Characterization of hemizygous deletions in Citrus using array-Comparative Genomic Hybridization and microsynteny comparisons with the poplar genome

<p>Abstract</p> <p>Background</p> <p>Many fruit-tree species, including relevant <it>Citrus </it>spp varieties exhibit a reproductive biology that impairs breeding and strongly constrains genetic improvements. In citrus, juvenility increases the generation time while sexual sterility, inbreeding depression and self-incompatibility prevent the production of homozygous cultivars. Genomic technology may provide citrus researchers with a new set of tools to address these various restrictions. In this work, we report a valuable genomics-based protocol for the structural analysis of deletion mutations on an heterozygous background.</p> <p>Results</p> <p>Two independent fast neutron mutants of self-incompatible clementine (<it>Citrus clementina </it>Hort. Ex Tan. cv. Clemenules) were the subject of the study. Both mutants, named 39B3 and 39E7, were expected to carry DNA deletions in hemizygous dosage. Array-based Comparative Genomic Hybridization (array-CGH) using a <it>Citrus </it>cDNA microarray allowed the identification of underrepresented genes in these two mutants. Subsequent comparison of citrus deleted genes with annotated plant genomes, especially poplar, made possible to predict the presence of a large deletion in 39B3 of about 700 kb and at least two deletions of approximately 100 and 500 kb in 39E7. The deletion in 39B3 was further characterized by PCR on available <it>Citrus </it>BACs, which helped us to build a partial physical map of the deletion. Among the deleted genes, <it>ClpC</it>-like gene coding for a putative subunit of a multifunctional chloroplastic protease involved in the regulation of chlorophyll <it>b </it>synthesis was directly related to the mutated phenotype since the mutant showed a reduced chlorophyll <it>a</it>/<it>b </it>ratio in green tissues.</p> <p>Conclusion</p> <p>In this work, we report the use of array-CGH for the successful identification of genes included in a hemizygous deletion induced by fast neutron irradiation on <it>Citrus clementina</it>. The study of gene content and order into the 39B3 deletion also led to the unexpected conclusion that microsynteny and local gene colinearity in this species were higher with <it>Populus trichocarpa </it>than with the phylogenetically closer <it>Arabidopsis thaliana</it>. This work corroborates the potential of <it>Citrus </it>genomic resources to assist mutagenesis-based approaches for functional genetics, structural studies and comparative genomics, and hence to facilitate citrus variety improvement.</p

The Altered Expression of microRNA408 Influences the Arabidopsis Response to Iron Deficiency

MicroRNAs contribute to the adaptation of plants to varying environmental conditions by affecting systemic mineral nutrient homeostasis. Copper and iron deficiencies antagonistically control the expression of Arabidopsis thaliana microRNA408 (miR408), which post-transcriptionally regulates laccase-like multicopper oxidase family members LAC3, LAC12, and LAC13. In this work, we used miR408 T-DNA insertion mutants (408-KO1 and 408-KO2) and a previously characterized transgenic line overexpressing miR408 (35S:408-14) to explore how miR408 influences copper- and iron-dependent metabolism. We observed that the altered expression of miR408 diminished plant performance and the activation of the iron-regulated genes under iron-deficient conditions. Consistently with the low expression of the miR408-target laccases, we showed that the vascular bundle lignification of the 35S:408-14 plants diminished. The decrease in the phenoloxidase and ferroxidase activities exhibited by wild-type plants under iron deficiency did not occur in the 408-KO1 plants, probably due to the higher expression of laccases. Finally, we observed that the hydrogen peroxide levels under iron starvation were altered in both the 408-KO1 and 35S:408-14 lines. Taken together, these results suggest that Arabidopsis plants with modified miR408 levels undergo multiple deregulations under iron-deficient conditions

Regulation of ovule initiation by gibberellins and brassinosteroids in tomato and Arabidopsis: two plant species, two molecular mechanisms

This is the peer reviewed version of the following article: Barro¿Trastoy, D., Carrera, E., Baños, J., Palau-Rodríguez, J., Ruiz-Rivero, O., Tornero, P., Alonso, J.M., López-Díaz, I., Gómez, M.D. and Pérez-Amador, M.A. (2020), Regulation of ovule initiation by gibberellins and brassinosteroids in tomato and Arabidopsis: two plant species, two molecular mechanisms. Plant J, 102: 1026-1041, which has been published in final form at https://doi.org/10.1111/tpj.14684. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] Ovule primordia formation is a complex developmental process with a strong impact on the production of seeds. In Arabidopsis this process is controlled by a gene network, including components of the signalling pathways of auxin, brassinosteroids (BRs) and cytokinins. Recently, we have shown that gibberellins (GAs) also play an important role in ovule primordia initiation, inhibiting ovule formation in both Arabidopsis and tomato. Here we reveal that BRs also participate in the control of ovule initiation in tomato, by promoting an increase on ovule primordia formation. Moreover, molecular and genetic analyses of the co-regulation by GAs and BRs of the control of ovule initiation indicate that two different mechanisms occur in tomato and Arabidopsis. In tomato, GAs act downstream of BRs. BRs regulate ovule number through the downregulation of GA biosynthesis, which provokes stabilization of DELLA proteins that will finally promote ovule primordia initiation. In contrast, in Arabidopsis both GAs and BRs regulate ovule number independently of the activity levels of the other hormone. Taken together, our data strongly suggest that different molecular mechanisms could operate in different plant species to regulate identical developmental processes even, as for ovule primordia initiation, if the same set of hormones trigger similar responses, adding a new level of complexity.We wish to thank B. Janssen (Horticulture and Food Research Institute, New Zealand) for the pBJ60 shuttle vector, C. Ferrandiz and M. Colombo (IBMCP, CSIC-UPV, Valencia, Spain) for their help in the generation of 35S:ANT lines and L.E.P. Peres (Universidade de Sao Paulo, Brazil) for the tomato mutant lines. Our thanks also go to C. Fuster for technical assistance. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness-FEDER (BIO2017-83138R) to MAPA and from NSF (DBI-0820755, MCB-1158181, and IOS-1444561) to JMA.Barro-Trastoy, D.; Carrera, E.; Baños, J.; Palau-Rodríguez, J.; Ruiz-Rivero, O.; Tornero Feliciano, P.; Alonso, JM.... (2020). 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Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

Background Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide.Methods A multimethods analysis was performed as part of the GlobalSurg 3 study-a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital.Findings Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3.85 [95% CI 2.58-5.75]; p&lt;0.0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63.0% vs 82.7%; OR 0.35 [0.23-0.53]; p&lt;0.0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer.Interpretation Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised

Hormonal regulation of tomato gibberellin 20-oxidase1 expressed in Arabidopsis

Gibberellin 20-oxidases, enzymes of gibberellin (GA) biosynthesis, play an important role in (GA) homeostasis. To investigate the regulation of tomato SlGA20ox1 expression a genomic clone was isolated, its promoter transcriptionally fused to the GUS reporter gene and the construct used to transform Arabidopsis. Expression was found in diverse vegetative (leaves and roots) and reproductive (flowers) organs. GUS staining was also localized in the columella of secondary roots. GA negative feed-back regulation of SlGA20ox1:GUS was shown to be active both in tomato and in transformed Arabidopsis. Auxin (indol-3-acetic acid, 2,4-dichlorophenoxyacetic acid and naphtaleneacetic acid), triiodobenzoic acid (an inhibitor of auxin transport) and benzyladenine (a cytokinin) treatment induced SlGA20ox1:GUS expression associated with increased auxin content and/or signalling , detected using DR5:GUS expression as a marker. Interestingly, SlGA20ox:GUS expression was induced by auxin and root excision in the hypocotyl, an organ not showing GUS staining in control seedlings. In etiolated seedlings, SlGA20ox1:GUS expression occurred in the elongating hypocotyl region of etiolated seedlings and was down-regulated upon transfer to light associated with decrease of growth rate elongation. Our results show that feed-back, auxin and light regulation of SlGA20ox1 expression depends on DNA elements contained within the first 834 bp of the 5´upstream promoter region. Putative DNA regulatory sequences involved in negative feed-back regulation and auxin response were identified in that promoter.This work was supported by Ministerio de Educación y Ciencia of Spain (grants BIO2003-00151 and BIO2006-13437 to J.L.G.-M.) and Consellería de Agricultura de la Generalitat Valenciana (fellowship to E. M.)Peer reviewe

New sesquiterpene synthases isolated from orange peel

La presente invención se relaciona con un enzima natural sesquiterpeno sintasa de naranjo (CsTPS3) con actividad δ-cadineno sintasa, así como con las variantes de dicha enzima modificadas por la substitución de al menos un aminoácido que altera su actividad catalítica, transformándolas preferentemente en enzimas con actividad elemol sintasa o germacreno D sintasa. Son asimismo objeto de la presente invención, el ácido nucleico que codifica el enzima natural CsTPS3 así como las versiones modificadas derivadas de éste, además de las construcciones génicas, vectores de expresión y cepas bacterianas que los comprenden. La invención también se refiere al uso de estas enzimas para sintetizar sesquiterpenos, preferentemente δ-cadineno, elemol y germacreno D, y proporciona un método para la obtención de los mismos.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Instituto Valenciano de Investigaciones AgrariasB1 Patente sin examen previ