Germinating Castor Oil Seeds Research Articles

Characterization of bacterial-type phosphoenolpyruvate carboxylase expressed in male gametophyte of higher plants

BackgroundPhosphoenolpyruvate carboxylase (PEPC) is a critical enzyme catalyzing the β-carboxylation of phosphoenolpyruvate (PEP) to oxaloacetate, a tricarboxylic acid (TCA) cycle intermediate. PEPC typically exists as a Class-1 PEPC homotetramer composed of plant-type PEPC (PTPC) polypeptides, and two of the subunits were reported to be monoubiquitinated in germinating castor oil seeds. By the large-scale purification of ubiquitin (Ub)-related proteins from lily anther, two types of PEPCs, bacterial-type PEPC (BTPC) and plant-type PEPC (PTPC), were identified in our study as candidate Ub-related proteins. Until now, there has been no information about the properties of the PEPCs expressed in male reproductive tissues of higher plants.ResultsExpression analyses showed that lily BTPC (LlBTPC) and Arabidopsis BTPC (AtBTPC) were significantly expressed in pollen. The fusion protein AtBTPC-Venus localized in the cytoplasm of the vegetative cell (VC). Both LlBTPC and AtBTPC expression initiated after the last mitosis before pollen germination. Lily PTPC (LlPTPC) and monoubiquitinated LlPTPC (Ub-LlPTPC) remained at constant levels during pollen development. In late bicellular pollen of lily, LlBTPC forms a hetero-octameric Class-2 PEPC complex with LlPTPC to express PEPC activity.ConclusionOur results suggest that an LlBTPC:Ub-LlPTPC:LlPTPC complex is formed in the VC cytoplasm during late pollen development. Both LlBTPC and AtBTPC expression patterns are similar to the patterns of the appearance of storage organelles during pollen development in lily and Arabidopsis, respectively. Therefore, BTPC is thought to accelerate the metabolic flow for the synthesis of storage substances during pollen maturation. Our study provides the first characterization of BTPC in pollen, the male gametophyte of higher plants.

Regulatory Monoubiquitination of Phosphoenolpyruvate Carboxylase in Germinating Castor Oil Seeds

Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated enzyme situated at the core of plant C-metabolism. Although its anaplerotic role and control by allosteric effectors, reversible phosphorylation, and oligomerization have been well documented in the endosperm of developing castor oil seeds (COS), relatively little is known about PEPC in germinating COS. The initial phase of COS germination was accompanied by elevated PEPC activity and accumulation of comparable amounts of pre-existing 107-kDa and inducible 110-kDa immunoreactive PEPC polypeptides (p107 and p110, respectively). A 440-kDa PEPC heterotetramer composed of an equivalent ratio of non-phosphorylated p110 and p107 subunits was purified from germinated COS. N-terminal microsequencing, mass spectrometry, and immunoblotting revealed that both subunits arose from the same gene (RcPpc3) that encodes the p107 subunit of a phosphorylated 410-kDa PEPC homotetramer in developing COS but that p110 is a monoubiquitinated form of p107. Tandem mass spectrometry sequencing of a diglycinated tryptic peptide identified Lys-628 as p110's monoubiquitination site. This residue is conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Incubation with a human deubiquitinating enzyme (USP-2 core) converted the p110:p107 PEPC heterotetramer into a p107 homotetramer while significantly reducing the enzyme's K(m)(PEP) and sensitivity to allosteric activators (hexose-Ps, glycerol-3-P) and inhibitors (malate, aspartate). Monoubiquitination is a non-destructive and reversible post-translational modification involved in the control of diverse processes such as transcription, endocytosis, and signal transduction. The current study demonstrates that tissue-specific monoubiquitination of a metabolic enzyme can also occur and that this modification influences its kinetic and regulatory properties.

Activity and concentration of non‐proteolyzed phosphoenolpyruvate carboxykinase in the endosperm of germinating castor oil seeds: effects of anoxia on its activity

Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.49) catalyses the reversible decarboxylation of oxaloacetate to phosphoenolpyruvate in the gluconeogenic production of sugars from storage lipids in germinating oil seeds. The enzyme is quite susceptible to limited proteolysis during extraction. Immunoblotting was used to diagnose unwanted in vitro proteolytic activity against PEPCK from germinating castor oil seeds (COS) by following the disappearance of its native 74‐kDa subunit and concomitant appearance of a truncated 64‐kDa polypeptide. Alkaline pH and the inclusion of thiol protease inhibitors effectively prevented COS PEPCK proteolysis during incubation of clarified COS extracts at 4°C. The carboxylating and decarboxylating activities and concentration of non‐proteolyzed COS PEPCK were investigated during germination. This is the first report in which both activities catalyzed by PEPCK were measured in vitro during a whole developmental process. Carboxylating activity and the level of immunoreactive 74‐kDa PEPCK polypeptides rapidly increased in parallel to maximal values by day 5 and then significantly declined over the subsequent 2 days. In contrast, decarboxylating PEPCK activity was much higher over the 7 days of growth examined. In addition, the effect on PEPCK activity while changing the endosperm from aerobic (when gluconeogenesis predominates in the tissue) to anaerobic conditions (where the tissue becomes glycolytic) was studied. While PEPCK decarboxylating activity remained almost constant, carboxylating activity declined to undetectable levels in response to anaerobiosis. These and the developmental profile results suggest that COS PEPCK may be subject to a mechanism of post‐translation control that selectively inhibits the carboxylating, but not the decarboxylating activity.

A fluorescence study of ligand-induced conformational changes in cytosolic fructose-1,6-bisphosphatase from germinating castor oil seeds

The intrinsic fluorescence of homogeneous castor oil seed cytosolic fructose-1,6-bisphosphatase (FBPase c) was used as an indicator of conformational changes due to ligand binding. Binding of the substrate and the inhibitor fructose-2,6-bisphosphate (F-2,6-P 2) was quantitatively compared to their respective kinetic effects on enzymatic activity. There are two distinct types of substrate interaction with FBPase c, corresponding to catalytic and inhibitory binding, respectively. Inhibitory substrate binding shares several characteristics with F-2,6-P 2 binding which indicates that both ligands bind at the same site. However, F-2,6-P 2 does not prevent fluorescence transitions attributed to catalytic substrate binding. The marked synergistic inhibition of FBPase c by AMP and F-2,6-P 2 appears to arise via AMP’s promotion of F-2,6-P 2 binding. Based on the X-ray crystal structure of porcine kidney FBPase our modelling studies suggest the existence of a distinct F-1,6-P 2/F-2,6-P 2 inhibitory binding site which partially overlaps with the enzyme’s catalytic site. We propose that a pronounced allosteric transition mediated by AMP binding increases access of F-1,6-P 2 and F-2,6-P 2 to this common inhibitory binding site.

Effect of polyethylene glycol on the activity, intrinsic fluorescence, and oligomeric structure of castor seed cytosolic fructose-1, 6-bisphosphatase

The effect of polyethylene glycol (PEG) on the activity, intrinsic fluorescence, and oligomeric structure of homogeneous cytosolic fructose-1, 6-bisphosphatase (FBPase c) from endosperm of germinating castor oil seeds has been examined. Increasing the PEG concentration in the FBPase c reaction mixture elicited a progressive 3-fold decrease in the enzyme'sK m for fructose-1, 6-P 2. The presence of PEG also: (i) increased the extent of FBPase c inhibition by high levels of fructose-1, 6-P 2, (ii) enhanced the intensity of the enzyme's fluorescence emission spectra, and (iii) prevents dissociation of the active tetrameric native enzyme into inactive lowerM r forms during gel filtration HPLC. It is concluded that the activity and structure of plant FBPase c is modified by extreme dilution, probably as a result of partial deaggregation of the native tetrameric enzyme.

Characterization of asparaginyl endopeptidase activity in endosperm of developing and germinating castor oil seeds

A spectrophotometric assay was devised to characterize the asparaginyl (Asn) endopeptidase activity from the endosperm of castor oil seeds. (Ricinus communis L. var. Baker 296). The assay measures the release of p‐nitroaniline from the hydrolysis of benzoyl‐l‐Asn‐p‐nitroanilide. Assay sensitivity was improved through diazotization of the reaction product with N(]‐napthy])‐ethylenediamine dihydrochloride: diazotized p‐nitroaniline was determined spectrophotometrically at 548 nm (ɛ548= 1.64 × 10−1M−1 cm−2). By using this assay. Asn endopeptidase activity was detected in endosperm extracts of developing, mature and germinating castor seeds. Comparison of the Asn endopeptidase activities of developing and germinating castor endosperms revealed that they: 1) have identical pH‐activity profiles with optimal activity occuring at pH 5.4: 2) are heat‐labile proteins displaying comparable thermal stability profiles, and 3) are activated and inhibited by dithiothreitol and thiol modifying reagents, respectively. Thus, the Asn endopeptidases of developing and germinating castor seeds are very similar, if not identical, cysteine proteases. The most significant increase in the activity of endosperm Asn endopeptidase occurs during the full coryledon to maturation stage of seed development, this period coincides with the most active phase of reserve protein accumulation by ripening castor oil seeds. Asn endopeptidase activity of fully mature (dry) castor seeds was about 2‐fold lower than that of muturation stage ripening castor oil seed. Asn endopeptidase activity showed a slight reduction over the inicial 2‐day period following seed imbibition, and then rapidly decreased over the next several days of germination. The results are compatible with the proposal that Asn endopeptidase functions both to process storage preproteins following their import into protein bodies of developing seeds, as well as to participate in the mobilization of storage proteins during the early phase of seed germination.

Characterization of asparaginyl endopeptidase activity in endosperm of developing and germinating castor oil seeds

Characterization of asparaginyl endopeptidase activity in endosperm of developing and germinating castor oil seeds

Copurification of Cytosolic Fructose-1,6-bisphosphatase and Cytosolic Aldolase from Endosperm of Germinating Castor Oil Seeds

The cytosolic isozymes of fructose-1,6-bisphosphatase (FBPase c) and aldolase (ALD c) from germinating castor oil seed endosperm (COS) ( Ricinus communis L.; cv Hale) were purified to homogeneity and final specific activities 49 and 2.8 (μmol product produced/min)/mg protein, respectively. Nondenaturing polyacrylamide gel electrophoresis of the final FBPase c preparation resolved a single protein-staining band which comigrated with FBPase activity. Two protein-staining bands of 41 and 39 kDa that occurred in an approximate 1:1 ratio were observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final FBPase c preparation. Rabbit anti-(FBPase c) immune serum immunoprecipitated the activities of FBPase c, but not that of the plastidic isozyme of FBPase from germinated COS. Immunoblot analysis utilizing affinity purified anti-(COS FBPase c) immunoglobulin G established that the 39-kDa subunit of FBPase c did not arise via proteolytic cleavage of the 41-kDa subunit during tissue extraction and enzyme purification. However, FBPase c was susceptible to degradation by endogenous protease(s) during incubation of an acidic (pH 5.9) clarified COS extract at 25°C. This proteolysis caused the production of a 32-kDa antigenic polypeptide and resulted in FBPase inactivation. Gel filtration indicated that purified FBPase c exists in at least 8 different oligomeric forms ranging in size from >2 million to <34 kDa. The majority of FBPase c, however, eluted as a 143-kDa heterotetramer. Sodium dodecyl sulfate gel electrophoresis of the final ALD c preparation yielded a single 40-kDa protein-staining polypeptide that cross-reacted with anti-(carrot ALD c) IgG. FBPase c copurified with ALD c through polyethylene glycol fractionation, Q-Sepharose, and phosphocellulose chromatographies, and the intensity of the fluorescence emission spectrum of ALD c was greatly reduced in the presence of COS FBPase c, but not rabbit muscle FBPase. These findings suggest that these two metabolically sequential enzymes might specifically interact in the cytosol of the highly gluconeogenic germinating COS. Our results also demonstrate that endogenous nonspecific acid phosphatase activity can interfere with the spectrophotometric assay for FBPase and can thus result in overestimations of FBPase activity in impure plant extracts.

Plant cytosolic pyruvate kinase: a kinetic study

The kinetic properties of cytosolic pyruvate kinase (PK c) from germinating castor oil seeds (COS) have been investigated. From experiments in which the free Mg 2+ concentration was varied at constant levels of either the complexed or free forms of the substrates it was determined that the true substrates are the free forms of both phospho enolpyruvate (PEP) and ADP. This conclusion is corroborated by the quenching of intrinsic PK c tryptophan fluorescence by free PEP and ADP. Mg 2+ is bound as the free bivalent cation but is likely released as MgATP. The fluorescence data, substrate interaction kinetics, and pattern of inhibition by products and substrate analogues (adenosine rm5′- O-(2-thiodiphosphate) for ADP and phenyl phosphate for PEP) are compatible with a sequential, compulsory-ordered, Tri-Bi type kinetic reaction mechanism. PEP is the leading substrate, and pyruvate the last product to abandon the enzyme. The dissociation constant and limiting Km for free PEP (8.2 to 22 and 38 μM, respectively) and the limiting K m for free ADP (2.9 μM) are considerably lower than those reported for the non-plant enzyme. The results indicate that COS PK c exists naturally in an activated state, similar to the fructose 1,6-bisphosphate-activated yeast enzyme. This deduction is consistent with a previous study (F.E. Podestá and W.C. Plaxton (1991) Biochem. J. 279, 495–501) that failed to identify any allosteric activators for the COS (PK c), but which proposed a regulatory mechanism based upon ATP levels and pH-dependent alterations in the enzyme's response to various metabolite inhibitors. As plant phosphofructokinases display potent inhibition by PEP, the overall rate of glycolytic flux from hexose 6-phosphate to pyruvate in the plant cytosol will ultimately depend upon variations in PEP levels brought about by the regulation of PK c.

Phosphoenolpyruvate Carboxylase Activity and Concentration in the Endosperm of Developing and Germinating Castor Oil Seeds

Monospecific polyclonal antibodies against maize leaf phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were utilized to examine the subunit composition and developmental profile of endosperm PEPC in developing and germinating castor oil seeds (Ricinus communis L. cv Baker 296). PEPC from developing endosperm consists of a single type of 100-kilodalton subunit, whereas the enzyme from 2- to 5-day germinated endosperm appears to contain equal proportions of immunologically related 103- and 108-kilodalton subunits. The maximal activity of PEPC in developing endosperms (2.67 micromoles oxaloacetate produced per minute per gram fresh weight) is approximately 20-fold and threefold greater than that of fully mature (dry seed) and germinating endosperms, respectively. The most significant increase in the activity and concentration of endosperm PEPC occurs during the middle cotyledon to full cotyledon stage of seed development; this period coincides with the most active phase of storage oil accumulation by ripening castor oil seeds. The data are compatible with the recent proposal (RG Smith, DA Gauthier, DT Dennis, DH Turpin [1992] Plant Physiol 1233-1238) that PEPC plays a fundamental role in vivo in the cytosolic production of an important substrate (malate) for fatty acid biosynthesis by developing castor oil seed leucoplasts. Immediately following seed imbibition, PEPC activity and concentration increase in parallel, with the greatest levels attained by the third day of germination. It is suggested that during this early phase of seed germination PEPC has a critical function to build up cellular dicarboxylic acid pools required to initiate significant activities of both the tricarboxylic acid and glyoxylate cycles.

Kinetic and regulatory properties of cytosolic pyruvate kinase from germinating castor oil seeds

The kinetic and regulatory properties of cytosolic pyruvate kinase (PKc) isolated from endosperm of germinating castor oil seeds (Ricinus communis L.) have been studied. Optimal efficiency in substrate utilization (in terms of Vmax/Km for phosphoenolpyruvate or ADP) occurred between pH 6.7 and 7.4. Enzyme activity was absolutely dependent on the presence of a bivalent and a univalent metal cation, with Mg2+ and K+ fulfilling this requirement. Mg2+ binding showed positive and negative co-operativity at pH 6.5 (h = 1.6) and pH 7.2 (h = 0.69) respectively. Hyperbolic saturation kinetics were observed with phosphoenolpyruvate (PEP) and K+, whereas ADP acted as a mixed-type inhibitor over 1 mM. Glycerol (10%, v/v) increased the S0.5(ADP) 2.3-fold and altered the pattern of nucleotide binding from hyperbolic (h = 1.0) to sigmoidal (h = 1.79) without modifying PEP saturation kinetics. No activators were identified. ATP, AMP, isocitrate, 2-oxoglutarate, malate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-phosphoglycerate, glycerol 3-phosphate and phosphoglycolate were the most effective inhibitors. These metabolites yielded additive inhibition when tested in pairs. ATP and 3-phosphoglycerate were mixed-type inhibitors with respect to PEP, whereas competitive inhibition was observed for other inhibitors. Inhibition by malate, 2-oxoglutarate, phosphorylated triose sugars or phosphoglycolate was far more pronounced at pH 7.2 than at pH 6.5. Although 32P-labelling studies revealed that extensive phosphorylation in vivo of soluble endosperm proteins occurred between days 3 and 5 of seed germination, no alteration in the 32P-labelling pattern of 5-day-germinated endosperm was observed after 30 min of anaerobiosis. Moreover, no evidence was obtained that PKc was a phosphoprotein in aerobic or anoxic endosperms. It is proposed that endosperm PKc activity of germinating castor seeds is enhanced after anaerobiosis through concerted decreases in ATP levels, cytosolic pH and concentrations of several key inhibitors.

Cloning and characterization of a cDNA for the cytosolic isozyme of plant pyruvate kinase: the relationship between the plant and non-plant enzyme

Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) is a key glycolytic enzyme that catalyzes the formation of pyruvate and ATP from phoshoenolpyruvate and ADP. The enzyme from several non-plant sources has been purified and studied extensively [8, 9, 12, 15, 18]. Most of these enzymes are homotetrameric with a native Mr of 190-250 kDa. In plants, pyruvate kinase occurs in both the plastid and cytosolic cellular compartments as kinetically and immunologically distinct isoforms [3, 6, 14]. The cytosolic isoform from germinating castor oil seeds has been purified to homogeneity and shown to be a heterotetramer of closely related 56 and 57 kDa subunits [13, 14]. The enzyme from developing castor endosperm has a native Mr of 240 kDa and is made up of four identical 56 kDa subunits [ 14]. The immunological relationship between all these subunits has been described in detail elsewhere [ 14]. Here we report the isolation and sequence of a cDNA clone for the cytosolic isozyme of pyruvate kinase and present a comparison of this with the enzyme from non-plant sources. Total and poly(A) + RNA was prepared from developing endosperm as described previously [ 1 ]. cDNA synthesis from this RNA and the construction of a library in 2gtl 1 was performed by Clontech Laboratories Inc., Palo Alto, California. A potato (Solanum tuberosum cv. Kennebec) 2ZAP cDNA library was kindly provided by N. Brisson, University of Montreal, Montreal, Quebec. Rabbit anti-(germinating castor bean endosperm cytosolic pyruvate kinase) IgG was prepared and affinity-purified against homogeneous plant cytosolic pyruvate kinase as described previously [14]. Both libraries were screened following the procedure outlined by Huynh [4], using an anti-rabbit alkaline phosphatase conjugate. Standard cloning procedures have been outlined elsewhere [ 11, 16]. Twentyfive clones with inserts that ranged in size from 0.5-2.0 kb for th 2ZAP potato and 0.6-2.3 kb for the 2gtl 1 castor libraries were isolated and those that contained single Eco RI inserts were selected. A single potato clone (2.0 kb) and a single castor clone (2.3 kb) were sequenced. The complete nucleotide sequence and derived amino acid sequence of the 2.0 kb potato cDNA clone is presented in Fig. 1. The nucleotide sequence contains an open reading frame (ORF) of 1530bp corresponding to a polypeptide of 510 amino acids. The identification of the translation start site was facilitated by the homology that this clone shares with the enzyme from mam-

Growth and metabolism of Ricinus communis endosperm in tissue culture

Endosperm tissue from both developing and germinating castor oil seeds (Ricinus communis L.) was grown as a callus in tissue culture. Callus growths were established from endosperm explants (without embryo) at all stages of seed development except from the quiescent unimbibed seed. Best tissue culture growth was observed with endosperm tissue obtained from seeds that had been germinated for 2 days.The lipid reserves diminished in all endosperm tissue which had been established on culture from germinating seeds but the rate of breakdown was much slower in cultures from 0-day germinated seeds. Glyoxylate cycle activity, as tested by acetate-1- and -2-14C incorporation, was not evident in long-term cultures.The process of lipid accumulation found in normal developing endosperm (24 to 36 days after fertilization) was not retained in culture but was replaced by a process resulting in the rapid loss of the already accumulated oil reserves.As far as could be determined the in vivo biochemical capabilities of the castor bean endosperm were not retained for a lengthy period in the tissue culture conditions used.