Isolation and characterization of a cDNA encoding a pea ornithine transcarbamoylase (argF) and comparison with other transcarbamoylases.

We used a PCR-based library screening method to isolate a 1.4 kb pea leaf cDNA encoding ornithine transcarbamoylase (OTCase). The cDNA contains a single major ORF of 375 amino acids whose deduced sequence exhibits a high degree of homology with other OTCases. The predicted molecular mass of 41361 Da for this protein is approximately the 40 kDa size of the polypeptide that is immunoprecipitated with OTCase antibody after in vitro translation of pea leaf mRNA. In vivo, OTCase occurs as a trimer of identical 36.5 kDa polypeptides, suggesting that this enzyme is synthesized as a cytosolic precursor protein. Southern blot analysis indicates that multiple OTCase genes occur in pea. An abundant 1.4 kb transcript is seen in northern blots of total RNA isolated from the leaves and roots of light- and dark-grown pea seedlings.

Molecular cloning and characterization of the pyrB1 and pyrB2 genes encoding aspartate transcarbamoylase in pea (Pisum sativum L.).

We cloned cDNAs encoding two different pea (Pisum sativum L.) aspartate transcarbamoylases (ATCases) by complementation of an Escherichia coli delta pyrB mutant. The two cDNAs, designated pyrB1 and pyrB2, encode polypeptides of 386 and 385 amino acid residues, respectively, both of which exhibit typical chloroplast transit peptide sequences. Wheat germ ATCase antibody recognizes a 36.5-kD polypeptide in pea leaf and root tissues that is similar in size to other plant ATCase polypeptides and to the catalytic polypeptides of bacterial ATCases. Northern analyses indicate that the pyrB1 and pyrB2 transcripts are 1.6 kb in size and are differentially expressed in pea tissues. The small transcript size and data from biochemical studies indicate that plant ATCases are simple homotrimers of 36- to 37-kD catalytic subunits, rather than part of a multifunctional enzyme containing glutamine-dependent carbamoylphosphate synthetase and dihydroorotase activities, as is seen in other eukaryotes. In the pea ATCases, the carbamoylphosphate- and aspartate-binding domains are highly homologous to those of other prokaryotic and eukaryotic ATCases and critical active-site residues are completely conserved. The pea ATCases also exhibit a putative pyrimidine-binding site, consistent with the known allosteric regulation of plant ATCases by UMP in vitro.

Effects of prenatal alcohol exposure on uncoupling protein in brown adipose tissue in neonatal rats.

Thermoregulatory deficits observed in neonatal rats exposed prenatally to alcohol may be due to peripheral and/or central dysfunction. One of the major mechanisms available to newborn mammals to generate heat is "nonshivering thermogenesis" in brown adipose tissue (BAT). In this study, the effects of prenatal alcohol exposure on the functional status of brown adipose tissue was assessed by immunoblot analysis of the content of mitochondrial uncoupling protein (UCP). BAT excised from 1- and 20-day-old male and female offspring from either alcohol-treated, pair-fed controls or standard control dams were analyzed. There were no effects of prenatal alcohol exposure on the UCP content. There was, however, a significant increase due to age. These results suggest that thermoregulatory deficits seen in alcohol-exposed offspring are not due to a deficiency in the concentration of mitochondrial UCP, and indicate a more central mechanism.

Molecular cloning and evidence for osmoregulation of the delta 1-pyrroline-5-carboxylate reductase (proC) gene in pea (Pisum sativum L.).

Several cDNA clones encoding delta 1-pyrroline-5-carboxylate reductase (P5CR, L-proline:NAD[P]+ 5-oxidoreductase, EC 1.5.1.2), which catalyzes the terminal step in proline biosynthesis, were isolated from a pea leaf library screened with a 32P-labeled Aval fragment of a soybean nodule P5CR cDNA (A.J. Delauney, D.P.S. Verma [1990] Mol Gen Genet 221: 299-305). DNA sequence analysis of one full-length 1.3-kb clone (pPPS3) indicated that the pea P5CR gene contains a single major open reading frame encoding a polypeptide of 28,242 Da. Genomic analysis suggested that two to three copies of the P5CR gene are present per haploid genome in pea. The primary structure of pea P5CR is 85% identical with that of soybean and exhibits significant homology to human, yeast, and Escherichia coli P5CR. The sequence of one of four highly conserved domains found in all prokaryotic and eukaryotic P5CRs is similar to the consensus sequence for the NAD(P)H-binding site of other enzymes. The pea P5CR cDNA hybridized to two transcripts, 1.3 and 1.1 kb in size, in polyadenylated RNA purified from leaf tissues of mature, light-grown plants (4 weeks old). Only the 1.3-kb transcript was detected in younger (1 week old) greened seedlings or in etiolated seedlings. In greened seedlings, steady-state levels of this 1.3-kb mRNA increased approximately 5-fold in root tissues within 6 h after plants were irrigated with 0.4 M NaCl, suggesting that expression of the P5CR gene is osmoregulated.

Electron-microscopic cytochemical localization of diamine and polyamine oxidases in pea and maize tissues.

An electron-microscopic cytochemical method was used to localize diamine oxidase (DAO) in pea and polyamine oxidase (PAO) in maize (Zea mays L.). The method, based on the precipitation of amine-oxidase-generated H2O2 by CeCl3, was shown to be specific for DAO and PAO and permitted their localization in plant tissues with a high degree of resolution. Both enzymes are localized exclusively in the cell wall. Both DAO- and PAO-activity staining is most intense in the middle lamellar region of the wall and in cells exhibiting highly lignified walls. The oxidases could provide H2O2 for peroxidase-mediated cross-linking reactions in the cell wall and may, in this capacity, play a role in the regulation of plant growth.

Purification and characterization of ornithine transcarbamylase from pea (Pisum sativum L.).

Pea (Pisum sativum) ornithine transcarbamylase (OTC) was purified to homogeneity from leaf homogenates in a single-step procedure, using delta-N-(phosphonacetyl)-L-ornithine-Sepharose 6B affinity chromatography. The 1581-fold purified OTC enzyme exhibited a specific activity of 139 micromoles citrulline per minute per milligram of protein at 37 degrees C, pH 8.5. Pea OTC represents approximately 0.05% of the total soluble protein in the leaf. The molecular weight of the native enzyme was approximately 108,200, as estimated by Sephacryl S-200 gel filtration chromatography. The purified protein ran as a single molecular weight band of 36,500 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the pea OTC is a trimer of identical subunits. The overall amino acid composition of pea OTC is similar to that found in other eukaryotic and prokaryotic OTCs, but the number of arginine residues is approximately twofold higher. The increased number of arginine residues probably accounts for the observed isoelectric point of 7.6 for the pea enzyme, which is considerably more basic than isoelectric point values that have been reported for other OTCs.

Immunological characterization of plant ornithine transcarbamylases.

Pea (Pisum sativum L.) ornithine transcarbamylase (OTC) antisera were used to investigate the immunological relatedness of several plant and animal OTC enzymes. The antisera immunoprecipitated OTC activity in all monocot and dicot species tested, and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of immunoprecipitated protein revealed monomeric proteins ranging from 35,200 to 36,800 daltons in size. Pea OTC antisera did not recognize mammalian OTC protein. OTC activity and protein levels detected on sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblots from homogenates of green leaf, etiolated epicotyl and cotyledon, and root tissues of pea were poorly correlated. This might result from differences in amounts of enzymatically active OTC protein in the homogenates. Alternatively, the antisera may fail to recognize different isozyme forms of OTC, which have been reported for some plant species. A putative cytosolic precursor OTC (pOTC) polypeptide exhibiting and Mr = 39,500 to 40,000 daltons was immunoprecipitated from in vitro translation mixtures of total pea leaf poly(A)+ RNA. The size of the pOTC polypeptide, as compared with mature OTC monomer (36,000 daltons), suggests that a 4 kilodalton N-terminal leader sequence, like that responsible for mitochondrial targeting of the mammalian enzyme, may be involved in organellar import of the plant enzyme.

Improved method for HPLC analysis of polyamines, agmatine and aromatic monoamines in plant tissue.

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucas carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.

DL-alpha-difluoromethyl[3,4-3H]arginine metabolism in tobacco and mammalian cells. Inhibition of ornithine decarboxylase activity after arginase-mediated hydrolysis of DL-alpha-difluoromethylarginine to DL-alpha-difluoromethylornithine.

DL-alpha-Difluoromethylarginine (DFMA) is an enzyme-activated irreversible inhibitor of arginine decarboxylase (ADC) in vitro. DFMA has also been shown to inhibit ADC activities in a variety of plants and bacteria in vivo. However, we questioned the specificity of this inhibitor for ADC in tobacco ovary tissues, since ornithine decarboxylase (ODC) activity was strongly inhibited as well. We now show that [3,4-3H]DFMA is metabolized to DL-alpha-difluoromethyl[3,4-3H]ornithine [( 3,4-3H]DFMO), the analogous mechanism-based inhibitor of ODC, by tobacco tissues in vivo. Both tobacco and mammalian (mouse, bovine) arginases (EC 3.5.3.1) hydrolyse DFMA to DFMO in vitro, suggesting a role for this enzyme in mediating the indirect inhibition of ODC by DFMA in tobacco. These results suggest that DFMA may have other effects, in addition to the inhibition of ADC, in tissues containing high arginase activities. The recent development of potent agmatine-based ADC inhibitors should permit selective inhibition of ADC, rather than ODC, in such tissues, since agmatine is not a substrate for arginase.

In vivo inhibition of polyamine biosynthesis and growth in tobacco ovary tissues.

Post fertilization growth of tobacco ovary tissues treated with inhibitors of polyamine (PA) biosynthesis was examined in relation to endogenous PA titers and the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17). DL-alpha-Difluoromethylornithine (DFMO) and DL-alpha-difluoromethylarginine (DFMA), specific, irreversible ("suicide") inhibitors of ODC and ADC in vitro, were used to modulate PA biosynthesis in excised flowers. ODC represented >99% of the total decarboxylase activity in tobacco ovaries. In vivo inhibition of ODC with DFMO resulted in a significant decrease in PA titers, ovary fresh weight and protein content. Simultaneous inhibition of both decarboxylases by DFMO and DFMA produced only a marginally greater depression in growth and PA titers, indicating that ODC activity is rate-limiting for PA biosynthesis in these tissues. Paradoxically, DFMA alone inhibited PA biosynthesis, not as a result of a specific inhibition of ADC, but primarily through the inactivation of ODC. In vivo inhibition of ODC by DFMA appears to result from arginase-mediated hydrolysis of this inhibitor to urea and DFMO, the suicide substrate for ODC. Putrescine conjugates in tobacco appear to function as a storage form of this amine which, upon hydrolysis, may contribute to Put homeostasis during growth.

Changes in polyamine biosynthesis associated with postfertilization growth and development in tobacco ovary tissues.

Polyamine (PA) titers and the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17), enzymes which catalyze rate-limiting steps in PA biosynthesis, were monitored during tobacco ovary maturation. In the period between anthesis and fertilization, the protein content of ovary tissues rapidly increased by about 40% and was accompanied by approximately a 3-fold increase in ODC activity, while ADC activity remained nearly constant. PA titers also remained relatively unchanged until fertilization, at which time they increased dramatically and the DNA content of ovary tissues doubled. This increase in PA biosynthesis was correlated with a further 3-fold increase in ODC activity, reaching a maximum 3 to 4 days after fertilization. During this time, ADC activity increased only slightly and accounted for approximately 1% of the total decarboxylase activity when ODC activity peaked. The postfertilization burst of biosynthetic activities slightly preceded a period of rapid ovary enlargement, presumably due to new cell division. During later stages of ovary development, DNA levels fell precipitously, while PA titers and decarboxylase activities decreased to preanthesis levels more slowly. In this period, growth producing a 300% increase in ovary fresh weight appears to be the result of cell enlargement. Synchronous changes in PA titers and in the rates of PA biosynthesis, macromolecular synthesis, and growth in the tobacco ovary suggest that PAs may play a role in the regulation of postfertilization growth and development of this reproductive organ.

Cytological and ultrastructural studies on root tissues.

The anatomy and fine structure of roots from oat and mung bean seedlings, grown under microgravity conditions for 8 days aboard the Space Shuttle, was examined and compared to that of roots from ground control plants grown under similar conditions. Roots from both sets of oat seedlings exhibited characteristic monocotyledonous tissue organization and normal ultrastructural features, except for cortex cell mitochondria, which exhibited a 'swollen' morphology. Various stages of cell division were observed in the meristematic tissues of oat roots. Ground control and flight-grown mung bean roots also showed normal tissue organization, but root cap cells in the flight-grown roots were collapsed and degraded in appearance, especially at the cap periphery. At the ultrastructural level, these cells exhibited a loss of organelle integrity and a highly-condensed cytoplasm. This latter observation perhaps suggests a differing tissue sensitivity for the two species to growth conditions employed in space flight. The basis for abnormal root cap cell development is not understood, but the loss of these putative gravity-sensing cells holds potential significance for long term plant growth orientation during space flight.

Cellular and subcellular localization of calcium in gravistimulated oat coleoptiles and its possible significance in the establishment of tropic curvature.

Light-and electron-microscopic studies of the distribution of calcium in gravitropically responding oat (Avena sativa L. cv. "Garry") coleoptiles are described. A modification of the antimonate precipitation procedure was used to localize tissue calcium in situ. An accumulation of Ca in the upper halves of horizontal, gravistimulated coleoptiles is seen within 10 min of stimulus onset. A pronounced redistribution of Ca to the upper side occurs within 30 min; although the localization of this cation is not uniform along the organ axis and in the apical region, Ca appears to accumulate along the lower side. The observed asymmetric distribution of Ca in these tissues precedes large-scale visible bending by 20-30 min, but is temporally well-correlated with differential growth responses in the coleoptile, as measured by more sensitive quantitative techniques. Gravitropic curvature is well developed by 3 h and is accompanied by further redistribution of Ca to tissues along the upper coleoptile half, centered around the bend. Ultrastructural localization studies indicate that Ca asymmetry results primarily from changes in the distribution of Ca within the apoplastic compartment. Large amounts of Ca accumulate at the cuticle in epidermal cell walls and in the walls of the underlying parenchyma cells at the upper side of the organ in the region of maximal bending. The differential growth response resulting in the establishment of gravitropic curvature may largely be the consequence of antagonistic effects of Ca on auxin-mediated cell wall loosening and elongation growth processes at the upper side of the organ.

An improved method for the subcellular localization of calcium using a modification of the antimonate precipitation technique.

A new variation of the antimonate precipitation technique, employing tannic acid in the primary aldehyde-antimonate fixative, is described for use in the subcellular localization of calcium in various tissues. Chelation studies and electron microscopic, X-ray microanalytical studies of antimonate precipitates in etiolated oat tissues indicate that calcium is the major cation localized using the present experimental protocol. Preservation of ultrastructural morphology in these tissues is greatly improved over that observed in tissues fixed with conventional antimonate-aldehyde or antimonate-osmium fixatives. The regularity and reproducibility of tissue precipitate patterns suggests that 1) penetration of the tissue by the fixative, and subsequent precipitation of calcium, is rapid and uniform and 2) ion displacement during sample preparation is negligible. Calcium appears to be immobilized efficiently in situ, with greater than 90% 45Ca retention in radiolabeled tissues prepared for electron microscopy. Quantitative aspects of calcium precipitation by antimonate in 45Ca-labeled CaCl2 solutions were examined over a wide range of calcium concentrations. Precipitation was essentially linear over the expected range of biological concentrations of calcium. Furthermore, the 3:1 antimonate to calcium ratio estimated for test tube precipitates was also established for Sb/Ca in tissue precipitates analyzed using energy dispersive x-ray microanalytical (EDX) techniques. These observations suggest that the present technique is potentially useful in the semiquantitative estimation of tissue calcium levels.

Phytochrome induces photoreversible calcium fluxes in a purified mitochondrial fraction from oats.

Previous studies have indicated that phytochrome regulates Ca(2+) fluxes across the plasma membrane of plant cells. In this study we investigated whether phytochrome can also regulate such fluxes across mitochondrial membranes, using the Ca(2+)-sensitive dye murexide to monitor the uptake and release of Ca(2+) by mitochondria. The results showed that Ca(2+) fluxes in these organelles could be photoreversibly altered, red light diminishing the net uptake rate and far-red light restoring this rate to its dark control level. Treatment of the mitochondria with ruthenium red blocked their Ca(2+) uptake. In the presence of this inhibitor, red light induced a net efflux of Ca(2+) from the mitochondria, and subsequent far-red light reduced this efflux to nearly zero, the dark control level. Light-induced rate changes in Ca(2+) flux, both with and without the inhibitor, persisted for several minutes in the dark and remained photoreversible through several irradiations for as long as 30 min. The purity of the mitochondrial preparation was judged to be about 80% by electron microscopic morphometry; most of the phytochrome present was localized on the mitochondria in the preparation by using immunocytochemical methods. Taken together with previous findings, the results suggest that red light activation of phytochrome would initiate an increase in the cytosolic Ca(2+) concentration. The results are integrated with the fact that calmodulin is a component of plant cell cytoplasms to construct a model postulating that phytochrome directs photomorphogenesis in part through its regulation of Ca(2+) and calmodulin-controlled enzyme activities.