Effects of methanol on the synthesis of aspartame precursor catalysed byPseudomonas aeruginosa elastase

Summary Pseudomonas elastase was found to be efficient in catalysing the reaction betweenN-benzyloxycarbonyl-L-aspartic acid and L-phenylalanine methyl ester producing the aspartame precursor in aqueous and aqueous methanolic solutions. 25% (v/v) methanol was most favourable for the synthesis where about 100% increase in yield was obtained compared to that in aqueous solution.Abbreviations N-cbz-L-Asp N-benzyloxycarbonyl-L-Aspartic acid - L-Phe-OMe L-phenyl alanine methyl ester - HPLC high performance liquid chromatography. All the % of methanol is a volume % in water unless otherwise specified     

Repeated batch and continuous syntheses of N-(benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester with immobilized thermolysin

Summary N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester was synthesized from N-(benzyloxycarbonyl)-l-phenylalanine and l-phenylalanine methyl ester in an aqueous solution (aqueous phasic reaction), in an aqueous/organic biphasic system (biphasic reaction), and in an organic solvent (organic phasic reaction) with immobilized thermolysin. In the aqueous phasic reaction with thermolysin immobilized on Amberlite XAD-7, the whole product was trapped inside the support; extraction with ethyl acetate was needed to recover the product, and the equilibrium yield was low (about 65%). With the biphasic and organic phasic reactions with ethyl acetate as an organic solvent, the yield was around 95%. Because of the high yield and feasibility of operation, repeated batch and continuous reactions were done in the biphasic and organic phasic systems, respectively. The half-lives of the activity for the immobilized enzyme used in the biphasic system at 40°C by repeated batch operation and in a plug flow reactor fed with substrate dissolved in ethyl acetate at 40°C and 30°C were estimated to be about 200 h (67 batches), 420 h, and 1100 h, respectively.     

Biotransformation of alkyl and aryl carbonates: enantioselective hydrolysis

Summary N-(Benzyloxycarbonyl)-l-asparty-l-phenylalanine methyl ester, the precursor of the synthetic sweetener aspartame, was continuously synthesized in an immobilized thermolysin plug-flow type reactor at 25° C with the substrates (N-benzyloxycarbonyl-l-aspartic acid and l-phenylalanine methyl ester) dissolved in ethyl acetate. The immobilized enzyme was quite stable in ethyl acetate containing 2.5% 0.01 M 2-(N-morpholino)ethanesulphonic acid-NaOH buffer, pH 6.0, and 20 mM CaCl₂ with or without the substrate at 25° C. By periodically washing the column, we could conduct a continuous reaction for over 500 h with an average yield of 95% and a space velocity of 1.85 h ^–1.Offprint requests to: K. Nakanishi     

Synthesis of kyotorphin precursor from eutectic mixtures catalysed by α-chymotrypsin

The kyotorphin precursor, N-carbobenzoxyl-l-tyrosine-l-arginine amide (N-CBZ-l-Tyr-l-ArgNH₂), was synthesized from N-CBZ-l-tyrosine ethyl ester (N-CBZ-l-TyrOEt) and l-arginine amide (l-ArgNH₂) by using -chymotrypsin. Eutectic mixtures were formed by mixing the substrates in the presence of water and/or organic solvents as adjuvants. The eutectic temperature was obtained with a 0.45 l-ArgNH₂ mole fraction. It was lowered to 23 °C by addition of 10% (v/w) water and 5% (v/w) dimethyl formamide, thus maintaining homogeneous liquid states at the reaction temperature of 30 °C. After 9 h the solutions became solidified and no further reaction took place. Approximately 90% (mol/mol) conversion was achieved from the substrate mixtures with substrate contents responsible for more than 80% (w/w) of the total mixture.     

Novel stabilization pattern of thermolysin due to the binding substrate induced by electrostatic change in enzyme active site caused by the temperature elevation

During the synthesis of the dipeptide, N-benzyloxycarbonyl-l-phenylalanyl-l-phenylalanine methyl ester, from N-benzyloxycarbonyl-l-phenylalanine and l-phenylalanine methyl ester by thermolysin, the enzyme was stabilized by 20°C up to 110°C. The stabilization was caused by the interaction of the enzyme with Phe-OMe, a counterpart of the substrate, which was bound at the enzyme active site due to the drop in pH and dielectric constant following the temperature elevation of the medium. The binding of the enzyme to Phe-OMe suggested the induction of the transition state formation at around 80°C based on the UV spectra, resulting in the increase in the stability in the higher temperature region. The fluorescence second-order derivative spectra suggested that the binding Phe-OMe interacted with Trp 115 at the active site of the enzyme. The phenomenon was considered to be a novel stabilization pattern of the enzyme resulting from the conduction due to the chemical modification by the binding substrate.     

On the Molecular Weight of Polypeptide Chain of Sweet Potato β-Amylase

A number of N-acyl-L-proline derivatives were synthesized and their biological activities were investigated by using lettuce (Lactuca sativa L. cv. Sacramento) seedling test. A wide variety of these compounds promoted root growth at 25°C both under light and in darkness. Of the compounds tested, N-(2-ftuorobenzoyl)-L-proline methyl ester (4) showed the highest activity and caused a 270% increase in the root elongation compared to the control. N-(2-Naphthoyl)-L-proline methyl ester (14) promoted the root growth, while N-(1-naphthoyl)-L-proline methyl ester inhibited it. L-Proline, benzoic acid, and 2-naphthoic acid had no significant effect on lettuce seedlings. Compounds 4 and 14, and N-(2-chlorobenzoyl)-L-proline methyl ester (7) reduced the inhibitory effect of 1 ppm ABA on the root growth, while the D-isomer of 4 was less activite than compound 4. Compounds 4, 7, and 14 did not show any rescue-activity for the complete inhibition of germination that was caused by treating 10 ppm of ABA.     

Mild, rapid and selective alcoholysis of terpyridine-appended amide substrates by Cu-catalysis: protonation state and reactivity of the complex

An amide bond of a terpyridine-appended substrate, 6-(L-phenylalanylamino)-2,2′:6′,2″-terpyridine (2a), was cleaved to yield phenylalanine methyl ester quantitatively in the presence of catalytic amounts of Cu²⁺ not only in methanol but also in aqueous methanol at 30 °C. The reaction proceeds via formation of an N₃O (three terpyridine nitrogens and one carbonyl oxygen) type 1:1 metal complex 2a-Cu²⁺. From spectral titration, the structure of the 2a-Cu²⁺ complex was confirmed to have three different protonation states, i.e., A (non-deprotonated amide with α-ammonium), D1 (deprotonated-amide with α-ammonium) and D2 (deprotonated-amide with α-amino) states. Among them, the complex in the D2 state was exclusively responsible for the observed mild, rapid and selective alcoholysis, showing the first-order rate constant of 6 × 10⁻³ s⁻¹ or half-decay time of 2 min in methanol at 30 °C. Acidity of the amide proton was found to be higher than that of the ammonium proton in the complex, allowing formation of the highly reactive amide-deprotonated D2 state in methanol even without addition of external bases. Factors contributing to the high reactivity of the complex were discussed.     

Evaluation of butanoic,pentanoic and hexanoic acids as internal standards for analysis of acidic fermentation end-products from Bacillus stearothermophilus by gas-liquid chromatography

The use of butanoic, pentanoic and hexanoic acids as internal standards for chromatographic analysis of complex mixtures of by-products from acidic fermentation, produced by Bacillus stearothermophilus grown in xylose-containing medium, was evaluated. After addition of the internal standards, the aqueous supernatant microbial fermentation fluids were submitted to the derivatisation process with methanol and sulphuric acid at 50° C. Injection of 1-l aliquots of the lower chloroform layer was carried out. The esterified compounds were separated in approximately 14 min by temperature programming on a glass column packed with 10% (w/w) diethyleneglycol adipate and 2% phosphoric acid, and analysed with a flame ionisation detector. Chromatograms of the methyl esters derivates have shown that both pentanoic and hexanoic acids can be used as internal standards for gas-chromatographic analysis of acidic fermentation end-products, since they are well separated and resolved. Under the experimental conditioss established, the methyl ester of butanoic acid was masked by the chloroform peak and so is not a convenient compound for further use.     

Purification and properties of ap-nitrobenzyl esterase fromBacillus subtilis

A procedure for purifying to homogeneity a microbially produced biocatalyst useful for deblocking intermediates in the manufacture of beta-lactam antibiotics is reported. In aqueous solution the purifiedp-nitrobenzyl (PNB) carboxy-esterase was soluble, monomeric (molecular weight: 54 000 by SDS-PAGE or by gel filtration) and exhibited an acidic pl, 4.1. The PNB carboxy-esterase catalyzed rapid ester hydrolysis for simple organic esters such as PNB-acetate, benzyl acetate and -naphthyl acetate and catalyzed deblocking (ester hydrolysis) of beta-lactam antibiotic PNB esters such as cephalexin-PNB and loracarbef-PNB. TheN-terminal amino acid sequence and the amino acid composition are reported. A serine residue is involved in ester hydrolysis: the PNB carboxy esterase was inhibited by phenylmethylsulfonyl fluoride and diethylp-nitrophenyl phosphate; one mole of diisopropyl fluorophosphate titration was required per mole of PNB carboxy-esterase for complete inhibition. When the [³H]-diisopropyl fluorophosphate-treated biocatalyst was digested with Lys C and the resulting peptides separated by HPLC, a single [³H]-labeled peptide was obtained; its amino acid sequence is reported. Inhibition of the PNB carboxy esterase by diethyl pyrocarbonate suggests that a histidinyl residue (or residues) is (are) also involved in the catalytic site of the esterase.Abbreviations used -ME -mercaptoethanol - Cf cefaclor - Cf nucleus-PNB - (6R, 7R) 7-amino-3-chloro-8-oxo-5-thia-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylic acid, (4-nitrophenyl)methyl ester - Cp cephalexin - Cp-PNB p-nitrobenzyl carboxy-ester of cephalexin - DEPC diethyl, pyrocarbonate - DFP diisopropyl fluorophosphate - DMSO dimethyl sulfoxide - DNP diethylp-nitrophenyl phosphate - EDTA ethylenediaminetetraacetic acid - EGTA ethylene, glycol-bis(aminoethyl ether) - N,N,NN tetracetic acid - Lc loracarbef - Lc-PNB p-nitrobenzyl carboxy-ester of loracarbef - Lc nucleus-PNB - (6R, 7S) 7-amino-3-chloro-8-oxo-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylic acid, (4-nitrophenyl)methyl ester - Lys C an endoproteinase specifically cleaving at C terminal lysine residues - MW_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - PNB p-nitrobenzyl - PNBCE p-nitrobenzyl carboxy-esterase - SDS sodium dodecyl sulfate     

Inhibition of photosynthesis by chilling in moderate light: a comparison of plants sensitive and insensitive to chilling

Photosynthetic activity, in leaf slices and isolated thylakoids, was examined at 25° C after preincubation of the slices at either 25° C or 4° C at a moderate photon flux density (PFD) of 450 mol·m^–2·s^–1, or at 4° C in the dark. The plants used wereSpinacia oleracea L.,Cucumis sativus L. andNerium oleander L. which was acclimated to growth at 20° C or 45° C. The plants were grown at a PFD of 550 mol·m^–2·s^–1. Photosynthesis, measured as CO₂-dependent O₂ evolution, was not inhibited in leaf slices from any plant after preincubation at 25° C at a moderate PFD or at 4° C in the dark. However, exposure to 4° C at a moderate PFD induced an inhibition of CO₂-dependent O₂ evolution within 1 h inC. sativus, a chilling-sensitive plant, and in 45° C-grownN. oleander. The inhibition in these plants after 5 h reached 80% and 40%, respectively, and was independent of the CO₂ concentration but was reduced at O₂ concentrations of less than 3%. Methyl-viologen-dependent O₂ exchange in leaf slices from these plants was not inhibited. There was no photoxidation of chlorophyll, in isolated thylakoids, or any inhibition of electron transport at photosystem (PS)II, PSI or through both photosystems which would account for the inhibition of photosynthesis. The conditions which inhibit photosynthesis in chilling-sensitive plants do not cause inhibition inS. oleracea, a chilling-insensitive plant, or in 20° C-grownN. oleander. The CO₂-dependent photosynthesis, measured at 5° C, was reduced to about 3% of that recorded at 25° C in chilling-sensitive plants but only to about 30% in the chilling-insensitive plants. Methyl-viologen-dependent O₂ exchange, measured at 5° C, was greater than 25% of the activity at 25° C in all the plants. The results indicate that the mechanism of the chilling-induced inhibition of photosynthesis does not involve damage to PSII. That inhibition of photosynthesis is observed only in the chilling-sensitive plants indicates it is related, in some way, to the disproportionate decrease in photosynthetic activity in these plants at chilling temperatures.Abbreviations Chl chlorophyll - DPIPH reduced form of 2,6-dichlorophenol-indophenol - DMQ 2,5-dimethyl-p-benzoquinone - MV methyl viologen - 20°-oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - PFD photon flux density (photon fluence rate) - PSI and PSII photosystem I and II, respectively     

Optimization of <Emphasis Type="Italic">Serratia marcescens</Emphasis> lipase production for enantioselective hydrolysis of 3-phenylglycidic acid ester

Lipase production and cell growth of Serratia marcescens ECU1010 were optimized in shake flasks, with lipase production being enhanced 9.5-fold (4,780 U/l) compared with the initial activity (500 U/l). Optimal carbon and nitrogen sources were Tween-80 and peptone, and the optimal ratio of Tween-80 to peptone was 1:3. The optimized cultivation conditions were 25°C and pH 6.5. Lipase activity, particularly specific activity, could be improved by decreasing the cultivation temperature from 35 to 25°C. Enzyme stability was significantly improved by simple immobilization with synthetic adsorption resin no. 8244. After five reaction cycles, enzyme activity decreased only very slightly, while enantioselectivity of the preparation remained constant, and the ee_s (enantiomeric excess of the remaining substrate) achieved in all cases was higher than 97%. The resin-8244-lipase preparation can be used for efficient enantioselective hydrolysis of trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM], a key intermediate in the synthesis of Diltiazem.     

Moisture content and CO2 exchange of lichens

Summary Net photosynthesis (10 klx light intensity, 150 E m^-2 s^-1 PAR) and dark respiration of the lichen Ramalina maciformis at different temperatures are measured in relation to thallus water content. Both first increase with increasing hydration. Dark respiration then remains constant with increased water content until thallus saturation. In contrast, a further increase in water content leads to a depression of net photosynthesis, as shown in previous studies, after a maximum of CO₂ uptake has been attained. However, the extent of this depression depends strongly on temperature. In saturated thalli (160% water content in relation to lichen dry weight) the depression amounts to about 15% and 63% of the maximum unsaturated rate at 5°C and 25°C thallus temperature, respectively. The moisture compensation-point of net photosynthesis is also decisively determined by temperature (for 0°C at 20% water content; for 25°C at 15%), and the water content that allows maximum rates of CO₂ uptake (for 0°C at 80%; for 25°C at less than 40% water content). An electrical analogue of CO₂ exchange in a lichen thallus is presented, and it is suggested that the experimental results may be interpreted in terms of temperature-dependent CO₂ diffusion resistances in imbibed lichen thalli.     

Differences in protein patterns of gill epithelial cells of the fish Gillichthys mirabilis after osmotic and thermal acclimation

Different protein patterns in gill epithelium of a euryhaline and eurythermal teleost fish (Gillichthys mirabilis, Family Gobiidae) in response to long-term (2 months) osmotic and thermal acclimation were found for the first time. Gill epithelial cells were isolated to remove extracellular proteins and quantify specialized cell types. Chloride cells were identified on the basis of size (>10 m) and bright appearance after [2-(p-dimethylaminostyryl)-1-methyl-pyridinium-iodine] staining. Small mitochondria-rich cells were <5 m in diameter and showed intermediate fluorescence. Abundance of chloride cells and small mitochondria-rich cells was significantly influenced by osmotic but not thermal acclimation (dilute seawater/25°C: 1.4±0.2% chloride cells, 11.9±4.6% small mitochondria-rich cells; seawater/25°C: 2.4±0.6% chloride cells, 2.2±1.3% small mitochondria-rich cells; seawater/10°C: 2.9±0.3% chloride cells, 1.2±0.7% small mitochondria-rich cells). Pavement cells, identified by low fluorescence and intermediate size (5–10 m), largely predominated under all conditions (>85% of cells). Thus, they represented the major protein source in gill epithelium. Differences in protein patterns were detectable using two-dimensional but not one-dimensional electrophoresis. Of 602 proteins identified by charge and molecular weight properties, only two were induced by high temperature (25°C) and three in response to cold acclimation (10°C). Nine proteins were induced in diluted seawater-acclimated fish, whereas no seawater-induced proteins were found. We hypothesize that proteins induced under dilute seawater conditions are important for the function of pavement cells in gills of hyper-osmoregulating G. mirabilis.Abbreviations BCA bicinchoninic acid - BSS balanced salt solution - CC chloride cells - CLB cell lysis buffer - DASPMI [2-(p-dimethylaminostryryl)-1-methylpyridinium-iodine] - DSW diluted sea water - DTT dithiothreitol - EDTA ethylene-diaminetetraacetate - FW fresh water - IEF isoelectric focusing - PC pavement cells - PDA diacrylpiperazine - pI isoelectric point(s) - PMSF phenylmethanesulphonylfluoride - SDS sodium dodecyl sulfate - SMRC small mitochondria-rich cells - SW sea water - TEMED tetramethylenediamine     

Improvement of<Emphasis Type="Italic"> Citrus clementina</Emphasis> Hort. ex Tan. microspore-derived embryoid induction and regeneration

An improvement of the protocol for haploid induction through anther culture of Citrus clementina Hort. ex Tan. cv. Nules was achieved following the evaluation of a number of the factors affecting androgenesis. The influence of thidiazuron (TDZ) and three temperature pre-treatments (4°C, 25°C, 32°C) on the floral buds with respect to anther culture of C. clementina Hort. ex Tan., cv. Nules was investigated. An increased embryoid production was induced in the medium supplemented with TDZ. Pre-treatment temperatures of 4°C and 25°C were more favorable for embryo production than 32°C. Regeneration of androgenic haploid plantlets from cv. SRA 63 of C. clementina is reported here for the first time.Abbreviations 6-BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - KI Kinetin - NAA -Naphthaleneacetic acid - TDZ Thidiazuron (N-phenyl-1,2,3,-thi-diazol-5-ylurea) - ZEA Zeatin Communicated by L. PeñaBoth authors have contributed equally to this article.     

Purification of an acid protease from Mucor rouxii that inactivates chitin synthetase

An acid protease was purified from the mycelial form of Mucor rouxii by a method which involved salt and acid precipitation, gel filtration and anion-exchange chromatography. The enzyme had a molecular mass of 16,000 Da. Its optimum pH was 4.0, maximal activity was obtained at 50°C, and it was inactivated at 70°C. It was not affected by leupeptin or N -p-tosyl-L-lysine chloromethyl ketone (TLCK) but diazoacetyl-DL-norleucine methyl ester (DNME) in the presence of Cu²⁺ and more noticeably pepstatin A, strongly inhibited the activity. This acid protease did not activate zymogenic chitin synthetase from the fungus, but brought about its inactivation even at low concentrations and after short periods of incubation time.Abbreviations TLCK N -p-tosyl-L-lysine chloromethyl ketone - DNME diazoacetyl-DL-norleucine methyl ester - TCA trichloroacetic acid - SDS sodium dodecyl sulfate     

Enzymatic synthesis of sialic acid derivative by immobilized lipase from Candida antarctica

The effects of important reaction parameters on the enhancement of sialic acid derivative lipophilic properties through the lipase-catalyzed esterification of N-acetyl neuraminic acid methyl ester are investigated in this study. It is found that the lipase Novozym 435 from Candida antarctica is particularly useful in the preparation of sialic acid methyl ester monononanoate (SAMEMN). The optimum temperature for the SAMEMN synthesis reaction using Novozym 435 is 60 °C, and nonanoic anhydride is found to be the best substrate among all acyl donors. The Novozym 435-catalyzed esterification of N-acetyl neuraminic acid methyl ester gave a maximum yield of 87.7% after 6 h in acetonitrile at 60 °C. Because the novel method developed is simple, yet effective, it could potentially be used industrially for the production of sialic acid derivatives.     

Purification and characterization of a type B feruloyl esterase (StFAE-A) from the thermophilic fungus<Emphasis Type="Italic"> Sporotrichum thermophile</Emphasis>

A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity. The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0±1.5 kDa, with a mass of 33±1 kDa on SDS-PAGE. The pI of the enzyme was estimated by cation-exchange chromatofocusing to be at pH 3.1. The enzyme activity was optimal at pH 6.0 and 55–60 °C. The purified esterase was stable at the pH range 5.0–7.0. The enzyme retained 70% of activity after 7 h at 50 °C and lost 50% of its activity after 45 min at 55 °C and after 12 min at 60 °C. Determination of k _cat/K _m revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively. No activity on methyl sinapinate was detected. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and it hydrolyzed 4-nitrophenyl 5-O-trans-feruloyl--l-arabinofuranoside (NPh-5-Fe-Araf) 2-fold more efficiently than NPh-2-Fe-Araf. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from S. thermophile (a maximum of 34% total ferulic acid released after 1 h incubation). StFAE-A by itself could release FA, but at a level almost 47-fold lower than that obtained in the presence of xylanase. The potential of StFAE-A for the synthesis of various phenolic acid esters was tested using a ternary water-organic mixture consisting of n-hexane, 1-butanol and water as a reaction system.     

Isolation and characterization of five serine proteases with trypsin-, chymotrypsin- and elastase-like characteristics from the gut of the lugworm Arenicola marina (L.) (Polychaeta)

Summary Five proteases were isolated from the digestive fluid of the lugworm, Arenicola marina L. The enzymes (molecular weight 24.0–24.6 kDa) were classified as serine proteases. Three enzymes showed a cleavage specificity corresponding to mammalian trypsin (E.C. 3.4.21.4). One protease possessed a chymotrypsin-like cleavage pattern (E.C. 3.4.21.1), and the fifth preferred cleavage behind short-chain amino acids like an elastase (E.C. 3.4.21.36). Detailed investigations revealed differences in molecular characteristics and cleavage patterns compared to mammalian proteases, especially in the chymotrypsin- and the elastase-like enzymes.Abbreviations APNE N-acetyl-d/l-Phe -naphthyl ester - BANA N-benzoyl-d/l-Arg -naphthylamide - BAPNA N-benzoyl-d/l-Arg-4-nitroanilide - BIGGANA N-benzoyl-l-Ile-l-Glu-Gly-l-Arg-4-nitroanilide - BLPNA N-benzoyl-d/l-Lys-4-nitroanilide - BTEE N-benzoyl-l-Tyr ethyl ester - enzyme T1/T2/T3 trypsin-like enzyme - enzyme ChT chymotrypsin-like enzyme - enzyme E elastase-like enzyme - GPANA N-glutaryl-l-Phe-4-nitroanilide - MUF 4-methylumbelliferryl - MW molecular weight - PMSF phenylmethylsulphonyl fluoride - SAAPPNA N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-4-nitroanilide - SBTI soybean trypsin inhibitor - SPPNA N-succinyl-l-Phe-4-nitroanilide - TAME N-tosyl-l-Arg methyl ester - TFA trifluoracetic acid - TLCK N-tosyl-l-Lys chloromethyl ketone - TPCK N-tosyl-l-Phe chloromethyl ketone - TRIS tris(hydroxymethyl)aminomethane     

Superoxide production by thylakoids during chilling and its implication in the susceptibility of plants to chilling-induced photoinhibition

Factors influencing the rate of superoxide (O ₂ ^- ) production by thylakoids were investigated to determine if increased production of the radical was related to injury induced by chilling at a moderate photon flux density (PFD). Plants used were Spinacia oleracea L., Cucumis sativus L. and Nerium oleander L. grown at either 200° C or 45° C. Superoxide production was determined by electron-spin-resonance spectroscopy of the (O ₂ ^- )-dependent rate of oxidation of 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) to the corresponding oxazolidinoxyl radical, OXANO ·. For all plants, the steady-state rate of O ₂ ^- production by thylakoids, incubated at 25° C and 350 mol photon · m^–2 · s^–1 (moderate PFD) with added ferredoxin and NADP, was between 7.5 and 12.5 mol · (mg chlorophyll)^–1 · h^–1. Incubation at 5° C and a moderate PFD, decreased the rate of O ₂ ^- production 40% and 15% by thylakoids from S. oleracea and 20° C-grown N. oleander, chillinginsensitive plants, but increased the rate by 56% and 5% by thylakoids from C. sativus and 45° C-grown N. oleander, chilling-sensitive plants. For all plants, the addition of either ferredoxin or methyl viologen increased the rate of O ₂ ^- -production at 25° C by 75–100%. With these electron acceptors, lowering the temperature to 5° C caused only a slight decrease in O ₂ ^- production. In the absence of added electron acceptors, thylakoids produced O ₂ ^- at a rate which was about 45% greater than that when ferredoxin and NADP were present. The addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced O ₂ ^- production under all conditions tested. The results show that the rate of O ₂ ^- production increases in thylakoids when the rate of electron transfer to NADP is reduced. This could explain differences in the susceptibility of thylakoids from chilling-sensitive and chilling-insensitive plants to chilling at a moderate PFD, and is consistent with the proposal that O ₂ ^- production is involved in the injury leading to the inhibition of photosynthesis induced under these conditions.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophen-yl)-1,1-dimethylurea - Fd ferredoxin - MV methyl viologen - 20°oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - OXANOH 2-ethyl-1-hydroxy-2,5,5-tri-methyl-3-oxazolidine - PFD photon flux density (photon fluence rate) - TEMED tetramethyl ethylenediamine We would like to thank R.T. Furbank, R.S.B.S., Australian National University, Canberra, A.C.T., and C.B. Osmond, now of Duke University, Durham, N.C., USA, for the gift of ferredoxin, R.A.J.H. was supported by a Commonwealth Postgraduate Research Award.