First RNA-seq approach to study fruit set and parthenocarpy in zucchini (Cucurbita pepo L.)

[EN] Background: Zucchini fruit set can be limited due to unfavourable environmental conditions in off-seasons crops that caused ineffective pollination/fertilization. Parthenocarpy, the natural or artificial fruit development without fertilization, has been recognized as an important trait to avoid this problem, and is related to auxin signalling. Nevertheless, differences found in transcriptome analysis during early fruit development of zucchini suggest that other complementary pathways could regulate fruit formation in parthenocarpic cultivars of this species. The development of next-generation sequencing technologies (NGS) as RNA-sequencing (RNA-seq) opens a new horizon for mapping and quantifying transcriptome to understand the molecular basis of pathways that could regulate parthenocarpy in this species. The aim of the current study was to analyze fruit transcriptome of two cultivars of zucchini, a non-parthenocarpic cultivar and a parthenocarpic cultivar, in an attempt to identify key genes involved in parthenocarpy. Results: RNA-seq analysis of six libraries (unpollinated, pollinated and auxin treated fruit in a non-parthenocarpic and parthenocarpic cultivar) was performed mapping to a new version of C. pepo transcriptome, with a mean of 92% success rate of mapping. In the non-parthenocarpic cultivar, 6479 and 2186 genes were differentially expressed (DEGs) in pollinated fruit and auxin treated fruit, respectively. In the parthenocarpic cultivar, 10,497 in pollinated fruit and 5718 in auxin treated fruit. A comparison between transcriptome of the unpollinated fruit for each cultivar has been performed determining that 6120 genes were differentially expressed. Annotation analysis of these DEGs revealed that cell cycle, regulation of transcription, carbohydrate metabolism and coordination between auxin, ethylene and gibberellin were enriched biological processes during pollinated and parthenocarpic fruit set. Conclusion: This analysis revealed the important role of hormones during fruit set, establishing the activating role of auxins and gibberellins against the inhibitory role of ethylene and different candidate genes that could be useful as markers for parthenocarpic selection in the current breeding programs of zucchini.Research worked is supported by the project RTA2014-00078 from the Spanish Institute of Agronomy Research INIA (Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria) and also PP.AVA.AVA201601.7, FEDER y FSE (Programa Operativo FSE de Andalucia 2007-2013 "Andalucia se mueve con Europa"). TPV is supported by a FPI scholarship from RTA2011-00044-C02-01/02 project of INIA. The funding agencies were not involved in the design of the study, collection, analysis, and interpretation of data and in writing the manuscript.Pomares-Viciana, T.; Del Rio-Celestino, M.; Roman, B.; Die, J.; Picó Sirvent, MB.; Gómez, P. (2019). First RNA-seq approach to study fruit set and parthenocarpy in zucchini (Cucurbita pepo L.). BMC Plant Biology. 19:1-20. https://doi.org/10.1186/s12870-019-1632-2S12019Varga A, Bruinsma J. Tomato. In: Monselise SP, editor. CRC Handbook of Fruit Set and Development. Boca Raton: CRC Press; 1986. p. 461–80.Nepi M, Cresti L, Guarnieri M, Pacini E. Effect of relative humidity on water content, viability and carbohydrate profile of Petunia hybrid and Cucurbita pepo pollen. Plant Syst Evol. 2010;284:57–64.Gustafson FG. Parthenocarpy: natural and artificial. Bot Rev. 1942;8:599–654.Robinson RW, Reiners S. Parthenocarpy in summer squash. Hortscience. 1999;34:715–7.Pomares-Viciana T, Die J, Del Río-Celestino M, Román B, Gómez P. Auxin signalling regulation during induced and parthenocarpic fruit set in zucchini. Mol Breeding. 2017;37:56.Ozga JA, Reinecke DM. Hormonal interactions in fruit development. J Plant Growth Regul. 2003;22:73–81.Kim IS, Okubo H, Fujieda K. Endogenous levels of IAA in relation to parthenocarpy in cucumber (Cucumis sativus L). Sci Hortic. 1992;52:1–8.Olimpieri I, Siligato F, Caccia R, Mariotti L, Ceccarelli N, Soressi GP, et al. Tomato fruit set driven by pollination or by the parthenocarpic fruit allele are mediated by transcriptionally regulated gibberellin biosynthesis. Planta. 2007;226:877–88.Cui L, Zhang T, Li J, Lou Q, Chen J. Cloning and expression analysis of Cs-TIR1/AFB2: the fruit development-related genes of cucumber (Cucumis sativus L.). Acta Physiol Plant. 2014;36:139–49.De Jong M, Wolters-Arts J, Feron R, Mariani C, Vriezen WH. The Solanum lycopersicum auxin response factor 7 (SlARF7) regulates auxin signalling during tomato fruit set and development. Plant J. 2009;57:160–70.Wang H, Jones B, Li Z, Frasse P, Delalande C, Regad F, Chaabouni S, Latché A, Pech JC, Bouzayen M. The tomato aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis. Plant Cell. 2005;17(10):2676–92.Goetz M, Vivian-Smith A, Johnson SD, Koltunow AM. AUXIN RESPONSE FACTOR 8 is a negative regulator of fruit initiation in Arabidopsis. Plant Cell. 2006;18(8):1873–86.Mazzucato A, Cellini F, Bouzayen M, Zouine M, Mila I, Minoia S et al. A TILLING allele of the tomato aux/IAA9 gene offers new insights into fruit set mechanisms and perspectives for breeding seedless tomatoes. Mol Breeding. 2015; 35(22):1-15.Blanca J, Cañizares J, Roig C, Ziarsolo P, Nuez F, Picó B. Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae). BMC Genomics. 2011;12:104.Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009;10(1):57–63.Da Fonseca RR, Albrechtsen A, Themudo GE, Ramos-Madrigal J, Sibbesen JA, Maretty L, et al. Next-generation biology: sequencing and data analysis approaches for non-model organisms. Mar Genomics. 2016;30:3–13.Conesa A, Madrigal P, Tarazona S, Gomez-Cabrero D, Cervera A, McPherson A, et al. A survey of best practices for RNA-seq data analysis. Genome Biol. 2016;17:13.Li J, Cui ZWJ, Zhang T, Guo Q, Xu J, Li J, et al. Transcriptome comparison of global distinctive features between pollination and parthenocarpic fruit set reveals transcriptional phytohormone cross-talk in cucumber (Cucumis sativus L). Plant Cell Physiol. 2014;55(7):1325–42.Fu L, Niu B, Zhu Z, Wu S, Li W. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics. 2012;28(23):3150–2.Montero-Pau J, Blanca J, Bombarely A, Ziarsolo P, Esteras C, Martí-Gómez C, et al. De novo assembly of the zucchini genome reveals a whole genome duplication associated with the origin of the Cucurbita genus. Plant Biotechnol J. 2017. https://doi.org/10.1111/pbi.12860 .Vriezen WH, Feron R, Maretto F, Keijman J, Mariani C. Changes in tomato ovary transcriptome demonstrate complex hormonal regulation of fruit set. New Phytol. 2008;177:60–76.Tang N, Deng W, Hu G, Hu N, Li Z. Transcriptome profiling reveals the regulatory mechanism underlying pollination dependent and parthenocarpic fruit set mainly mediated by auxin and gibberellin. PLoS One. 2015;10(4):e0125355.Li J, Yan S, Yang W, Li Y, Xia M, Chen Z, et al. Transcriptomic analysis reveals the roles of microtubule-related genes and transcription factors in fruit length regulation in cucumber (Cucumis sativus L.). Sci Rep. 2015;26(5):8031.Mironov V, De Veylder L, Van Montagu M, Inze D. Cyclin-dependent kinases and cell division in plants- the nexus. Plant Cell. 1999;11(4):509–22.Perrot-Rechenmann C. Cellular responses to auxin: division versus expansion. Cold Spring Harb Perspect Biol. 2010;2(5):a001446.De Veylder L, Beeckman T, Beemster GT, Krols L, Terras F, Landrieu I, et al. Functional analysis of cyclin-dependent kinase inhibitors of Arabidopsis. Plant Cell. 2001;13:1653–68.Nieuwland J, Menges M, Murray JAH. The plant cyclins. In: Inze D, editor. Cell cycle control and plant development, vol. 2007. Oxford: Wiley-Blackwell Publishing; 2007. p. 33–61.Menges M, Samland AK, Planchais S, Murray JA. The D-type cyclin CYCD3;1 is limiting for the G1-to-S-phasetransition in Arabidopsis. Plant Cell. 2006;18:893–906.Boruc J, Mylle E, Duda M, De Clercq R, Rombauts S, Geelen D, et al. Systematic localization of the Arabidopsis core cell cycle proteins reveals novel cell division complexes. Plant Physiol. 2010;152(2):553–65.Sampedro J, Cosgrove DJ. The expansin superfamily. Genome Biol. 2005;6:242.Esmon CA, Tinsley AG, Ljung K, Sandberg G, Hearne LB, Liscum E. A gradient of auxin and auxin-dependent transcription precedes tropic growth responses. Proc Natl Acad Sci. 2006;103:236–41.De Folter S, Busscher J, Colombo L, Losa A, Angenent GC. Transcript profiling of transcription factors genes during siliques development in Arabidopsis. Plant Mol Bio. 2004;56:351–3662004.Son O, Cho HY, Kim MR, Lee H, Lee MS, Song E, et al. Induction of a homeodomain-leucine zipper gene by auxin is inhibited by cytokinin in Arabidopsis roots. Biochem Biophys Res Commun. 2005;326(1):203–9.Olsson ASB, Engstroem P, Seoderman E. The homeobox genes ATHB12 and ATHB7 encode potential regulators of growth in response to water deficit in Arabidopsis. Plant Mol Biol. 2004;55:663–77.Merrow SB, Hopp RJ. Storage effects on winter squashes. Associations between the sugar and starch content of and the degree of preference for winter squashes. J Agric Food Chem. 1961;9:321–6.Berg JM, Tymoczko JL, Stryer L. Carbohydrates. In: Freeman WH, editor. Biochemistry. 5th ed. New York: W H Freeman; 2002.Prabhakar V, Löttgert T, Gigolashvili T, Bell K, Flügge UI, Häusler RE. Molecular and functional characterization of the plastid-localized phosphoenolpyruvate enolase (ENO1) from Arabidopsis thaliana. FEBS Lett. 2009;583(6):983–91.Rius SP, Casati P, Iglesias AA, Gomez-Casati DF. Characterization of Arabidopsis lines deficient in GAPC-1, a cytosolic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. Plant Physiol. 2008;148(3):1655–67.Van der Linde K, Gutsche N, Leffers HM, Lindermayr C, Müller B, Holtgrefe S, et al. Regulation of plant cytosolic aldolase functions by redox-modifications. Plant Physiol Biochem. 2011;49(9):946–57.Lim H, Cho MH, Jeon JS, Bhoo SH, Kwon YK, Hahn TR. Altered expression of pyrophosphate: fructose-6-phosphate 1-phosphotransferase affects the growth of transgenic Arabidopsis plants. Mol Cells. 2009;27(6):641–9.Baud S, Wuillème S, Dubreucq B, De Almeida A, Vuagnat C, Lepiniec L, et al. Function of plastidial pyruvate kinases in seeds of Arabidopsis thaliana. Plant J. 2007;52:405–19.De Jong M, Mariani C, Vriezen WH. The role of auxin and gibberellin in tomato fruit set. J Exp Bot. 2009;60(5):1523–32.Martínez C, Manzano S, Megías Z, Garrido D, Picó B, Jamilena M. Involvement of ethylene biosynthesis and signalling in fruit set and early fruit development in zucchini squash (Cucurbita pepo L.). BMC Plant Biol. 2013;13:139.Serrani JC, Fos M, Atarés A, Garcia-martinez JL. Effect of gibberellin and auxin on parthenocarpic fruit growth induction in the cv. micro-tom of tomato. J Plant Growth Regul. 2007;26:211–21.Mapelli S. Changes in cytokinin in the fruits of parthenocarpic and normal tomatoes. Plant Sci Lett. 1981;22:227–33.Ulmasov T, Hagen G, Guilfoyle TJ. Activation and repression of transcription by auxin-response factors. Proc Natl Acad Sci U S A. 1999;96:5844–9.Ulmasov T, Hagen G, Guilfoyle TJ. Dimerization and DNA binding of auxin response factors. Plant J. 1999;19:309–19.Tiwari SB, Hagen G, Guilfoyle TJ. Aux/IAA proteins contain a potent transcriptional repression domain. Plant Cell. 2004;16:533–43.Switzenberg JA, Beaudry RM, Grumet R. Effect of CRC:: etr1-1 transgene expression on ethylene production, sex expression, fruit set and fruit ripening in transgenic melon (Cucumis melo L.). Transgenic Res. 2015;24(3):497-507.Nitsch LM, Oplaat C, Feron R, Ma Q, Wolters-Arts M, Hedden P, et al. Abscisic acid levels in tomato ovaries are regulated by LeNCED1 and SlCYP707A1. Planta. 2009;229(6):1335–46.Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-seq. Nat Methods. 2008;5(7):621–8.Robinson MD, McCarthy DJ, Smyth GK. Edger: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2008;26(1):139–40.Raza K, Mishra A. A novel anticlustering filtering algorithm for the prediction of genes as a drug target. Am J Bio Engi. 2012;2(5):206–11.Van Iterson M, Boer JM, Menezes RX. Filtering, FDR and power. BMCBioinformatics. 2010;11:450.Conesa A, Götz S, Garcia-Gomez JM, Terol J, Talon M, Robles M. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics. 2005;21:3674–6.Berardini TZ, Reiser L, Li D, Mezheritsky Y, Muller R, Strait E, Huala E. The Arabidopsis information resource: making and mining the “gold standard” annotated reference plant genome. Genesis. 2015. https://doi.org/10.1002/dvg.22877 .Bairoch A, Apweiler R. The SWISS-PROT protein sequence database and its supplement TrEMBL. Nucleic Acids Res. 2000;28(1):45–8.Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL. NCBI BLAST: a better web interface. Nucleic Acids Res. 2008;36:W5–9.Wyatt LE, Strickler SR, Mueller LA, Mazourek M. An acorn squash (Cucurbita pepo ssp. ovifera) fruit and seed transcriptome as a resource for the study of fruit traits in Cucurbita. Hortic Res. 2015;2:14070. https://doi.org/10.1038/hortres.2014.70 .Zhang A, Ren GA, Sun YA, Guo H, Zhang SA, Zhang FA, et al. A high-density genetic map for anchoring genome sequences and identifying QTLs associated with dwarf vine in pumpkin (Cucurbita maxima Duch.). BMC Genomics. 2015;16:1101.Finn RD, Attwood TK, Babbit AB, Bork P, Bridge AJ, Chang HY. InterPro in 2017-beyond protein family and domain annotations. Nucleic Acids Res. https://doi.org/10.1093/nar/gkw1107 .Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Sherlock G. Gene ontology: tool for the unification of biology. Nat Genet. 2000;25(1):25–9.Kanehisa M, Araki M, Goto S, et al. KEGG for linking genomes to life and the environment. Nucleic Acids Res. 2008;36:480–4

Tomato fruit set driven by pollination or by the parthenocarpic fruit allele are mediated by transcriptionally regulated gibberellin biosynthesis

We investigated the role of gibberellins (GAs) in the phenotype of parthenocarpic fruit (pat), a recessive mutation conferring parthenocarpy in tomato (Solanum lycopersicum L.). Novel phenotypes that parallel those reported in plants repeatedly treated with gibberellic acid or having a GA-constitutive response indicate that the pat mutant probably expresses high levels of GA. The retained sensitivity to the GA-biosynthesis inhibitor paclobutrazol reveals that this condition is dependent on GA biosynthesis. Expression analysis of genes encoding key enzymes involved in GA biosynthesis shows that in normal tomato ovaries, the GA20ox1 transcript is in low copy number before anthesis and only pollination and fertilization increase its transcription levels and, thus, GA biosynthesis. In the unpollinated ovaries of the pat mutant, this mechanism is de-regulated and GA20ox1 is constitutively expressed, indicating that a high GA concentration could play a part in the parthenocarpic phenotype. The levels of endogenous GAs measured in the Xoral organs of the pat mutant support such a hypothesis. Collectively, the data indicate that transcriptional regulation of GA20ox1 mediates pollination-induced fruit set in tomato and that parthenocarpy in pat results from the mis-regulation of this mechanism. As genes involved in the control of GA synthesis (LeT6, LeT12 and LeCUC2) and response (SPY) are also altered in the pat ovary, it is suggested that the pat mutation aVects a regulatory gene located upstream of the control of fruit set exerted by GAs

Novel pyrazole derivatives as neutral CB1 antagonists with significant activity towards food intake.

In spite of rimonabant’s withdrawal from the European market due to its adverse effects, interest in the development of drugs based on CB1 antagonists is revamping on the basis of the peculiar properties of this class of compounds. In particular, new strategies have been proposed for the treatment of obesity and/or related risk factors through CB1 antagonists, i.e. by the development of selectively peripherally acting agents or by the identification of neutral CB1 antagonists. New compounds based on the lead CB1 antagonist/inverse agonist rimonabant have been synthesized with focus on obtaining neutral CB1 antagonists. Amongst the new derivatives described in this paper, the mixture of the two enantiomers (#1;)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-(2-cyclohexyl-1-hydroxyethyl)-4-methyl-1H-pyrazole ((#1;)-5), and compound 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-[(Z)-2-cyclohexyl-1-fluorovinyl]-4- methyl-1H-pyrazole ((Z)-6), showed interesting pharmacological profiles. According to the preliminary pharmacological evaluation, these novel pyrazole derivatives showed in fact both neutral CB1 antagonism behaviour and significant in vivo activity towards food intake

Combined host- and pathogen-directed therapy for the control of mycobacterium abscessus infection

Mycobacterium abscessus is the etiological agent of severe pulmonary infections in vulnerable patients, such as those with cystic fibrosis (CF), where it represents a relevant cause of morbidity and mortality. Treatment of pulmonary infections caused by M. abscessus remains extremely difficult, as this species is resistant to most classes of antibiotics, including macrolides, aminoglycosides, rifamycins, tetracyclines, and β-lactams. Here, we show that apoptotic body like liposomes loaded with phosphatidylinositol 5-phosphate (ABL/PI5P) enhance the antimycobacterial response, both in macrophages from healthy donors exposed to pharmacological inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) and in macrophages from CF patients, by enhancing phagosome acidification and reactive oxygen species (ROS) production. The treatment with liposomes of wild-type as well as CF mice, intratracheally infected with M. abscessus, resulted in about a 2-log reduction of pulmonary mycobacterial burden and a significant reduction of macrophages and neutrophils in bronchoalveolar lavage fluid (BALF). Finally, the combination treatment with ABL/PI5P and amikacin, to specifically target intracellular and extracellular bacilli, resulted in a further significant reduction of both pulmonary mycobacterial burden and inflammatory response in comparison with the single treatments. These results offer the conceptual basis for a novel therapeutic regimen based on antibiotic and bioactive liposomes, used as a combined host- and pathogen-directed therapeutic strategy, aimed at the control of M. abscessus infection, and of related immunopathogenic responses, for which therapeutic options are still limited. IMPORTANCE Mycobacterium abscessus is an opportunistic pathogen intrinsically resistant to many antibiotics, frequently linked to chronic pulmonary infections, and representing a relevant cause of morbidity and mortality, especially in immunocompromised patients, such as those affected by cystic fibrosis. M. abscessus-caused pulmonary infection treatment is extremely difficult due to its high toxicity and long-lasting regimen with life-impairing side effects and the scarce availability of new antibiotics approved for human use. In this context, there is an urgent need for the development of an alternative therapeutic strategy that aims at improving the current management of patients affected by chronic M. abscessus infections. Our data support the therapeutic value of a combined host- and pathogen-directed therapy as a promising approach, as an alternative to single treatments, to simultaneously target intracellular and extracellular pathogens and improve the clinical management of patients infected with multidrug-resistant pathogens such as M. abscessus