S Robinson, J. T., Thorvaldsdóttir, H., Winckler, W., Guttman, M., Lander, E. S., Getz, G., & Mesirov, J. P. (2011). Integrative genomics viewer. Nature Biotechnology, 29(1), 24-26. doi:10.1038/nbt.1754

[EN] A network of environmental inputs and internal signaling controls plant growth, development and organ elongation. In particular, the growth-promoting hormone gibberellin (GA) has been shown to play a significant role in organ elongation. The use of tomato as a model organism to study elongation presents an opportunity to study the genetic control of internode-specific elongation in a eudicot species with a sympodial growth habit and substantial internodes that can and do respond to external stimuli. To investigate internode elongation, a mutant with an elongated hypocotyl and internodes but wild-type petioles was identified through a forward genetic screen. In addition to stem-specific elongation, this mutant, named tomato internode elongated -1 (tie-1) is more sensitive to the GA biosynthetic inhibitor paclobutrazol and has altered levels of intermediate and bioactive GAs compared with wild-type plants. The mutation responsible for the internode elongation phenotype was mapped to GA2oxidase 7, a class III GA 2-oxidase in the GA biosynthetic pathway, through a bulked segregant analysis and bioinformatic pipeline, and confirmed by transgenic complementation. Furthermore, bacterially expressed recombinant TIE protein was shown to have bona fide GA 2-oxidase activity. These results define a critical role for this gene in internode elongation and are significant because they further the understanding of the role of GA biosynthetic genes in organ-specific elongation. This work used the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH S10 Instrumentation Grants S10RR029668 and S10RR027303. We thank the Tomato Genetics Resource Center for providing seed of the M82 and Heinz cultivars. The material was developed by and/or obtained from the UC Davis/C M Rick Tomato Genetics Resource Center and maintained by the Department of Plant Sciences, University of California, Davis, CA 95616, USA. We thank Anthony Bolger, Alisdair Fernie and Bjorn Usadel for providing us with access to pre-publication genomic reads of the S. lycopersicum cultivar M82, and Cristina Urbez and Noel Blanco-Tourinan (IBMCP, Spain) for technical help with in vitro production of TIE1. This work was supported in part by the Elsie Taylor Stocking Memorial Fellowship awarded to ASL in 2013, by NSF grant IOS-0820854, by USDA National Institute of Food and Agriculture project CA-D-PLB-2465-H, by internal UC Davis funds, and by Spanish Ministry of Economy and Competitiveness grant BFU2016-80621-P. Lavelle, A.; Gath, N.; Devisetty, U.; Carrera Bergua, E.; Lopez Diaz, I.; Blazquez Rodriguez, MA.; Maloof, J. (2018). The role of a class III gibberellin 2-oxidase in tomato internode elongation. The Plant Journal. https://doi.org/10.1111/tpj.14145 Andrés, F., Porri, A., Torti, S., Mateos, J., Romera-Branchat, M., García-Martínez, J. L., … Coupland, G. (2014). SHORT VEGETATIVE PHASE reduces gibberellin biosynthesis at theArabidopsisshoot apex to regulate the floral transition. Proceedings of the National Academy of Sciences, 111(26), E2760-E2769. doi:10.1073/pnas.1409567111 Bolger, A., Scossa, F., Bolger, M. E., Lanz, C., Maumus, F., Tohge, T., … Fernie, A. R. (2014). The genome of the stress-tolerant wild tomato species Solanum pennellii. Nature Genetics, 46(9), 1034-1038. doi:10.1038/ng.3046 Bowen, M. E., Henke, K., Siegfried, K. R., Warman, M. L., & Harris, M. P. (2011). Efficient Mapping and Cloning of Mutations in Zebrafish by Low-Coverage Whole-Genome Sequencing. Genetics, 190(3), 1017-1024. doi:10.1534/genetics.111.136069 Burset, M. (2000). Analysis of canonical and non-canonical splice sites in mammalian genomes. Nucleic Acids Research, 28(21), 4364-4375. doi:10.1093/nar/28.21.4364 Chen, W., Yao, J., Chu, L., Yuan, Z., Li, Y., & Zhang, Y. (2015). Genetic mapping of the nulliplex-branch gene (gb_nb1) in cotton using next-generation sequencing. Theoretical and Applied Genetics, 128(3), 539-547. doi:10.1007/s00122-014-2452-2 Cingolani, P., Platts, A., Wang, L. L., Coon, M., Nguyen, T., Wang, L., … Ruden, D. M. (2012). A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff. Fly, 6(2), 80-92. doi:10.4161/fly.19695 Cuperus, J. T., Montgomery, T. A., Fahlgren, N., Burke, R. T., Townsend, T., Sullivan, C. M., & Carrington, J. C. (2009). Identification of MIR390a precursor processing-defective mutants in Arabidopsis by direct genome sequencing. Proceedings of the National Academy of Sciences, 107(1), 466-471. doi:10.1073/pnas.0913203107 Curtis, M. D., & Grossniklaus, U. (2003). A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta. Plant Physiology, 133(2), 462-469. doi:10.1104/pp.103.027979 Devisetty, U. K., Covington, M. F., Tat, A. V., Lekkala, S., & Maloof, J. N. (2014). Polymorphism Identification and Improved Genome Annotation ofBrassica rapaThrough Deep RNA Sequencing. G3: Genes|Genomes|Genetics, 4(11), 2065-2078. doi:10.1534/g3.114.012526 Eckardt, N. A. (2007). GA Perception and Signal Transduction: Molecular Interactions of the GA Receptor GID1 with GA and the DELLA Protein SLR1 in Rice. The Plant Cell, 19(7), 2095-2097. doi:10.1105/tpc.107.054916 Ernst, H. A., Lo Leggio, L., Willemoës, M., Leonard, G., Blum, P., & Larsen, S. (2006). Structure of the Sulfolobus solfataricus α-Glucosidase: Implications for Domain Conservation and Substrate Recognition in GH31. Journal of Molecular Biology, 358(4), 1106-1124. doi:10.1016/j.jmb.2006.02.056 Fillatti, J. J., Kiser, J., Rose, R., & Comai, L. (1987). Efficient Transfer of a Glyphosate Tolerance Gene into Tomato Using a Binary Agrobacterium Tumefaciens Vector. Nature Biotechnology, 5(7), 726-730. doi:10.1038/nbt0787-726 Garrison , E. Marth , G. 2012 Haplotype-based variant detection from short-read sequencing Hedden, P., & Graebe, J. E. (1985). Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates ofCucurbita maxima endosperm andMalus pumila embryos. Journal of Plant Growth Regulation, 4(1-4), 111-122. doi:10.1007/bf02266949 Kimura, S., & Sinha, N. (2008). Tomato (Solanum lycopersicum): A Model Fruit-Bearing Crop. Cold Spring Harbor Protocols, 2008(12), pdb.emo105-pdb.emo105. doi:10.1101/pdb.emo105 Koenig, D., Jimenez-Gomez, J. M., Kimura, S., Fulop, D., Chitwood, D. H., Headland, L. R., … Maloof, J. N. (2013). Comparative transcriptomics reveals patterns of selection in domesticated and wild tomato. Proceedings of the National Academy of Sciences, 110(28), E2655-E2662. doi:10.1073/pnas.1309606110 Li, H., & Durbin, R. (2009). Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 25(14), 1754-1760. doi:10.1093/bioinformatics/btp324 Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., … Homer, N. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics, 25(16), 2078-2079. doi:10.1093/bioinformatics/btp352 Li, J., Sima, W., Ouyang, B., Wang, T., Ziaf, K., Luo, Z., … Ye, Z. (2012). Tomato SlDREB gene restricts leaf expansion and internode elongation by downregulating key genes for gibberellin biosynthesis. Journal of Experimental Botany, 63(18), 6407-6420. doi:10.1093/jxb/ers295 Lorrain, S., & Fankhauser, C. (2012). Plant Development: Should I Stop or Should I Grow? Current Biology, 22(16), R645-R647. doi:10.1016/j.cub.2012.06.054 Menda, N., Semel, Y., Peled, D., Eshed, Y., & Zamir, D. (2004). In silicoscreening of a saturated mutation library of tomato. The Plant Journal, 38(5), 861-872. doi:10.1111/j.1365-313x.2004.02088.x Michelmore, R. W., Paran, I., & Kesseli, R. V. (1991). Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proceedings of the National Academy of Sciences, 88(21), 9828-9832. doi:10.1073/pnas.88.21.9828 Pimenta Lange, M. J., Liebrandt, A., Arnold, L., Chmielewska, S.-M., Felsberger, A., Freier, E., … Lange, T. (2013). Functional characterization of gibberellin oxidases from cucumber, Cucumis sativus L. Phytochemistry, 90, 62-69. doi:10.1016/j.phytochem.2013.02.006 Raskin, I., & Kende, H. (1984). Role of Gibberellin in the Growth Response of Submerged Deep Water Rice. Plant Physiology, 76(4), 947-950. doi:10.1104/pp.76.4.947 Reinecke, D. M., Wickramarathna, A. D., Ozga, J. A., Kurepin, L. V., Jin, A. L., Good, A. G., & Pharis, R. P. (2013). Gibberellin 3-oxidase Gene Expression Patterns Influence Gibberellin Biosynthesis, Growth, and Development in Pea. PLANT PHYSIOLOGY, 163(2), 929-945. doi:10.1104/pp.113.225987 Robinson, M. D., & Oshlack, A. (2010). A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biology, 11(3), R25. doi:10.1186/gb-2010-11-3-r25 Robinson, M. D., McCarthy, D. J., & Smyth, G. K. (2009). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 26(1), 139-140. doi:10.1093/bioinformatics/btp616 Schneeberger, K., Ossowski, S., Lanz, C., Juul, T., Petersen, A. H., Nielsen, K. L., … Andersen, S. U. (2009). SHOREmap: simultaneous mapping and mutation identification by deep sequencing. Nature Methods, 6(8), 550-551. doi:10.1038/nmeth0809-550 Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9(7), 671-675. doi:10.1038/nmeth.2089 Schomburg, F. M., Bizzell, C. M., Lee, D. J., Zeevaart, J. A. D., & Amasino, R. M. (2002). Overexpression of a Novel Class of Gibberellin 2-Oxidases Decreases Gibberellin Levels and Creates Dwarf Plants. The Plant Cell, 15(1), 151-163. doi:10.1105/tpc.005975 Seo, M., Jikumaru, Y., & Kamiya, Y. (2011). Profiling of Hormones and Related Metabolites in Seed Dormancy and Germination Studies. Methods in Molecular Biology, 99-111. doi:10.1007/978-1-61779-231-1_7 Sun, T. (2011). The Molecular Mechanism and Evolution of the GA–GID1–DELLA Signaling Module in Plants. Current Biology, 21(9), R338-R345. doi:10.1016/j.cub.2011.02.036 Sun, T., & Gubler, F. (2004). MOLECULAR MECHANISM OF GIBBERELLIN SIGNALING IN PLANTS. Annual Review of Plant Biology, 55(1), 197-223. doi:10.1146/annurev.arplant.55.031903.141753 Thorvaldsdottir, H., Robinson, J. T., & Mesirov, J. P. (2012). Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Briefings in Bioinformatics, 14(2), 178-192. doi:10.1093/bib/bbs017 (2012). The tomato genome sequence provides insights into fleshy fruit evolution. Nature, 485(7400), 635-641. doi:10.1038/nature11119 Trapnell, C., Pachter, L., & Salzberg, S. L. (2009). TopHat: discovering splice junctions with RNA-Seq. Bioinformatics, 25(9), 1105-1111. doi:10.1093/bioinformatics/btp120 Trapnell, C., Roberts, A., Goff, L., Pertea, G., Kim, D., Kelley, D. R., … Pachter, L. (2012). Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature Protocols, 7(3), 562-578. doi:10.1038/nprot.2012.016 Tsai, H., Howell, T., Nitcher, R., Missirian, V., Watson, B., Ngo, K. J., … Comai, L. (2011). Discovery of Rare Mutations in Populations: TILLING by Sequencing. Plant Physiology, 156(3), 1257-1268. doi:10.1104/pp.110.169748 Ueguchi-Tanaka, M., Nakajima, M., Katoh, E., Ohmiya, H., Asano, K., Saji, S., … Matsuoka, M. (2007). Molecular Interactions of a Soluble Gibberellin Receptor, GID1, with a Rice DELLA Protein, SLR1, and Gibberellin. The Plant Cell, 19(7), 2140-2155. doi:10.1105/tpc.106.043729 Wickham, H. (2016). ggplot2. Use R! doi:10.1007/978-3-319-24277-4 Winter, D., Vinegar, B., Nahal, H., Ammar, R., Wilson, G. V., & Provart, N. J. (2007). An «Electronic Fluorescent Pictograph» Browser for Exploring and Analyzing Large-Scale Biological Data Sets. PLoS ONE, 2(8), e718. doi:10.1371/journal.pone.0000718 Xu, H., Liu, Q., Yao, T., & Fu, X. (2014). Shedding light on integrative GA signaling. Current Opinion in Plant Biology, 21, 89-95. doi:10.1016/j.pbi.2014.06.010 Yamaguchi, S. (2008). Gibberellin Metabolism and its Regulation. Annual Review of Plant Biology, 59(1), 225-251. doi:10.1146/annurev.arplant.59.032607.092804

The role of a class III gibberellin 2-oxidase in tomato internode elongation

Abstract