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

Isolation and Functional Analysis of a PISTILLATA-like MADS-Box Gene from Argan Tree (Argania spinosa)

Argan trees (Argania spinosa) belong to a species native to southwestern Morocco, playing an important role in the environment and local economy. Argan oil extracted from kernels has a unique composition and properties. Argan trees were introduced in Tunisia, where hundreds of trees can be found nowadays. In this study, we examined reproductive development in Argan trees from four sites in Tunisia and carried out the functional characterization of a floral homeotic gene in this non-model species. Despite the importance of reproductive development, nothing is known about the genetic network controlling flower development in Argania spinosa. Results obtained in several plant species established that floral organ development is mostly controlled by MADS-box genes and, in particular, APETALA3 (AP3) and PISTILLATA (PI) homologs are required for proper petal and stamen identity. Here, we describe the isolation and functional characterization of a MADS-box gene from Argania spinosa. Phylogenetic analyses showed strong homology with PI-like proteins, and the expression of the gene was found to be restricted to the second and third whorls. Functional homology with Arabidopsis PI was demonstrated by the ability of AsPI to confer petal and stamen identity when overexpressed in a pi-1 mutant background. The identification and characterization of this gene support the strong conservation of PI homologs among distant angiosperm plants.</jats:p

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.</jats:p

SUPERMAN genes: uncovering a new function in the development of complex inflorescences

The Arabidopsis SUPERMAN (SUP) gene and its orthologs in eudicots are crucial in regulating the number of reproductive floral organs. In Medicago truncatula, in addition to this function, a novel role in controlling meristem activity during compound inflorescence development was assigned to the SUP-ortholog (MtSUP). These findings led us to investigate whether the role of SUP genes in inflorescence development was legume-specific or could be extended to other eudicots. To assess that, we used Solanum lycopersicum as a model system with a cymose complex inflorescence and Arabidopsis thaliana as the best-known example of simple inflorescence. We conducted a detailed comparative expression analysis of SlSUP and SUP from vegetative stages to flower transition. In addition, we performed an exhaustive phenotypic characterisation of two different slsup and sup mutants during the plant life cycle. Our findings reveal that SlSUP is required for precise regulation of the meristems that control shoot and inflorescence architecture in tomato. In contrast, in Arabidopsis, SUP performs no meristematic function, but we found a role of SUP in floral transition. Our findings suggest that the functional divergence of SUP-like genes contributed to the modification of inflorescence architecture during angiosperm evolution