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1.  Draft genome sequence of the ricin-producing oilseed castor bean 
Nature biotechnology  2010;28(9):951-956.
Castor bean (Ricinus communis) is an oil crop that belongs to the spurge (Euphorbiaceae) family. Its seeds are the source of castor oil, used for the production of high-quality lubricants due to its high proportion of the unusual fatty acid ricinoleic acid. Castor bean seeds also produce ricin, a highly toxic ribosome inactivating protein, making castor bean relevant for biosafety. We report here the 4.6X draft genome sequence of castor bean, representing the first reported Euphorbiaceae genome sequence. Our analysis shows that most key castor oil metabolism genes are single-copy while the ricin gene family is larger than previously thought. Comparative genomics analysis suggests the presence of an ancient hexaploidization event that is conserved across the dicotyledonous lineage.
doi:10.1038/nbt.1674
PMCID: PMC2945230  PMID: 20729833
2.  Genome-wide survey and expression profiles of the AP2/ERF family in castor bean (Ricinus communis L.) 
BMC Genomics  2013;14(1):785.
Background
The AP2/ERF transcription factor, one of the largest gene families in plants, plays a crucial role in the regulation of growth and development, metabolism, and responses to biotic and abiotic stresses. Castor bean (Ricinus communis L., Euphobiaceae) is one of most important non-edible oilseed crops and its seed oil is broadly used for industrial applications. The available genome provides a great chance to identify and characterize the global information on AP2/ERF transcription factors in castor bean, which might provide insights in understanding the molecular basis of the AP2/ERF family in castor bean.
Results
A total of 114 AP2/ERF transcription factors were identified based on the genome in castor bean. According to the number of the AP2/ERF domain, the conserved amino acid residues within AP2/ERF domain, the conserved motifs and gene organization in structure, and phylogenetical analysis, the identified 114 AP2/ERF transcription factors were characterized. Global expression profiles among different tissues using high-throughput sequencing of digital gene expression profiles (DGEs) displayed diverse expression patterns that may provide basic information in understanding the function of the AP2/ERF gene family in castor bean.
Conclusions
The current study is the first report on identification and characterization of the AP2/ERF transcription factors based on the genome of castor bean in the family Euphobiaceae. Results obtained from this study provide valuable information in understanding the molecular basis of the AP2/ERF family in castor bean.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-14-785) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-14-785
PMCID: PMC4046667  PMID: 24225250
3.  Gene-based microsatellites for cassava (Manihot esculenta Crantz): prevalence, polymorphisms, and cross-taxa utility 
BMC Plant Biology  2009;9:118.
Background
Cassava (Manihot esculenta Crantz), a starchy root crop grown in tropical and subtropical climates, is the sixth most important crop in the world after wheat, rice, maize, potato and barley. The repertoire of simple sequence repeat (SSR) markers for cassava is limited and warrants a need for a larger number of polymorphic SSRs for germplasm characterization and breeding applications.
Results
A total of 846 putative microsatellites were identified in silico from an 8,577 cassava unigene set with an average density of one SSR every 7 kb. One hundred and ninety-two candidate SSRs were screened for polymorphism among a panel of cassava cultivars from Africa, Latin America and Asia, four wild Manihot species as well as two other important taxa in the Euphorbiaceae, leafy spurge (Euphorbia esula) and castor bean (Ricinus communis). Of 168 markers with clean amplification products, 124 (73.8%) displayed polymorphism based on high resolution agarose gels. Of 85 EST-SSR markers screened, 80 (94.1%) amplified alleles from one or more wild species (M epruinosa, M glaziovii, M brachyandra, M tripartita) whereas 13 (15.3%) amplified alleles from castor bean and 9 (10.6%) amplified alleles from leafy spurge; hence nearly all markers were transferable to wild relatives of M esculenta while only a fraction was transferable to the more distantly related taxa. In a subset of 20 EST-SSRs assessed by fluorescence-based genotyping the number of alleles per locus ranged from 2 to 10 with an average of 4.55 per locus. These markers had a polymorphism information content (PIC) from 0.19 to 0.75 with an average value of 0.55 and showed genetic relationships consistent with existing information on these genotypes.
Conclusion
A set of 124 new, unique polymorphic EST-SSRs was developed and characterized which extends the repertoire of SSR markers for cultivated cassava and its wild relatives. The markers show high PIC values and therefore will be useful for cultivar identification, taxonomic studies, and genetic mapping. The study further shows that mining ESTs is a highly efficient strategy for polymorphism detection within the cultivated cassava gene pool.
doi:10.1186/1471-2229-9-118
PMCID: PMC2758884  PMID: 19747391
4.  Identification of Critical Amino Acids in the IgE Epitopes of Ric c 1 and Ric c 3 and the Application of Glutamic Acid as an IgE Blocker 
PLoS ONE  2011;6(6):e21455.
Background
The allergenicity of Ricinus communis L. (castor bean, Euphorbiaceae) is associated with components of its seeds and pollen. Castor bean allergy has been described not only in laboratory workers, but also in personnel working in oil processing mills, fertilizer retail, the upholstery industry and other industrial fields. In the present study, we describe the critical amino acids in the IgE-binding epitopes in Ric c 1 and Ric c 3, two major allergens of R. communis. In addition, we also investigate the cross-reactivity between castor bean and some air and food allergen extracts commonly used in allergy diagnosis.
Methodology/Principal Findings
The IgE reactivity of human sera from atopic patients was screened by immune-dot blot against castor bean allergens. Allergenic activity was evaluated in vitro using a rat mast cell activation assay and by ELISA. Cross-reactivity was observed between castor bean allergens and extracts from shrimp, fish, gluten, wheat, soybean, peanut, corn, house dust, tobacco and airborne fungal allergens. We observed that treatment of rat and human sera (from atopic patients) with glutamic acid reduced the IgE-epitope interaction.
Conclusions/Significance
The identification of glutamic acid residues with critical roles in IgE-binding to Ric c 3 and Ric c 1 support the potential use of free amino acids in allergy treatment.
doi:10.1371/journal.pone.0021455
PMCID: PMC3124516  PMID: 21738671
5.  CastorDB: a comprehensive knowledge base for Ricinus communis 
BMC Research Notes  2011;4:356.
Background
Ricinus communis is an industrially important non-edible oil seed crop, native to tropical and subtropical regions of the world. Although, R. communis genome was assembled in 4X draft by JCVI, and is predicted to contain 31,221 proteins, the function of most of the genes remains to be elucidated. A large amount of information of different aspects of the biology of R. communis is available, but most of the data are scattered one not easily accessible. Therefore a comprehensive resource on Castor, Castor DB, is required to facilitate research on this important plant.
Findings
CastorDB is a specialized and comprehensive database for the oil seed plant R. communis, integrating information from several diverse resources. CastorDB contains information on gene and protein sequences, gene expression and gene ontology annotation of protein sequences obtained from a variety of repositories, as primary data. In addition, computational analysis was used to predict cellular localization, domains, pathways, protein-protein interactions, sumoylation sites and biochemical properties and has been included as derived data. This database has an intuitive user interface that prompts the user to explore various possible information resources available on a given gene or a protein.
Conclusion
CastorDB provides a user friendly comprehensive resource on castor with particular emphasis on its genome, transcriptome, and proteome and on protein domains, pathways, protein localization, presence of sumoylation sites, expression data and protein interacting partners.
doi:10.1186/1756-0500-4-356
PMCID: PMC3184282  PMID: 21914200
6.  Transcriptome-Wide Identification and Characterization of MicroRNAs from Castor Bean (Ricinus communis L.) 
PLoS ONE  2013;8(7):e69995.
Background
MicroRNAs (miRNAs) are endogenously encoded small RNAs that post-transcriptionally regulate gene expression and play essential roles in numerous developmental and physiological processes. Currently, little information on the transcriptome and tissue-specific expression of miRNAs is available in the model non-edible oilseed crop castor bean (Ricinus communis L.), one of the most important non-edible oilseed crops cultivated worldwide. Recent advances in sequencing technologies have allowed the identification of conserved and novel miRNAs in many plant species. Here, we used high-throughput sequencing technologies to identify and characterize the miRNAs in castor bean.
Results
Five small RNA libraries were constructed for deep sequencing from root tips, leaves, developing seeds (at the initial stage, seed1; and at the fast oil accumulation stage, seed2) and endosperms in castor bean. High-throughput sequencing generated a large number of sequence reads of small RNAs in this study. In total, 86 conserved miRNAs were identified, including 63 known and 23 newly identified. Sixteen miRNA isoform variants in length were found from the conserved miRNAs of castor bean. MiRNAs displayed diverse organ-specific expression levels among five libraries. Combined with criteria for miRNA annotation and a RT-PCR approach, 72 novel miRNAs and their potential precursors were annotated and 20 miRNAs newly identified were validated. In addition, new target candidates for miRNAs newly identified in this study were proposed.
Conclusions
The current study presents the first high-throughput small RNA sequencing study performed in castor bean to identify its miRNA population. It characterizes and increases the number of miRNAs and their isoforms identified in castor bean. The miRNA expression analysis provides a foundation for understanding castor bean miRNA organ-specific expression patterns. The present study offers an expanded picture of miRNAs for castor bean and other members in the family Euphorbiaceae.
doi:10.1371/journal.pone.0069995
PMCID: PMC3722108  PMID: 23894571
7.  Intercropping for Management of Insect Pests of Castor, Ricinus communis, in the Semi—Arid Tropics of India 
Intercropping is one of the important cultural practices in pest management and is based on the principle of reducing insect pests by increasing the diversity of an ecosystem. On—farm experiments were conducted in villages of semi—arid tropical (SAT) India to identify the appropriate combination of castor (Ricinus communis L.) (Malpighiales: Euphorbiaceae) and intercropping in relation to pest incidence. The diversity created by introducing cluster bean, cowpea, black gram, or groundnut as intercrops in castor (1:2 ratio proportions) resulted in reduction of incidence of insect pests, namely semilooper (Achaea janata L.), leaf hopper (Empoasca flavescens Fabricius), and shoot and capsule borer (Conogethes punctiferalis Guenee). A buildup of natural enemies (Microplitis, coccinellids, and spiders) of the major pests of castor was also observed in these intercropping systems and resulted in the reduction of insect pests. Further, these systems were more efficient agronomically and economically, and were thus more profitable than a castor monocrop.
doi:10.1673/031.012.1401
PMCID: PMC3467089  PMID: 22934569
cultural practices; ecosystem diversity; natural enemies; agronomical evaluation; gross margin
8.  Castor Bean Organelle Genome Sequencing and Worldwide Genetic Diversity Analysis 
PLoS ONE  2011;6(7):e21743.
Castor bean is an important oil-producing plant in the Euphorbiaceae family. Its high-quality oil contains up to 90% of the unusual fatty acid ricinoleate, which has many industrial and medical applications. Castor bean seeds also contain ricin, a highly toxic Type 2 ribosome-inactivating protein, which has gained relevance in recent years due to biosafety concerns. In order to gain knowledge on global genetic diversity in castor bean and to ultimately help the development of breeding and forensic tools, we carried out an extensive chloroplast sequence diversity analysis. Taking advantage of the recently published genome sequence of castor bean, we assembled the chloroplast and mitochondrion genomes extracting selected reads from the available whole genome shotgun reads. Using the chloroplast reference genome we used the methylation filtration technique to readily obtain draft genome sequences of 7 geographically and genetically diverse castor bean accessions. These sequence data were used to identify single nucleotide polymorphism markers and phylogenetic analysis resulted in the identification of two major clades that were not apparent in previous population genetic studies using genetic markers derived from nuclear DNA. Two distinct sub-clades could be defined within each major clade and large-scale genotyping of castor bean populations worldwide confirmed previously observed low levels of genetic diversity and showed a broad geographic distribution of each sub-clade.
doi:10.1371/journal.pone.0021743
PMCID: PMC3131294  PMID: 21750729
9.  Homologous electron transport components fail to increase fatty acid hydroxylation in transgenic Arabidopsis thaliana  
F1000Research  2013;2:203.
Ricinoleic acid, a hydroxylated fatty acid (HFA) present in castor ( Ricinus communis) seeds, is an important industrial commodity used in products ranging from inks and paints to polymers and fuels. However, due to the deadly toxin ricin and allergens also present in castor, it would be advantageous to produce ricinoleic acid in a different agricultural crop. Unfortunately, repeated efforts at heterologous expression of the castor fatty acid hydroxylase (RcFAH12) in the model plant Arabidopsis thaliana have produced only 17-19% HFA in the seed triacylglycerols (TAG), whereas castor seeds accumulate up to 90% ricinoleic acid in the endosperm TAG. RcFAH12 requires an electron supply from NADH:cytochrome b5 reductase (CBR1) and cytochrome b5 (Cb5) to synthesize ricinoleic acid. Previously, our laboratory found a mutation in the Arabidopsis CBR1 gene, cbr1-1, that caused an 85% decrease in HFA levels in the RcFAH12 Arabidopsis line. These results raise the possibility that electron supply to the heterologous RcFAH12 may limit the production of HFA. Therefore, we hypothesized that by heterologously expressing RcCb5, the reductant supply to RcFAH12 would be improved and lead to increased HFA accumulation in Arabidopsis seeds. Contrary to this proposal, heterologous expression of the top three RcCb5 candidates did not increase HFA accumulation. Furthermore, coexpression of RcCBR1 and RcCb5 in RcFAH12 Arabidopsis also did not increase in HFA levels compared to the parental lines. These results demonstrate that the Arabidopsis electron transfer system is supplying sufficient reductant to RcFAH12 and that there must be other bottlenecks limiting the accumulation of HFA.
doi:10.12688/f1000research.2-203.v1
PMCID: PMC3893003  PMID: 24555099
10.  Homologous electron transport components fail to increase fatty acid hydroxylation in transgenic Arabidopsis thaliana  
F1000Research  2013;2:203.
Ricinoleic acid, a hydroxylated fatty acid (HFA) present in castor ( Ricinus communis) seeds, is an important industrial commodity used in products ranging from inks and paints to polymers and fuels. However, due to the deadly toxin ricin and allergens also present in castor, it would be advantageous to produce ricinoleic acid in a different agricultural crop. Unfortunately, repeated efforts at heterologous expression of the castor fatty acid hydroxylase (RcFAH12) in the model plant Arabidopsis thaliana have produced only 17-19% HFA in the seed triacylglycerols (TAG), whereas castor seeds accumulate up to 90% ricinoleic acid in the endosperm TAG. RcFAH12 requires an electron supply from NADH:cytochrome b5 reductase (CBR1) and cytochrome b5 (Cb5) to synthesize ricinoleic acid. Previously, our laboratory found a mutation in the Arabidopsis CBR1 gene, cbr1-1, that caused an 85% decrease in HFA levels in the RcFAH12 Arabidopsis line. These results raise the possibility that electron supply to the heterologous RcFAH12 may limit the production of HFA. Therefore, we hypothesized that by heterologously expressing RcCb5, the reductant supply to RcFAH12 would be improved and lead to increased HFA accumulation in Arabidopsis seeds. Contrary to this proposal, heterologous expression of the top three RcCb5 candidates did not increase HFA accumulation. Furthermore, coexpression of RcCBR1 and RcCb5 in RcFAH12 Arabidopsis also did not increase in HFA levels compared to the parental lines. These results demonstrate that the Arabidopsis electron transfer system is supplying sufficient reductant to RcFAH12 and that there must be other bottlenecks limiting the accumulation of HFA.
doi:10.12688/f1000research.2-203.v2
PMCID: PMC3893003  PMID: 24555099
11.  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 × 105 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.
doi:10.1590/S0100-879X2012007500132
PMCID: PMC3854158  PMID: 22911344
Ricinus communis; 2S albumin; Ricin; Biodiesel fuel; Solid-state fermentation; Vero cells
12.  Tissue-Specific Whole Transcriptome Sequencing in Castor, Directed at Understanding Triacylglycerol Lipid Biosynthetic Pathways 
PLoS ONE  2012;7(2):e30100.
Background
Storage triacylglycerols in castor bean seeds are enriched in the hydroxylated fatty acid ricinoleate. Extensive tissue-specific RNA-Seq transcriptome and lipid analysis will help identify components important for its biosynthesis.
Methodology/Findings
Storage triacylglycerols (TAGs) in the endosperm of developing castor (Ricinus communis) seeds are highly enriched in ricinoleic acid (18:1-OH). We have analysed neutral lipid fractions from other castor tissues using TLC, GLC and mass spectrometry. Cotyledons, like the endosperm, contain high levels of 18:1-OH in TAG. Pollen and male developing flowers accumulate TAG but do not contain 18:1-OH and leaves do not contain TAG or 18:1-OH. Analysis of acyl-CoAs in developing endosperm shows that ricinoleoyl-CoA is not the dominant acyl-CoA, indicating that either metabolic channelling or enzyme substrate selectivity are important in the synthesis of tri-ricinolein in this tissue. RNA-Seq transcriptomic analysis, using Illumina sequencing by synthesis technology, has been performed on mRNA isolated from two stages of developing seeds, germinating seeds, leaf and pollen-producing male flowers in order to identify differences in lipid-metabolic pathways and enzyme isoforms which could be important in the biosynthesis of TAG enriched in 18:1-OH. This study gives comprehensive coverage of gene expression in a variety of different castor tissues. The potential role of differentially expressed genes is discussed against a background of proteins identified in the endoplasmic reticulum, which is the site of TAG biosynthesis, and transgenic studies aimed at increasing the ricinoleic acid content of TAG.
Conclusions/Significance
Several of the genes identified in this tissue-specific whole transcriptome study have been used in transgenic plant research aimed at increasing the level of ricinoleic acid in TAG. New candidate genes have been identified which might further improve the level of ricinoleic acid in transgenic crops.
doi:10.1371/journal.pone.0030100
PMCID: PMC3272049  PMID: 22319559
13.  Repertoire of SSRs in the Castor Bean Genome and Their Utilization in Genetic Diversity Analysis in Jatropha curcas 
Castor bean and Jatropha contain seed oil of industrial importance, share taxonomical and biochemical similarities, which can be explored for identifying SSRs in the whole genome sequence of castor bean and utilized in Jatropha curcas. Whole genome analysis of castor bean identified 5,80,986 SSRs with a frequency of 1 per 680 bp. Genomic distribution of SSRs revealed that 27% were present in the non-genic region whereas 73% were also present in the putative genic regions with 26% in 5′UTRs, 25% in introns, 16% in 3′UTRs and 6% in the exons. Dinucleotide repeats were more frequent in introns, 5′UTRs and 3′UTRs whereas trinucleotide repeats were predominant in the exons. The transferability of randomly selected 302 SSRs, from castor bean to 49 J. curcas genotypes and 8 Jatropha species other than J. curcas, showed that 211 (∼70%) amplified on Jatropha out of which 7.58% showed polymorphisms in J. curcas genotypes and 12.32% in Jatropha species. The higher rate of transferability of SSR markers from castor bean to Jatropha coupled with a good level of PIC (polymorphic information content) value (0.2 in J. curcas genotypes and 0.6 in Jatropha species) suggested that SSRs would be useful in germplasm analysis, linkage mapping, diversity studies and phylogenetic relationships, and so forth, in J. curcas as well as other Jatropha species.
doi:10.1155/2011/286089
PMCID: PMC3115337  PMID: 21687555
14.  Conservation and divergence of microRNAs and their functions in Euphorbiaceous plants 
Nucleic Acids Research  2009;38(3):981-995.
MicroRNAs (miRNAs) are ∼21 nt non-coding RNAs which regulate post-transcriptional gene expression. miRNAs are key regulators of nearly all essential biological processes. Aiming at understanding miRNA’s functions in Euphorbiaceae, a large flowering plant family, we performed a genome-scale systematic study of miRNAs in Euphorbiaceae, by combining computational prediction and experimental analysis to overcome the difficulty of lack of genomes for most Euphorbiaceous species. Specifically, we predicted 85 conserved miRNAs in 23 families in the Castor bean (Ricinus communis), and experimentally verified and characterized 58 (68.2%) of the 85 miRNAs in at least one of four Euphorbiaceous species, the Castor bean, the Cassava (Manihot esculenta), the Rubber tree (Hevea brasiliensis) and the Jatropha (Jatropha curcas) during normal seedling development. To elucidate their function in stress response, we verified and profiled 48 (56.5%) of the 85 miRNAs under cold and drought stresses as well as during the processes of stress recovery. The results revealed some species- and condition-specific miRNA expression patterns. Finally, we predicted 258 miRNA:target partners, and identified the cleavage sites of six out of ten miRNA targets by a modified 5′ RACE. This study produced the first collection of miRNAs and their targets in Euphorbiaceae. Our results revealed wide conservation of many miRNAs and diverse functions in Euphorbiaceous plants during seedling growth and in response to abiotic stresses.
doi:10.1093/nar/gkp1035
PMCID: PMC2817462  PMID: 19942686
15.  Single nucleotide polymorphisms for assessing genetic diversity in castor bean (Ricinus communis) 
BMC Plant Biology  2010;10:13.
Background
Castor bean (Ricinus communis) is an agricultural crop and garden ornamental that is widely cultivated and has been introduced worldwide. Understanding population structure and the distribution of castor bean cultivars has been challenging because of limited genetic variability. We analyzed the population genetics of R. communis in a worldwide collection of plants from germplasm and from naturalized populations in Florida, U.S. To assess genetic diversity we conducted survey sequencing of the genomes of seven diverse cultivars and compared the data to a reference genome assembly of a widespread cultivar (Hale). We determined the population genetic structure of 676 samples using single nucleotide polymorphisms (SNPs) at 48 loci.
Results
Bayesian clustering indicated five main groups worldwide and a repeated pattern of mixed genotypes in most countries. High levels of population differentiation occurred between most populations but this structure was not geographically based. Most molecular variance occurred within populations (74%) followed by 22% among populations, and 4% among continents. Samples from naturalized populations in Florida indicated significant population structuring consistent with local demes. There was significant population differentiation for 56 of 78 comparisons in Florida (pairwise population ϕPT values, p < 0.01).
Conclusion
Low levels of genetic diversity and mixing of genotypes have led to minimal geographic structuring of castor bean populations worldwide. Relatively few lineages occur and these are widely distributed. Our approach of determining population genetic structure using SNPs from genome-wide comparisons constitutes a framework for high-throughput analyses of genetic diversity in plants, particularly in species with limited genetic diversity.
doi:10.1186/1471-2229-10-13
PMCID: PMC2832895  PMID: 20082707
16.  An analysis of expressed sequence tags of developing castor endosperm using a full-length cDNA library 
BMC Plant Biology  2007;7:42.
Background
Castor seeds are a major source for ricinoleate, an important industrial raw material. Genomics studies of castor plant will provide critical information for understanding seed metabolism, for effectively engineering ricinoleate production in transgenic oilseeds, or for genetically improving castor plants by eliminating toxic and allergic proteins in seeds.
Results
Full-length cDNAs are useful resources in annotating genes and in providing functional analysis of genes and their products. We constructed a full-length cDNA library from developing castor endosperm, and obtained 4,720 ESTs from 5'-ends of the cDNA clones representing 1,908 unique sequences. The most abundant transcripts are genes encoding storage proteins, ricin, agglutinin and oleosins. Several other sequences are also very numerous, including two acidic triacylglycerol lipases, and the oleate hydroxylase (FAH12) gene that is responsible for ricinoleate biosynthesis. The role(s) of the lipases in developing castor seeds are not clear, and co-expressing of a lipase and the FAH12 did not result in significant changes in hydroxy fatty acid accumulation in transgenic Arabidopsis seeds. Only one oleate desaturase (FAD2) gene was identified in our cDNA sequences. Sequence and functional analyses of the castor FAD2 were carried out since it had not been characterized previously. Overexpression of castor FAD2 in a FAH12-expressing Arabidopsis line resulted in decreased accumulation of hydroxy fatty acids in transgenic seeds.
Conclusion
Our results suggest that transcriptional regulation of FAD2 and FAH12 genes maybe one of the mechanisms that contribute to a high level of ricinoleate accumulation in castor endosperm. The full-length cDNA library will be used to search for additional genes that affect ricinoleate accumulation in seed oils. Our EST sequences will also be useful to annotate the castor genome, which whole sequence is being generated by shotgun sequencing at the Institute for Genome Research (TIGR).
doi:10.1186/1471-2229-7-42
PMCID: PMC1950504  PMID: 17672910
17.  Potential of Trap Crops for Integrated Management of the Tropical Armyworm, Spodoptera litura in Tobacco 
The tropical armyworm, Spodoptera litura (F.) (Lepidoptera: Noctuidae), is an important pest of tobacco, Nicotiana tabacum L. (Solanales: Solanaceae), in South China that is becoming increasingly resistant to pesticides. Six potential trap crops were evaluated to control S. litura on tobacco. Castor bean, Ricinus communis L. (Malpighiales: Euphorbiaceae), and taro, Colocasia esculenta (L.) Schott (Alismatales: Araceae), hosted significantly more S. litura than peanut, Arachis hypogaea L. (Fabales: Fabaceae), sweet potato, Ipomoea batata Lam. (Solanales: Convolvulaceae) or tobacoo in a greenhouse trial, and tobacco field plots with taro rows hosted significantly fewer S. litura than those with rows of other trap crops or without trap crops, provided the taro was in a fast-growing stage. When these crops were grown along with eggplant, Solanum melongena L. (Solanales: Solanaceae), and soybean, Glycines max L. (Fabales: Fabaceae), in separate plots in a randomized matrix, tobacco plots hosted more S. litura than the other crop plots early in the season, but late in the season, taro plots hosted significantly more S. litura than tobacco, soybean, sweet potato, peanut or eggplant plots. In addition, higher rates of S. litura parasitism by Microplitis prodeniae Rao and Chandry (Hymenoptera: Bracondidae) and Campoletis chlorideae Uchida (Ichnumonidae) were observed in taro plots compared to other crop plots. Although taro was an effective trap crop for managing S. litura on tobacco, it did not attract S. litura in the seedling stage, indicating that taro should either be planted 20–30 days before tobacco, or alternative control methods should be employed during the seedling stage.
doi:10.1673/031.010.11701
PMCID: PMC3016987  PMID: 20874598
Colocasia esculenta; cultural control; trap crop; oviposition preference; attraction
18.  Effect of the Extracts of the Spiderflower, Cleome arabica, on Feeding and Survival of Larvae of the Cotton Leafworm, Spodoptera littoralis 
Aqueous and organic (hexane, chloroform, and methanol) extracts of siliquae, stems and leaves, and seeds of Cleome arabica L. (Brassicales: Capparidaceae) were evaluated in the laboratory for their antifeeding and insecticidal effect on larvae of the cotton leafworm, Spodoptera littoralis (Boisduval) (Lepidoptera, Noctuidae), using a leaf dipping bioassay with castor bean, Ricinus communis L. (Malpighiales: Euphorbiaceae), leaf discs. The polar extracts caused significant mortality. At the highest dose, C. arabica extracts exhibited significant antifeeding and phagostimulating activities against S. littoralis larvae. Under no-choice conditions, the methanol extract of siliquae was the most active, and the antifeedant index calculated over 24 hr for 3rd instar larvae varied significantly from 16 to 37%. Using nutritional indices, it was established that there was a significant decrease in growth rate concomitant with a reduction in consumption. These results suggest the presence of anti-feeding and/or toxic substances in the extracts that may be useful in developing bio-insecticides based on C. arabica extracts for use in integrated pest management of leafworm and other agricultural pests.
doi:10.1673/031.013.6101
PMCID: PMC3740824  PMID: 23906290
antifeeding; bio-inesceticides; botanicals extracts; nutritional indices; toxicity
19.  Ectopic overexpression of castor bean LEAFY COTYLEDON2 (LEC2) in Arabidopsis triggers the expression of genes that encode regulators of seed maturation and oil body proteins in vegetative tissues☆ 
FEBS Open Bio  2013;4:25-32.
The LEAFY COTYLEDON2 (LEC2) gene plays critically important regulatory roles during both early and late embryonic development. Here, we report the identification of the LEC2 gene from the castor bean plant (Ricinus communis), and characterize the effects of its overexpression on gene regulation and lipid metabolism in transgenic Arabidopsis plants. LEC2 exists as a single-copy gene in castor bean, is expressed predominantly in embryos, and encodes a protein with a conserved B3 domain, but different N- and C-terminal domains to those found in LEC2 from Arabidopsis. Ectopic overexpression of LEC2 from castor bean under the control of the cauliflower mosaic virus (CaMV) 35S promoter in Arabidopsis plants induces the accumulation of transcripts that encodes five major transcription factors (the LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON1-LIKE (L1L), FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE 3 (ABI3) transcripts for seed maturation, and WRINKELED1 (WRI1) transcripts for fatty acid biosynthesis), as well as OLEOSIN transcripts for the formation of oil bodies in vegetative tissues. Transgenic Arabidopsis plants that express the LEC2 gene from castor bean show a range of dose-dependent morphological phenotypes and effects on the expression of LEC2-regulated genes during seedling establishment and vegetative growth. Expression of castor bean LEC2 in Arabidopsis increased the expression of fatty acid elongase 1 (FAE1) and induced the accumulation of triacylglycerols, especially those containing the seed-specific fatty acid, eicosenoic acid (20:1Δ11), in vegetative tissues.
Graphical abstract
Highlights
•Castor bean LEC2 is single copy and shows seed-specific expression.•Over-expression of castor LEC2 induces genes involved in seed maturation in leaves.•Castor LEC2 induces the accumulation of triacylglycerols and 20:1 fatty acids in leaves.•Ectopic expression of castor LEC2 in Arabidopsis affects plant growth.
doi:10.1016/j.fob.2013.11.003
PMCID: PMC3863707  PMID: 24363987
Castor bean; Eicosenoic acid; LEAFY COTYLEDON2; Seed maturation; Transcription factor; Triacylglycerol; ABI3-VP1, abscisic acid-insensitive 3-viviparous 1; CaMV, cauliflower mosaic virus; cDNA, complementary DNA; DHA, docosahexaenoic acid; DIG, digoxigenin; FAE1, fatty acid elongase 1; GC, gas chromatography; ORF, open reading frame; RT-PCR, reverse transcription polymerase chain reaction; SSC, sodium chloride-sodium citrate; TAG, triacylglycerol; TF, transcription factor; TLC, thin-layer chromatography
20.  Ricin Trafficking in Plant and Mammalian Cells 
Toxins  2011;3(7):787-801.
Ricin is a heterodimeric plant protein that is potently toxic to mammalian and many other eukaryotic cells. It is synthesized and stored in the endosperm cells of maturing Ricinus communis seeds (castor beans). The ricin family has two major members, both, lectins, collectively known as Ricinus communis agglutinin ll (ricin) and Ricinus communis agglutinin l (RCA). These proteins are stored in vacuoles within the endosperm cells of mature Ricinus seeds and they are rapidly broken down by hydrolysis during the early stages of post-germinative growth. Both ricin and RCA traffic within the plant cell from their site of synthesis to the storage vacuoles, and when they intoxicate mammalian cells they traffic from outside the cell to their site of action. In this review we will consider both of these trafficking routes.
doi:10.3390/toxins3070787
PMCID: PMC3202855  PMID: 22069740
ricin biosynthesis; anterograde transport; retrograde transport; endoplasmic reticulum; retrotranslocation
21.  Ricinus communis Intoxications in Human and Veterinary Medicine-A Summary of Real Cases 
Toxins  2011;3(10):1332-1372.
Accidental and intended Ricinus communis intoxications in humans and animals have been known for centuries but the causative agent remained elusive until 1888 when Stillmark attributed the toxicity to the lectin ricin. Ricinus communis is grown worldwide on an industrial scale for the production of castor oil. As by-product in castor oil production ricin is mass produced above 1 million tons per year. On the basis of its availability, toxicity, ease of preparation and the current lack of medical countermeasures, ricin has gained attention as potential biological warfare agent. The seeds also contain the less toxic, but highly homologous Ricinus communis agglutinin and the alkaloid ricinine, and especially the latter can be used to track intoxications. After oil extraction and detoxification, the defatted press cake is used as organic fertilizer and as low-value feed. In this context there have been sporadic reports from different countries describing animal intoxications after uptake of obviously insufficiently detoxified fertilizer. Observations in Germany over several years, however, have led us to speculate that the detoxification process is not always performed thoroughly and controlled, calling for international regulations which clearly state a ricin threshold in fertilizer. In this review we summarize knowledge on intended and unintended poisoning with ricin or castor seeds both in humans and animals, with a particular emphasis on intoxications due to improperly detoxified castor bean meal and forensic analysis.
doi:10.3390/toxins3101332
PMCID: PMC3210461  PMID: 22069699
ricin; poisoning; animal intoxication; human intoxication; fertilizer
22.  Tissue-specific expression and post-translational modifications of plant- and bacterial-type phosphoenolpyruvate carboxylase isozymes of the castor oil plant, Ricinus communis L. 
Journal of Experimental Botany  2011;62(15):5485-5495.
This study employs transcript profiling together with immunoblotting and co-immunopurification to assess the tissue-specific expression, protein:protein interactions, and post-translational modifications (PTMs) of plant- and bacterial-type phosphoenolpyruvate carboxylase (PEPC) isozymes (PTPC and BTPC, respectively) in the castor plant, Ricinus communis. Previous studies established that the Class-1 PEPC (PTPC homotetramer) of castor oil seeds (COS) is activated by phosphorylation at Ser-11 and inhibited by monoubiquitination at Lys-628 during endosperm development and germination, respectively. Elimination of photosynthate supply to developing COS by depodding caused the PTPC of the endosperm and cotyledon to be dephosphorylated, and then subsequently monoubiquitinated in vivo. PTPC monoubiquitination rather than phosphorylation is widespread throughout the castor plant and appears to be the predominant PTM of Class-1 PEPC that occurs in planta. The distinctive developmental patterns of PTPC phosphorylation versus monoubiquitination indicates that these two PTMs are mutually exclusive. By contrast, the BTPC: (i) is abundant in the inner integument, cotyledon, and endosperm of developing COS, but occurs at low levels in roots and cotyledons of germinated COS, (ii) shows a unique developmental pattern in leaves such that it is present in leaf buds and young expanding leaves, but undetectable in fully expanded leaves, and (iii) tightly interacts with co-expressed PTPC to form the novel and allosterically-desensitized Class-2 PEPC heteromeric complex. BTPC and thus Class-2 PEPC up-regulation appears to be a distinctive feature of rapidly growing and/or biosynthetically active tissues that require a large anaplerotic flux from phosphoenolpyruvate to replenish tricarboxylic acid cycle C-skeletons being withdrawn for anabolism.
doi:10.1093/jxb/err225
PMCID: PMC3223045  PMID: 21841182
Enzyme phosphorylation; metabolic control; monoubiquitination; phosphoenolpyruvate carboxylase; post-translational modification; protein:protein interactions; tissue-specific gene expression
23.  Analysis of castor bean ribosome-inactivating proteins and their gene expression during seed development 
Genetics and Molecular Biology  2013;36(1):74-86.
Ribosome-inactivating proteins (RIPs) are enzymes that inhibit protein synthesis after depurination of a specific adenine in rRNA. The RIP family members are classified as type I RIPs that contain an RNA-N-glycosidase domain and type II RIPs that contain a lectin domain (B chain) in addition to the glycosidase domain (A chain). In this work, we identified 30 new plant RIPs and characterized 18 Ricinus communis RIPs. Phylogenetic and functional divergence analyses indicated that the emergence of type I and II RIPs probably occurred before the monocot/eudicot split. We also report the expression profiles of 18 castor bean genes, including those for ricin and agglutinin, in five seed stages as assessed by quantitative PCR. Ricin and agglutinin were the most expressed RIPs in developing seeds although eight other RIPs were also expressed. All of the RIP genes were most highly expressed in the stages in which the endosperm was fully expanded. Although the reason for the large expansion of RIP genes in castor beans remains to be established, the differential expression patterns of the type I and type II members reinforce the existence of biological functions other than defense against predators and herbivory.
doi:10.1590/S1415-47572013005000005
PMCID: PMC3615529  PMID: 23569411
agglutinin; evolution; lipase; Ricinus communis; ricin; RIPs
24.  Genomic imprinting, methylation and parent-of-origin effects in reciprocal hybrid endosperm of castor bean 
Nucleic Acids Research  2014;42(11):6987-6998.
Genomic imprinting often results in parent-of-origin specific differential expression of maternally and paternally inherited alleles. In plants, the triploid endosperm is where gene imprinting occurs most often, but aside from studies on Arabidopsis, little is known about gene imprinting in dicotyledons. In this study, we inspected genomic imprinting in castor bean (Ricinus communis) endosperm, which persists throughout seed development. After mapping out the polymorphic SNP loci between accessions ZB306 and ZB107, we generated deep sequencing RNA profiles of F1 hybrid seeds derived from reciprocal crosses. Using polymorphic SNP sites to quantify allele-specific expression levels, we identified 209 genes in reciprocal endosperms with potential parent-of-origin specific expression, including 200 maternally expressed genes and 9 paternally expressed genes. In total, 57 of the imprinted genes were validated via reverse transcriptase-polymerase chain reaction sequencing, and analysis of the genomic DNA methylation distribution between embryo and endosperm tissues showed significant hypomethylation in the endosperm and an enrichment of differentially methylated regions around the identified genes. Curiously, the expression of the imprinted genes was not tightly linked to DNA methylation. These results largely extended gene imprinting information existing in plants, providing potential directions for further research in gene imprinting.
doi:10.1093/nar/gku375
PMCID: PMC4066788  PMID: 24799438
25.  Bayesian Integration of Isotope Ratio for Geographic Sourcing of Castor Beans 
Recent years have seen an increase in the forensic interest associated with the poison ricin, which is extracted from the seeds of the Ricinus communis plant. Both light element (C, N, O, and H) and strontium (Sr) isotope ratios have previously been used to associate organic material with geographic regions of origin. We present a Bayesian integration methodology that can more accurately predict the region of origin for a castor bean than individual models developed independently for light element stable isotopes or Sr isotope ratios. Our results demonstrate a clear improvement in the ability to correctly classify regions based on the integrated model with a class accuracy of 60.9 ± 2.1% versus 55.9 ± 2.1% and 40.2 ± 1.8% for the light element and strontium (Sr) isotope ratios, respectively. In addition, we show graphically the strengths and weaknesses of each dataset in respect to class prediction and how the integration of these datasets strengthens the overall model.
doi:10.1155/2012/450967
PMCID: PMC3418698  PMID: 22919270

Results 1-25 (120239)