, and leaf diameter and reduced oxidative stress manifested by the lesser production of proline, hydrogen peroxide (H2O2), and malondialdehyde (MDA) with respect to control plants. Soil usage of PJC at 10% decreased the Pb accumulation by 61%, whereas BWC decreased Pb concentration in roots by 56% concerning control. Field emission scanning electron microscope (FE-SEM) coupled with energy-dispersive X-ray spectroscopy (EDS) showed high macro and microspores on the surface of charcoal while banana compost showed significant raise in the nutrient content (N, P, K, Zn, Ca, Fe, and Mg). Thermogravimetric (TG) and Fourier-transform infrared spectroscopy (FTIR) analysis of banana compost showed enhanced molar convolution of carbohydrate composites and nitrogen content. These findings pave a clear understanding that PJC and BWC are recalcitrant for Pb phytotoxicity and can also be used as nutrient-rich composites for increased crop production.

Effects of iron oxide nanoparticles on Fe and heavy metal accumulation in castor (Ricinus communis L.) plants and the soil aggregate.

In this study, the effects of iron (Fe) nanoparticles (NPs) on Fe and heavy metal accumulations by castor (Ricinus communis L.) plants were investigated. The castor cultivar was planted in the soil contaminated with Pb and Zn for 48 days with Fe2O3 NPs treatments. The Fe and heavy metal concentrations in the plant tissues, the plant tissues' ultrastructures, and the Fe and heavy metal distributions in the soil aggregate were analyzed. The results of this study indicate that there is a mutual promotion relationship between Fe and heavy metals (Pb and Zn). The scanning electron microscopy (SEM) revealed ultrastructural differences in the xylem and phloem with Fe2O3 NPs addition, and the presence of Fe2O3 NPs may influence the synthesis of starch granules in response to heavy metal stress. Based on the analysis of the soil aggregate, α-Fe2O3 NPs and γ-Fe2O3 NPs changed the size distribution of the soil aggregate, that is, the macro-aggregate and the clay fraction contents increased and the micro-aggregate content decreased. Moreover, in the different size fractions of the soil aggregate, Fe2O3 NPs can change the Zn and Fe enrichment and migration between the macro-aggregate and clay fractions, and there is a synergistic effect between the Fe and Zn migration. In addition, in the castor organs (roots and shoots), the Zn accumulation was mainly determined by the Zn concentration of the macro-aggregate fraction, while the Fe accumulation was mainly determined by the Fe concentration of the micro-aggregate fraction. Overall, these direct observations help improve our understanding of the migration and transport characteristics of Fe and heavy metals in soil-plant systems when Fe nanoparticles are added to metal-contaminated soils.

Variations of dissolved organic matter and Cu fractions in rhizosphere soil induced by the root activities of castor bean.

The root soil interaction affects metal bioavailability in the rhizosphere, thus impacting the uptake and accumulation of metals by plants. In this study, a greenhouse experiment using a root-bag technique for castor bean plants was conducted to determine the i) rhizosphere effect on the fractions of Cu, and ii) the characteristics of dissolved organic matter (DOM) in the rhizosphere soil. Results showed that the Cu concentration in the leaves, stems, and roots was 15.41, 6.71, and 47.85 mg kg-1, respectively, in the control and reached up to 96.5, 254.9, and 3204 mg kg-1 in Cu400 treatment, respectively. After cultivating castor bean plants, the concentration of acid exchangeable Cu in rhizosphere soil was higher than that in the bulk soil for the same Cu addition, whereas the concentrations of reducible Cu, oxidizable Cu, and residual Cu in the rhizosphere soil were all lower than those in the bulk soil, respectively. In comparison to the bulk soil, the pH decreased while the total nitrogen and total carbon concentrations both increased in the rhizosphere soil. Moreover, the concentrations of total low molecular weight organic acids (LMWOAs) and total amino acids in the rhizosphere soil of the Cu treatments increased by between 15.18% to 47.17% and 36.35%-200%, respectively with respect to the control. The less complex DOM with a high LMWOAs concentration in the rhizosphere soil shifted the soil Cu from a relative stable fraction to available fractions.

Phytoremediation of soil heavy metals (Cd and Zn) by castor seedlings: Tolerance, accumulation and subcellular distribution.

Cd and Zn pollution was observed to often occur simultaneously in soils. However, previous studies focused on single heavy metal instead of Cd and Zn combined pollution. Castor (Ricinus communis) is considered to have great potential for contaminated soil remediation. The resistance of castor seedlings to heavy metals and the mechanism behind it remain unknown. In this study, the tolerance and accumulation ability of castor seedlings to Cd and Zn were investigated, and the accumulation mechanism involving the subcellular distribution in different tissues was further explored. The results on biomass and chlorophyll revealed that castor seedlings have good tolerance to the pollution with 0-5 mg/kg Cd and 380 mg/kg Zn, while not to the heavy pollution with 25 mg/kg Cd and 380 mg/kg Zn. The maximum accumulation concentrations of Cd and Zn, 175.3 mg Cd/kg and 386.8 mg/kg Zn, appeared in castor seedling root instead of stem and leaf, indicating that root played a significant part in accumulating Zn and Cd. The relative low dosage of Cd (0-5 mg/kg) promoted the accumulation of Zn in the subcellular component, while high dosage (25 mg/kg) inhibited the accumulation of Zn. In subcellular accumulation and distribution of castor seedlings, Cd (27.1%-69.4%) and Zn (39.6%-66.6%) in the cell wall was the highest. With the increase of Cd addition, the accumulation of Cd increased in cell wall while decreased in organelle and soluble fraction. Hydroxyl, amino, amides and carboxyl functional groups on cell wall might provided the main binding sites for Cd and Zn.

Coupling phytoremediation efficiency and detoxification to assess the role of P in the Cu tolerant Ricinus communis L.

Phosphorous (P) fertilization is an important agronomic practice, but its role in enhancing phytoremediation efficacy and mediating detoxification has rarely been reported in environmental remediation studies. In this study, a pot experiment was undertaken to assess: firstly, the effect of P on phytoextraction of Cu by Ricinus communis L.; secondly, the potential mechanisms by differentiating the effects of the plant from that of P fertilizer (Ca(H2PO4)2); and thirdly, the role of P in physiological detoxification. Results showed that the application of P fertilizer significantly (p ≤ 0.05) increased the plant biomass as well as the Cu concentrations in plant tissues. This enhanced the phytoremediation efficiency represented by the total Cu extraction (up to 121.3 µg Cu plant-1). Phosphorous (P) fertilizer led to a negligible decline in soil pH (0.2 units) but significantly (p ≤ 0.05) reduced the concentrations of soil available in Cu and Fe, due to the formation of insoluble Cu/Fe-phosphate precipitates. Nevertheless, P fertilizer still improved the accumulation and extraction of Cu by R. communis, most likely attributable to the Fe-deficiency induced by applied P fertilizer. Moreover, the application of P fertilizer revealed a significant reduction in MDA, and a profound (p ≤ 0.05) elevation in the amount of photosynthetic pigments, GSH and AsA, along with the enhanced activities of antioxidative enzymes (SOD, POD, and CAT). In this way, Cu toxicity was alleviated. P fertilizers not only enhance the phytoremediation efficiency of Cu-contaminated soils by R. communis, but they also facilitate detoxification, which improves our understanding of the role of P in phytoremediation technologies.

Castor Stearoyl-ACP Desaturase Can Synthesize a Vicinal Diol by Dioxygenase Chemistry.

In previous work, we identified a triple mutant of the castor (Ricinus communis) stearoyl-Acyl Carrier Protein desaturase (T117R/G188L/D280K) that, in addition to introducing a double bond into stearate to produce oleate, performed an additional round of oxidation to convert oleate to a trans allylic alcohol acid. To determine the contributions of each mutation, in this work we generated individual castor desaturase mutants carrying residue changes corresponding to those in the triple mutant and investigated their catalytic activities. We observed that T117R, and to a lesser extent D280K, accumulated a novel product, namely erythro-9,10-dihydroxystearate, that we identified via its methyl ester through gas chromatography-mass spectrometry and comparison with authentic standards. The use of 18O2 labeling showed that the oxygens of both hydroxyl moieties originate from molecular oxygen rather than water. Incubation with an equimolar mixture of 18O2 and 16O2 demonstrated that both hydroxyl oxygens originate from a single molecule of O2, proving the product is the result of dioxygenase catalysis. Using prolonged incubation, we discovered that wild-type castor desaturase is also capable of forming erythro-9,10-dihydroxystearate, which presents a likely explanation for its accumulation to ∼0.7% in castor oil, the biosynthetic origin of which had remained enigmatic for decades. In summary, the findings presented here expand the documented constellation of di-iron enzyme catalysis to include a dioxygenase reactivity in which an unactivated alkene is converted to a vicinal diol.