Quinones as plant growth regulators

The effects of benzoquinone, naphthaquinone and anthraquinone on the growth of tomato callus, whole plants of tomato and on rooting of mungbean cuttings were studied. Naphthaquinone effects on some oxidases and on the isozyme patterns of peroxidases in all the three systems were also observed. Quinones increased callus growth, the number of roots initiated in mungbean cuttings and the growth of tomato plants, significant increases being obtained with 10^–5 M naphthaquinone. Naththaquinone also decreased the activities of IAA oxidase, ascorbic acid oxidase and polyphenol oxidase, and led to the disappearance of one of the isozyme of peroxidase in all systems.     

Genomic,molecular evolution,and expression analysis of NOX genes in soybean (Glycine max)

Reactive oxygen species (ROS) are versatile signaling molecules in sensing stresses and play critical roles in signaling and development. Plasma membrane NADPH oxidases (NOXs) are key producers of ROS, and play important roles in the regulation of plant-pathogen interactions. Here, we performed a comprehensive analysis of the NOX gene family in the soybean genome (Glycine max) and 17 NOX (GmNOX) genes were identified. Structural analysis revealed that the GmNOX proteins in soybean were as conserved as those in other plants. 8 duplicated gene pairs were formed by a Glycine-specific whole-genome duplication (WGD) event approximately 13 million years ago (Mya). The Ka/Ks ratios of GmNOX genes ranged from 0.04 to 0.28, suggesting that the GmNOX family had undergone purifying selection in soybean. Gene expression patterns showed different expression of these duplicate genes, suggesting that the GmNOXs were retained by substantial subfunctionalization during the soybean evolutionary processes. Subsequently, the expression of GmNOXs in response to drought and phytohormones were characterized via qPCR. Importantly, four GmNOXs showed strong expression in nodules, pointing to their probable involvement in nodulation. Thus, our results shed light on the evolutionary history of this family in soybean and contribute to the functional characterization of GmNOX genes in soybean.     

Roles for heme–copper oxidases in extreme high-light and oxidative stress response in the cyanobacterium Synechococcus sp. PCC 7002

The ctaCIDIEI and ctaCIIDIIEII gene clusters that encode heme–copper cytochrome oxidases have been characterized in the marine cyanobacterium Synechococcus sp. PCC 7002 and the inactivation of ctaDI was shown to affect high-light adaptation. In this study, Synechococcus sp. PCC 7002 wild-type, ctaDI, ctaDII, and ctaDI–ctaDII double mutants were grown under extreme high-light and oxidative stress to further assess the roles of cytochrome oxidases in cyanobacteria. Cells of the ctaDI mutant strain barely grew under extreme high-light illumination of 4.5 mE m⁻² s⁻¹, suggesting that CtaDI is required for high-light acclimation in Synechococcus sp. PCC 7002. The ctaDI–ctaDII double mutant cells unexpectedly tolerated extreme high-light intensity, indicating that the disruption of ctaDII gene suppresses the high-light sensitivity phenotype of the ctaDI single mutant. The ctaDII mutant cells also exhibited higher tolerance to the oxidative stress compound, methyl viologen, in the growth media. The ctaDII mutant and the ctaDI–ctaDII double mutant cells had approximately twofold higher levels of superoxide dismutase (SOD) activity, indicating that the disruption of ctaDII gene increased the capacity to decompose active oxygen species. These results suggest that the CtaII cytochrome oxidase may be involved with the oxidative stress response, including the control of SOD expression.     

Molecular structure and catechol oxidase activity of a new copper(I) complex with sterically crowded monodentate N-donor ligand

The attempted alkylation of 1,3-bis(2′-pyridylimino)isoindoline (indH) by the use of n-BuLi and subsequent alkyl halides led to quaternization of the pyridine nitrogens and the zwitterionic monodentate N-ligand (Me₂ind)I was formed. By the use of the ligand the copper(I) complex [Cu^I(Me₂ind)I₂] was prepared and its structure determined. It was found to be good catalyst for the oxidation of 3,5-di-tert-butylcatechol (DTBCH₂) to 3,5-di-tert-butyl-1,2-benzoquinone (DTBQ) and H₂O₂ by dioxygen. Detailed kinetic studies revealed first-order dependence on the catalyst and dioxygen concentration and saturation type behavior with respect to the substrate.     

Involvement of Hydrogen Peroxide Generated by Polyamine Oxidative Degradation in the Development of Lateral Roots in Soybean

In order to determine whether hydrogen peroxide （H2O2） generated by polyamlne oxidative degradation Is Involved In the development of lateral roots In soybean, the length and the number of lateral roots, the actlvltlea of polyamlne oxldases and dlamlne oxldases, and the endogenous free polyamlne and H2O2 content were analyzed In soybean （Giycine max （Linn.） Merr.） main roots of 2-d-old seedlings after treatments for 2 d with exogenous β-hydroxyethylhydrazine （an Inhibitor of polyamlne oxldases）, H202, putresclne, cyclohexylamlne （an Inhibitor of spermidine synthase） or N,N＇-dimethylthlourea （a scavenger of hydrogen peroxide）.β-hydroxyethylhydrazlne treatment strongly Inhibited the development of lateral roots In soybean seedlings, reduced the activities of polyamine oxldases and dlamlne oxidases, decreased H2O2 levels, and led to the accumulation of endogenous polyamlnes In the main roots. The inhibitory effect of β-hydroxyethylhydrazlne on root development could be alleviated by exogenously applied 10 μmol/L H2O2 （a major product of polyamlne oxidation）. Treatment with cyclohexylamlne and putresclne promoted root growth slightly, but treatment with cyclohexylamlne plus N,N＇dlmethylthlourea or putresclne plus N,N＇-dlmethylthlourea prevented the development of soybean lateral roots. The effects of these treatments on the development of soybean lateral roots were consistent with the changes In endogenous H2O2 levels. These results suggest that the development of soybean lateral roots Is associated with the oxidative degradation of polyamlnes, and that their products, especially H2O2, are likely to play an Important role In the growth of soybean lateral roots.     

Structural roles of the active site iron(III) ions in catechol 1,2-dioxygenases and differential secondary structure changes in isoenzymes A and B from Acinetobacter radioresistens S13

The reversible active site metal ion removal process for two catechol 1,2-dioxygenase isoenzymes (IsoA and IsoB) isolated from Acinetobacter radioresistens S13 has been monitored using circular dichroism and fluorescence spectroscopic techniques. IsoA and IsoB are homodimers, containing one iron(III) ion per subunit. Their amino acid sequence identity is 48.4%. Previous experiments suggested that structural diversities could be responsible for the differential thermal and pH stabilities of the two isoenzymes and of their distinct demetallation kinetics. The far-UV CD spectra of IsoA and IsoB catechol 1,2-dioxygenases from A. radioresistens S13 provide information on their secondary structures. IsoB appears to have a content of alpha-helices higher than IsoA. Upon metal ion removal, both proteins reversibly lose part of their secondary structure following distinct pathways. CD spectra simulations allowed us to estimate the content of alpha-helices, beta-sheets, and turns for each isoenzyme and to monitor the secondary structure rearrangements. The metal ion withdrawal has large influence on the secondary structure: in particular a significant reduction of alpha-helices content is observed for both isoenzymes. Intrinsic fluorescence emission spectra clearly support such results, adding information on the local environment changes of the tryptophan residues. The positioning of Trp250 in IsoB has been shown to be of particular interest for monitoring the local structure changes occurring upon metal ion removal. For the first time these studies allow to underline the role of active site iron ions on dioxygenases folding and stability, further evidencing the differences in structural assembling between the two isoenzymes from A. radioresistens S13.     

An examination of the peroxidases from Lupinus albus L. hypocotyls

Peroxidases (EC 1.11.1.7) from hypocotyls of Lupinus albus L. cv. Rio Maior have been characterised using one- and two-dimensional, native electrophoretic techniques. Data are presented showing the complexity in charge and molecular size or shape of these peroxidases. We report the finding of a new acidic peroxidase and several new basic peroxidases in these hypocotyls, and of their stability to treatments considered to break ligand-induced variants and conformational variants derived from differences in polypeptide folding. Densitometric data demonstrate that these new peroxidases contribute up to 60 of the total peroxidase activity in hypocotyls. Studies of intercellular fluid, cell-wall and soluble fractions, with assays of purity were conducted in an attempt to define the subcellular locations of these additional peroxidases. The acidic form (pI 4.1) is greatly enriched in soluble fractions, three of the basic peroxidases (pIs 9.5, 9.7 and >9.7) are strongly associated to the cell wall, ad a minor, basic component (pI 9.7) is enriched in the intercellular fluid. Individual peroxidase activities with the substrates coniferyl alcohol, ferulic acid or indole acetic acid were compared by densitometric analysis of zymograms with those for guaiacol, and notable differences between these peroxidases in their capacity to oxidise indole acetic acid in vitro were identified. The possible functions of these peroxidases in vivo and their implications to current understanding of peroxidases in L. albus are discussed.Abbreviations APAGE anionic polyacrylamide gel electrophoresis - CA coniferyl alcohol - CPAGE cationic polyacrylamide gel electrophoresis - IEF isoelectric focusin - NEIEF non-equilibrated isoelectric focusing - 2D two dimensional - pI isoelectric point - RCPAGE reversed current polyacrylamide gel electrophoresis     

Substrate, substrate analogue, and inhibitor interactions with the ferrous active site of catechol 2,3-dioxygenase monitored through XAS studies

The interactions of catechol (substrate), 2-hydroxy-pyridine-N-oxide (substrate analogue) and 2-bromophenol (inhibitor) with the extradiol cleaving catechol-2,3-dioxygenase from Pseudomonas putida mt-2 have been monitored through X-ray absorption spectroscopy (XAS). The analysis of the data provides details about the mode of coordination of the substrate and of the inhibitors to the active site of the enzyme.     

Immunological similarities of mammalian xanthine oxidases

The degree of relatedness among mammalian xanthine oxidases (XO) was determined by microcomplement fixation. Rabbit anti bovine milk XO serum was tested against xanthine oxidase (homologous protein) and against the heterologous proteins of bovine liver, monkey liver, rat liver, lactating cow serum, nonlactating cow serum, and steer serum. The indices of dissimilarity for the heterologous proteins were expressed as units of immunological distance and the percent sequence differences among these proteins inferred from the y=5x relationship where y is immunological distance and x is percent sequence difference. Rat liver XO differed by approximately 27% in amino acid sequence from bovine milk XO. In order of increasing immunological distance from bovine milk XO, the sources of XO ranked as follows: lactating cow serum < nonlactating cow serum < steer serum = beef liver < monkey liver < rat liver. The monkey ranked much closer than the rat in order of phylogenetic kinship to the cow. Starch gel electrophoresis of liver, milk, and serum showed that the milk and the serum contained only cationic forms of xanthine oxidase while all the liver samples tested contained cationic as well as anionic forms of the enzyme. The electrophoretic mobility properties of xanthine oxidase confirmed the polymorphic nature of the enzyme as revealed by the immunological data.This investigation was supported by the National Dairy Council, Chicago, Illinois, and by USPHS Grant AM16726.