The genome of Ricinus communis encodes a single glycolate oxidase with different functions in photosynthetic and heterotrophic organs.

MAIN CONCLUSION: The biochemical characterization of glycolate oxidase in Ricinus communis hints to different physiological functions of the enzyme depending on the organ in which it is active. Enzymatic activities of the photorespiratory pathway are not restricted to green tissues but are present also in heterotrophic organs. High glycolate oxidase (GOX) activity was detected in the endosperm of Ricinus communis. Phylogenetic analysis of the Ricinus L-2-hydroxy acid oxidase (Rc(L)-2-HAOX) family indicated that Rc(L)-2-HAOX1 to Rc(L)-2-HAOX3 cluster with the group containing streptophyte long-chain 2-hydroxy acid oxidases, whereas Rc(L)-2-HAOX4 clusters with the group containing streptophyte GOX. Rc(L)-2-HAOX4 is the closest relative to the photorespiratory GOX genes of Arabidopsis. We obtained Rc(L)-2-HAOX4 as a recombinant protein and analyze its kinetic properties in comparison to the Arabidopsis photorespiratory GOX. We also analyzed the expression of all Rc(L)-2-HAOXs and conducted metabolite profiling of different Ricinus organs. Phylogenetic analysis indicates that Rc(L)-2-HAOX4 is the only GOX encoded in the Ricinus genome (RcGOX). RcGOX has properties resembling those of the photorespiratory GOX of Arabidopsis. We found that glycolate, the substrate of GOX, is highly abundant in non-green tissues, such as roots, embryo of germinating seeds and dry seeds. We propose that RcGOX fulfills different physiological functions depending on the organ in which it is active. In autotrophic organs it oxidizes glycolate into glyoxylate as part of the photorespiratory pathway. In fast growing heterotrophic organs, it is most probably involved in the production of serine to feed the folate pathway for special demands of those tissues.

Application of a high-resolution genetic map for chromosome-scale genome assembly and fine QTLs mapping of seed size and weight traits in castor bean.

Castor bean (Ricinus communis L., Euphorbiaceae) is a critical biodiesel crop and its seed derivatives have important industrial applications. Due to lack of a high-density genetic map, the breeding and genetic improvement of castor bean has been largely restricted. In this study, based on a recombinant inbred line (RIL) population consisting of 200 individuals, we generated 8,896 high-quality genomic SNP markers and constructed a high-resolution genetic map with 10 linkage groups (LGs), spanning 1,852.33 centiMorgan (cM). Based on the genetic map, 996 scaffolds from the draft reference genome were anchored onto 10 pseudo-chromosomes, covering 84.43% of the castor bean genome. Furthermore, the quality of the pseudo-chromosome scale assembly genome was confirmed via genome collinearity analysis within the castor bean genome as well as between castor bean and cassava. Our results provide new evidence that the phylogenetic position of castor bean is relatively solitary from other taxa in the Euphorbiaceae family. Based on the genetic map, we identified 16 QTLs that control seed size and weight (covering 851 candidate genes). The findings will be helpful for further research into potential new mechanisms controlling seed size and weight in castor bean. The genetic map and improved pseudo-chromosome scale genome provide crucial foundations for marker-assisted selection (MAS) of QTL governing important agronomic traits, as well as the accelerated molecular breeding of castor bean in a cost-effective pattern.

Lethal Injection of a Castor Bean Extract: Ricinine Quantification as a Marker for Ricin Exposure Using a Validated LC-MS/MS Method.

Ricin is a highly toxic agent derived from the castor bean plant (Ricinus communis). Poisoning occurs commonly by oral ingestion of the beans. Injection of ricin is believed to be more lethal. Ricin is a large glycosylated protein difficult to detect in clinical samples. Instead, ricinine, a small alkaloid found in the same beans, is used as surrogate marker for ricin exposure. We describe a simple LC-MS/MS method for the detection of ricinine in serum, blood and urine, validated according to EMA guidelines and successfully applied to patient samples of a suicidal death after injection of a castor bean extract. A 26-year-old man self-presented to the emergency department with severe abdominal cramps and nausea after injection of a castor bean extract. Due to rapid deterioration of his hemodynamic function despite early aggressive fluid resuscitation, he was transferred to ICU. Abdominal cramps worsened and a fulminant diarrhea developed, resulting in hypovolemic shock and cardiorespiratory collapse. Despite full supportive therapy, the patient died approximately 10 hours after injection due to multiple organ failure. Ricinine was quantified by LC-MS/MS after LLE with diethyl ether using ricinine-D3 as internal standard. Six hours after injection, ricinine concentrations in serum and blood were 16.5 and 12.9 ng/mL, respectively, which decreased to 12.4 and 10.6 ng/mL, 4 hours later. The urinary concentration was 81.1 ng/mL 7 hours after injection, which amply exceeded the levels previously reported in similar cases with lethal outcome. Concentrations of ricinine, compatible with a lethal exposure to castor beans, were detected in serum, blood and urine. Ricinine was also found in bile and liver tissue.

Genome-Wide Identification, Evolutionary Analysis, and Stress Responses of the GRAS Gene Family in Castor Beans.

Plant-specific GRAS transcription factors play important roles in regulating growth, development, and stress responses. Castor beans (Ricinus communis) are important non-edible oilseed plants, cultivated worldwide for its seed oils and its adaptability to growth conditions. In this study, we identified and characterized a total of 48 GRAS genes based on the castor bean genome. Combined with phylogenetic analysis, the castor bean GRAS members were divided into 13 distinct groups. Functional divergence analysis revealed the presence of mostly Type-I functional divergence. The gene structures and conserved motifs, both within and outside the GRAS domain, were characterized. Gene expression analysis, performed in various tissues and under a range of abiotic stress conditions, uncovered the potential functions of GRAS members in regulating plant growth development and stress responses. The results obtained from this study provide valuable information toward understanding the potential molecular mechanisms of GRAS proteins in castor beans. These findings also serve as a resource for identifying the genes that allow castor beans to grow in stressful conditions and to enable further breeding and genetic improvements in agriculture.

Liquid chromatography-mass spectrometry and chemometric analysis of Ricinus communis extracts for cultivar identification.

INTRODUCTION: Seeds of Ricinus communis contain the toxic protein ricin, a 64 kD heterodimeric type II ribosome-inactivating protein that has been used in several high-profile poisoning incidents. The ability to determine which cultivar the toxin was isolated from via an LC-MS method would be of significant use to law enforcement and forensic agencies. OBJECTIVE: To analyse via LC-MS and chemometrics (principal components analysis (PCA), orthogonal partial-least-squares discriminant analysis (OPLS-DA)) extracts of R. communis to identify compounds specific to a particular cultivar. METHODS: Seeds from eight specimens of six cultivars of R. communis ('carmencita', 'dehradun', 'gibsonii', 'impala', 'sanguineus' and 'zanzibariensis') were extracted using a standard methodology. These extracts were analysed by LC-MS then subjected to chemometric analysis (PCA and OPLS-DA). Identified compounds of importance were subjected to high-resolution Fourier transform (HRFT) MS and MS/MS to elucidate their structures. RESULTS: This analysis identified 17 ions as potential cultivar determinators. Through accurate mass measurement and MS/MS, molecular formulae for 13 ions were determined, including two known and 11 new peptides. CONCLUSION: Unique ions in extracts of 'carmencita', 'dehradun', 'gibsonii', 'impala' and 'zanzibariensis' were identified that would allow an individual cultivar to be distinguished from other cultivars in this study. Although 'sanguineus' extracts contained no unique compounds, a unique LC-MS profile would allow for cultivar assignment.

Genus identification of toxic plant by real-time PCR.

Some plants have toxicities that are dangerous for humans. In the case of poisoning by toxic plants, a rapid and easy screening test is required for accurate medical treatment or forensic investigation. In this study, we designed specific primer pairs for identification of toxic plants, such as subgenus Aconitum, genus Ricinus, genus Illicium, and genus Scopolia, by internal transcribed spacer sequences of nuclear ribosomal DNA. Allied species of target plants, foods, and human DNA were not detected, but each primer pair provided a specific PCR product from the target plant using real-time PCR. This method can detect the subgenus Aconitum, genus Ricinus, and genus Scopolia with template DNA of 10 pg, respectively, and genus Illicium with 1 pg. Furthermore, each primer pair provided the exact PCR product from digested target plants in artificial gastric fluid. When a trace unknown plant sample in forensic investigation is collected from stomach contents, this PCR assay may be useful for screening toxic plants.

Characterization of the 11S globulin gene family in the castor plant Ricinus communis L.

The 11S globulin (legumin) gene family has been characterized in the castor plant Ricinus communis L. Phylogenetic analysis reveals the presence of two diverged subfamilies (RcLEG1 and RcLEG2) comprising a total of nine genes and two putative pseudogenes. The expression of castor legumin genes has been studied, indicating that it is seed specific and developmentally regulated, with a maximum at the stage when cellular endosperm reaches its full expansion (around 40-45 DAP). However, conspicuous differences are appreciated in the expression timing of individual genes. A characterization of the 5'-proximal regulatory regions for two genes, RcLEG1-1 and RcLEG2-1, representative of the two legumin subfamilies, has also been performed by fusion to the GUS reporter gene. The results obtained from heterologous expression in tobacco and transient expression in castor, indicating seed-specific regulation, support the possible utility of these promoters for biotechnological purposes.

Ricin A Chain from Ricinus sanguineus: DNA sequence, structure and toxicity.

The gene coding for the ricin A-chain from Ricinus sanguineus (RsTA) was isolated and sequenced (GB: DQ661048). Comparison of RsTA with the ricin A-chain from Ricinus communis (RTA) revealed the presence of five differences in the gene sequence. At the protein level only two differences were noticed, the replacement of Asn136 by Ser (N136S) and Ile173 by Val (I174 V). From the RsTA structure model (PMDB: PM0074652), the N136S mutation was predicted to have no effect on RsTA structure. The I174V mutation is believed to have no effect on the RsTA structure or on toxicity since this replacement was found in the Ricinus agglutinin's A-chain and the latter has a comparable toxicity to RTA. The Ser221 of the putative lipase active site was found. Toxicity experiments showed that RsTA and RTA have similar toxicities. This finding proves that the N136S and I174 V mutations have no effect on RsTA toxicity.