Ethylene is Involved in Symptom Development and Ribosomal Stress of Tomato Plants upon Citrus Exocortis Viroid Infection

[EN] Citrus exocortis viroid (CEVd) is known to cause different symptoms in citrus trees, and its mechanism of infection has been studied in tomato as an experimental host, producing ribosomal stress on these plants. Some of the symptoms caused by CEVd in tomato plants resemble those produced by the phytohormone ethylene. The present study is focused on elucidating the relationship between CEVd infection and ethylene on disease development. To this purpose, the ethylene insensitive Never ripe (Nr) tomato mutants were infected with CEVd, and several aspects such as susceptibility to infection, defensive response, ethylene biosynthesis and ribosomal stress were studied. Phenotypic characterization revealed higher susceptibility to CEVd in these mutants, which correlated with higher expression levels of both defense and ethylene biosynthesis genes, as well as the ribosomal stress marker SlNAC082. In addition, Northern blotting revealed compromised ribosome biogenesis in all CEVd infected plants, particularly in Nr mutants. Our results indicate a higher ethylene biosynthesis in Nr mutants and suggest an important role of this phytohormone in disease development and ribosomal stress caused by viroid infection.Vázquez Prol, F.; López-Gresa, MP.; Rodrigo Bravo, I.; Belles Albert, JM.; Lisón, P. (2020). Ethylene is Involved in Symptom Development and Ribosomal Stress of Tomato Plants upon Citrus Exocortis Viroid Infection. Plants. 9(5):1-15. https://doi.org/10.3390/plants9050582S11595Flores, R., Hernández, C., Alba, A. E. M. de, Daròs, J.-A., & Serio, F. D. (2005). Viroids and Viroid-Host Interactions. Annual Review of Phytopathology, 43(1), 117-139. doi:10.1146/annurev.phyto.43.040204.140243Adkar‐Purushothama, C. R., & Perreault, J. (2019). Current overview on viroid–host interactions. WIREs RNA, 11(2). doi:10.1002/wrna.1570Di Serio, F., Flores, R., Verhoeven, J. T. J., Li, S.-F., Pallás, V., Randles, J. W., … Owens, R. A. (2014). Current status of viroid taxonomy. Archives of Virology, 159(12), 3467-3478. doi:10.1007/s00705-014-2200-6Verhoeven, J. th. j., Jansen, C. C. C., Willemen, T. M., Kox, L. F. F., Owens, R. A., & Roenhorst, J. W. (2004). Natural infections of tomato by Citrus exocortis viroid, Columnea latent viroid, Potato spindle tuber viroid and Tomato chlorotic dwarf viroid. European Journal of Plant Pathology, 110(8), 823-831. doi:10.1007/s10658-004-2493-5López-Gresa, M. P., Lisón, P., Yenush, L., Conejero, V., Rodrigo, I., & Bellés, J. M. (2016). Salicylic Acid Is Involved in the Basal Resistance of Tomato Plants to Citrus Exocortis Viroid and Tomato Spotted Wilt Virus. PLOS ONE, 11(11), e0166938. doi:10.1371/journal.pone.0166938Wang, Y., Wu, J., Qiu, Y., Atta, S., Zhou, C., & Cao, M. (2019). Global Transcriptomic Analysis Reveals Insights into the Response of ‘Etrog’ Citron (Citrus medica L.) to Citrus Exocortis Viroid Infection. Viruses, 11(5), 453. doi:10.3390/v11050453Jia, C., Zhang, L., Liu, L., Wang, J., Li, C., & Wang, Q. (2013). Multiple phytohormone signalling pathways modulate susceptibility of tomato plants to Alternaria alternata f. sp. lycopersici. Journal of Experimental Botany, 64(2), 637-650. doi:10.1093/jxb/ers360Van Loon, L. C., Geraats, B. P. J., & Linthorst, H. J. M. (2006). Ethylene as a modulator of disease resistance in plants. Trends in Plant Science, 11(4), 184-191. doi:10.1016/j.tplants.2006.02.005Bellés, J. M., & Conejero, V. (1989). Ethylene Mediation of the Viroid-Like Syndrome Induced by Ag+Ions inGynura aurantiacaDC Plants. Journal of Phytopathology, 124(4), 275-284. doi:10.1111/j.1439-0434.1989.tb04924.xDubois, M., Van den Broeck, L., & Inzé, D. (2018). The Pivotal Role of Ethylene in Plant Growth. Trends in Plant Science, 23(4), 311-323. doi:10.1016/j.tplants.2018.01.003Yang, S. F., & Hoffman, N. E. (1984). Ethylene Biosynthesis and its Regulation in Higher Plants. Annual Review of Plant Physiology, 35(1), 155-189. doi:10.1146/annurev.pp.35.060184.001103Wang, K. L.-C., Li, H., & Ecker, J. R. (2002). Ethylene Biosynthesis and Signaling Networks. The Plant Cell, 14(suppl 1), S131-S151. doi:10.1105/tpc.001768Han, L., Li, G.-J., Yang, K.-Y., Mao, G., Wang, R., Liu, Y., & Zhang, S. (2010). Mitogen-activated protein kinase 3 and 6 regulate Botrytis cinerea-induced ethylene production in Arabidopsis. The Plant Journal, no-no. doi:10.1111/j.1365-313x.2010.04318.xBellés, J. M., Granell, A., Durán-vila, N., & Conejero, V. (1989). ACC Synthesis as the Activated Step Responsible for the Rise of Ethylene Production Accompanying Citrus Exocortis Viroid Infection in Tomato Plants. Journal of Phytopathology, 125(3), 198-208. doi:10.1111/j.1439-0434.1989.tb01061.xBellés, J. M., Vera, P., Durán-Vila, N., & Conejero, V. (1989). Ethylene production in tomato cultures infected with citrus exocortis viroid (CEV). Canadian Journal of Plant Pathology, 11(3), 256-262. doi:10.1080/07060668909501109Bellés, J. M., & Conejero, V. (1989). Evolution of Ethylene Production, ACC and Conjugated ACC Levels Accompanying Symptom Development in Tomato and Gynura aurantiaca DC Leaves Infected with Citrus Exocortis Viroid (CEV). Journal of Phytopathology, 127(1), 81-85. doi:10.1111/j.1439-0434.1989.tb04506.xBellés, J. M., & Conejero, V. (1991). Suppression by Citrus Exocortis Viroid Infection of the Naturally Occurring Inhibitor of the Conversion of 1-aminocyclopropane-1-carboxylic Acid to Ethylene by Tomato Microsomes. Journal of Phytopathology, 132(3), 245-250. doi:10.1111/j.1439-0434.1991.tb00117.xJu, C., Yoon, G. M., Shemansky, J. M., Lin, D. Y., Ying, Z. I., Chang, J., … Chang, C. (2012). CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis. Proceedings of the National Academy of Sciences, 109(47), 19486-19491. doi:10.1073/pnas.1214848109Aloni, R., Wolf, A., Feigenbaum, P., Avni, A., & Klee, H. J. (1998). The Never ripe Mutant Provides Evidence That Tumor-Induced Ethylene Controls the Morphogenesis ofAgrobacterium tumefaciens-Induced Crown Galls on Tomato Stems1,2. Plant Physiology, 117(3), 841-849. doi:10.1104/pp.117.3.841Klee, H. J. (2004). Ethylene Signal Transduction. Moving beyond Arabidopsis. Plant Physiology, 135(2), 660-667. doi:10.1104/pp.104.040998Chen, Y., Rofidal, V., Hem, S., Gil, J., Nosarzewska, J., Berger, N., … Chervin, C. (2019). Targeted Proteomics Allows Quantification of Ethylene Receptors and Reveals SlETR3 Accumulation in Never-Ripe Tomatoes. Frontiers in Plant Science, 10. doi:10.3389/fpls.2019.01054HU, X., NIE, X., SONG, Y., XIONG, X., & Tai, H. (2011). Ethylene is Involved but Plays a Limited Role in Tomato Chlorotic Dwarf Viroid-Induced Symptom Development in Tomato. Agricultural Sciences in China, 10(4), 544-552. doi:10.1016/s1671-2927(11)60035-7Dı́az, J., ten Have, A., & van Kan, J. A. L. (2002). The Role of Ethylene and Wound Signaling in Resistance of Tomato to Botrytis cinerea  . Plant Physiology, 129(3), 1341-1351. doi:10.1104/pp.001453Lund, S. T., Stall, R. E., & Klee, H. J. (1998). Ethylene Regulates the Susceptible Response to Pathogen Infection in Tomato. The Plant Cell, 10(3), 371-382. doi:10.1105/tpc.10.3.371Tsolakidou, M.-D., Pantelides, lakovos S., Tzima, A. K., Kang, S., Paplomatas, E. J., & Tsaltas, D. (2019). Disruption and Overexpression of the Gene Encoding ACC (1-Aminocyclopropane-1-Carboxylic Acid) Deaminase in Soil-Borne Fungal Pathogen Verticillium dahliae Revealed the Role of ACC as a Potential Regulator of Virulence and Plant Defense. Molecular Plant-Microbe Interactions®, 32(6), 639-653. doi:10.1094/mpmi-07-18-0203-rWięsyk, A., Iwanicka-Nowicka, R., Fogtman, A., Zagórski-Ostoja, W., & Góra-Sochacka, A. (2018). Time-Course Microarray Analysis Reveals Differences between Transcriptional Changes in Tomato Leaves Triggered by Mild and Severe Variants of Potato Spindle Tuber Viroid. Viruses, 10(5), 257. doi:10.3390/v10050257Eiras, M., Nohales, M. A., Kitajima, E. W., Flores, R., & Daròs, J. A. (2010). Ribosomal protein L5 and transcription factor IIIA from Arabidopsis thaliana bind in vitro specifically Potato spindle tuber viroid RNA. Archives of Virology, 156(3), 529-533. doi:10.1007/s00705-010-0867-xDubé, A., Bisaillon, M., & Perreault, J.-P. (2009). Identification of Proteins from Prunus persica That Interact with Peach Latent Mosaic Viroid. Journal of Virology, 83(23), 12057-12067. doi:10.1128/jvi.01151-09Lisón, P., Tárraga, S., López-Gresa, P., Saurí, A., Torres, C., Campos, L., … Rodrigo, I. (2013). A noncoding plant pathogen provokes both transcriptional and posttranscriptional alterations in tomato. PROTEOMICS, 13(5), 833-844. doi:10.1002/pmic.201200286Cottilli, P., Belda-Palazón, B., Adkar-Purushothama, C. R., Perreault, J.-P., Schleiff, E., Rodrigo, I., … Lisón, P. (2019). Citrus exocortis viroid causes ribosomal stress in tomato plants. Nucleic Acids Research, 47(16), 8649-8661. doi:10.1093/nar/gkz679Ohbayashi, I., Lin, C.-Y., Shinohara, N., Matsumura, Y., Machida, Y., Horiguchi, G., … Sugiyama, M. (2017). Evidence for a Role of ANAC082 as a Ribosomal Stress Response Mediator Leading to Growth Defects and Developmental Alterations in Arabidopsis. The Plant Cell, 29(10), 2644-2660. doi:10.1105/tpc.17.00255Mayer, C., & Grummt, I. (2005). Cellular Stress and Nucleolar Function. Cell Cycle, 4(8), 1036-1038. doi:10.4161/cc.4.8.1925Weis, B. L., Kovacevic, J., Missbach, S., & Schleiff, E. (2015). Plant-Specific Features of Ribosome Biogenesis. Trends in Plant Science, 20(11), 729-740. doi:10.1016/j.tplants.2015.07.003Palm, D., Streit, D., Shanmugam, T., Weis, B. L., Ruprecht, M., Simm, S., & Schleiff, E. (2018). Plant-specific ribosome biogenesis factors in Arabidopsis thaliana with essential function in rRNA processing. Nucleic Acids Research, 47(4), 1880-1895. doi:10.1093/nar/gky1261Christoffersen, R. E., & Laties, G. G. (1982). Ethylene regulation of gene expression in carrots. Proceedings of the National Academy of Sciences, 79(13), 4060-4063. doi:10.1073/pnas.79.13.4060Marei, N., & Romani, R. (1971). Ethylene-stimulated Synthesis of Ribosomes, Ribonucleic Acid, and Protein in Developing Fig Fruits. Plant Physiology, 48(6), 806-808. doi:10.1104/pp.48.6.806Spiers, J., Brady, C., Grierson, D., & Lee, E. (1984). Changes in Ribosome Organization and Messenger RNA Abundance in Ripening Tomato Fruits. Functional Plant Biology, 11(3), 225. doi:10.1071/pp9840225Merchante, C., Brumos, J., Yun, J., Hu, Q., Spencer, K. R., Enríquez, P., … Alonso, J. M. (2015). Gene-Specific Translation Regulation Mediated by the Hormone-Signaling Molecule EIN2. Cell, 163(3), 684-697. doi:10.1016/j.cell.2015.09.036Tornero, P., Rodrigo, I., Conejero, V., & Vera, P. (1993). Nucleotide Sequence of a cDNA Encoding a Pathogenesis-Related Protein, P1-p14, from Tomato (Lycopersicon esculentum). Plant Physiology, 102(1), 325-325. doi:10.1104/pp.102.1.325Granell, A., Bellés, J. M., & Conejero, V. (1987). Induction of pathogenesis-related proteins in tomato by citrus exocortis viroid, silver ion and ethephon. Physiological and Molecular Plant Pathology, 31(1), 83-90. doi:10.1016/0885-5765(87)90008-7Mehari, Z. H., Elad, Y., Rav-David, D., Graber, E. R., & Meller Harel, Y. (2015). Induced systemic resistance in tomato (Solanum lycopersicum) against Botrytis cinerea by biochar amendment involves jasmonic acid signaling. Plant and Soil, 395(1-2), 31-44. doi:10.1007/s11104-015-2445-1Nakatsuka, A., Murachi, S., Okunishi, H., Shiomi, S., Nakano, R., Kubo, Y., & Inaba, A. (1998). Differential Expression and Internal Feedback Regulation of 1-Aminocyclopropane-1-Carboxylate Synthase, 1-Aminocyclopropane-1-Carboxylate Oxidase, and Ethylene Receptor Genes in Tomato Fruit during Development and Ripening. Plant Physiology, 118(4), 1295-1305. doi:10.1104/pp.118.4.1295Katsarou, K., Wu, Y., Zhang, R., Bonar, N., Morris, J., Hedley, P. E., … Hornyik, C. (2016). Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection. PLOS ONE, 11(3), e0150711. doi:10.1371/journal.pone.0150711Bellés, J. M., Carbonell, J., & Conejero, V. (1991). Polyamines in Plants Infected by Citrus Exocortis Viroid or Treated with Silver Ions and Ethephon. Plant Physiology, 96(4), 1053-1059. doi:10.1104/pp.96.4.1053O’Donnell, P. J., Jones, J. B., Antoine, F. R., Ciardi, J., & Klee, H. J. (2001). Ethylene-dependent salicylic acid regulates an expanded cell death response to a plant pathogen. The Plant Journal, 25(3), 315-323. doi:10.1046/j.1365-313x.2001.00968.xGómez, G., Martínez, G., & Pallás, V. (2008). Viroid-Induced Symptoms in Nicotiana benthamiana Plants Are Dependent on RDR6 Activity  . Plant Physiology, 148(1), 414-423. doi:10.1104/pp.108.120808Li, G., Meng, X., Wang, R., Mao, G., Han, L., Liu, Y., & Zhang, S. (2012). Dual-Level Regulation of ACC Synthase Activity by MPK3/MPK6 Cascade and Its Downstream WRKY Transcription Factor during Ethylene Induction in Arabidopsis. PLoS Genetics, 8(6), e1002767. doi:10.1371/journal.pgen.1002767Berrocal-Lobo, M., Molina, A., & Solano, R. (2002). Constitutive expression ofETHYLENE-RESPONSE-FACTOR1inArabidopsisconfers resistance to several necrotrophic fungi. The Plant Journal, 29(1), 23-32. doi:10.1046/j.1365-313x.2002.01191.xChowdhury, S., Basu, A., & Kundu, S. (2017). Biotrophy-necrotrophy switch in pathogen evoke differential response in resistant and susceptible sesame involving multiple signaling pathways at different phases. Scientific Reports, 7(1). doi:10.1038/s41598-017-17248-7Shin, S., Lv, J., Fazio, G., Mazzola, M., & Zhu, Y. (2014). Transcriptional regulation of ethylene and jasmonate mediated defense response in apple (Malus domestica) root during Pythium ultimum infection. Horticulture Research, 1(1). doi:10.1038/hortres.2014.53Glazebrook, J. (2005). Contrasting Mechanisms of Defense Against Biotrophic and Necrotrophic Pathogens. Annual Review of Phytopathology, 43(1), 205-227. doi:10.1146/annurev.phyto.43.040204.135923McDowell, J. M., & Dangl, J. L. (2000). Signal transduction in the plant immune response. Trends in Biochemical Sciences, 25(2), 79-82. doi:10.1016/s0968-0004(99)01532-7Heck, S., Grau, T., Buchala, A., Metraux, J.-P., & Nawrath, C. (2003). Genetic evidence that expression of NahG modifies defence pathways independent of salicylic acid biosynthesis in the Arabidopsis-Pseudomonas syringae pv. tomato interaction. The Plant Journal, 36(3), 342-352. doi:10.1046/j.1365-313x.2003.01881.xConejero, V., & Granell, A. (1986). Stimulation of a viroid-like syndrome and the impairment of viroid infection in Gynura aurantiaca plants by treatment with silver ions. Physiological and Molecular Plant Pathology, 29(3), 317-323. doi:10.1016/s0048-4059(86)80048-0Yan, S., & Dong, X. (2014). Perception of the plant immune signal salicylic acid. Current Opinion in Plant Biology, 20, 64-68. doi:10.1016/j.pbi.2014.04.006Fu, Z. Q., Yan, S., Saleh, A., Wang, W., Ruble, J., Oka, N., … Dong, X. (2012). NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants. Nature, 486(7402), 228-232. doi:10.1038/nature11162Schott-Verdugo, S., Müller, L., Classen, E., Gohlke, H., & Groth, G. (2019). Structural Model of the ETR1 Ethylene Receptor Transmembrane Sensor Domain. Scientific Reports, 9(1). doi:10.1038/s41598-019-45189-wClark, D. G., Gubrium, E. K., Barrett, J. E., Nell, T. A., & Klee, H. J. (1999). Root Formation in Ethylene-Insensitive Plants. Plant Physiology, 121(1), 53-60. doi:10.1104/pp.121.1.53Rodrı́guez, F. I., Esch, J. J., Hall, A. E., Binder, B. M., Schaller, G. E., & Bleecker, A. B. (1999). A Copper Cofactor for the Ethylene Receptor ETR1 from Arabidopsis. Science, 283(5404), 996-998. doi:10.1126/science.283.5404.996Schaller, G. E., Ladd, A. N., Lanahan, M. B., Spanbauer, J. M., & Bleecker, A. B. (1995). The Ethylene Response Mediator ETR1 from Arabidopsis Forms a Disulfide-linked Dimer. Journal of Biological Chemistry, 270(21), 12526-12530. doi:10.1074/jbc.270.21.12526Gao, Z., & Schaller, G. E. (2009). The role of receptor interactions in regulating ethylene signal transduction. Plant Signaling & Behavior, 4(12), 1152-1153. doi:10.4161/psb.4.12.9943Gao, Z., Wen, C.-K., Binder, B. M., Chen, Y.-F., Chang, J., Chiang, Y.-H., … Schaller, G. E. (2008). Heteromeric Interactions among Ethylene Receptors Mediate Signaling in Arabidopsis. Journal of Biological Chemistry, 283(35), 23801-23810. doi:10.1074/jbc.m800641200Grefen, C., Städele, K., Růžička, K., Obrdlik, P., Harter, K., & Horák, J. (2008). Subcellular Localization and In Vivo Interactions of the Arabidopsis thaliana Ethylene Receptor Family Members. Molecular Plant, 1(2), 308-320. doi:10.1093/mp/ssm015Kim, H. J., Park, J.-H., Kim, J., Kim, J. J., Hong, S., Kim, J., … Hwang, D. (2018). Time-evolving genetic networks reveal a NAC troika that negatively regulates leaf senescence in Arabidopsis. Proceedings of the National Academy of Sciences, 115(21), E4930-E4939. doi:10.1073/pnas.1721523115Semancik, J. S., Roistacher, C. N., Rivera-Bustamante, R., & Duran-Vila, N. (1988). Citrus Cachexia Viroid, a New Viroid of Citrus: Relationship to Viroids of the Exocortis Disease Complex. Journal of General Virology, 69(12), 3059-3068. doi:10.1099/0022-1317-69-12-3059Campos, L., Granell, P., Tárraga, S., López-Gresa, P., Conejero, V., Bellés, J. M., … Lisón, P. (2014). Salicylic acid and gentisic acid induce RNA silencing-related genes and plant resistance to RNA pathogens. Plant Physiology and Biochemistry, 77, 35-43. doi:10.1016/j.plaphy.2014.01.016Adkar-Purushothama, C. R., Brosseau, C., Giguère, T., Sano, T., Moffett, P., & Perreault, J.-P. (2015). Small RNA Derived from the Virulence Modulating Region of the Potato spindle tuber viroid Silences callose synthase Genes of Tomato Plants. The Plant Cell, 27(8), 2178-2194. doi:10.1105/tpc.15.00523LAEMMLI, U. K. (1970). Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 227(5259), 680-685. doi:10.1038/227680a

Salicylic Acid Is Involved in the Basal Resistance of Tomato Plants to Citrus Exocortis Viroid and Tomato Spotted Wilt Virus

[EN] Tomato plants expressing the NahG transgene, which prevents accumulation of endogenous salicylic acid (SA), were used to study the importance of the SA signalling pathway in basal defence against Citrus Exocortis Viroid (CEVd) or Tomato Spotted Wilt Virus (TSWV). The lack of SA accumulation in the CEVd- or TSWV-infected NahG tomato plants led to an early and dramatic disease phenotype, as compared to that observed in the corresponding parental Money Maker. Addition of acibenzolar-S-methyl, a benzothiadiazole (BTH), which activates the systemic acquired resistance pathway downstream of SA signalling, improves resistance of NahG tomato plants to CEVd and TSWV. CEVd and TSWV inoculation induced the accumulation of the hydroxycinnamic amides p-coumaroyltyramine, feruloyltyramine, caffeoylputrescine, and feruloylputrescine, and the defence related proteins PR1 and P23 in NahG plants earlier and with more intensity than in Money Maker plants, indicating that SA is not essential for the induction of these plant defence metabolites and proteins. In addition, NahG plants produced very high levels of ethylene upon CEVd or TSWV infection when compared with infected Money Maker plants, indicating that the absence of SA produced additional effects on other metabolic pathways. This is the first report to show that SA is an important component of basal resistance of tomato plants to both CEVd and TSWV, indicating that SA-dependent defence mechanisms play a key role in limiting the severity of symptoms in CEVd- and TSWV-infected NahG tomato plants.This work was supported by grant BIO2012-33419 from the Spanish Ministry of Economy and Competitiveness received by JMB. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.López-Gresa, MP.; Lisón, P.; Yenush, L.; Conejero Tomás, V.; Rodrigo Bravo, I.; Belles Albert, JM. (2016). Salicylic Acid Is Involved in the Basal Resistance of Tomato Plants to Citrus Exocortis Viroid and Tomato Spotted Wilt Virus. PLoS ONE. 11(11). https://doi.org/10.1371/journal.pone.0166938S111

Tomato glycosyltransferase Twi1 plays a role in flavonoid glycosylation and defence against virus

[EN] Background: Secondary metabolites play an important role in the plant defensive response. They are produced as a defence mechanism against biotic stress by providing plants with antimicrobial and antioxidant weapons. In higher plants, the majority of secondary metabolites accumulate as glycoconjugates. Glycosylation is one of the commonest modifications of secondary metabolites, and is carried out by enzymes called glycosyltransferases. Results: Here we provide evidence that the previously described tomato wound and pathogen-induced glycosyltransferase Twi1 displays in vitro activity toward the coumarins scopoletin, umbelliferone and esculetin, and the flavonoids quercetin and kaempferol, by uncovering a new role of this gene in plant glycosylation. To test its activity in vivo, Twi1-silenced transgenic tomato plants were generated and infected with Tomato spotted wilt virus. The Twi1- silenced plants showed a differential accumulation of Twi1 substrates and enhanced susceptibility to the virus. Conclusions: Biochemical in vitro assays and transgenic plants generation proved to be useful strategies to assign a role of tomato Twi1 in the plant defence response. Twi1 glycosyltransferase showed to regulate quercetin and kaempferol levels in tomato plants, affecting plant resistance to viral infection.This work was supported by grant BIO2012-33419 from the Direccion General de Programas y Transferencia de Conocimiento, Spanish Ministry of Science and Innovation, and grant AICO/2017/048 from the Valencian Local Government (Generalitat Valenciana, Spain). LC was supported by a predoctoral fellowship (ACIF/2010/231) from the Valencian Local Government (Generalitat Valenciana, Spain). None of the funding bodies was involved in the design of the study, the collection, analysis, and interpretation of the data, nor in the writing of the manuscript, which was made entirely by the authors.Campos Beneyto, L.; López-Gresa, MP.; Fuertes, D.; Belles Albert, JM.; Rodrigo Bravo, I.; Lisón, P. (2019). Tomato glycosyltransferase Twi1 plays a role in flavonoid glycosylation and defence against virus. BMC Plant Biology. 19:1-17. https://doi.org/10.1186/s12870-019-2063-9S11719Kliebenstein DJ. Secondary metabolites and plant/environment interactions: a view through Arabidopsis thaliana tinged glasses. Plant Cell Environ. 2004;27(6):675–84.Yang L, Wen K-S, Ruan X, Zhao Y-X, Wei F, Wang Q. Response of plant secondary metabolites to environmental factors. Molecules (Basel, Switzerland). 2018;23(4):762.D'Auria JC, Gershenzon J. The secondary metabolism of Arabidopsis thaliana: growing like a weed. Curr Opin Plant Biol. 2005;8(3):308–16.Dixon RA, Paiva NL. Stress-induced phenylpropanoid metabolism. Plant Cell. 1995;7(7):1085–97.Kefeli VI, Kalevitch M, Borsari B. Phenolic cycle in plants and environment. J Cell Mol Biol. 2003;2:13–8.Klessig DF, Choi HW, Dempsey DA. Systemic acquired resistance and salicylic acid: past, present, and future. Mol Plant-Microbe Interact. 2018;31(9):871–88.He XZ, Dixon RA. Genetic manipulation of isoflavone 7-O-methyltransferase enhances biosynthesis of 4′-O-methylated isoflavonoid phytoalexins and disease resistance in alfalfa. Plant Cell. 2000;12(9):1689–702.Rice-Evans C, Miller N, Paganga G. Antioxidant properties of phenolic compounds. Trends Plant Sci. 1997;2(4):152–9.Gnonlonfin GJB, Sanni A, Brimer L. Review Scopoletin – a coumarin phytoalexin with medicinal properties. Crit Rev Plant Sci. 2012;31(1):47–56.Mierziak J, Kostyn K, Kulma A. Flavonoids as important molecules of plant interactions with the environment. Molecules (Basel, Switzerland). 2014;19(10):16240–65.Liu X, Lin C, Ma X, Tan Y, Wang J, Zeng M. Functional characterization of a flavonoid glycosyltransferase in sweet orange (Citrus sinensis). Front Plant Sci. 2018;9:166.Baidez AG, Gomez P, Del Rio JA, Ortuno A. Dysfunctionality of the xylem in Olea europaea L. plants associated with the infection process by Verticillium dahliae Kleb. Role of phenolic compounds in plant defense mechanism. J Agric Food Chem. 2007;55(9):3373–7.El Hadrami A, Adam LR, Daayf F. Biocontrol treatments confer protection against Verticillium dahliae infection of potato by inducing antimicrobial metabolites. Mol Plant-Microbe Interact. 2011;24(3):328–35.Hernandez I, Alegre L, Van Breusegem F, Munne-Bosch S. How relevant are flavonoids as antioxidants in plants? Trends Plant Sci. 2009;14(3):125–32.Agati G, Azzarello E, Pollastri S, Tattini M. Flavonoids as antioxidants in plants: location and functional significance. Plant Sci. 2012;196:67–76.Wang J, Hou B. Glycosyltransferases: key players involved in the modification of plant secondary metabolites. Front Biol China. 2009;4(1):39–46.Bowles D, Isayenkova J, Lim EK, Poppenberger B. Glycosyltransferases: managers of small molecules. Curr Opin Plant Biol. 2005;8(3):254–63.Lim EK, Bowles DJ. A class of plant glycosyltransferases involved in cellular homeostasis. EMBO J. 2004;23(15):2915–22.Gachon CM, Langlois-Meurinne M, Saindrenan P. Plant secondary metabolism glycosyltransferases: the emerging functional analysis. Trends Plant Sci. 2005;10(11):542–9.Ghose K, Selvaraj K, McCallum J, Kirby CW, Sweeney-Nixon M, Cloutier SJ, Deyholos M, Datla R, Fofana B. Identification and functional characterization of a flax UDP-glycosyltransferase glucosylating secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG). BMC Plant Biol. 2014;14:82.Li Y, Baldauf S, Lim EK, Bowles DJ. Phylogenetic analysis of the UDP-glycosyltransferase multigene family of Arabidopsis thaliana. J Biol Chem. 2001;276(6):4338–43.Vogt T, Jones P. Glycosyltransferases in plant natural product synthesis: characterization of a supergene family. Trends Plant Sci. 2000;5(9):380–6.Le Roy J, Huss B, Creach A, Hawkins S, Neutelings G. Glycosylation is a major regulator of phenylpropanoid availability and biological activity in plants. Front Plant Sci. 2016;7:735.Lim EK, Doucet CJ, Li Y, Elias L, Worrall D, Spencer SP, Ross J, Bowles DJ. The activity of Arabidopsis glycosyltransferases toward salicylic acid, 4-hydroxybenzoic acid, and other benzoates. J Biol Chem. 2002;277(1):586–92.Horvath DM, Chua NH. Identification of an immediate-early salicylic acid-inducible tobacco gene and characterization of induction by other compounds. Plant Mol Biol. 1996;31(5):1061–72.Fraissinet-Tachet L, Baltz R, Chong J, Kauffmann S, Fritig B, Saindrenan P. Two tobacco genes induced by infection, elicitor and salicylic acid encode glucosyltransferases acting on phenylpropanoids and benzoic acid derivatives, including salicylic acid. FEBS Lett. 1998;437(3):319–23.Chong J, Baltz R, Schmitt C, Beffa R, Fritig B, Saindrenan P. Downregulation of a pathogen-responsive tobacco UDP-Glc:phenylpropanoid glucosyltransferase reduces scopoletin glucoside accumulation, enhances oxidative stress, and weakens virus resistance. Plant Cell. 2002;14(5):1093–107.Langlois-Meurinne M, Gachon CMM, Saindrenan P. Pathogen-responsive expression of glycosyltransferase genes UGT73B3 and UGT73B5 is necessary for resistance to Pseudomonas syringae pv tomato in Arabidopsis. Plant Physiol. 2005;139(4):1890–901.Lee BJ, Kim SK, Choi SB, Bae J, Kim KJ, Kim YJ, Paek KH. Pathogen-inducible CaUGT1 is involved in resistance response against TMV infection by controlling salicylic acid accumulation. FEBS Lett. 2009;583(13):2315–20.Simon C, Langlois-Meurinne M, Didierlaurent L, Chaouch S, Bellvert F, Massoud K, Garmier M, Thareau V, Comte G, Noctor G, et al. The secondary metabolism glycosyltransferases UGT73B3 and UGT73B5 are components of redox status in resistance of Arabidopsis to Pseudomonas syringae pv. tomato. Plant Cell Environ. 2014;37(5):1114–29.Huang XX, Zhu GQ, Liu Q, Chen L, Li YJ, Hou BK. Modulation of plant salicylic acid-associated immune responses via glycosylation of dihydroxybenzoic acids. Plant Physiol. 2018;176(4):3103–19.Gachon C, Baltz R, Saindrenan P. Over-expression of a scopoletin glucosyltransferase in Nicotiana tabacum leads to precocious lesion formation during the hypersensitive response to tobacco mosaic virus but does not affect virus resistance. Plant Mol Biol. 2004;54(1):137–46.Langenbach C, Campe R, Schaffrath U, Goellner K, Conrath U. UDP-glucosyltransferase UGT84A2/BRT1 is required for Arabidopsis nonhost resistance to the Asian soybean rust pathogen Phakopsora pachyrhizi. New Phytol. 2013;198(2):536–45.Song C, Gu L, Liu J, Zhao S, Hong X, Schulenburg K, Schwab W. Functional characterization and substrate promiscuity of UGT71 glycosyltransferases from strawberry (Fragaria x ananassa). Plant Cell Physiol. 2015;56(12):2478–93.Tárraga S, Lisón P, López-Gresa MP, Torres C, Rodrigo I, Bellés JM, Conejero V. Molecular cloning and characterization of a novel tomato xylosyltransferase specific for gentisic acid. J Exp Bot. 2010;61(15):4325–38.von Saint PV, Zhang W, Kanawati B, Geist B, Faus-Kessler T, Schmitt-Kopplin P, Schaffner AR. The Arabidopsis glucosyltransferase UGT76B1 conjugates isoleucic acid and modulates plant defense and senescence. Plant Cell. 2011;23(11):4124–45.Kim JH, Kim BG, Ko JH, Lee Y, Hur H-G, Lim Y, Ahn J-H. Molecular cloning, expression, and characterization of a flavonoid glycosyltransferase from Arabidopsis thaliana. Plant Sci. 2006;170(4):897–903.Griesser M, Vitzthum F, Fink B, Bellido ML, Raasch C, Munoz-Blanco J, Schwab W. Multi-substrate flavonol O-glucosyltransferases from strawberry (Fragaria x ananassa) achene and receptacle. J Exp Bot. 2008;59(10):2611–25.Rehman HM, Nawaz MA, Shah ZH, Ludwig-Müller J, Chung G, Ahmad MQ, Yang SH, Lee SI. Comparative genomic and transcriptomic analyses of Family-1 UDP glycosyltransferase in three Brassica species and Arabidopsis indicates stress-responsive regulation. Sci Rep. 2018;8(1):1875.Sun Y, Ji K, Liang B, Du Y, Jiang L, Wang J, Kai W, Zhang Y, Zhai X, Chen P, et al. Suppressing ABA uridine diphosphate glucosyltransferase (SlUGT75C1) alters fruit ripening and the stress response in tomato. Plant J. 2017;91(4):574–89.O'Donnell PJ, Truesdale MR, Calvert CM, Dorans A, Roberts MR, Bowles DJ. A novel tomato gene that rapidly responds to wound- and pathogen-related signals. Plant J. 1998;14(1):137–42.Lee HI, Raskin I. Purification, cloning, and expression of a pathogen inducible UDP-glucose: Salicylic acid glucosyltransferase from tobacco. J Biol Chem. 1999;274(51):36637–42.Taguchi G, Imura H, Maeda Y, Kodaira R, Hayashida N, Shimosaka M, Okazaki M. Purification and characterization of UDP-glucose:hydroxycoumarin 7-O-glucosyltransferase, with broad substrate specificity from tobacco cultured cells. Plant Sci. 2000;157(1):105–12.Jackson RG, Lim EK, Li Y, Kowalczyk M, Sandberg G, Hoggett J, Ashford DA, Bowles DJ. Identification and biochemical characterization of an Arabidopsis indole-3-acetic acid glucosyltransferase. J Biol Chem. 2001;276(6):4350–6.Isayenkova J, Wray V, Nimtz M, Strack D, Vogt T. Cloning and functional characterisation of two regioselective flavonoid glucosyltransferases from Beta vulgaris. Phytochemistry. 2006;67(15):1598–612.Seto Y, Hamada S, Matsuura H, Matsushige M, Satou C, Takahashi K, Masuta C, Ito H, Matsui H, Nabeta K. Purification and cDNA cloning of a wound inducible glucosyltransferase active toward 12-hydroxy jasmonic acid. Phytochemistry. 2009;70(3):370–9.Li X, Svedin E, Mo H, Atwell S, Dilkes BP, Chapple C. Exploiting natural variation of secondary metabolism identifies a gene controlling the glycosylation diversity of dihydroxybenzoic acids in Arabidopsis thaliana. Genetics. 2014;198(3):1267–76.Dewitte G, Walmagh M, Diricks M, Lepak A, Gutmann A, Nidetzky B, Desmet T. Screening of recombinant glycosyltransferases reveals the broad acceptor specificity of stevia UGT-76G1. J Biotechnol. 2016;233:49–55.Garcia D, Sanier C, Macheix JJ, D'Auzac J. Accumulation of scopoletin in Hevea brasiliensis infected by Microcyclus ulei (P. Henn.) V. ARX and evaluation of its fungitoxicity for three leaf pathogens of rubber tree. Physiol Mol Plant Pathol. 1995;47(4):213–23.Baillieul F, de Ruffray P, Kauffmann S. Molecular cloning and biological activity of alpha-, beta-, and gamma-megaspermin, three elicitins secreted by Phytophthora megasperma H20. Plant Physiol. 2003;131(1):155–66.Shimizu B, Miyagawa H, Ueno T, Sakata K, Watanabe K, Ogawa K. Morning glory systemically accumulates scopoletin and scopolin after interaction with Fusarium oxysporum. Z Naturforsch C. 2005;60(1–2):83–90.Ogawa K. Studies on Fusarium wilt of sweet potato (Ipomoea batatas L.). Bull Natl Agri Res Center Jpn. 1988;10:127–61.Tanguy J, Martin C. Phenolic compounds and the hypersensitivity reaction in Nicotiana tabacum infected with tobacco mosaic virus. Phytochemistry. 1972;11(1):19–28.Churngchow N, Rattarasarn M. The elicitin secreted by Phytophthora palmivora, a rubber tree pathogen. Phytochemistry. 2000;54(1):33–8.Goy PA, Signer H, Reist R, Aichholz R, Blum W, Schmidt E, Kessmann H. Accumulation of scopoletin is associated with the high disease resistance of the hybrid Nicotiana glutinosa x Nicotiana debneyi. Planta. 1993;191(2):200–6.Sun H, Wang L, Zhang B, Ma J, Hettenhausen C, Cao G, Sun G, Wu J, Wu J. Scopoletin is a phytoalexin against Alternaria alternata in wild tobacco dependent on jasmonate signalling. J Exp Bot. 2014;65(15):4305–15.Kai K, Shimizu B, Mizutani M, Watanabe K, Sakata K. Accumulation of coumarins in Arabidopsis thaliana. Phytochemistry. 2006;67(4):379–86.Sim M-O, Lee H-I, Ham JR, Seo K-I, Lee M-K. Long-term supplementation of esculetin ameliorates hepatosteatosis and insulin resistance partly by activating AdipoR2–AMPK pathway in diet-induced obese mice. J Funct Foods. 2015;15:160–71.Mazimba O. Umbelliferone: sources, chemistry and bioactivities review. Bull Faculty Pharmacy Cairo Univ. 2017;55(2):223–32.Carpinella MC, Ferrayoli CG, Palacios SM. Antifungal synergistic effect of scopoletin, a hydroxycoumarin isolated from Melia azedarach L. fruits. J Agric Food Chem. 2005;53(8):2922–7.Chen T, Guo Q, Wang H, Zhang H, Wang C, Zhang P, Meng S, Li Y, Ji H, Yan T. Effects of esculetin on lipopolysaccharide (LPS)-induced acute lung injury via regulation of RhoA/Rho Kinase/NF-кB pathways in vivo and in vitro. Free Radic Res. 2015;49(12):1459–68.Prabakaran D, Ashokkumar N. Protective effect of esculetin on hyperglycemia-mediated oxidative damage in the hepatic and renal tissues of experimental diabetic rats. Biochimie. 2013;95(2):366–73.Sheyn U, Rosenwasser S, Ben-Dor S, Porat Z, Vardi A. Modulation of host ROS metabolism is essential for viral infection of a bloom-forming coccolithophore in the ocean. ISME J. 2016;10(7):1742–54.Bellés JM, Garro R, Fayos J, Navarro P, Primo J, Conejero V. Gentisic acid as a pathogen-inducible signal, additional to salicylic acid for activation of plant defenses in tomato. Mol Plant-Microbe Interact. 1999;12(3):227–35.Fayos J, Bellés JM, López-Gresa MP, Primo J, Conejero V. Induction of gentisic acid 5-O-beta-D-xylopyranoside in tomato and cucumber plants infected by different pathogens. Phytochemistry. 2006;67(2):142–8.Bartsch M, Bednarek P, Vivancos PD, Schneider B, von Roepenack-Lahaye E, Foyer CH, Kombrink E, Scheel D, Parker JE. Accumulation of isochorismate-derived 2,3-dihydroxybenzoic 3-O-beta-D-xyloside in arabidopsis resistance to pathogens and ageing of leaves. J Biol Chem. 2010;285(33):25654–65.Dachineni R, Kumar DR, Callegari E, Kesharwani SS, Sankaranarayanan R, Seefeldt T, Tummala H, Bhat GJ. Salicylic acid metabolites and derivatives inhibit CDK activity: Novel insights into aspirin's chemopreventive effects against colorectal cancer. Int J Oncol. 2017;51(6):1661–73.Lorenc-Kukula K, Zuk M, Kulma A, Czemplik M, Kostyn K, Skala J, Starzycki M, Szopa J. Engineering flax with the GT family 1 Solanum sogarandinum glycosyltransferase SsGT1 confers increased resistance to Fusarium infection. J Agric Food Chem. 2009;57(15):6698–705.Song JT, Koo YJ, Seo HS, Kim MC, Choi YD, Kim JH. Overexpression of AtSGT1, an Arabidopsis salicylic acid glucosyltransferase, leads to increased susceptibility to Pseudomonas syringae. Phytochemistry. 2008;69(5):1128–34.Hirade Y, Kotoku N, Terasaka K, Saijo-Hamano Y, Fukumoto A, Mizukami H. Identification and functional analysis of 2-hydroxyflavanone C-glucosyltransferase in soybean (Glycine max). FEBS Lett. 2015;589(15):1778–86.Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr Rev. 1998;56(11):317–33.Ross JA, Kasum CM. Dietary flavonoids: bioavailability, metabolic effects, and safety. Annu Rev Nutr. 2002;22:19–34.Wang Y, Chen S, Yu O. Metabolic engineering of flavonoids in plants and microorganisms. Appl Microbiol Biotechnol. 2011;91(4):949–56.Treutter D. Significance of flavonoids in plant resistance: a review. Environ Chem Lett. 2006;4(3):147.Bollina V, Kushalappa AC. In vitro inhibition of trichothecene biosynthesis in Fusarium graminearum by resistance-related endogenous metabolites identified in barley. Mycology. 2011;2(4):291–6.Bilska K, Stuper-Szablewska K, Kulik T, Busko M, Zaluski D, Jurczak S, Perkowski J. Changes in phenylpropanoid and trichothecene production by Fusarium culmorum and F. graminearum sensu stricto via exposure to flavonoids. Toxins. 2018;10(3):110–22.French CJ, Elder M, Leggett F, Ibrahim RK, Neil Towers GH. Flavonoids inhibit infectivity of tobacco mosaic virus. Can J Plant Pathol. 1991;13(1):1–6.Ko CH, Shen SC, Hsu CS, Chen YC. Mitochondrial-dependent, reactive oxygen species-independent apoptosis by myricetin: roles of protein kinase C, cytochrome c, and caspase cascade. Biochem Pharmacol. 2005;69(6):913–27.López-Gresa MP, Torres C, Campos L, Lisón P, Rodrigo I, Bellés JM, Conejero V. Identification of defence metabolites in tomato plants infected by the bacterial pathogen Pseudomonas syringae. Environ Exp Bot. 2011;74:216–28.Chitarrini G, Nobili C, Pinzari F, Antonini A, De Rossi P, Del Fiore A, Procacci S, Tolaini V, Scala V, Scarpari M, et al. Buckwheat achenes antioxidant profile modulates Aspergillus flavus growth and aflatoxin production. Int J Food Microbiol. 2014;189:1–10.Bollina V, Kumaraswamy GK, Kushalappa AC, Choo TM, Dion Y, Rioux S, Faubert D, Hamzehzarghani H. Mass spectrometry-based metabolomics application to identify quantitative resistance-related metabolites in barley against Fusarium head blight. Mol Plant Pathol. 2010;11(6):769–82.Gunnaiah R, Kushalappa AC, Duggavathi R, Fox S, Somers DJ. Integrated metabolo-proteomic approach to decipher the mechanisms by which wheat QTL (Fhb1) contributes to resistance against Fusarium graminearum. PLoS One. 2012;7(7):e40695.Huang F-C, Giri A, Daniilidis M, Sun G, Härtl K, Hoffmann T, Schwab W. Structural and functional analysis of UGT92G6 suggests an evolutionary link between mono- and disaccharide glycoside-forming transferases. Plant Cell Physiol. 2018;59(4):862–75.Dhaubhadel S, Farhangkhoee M, Chapman R. Identification and characterization of isoflavonoid specific glycosyltransferase and malonyltransferase from soybean seeds. J Exp Bot. 2008;59(4):981–94.Bellés JM, Garro R, Pallás V, Fayos J, Rodrigo I, Conejero V. Accumulation of gentisic acid as associated with systemic infections but not with the hypersensitive response in plant-pathogen interactions. Planta. 2006;223(3):500–11.Gu YQ, Yang C, Thara VK, Zhou J, Martin GB. Pti4 is induced by ethylene and salicylic acid, and its product is phosphorylated by the Pto kinase. Plant Cell. 2000;12(5):771–86.Ntoukakis V, Mucyn TS, Gimenez-Ibanez S, Chapman HC, Gutierrez JR, Balmuth AL, Jones AM, Rathjen JP. Host inhibition of a bacterial virulence effector triggers immunity to infection. Science. 2009;324(5928):784–7.López-Gresa MP, Lisón P, Campos L, Rodrigo I, Rambla JL, Granell A, Conejero V, Bellés JM. A non-targeted metabolomics approach unravels the VOCs associated with the tomato immune response against Pseudomonas syringae. Front Plant Sci. 2017;8:1188.Soler S, Díez MJ, Roselló S, Nuez F. Movement and distribution of tomato spotted wilt virus in resistant and susceptible accessions of Capsicum spp. Can J Plant Pathol. 1999;21(4):317–25.Campos L, Granell P, Tárraga S, López-Gresa P, Conejero V, Bellés JM, Rodrigo I, Lisón P. Salicylic acid and gentisic acid induce RNA silencing-related genes and plant resistance to RNA pathogens. Plant Physiol Biochem. 2014;77:35–43.Nakagawa T, Suzuki T, Murata S, Nakamura S, Hino T, Maeo K, Tabata R, Kawai T, Tanaka K, Niwa Y, et al. Improved Gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci Biotechnol Biochem. 2007;71(8):2095–100.Helliwell C, Waterhouse P. Constructs and methods for high-throughput gene silencing in plants. Methods. 2003;30(4):289–95.Lakatos L, Szittya G, Silhavy D, Burgyan J. Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses. EMBO J. 2004;23(4):876–84.Ellul P, Garcia-Sogo B, Pineda B, Rios G, Roig LA, Moreno V. The ploidy level of transgenic plants in Agrobacterium-mediated transformation of tomato cotyledons (Lycopersicon esculentum Mill.) is genotype and procedure dependent. Theor Appl Genet. 2003;106(2):231–8.Yalpani N, Schulz M, Davis MP, Balke NE. Partial purification and properties of an inducible uridine 5′-diphosphate-glucose-salicylic acid glucosyltransferase from oat roots. Plant Physiol. 1992;100(1):457–63.Campos L, Lisón P, López-Gresa MP, Rodrigo I, Zacarés L, Conejero V, Bellés JM. Transgenic tomato plants overexpressing tyramine N-hydroxycinnamoyltransferase exhibit elevated hydroxycinnamic acid amide levels and enhanced resistance to Pseudomonas syringae. Mol Plant-Microbe Interact. 2014;27(10):1159–69.Conejero V, Semancik J. Analysis of the proteins in crude plant extracts by polyacrylamide gel electrophoresis. Phytopathology. 1977;67:1424–6.Yalpani N, Leon J, Lawton

(Z)-3-Hexenyl Butyrate induces stomata closure and ripening in Vitis vinifera

[EN] Agronomy solutions for modifying pre-harvest grape ripening are needed for a more sustainable viticulture. Field experiments were performed inVitis viniferaL. vines to study the effect of the previously described stomata-closing compound (Z)-3-hexenyl butyrate (HB). Exogenous treatments at different doses were periodically carried out using a randomized block design. Firstly, we observed that HB was able to induce stomatal closure in grapevine plants. Under field conditions, the application of HB around veraison induced a higher color intensity in berries, and vines treated at higher doses reached this stage earlier than the un-treated controls. There was also a clear increase in both grape anthocyanin concentration and total soluble solids without having a negative impact on total yield. We therefore, confirm the role of HB as a universal natural stomatal closure compound and propose a new use for HB in viticulture as a ripening inducer, by accelerating anthocyanin accumulation.This research was funded by Grant INNVAL10/18/005 from the Agencia Valenciana de la Innovacio (Spain). C.P. was a recipient of a predoctoral contract of the Generalitat Valenciana (ACIF/2019/187). D.S.I. is supported by AEI-FEDER grant AGL2017-83738-C3-3-R.Payá Montes, C.; López-Gresa, MP.; Intrigliolo, DS.; Rodrigo Bravo, I.; Belles Albert, JM.; Lisón, P. (2020). (Z)-3-Hexenyl Butyrate induces stomata closure and ripening in Vitis vinifera. Agronomy. 10(8):1-12. https://doi.org/10.3390/agronomy10081122S112108Zoccatelli, G., Zenoni, S., Savoi, S., Dal Santo, S., Tononi, P., Zandonà, V., … Tornielli, G. B. (2013). Skin pectin metabolism during the postharvest dehydration of berries from three distinct grapevine cultivars. Australian Journal of Grape and Wine Research, 19(2), 171-179. doi:10.1111/ajgw.12014Lund, S. T., & Bohlmann, J. (2006). The Molecular Basis for Wine Grape Quality-A Volatile Subject. Science, 311(5762), 804-805. doi:10.1126/science.1118962Kuhn, N., Guan, L., Dai, Z. W., Wu, B.-H., Lauvergeat, V., Gomès, E., … Delrot, S. (2013). Berry ripening: recently heard through the grapevine. Journal of Experimental Botany, 65(16), 4543-4559. doi:10.1093/jxb/ert395Gerós, H., Chaves, M. M., Gil, H. M., & Delrot, S. (Eds.). (2015). Grapevine in a Changing Environment. doi:10.1002/9781118735985Palliotti, A., Tombesi, S., Silvestroni, O., Lanari, V., Gatti, M., & Poni, S. (2014). Changes in vineyard establishment and canopy management urged by earlier climate-related grape ripening: A review. Scientia Horticulturae, 178, 43-54. doi:10.1016/j.scienta.2014.07.039van Leeuwen, Destrac-Irvine, Dubernet, Duchêne, Gowdy, Marguerit, … Ollat. (2019). An Update on the Impact of Climate Change in Viticulture and Potential Adaptations. Agronomy, 9(9), 514. doi:10.3390/agronomy9090514Schultz, H. R. (2010). Climate Change and Viticulture: Research Needs for Facing the Future. Journal of Wine Research, 21(2-3), 113-116. doi:10.1080/09571264.2010.530093Fraga, H., Malheiro, A. C., Moutinho‐Pereira, J., & Santos, J. A. (2012). An overview of climate change impacts on European viticulture. Food and Energy Security, 1(2), 94-110. doi:10.1002/fes3.14Zhang, Y., Mechlin, T., & Dami, I. (2011). Foliar Application of Abscisic Acid Induces Dormancy Responses in Greenhouse-grown Grapevines. HortScience, 46(9), 1271-1277. doi:10.21273/hortsci.46.9.1271Jung, C. J., Hur, Y. Y., Yu, H.-J., Noh, J.-H., Park, K.-S., & Lee, H. J. (2014). Gibberellin Application at Pre-Bloom in Grapevines Down-Regulates the Expressions of VvIAA9 and VvARF7, Negative Regulators of Fruit Set Initiation, during Parthenocarpic Fruit Development. PLoS ONE, 9(4), e95634. doi:10.1371/journal.pone.0095634Deytieux-Belleau, C., Gagne, S., L’Hyvernay, A., Donèche, B., & Geny, L. (2007). Possible roles of both abscisic acid and indol-acetic acid in controlling grape berry ripening process. OENO One, 41(3), 141. doi:10.20870/oeno-one.2007.41.3.844El-kenawy, M. (2017). Effect of Chitosan, Salicylic Acid and Fulvic Acid on Vegetative Growth, Yield and Fruit Quality of Thompson Seedless Grapevines. Egyptian Journal of Horticulture, 44(1), 45-59. doi:10.21608/ejoh.2017.1104.1007Becatti, E., Genova, G., Ranieri, A., & Tonutti, P. (2014). Postharvest treatments with ethylene on Vitis vinifera (cv Sangiovese) grapes affect berry metabolism and wine composition. Food Chemistry, 159, 257-266. doi:10.1016/j.foodchem.2014.02.169MENG, J.-F., YU, Y., SHI, T.-C., FU, Y.-S., ZHAO, T., & ZHANG, Z.-W. (2019). Melatonin treatment of pre-veraison grape berries modifies phenolic components and antioxidant activity of grapes and wine. Food Science and Technology, 39(1), 35-42. doi:10.1590/1678-457x.24517Mirdehghan, S. H., & Rahimi, S. (2016). Pre-harvest application of polyamines enhances antioxidants and table grape ( Vitis vinifera L.) quality during postharvest period. Food Chemistry, 196, 1040-1047. doi:10.1016/j.foodchem.2015.10.038Mencarelli, F., & Bellincontro, A. (2018). Recent advances in postharvest technology of the wine grape to improve the wine aroma. Journal of the Science of Food and Agriculture, 100(14), 5046-5055. doi:10.1002/jsfa.8910BELLINCONTRO, A., FARDELLI, A., SANTIS, D. D., BOTONDI, R., & MENCARELLI, F. (2006). Postharvest ethylene and 1-MCP treatments both affect phenols, anthocyanins, and aromatic quality of Aleatico grapes and wine. Australian Journal of Grape and Wine Research, 12(2), 141-149. doi:10.1111/j.1755-0238.2006.tb00054.xBotondi, R., Lodola, L., & Mencarelli, F. (2011). Postharvest ethylene treatment affects berry dehydration, polyphenol and anthocyanin content by increasing the activity of cell wall enzymes in Aleatico wine grape. European Food Research and Technology, 232(4), 679-685. doi:10.1007/s00217-011-1437-5Lovisolo, C., Hartung, W., & Schubert, A. (2002). Whole-plant hydraulic conductance and root-to-shoot flow of abscisic acid are independently affected by water stress in grapevines. Functional Plant Biology, 29(11), 1349. doi:10.1071/fp02079Flexas, J., Barón, M., Bota, J., Ducruet, J.-M., Gallé, A., Galmés, J., … Medrano, H. (2009). Photosynthesis limitations during water stress acclimation and recovery in the drought-adapted Vitis hybrid Richter-110 (V. berlandieri×V. rupestris). Journal of Experimental Botany, 60(8), 2361-2377. doi:10.1093/jxb/erp069Yu, D. J., Kim, S. J., & Lee, H. J. (2009). Stomatal and non-stomatal limitations to photosynthesis in field-grown grapevine cultivars. Biologia plantarum, 53(1), 133-137. doi:10.1007/s10535-009-0019-xWong, S. C., Cowan, I. R., & Farquhar, G. D. (1979). Stomatal conductance correlates with photosynthetic capacity. Nature, 282(5737), 424-426. doi:10.1038/282424a0Franks, P. J., & Farquhar, G. D. (2006). The Mechanical Diversity of Stomata and Its Significance in Gas-Exchange Control. Plant Physiology, 143(1), 78-87. doi:10.1104/pp.106.089367López-Gresa, M. P., Lisón, P., Campos, L., Rodrigo, I., Rambla, J. L., Granell, A., … Bellés, J. M. (2017). A Non-targeted Metabolomics Approach Unravels the VOCs Associated with the Tomato Immune Response against Pseudomonas syringae. Frontiers in Plant Science, 8. doi:10.3389/fpls.2017.01188López-Gresa, M. P., Payá, C., Ozáez, M., Rodrigo, I., Conejero, V., Klee, H., … Lisón, P. (2018). A New Role For Green Leaf Volatile Esters in Tomato Stomatal Defense Against Pseudomonas syringe pv. tomato. Frontiers in Plant Science, 9. doi:10.3389/fpls.2018.01855Meyers, K. J., Watkins, C. B., Pritts, M. P., & Liu, R. H. (2003). Antioxidant and Antiproliferative Activities of Strawberries. Journal of Agricultural and Food Chemistry, 51(23), 6887-6892. doi:10.1021/jf034506nGarcía-Hurtado, N., Carrera, E., Ruiz-Rivero, O., López-Gresa, M. P., Hedden, P., Gong, F., & García-Martínez, J. L. (2012). The characterization of transgenic tomato overexpressing gibberellin 20-oxidase reveals induction of parthenocarpic fruit growth, higher yield, and alteration of the gibberellin biosynthetic pathway. Journal of Experimental Botany, 63(16), 5803-5813. doi:10.1093/jxb/ers229Fernández-López, J. A., Almela, L., Muñoz, J. A., Hidalgo, V., & Carreño, J. (1998). Dependence between colour and individual anthocyanin content in ripening grapes. Food Research International, 31(9), 667-672. doi:10.1016/s0963-9969(99)00043-5KENNEDY, J. A., TROUP, G. J., PILBROW, J. R., HUTTON, D. R., HEWITT, D., HUNTER, C. R., … JONES, G. P. (2000). Development of seed polyphenols in berries from Vitis vinifera L. cv. Shiraz. Australian Journal of Grape and Wine Research, 6(3), 244-254. doi:10.1111/j.1755-0238.2000.tb00185.xGupta, A., Rico-Medina, A., & Caño-Delgado, A. I. (2020). The physiology of plant responses to drought. Science, 368(6488), 266-269. doi:10.1126/science.aaz7614Jordan, W. R., Brown, K. W., & Thomas, J. C. (1975). Leaf Age as a Determinant in Stomatal Control of Water Loss from Cotton during Water Stress. Plant Physiology, 56(5), 595-599. doi:10.1104/pp.56.5.595Whitehead, D., Barbour, M. M., Griffin, K. L., Turnbull, M. H., & Tissue, D. T. (2011). Effects of leaf age and tree size on stomatal and mesophyll limitations to photosynthesis in mountain beech (Nothofagus solandrii var. cliffortiodes). Tree Physiology, 31(9), 985-996. doi:10.1093/treephys/tpr021Tombesi, S., Nardini, A., Frioni, T., Soccolini, M., Zadra, C., Farinelli, D., … Palliotti, A. (2015). Stomatal closure is induced by hydraulic signals and maintained by ABA in drought-stressed grapevine. Scientific Reports, 5(1). doi:10.1038/srep12449Gamm, M., Héloir, M.-C., & Adrian, M. (2015). Trehalose and trehalose-6-phosphate induce stomatal movements and interfere with ABA-induced stomatal closure in grapevine. OENO One, 49(3), 165. doi:10.20870/oeno-one.2015.49.3.84Di Vaio, C., Marallo, N., Di Lorenzo, R., & Pisciotta, A. (2019). Anti-Transpirant Effects on Vine Physiology, Berry and Wine Composition of cv. Aglianico (Vitis vinifera L.) Grown in South Italy. Agronomy, 9(5), 244. doi:10.3390/agronomy9050244Di Vaio, C., Villano, C., Lisanti, M. T., Marallo, N., Cirillo, A., Di Lorenzo, R., & Pisciotta, A. (2020). Application of Anti-Transpirant to Control Sugar Accumulation in Grape Berries and Alcohol Degree in Wines Obtained from Thinned and Unthinned Vines of cv. Falanghina (Vitis vinifera L.). Agronomy, 10(3), 345. doi:10.3390/agronomy10030345Medrano, H., Tomás, M., Martorell, S., Escalona, J.-M., Pou, A., Fuentes, S., … Bota, J. (2014). Improving water use efficiency of vineyards in semi-arid regions. A review. Agronomy for Sustainable Development, 35(2), 499-517. doi:10.1007/s13593-014-0280-zKALLITHRAKA, S., BAKKER, J., & CLIFFORD, M. N. (1997). EVALUATION OF BITTERNESS AND ASTRINGENCY OF (+)-CATECHIN AND (-)-EPICATECHIN IN RED WINE AND IN MODEL SOLUTION. Journal of Sensory Studies, 12(1), 25-37. doi:10.1111/j.1745-459x.1997.tb00051.xBrillante, L., Belfiore, N., Gaiotti, F., Lovat, L., Sansone, L., Poni, S., & Tomasi, D. (2016). Comparing Kaolin and Pinolene to Improve Sustainable Grapevine Production during Drought. PLOS ONE, 11(6), e0156631. doi:10.1371/journal.pone.0156631Palliotti, A., Panara, F., Famiani, F., Sabbatini, P., Howell, G. S., Silvestroni, O., & Poni, S. (2013). Postveraison Application of Antitranspirant Di-1- p -Menthene to Control Sugar Accumulation in Sangiovese Grapevines. American Journal of Enology and Viticulture, 64(3), 378-385. doi:10.5344/ajev.2013.13015Fahey, D. J., & Rogiers, S. Y. (2018). Di-1-p -menthene reduces grape leaf and bunch transpiration. Australian Journal of Grape and Wine Research, 25(1), 134-141. doi:10.1111/ajgw.12371Martín, P., Delgado, R., González, M. R., & Gallegos, J. I. (2004). COLOUR OF «TEMPRANILLO» GRAPES AS AFFECTED BY DIFFERENT NITROGEN AND POTASSIUM FERTILIZATION RATES. Acta Horticulturae, (652), 153-160. doi:10.17660/actahortic.2004.652.18Fortes, A., Teixeira, R., & Agudelo-Romero, P. (2015). Complex Interplay of Hormonal Signals during Grape Berry Ripening. Molecules, 20(5), 9326-9343. doi:10.3390/molecules20059326Griesser, M., Savoi, S., Supapvanich, S., Dobrev, P., Vankova, R., & Forneck, A. (2020). Phytohormone profiles are strongly altered during induction and symptom development of the physiological ripening disorder berry shrivel in grapevine. Plant Molecular Biology, 103(1-2), 141-157. doi:10.1007/s11103-020-00980-6Portu, J., López, R., Baroja, E., Santamaría, P., & Garde-Cerdán, T. (2016). Improvement of grape and wine phenolic content by foliar application to grapevine of three different elicitors: Methyl jasmonate, chitosan, and yeast extract. Food Chemistry, 201, 213-221. doi:10.1016/j.foodchem.2016.01.086Gómez-Plaza, E., Bautista-Ortín, A. B., Ruiz-García, Y., Fernández-Fernández, J. I., & Gil-Muñoz, R. (2016). Effect of elicitors on the evolution of grape phenolic compounds during the ripening period. Journal of the Science of Food and Agriculture, 97(3), 977-983. doi:10.1002/jsfa.7823Silva, V., Singh, R. K., Gomes, N., Soares, B. G., Silva, A., Falco, V., … Poeta, P. (2020). Comparative Insight upon Chitosan Solution and Chitosan Nanoparticles Application on the Phenolic Content, Antioxidant and Antimicrobial Activities of Individual Grape Components of Sousão Variety. Antioxidants, 9(2), 178. doi:10.3390/antiox9020178El-Kereamy, A., Chervin, C., Roustan, J.-P., Cheynier, V., Souquet, J.-M., Moutounet, M., … Bouzayen, M. (2003). Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berries. Physiologia Plantarum, 119(2), 175-182. doi:10.1034/j.1399-3054.2003.00165.xAllègre, M., Héloir, M.-C., Trouvelot, S., Daire, X., Pugin, A., Wendehenne, D., & Adrian, M. (2009). Are Grapevine Stomata Involved in the Elicitor-Induced Protection Against Downy Mildew? Molecular Plant-Microbe Interactions®, 22(8), 977-986. doi:10.1094/mpmi-22-8-097

Induction of cinnamate 4-hydroxylase and phenylpropanoids in virus-infected cucumber and melon plants

[EN] In the present work, we have looked for the nature of the phenylpropanoids biosynthesized during the plant-pathogen reaction of two systems, Cucumis sativus and Cucumis melo infected with either prunus necrotic ringspot virus (PNRSV) or melon necrotic spot virus (MNSV), respectively. An accumulation of p-coumaric, caffeic and/or ferulic acids was observed in infected plant extracts hydrolysed with P-glucosidase or esterase. Analysis of undigested samples by HPLC/ESI revealed that these compounds are mainly forming esters with glucose: 1-O-coumaroyl-beta-glucose, 1-O-caffeoyl-beta-glucose, and 1-O-fer-uloyl-beta-glucose. Cinnamic acid 4-hydroxylase (C4H, EC 1.14.13.11), the second enzyme of the plant phenylpropanoid pathway, plays a pivotal role in the synthesis of these hydroxycinnamic acids. Thus, we have isolated and characterised a cDNA clone encoding this enzyme from PNRSV-infected cucumber, and a partial cDNA from MNSV-infected melon leaves. The deduced amino acid sequence revealed a notable degree of identity with homologous C4H enzymes from other plant species. In agreement with the induction of the phenylpropanoids presently described, it is reported that in cucumber and melon leaves, both viral infections studied induced C4H mRNA expression. A similar induction was observed for the first pherylpropanoid biosynthetic enzyme, phyenylalanine ammonia-lyase (PAL, EC 4.3.1.5), and for chitinase and peroxidase defence-related genes.We gratefully acknowledge Dr. Lynne Yenush for critical reading of the manuscript and helpful discussions, and L. Latorre, and L. Coracha´n for the technical support in the greenhouse. The authors want to acknowledge the detailed suggestions of the referees. This work was supported by Grant BMC2000-1136 from Comisio´n Interministerial de Ciencia y Tecnologı´a, Spanish Ministry of Science and TechnologyS52453317

Bacillus subtilis IAB/BS03 as a potential biological control agent

[EN] We describe the efficacy of Bacillus subtilis strain IAB/BS03 in reducing disease incidence of B. subtilis IAB/BS03 as a foliar treatment against Botrytis cinerea and Pseudomonas syringae on greenhouse-grown tomato (Solanum lycopersicon) plants. We also tested the effect of foliar treatments on lettuce (Lactuca sativa) against lettuce downy mildew caused by Bremia lactucae in multiple trials under different field conditions. All the assays indicated that B. subtilis IAB/BS03 reduced disease. To ascertain the mechanism of action, the induction of pathogenesis-related (PR) proteins, the accumulation of salicylic acid and the activation of peroxidase caused by foliar or root treatments with B. subtilis IAB/BS03 were studied in tomato. A salicylic acid-independent induction of the antifungal protein PR1 was observed after treatment with B. subtilis IAB/BS03, with the strongest induction due to root treatment compared with foliar application. A metabolic analysis of B. subtilis IAB/BS03 culture broth using Ultra Performance Liquid Chromatography coupled with ultraviolet and mass spectrometric detection determined surfactin and iturin A isomers. These compounds have been described as antifungal and antibiotic lipopeptides. The results indicated that B. subtilis IAB/BS03 could be effectively used as a biocontrol agent.This work was funded by IAB S. L. (Investigaciones y Aplicaciones Biotecnologicas, S. L.), and by grant BIO2012-33419 from the Spanish Ministry of Economy and Competitiveness. Mayte Castellano was the recipient of a research grant also funded by IAB S. L. The authors would like to thank Cristina Torres (IBMCP, UPV-CSIC) for her excellent technical assistance.Hinarejos, E.; Castellano Pérez, M.; Rodrigo Bravo, I.; Belles Albert, JM.; Conejero Tomás, V.; López-Gresa, MP.; Lisón, P. (2016). Bacillus subtilis IAB/BS03 as a potential biological control agent. European Journal of Plant Pathology. 146(3):597-608. https://doi.org/10.1007/s10658-016-0945-3S5976081463Abbott, W. S. (1925). A method for computing the effectiveness of an insecticide. Journal Economic Entomology, 18, 265–267.Chen, H., Wang, L., Su, C.X., Gong, G. H., Wang, P., Yu, Z. L. (2008). Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis. Letters in Applied Microbiology. 47, 180–186.Cho, S. J., Lee, S. K., Cha, B. J., Kim, Y. H., & Shin, K. S. (2003). Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin a from Bacillus subtilis strain KS03. FEMS Microbiology Letters, 223, 47–51.Choudhary, D. K., & Johri, B. N. (2009). Interactions of Bacillus spp. and plants with special reference to induced systemic resistance (ISR). Microbiology Research, 164, 493–513.Coego, A., Ramírez, V., Ellul, P., Mayda, E., & Vera, P. (2005). The H2O2-regulated Ep5C gene encodes a peroxidase required for bacterial speck susceptibility in tomato. The Plant Journal, 42, 283–293.Conrath, U., Pieterse, C. M. J., & Mauch-Mani, B. (2002). Priming in plant-pathogen interactions. Trends in Plant Science, 7, 210–216.Fleming, A. J., Mandel, T., Roth, I., & Kuhlemier, C. (1993). The patterns of gene expression in the tomato shoot apical meristem. The Plant Cell, 5, 297–309.Fravel, D. R. (2005). Commercialization and implementation of biocontrol. Annual Review of Phytopathology, 43, 337–359.Hammerschmidt, R., Nuckles, E. M., & Kuc, J. (1982). Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiological Plant Pathology, 20, 73–76.Kawagoe, Y., Shiraishi, S., Kondo, H., Yamamoto, S., Aoki, Y., & Suzuki, S. (2015). Cyclic lipopeptide iturin a structure-dependently induces defense response in Arabidopsis plants by activating SA and JA signaling pathways. Biochemical and Biophysical Research Communications, 460, 1015–1020.Kloepper, J. W., Ryu, C. M., & Zhang, S. A. (2004). Induced systemic resistance and promotion of plant growth by Bacillus spp. Phytopathology, 94, 1259–1266.Liu, H.-X., Li, S.-M., Luo, Y.-M., Luo, L.-X., Li, J.-Q., & Guo, J.-H. (2014). Biological control of Ralstonia wilt, Phytophthora blight, Meloidogyne root-knot on bell pepper by the combination of Bacillus subtilis AR12, Bacillus subtilis SM21 and Chryseobacterium sp. R89. European Journal of Plant Pathology, 139, 107–116.Mohammadi, M., & Kazemi, H. (2002). Changes in peroxidase and polyphenol oxidase activities in susceptible and resistant wheat heads inoculated with Fusarium graminearum and induced resistance. Plant Science, 162, 491–498.Niderman, T., Genetet, I., Bruyere, T., Gees, R., Stintzi, A., Legrand, M., et al. (1995). Pathogenesis-related PR-1 proteins are antifungal - isolation and characterization of 3 14-Kilodalton proteins of tomato and of a basic PR-1 of tobacco with inhibitory activity against Phytophthora infestans. Plant Physiology, 108, 17–27.Ohno, A., Ano, T., & Shoda, M. (1995). Effect of temperature on production of lipopeptide antibiotics, iturin a and surfactin by a dual producer, Bacillus subtilis Rb14, in solid-state fermentation. Journal of Fermentation and Bioengineering, 80, 517–519.Ongena, M., & Jacques, P. (2008). Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends in Microbiology, 16, 115–125.Pérez-García, A., Romero, D., & De Vicente, A. (2011). Plant protection and growth stimulation by microorganisms: biotechnological applications of bacilli in agriculture. Current Opinion in Biotechnology, 22, 187–193.Phister, T. G., O’Sullivan, D. J., & McKay, L. L. (2004). Identification of bacilysin, chlorotetaine, and iturin a produced by Bacillus sp strain CS93 isolated from pozol, a Mexican fermented maize dough. Applied Environmental Microbiology, 70, 631–634.Pieterse, C. M. J., vanWees, S. C. M., Hoffland, E., Van Pelt, J. A., & Van Loon, L. C. (1996). Systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis-related gene expression. The Plant Cell, 8, 1225–1237.Pryor, S. W., Gibson, D. M., Krasnoff, S. B., & Walker, L. P. (2006). Identification of antifungal compounds in a biological control product using a microplate inhibition bioassay. Transactions of the ASAE, 49, 1643–1649.Robert-Seilaniantz, A., Navarro, L., Bari, R., & Jones, J. D. (2007). Pathological hormone imbalances. Current Opinion in Plant Biology, 10, 372–379.Rudrappa, T., Biedrzycki, M. L., Kunjeti, S. G., Donofrio, N. M., Czymmek, K. J., Paul W, P., et al. (2010). The rhizobacterial elicitor acetoin induces systemic resistance in Arabidopsis thaliana. Communicative and Integrative Biology, 3, 130–138.Summermatter, K., Sticher, L., & Métraux, J. P. (1995). Systemic responses in Arabidopsis thaliana infected and challenged with Pseudomonas syringae pv syringae. Plant Physiology, 108, 1379–1385.Tang, J. S., Zhao, F., Gao, H., Dai, Y., Yao, Z. H., Hong, K., et al. (2010). Characterization and online detection of surfactin isomers based on HPLC-MSn analyses and their inhibitory effects on the overproduction of nitric oxide and the release of TNF-α and IL-6 in LPS induced macrophages. Marine Drugs, 8, 2605–2618.Tornero, P., Gadea, J., Conejero, V., & Vera, P. (1997). Two PR-1 genes from tomato are differentially regulated and reveal a novel mode of expression for a pathogenesis-related gene during the hypersensitive response and development. Molecular Plant-Microbe Interactions, 10, 624–634.Tsavkelova, E. A., Klimova, S. Y., Cherdyntseva, T. A., & Netrusov, A. I. (2006). Microbial producers of plant growth stimulators and their practical use: a review. Applied Biochemistry and Microbiology, 42, 117–126.Van Loon, L. C. (2007). Plant responses to plant growth-promoting rhizobacteria. European Journal of Plant Pathology, 119, 243–254.Verhagen, B. W. M., Glazebrook, J., Zhu, T., Chang, H. S., Van Loon, L. C., & Pieterse, C. M. J. (2004). The transcriptome of rhizobacteria-induced systemic resistance in Arabidopsis. Molecular Plant-Microbe Interactions, 17, 895–908.Wulff, B. B. H., Horvath, D. M., & Ward, E. R. (2011). Improving immunity in crops: new tactics in an old game. Current Opinion in Plant Biology, 14, 468–476.Yáñez-Mendizábal, V., Zeriouh, H., Viñas, I., Torres, R., Usall, J., de Vicente, A., et al. (2012). Biological control of peach brown rot (Monilinia spp.) by Bacillus subtilis CPA-8 is based on production of fengycin-like lipopeptides. European Journal of Plant Pathology, 132, 609–619.Zacarés, L., López-Gresa, M. P., Fayos, J., Primo, J., Bellés, J. M., & Conejero, V. (2007). Induction of p-coumaroyldopamine and feruloyldopamine, two novel metabolites, in tomato by the bacterial pathogen Pseudomonas syringae. Molecular Plant-Microbe Interactions, 20, 1439–1448.Zadoks, J. C., Chang, T. T., & Konzak, C. F. (1974). A decimal code for the growth stages of cereals. Weed Research, 14, 415–421.Zeriouh, H., Romero, D., García-Gutiérrez, L., Cazorla, F. M., De Vicente, A., & Pérez-García, A. (2011). The iturin-like lipopeptides are essential components in the biological control arsenal of Bacillus subtilis against bacterial diseases of cucurbits. Molecular Plant-Microbe Interactions, 24, 1540–1552

Aceites esenciales: productos antimicrobianos y antioxidantes naturales en la industria agroalimentaria

[ES] Los consumidores son conscientes del peligro derivado del uso de antioxidantes y antimicrobianos sintéticos en la industria agroalimentaria, demandando alternativas más seguras y ecológicas. En este estudio, se ha determinado la actividad antioxidante de aceites esenciales comerciales mediante el método DPPH y su efecto antimicrobiano frente a la bacteria Pseudomonas syringae y el hongo fitopatógeno Fusarium oxysporum a través del empleo del método estandarizado de disco. Los aceites esenciales de clavo, ajedrea, canela y orégano, así como carvacrol, mostraron la máxima actividad antioxidante, comparable a antioxidantes establecidos. El aceite esencial de gaulteria fue el más potente inhibidor del crecimiento de P. syringae en las dosis más altas (20 y 10 µL) ensayadas. El aceite esencial de orégano, así como su componente principal carvacrol, detuvieron el crecimiento de la bacteria incluso a la dosis más baja ensayada (1 µL). Los aceites esenciales de canela, orégano y menta inhibieron el desarrollo de F. oxysporum en todas las dosis (20, 10 y 5 µL) aplicadas. En general, la mayoría de aceites esenciales mostraron más actividad antifúngica que antibacteriana y antioxidante.[EN] Consumers are aware of the dangers arising from the use of synthetic antioxidants and antimicrobials in the agrifood industry, demanding safer and "greener" alternatives. In this study, the antioxidant activity of commercial essential oils through DPPH method, their antimicrobial effects against the bacterium Pseudomonas syringae and the phytopathogenic fungus Fusarium oxysporum by means of the standardized disk method were determined. Clove along with winter savory, cinnamon and oregano essential oils as well as carvacrol showed the highest antioxidant activity comparable to reference standards. Wintergreen essential oil was the most potent inhibitor against P. syringae growth at the highest doses (20 and 10 µL). Oregano essential oil and its main component carvacrol were able to stop the bacterium growth even at the lowest treatment (1 µL). Cinnamon, oregano and peppermint essential oils inhibited F. oxysporum development at all doses (20, 10 and 5 µL) assayed. In general, most of the essential oils displayed more antifungal than antibacterial and antioxidant activities.Ibáñez, MD.; López-Gresa, MP.; Lisón, P.; Rodrigo Bravo, I.; Belles Albert, JM.; González-Mas, MC.; Blázquez, MA. (2020). Essential oils as natural antimicrobial and antioxidant products in the Agrifood Industry. Nereis. Revista Iberoamericana Interdisciplinar de Métodos, Modelización y Simulación. (12):55-69. https://doi.org/10.46583/nereis_2020.12.585S55691

A new role for green leaf volatile esters in tomato stomatal defense against Pseudomonas syringe pv. tomato

[EN] The volatile esters of (Z)-3-hexenol with acetic, propionic, isobutyric, or butyric acids are synthesized by alcohol acyltransferases (AAT) in plants. These compounds are differentially emitted when tomato plants are efficiently resisting an infection with Pseudomonas syringae pv. tomato. We have studied the defensive role of these green leaf volatile (GLV) esters in the tomato response to bacterial infection, by analyzing the induction of resistance mediated by these GLVs and the phenotype upon bacterial infection of tomato plants impaired in their biosynthesis. We observed that treatments of plants with (Z)-3-hexenyl propionate (HP) and, to a greater extent with (Z)-3-hexenyl butyrate (HB), resulted in stomatal closure, PR gene induction and enhanced resistance to the bacteria. HB-mediated stomatal closure was also effective in several plant species belonging to Nicotiana, Arabidopsis, Medicago, Zea and Citrus genus, and both stomatal closure and resistance were induced in HB-treated NahG tomato plants, which are deficient in salicylic acid (SA) accumulation. Transgenic antisense AAT1 tomato plants, which displayed a reduction of ester emissions upon bacterial infection in leaves, exhibited a lower ratio of stomatal closure and were hyper-susceptible to bacterial infection. Our results confirm the role of GLV esters in plant immunity, uncovering a SA-independent effect of HB in stomatal defense. Moreover, we identified HB as a natural stomatal closure compound with potential agricultural applications.This work was funded by Grant AICO/2017/048 from the Generalitat Valenciana and by Grant INNVAL10/18/005 from the Agencia Valenciana de la Innovacio (Spain). We would like to thank the Metabolomics Service of the IBMCP (Valencia, Spain), especially to Teresa Caballero for her excellent technical support in the VOCs quantification. We also thank Eduardo Moya for technical assistance.López-Gresa, MP.; Payá, C.; Ozáez-Martínez, M.; Rodrigo Bravo, I.; Conejero Tomás, V.; Klee, H.; Belles Albert, JM.... (2018). A new role for green leaf volatile esters in tomato stomatal defense against Pseudomonas syringe pv. tomato. Frontiers in Plant Science. 9:1-12. https://doi.org/10.3389/fpls.2018.01855S112

Citrus exocortis viroid causes ribosomal stress in tomato plants

[EN] Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress.Spanish Ministry of Science, Innovation and Universities [BIO2009-11818, BIO2015-70483-R to A.F.]; Spanish Ministry of Science, Innovation and Universities [BFU2009-11958]; Generalitat Valenciana (Valencia, Spain) [AICO/2017/048]; Natural Sciences and Engineering Research Council of Canada [155219-17 to J.-P.P.]; The RNA group is supported by a grant from the Universite de Sherbrooke; J.-P.P. holds the Research Chair of the Universite de Sherbrooke in RNA Structure and Genomics, and is a member of the Centre de Recherche du CHUS; B.B.-P. was a recipient of a VALi+d postdoctoral contract of the Generalitat Valenciana [APOSTD/2017/039]; Schleiff group is funded through the Deutsche Forschungsgemeinschaft [SFB 902]. Funding for open access charge: Spanish Ministry of Science, Innovation and Universities.Cottilli, P.; Belda-Palazón, B.; Adkar-Purushothama, CR.; Perreault, J.; Schleiff, E.; Rodrigo Bravo, I.; Ferrando Monleón, AR.... (2019). Citrus exocortis viroid causes ribosomal stress in tomato plants. Nucleic Acids Research. 47(16):8649-8661. https://doi.org/10.1093/nar/gkz679S86498661471

A Non-targeted Metabolomics Approach Unravels the VOCs Associated with the Tomato Immune Response against Pseudomonas syringae

[EN] Volatile organic compounds (VOCs) emitted by plants are secondary metabolites that mediate the plant interaction with pathogens and herbivores. These compounds may perform direct defensive functions, i. e., acting as antioxidant, antibacterial, or antifungal agents, or indirectly by signaling the activation of the plant's defensive responses. Using a non-targeted GC-MS metabolomics approach, we identified the profile of the VOCs associated with the differential immune response of the Rio Grande tomato leaves infected with either virulent or avirulent strains of Pseudomonas syringae DC3000 pv. tomato. The VOC profile of the tomato leaves infected with avirulent bacteria is characterized by esters of (Z)-3-hexenol with acetic, propionic, isobutyric or butyric acids, and several hydroxylated monoterpenes, e. g., linalool, a -terpineol, and 4-terpineol, which defines the profile of an immunized plant response. In contrast, the same tomato cultivar infected with the virulent bacteria strain produced a VOC profile characterized by monoterpenes and SA derivatives. Interestingly, the differential VOCs emission correlated statistically with the induction of the genes involved in their biosynthetic pathway. Our results extend plant defense system knowledge and suggest the possibility for generating plants engineered to over-produce these VOCs as a complementary strategy for resistance.This work was funded by Grant BIO2012-33419 from the Spanish Ministry of Economy and Competitiveness.López-Gresa, MP.; Lisón, P.; Campos Beneyto, L.; Rodrigo Bravo, I.; Rambla Nebot, JL.; Granell Richart, A.; Conejero Tomás, V.... (2017). A Non-targeted Metabolomics Approach Unravels the VOCs Associated with the Tomato Immune Response against Pseudomonas syringae. Frontiers in Plant Science. 8. doi:10.3389/fpls.2017.01188S