Bacterial- and plant-type phosphoenolpyruvate carboxylase isozymes from developing castor oil seeds interact in vivo and associate with the surface of mitochondria.

Phosphoenolpyruvate carboxylase (PEPC) from developing castor oil seeds (COS) exists as two distinct oligomeric isoforms. The typical class-1 PEPC homotetramer consists of 107-kDa plant-type PEPC (PTPC) subunits, whereas the allosterically desensitized 910-kDa class-2 PEPC hetero-octamer arises from the association of class-1 PEPC with 118-kDa bacterial-type PEPC (BTPC) subunits. The in vivo interaction and subcellular location of COS BTPC and PTPC were assessed by imaging fluorescent protein (FP)-tagged PEPCs in tobacco suspension-cultured cells. The BTPC-FP mainly localized to cytoplasmic punctate/globular structures, identified as mitochondria by co-immunostaining of endogenous cytochrome oxidase. Inhibition of respiration with KCN resulted in proportional decreases and increases in mitochondrial versus cytosolic BTPC-FP, respectively. The FP-PTPC and NLS-FP-PTPC (containing an appended nuclear localization signal, NLS) localized to the cytosol and nucleus, respectively, but both co-localized with mitochondrial-associated BTPC when co-expressed with BTPC-FP. Transmission electron microscopy of immunogold-labeled developing COS revealed that BTPC and PTPC are localized at the mitochondrial (outer) envelope, as well as the cytosol. Moreover, thermolysin-sensitive BTPC and PTPC polypeptides were detected on immunoblots of purified COS mitochondria. Overall, our results demonstrate that: (i) COS BTPC and PTPC interact in vivo as a class-2 PEPC complex that associates with the surface of mitochondria, (ii) BTPC's unique and divergent intrinsically disordered region mediates its interaction with PTPC, whereas (iii) the PTPC-containing class-1 PEPC is entirely cytosolic. We hypothesize that mitochondrial-associated class-2 PEPC facilitates rapid refixation of respiratory CO(2) while sustaining a large anaplerotic flux to replenish tricarboxylic acid cycle C-skeletons withdrawn for biosynthesis.

Nonspecific lipid transfer protein in castor bean cotyledon cells: subcellular localization and a possible role in lipid metabolism.

The subcellular localization and several biochemical activities of nonspecific lipid transfer protein (nsLTP) were investigated. A section of a castor bean cotyledon cell was labeled with anti-nsLTP serum followed by protein A-gold. Gold particles were more abundant in the glyoxysome matrix and the vessel cell wall than in other areas. Cell fractionation analysis of 6-day-old castor bean cotyledons by sucrose density gradient centrifugation demonstrated that 13% of nsLTP was distributed in the glyoxysomal fraction, identified on the basis of catalase as a marker, and 87% in the soluble fraction near the top of the gradient. The location of castor bean nsLTP in glyoxysomes was further confirmed by in vitro import experiments. The synthesized precursor of nsLTP (pro-nsLTP-C) was incorporated into intact castor bean glyoxysomes and processed to the mature form after import into the glyoxysomes, but it was not imported into canine pancreatic microsomes. Castor bean nsLTP-A was found to possess the ability to bind oleic acid and oleoyl-CoA by means of a method involving Lipidex 1000. The dissociation constants (Kd) for oleic acid and oleoyl-CoA binding to nsLTP-A were 4.8 and 5.0 microM, respectively. The saturated binding capacities (Bmax) for oleic acid and oleoyl-CoA per mol of nsLTP-A were 1.1 and 1.2 mol, respectively. When acyl-CoA oxidase activity was assayed in the glyoxysomal fraction, marked enhancement of the activity was observed in the presence of nsLTP. These results suggest the possibility that nsLTP regulates fatty acid beta-oxidation through the enhancement of acyl-CoA oxidase activity in glyoxysomes. The occurrence of castor bean nsLTP in the vessel wall was discussed.

Molecular species of PC and PE formed during castor oil biosynthesis.

As part of a program to elucidate castor oil biosynthesis, we have identified 36 molecular species of PC and 35 molecular species of PE isolated from castor microsomes after incubations with [14C]-labeled FA. The six [14C]FA studied were ricinoleate, stearate, oleate, linoleate, linolenate, and palmitate, which were the only FA identified in castor microsomal incubations. The incorporation of each of the six FA into PC was better than that into PE. The [14C]FA were incorporated almost exclusively into the sn-2 position of both PC and PE. The incorporation of [14C]stearate and [14C]palmitate into 2-acyl-PC was slower compared to the other four [14C]FA. The incorporation does not show any selectivity for the various lysoPC molecular species. The level of incorporation of [14C]FA in PC was in the order of: oleate > linolenate > palmitate > linoleate > stearate > ricinoleate, and in PE: linoleate > linolenate > oleate > palmitate > stearate > ricinoleate. In general, at the sn-1 position of both PC and PE, linoleate was the most abundant FA, palmitate was the next, and oleate, linolenate, stearate, and ricinoleate were minor FA. The activities of oleoyl-12-hydroxylase, oleoyl-12-desaturase seem unaffected by the FA at the sn-1 position of 2-oleoyl-PC. The FA in the sn-1 position of PC does not significantly affect the activity of phospholipase A2, whereas ricinoleate is preferentially removed from the sn-2 position of PC. The results show that (i) [14C]oleate is most actively incorporated to form 2-oleoyl-PC, the immediate substrate of oleoyl-12-hydroxylase; (ii) 2-ricinoleoyl-PC is formed mostly by the hydroxylation of 2-oleoyl-PC, not from the incorporation of ricinoleate into 2-ricinoleoyl-PC; and (iii) 2-oleoyl-PE is less actively formed than 2-oleoyl-PC.

Graviresponsiveness and the development of columella tissue in primary and lateral roots of Ricinus communis.

Half-tipped primary and lateral roots of Ricinus communis cv Hale bend toward the side of the root on which the intact half-tip remains. Therefore, the minimal graviresponsiveness of lateral roots is not due to the inability of their caps to produce growth effectors (presumably inhibitors). The columella tissues of primary (i.e. graviresponsive) roots are (a) 4.30 times longer, (b) 2.95 times wider, (c) 37.4 times more voluminous, and (d) composed of 17.2 times more cells than those of lateral roots. The onset of positive gravitropism by lateral roots is positively correlated with a (a) 2.99-fold increase in length, (b) 2.63-fold increase in width, (c) 20.7-fold increase in volume of their columella tissues. We propose that the minimal graviresponsiveness of lateral roots is due to the small size of their columella tissues, which results in their caps being unable to (a) establish a concentration gradient of the effector sufficient to induce gravicurvature and (b) produce as much as the effector as caps of graviresponsive roots.

Programmed cell death in castor bean endosperm is associated with the accumulation and release of a cysteine endopeptidase from ricinosomes.

The cells of the endosperm of castor bean seeds (Ricinus communis) undergo programmed cell death during germination, after their oil and protein reserves have been mobilized. Nuclear DNA fragmentation first was observed at day 3 in the endosperm cells immediately adjacent to the cotyledons and progressed across to the outermost cell layers by day 5. We also detected the accumulation of small organelles known as ricinosomes, by using an antibody against a cysteine endoprotease. By the time the nuclear DNA was susceptible to heavy label by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, the ricinosomes had released into the cytoplasm their content of cysteine endoprotease, which became activated because of the cleavage of its propeptide. The cysteine endoprotease is distinguished by a C-terminal KDEL sequence, although it is not retained in the lumen of the endoplasmic reticulum and is a marker for ricinosomes. Homologous proteases are found in the senescing tissues of other plants, including the petals of the daylily. Ricinosomes were identified in this tissue by electron microscopy and immunocytochemistry. It seems that ricinosomes are not unique to Ricinus and play an important role in the degradation of plant cell contents during programmed cell death.

Graviresponsiveness and columella cell structure in primary and secondary roots of Ricinus communis.

In order to determine what structural changes are associated with the onset of graviresponsiveness by plant roots, we have monitored the quantitative ultrastructures of columella (i.e., graviperceptive) cells in primary and secondary roots of Ricinus communis. The relative volumes of cellular components in lateral (i.e., minimally graviresponsive) roots were not significantly different from those of primary roots. The relative volumes of cellular components in secondary roots growing laterally were not significantly different from those of graviresponsive secondary roots. Therefore, the onset of graviresponsiveness by secondary roots of R. communis is not correlated with changes in organellar concentrations in columella cells. These results are discussed relative to a model for the differential graviresponsiveness of plant roots.

A cysteine endopeptidase with a C-terminal KDEL motif isolated from castor bean endosperm is a marker enzyme for the ricinosome, a putative lytic compartment.

A papain-type cysteine endopeptidase with a molecular mass of 35 kDa for the mature enzyme, was purified from germinating castor bean (Ricinus communis L.) endosperm by virtue of its capacity to process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro (C. Gietl et al., 1997, Plant Physiol 113: 863-871). The cDNA clones from endosperm of germinating seedlings and from developing seeds were isolated and sequence analysis revealed that a very similar or identical peptidase is synthesised in both tissues. Sequencing established a presequence for co-translational targeting into the endoplasmic reticulum, an N-terminal propeptide and a C-terminal KDEL motif for the castor bean cysteine endopeptidase precursor. The 45-kDa pro-enzyme stably present in isolated organelles was enzymatically active. Immunocytochemistry with antibodies raised against the purified cysteine endopeptidase revealed highly specific labelling of ricinosomes, organelles which co-purify with glyoxysomes from germinating Ricinus endosperm. The cysteine endopeptidase from castor bean endosperm, which represents a senescing tissue, is homologous to cysteine endopeptidases from other senescing tissues such as the cotyledons of germinating mung bean (Vigna mungo) and vetch (Vicia sativa), the seed pods of maturing French bean (Phaseolus vulgaris) and the flowers of daylily (Hemerocallis sp.).