Electron-microscopic cytochemical localization of diamine and polyamine oxidases in pea and maize tissues

An electron-microscopic cytochemical method was used to localize diamine oxidase (DAO) in pea and polyamine oxidase (PAO) in maize (Zea mays L.). The method, based on the precipitation of amine-oxidase-generated H₂O₂ by CeCl₃, was shown to be specific for DAO and PAO and permitted their localization in plant tissues with a high degree of resolution. Both enzymes are localized exclusively in the cell wall. Both DAO- and PAO-activity staining is most intense in the middle lamellar region of the wall and in cells exhibiting highly lignified walls. The oxidases could provide H₂O₂ for peroxidase-mediated cross-linking reactions in the cell wall and may, in this capacity, play a role in the regulation of plant growth.Abbreviations AG 1-aminoguanidine - AT 3-amino-1,2,4-triazole - -HEH -hydroxyethylhydrazine - DAO(s) diamine oxidase(s) - PAO(s) polyamine oxidase(s) - Put putrescine - Spd spermidine - Spm spermine The authors wish to thank Nancy Piatczyc for the technical assistance with electron-microscopy studies. We are grateful to Dr. Stanley J. Roux, University of Texas at Austin, for providing us with samples of maize cell-wall exudates. This work was supported by grants to R.D.S from the National Aeronautics and Space Administration (NAGW-1049 and NAGW-1382).     

The polyamines and their catabolic products are significant players in the turnover of nitrogenous molecules in plants

Polyamines (PAs) are nitrogenous molecules which play a well-established role in most cellular processes during growth and development under physiological or biotic/abiotic stress conditions. The molecular mode(s) of PA action have only recently started to be unveiled, and comprehensive models for their molecular interactions have been proposed. Their multiple roles are exerted, at least partially, through signalling by hydrogen peroxide (H(2)O(2)), which is generated by the oxidation/back-conversion of PAs by copper amine oxidases and PA oxidases. Accumulating evidence suggests that in plants the cellular titres of PAs are affected by other nitrogenous compounds. Here, we discuss the state of the art on the possible nitrogen flow in PAs, their interconnection with nitrogen metabolism, as well as the signalling roles of PA-derived H(2)O(2) during some developmental processes and stress responses.     

Distribution of polyamines and their related catabolic enzyme in etiolated and light-grown leguminosae seedlings

Diamine-oxidase (DAO; EC 1.4.3.6) activity and di-and polyamine levels were estimated along the epicotyl and root of light-grown and etiolated lentil (Lens culinaris Medicus) and pea (Pisum sativum L.) seedlings. The activity of DAO was higher in etiolated epicotyls than in lightgrown ones. In both species there was a positive correlation between DAO activity and the diamine (putrescine and cadaverine) levels along the whole epicotyl and root. Polyamine (spermine and spermidine) distribution seemed to be associated with the meristematic and elongating zone of the epicotyl and root. The physiological function of DAO is discussed in relation to its possible role in providing hydrogen peroxide to peroxidase-dependent reactions occurring in the cell wall.Abbreviations CAD cadaverine - DA diamine - DAO diamine oxidase - PA polyamine - PUT putrescine - SPD spermidine - SPM spermine     

Does polyamine catabolism influence root development and xylem differentiation under stress conditions?

Amine oxidases (AOs) oxidize polyamines (PAs) to aldehydes, simultaneously producing the removed amine moiety and hydrogen peroxide (H₂O₂). AOs, which include copper-containing amine oxidases (CuAOs) and flavin-containing amine oxidases (PAOs), are stress-inducible enzymes involved in both PA homeostasis and H₂O₂ production. Here, we suggest that H₂O₂ derived from PAO-mediated PA catabolism has a role in inducing root xylem differentiation during plant stress responses, whereas its involvement in this event during plant development under physiological conditions is not suitably supported by the currently available data. Moreover, we show that spermidine (Spd) supply leads to a higher induction of cell death in wild-type (WT) tobacco (Nicotiana tabacum) plants as compared to tobacco plants over-expressing maize (Zea mays) PAO (S-ZmPAO) in the cell wall, in apparent contradiction with the already reported results obtained by the analysis of the corresponding WT and S-ZmPAO Spd-untreated plants. Considering this last observation, we propose that PAs  diversely affect plant development and stress responses depending on the expression levels of AOs, which in turn may lead to different plant responses by altering the PAs/H₂O₂ balance.     

Developmental aspects of polyamine-oxidizing enzyme activities in the mouse kidney. Effects of testosterone

Summary In the present study developmental patterns of renal polyamineoxidizing enzymes polyamine oxidase (PAO) and diamine oxidase (DAO) in male and female ICR mice were demonstrated. The effects of testosterone (10g/100g body weight) on renal PAO and DAO activities were also studied. The differences between sexes in both PAO and DAO activities were most clearly expressed in the immature kidney. At the age of 20 days PAO and DAO activities were 1.52 fold (p < 0.01) and 1.75 (p < 0.02) respectively higher in male mouse kidney than in female. Maturational processes reflected in significant increases in polyamine- oxidizing enzyme activities mainly in female mouse kidney, comparable with the gain in the kidney wet weight. Our data show that testosterone is able to influence renal PAO and DAO activities in addition to the well-known stimulation of polyamine biosynthesis. The hormonal effects were sex and age dependent. The influence of testosterone on renal PAO activity was mainly age dependent. The slight stimulation of renal PAO activity observed in 20- and 50-day old mice, 24h after testosterone administration, change with a decrease in the enzyme activity at the age of 70 days. The effects of testosterone on renal DAO activity were mainly sex dependent. Testosterone caused stimulation of DAO activity with a very close magnitude (nearly twice) in female mouse kidney, independently of the age of mice. In contrast, in male mice the hormone treatment resulted in a statistically significant increase in renal DAO activity at the age of 70 days (.1.3 fold, p < 0.05) only. It could be suggested that our data indicate the different contribution of renal PAO and DAO in androgen regulation of polyamine levels, depending on sex and the stage of the postnatal development.     

Quantitative analysis of plant polyamines including thermospermine during growth and salinity stress

Arabidopsis thaliana was thought to contain two spermine synthase genes, ACAULIS 5 (ACL5) and SPMS. Recent investigations, however, revealed that the ACL5 gene encodes thermospermine synthase. In this study, we have established a simple method to separate two isomers of tetraamine, spermine and thermospermine, in extracts from plant tissues of less than 500 mg. Polyamines (PAs) extracted from plant tissues were benzoylated, and the derivatives were completely resolved by high-performance liquid chromatography on a C₁₈ reverse-phase column, by eluting with 42% (v/v) acetonitrile in water in an isocratic manner at 30 °C and monitoring at 254 nm. The relevance of the method was confirmed by co-chromatography with respective PAs and by the PA analysis of the single- and double-mutants of acl5 and spms, which could not synthesize thermospermine and/or spermine, respectively. Furthermore, with this method, we monitored the thermospermine contents in various tissues of A. thaliana and found that stems and flowers contain two- to three-fold more thermospermine compared to whole seedlings and mature leaves. The presence of thermospermine was confirmed in Oryza sativa and Lycopersicon pesculentum. Finally we addressed whether salinity stress changes the contents of PAs including thermospermine in Arabidopsis.     

Polyamine metabolism and physiological responses of <Emphasis Type="Italic">Potamogeton crispus</Emphasis> leaves under lead stress

Aquatic macrophytes were found to be the potential scavengers of heavy metals from aquatic environment. In this study, influences of ladder concentration of lead (Pb) on the leaves of Potamogeton crispus Linn were studied after 7 days of treatment. The accumulation of Pb, nutrient element contents, the generation rate of superoxide radical (O₂^·−), MDA, proline, and polyamine (PAs) contents, as well as the activities of diamine oxidases (DAO), polyamine oxidases (PAO), arginine decarboxylase (ADC), and ornithine decarboxylase (ODC) in P. crispus leaves were investigated. The result indicated that Pb treatment decreased the activity of DAO, whereas the proline content, MDA content, the generation rate of O₂^·− and the activity of ODC increased in different degrees. Meantime, Pb treatment significantly increased the free putrescine (Put) level and made other PAs levels dynamic changes. The activities of PAO and ADC were declined firstly and then enhanced with the increase in the Pb concentration.     

多胺对荔枝胚性愈伤组织增殖与体胚发生的影响

为探讨外源多胺(PAs)对荔枝胚性愈伤组织(EC)增殖及体胚发生的影响机制,该研究以“妃子笑”荔枝EC为材料,采用单因素法在增殖培养基中添加腐胺(Put)、亚精胺(Spd)及精胺(Spm),分析了不同PAs处理后EC的形态、结构、内源PAs含量及相关酶指标的变化。结果表明:(1)外源Put、Spd和Spm处理均显著提高了EC增殖率,减少了体胚诱导及萌发数量。经外源PAs处理增殖的EC胚性细胞大小较一致,染色深且均匀,多细胞原胚减少,可见已经分化完全的早期子叶胚。(2)外源PAs处理均显著提高了EC中内源PAs含量,其中Put处理的EC中各类内源PAs及总PAs含量最高; 当在含外源PAs培养基上增殖的EC转入不含外源PAs的培养基上增殖时(恢复培养),EC中的Put含量仍然显著高于对照,内源Spd和Spm则显著降低。(3)外源Put处理显著提高了EC中的鸟氨酸脱羧酶(ODC)、精氨酸脱羧酶(ADC)和二胺氧化酶(DAO)活性,而外源Spd、Spm处理显著降低了EC中的ODC及ADC活性,外源Spd显著提高了多胺氧化酶(PAO)活性; 恢复培养后,EC中ADC和DAO活性比恢复培养前显著降低,ODC和PAO无显著性差异。综上认为,外源PAs可以通过调节PAs代谢相关酶活性影响内源PAs含量,进而影响荔枝EC增殖和体胚诱导。该研究结果为进一步研究PAs调节荔枝体胚发生机制及提高荔枝离体再生效率提供了基础。     

干旱胁迫下小麦幼苗根、叶多胺含量和多胺氧化酶活性的变化

 以抗旱性不同的两个小麦品种（‘晋麦33’和‘温麦8’）（Triticum aestivum cv. Jinmai 33 and Wenmai 8）为材料,研究了干旱胁迫下多胺含量和多胺氧化酶活性的变化。结果表明：旱过程中,幼苗根、叶中腐胺（Put）、亚精胺（Spd）、精胺（Spm）3种多胺含量和多胺氧化酶（PAO）活性先迅速升高,而后下降。与抗旱性弱的‘晋麦33’相比,抗旱性强的品种‘温麦8’幼苗根、叶中Spd、Spm 含量初期升高幅度大,之后下降速率减慢;PAO活性的变化与之相反,‘晋麦33’的PAO活性提高的幅度大于‘温麦8号’。多胺含量和PAO活性在小麦幼苗的根与叶之间呈极显著正相关。干旱初期,小麦根、叶中多胺迅速积累可能是干旱胁迫反应的一个信号,随后较高的Spd、Spm 水平有利于增强小麦幼苗的抗旱性。     

Spermidine alleviates the growth of saline-stressed ginseng seedlings through antioxidative defense system

Protective effects of exogenous spermidine (Spd), activity of antioxygenic enzymes, and levels of free radicals in a well-known medicinal plant, Panax ginseng was examined. Seedlings grown in salinized nutrient solution (150 mM NaCl) for 7 d exhibited reduced relative water content, plant growth, increased free radicals, and showing elevated lipid peroxidation. Application of Spd (0.01, 0.1, and 1 mM) to the salinized nutrient solution showed increased plant growth by preventing chlorophyll degradation and increasing PA levels, as well as antioxidant enzymes such as CAT, APX, and GPX activity in the seedlings of ginseng. During salinity stress, Spd was effective for lowering the accumulation of putrescine (Put), with a significant increase in the spermidine (Spd) and spermine (Spm) levels in the ginseng seedlings. A decline in the Put level ran parallel to the higher accumulation of proline (Pro), and exogenous Spd also resulted in the alleviation of Pro content under salinity. Hydrogen peroxide (H₂O₂) and superoxide (O₂⁻) production rates were also reduced in stressed plants after Spd treatment. Furthermore, the combined effect of Spd and salt led to a significant increase in diamine oxidase (DAO), and subsequent decline in polyamine oxidase (PAO). These positive effects were observed in 0.1 and 1 mM Spd concentrations, but a lower concentration (0.01 mM) had a very limited effect. In summary, application of exogenous Spd could enhance salt tolerance of P. ginseng by enhancing the activities of enzyme scavenging system, which influence the intensity of oxidative stress.     

Correlation of gibberellin-induced growth, polyamine levels and amine oxidases in epicotyl, root and leaf blade of barley seedlings

The potential role of diamine oxidase (DAO) and polyamine oxidase (PAO) in relation to polyamines was investigated in epicotyls, roots and leaf blades at 3 and 6 days after gibberellic acid (GA) application in barley (Hordeum vulgare L.) seedlings of cvs. Maythorpe (non-mutant parent) and Golden Promise (semi-dwarf mutant). There was a significant increase in epicotyl and leaf-blade elongation rates in GA-treated seedlings of cv. Maythorpe as compared to cv. Golden Promise. DAO and PAO were detectable in all segments of the leaf blade, but the highest activities were present in basal segments. These enzymes, which are thought to have a role in the elimination of cellular polyamines, increased in activity following GA application compared to controls. Application of 10⁻⁶ M GA to the first leaf, significantly increased endogenous bound putrescine (Put) levels in both the epicotyl and leaf blade of cv. Maythorpe. In contrast, there was only a slight increase in cv. Golden Promise. Levels of soluble Put increased in roots and leaf blades of both cultivars following GA treatment but the effect was greatest in leaves of cv. Maythorpe. It is suggested that polyamines may play a role in GA-induced epicotyl and leaf-blade elongation in barley.     

cbb3-Type Cytochrome c Oxidases,Aerobic Respiratory Enzymes,Impact the Anaerobic Life of Pseudomonas aeruginosa PAO1

For bacteria, many studies have focused on the role of respiratory enzymes in energy conservation; however, their effect on cell behavior is poorly understood. Pseudomonas aeruginosa can perform both aerobic respiration and denitrification. Previous studies demonstrated that cbb₃-type cytochrome c oxidases that support aerobic respiration are more highly expressed in P. aeruginosa under anoxic conditions than are other aerobic respiratory enzymes. However, little is known about their role under such conditions. In this study, it was shown that cbb₃ oxidases of P. aeruginosa PAO1 alter anaerobic growth, the denitrification process, and cell morphology under anoxic conditions. Furthermore, biofilm formation was promoted by the cbb₃ oxidases under anoxic conditions. cbb₃ oxidases led to the accumulation of nitric oxide (NO), which is produced during denitrification. Cell elongation induced by NO accumulation was reported to be required for robust biofilm formation of P. aeruginosa PAO1 under anoxic conditions. Our data show that cbb₃ oxidases promote cell elongation by inducing NO accumulation during the denitrification process, which further leads to robust biofilms. Our findings show that cbb₃ oxidases, which have been well studied as aerobic respiratory enzymes, are also involved in denitrification and influence the lifestyle of P. aeruginosa PAO1 under anoxic conditions.     

A novel enzyme with spermine oxidase properties in bovine liver mitochondria: Identification and kinetic characterization

The uptake of spermine into mammalian mitochondria indicated the need to identify its catabolic pathway in these organelles. Bovine liver mitochondria were therefore purified and their capacity for natural polyamine uptake was verified. A kinetic approach was then used to determine the presence of an MDL 72527-sensitive enzyme with spermine oxidase activity in the matrix of bovine liver mitochondria. Western blot analysis of mitochondrial fractions and immunogold electron microscopy observations of purified mitochondria unequivocally confirmed the presence of a protein recognized by anti-spermine oxidase antibodies in the mitochondrial matrix. Preliminary kinetic characterization showed that spermine is the preferred substrate of this enzyme; lower activity was detected with spermidine and acetylated polyamines. Catalytic efficiency comparable to that of spermine was also found for 1-aminododecane. The considerable effect of ionic strength on the V_max/K_M ratio suggested the presence of more than one negatively charged zone inside the active site cavity of this mitochondrial enzyme, which is probably involved in the docking of positively charged substrates. These findings indicate that the bovine liver mitochondrial matrix contains an enzyme belonging to the spermine oxidase class. Because H₂O₂ is generated by spermine oxidase activity, the possible involvement of the latter as an important signaling transducer under both physiological and pathological conditions should be considered.     

Pharmacological and genetical evidence supporting nitric oxide requirement for 2,4-epibrassinolide regulation of root architecture in Arabidopsis thaliana

Brassinosteroids (BRs) regulate various physiological processes, such as tolerance to stresses and root growth. Recently, a connection was reported between BRs and nitric oxide (NO) in plant responses to abiotic stress. Here we present evidence supporting NO functions in BR signaling during root growth process. Arabidopsis seedlings treated with BR 24-epibrassinolide (BL) show increased lateral roots (LR) density, inhibition of primary root (PR) elongation and NO accumulation. Similar effects were observed adding the NO donor GSNO to BR-receptor mutant bri1-1. Furthermore, BL-induced responses in the root were abolished by the specific NO scavenger c-PTIO. The activities of nitrate reductase (NR) and nitric oxide synthase (NOS)-like, two NO generating enzymes were involved in BR signaling. These results demonstrate that BR increases the NO concentration in root cells, which is required for BR-induced changes in root architecture.