Differences between Indica and Japonica rice varieties in CO2 exchange rates in response to leaf nitrogen and temperature

Four Indica and five Japonica varieties of rice (Oryza sativa L.) were examined to elucidate their differences in photosynthetic activity and dark respiratory rate as influenced by leaf nitrogen levels and temperatures. The photosynthetic rates of single leaf showed correlations with total nitrogen and soluble protein contents in the leaves. Respiratory rate was also positively correlated with the leaf nitrogen content. When compared at the same level of leaf nitrogen or soluble protein content, the four Indica varieties and one of Japonica varieties, Tainung 67, which have some Indica genes derived from one of its parents, showed higher photosynthetic rates than the remaining four Japonica varieties. At the same photosynthetic rate, the Indica varieties showed lower respiratory rate than Japonica varieties. When the leaf temperature rose from 20°C to 30°C, the photosynthetic rate increased by 18 to 41%, whereas the respiratory rate increased by 100 to 150%. These increasing rates in response to temperature were higher in the Japonica than in the Indica varieties. In this respect, Tainung 67 showed the same behavior as of the other four Japonica varieties.Abbreviations 30/20 ratios the ratios of photosynthetic and respiratory rates at 30°C to those at 20°C     

Nitrosyl cytochrome c oxidase. Formation and properties of mixed valence enzyme

We report the first resonance Raman scattering studies of NO-bound cytochrome c oxidase. Resonance Raman scattering and optical absorption spectra have been obtained on the fully reduced enzyme (a2+, a2+(3) NO) and the mixed valence enzyme (a3+, a2+(3) NO). Clear vibrational frequency shifts are detected in the lines associated with cytochrome a in comparing the two redox states. With 441.6 nm excitation the fully reduced preparation yields a spectrum similar to that of carbon monoxide-bound cytochrome c oxidase and is dominated by the spectrum of reduced cytochrome a. In contrast, in the mixed valence preparation no contributions from reduced cytochrome a are evident in the spectrum, verifying that this heme is no longer in the Fe2+ state. In the mixed valence NO-bound samples, a line appears at approximately 545 cm-1, a frequency similar to that found in NO-bound hemoglobin and myoglobin and assigned as an Fe-N-O-bending mode in those proteins. We do not detect this line in the spectrum of the fully reduced NO-bound enzyme. The carbonyl line of the cytochrome a3 heme formyl group in the fully reduced NO-bound enzyme appears at approximately equal to 1666 cm-1 in the resonance Raman spectrum. In the mixed valence NO-bound preparation the frequency of the carbonyl line increases by 1.2 cm-1 to approximately equal to 1667 cm-1. Thus, modes in cytochrome a2+(3) NO are sensitive to the redox state of the cytochrome a and/or CuA centers. We propose that the redox sensitivity of the formyl mode and the Fe-N-O mode results from an interaction between cytochrome a2+(3) (NO) and the cytochrome a-CuA pair, and is linked to the cytochrome a3 (NO) by the coupling between CuB and the NO-bound cytochrome a3 heme.     

Resonance Raman spectral isolation of the a and a3 chromophores in cytochrome oxidase.

Resonance Raman spectra of reduced CO-bound cytochrome oxidase obtained at two different excitation frequencies (441.6 and 413.1 nm) are compared with the spectra of the fully reduced enzyme. In the spectra of the CO-bound complex only the cytochrome a modes are strongly enhanced with 441.6 nm excitation and only the modes of the CO-bound cytochrome a3 heme are strongly enhanced with 413.1-nm excitation. In the fully reduced complex with both excitation frequencies, modes of both cytochrome a and a3 are enhanced. By subtraction we are able to uncover the complete spectrum of the fully reduced ligand-free cytochrome a3 heme. Thus, we report the discrete resonance Raman spectra of cytochromes a2+, a2+3, and a2+3 (CO). The spectra of fully reduced cytochrome a and ligand-free cytochrome a3 are very different especially in the low frequency region. Binding CO to ferrous cytochrome a3 results in electronic structure changes in the heme analogous to those in hemoglobin and myoglobin, from which we conclude that there is nothing electronically unique in the ferrous cytochrome a3 heme to account for its catalytic properties.     

Identification of functional murine adenosine deaminase cDNA clones by complementation in Escherichia coli

Total poly(A+) RNA derived from a mouse cell line with amplified adenosine deaminase genes was used as template to synthesize double-stranded cDNA. The cDNAs were inserted into the PstI site of the beta-lactamase gene in plasmid pBR322 following G-C tailing. After transformation into adenosine deaminase-deficient Escherichia coli hosts, recombinant plasmids containing functional murine adenosine deaminase cDNAs were identified by selecting for functional complementation. Analysis of plasmids containing functional adenosine deaminase cDNA sequences strongly suggested that adenosine deaminase expression resulted mainly from beta-lactamase/adenosine deaminase fusion proteins even when the adenosine deaminase codons were out-of-frame with respect to the beta-lactamase gene codons upstream. The nucleotide sequence of a 1.65-kilobase pair cDNA insert in one of the functional recombinant clones was determined and found to contain a 1056-nucleotide open reading frame. When this 1056-nucleotide open reading frame was inserted into a mammalian expression vector and introduced into monkey kidney cells, a high level of authentic mouse adenosine deaminase was produced. Nucleic acid blot analysis using a full-length adenosine deaminase cDNA clone as probe revealed that the mouse adenosine deaminase structural gene was at least 21 kilobase pairs in size and encoded three polyadenylated mRNAs. Analysis of the cDNA library from which the functional clones were isolated suggested that this approach of cloning functional mammalian adenosine deaminase cDNA clones by genetic complementation of enzyme-deficient bacteria could be accomplished even if the abundance of the adenosine deaminase mRNA sequences were as low as approximately 0.001%.     

Evolution of the lipoprotein gene in the enterobacteriaceae. Cloning and DNA sequence of the lpp gene from Proteus mirabilis

We cloned the lipoprotein gene from Proteus mirabilis and determined its DNA sequence. Comparison with the lpp genes from Escherichia coli, Serratia marcescens, Erwinia amylovora and Morganella morganii revealed several unique features of the evolution of the lpp gene in the Enterobacteriaceae and enabled us to establish phylogenetic relationships between these bacteria.     

Mutants with Altered Ca2+-Channel Properties in PARAMECIUM TETRAURELIA: Isolation, Characterization and Genetic Analysis

Dancers are a group of mutants in Paramecium tetraurelia whose Ca²⁺ current inactivates poorly and are likely to be defective in the structure of their Ca²⁺ channels. These mutants show prolonged backward swimming in response to K⁺ and Ba ²⁺ in the medium and were selected by this property in a galvanotactic trough. The dancer mutants are semidominant, and all isolated mutants belong to one complementation group; they are not allelic to any of the previously isolated behavioral mutants of P. tetraurelia. The phenotypic change from the homozygous parent to heterozygous F₁ generation takes three to five fissions. There is no evidence of a cytoplasmic factor capable of converting the dancer to the wild-type phenotype, as has been demonstrated in the mutants pawn and cnr. We suggest that the dancer locus is a structural gene for the Ca²⁺ channel.     

Microbial oxidation of methanol: Purification and properties of formaldehyde dehydrogenase from a Pichia sp. NRRL-Y-11328

An NAD⁺-linked, reduced glutathione-dependent formaldehyde dehydrogenase was purified to homogeneity from soluble extracts of methanol-grown yeast, Pichia sp. Formaldehyde and methylglyoxal are oxidized in the presence of NAD⁺ as an electron acceptor. NADP⁺ could not replace NAD⁺. Other straight chain aldehydes (C₂–C₆ tested), branched-chain aldehydes (e.g., isobutyaldehyde), aromatic aldehydes (e.g., salicylal-dehyde, benzaldehyde), glutyraldehyde, glyceraldehyde, glycoaldehyde, and glyoxal-dehyde tested were not oxidized by the purified formaldehyde dehydrogenase. The product of formaldehyde oxidation by purified enzyme was demonstrated to be S-for-mylglutathione by measuring the absorption at 240 nm due to the formation of thioester of formaldehyde and reduced glutathione. The K_m values for NAD⁺, formaldehyde, and reduced glutathione were 0.12, 0.31, and 0.16 mm, respectively, for the forward reaction at pH 8.0. The purified formaldehyde dehydrogenase also catalyzed the reduction of S-formylglutathione in the presence of NADH. Formate was not reduced by the purified enzyme. The K_m values for S-formylglutathione and NADH were 0.60 and 0.25 mm, respectively, for the reverse reaction at pH 6.0. Formaldehyde dehydrogenase has a molecular weight of 84,000 as determined by gel filtration and subunit molecular weight of 41,000 as determined by sodium dodecyl sulfate-gel electrophoresis. S-Formylglutathione, a product of formaldehyde oxidation, was oxidized by the partially purified formate dehydrogenase from Pichia sp. Formate dehydrogenase has a higher affinity toward S-formylglutathione (K_m value 1.8 mm) than toward formate (K_m value 25 mm). Antiserum prepared against the purified formaldehyde dehydrogenase from Pichia sp. NRRL-Y-11328 forms strong precipitin bands with isofunctional enzymes from methanol-grown Pichia pastoris NRRL-Y-7556 and Torulopsis candida Y-11419 and weak precipitin bands with Hansenula polymorpha NRRL-Y-2214. No cross-reaction was observed with isofunctional enzyme derived from methanol-grown Kloeckera sp.     

Epoxidation of Short-Chain Alkenes by Resting-Cell Suspensions of Propane-Grown Bacteria

Sixteen new cultures of propane-utilizing bacteria were isolated from lake water from Warinanco Park, Linden, N.J. and from lake and soil samples from Bayway Refinery, Linden, N.J. In addition, 19 known cultures obtained from culture collections were also found to be able to grow on propane as the sole carbon and energy source. In addition to their ability to oxidize n-alkanes, resting-cell suspensions of both new cultures and known cultures grown on propane oxidize short-chain alkenes to their corresponding 1,2-epoxides. Among the substrate alkenes, propylene was oxidized at the highest rate. In contrast to the case with methylotrophic bacteria, the product epoxides are further metabolized. Propane and other gaseous n-alkanes inhibit the epoxidation of propylene. The optimum conditions for in vivo epoxidation are described. Results from inhibition studies indicate that a propane monooxygenase system catalyzes both the epoxidation and hydroxylation reactions. Experiments with cell-free extracts show that both hydroxylation and epoxidation activities are located in the soluble fraction obtained after 80,000 × g centrifugation.     

Production of Methyl Ketones from Secondary Alcohols by Cell Suspensions of C2 to C4n-Alkane-Grown Bacteria

Nineteen new C₂ to C₄n-alkane-grown cultures were isolated from lake water from Warinanco Park, Linden, N.J., and from lake and soil samples from Bayway Refinery, Linden, N.J. Fifteen known liquid alkane-utilizing cultures were also found to be able to grow on C₂ to C₄n-alkanes. Cell suspensions of these C₂ to C₄n-alkane-grown bacteria oxidized 2-alcohols (2-propanol, 2-butanol, 2-pentanol, and 2-hexanol) to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Cells grown on 1-propanol or 2-propanol oxidized both primary and secondary alcohols. In addition, the activity for production of methyl ketones from secondary alcohols was found in cells grown on either alkanes, alcohols, or alkylamines, indicating that the enzyme(s) responsible for this reaction is constitutive. The optimum conditions for in vivo methyl ketone formation from secondary alcohols were compared among selected strains: Brevibacterium sp. strain CRL56, Nocardia paraffinica ATCC 21198, and Pseudomonas fluorescens NRRL B-1244. The rates for the oxidation of secondary alcohols were linear for the first 3 h of incubation. Among secondary alcohols, 2-propanol and 2-butanol were oxidized at the highest rate. A pH around 8.0 to 9.0 was found to be the optimum for acetone or 2-butanone formation from 2-alcohols. The temperature optimum for the production of acetone or 2-butanone from 2-propanol or 2-butanol was rather high at 60°C, indicating that the enzyme involved in the reaction is relatively thermally stable. Metal-chelating agents inhibit the production of methyl ketones, suggesting the involvement of a metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble fraction; this activity requires a cofactor, specifically NAD. Propane monooxygenase activity was also found in the cell-free soluble fraction. It is a nonspecific enzyme catalyzing both terminal and subterminal oxidation of n-alkanes.