Abstract
Key message
Seedlessness, one of the most desired traits in fleshy fruits, can be obtained altering solely AGL11 gene, a D -class MADS-box. Opposite to overlap** functions described for ovule identity.
Abstract
AGAMOUS like-11 (AGL11) is a D-class MADS-box gene that determines ovule identity in model species. In grapevine, VviAGL11 has been proposed as the main candidate gene responsible for seedlessness because ovules develop into seeds after fertilization. Here, we demonstrate that AGL11 has a direct role in the determination of the seedless phenotype. In grapevine, broad expression analysis revealed very low expression levels of the seedless allele compared to the seeded allele at the pea-size berry stage. Heterozygous genotypes have lower transcript accumulation than expected considering the diploid nature of grapevine, thereby revealing that the dominant phenotype previously described for seedlessness is based on its expression level. In a seeded somatic variant of Sultanina (Thompson Seedless) that has well-developed seeds, Sultanine Monococco, structural differences were identified in the regulatory region of VviAGL11. These differences affect transcript accumulation levels and explain the phenotypic differences between the two varieties. Functional experiments in tomato demonstrated that SlyAGL11 gene silencing produces seedless fruits and that the degree of seed development is proportional to transcript accumulation levels. Furthermore, the genes involved in seed coat development, SlyVPE1 and SlyVPE2 in tomato and VviVPE in grapevine, that are putatively controlled by SlyAGL11 and VviAGL11, respectively, are expressed at lower levels in silenced tomato lines and in seedless grapevine genotypes. In conclusion, this work provides evidence that the D-class MADS-box AGL11 plays a major and direct role in seed development in fleshy fruits, providing a valuable tool for further analysis of fruit development.
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs00299-015-1882-x/MediaObjects/299_2015_1882_Fig1_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs00299-015-1882-x/MediaObjects/299_2015_1882_Fig2_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs00299-015-1882-x/MediaObjects/299_2015_1882_Fig3_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs00299-015-1882-x/MediaObjects/299_2015_1882_Fig4_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs00299-015-1882-x/MediaObjects/299_2015_1882_Fig5_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs00299-015-1882-x/MediaObjects/299_2015_1882_Fig6_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs00299-015-1882-x/MediaObjects/299_2015_1882_Fig7_HTML.gif)
Similar content being viewed by others
References
Alvarez-Buylla ER, Liljegren SJ, Pelaz S et al (2000) MADS-box gene evolution beyond flowers: expression in pollen, endosperm, guard cells, roots and trichomes. Plant J Cell Mol Biol 24:457–466
Antliff AJ, Webster WJ (1962) Bruce’s sport—a mutant of the sultana. Aust J Agric Anim Husb 2:97–100
Battaglia R, Brambilla V, Colombo L, Stuitje AR, Kater MM (2006) Functional analysis of MADS-box genes controlling ovule development in Arabidopsis using the ethanol-inducible alc gene-expression system. Mech Dev 123:267–276. doi:10.1016/j.mod.2006.01.002
Becker A, Theissen G (2003) The major clades of MADS-box genes and their role in the development and evolution of flowering plants. Mol Phylogenet Evol 29:464–489
Boss PK, Vivier M, Matsumoto S, Dry IB, Thomas MR (2001) A cDNA from grapevine (Vitis vinifera L.), which shows homology to AGAMOUS and SHATTERPROOF, is not only expressed in flowers but also throughout berry development. Plant Mol Biol 45:541–553
Boss PK, Sensi E, Hua C, Davies C, Thomas MR (2002) Cloning and characterisation of grapevine (Vitis vinifera L.) MADS-box genes expressed during inflorescence and berry development. Plant Sci 162:887–895. doi:10.1016/S0168-9452(02)00034-1
Bouquet A, Danglot Y (1996) Inheritance of seedlessness in grapevine (Vitis vinifera L.). Vitis 35:35–42
Branzei D, Foiani M (2007) Template switching: from replication fork repair to genome rearrangements. Cell 131:1228–1230. doi:10.1016/j.cell.2007.12.007
Brukhin V, Hernould M, Gonzalez N, Chevalier C, Mouras A (2003) Flower development schedule in tomato Lycopersicon esculentum cv. sweet cherry. Sex Plant Reprod 15:311–320. doi:10.1007/s00497-003-0167-7
Busi MV, Bustamante C, D’Angelo C, Hidalgo-Cuevas M, Boggio SB, Valle EM, Zabaleta E (2003) MADS-box genes expressed during tomato seed and fruit development. Plant Mol Biol 52:801–815
Cabezas JA, Cervera MT, Ruiz-Garcia L, Carreno J, Martinez-Zapater JM (2006) A genetic analysis of seed and berry weight in grapevine. Genome Natl Res Counc Can Genome Cons Natl de Rech Can 49:1572–1585. doi:10.1139/g06-122
Cain DW, Emershad RL, Tarailo RE (1983) In-ovulo embryo culture and seedling development of seeded and seedless grapes (Vitis vinifera L.). Vitis 22:9–14
Carmona MJ, Chaib J, Martinez-Zapater JM, Thomas MR (2008) A molecular genetic perspective of reproductive development in grapevine. J Exp Bot 59:2579–2596. doi:10.1093/jxb/ern160
Coen ES, Meyerowitz EM (1991) The war of the whorls: genetic interactions controlling flower development. Nature 353:31–37. doi:10.1038/353031a0
Colombo L, Franken J, Koetje E, van Went J, Dons HJ, Angenent GC, van Tunen AJ (1995) The petunia MADS box gene FBP11 determines ovule identity. Plant Cell 7:1859–1868
Colombo L, Franken J, Van der Krol AR, Wittich PE, Dons HJ, Angenent GC (1997) Downregulation of ovule-specific MADS box genes from petunia results in maternally controlled defects in seed development. Plant Cell 9:703–715
Coombe BG (1995) Growth stages of the grapevine: adoption of a system for identifying grapevine growth stages. Aust J Grape Wine Res 1:104–110. doi:10.1111/j.1755-0238.1995.tb00086.x
Costantini L, Battilana J, Lamaj F, Fanizza G, Grando MS (2008) Berry and phenology-related traits in grapevine (Vitis vinifera L.): from quantitative trait loci to underlying genes. BMC Plant Biol 8:38. doi:10.1186/1471-2229-8-38
Daminato M, Masiero S, Resentini F, Lovisetto A, Casadoro G (2014) Characterization of TM8, a MADS-box gene expressed in tomato flowers. BMC Plant Biol 14:319. doi:10.1186/s12870-014-0319-y
de Folter S, Angenent GC (2006) Trans meets cis in MADS science. Trends Plant Sci 11:224–231. doi:10.1016/j.tplants.2006.03.008
Dhekney SA, Li ZT, Dutt M, Gray DJ (2012) Initiation and transformation of grapevine embryogenic cultures. Methods Mol Biol 847:215–225. doi:10.1007/978-1-61779-558-9_18
Diaz-Riquelme J, Lijavetzky D, Martinez-Zapater JM, Carmona MJ (2009) Genome-wide analysis of MIKCC-type MADS box genes in grapevine. Plant Physiol 149:354–369. doi:10.1104/pp.108.131052
Diaz-Riquelme J, Martinez-Zapater JM, Carmona MJ (2014) Transcriptional analysis of tendril and inflorescence development in grapevine (Vitis vinifera L.). PLoS One 9:e92339. doi:10.1371/journal.pone.0092339
Doligez A, Bouquet A, Danglot Y et al (2002) Genetic map** of grapevine (Vitis vinifera L.) applied to the detection of QTLs for seedlessness and berry weight. TAG Theor Appl Genet Theor und Angew Genet 105:780–795. doi:10.1007/s00122-002-0951-z
Doligez A, Bertrand Y, Farnos M et al (2013) New stable QTLs for berry weight do not colocalize with QTLs for seed traits in cultivated grapevine (Vitis vinifera L.). BMC Plant Biol 13:217. doi:10.1186/1471-2229-13-217
Dong T, Hu Z, Deng L, Wang Y, Zhu M, Zhang J, Chen G (2013) A tomato MADS-box transcription factor, SlMADS1, acts as a negative regulator of fruit ripening. Plant Physiol 163:1026–1036. doi:10.1104/pp.113.224436
Exposito-Rodriguez M, Borges AA, Borges-Perez A, Perez JA (2008) Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol 8:131. doi:10.1186/1471-2229-8-131
Favaro R, Pinyopich A, Battaglia R et al (2003) MADS-box protein complexes control carpel and ovule development in Arabidopsis. Plant Cell 15:2603–2611. doi:10.1105/tpc.015123
Fernandez-Pozo N, Menda N, Edwards JD et al (2015) The Sol Genomics Network (SGN)–from genotype to phenotype to breeding. Nucleic Acids Res 43:D1036–D1041. doi:10.1093/nar/gku1195
Franks T, Botta R, Thomas MR, Franks J (2002) Chimerism in grapevines: implications for cultivar identity, ancestry and genetic improvement. TAG Theor Appl Genet Theor und Angew Genet 104:192–199. doi:10.1007/s001220100683
Giovannoni JJ (2004) Genetic regulation of fruit development and ripening. Plant Cell 16(Suppl):S170–S180. doi:10.1105/tpc.019158
Golubov A, Yao Y, Maheshwari P, Bilichak A, Boyko A, Belzile F, Kovalchuk I (2010) Microsatellite instability in Arabidopsis increases with plant development. Plant Physiol 154:1415–1427. doi:10.1104/pp.110.162933
Gonzalez-Aguero M, Garcia-Rojas M, Di Genova A, Correa J, Maass A, Orellana A, Hinrichsen P (2013) Identification of two putative reference genes from grapevine suitable for gene expression analysis in berry and related tissues derived from RNA-Seq data. BMC Genom 14:878. doi:10.1186/1471-2164-14-878
Gruis DF, Selinger DA, Curran JM, Jung R (2002) Redundant proteolytic mechanisms process seed storage proteins in the absence of seed-type members of the vacuolar processing enzyme family of cysteine proteases. Plant Cell 14:2863–2882
Heijmans K, Ament K, Rijpkema AS, Zethof J, Wolters-Arts M, Gerats T, Vandenbussche M (2012) Redefining C and D in the petunia ABC. Plant Cell 24:2305–2317. doi:10.1105/tpc.112.097030
Helliwell C, Waterhouse P (2003) Constructs and methods for high-throughput gene silencing in plants. Methods 30:289–295
Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database. Nucleic Acids Res 27:297–300. doi:10.1093/nar/27.1.297
Hofgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16:9877. doi:10.1093/nar/16.20.9877
Honma T, Goto K (2001) Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature 409:525–529. doi:10.1038/35054083
Jaillon O, Aury JM, Noel B et al (2007) The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449:463–467. doi:10.1038/nature06148
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907
Kaufmann K, Melzer R, Theissen G (2005) MIKC-type MADS-domain proteins: structural modularity, protein interactions and network evolution in land plants. Gene 347:183–198. doi:10.1016/j.gene.2004.12.014
Kearse M, Moir R, Wilson A et al (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649. doi:10.1093/bioinformatics/bts199
Kramer EM, Jaramillo MA, Di Stilio VS (2004) Patterns of gene duplication and functional evolution during the diversification of the AGAMOUS subfamily of MADS box genes in angiosperms. Genetics 166:1011–1023
Liljegren SJ, Ditta GS, Eshed Y, Savidge B, Bowman JL, Yanofsky MF (2000) SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis. Nature 404:766–770. doi:10.1038/35008089
Lodhi M, Ye G-N, Weeden N, Reisch B (1994) A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Mol Biol Rep 12:6–13. doi:10.1007/BF02668658
Mejia N, Soto B, Guerrero M et al (2011) Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine. BMC Plant Biol 11:57. doi:10.1186/1471-2229-11-57
Mejía N, Gebauer M, Muñoz L, Hewstone N, Muñoz C, Hinrichsen P (2007) Identification of QTLs for seedlessness, berry size, and ripening date in a seedless × seedless table grape progeny. Am J Enol Vitic 58:499–507
Mizzotti C, Ezquer I, Paolo D et al (2014) SEEDSTICK is a master regulator of development and metabolism in the Arabidopsis seed coat. PLoS Genet 10:e1004856. doi:10.1371/journal.pgen.1004856
Molesini B, Rotino GL, Spena A, Pandolfini T (2009) Expression profile analysis of early fruit development in iaaM-parthenocarpic tomato plants. BMC Res Notes 2:143. doi:10.1186/1756-0500-2-143
Mounet F, Moing A, Kowalczyk M et al (2012) Down-regulation of a single auxin efflux transport protein in tomato induces precocious fruit development. J Exp Bot 63:4901–4917. doi:10.1093/jxb/ers167
Mueller LA, Solow TH, Taylor N et al (2005) The SOL Genomics Network: a comparative resource for Solanaceae biology and beyond. Plant Physiol 138:1310–1317. doi:10.1104/pp.105.060707
Nakagawa T, Suzuki T, Murata S et al (2007) Improved gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci Biotechnol Biochem 71:2095–2100. doi:10.1271/bbb.70216
Nakaune S, Yamada K, Kondo M, Kato T, Tabata S, Nishimura M, Hara-Nishimura I (2005) A vacuolar processing enzyme, δVPE, is involved in seed coat formation at the early stage of seed development. Plant Cell 17:876–887. doi:10.1105/tpc.104.026872
Olimpieri I, Caccia R, Picarella ME, Pucci A, Santangelo E, Soressi GP, Mazzucato A (2011) Constitutive co-suppression of the GA 20-oxidase1 gene in tomato leads to severe defects in vegetative and reproductive development. Plant Sci 180:496–503. doi:10.1016/j.plantsci.2010.11.004
Olmo HP (1934) Bud mutation in the Vinifera grape. I. “Parthenocarpic” Sultanina. Proc Am Soc Hort Sci 31:119–121
Olmo HP (1935) Bud mutation in the Vinifera grape. II. Sultanina gigas. Proc Am Soc Hort Sci 33:437–439
Pan IL, McQuinn R, Giovannoni JJ, Irish VF (2010) Functional diversification of AGAMOUS lineage genes in regulating tomato flower and fruit development. J Exp Bot 61:1795–1806. doi:10.1093/jxb/erq046
Parenicova L, de Folter S, Kieffer M et al (2003) Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: new openings to the MADS world. Plant Cell 15:1538–1551
Pelsy F, Dumas V, Bevilacqua L, Hocquigny S, Merdinoglu D (2015) Chromosome replacement and deletion lead to clonal polymorphism of berry color in grapevine. PLoS Genet 11:e1005081. doi:10.1371/journal.pgen.1005081
Peng JC, Karpen GH (2008) Epigenetic regulation of heterochromatic DNA stability. Curr Opin Genet Dev 18:204–211. doi:10.1016/j.gde.2008.01.021
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45
Pinyopich A, Ditta GS, Savidge B, Liljegren SJ, Baumann E, Wisman E, Yanofsky MF (2003) Assessing the redundancy of MADS-box genes during carpel and ovule development. Nature 424:85–88. doi:10.1038/nature01741
Poupin MJ, Federici F, Medina C, Matus JT, Timmermann T, Arce-Johnson P (2007) Isolation of the three grape sub-lineages of B-class MADS-box TM6, PISTILLATA and APETALA3 genes which are differentially expressed during flower and fruit development. Gene 404:10–24. doi:10.1016/j.gene.2007.08.005
Riechmann JL, Meyerowitz EM (1997a) Determination of floral organ identity by Arabidopsis MADS domain homeotic proteins AP1, AP3, PI, and AG is independent of their DNA-binding specificity. Mol Biol Cell 8:1243–1259
Riechmann JL, Meyerowitz EM (1997b) MADS domain proteins in plant development. Biol Chem 378:1079–1101
Riechmann JL, Wang M, Meyerowitz EM (1996) DNA-binding properties of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA and AGAMOUS. Nucleic Acids Res 24:3134–3141
Rounsley SD, Ditta GS, Yanofsky MF (1995) Diverse roles for MADS box genes in Arabidopsis development. Plant Cell 7:1259–1269. doi:10.1105/tpc.7.8.1259
Scott JW, Harbaugh BK (1989) Micro-Tom a miniature dwarf tomato. Fla Agr Expt Sta Circ 370:1–6
Seymour GB, Fray RG, Hill P, Tucker GA (1993) Down-regulation of two non-homologous endogenous tomato genes with a single chimaeric sense gene construct. Plant Mol Biol 23:1–9
Shimada T, Yamada K, Kataoka M et al (2003) Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana. J Biol Chem 278:32292–32299. doi:10.1074/jbc.M305740200
Simonini S, Roig-Villanova I, Gregis V, Colombo B, Colombo L, Kater MM (2012) Basic pentacysteine proteins mediate MADS domain complex binding to the DNA for tissue-specific expression of target genes in Arabidopsis. Plant Cell 24:4163–4172. doi:10.1105/tpc.112.103952
Sun HJ, Uchii S, Watanabe S, Ezura H (2006) A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant Cell Physiol 47:426–431. doi:10.1093/pcp/pci251
Symington LS (2002) Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol Mol Biol Rev MMBR 66:630–670 (table of contents)
Tanabata T, Shibaya T, Hori K, Ebana K, Yano M (2012) SmartGrain: high-throughput phenoty** software for measuring seed shape through image analysis. Plant Physiol 160:1871–1880. doi:10.1104/pp.112.205120
Theissen G (2001) Development of floral organ identity: stories from the MADS house. Curr Opin Plant Biol 4:75–85
Theissen G, Saedler H (2001) Plant biology. Floral quartets. Nature 409:469–471. doi:10.1038/35054172
Theissen G, Becker A, Di Rosa A et al (2000) A short history of MADS-box genes in plants. Plant Mol Biol 42:115–149
Torregrosa L, Fernandez L, Bouquet A, Boursiquot JM, Pelsy F, Martínez-Zapater JM (2011) Origins and consequences of somatic variation in grapevine. In: Adam-Blondon A-F, Martinez-Zapater J-M, Kole C (eds) Genetics, genomics, and breeding of grapes. Science Publishers, Jersey, British Isles Enfield, New Hampshire, pp 68–92. doi:10.1201/b10948-4
Tzeng TY, Chen HY, Yang CH (2002) Ectopic expression of carpel-specific MADS box genes from lily and lisianthus causes similar homeotic conversion of sepal and petal in Arabidopsis. Plant Physiol 130:1827–1836. doi:10.1104/pp.007948
Vitulo N, Forcato C, Carpinelli EC et al (2014) A deep survey of alternative splicing in grape reveals changes in the splicing machinery related to tissue, stress condition and genotype. BMC Plant Biol 14:99. doi:10.1186/1471-2229-14-99
Wang S, Lu G, Hou Z et al (2014) Members of the tomato FRUITFULL MADS-box family regulate style abscission and fruit ripening. J Exp Bot 65:3005–3014. doi:10.1093/jxb/eru137
Wierdl M, Dominska M, Petes TD (1997) Microsatellite instability in yeast: dependence on the length of the microsatellite. Genetics 146:769–779
Wyman C, Kanaar R (2006) DNA double-strand break repair: all’s well that ends well. Annu Rev Genet 40:363–383. doi:10.1146/annurev.genet.40.110405.090451
Wyman C, Ristic D, Kanaar R (2004) Homologous recombination-mediated double-strand break repair DNA repair 3:827–833. doi:10.1016/j.dnarep.2004.03.037
Xu X, Peng M, Fang Z, Xu X (2000) The direction of microsatellite mutations is dependent upon allele length. Nat Genet 24:396–399
Acknowledgments
This research was supported by FONDECYT grant 1120532, FONDEF grant G09i1007 and the BIOFRUTALES consortium. We are very grateful to Loïc Le Cunff and colleagues from the grapevine team INRA Montpellier for providing assistance and precious samples.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no competing interests.
Additional information
Communicated by P. Puigdomenech.
Electronic supplementary material
Below is the link to the electronic supplementary material.
299_2015_1882_MOESM1_ESM.pdf
Supplementary material 1: Primers used in this study. Target name, target accession number, primers name, primers sequence and use are listed (PDF 84 kb)
299_2015_1882_MOESM2_ESM.jpg
Supplementary material 2: The somatic variant of Sultanina, Sultanine Monococco, is a chimera with mutations derived from mitotic recombination events in the regulatory region of the VviAGL11 MADS-Box gene. Multiple alignment of eight alleles isolated from genomic DNA from Sultanine Monococco, Sultanina and reference Pinot Noir (PN44024, (Jaillon et al. 2007)). Sultanina alleles are indicated as seeded (188) and seedless (198) alleles based on genotype by marker p3_VvAGL11 (Mejia et al. 2011). Four recombinant alleles (SuMo 1 2, 3, and 5) and two original alleles (SuMo 4 and 6) are represented by green or orange fragments to denote seedless and seeded alleles, respectively (JPEG 1244 kb)
299_2015_1882_MOESM3_ESM.jpg
Supplementary material 3: Spatio-temporal expression of SlyAGL11 analyzed by histochemical GUS assay. Transgenic lines expressing GUS reporter gene under the control of SlyAGL11 promoter (pSlyAGL11 of 1568 bp) were used to analyze patterns of expression between pre and post anthesis stages: Stage nine (petals approaching top of sepals and develo** ovules) according to Brukhin et al. (2003), anthesis, eight DPA (where a peak of expression was detected by RT-qPCR), 18 and 30 DPA (JPEG 14532 kb)
Rights and permissions
About this article
Cite this article
Ocarez, N., Mejía, N. Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits. Plant Cell Rep 35, 239–254 (2016). https://doi.org/10.1007/s00299-015-1882-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-015-1882-x