Evaluation of supplemented protein-L-isoaspartate-O-methyltransferase (PIMT) gene of Carica papaya and Ricinus communis in stress survival of Escherichia coli BL21(DE3) cells.

In growing plant population, effect of stress is a perturb issue affecting its physiological, biochemical, yield loss and developmental growth. Protein-L-isoaspartate-O-methyltransferase (PIMT) is a broadly distributed protein repair enzyme which actuate under stressful environment or aging. Stress can mediate damage converting protein bound aspartate (Asp) residues to isoaspartate (iso-Asp). This spontaneous and deleterious conversion occurs at an elevated state of stress and aging. Iso-Asp formation is associated with protein inactivation and compromised cellular survival. PIMT can convert iso-Asp back to Asp, thus repairing and contributing to cellular survival. The present work describes the isolation, cloning, sequencing and expression of PIMT genes of Carica papaya (Cp pimt) and Ricinus communis (Rc pimt) Using gene specific primers, both the pimts were amplified from their respective cDNAs and subsequently cloned in prokaryotic expression vector pProEXHTa. BL21(DE3) strain of E. coli cells were used as expression host. The expression kinetics of both the PIMTs were studied with various concentrations of IPTG and at different time points. Finally, the PIMT supplemented BL21(DE3) cells were evaluated against different stresses in comparison to their counterparts with the empty vector control.

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.

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

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

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

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

An efficient method for protoplast-mediated production of transformed castor bean (Ricinus communis) lines.

OBJECTIVE: The purpose of this study was to develop a method for the isolation, culture, and PEG-mediated protoplast transfection from leaves of in vitro-grown plants of Ricinus communis. RESULTS: Factors such as the enzymatic composition and the incubation time were evaluated. The enzymatic solution, containing 1.6% Cellulase-R10 and 0.8% Macerozyme-R10, with 16 h of incubation, was the best condition to achieve a high protoplast yield (481.16 × 104 protoplasts/g FW) with a high percentage of viability (95%). The combination and concentration of enzymes have been shown to affect the protoplast isolation efficiency significantly. Furthermore, we found that a higher number of protoplasts (8.5 × 105 protoplast/g FW) was obtained at a longer incubation time, but their viability decreased. We obtained a simple and efficient protocol to isolate protoplast from Ricinus communis leaves and culture. A PEG-mediated protoplast transfection protocol was also established to introduce plasmid DNA into Ricinus communis genotypes cultivated in Colombia. Thus, strengthening advances in the genetic improvement processes for this crop are presented.

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

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

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.

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.

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.

Characterization of a novel sn1,3 lipase from Ricinus communis L. suitable for production of oleic acid-palmitic acid-glycerol oleate.

The hydrolysis properties of lipase in castor was evaluated using two different substrate forms (tripalmitic glycerides and trioleic glycerides) to catalyze the reaction under different operational conditions. RcLipase was obtained from castor seeds and results show that RcLipase is a conservative serine lipase with a conserved catalytic center (SDH) and a conserved pentapeptide (GXSXG). This enzyme exhibited the greatest activity and tolerance to chloroform and toluene when it was expressed in Pichia pastoris GS115 at 40 ℃ and pH 8.0. Zn and Cu ions exerted obvious inhibitory effects on the enzyme, and displayed good hydrolytic activity for long-chain natural and synthetic lipids. HPLC analysis showed that this enzyme has 1,3 regioselectivity when glycerol tripalmitate and oleic acid are used as substrates. The fatty acid composition in the reaction product was 21.3% oleic acid and 79.1% sn-2 palmitic acid.

Effect of arsenate toxicity on antioxidant enzymes and expression of nicotianamine synthase in contrasting genotypes of bioenergy crop Ricinus communis.

Arsenic (As) is a toxic environmental pollutant. Growing Ricinus communis (castor) on As-contaminated land has the advantage that in addition to revegetation of contaminated land, it can produce bioenergy. To date, As tolerance mechanisms of this plant are not fully understood. In our previous study, we screened tolerant and sensitive genotypes of castor and reported higher total As concentration, enhanced reactive oxygen species (ROS) generation, and oxidative stress in sensitive genotypes of castor GCH 2 and GCH 4 in comparison to tolerant genotypes WM and DCH 177. In the present study, we compared the activity, isoenzyme profile, and gene expression of ROS-scavenging enzymes, proline content, and expression of nicotianamine synthase genes (RcNAS1, RcNAS2, and RcNAS3) in As-tolerant and As-sensitive genotypes of castor. SOD and GPX activity increased significantly in roots of tolerant genotype WM but remained the same or decreased in sensitive genotype GCH 2 and GCH 4 at 200 µM arsenate [As(V)] treatment indicating their important role in As tolerance in castor. CAT activity and proline content increased in sensitive genotypes but remained the same in tolerant genotypes due to As(V) treatment. APX activity showed no significant change in roots and leaves of both tolerant and sensitive genotypes. NAS genes (RcNAS1, RcNAS2, and RcNAS3) encode enzymes that catalyze trimerization of S-adenosylmethionine to form nicotianamine and are critical for metal chelation and heavy metal tolerance. Differential responses of RcNAS1, RcNAS2, and RcNAS3 genes in WM and GCH 2 due to As(V) treatment suggest their role in As(V) tolerance.

Transcriptome analysis of salt stress responsiveness in the seedlings of wild and cultivated Ricinus communis L.

Soil salinity is one of the major environmental factors, influencing agricultural productivity of crops. As a non-edible and ideal oilseed crop, castor (Ricinus communis L.) has great industrial value in biofuel, but molecular mechanisms of salt stress regulation are still unknown. In this study, the differentially expressed genes (DEGs) for differential salt tolerance in two castor cultivar (wild castor : Y, cultivated castor 'Tongbi 5': Z) were identified. 12 libraries were sampled for Illumina high-throughput sequencing to consider 132,426 nonredundant unigenes and 31,221 gene loci. Multiple phytohormones and transcription factors (TFs) were correlated with salt-tolerance and differently enriched in these two genotypes. The type 2C protein phosphatases (PP2C) homologs were all upregulated under salt stress. Importantly, IAA (1), DELLA (1) and Jasmonate zim domain (JAZ) (1) were also identified and found to be differentially expressed. Based on the co-expressed module by regulatory networks and heatmap analysis, ERF/AP2, WRKY and bHLH families were prominently participate in high salt stress response of wild and cultivated castor. Finally, these results highlight that the hub DEGs and families were more accumulated in cultivated castor than those in wild castor, providing novel insights into the salinity adaptive mechanisms and genetic improvement in castor.

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.

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.

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.

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.

Selection and validation of castor bean (Ricinus communis) reference genes for quantitative PCR (RT-qPCR) in developing and germinating seeds and expression pattern of four ricin-family genes.

This study aims to expand the set of internal control genes used for RT-qPCR experiments with Castor bean (Ricinus communis) seeds by evaluating candidate genes across several seed tissues and developmental stages. Nine reference genes were selected, including actin-11 (ACT11), tubulin alpha-2 (Tα2), elongation factor 1-alpha (EF1-α), protein phosphatase 2A-2 (PP2A2), polyubiquitin-3 (PUB3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Biological samples consisted of R. communis seeds in 15 stages of maturation and germination. We demonstrate that PP2A2, PUB3 and EF1-α are the most stably expressed genes across the tested conditions and therefore appropriate for RT-qPCR. Subsequently, those reference genes were used for the analysis of the expression of four R. communis ricin-family genes. In developing seeds, the highest ricin expression levels was seen in the nucellus and in the endosperm, whereas in germinating seeds a peak expression occurs 4-6 days after germination. The four tested ricin isoforms exhibited differential expression patterns across tissues and seed developmental stages, which may indicate distinct biological roles for each ricin gene.

WRINKLED1 homologs highly and functionally express in oil-rich endosperms of oat and castor.

Oat (Avena sativa) and castor (Ricinus communis) accumulate a large amount of lipids in their endosperms, however the molecular mechanism remains unknown. In this study, differences in oil regulators between oat and wheat (Triticum aestivum) as well as common features between oat and castor were tested by analyzing their transcriptomes with further q-PCR analysis. Results indicated that WRINKLED1 (WRI1) homologs and their target genes highly expressed in the endosperms of oat and castor, but not in the starchy endosperms of wheat. Expression pattern of WRI1s was in agreement with that of oil accumulation. Three AsWRI1s (AsWRI1a, AsWRI1b and AsWRI1c) and one RcWRI1 were identified in the endosperms of oat and castor, respectively. AsWRI1c lacks VYL motif, which is different from the other three WRI1s. Expressions of these four WRI1s all complemented the phenotypes of Arabidopsis wri1-1 mutant. Overexpression of these WRI1s in Arabidopsis and tobacco BY2 cells increased oil contents of seeds and total fatty acids of the cells, respectively. Moreover, this overexpression also resulted in up-regulations of WRI1 target genes, such as PKp-ß1. Taken together, our results suggest that high and functional expression of WRI1 play a key role in the oil-rich endosperms and the VYL motif is dispensable for WRI1 function.

Sequencing of Chinese castor lines reveals genetic signatures of selection and yield-associated loci.

Oil produced by castor (Ricinus communis) has broad industrial applications. However, knowledge on the genetic diversity, especially genetic alterations that occurred during domestication and subsequent traits selection, of this oil crop is limited. Here, our population genomics analyses show that the Chinese castors have developed a geographic pattern, classified into the southern-, the middle-, and the northern-China groups. We detect a number of candidate genomic loci that are associated with the selection signals during the geographical differentiation and domestication. Using genome-wide association analysis, we identify candidate genes associated with nine agronomically important traits. One of the candidate genes encoding a glycosyltransferase related to cellulose and lignin biosynthesis is associated with both capsule dehiscence and endocarp thickness. We hypothesize that the abundance of cellulose or lignin in endocarp is an important factor for capsule dehiscence. Our results provide foundation for castor breeding and genetic study.

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.