Effect of L-Pipecolic Acid on Flowering in Lemna paucicostata and Lemna gibba

L-Pipecolic acid was found to be effective in inducing floweringof Lemna paucicostata 151, 381, 441 and 6746, and of Lemna gibbaG3. When the plants were grown on half-strength Hutner's medium,L-pipecolic acid caused profuse flowering of L. paucicostata151 maintained under 9 and 10 h of light daily. In L. paucicostata441 and 6746, L-pipecolic acid had a strong flower-promotingeffect under a near critical photoperiod. In L. paucicostata381, by contrast, L-pipecolic acid had only a very small effecton flowering. In L. gibba G3 substantial promotion of floweringwas observed under continuous light. When one-twentieth-strengthHutner's medium was used as the basic medium, L-pipecolic acidstimulated flowering in all strains of Lemna examined, evenunder continuous light. When L. paucicostata 151 was grown on one-tenth-strength M mediumor one-twentieth-strength Hutner's medium, the flower-inducingactivity of L-pipecolic acid was greatly enhanced by cytokininunder continuous light. However, when this strain was grownwith 9 h of illumination daily, this synergistic effect of cytokininwas only slight. A short-term (even 1-h) treatment with L-pipecolicacid resulted in flowering, suggesting that L-pipecolic acidis involved in the induction of flowering, rather than its evocation.D-Pipecolic acid also had flower-inducing activity, but itsactivity was 50 times lower than that of the L-isomer. (Received January 23, 1992; Accepted March 9, 1992)     

Isolation and Identification of L-Pipecolic Acid and Nicotinamide as Flower-Inducing Substances in Lemna

Flower-inducing factors in extracts of flowering Lemna gibbaG3 were investigated using Lemna paucicostata 151 as the bioassayplant. Fractions with flower-inducing activity were obtainedafter several purification steps. Two of the active substanceswere identified as L-pipecolic acid and nicotinamide by MS andNMR analyses. Both L-pipecolic acid and nicotinamide exhibited flower-inducingactivity in L. paucicostata 151 grown on one-tenth-strengthM medium containing benzyladenine, the former being ten timesas active as the latter. L-Pipecolic acid was active even at0.01 ppm (7.8 ? 10^–8 M). The effect of L-pipecolic acidon flowering strongly depended upon the presence of exogenouscytokinin. The coexistence of cytokinin seemed to be essentialfor L-pipecolic acid to exhibit flower-inducing activity. Incontrast, the effect of nicotinamide on flowering was basicallythe same as that of benzoic acid or nicotinic acid. (Received February 9, 1987; Accepted May 21, 1987)     

Interaction between L-Pipecolic Acid and Water Extracts of Various Plant Species in Floral Induction of Lemna paucicostata

The flower-inducing activity of L-pipecolic acid was synergisticallyenhanced by simultaneous application of the water extracts ofLemna paucicostata and Pharbitis nil, but suppressed by thewater extracts of all other plants we examined. Simultaneousapplication of the water extract of Lemna enhanced the flower-inducingactivity of all plant water extracts. (Received June 6, 1990; Accepted July 7, 1990)     

L-pipecolic acid metabolism in human liver: detection of L-pipecolate oxidase and identification of its reaction product

L-Pipecolate oxidase, an enzyme that oxidizes L-pipecolic acid in the human liver has been demonstrated in the peroxisomal preparation. This enzyme oxidizes L-pipecolic acid with concomitant production of H2O2 in the peroxisome of the normal human liver. The immediate product of L-pipecolic acid oxidation has been identified as L-alpha-aminoadipate delta-semialdehyde. This reaction product was directly, and also after conversion to pipecolic acid by NaBH4 reduction, characterized by use of an amino acid analyzer and thin-layer chromatography. The pit fall of an indirect assay of L-pipecolate oxidase by means of the assay of alpha-aminoadipic acid formation was discussed.     

A 15N NMR study on D-lysine metabolism in Neurospora crassa

The metabolic relationship of D-lysine, L-lysine, and L-pipecolic acid has been investigated in Neurospora crassa. Kinetic experiments show that radioactivity from D-lysine is efficiently incorporated into L-pipecolic acid and that this metabolite is converted to L-lysine. The alpha-amino group from D-[alpha-15N]lysine is lost in the course of its conversion to L-pipecolic acid and is trapped by pyruvate and alpha-keto glutarate to give L-[alpha-15N]alanine and L-[alpha-15N]glutamic acid. These amino acids are devoid of any label, however, when D-[epsilon-15N]lysine is applied to the fungus. As determined by mass and 15N NMR spectrometry the label from D-[epsilon-15N]lysine migrate via L-pipecolic acid into the alpha position of L-lysine, i.e. D-[epsilon-15N]lysine is converted to L-[alpha-15N]lysine. L-Pipecolic acid functions as an intermediate in this conversion.     

Enzymatic synthesis of L-pipecolic acid by Delta1-piperideine-2-carboxylate reductase from Pseudomonas putida

L-Pipecolic acid is a chiral pharmaceutical intermediate. An enzymatic system for the synthesis of L-pipecolic acid from L-lysine by commercial L-lysine alpha-oxidase from Trichoderma viride and an extract of recombinant Escherichia coli cells coexpressing Delta1-piperideine-2-carboxylate reductase from Pseudomonas putida and glucose dehydrogenase from Bacillus subtilis is described. A laboratory-scale process provided 27 g/l of L-pipecolic acid in 99.7% e.e.     

Effect on brain monoamines in the rat of substituted and protected analogues of the oxytocin fragment, prolyl-leucyl-glycinamide following N-terminal substitution by D- and L-pipecolic acid

The effect of modified and substituted analogues of prolyl-leucyl-glycinamide (PLG, MIF-I) was investigated on the steady-state level of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) in various brain regions. Proline was replaced by D- or L-pipecolic acid (D- or L-Pip), which analogues in turn were protected by benzoxy-carbonyl (Z) group. Substitution by D- or L-pipecolic acid caused opposite changes in the DA level of the dorsal hippocampus. These effects were absent it the N-terminal of either analogues was protected by Z-group. Following the above mentioned N-terminal modification, the amino group of the C-terminal glycine was also substituted by methyl-esther (Gly-OMe), Z-D-Pip-Leu-Gly-OMe decreased the mesencephalic DA level, while Z-L-Pip-Leu-Gly-OMe increased the 5-HT content of the mesencephalon and striatum. In general, N-terminal substitution by D-pipecolic acid decreased, whereas that by L-pipecolic acid increased the monoamine level in the brain.     

dad-1, A Putative Programmed Cell Death Suppressor Gene in Rice

The human dad-1 cDNA homolog was isolated from rice plants.The amino acid sequence of the predicted protein product iswell conserved in both animals and plants. This rice dad-1 homologcan rescue the temperature-sensitive dad-1 mutants of hamstercells from apoptotic death, suggesting that the rice dad-1 homologalso functions as a suppressor for programmed cell death. (Received December 24, 1997; Accepted January 27, 1997)     

L-pipecolic acid oxidation in the rabbit and cynomolgus monkey. Evidence for differing organellar locations and cofactor requirements in each species

L-Pipecolic acid oxidation was studied in the rabbit and cynomolgus monkey. Tissue homogenates from both species incubated with L-[2,3,4,5,6-3H]pipecolic acid produced a single radioactive product identified as alpha-aminoadipic acid. In the rabbit, L-pipecolic acid oxidation was greatest in kidney cortex with progressively lesser specific activities in liver, heart, and brain. When rabbit kidney cortex was fractionated by differential centrifugation or on Percoll gradients, activity paralleled that of the mitochondrial marker, glutamate dehydrogenase. In sonicated mitochondria, 92% of the activity was in the soluble fraction. Activity was inhibited by both rotenone and antimycin A and was maximal when FAD, phenazine ethosulfate, and glycerol were included in the assay; Km,app was 0.74 +/- 0.16 mM. Nipecotic acid, piperidine, and cis-2,4-piperidine dicarboxylic acid did not inhibit L-pipecolic acid oxidation, while L-proline had a Ki greater than or equal to 10 mM. D-Alanine and kojic acid, substrate and inhibitor of D-amino acid oxidase, respectively, were also not inhibitory. When monkey kidney cortex was fractionated on Percoll gradients, L-pipecolic acid oxidation activity paralleled that of the peroxisomal marker, catalase. After organellar subfractionation, the activity was membrane-associated and maximal at pH 8.5; Km,app was 4.22 +/- 0.30 mM. L-Pipecolic acid oxidation produced hydrogen peroxide, suggesting involvement of an oxidase in alpha-aminoadipic acid formation. Antimycin A did not inhibit the reaction. No specific cofactor requirements were identified and phenazine ethosulfate inhibited the reaction. D-Pipecolic acid, L-proline, and the other compounds cited above did not significantly inhibit the activity.     

Purification and characterization of peroxisomal L-pipecolic acid oxidase from monkey liver

L-Pipecolic acid oxidase has been purified to near homogeneity from Rhesus monkey liver. The protein, a yellow monomer, has a molecular weight of 46,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 8.9. It contains a covalently bound flavin with absorption maxima at 457 and 383 nm and a shoulder at 480 nm. The purified enzyme is most reactive toward L-pipecolic acid, with lesser reactivities toward L-proline and sarcosine. The enzyme has no significant reactivity toward the D-enantiomer of pipecolic acid or toward any other amino acid tested. Benzoic acid is a competitive inhibitor of the enzyme with a Ki of 750 microM. The Km of the purified enzyme is 3.7 mM for L-pipecolic acid. With less purified preparations, the reaction product is alpha-aminodipic acid. The purified enzyme, however, produces an intermediate which reacts with ortho-aminobenzaldehyde to form an alpha-aminoadipic acid semialdehyde adduct. Thus, the formation of alpha-aminoadipic acid requires at least two enzymes.     

Nucleotide Sequences of Genes Coding for Photosynthetic Reaction Centers and Light-Harvesting Proteins of Acidiphilium rubrum and Related Aerobic Acidophilic Bacteria

The nucleotide sequences of the puf operons of the Zn-bacteriochlorophylla(Zn-BChl a)-containing photosynthetic aerobic bacteria, Acidiphiliumrubrum and Acidiphilium angustum, were determined. The nucleotidesequences of the pufL and –M of Acidiphilium cryptum,Acidiphilium multivorum, and Acidiphilium organovorum were alsodetermined. The puf operons of A. rubrum and A. angustum containedpufB, –A,–L, –M, and –C as seen in otherpurple bacteria with an unknown gene directly upstream of pufB.Comparing the deduced amino acid sequences of the puf genesof the Acidiphilium species with those of other purple bacteriashowed that His L168, which is highly conserved in other bacteria,is replaced by a glu-tamic acid in the Acidiphilium species.The three-dimensional structures of the reaction centers ofBlastochloris (Rhodopseudomonas) viridis and Rhodobacter sphaeroidessuggest that this residue locates closely to a special pairof bacteriochlorophylls and may be involved in the stabilizationand function of "Zn-BChl a". The relative content of chargedamino acid residues in the L and M subunit is a little lowerin A. rubrum (10%of total) than in B. viridis (12%), and thetendency is more pronounced in the cyto-chrome subunit: 12.5%in A. rubrum and 18.8% in B. viridis. (Received July 24, 1997; Accepted September 9, 1997)     

Molecular Cloning of Plant Spermidine Synthases

Four cDNAs for spermidine synthase (SPDS), which converts thediamine putrescine to the higher polyamine spermidine usingdecarboxylated S-adenosylmethionine as the co-factor, were isolatedfrom Nicotiana sylvestris, Hyoscyamus niger, and Arabidopsisthaliana. When the N. sylvestris SPDS cDNA was expressed ina SPDS-deficient E. coli mutant, the recombinant protein showedhigh SPDS activity, but did not have any spermine synthase activity.The plant SPDSs have molecular masses of about 34 kDa, possessthe co-factor binding motifs which have been proposed for S-adenosylmethionine,and are more homologous in amino acid sequence to tobacco putrescineN-methyltransferase (PMT) than to SPDSs from mammals and E.coli. The SPDS gene is expressed in root, stem, and leaf inN. sylvestris, whereas the PMT gene is expressed only in root.The potential evolution of plant SPDS and PMT, and their evolutionaryrelationships with animal SPDS are discussed. (Received September 3, 1997; Accepted November 5, 1997)     

The Spirulina platensis Adenylate Cyclase Gene, cyaC, Encodes a Novel Signal Transduction Protein

A cyaC gene encoding an adenylate cyclase of the filamentouscyanobacterium Spirulina platensis was se-quenced. The predictedamino acid sequence of the C-ter-minal region of cyaC is similarto the catalytic domains of adenylate cyclases in other cyanobacteriaand eukaryotes. The sequences of other regions are similar tothose of proteins consisting of the bacterial two-componentsignal transduction system: the sensory kinase and the responseregulator. The predicted gene product of cyaC contains, fromthe N-terminal end, a receiver domain of the response regulatorprotein (Rl), a domain similar to the ETR1 of Arabi-dopsis thaliana,a transmitter domain of the sensory kinase protein, a receiverdomain of the response regulator protein (R2), and a catalyticdomain of adenylate cyclase. The cyaC gene was expressed asan affinity-tagged protein in Escherichia coli, and the recombinantprotein was purified. The purified protein had adenylate cyclaseactivity which was activated by Mn²+. The results of Westernblotting using an anti-CyaC antiserum and the S. platensis cellextract confirmed that cyaC gene is expressed in S. platensis (Received February 27, 1997; Accepted April 26, 1997)     

Pectin Depolymerase Activities Associated with Cell Walls from Cicer arietinum L. Epicotyl

The hydrolytic activity of the proteins extracted with 3 M LiClfrom chick-pea (Cicer arietinum) cell walls to pec-tic fractionsextracted with 50 mM trans-l,2-diaminocy-clohexane-N,N,N,N,-tetraaceticacid (CDTA) and 50 mM sodium carbonate was studied. The pecticfractions contained acidic polysaccharides with high molecularmass (higher than 5 x 10³ kDa), mainly composed of uronic acids,galac-tose, arabinose and rhamnose. The extracted proteins depo-lymerizedthe pectic polysaccharides and also a commercial preparationof polygalacturonic acid from citrus, detected by a decreasein their viscosity and a shift of their molecular mass distribution.The extract was able to depolymerize a uronic acid-rich componentin all the cases, although in different extent. Also, with regardto the CDTA-soluble pectins, a degradation of polyuronide anda shift of the molecular mass distribution of arabinogalactanwas observed. (Received March 11, 1997; Accepted September 10, 1997)     

Expression of Plasma Membrane Water Channel Genes under Water Stress in Nicotiana excelsior

Deduced amino acid sequences encoded by the cDNAs related tothe MIP gene family from Nicotiana excelsior were characterized.Phylogenetic characterization of the products of correspondinggenes named NeMip1, NeMip2, and NeMip3 strongly suggested thatthey are water channel proteins localized in the plasma membrane.Organ specificity of the gene expression was examined in leaves,roots, and reproductive organs. NeMip1 was expressed in rootsand reproductive organs; however, it was hardly detectable inleaves. Two other genes, NeMip2 and NeMip3, were expressed inall of organs examined. mRNA accumulation from the genes wasinvestigated in leaves under salt- and drought-stresses. Theresults demonstrated that mRNA accumulation from all three genesincreased under salt- and drought-stresses within one day. However,they showed different accumulation patterns. In addition totheir up-reg-ulation under salt- and drought-stresses, dailychanges in NeMip2 and NeMip3 mRNA accumulation was observedunder unstressed conditions in leaves. (Received May 2, 1997; Accepted September 3, 1997)     

Involvement of Calyculin A- and Okadaic Acid-sensitive Protein Phosphatase in the Blue Light Response of Stomatal Guard Cells

Calyculin A (CA) and okadaic acid (OA), inhibitors of proteinphosphatases, inhibited blue light (BL)-dependent H⁺pumpingin Vicia guard cell protoplasts at half-inhibitory concentrationsof 4.5 nM and 400 nM, respectively. Light-induced stomatal openingin Viciaepidermis was completely suppressed by CA at 100 nMand by OA at 1 µM. These results suggest that CA- andOA-sensitive protein phosphatase is involved in the BL responseof stomatal guard cells. (Received June 27, 1997; Accepted September 2, 1997)     

Specific Secretion of Citric Acid Induced by Al Stress in Cassia tora L.

A rapid and sensitive assay method for Al-chelating activitywas established to screen Al-chelating substances secreted fromroots of Al-resistant species in response to Al stress. Fromone Al-resistant species, Cassia tora L., an Al-chelating substancewas detected in the root exudates when they were exposed toSO µM Al in 0.5 mM CaCl₂ solution at pH 4.5; the dominantcomponent was identified as citric acid. The secretion of citricacid was very low during the first 4 h after initiation of Altreatment, but increased markedly thereafter. A 3-h pulse with50 µM Al also induced significant secretion of citricacid after 6 h. The lag between Al addition and secretion ofcitric acid suggests that inducible processes are involved.A dose-response experiment showed that the amount of secretedcitric acid increased with increasing external concentrationsof Al. Eight-d treatment of P deficiency did not induce thesecretion of citric acid. Exposure to 50µM of either lanthanum(La³⁺) or ytterbium (Yb³⁺) did not induce the secretion of citricacid either. These findings indicate that the secretion of citricacid is a response specific to Al stress in .C. toraand constitutesa mechanism of Al resistance. (Received April 23, 1997; Accepted June 25, 1997)     

Cloning and Characterization of High-CO2-Specific cDNAs from a Marine Microalga, Chlorococcum littorale, and Effect of CO2 Concentration and Iron Deficiency on the Gene Expression

Two cDNA clones exclusively induced under an extremely high-CO₂concentration (20%) were isolated from Chlorococcum littoraleby differential screening and named HCR (high-CO₂ response)1 and 2, respectively. The amino acid sequence of the proteinencoded by HCR2 exhibited homology to the gp91-phox protein,a critical component of a human phagocyte oxidoreductase, andto the yeast ferric reductases, Saccharomyces cerevisiae FRE1and FRE2 and Schizosaccharomyces pombe Frpl. The induction ofboth HCR mRNAs required extremely high-CO₂ conditions and irondeficiency, being suppressed under air conditions and by ironsufficiency, suggesting that the expression of these two HCRgenes required extremely high-CO₂ conditions and iron deficiencyin combination. The HCR2 protein was detected in the membranefractions of cells grown under conditions which would favorthe induction of HCR2-mRNA and the protein level was loweredwhen the cells were transferred from iron deficient to 10 µMFeSO₄ conditions (with 20% CO²). (Received September 10, 1997; Accepted November 14, 1997)     

Interannual variability in a predator-prey interaction: climate, chaetognaths and copepods in the southeastern Bering Sea

The interaction of the chaetognath Sagitta elegans with thecopepod community of the southeast Bering Sea middle shelf wasexamined in relation to environmental conditions during 1995–1999.Predation impact was estimated for 2 years, 1995 and 1997, usinggut content analysis, experimentally derived digestion time(DT) and abundances of chaetognaths and prey. Pseudocalanusconcentrations correlated with water temperature and Calanusmarshallae with sea ice extent. Sagitta elegans were less abundantbut individuals were larger in 1995, when C. marshallae predominated,compared to 1997, when Pseudocalanus and Acartia were the primaryprey. Predation by S. elegans removed <1% standing stockday^–1 of Pseudocalanus or C. marshallae in 1995 and 1.7to 2.3% of Pseudocalanus in 1997. The percent of the copepodcommunity biomass required by chaetognaths was estimated tobe <1% in 1995 compared with 8–12% in 1997. Calanusmarshallae may be more vulnerable than Pseudocalanus to cumulativepredation effects because of its reproductive strategy. Theeffect of chaetognath predation on the copepod community dependson which copepod species is predominant and its susceptibilityto cumulative predation effects, as well as on daily predationimpact, both of which varied between years with different climaticconditions.