Simulaciones de Monte Carlo-Metropolis del almacenamiento de hidrógeno en COFs (Covalent Organic Frameworks)

La reducción de la emisión de los gases de efecto invernadero es uno de los temas que más preocupa a la sociedad en este siglo. Ante esta situación, el vehículo de hidrógeno se erige como una propuesta que puede convertirse en una opción de presente y futuro. Uno de los principales problemas que presenta el vehículo de hidrógeno es el almacenamiento de este gas. Una de las formas que está ofreciendo resultados más prometedores es el almacenamiento en materiales nanoporosos mediante fisisorción. En concreto, el uso de los COFs como material es una de las opciones más atractivas, gracias a sus grandes cualidades de porosidad, lo que contribuye al almacenamiento del hidrógeno. En este trabajo, se estudia el material COF-102, calculando mediante simulaciones de Monte Carlo la capacidad de almacenamiento de este, a la temperatura de 77 K y a bajas y medias presiones. Los resultados obtenidos se compararon con datos experimentales y simulados obtenidos en otras simulaciones.Reducing the emission of greenhouse gases is one of the issues that most concerns society in this century. In this situation, the hydrogen vehicle stands as a proposal that can become an option for the present and the future. One of the main problems that the hydrogen vehicle presents is the storage of this gas. One of the ways that is o ering the most promising results is storage in nanoporous materials by physisorption. Speci cally, the use of COFs as a material is one of the most attractive options, due to its great porosity qualities, which contribute to the storage of hydrogen. In this work, COF-102 material is studied. Its storage capacity through Monte Carlo simulations was calculated, at a temperature of 77 K and at low and medium pressures. The results obtained were compared with experimental and simulated data obtained in other simulations.Grado en Físic

Enzymatic Assays and Enzyme Histochemistry of Tuta absoluta Feeding on Tomato Leaves

[EN] Enzymes play a key role in insect-plant relationships. For a better understanding of these interactions, we analyzed Tuta absoluta digestive enzymes. Here, we describe a detailed protocol for the detection of trypsin and papain-like enzymes in Tuta absoluta larvae by enzyme histochemistry. This assay uses frozen and unfixed samples to avoid the loss of enzymatic activity. We also describe a protocol for the quantification of trypsin and papain-like enzymes in the larvae of Tuta absoluta at different developmental instars.Rim Hamza acknowledges fellowships from the Tunisian Ministry for Higher Education and Scientific Research and from the Erasmus Mundus EMMAG program of the European Union. This work was partly supported by grants BIO2013-40747-R and AGL2014-55616-C3 from the Spanish Ministry of Economy and Competitiveness (MINECO). We wish to thank Drs. Alberto Urbaneja and Meritxell Perez-Hedo (Instituto Valenciano de Investigaciones Agrarias, Centro de Proteccion Vegetal y Biotecnologia, Unidad Asociada de Entomologia, UJI-IVIA) for providing Tuta absoluta larvae and to Marisol Gascon (IBMCP) for technical assistance in sample processing. This protocol has been described in the publication: Hamza et al. (2018).Hamza, R.; Beltran Porter, JP.; Cañas Clemente, LA. (2018). Enzymatic Assays and Enzyme Histochemistry of Tuta absoluta Feeding on Tomato Leaves. Bio-protocol. 8(17). https://doi.org/10.21769/BioProtoc.2993S81

Pensamiento crítico para el Pensamiento gráfico

PASSMORE (1967) define el Pensamiento Crítico como un proceso que es a la vez reflexivo e imaginativo, cualidades imprescindibles en todo proceso de diseño. En este artículo nos centramos en la utilización del Pensamiento Crítico para mejorar lo que se ha dado en llamar Pensamiento Gráfico. El trabajo se divide en dos partes complementarias. En la primera, se formula un marco teórico en torno a los conceptos de Pensamiento Crítico y Pensamiento Gráfico, para proponer una metodología de enseñanza de la ingeniería que relacione ambos conceptos. En la segunda, se aplican dichos aspectos al estudio de una herramienta esencial dentro del proceso de diseño, el diagrama, y a la manera de proyectar en la contemporaneidad. - Critical thinking is defined by PASSMORE (1967) as a process that is both reflexive and imaginative, essencial aspects of the design process. This paper focuses on the use of Critical Thinking to improve what we call Graphic Thinking. The content is two fold. The first part establishes a theoretical framework around the concepts of Critical Thinking and Graphic Thinking, in order to propose a methodology for engineering education through the combination of both concepts. The second one deals with those concepts, which are applied to one of the basic tools within the design process, the diagram, analysing through it the contemporary way of designing

Engineered Male Sterility by Early Anther Ablation Using the Pea Anther-Specific Promoter PsEND1

[EN] Genetic engineered male sterility has different applications, ranging from hybrid seed production to bioconfinement of transgenes in genetic modified crops. The impact of this technology is currently patent in a wide range of crops, including legumes, which has helped to deal with the challenges of global food security. Production of engineered male sterile plants by expression of a ribonuclease gene under the control of an anther- or pollen-specific promoter has proven to be an efficient way to generate pollen-free elite cultivars. In the last years, we have been studying the genetic control of flower development in legumes and several genes that are specifically expressed in a determinate floral organ were identified. Pisum sativum ENDOTHECIUM 1 (PsEND1) is a pea anther-specific gene displaying very early expression in the anther primordium cells. This expression pattern has been assessed in both model plants and crops (tomato, tobacco, oilseed rape, rice, wheat) using genetic constructs carrying the PsEND1 promoter fused to the uidA reporter gene. This promoter fused to the barnase gene produces full anther ablation at early developmental stages, preventing the production of mature pollen grains in all plant species tested. Additional effects produced by the early anther ablation in the PsEND1::barnase-barstar plants, with interesting biotechnological applications, have also been described, such as redirection of resources to increase vegetative growth, reduction of the need for deadheading to extend the flowering period, or elimination of pollen allergens in ornamental plants (Kalanchoe, Pelargonium). Moreover, early anther ablation in transgenic PsEND1::barnase-barstar tomato plants promotes the developing of the ovaries into parthenocarpic fruits due to the absence of signals generated during the fertilization process and can be considered an efficient tool to promote fruit set and to produce seedless fruits. In legumes, the production of new hybrid cultivars will contribute to enhance yield and productivity by exploiting the hybrid vigor generated. The PsEND1::barnase-barstar construct could be also useful to generate parental lines in hybrid breeding approaches to produce new cultivars in different legume species.This work was funded by grants BIO2000-0940, BIO2000-0940, BIO2003-01171, BIO2006-09374, PTR95-0979-OP-03-01, RYC-2007-00627, AGL2009-13388-C03-01, AGL2009-07617, BIO2009-08134, AGL2015-64991-C3-3-R, and BIO2016-75485-R from the Spanish Ministry of Economy and Competitiveness (MINECO).Roque Mesa, EM.; Gómez Mena, MC.; Hamza, R.; Beltran Porter, JP.; Cañas Clemente, LA. (2019). Engineered Male Sterility by Early Anther Ablation Using the Pea Anther-Specific Promoter PsEND1. Frontiers in Plant Science. 10:1-9. https://doi.org/10.3389/fpls.2019.00819S1910Beals, T. P., & Goldberg, R. B. (1997). A novel cell ablation strategy blocks tobacco anther dehiscence. The Plant Cell, 9(9), 1527-1545. doi:10.1105/tpc.9.9.1527Canales, C., Bhatt, A. M., Scott, R., & Dickinson, H. (2002). EXS, a Putative LRR Receptor Kinase, Regulates Male Germline Cell Number and Tapetal Identity and Promotes Seed Development in Arabidopsis. Current Biology, 12(20), 1718-1727. doi:10.1016/s0960-9822(02)01151-xCa�as, L. A., Essid, R., G�mez, M. D., & Beltr�n, J. (2002). Monoclonal antibodies as developmental markers to characterize pea floral homeotic transformations. Sexual Plant Reproduction, 15(3), 141-152. doi:10.1007/s00497-002-0148-2Christensen, B., Sriskandarajah, S., Serek, M., & Müller, R. (2008). Transformation of Kalanchoe blossfeldiana with rol-genes is useful in molecular breeding towards compact growth. Plant Cell Reports, 27(9), 1485-1495. doi:10.1007/s00299-008-0575-0Block, M. D., Debrouwer, D., & Moens, T. (1997). The development of a nuclear male sterility system in wheat. Expression of the barnase gene under the control of tapetum specific promoters. Theoretical and Applied Genetics, 95(1-2), 125-131. doi:10.1007/s001220050540Denis, M., Delourme, R., Gourret, J. P., Mariani, C., & Renard, M. (1993). Expression of Engineered Nuclear Male Sterility in Brassica napus (Genetics, Morphology, Cytology, and Sensitivity to Temperature). Plant Physiology, 101(4), 1295-1304. doi:10.1104/pp.101.4.1295Dutt, M., Dhekney, S. A., Soriano, L., Kandel, R., & Grosser, J. W. (2014). Temporal and spatial control of gene expression in horticultural crops. Horticulture Research, 1(1). doi:10.1038/hortres.2014.47García-Sogo, B., Pineda, B., Castelblanque, L., Antón, T., Medina, M., Roque, E., … Cañas, L. A. (2009). Efficient transformation of Kalanchoe blossfeldiana and production of male-sterile plants by engineered anther ablation. Plant Cell Reports, 29(1), 61-77. doi:10.1007/s00299-009-0798-8García-Sogo, B., Pineda, B., Roque, E., Antón, T., Atarés, A., Borja, M., … Cañas, L. (2012). Production of engineered long-life and male sterile Pelargonium plants. BMC Plant Biology, 12(1), 156. doi:10.1186/1471-2229-12-156Gardner, N., Felsheim, R., & Smith, A. G. (2009). Production of male- and female-sterile plants through reproductive tissue ablation. Journal of Plant Physiology, 166(8), 871-881. doi:10.1016/j.jplph.2008.10.002Goldberg, A., Confino-Cohen, R., & Waisel, Y. (1998). Allergic responses to pollen of ornamental plants: High incidence in the general atopic population and especially among flower growers. Journal of Allergy and Clinical Immunology, 102(2), 210-214. doi:10.1016/s0091-6749(98)70088-0G�mez, M. D., Beltr�n, J.-P., & Ca�as, L. A. (2004). The pea END1 promoter drives anther-specific gene expression in different plant species. Planta, 219(6), 967-981. doi:10.1007/s00425-004-1300-zHarris, N., & Croy, R. R. D. (1985). The major albumin protein from pea (Pisum sativum L.). Planta, 165(4), 522-526. doi:10.1007/bf00398098Hartley, R. W. (1988). Barnase and barstar. Journal of Molecular Biology, 202(4), 913-915. doi:10.1016/0022-2836(88)90568-2Higgins, T. J. V., Beach, L. R., Spencer, D., Chandler, P. M., Randall, P. J., Blagrove, R. J., … Guthrie, R. E. (1987). cDNA and protein sequence of a major pea seed albumin (PA 2 : Mr?26 000). Plant Molecular Biology, 8(1), 37-45. doi:10.1007/bf00016432Hird, D. L., Worrall, D., Hodge, R., Smartt, S., Paul, W., & Scott, R. (1993). The anther-specific protein encoded by the Brassica napus and Arabidopsis thaliana A6 gene displays similarity to beta-1,3-glucanases. The Plant Journal, 4(6), 1023-1033. doi:10.1046/j.1365-313x.1993.04061023.xHuang, J., Smith, A. R., Zhang, T., & Zhao, D. (2016). Creating Completely Both Male and Female Sterile Plants by Specifically Ablating Microspore and Megaspore Mother Cells. Frontiers in Plant Science, 7. doi:10.3389/fpls.2016.00030Skinner, J. S., Meilan, R., Brunner, A. M., & Strauss, S. H. (2000). Options for Genetic Engineering of Floral Sterility in Forest Trees. Forestry Sciences, 135-153. doi:10.1007/978-94-017-2311-4_5Jenne, D. (1991). Homology of placental protein 11 and pea seed albumin 2 with vitronectin. Biochemical and Biophysical Research Communications, 176(3), 1000-1006. doi:10.1016/0006-291x(91)90381-gKoltunow, A. M., Truettner, J., Cox, K. H., Wallroth, M., & Goldberg, R. B. (1990). Different Temporal and Spatial Gene Expression Patterns Occur during Anther Development. The Plant Cell, 1201-1224. doi:10.1105/tpc.2.12.1201Lee, Y.-H., Chung, K.-H., Kim, H.-U., Jin, Y.-M., Kim, H.-I., & Park, B.-S. (2003). Induction of male sterile cabbage using a tapetum-specific promoter from Brassica campestris L. ssp. pekinensis. Plant Cell Reports, 22(4), 268-273. doi:10.1007/s00299-003-0688-4Ma, H. (2005). MOLECULAR GENETIC ANALYSES OF MICROSPOROGENESIS AND MICROGAMETOGENESIS IN FLOWERING PLANTS. Annual Review of Plant Biology, 56(1), 393-434. doi:10.1146/annurev.arplant.55.031903.141717Mariani, C., Beuckeleer, M. D., Truettner, J., Leemans, J., & Goldberg, R. B. (1990). Induction of male sterility in plants by a chimaeric ribonuclease gene. Nature, 347(6295), 737-741. doi:10.1038/347737a0Mariani, C., Gossele, V., Beuckeleer, M. D., Block, M. D., Goldberg, R. B., Greef, W. D., & Leemans, J. (1992). A chimaeric ribonuclease-inhibitor gene restores fertility to male sterile plants. Nature, 357(6377), 384-387. doi:10.1038/357384a0Medina, M., Roque, E., Pineda, B., Cañas, L., Rodriguez-Concepción, M., Beltrán, J. P., & Gómez-Mena, C. (2013). Early anther ablation triggers parthenocarpic fruit development in tomato. Plant Biotechnology Journal, 11(6), 770-779. doi:10.1111/pbi.12069Millwood, R. J., Moon, H. S., Poovaiah, C. R., Muthukumar, B., Rice, J. H., Abercrombie, J. M., … Stewart, C. N. (2015). Engineered selective plant male sterility through pollen-specific expression of theEcoRI restriction endonuclease. Plant Biotechnology Journal, 14(5), 1281-1290. doi:10.1111/pbi.12493Mishra, S., & Kumari, V. (2018). A Review on Male Sterility-Concepts and Utilization in Vegetable Crops. International Journal of Current Microbiology and Applied Sciences, 7(2), 3016-3034. doi:10.20546/ijcmas.2018.702.367Nonomura, K.-I., Miyoshi, K., Eiguchi, M., Suzuki, T., Miyao, A., Hirochika, H., & Kurata, N. (2003). The MSP1 Gene Is Necessary to Restrict the Number of Cells Entering into Male and Female Sporogenesis and to Initiate Anther Wall Formation in Rice. The Plant Cell, 15(8), 1728-1739. doi:10.1105/tpc.012401Paul, W., Hodge, R., Smartt, S., Draper, J., & Scott, R. (1992). The isolation and characterisation of the tapetum-specific Arabidopsis thaliana A9 gene. Plant Molecular Biology, 19(4), 611-622. doi:10.1007/bf00026787Pedroche, J., Yust, M. M., Lqari, H., Megías, C., Girón-Calle, J., Alaiz, M., … Vioque, J. (2005). Chickpea pa2 albumin binds hemin. Plant Science, 168(4), 1109-1114. doi:10.1016/j.plantsci.2004.12.011Pistón, F., García, C., de la Viña, G., Beltran, J. P., Cañas, L. A., & Barro, F. (2007). The pea PsEND1 promoter drives the expression of GUS in transgenic wheat at the binucleate microspore stage and during pollen tube development. Molecular Breeding, 21(3), 401-405. doi:10.1007/s11032-007-9133-7Roberts, M., Boyes, E., & Scott, R. (1995). An investigation of the role of the anther tapetum during microspore development using genetic cell ablation. Sexual Plant Reproduction, 8(5). doi:10.1007/bf00229387Rojas-Gracia, P., Roque, E., Medina, M., López-Martín, M. J., Cañas, L. A., Beltrán, J. P., & Gómez-Mena, C. (2019). The DOF Transcription Factor SlDOF10 Regulates Vascular Tissue Formation During Ovary Development in Tomato. Frontiers in Plant Science, 10. doi:10.3389/fpls.2019.00216Rojas-Gracia, P., Roque, E., Medina, M., Rochina, M., Hamza, R., Angarita-Díaz, M. P., … Gómez-Mena, C. (2017). The parthenocarpichydramutant reveals a new function for aSPOROCYTELESS-like gene in the control of fruit set in tomato. New Phytologist, 214(3), 1198-1212. doi:10.1111/nph.14433Roque, E., Gómez, M. D., Ellul, P., Wallbraun, M., Madueño, F., Beltrán, J.-P., & Cañas, L. A. (2006). The PsEND1 promoter: a novel tool to produce genetically engineered male-sterile plants by early anther ablation. Plant Cell Reports, 26(3), 313-325. doi:10.1007/s00299-006-0237-zRosellini, D., Pezzotti, M., & Veronesi, F. (2001). Euphytica, 118(3), 313-319. doi:10.1023/a:1017568201732Sanikhani, M., Mibus, H., Stummann, B. M., & Serek, M. (2007). Kalanchoe blossfeldiana plants expressing the Arabidopsis etr1-1 allele show reduced ethylene sensitivity. Plant Cell Reports, 27(4), 729-737. doi:10.1007/s00299-007-0493-6Saxena, K. B., & Hingane, A. J. (2015). Male Sterility Systems in Major Field Crops and Their Potential Role in Crop Improvement. Plant Biology and Biotechnology, 639-656. doi:10.1007/978-81-322-2286-6_25Schiefthaler, U., Balasubramanian, S., Sieber, P., Chevalier, D., Wisman, E., & Schneitz, K. (1999). Molecular analysis of NOZZLE, a gene involved in pattern formation and early sporogenesis during sex organ development in Arabidopsis thaliana. Proceedings of the National Academy of Sciences, 96(20), 11664-11669. doi:10.1073/pnas.96.20.11664Shull, G. H. (1908). The Composition of a Field of Maize. Journal of Heredity, os-4(1), 296-301. doi:10.1093/jhered/os-4.1.296Thirukkumaran, G., Khan, R. S., Chin, D. P., Nakamura, I., & Mii, M. (2009). Isopentenyl transferase gene expression offers the positive selection of marker-free transgenic plant of Kalanchoe blossfeldiana. Plant Cell, Tissue and Organ Culture (PCTOC), 97(3), 237-242. doi:10.1007/s11240-009-9519-9Topp, S. H., Rasmussen, S. K., & Sander, L. (2008). Alcohol induced silencing of gibberellin 20-oxidases in Kalanchoe blossfeldiana. Plant Cell, Tissue and Organ Culture, 93(3), 241-248. doi:10.1007/s11240-008-9368-yVigeolas, H., Chinoy, C., Zuther, E., Blessington, B., Geigenberger, P., & Domoney, C. (2007). Combined Metabolomic and Genetic Approaches Reveal a Link between the Polyamine Pathway and Albumin 2 in Developing Pea Seeds. Plant Physiology, 146(1), 74-82. doi:10.1104/pp.107.111369Yang, W.-C., Ye, D., Xu, J., & Sundaresan, V. (1999). The SPOROCYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein. Genes & Development, 13(16), 2108-2117. doi:10.1101/gad.13.16.2108Yue, Y., Yin, C., Guo, R., Peng, H., Yang, Z., Liu, G., … Hu, H. (2017). An anther-specific gene PhGRP is regulated by PhMYC2 and causes male sterility when overexpressed in petunia anthers. Plant Cell Reports, 36(9), 1401-1415. doi:10.1007/s00299-017-2163-7Zhan, X., Wu, H., & Cheung, A. (1996). Nuclear male sterility induced by pollen-specific expression of a ribonuclease. Sexual Plant Reproduction, 9(1). doi:10.1007/bf0023036

Efficient evaluation of a gene containment system for poplar through early flowering induction

[EN] Key message The early flowering system HSP::AtFT allowed a fast evaluation of a gene containment system based on the construct PsEND1::barnase-barstar for poplar. Transgenic lines showed disturbed pollen development and sterility. Vertical gene transfer through pollen flow from transgenic or non-native plant species into their crossable natural relatives is a major concern. Gene containment approaches have been proposed to reduce or even avoid gene flow among tree species. However, evaluation of genetic containment strategies for trees is very difficult due to the long-generation times. Early flowering induction would allow faster evaluation of genetic containment in this case. Although no reliable methods were available for the induction of fertile flowers in poplar, recently, a new early flowering approach was developed. In this study, early flowering poplar lines containing the gene construct PsEND1::barnase-barstar were obtained. The PsEND1 promoter was chosen due to its early expression pattern, its versality and efficiency for generation of male-sterile plants fused to the barnase gene. RT-PCRs confirmed barnase gene activity in flowers, and pollen development was disturbed, leading to sterile flowers. The system developed in this study represents a valuable tool for gene containment studies in forest tree species.Open Access funding provided by Projekt DEAL. This work was funded with a scholarship by the Deutscher Akademischer Austauschdienst (DAAD). We thank S. Bein, D. Ebbinghaus, and A. Worm for helpful technical assistance in the laboratory, and the greenhouse staff (M. Hunger, G. Wiemann, R. Ebbinghaus, and M. Spauszus) for plant cultivation.Briones, MV.; Hoenicka, H.; Cañas Clemente, LA.; Beltran Porter, JP.; Hanelt, D.; Sharry, S.; Fladung, M. (2020). Efficient evaluation of a gene containment system for poplar through early flowering induction. Plant Cell Reports. 39(5):577-587. https://doi.org/10.1007/s00299-020-02515-1S577587395Al-Ahmad H (2018) Biotechnology for bioenergy dedicated trees: meeting future energy demands. Z Naturforsch 73:15–32. https://doi.org/10.1515/znc-2016-0185Beltrán JP, Roque E, Medina M, Madueño F, Gómez MD, Cañas LA (2007) Androesterilidad inducida mediante ingeniería genética. Fundamentos y aplicaciones biotecnológicas. An R Acad Nac Farm 73:1237–1264Böhlenius H, Huang T, Charbonnel-Campaa L, Brunner AM, Jansson S, Strauss SH, Nilsson O (2006) CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science 312:1040–1043Brunner AM, Varkonyi-Gasic E, Jones RC (2017) Phase change and phenology in trees. In: Groover A, Cronk Q (eds) Comparative and evolutionary genomics of Angiosperm trees. Springer, Cham, pp 227–274. https://doi.org/10.1007/7397_2016_30Chang YY, Wang AY, Cronan JE (1993) Molecular cloning, DNA sequencing, and biochemical analyses of Escherichia coli glyoxylate carboligase. An enzyme of the acetohydroxy acid synthase-pyruvate oxidase family. J Biol Chem 268:3911–3919Dumolin S, Demesure B, Petit RJ (1995) Inheritance of chloroplast and mitochondrial genomes in pedunculate oak investigated with an efficient PCR method. Theor Appl Genet 91:1253–1256Elorriaga E, Meilan R, Ma C, Skinner JS, Etherington E, Brunner AM, Strauss SH (2014) A tapetal ablation transgene induces stable male sterility and slows field growth in Populus. Tree Gen Gen 10:1583–1593Elorriaga E, Klocko AL, Ma C, Strauss SH (2018) Variation in mutation spectra among CRISPR/Cas9 mutagenized poplars. Front Plant Sci 9:594. https://doi.org/10.3389/fpls.2018.00594Fladung M, Ahuja M (1995) Sandwich method for non-radioactive hybridisations. Biotechniques 18:800–802Fladung M, Muhs HJ, Ahuja MR (1996) Morphological changes observed in transgenic Populus carrying the rolC gene from Agrobacterium rhizogenes. Silv Genet 45:349–354Fladung M, Kumar S, Ahuja MR (1997) Genetic transformation of Populus genotypes with different chimaeric gene constructs: transformation efficiency and molecular analysis. Transgenic Res 6:111–121Fritsche S, Klocko AL, Boron A, Brunner AM, Thorlby G (2018) Strategies for engineering reproductive sterility in plantation forests. Front Plant Sci 9:1671. https://doi.org/10.3389/fpls.2018.01671García-Sogo B, Pineda B, Castelblanque L, Antón T, Medina M, Roque E, Torresi C, Beltrán JP, Moreno V, Cañas LA (2010) Efficient transformation of Kalanchoe blossfeldiana and production of male-sterile plants by engineered anther ablation. Plant Cell Rep 29:61–77García-Sogo B, Pineda B, Roque E, Atarés A, Antón T, Beltrán JP, Moreno V, Cañas LA (2012) Production of engineered long-life and male sterile Pelargonium plants. BMC Plant Biol 12:156–162Gardner N, Felsheim R, Smith AG (2009) Production of male- and female-sterile plants through reproductive tissue ablation. J Plant Physiol 166(8):871–881Gómez MD, Beltrán JP, Cañas LA (2004) The pea END1 promoter drives anther-specific gene expression in different plant species. Planta 219:967–981Hartley RW (1988) Barnase and barstar expression of its cloned inhibitor permits expression of a cloned ribonuclease. J Mol Biol 202:913–915Hoenicka H, Fladung M (2006) Biosafety in Populus spp. and other forest trees: from non-native species to taxa derived from traditional breeding and genetic engineering. Trees 20:131–144Hoenicka H, Nowitzki O, Debener T, Fladung M (2006) Faster evaluation of sterility strategies in transgenic trees. Silv Genet 55:285–291Hoenicka H, Lehnhardt D, Polak O, Fladung M (2012) Early flowering and genetic containment studies in transgenic poplar. iForest 5:138–146Hoenicka H, Lehnhardt D, Nilsson O, Hanelt D, Fladung M (2014) Successful crossings with early flowering transgenic poplar: interspecific crossings, but not transgenesis, promoted aberrant phenotypes in offspring. Plant Biotechnol J 12:1066–1074Hoenicka H, Lehnhardt D, Briones V, Nilsson O, Fladung M (2016) Low temperatures are required to induce the development of fertile flowers in transgenic male and female early flowering poplar (Populus tremula L.). Tree Physiol 36:667–677. https://doi.org/10.1093/treephys/tpw015Hsu CY, Liu YX, Luthe DS, Yuceer C (2006) Poplar FT2 shortens the juvenile phase and promotes seasonal flowering. Plant Cell 18:1846–1861Klocko AL, Brunner AM, Huang J, Meilan R, Lu H, Strauss SH (2016) Containment of transgenic trees by suppression of LEAFY. Nat Biotechnol 34:918–922. https://doi.org/10.1038/nbt.3636Klocko AL, Lu H, Magnuson A, Brunner AM, Ma C, Strauss SH (2018) Phenotypic expression and stability in a large-scale field study of genetically engineered poplars containing sexual containment transgenes. Front Bioeng Biotechnol 6:100. https://doi.org/10.3389/fbioe.2018.00100Lännenpää M, Hassinen M, Ranki A, Hölttä-Vuori M, Lemmetyinen J, Keinonen K, Sopanen T (2005) Prevention of flower development in birch and other plants using a BpFULL1:BARNASE construct. Plant Cell Rep 24(2):69–78Lloyd G, McCown B (1980) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Comb Proc Int Plant Prop Soc 30:421–427Lu S, Yi S, Zhang J, Liu L, Bao M, Liu G (2019) Isolation and functional characterization of the promoter of SEPALLATA3 gene in London plane and its application in genetic engineering of sterility. Plant Cell Tiss Org Cult 136:109–121Mariani C, Gossele V, De Beuckeleer M, De Block M, Goldberg RB, De Greef W, Leemans J (1992) A chimaeric ribonuclease inhibitor gene restores fertility to male sterile plants. Nature 357:384–387Medina M, Roque E, Pineda B, Cañas LA, Rodríguez-Concepción M, Beltrán JP, Gómez-Mena C (2013) Early anther ablation triggers parthenocarpic fruit development in tomato plants. Plant Biotechnol J 11:770–779Meilan R, Brunner A, Skinner J, Strauss SH (2001) Modification of flowering in transgenic trees. In: Komamine A, Morohoshi N (eds) Molecular breeding of woody plants. Elsevier Science BV, Amsterdam, pp 247–256Paddon CJ, Hartley RW (1986) Cloning, sequencing and transcription of an inactivated copy of Bacillus amyloliquefaciens extracellular ribonuclease (barnase). Gene 40(2–3):231–239. https://doi.org/10.1016/0378-1119(85)90045-9Parmentier-Line CM, Coleman GD (2016) Constitutive expression of the poplar FD-like basic leucine zipper transcription factor alters growth and bud development. Plant Biotechnol J 14:260–270Pistón F, García C, de la Viña G, Beltrán JP, Cañas LA, Barro F (2008) The pea PsEND1 promoter drives the expression of GUS in transgenic wheat at the binucleate microspores stage and during pollen tube development. Mol Breed 21:401–405Rojas-Gracia P, Roque E, Medina M, Rochina M, Hamza R, Angarita-Díaz P, Moremo V, Pérez-Martín F, Lozano R, Cañas LA, Beltrán JP, Gómez-Mena C (2017) The parthenocarpic hydra mutant reveals a new function for a SPOROCYTELESS-like gene in the control of fruit set in tomato. New Phytol 214:1198–1212Rojas-Gracia P, Roque E, Medina M, López-Martín MJ, Cañas LA, Beltrán JP, Gómez-Mena C (2019) The DOF transcription factor SlDOF10 regulates vascular tissue formation during ovary development in tomato. Front Plant Sci 10:216Roque E, Gómez MD, Ellul P, Wallbraun M, Madueño F, Beltrán JP, Cañas LA (2007) The PsEND1 promoter: a novel tool to produce genetically engineered male-sterile plants by early anther ablation. Plant Cell Rep 26:313–325Roque E, Gómez-Mena C, Hamza R, Beltrán JP, Cañas LA (2019) Engineered male sterility by early anther ablation using the Pea anther-specific promoter PsEND1. Front Plant Sci 10:819Rottmann WH, Meilan R, Sheppard LA, Brunner AM, Skinner JS, Cheng S, Jouanin L, Pilate G, Strauss SH (2000) Diverse effects of overexpression of LEAFY and PTLF, a poplar (Populus) homolog of LEAFY/FLORICAULA, in transgenic poplar and Arabidopsis. Plant J 22:235–245Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386Sanders PM, Bui AQ, Weterings K, McIntire KN, Hsu YC, Lee PY, Truong MT, Beals TP, Goldberg RB (1999) Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sex Plant Reprod 11:297–322Shen L, Chen Y, Su X, Zhang S, Pan H, Huang M (2012) Two FT orthologs from Populus simonii Carrière induce early flowering in Arabidopsis and poplar trees. Plant Cell Tissue Org Cult 108:371–379Skinner JS, Meilan R, Ma C, Strauss SH (2003) The Populus PTD promoter imparts floral-predominant expression and enables high levels of floral–organablation in Populus, Nicotiana, and Arabidopsis. Mol Breed 12:119–132Smith EF, Townsend CO (1907) A plant tumor of bacterial origin. Science 25:671473Tränkner C, Lehmann S, Hoenicka H, Hanke M-V, Fladung M, Lehnhardt D, Dunemann F, Gau A, Schlangen K, Malnoy M, Flachowsky H (2010) Over-expression of an FT-homologous gene of apple induces early flowering in annual and perennial plants. Planta 232:1309–1324Weigel D, Nilsson O (1995) A developmental switch sufficient for flower initiation in diverse plants. Nature 377:495–500Wildholm JM (1972) The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol 47:189–194WWF (2015) WWF living forests report: chapter 5: saving forests at risk. Gland: WWF- WorldWide Fund for Nature. https://www.worldwildlife.org/publications/living-forests-report-chapter-5-saving-forests-at-risk. Accessed Jan 2020Xiaoming J, Huanling Z (2014) FT gene with a CaMV35S promoter to control early flowering of transgenic poplar. J Zhejiang A&F 31:404–409Xiaoming J, Hualing Z, Jungfeng F (2011) System optimization of precociously flowering of poplar induced by FT gene controlled by a heat shock promoter. Sci Silvae Sin 47:37–43Zhang H, Harry DE, Yuceer C, Hsu CY, Vikram V, Shevchenko O, Etherington E, Strauss SH (2010) Precocious flowering in trees: the FLOWERING LOCUS T gene as a research and breeding tool in Populus. J Exp Bot 61:2549–256

PsPMEP, a pollen specific pectin methylesterase of pea (Pisum sativum L.)

[EN] Pectin methylesterases (PMEs) are a family of enzymes involved in plant reproductive processes such as pollen development and pollen tube growth. We have isolated and characterized PsPMEP, a pea (Pisum sativum L.) pollen-specific gene that encodes a protein with homology to PMEs. Sequence analysis showed that PsPMEP belongs to group 2 PMEs, which are characterized by the presence of a processable amino-terminal PME inhibitor domain followed by the catalytic PME domain. Moreover, PsPMEP contains several motifs highly conserved among PMEs with the essential amino acid residues involved in enzyme substrate binding and catalysis. Northern blot and in situ hybridization analyses showed that PsPMEP is expressed in pollen grains from 4 days before anthesis till anther dehiscence and in pollinated carpels. In the PsPMEP promoter region, we have identified several conserved cis-regulatory elements that have been associated with gene pollen-specific expression. Expression analysis of PsPMEP promoter fused to the uidA reporter gene in Arabidopsis thaliana plants showed a similar expression pattern when compared with pea, indicating that this promoter is also functional in a non-leguminous plant. GUS expression was detected in mature pollen grains, during pollen germination, during pollen tube elongation along the transmitting tract, and when the pollen tube reaches the embryo sac in the ovule.This work was funded by grants BIO2006-09374 and BIO2009-08134 from the Spanish Ministry of Science and Innovation (MICINN). The collaboration and assistance of Julia Marin-Navarro in the catalytic activity assays of PsPMEP in yeast and Rafael Martinez-Pardo in the greenhouse is gratefully acknowledged. We would like to thank the HAPRECI consortium (COST Action FA0903) to bring us the opportunity to collaborate with other European research groups working in the field of Plant Reproduction and to select our manuscript to be published in this special issue.Gómez Jiménez, MD.; Renau Morata, B.; Roque Mesa, EM.; Polaina, J.; Beltran Porter, JP.; Cañas Clemente, LA. (2013). PsPMEP, a pollen specific pectin methylesterase of pea (Pisum sativum L.). Plant Reproduction. 26(3):245-254. https://doi.org/10.1007/s00497-013-0220-0S245254263Bate N, Twell D (1998) Functional architecture of a late pollen promoter: pollen specific transcription is developmentally regulated by multiple stage-specific and co-dependent activator elements. Plant Mol Biol 37:859–869Bechtold N, Ellis J, Pelletier G (1993) In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci 316:1194–1199Bosch M, Hepler PK (2005) Pectin methylesterases and pectin dynamics in pollen tubes. Plant Cell 17:3219–3226Bosch M, Hepler PK (2006) Silencing of the tobacco pollen pectin methylesterase NtPPME1 results in retarded in vivo pollen tube growth. Planta 233:736–745Camardella L (2000) Kiwi protein inhibitor of pectin methylesterase–amino-acid sequence and structural importance of two disulfide bridges. Eur J Biochem 267:4561–4565Carpenter JL, Ploense SE, Snustad DP, Silflow CD (1992) Preferential expression of an alpha-tubulin gene of Arabidopsis in pollen. Plant Cell 4:557–571Combet C, Blanchet C, Geourjon C, Deléage G (2000) NPS@: network protein sequence analysis. Trend Biochem Sci 25:147–150De Almeida Engler J, Van Montagu M, Engler G (1998) Whole-mount in situ hybridization in plants. Methods Mol Biol (Clifton, NJ) 82:373–384De Caroli M, Lenucci MS, Di Sansebastiano GP, Dalessandro G, De Lorenzo G, Piro G (2011) Protein trafficking to the cell wall occurs through mechanisms distinguishable from default sorting in tobacco. Plant J 65(2):295–308Francis KE, Lam SY, Copenhaver GP (2006) Separation of Arabidopsis pollen tetrads is regulated by QUARTET1, a pectin methylesterase gene. Plant Physiol 142:1004–1013Futamura N, Mori H, Kouchi H, Shinohara K (2000) Male flower-specific expression of genes for polygalacturonase, pectin methylesterase and β-1,3-glucanase in a dioecious willow (Salix gilgliana Seemen). Plant Cell Physiol 41:16–26García-Sogo B, Pineda B, Castelblanque L, Antón T, Medina M, Roque E, Torresi C, Beltrán JP, Moreno V, Cañas LA (2010) Efficient transformation of Kalanchoe blossfeldiana and production of male sterile plants by engineered anther ablation. Plant Cell Rep 29:61–77García-Sogo B, Pineda B, Roque E, Antón T, Atarés A, Borja M, Beltrán JP, Moreno V, Cañas LA (2012) Production of engineered long-life and male sterile Pelargonium plants. BMC Plant Biol 12:156–162Gómez MD, Beltrán JP, Cañas LA (2004) The pea END1 promoter drives anther-specific gene expression in different plant species. Planta 219:967–981Gummadi SN, Manoj N, Kumar DS (2007) Structure and biochemical properties of pectinases. In: Polaina J, MacCabe AP (eds) Industrial enzymes: structure, function and applications. Springer, Berlin, pp 99–115. ISBN 9781402053764Gurgu L, Lafraya A, Polaina J, Marín-Navarro J (2011) Fermentation of cellobiose to ethanol by industrial Saccharomyces strains carrying the β-glucosidase gene (BGL1) from Saccharomycopsis fibuligera. Bioresour Technol 102:5229–5236Hamilton DA, Schwarz YH, Mascarenhas JP (1998) A monocot pollen-specific promoter contains separable pollen-specific and quantitative elements. Plant Mol Biol 38:663–669Hewitt EJ (1966) Hoagland No. 1 solution with oligoelements. In: Sand and water culture methods used in the study of plant nutrition, 2nd edn, Technical Communication No. 22. Commonwealth Agricultural Bureaux, Farnham Royal, EnglandHothorn M, Wolf S, Aloy P, Greiner S, Scheffzek K (2004) Structural insights into the target specificity of plant invertase and pectin methylesterase inhibitory proteins. Plant Cell 16:3437–3447Jiang LX, Yang SL, Xie LF, Puah CS, Zhang XQ, Yang WC, Sundaresan V, Ye D (2005) VANGUARD1 encodes a pectin methylesterase that enhances pollen tube growth in the Arabidopsis style and transmitting tract. Plant Cell 17:584–596Johansson K, El-Ahmad M, Friemann R, Jörnvall H, Markovic O, Eklund H (2002) Crystal structure of plant pectin methylesterase. FEBS Lett 514:243–249Jolie RP, Duvetter T, Van Loey AM, Hendrickx ME (2010) Pectin methylesterase and its proteinaceous inhibitor: a review. Carbohydr Res 345:2583–2595Kononowicz AK, Nelson DE, Singh NK, Hasegawa PM, Bressan RA (1992) Regulation of the osmotin gene promoter. Plant Cell 4:513–524Li YQ, Mareck A, Faleric C, Moscatelli A, Liu Q, Cresti M (2002) Detection and localization of pectin methylesterase isoforms in pollen tubes of Nicotiana tabacum L. Planta 214:734–740Marín-Navarro J, Gurgu L, Alamar S, Polaina J (2011) Structural and functional analysis of hybrid enzymes generated by domain shuffling between Saccharomyces cerevisiae (var. diastaticus) Sta1 glucoamylase and Saccharomycopsis fibuligera Bgl1 β-glucosidase. Appl Microbiol Biotechnol 89:121–130Markovic O, Janecek S (2004) Pectin methylesterases: sequence-structural features and phylogenetic relationships. Carbohydr Res 339:2281–2295Micheli F (2001) Pectin methylesterases: cell wall enzymes with important roles in plant physiology. Trends Plant Sci 6:414–419Mitsuda N, Takeyasu K, Sato MH (2001) Pollen-specific regulation of vacuolar H+-PPase expression by multiple cis-acting elements. Plant Mol Biol 46:185–192Okada T, Zhang Z, Russell RD, Toriyama K (1999) Localization of Bra r 1, a Ca2+-binding protein of Brassica rapa, in anthers and pollen tubes. Plant Cell Physiol 40:1243–1252Palin R, Geitmann A (2012) The role of pectin in plant morphogenesis. Bio Syst 109:397–402Pelloux J, Rustérucci C, Mellerowicz EJ (2007) New insights into pectin methylesterase structure and function. Trends Plant Sci 12:267–277Pistón F, García C, de la Viña G, Beltrán JP, Cañas LA, Barro F (2008) The pea PsEND1 promoter drives the expression of GUS in transgenic wheat at the binucleate microspores stage and during pollen tube development. Mol Breed 21:401–405Qiu X, Erickson L (1995) A pollen-specific cDNA (P65, accession no. U28148) encoding a putative pectin esterase in alfalfa (PGR95-094). Plant Physiol 109:1127Rodríguez-Llorente ID, Pérez-Hormaeche J, El Mounadi K, Dary M, Caviedes MA, Cosson V, Kondorosi A, Ratet P, Palomares AJ (2004) From pollen tubes to infection threads: recruitment of Medicago floral pectic genes for symbiosis. Plant J 39:587–598Roque E, Gómez MD, Ellull P, Wallbraun M, Madueño F, Beltrán JP, Cañas LA (2007) The PsEND1 promoter: a novel tool to produce genetically engineered male-sterile plants by early anther ablation. Plant Cell Rep 26:313–325Sambrook J, Fritsch EF, Maniatis T (1989) Hybridization of radiolabeled probes to immobilized nucleic acids. In: Ford N, Nolan C, Ferguson M (eds) Molecular cloning: a laboratory manual, 2nd edn, chap 9. Cold Spring Harbor Laboratory Press, New York, USA, pp 47–48Scognamiglio MA, Ciardiello MA, Tamburrini M, Carratore V, Rausch T, Camardella L (2003) The plant invertase inhibitor shares structural properties and disulfide bridges arrangement with the pectin methylesterase inhibitor. J Protein Chem 22:363–369Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599Tang W, Perry SE (2003) Binding site selection for the plant MADS domain protein AGL15: an in vitro and in vivo study. J Biol Chem 278:28154–28159Tian G-W, Chen M-H, Zaltsman A, Citovsky V (2006) Pollen-specific pectin methylesterase involved in pollen tube growth. Dev Biol 294:83–91Twell D, Klein TM, Fromm ME, McCormick S (1989) Transient expression of chimeric genes delivered into pollen by microprojectile bombardment. Plant Physiol 91:1270–1274Twell D, Yamaguchi J, McCormick S (1990) Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis. Development 109:705–713Twell D, Yamaguchi J, Wing RA, Ushiba J, McCormick S (1991) Promoter analysis of genes that are co-ordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements. Genes Dev 5:496–507Wakeley PR, Rogers HJ, Rozychk M, Greenland AJ, Hussey PJ (1998) A maize pectin methylesterase-gene, ZmC5, specifically expressed in pollen. Plant Mol Biol 37:187–192Wen F, Zhu Y, Hawes MC (1999) Effect of pectin methylesterase gene expression on pea root development. Plant Cell 11:1129–1140Weterings K, Schrauwen J, Wullems G, Twell D (1995) Functional dissection of the promoter of the pollen-specific gene NTP303 reveals a novel pollen-specific, and conserved cis-regulatory element. Plant J 8:55–63Wolf S, Rausch T, Greiner S (2009) The N-terminal pro region mediates retention of unprocessed type-I PME in the Golgi apparatus. Plant J 58:361–375Woriedh M, Wolf S, Márton ML, Hinze A, Gahrtz M, Becker D, Dresselhaus T (2013) External application of gametophyte-specific ZmPMEI1 induces pollen tube burst in maize. Plant Reprod. doi: 10.1007/s00497-013-0221-zZhu C, Perry SE (2005) Control of expression and autoregulation of AGL15, a member of the MADS-box family. Plant J 41:583–59

Functional specialization of duplicated AP3-like genes in Medicago truncatula

This is the accepted version of the following article: Roque, E., Serwatowska, J., Cruz Rochina, M., Wen, J., Mysore, K. S., Yenush, L., Beltrán, J. P. and Cañas, L. A. (2013), Functional specialization of duplicated AP3-like genes in Medicago truncatula. Plant J, 73: 663–675 , which has been published in final form at http://dx.doi.org/10.1111/tpj.12068The Bclass of MADS box genes has been studied in a wide range of plant species, but has remained largely uncharacterized in legumes. Here we investigate the evolutionary fate of the duplicated AP3-like genes of a legume species. To obtain insight into the extent to which B-class MADS box gene functions are conserved or have diversified in legumes, we isolated and characterized the two members of the AP3 lineage in Medicago truncatula: MtNMH7 and MtTM6 (euAP3 and paleoAP3 genes, respectively). A non-overlapping and complementary expression pattern of both genes was observed in petals and stamens. MtTM6 was expressed predominantly in the outer cell layers of both floral organs, and MtNMH7 in the inner cell layers of petals and stamens. Functional analyses by reverse genetics approaches (RNAi and Tnt1 mutagenesis) showed that the contribution of MtNMH7 to petal identity is more important than that of MtTM6, whereas MtTM6 plays a more important role in stamen identity than its paralog MtNMH7. Our results suggest that the M.truncatula AP3-like genes have undergone a functional specialization process associated with complete partitioning of gene expression patterns of the ancestral gene lineage. We provide information regarding the similarities and differences in petal and stamen development among core eudicots.This work was funded by grants BIO2006-09374 and BIO2009-08134 from the Spanish Ministry of Science and Innovation. We are gratefully to Mario A. Fares and Santiago F. Elena (Instituto de Biologia Molecular y Celular de Plantas, Valencia, Spain) for helpful comments and bioinformatics support. The collaboration and assistance of Rafael Martinez-Pardo in the greenhouse is gratefully acknowledged.Roque Mesa, EM.; Serwatowska, J.; Rochina Peñalver, MC.; Wen, J.; Mysore, KS.; Yenush, L.; Beltran Porter, JP.... (2013). Functional specialization of duplicated AP3-like genes in Medicago truncatula. The Plant Journal. 73(4):663-675. doi:10.1111/tpj.12068S663675734Altschul, S. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25(17), 3389-3402. doi:10.1093/nar/25.17.3389Aoki, S., Uehara, K., Imafuku, M., Hasebe, M., & Ito, M. (2004). Phylogeny and divergence of basal angiosperms inferred from APETALA3- and PISTILLATA-like MADS-box genes. Journal of Plant Research, 117(3). doi:10.1007/s10265-004-0153-7Baum, D. (2002). Response: Missing links: the genetic architecture of flower and floral diversification. Trends in Plant Science, 7(1), 31-34. doi:10.1016/s1360-1385(01)02181-1A., B., K., K., A., F., C., V., M.-A., L., H., S., & G., T. (2002). A novel MADS-box gene subfamily with a sister-group relationship to class B floral homeotic genes. Molecular Genetics and Genomics, 266(6), 942-950. doi:10.1007/s00438-001-0615-8Benlloch, R., d’ Erfurth, I., Ferrandiz, C., Cosson, V., Beltrán, J. P., Cañas, L. A., … Ratet, P. (2006). Isolation of mtpim Proves Tnt1 a Useful Reverse Genetics Tool in Medicago truncatula and Uncovers New Aspects of AP1-Like Functions in Legumes. Plant Physiology, 142(3), 972-983. doi:10.1104/pp.106.083543Benlloch, R., Roque, E., Ferrándiz, C., Cosson, V., Caballero, T., Penmetsa, R. V., … Madueño, F. (2009). Analysis of B function in legumes: PISTILLATA proteins do not require the PI motif for floral organ development inMedicago truncatula. The Plant Journal, 60(1), 102-111. doi:10.1111/j.1365-313x.2009.03939.xBerbel, A., Navarro, C., Ferrándiz, C., Cañas, L. A., Beltrán, J.-P., & Madueño, F. (2005). Functional Conservation of PISTILLATA Activity in a Pea Homolog Lacking the PI Motif. Plant Physiology, 139(1), 174-185. doi:10.1104/pp.104.057687Bowman, J. L., Smyth, D. R., & Meyerowitz, E. M. (1989). Genes directing flower development in Arabidopsis. The Plant Cell, 1(1), 37-52. doi:10.1105/tpc.1.1.37Broholm, S. K., Pöllänen, E., Ruokolainen, S., Tähtiharju, S., Kotilainen, M., Albert, V. A., … Teeri, T. H. (2009). Functional characterization of B class MADS-box transcription factors in Gerbera hybrida. Journal of Experimental Botany, 61(1), 75-85. doi:10.1093/jxb/erp279Cheng, X., Wen, J., Tadege, M., Ratet, P., & Mysore, K. S. (2010). Reverse Genetics in Medicago truncatula Using Tnt1 Insertion Mutants. Plant Reverse Genetics, 179-190. doi:10.1007/978-1-60761-682-5_13Coen, E. S., & Meyerowitz, E. M. (1991). The war of the whorls: genetic interactions controlling flower development. Nature, 353(6339), 31-37. doi:10.1038/353031a0Coronado, C., Zuanazzi, J., Sallaud, C., Quirion, J. C., Esnault, R., Husson, H. P., … Ratet, P. (1995). Alfalfa Root Flavonoid Production Is Nitrogen Regulated. Plant Physiology, 108(2), 533-542. doi:10.1104/pp.108.2.533Dellaporta, S. L., Wood, J., & Hicks, J. B. (1983). A plant DNA minipreparation: Version II. Plant Molecular Biology Reporter, 1(4), 19-21. doi:10.1007/bf02712670Drea, S., Hileman, L. C., de Martino, G., & Irish, V. F. (2007). Functional analyses of genetic pathways controlling petal specification in poppy. Development, 134(23), 4157-4166. doi:10.1242/dev.013136D’ Erfurth, I., Cosson, V., Eschstruth, A., Lucas, H., Kondorosi, A., & Ratet, P. (2003). Efficient transposition of theTnt1tobacco retrotransposon in the model legumeMedicago truncatula. The Plant Journal, 34(1), 95-106. doi:10.1046/j.1365-313x.2003.01701.xFerr�ndiz, C., Navarro, C., G�mez, M. D., Ca�as, L. A., & Beltr�n, J. P. (1999). Flower development inPisum sativum: From the war of the whorls to the battle of the common primordia. Developmental Genetics, 25(3), 280-290. doi:10.1002/(sici)1520-6408(1999)25:33.0.co;2-3Geuten, K., & Irish, V. (2010). Hidden Variability of Floral Homeotic B Genes in Solanaceae Provides a Molecular Basis for the Evolution of Novel Functions. The Plant Cell, 22(8), 2562-2578. doi:10.1105/tpc.110.076026Goto, K., & Meyerowitz, E. M. (1994). Function and regulation of the Arabidopsis floral homeotic gene PISTILLATA. Genes & Development, 8(13), 1548-1560. doi:10.1101/gad.8.13.1548Heard, J., & Dunn, K. (1995). Symbiotic induction of a MADS-box gene during development of alfalfa root nodules. Proceedings of the National Academy of Sciences, 92(12), 5273-5277. doi:10.1073/pnas.92.12.5273Hecht, V., Foucher, F., Ferrándiz, C., Macknight, R., Navarro, C., Morin, J., … Weller, J. L. (2005). Conservation of Arabidopsis Flowering Genes in Model Legumes. Plant Physiology, 137(4), 1420-1434. doi:10.1104/pp.104.057018The evolution of functionally novel proteins after gene duplication. (1994). Proceedings of the Royal Society of London. Series B: Biological Sciences, 256(1346), 119-124. doi:10.1098/rspb.1994.0058Irish, V. F. (2006). Duplication, Diversification, and Comparative Genetics of Angiosperm MADS‐Box Genes. Advances in Botanical Research, 129-161. doi:10.1016/s0065-2296(06)44003-9Jack, T., Fox, G. L., & Meyerowitz, E. M. (1994). Arabidopsis homeotic gene APETALA3 ectopic expression: Transcriptional and posttranscriptional regulation determine floral organ identity. Cell, 76(4), 703-716. doi:10.1016/0092-8674(94)90509-6Kim, S., Yoo, M.-J., Albert, V. A., Farris, J. S., Soltis, P. S., & Soltis, D. E. (2004). Phylogeny and diversification of B-function MADS-box genes in angiosperms: evolutionary and functional implications of a 260-million-year-old duplication. American Journal of Botany, 91(12), 2102-2118. doi:10.3732/ajb.91.12.2102Kramer, E. M., & Irish, V. F. (2000). Evolution of the Petal and Stamen Developmental Programs: Evidence from Comparative Studies of the Lower Eudicots and Basal Angiosperms. International Journal of Plant Sciences, 161(S6), S29-S40. doi:10.1086/317576Kramer, E. M., Di Stilio, V. S., & Schlüter, P. M. (2003). Complex Patterns of Gene Duplication in the APETALA3 and PISTILLATA Lineages of the Ranunculaceae. International Journal of Plant Sciences, 164(1), 1-11. doi:10.1086/344694Kramer, E. M., Su, H.-J., Wu, C.-C., & Hu, J.-M. (2006). BMC Evolutionary Biology, 6(1), 30. doi:10.1186/1471-2148-6-30Lamb, R. S., & Irish, V. F. (2003). Functional divergence within the APETALA3/PISTILLATA floral homeotic gene lineages. Proceedings of the National Academy of Sciences, 100(11), 6558-6563. doi:10.1073/pnas.0631708100Liu, Y., Nakayama, N., Schiff, M., Litt, A., Irish, V. F., & Dinesh-Kumar, S. P. (2004). Virus Induced Gene Silencing of a DEFICIENS Ortholog in Nicotiana Benthamiana. Plant Molecular Biology, 54(5), 701-711. doi:10.1023/b:plan.0000040899.53378.83De Martino, G., Pan, I., Emmanuel, E., Levy, A., & Irish, V. F. (2006). Functional Analyses of Two Tomato APETALA3 Genes Demonstrate Diversification in Their Roles in Regulating Floral Development. The Plant Cell, 18(8), 1833-1845. doi:10.1105/tpc.106.042978Ohno, S. (1970). Evolution by Gene Duplication. doi:10.1007/978-3-642-86659-3Páez-Valencia, J., Sánchez-Gómez, C., Valencia-Mayoral, P., Contreras-Ramos, A., Hernández-Lucas, I., Orozco-Segovia, A., & Gamboa-deBuen, A. (2008). Localization of the MADS domain transcriptional factor NMH7 during seed, seedling and nodule development of Medicago sativa. Plant Science, 175(4), 596-603. doi:10.1016/j.plantsci.2008.06.008Pnueli, L., Abu-Abeid, M., Zamir, D., Nacken, W., Schwarz-Sommer, Z., & Lifschitz, E. (1991). The MADS box gene family in tomato: temporal expression during floral development, conserved secondary structures and homology with homeotic genes fromAntirrhinumandArabidopsis. The Plant Journal, 1(2), 255-266. doi:10.1111/j.1365-313x.1991.00255.xRiechmann, J. L., Krizek, B. A., & Meyerowitz, E. M. (1996). Dimerization specificity of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA, and AGAMOUS. Proceedings of the National Academy of Sciences, 93(10), 4793-4798. doi:10.1073/pnas.93.10.4793Rijpkema, A. S., Royaert, S., Zethof, J., van der Weerden, G., Gerats, T., & Vandenbussche, M. (2006). Analysis of the Petunia TM6 MADS Box Gene Reveals Functional Divergence within the DEF/AP3 Lineage. The Plant Cell, 18(8), 1819-1832. doi:10.1105/tpc.106.042937Schwarz-Sommer, Z., Hue, I., Huijser, P., Flor, P. J., Hansen, R., Tetens, F., … Sommer, H. (1992). Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens: evidence for DNA binding and autoregulation of its persistent expression throughout flower development. The EMBO Journal, 11(1), 251-263. doi:10.1002/j.1460-2075.1992.tb05048.xSoltis, P. S., Brockington, S. F., Yoo, M.-J., Piedrahita, A., Latvis, M., Moore, M. J., … Soltis, D. E. (2009). Floral variation and floral genetics in basal angiosperms. American Journal of Botany, 96(1), 110-128. doi:10.3732/ajb.0800182Sommer, H., Beltrán, J. P., Huijser, P., Pape, H., Lönnig, W. E., Saedler, H., & Schwarz-Sommer, Z. (1990). Deficiens, a homeotic gene involved in the control of flower morphogenesis in Antirrhinum majus: the protein shows homology to transcription factors. The EMBO Journal, 9(3), 605-613. doi:10.1002/j.1460-2075.1990.tb08152.xStellari, G. M., Jaramillo, M. A., & Kramer, E. M. (2004). Evolution of the APETALA3 and PISTILLATA Lineages of MADS-Box–Containing Genes in the Basal Angiosperms. Molecular Biology and Evolution, 21(3), 506-519. doi:10.1093/molbev/msh044Tadege, M., Ratet, P., & Mysore, K. S. (2005). Insertional mutagenesis: a Swiss Army knife for functional genomics of Medicago truncatula. Trends in Plant Science, 10(5), 229-235. doi:10.1016/j.tplants.2005.03.009Tadege, M., Wen, J., He, J., Tu, H., Kwak, Y., Eschstruth, A., … Mysore, K. S. (2008). Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula. The Plant Journal, 54(2), 335-347. doi:10.1111/j.1365-313x.2008.03418.xTamura, K., Dudley, J., Nei, M., & Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0. Molecular Biology and Evolution, 24(8), 1596-1599. doi:10.1093/molbev/msm092Taylor, S., Hofer, J., & Murfet, I. (2001). Stamina pistilloida, the Pea Ortholog of Fim and UFO, Is Required for Normal Development of Flowers, Inflorescences, and Leaves. The Plant Cell, 13(1), 31-46. doi:10.1105/tpc.13.1.31Theissen, G., & Melzer, R. (2007). Molecular Mechanisms Underlying Origin and Diversification of the Angiosperm Flower. Annals of Botany, 100(3), 603-619. doi:10.1093/aob/mcm143Tröbner, W., Ramirez, L., Motte, P., Hue, I., Huijser, P., Lönnig, W. E., … Schwarz-Sommer, Z. (1992). GLOBOSA: a homeotic gene which interacts with DEFICIENS in the control of Antirrhinum floral organogenesis. The EMBO Journal, 11(13), 4693-4704. doi:10.1002/j.1460-2075.1992.tb05574.xTucker, S. C. (2003). Floral Development in Legumes. Plant Physiology, 131(3), 911-926. doi:10.1104/pp.102.017459Urbanus, S. L., de Folter, S., Shchennikova, A. V., Kaufmann, K., Immink, R. G., & Angenent, G. C. (2009). In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana. BMC Plant Biology, 9(1), 5. doi:10.1186/1471-2229-9-5Vandenbussche, M., Zethof, J., Royaert, S., Weterings, K., & Gerats, T. (2004). The Duplicated B-Class Heterodimer Model: Whorl-Specific Effects and Complex Genetic Interactions in Petunia hybrida Flower Development. The Plant Cell, 16(3), 741-754. doi:10.1105/tpc.019166Wesley, S. V., Helliwell, C. A., Smith, N. A., Wang, M., Rouse, D. T., Liu, Q., … Waterhouse, P. M. (2001). Construct design for efficient, effective and high-throughput gene silencing in plants. The Plant Journal, 27(6), 581-590. doi:10.1046/j.1365-313x.2001.01105.xWu, C., Ma, Q., Yam, K.-M., Cheung, M.-Y., Xu, Y., Han, T., … Chong, K. (2005). In situ expression of the GmNMH7 gene is photoperiod-dependent in a unique soybean (Glycine max [L.] Merr.) flowering reversion system. Planta, 223(4), 725-735. doi:10.1007/s00425-005-0130-

Vitamin C activates pyruvate dehydrogenase (PDH) targeting the mitochondrial tricarboxylic acid (TCA) cycle in hypoxic KRAS mutant colon cancer

Background: In hypoxic tumors, positive feedback between oncogenic KRAS and HIF-1α involves impressive metabolic changes correlating with drug resistance and poor prognosis in colorectal cancer. Up to date, designed KRAS-targeting molecules do not show clear benefits in patient overall survival (POS) so pharmacological modulation of aberrant tricarboxylic acid (TCA) cycle in hypoxic cancer has been proposed as a metabolic vulnerability of KRAS-driven tumors. Methods: Annexin V-FITC and cell viability assays were carried out in order to verify vitamin C citotoxicity in KRAS mutant SW480 and DLD1 as well as in Immortalized Human Colonic Epithelial Cells (HCEC). HIF1a expression and activity were determined by western blot and functional analysis assays. HIF1a direct targets GLUT1 and PDK1 expression was checked using western blot and qRT-PCR. Inmunohistochemical assays were perfomed in tumors derived from murine xenografts in order to validate previous observations in vivo. Vitamin C dependent PDH expression and activity modulation were detected by western blot and colorimetric activity assays. Acetyl-Coa levels and citrate synthase activity were assessed using colorimetric/fluorometric activity assays. Mitochondrial membrane potential (Δψ) and cell ATP levels were assayed using fluorometric and luminescent test. Results: PDK-1 in KRAS mutant CRC cells and murine xenografts was downregulated using pharmacological doses of vitamin C through the proline hydroxylation (Pro402) of the Hypoxia inducible factor-1(HIF-1)α, correlating with decreased expression of the glucose transporter 1 (GLUT-1) in both models. Vitamin C induced remarkable ATP depletion, rapid mitochondrial Δψ dissipation and diminished pyruvate dehydrogenase E1-α phosphorylation at Serine 293, then boosting PDH and citrate synthase activity. Conclusion: We report a striking and previously non reported role of vitamin C in the regulation of the pyruvate dehydrogenase (PDH) activity, then modulating the TCA cycle and mitochondrial metabolism in KRAS mutant colon cancer. Potential impact of vitamin C in the clinical management of anti-EGFR chemoresistant colorectal neoplasias should be further considered.This research is supported by Ministerio de Ciencia e Innovación, Centro para el Desarrollo Tecnológico Industrial (CDTI) and Universidad Católica San Antonio (Murcia

The parthenocarpic hydra mutant reveals a new function for a SPOROCYTELESS-like gene in the control of fruit set in tomato

[EN] Fruit set is an essential process to ensure successful sexual plant reproduction. The development of the flower into a fruit is actively repressed in the absence of pollination. However, some cultivars from a few species are able to develop seedless fruits overcoming the standard restriction of unpollinated ovaries to growth. We report here the identification of the tomato hydra mutant that produces seedless (parthenocarpic) fruits. Seedless fruit production in hydra plants is linked to the absence of both male and female sporocyte development. The HYDRA gene is therefore essential for the initiation of sporogenesis in tomato. Using positional cloning, virus-induced gene silencing and expression analysis experiments, we identified the HYDRA gene and demonstrated that it encodes the tomato orthologue of SPOROCYTELESS/NOZZLE (SPL/NZZ) of Arabidopsis. We found that the precocious growth of the ovary is associated with changes in the expression of genes involved in gibberellin (GA) metabolism. Our results support the conservation of the function of SPL-like genes in the control of sporogenesis in plants. Moreover, this study uncovers a new function for the tomato SlSPL/HYDRA gene in the control of fruit initiation.This work was supported by grants from the Spanish Ministerio de Ciencia e Innovaci on (MICINN; AGL2009-07617 to C.G-M.; AGL2015-64991-C3-3-R to V.M.; and AGL2015-64991-C3-1-R to R.L.) and the Ram on y Cajal Program (RYC-2007-00627). We thank Rafael Martinez and Primitivo Murias for expert plant care; Marisol Gasc on for technical assistance with the microscope; and Dr Cristina Ferrandiz for critical reading of the manuscript. The authors declare no conflicts of interest.This work was supported by grants from the Spanish Ministerio de Ciencia e Innovaci on (MICINN; AGL2009-07617 to C.G-M.; AGL2015-64991-C3-3-R to V.M.; and AGL2015-64991-C3-1-R to R.L.) and the Ram on y Cajal Program (RYC-2007-00627). We thank Rafael Martinez and Primitivo Murias for expert plant care; Marisol Gasc on for technical assistance with the microscope; and Dr Cristina Ferrandiz for critical reading of the manuscript. The authors declare no conflicts of interest.Rojas-Gracia, P.; Roque Mesa, EM.; Medina Herranz, M.; Rochina Peñalver, MC.; Hamza, R.; Angarita-Diaz, MP.; Moreno Ferrero, V.... (2017). The parthenocarpic hydra mutant reveals a new function for a SPOROCYTELESS-like gene in the control of fruit set in tomato. New Phytologist. 214(3):1198-1212. https://doi.org/10.1111/nph.14433S11981212214

El desarrollo municipal, factor estratégico en el posicionamiento de México en los escenarios políticos y sociales del siglo XXI

LA DEMOCRACIA COMO GOBERNABILIDAD IMPLICA, EN UN PRIMER MOMENTO, establecer una revisión periódica del papel interventor del Estado, por ser éste el principal factor de estabilidad y desarrollo democrático. En un segundo punto, de forma simultánea al estudio del papel del Estado en la conformación de un ambiente de estabilidad, crecimiento, desarrollo, orden y gobernabilidad, merece especial atención el papel y funciones cumplidas tradicionalmente por sus ámbitos de gobierno, como instancias que son fundamentales para la transición, democratización, liberalización y para la propia gobernabilidad