Bulked segregant analysis reveals candidate genes responsible for dwarf formation in woody oilseed crop castor bean.

Plant dwarfism is a desirable agronomic trait in non-timber trees, but little is known about the physiological and molecular mechanism underlying dwarfism in woody plants. Castor bean (Ricinus communis) is a typical woody oilseed crop. We performed cytological observations within xylem, phloem and cambia tissues, revealing that divergent cell growth in all tissues might play a role in the dwarf phenotype in cultivated castor bean. Based on bulked segregant analyses for a F2 population generated from the crossing of a tall and a dwarf accession, we identified two QTLs associated with plant height, covering 325 candidate genes. One of these, Rc5NG4-1 encoding a putative IAA transport protein localized in the tonoplast was functionally characterized. A non-synonymous SNP (altering the amino acid sequence from Y to C at position 218) differentiated the tall and dwarf plants and we confirmed, through heterologous yeast transformation, that the IAA uptake capacities of Rc5NG4-1Y and Rc5NG4-1C were significantly different. This study provides insights into the physiological and molecular mechanisms of dwarfing in woody non-timber economically important plants, with potential to aid in the genetic breeding of castor bean and other related crops.

Comparative proteomic and transcriptomic analyses provide new insight into the formation of seed size in castor bean.

BACKGROUND: Little is known about the molecular basis of seed size formation in endospermic seed of dicotyledons. The seed of castor bean (Ricinus communis L.) is considered as a model system in seed biology studies because of its persistent endosperms throughout seed development. RESULTS: We compared the size of endosperm and endospermic cells between ZB107 and ZB306 and found that the larger seed size of ZB107 resulted from a higher cell count in the endosperm, which occupy a significant amount of the total seed volume. In addition, fresh weight, dry weight, and protein content of seeds were remarkably higher in ZB107 than in ZB306. Comparative proteomic and transcriptomic analyses were performed between large-seed ZB107 and small-seed ZB306, using isobaric tags for relative and absolute quantification (iTRAQ) and RNA-seq technologies, respectively. A total of 1416 protein species were identified, of which 173 were determined as differentially abundant protein species (DAPs). Additionally, there were 9545 differentially expressed genes (DEGs) between ZB306 and ZB107. Functional analyses revealed that these DAPs and DEGs were mainly involved in cell division and the metabolism of carbohydrates and proteins. CONCLUSIONS: These findings suggest that both cell number and storage-component accumulation are critical for the formation of seed size, providing new insight into the potential mechanisms behind seed size formation in endospermic seeds.

The signal metabolite trehalose-6-phosphate inhibits the sucrolytic activity of sucrose synthase from developing castor beans.

In plants, trehalose 6-phosphate (T6P) is a key signaling metabolite that functions as both a signal and negative feedback regulator of sucrose levels. The mode of action by which T6P senses and regulates sucrose is not fully understood. Here, we demonstrate that the sucrolytic activity of RcSUS1, the dominant sucrose synthase isozyme expressed in developing castor beans, is allosterically inhibited by T6P. The feedback inhibition of SUS by T6P may contribute to the control of sink strength and sucrolytic flux in heterotrophic plant tissues.

Transcriptomic analyses reveal complex and interconnected sucrose signaling cascades in developing seeds of castor bean.

Seeds are highly specific organs that strongly sink sucrose resources from leaf and stem tissues to trigger seed metabolism and development. In particular, for heterotrophic non-green seeds, the potential molecular mechanism underlying sucrose-driven seed development remains unanswered. Castor bean (Ricinus communis L.), a typical non-green seed, has been considered as a model plant for seed biology study in dicotyledonous plants due to its heterotrophic seeds with persistent endosperms. In the present study, the fast-developing castor bean seeds were treated with exogenous sucrose and mannitol for four hours. The global transcriptomic data were obtained by high-throughput RNA-seq technique, resulting in 468 differentially expressed genes (DGEs). Further analyses revealed that sucrose functioned as both metabolic substrates and signal molecules. Specifically, 73 DGEs involved in carbohydrate and nitrogen metabolism, 42 differentially expressed transcription factors, and 35 DGEs involved in diverse signaling pathways such as auxin, brassinosteroid, ethelyene, cytokinin, gibberellin, and calcium signals, were identified, suggesting that the sucrose signaling pathway might have complex and multi-connected cross-talks with other signals to regulate castor bean seed development. Taken together, this study provides novel data to improve understanding of the potential molecular mechanisms of sucrose in regulating non-green seed development and storage reservoir accumulation during seed development.

Influence of phosphorous fertilization on copper phytoextraction and antioxidant defenses in castor bean (Ricinus communis L.).

Application of fertilizers to supply appropriate nutrients has become an essential agricultural strategy for enhancing the efficiency of phytoremediation in heavy metal contaminated soils. The present study was conducted to investigate the beneficial effects of three types of phosphate fertilizers (i.e., oxalic acid-activated phosphate rock (APR), Ca(H2PO4)2, and NaH2PO4) in the range of 0-600 mg P kg-1 soil, on castor bean growth, antioxidants [antioxidative enzymes and glutathione (GSH)], and Cu uptake. Results showed that with the addition of phosphorus fertilizers, the dry weight of castor bean and the Cu concentration in roots increased significantly, resulting in increased Cu extraction. The phosphorus concentration in both shoots and roots was increased as compared with the control, and the Ca(H2PO4)2 treatment had the greatest effect. Application of APR, NaH2PO4, and Ca(H2PO4)2 reduced the malondialdehyde (MDA) content, and the activity of the two antioxidant enzymes superoxide dismustase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) in the leaves of castor bean. GSH concentration in leaves increased with the increasing levels of phosphorus applied to soil as well as the accumulation of phosphorus in shoots, compared to the control. These results demonstrated that the addition of phosphorus fertilizers can enhance the resistance of castor bean to Cu and increase the Cu extraction efficiency of the plant from contaminated soils.

Ricinus communis L. (castor bean) as a potential candidate for revegetating industrial waste contaminated sites in peri-urban Greater Hyderabad: remarks on seed oil.

Ricinus communis L. (castor bean or castor oil plant) was found growing on metal-contaminated sites (4) of peri-urban Greater Hyderabad comprises of erstwhile industrial areas viz Bollaram, Patancheru, Bharatnagar, and Kattedan industrial areas. During 2013-2017, about 60 research papers have appeared focusing the role of castor bean in phytoremediation of co-contaminated soils, co-generation of biomaterials, and environmental cleanup, as bioenergy crop and sustainable development. The present study is focused on its use as a multipurpose phytoremediation crop for phytostabilization and revegetation of waste disposed peri-urban contaminated soils. To determine the plant tolerance level, metal accumulation, chlorophyll, protein, proline, lipid peroxidation, oil content, and soil properties were characterized. It was noticed that the castor plant and soils have high concentration of metals such as cadmium (Cd), lead (Pb), iron (Fe), manganese (Mn), and zinc (Zn). The soils have high phosphorous (P), adequate nitrogen (N), and low concentration of potassium (K). Iron (Fe) concentrations ranged from1672±50.91 to 2166±155.78 mg kg-1 in the soil. The trend of metal accumulation Fe>Zn>Mn>Pb>Cd was found in different plant parts at polluted sites. The translocation of Cd and Pb showed values more than one in industrial areas viz Bollaram, Kattedan, and Bharatnagar indicating the plants resistance to metal toxicity. Chlorophyll and protein content reduced while proline and malondialdehyde increased due to its tolerance level under metal exposure. The content of ricinoleic acid was higher, and the fatty acids composition of polluted areas was almost similar to that of the control area. Thus, R. communis L. can be employed for reclamation of heavy metal contaminated soils.

Differential Contribution of Malic Enzymes during Soybean and Castor Seeds Maturation.

Malic enzymes (ME) catalyze the decarboxylation of malate generating pyruvate, CO2 and NADH or NADPH. In some organisms it has been established that ME is involved in lipids biosynthesis supplying carbon skeletons and reducing power. In this work we studied the MEs of soybean and castor, metabolically different oilseeds. The comparison of enzymatic activities, transcript profiles and organic acid contents suggest different metabolic strategies operating in soybean embryo and castor endosperm in order to generate precursors for lipid biosynthesis. In castor, the malate accumulation pattern agrees with a central role of this metabolite in the provision of carbon to plastids, where the biosynthesis of fatty acids occurs. In this regard, the genome of castor possesses a single gene encoding a putative plastidic NADP-ME, whose expression level is high when lipid deposition is active. On the other hand, NAD-ME showed an important contribution to the maturation of soybean embryos, perhaps driving the carbon relocation from mitochondria to plastids to support the fatty acids synthesis in the last stages of seed filling. These findings provide new insights into intermediary metabolism in oilseeds and provide new biotechnological targets to improve oil yields.

Tolerance of Ricinus communis L. to Cd and screening of high Cd accumulation varieties for remediation of Cd contaminated soils.

Response of castor (Ricinus communis L.) to cadmium (Cd) was assessed by a seed-suspending seedbed approach. Length of total radicle was the most sensitive indicator of Cd tolerance among the tested germination and growth characters. The ED50 value for Cd was 11.87 mg L(-1), indicating high Cd tolerance in castor. A pot experiment was conducted by growing 46 varieties of castor under CK (without Cd) and Cd1 (10 mg kg(-1) of Cd) and Cd2 (50 mg kg(-1) of Cd) treatments to investigate genotype variations in growth response and Cd accumulation of castor under different Cd exposures. Castor possessed high Cd accumulation ability; average shoot and root Cd concentrations of the 46 tested varieties were 21.83 and 185.43 mg kg(-1), and 174.99 and 1181.96 mg kg(-1) under Cd1 and Cd2, respectively. Great variation in Cd accumulation was observed among varieties, and Cd concentration of castor was genotype dependent. The correlation between biomass and Cd accumulation was significantly positive, while no significant correlation was observed between Cd concentration and Cd accumulation, which indicated that biomass performance is the dominant factor in determining Cd accumulation ability.

Distribution and chemical forms of copper in the root cells of castor seedlings and their tolerance to copper phytotoxicity in hydroponic culture.

The subcellular localization and chemical forms of copper in castor (Ricinus communis L.) seedlings grown in hydroponic nutrient solution were identified by chemical extraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. The wild castor seeds were harvested from an abandoned copper mine in Tonglu Mountain, Daye City of Hubei Province, China. The results revealed that (1) the seedlings grew naturally in MS liquid medium with 40.00 mg kg(-1) CuSO4, in which the seedling growth rate and biomass index were 0.14 and 1.23, respectively, which were the highest values among all the treatments. The copper content in castor seedlings increased along with elevated CuSO4 concentration in the medium, reaching a maximum value of 16 570.12 mg kg(-1)(DW) when exposed to 60.00 mg L(-1) CuSO4, where 91.31% of the copper was accumulated in roots. (2) The copper existed in various chemical forms in the roots of the castor seedlings. Copper of 67.66% was extracted from the components of cell walls, such as exchangeable acidic polar compounds, cellulose and lignin, protein and pectin, and less concentrated in cell cytoplasm and nuclei. (3) Furthermore, the root cell walls were thickened when the castor seedlings exposed to CuSO4, with a large amount of high-density electron bodies, attached to the thickened cell walls. In the cell walls, most copper was bound to the carboxyl (-COOH) and hydroxyl (-OH) groups of acidic polar compounds, cellulose, hemicellulose, and polysaccharides. The conclusion showed that castor exhibited a strong tolerance to copper, the copper were accumulated mainly in the root cell, the root cell walls of castor were the major location of patience and detoxification in copper stress.

Evaluation of castor bean genotypes sown in winter and summer at a low altitude.

Current demands for the genetic breeding of castor bean include the adaptation of genotypes to low altitudes to expand the potential areas of cultivation. This study was conducted to evaluate the response of different genotypes of castor bean that were sown in winter and summer at a low altitude. The study was conducted in northwestern Rio de Janeiro State at an altitude of 60 m. The treatments consisted of genotypes IAC 80, AL Guarany 2002, BRS 149 Nordestina, BRS 188 Paraguaçu, Savana, Lyra, Mirante 10, IAC 226, Cafelista, G1, V1, and T1 with spaces of 2.0 m between rows, 1.0 m between plants, and one plant per hole. The plant height, number and length of racemes, number of fruits per raceme, seed yield, and incidence of gray mold were determined. The highest yields in both seasons were obtained by the genotypes BRS 149 Nordestina, G1, BRS 188 Paraguaçu, and IAC 226. The genotypes Savannah and Lyra had the lowest yields in both cultivation seasons. The genotypes responded differently to the environmental conditions depending on the growing season, and the spring-summer period was found to be more favorable at the low altitude of the study site.

Cadmium accumulation and tolerance of two castor cultivars in relation to antioxidant systems.

To investigate the effects of Cd on tolerance and antioxidant activities of castor, two different castor (Ricinus communis L.) cultivars (Zibo No. 5 and Zibo No. 8) were used for a hydroponic experiment (0, 1 and 2mg/L Cd) and a pot experiment using Cd-contaminated soil (34mg/kg) with the addition of ethylenedinitrilotetraacetic acid (EDTA). The results indicated that there were significant differences between the two cultivars with respect to Cd uptake in shoots (113-248mg/kg for Zibo No. 5 and 130-288mg/kg Zibo No. 8), biomass tolerance indexes (64.9%-74.6% for Zibo No. 5 and 80.1%-90.9% for Zibo No. 8) in the hydroponic experiment and survival rates (0% for Zibo No. 5 and 100% for Zibo No. 8) determined by the addition of EDTA in the pot experiment, suggesting that Zibo No. 8 has higher tolerance than Zibo No. 5. Moreover, the castor cultivars have low bioconcentration factors (4.80% for Zibo No. 5 and 5.43% for Zibo No. 8) and low translocation factors (<1%). Consequently, Zibo No. 8 can participate in Cd phytostabilization in highly Cd-polluted areas. The results indicated that glutathione (GSH) as a non-enzymatic antioxidant, and antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPX), were cultivar- and dose-dependent. The higher tolerance of Zibo No. 8 compared with Zibo No. 5 can be attributed to the higher GSH levels in the root and higher GPX activity in the leaf.

Seeds' physicochemical traits and mucilage protection against aluminum effect during germination and root elongation as important factors in a biofuel seed crop (Ricinus communis).

We determined the length, volume, dry biomass, and density in seeds of five castor bean cultivars and verified notable physicochemical trait differences. Seeds were then subjected to different toxic aluminum (Al) concentrations to evaluate germination, relative root elongation, and the role of root apices' rhizosphere mucilage layer. Seeds' physicochemical traits were associated with Al toxicity responses, and the absence of Al in cotyledons near to the embryo was revealed by Al-hematoxylin staining, indicating that Al did not induce significant germination reduction rates between cultivars. However, in the more sensitive cultivar, Al was found around the embryo, contributing to subsequent growth inhibition. After this, to investigate the role of mucilage in Al tolerance, an assay was conducted using NH4Cl to remove root mucilage before or after exposure to different Al concentrations. Sequentially, the roots were stained with hematoxylin and a quantitative analysis of staining intensity was obtained. These results revealed the significant contribution of the mucilage layer to Al toxicity responses in castor bean seedlings. Root growth elongation under Al toxicity confirmed the role of the mucilage layer, which jointly indicated the differential Al tolerance between cultivars and an efficient Al-exclusion mechanism in the tolerant cultivar.

Molecular and biochemical identification of inositol 1,3,4,5,6-pentakisphosphate 2-kinase encoding mRNA variants in castor bean (Ricinus communis L.) seeds.

During seed development, phytic acid (PA) associated with mineral cations is stored as phytin and mobilized following germination in support of seedling growth. Two parallel biosynthetic pathways for PA have been proposed; yet the pathway is still poorly understood in terms of its regulation and the enzymes involved. Here, the castor bean (Ricinus communis L.) gene for inositol 1,3,4,5,6-pentakisphosphate 2-kinase (RcIPK1) has been identified. This encodes the enzyme implicated in catalyzing the final reaction in PA biosynthesis, and its expression is enhanced in isolated germinated embryos by application of phosphate and myo-inositol (Ins). Even though only one copy of the RcIPK1 gene is present in the genome, numerous RNA variants are present, most likely due to alternative splicing. These are translated into six closely related protein isoforms according to in silico analysis. Functional analyses using yeast ipk1Δ revealed that only three of the mRNA variants can rescue a temperature-sensitive growth phenotype of this strain. High-performance liquid chromatography (HPLC) analysis of the synthesized inositol phosphates demonstrated that the ability to complement the missing yeast IPK1 enzyme is associated with the production of enzyme activity. The three active isoforms possess unique conserved motifs important for IPK1 catalytic activity.

Genomic surveys and expression analysis of bZIP gene family in castor bean (Ricinus communis L.).

The basic leucine zipper (bZIP) transcription factors comprise a family of transcriptional regulators present extensively in plants, involved in regulating diverse biological processes such as flower and vascular development, seed maturation, stress signaling and pathogen defense. Castor bean (Ricinus communis L. Euphorbiaceae) is one of the most important non-edible oilseed crops and its seed oil is broadly used for industrial applications. We performed a comprehensive genome-wide identification and analysis of the bZIP transcription factors that exist in the castor bean genome in this study. In total, 49 RcbZIP transcription factors were identified, characterized and categorized into 11 groups (I-XI) based on their gene structure, DNA-binding sites, conserved motifs, and phylogenetic relationships. The dimerization properties of 49 RcbZIP proteins were predicted on the basis of the characteristic features in the leucine zipper. Global expression profiles of 49 RcbZIP genes among different tissues were examined using high-throughput sequencing of digital gene expression profiles, and resulted in diverse expression patterns that may provide basic information to further reveal the function of the 49 RcbZIP genes in castor bean. The results obtained from this study would provide valuable information in understanding the molecular basis of the RcbZIP transcription factor family and their potential function in regulating the growth and development, particularly in seed filling of castor bean.

Phytoextraction of metals and rhizoremediation of PAHs in co-contaminated soil by co-planting of Sedum alfredii with ryegrass (Lolium perenne) or castor (Ricinus communis).

A pot experiment was conducted to investigate the potential for phytoextraction of heavy metals and rhizoremediation of polycyclic aromatic hydrocarbons (PAHs) in co-contaminated soil by co-planting a cadmium/zinc (Cd/Zn) hyperaccumulator and lead (Pb) accumulator Sedum alfredii with ryegrass (Lolium perenne) or castor (Ricinus communis). Co-planting with castor decreased the shoot biomass of S. alfredii as compared to that in monoculture. Cadmium concentration in S. alfredii shoot significantly decreased when grown with ryegrass or castor as compared to that in monoculture. However, no reduction of Zn or Pb concentration in S. alfredii shoot was detected in co-planting treatments. Total removal of either Cd, Zn, or Pb by plants was similar across S. alfredii monoculture or co-planting with ryegrass or castor, except enhanced Pb removal in S. alfredii and ryegrass co-planting treatment. Co-planting of S. alfredii with ryegrass or castor significantly enhanced the pyrene and anthracene dissipation as compared to that in the bare soil or S. alfredii monoculture. This appears to be due to the increased soil microbial population and activities in both co-planting treatments. Co-planting of S. alfredii with ryegrass or castor provides a promising strategy to mitigate both metal and PAH contaminants from co-contaminated soils.

The multigene family of lysophosphatidate acyltransferase (LPAT)-related enzymes in Ricinus communis: cloning and molecular characterization of two LPAT genes that are expressed in castor seeds.

The multigene family encoding proteins related to lysophosphatidyl-acyltransferases (LPATs) has been analyzed in the castor plant Ricinus communis. Among them, two genes designated RcLPAT2 and RcLPATB, encoding proteins with LPAT activity and expressed in the developing seed, have been cloned and characterized in some detail. RcLPAT2 groups with well characterized members of the so-called A-class LPATs and it shows a generalized expression pattern in the plant and along seed development. Enzymatic assays of RcLPAT2 indicate a preference for ricinoleoyl-CoA over other fatty acid thioesters when ricinoleoyl-LPA is used as the acyl acceptor, while oleoyl-CoA is the preferred substrate when oleoyl-LPA is employed. RcLPATB groups with B-class LPAT enzymes described as seed specific and selective for unusual fatty acids. However, RcLPATB exhibit a broad specificity on the acyl-CoAs, with saturated fatty acids (12:0-16:0) being the preferred substrates. RcLPATB is upregulated coinciding with seed triacylglycerol accumulation, but its expression is not restricted to the seed. These results are discussed in the light of a possible role for LPAT isoenzymes in the channelling of ricinoleic acid into castor bean triacylglycerol.

Growth, tolerance efficiency and phytoremediation potential of Ricinus communis (L.) and Brassica juncea (L.) in salinity and drought affected cadmium contaminated soil.

We have previously reported that Ricinus communis (castor) is more tolerant to soil cadmium (Cd) and more efficient for Cd phytoremediation than Brassica juncea (Indian mustard) (Bauddh and Singh, 2012). In the present study, R. communis was found more tolerant to salinity and drought in presence of Cd and removed more Cd in a given time than Indian mustard. R. communis produced 23 and twelve folds higher biomass in terms of fresh weight and dry weight, respectively than that in B. juncea during three months when grown in Cd contaminated soil in presence of 100mM NaCl salinity and ten day water withdrawal based drought at 90 day after sowing (DAS). Castor plants showed stronger self-protection ability in form of proline bioaccumulation (r(2)=0.949) than Indian mustard (r(2)=0.932), whereas a lower r(2) for malondialdehyde (MDA) and total soluble protein in R. communis (r(2)=0.914 and r(2)=0.915, respectively) than that of B. juncea (r(2)=0.947 and r(2)=0.927, respectively) indicated a greater damage to cell membrane in Indian mustard during the multiple stress conditions. Though, the amount of Cd accumulated in the roots and shoots of Indian mustard was higher as per unit biomass than that in castor, total removal of the metal from soil was much higher in castor on per plant basis in the same period in presence of the stresses. R. communis accumulated about seventeen and 1.5 fold higher Cd in their roots and shoots, respectively than that of B. juncea in 90 DAS under the multiple stresses. Salinity alone enhanced Cd uptake, whereas drought stress reduced its uptake in both the plants.

Apis mellifera pollination improves agronomic productivity of anemophilous castor bean (Ricinus communis).

Castor bean (Ricinus communis L.) is cultivated mainly for biodiesel production because of its oil-rich seeds; it is assumed to be an anemophylous species. But pollination deficit can lead to low productivity often attributed to other reasons. In this paper, we investigated pollination requirements, pollination mechanism, occurrence of pollination deficit, and the role of biotic pollinators in a large commercial plantation of castor bean. Our results show that R. communis bears a mixed breeding system favoring selfing by geitonogamy, although the wind promotes mostly outcrossing. We also found that the honey bee (Apis mellifera L.) foraging on castor bean can both transfer pollen from male to female flowers within the same raceme and boost the release of airborne pollen by male flowers. Both situations increase geitonogamy rates, raising significantly fruit set and seed yield. This is the first report of an animal foraging activity increasing seed yield in an anemophilous and geitonogamous crop and elucidates the role of biotic pollinators in castor bean reproduction.

Changes in the diversity of soil arbuscular mycorrhizal fungi after cultivation for biofuel production in a Guantanamo (Cuba) tropical system.

The arbuscular mycorrhizal fungi (AMF) are a key, integral component of the stability, sustainability and functioning of ecosystems. In this study, we characterised the AMF biodiversity in a native vegetation soil and in a soil cultivated with Jatropha curcas or Ricinus communis, in a tropical system in Guantanamo (Cuba), in order to verify if a change of land use to biofuel plant production had any effect on the AMF communities. We also asses whether some soil properties related with the soil fertility (total N, Organic C, microbial biomass C, aggregate stability percentage, pH and electrical conductivity) were changed with the cultivation of both crop species. The AM fungal small sub-unit (SSU) rRNA genes were subjected to PCR, cloning, sequencing and phylogenetic analyses. Twenty AM fungal sequence types were identified: 19 belong to the Glomeraceae and one to the Paraglomeraceae. Two AMF sequence types related to cultured AMF species (Glo G3 for Glomus sinuosum and Glo G6 for Glomus intraradices-G. fasciculatum-G. irregulare) did not occur in the soil cultivated with J. curcas and R. communis. The soil properties (total N, Organic C and microbial biomass C) were higher in the soil cultivated with the two plant species. The diversity of the AMF community decreased in the soil of both crops, with respect to the native vegetation soil, and varied significantly depending on the crop species planted. Thus, R. communis soil showed higher AMF diversity than J. curcas soil. In conclusion, R. communis could be more suitable for the long-term conservation and sustainable management of these tropical ecosytems.

Proteomic profile of the nucellus of castor bean (Ricinus communis L.) seeds during development.

In this study, we performed a proteomic analysis of nucellus from two developmental stages of Ricinus communis seeds by a GeLC-MS/MS approach, using of a high resolution orbitrap mass spectrometer, which resulted in the identification of a total of 766 proteins that were grouped into 553 protein groups. The distribution of the identified proteins in stages III and IV into different Gene Ontology categories was similar, with a remarkable abundance of proteins associated with the protein synthesis machinery of cells, as well as several classes of proteins involved in protein degradation, particularly of peptidases associated with programmed cell death. Consistent with the role of the nucellus in mediating nutrient transfer from maternal tissues to the endosperm and embryo, a significant proportion of the identified proteins are related to amino acid metabolism, but none of the identified proteins are known to have a role as storage proteins. Moreover for the first time, ricin isoforms were identified in tissues other than seed endosperm. Results are discussed in the context of the spatial and temporal distribution of the identified proteins within the nucellar cell layers.