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It is well known that plants respond to various stresses by releasing volatile organic compounds (VOCs). Green leaf volatiles (GLVs), which are commonly produced across different plant species after tissue damage, comprise an important group within VOCs. Among them, (Z)-3-hexenyl butyrate (HB) was described as a natural stomatal closure inducer, playing an important role in stomatal immunity, although its mechanism of action is still unknown. Here, through different genetic, pharmacological, and biochemical approaches we uncover that HB perception initiates different defense signaling events such as activation of Ca2+ permeable channels, mitogen-activated protein kinases (MPKs) and production of NADPH oxidase-mediated reactive oxygen species (ROS). Furthermore, HB-mediated stomata closure resulted to be independent of abscisic acid (ABA) biosynthesis and signaling. Additionally, exogenous treatments with this volatile alleviate water stress and improve fruit productivity in tomato plants. HB efficacy was also tested under open field conditions, leading to enhanced resistance against Phytophthora spp. and Pseudomonas syringae infection in potato and tomato plants, respectively. Collectively, our results provide insights into HB signaling transduction pathway, confirming its role in stomatal closure and plant immune system activation, and proposing HB as a new phytoprotector for the sustainable control of biotic and abiotic stresses in agriculture.

SummaryCarotenoids are photoprotectant pigments and precursors of the hormones abscisic acid (ABA) and strigolactones (SL). Carotenoids are produced in plastids from geranylgeranyl diphosphate (GGPP), which is diverted to the carotenoid pathway by phytoene synthase (PSY). In tomato (Solanum lycopersicum), 3 genes encode plastid-targeted GGPP synthases (SlG1 to 3) and 3 genes encode PSY isoforms (PSY1 to 3).Here we investigated the function of SlG1 by generating loss-of-function lines and combining their metabolic and physiological phenotypes with gene co-expression and co-immunoprecipitation analyses.Leaves and fruits ofslg1lines showed a wild-type phenotype in terms of isoprenoid accumulation, photosynthesis and development. Consistently,SlG1is co-expressed withPSY3and other genes involved in the production of carotenoids and SL (but not ABA) only in roots. SlG1 was also found to physically interact with the root-specific PSY3 isoform (and not with PSY1 and PSY2). Root SL (but not ABA) levels were reduced inslg1lines.Our results confirm a specific role of SlG1 in SL production in combination with PSY3. This role appears to be restricted to roots asslg1plants do not exhibit the shoot phenotype displayed by other SL-deficient mutants.

Cutin and suberin are lipid polyesters deposited in specific apoplastic compartments. Their fundamental roles in plant biology include controlling the movement of gases, water and solutes, and conferring pathogen resistance. Both cutin and suberin have been shown to be present in the Arabidopsis seed coat where they regulate seed dormancy and longevity. In this study, we use accelerated and natural ageing seed assays, glutathione redox potential measures, optical and transmission electron microscopy and gas chromatography-mass spectrometry to demonstrate that increasing the accumulation of lipid polyesters in the seed coat is the mechanism by which the AtHB25 transcription factor regulates seed permeability and longevity. Chromatin immunoprecipitation during seed maturation revealed that the lipid polyester biosynthetic gene long-chain acyl-CoA synthetase 2 (LACS2) is a direct AtHB25 binding target. Gene transfer of this transcription factor to wheat and tomato demonstrated the importance of apoplastic lipid polyesters for the maintenance of seed viability. Our work establishes AtHB25 as a trans-species regulator of seed longevity and has identified the deposition of apoplastic lipid barriers as a key parameter to improve seed longevity in multiple plant species.

Summary RNA interference (RNAi)‐based tools are used in multiple organisms to induce antiviral resistance through the sequence‐specific degradation of target RNAs by complementary small RNAs. In plants, highly specific antiviral RNAi‐based tools include artificial microRNAs (amiRNAs) and synthetic trans‐acting small interfering RNAs (syn‐tasiRNAs). syn‐tasiRNAs have emerged as a promising antiviral tool allowing for the multi‐targeting of viral RNAs through the simultaneous expression of several syn‐tasiRNAs from a single precursor. Here, we compared in tomato plants the effects of an amiRNA construct expressing a single amiRNA and a syn‐tasiRNA construct expressing four different syn‐tasiRNAs against Tomato spotted wilt virus (TSWV), an economically important pathogen affecting tomato crops worldwide. Most of the syn‐tasiRNA lines were resistant to TSWV, whereas the majority of the amiRNA lines were susceptible and accumulated viral progenies with mutations in the amiRNA target site. Only the two amiRNA lines with higher amiRNA accumulation were resistant, whereas resistance in syn‐tasiRNA lines was not exclusive of lines with high syn‐tasiRNA accumulation. Collectively, these results suggest that syn‐tasiRNAs induce enhanced antiviral resistance because of the combined silencing effect of each individual syn‐tasiRNA, which minimizes the possibility that the virus simultaneously mutates all different target sites to fully escape each syn‐tasiRNA., Significance Statement This work represents the successful application of syn‐tasiRNAs in a crop to induce high resistance against an economically important virus. Syn‐tasiRNAs outcompete amiRNAs in antiviral resistance: the possibility of co‐expressing multiple syn‐tasiRNAs from a single precursor allows for the simultaneous multi‐targeting of viral RNAs, which limits the ability of the virus to mutate target sites.

[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.

New strategies of control need to be developed with the aim of economic and environmental sustainability in plant and crop protection. Metabolomics is an excellent platform for both understanding the complex plant–pathogen interactions and unraveling new chemical control strategies. GC-MS-based metabolomics, along with a phytohormone analysis of a compatible and incompatible interaction between tomato plants and Fusarium oxysporum f. sp. lycopersici, revealed the specific volatile chemical composition and the plant signals associated with them. The susceptible tomato plants were characterized by the over-emission of methyl- and ethyl-salicylate as well as some fatty acid derivatives, along with an activation of salicylic acid and abscisic acid signaling. In contrast, terpenoids, benzenoids, and 2-ethylhexanoic acid were differentially emitted by plants undergoing an incompatible interaction, together with the activation of the jasmonic acid (JA) pathway. In accordance with this response, a higher expression of several genes participating in the biosynthesis of these volatiles, such as MTS1, TomloxC,TomloxD, and AOS, as well as JAZ7, a JA marker gene, was found to be induced by the fungus in these resistant plants. The characterized metabolome of the immune tomato plants could lead to the development of new resistance inducers against Fusarium wilt treatment.

[EN] Viroids are small, circular, highly structured pathogens that infect a broad range of plants, causing economic losses. Since their discovery in the 1970s, they have been considered as non-coding pathogens. In the last few years, the discovery of other RNA entities, similar in terms of size and structure, that were shown to be translated (e.g., cirRNAs, precursors of miRNA, RNA satellites) as well as studies showing that some viroids are located in ribosomes, have reignited the idea that viroids may be translated. In this study, we used advanced bioinformatic analysis, in vitro experiments and LC-MS/MS to search for small viroid peptides of the PSTVd. Our results suggest that in our experimental conditions, even though the circular form of PSTVd is found in ribosomes, no produced peptides were identified. This indicates that the presence of PSTVd in ribosomes is most probably not related to peptide production but rather to another unknown function that requires further study., Katsarou K. and Kalantidis K. are supported in part by the grant Emblematic Action for Research in the Cretan Agrofood sector: Four Institutions, Four References' (AGRO4CRETE- 2018SigmaE01300000) held by the General Secretary for Research and Technology of Greece as well as the National Flagship Initiative "The paths of Grapevine¿ of the public investments programs of the GSRT:2018SigmaE0100000.Proteomic platform was supported by the project "The Greek Research Infrastructure for Personalized Medicine (pMED-GR)" (MIS 5002802) which is implemented under the Action "Reinforcement of the Research and Innovation Infrastructure", funded by the Operational Program "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund). J.-P.P. was funded by the Natural Sciences and Engineering Research Council of Canada [155219-17 to J.-P.P.]; The RNA group is supported by a grant from the Université de Sherbrooke. J.-P.P. holds the Research Chair of the Université de Sherbrooke in RNA Structure and Genomics and is a member of the Centre de Recherche du CHUS

[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.

© 2020 by the authors., Agronomy solutions for modifying pre-harvest grape ripening are needed for a more sustainable viticulture. Field experiments were performed in Vitis vinifera L. 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 Agència Valenciana de la Innovació (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.

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.

Capítulo 10., Many hypotheses have been advanced to explain how viroid infection might alter host gene expression to produce the symptoms seen on diseased plants. Only recently, however, has a series of technological advances allowed these hypotheses to be tested experimentally. The first section of this chapter describes changes in the host proteome following viroid infection. Next, we describe changes at the transcriptional level as measured by microarray analysis and other genomics-based approaches and evidence for both pre- and posttranscriptional regulation. Interactions between proteins and nucleic acids result in formation of molecular networks, and identification of these “interactomes” allows integration of results obtained by transcriptomic and proteomic approaches. The final section describes interactions between viroids and several host proteins, outlining possible directions for future systems-based research.

Proteinaceous aspartic proteinase inhibitors are rare in nature and are described in only a few plant species. One of them corresponds to a family of cathepsin D inhibitors (CDIs) described in potato (Solanum tuberosum), involving up to 15 isoforms with a high sequence similarity. In this work, we describe a tomato (Solanum lycopersicum) wound-inducible protein called jasmonic-induced protein 21 (JIP21). Sequence analysis of its cDNA predicted a putative function as a CDI. The JIP21 gene, whose protein has been demonstrated to be glycosylated, is constitutively expressed in flowers, stem, and fruit, and is inducible to high levels by wounding and methyl jasmonate in leaves of tomato plants. The genomic sequence of JIP21 shows that the gene is intronless and reveals the presence of both a methyl jasmonate box (TGACT) and a G-box (CACGT) in the promoter. In contrast to the presumed role of JIP21 based on sequence analysis, a detailed biochemical characterization of the purified protein uncovers a different function as a strong chymotrypsin inhibitor, which questions the previously predicted inhibitory activity against aspartic proteinases. Moreover, Egyptian cotton worm (Spodoptera littoralis) larvae fed on transgenic tomato plants overexpressing JIP21 present an increase in mortality and a delay in growth when compared with larvae fed on wild-type plants. These larvae belong to the Lepidoptera family whose main digestive enzymes have been described as being Ser proteases. All these results support the notion that tomato JIP21 should be considered as a chymotrypsin inhibitor belonging to the Ser proteinase inhibitors rather than a CDI. Therefore, we propose to name this protein tomato chymotrypsin inhibitor 21 (TCI21).

A proteomic analysis of buds from mandarin trees with contrasting fruit load (on- and off-crop trees) was carried out during the onset of low-temperature induction. The aim of the study was to find out more about the molecular mechanism relating to alternate bearing in Citrus and its relationship with flowering. The 'Moncada' variety (Clementine 'Oroval' x 'Kara' mandarin), displaying remarkable behaviour in alternate production, was used in this study. From 2D DICE gel, 192 spots were isolated: 97 showed increased expression in the off-crop buds as compared to the on-crop buds, while 95 exhibited enhanced expression in the on-crop buds versus the off-crop buds. These spots were identified by MALDI-MS or LC -MS-MS. The largest groups of proteins up-expressed in the off-crop buds were the proteins involved in carbohydrate and amino acid metabolism, and the proteins expressed in response to stimuli such as reactive oxygen species. The largest groups of proteins up-expressed in the on-crop buds were related to primary metabolism, oxidative stress and defence responses. Depending on their function, some of these proteins can stimulate the flowering, such as fructose-bisphosphate aldolase or leucine-rich repeat transmembrane protein kinase, while others can inhibit it, such as cytochrome c oxidase subunit II. Twenty-two other proteins with unknown functions were up-expressed in the on- or off-crop buds. (C) 2013 Elsevier Masson SAS. All rights reserved., M.C. Gonzalez-Mas was the recipient of a contract by the Fundacion AgroAlimed (Conselleria d'Agricultura, Pesca i Alimentacio, Generalitat Valenciana). This work has been supported by grants from the Instituto Nacional de Investigaciones Agrarias, Spain (RTA2009-00147, RTA2011-00114-00-00). The DIGE were performed by the Proteomic Service of the Instituto de Biologia Molecular y Celular de Plantas (UPV-Valencia). Mass analyses were carried out in the Proteomic Laboratory of CIPF (a ProteoRed_ISCIII member; the Spanish Proteomics Network); special thanks are due to Dr. L. Valero and E. Dionis for assistance and advice on mass analyses.

Viroids are single-stranded, circular, noncoding RNAs that infect plants, causing devastating diseases. In this work, we employed 2D DIGE, followed by MS identification, to analyze the response of tomato plants infected by Citrus exocortis viroid (CEVd). Among the differentially expressed proteins detected, 45 were successfully identified and classified into different functional categories. Validation results by RT-PCR allowed us to classify the proteins into two expression groups. First group included genes with changes at the transcriptional level upon CEVd infection, such as an endochitinase, a β-glucanase, and pathogenesis-related proteins, PR10 and P69G. All these defense proteins were also induced by gentisic acid, a pathogen-induced signal in compatible interactions. The second group of proteins showed no changes at the transcriptional level and included several ribosomal proteins and translation factors, such as the elongation factors 1 and 2 and the translation initiation factor 5-alpha. These results were validated by 2D Western blot, and possible PTMs caused by CEVd infection were detected. Moreover, an interaction between eukaryotic elongation factor 1 and CEVd was observed by 2D Northwestern. The present study provides new protein-related information on the mechanisms of plant resistance to pathogens., We would like to thank the Proteomic Service of the IBMCP (Instituto de Biologia Molecular y Celular de Plantas, Valencia, Spain) for the technical assistance. We also thank Dr. Alejandro Ferrando (Instituto de Biologia Molecular y Celular de Plantas UPV-CSIC) for critical reading of the manuscript, discussions, and for kindly providing us with the eIF5A antisera. We are also grateful to Dr. Brenda Hunter (University of Arizona) for both eEF1A and eEF2 antiserum. This work was supported by Grant BFU2009-11958 from Direccion General de Programas y Transferencia de Conocimiento, from Spanish Ministry of Science and Innovation. Laura Campos was the recipient of a predoctoral fellowship ACIF/2010/231 from Generalitat Valenciana (Spain). M. Pilar Lopez-Gresa held a postdoctoral fellowship JAEDoc_08_ 00402 from the Consejo Superior de Investigaciones Cientificas (Spain).