Polyamine oxidase 2 is involved in regulating excess spermidine contents during seed germination and early seedling development in Arabidopsis thaliana.

To understand the physiological functions of polyamine oxidases (PAOs) in plants, we analyzed the effects of exogenous polyamines during seed germination and early seedling development, using Arabidopsis thaliana lines independently harboring T-DNA insertions in each PAO gene. Spermine caused seedling growth inhibition but did not affect the germination in all lines including wild-type Col-0. However, an AtPAO2 knockout mutant, -pao2, could not germinate under excess spermidine (Spd) conditions. The root growth rates of post-germination -pao2 seedlings were also strongly inhibited by the Spd treatment compared with the wild-type plants. AtPAO2 has a conserved peroxisome-targeting signal sequence at its C-terminus. We prepared two types of AtPAO2 expression plants in a -pao2 background. In -pao2/PAO2 plants a 5.8-kbp genomic fragment containing the complete coding sequence of AtPAO2 was introduced, while in -pao2/PAO2ΔC plants the same fragment lacking the peroxisome-targeting signal was introduced. The Spd-sensitive phenotypes observed in -pao2 were completely recovered in both of the transgenic complementation lines. Thus, AtPAO2 appears to be involved in excess Spd catabolism during seed germination and early seedling development irrespective of subcellular localization.

Mechanism of Flavoprotein l-6-Hydroxynicotine Oxidase: pH and Solvent Isotope Effects and Identification of Key Active Site Residues.

The flavoenzyme l-6-hydroxynicotine oxidase is a member of the monoamine oxidase family that catalyzes the oxidation of (S)-6-hydroxynicotine to 6-hydroxypseudooxynicotine during microbial catabolism of nicotine. While the enzyme has long been understood to catalyze oxidation of the carbon-carbon bond, it has recently been shown to catalyze oxidation of a carbon-nitrogen bond [Fitzpatrick, P. F., et al. (2016) Biochemistry 55, 697-703]. The effects of pH and mutagenesis of active site residues have now been utilized to study the mechanism and roles of active site residues. Asn166 and Tyr311 bind the substrate, while Lys287 forms a water-mediated hydrogen bond with flavin N5. The N166A and Y311F mutations result in ∼30- and ∼4-fold decreases in kcat/Km and kred for (S)-6-hydroxynicotine, respectively, with larger effects on the kcat/Km value for (S)-6-hydroxynornicotine. The K287M mutation results in ∼10-fold decreases in these parameters and a 6000-fold decrease in the kcat/Km value for oxygen. The shapes of the pH profiles are not altered by the N166A and Y311F mutations. There is no solvent isotope effect on the kcat/Km value for amines. The results are consistent with a model in which both the charged and neutral forms of the amine can bind, with the former rapidly losing a proton to a hydrogen bond network of water and amino acids in the active site prior to the transfer of hydride to the flavin.

Polyamine oxidase 5 loss-of-function mutations in Arabidopsis thaliana trigger metabolic and transcriptional reprogramming and promote salt stress tolerance.

The family of polyamine oxidases (PAO) in Arabidopsis (AtPAO1-5) mediates polyamine (PA) back-conversion, which reverses the PA biosynthetic pathway from spermine and its structural isomer thermospermine (tSpm) into spermidine and then putrescine. Here, we have studied the involvement of PA back-conversion in Arabidopsis salinity tolerance. AtPAO5 is the Arabidopsis PAO gene member most transcriptionally induced by salt stress. Two independent loss-of-function mutants (atpao5-2 and atpao5-3) were found to exhibit constitutively higher tSpm levels, with associated increased salt tolerance. Using global transcriptional and metabolomic analyses, the underlying mechanisms were studied. Stimulation of abscisic acid and jasmonate (JA) biosynthesis and accumulation of important compatible solutes, such as sugars, polyols and proline, as well as TCA cycle intermediates were observed in atpao5 mutants under salt stress. Expression analyses indicate that tSpm modulates the transcript levels of several target genes, including many involved in the biosynthesis and signalling of JA, some of which are already known to promote salinity tolerance. Transcriptional modulation by tSpm is isomer-dependent, thus demonstrating the specificity of this response. Overall, we conclude that tSpm triggers metabolic and transcriptional reprogramming that promotes salt stress tolerance in Arabidopsis.

The Arabidopsis polyamine oxidase/dehydrogenase 5 interferes with cytokinin and auxin signaling pathways to control xylem differentiation.

In plants, the polyamines putrescine, spermidine, spermine (Spm), and thermospermine (Therm-Spm) participate in several physiological processes. In particular, Therm-Spm is involved in the control of xylem differentiation, having an auxin antagonizing effect. Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. In Arabidopsis, five PAOs are present, among which AtPAO5 catalyzes the back-conversion of Spm, Therm-Spm, and N1-acetyl-Spm to spermidine. In the present study, it is shown that two loss-of-function atpao5 mutants and a 35S::AtPAO5 Arabidopsis transgenic line present phenotypical differences from the wild-type plants with regard to stem and root elongation, differences that are accompanied by changes in polyamine levels and the number of xylem vessels. It is additionally shown that cytokinin treatment, which up-regulates AtPAO5 expression in roots, differentially affects protoxylem differentiation in 35S::AtPAO5, atpao5, and wild-type roots. Together with these findings, Therm-Spm biosynthetic genes, as well as auxin-, xylem-, and cytokinin-related genes (such as ACL5, SAMDC4, PIN1, PIN6, VND6, VND7, ATHB8, PHB, CNA, PXY, XTH3, XCP1, and AHP6) are shown to be differentially expressed in the various genotypes. These data suggest that AtPAO5, being involved in the control of Therm-Spm homeostasis, participates in the tightly controlled interplay between auxin and cytokinins that is necessary for proper xylem differentiation.

Bacterial cyanogenesis: impact on biotic interactions.

The ability of bacteria to influence organisms that they associate with via metabolite production is one of the hallmarks of microbial interactions. One metabolite of interest is the metabolic poison cyanide. Production of this metabolite is an unique characteristic of certain bacteria that inhabit a wide array of habitats ranging from the human body to the rhizosphere. This review focuses on four targets of cyanogenic bacteria: the human lung, plant pathogens, plants and invertebrates. For a number of cyanogenic bacteria, the contribution of cyanide to the interaction has been rigorously tested using mutants altered in cyanide production. Both deleterious and stimulatory effects of cyanogenic bacteria on other organisms have been documented. In addition, the HCN synthase-encoding gene cluster hcnABC has served as a marker of cyanogenic capability in the soil environment revealing both genetic diversity at this locus and regulatory influences by other organisms. The pervasive nature of cyanogenesis in a number of different ecological contexts encourages exploration of this bacterial ability and its possible optimization for improving human health, crop production and pest control.

Decrease of Arabidopsis PAO activity entails increased RBOH activity, ROS content and altered responses to Pseudomonas.

Polyamines (PAs) are small aliphatic amines with important regulatory activities in plants. Biotic stress results in changes in PA levels due to de novo synthesis and PA oxidation. In Arabidopsis thaliana five FAD-dependent polyamine oxidase enzymes (AtPAO1-5) participate in PA back-conversion and degradation. PAO activity generates H2O2, an important molecule involved in cell signaling, elongation, programmed cell death, and defense responses. In this work we analyzed the role of AtPAO genes in the Arabidopsis thaliana-Pseudomonas syringae pathosystem. AtPAO1 and AtPAO2 genes were transcriptionally up-regulated in infected plants. Atpao1-1 and Atpao2-1 single mutant lines displayed altered responses to Pseudomonas, and an increased susceptibility was found in the double mutant Atpao1-1 x Atpao2-1. These polyamine oxidases mutant lines showed disturbed contents of ROS (H2O2 and O2-) and altered activities of RBOH, CAT and SOD enzymes both in infected and control plants. In addition, changes in the expression levels of AtRBOHD, AtRBOHF, AtPRX33, and AtPRX34 genes were also noticed. Our data indicate an important role for polyamine oxidases in plant defense and ROS homeostasis.

Genetic diversity and biocontrol potential of fluorescent pseudomonads producing phloroglucinols and hydrogen cyanide from Swiss soils naturally suppressive or conducive to Thielaviopsis basicola-mediated black root rot of tobacco.

Pseudomonas populations producing the biocontrol compounds 2,4-diacetylphloroglucinol (Phl) and hydrogen cyanide (HCN) were found in the rhizosphere of tobacco both in Swiss soils suppressive to Thielaviopsis basicola and in their conducive counterparts. In this study, a collection of Phl+ HCN+Pseudomonas isolates from two suppressive and two conducive soils were used to assess whether suppressiveness could be linked to soil-specific properties of individual pseudomonads. The isolates were compared based on restriction analysis of the biocontrol genes phlD and hcnBC, enterobacterial repetitive intergenic consensus (ERIC)-PCR profiling and their biocontrol ability. Restriction analyses of phlD and hcnBC yielded very concordant relationships between the strains, and suggested significant population differentiation occurring at the soil level, regardless of soil suppressiveness status. This was corroborated by high strain diversity (ERIC-PCR) within each of the four soils and among isolates harboring the same phlD or hcnBC alleles. No correlation was found between the origin of the isolates and their biocontrol activity in vitro and in planta. Significant differences in T. basicola inhibition were however evidenced between the isolates when they were grouped according to their biocontrol alleles. Moreover, two main Pseudomonas lineages differing by the capacity to produce pyoluteorin were evidenced in the collection. Thus, Phl+ HCN+ pseudomonads from suppressive soils were not markedly different from those from nearby conducive soils. Therefore, as far as biocontrol pseudomonads are concerned, this work yields the hypothesis that the suppressiveness of Swiss soils may rely on the differential effects of environmental factors on the expression of key biocontrol genes in pseudomonads rather than differences in population structure of biocontrol Pseudomonas subcommunities or the biocontrol potential of individual Phl+ HCN+ pseudomonad strains.

Arabidopsis Polyamine oxidase-2 uORF is required for downstream translational regulation.

In eukaryotic mRNAs, small upstream open reading frames (uORFs) located in the 5'-untranslated region control the translation of the downstream main ORF. Polyamine oxidase (PAO) enzymes catalyze the oxidation of higher polyamines such as spermidine and spermine, and therefore contribute to the maintenance of intracellular polyamine content and to the regulation of physiological processes through their catabolic products. Recently, we reported that the Arabidopsis thaliana Polyamine Oxidase 2 (AtPAO2) is post-transcriptionally regulated by its 5'-UTR region through an uORF. In the present study, we analyzed whether the translation of the uORF is needed for the translational repression of the main ORF, and whether the inactivation of the uORF had an effect on the translational control mediated by polyamines. To this aim, we generated diverse single mutations in the uORF sequence; these mutant 5'-UTRs were fused to the GUS reporter gene, and tested in onion monolayer cells and A. thaliana transgenic seedlings. Removal of the start codon or introduction of a premature stop codon in the AtPAO2 uORF sequence abolished the negative regulation of the GUS expression exerted by the wild-type AtPAO2 uORF. An artificial uORF (32 amino acids in length) generated by the addition of a single nucleotide in AtPAO2 uORF proved to be less repressive than the wild-type uORF. Thus, our findings suggest that translation of the AtPAO2 uORF is necessary for the translational repression of the main ORF.

A novel method to perform genomic walks using a combination of single strand DNA circularization and rolling circle amplification.

Characterization of regions flanking a known sequence within a genome, known as genome walking, is a cornerstone technique in modern genetic analysis. In the present work we have developed a new PCR-dependent, directional genome walking protocol based on the unique circularization property of a novel DNA ligase, CircLigase. In the first step, PCR based primer extension is performed using a phosphorylated primer, designed to extend from the boundary of the known sequence, into the flanking region. This linear amplification results in the generation of single-stranded (ss) DNA, which is then circularized using CircLigase. Using the hyperbranching activity of Phi29 DNA polymerase, the circular ssDNA is then linearized by rolling circle amplification, resulting in copious amounts of double stranded concatameric DNA. Nested primers are used to amplify the flanking sequence using inverse PCR. The products are resolved on an agarose gel and the bands whose mobility change due to the nested location of the primer combination used are identified, extracted, and cloned into a plasmid vector for sequencing. Empirical proof for this concept was generated on two antimicrobial biosynthetic genes in Pseudomonas sp. LBUM300. Using the hcnB and phlD genes as starting points, ca 1 kb of flanking sequences were successfully isolated. The use of locus specific primers ensured both directionality and specificity of the walks, alleviating the generation of spurious amplicons, typically observed in randomly primed walking protocols. The presented genome walking protocol could be applied to any microbial genome and requires only 100-150 bp of prior sequence information. The proposed methodology does not entail laborious testing of restriction enzymes or adaptor ligation. This is the first report of a successful application of the novel ligase enzyme, CircLigase for genomic walking purposes.

Effect of long-term vineyard monoculture on rhizosphere populations of pseudomonads carrying the antimicrobial biosynthetic genes phlD and/or hcnAB.

The impact of repeated culture of perennial plants (i.e. in long-term monoculture) on the ecology of plant-beneficial bacteria is unknown. Here, the influence of extremely long-term monocultures of grapevine (up to 1603 years) on rhizosphere populations of fluorescent pseudomonads carrying the biosynthetic genes phlD for 2,4-diacetylphloroglucinol and/or hcnAB for hydrogen cyanide was determined. Soils from long-term and adjacent short-term monoculture vineyards (or brushland) in four regions of Switzerland were baited with grapevine or tobacco plantlets, and rhizosphere pseudomonads were studied by most probable number (MPN)-PCR. Higher numbers and percentages of phlD(+) and of hcnAB(+) rhizosphere pseudomonads were detected on using soil from long-term vineyards. On focusing on phlD, restriction fragment length polymorphism profiling of the last phlD-positive MPN wells revealed seven phlD alleles (three exclusively on tobacco, thereof two new ones). Higher numbers of phlD alleles coincided with a lower prevalence of the allele displayed by the well-studied biocontrol strain Pseudomonas fluorescens F113. The prevalence of this allele was 35% for tobacco in long-term monoculture soils vs. >60% in the other three cases. We conclude that soils from long-term grapevine monocultures represent an untapped resource for isolating novel biocontrol Pseudomonas strains when tobacco is used as bait.

Mechanism of hcnA mRNA recognition in the Gac/Rsm signal transduction pathway of Pseudomonas fluorescens.

In the plant-beneficial bacterium Pseudomonas fluorescens CHA0, the expression of antifungal exoproducts is controlled by the GacS/GacA two-component system. Two RNA binding proteins (RsmA, RsmE) ensure effective translational repression of exoproduct mRNAs. At high cell population densities, GacA induces three small RNAs (RsmX, RsmY, RsmZ) which sequester both RsmA and RsmE, thereby relieving translational repression. Here we systematically analyse the features that allow the RNA binding proteins to interact strongly with the 5' untranslated leader mRNA of the P. fluorescens hcnA gene (encoding hydrogen cyanide synthase subunit A). We obtained evidence for three major RsmA/RsmE recognition elements in the hcnA leader, based on directed mutagenesis, RsmE footprints and toeprints, and in vivo expression data. Two recognition elements were found in two stem-loop structures whose existence in the 5' leader region was confirmed by lead(II) cleavage analysis. The third recognition element, which overlapped the hcnA Shine-Dalgarno sequence, was postulated to adopt either an open conformation, which would favour ribosome binding, or a stem-loop structure, which may form upon interaction with RsmA/RsmE and would inhibit access of ribosomes. Effective control of hcnA expression by the Gac/Rsm system appears to result from the combination of the three appropriately spaced recognition elements.

Hfq-dependent alterations of the transcriptome profile and effects on quorum sensing in Pseudomonas aeruginosa.

The Pseudomonas aeruginosa quorum-sensing (QS) systems, Las and Rhl, control the production of several virulence factors and other proteins, which are important to sustain adverse conditions. A comparative transcriptome analysis of a rpoS (-) and a rpoS(-)hfq( -) strain indicated that the Sm-like RNA-binding protein Hfq affects approximately 5% of the P. aeruginosa O1 transcripts. Among these transcripts 72 were identified to be QS regulated. Expression studies revealed that Hfq does not control the master regulators of the Las system, LasR and LasI. Upon entry into stationary phase, Hfq exerted a moderate stimulatory effect on translation of the rhlR gene and on the qscR gene, encoding a LasR/RhlR homologue. However, Hfq considerably stimulated translation of the rhlI gene, encoding the synthetase of the autoinducer N-Butyryl-homoserine lactone (C4-HSL). Correspondingly, the C4-HSL levels were reduced in a hfq(-) strain. To elucidate the stimulatory effect of Hfq on rhlI expression we asked whether Hfq affects the stability of the regulatory RNAs RsmY and RsmZ, which have been implicated in sequestration of the translational repressor RsmA, which in turn is known to negatively regulate RhlI synthesis. We demonstrate that Hfq binds to and stabilizes the regulatory RNA RsmY, which is further shown to bind to the regulatory protein RsmA. A model for the Hfq regulatory network is presented, wherein an alleviation of the negative effect of RsmA accounts for the observed stimulation of rhlI expression by Hfq. The model is corroborated by the observation that a rsmY(-) mutant mimics the hfq(-) phenotype with regard to rhlI expression.

Cloning and characterization of Arabidopsis and Brassica juncea flavin-containing amine oxidases.

Polyamines (PAs) are low molecular weight metabolites involved in various physiological and developmental processes in eukaryotic and prokaryotic cells. The cellular PA level is regulated in part by the action of amine oxidases (AOs) including copper diamine oxidases (DAOs) and flavoprotein polyamine oxidases (PAOs). In this study, the isolation and characterization of flavin amine oxidases (FAOs) from Brassica juncea (BJFAO) and Arabidopsis (ATFAO1) are reported that were clustered in the same group as polyamine oxidases from maize (MPAO) and barley (BPAO1) and monoamine oxidases from mammalian species. ATFAO1 was temporally and spatially regulated in Arabidopsis and showed distinct expression patterns in response to different stress treatments. To investigate the in vivo function of FAO, transgenic Arabidopsis plants expressing sense, antisense, and double-stranded BJFAO RNAs were generated and those with altered activity of FAOs were selected for further characterization. It was found that the shoot regeneration response in transgenic plants was significantly affected by the modulated PA levels corresponding to FAO activities. Tissues that originated from transgenic plants with down-regulated FAO activity were highly regenerative, while those from transgenic plants with upregulated FAO activity were poorly regenerative. The shoot regeneration capacity in these transgenic plants was related to the levels of individual PAs, suggesting that FAO affects shoot regeneration by regulating cellular PAs. Furthermore, it was found that the effect of FAO activity on shoot regeneration was exerted downstream of the Enhancer of Shoot Regeneration (ESR1) gene, which may function in a branch of the cytokinin signalling pathway.