Análisis funcional y aplicaciones biotecnológicas del promotor del gen END1 de guisante (Pisum sativum L.)

[EN] END1 is an anther-specific gene of pea (Pisum sativum. L) that displays specific expression in the cell lines that will develop the epidermis, connective, middlle layer and endothecium tissues from very early stages (anther primordium) to late stages of the anther development. The END1 promoter region drives the uidA (GUS) gene expression specifically to the anthers of Arabidopsis thaliana, Nicotiana tabacum and Lycopersicon esculentum. The chimaeric END1::GUS gene is regulated exactly like the endogenous END1. The spatial and temporal expression pattern of END1 and the functionality of its promoter region in different plant species suggested us the possibility to use its promoter region for biotechnological applications. We fused the 2’7 kb END1 5’ region with the ribonuclease barnase gene. The barnase is a natural ribonuclease isolated from Bacillus amyloliquefaciens. The chimaeric END1::barnasa gene was introduced into Arabidopsis, tobacco and tomato plants. The expression of the chimaeric gene inhibited anther morphogenesis and efficiently produced male-sterile transgenic plants. A detailed histological study of the development of both, WT and transgenic anthers, showed severe morphological differences from very early stages of anther development. The effects of barnase in transgenic anthers appeared when the anther was at the stage of anther primordium and it was composed by undifferentiated cells. Our results show that the END1 promoter might be a biotechnological tool to generate male-sterile plants for the production of hybrid crop plants. Tomato male-sterile transgenic plants produced parthenocarpic fruits. This observation suggests that male-sterility in tomato is related to the induction of parthenocarpic fruits. To identify and characterize cis-regulatory elements involved in the promoter strength and specificity, expression analysis were performed using constructs containing the END1 promoter, modified by deleting different nucleotide sequences, and the β-glucuronidase gene. Our results reveal that the 5’ region -336/-6 is sufficient to direct properly the spatial and temporal END1 gene expression and also, suggest that the END1 might be a target gene of floral organ identity genes of classes B and C.[ES] END1 es un gen de guisante (Pisum sativum L.) específico de antera que comienza a expresarse desde estadios tempranos del desarrollo (primordio de antera) en aquellas líneas celulares que darán lugar a los tejidos epidermis, conectivo, capa intermedia y endotecio, y en estos tejidos una vez desarrollados. La región promotora de dicho gen es capaz de dirigir la expresión específica del gen uidA (GUS) a las anteras de plantas de diferentes especies (Arabidopsis thaliana, Nicotiana tabacum y Lycopersicon esculentum). El patrón de expresión del gen GUS en esas plantas es similar al del gen END1 en guisante. Esta especificidad que confiere el promotor de END1 para la expresión de un gen foráneo en las anteras ofrecía la posibilidad de utilizarlo, fusionándolo a un gen citotóxico, como una herramienta biotecnológica para la obtención de plantas transgénicas androestériles. En este trabajo, hemos fusionado la región 5’ completa (-2736/-6) del promotor de END1 a la secuencia codificante de la ribonucleasa extracelular producida por el Bacillus amyloliquefaciens, barnasa. Con esta construcción hemos transformado dos plantas modelo, Arabidopsis y tabaco y una planta de interés agronómico, el tomate. La expresión del gen citotóxico barnasa donde END1 es activo, trajo como consecuencia una degeneración de tejidos de la antera que inhibió el desarrollo de los granos de polen. Las plantas transgénicas resultantes fueron androestériles. Los estudios a nivel histológico de las anteras transgénicas mostraron que los efectos de la barnasa comienzan a observarse muy pronto en el primordio de antera, cuando este está constituido sólo por células indiferenciadas, y continúa observándose a lo largo del desarrollo de la misma. De manera general, al final del programa de desarrollo, las anteras transgénicas eran de menor tamaño, su morfología era distinta de las de las anteras silvestres correspondientes, y en su interior se observaba el tejido conectivo colapsado y una estructura amorfa en lugar de granos de polen viable. La posible pérdida de las células parietales primarias por la acción ribonucleasa podría estar afectando la diferenciación de las células esporógenas, contiguas en el territorio del futuro microsporangio. Estos resultados muestran que el promotor de END1 podría ser una herramienta biotecnológica útil en los programas de obtención de semillas híbridas de diferentes cultivos de interés agronómico. En el caso particular del tomate, todas las plantas transgénicas androestériles produjeron frutos partenocárpicos. Este resultado muestra que existe una relación entre la androesterilidad y el desarrollo autónomo del ovario en esa especie. Por otra parte, se ha realizado un análisis del promotor del gen END1. Para este análisis se realizaron delecciones sucesivas de la región 5’ del gen y los fragmentos resultantes se fusionaron transcripcionalmente al gen delator uidA (GUS). Con estas construcciones se transformaron plantas de A. thaliana y se estudió la expresión del gen uidA mediante el ensayo histoquímico de la actividad de la β-glucuronidasa. Hemos visto que el fragmento de 366/-6 de la región 5’ es la secuencia mínima con capacidad para dirigir la correcta expresión espacial y temporal del gen END1. La pérdida de la expresión de GUS en las anteras de las plantas de Arabidopsis thaliana transformadas con el fragmento de la región 5’ donde se elimina el motivo CArG2, apoya la hipótesis de que END1 podría estar regulado directamente por los genes de identidad de órgano de clase B y C.[CA] END 1 és un gen de pèsol (Pisum sativum L.) específic d'antera que comença a expressar-se des d'estadis primerencs del desenvolupament (primordi d'antera) en aquelles línies cel·lulars que donaran lloc als teixits epidèrmics, connectiu, capa intermèdia i endoteci, i en aquests teixits una vegada desenvolupats. La regió promotora d'aquest gen és capaç de dirigir l'expressió específica del gen uidA (GUS) a les anteres de plantes de diferents espècies (Arabidopsis thaliana, Nicotiana tabacum i Lycopersicon esculentum). El patró d'expressió del gen GUS en aquestes plantes és similar al del gen END1 en pèsol. Aquesta especificitat que confereix el promotor d' END 1 per a l'expressió d'un gen forà les anteres oferia la possibilitat d'utilitzar-lo, fusionant-lo a un gen citotòxic, com una ferramenta biotecnològica per a l'obtenció de plantes transgèniques androestèrils. En aquest treball, hem fusionat la regió 5' completa (-2736/-6) del promotor d' END1 a la seqüència codificant de la ribonucleasa extracel·lular produïda per Bacillus amyloliquefaciens, barnasa. Amb aquesta construcció hem transformat dos plantes model, Arabidopsis i tabac i una planta d'interés agronòmic, la tomata. L'expressió del gen citotòxic barnasa a on END1 és actiu, porta com a conseqüència una degeneració de teixits de l'antera que va inhibir el desenvolupament dels grans de pol·len. Les plantes transgèniques resultants foren androestèrils. Els estudis a nivell histològic de les anteres transgèniques mostraren que els efectes de la barnasa comencen a observar-se molt prompte en el primordi d'antera, quant aquest está constituït només per cèl·lules indiferenciades, i continua observant-se al llarg del desenvolupament de la mateixa. De forma general, al final del programa de desenvolupament, les anteres transgèniques eren de menor mida , la seua morfologia era diferent de la de les anteres silvestres corresponents , i al seu interior s'observava el teixit connectiu col lapsat i una estructura amorfa en lloc de grans de pol·len viable. La possible pèrdua de les cèl·lules parietals primàries per l'acció ribonucleasa podria estar afectant la diferenciació de les cèl·lules esporògenes,contigües en el territori del futur microesporangi. Aquests resultats mostren que el promotor d' END1 podria ser una ferramenta biotecnològica útil en els programes d'obtenció de llavors híbrides de diferents cultius d'interès agronòmic. En el cas particular de la tomata, totes les plantes transgèniques androestèrils produïren fruits partenocàrpics. Aquestresultat mostra que existeix una relació entre l'androstèrilitat i el desenvolupament atònom de l'ovari en eixa espècie. D' altra banda, s' ha realitzat una anàlisi del promotor del gen END1. Per a aquesta anàlisi és realitzaren delecions successives de la regió 5' del gen i els fragments resultants es fusionaren transcripcionalment al gen delator uidA (GUS). Amb estes construccions és transformaren plantes de A. thaliana i es va estudiar l'expressió del gen uidA mitjançant l'assaig histoquímic de l'activitat de la beta-glucuronidasa. Hem vist que el fragment de 366/-6 de la regió 5' ès la seqüència mínima amb capacitat per a dirigir la correcta expressió espacial i temporal de gen END1. La pèrdua de l'expressió de GUS en les anteres de les plantes d'Arabidopsis thaliana transformades amb el fragment de la regió 5' a on és elimina el motiu CArG2, recolza la hipòtesi que END1 podria estar regulat directament pels gens d'identitat d'òrgan de la classe B i C.Este trabajo ha sido financiado por el Ministerio de Ciencia y Tecnología mediante los proyectos BIO2000-0940 y BIO2003-01171. Ha sido posible la realización del mismo gracias a la beca otorgada por las Cortes Valencianas en su programa de ayuda a los países del tercer mundo, a las becas de acción social otorgadas por el Rector de la Universidad Politécnica de Valencia Justo Nieto Nieto y a los contratos de trabajo adjudicados a los proyectos antes mencionados.Roque Mesa, EM. (2004). Análisis funcional y aplicaciones biotecnológicas del promotor del gen END1 de guisante (Pisum sativum L.) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/114847TESI

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

[EN] 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.This work was funded by the grants PID2019-106060RB-I00 and RTI2018-094280-100 from the Spanish Ministry of Science and Innovation (MICINN). We acknowledge the support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)Hamza, R.; Gómez Mena, MC.; Roque Mesa, EM.; Madueño Albi, F.; Beltran Porter, JP.; Cañas Clemente, LA. (2021). PsEND1 Is a Key Player in Pea Pollen Development Through the Modulation of Redox Homeostasis. Frontiers in Plant Science. 12:1-15. https://doi.org/10.3389/fpls.2021.7652771151

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). 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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

SUPERMAN strikes again in legumes

[EN] The SUPERMAN (SUP) gene was described in Arabidopsis thaliana over 30 years ago. SUP was classified as a cadastral gene required to maintain the boundaries between reproductive organs, thus controlling stamen and carpel number in flowers. We summarize the information on the characterization of SUP orthologs in plant species other than Arabidopsis, focusing on the findings for the MtSUP, the ortholog in the legume Medicago truncatula. M. truncatula has been widely used as a model system to study the distinctive developmental traits of this family of plants, such as the existence of compound inflorescence and complex floral development. MtSUP participates in the complex genetic network controlling these developmental processes in legumes, sharing conserved functions with SUP. However, transcriptional divergence between SUP and MtSUP provided context-specific novel functions for a SUPERMAN ortholog in a legume species. MtSUP controls the number of flowers per inflorescence and the number of petals, stamens and carpels regulating the determinacy of ephemeral meristems that are unique in legumes. Results obtained in M. truncatula provided new insights to the knowledge of compound inflorescence and flower development in legumes. Since legumes are valuable crop species worldwide, with high nutritional value and important roles in sustainable agriculture and food security, new information on the genetic control of their compound inflorescence and floral development could be used for plant breeding.This work was supported by a grant from the Spanish Ministry of Science and Innovation (PID2019-106060RB-I00). AR acknowledges a Santiago Grisolía fellowship (GRISOLIA 2017/168) from the Generalitat Valenciana.Rodas, AL.; Roque Mesa, EM.; Hamza, R.; Gómez Mena, MC.; Beltran Porter, JP.; Cañas Clemente, LA. (2023). SUPERMAN strikes again in legumes. Frontiers in Plant Science. 14. https://doi.org/10.3389/fpls.2023.11203421

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

[EN] 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.This research was supported by the Tunisian Ministry of Higher Education, Scientific Research and by a grant from the Spanish Ministry of Science, Innovation and Universities (RTI2018-094280-B-100).Louati, M.; Salazar-Sarasúa, B.; Roque Mesa, EM.; Beltran Porter, JP.; Salhi Hannachi, A.; Gómez Mena, MC. (2021). Isolation and Functional Analysis of a PISTILLATA-like MADS-Box Gene from Argan Tree (Argania spinosa). Plants. 10(8):1-14. https://doi.org/10.3390/plants10081665S11410

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

[EN] 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.This work was supported by grant RTI2018-094280-B-I00 funded by Ministerio de Ciencia e Innovacion (MCIN)/Agencia Estatal de Investigacion (AEI)/10.13039/501100011033 and by Fondo Europeo de Desarrollo Regional (FEDER) `A way of making Europe'. We thank Aureliano Bombarely for his help in the conversion of gene IDs into Solyc identifiers, Diego Orzaez for providing GoldenBraid parts and Maricruz Rochina for expert technical assistance during the project.Salazar-Sarasua, B.; López-Martín, MJ.; Roque Mesa, EM.; Hamza, R.; Cañas Clemente, LA.; Beltran Porter, JP.; Gómez Mena, MC. (2022). The tapetal tissue is essential for the maintenance of redox homeostasis during microgametogenesis in tomato. The Plant Journal. 112(5):1281-1297. https://doi.org/10.1111/tpj.1601412811297112

MtSUPERMAN plays a key role in compound inflorescence and flower development in Medicago truncatula

[EN] Legumes have unique features, such as compound inflorescences and a complex floral ontogeny. Thus, the study of regulatory genes in these species during inflorescence and floral development is essential to understand their role in the evolutionary origin of developmental novelties. The SUPERMAN (SUP) gene encodes a C2H2 zinc-finger transcriptional repressor that regulates the floral organ number in the third and fourth floral whorls of Arabidopsis thaliana. In this work, we present the functional characterization of the Medicago truncatula SUPERMAN (MtSUP) gene based on gene expression analysis, complementation and overexpression assays, and reverse genetic approaches. Our findings provide evidence that MtSUP is the orthologous gene of SUP in M. truncatula. We have unveiled novel functions for a SUP-like gene in eudicots. MtSUP controls not only the number of floral organs in the inner two whorls, but also in the second whorl of the flower. Furthermore, MtSUP regulates the activity of the secondary inflorescence meristem, thus controlling the number of flowers produced. Our work provides insight into the regulatory network behind the compound inflorescence and flower development in this angiosperm family.This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness (MINECO; BIO2016-75485-R). A.L.R. acknowledges a Santiago Grisolia fellowship (GRISOLIA 2017/168) from the Generalitat Valenciana. We thank Dr Beatriz Sabater (IBMCP, Valencia) for valuable suggestions and comments regarding evolutionary biology and Dr Javier Forment, head of the Bioinformatics Core Service at the IBMCP, for providing data from the OrthoVenn web platform. We thank Dr Lynne Yenush (IBMCP, Valencia) for English correction, and Dr Miguel A. Blazquez (IBMCP, Valencia) and Dr Javier Paz-Ares (CNB, Madrid) for critical reading of the manuscript. We would also like to acknowledge the technical assistance of Maria Victoria Palau in the glasshouse and Marisol Gascon in microscopy. DocumentRodas-Méndez, AL.; Roque Mesa, EM.; Hamza, R.; Gómez Mena, MC.; Minguet, E.; Wen, J.; Mysore, KS.... (2021). MtSUPERMAN plays a key role in compound inflorescence and flower development in Medicago truncatula. The Plant Journal. 105(3):816-830. https://doi.org/10.1111/tpj.15075S816830105