Effect of low concentration of hydrogen peroxide on indoleacetate oxidase zymogram in Pharbitis nil

Indoleacetate oxidase activity in Pharbitis nil, Japanese morningglory, was zymographically examined. Some isozymes appearedonly after treatment with a low concentration of hydrogen peroxide.This phenomenon was assumed to be due to the overlapping ofboth isozymes and natural inhibitor substances on the zymograms. ¹Present address: Biological Institute, Department of LiberalArts, Shizuoka University, Ooya 836, Shizuoka, Japan. (Received September 30, 1968; )     

Old-Culture Flowering of Lemna paucicostata 6746 in Aged Hutner's Medium

Lemna paucicostata 6746, a short-day plant, flowers in agedHutner's medium even under continuous light, when the endogenousnitrogen level decreases to below 1.6 µmg fr wt. At thesenitrogen levels, daylength-independent flowering of the plantcan be induced even in fresh Hutner's medium. Thus, old-cultureflowering in Hutner's medium is due to nitrogen deficiency inthe plants. ¹Present address: Biological Institute, Faculty of Science,Shizuoka University, Shizuoka 422, Japan. (Received February 12, 1987; Accepted August 28, 1987)     

Modification of Sakami's Method for Degradation of 14C-3-Phosphoglyceric Acid and 14C-Serine

Sakami's procedure for determination of intramolecular radio-tracerdistribution in 3-phosphoglyceric acid or serine is modified.This new procedure uses ion exchange resin chromatography toseparate formic acid and formaldehyde, minimizing cross contaminationbetween C-2 and C-3 position. Specific activity was determinedby initial weighing of Ba^l4CO₃ on a fiberglass filter, followedby scintillation counting. The modified procedure is simplerand shorter than the original one, requiring much less startingmaterial and it permits the parallel handling of multiple samples. ³Present address: National Research Inst. of Tea, 2769 Kanaya,Kanaya-cho, Kashiwabara-gun, Shizuoka 428, Japan. (Received August 30, 1980; Accepted December 2, 1980)     

Purification and Characterization of Two Isozymes of Chlorophyllase from Mature Leaves of Chenopodium album

Chlorophyllase (Chlase) was purified from mature leaves of Chenopodiumalbum, and its enzymatic properties were investigated. Chlasewas extracted from acetone powder of C. album and purified bythe following chroma-tographic procedures: hydrophobic chromatography,Con A Sepharose, Heparin affinity chromatography, Mono Q ion-exchangechromatography, and gel-filtration. Con A Sepharose affinitychromatography and gel-filtration were the most effective stepson the purification. On Mono Q chromatography, the Chlase preparationseparated into two major and one minor fractions that exhibitedChlase activity. The two major Chlases were purified to homogeneity.Their molecular masses were estimated as 41.3 kDa and 40.2 kDaby SDS-PAGE. The optimum pH and K_m values of these two Chlaseswere similar. Their N-terminal amino acid sequences were almostidentical except for a deletion in the tenth amino acid residuein one of the Chlase; there was no homologous protein detectedby database search. ³Present address: Department of Biology and Geoscience, Facultyof Science, Shizuoka University, 836 Ohya, Shizuoka, 422 Japan.     

Enzymatic Conversion of Pheophorbide a to the Precursor of Pyropheophorbide a in Leaves of Chenopodium album

Soluble proteins extracted from leaves of Chenopodium albumcatalyzed the conversion of pheophorbide a to a precursor ofpyropheophorbide a, putatively identified as C-13²-carboxyl-pyropheophorbidea. The precursor was then decarboxylated non-enzymatically toyield pyropheophorbide a. Soluble proteins and pheophorbidea, as the substrate, were required for the formation of theprecursor, and boiled proteins were enzymatically inactive.The maximum rate of conversion of pheophorbide a to the precursoroccurred at pH 7.5. The K_m for pheophorbide a was 12.5 µMat pH 7.0. Both pheophorbide b and bacteriopheophorbide a couldserve as substrates, but protopheophorbide a could not. Formationof methanol was detected during the enzymatic reaction, an indicationthat the enzyme is an esterase. Among seven alcohol analogstested, only methanol inhibited the enzymatic activity uncompetitively,with a K₁ of 71.6 mM. Mass-spectrometric (MS) analysis of theprecursor yield a peak at m/z 579 that indicated the releaseof a methyl group from pheophorbide a. It appears thereforethat the enzyme catalyzes the demethylation of the carbomethoxygroup at C-13² of pheophorbide a by hydrolysis to yield methanoland the precursor, C-13²-carboxyl-pyropheophorbide a, whichis converted to pyropheophorbide a by spontaneous decarboxylation.We have tentatively designated the enzyme "pheophorbidase".The presence of the enzyme was dependent on plant species andit was expressed constitutively. ¹Present address: Faculty of Science, Shizuoka University, Ohya,Shizuoka, 422 Japan     

Studies on Microbody Development in Wounded Sweet Potato Root Tissue: Proposal for the Post-Translational Transport of Catalase into Preexisting Microbodies

Pure microbody fractions could be prepared in considerable yieldsfrom sweet potato root tissue slices incubated for 16 hr and3 days. The ratio of catalase activity to phospholipid contentin the fraction from slices incubated for 3 days was about 3times that from slices incubated for 16 hr. Total catalase activityin the former slices was about twice that in the latter. Thissuggests that catalase synthesized during incubation of theslices is transported into microbodies preexisting in intacttissue. ¹ Present address: Laboratory of Food Technology, Faculty ofApplied Biological Science, Hiroshima University, Fukuyama,Hiroshima 720, Japan. ² Present address: Terumo Co., Ltd., Omiya, Fujinomiya, Shizuoka418, Japan. (Received July 1, 1982; Accepted September 24, 1982)     

Studies on Plastid-Nuclei (Nucleoids) in Nicotiana tabacum L. I. Isolation of Proplastid-Nuclei from Cultured Cells and Identification of Proplastid-Nuclear Proteins

We have developed a method of isolating morphologically intactproplastid-nuclei (nucleoids) in large quantities from Nicotianatabacum cultured cells (line BY-2) without contamination bymitochondria and cell-nuclei. Fluorescence microscopy using 4',6-diamidino-2-phenylindole(DAPI) revealed that the compact structure of the isolated proplastid-nuclei(pp-nuclei) was disorganized by DNase I, micrococcal nuclease,proteinase K, 2 M NaCl and 2 M KC1, but was not affected byRNase A, suggesting that the pp-nuclei are compactly organizedby an electrostatic interaction between the proplastid- DNA(pp-DNA) and some protein(s). Although SDS-polyacrylamide gelelectrophoresis showed that the isolated pp-nuclear fractionstill contained a number of polypeptides, only four of them(mol wt: 69 kDa, 31 kDa, 30 kDa and 14 kDa) were found to besolubilized by treatments of the pelletable pp-nuclear fractionwith DNase I, micrococcal nuclease and 2 M NaCl. Furthermore,when the supernatant of the pp-nuclei treated with DNase I wasapplied onto a denatured or a native DNA-cellulose affinitycolumn, these four polypeptides were bound to both DNAs andeluted by raising the NaCl concentration. These findings, taken together, show that these proteins areproplastid DNA-binding proteins and strongly suggest that thepp-nuclei are compactly organized by interaction between thepp-DNA and these proteins. ⁴Present address: Department of Biology, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. ⁵Present address: School of Food and Nutritional Sciences, Universityof Shizuoka, Yata, Shizuoka 422, Japan. (Received August 13, 1987; Accepted November 9, 1987)     

The possibility of photo-induced induction of nitrate reductase in rice seedlings

Nitrate reductase activity in rice seedlings showed daily fluctuations.Seedlings placed in the dark slowly lost activity and quicklyregained it when exposed to sunlight. Etiolated seedlings alsoshowed rapid increases in activity when transferred to sunlight.This increase in activity by sunlight was inhibited by bothchloramphenicol and ethionine. Ethionine inhibition was reversedby methionine. Purification of nitrate reductase was carriedout by ammonium sulfate fractionation and DEAE-cellulose columnchromatography. Nitrate reductase was purified about 40-foldfrom plants placed in the dark and in sunlight. Incorporationof mediionine-S-¹⁴CH₃ into the nitrate reductase fraction wasstudied. In sunlight, the specific radioactivity of the nitratereductase fraction from the DEAE-cellulose column increased2-fold as compared with that of the crude extracts. Specificradioactivity did not increase in the dark. ¹Present address: Asahi Kasei Chemicals Industry Co., Ltd.,Sameshima 2-1, Fuji, Shizuoka, Japan (Received December 3, 1968; )     

Resistance to Oxidative Injury in Submerged Rice Seedlings after Exposure to Air

Rice (Oryza sativa L.) seedlings were germinated under waterin darkness for 5 or 6 days (submerged seedlings) and then inair for 1 day. Control seedlings were germinated in air, indarkness, for 5 or 6 days (aerobic seedlings). Changes in levelsof antioxidants and in the extent of oxidative damage afterexposure of submerged seedlings to air were studied. -Tocopherol,which inhibits lipid peroxidation, was present in submergedseedlings at about 3 times the level found in aerobically growncontrols, and higher levels than in controls were maintainedfor 24 h after transfer of the seedlings to air. Products oflipid peroxidation were present at a one-third of the levelsfound in aerobic controls, and their levels increased aftertransfer to air. However, these levels remained lower than thosein aerobic controls even after 24 h of contact with air. Carotenoids,which are considered to protect chlorophyllous compounds againstphotooxidation, were not found in submerged seedlings, but theirlevels increased after exposure of the seedlings to air. Lightat an intensity that did not cause photooxidative damage tochlorophyllous compounds in aerobic controls induced photobleachingof these compounds in submerged seedlings during the early stagesof adaptation to air. However, the extent of photobleachingdiminished as adaptation to air proceeded, and photobleachingwas no longer detected after 24 h of adaptation to air. Thus,the system for protection of cellular membranes from lipid peroxidationin the post-hypoxic phase appeared already to exist in submergedseedlings. However, the system for protection of pigments fromphotobleaching was poorly developed in submerged seedlings andwas fully active only after 24 h of adaptation to air. ¹Present address: Department of Biology, Faculty of Science,Shizuoka University Shizuoka, 422 Japan ²Present address: Research Institute for Bioresources, OkayamaUniversity, Kurashiki, 710 Japan     

Metabolic conversion of N-methyl carbon of {gamma}-glutamylmethylamide to caffeine in tea plants

Tea seedlings were treated with ¹⁴C-methylamine to cause synthesisof ¹⁴C--glutamylmethylamide (N-methyl-¹⁴C). The metabolic conversionof -glutamylmethylamide was studied by tracing ¹⁴C. ¹⁴C--Glutamylmethylamide (N-methyl-¹⁴C) translocated from rootsand cotyledons to shoots of tea seedlings, was converted almostentirely into caffeine. Conversion was greater in light-exposedsamples. For those grown in the dark, the converted amount didnot correspond to the total caffeine produced. More ¹⁴C--glutamylmethylamidewas present in stems than in leaves, but with ¹⁴C-caffeine,the opposite was found. When ¹⁴C--glutamylmethylamide or ¹⁴C-methylamine was appliedto leaf disks, ¹⁴C-caffeine was biosynthesized from both substances. ¹ Present address: Department of Agricultural Chemistry, ShizuokaUniversity, Iwata, Shizuoka 438, Japan. (Received September 25, 1971; )     

Activities of RNases in different cell compartments in different regions of pea roots

RNase activity in three different regions of pea roots—thetip, middle and basal regions—was analyzed. There werethree types of RNases differing from each other in their intracellularlocalization; the enzymes in a soluble form and two bound formsassociated with unknown, small particles or ribosomes and withthe microsomal membrane. The top region showed a high activityper DNA content of RNase in the microsomal membrane and lowactivities for the other two RNases, as compared with the otherregions. The middle region contained a low amount of RNA perDNA and showed a higher activity per DNA content of RNase inthe unknown particles or ribosomes than in the basal region.The activity of RNase in the unknown particles or ribosomesvaried greatly among the regions, but that in microsomal membranevaried only slightly. ¹ Present address: Okitsu Branch, Fruit Tree Research Station,Okitsu, Shizuoka, Japan. ² Present address: Asahi Denka Co. Ltd., 7–1 Higashiogu,Arakawa, Tokyo, Japan. (Received July 25, 1974; )     

The occurrence and properties of methenyltetrahydrofolate cyclohydrolase in plants

Methenyltetrahydrofolate cyclohydrolase (E.C. 3.5.4.9 [EC] ), whichis responsible for the enzymatic conversion of 5,10-methenyl-H₄FAto 10-formyl-H₄FA, has been found in various plant tissues.The enzyme was partially purified from pea seedlings and someof its properties were investigated. It was unstable, but wasstabilized by the addition of 25% glycerol. The enzyme was purifiedabout 60-fold by fractionation with ammonium sulfate and columnchromatography on DEAE-cellulose in the presence of 25% glycerol.Optimum pH for the reaction was 7.7. Michaelis constants for5,10-methenyl-H₄FA in the forward reaction, and for 10-formyl-H₄FAin the reverse reaction were 4x10^–5M and 2x10^–4M,respectively. The apparent equilibrium constant for the reactionwas calculated as 50. Enzyme activity was greatly inhibitedby the reduced forms of folate derivatives. The probable participationof this enzyme in the regulation of folate coenzyme levels inplant tissues has been suggested. ¹ Studies on the enzymatic synthesis and metabolism of folatecoenzymes in plants, VI. (For Part V, see Reference (5) ). Partof this paper was presented at the 22nd annual meeting of theJapan Vitamin Society held at Hiroshima on October 14, 1970. ² Present address: Sizuoka Eiwa Junior College, Ikeda, Shizuoka. (Received September 9, 1972; )     

Cellular localization of particulate-bound polyphenol oxidase in tea leaves

Cellular localization of polyphenol oxidase in tea leaves wasinvestigated using Polyclar AT as an adsorbent of polyphenolsexisting in large amounts in the leaves. Two polyphenol oxidasefractions from the particulate fraction were separated fromeach other by sucrose density gradient centrifugation and calledthe heavy and the light fraction. The centrifugal pattern inthe gradient indicated that the position of polyphenol oxidasein the heavy fraction coincided with those of the markers ofperoxisomes, catalase and malate dehydrogenase, but not withthose of mitochondria and chloroplasts, cytochrome c oxidaseand chlorophyll. The peak of activity in the heavy fractionshifted toward lower density concomitantly with the increaseof Polyclar AT content in the homogenizing medium. The heavyfraction was broken and disappeared at low pH condition, butthe light fraction remained unchanged. The light fraction seemedto be a fragment of the heavy fraction. About 30% of the polyphenoloxidase activity was retained in the 10,000xg pellet involvingperoxisomes, even after repeated washings with the suspendingmedium containing 1 M KCl. ¹This work was supported in part by a grant from the Ministryof Agriculture and Forestry. ²Tea Research Station, Ministry of Agriculture and Forestry,Kanaya, Shizuoka 428, Japan. (Received February 12, 1976; )     

Suppression of Acid Invertase Activity by Antisense RNA Modifies the Sugar Composition of Tomato Fruit

cDNA for an acid invertase (EC 3.2.1.26 [EC] ) of tomato (Lycopersiconesculentum Mill.) fruit was introduced into tomato plants underthe control of the cauliflower mosaic virus 35S promoter inthe antisense orientation. The antisense gene effectively suppressedthe invertase activity in soluble and cell wall fractions fromripening fruits. The sucrose content of fruits of the transformantswas markedly increased, while the hexose content was reduced.These results indicate that acid invertase is one of main determinantsof the sugar composition of tomato fruit. The invertase activityin the cell wall fraction of the leaf tissues of the transformantswas not suppressed to the same extent as that in the solublefraction. Wounding of the control leaf tissues induced invertaseactivity in both soluble and cell wall fractions. The inductionof activity in the soluble fraction was suppressed by the antisensegene, while that in the cell wall fraction was unaffected. Thesefindings suggest that mRNA for some other invertase, in particular,the mRNA for a cell wall-bound invertase, was present in leaves. ¹Present address: Plant Breeding and Genetics Research Laboratory,Japan Tobacco Inc., 700 Higashibara, Toyoda, Iwata, Shizuoka,438 Japan. ²Present address: National Institute of Agrobiological Resources,Kannondai, Tsukuba, Ibaraki, 305 Japan.     

Transit Peptides of Thylakoid Luminal Proteins: the Sites of Stromal Processing are Conserved among Higher Plants

The amino terminal of the transport intermediate of the 23-kDaprotein of PSII, which was generated by stromal processing,was determined by radiosequencing in order to identify the siteof processing of its precursor. The site was located in thecentral region of the transit peptide. The primary structureof the processing site included a motif that is common to theprocessing sites of transit peptides of various stromal proteins.This common motif was also found in the transit peptides ofother thylakoid luminal proteins, an indication of its conservationamong higher plants. A common secondary structure, rßturn/rß-sheet/-helix,was predicted from the amino acid sequence around the consensusmotif. This structural similarity between stromal and thylakoidluminal proteins suggests that the sequences are under a commonselective pressure to maintain these sites of cleavage by astromal processing peptidase. ³Present address: Department of Food Science and Technology,Faculty of Agriculture, Kyoto University, Kyoto, 606-01 Japan ⁴Present address: Plant Breeding and Genetics Research Laboratory,Japan Tobacco Company, Toyota-cho, Iwata, Shizuoka, 438 Japan     

Localization of the carbon in caffeine biosynthesized from N-methyl carbon of {gamma}-glutamylmethylamide in tea plants

1. Localization of carbon in caffeine molecule biosynthesizedfrom the N-methyl carbon of -glutamylmethylamide in tea plantswas observed. ¹⁴C-Caffeine produced from ¹⁴C--glutamylmethylamidewas isolated and degraded. Approximately 26–55% of the¹⁴C was observed in the three methyl carbons in caffeine, withonly 2–3% at the C-2 carbon, 3–7% at the C-8 carbonposition. The amount of ¹⁴C at the C-4, C-5 and C-6 positionswas calculated from the results obtained. 2. The role of the N-methyl carbon of -glutamylmethylamide inthe formation of RNA in tea plants was examined. Incorporationof the N-methyl-¹⁴C of ¹⁴C--glutamylmethylamide into AMP andGMP in RNA was found. These facts indicate that in tea plants, -glutamylmethylamideis metabolized and most of its N-methyl carbon is utilized asa precursor for caffeine formation and little, if any, as aprecursor for nucleic acid formation. ¹ Present address: Department of Agricultural Chemistry, ShizuokaUniversity, Iwata, Shizuoka 438, Japan. (Received February 2, 1972; )     

Differential Expression of Genes Involved in the Biosynthesis and Perception of Ethylene during Ripening of Passion Fruit (Passiflora edulis Sims)

An increase in the enzyme activity of 1-aminocyclopropane-1-carboxylicacid (ACC) synthase and ACC oxidase induces the evolution ofethylene during the ripening of passion fruit. A much higherlevel of ethylene is produced in arils than in seeds or peelsduring ripening. The pattern of expression of two ACC synthasegenes (PE-ACS1 and PE-ACS2), one ACC oxidase gene (PE-ACO1),and two ethylene receptor genes (PE-ETR1 and PE-ERS1) revealedthat the expression of these genes is differentially regulated.Expression of PE-ACS1 and PE-ACO1 was enhanced during ripeningand after ethylene treatment. However, prominent expressionof PE-ACS1 was delayed compared to that of PE-ACO1. Much largerquantities of PE-ACS1 mRNA and PE-ACO1 mRNA were seen in arilsthan in seeds; this corresponds well with an increase in theamount of ethylene produced by the plant tissue itself. Thelevel of PE-ACS2 mRNA was detectable in arils of the preclimactericfruit, although it decreased during ripening. These resultssuggest that expression of PE-ACS1 and PE-ACO1 is required toincrease the activity of ethylene biosynthetic enzymes duringripening. The level of expression of PE-ETR1 and PE-ERS1 didnot significantly change over the course of ripening; however,the mRNA levels of PE-ETR1 and PE-ERS1 were much higher in arilsthan in seeds. ⁴Present address: Center forMolecular Genetics Research, Shizuoka University, Shizuoka, 422-8529 Japan.     

Dark Hydrogen Evolution and Adenine Nucleotides of Subtropical Marine Unicellular Green Algae during Anaerobic Incubation

The relation between dark anaerobic hydrogen (H₂) evolutionof marine unicellular green algae and the energetic state ofthe cells, as revealed by adenine nucleotide (AN) levels, wasstudied. One of the 4 investigated strains produced H₂ continuouslyfor 48 h and maintained its adenylate energy charge (EC) atabout 0.6, whereas the H₂ evolution of the other strains stoppedwithin 24 h and their EC decreased to low values. Prolongedaerobic preincubation resulted in reduced H₂ evolution and astronger decrease in EC during subsequent anaerobiosis. Amongcultures of the same strain, H₂ evolution was correlated withthe initial pool levels of total AN and chlorophyll and withthe ATP level and EC during anaerobiosis. These results indicatea dependence between H₂ evolution and capacity for starvation.EC was not affected by complete inhibition of H₂ evolution bycarbon monoxide in one strain but was significantly loweredin another strain. This difference between the two strains isinterpreted as reflecting a different dependence on, or utilizationof, alternative pathways for disposal of electrons during fermentation,although H₂ evolution apparently played a minor and non-essentialrole in the dark anaerobic fermentation. ¹ Present address: Institute of Oceanic Research and Development,Tokai University, Orido, Shimizu, Shizuoka 424, Japan. ² Present address: Institute of Marine Research, Directorateof Fisheries, P.O. Box 1870, N-5011 Bergen-Nordnes, Norway. (Received November 19, 1986; Accepted March 17, 1987)     

Auxin- and Acid-Induced Changes in the Mechanical Properties of the Cell Wall

Auxin- and acid-induced changes in the mechanical propertiesof the cell wall were analyzed by measuring the creep of thecell wall using pumpkin (Cucurbita moschata Duch cv. Shirakikuza)hypocotyl segments. Hypocotyl segments were treated with orwithout IAA and stored in 50% glycerol at –15°C formore than 2 weeks before measurements. Creep rate increasedwith the increase in the load. The increase was first very slowup to the phase shift point (yield threshold, y), and afterthat, it was steep. The rate of the creep rate increase (creepcoefficient, Cm) was larger and y was smaller at pH 4.5 thanpH 6.8. This indicates cell wall loosening was facilitated underacidic conditions. IAA-pretreatment of the segments resultedin the lowering of y at pH 6.8 only. Around pH 5 and 45°C,Cm was highest and y was lowest. Boiling in distilled wateralmost lost the differential effect of Cm and y on pH and IAA.The differential effect of pH on Cm and y were recovered bythe addition of a crude extract of cell wall-bound proteins.It is implied that some enzymatic processes are involved inthe control of acid-induced cell wall extension. ¹Present address: Nihon Shokuhin Kako Co., Ltd. 30 Tajima, Fuji-City,Shizuoka, 417-8530 Japan. ²Present address: Graduate School of Environmental Earth Science,Hokkaido University, Sapporo, 060 Japan.     

The Effects of Auxin on the Mechanical Properties in Vivo of Cell Wall in Hypocotyl Segments from Gibberellin-Deficient Cowpea Seedlings

Auxin-induced changes in the mechanical properties of cell wallwere examined by both positive and negative pressure jump methodsusing hypocotyl segments excised from the 3-day-old seedlingsof cowpea that has been treated with uniconazole, a potent inhibitorof the biosynthesis of gibberellins. In such segments (U-segments)that were deficient in endogenous gibberellin, auxin increasedonly the effective turgor (Pⁱ–Y) and did not change theextensibility () of cell wall. As a result, the extent of theauxin-induced promotion of growth was halved. However, auxinwas able to increase of U-segments that has been pretreatedfor two hours with GA₃ prior to the application of IAA. Measurementof intracellular pressure (P_i) with a pressure probe revealedthat auxin did not change Pⁱ in either U-segments or GA₃-pretreatedsegments. The results suggest that auxin can decrease the yieldthreshold of the cell wall (Y) independently of gibberellinbut can increase only in the presence of gibberellin. The differencebetween and Y in terms of their requirement for gibberellinto respond to auxin suggests that they are mutually separablemechanical properties that originate from different molecularprocesses that occur in the architecture of yielding cell walls. ³Present address: Ohishi, Enden, Mori-machi, Shuchi-gun, Shizuoka,437-02 Japan