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.

Scaling of leaf area with biomass in trees reconsidered: constant metabolically active sapwood volume per unit leaf area with height growth.

Hypoallometric (slope<1) scaling between metabolic rate and body mass is often regarded as near-universal across organisms. However, there are compelling reasons to question hypoallometric scaling in woody plants, where metabolic rate is directly proportional to leaf area. This leaf area must provide carbon to the volume of the metabolically active sapwood (VMASW). Within populations of a species, variants in which VMASW increases per unit leaf area with height growth (e.g. ⅔ or ¾ scaling) would have proportionally less carbon for growth and reproduction as they grow taller. Therefore, selection should favor individuals in which, as they grow taller, leaf area scales isometrically with shoot VMASW (slope=1). Using tetrazolium staining, we measured total VMASW and total leaf area (LAtot) across 22 individuals of Ricinus communis and confirmed that leaf area scales isometrically with VMASW, and that VMASW is much smaller than total sapwood volume. With the potential of the LAtot-VMASW relationship to shape factors as diverse as the crown area-stem diameter relationship, conduit diameter scaling, reproductive output, and drought-induced mortality, our work indicates that the notion that sapwood increases per unit leaf area with height growth requires revision.

Deciphering the Molecular Mechanism of the Intermediate Secondary Growth and Internode Elongation of the Castor Bean (Ricinus communis L.) by the Combined Analysis of the Transcriptome and Metabolome.

The length of internodes plays a crucial role in determining the height of the castor plant (Ricinus communis L.). However, the specific mechanisms underlying internode elongation, particularly in the main stem of the castor plant, remain uncertain. To further investigate this, we conducted a study focusing on the internode tissue of the dwarf castor variety 071113, comparing it with the control high-stalk Zhuansihao. Our study included a cytological observation, physiological measurement, transcriptome sequencing, and metabolic determination. Our integrated findings reveal that the dwarf variety 071113 undergoes an earlier lignification development in the main stem and has a more active lignin synthesis pathway during internode intermediate development. In addition, the dwarf variety exhibited lower levels of the plant hormone indole-3-acetic acid (IAA), which had an impact on the development process. Furthermore, we identified specific enzymes and regulators that were enriched in the pathways of the cell cycle, auxin signal transduction, and secondary cell wall synthesis. Using these findings, we developed a model that explained the intermediate secondary growth observed in castor internode elongation and enhanced our comprehension of the dwarfing mechanism of the 071113 variety. This research provides a theoretical groundwork for the future breeding of dwarf castor varieties.

Pollution pressure drives microbial assemblages that improve the phytoremediation potential of heavy metals by Ricinus communis.

Due to the rapid expansion of industrial activity, soil pollution has intensified. Plants growing in these polluted areas have developed a rhizobiome uniquely and specially adapted to thrive in such environments. However, it remains uncertain whether pollution acts as a sufficiently selective force to shape the rhizobiome, and whether these adaptations endure over time, potentially aiding in long-term phytoremediation. Therefore, in the present study, we aimed to compare whether the microbiome associated with roots from plants germinated in polluted riverbanks will improve the phytoremediation of Cd and Pb under mesocosm experiments compared with plants germinating in a greenhouse. The experimental design was a factorial 2 × 2, i.e., the origin of the plant and the presence or absence of 100 mg/L of Cd and 1000 mg/L of Pb. Our results showed that plants germinated in polluted riverbanks have the capacity to accumulate twice the amount of Pb and Cd during mesocosm experiments. The metagenomic analysis showed that plants from the river exposed to heavy metals at the end of mesocosm experiments were rich in Rhizobium sp. AC44/96 and Enterobacter sp. EA-1, Enterobacter soli, Pantoea rwandensis, Pantoea endophytica. In addition, those plants were uniquely associated with Rhizobium grahamii, which likely contributed to the differences in the levels of phytoremediation achieved. Furthermore, the functional analysis revealed an augmented functional potential related to hormones, metallothioneins, dismutases, and reductases; meanwhile, the plants germinated in the greenhouse showed an unspecific strategy to exceed heavy metal stress. In conclusion, pollution pressure drives stable microbial assemblages, which could be used in future phytostabilization and phytoremediation experiments.

Phosphatidylcholine:diacylglycerol cholinephosphotransferase's unique regulation of castor bean oil quality.

Castor bean (Ricinus communis) seed oil (triacylglycerol [TAG]) is composed of ∼90% of the industrially important ricinoleoyl (12-hydroxy-9-octadecenoyl) groups. Here, phosphatidylcholine (PC):diacylglycerol (DAG) cholinephosphotransferase (PDCT) from castor bean was biochemically characterized and compared with camelina (Camelina sativa) PDCT. DAGs with ricinoleoyl groups were poorly used by Camelina PDCT, and their presence inhibited the utilization of DAG with "common" acyl groups. In contrast, castor PDCT utilized DAG with ricinoleoyl groups similarly to DAG with common acyl groups and showed a 10-fold selectivity for DAG with one ricinoleoyl group over DAG with two ricinoleoyl groups. Castor DAG acyltransferase2 specificities and selectivities toward different DAG and acyl-CoA species were assessed and shown to not acylate DAG without ricinoleoyl groups in the presence of ricinoleoyl-containing DAG. Eighty-five percent of the DAG species in microsomal membranes prepared from developing castor endosperm lacked ricinoleoyl groups. Most of these species were predicted to be derived from PC, which had been formed by PDCT in exchange with DAG with one ricinoleoyl group. A scheme of the function of PDCT in castor endosperm is proposed where one ricinoleoyl group from de novo-synthesized DAG is selectivity transferred to PC. Nonricinoleate DAG is formed and ricinoleoyl groups entering PC are re-used either in de novo synthesis of DAG with two ricinoleoyl groups or in direct synthesis of triricinoleoyl TAG by PDAT. The PC-derived DAG is not used in TAG synthesis but is proposed to serve as a substrate in membrane lipid biosynthesis during oil deposition.

Molecular genetics, seed morphology and fatty acids diversity in castor (Ricinus communis L., Euphorbiaceae) Iranian populations.

BACKGROUND: Castor (Ricinus communis L.) seeds contain a large amount of oil that has several biological activities. In the current research, phytogeographic distribution, seed morphological characteristics, molecular genetic diversity and structure, and fatty acid composition were investigated in nine Iranian castor populations. METHODS AND RESULTS: The cetyltrimethylammonium bromide (CTAB) protocol was used to extract the nuclear genomes. These were later amplified using 13 SCoT molecular primers. The phytogeographic distribution was determined based on the Zohary mapping, GC apparatus determined the fatty acid composition of the seeds. GenAlex, STRUCTURE, GenoDive, PopGene, and PopART software were used for the statistical analyzes. On phytogeographic mapping, the harvested populations belonged to different districts of the Euro-Siberian and Irano-Turanian regions (Holarctic kingdom). Most of the quantitative morphological traits of the seeds differed significantly (P ≤ 0.05) between the populations. The AMOVA test demonstrated a large proportion of significant genetic diversity assigned among populations, which were approved by some estimated parameters of genetic diversity such as Nm, Ht, Hs, and Gst. Nei's genetic distance and structure analysis confirmed the existence of two main genotype groups and some intermediates. However, there was no isolation by distance between the genotypes. Unsaturated fatty acids were detected as the main component of seed oil with linoleic and ricinoleic acids. Significant correlations were detected between the main fatty acids of seed oil with seed morphological traits, geographic distance and the geographic parameters of habitats. According to the composition of the seed fatty acids, four chemotypes groups were detected. CONCLUSIONS: The classification patterns of the populations based on molecular genetic data, fatty acid composition, and phytogeographic mapping were not identical. These findings indicated that Iranian castor populations had unusual seed fatty acid composition which strongly depended on habitat geographic factors and seed morphological traits. However, the identified chemotypes and genotypes can be used in future breeding programs.

Evaluation of molluscicidal activity on Biomphalaria glabrata (Say, 1818) and phytochemical characterization of hydroalcoholic extract of leaves of Ricinus communis L. (EUPHORBIACEAE).

Schistosomiasis is a parasitic infection of great prevalence worldwide, affecting 250 million people in 78 countries. Faced with this problem, studies that seek to analyze molluscicidal activity from plant extracts have stood out. The present work aimed to obtain the phytochemical characterization and investigate the molluscicidal activity in the hydroalcoholic extract of Ricinus communis leaves on Biomphalaria glabrata. The hydroalcoholic extract was prepared by macerated with solvent ethanol P.A 96%, followed by filtration and concentration in rotary evaporator. Next, five groups of snails with 10 animals each, one being the negative control group, were submitted to treatments with four concentrations of 25, 50, 75 and 100 mg/L of hydroalcoholic extract of R. communis. The parameters mortality, physiological and behavioral aspects of mollusks were analyzed during 96h. The chemical characterization of the extract was performed by high-performance liquid chromatography coupled to mass spectrometry (LC-MS). Chemical characterization revealed the presence of tannins, flavonoids and ricinin alkaloid, but under the conditions analyzed, the presence of saponins was not observed. There was no significant molluscicidal activity of the extract. However, a greater influence was observed in the diet, in addition to the motility and physiological state of the snails (alteration of cephalopodal mass and oviposition). The toxicity test was performed with Artemia salina and no toxicity was observed for this microcrustacean. It is expected that the results obtained contribute to the fight against the expansion of schistosomiasis and that they make room for other studies that investigate the molluscicidal action of plant extracts.

Effect of phosphorus fertilizer on phytoextraction using Ricinus communis L. in Cu and Cd co-contaminated soil.

Mining activities have led to Cu and Cd contaminated of surrounding agricultural soil. To decrease the Cu and Cd accumulation in crops, the Ricinus communis L. (castor) has been used for phytoremediation. A pot experiment was served to investigate the effect of phosphate fertilizer (Ca(H2PO4)2) on the growth and Cu/Cd uptake of castor in contaminated soil. The results showed that the application of P fertilizer improved the leaf cell morphology, decreased the malonaldehyde (MDA) content of castor leaves, and increased the plant biomass (28.2-34.2%). Besides, phosphate fertilizer still facilitated accumulation Cu and Cd by castor. The addition of phosphate fertilizer increased the contents of Cu in the root of castor, improved the bioconcentration factor (BCF) of Cu, and observably enhanced the accumulation of Cu (up to 201 µg/plant) in castor. Applying phosphorus increased the percentage of residual Cd, diminished the percentage of acid extractable Cd in soil, and the accumulation of Cd in castor was not significantly increased. These results suggest that phosphorus alleviated the stress of heavy metals on castor leaves and enhanced the accumulation of Cu and Cd in castor by promoting the growth of castor.

Applying phosphate fertilizer effectively alleviated the stress of heavy metals on castor and significantly increased the biomass of castor.The reason of applying phosphorus enhanced the castor uptake Cu and Cd was that phosphorus promoted the growth of castor.Applying phosphorus markedly increased the percentage of residual Cd but diminished the percentage of acid extractable Cd in soil.

Analysis of the Physiological Response and Reactive Oxygen Species in Castor Oil Plant (Ricinus Communis) in the Phytoremediation Processes with Plant Growth Promoter Bacteria (PGPB).

In the phytoremediation processes of mine tailings with Ricinus communis inoculated with PGPB, it was found that the Serratia K120 bacterium favors the translocation of Al, As, Cu, Pb, Cr, Cd, and Mn to the aerial part of the plant, with a significant difference (p < 0.05) concerning for the control. The bioaccumulation factor (BF) was > 1 in Al with all the bacteria, Pb, Serratia K120, Fe, Pantoea 113, Cu, Pb, Cd, Mn in Serratia MC119 and Serratia K120, Fe and As in Serratia K120 and Pantoea 134, indicating that Ricinus communis inoculated with PGPB functions as a hyper accumulating plant. The PGPB help to reduce the stress in the plants generated by the heavy metals, decreasing the H2O2, and increasing the activity of the enzymes SOD, CAT, APX, POX, and GR, for which the bacteria Serratia K120 and Pantoea 113 can be used as bioinoculants to favor phytoremediation processes.

Physaria fendleri and Ricinus communis lecithin:cholesterol acyltransferase-like phospholipases selectively cleave hydroxy acyl chains from phosphatidylcholine.

Production of hydroxy fatty acids (HFAs) in transgenic crops represents a promising strategy to meet our demands for specialized plant oils with industrial applications. The expression of Ricinus communis (castor) OLEATE 12-HYDROXYLASE (RcFAH12) in Arabidopsis has resulted in only limited accumulation of HFAs in seeds, which probably results from inefficient transfer of HFAs from their site of synthesis (phosphatidylcholine; PC) to triacylglycerol (TAG), especially at the sn-1/3 positions of TAG. Phospholipase As (PLAs) may be directly involved in the liberation of HFAs from PC, but the functions of their over-expression in HFA accumulation and distribution at TAG in transgenic plants have not been well studied. In this work, the functions of lecithin:cholesterol acyltransferase-like PLAs (LCAT-PLAs) in HFA biosynthesis were characterized. The LCAT-PLAs were shown to exhibit homology to LCAT and mammalian lysosomal PLA2 , and to contain a conserved and functional Ser/His/Asp catalytic triad. In vitro assays revealed that LCAT-PLAs from the HFA-accumulating plant species Physaria fendleri (PfLCAT-PLA) and castor (RcLCAT-PLA) could cleave acyl chains at both the sn-1 and sn-2 positions of PC, and displayed substrate selectivity towards sn-2-ricinoleoyl-PC over sn-2-oleoyl-PC. Furthermore, co-expression of RcFAH12 with PfLCAT-PLA or RcLCAT-PLA, but not Arabidopsis AtLCAT-PLA, resulted in increased occupation of HFA at the sn-1/3 positions of TAG as well as small but insignificant increases in HFA levels in Arabidopsis seeds compared with RcFAH12 expression alone. Therefore, PfLCAT-PLA and RcLCAT-PLA may contribute to HFA turnover on PC, and represent potential candidates for engineering the production of unusual fatty acids in crops.

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.

Reactivity Analysis of New Multivalent Electrophilic Probes for Affinity Labeling of Carbohydrate Binding Proteins.

We have designed and synthesized six different multivalent electrophiles as carbohydrate affinity labeling probes. Evaluation of the reactivity of the electrophiles against peanut agglutinin (PNA) and Ricinus communis agglutinin (RCA) showed that p- and m-aryl sulfonyl fluoride are effective protein reactive groups that label carbohydrate binding lectins in a ligand-dependent fashion at a nanomolar probe concentration. Analysis of the selectivity of affinity labeling in the presence of excess BSA as a nonspecific protein indicated that m-arylsulfonyl fluoride is a more selective protein-reactive group, albeit with attenuated reactivity. Further analysis showed that the labeling efficiency of the multivalent electrophilic probes can be improved by employing reaction conditions involving 25 °C instead of typically employed 4 °C. Both isomers of arylsulfonyl fluoride groups together represent promising affinity labels for target identification studies that could serve as more efficient alternatives to photoreactive groups.

Phloem turgor is maintained during severe drought in Ricinus communis.

The phloem is a key player in whole plant functioning-transporting carbon from sites of production to sites of demand-and is likely influenced by drought due to its dependence on water for generating pressure-driven bulk flow transport. Yet, phloem functioning during drought remains largely unknown due to a lack of experimental studies. Here, we use a phloem-bleeding species, Ricinus communis, to investigate phloem loss-of-function in the context of leaf physiological processes, the mechanisms of phloem turgor maintenance during drought, and the role of turgor in phloem loss-of-function. We found that the solute concentration in the phloem sap doubled over the drought, which allowed phloem turgor to be maintained past the point at which leaves have reached permanent stomatal closure. We also found that phloem turgor did not decline before bleeding ceased, which suggests that phloem bleeding ceassation (interpreted as the cessation of transport) occurred when the phloem still had turgor. In sum, our findings highlight the robustness of phloem functioning, with important implications for forecasting whole-plant carbon dynamics and drought-induced tree mortality.

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.

The effects of plant growth substances on the oil content and fatty acid composition of Ricinus communis L.: an in vitro study.

BACKGROUND: Ricinus communis L. (castor bean) is valued for its oil and the performance of oil is closely related to its fatty acid composition. Thus, producing oil in vitro with favored fatty acid profiles is a promising research area and may also offer industrial opportunities. MATERIAL AND METHOD: In line with this, the total amount of oil and the fatty acid composition of the samples, which were endosperm and calli obtained by treatment of various doses of plant growth regulators were determined. RESULTS: Results showed that the type and amount of the plant growth regulator used in the media affect the fatty acid composition. In detail, the biggest change was shown by Indole-3-Acetic Acid (IAA), in general, using the plant growth regulators at 5 mg L-1, instead of 20 mg L-1, was found to have induced larger differentiations. The effect of a natural plant growth regulator (IAA) on fatty acid profiles was bigger than the synthetic ones (NAA, 1-Naphthaleneacetic acid, and 2,4 D, 2,4-Dichlorophenoxyacetic acid). The media containing 5 mg L-1 of NAA, 20 mg L-1 of NAA, 20 mg L-1 of 2,4 D, or 5 mg L-1 of 2,4 D gave similar results.

Alleviating lead-induced phytotoxicity and enhancing the phytoremediation of castor bean (Ricinus communis L.) by glutathione application: new insights into the mechanisms regulating antioxidants, gas exchange and lead uptake.

Heavy metals pollution represents a serious issue for cultivable lands and ultimately threatens the worldwide food security. Lead (Pb) is a menacing metal which induces toxicity in plants and humans. Lead toxicity reduces the photosynthesis in plants, resulting in the reduction of plant growth and biomass. The excessive concentration of Pb in soil accumulates in plants body and enters into food chain, resulting in health hazards in humans. The phytoremediation is eco-friendly and cost-efficient technique to clean up the polluted soils. However, to the best of our Knowledge, there are very few reports addressing the enhancement of the phytoremediation potential of castor bean plants. Therefore, the present study aimed to investigate the potential role of glutathione (GSH), as a promising plant growth regulator, in enhancing the lead stress tolerance and phytoremediation potential of castor bean plants grown under lead stress conditions. The results indicated that Pb stress reduced the growth, biomass, chlorophyll pigments and gas exchange attributes of castor bean plants, causing oxidative damage in plants. Pb stress induced the oxidative stress markers and activities of antioxidant enzymes. On the other hand, the application of GSH reduced oxidative stress markers, but enhanced the growth, biomass, photosynthetic pigments, gas exchange attributes, Pb accumulation and antioxidant enzymes activities of lead-stressed castor bean plants. Both Pb uptake and Pb accumulation were increased by increasing concentrations of Pb in a dose-additive manner. However, at high dose of exogenous GSH (25 mg L-1) further enhancements were recorded in the Pb uptake in shoot by 48% and in root by 46%; Pb accumulation was further enhanced in shoot by 98% and in root by 101% in comparison with the respective control where no GSH was applied. Taken together, the findings revealed the promising role of GSH in enhancing the lead stress tolerance and phytoremediation potential of castor bean (Ricinus communis) plants cultivated in Pb-polluted soils through regulating leaf gas exchange, antioxidants machinery, and metal uptake.

The excessive concentration of Lead (Pb) in soil accumulates in plants body and enters into food chain, resulting in health hazards in humans. Phytoremediation is eco-friendly and cost-efficient technique to clean up the polluted soils. However, to the best of our knowledge, there are very few reports addressing the enhancement of the phytoremediation potential of castor bean plants. Therefore, the novelty of this research is that this research studied the potential role of glutathione (GSH), as a promising plant growth regulator, in enhancing the lead stress tolerance and phytoremediation potential of castor bean plants grown under lead stress conditions. The results indicated that Pb stress reduced the growth, biomass, chlorophyll pigments and gas exchange attributes of castor bean plants, causing oxidative damage in plants. Pb stress induced the oxidative stress markers and activities of antioxidant enzymes. On the other hand, the application of GSH reduced oxidative stress markers, but enhanced the growth, biomass, photosynthetic pigments, gas exchange attributes, Pb accumulation and antioxidant enzymes activities of lead-stressed castor bean plants. Taken together, the findings revealed the promising role of GSH in enhancing the lead stress tolerance and phytoremediation potential of castor bean plants cultivated in lead-polluted soils.

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.

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.

Castor LPCAT and PDAT1A Act in Concert to Promote Transacylation of Hydroxy-Fatty Acid onto Triacylglycerol.

Oilseeds produce abundant triacylglycerol (TAG) during seed maturation to fuel the establishment of photoautotrophism in the subsequent generation. Commonly, TAG contains 18-carbon polyunsaturated fatty acids (FA), but plants also produce oils with unique chemical properties highly desirable for industrial processes. Unfortunately, plants that produce such oils are poorly suited to agronomic exploitation, leading to a desire to reconstitute novel oil biosynthesis in crop plants. Here, we studied the production and incorporation of hydroxy-fatty acids (HFA) onto TAG in Arabidopsis (Arabidopsis thaliana) plants expressing the castor (Ricinus communis) FAH12 hydroxylase. One factor limiting HFA accumulation in these plants is the inefficient removal of HFA from the site of synthesis on phosphatidylcholine (PC). In Arabidopsis, lysophosphatidic acid acyltransferase (LPCAT) cycles FA to and from PC for modification. We reasoned that the castor LPCAT (RcLPCAT) would preferentially remove HFA from PC, resulting in greater incorporation onto TAG. However, expressing RcLPCAT in Arabidopsis expressing FAH12 alone (line CL37) or together with castor acyl:coenzyme A:diacylglycerol acyltransferase2 reduced HFA and total oil yield. Detailed analysis indicated that RcLPCAT reduced the removal of HFA from PC, possibly by competing with the endogenous LPCAT isozymes. Significantly, coexpressing RcLPCAT with castor phospholipid:diacylglycerol acyltransferase increased novel FA and total oil contents by transferring HFA from PC to diacylglycerol. Our results demonstrate that a detailed understanding is required to engineer modified FA production in oilseeds and suggest that phospholipase A2 enzymes rather than LPCAT mediate the highly efficient removal of HFA from PC in castor seeds.

Detection of ricin activity and structure by using novel galactose-terminated magnetic bead extraction coupled with mass spectrometric detection.

Ricin is a toxic protein derived from the castor bean plant (Ricinus communis) and has potential for bioterrorism or criminal use. Therefore, sensitive and rapid analytical methods are needed for its confirmatory detection in environmental samples. Our laboratory previously reported on the development of a confirmatory method to detect ricin involving antibody capture of ricin followed by mass spectrometric detection of ricin's enzymatic activity and of tryptic fragments unique to ricin. Here, we describe a novel ricin capture method of magnetic beads coated with 4-aminophenyl-1-thiol-ß-galactopyranoside, using ricin's lectin characteristics. The assay has been adapted for use on a simple, benchtop MALDI-TOF MS mass spectrometer common in clinical microbiology laboratories. Validation of the novel assay includes establishment of a limit of detection, and an examination of assay selectivity. The limit of detection of the enzymatic activity method is 8 ng/mL and 500 ng/mL for the confirmatory tryptic fragment assay. The assay is highly selective with no cross-reactivity from near neighbors and highly specific with a panel of 19 cultivars all testing positive. Additionally, there were no interferences found during testing of a panel of white powders. This allows for a confirmatory detection method for ricin in laboratories lacking expensive, sophisticated mass spectrometers.