Transcriptional Activity of the MADS Box ARLEQUIN/TOMATO AGAMOUS-LIKE1 Gene Is Required for Cuticle Development of Tomato Fruit

[EN] Fruit development and ripening entail key biological and agronomic events, which ensure the appropriate formation and dispersal of seeds and determine productivity and yield quality traits. The MADS box gene ARLEQUIN/TOMATO AGAMOUS-LIKE1 (hereafter referred to as TAGL1) was reported as a key regulator of tomato (Solanum lycopersicum) reproductive development, mainly involved in flower development, early fruit development, and ripening. It is shown here that silencing of the TAGL1 gene (RNA interference lines) promotes significant changes affecting cuticle development, mainly a reduction of thickness and stiffness, as well as a significant decrease in the content of cuticle components (cutin, waxes, polysaccharides, and phenolic compounds). Accordingly, overexpression of TAGL1 significantly increased the amount of cuticle and most of its components while rendering a mechanically weak cuticle. Expression of the genes involved in cuticle biosynthesis agreed with the biochemical and biomechanical features of cuticles isolated from transgenic fruits; it also indicated that TAGL1 participates in the transcriptional control of cuticle development mediating the biosynthesis of cuticle components. Furthermore, cell morphology and the arrangement of epidermal cell layers, on whose activity cuticle formation depends, were altered when TAGL1 was either silenced or constitutively expressed, indicating that this transcription factor regulates cuticle development, probably through the biosynthetic activity of epidermal cells. Our results also support cuticle development as an integrated event in the fruit expansion and ripening processes that characterize fleshy-fruited species such as tomato.This work was supported by the Ministerio de Ciencia e Innovacion (grant nos. BIO2009-11484, AGL2012-32613, AGL2012-40150-C03-01, and AGL2012-40150-C03-02) and by the European Commission through the Junta para la Ampliacion de Estudios-Doc program of the Consejo Superior de Investigaciones Cientificas (to B.P.).Giménez Caminero, ME.; Domínguez, E.; Pineda Chaza, BJ.; Heredia, A.; Moreno Ferrero, V.; Lozano, R.; Angosto, T. (2015). Transcriptional Activity of the MADS Box ARLEQUIN/TOMATO AGAMOUS-LIKE1 Gene Is Required for Cuticle Development of Tomato Fruit. Plant Physiology. 168(3):1036-1048. doi:10.1104/pp.15.00469S10361048168

TOMATO AGAMOUS1 and ARLEQUIN/TOMATO AGAMOUS-LIKE1 MADS-box genes have redundant and divergent functions required for tomato reproductive development

[EN] Within the tomato MADS-box gene family, TOMATO AGAMOUS1 (TAG1) and ARLEQUIN/TOMATO AGAMOUS LIKE1 (hereafter referred to as TAGL1) are, respectively, members of the euAG and PLE lineages of the AGAMOUS clade. They perform crucial functions specifying stamen and carpel development in the flower and controlling late fruit development. To gain insight into the roles of TAG1 and TAGL1 genes and to better understand their functional redundancy and diversification, we characterized single and double RNAi silencing lines of these genes and analyzed expression profiles of regulatory genes involved in reproductive development. Double RNAi lines did show cell abnormalities in stamens and carpels and produced extremely small fruit-like organs displaying some sepaloid features. Expression analyses indicated that TAG1 and TAGL1 act together to repress fourth whorl sepal development, most likely through the MACROCALYX gene. Results also proved that TAG1 and TAGL1 have diversified their functions in fruit development: while TAG1 controls placenta and seed formation, TAGL1 participates in cuticle development and lignin biosynthesis inhibition. It is noteworthy that both TAG1 and double RNAi plants lacked seed development due to abnormalities in pollen formation. This seedless phenotype was not associated with changes in the expression of B-class stamen identity genes Tomato MADS-box 6 and Tomato PISTILLATA observed in silencing lines, suggesting that other regulatory factors should participate in pollen formation. Taken together, results here reported support the idea that both redundant and divergent functions of TAG1 and TAGL1 genes are needed to control tomato reproductive development.This work was supported by the Spanish Ministry of Economy and Competitiveness (Grant Numbers AGL2012-40150-C03-01, AGL2012-40150-C03-02 and AGL2015-64991-C3-1-R); and the European Commission through the JAE-Doc Program of the Spanish National Research Council (CSIC) (Grant Number AGL2012-40150-C03-01 to B.P.).Giménez Caminero, ME.; Castañeda, L.; Pineda Chaza, BJ.; Pan, IL.; Moreno Ferrero, V.; Angosto, T.; Lozano, R. (2016). TOMATO AGAMOUS1 and ARLEQUIN/TOMATO AGAMOUS-LIKE1 MADS-box genes have redundant and divergent functions required for tomato reproductive development. Plant Molecular Biology. 91(4-5):513-531. https://doi.org/10.1007/s11103-016-0485-4S513531914-

Alq mutation increases fruit set rate and allows the maintenance of fruit yield under moderate saline conditions

[EN] Arlequin (Alq) is a gain-of-function mutant whose most relevant feature is that sepals are able to become fruit-like organs due to the ectopic expression of the ALQ-TAGL1 gene. The role of this gene in tomato fruit ripening was previously demonstrated. To discover new functional roles for ALQ-TAGL1, and most particularly its involvement in the fruit set process, a detailed characterization of Alq yield-related traits was performed. Under standard conditions, the Alq mutant showed a much higher fruit set rate than the wild type. A significant percentage of Alq fruits were seedless. The results showed that pollination-independent fruit set in Alq is due to early transition from flower to fruit. Analysis of endogenous hormones in Alq suggests that increased content of cytokinins and decreased level of abscisic acid may account for precocious fruit set. Comparative expression analysis showed relevant changes of several genes involved in cell division, gibberellin metabolism, and the auxin signalling pathway. Since pollination-independent fruit set may be a very useful strategy for maintaining fruit production under adverse conditions, fruit set and yield in Alq plants under moderate salinity were assessed. Interestingly, Alq mutant plants showed a high yield under saline conditions, similar to that of Alq and the wild type under unstressed conditions.This work was supported by the research grants AGL2015-64991-C3-3-R and AGL2015-64991-C3-1-R from the Spanish Ministry of Economy and Competitiveness (MINECO/FEDER). The PhD grant to CRA (BES-2013-063778) was funded by the Spanish Ministry of Economy and Competitiveness.The authors thank Dr Isabel Lopez-Diaz and Dr Esther Carrera for their help in hormone quantification carried out at the Plant Hormone Quantification Service, IBMCP,Valencia, Spain. The authors thank David Harry Rhead for reviewing the manuscript in the English language.Ribelles Alfonso, C.; García Sogo, B.; Yuste-Lisbona, FJ.; Atarés Huerta, A.; Castañeda, L.; Capel, C.; Lozano, R.... (2019). Alq mutation increases fruit set rate and allows the maintenance of fruit yield under moderate saline conditions. Journal of Experimental Botany. 70(20):5731-5744. https://doi.org/10.1093/jxb/erz342S57315744702

The sodium transporter encoded by the HKT1;2 gene modulates sodium/potassium homeostasis in tomato shoots under salinity

[EN] Excessive soil salinity diminishes crop yield and quality. In a previous study in tomato, we identified two closely linked genes encoding HKT1-like transporters, HKT1;1 and HKT1;2, as candidate genes for a major quantitative trait locus (kc7.1) related to shoot Na+/K+ homeostasis - a major salt tolerance trait - using two populations of recombinant inbred lines (RILs). Here, we determine the effectiveness of these genes in conferring improved salt tolerance by using two near-isogenic lines (NILs) that were homozygous for either the Solanum lycopersicum allele (NIL17) or for the Solanum cheesmaniae allele (NIL14) at both HKT1 loci; transgenic lines derived from these NILs in which each HKT1;1 and HKT1;2 had been silenced by stable transformation were also used. Silencing of ScHKT1;2 and SlHKT1;2 altered the leaf Na+/K+ ratio and caused hypersensitivity to salinity in plants cultivated under transpiring conditions, whereas silencing SlHKT1;1/ScHKT1;1 had a lesser effect. These results indicate that HKT1;2 has the more significant role in Na+ homeostasis and salinity tolerance in tomato.We thank Dr Espen Granum for critically reading the manuscript, Maria Isabel Gaspar Vidal and Elena Sanchez Romero for technical assistance, the Instrumental Technical Service at EEZ-CSIC for DNA sequencing and ICP-OES mineral analysis and Michael O'Shea for proofreading the text. In addition, we thank Dr Ana P. Ortega who assisted in preliminary experiments. This work was supported by ERDF-cofinanced grants, AGL2010-17090 and AGL2013-41733-R (A.B.), AGL2015-64991-C3-3-R (V.M.) and AGL2014-56675-R (M.J.A.) from the Spanish "Ministerio de Economia, Industria y Competitividad'; CVI-7558, Proyecto de Excelencia, from Junta de Andalucia (A.B); and the Australian Research Council (ARC) for Centre of Excellence (CE14010008) and Future Fellowship (FT130100709) funding (M.G.). N.J-P. was supported by an FPI program BES-2011-046096 and her stay in M.G.'s lab by a short-stay EEBB-I-14-08682, both from the Spanish from "Ministerio de Economia Industria y Competitividad'. The authors have no conflict of interest to declare.Jaime-Perez, N.; Pineda Chaza, BJ.; García Sogo, B.; Atarés Huerta, A.; Athman, A.; Byrt, CS.; Olias, R.... (2017). The sodium transporter encoded by the HKT1;2 gene modulates sodium/potassium homeostasis in tomato shoots under salinity. Plant Cell & Environment. 40(5):658-671. https://doi.org/10.1111/pce.12883S65867140

Nuevo cultivar con frutos y sépalos convertidos en frutos de alto interés para su consumo fresco y procesado industrial

Número de publicación: 2 341 527 21 Número de solicitud: 200900003 51 Int. Cl.: C12N 15/82 (2006.01) A01H 5/00 (2006.01Nuevo cultivar con frutos y sépalos convertidos en frutos de alto interés para su consumo fresco y procesado industrial. En la presente invención se describen secuencias de nucleótidos capaces de incrementar la expresión de un gen de desarrollo reproductivo lo que tiene como resultado la generación de cultivares con un fruto de alto interés para su consumo fresco y procesado industrial caracterizado por poseer características mejoradas respecto de los cultivares conocidos de variedades comerciales. Estos nuevos cultivares tienen el cáliz de la flor carnoso y convertido en fruto. El fruto verdadero y el cáliz tienen mayores niveles de azúcares y licopeno y un mayor contenido en grados Brix. Además, exhiben una mayor tasa de cuajado de fruto y tienen inhibida la zona de abscisión del fruto, lo que facilita la recolección mecánica.Universidad de Almerí

ENO regulates tomato fruit size through the floral meristem development network

A dramatic evolution of fruit size has accompanied the domestication and improvement of fruit-bearing crop species. In tomato (Solanum lycopersicum), naturally occurring cis-regulatory mutations in the genes of the CLAVATA-WUSCHEL signaling pathway have led to a significant increase in fruit size generating enlarged meristems that lead to flowers with extra organs and bigger fruits. In this work, by combining mapping-by-sequencing and CRISPR/Cas9 genome editing methods, we isolated EXCESSIVE NUMBER OF FLORAL ORGANS (ENO), an AP2/ERF transcription factor which regulates floral meristem activity. Thus, the ENO gene mutation gives rise to plants that yield larger multilocular fruits due to an increased size of the floral meristem. Genetic analyses indicate that eno exhibits synergistic effects with mutations at the LOCULE NUMBER (encoding SlWUS) and FASCIATED (encoding SlCLV3) loci, two central players in the evolution of fruit size in the domestication of cultivated tomatoes. Our findings reveal that an eno mutation causes a substantial expansion of SlWUS expression domains in a flower-specific manner. In vitro binding results show that ENO is able to interact with the GGC-box cis-regulatory element within the SlWUS promoter region, suggesting that ENO directly regulates SlWUS expression domains to maintain floral stem-cell homeostasis. Furthermore, the study of natural allelic variation of the ENO locus proved that a cis-regulatory mutation in the promoter of ENO had been targeted by positive selection during the domestication process, setting up the background for significant increases in fruit locule number and fruit size in modern tomatoes

Approaching the genetic dissection of indirect adventitious organogenesis process in tomato explants

[EN] The screening of 862 T-DNA lines was carried out to approach the genetic dissection of indirect adventitious organogenesis in tomato. Several mutants defective in different phases of adventitious organogenesis, namely callus growth (tdc-1), bud differentiation (tdb-1,-2,-3) and shoot-bud development (tds-1) were identified and characterized. The alteration of the TDC-1 gene blocked callus proliferation depending on the composition of growth regulators in the culture medium. Calli from tds-1 explants differentiated buds but did not develop normal shoots. Histological analysis showed that their abnormal development is due to failure in the organization of normal adventitious shoot meristems. Interestingly, tdc-1 and tds-1 mutant plants were indistinguishable from WT ones, indicating that the respective altered genes play specific roles in cell proliferation from explant cut zones (TDC-1 gene) or in the organization of adventitious shoot meristems (TDS-1 gene). Unlike the previous, plants of the three mutants defective in the differentiation of adventitious shoot-buds (tdb-1,-2,-3) showed multiple changes in vegetative and reproductive traits. Cosegregation analyses revealed the existence of an association between the phenotype of the tdb-3 mutant and a T-DNA insert, which led to the discovery that the SlMAPKKK17 gene is involved in the shoot-bud differentiation process.Vicente Moreno and Rafael Lozano thank the Ministry of Science and Innovation (State Innovation Agency) for granting the projects PID2019-110833RB-C32 and PID2019-110833RB-C31. Benito Pineda's work in the context of this article has been funded by 'Aid for First Research Projects (PAID-06-18)' by the Vicerrectorado de Investigacion, Innovacion y Transferencia de la Universitat Politecnica de Valencia (UPV), Valencia, Spain'. The PhD fellowship for Jorge Sanchez-Lopez and Marybel Jaquez-Gutierrez were funded by the Universidad de Sinaloa and the CONACYT of Mexico.Sanchez-Lopez, J.; Atarés Huerta, A.; Jaquez-Gutierrez, M.; Ortiz-Atienza, A.; Capel, C.; Pineda Chaza, BJ.; García Sogo, B.... (2021). Approaching the genetic dissection of indirect adventitious organogenesis process in tomato explants. Plant Science. 302:1-14. https://doi.org/10.1016/j.plantsci.2020.110721S11430

The tomato mutant ars1 (altered response to salt stress 1) identifies an R1-type MYB transcription factor involved in stomatal closure under salt acclimation

[EN] A screening under salt stress conditions of a T-DNA mutant collection of tomato (Solanum lycopersicum L.) led to the identification of the altered response to salt stress 1 (ars1) mutant, which showed a salt-sensitive phenotype. Genetic analysis of the ars1 mutation revealed that a single T-DNA insertion in the ARS1 gene was responsible of the mutant phenotype. ARS1 coded for an R1-MYB type transcription factor and its expression was induced by salinity in leaves. The mutant reduced fruit yield under salt acclimation while in the absence of stress the disruption of ARS1 did not affect this agronomic trait. The stomatal behaviour of ars1 mutant leaves induced higher Na+ accumulation via the transpiration stream, as the decreases of stomatal conductance and transpiration rate induced by salt stress were markedly lower in the mutant plants. Moreover, the mutation affected stomatal closure in a response mediated by abscisic acid (ABA). The characterization of tomato transgenic lines silencing and overexpressing ARS1 corroborates the role of the gene in regulating the water loss via transpiration under salinity. Together, our results show that ARS1 tomato gene contributes to reduce transpirational water loss under salt stress. Finally, this gene could be interesting for tomato molecular breeding, because its manipulation could lead to improved stress tolerance without yield penalty under optimal culture conditions.This work was funded by a research project (AGL2012-40150-C01/C02/C03) from the Spanish Ministry of Economy and Competitiveness (MINECO). This work was also supported by grant RYC2010-06369 (Ramon y Cajal Programme) from the MINECO to NF-G and grant E-30-2011-0443170 (JAE-Doc Programme) from the Spanish Council of Scientific Research (CSIC) to IE and BP. The authors have no conflict of interests to declareCampos, JF.; Cara, B.; Perez-Martin, F.; Pineda Chaza, BJ.; Egea, I.; Flores, FB.; Fernandez-Garcia, N.... (2016). The tomato mutant ars1 (altered response to salt stress 1) identifies an R1-type MYB transcription factor involved in stomatal closure under salt acclimation. Plant Biotechnology Journal. 14(6):1345-1356. https://doi.org/10.1111/pbi.124981345135614

Albino T-DNA tomato mutant reveals a key function of 1-deoxy-D-xylulose-5-phosphate synthase (DXS1) in plant development and survival

[EN] Photosynthetic activity is indispensable for plant growth and survival and it depends on the synthesis of plastidial isoprenoids as chlorophylls and carotenoids. In the non-mevalonate pathway (MEP), the 1-deoxy-D-xylulose-5-phosphate synthase 1 (DXS1) enzyme has been postulated to catalyze the ratelimiting step in the formation of plastidial isoprenoids. In tomato, the function of DXS1 has only been studied in fruits, and hence its functional relevance during plant development remains unknown. Here we report the characterization of the wls-2297 tomato mutant, whose severe deficiency in chlorophylls and carotenoids promotes an albino phenotype. Additionally, growth of mutant seedlings was arrested without developing vegetative organs, which resulted in premature lethality. Gene cloning and silencing experiments revealed that the phenotype of wls-2297 mutant was caused by 38.6 kb-deletion promoted by a single T-DNA insertion affecting the DXS1 gene. This was corroborated by in vivo and molecular complementation assays, which allowed the rescue of mutant phenotype. Further characterization of tomato plants overexpressing DXS1 and comparative expression analysis indicate that DXS1 may play other important roles besides to that proposed during fruit carotenoid biosynthesis. Taken together, these results demonstrate that DXS1 is essentially required for the development and survival of tomato plants.This work was supported by research grants from the Spanish Ministry of Economy and Competitiveness and the UE-European Regional Development Fund (AGL2015-64991-C3-1-R, and AGL2015-64991-C3-3-R), and Junta de Andalucia (P12-AGR-1482). PhD fellowship to M.G.-A. was funded by the FPU Programme of the Spanish Ministry of Science and Innovation. The authors thank research facilities provided by the Campus de Excelencia Internacional Agroalimentario (CeiA3).Garcia-Alcazar, M.; Giménez Caminero, ME.; Pineda Chaza, BJ.; Capel, C.; García Sogo, B.; Sánchez Martín-Sauceda, S.; Yuste-Lisbona, FJ.... (2017). Albino T-DNA tomato mutant reveals a key function of 1-deoxy-D-xylulose-5-phosphate synthase (DXS1) in plant development and survival. Scientific Reports. 7:1-12. https://doi.org/10.1038/srep45333112

A highly efficient organogenesis protocol based on zeatin riboside for in vitro regeneration of eggplant

[EN] Background Efficient organogenesis induction in eggplant (Solanum melongena L.) is required for multiple in vitro culture applications. In this work, we aimed at developing a universal protocol for efficient in vitro regeneration of eggplant mainly based on the use of zeatin riboside (ZR). We evaluated the effect of seven combinations of ZR with indoleacetic acid (IAA) for organogenic regeneration in five genetically diverse S. melongena and one S. insanum L. accessions using two photoperiod conditions. In addition, the effect of six different concentrations of indolebutyric acid (IBA) in order to promote rooting was assessed to facilitate subsequent acclimatization of plants. The ploidy level of regenerated plants was studied. Results In a first experiment with accessions MEL1 and MEL3, significant (p < 0.05) differences were observed for the four factors evaluated for organogenesis from cotyledon, hypocotyl and leaf explants, with the best results obtained (9 and 11 shoots for MEL1 and MEL3, respectively) using cotyledon tissue, 16 h light / 8 h dark photoperiod conditions, and medium E6 (2 mg/L of ZR and 0 mg/L of IAA). The best combination of conditions was tested in the other four accessions and confirmed its high regeneration efficiency per explant when using both cotyledon and hypocotyl tissues. The best rooting media was R2 (1 mg/L IBA). The analysis of ploidy level revealed that between 25 and 50% of the regenerated plantlets were tetraploid. Conclusions An efficient protocol for organogenesis of both cultivated and wild accessions of eggplant, based on the use of ZR, is proposed. The universal protocol developed may be useful for fostering in vitro culture applications in eggplant requiring regeneration of plants and, in addition, allows developing tetraploid plants without the need of antimitotic chemicals.This research was funded by the Spanish Ministerio de Ciencia, Innovacion y Universidades, Agencia Estatal de Investigacion and Fondo Europeo de Desarrollo Regional (grant RTI-2018-094592-B-100 from MCIU/AEI/FEDER, UE) and by Universitat Politecnica de Valencia. The Spanish Ministerio de Educacion, Cultura y Deporte funded a predoctoral fellowship granted to Edgar Garcia-Fortea (FPU17/02389). The Generalitat Valenciana and Fondo Social Europeo funded a post-doctoral fellowship granted to Mariola Plazas (APOSTD/2018/014). The Japan Society for the Promotion of Science funded a post-doctoral fellowship granted to Pietro Gramazio (FY 2019 Postdoctoral Fellowship for Research in Japan [Standard]). 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