Photoinactivation of δ-aminolevulinic acid dehydratase from maize by flavin mononuclotide

The -aminolevulinic acid dehydratase activity was irreversibly inactivated by irradiation of the enzyme in presence of flavin mononucleotide. The loss of enzyme activity was dependent on time of irradiation, concentration of FMN and intensity of irradiance. It required oxygen and was markedly enhanced in heavy water. The presence of levulinic acid (a competitive inhibitor of -ALAD) during irradiation prevented the inactivation considerably indicating photooxidative damage at or near the active site. Superoxide dismutase, sodium benzoate and sodium formate offered no protection, but singlet oxygen quenchers like azide and tryptophan were effective. NADH, electron donor to excited flavins, also prevented the loss of enzyme activity. These results indicate that singlet oxygen produced by light absorption of FMN was responsible for the photooxidative inhibition of the enzyme.Abbreviations ALAD -aminolevulinic acid dehydratase - FMN flavin mononucleotide - O₂ ^- superoxide - H₂O₂ hydrogen peroxide - 10₂ singlet oxygen - LA levulinic acid - PBG porphobilinogen - BSA bovine serum albumin - BME 2-mercaptoethanol - SOD superoxide dismutase - pHMB para-hydroxymercuribenzoate - DTT dithiothreitol - FAD flavin adenine dinucleotide - NADH nicotinamide adenine dinucleotide     

Altitudinal changes in the incidence of crassulacean acid metabolism in vascular epiphytes and related life forms in Papua New Guinea

Summary The occurrence of Crassulacean acid metabolism (CAM), as judged from ¹³C values, was investigated in epiphytes and some related plant species at a series of sites covering the approximate altitudinal range of epiphytes in Papua New Guinea. Comprehensive collections were made at each site and the occurrence of water storage tissue and blade thickness was also determined. Some 26% of epiphytic orchids from a lowland rainforest (2–300 m.a.s.l) showed ¹³C values typical of obligate CAM and possessed leaves thicker than 1 mm. A second group of orchids, mostly with succulent leaves, possessed intermediate ¹³C values between -23 and -26% and accounted for 25% of the total species number. Some species of this group may exhibit weak CAM or be facultative CAM plants. The remainder of the lowland rainforest species appeared to be C₃ plants with ¹³C values between -28 and -35%. and generally possessed thin leaves. Obligate CAM species of orchids from a lower montane rainforest (1175 m.a.s.l) comprised 26% of the species total and mostly possessed thick leaves. The remainder of the species were generally thin-leaved with ¹³C values between -26 and -35%. largely indicative of C₃ photosynthesis. Orchids with intermediate ¹³C values were not found in the lower montane rainforest. Obligate CAM appeared to be lacking in highland epiphytes from an upper montane rainforest and subalpine rainforest (2600–3600 m.a.s.l). However the fern, Microsorium cromwellii had a ¹³C value of -21.28%. suggesting some measure of CAM activity. Other highland ferns and orchids showed more negative °¹³C values, up to-33%., typical of C₃ photosynthesis. The highland epiphytic orchids possessed a greater mean leaf thickness than their lowland C₃ counterparts due to the frequent occurrence of water storage tissue located on the adaxial side of the leaf. It is suggested that low daytime temperatures in the highland microhabitats is a major factor in explaining the absence of CAM. The increased frequency of water storage tissue in highland epiphytes may be an adaptation to periodic water stress events in the dry season and/or an adaptation to increased levels of UV light in the tropicalpine environment.     

Activation of fatty acids by non-glyoxysomal peroxisomes

Ethylene treatment (approx. 20 l ·1^-1 in air for 2 d) of tobacco (Nicotiana tabacum L. cv. Havana 425) plants markedly increases the endo--1,3-glucanase (EC 3.2.1.39) content of leaves. The antigenic form of the enzyme induced is the same one whose production is blocked by treating cultured cells with combinations of auxin (1.1 · 10^-5 M -naphthaleneacetic acid) and cytokinin (1.4 · 10^-6 M kinetin). Evidence is presented that cultured tobacco cells require ethylene for -1,3-glucanase accumulation: i) ethylene treatment increased the accumulation of \-1,3-glucanase in callus tissues >10 d after subculturing and in cell-suspension cultures; ii) callus tissues can produce ethylene; iii) conditions known to inhibit ethylene production (1 mM CoCl₂; 33° C treatment) or ethylene action (approx. 1.6 mmol · 1^-1 norbornadiene in air) inhibited -1,3-glucanase accumulation by callus tissues treated for 4 d following subculturing; and, these inhibitory effects were prevented by exogenous ethylene. Combinations of auxin and cytokinin blocked ethylene-induced accumulation of -1,3-glucanase by cell-suspension cultures. The results favor a model in which ethylene induces results favor a model in which ethylene induces 1,3-glucanase accumulation, and auxin and cytokinin inhibit this induction process.Abbreviations NAA -naphthaleneacetic acid - NDE norbornadiene     

Large non-homology in heteroduplex DNA is processed differently than single base pair mismatches

Summary Unmethylated DNA heteroduplexes with a large single stranded loop in one strand have been prepared from separated strands of DNA from two different strains of bacteriophage , one of which has a 800 base pair IS1 insertion in the cI gene. The results of transfections with these heteroduplexes into wild-type and mismatch repair deficient bacteria indicate that such large non-homologies are not repaired by the Escherichia coli mismatch repair system. However, the results do suggest that some process can act to repair such large non-homologies in heteroduplex DNA. Transfections of a series of recombination and excision repair deficient mutants suggest that known excision or recombination repair systems of E. coli are not responsible for the repair. Repair of large non-homologies may play a role in gene conversion involving large insertion or deletion mutations.     

An endogenous circadian rhythm of transpiration in Tamarix aphylla

An endogenous circadian rhythm in the transpiration of Tamarix aphylla (L.) Karst. was found for plants grown in continuous light under laboratory conditions. The mean period (±SD) was 21.7±2.3 h (n = 121). No such rhythm was observed in continuous darkness, except for one small hump at the time of the first cycle. The influence of NaCl, Cd(NO₃)₂ and LiCi on the rhythmic behaviour of young T. aphylla plants was investigated. NaCl concentrations of up to 150 m M reduced the overall transpiration rates of the plants, but did not change the period of the rhythm. The amplitude and the mesor of the oscillations were inversely correlated with the NaCl concentration. A similar influence was found for Cd(NO₃)₂, but with concentrations that were approximately three orders of magnitude smaller than those of the NaCl treatments. The rhythmic behaviour of the plants was not altered by 10 m M LiCl. It is suggested that the described rhythm of transpiration may have a dual effect: (a) it might cause a partial closure of the stomates during midday hours and (b) it might serve as a possible synchronizer ("master clock") for other rhythmic phenomena in the plants.     

Evaluation of N2-fixation and nitrogen economy of a maize/cowpea intercrop system using15N dilution methods

Yields of above ground biomass and total N were determined in summer-grown maize and cowpea as sole crops or intercrops, with or without supplementary N fertilizer (25 kg N ha⁻¹, urea) at an irrigated site in Waroona, Western Australia over the period 1982–1985. Good agreement was obtained between estimates of N₂ fixation of sole or intercrop cowpea (1984/85 season) based on the¹⁵N natural abundance and¹⁵N fertilizer dilution techniques, both in the field and in a glasshouse pot study. Field-grown cowpea was estimated to have received 53–69% of its N supply from N₂-fixation, with N₂-fixation onlyslightly affected by intercropping or N fertilizer application. Proportional reliance on N₂-fixation of cowpea in glasshouse culture was lower (36–66%) than in the field study and more affected by applied N. Budgets for N were drawn up for the field intercrops, based on above-ground seed yields, return of crop residues, inputs of fixed N and fertilizer N. No account was taken of possible losses of N through volatilization, denitrification and leaching or gains of N in the soil from root biomass. N₂-fixation was estimated tobe 59 kg N ha⁻¹ in the plots receiving no fertilizer N, and 73 kg N ha⁻¹ in plots receiving 25 kg N ha⁻¹ as urea. Comparable fixation by sole cowpea was higher (87 and 82 kg N ha⁻¹ respectively) but this advantage was outweighed by greater land use efficiency by the intercrop than sole crops.     

Spinach ferredoxin is a calcium-binding protein

Spinach-leaf ferredoxin was identified as a calcium-binding protein by ⁴⁵Ca autoradiography on nitrocellulose membranes and with the cationic carbocyanine dye 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl] naphtho[1,2-d]thiazolium bromide (stains-all). Binding of ⁴⁵Ca was observed at pH 6.8 and pH 7.8 and in the presence of 5 mM and 20 mM MgCl₂. At the higher MgCl₂ concentration the Ca²⁺-binding capacity is reduced. Only micromolar concentrations of LaCl₃, however, are required to achieve a similar effect. Both the oxidized and reduced forms of ferredoxin bind calcium.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - stains-all 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl] naptho[1,2-d]thiazolium bromide     

Expression of the β-subunit of β-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein -conglycinin, encoded by a multigene family. -Conglycinin consists of three subunits; , , and . A genomic clone for a -subunit of -conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this -subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The -subunit expressed in seeds of petunia and tobacco was recognized by anti--conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the -subunit were produced. There was approximately a twofold variation in the accumulation of the -subunit protein in the mature seeds of transgenic petunia plants, each containing a single -subunit gene. However, the level of protein accumulation in mature seeds and the amount of -subunit mRNA in developing seeds was not correlated. Accumulation of the -subunit protein in transgenic seeds was less than the -subunit protein that accumulated in transgenic petunia seeds containing a single -subunit gene and less than the amount of the -subunit in mature soybean seeds which contain 8–13 -subunit genes. In transgenic tobacco plants, the accumulation of the -subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis