Arabidopsis thaliana seedling growth with pure oxylipins resulted in root waving, loss of root apical dominance, and decreased root elongation. 9-Hydroxyoctadecatrienoic acid (9-HOT) was a potent inducer of root waving. Studies with noxy2 (for nonresponding to oxylipins2), a new 9-HOT-insensitive mutant, and coronatine insensitive1-1 (jasmonateinsensitive) revealed at least three signaling cascades mediating the oxylipin actions. Treatment with 9-HOT resulted in a reduction in lateral roots and an increase in stage V primordia. Roots showed strong 9-lipoxygenase (9-LOX) activity, and root primordia expressed 9-LOX genes. These results, along with findings that noxy2 and mutants with defective 9-LOX activity showed increased numbers of lateral roots, suggest that 9-HOT, or a closely related 9-LOX product, is an endogenous modulator of lateral root formation. Histochemical and molecular analyses revealed that 9-HOT activated events common to development and defense responses. A subset of 9-HOT-responding root genes was also induced in leaves after 9-HOT treatment or pathogen inoculation. The results that noxy2 displayed altered root development, enhanced susceptibility to Pseudomonas, and reduced the activation of 9-HOT-responding genes are consistent with mechanistic links among these processes. The nature of the changes detected suggests that oxylipins from the 9-LOX pathway function in cell wall modifications required for lateral root development and pathogen arrest.

Lipoxygenases (LOX) catalyze the oxygenation of polyunsaturated fatty acids, the first step in the biosynthesis of a large group of biologically active fatty acid metabolites collectively named oxylipins. In the present study we report the characterization of the enzymatic activity of the six lipoxygenases found in the genome of the model plant Arabidopsis thaliana. Recombinant expressed AtLOX-1 and AtLOX-5 had comparable oxygenase activity with either linoleic acid or linolenic acid. AtLOX-2, AtLOX-3, AtLOX-4 and AtLOX-6 displayed a selective oxygenation of linolenic acid. Analyses by high-performance liquid chromatography and gas chromatography-mass spectrometry demonstrated that AtLOX-1 and AtLOX-5 are 9S-lipoxygenases, and AtLOX-2, AtLOX-3, AtLOX-4 and AtLOX-6 are 13S-lipoxygenases. None of the enzymes had dual positional specificity. The determined activities correlated with that predicted by their phylogenetic relationship to other biochemically-characterized plant lipoxygenases.

No abstract

SUMMARY9-lipoxygenases (9-LOXs) initiate fatty acid oxygenation in plant tissues, with formation of 9-hydroxy-10,12,15-octadecatrienoic acid (9-HOT) from linolenic acid. A lox1 lox5 mutant, which is deficient in 9-LOX activity, and two mutants noxy6 and noxy22 (non-responding to oxylipins), which are insensitive to 9-HOT, have been used to investigate 9-HOT signalling. Map-based cloning indicated that the noxy6 and noxy22 mutations are located at the CTR1 (CONSTITUTIVE ETHYLENE RESPONSE1) and ETO1 (ETHYLENE-OVERPRODUCER1) loci, respectively. In agreement, the noxy6 and noxy22 mutants, renamed as ctr1-15 and eto1-14, respectively, showed enhanced ethylene (ET) production. The correlation between increased ET production and reduced 9-HOT sensitivity indicated by these results was supported by experiments in which exogenously added ethylene precursor ACC (1-aminocyclopropane-1-carboxylic acid) impaired the responses to 9-HOT. Moreover, a reciprocal interaction between ET and 9-HOT signalling was indicated by results showing that the effect of ACC was reduced in the presence of 9-HOT. We found that the 9-LOX and ET pathways regulate the response to the lipid peroxidation-inducer singlet oxygen. Thus, the massive transcriptional changes seen in wild-type plants in response to singlet oxygen were greatly affected in the lox1 lox5 and eto1-14 mutants. Furthermore, these mutants displayed enhanced susceptibility to both singlet oxygen and Pseudomonas syringae pv. tomato, in the latter case leading to increased accumulation of the lipid peroxidation product malondialdehyde. These findings demonstrate an antagonistic relationship between products of the 9-LOX and ET pathways, and suggest a role for the 9-LOX pathway in modulating oxidative stress, lipid peroxidation and plant defence.

Rhizobium leguminosarum bv. viciae expresses an uptake hydrogenase in symbiosis with peas (Pisum sativum) but, unlike all other characterized hydrogenoxidizing bacteria, cannot express it in free-living conditions. The hydrogenase-specific transcriptional activator gene hoxA described in other species was shown to have been inactivated in R. leguminosarum by accumulation of frameshift and deletion mutations. Symbiotic transcription of hydrogenase structural genes hupSL originates from a ؊24͞؊12 type promoter (hupS p ). A regulatory region located in the ؊173 to ؊88 region was essential for promoter activity in R. leguminosarum. Activation of hupS p was observed in Klebsiella pneumoniae and Escherichia coli cells expressing the K. pneumoniae nitrogen fixation regulator NifA, and in E. coli cells expressing R. meliloti NifA. This activation required direct interaction of NifA with the essential ؊173 to ؊88 regulatory region. However, no sequences resembling known NifA-binding sites were found in or around this region. NifA-dependent activation was also observed in R. etli bean bacteroids. NifAdependent hupS p activity in heterologous hosts was also absolutely dependent on the RpoN -factor and on integration host factor. Proteins immunologically related to integration host factor were identified in R. leguminosarum. The data suggest that hupS p is structurally and functionally similar to nitrogen fixation promoters. The requirement to coordinate nitrogenase-dependent H 2 production and H 2 oxidation in nodules might be the reason for the loss of HoxA in R. leguminosarum and the concomitant NifA control of hup gene expression. This evolutionary acquired control would ensure regulated synthesis of uptake hydrogenase in the most common H 2 -rich environment for rhizobia, the legume nodule.

Motivation Recent technological advances and computational developments have allowed the reconstruction of Cryo-Electron Microscopy (cryo-EM) maps at near-atomic resolution. On a typical workflow and once the cryo-EM map has been calculated, a sharpening process is usually performed to enhance map visualization, a step that has proven very important in the key task of structural modeling. However, sharpening approaches, in general, neglects the local quality of the map, which is clearly suboptimal. Results Here, a new method for local sharpening of cryo-EM density maps is proposed. The algorithm, named LocalDeblur, is based on a local resolution-guided Wiener restoration approach of the original map. The method is fully automatic and, from the user point of view, virtually parameter-free, without requiring either a starting model or introducing any additional structure factor correction or boosting. Results clearly show a significant impact on map interpretability, greatly helping modeling. In particular, this local sharpening approach is especially suitable for maps that present a broad resolution range, as is often the case for membrane proteins or macromolecules with high flexibility, all of them otherwise very suitable and interesting specimens for cryo-EM. To our knowledge, and leaving out the use of local filters, it represents the first application of local resolution in cryo-EM sharpening. Availability and implementation The source code (LocalDeblur) can be found at https://github.com/I2PC/xmipp and can be run using Scipion (http://scipion.cnb.csic.es) (release numbers greater than or equal 1.2.1). Supplementary information Supplementary data are available at Bioinformatics online.

Plant a-dioxygenases initiate the synthesis of oxylipins by catalyzing the incorporation of molecular oxygen at the a-methylene carbon atom of fatty acids. Previously, a-DOX1 has been shown to display a-dioxygenase activity and to be implicated in plant defense. In this study, we investigated the function of a second a-dioxygenase isoform, a-DOX2, in tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). Recombinant Sla-DOX2 and Ata-DOX2 proteins catalyzed the conversion of a wide range of fatty acids into 2(R)-hydroperoxy derivatives. Expression of Sla-DOX2 and Ata-DOX2 was found in seedlings and increased during senescence induced by detachment of leaves. In contrast, microbial infection, earlier known to increase the expression of a-DOX1, did not alter the expression of Sla-DOX2 or Ata-DOX2. The tomato mutant divaricata, characterized by early dwarfing and anthocyanin accumulation, carries a mutation at the Sla-DOX2 locus and was chosen for functional studies of a-DOX2. Transcriptional changes in such mutants showed the up-regulation of genes playing roles in lipid and phenylpropanoid metabolism, the latter being in consonance with the anthocyanin accumulation. Transgenic expression of Ata-DOX2 and Sla-DOX2 in divaricata partially complemented the compromised phenotype in mature plants and fully complemented it in seedlings, thus indicating the functional exchangeability between a-DOX2 from tomato and Arabidopsis. However, deletion of Ata-DOX2 in Arabidopsis plants did not provoke any visible phenotypic alteration indicating that the relative importance of a-DOX2 in plant physiology is species specific.Plants have evolved elaborate signaling systems to regulate a variety of physiological responses to the environment and to facilitate intercellular cross talk in development and reproduction. Oxylipins comprise a large class of oxygenated fatty acid-derived lipid mediators that contribute to such signaling circuits (Weber, 2002;Farmer et al., 2003). A variety of functions have been ascribed to plant oxylipins, including critical roles in plant defense against microbial pathogens, as well as in reproduction and tissue development (Howe and Schilmiller, 2002;Browse, 2005;Kachroo and Kachroo 2009).The biosynthesis of oxylipins is initiated by hydroperoxide formation catalyzed by fatty acid oxygenases, among which the 9-and 13-lipoxygenases have been studied most intensively (Shibata and Axelrod, 1995;Feussner and Wasternack, 2002). a-Dioxygenase, first encountered about 10 years ago, also catalyzes primary fatty acid oxygenation. This enzyme was first identified in Nicotiana tabacum plants as a pathogeninduced protein showing homology to mammalian prostaglandin endoperoxide synthases (Sanz et al., 1998). Studies of the catalytic function of the recombinant tobacco protein and of a homologous protein from Arabidopsis (Arabidopsis thaliana) revealed that these plant enzymes, designated as a-dioxygenase-1 or a-DOX1, catalyze the incorporation of molecular oxygen at the a-methylene carbon atom of fatty ...

Single-particle analysis by electron microscopy is a well established technique for analyzing the three-dimensional structures of biological macromolecules. Besides its ability to produce high-resolution structures, it also provides insights into the dynamic behavior of the structures by elucidating their conformational variability. Here, the different image-processing methods currently available to study continuous conformational changes are reviewed.