Analysis of the Arabidopsis venosa4-0 mutant supports the role of VENOSA4 in dNTP metabolism

Human Sterile alpha motif and histidine-aspartate domain containing protein 1 (SAMHD1) functions as a dNTPase to maintain dNTP pool balance. In eukaryotes, the limiting step in de novo dNTP biosynthesis is catalyzed by RIBONUCLEOTIDE REDUCTASE (RNR). In Arabidopsis, the RNR1 subunit of RNR is encoded by CRINKLED LEAVES 8 (CLS8), and RNR2 by three paralogous genes, including TSO MEANING 'UGLY' IN CHINESE 2 (TSO2). In plants, DIFFERENTIAL DEVELOPMENT OF VASCULAR ASSOCIATED CELLS 1 (DOV1) catalyzes the first step of the de novo biosynthesis of purines. Here, to explore the role of VENOSA4 (VEN4), the most likely Arabidopsis ortholog of human SAMHD1, we studied the ven4‐0 point mutation, whose leaf phenotype was stronger than those of its insertional alleles. Structural predictions suggested that the E249L substitution in the mutated VEN4-0 protein rigidifies its 3D structure. The morphological phenotypes of the ven4, cls8, and dov1 single mutants were similar, and those of the ven4 tso2 and ven4 dov1 double mutants were synergistic. The ven4‐0 mutant had reduced levels of four amino acids related to dNTP biosynthesis, including glutamine and glycine, which are precursors in the de novo purine biosynthesis. Our results reveal high functional conservation between VEN4 and SAMHD1 in dNTP metabolism

High serum cyclophilin C levels as a risk factor marker for coronary artery disease

Cyclophilins (Cyps) are ubiquitous proteins that belong to the immunophilins family consistently associated with infammatory and cardiovascular diseases. While levels of CypA have been extensively studied, less data are available for other Cyps. The purpose of this case-control study was to determine the relationship of Cyps (A, B, C and D) with coronary artery disease (CAD) and eight infammation markers. Serum levels of Cyps, interleukins and metalloproteinases were measured in serum collected from 84 subjects. Participants were divided into two sub-groups based on CAD diagnosis: 40 CAD patients and 44 control volunteers. Serum levels of CypA, CypB and CypC, IL-1β and IL-6 were signifcantly higher in CAD patients. Bivariate correlation analysis revealed a signifcant positive correlation between Cyps and several blood and biochemical parameters. When the ability of Cyps levels for CAD diagnosis was evaluated, higher sensitivity and selectivity values were obtained with CypC (c-statistic 0.891, p<0.001) indicating that it is a good marker of CAD disease, while less conclusive results were obtained with CypA (c-statistic 0.748, p<0.001) and CypB (c-statistic 0.655, p<0.014). In addition, signifcant correlations of traditional CAD risk factors and CypC were observed. In summary, high levels of CypC are a risk factor for CAD and therefore it can be proposed as a new biomarker for this diseaseThe research leading to these results has received funding from the following FEDER cofunded-grants. From Conselleria de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830, ISCIII/PI16/01816 and RTC-2016-5507-2, ITC-20161072. From European Union POCTEP 0161-Nanoeaters -1-E-1, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX. Sandra Gegunde was supported by a fellowship from FIDIS, SpainS

Caracterización genética y molecular de los genes RE e ICU2 de Arabidopsis thaliana

Con el objetivo de contribuir a la disección genética de la ontogenia foliar, hemos obtenido y estudiado varias estirpes mutantes de Arabidopsis thaliana que presentan alteraciones en la morfología de sus hojas vegetativas, a las que hemos denominado venosa (ven) e incurvata2 (icu2). Las razones de nuestro interés inicial por estos mutantes fueron las siguientes: Las hojas vegetativas de los mutantes ven presentan una red vascular intensamente verde, que destaca sobre un limbo pálido, lo que sugiere que sufren alteraciones en la biogénesis de los cloroplastos, la diferenciación del mesófilo, o ambas; El margen de las hojas vegetativas del mutante icu2-1 está ligeramente recurvado hacia el haz y en su epidermis se observan grupos de células de tamaño reducido, lo que podría deberse a perturbaciones en la especificación de la dorsoventralidad o en los mecanismos de coordinación del crecimiento de los tejidos dorsales y ventrales de este órgano. En esta Tesis Doctoral hemos caracterizado seis alelos recesivos del gen RETICULATA (RE; VEN2) y demostrado que su fenotipo foliar reticulado se debe a una gran reducción en la densidad de las células del mesófilo intervenal. Las hojas de las plantas re/re presentan una ligera disminución en tamaño y una forma casi normal, a pesar de que les faltan numerosas células del mesófilo, que son sustituidas por espacios aéreos intercelulares, lo que sugiere que el correcto desarrollo de los tejidos internos incide poco en la forma final del órgano, que parece depender en mayor medida de la epidermis. También apoya esta hipótesis la observación de que las células de la epidermis foliar de los mutantes re son aparentemente normales en tamaño y morfología. Hemos clonado posicionalmente el gen RE y caracterizado molecularmente sus alelos, varios de los cuales parecen nulos. RE es LCD1, que había sido identificado por otros autores en base a la sensibilidad al ozono y a Pseudomonas syringae causada por lcd1-1, su único alelo conocido previamente (Barth y Conklin, 2003), y codifica una proteína de función desconocida y expresión ubicua. Nuestros resultados indican que los alelos re hipomorfos o nulos perturban específicamente la división de las células del mesófilo en estadios tempranos de la organogénesis foliar. Hemos demostrado además que la reticulación foliar es un rasgo externo útil como criterio selectivo para el aislamiento de mutantes con anomalías en la arquitectura interna de la hoja. Hemos estudiado las interacciones genéticas entre RE y su parálogo más cercano, al que hemos denominado RE2, cuyo alelo nulo re2-1 no tiene manifestación fenotípica. El análisis de la progenie de varios cruzamientos re/re re2-1/re2-1 indica que los genotipos re/re;re2-1/re2-1 y RE/re;re2-1/re2-1 son letales, mientras que re/re;RE2/re2-1 causa sinergia fenotípica, lo que sugiere que RE y RE2 son redundantes y necesarios no sólo para la organogénesis foliar sino también para el desarrollo reproductivo y embrionario. La obtención del doble mutante cue1-5/cue1-5;re-3/re-3 nos ha permitido establecer que cue1-5 es epistático sobre re-3, lo que indica que RE participa en la ruta del shikimato. Hemos clonado posicionalmente otros dos genes cuya insuficiencia de función provoca un fenotipo reticulado, comprobando que VEN4 codifica una fosfohidrolasa y VEN5, otro parálogo de RE, una proteína de la envuelta del cloroplasto, ambas de función desconocida. Estamos intentando determinar la naturaleza molecular de otros tres genes VEN (VEN1, VEN3 y VEN6) mediante un abordaje posicional similar al que nos ha permitido identificar RE, VEN4 y VEN5. Además de su fenotipo foliar, el mutante icu2-1 muestra floración temprana y transformaciones homeóticas parciales entre órganos florales, similares a las causadas por la insuficiencia de función del gen de identidad de órgano floral AP2, que a su vez causa la desrepresión ectópica de AG. Hemos comprobado mediante RT-PCR cuantitativa que en las hojas del mutante icu2-1 se expresan ectópicamente, entre otros, los genes AG, AP1, AP3, PI, SEP3, CAL, FUL y FT, todos los cuales codifican factores de transcripción. La desrepresión de AG causa el fenotipo foliar del mutante icu2-1, y la de FT su floración temprana, ya que las mutaciones de insuficiencia de función ag-1 y ft-1, respectivamente, suprimen dichos rasgos. Hemos clonado posicionalmente el gen ICU2, que codifica la subunidad catalítica de la ADN polimerasa . El alelo icu2-1 es hipomorfo y probablemente modifica la estructura tridimensional de la proteína ICU2, pero no parece reducir su actividad polimerasa. Los alelos insercionales y aparentemente nulos icu2-2 e icu2-3 causan letalidad gamética y embrionaria, tal como cabe esperar del papel esencial de la ADN polimerasa en la replicación. La obtención de dobles mutantes nos ha permitido establecer que las mutaciones clf, tfl2 y emf2 interaccionan sinérgicamente con icu2-1. Este análisis de interacciones sugiere la existencia en Arabidopsis thaliana de un mecanismo de silenciamiento génico mediado por la cromatina similar al que se ha descrito para algunas levaduras y animales, en el que las marcas epigenéticas y el ADN se replican simultáneamente. Proponemos que la polimerasa ICU2 participa en la unión de TFL2 a las histonas, contribuyendo así al marcaje epigenético de la cromatina de manera casi simultánea a la replicación

INCURVATA2 Encodes the Catalytic Subunit of DNA Polymerase α and Interacts with Genes Involved in Chromatin-Mediated Cellular Memory in Arabidopsis thaliana

Cell type–specific gene expression patterns are maintained by the stable inheritance of transcriptional states through mitosis, requiring the action of multiprotein complexes that remodel chromatin structure. Genetic and molecular interactions between chromatin remodeling factors and components of the DNA replication machinery have been identified in Schizosaccharomyces pombe, indicating that some epigenetic marks are replicated simultaneously to DNA with the participation of the DNA replication complexes. This model of epigenetic inheritance might be extended to the plant kingdom, as we report here with the positional cloning and characterization of INCURVATA2 (ICU2), which encodes the putative catalytic subunit of the DNA polymerase α of Arabidopsis thaliana. The strong icu2-2 and icu2-3 insertional alleles caused fully penetrant zygotic lethality when homozygous and incompletely penetrant gametophytic lethality, probably because of loss of DNA polymerase activity. The weak icu2-1 allele carried a point mutation and caused early flowering, leaf incurvature, and homeotic transformations of sepals into carpels and of petals into stamens. Further genetic analyses indicated that ICU2 interacts with TERMINAL FLOWER2, the ortholog of HETEROCHROMATIN PROTEIN1 of animals and yeasts, and with the Polycomb group (PcG) gene CURLY LEAF. Another PcG gene, EMBRYONIC FLOWER2, was found to be epistatic to ICU2. Quantitative RT-PCR analyses indicated that a number of regulatory genes were derepressed in the icu2-1 mutant, including genes associated with flowering time, floral meristem, and floral organ identity

Enhanced Conjugation of Auxin by GH3 Enzymes Leads to Poor Adventitious Rooting in Carnation Stem Cuttings

Commercial carnation (Dianthus caryophyllus) cultivars are vegetatively propagated from axillary stem cuttings through adventitious rooting; a process which is affected by complex interactions between nutrient and hormone levels and is strongly genotype-dependent. To deepen our understanding of the regulatory events controlling this process, we performed a comparative study of adventitious root (AR) formation in two carnation cultivars with contrasting rooting performance, “2101–02 MFR” and “2003 R 8”, as well as in the reference cultivar “Master”. We provided molecular evidence that localized auxin response in the stem cutting base was required for efficient adventitious rooting in this species, which was dynamically established by polar auxin transport from the leaves. In turn, the bad-rooting behavior of the “2003 R 8” cultivar was correlated with enhanced synthesis of indole-3-acetic acid conjugated to aspartic acid by GH3 proteins in the stem cutting base. Treatment of stem cuttings with a competitive inhibitor of GH3 enzyme activity significantly improved rooting of “2003 R 8”. Our results allowed us to propose a working model where endogenous auxin homeostasis regulated by GH3 proteins accounts for the cultivar dependency of AR formation in carnation stem cuttings

Measurements of PBGD activity and accumulation of PBG in <i>rug1</i> and L<i>er</i>.

<p>(a) PBGD activity in enzyme units per milligram of protein and (b) PBG accumulation in micrograms per gram of fresh weight in L<i>er</i> and <i>rug1</i> plants grown under long day conditions (16-h light/8-h dark) or continuous light. Asterisks indicate <i>rug1</i> values significantly different from those of the wild type (Students t-test, P<0.01).</p

<em>PORPHOBILINOGEN DEAMINASE</em> Deficiency Alters Vegetative and Reproductive Development and Causes Lesions in Arabidopsis

<div><p>The Arabidopsis <em>rugosa1</em> (<em>rug1</em>) mutant has irregularly shaped leaves and reduced growth. In the absence of pathogens, leaves of <em>rug1</em> plants have spontaneous lesions reminiscent of those seen in lesion-mimic mutants; <em>rug1</em> plants also express cytological and molecular markers associated with defence against pathogens. These <em>rug1</em> phenotypes are made stronger by dark/light transitions. The <em>rug1</em> mutant also has delayed flowering time, upregulation of the floral repressor <em>FLOWERING LOCUS C</em> (<em>FLC</em>) and downregulation of the flowering promoters <em>FT</em> and <em>SOC1/AGL20</em>. Vernalization suppresses the late flowering phenotype of <em>rug1</em> by repressing <em>FLC</em>. Microarray analysis revealed that 280 nuclear genes are differentially expressed between <em>rug1</em> and wild type; almost a quarter of these genes are involved in plant defence. In <em>rug1</em>, the auxin response is also affected and several auxin-responsive genes are downregulated. We identified the <em>RUG1</em> gene by map-based cloning and found that it encodes porphobilinogen deaminase (PBGD), also known as hydroxymethylbilane synthase, an enzyme of the tetrapyrrole biosynthesis pathway, which produces chlorophyll, heme, siroheme and phytochromobilin in plants. PBGD activity is reduced in <em>rug1</em> plants, which accumulate porphobilinogen. Our results indicate that Arabidopsis PBGD deficiency impairs the porphyrin pathway and triggers constitutive activation of plant defence mechanisms leading to leaf lesions and affecting vegetative and reproductive development.</p> </div

Lesion phenotype in the <i>rug1</i> mutant.

<p>(a, d) Three-week-old rosettes of the <i>rug1</i> mutant and the wild-type L<i>er</i>. (b, e) Close-up views of third-node vegetative leaves from the plants shown in panels (a) and (d). (c, f, h, i) Confocal micrographs showing fluorescing chlorophyll within mesophyll cells of (c, f) whole third-node leaves [those shown in (b) and (e)] and (h) details of the subepidermal layer of mesophyll cells of L<i>er</i> and (i) the boundary between a green and a pale sector in a <i>rug1</i> leaf. (g) 45-day-old plants grown in soil. (j, k) Transverse sections of third leaves. Bars = (a–f) 1 mm, (g) 1 cm, (h, i) 250 µm, and (j, k) 50 µm.</p

Conservation of PBGD and structure of the <i>RUG1</i> gene.

<p>Alignment of the predicted amino acids of the Arabidopsis RUG1 (NP_196445) protein with those of its putative orthologues from <i>Pisum sativum</i> (Q43082), <i>Triticum aestivum</i> (AAL12220), <i>Oryza sativa</i> (NP_001046017), <i>Escherichia coli</i> (YP_001460596), <i>Homo sapiens</i> (NP_000181), <i>Mus musculus</i> (AAH03861), <i>Danio rerio</i> (NP_957448) and <i>Saccharomyces cerevisiae</i> (NP_010076). Residues identical across all the sequences are shaded black; residues with similar chemical properties conserved across all five sequences are shaded grey. Numbers correspond to amino acid positions. Continuous lines indicate the N-terminal chloroplast transit peptide (as identified by the TargetP v1.0 program; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053378#pone.0053378-Emanuelsson1" target="_blank">[82]</a>; <a href="http://www.cbs.dtu.dk/services/TargetP/" target="_blank">http://www.cbs.dtu.dk/services/TargetP/</a>). The alignment was obtained using ClustalX v 1.5b. The highly conserved amino acid that is changed in the <i>rug1</i> mutant is indicated by an asterisk. A schematic representation of the <i>RUG1</i> gene is also shown, with indication of the position of the <i>rug1</i> mutation. Exons and introns are represented by boxes and lines, respectively. White boxes correspond to the 5′ and 3′ untranslated regions. The predicted translation start (ATG) and stop (TGA) codons are indicated. Horizontal arrows, not drawn to scale, indicate the oligonucleotides used to characterize the structure of <i>RUG1</i>.</p

Lesion histology.

<p>(a–c) 21-day-old rosettes of (a) L<i>er</i> and (b) <i>rug1</i>, stained with toluidine blue, and (c) a third-node leaf from the plant shown in (b). Arrows in (c) indicate defective cuticle in a necrotic sector. (d–g) Trypan blue staining of (d) L<i>er</i> and (f) <i>rug1</i> third-node leaves and (e, g) close-up views of the leaves shown in (d) and (f), revealing dead cells in <i>rug1</i>. (h–o) (h, j, l, n) Rosettes of the genotypes indicated and (i, k, m, o) visualization of H₂O₂ accumulation by means of DAB staining of (i, k, m) one or (o) all of their leaves. Plants were grown under (a–k) continuous light or (l–o) long day conditions (16-h light/8-h dark). Bars = (a, b, h, j, l) 5 mm, (c, d, f, i, k, m–o) 1 mm, and (e, g) 200 µm.</p