Adenosine stimulates anabolic metabolism in developing castor bean (Ricinus communis L.) cotyledons

In previous experiments it was shown that Castor-bean (Ricinus communis) endosperm releases carbohydrates, amino acids and nucleoside derivatives, which are subsequently imported into the developing cotyledons (Kombrink and Beevers in Plant Physiol 73:370-376, 1983). To investigate the importance of the most prominent nucleoside adenosine for the metabolism of growing Ricinus seedlings, we supplied adenosine to cotyledons of 5-days-old seedlings after removal of the endosperm. This treatment led to a 16% increase in freshweight of intact seedlings within 16 h, compared to controls. Using detached cotyledons, we followed uptake of radiolabelled adenosine and identified 40% of label in solubles (mostly ATP and ADP), 46% incorporation in RNA and 2.5% in DNA, indicating a highly active salvage pathway. About 7% of freshly imported adenosine entered the phloem, which indicates a major function of adenosine for cotyledon metabolism. Import and conversion of adenosine improved the energy content of cotyledons as revealed by a substantially increased ATP/ADP ratio. This effect was accompanied by slight increases in respiratory activity, decreased levels of hexose phosphates and increased levels of fructose-1,6-bisphosphate and triose phosphates. These alterations indicate a stimulation of glycolytic flux by activation of phosphofructokinase, and accordingly we determined a higher activity of this enzyme. Furthermore the rate of [(14)C]-sucrose driven starch biosynthesis in developing castor-bean is significantly increased by feeding of adenosine. In conclusion, our data indicate that adenosine imported from mobilizing endosperm into developing castor-bean cotyledons fulfils an important function as it promotes anabolic reactions in this rapidly developing tissue.     

Remodelling of triacylglycerols in microsomal preparations from developing castor bean (Ricinus communis L.) endosperm

Microsomal preparations from developing castor bean (Ricinus communis L.) endosperm catalyzed remodelling of in-situ-formed triacylglycerol (TAG) species. Castor bean microsomal membranes synthesized [¹⁴C]TAGs from either glycerol 3-phosphate and [¹⁴C]ricinoleoyl-CoA or [¹⁴C]glycerol 3-phosphate and ricinoleoyl-CoA. Upon repelleting and subsequent incubation of the microsomes a redistribution occurred of both the [¹⁴C]glycerol and [¹⁴C]ricinoleoyl moieties of the in-situ-synthesized [¹⁴C]TAGs. Radioactivity was transferred from TAG species with three (3HO-TAG) or two (2HO-TAG)ricinoleoyl groups into species with two or one (HO-TAG) ricinoleoyl groups. Mass analysis of the lipid and fatty acid movements in the membranes showed that a net synthesis of TAGs with no, one and two ricinoleoyl groups occurred at the expense of 3HO-TAG and polar lipids. Thus, the non-hydroxylated acyl groups from polar lipids were used in the remodelling of TAGs. In-vivo feeding of [¹⁴C]ricinoleic acid to slices of castor bean endosperm demonstrated the presence of two radioactive pools of TAGs one in the oil bodies, which was rich in [¹⁴C]3HO-TAG, and one associated with the microsomal membranes, which was dominated by radioactive 1HO-TAG and 2HO-TAG. The microsomal TAG pool was remodelled in vivo in a similar way as in the in-vitro experiments with microsomal membranes. Received: 8 November 1996 / Accepted: 5 February 1997     

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.     

Bioethanol production with carboxymethylcellulase of Pseudomonas poae using castor bean (Ricinus communis L.) cake

Increased consumption of fossil fuels is an emerging problem. Scientists look for the existence of other alternatives to fossil fuels, including so-called renewable energy. Accordingly, we report the production of bio-ethanol from the remnants of castor oil bean seed cake (CBC) by the carboxymethylcellulase enzyme (CMCase). A bacterial strain isolated from rice straw showing higher CMCase activity was identified. The 16S rRNA result showed a 93% homology with the 16SrRNA gene sequences of Pseudomonas poae RE11-1-14, the strain was identified as Pseudomonas poae AB3. In addition, our results showed that the highest enzyme activity was achieved after 48 h and inoculum size of 3.7 × 10⁵ CFU. The optimum temperature, pH and Carboxymethylcellulose (CMC) concentration for the highest enzyme activity was 25 °C, pH 7 and 10 g/l respectively. Furthermore, The CMCase was purified by ammonium sulphate at a concentration of 60%. The SDS-PAGE of the purified enzyme showed a molecular weight of 88 kDa. Additionally, the (CBC) was hydrolyzed by the purified CMCase at the enzyme optimum conditions. The results showed the liberation of 5.2 g/L of reducing sugar by using dinitrosalicylic acid (DNS) assay. Finally, the total sugar produces 35 g/L after 48 h when Saccharomyces cerevisiae was used as a fermentation agent. Hence for the first time, we have been successfully able to produce bioethanol from CBC with CMCase of Pseudomonas poae.     

Genetic diversity of castor bean (Ricinus communis L.) in Northeast China revealed by ISSR markers

Inter-Simple Sequence Repeat (ISSR) markers were employed to analyze the genetic diversity of Ricinus communis L. in northeastern China plants. We selected ten primers that produced clear, reproducible and multiple bands for these experiments and 179 bands were obtained across 39 genotypes. Polymorphic band ratios ranged from 100% to a minimum of 78.9% with an average of 96.4% while band numbers were comprised between 13 (UBC823) and 23 (UBC856). The results obtained from UPGMA clustering dendrogram and PCoA lead to 39 distinct castor bean accessions belonging to four major groups. We found that all groups shared a common node with 66% similarity while Jaccard's similarity coefficient ranged from 0.58 to 0.92. Compatible inference was also observed from the high values of heterozygosity (Ht = 0.3378 ± 0.0218), Nei's genetic diversity (H = 0.1765 ± 0.2090), and Shannon's information index (I = 0.4942 ± 0.1872). In addition, our data reveal a Nei's genetic differentiation index (G_ST) of 0.3452 and estimated the gene flow (Nm) at 0.9482. These findings clearly suggest a genetic diversity in castor bean germplasms from various geographic origins and contribute to our understanding of breeding and conservation of castor beans.     

Development of an efficient protoplast isolation and transfection system for castor bean (Ricinus communis L.)

Plant Cell, Tissue and Organ Culture (PCTOC) - Castor bean is an oil crop plant (Euphorbiaceae) found across the tropical, subtropical, and temperate regions. Despite its important oil properties...     

Characterization of the activity of fatty-acid epoxide hydrolase in seeds of castor bean (Ricinus communis L.)

Epoxide hydrolase (EC 3.3.2.3) activity was measured with [1-¹⁴C]cis-9,10-epoxystearic acid as the substrate. Homogenates were prepared from the endosperm tissue of germinating seeds of castor bean (Ricinus communis L. zanzibariensis). The activity of fatty-acid epoxide hydrolase was characterized with respect to dependence on time, amount of protein, pH and temperature. Analyses of enzyme distribution in endosperm, cotyledons, root and hypocotyl showed the highest total activity in the endosperm, less in the cotyledons and low activity in the root and hypocotyl. The specific activity was similar for cotyledons and endosperm. Analysis of the temporal expression of the enzyme in the endosperm during germination revealed high activity already in the imbibed seed. Activity was maximal between days four to six and then decreased at the end of one week. Subcellular fractionation of endosperm revealed a dual distribution of activity between the glyoxysomal and the cytosolic fractions.     

Diurnal changes in assimilate concentrations and fluxes in the phloem of castor bean (Ricinus communis L.) and tansy (Tanacetum vulgare L.)

Reports about diurnal changes of assimilates in phloem sap are controversial. We determined the diurnal changes of sucrose and amino acid concentrations and fluxes in exudates from cut aphid stylets on tansy leaves (Tanacetum vulgare), and sucrose, amino acid and K(+) concentrations and fluxes in bleeding sap of castor bean pedicel (Ricinus communis). Approximately half of the tansy sieve tubes exhibited a diurnal cycle of sucrose concentrations and fluxes in phloem sap. Data from many tansy plants indicated an increased sucrose flux in the phloem during daytime in case of low N-nutrition, not at high N-nutrition. The sucrose concentration in phloem sap of young Ricinus plants changed marginally between day and night, whereas the sucrose flux increased 1.5-fold during daytime (but not in old Ricinus plants). The amino acid concentrations and fluxes in tansy sieve tubes exhibited a similar diurnal cycle as the sucrose concentrations and fluxes, including their dependence on N-nutrition. The amino acid fluxes, but not the concentrations, in phloem sap of Ricinus were higher at daytime. The sucrose/amino acid ratio showed no diurnal cycle neither in tansy nor in Ricinus. The K(+)-concentrations in phloem sap of Ricinus, but not the K(+) fluxes, decreased slightly during daytime and the sucrose/K(+)-ratio increased. In conclusion, a diurnal cycle was observed in sucrose, amino acid and K(+) fluxes, but not necessarily in concentrations of these assimilates. Because of the large variations between different sieve tubes and different plants, the nutrient delivery to sink tissues is not homeostatic over time.     

Endogenous abscisic acid signaling towards storage reserve filling in developing seed tissues of castor bean (Ricinus communis L.)

Abscisic acid (ABA; free form) is a naturally occurring physiological growth hormone of higher plants. A detailed study involving the time course growth of developing seed tissues associated with endogenous levels of free ABA were investigated using a novel enzyme-linked immunosorbent assay. Seed filling in castor (Ricinuc communis L.) endosperm, embryo, and pod is marked with a rapid increase in fresh weight during the mid-developmental stages [21–42 days after pollination (DAP)], followed by a steady decline at the maturation stages (42–63 DAP) accompanied with a rapid lipid synthesis (in endosperm and embryo) during the same period, except for in pod. Endogenous ABA levels in endosperm (0.001–0.32 μg/g) and embryo (0.003–0.13 μg/g) followed a concurrent pattern with seed reserve filling, showing a rapid increase during the mid-developmental stages 21–42 DAP, whereas ABA levels in seed pod (0.2–22.9 μg/g) showed a different accumulation pattern with rapid increase and decline during the early-mid developmental stages, preceded by the maximal increase during the maturation stage (63 DAP). Together, our results provide evidence for the association of endogenous ABA in seed filling as well as in reserve deposition and provides clue for the effective usage of exogenous ABA concentrations in developing seeds with a focus, on improving seed reserve complex in castor.     

Development of EST-SSR markers in castor bean (Ricinus communis L.) and their utilization for genetic purity testing of hybrids

Expressed sequence tag (EST) databases offer opportunity for the rapid development of simple sequence repeat (SSR) markers in crops. Sequence assembly and clustering of 57?895 ESTs of castor bean resulted in the identification of 10?960 unigenes (6459 singletons and 4501 contigs) having 7429 SSRs. On an average, the unigenes contained 1 SSR for every 1.23?kb of unigene sequence. The identified SSRs mostly consisted of dinucleotide (62.4%) and trinucleotide (33.5%) repeats. The AG class was the most common among the dinucleotide motifs (68.9%), whereas the AAG class (25.9%) was predominant among the trinucleotide motifs. A total of 611 primer pairs were designed for the SSRs, having repeat length more than or equal to 20 nucleotides, of which a set of 130 markers were tested and 92 of these yielding robust amplicons were analyzed for their utility in genetic purity assessment of castor bean hybrids. Nine markers were able to detect polymorphism between the parental lines of nine commercial castor bean hybrids (DCH-32, DCH-177, DCH-519, GCH-2, GCH-4, GCH-5, GCH-6, GCH-7, and RHC-1), and their utility in genetic purity testing was demonstrated. These novel EST-SSR markers would be a valuable addition to the growing molecular marker resources that could be used in genetic improvement programmes of castor bean.     

The amino acid sequence of sesame (Sesamum indicum L.) and castor (Ricinus communis L.) cytochrome c

The amino acid sequences of sesame (Sesamum indicum L.) and castor (Ricinus communis L.) cytochrome c were determined by using 1.5mumol of protein from each species. Both molecules consist of a single chain of 111 amino acid residues and are homologous with other mitochondrial cytochrome c molecules. Both have an N-acetylated ;tail' of eight amino acids and two in-N-trimethyl-lysine residues, as also reported for wheat germ (Delange, Glazer & Smith, 1969) and mung-bean cytochrome c (Thompson, Laycock, Ramshaw & Boulter, 1970). Two different preparations of castor cytochrome c differed by one residue. This was glutamic acid for glutamine in position 100. The results for sesame and castor cytochrome c led to a re-examination and subsequent correction to the N-terminal region of the mung-bean cytochrome c sequence, as given by Thompson et al. (1970).     

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.     

Role of biotechnological interventions in the improvement of castor (Ricinus communis L.) and Jatropha curcas L

Castor and Jatropha belong to the Euphorbiaceae family. This review highlights the role of biotechnological tools in the genetic improvement of castor and jatropha. Castor is monotypic and breeding programmes have mostly relied on the variability available in the primary gene pool. The major constraints limiting profitable cultivation are: vulnerability to insect pests and diseases, and the press cake is toxic which restrict its use as cattle feed. Conventional breeding techniques have limited scope in improvement of resistance to biotic stresses and in quality improvement owing to low genetic variability for these traits. Genetic diversity was assessed using protein based markers while use of molecular markers is at infancy. In vitro studies in castor have been successful in shoot proliferation from meristematic explants, but not callus-mediated regeneration. Genetic transformation experiments have been initiated for development of insect resistant and ricin-free transgenics with very low transformation frequency. In tropical and subtropical countries jatropha is viewed as a potential biofuel crop. The limitations in available germplasm include; lack of knowledge of the genetic base, poor yields, low genetic diversity and vulnerability to a wide array of insects and diseases. Great scope exists for genetic improvement through conventional methods, induced mutations, interspecific hybridization and genetic transformation. Reliable and highly efficient tissue culture protocols for direct and callus-mediated shoot regeneration and somatic embryogenesis are established for jatropha which indicates potential for widening the genetic base through biotechnological tools. Assessment of genetic diversity using molecular markers disclosed low interaccessional variability in local Jatropha curcas germplasm. The current status and future prospects of in vitro regeneration, genetic transformation and the role of molecular tools in the genetic enhancement of the two-oilseed crops are discussed.     

Effects of P deficiency on assimilation and transport of nitrate and phosphate in intact plants of castor bean (Ricinus communis L.)

An experimentally-based modelling technique was applied to describequantitatively the uptake, translocation, storage, and assimilationof     

Defense response of resistant and susceptible genotypes of castor (Ricinus communis L.) to wilt disease

The biochemical basis of resistance in castor (Ricinus communis L.) to Fusarium wilt, caused by the pathogen Fusarium oxysporum f. sp. ricini, was investigated. Induction of plant defence against pathogen attack is regulated by a complex network of different signals. Thus changes in various biochemical defenses including antioxidant enzymes, phenolic compounds and pathogenesis related (PR) proteins were investigated in the roots of resistant and susceptible genotypes of castor at 0, 24, 48 and 72 h.a.i. Infection by F. oxysporum significantly increased the superoxide dismutase (SOD) and peroxidase (POX) activities in the roots of susceptible genotypes, while the catalase (CAT) activities were appreciably higher in the roots of resistant genotypes at different stages. Constitutive levels of ascorbate peroxidase (APX) and polyphenol oxidase (PPO) were higher in the resistant genotypes. Also, the activities of phenylalanine ammonia lyase (PAL) and β 1, 3 glucanase significantly increased in the roots of the resistant genotypes after infections. The rate of increment of thiobarbituric acid reactive substances (TBARS) was higher in resistant genotypes after infection. Analysis of isozyme banding pattern of SOD, POX, PPO and esterase on native PAGE electrophoresis revealed that interaction between plant and fungi invoked various isozymes at 48 h of infection. SOD 3 was observed only in resistant genotypes at 24 h.a.i. except Geeta. Similarly induction of POX 5 was observed only in resistant genotypes at 48 h of infection, though the intensity of POX 5 was very less.