Expression of a Soybean Gene Encoding the Tetrapyrrole-Synthesis Enzyme Glutamyl-tRNA Reductase in Symbiotic Root Nodules1

Heme and chlorophyll accumulate to high levels in legume root nodules and in photosynthetic tissues, respectively, and they are both derived from the universal tetrapyrrole precursor δ-aminolevulinic acid (ALA). The first committed step in ALA and tetrapyrrole synthesis is catalyzed by glutamyl-tRNA reductase (GTR) in plants. A soybean (Glycine max) root-nodule cDNA encoding GTR was isolated by complementation of an Escherichia coli GTR-defective mutant for restoration of ALA prototrophy. Gtr mRNA was very low in uninfected roots but accumulated to high levels in root nodules. The induction of Gtr mRNA in developing nodules was subsequent to that of the gene Enod2 (early nodule) and coincided with leghemoglobin mRNA accumulation. Genomic analysis revealed two Gtr genes, Gtr1 and a 3′ portion of Gtr2, which were isolated from the soybean genome. RNase-protection analysis using probes specific to Gtr1 and Gtr2 showed that both genes were expressed, but Gtr1 mRNA accumulated to significantly higher levels. In addition, the qualitative patterns of expression of Gtr1 and Gtr2 were similar to each other and to total Gtr mRNA in leaves and nodules of mature plants and etiolated plantlets. The data indicate that Gtr1 is universal for tetrapyrrole synthesis and that a Gtr gene specific for a tissue or tetrapyrrole is unlikely. We suggest that ALA synthesis in specialized root nodules involves an altered spatial expression of genes that are otherwise induced strongly only in photosynthetic tissues of uninfected plants.

Feedback Control and Diurnal Regulation of Gibberellin 20-Oxidase Transcript Levels in Potato1

Tuber formation in potato (Solanum tuberosum) is promoted by short photoperiods and is inhibited by gibberellins (GAs). Endogenous levels of GA1 were shown to decrease in stolons and leaves of potato plants induced to tuberize, which suggests that photoperiodic regulation of GA biosynthesis may play a role in tuber induction. We report the isolation of three potato cDNA clones (StGA20ox1–3) encoding GA 20-oxidase, a key regulatory enzyme in the GA-biosynthetic pathway. Using northern analysis, we detected a differential pattern of tissue-specific expression of the mRNAs corresponding to these clones. StGA20ox mRNAs were also very abundant in leaves of the potato ga1 mutant, which is blocked in the 13-hydroxylation step, and were strongly down-regulated by gibberellic acid, suggesting a feedback regulation of these genes. In plants grown in short-day (inductive) conditions, levels of the StGA20ox transcripts in leaves fluctuated during a 24-h period, with a peak of accumulation observed about 4 h after the lights were turned off. Interruption of the night with a 30-min “night break” of light (noninductive conditions) did not have a marked effect on the levels of accumulation of the three GA 20-oxidase mRNAs during the day, but it induced a second peak of expression of StGA20ox1 and StGA20ox3 transcripts late in the night. This observation, together with the finding that StGA20ox1 mRNA is expressed at high levels in leaves, suggests that night-break induction of this gene might play a role in the control of tuberization by regulating endogenous levels of GAs in response to daylength conditions.

Purification and Molecular Genetic Characterization of ZPU1, a Pullulanase-Type Starch-Debranching Enzyme from Maize1

This study identified and purified specific isoamylase- and pullulanase-type starch-debranching enzymes (DBEs) present in developing maize (Zea mays L.) endosperm. The cDNA clone Zpu1 was isolated based on its homology with a rice (Oryza sativa L.) cDNA coding for a pullulanase-type DBE. Comparison of the protein product, ZPU1, with 18 other DBEs identified motifs common to both isoamylase- and pullulanase-type enzymes, as well as class-specific sequence blocks. Hybridization of Zpu1 to genomic DNA defined a single-copy gene, zpu1, located on chromosome 2. Zpu1 mRNA was abundant in endosperm throughout starch biosynthesis, but was not detected in the leaf or the root. Anti-ZPU1 antiserum specifically recognized the approximately 100-kD ZPU1 protein in developing endosperm, but not in leaves. Pullulanase- and isoamylase-type DBEs were purified from extracts of developing maize kernels. The pullulanase-type activity was identified as ZPU1 and the isoamylase-type activity as SU1. Mutations of the sugary1 (su1) gene are known to cause deficiencies of SU1 isoamylase and a pullulanase-type DBE. ZPU1 activity, protein level, and electrophoretic mobility were altered in su1-mutant kernels, indicating that it is the affected pullulanase-type DBE. The Zpu1 transcript levels were equivalent in nonmutant and su1-mutant kernels, suggesting that coordinated regulation of ZPU1 and SU1 occurs posttranscriptionally.

Purification and Characterization of NADP+-Linked Isocitrate Dehydrogenase from Scots Pine1 : Evidence for Different Physiological Roles of the Enzyme in Primary Development

NADP+-isocitrate dehydrogenase (NADP+-IDH; EC 1.1.1.42) is involved in the supply of 2-oxoglutarate for ammonia assimilation and glutamate synthesis in higher plants through the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Only one NADP+-IDH form of cytosolic localization was detected in green cotyledons of pine (Pinus spp.) seedlings. The pine enzyme was purified and exhibited molecular and kinetic properties similar to those described for NADP+-IDH from angiosperm, with a higher catalytic efficiency (105 m−1 s−1) than the deduced efficiencies for GS and GOGAT in higher plants. A polyclonal antiserum was raised against pine NADP+-IDH and used to assess protein expression in the seedlings. Steady-state levels of NADP+-IDH were coordinated with GS during seed germination and were associated with GS/GOGAT enzymes during chloroplast biogenesis, suggesting that NADP+-IDH is involved in the provision of carbon skeletons for the synthesis of nitrogen-containing molecules. However, a noncoordinated pattern of NADP+-IDH and GS/GOGAT was observed in advanced stages of cotyledon development and in the hypocotyl. A detailed analysis in hypocotyl sections revealed that NADP+-IDH abundance was inversely correlated with the presence of GS, GOGAT, and ribulose-1,5-bisphosphate carboxylase/oxygenase but was associated with the differentiation of the organ. These results cannot be explained by the accepted role of the enzyme in nitrogen assimilation and strongly suggest that NADP+-IDH may have other, as-yet-unknown, biological functions.

Gibberellin Dose-Response Regulation of GA4 Gene Transcript Levels in Arabidopsis1

The gibberellins (GAs) are a complex family of diterpenoid compounds, some of which are potent endogenous regulators of plant growth. As part of a feedback control of endogenous GA levels, active GAs negatively regulate the abundance of mRNA transcripts encoding GA biosynthesis enzymes. For example, Arabidopsis GA4 gene transcripts encode GA 3β-hydroxylase, an enzyme that catalyzes the conversion of inactive to active GAs. Here we show that active GAs regulate GA4 transcript abundance in a dose-dependent manner, and that down-regulation of GA4 transcript abundance is effected by GA4 (the product of 3β-hydroxylation) but not by its immediate precursor GA9 (the substrate). Comparison of several different GA structures showed that GAs active in promoting hypocotyl elongation were also active in regulating GA4 transcript abundance, suggesting that similar GA:receptor and subsequent signal transduction processes control these two responses. It is interesting that these activities were not restricted to 3β-hydroxylated GAs, being also exhibited by structures that were not 3β-hydroxylated but that had another electronegative group at C-3. We also show that GA-mediated control of GA4 transcript abundance is disrupted in the GA-response mutants gai and spy-5. These observations define a sensitive homeostatic mechanism whereby plants may regulate their endogenous GA levels.

In Vivo Ubiquinone Reduction Levels during Thermogenesis in Araceae1

In vivo ubiquinone (UQ) reduction levels were measured during the development of the inflorescences of Arum maculatum and Amorphophallus krausei. Thermogenesis in A. maculatum spadices appeared not to be confined to a single developmental stage, but occurred during various stages. The UQ pool in both A. maculatum and A. krausei appendices was approximately 90% reduced during thermogenesis. Respiratory characteristics of isolated appendix mitochondria did not change in the period around thermogenesis. Apparently, synthesis of the required enzyme capacity is regulated via a coarse control upon which a fine control of metabolism that regulates the onset of thermogenesis is imposed.

Biosynthesis of the Monoterpenes Limonene and Carvone in the Fruit of Caraway1 : I. Demonstration of Enzyme Activities and Their Changes with Development

The biosynthesis of the monoterpenes limonene and carvone in the fruit of caraway (Carum carvi L.) proceeds from geranyl diphosphate via a three-step pathway. First, geranyl diphosphate is cyclized to (+)-limonene by a monoterpene synthase. Second, this intermediate is stored in the essential oil ducts without further metabolism or is converted by limonene-6-hydroxylase to (+)-trans-carveol. Third, (+)-trans-carveol is oxidized by a dehydrogenase to (+)-carvone. To investigate the regulation of monoterpene formation in caraway, we measured the time course of limonene and carvone accumulation during fruit development and compared it with monoterpene biosynthesis from [U-14C]Suc and the changes in the activities of the three enzymes. The activities of the enzymes explain the profiles of monoterpene accumulation quite well, with limonene-6-hydroxylase playing a pivotal role in controlling the nature of the end product. In the youngest stages, when limonene-6-hydroxylase is undetectable, only limonene was accumulating in appreciable levels. The appearance of limonene-6-hydroxylase correlates closely with the onset of carvone accumulation. At later stages of fruit development, the activities of all three enzymes declined to low levels. Although this correlates closely with a decrease in monoterpene accumulation, the latter may also be the result of competition with other pathways for substrate.

Uridine 5′-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia1

To understand the regulation and expression of pyrimidine biosynthesis in plants, we have examined the effect of the metabolic inhibitor 5-fluoroorotic acid (FOA) on uridine-5′-monophosphate synthase (UMPSase) expression in cell cultures of Nicotiana plumbaginifolia. UMPSase is the rate-limiting step of pyrimidine biosynthesis in plants. Addition of FOA causes an up-regulation of UMPSase enzyme activity in cell cultures after a lag phase of several days. Western-blot analysis demonstrated that the up-regulation in enzyme activity was caused by increased expression of the UMPSase protein. Northern-blot analysis demonstrated a higher level of UMPSase mRNA in the FOA-induced tissues than in control tissues. Run-on transcriptional assays showed that the UMPSase gene was transcriptionally activated after FOA treatment. The mechanism of toxicity of FOA is through thymine starvation. We found that addition of thymine abrogated the FOA-mediated up-regulation of UMPSase. In addition, methotrexate and aminopterin, which affect thymine levels by inhibiting dihydrofolate reductase, also up-regulate UMPSase in N. plumbaginifolia cells.

Activation of an interleukin 1 converting enzyme-dependent apoptosis pathway by granzyme B.

Cytotoxic T lymphocytes (CTL) can induce apoptosis through a granzyme B-based killing mechanism. Here we show that in cells undergoing apoptosis by granzyme B, both p45 pro-interleukin 1 beta converting enzyme (ICE) and pro-CPP32 are processed. Using ICE deficient (ICE -/-) mice, embryonic fibroblasts exhibit high levels of resistance to apoptosis by granzyme B or granzyme 3, while B lymphoblasts are granzyme B-resistant, thus identifying an ICE-dependent apoptotic pathway that is activated by CTL granzymes. In contrast, an alternative ICE-independent pathway must also be activated as ICE -/- thymocytes remain susceptible to apoptosis by both granzymes. In ICE -/- B cells or HeLa cells transfected with mutant inactive ICE or Ich-1S that exhibit resistance to granzyme B, CPP32 is processed to p17 and poly(ADP-ribose) polymerase is cleaved indicating that this protease although activated was not associated with an apoptotic nuclear phenotype. Using the peptide inhibitor Ac-DEVD-CHO, apoptosis as well as p45 ICE hydrolysis are suppressed in HeLa cells, suggesting that a CPP32-like protease is upstream of ICE. In contrast, p34cdc2 kinase, which is required for granzyme B-induced apoptosis, remains inactive in ICE -/- B cells indicating it is downstream of ICE. We conclude that granzyme B activates an ICE-dependent cell death pathway in some cell types and requires a CPP32-like Ac-DEVD-CHO inhibitable protease acting upstream to initiate apoptosis.

Increased mRNA levels for components of the lysosomal, Ca2+-activated, and ATP-ubiquitin-dependent proteolytic pathways in skeletal muscle from head trauma patients.

The cellular mechanisms responsible for enhanced muscle protein breakdown in hospitalized patients, which frequently results in lean body wasting, are unknown. To determine whether the lysosomal, Ca2+-activated, and ubiquitin-proteasome proteolytic pathways are activated, we measured mRNA levels for components of these processes in muscle biopsies from severe head trauma patients. These patients exhibited negative nitrogen balance and increased rates of whole-body protein breakdown (assessed by [13C]leucine infusion) and of myofibrillar protein breakdown (assessed by 3-methylhistidine urinary excretion). Increased muscle mRNA levels for cathepsin D, m-calpain, and critical components of the ubiquitin proteolytic pathway (i.e., ubiquitin, the 14-kDa ubiquitin-conjugating enzyme E2, and proteasome subunits) paralleled these metabolic adaptations. The data clearly support a role for multiple proteolytic processes in increased muscle proteolysis. The ubiquitin proteolytic pathway could be activated by altered glucocorticoid production and/or increased circulating levels of interleukin 1beta and interleukin 6 observed in head trauma patients and account for the breakdown of myofibrillar proteins, as was recently reported in animal studies.

Mammalian ubiquitin-conjugating enzyme Ubc9 interacts with Rad51 recombination protein and localizes in synaptonemal complexes.

Hsubc9, a human gene encoding a ubiquitin-conjugating enzyme, has been cloned. The 18-kDa HsUbc9 protein is homologous to the ubiquitin-conjugating enzymes Hus5 of Schizosaccharomyces pombe and Ubc9 of Saccharomyces cerevisiae. The Hsubc9 gene complements a ubc9 mutation of S. cerevisiae. It has been mapped to chromosome 16p13.3 and is expressed in many human tissues, with the highest levels in testis and thymus. According to the Ga14 two-hybrid system analysis, HsUbc9 protein interacts with human recombination protein Rad51. A mouse homolog, Mmubc9, encodes an amino acid sequence that is identical to the human protein. In mouse spermatocytes, MmUbc9 protein, like Rad51 protein, localizes in synaptonemal complexes, which suggests that Ubc9 protein plays a regulatory role in meiosis.

Interleukin 1 beta suppresses transforming growth factor-induced inorganic pyrophosphate (PPi) production and expression of the PPi-generating enzyme PC-1 in human chondrocytes.

Articular cartilage chondrocytes have the unique ability to elaborate large amounts of extracellular pyrophosphate (PPi), and transforming growth factor beta (TGF beta) appears singular among cartilage regulatory factors in stimulating PPi production. TGF beta caused a time and dose-dependent increase in intracellular and extracellular PPi in human articular chondrocyte cultures. TGF beta and interleukin 1 beta (IL-1 beta) antagonistically regulate certain chondrocyte functions. IL-1 beta profoundly inhibited basal and TGF beta-induced PPi elaboration. To address mechanisms involved with the regulation of PPi synthesis by IL-1 beta and TGF beta, we analyzed the activity of the PPi-generating enzyme NTP pyrophosphohydrolase (NTPPPH) and the PPi-hydrolyzing enzyme alkaline phosphatase. Human chondrocyte NTPPPH activity was largely attributable to plasma cell membrane glycoprotein 1, PC-1. Furthermore, TGF beta induced comparable increases in the activity of extracellular PPi, intracellular PPi, and cellular NTPPPH and in the levels of PC-1 protein and mRNA in chondrocytes as well as a decrease in alkaline phosphatase. All of these TGF beta-induced responses were completely blocked by IL-1 beta. Thus, IL-1 beta may be an important regulator of mineralization in chondrocytes by inhibiting TGF beta-induced PPi production and PC-1 expression.

Selenophosphate synthetase: detection in extracts of rat tissues by immunoblot assay and partial purification of the enzyme from the archaean Methanococcus vannielii.

In Escherichia coli and Salmonella typhimurium it has been shown that selenophosphate serves as the selenium donor for the conversion of seryl-tRNA to selenocysteyl-tRNA and for the synthesis of 2-selenouridine, a modified nucleoside present in tRNAs. Although selenocysteyl-tRNA also is formed in eukaryotes and is used for the specific insertion of selenocysteine into proteins, the precise mechanism of its biosynthesis from seryl-tRNA in these systems is not known. Because selenophosphate is extremely oxygen labile and difficult to identify in biological systems, we used an immunological approach to detect the possible presence of selenophosphate synthetase in mammalian tissues. With antibodies elicited to E. coli selenophosphate synthetase the enzyme was detected in extracts of rat brain, liver, kidney, and lung by immunoblotting. Especially high levels were detected in Methanococcus vannielii, a member of the domain Archaea, and the enzyme was partially purified from this source. It seems likely that the use of selenophosphate as a selenium donor is widespread in biological systems.

Complete congruence between gene diversity estimates derived from genotypic data at enzyme and random amplified polymorphic DNA loci in black spruce.

Controversy still exists over the adaptive nature of variation of enzyme loci. In conifers, random amplified polymorphic DNAs (RAPDs) represent a class of marker loci that is unlikely to fall within or be strongly linked to coding DNA. We have compared the genetic diversity in natural populations of black spruce [Picea mariana (Mill.) B.S.P.] using genotypic data at allozyme loci and RAPD loci as well as phenotypic data from inferred RAPD fingerprints. The genotypic data for both allozymes and RAPDs were obtained from at least six haploid megagametophytes for each of 75 sexually mature individuals distributed in five populations. Heterozygosities and population fixation indices were in complete agreement between allozyme loci and RAPD loci. In black spruce, it is more likely that the similar levels of variation detected at both enzyme and RAPD loci are due to such evolutionary forces as migration and the mating system, rather than to balancing selection and overdominance. Furthermore, we show that biased estimates of expected heterozygosity and among-population differentiation are obtained when using allele frequencies derived from dominant RAPD phenotypes.