Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds.

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds (Ricinus communis) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca2+-dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC's BTPC subunit's at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca2+-dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: (i) a pair of Ca2+ binding sites with identical dissociation constants of 5.03 µM, (ii) a Ca2+-dependent electrophoretic mobility shift, and (iii) a marked Ca2+-independent hydrophobicity. Pull-down experiments established the Ca2+-dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca2+-dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis (Arabidopsis thaliana) CPK4 and soybean (Glycine max) CDPKß are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca2+ signaling and the posttranslational control of respiratory CO2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS.

Components of complex lipid biosynthetic pathways in developing castor (Ricinus communis) seeds identified by MudPIT analysis of enriched endoplasmic reticulum.

Ricinoleic acid is a feedstock for nylon-11 (N11) synthesis which is currently obtained from castor (Ricinus communis) oil. Production of this fatty acid in a temperate oilseed crop is of great commercial interest, but the highest reported level in transgenic plant oils is 30%, below the 90% observed in castor and insufficient for commercial exploitation. To identify castor oil-biosynthetic enzymes and inform strategies to improve ricinoleic acid yields, we performed MudPIT analysis on endoplasmic reticulum (ER) purified from developing castor bean endosperm. Candidate enzymes for all steps of triacylglycerol synthesis were identified among 72 proteins in the data set related to complex-lipid metabolism. Previous reported proteomic data from oilseeds had not included any membrane-bound enzyme that might incorporate ricinoleic acid into oil. Analysis of enriched ER enabled determination of which protein isoforms for these enzymes were in developing castor seed. To complement this data, quantitative RT-PCR experiments with castor seed and leaf RNA were performed for orthologues of Arabidopsis oil-synthetic enzymes, determining which were highly expressed in the seed. These data provide important information for further manipulation of ricinoleic acid content in oilseeds and peptide data for future quantification strategies.

Removal of Ni (II) from aqueous solutions by adsorption onto Ricinus communis seed shell activated carbons.

The adsorption studies on the removal of Ni(II) from aqueous solution using Ricinus communis seed shells activated carbon and polypyrrole coated Ricinus communis seed shells activated carbon were carried out under various experimental conditions. The effects of various process parameters have been investigated by following the batch adsorption technique. Adsorption data was modeled with Freundlich, Langmuir and tempkin adsorption isotherms. Thermodynamics parameters such as DeltaH0, DeltaS0, and DeltaG0 were calculated indicating that the adsorption was spontaneous and endothermic nature. A mechanism, involving intra particle diffusion and surface adsorption, has been proposed for the adsorption of Ni(II) onto the adsorbent. Adsorbent used in this study is characterized by FTIR and SEM before and after the adsorption of metal ions.

Bacterial- and plant-type phosphoenolpyruvate carboxylase polypeptides interact in the hetero-oligomeric Class-2 PEPC complex of developing castor oil seeds.

Two classes of phosphoenolpyruvate carboxylase (PEPC) sharing the same 107-kDa catalytic subunit (p107) were previously purified from developing castor oil seed (COS) endosperm. The association of p107 with an immunologically unrelated 64-kDa polypeptide (p64) causes pronounced physical and kinetic differences between the Class-1 PEPC p107 homotetramer and Class-2 PEPC p107/p64 hetero-octamer. Tryptic peptide sequencing matched p64 to the deduced C-terminal half of several bacterial-type PEPCs (BTPCs) of vascular plants. Immunoblots probed with anti-(COS p64 peptide or p107)-IgG established that: (i) BTPC exists in vivo as an approximately 118-kDa polypeptide (p118) that is rapidly truncated to p64 by an endogenous cysteine endopeptidase during incubation of COS extracts on ice, and (ii) mature and germinated COS contain Class-1 PEPC and p107, but no detectable Class-2 PEPC nor p118. Non-denaturing PAGE, in-gel PEPC activity staining and immunoblotting of developing COS extracts demonstrated that p118 and p107 are subunits of the non-proteolysed approximately 910-kDa Class-2 PEPC complex. As total PEPC activity of clarified COS extracts was unaffected following p118 truncation to p64, the BTPC p118 may function as a regulatory rather than catalytic subunit of the Class-2 PEPC. Moreover, recombinant AtPPC3 and AtPPC4 (Arabidopsis orthologs of COS p107 and p118) expressed as active and inactive PEPCs, respectively. Cloning of cDNAs encoding p118 (RcPpc4) and p107 (RcPpc3) confirmed their respective designation as bacterial- and plant-type PEPCs. Levels of RcPpc3 and RcPpc4 transcripts generally mirrored the respective amounts of p107 and p118. The collective findings provide insights into the molecular features and functional significance of vascular plant BTPCs.

Stable genetic transformation of castor (Ricinus communis L.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds.

A protocol for the transformation of castor embryo axes using the pCAMBIA vector 1304 in disarmed Agrobacterium tumefaciens strain EHA105 is presented. Co-cultivated explants were initially subjected to expansion and proliferation on MS medium with 0.5 mg l(-1) TDZ followed by three cycles of selection on medium with 0.5 mg l(-1) BA and increasing concentrations of hygromycin (20-40-60 mg l(-1)). Selected shoot clusters were transferred to medium with 0.5 mg l(-1) BA for proliferation and 0.2 mg l(-1) BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium with 2.0 mg l(-1) NAA. The presence and stable integration of the hpt gene was confirmed through PCR, RT-PCR, PCR-Southern blot, sequence analysis, Southern blot analysis and PCR analysis of progeny. Southern blot analysis of the primary transformants showed single copy integration and progeny analysis revealed monogenic inheritance of the introduced gene. This paper reports the first successful attempt at producing transgenic castor.

A metal-binding member of the late embryogenesis abundant protein family transports iron in the phloem of Ricinus communis L.

The transport of metal micronutrients to developing organs in a plant is mediated primarily by the sieve elements. Ligands are thought to form complexes with the free ions in order to prevent cellular damage, but no binding partners have been unequivocally identified from plants so far. This study has used the phloem-mediated transport of micronutrients during the germination of the castor bean seedling to identify an iron transport protein (ITP). It is demonstrated that essentially all (55)Fe fed to seedlings is associated with the protein fraction of phloem exudate. It is shown that ITP carries iron in vivo and binds additional iron in vitro. ITP was purified to homogeneity from minute amounts of phloem exudate using immobilized metal ion affinity chromatography. It preferentially binds to Fe(3+) but not to Fe(2+) and also complexes Cu(2+), Zn(2+), and Mn(2+) in vitro. The corresponding cDNA of ITP was cloned using internal peptide fragments. The deduced protein of 96 amino acids shows high similarity to the stress-related family of late embryogenesis abundant proteins. Its predicted characteristics and its RNA expression pattern are consistent with a function in metal ion binding. The ITP from Ricinus provides the first identified micronutrient binding partner for phloem-mediated long distance transport in plants and is the first member of the late embryogenesis abundant protein family shown to have such a function.

Ricinoleic acid biosynthesis and triacylglycerol assembly in microsomal preparations from developing castor-bean (Ricinus communis) endosperm.

Microsomal membrane preparations from the developing endosperm of castor bean (Ricinus communis) catalysed the transfer of oleate from [14C]oleoyl-CoA to phosphatidylcholine (PtdCho). In the presence of NADH, radioactive ricinoleate (12-hydroxyoctadec-9-enoate) was synthesized from [14C]oleate, and this was largely recovered in PtdCho and as free fatty acid. The addition of unlabelled ricinoleoyl-CoA to these incubation mixtures did not increase the low [14C]ricinoleate concentration found in the acyl-CoA fraction nor decrease the [14C]ricinoleate concentration in PtdCho and free fatty acid, and thus no evidence was obtained for a hydroxylation with oleoyl-CoA as a substrate. The addition of NADH, necessary for the formation of ricinoleate, caused a decrease of the total radioactivity in PtdCho with a corresponding increase in the amount of label in free ricinoleic acid. This increase was due to the action of a phospholipase A, which released ricinoleic acid but not oleic acid from PtdCho. Such a phospholipase activity, attacking ricinoleoyl-PtdCho but not oleoyl-PtdCho, was also demonstrated in microsomal preparations from developing cotyledons of safflower and oil-seed rape. An analysis of the acyl groups at different positions in microsomal PtdCho of castor bean showed that ricinoleate was almost entirely associated with position sn-2. Likewise the [14C]ricinoleate in [14C]PtdCho formed after incubations with microsomal preparations with NADH and [14C]oleoyl-CoA resided in position sn-2 with none in position sn-1. In contrast, the [14C]linoleate formed by desaturation of [14C]oleoyl-PtdCho was present at both positions. In the presence of ATP, CoA and Mg2+, the ricinoleate acid released from PtdCho was activated to ricinoleoyl-CoA. The ricinoleoyl-CoA was an efficient acyl donor in the acylation of glycerol 3-phosphate (Gro3P) to yield phosphatidic acid and triacylglycerols. In microsomal preparations incubated with an equimolar mixture of [14C]oleoyl-CoA and [14C]ricinoleoyl-CoA in the presence of Gro3P, only a minor amount of [14C]ricinoleate entered PtdCho, and this was believed to be via the exchange of phosphocholine groups between a diacylglycerol pool and the PtdCho. On the basis of our results, a scheme of ricinoleate formation and its incorporation into triacylglycerols in castor-bean endosperm is proposed.