Abstract
Main conclusion
Simultaneous knockdown or knockout of Torenia fournieri PLENA (TfPLE) and FALINELLI (TfFAR) genes with RNAi or genome-editing technologies generated a multi-petal phenotype in torenia.
Abstract
The MADS-box gene AGAMOUS (AG) is well known to play important roles in the development of stamens and carpels in Arabidopsis. Mutations in AG cause the morphological transformation of stamens and carpels into petaloid organs. In contrast, torenia (Torenia fournieri Lind.) has two types of class-C MADS-box genes, PLENA (PLE) and FALINELLI (FAR); however, their functions were previously undetermined. To examine the function of TfPLE and TfFAR in torenia, we used RNAi to knockdown expression of these two genes. TfPLE and TfFAR double-knockdown transgenic torenia plants had morphologically altered stamens and carpels that developed into petaloid organs. TfPLE knockdown transgenic plants also exhibited morphological transformations that included shortened styles, enlarged ovaries, and absent stigmata. Furthermore, simultaneous disruption of TfPLE and TfFAR genes by CRISPR/Cas9-mediated genome editing also resulted in the conversion of stamens and carpels into petaloid organs as was observed in the double-knockdown transgenic plants mediated by RNAi. In addition, the carpels of one TfPLE knockout mutant had the same morphological abnormalities as TfPLE knockdown transgenic plants. TfFAR knockdown genome-edited mutants had no morphological changes in their floral organs. These results clearly show that TfPLE and TfFAR cooperatively play important roles in the development of stamens and carpels. Simultaneous disruption of TfPLE and TfFAR functions caused a multi-petal phenotype, which is expected to be a highly valuable commercial floral trait in horticultural flowers.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- AG :
-
AGAMOUS
- ANS :
-
Anthocyanin synthase
- CaMV:
-
Cauliflower mosaic virus
- CRISPR/Cas9:
-
Clustered regularly interspaced short palindromic repeats/associated protein 9
- DSB:
-
Double-strand break
- DP :
-
DUPLICATED
- FAR :
-
FARINELLI
- F3'5'H :
-
Flavonoid 3′5′-hydroxylase
- KD:
-
Knockdown
- PAM:
-
Protospacer-adjacent motif
- PLE :
-
PLENA
- RNAi:
-
RNA interference
- RT-PCR:
-
Reverse transcription polymerase chain reaction
- SEM:
-
Scanning electron microscope
- sgRNA:
-
Single-guide RNA
- Ttf1 :
-
TransposonT. fournieri 1
References
Aida R (2008) Torenia fournieri (torenia) as a model plant for transgenic studies. Plant Biotechnol 25:541–545. https://doi.org/10.5511/plantbiotechnology.25.541
Aida R, Shibata M (1995) Agrobacterium-mediated Transformation of Torenia (Torenia fournieri). Breed Sci 45:71–74. https://doi.org/10.1270/jsbbs1951.45.71
Bomblies K, Dagenais N, Weigel D (1999) Redundant enhancers mediate transcriptional repression of AGAMOUS by APETALA2. Dev Biol 216:260–264. https://doi.org/10.1006/dbio.1999.9504
Bowman JL, Smyth DR, Meyerowitz EM (1989) Genes directing flower development in Arabidopsis. Plant Cell 1:37–52. https://doi.org/10.1105/tpc.1.1.37
Busch MA, Bomblies K, Weigel D (1999) Activation of a floral homeotic gene in Arabidopsis. Science 285:585–587. https://doi.org/10.1126/science.285.5427.585
Causier B, Castillo R, Zhou J, Ingram R, Xue Y, Schwarz-Sommer Z, Davies B (2005) Evolution in action: following function in duplicated floral homeotic genes. Curr Biol 15:1508–1512. https://doi.org/10.1016/j.cub.2005.07.063
Causier B, Bradley D, Cook H, Davies B (2009) Conserved intragenic elements were critical for the evolution of the floral C-function. Plant J 58:41–52. https://doi.org/10.1111/j.1365-313X.2008.03759.x
Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 18:757–761. https://doi.org/10.1534/genetics.110.120717
Davies B, Motte P, Keck E, Saedler H, Sommer H, Schwarz-Sommer Z (1999) PLENA and FARINELLI: redundancy and regulatory interactions between two Antirrhinum MADS-box factors controlling flower development. EMBO J 15:4023–4034. https://doi.org/10.1093/emboj/18.14.4023
Fauser F, Schiml S, Puchta H (2014) Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J 79:348–359. https://doi.org/10.1111/tpj.12554
He XJ, Chen T, Zhu JK (2011) Regulation and function of DNA methylation in plants and animals. Cell Res 21:442–465. https://www.nature.com/articles/cr201123
Hiratsu K, Ohta M, Matsui K, Ohme-Takagi M (2002) The SUPERMAN protein is an active repressor whose carboxy-terminal repression domain is required for the development of normal flowers. FEBS Lett 514:351–354. https://doi.org/10.1016/S0014-5793(02)02435-3
Hiratsu K, Matsui K, Koyama T, Ohme-Takagi M (2003) Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. Plant J 34:733–739. https://doi.org/10.1046/j.1365-313X.2003.01759.x
Hraška M, Rakouský S, Čurn V (2008) Tracking of the CaMV-35S promoter performance in GFP transgenic tobacco, with a special emphasis on flowers and reproductive organs, confirmed its predominant activity in vascular tissues. Plant Cell Tissue Organ Cult 94:239–251. https://doi.org/10.1007/s11240-007-9312-6
Kennerdell JR, Carthew RW (2000) Heritable gene silencing in Drosophila using double-stranded RNA. Nat Biotechnol 18:896–898. https://www.nature.com/articles/nbt0800_896
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 166:1011–1123. https://doi.org/10.1534/genetics.166.2.1011
Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11:204–220
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. https://www.nature.com/articles/35008089
Lohmann JU, Hong RL, Hobe M, Busch MA, Parcy F, Simon R, Weigel D (2001) A molecular link between stem cell regulation and floral patterning in Arabidopsis. Cell 105:793–803. https://www.nature.com/articles/nrg2719
Marzougui S, Sugimoto K, Yamanouchi U, Shimono M, Hoshino T, Hori K, Kobayashi M, Ishiyama K, Yano M (2012) Mapping and characterization of seed dormancy QTLs using chromosome segment substitution lines in rice. Theor Appl Genet 124:893–902. https://doi.org/10.1007/s00122-011-1753-y
Matzke MA, Mosher RA (2014) RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat Rev Genet 15:394–408. https://www.nature.com/articles/nrg3683
Mitsuda N, Hiratsu K, Todaka D, Nakashima K, Yamaguchi-Shinozaki K, Ohme-Takagi M (2006) Efficient production of male and female sterile plants by expression of a chimeric repressor in Arabidopsis and rice. Plant Biotechnol J 4:325–332. https://doi.org/10.1111/j.1467-7652.2006.00184.x
Murovec J, Gucek J, Bohanec B, Avbelj M, Jerala R (2018) DNA-free genome editing of Brassica oleracea and B. rapa protoplasts using CRISPR-Cas9 ribonucleoprotein complexes. Front Plant Sci 9:1594. https://doi.org/10.3389/fpls.2018.01594
Movahedi A, Sun W, Zhang J, Wu X, Mousavi M, Mohammadi K, Yin T, Zhuge Q (2015) RNA-directed DNA methylation in plants. Plant Cell Rep 34:1857–1862. https://doi.org/10.1007/s00299-015-1839-0
Nadakuduti SS, Buell CR, Voytas DF, Starker CG, Douches DS (2018) Genome editing for crop improvement—applications in clonally propagated polyploids with a focus on potato (Solanum tuberosum L.). Front Plant Sci 9:1607. https://doi.org/10.3389/fpls.2018.01607
Nakatsuka T, Saito M, Yamada E, Fujita K, Yamagishi N, Yoshikawa N, Nishihara M (2015) Isolation and characterization of the C-class MADS-box gene involved in the formation of double flowers in Japanese gentian. BMC Plant Biol 15:182. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4504037/
Narumi T, Aida R, Niki T, Nishijima T, Mitsuda N, Hiratsu K, Ohme-Takagi M, Ohtsubo N (2008) Chimeric AGAMOUS repressor induces serrated petal phenotype in Torenia fournieri similar to that induced by cytokinin application. Plant Biotechnol 25:45–54. https://doi.org/10.5511/plantbiotechnology.25.45
Nishihara M, Higuchi A, Watanabe A, Tasaki K (2018) Application of the CRISPR/Cas9 system for modification of flower color in Torenia fournieri. BMC Plant Biol 18:331. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280492/
Nishijima T, Morita Y, Sasaki K, Nakayama M, Yamaguchi H, Ohtsubo N, Niki T, Niki T (2013) A torenia (Torenia fournieri Lind. ex Fourn.) novel mutant ‘flecked’ produces variegated flowers by insertion of a DNA transposon into an R2R3-MYB gene. J Jpn Soc Hort Sci 82:39–50. https://doi.org/10.2503/jjshs1.82.39
Nishijima T, Niki T, Niki T (2016) A novel, “petaloid” mutant of torenia (Torenia fournieri Lind. ex Fourn.) bears double flowers through insertion of the DNA transposon Ttf1 into a C-class floral homeotic gene. Hortic J 85:272–283. https://doi.org/10.2503/hortj.MI-108
Nitasaka E (2003) Insertion of an En/Spm-related transposable element into a floral homeotic gene DUPLICATED causes a double flower phenotype in the Japanese morning glory. Plant J 36:522–531. https://doi.org/10.1046/j.1365-313X.2003.01896.x
Ó MaoiléidighGracietWellmer DSEF (2014) Gene networks controlling Arabidopsis thaliana flower development. New Phytol 201:16–30. https://doi.org/10.1111/nph.12444
Pecinka A, Abdelsamad A, Vu GT (2013) Hidden genetic nature of epigenetic natural variation in plants. Trends Plant Sci 18:625–632. https://doi.org/10.1016/j.tplants.2013.07.005
Sage-Ono K, Ozeki Y, Hiyama S, Higuchi Y, Kamada H, Mitsuda N, Ohme-Takagi M, Ono M (2011) Induction of double flowers in Pharbitis nil using a class-C MADS-box transcription factor with Chimeric REpressor gene-silencing technology. Plant Biotechnol 28:153–165. https://doi.org/10.5511/plantbiotechnology.11.0119a
Sasaki K, Aida R, Yamaguchi H, Shikata M, Niki T, Nishijima T, Ohtsubo N (2010) Functional divergence within class B MADS-box genes TfGLO and TfDEF in Torenia fournieri Lind. Mol Genet Genomics 284:399–414. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2955243/
Sasaki K, Yamaguchi H, Nakayama M, Aida R, Ohtsubo N (2014) Co-modification of class B genes TfDEF and TfGLO in Torenia fournieri Lind. alters both flower morphology and inflorescence architecture. Plant Mol Biol 86:319–334. https://doi.org/10.1007/s11103-014-0231-8
Sasaki K, Yamaguchi H, Kasajima I, Narumi T, Ohtsubo N (2016) Generation of novel floral traits using a combination of floral organ-specific promoters and a chimeric repressor in Torenia fournieri Lind. Plant Cell Physiol 57:1319–1331. https://doi.org/10.1093/pcp/pcw081
Sieburth LE, Meyerowitz EM (1997) Molecular dissection of the AGAMOUS control region shows that cis elements for spatial regulation are located intragenically. Plant Cell 9:355–365. https://doi.org/10.1105/tpc.9.3.355
Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407:319–320. https://www.nature.com/articles/35030305
Soltis DE, Ma H, Frohlich MW, Soltis PS, Albert VA, Oppenheimer DG, Altman NS, dePamphilis C, Leebens-Mack J (2007) The floral genome: an evolutionary history of gene duplication and shifting patterns of gene expression. Trends Plant Sci 12:358–367. https://doi.org/10.1016/j.tplants.2007.06.012
Su S, Xiao W, Guo W, Yao X, Xiao J, Ye Z, Wang N, Jiao K, Lei M, Peng Q, Hu X, Huang X, Luo D (2017) The CYCLOIDEA-RADIALIS module regulates petal shape and pigmentation, leading to bilateral corolla symmetry in Torenia fournieri (Linderniaceae). New Phytol 215:1582–1593. https://doi.org/10.1111/nph.14673
Tanaka Y, Oshima Y, Yamamura T, Sugiyama M, Mitsuda N, Ohtsubo N, Ohme-Takagi M, Terakawa T (2013) Multi-petal cyclamen flowers produced by AGAMOUS chimeric repressor expression. Sci Rep 3:2641. https://www.nature.com/articles/srep02641
Theißen G (2001) Development of floral organ identity: stories from the MADS house. Curr Opin Plant Biol 4:75–85. https://doi.org/10.1016/S1369-5266(00)00139-4
Theißen G, Melzer R, Rümpler F (2016) MADS-domain transcription factors and the floral quartet model of flower development: linking plant development and evolution. Development 143:3259–3271. https://dev.biologists.org/content/143/18/3259.long
Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482:331–338. https://www.nature.com/articles/nature10886
Wilkinson JE, Twell D, Lindsey K (1997) Activities of CaMV 35S and nos promoters in pollen: implications for field release of transgenic plants. J Exp Bot 48:265–275. https://academic.oup.com/jxb/article/48/2/265/652834
Yamaguchi H, Sasaki K, Shikata M, Aida R, Ohtsubo N (2011) Trehalose drastically extends the in vitro vegetative culture period and facilitates maintenance of Torenia fournieri plants. Plant Biotechnol 28:263–266. https://doi.org/10.5511/plantbiotechnology.11.0124c
Yan W, Chen D, Kaufmann K (2016) Molecular mechanisms of floral organ specification by MADS domain proteins. Curr Opin Plant Biol 29:154–162. https://doi.org/10.1016/j.pbi.2015.12.004
Yanofsky MF, Ma H, Bowman JL, Drews GN, Feldmann KA, Meyerowitz EM (1990) The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature 346:35–39. https://doi.org/10.1016/j.pbi.2015.12.004
Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–771. https://doi.org/10.1016/j.cell.2015.09.038
Acknowledgements
We thank Dr. Mily Ron and Prof. Anne Britt (U. C. Davis) for permission to use pDeCas9_Kan, pMR203, pMR204 and pMR205; Dr. Friedrich Fauser (Carnegie Institution for Science), Mr. Simon Schiml, and Dr. Holger Puchta (University of Karlsruhe) for pDe-Cas9; Dr. Masaki Endo, Dr. Seiichi Toki for providing genome-editing plasmids; Ms. Satoko Ohtawa, Ms. Miyuki Tsuruoka, Ms. Yuko Namekawa, and Ms. Yoshiko Kashiwagi for generating and maintaining the transgenic torenia plants, Ms. Yasuko Taniji, Ms. Hiroko Yamada, Ms. Miho Seki, and Ms. Mayumi Takimoto for assistance with the molecular biological work as well as for maintaining the torenia plants used in the study; and Dr. Takaaki Nishijima for helpful discussions.
Funding
This work was partially supported by the Scientific Technique Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry (Japan).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Communicated by Anastasios Melis.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Fig. S1.
Nucleotide sequences of the RNAi construct for TfFAR. Fig. S2. Nucleotide sequences of the RNAi construct for TfPLE. Fig. S3. Nucleotide sequences of the RNAi construct for TfPLE and TfFAR. Fig. S4. Expression of putative class-C genes in torenia. Fig. S5. Confirmation of introduction of transgenes in TfFAR/TfPLE-IR transgenic plants. Fig. S6. Generation of TfPLE-KD transgenic torenia plants. Fig. S7. Mutations in sequences of TfPLE and TfFAR in the genome-edited torenia line#2. Fig. S8. Mutations in sequences of TfPLE and TfFAR in the genome-edited torenia line#16. Fig S9. Generation of TfPLE genome-edited torenia line#53. Fig. S10. Mutations in sequences of TfPLE in the genome-edited torenia line#53. Fig. S11. Generation of TfFAR genome-edited torenia plants. Fig. S12. Mutations in sequences of TfFAR in the genome-edited torenia line#34. Fig. S13. Mutations in sequences of TfFAR in the genome-edited torenia line#91. Fig. S14. Mutations in sequences of TfFAR in the genome-edited torenia line#96. Fig. S15. Mutations in sequences of TfFAR in the genome-edited torenia line#126. Fig. S16. Observation of epidermal cell shapes with SEM in the petaloid-organs of genome-edited torenia plants (PDF 867 kb)
Table S1.
Primer sequences used for RNAi construction. Table S2. Sequences used for construction of CRISPR/Cas9 vector. Table S3. Sequences used for construction of genome editing plasmids. Table S4. Primer sequences used for expression analysis in torenia. Table S5. Sequences used for assessment in vitro cleavage efficiency. Table S6. Primer sequences used for amplification of cleavage templates of TfPLE and TfFAR. Table S7. Primer sequences used for genomic PCR to check introduction of the transgenes used in this study. Table S8. Primer sequences used for screening the mutation by genome editing (XLSX 22 kb)
Rights and permissions
About this article
Cite this article
Sasaki, K., Ohtsubo, N. Production of multi-petaled Torenia fournieri flowers by functional disruption of two class-C MADS-box genes. Planta 251, 101 (2020). https://doi.org/10.1007/s00425-020-03393-3
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00425-020-03393-3