Estudio de distintos métodos de conservación de polen de calabacín (Curcubita pepo L.)

Breeding programmes in zucchini (Cucurbita pepo) are focused in obtaining genetic variability by mutant population induced with chemical agent as ethyl methanesulfonate. This chemical agent causes morphological and physiological changes that may alter flowering pattern of C. pepo, preventing selfing. Pollen storage may allow artificially selfing in mutant plants with abnormal flowering pattern. Cucurbita pepo pollen has a short longevity and nowadays there are few studies on storage methods. In this work, we have compared different storage conditions (temperature, humidity and immersion in organic solvents) of pollen collected in two development stages, anthesis and anthesis-1 (one day before anthesis), with the aim of extending pollen viability. Viability of stored pollen was assessed by histochemical test TTC and fruit set and seed production. Storage of complete flowers in anthesis stage at 4ºC maintained pollen viability up 96 hours, however, only stored pollen up 48 hours was able to produce seeded fruits. Pollen from flower in a stage previous to anthesis, set fruits, but these fruits had not seeds

First RNA-seq approach to study fruit set and parthenocarpy in zucchini (Cucurbita pepo L.)

[EN] Background: Zucchini fruit set can be limited due to unfavourable environmental conditions in off-seasons crops that caused ineffective pollination/fertilization. Parthenocarpy, the natural or artificial fruit development without fertilization, has been recognized as an important trait to avoid this problem, and is related to auxin signalling. Nevertheless, differences found in transcriptome analysis during early fruit development of zucchini suggest that other complementary pathways could regulate fruit formation in parthenocarpic cultivars of this species. The development of next-generation sequencing technologies (NGS) as RNA-sequencing (RNA-seq) opens a new horizon for mapping and quantifying transcriptome to understand the molecular basis of pathways that could regulate parthenocarpy in this species. The aim of the current study was to analyze fruit transcriptome of two cultivars of zucchini, a non-parthenocarpic cultivar and a parthenocarpic cultivar, in an attempt to identify key genes involved in parthenocarpy. Results: RNA-seq analysis of six libraries (unpollinated, pollinated and auxin treated fruit in a non-parthenocarpic and parthenocarpic cultivar) was performed mapping to a new version of C. pepo transcriptome, with a mean of 92% success rate of mapping. In the non-parthenocarpic cultivar, 6479 and 2186 genes were differentially expressed (DEGs) in pollinated fruit and auxin treated fruit, respectively. In the parthenocarpic cultivar, 10,497 in pollinated fruit and 5718 in auxin treated fruit. A comparison between transcriptome of the unpollinated fruit for each cultivar has been performed determining that 6120 genes were differentially expressed. Annotation analysis of these DEGs revealed that cell cycle, regulation of transcription, carbohydrate metabolism and coordination between auxin, ethylene and gibberellin were enriched biological processes during pollinated and parthenocarpic fruit set. Conclusion: This analysis revealed the important role of hormones during fruit set, establishing the activating role of auxins and gibberellins against the inhibitory role of ethylene and different candidate genes that could be useful as markers for parthenocarpic selection in the current breeding programs of zucchini.Research worked is supported by the project RTA2014-00078 from the Spanish Institute of Agronomy Research INIA (Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria) and also PP.AVA.AVA201601.7, FEDER y FSE (Programa Operativo FSE de Andalucia 2007-2013 "Andalucia se mueve con Europa"). TPV is supported by a FPI scholarship from RTA2011-00044-C02-01/02 project of INIA. 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First TILLING Platform in Cucurbita pepo: A New Mutant Resource for Gene Function and Crop Improvement

Although the availability of genetic and genomic resources for Cucurbita pepo has increased significantly, functional genomic resources are still limited for this crop. In this direction, we have developed a high throughput reverse genetic tool: the first TILLING (Targeting Induced Local Lesions IN Genomes) resource for this species. Additionally, we have used this resource to demonstrate that the previous EMS mutant population we developed has the highest mutation density compared with other cucurbits mutant populations. The overall mutation density in this first C. pepo TILLING platform was estimated to be 1/133 Kb by screening five additional genes. In total, 58 mutations confirmed by sequencing were identified in the five targeted genes, thirteen of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was studied in a peroxidase gene, revealing that the phenotype of seedling homozygous for one of the isolated mutant alleles was albino. These results indicate that the TILLING approach in this species was successful at providing new mutations and can address the major challenge of linking sequence information to biological function and also the identification of novel variation for crop breeding.Financial support was provided by the Spanish Project INIA (Instituto Nacional de Investigacion y Tecnologia Agraria y Almentaria) RTA2011-00044C02-01, the ANR MELODY (ANR-11-BSV7-0024), the European Research Council (ERCSEXYPARTH), FEDER, and FSE funds. NVD has been awarded a grant by the Andalusian Institute of Agronomy Research IFAPA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Vicente-Dolera, N.; Troadec, C.; Moya, M.; Río-Celestino, MD.; Pomares-Viciana, T.; Bendahmane, A.; Picó Sirvent, MB.... (2014). First TILLING Platform in Cucurbita pepo: A New Mutant Resource for Gene Function and Crop Improvement. PLoS ONE. 9(11):112743-112743. https://doi.org/10.1371/journal.pone.0112743S112743112743911Paris, H. S., Yonash, N., Portnoy, V., Mozes-Daube, N., Tzuri, G., & Katzir, N. (2002). Assessment of genetic relationships in Cucurbita pepo (Cucurbitaceae) using DNA markers. Theoretical and Applied Genetics, 106(6), 971-978. doi:10.1007/s00122-002-1157-0Parry, M. A. J., Madgwick, P. J., Bayon, C., Tearall, K., Hernandez-Lopez, A., Baudo, M., … Phillips, A. L. (2009). Mutation discovery for crop improvement. Journal of Experimental Botany, 60(10), 2817-2825. doi:10.1093/jxb/erp189Gilchrist, E., & Haughn, G. (2010). Reverse genetics techniques: engineering loss and gain of gene function in plants. Briefings in Functional Genomics, 9(2), 103-110. doi:10.1093/bfgp/elp059McCallum, C. M., Comai, L., Greene, E. A., & Henikoff, S. (2000). Targeting Induced LocalLesions IN Genomes (TILLING) for Plant Functional Genomics. Plant Physiology, 123(2), 439-442. doi:10.1104/pp.123.2.439Colbert, T., Till, B. J., Tompa, R., Reynolds, S., Steine, M. N., Yeung, A. T., … Henikoff, S. (2001). High-Throughput Screening for Induced Point Mutations. Plant Physiology, 126(2), 480-484. doi:10.1104/pp.126.2.480Wang, T. L., Uauy, C., Robson, F., & Till, B. (2012). TILLINGin extremis. Plant Biotechnology Journal, 10(7), 761-772. doi:10.1111/j.1467-7652.2012.00708.xDong, C., Dalton-Morgan, J., Vincent, K., & Sharp, P. (2009). A Modified TILLING Method for Wheat Breeding. The Plant Genome Journal, 2(1), 39. doi:10.3835/plantgenome2008.10.0012Uauy, C., Paraiso, F., Colasuonno, P., Tran, R. K., Tsai, H., Berardi, S., … Dubcovsky, J. (2009). A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC Plant Biology, 9(1), 115. doi:10.1186/1471-2229-9-115Kumar, A. P., Boualem, A., Bhattacharya, A., Parikh, S., Desai, N., Zambelli, A., … Bendahmane, A. (2013). SMART -- Sunflower Mutant population And Reverse genetic Tool for crop improvement. BMC Plant Biology, 13(1), 38. doi:10.1186/1471-2229-13-38Kurowska, M., Daszkowska-Golec, A., Gruszka, D., Marzec, M., Szurman, M., Szarejko, I., & Maluszynski, M. (2011). TILLING - a shortcut in functional genomics. Journal of Applied Genetics, 52(4), 371-390. doi:10.1007/s13353-011-0061-1Rigola, D., van Oeveren, J., Janssen, A., Bonné, A., Schneiders, H., van der Poel, H. J. A., … van Eijk, M. J. T. (2009). High-Throughput Detection of Induced Mutations and Natural Variation Using KeyPoint™ Technology. PLoS ONE, 4(3), e4761. doi:10.1371/journal.pone.0004761González, M., Xu, M., Esteras, C., Roig, C., Monforte, A. J., Troadec, C., … Picó, B. (2011). Towards a TILLING platform for functional genomics in Piel de Sapo melons. BMC Research Notes, 4(1). doi:10.1186/1756-0500-4-289Elias, R., Till, B. J., Mba, C., & Al-Safadi, B. (2009). Optimizing TILLING and Ecotilling techniques for potato (Solanum tuberosum L). BMC Research Notes, 2(1), 141. doi:10.1186/1756-0500-2-141Dahmani-Mardas, F., Troadec, C., Boualem, A., Lévêque, S., Alsadon, A. A., Aldoss, A. A., … Bendahmane, A. (2010). Engineering Melon Plants with Improved Fruit Shelf Life Using the TILLING Approach. PLoS ONE, 5(12), e15776. doi:10.1371/journal.pone.0015776Boualem, A., Fleurier, S., Troadec, C., Audigier, P., Kumar, A. P. K., Chatterjee, M., … Bendahmane, A. (2014). Development of a Cucumis sativus TILLinG Platform for Forward and Reverse Genetics. 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The yeast Ptc7p mitochondrial phosphatase, the crossroad of coenzyme Q biosynthesis, mitophagy activation and chronological life span extension

Resumen del póster presentado al 22nd IUBMB & 37th FEBS Congress, celebrado en Sevilla (España) del 4 al 9 de septiembre de 2012.-- et al.Coenzyme Q (CoQ or Q) is an essential isoprenylated benzoquinone component of mitochondria, which functions mainly as an electron carrier from complex I, or II to complex III at the inner membrane, and as an antioxidant particularly on lipoproteins and plasma membrane. CoQ biosynthesis is a highly regulated process driven by a multi-protein complex that catalyzes the modifications of the benzene ring. Coq7p/Cat5p (Coq7p) catalyzes one of the latest steps required for the final conversion of the late intermediate demethoxy-Q6 (DMQ6) to Q6, which also represents a key regulatory step in this pathway. Coq7p dephosphorylation was produced by the mitochondrial Ptc7 protein Ser/Thr phosphatase that activates aerobic yeast metabolism by regulating coenzyme Q (CoQ) biosynthesis. Yeast lacking PTC7 (YHR076w) gene exhibited decreased of both mitochondrial function and oxidative stress defenses, leading to increased protein carbonylation damage. CoQ content was decreased in PTC7 deleted strain, suggesting that during respiratory metabolism Ptc7p activates the CoQ6 biosynthesis. Ptc7p dephosphorylates Coq7p in both in vivo and in vitro assays. PTC7 null mutant exhibited increased Coq7p phosphorylation when CoQ biosynthesis was induced. Chronological life span (CLS) is defined as a survival mechanism that depends on metabolic and stress adaptations to environment. PTC7 strain showed a decreased CLS that was not rescued by exogenous CoQ6. Rescue of CLS required Ptc7p that also activated mitophagy but not macroautophagy. These results led us to propose that Ptc7p links homeostasis of CoQ by regulating its biosynthesis through the phosphorylation stage of Coq7p and mitochondrial recycling as an adaptation mechanism to both stress and nutritional environment changes to promote CLS.Peer reviewe

Regulation of coenzyme Q biosynthesis in yeast: A new complex in the block

Coenzyme Q (CoQ) is an isoprenylated benzoquinone found in mitochondria, which functions mainly as an electron carrier from complex I or II to complex III in the inner membrane. CoQ is also an antioxidant that specifically prevents the oxidation of lipoproteins and the plasma membrane. Most of the information about the synthesis of CoQ comes from studies performed in Saccharomyces cerevisiae. CoQ biosynthesis is a highly regulated process of sequential modifications of the benzene ring. There are three pieces of evidence supporting the involvement of a multienzymatic complex in yeast CoQ6 biosynthesis: (a) the accumulation of a unique early precursor in all null mutants of the COQ genes series, 4-hydroxy-3-hexaprenyl benzoate (HHB), (b) the lack of expression of several Coq proteins in COQ null mutants, and (c) the restoration of CoQ biosynthesis complex after COQ8 overexpression. The model we propose based on the formation of a multiprotein complex should facilitate a better understanding of CoQ biosynthesis. According to this model, the complex assembly requires the synthesis of a precursor such as HHB by Coq2p that must be recognized by the regulatory protein Coq4p to act as the core component of the complex. The phosphorylation of Coq3p and Coq5p by the kinase Coq8p facilitates the formation of an initial precomplex of 700 kDa that contains all Coq proteins with the exception of Coq7p. The precomplex is required for the synthesis of 5-demethoxy-Q6, the substrate of Coq7p. When cells require de novo CoQ6 synthesis, Coq7p is dephosphorylated by Ptc7p, a mitochondrial phosphatase that activates the synthesis of CoQ6. This event allows for the full assembly of a complex of 1,300 kDa that is responsible for the final product of the pathway, CoQ6.The work was supported by the Spanish Ministerio de Ciencia y Tecnología, Spanish PI11/00078, Junta de Andalucía P08-CTS-03988, and by the International Q10 Association “Phosphorylation based regulation of coenzyme Q biosynthesis in yeast.” A.M.M. received a predoctoral fellowship from the Consejería de Innovación Ciencia y Empresa, Junta de Andalucía (Spain). I.G.M. received a predoctoral fellowship from the Plan Propio of the Universidad Pablo de Olavide de Sevilla. T.P.V. and P.G.D. received a predoctoral fellowship from the CIBERER-ISCIII (Spain).Peer Reviewe

The regulation of coenzyme Q biosynthesis in eukaryotic cells: All that yeast can tell us

Coenzyme Q (CoQ) is a mitochondrial lipid, which functions mainly as an electron carrier from complex I or II to complex III at the mitochondrial inner membrane, and also as antioxidant in cell membranes. CoQ is needed as electron acceptor in β-oxidation of fatty acids and pyridine nucleotide biosynthesis, and it is responsible for opening the mitochondrial permeability transition pore. The yeast model has been very useful to analyze the synthesis of CoQ, and therefore, most of the knowledge about its regulation was obtained from the Saccharomyces cerevisiae model. CoQ biosynthesis is regulated to support 2 processes: the bioenergetic metabolism and the antioxidant defense. Alterations of the carbon source in yeast, or in nutrient availability in yeasts or mammalian cells, upregulate genes encoding proteins involved in CoQ synthesis. Oxidative stress, generated by chemical or physical agents or by serum deprivation, modifies specifically the expression of some COQ genes by means of stress transcription factors such as Msn2/4p, Yap1p or Hsf1p. In general, the induction of COQ gene expression produced by metabolic changes or stress is modulated downstream by other regulatory mechanisms such as the protein import to mitochondria, the assembly of a multi-enzymatic complex composed by Coq proteins and also the existence of a phosphorylation cycle that regulates the last steps of CoQ biosynthesis. The CoQ biosynthetic complex assembly starts with the production of a nucleating lipid such as HHB by the action of the Coq2 protein. Then, the Coq4 protein recognizes the precursor HHB acting as the nucleus of the complex. The activity of Coq8p, probably as kinase, allows the formation of an initial pre-complex containing all Coq proteins with the exception of Coq7p. This pre-complex leads to the synthesis of 5-demethoxy-Q6 (DMQ6), the Coq7p substrate. When de novo CoQ biosynthesis is required, Coq7p becomes dephosphorylated by the action of Ptc7p increasing the synthesis rate of CoQ6. This critical model is needed for a better understanding of CoQ biosynthesis. Taking into account that patients with CoQ10 deficiency maintain to some extent the machinery to synthesize CoQ, new promising strategies for the treatment of CoQ 10 deficiency will require a better understanding of the regulation of CoQ biosynthesis in the future.The work was supported by the Spanish Ministerio de Ciencia y Tecnología, Spanish PI11/00078, Junta de Andalucía P08-CTS-03988 and by the International Q10 Association ‘Phosphorylation based regulation of coenzyme Q biosynthesis in yeast’. A.M.-M. received a predoctoral fellowship from the Consejería de Innovación Ciencia y Empresa, Junta de Andalucía (Spain). I.G.-M. received a predoctoral fellowship from the Plan Propio of the Universidad Pablo de Olavide de Sevilla. T.P.V. and P.G.D. received a predoctoral fellowship from the CIBERER-ISCIII (Spain).Peer Reviewe

The mitochondrial phosphatase PPTC7 orchestrates mitochondrial metabolism regulating coenzyme Q10 biosynthesis

Coenzyme Q10 (CoQ10) is a redox molecule critical for the proper function of energy metabolism and antioxidant defenses. Despite its essential role in cellular metabolism, the regulation of CoQ10 biosynthesis in humans remains mostly unknown. Herein, we determined that PPTC7 is a regulatory protein of CoQ10 biosynthesis required for human cell survival. We demonstrated by in vitro approaches that PPTC7 is a bona fide protein phosphatase that dephosphorylates the human COQ7. Expression modulation experiments determined that human PPTC7 dictates cellular CoQ10 content. Using two different approaches (PPTC7 over-expression and caloric restriction), we demonstrated that PPTC7 facilitates and improves the human cell adaptation to respiratory conditions. Moreover, we determined that the physiological role of PPTC7 takes place in the adaptation to starvation and pro-oxidant conditions, facilitating the induction of mitochondrial metabolism while preventing the accumulation of ROS. Here we unveil the first post-translational mechanism regulating CoQ10 biosynthesis in humans and propose targeting the induction of PPTC7 activity/expression for the treatment of CoQ10-related mitochondrial diseases.The research group is funded by the Andalusian Government as the BIO177 group through FEDER funds (European Commission), by the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (FIS PI14/01962 and FIS PI17/01286)

Tilled genes and mutation frequency in the <i>C. pepo</i> mutant collection with 768 M2 families screened.

a<p>nucleic acid transition is a non-synonymous mutation and induce amino acid change in the translated protein.</p>b<p>nucleic acid transition produces a stop codon and may induce a truncated protein.</p>c<p>nucleic acid transition is located in splicing motif.</p>d<p>nucleic acid transition induces a synonymous mutation and then no change in the translated protein.</p>e<p>nucleid acid transition is located in an intronic or promotor region.</p><p>Tilled genes and mutation frequency in the <i>C. pepo</i> mutant collection with 768 M2 families screened.</p

Examples of morphological mutants observed within the <i>C. pepo</i> TILLING population.

<p>(a) Plant affected in cotyledon colour, albino. (b) Plant affected in cotyledon colour, chlorotic. (c) Plant affected in cotyledon number. (d–e) Female flowers with abnormal stigma. (f–h) Different coloured fruits. (i–k) Semi-dwarf plants with bushy and hyper compact architecture. (l) Albino dwarf plant. (m) Different size and colour leaves.</p

Summary of the <i>Cucurbita pepo</i> mutant collection development.

<p>Summary of the <i>Cucurbita pepo</i> mutant collection development.</p