Androesterilidad inducida mediante ingeniería genética en plantas: fundamentos y aplicaciones biotecnológicas

The availability of male-sterile plant varieties is relevant for obtaining of hybrid plant lines which are more vigorous than the corresponding parental pure lines because the phenomenon known as heterosis. Moreover, the use of male-sterile plants prevents undesirable horizontal gene transfer. We have developed biotechnological tools to obtain androsteryle lines of plants with agronomic interest such as tomato, tobacco, rape seed and wheat. This is accomplished by the use of the promoter region of the PsEND1 gene to drive the expression of cytotoxic agents, such as barnase, to the structural tissues of the anthers. The male-sterile transgenic plants obtained live longer and show a higher number of branches and flowers than the corresponding wild type plants. This will allow plant breeders to incorporate those valuable characteristics to increase the number of flowers of plants already displaying new colours, shapes and fragrances. These new ornamental plants are environmental friendly since horizontal gene transfer can not take place.La disponibilidad de genotipos de plantas androestériles es crucial para la obtención de semillas híbridas y abre la posibilidad del manejo de las plantas de forma más respetuosa con el medio ambiente. Nosotros hemos desarrollado herramientas biotecnológicas para la producción de plantas androestériles de interés agronómico (tomate, colza, tabaco) mediante el uso de la región promotora del gen PsEND1 de guisante para dirigir la expresión de agentes citotóxicos específicamente a los tejidos estructurales de las anteras para producir su ablación genética. En las plantas androestériles obtenidas mediante ingeniería genética, hemos observado que se produce un mayor número de ramas y consecuentemente una mayor producción de flores. Además, la vida útil de estas plantas se prolonga de forma notable. Estas características son de interés para el sector de la floricultura, ya que actualmente se están produciendo híbridos mediante mejora convencional con flores muy vistosas y colores novedosos, pero con escasa producción de flores por planta. Por otra parte, la introducción en la planta modificada genéticamente de un gen que le confiera androesterilidad, es otra característica deseable en el campo de las plantas ornamentales ya que evitaría la transferencia horizontal de transgenes al medio ambiente y a especies sexualmente compatibles

Production of engineered long-life and male sterile Pelargonium plants

[EN] Background: Pelargonium is one of the most popular garden plants in the world. Moreover, it has a considerable economic importance in the ornamental plant market. Conventional cross-breeding strategies have generated a range of cultivars with excellent traits. However, gene transfer via Agrobacterium tumefaciens could be a helpful tool to further improve Pelargonium by enabling the introduction of new genes/traits. We report a simple and reliable protocol for the genetic transformation of Pelargonium spp. and the production of engineered long-life and male sterile Pelargonium zonale plants, using the pSAG12::ipt and PsEND1::barnase chimaeric genes respectively. Results: The pSAG12::ipt transgenic plants showed delayed leaf senescence, increased branching and reduced internodal length, as compared to control plants. Leaves and flowers of the pSAG12::ipt plants were reduced in size and displayed a more intense coloration. In the transgenic lines carrying the PsEND1::barnase construct no pollen grains were observed in the modified anther structures, which developed instead of normal anthers. The locules of sterile anthers collapsed 3¿4 days prior to floral anthesis and, in most cases, the undeveloped anther tissues underwent necrosis. Conclusion: The chimaeric construct pSAG12::ipt can be useful in Pelargonium spp. to delay the senescence process and to modify plant architecture. In addition, the use of engineered male sterile plants would be especially useful to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals and related plant species. These characteristics could be of interest, from a commercial point of view, both for pelargonium producers and consumers.This work was funded by grants AGL2009-13388-C03-01 and BIO2009-08134 from the Spanish Ministry of Science and Innovation (MICINN). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). In the past five years we have received funding from the Spanish Ministry of Science and Innovation (MICINN) and the article-processing charge will be pay with funds from two granted projects. The authors received salaries from two different institutions: The Polytechnic University of Valencia (UPV) or the High Spanish Council of Scientific Research (CSIC). We are not currently applying for a patent related with the content of this manuscript. All the mentioned organisms/institutions do not gain or lose financially from the publication of this manuscript either now or in the future.García Sogo, B.; Pineda Chaza, BJ.; Roque Mesa, EM.; Antón Martínez, MT.; Atarés Huerta, A.; Borja, M.; Beltran Porter, JP.... (2012). Production of engineered long-life and male sterile Pelargonium plants. BMC Plant Biology. 12:156-171. https://doi.org/10.1186/1471-2229-12-1561561711

The DOF Transcription Factor SlDOF10 Regulates Vascular Tissue Formation During Ovary Development in Tomato

The formation of fruits is an important step in the life cycle of flowering plants. The process of fruit development is highly regulated and involves the interaction of a complex regulatory network of genes in both space and time. To identify regulatory genes involved in fruit initiation in tomato we analyzed the transcriptomic profile of ovaries from the parthenocarpic PsEND1:barnase transgenic line. This line was generated using the cytotoxic gene barnase targeted to the anthers with the PsEND1 anther-specific promoter from pea. Among the differentially expressed genes we identified SlDOF10, a gene coding a DNA-binding with one finger (DOF) transcription factor which is activated in unpollinated ovaries of the parthenocarpic plants. SlDOF10 is preferentially expressed in the vasculature of the cotyledons and young leaves and in the root tip. During floral development, expression is visible in the vascular tissue of the sepals, the flower pedicel and in the ovary connecting the placenta with the developing ovules. The induction of the gene was observed in response to exogenous gibberellins and auxins treatments. To evaluate the gene function during reproductive development, we have generated SlDOF10 overexpressing and silencing stable transgenic lines. In particular, down-regulation of SlDOF10 activity led to a decrease in the area occupied by individual vascular bundles in the flower pedicel. Associated with this phenotype we observed induction of parthenocarpic fruit set. In summary, expression and functional analyses revealed a role for SlDOF10 gene in the development of the vascular tissue specifically during reproductive development highlighting the importance of this tissue in the process of fruit set

Evolution by gene duplication of Medicago truncatula PISTILLATA-like transcription factors

[EN] PISTILLATA (PI) is a member of the B-function MADS-box gene family, which controls the identity of both petals and stamens in Arabidopsis thaliana. In Medicago truncatula (Mt), there are two PI-like paralogs, known as MtPI and MtNGL9. These genes differ in their expression patterns, but it is not known whether their functions have also diverged. Describing the evolution of certain duplicated genes, such as transcription factors, remains a challenge owing to the complex expression patterns and functional divergence between the gene copies. Here, we report a number of functional studies, including analyses of gene expression, protein-protein interactions, and reverse genetic approaches designed to demonstrate the respective contributions of each M. truncatula PI-like paralog to the B-function in this species. Also, we have integrated molecular evolution approaches to determine the mode of evolution of Mt PI-like genes after duplication. Our results demonstrate that MtPI functions as a master regulator of B-function in M. truncatula, maintaining the overall ancestral function, while MtNGL9 does not seem to have a role in this regard, suggesting that the pseudogenization could be the functional evolutionary fate for this gene. However, we provide evidence that purifying selection is the primary evolutionary force acting on this paralog, pinpointing the conservation of its biochemical function and, alternatively, the acquisition of a new role for this gene.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO; BIO2009-08134 and BIO2012-39849-CO2-01). MAF was supported by a grant from the MINECO (BFU2012-36346). We wish to thank Drs Santiago F. Elena Fito (IBMCP, CSIC-UPV), Jose V. Gimeno Alcaniz (IATA, CSIC), Javier Paz-Ares (CNB, CSIC), and Carlos Alonso-Blanco (CNB, CSIC) for valuable suggestions and comments in the initial stages of this work. The technical assistance of Rafael Martinez-Pardo in the greenhouse is gratefully acknowledged.Roque Mesa, EM.; Fares Riaño, MA.; Yenush, L.; Rochina Peñalver, MC.; Wen, J.; Mysore, KS.; Gómez Mena, MC.... (2016). Evolution by gene duplication of Medicago truncatula PISTILLATA-like transcription factors. Journal of Experimental Botany. 67(6):1805-1817. https://doi.org/10.1093/jxb/erv571S1805181767

Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp

[EN] Proanthocyanidins (PAs), or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila) to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR) to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (-) catechin/g FW and 228.5 nmol (-) epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA) method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce "bloatsafe" plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass) are discussed.This work was supported by grants BIO2012-39849-C02-01 and BIO2016-75485-R from the Spanish Ministry of Economy and Competitiveness (MINECO) (http://www.idi.mineco.gob.es/portal/site/MICINN) to LAC and a fellowship of the JAE-CSIC program to SF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Fresquet-Corrales, S.; Roque Mesa, EM.; Sarrión-Perdigones, A.; Rochina, M.; López-Gresa, MP.; Díaz-Mula, HM.; Belles Albert, JM.... (2017). Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp. PLoS ONE. 12(9). https://doi.org/10.1371/journal.pone.0184839Se018483912

Two euAGAMOUS genes control C-function in Medicago truncatula

[EN] C-function MADS-box transcription factors belong to the AGAMOUS (AG) lineage and specify both stamen and carpel identity and floral meristem determinacy. In core eudicots, the AG lineage is further divided into two branches, the euAG and PLE lineages. Functional analyses across flowering plants strongly support the idea that duplicated AG lineage genes have different degrees of subfunctionalization of the C-function. The legume Medicago truncatula contains three C-lineage genes in its genome: two euAG genes (MtAGa and MtAGb) and one PLENA-like gene (MtSHP). This species is therefore a good experimental system to study the effects of gene duplication within the AG subfamily. We have studied the respective functions of each euAG genes in M. truncatula employing expression analyses and reverse genetic approaches. Our results show that the M. truncatula euAG- and PLENA-like genes are an example of subfunctionalization as a result of a change in expression pattern. MtAGa and MtAGb are the only genes showing a full C-function activity, concomitant with their ancestral expression profile, early in the floral meristem, and in the third and fourth floral whorls during floral development. In contrast, MtSHP expression appears late during floral development suggesting it does not contribute significantly to the C-function. Furthermore, the redundant MtAGa and MtAGb paralogs have been retained which provides the overall dosage required to specify the C-function in M. truncatula.This work was funded by grants BIO2009-08134 and BIO2012-39849-C02-01 from the Spanish Ministry of Economy and Competitiveness and the Ramon y Cajal Program (RYC-2007-00627 to CGM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Serwatowska, J.; Roque Mesa, EM.; Gómez Mena, MC.; Constantin, GD.; Wen, J.; Mysore, KS.; Lund, OS.... (2014). Two euAGAMOUS genes control C-function in Medicago truncatula. 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Tomates partenocárpicos y procedimiento para su producción

Referencia OEPM: P200401761.-- Fecha de solicitud: 17/07/2004.-- Titulares: Newbiotechnic, S.A., Consejo Superior de Investigaciones Científicas (CSIC).Tomates partenocárpicos y procedimiento para su producción. Los tomates (Lycopersicon esculentum Mill.) partenocárpicos carecen de semillas, son de menor tamaño que los silvestres y, en estado maduro, muestran un color rojo más intenso que los tomates silvestres. Dichos tomates se obtienen cultivando una planta de tomate transgénica productora de dichos tomates partenocárpicos obtenida mediante un procedimiento que comprende (a) introducir en una célula o tejido de una planta de tomate una construcción de DNA que comprende (i) el promotor del gen END1 de guisante o un fragmento funcional del mismo y (ii) un gen citotóxico, operativamente unido a dicho promotor o fragmento del mismo; y (b) regenerar dicha célula o tejido de planta de tomate transformado de la etapa (a) para producir una planta de tomate transgénica productora de tomates partenocárpicos. De aplicación en alimentación e industrias agroalimentarias.Peer reviewe

PsEND1 Is a Key Player in Pea Pollen Development Through the Modulation of Redox Homeostasis

Redox homeostasis has been linked to proper anther and pollen development. Accordingly, plant cells have developed several Reactive Oxygen Species (ROS)-scavenging mechanisms to maintain the redox balance. Hemopexins constitute one of these mechanisms preventing heme-associated oxidative stress in animals, fungi, and plants. Pisum sativum ENDOTHECIUM 1 (PsEND1) is a pea anther-specific gene that encodes a protein containing four hemopexin domains. We report the functional characterization of PsEND1 and the identification in its promoter region of cis-regulatory elements that are essential for the specific expression in anthers. PsEND1 promoter deletion analysis revealed that a putative CArG-like regulatory motif is necessary to confer promoter activity in developing anthers. Our data suggest that PsEND1 might be a hemopexin regulated by a MADS-box protein. PsEND1 gene silencing in pea, and its overexpression in heterologous systems, result in similar defects in the anthers consisting of precocious tapetum degradation and the impairment of pollen development. Such alterations were associated to the production of superoxide anion and altered activity of ROS-scavenging enzymes. Our findings demonstrate that PsEND1 is essential for pollen development by modulating ROS levels during the differentiation of the anther tissues surrounding the microsporocytes

The tapetal tissue is essential for the maintenance of redox homeostasis during microgametogenesis in tomato

The tapetum is a specialized layer of cells within the anther, adjacent to the sporogenous tissue. During its short life, it provides nutrients, molecules and materials to the pollen mother cells and microsporocytes, being essential during callose degradation and pollen wall formation. The interaction between the tapetum and sporogenous cells in Solanum lycopersicum (tomato) plants, despite its importance for breeding purposes, is poorly understood. To investigate this process, gene editing was used to generate loss-of-function mutants that showed the complete and specific absence of tapetal cells. These plants were obtained targeting the previously uncharacterized Solyc03g097530 (SlTPD1) gene, essential for tapetum specification in tomato plants. In the absence of tapetum, sporogenous cells developed and callose deposition was observed. However, sporocytes failed to undergo the process of meiosis and finally degenerated, leading to male sterility. Transcriptomic analysis conducted in mutant anthers lacking tapetum revealed the downregulation of a set of genes related to redox homeostasis. Indeed, mutant anthers showed a reduction in the accumulation of reactive oxygen species (ROS) at early stages and altered activity of ROS-scavenging enzymes. The results obtained highlight the importance of the tapetal tissue in maintaining redox homeostasis during male gametogenesis in tomato plants.Peer reviewe

Tomates partenocárpicos y procedimiento para su producción.

Fecha de presentación internacional: 14.07.2005.- Titulares: Consejo Superior de Investigaciones Científicas (CSIC).- Newbiotechnic, S.A.[EN]The invention relates to parthenocarpic tomatoes (Lycopersicon esculentum Mili.) which are grown from seeds, which are smaller than wild tomatoes and which, when ripe, display a more intense red colour than wild tomatoes. The tomatoes are obtained by cultivating a transgenic tomato plant which produces said parthenocarpic tomatoes and which is obtained using a method consisting in: (a) introducing a DNA construction into a cell or tissue of a tomato plant, said construction comprising (i) the pea END1 gene promoter or a functional fragment of same and (ii) a cytotoxic gene which is functionally bound to the aforementioned promoter or fragment of same; and (b) regenerating the tomato plant cell or tissue transformed in step (a) in order to produce a transgenic tomato plant that produces parthenocarpic tomatoes. The invention is suitable for use in food and agrifood industries.[ES]Los tomates (Lycopersicon esculentum Mili.) partenocárpicos carecen de semillas, son de menor tamaño menor que los silvestres y, en estado maduro, muestran un color rojo más intenso que los tomates silvestres. Dichos tomates se obtienen cultivando una planta de tomate transgénica productora de dichos tomates partenocárpicos obtenida mediante un procedimiento que comprende (a) introducir en una célula o tejido de una planta de tomate una construcción de DNA que comprende (i) el promotor del gen ENDl de guisante o un fragmento funcional del mismo y (ii) un gen citotóxico, operativamente unido a dicho promotor o fragmento del mismo; y (b) regenerar dicha célula o tejido de planta de tomate transformado de la etapa (a) para producir una planta de tomate transgénica productora de tomates partenocárpicos. De aplicación en alimentación e industrias agroalimentarias.Peer reviewe