Abstract
The genus Viola is among the largest genera in angiosperm, which includes many ornamental crops and wild herbs broadly consumed in human society. The members of Viola are one of very few taxonomic groups that produce cyclotide, a novel molecular scaffold for drug discovery, and various unique adaptive behaviors, such as dimorphic pollination strategies and mutualistic interaction with insect in seed dispersal, are also widespread across Viola species. However, the lack of effective model species that are genetically accessible and insufficiently cataloged genetic engineering methods have rendered the genus remaining largely unexplored. Here, using V. philippica as a central model organism, our study constructs the satellite genetic system that enables versatile and robust comparative gene function studies in Viola. In V. philippica, in vitro whole plant regeneration from various somatic tissues was achieved through the relayed actions of two cytokinin analogs, thidiazuron (TDZ) and 6-benzylaminopurine (BAP), and the optimized de novo plant regeneration led to highly efficient and heritable Agrobacterium-dependent genetic transformation being constructed. We further demonstrate CRISPR/Cas9-mediated site-directed genome editing that resulted in induced polymorphisms at the selected genomic loci and tobacco rattle virus-based virus-induced gene silencing (TRV-VIGS) that elicited the targeted and systemic host gene silencing, thereby presenting multiple routes to manipulating gene functions in the model Viola species. Furthermore, by applying the techniques established in V. philippica to multiple other Viola species that differ markedly in their evolutionary behaviors, the potent comparative study system was verified. Our study therefore provides a methodological framework instigating molecular dissection and engineering of novel genetic traits in phytochemistry, body architecture, and organ differentiation that evolved during the diversification of Viola species.
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The data that support the findings of this study are contained within the article and available from the corresponding author upon reasonable request.
References
Ahmad N, Faisal M (2018) Thidiazuron: from urea derivative to plant growth regulator, Ed 1st, Vol Springer, Singapore: Imprint: Springer, Singapore
Ballard HE, Sytsma KJ, Kowal RR (1998) Shrinking the violets: Phylogenetic relationships of infrageneric groups in Viola (Violaceae) based on internal transcribed spacer DNA sequences. Syst Bot 23(4):439–458
Beattie AJ, Lyons N (1975) Seed dispersal in viola (violaceae) - adaptations and strategies. Am J Bot 62(7):714–722
Bhardwaj G, O'Connor J, Rettie S, Huang YH, Ramelot TA, Mulligan VK, Alpkilic GG, Palmer J, Bera AK, Bick MJ, Di Piazza M, Li X, Hosseinzadeh P, Craven TW, Tejero R, Lauko A, Choi R, Glynn C, Dong L, Griffin R, van Voorhis WC, Rodriguez J, Stewart L, Montelione GT, Craik D, Baker D (2022) Accurate de novo design of membrane-traversing macrocycles. Cell 185(19):3520–3532 e3526
Burch-Smith TM, Anderson JC, Martin GB, Dinesh-Kumar SP (2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J 39(5):734–746
Camarero JA, Campbell MJ (2019) The potential of the cyclotide scaffold for drug development. Biomedicines 7(2):31
Chang C, Bowman JL, Meyerowitz EM (2016) Field guide to plant model systems. Cell 167(2):325–339
Chatfield JM, Armstrong DJ (1986) Regulation of cytokinin oxidase activity in callus tissues of phaseolus vulgaris L. cv great Northern. Plant Physiol 80(2):493–499
Cheng ZM, Schnurr JA, Kapaun JA (1998) Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation. Plant Cell Rep 17(8):646–649
Culley TM, Klooster MR (2007) The Cleistogamous breeding system: a review of its frequency, evolution, and ecology in angiosperms. Bot Rev 73(1):1–30
Dai C, Li Y, Li L, Du Z, Lin S, Tian X, Li S, Yang B, Yao W, Wang J (2020) An efficient Agrobacterium-mediated transformation method using hypocotyl as explants for Brassica napus. Mol Breeding 40(10):1–13
De Saeger J, Park J, Chung HS, Hernalsteens JP, Van Lijsebettens M, Inze D, Van Montagu M, Depuydt S (2021) Agrobacterium strains and strain improvement: Present and outlook. Biotechnol Adv 53:107677
Fernie AR, Yan J (2019) De Novo domestication: an alternative route toward new crops for the future. Mol Plant 12(5):615–631
Feyzabadi Z, Ghorbani F, Vazani Y, Zarshenas MM (2017) A critical review on phytochemistry, pharmacology of Viola odorata L. and Related multipotential products in traditional persian medicine. Phytother Res 31(11):1669–1675
Gao J, Luo M, Zhu Y, He Y, Wang Q, Zhang C (2015) Transcriptome sequencing and differential gene expression analysis in Viola yedoensis Makino (Fam. Violaceae) responsive to cadmium (Cd) pollution. Biochem Biophys Res Commun 459(1):60–65
Goransson U, Malik S, Slazak B (2015) Cyclotides in the Violaceae. Adv Bot Res 76:15–49
Hare PD, Vanstaden J (1994) Inhibitory effect of thidiazuron on the activity of cytokinin oxidase isolated from Soybean Callus. Plant Cell Physiol 35(8):1121–1125
Hyun Y, Kim J, Cho S, Choi Y, Kim JS, Coupland G (2015) Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles. Planta 241(1):271–284
Ireland DC, Colgrave ML, Craik DJ (2006) A novel suite of cyclotides from Viola odorata: sequence variation and the implications for structure, function and stability. Biochem J 400:1–12
Kelly RO, Schoellhorn RK, Deng ZN, Harbaugh BK (2005) Evaluation of pansy cultivars as bedding plants to select the best-of-class. HortTechnology 15(3):706–715
Kim H, Kim ST, Ryu J, Choi MK, Kweon J, Kang BC, Ahn HM, Bae S, Kim J, Kim JS, Kim SG (2016) A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system. J Integr Plant Biol 58(8):705–712
Labun K, Montague TG, Krause M, Torres Cleuren YN, Tjeldnes H, Valen E (2019) CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res 47(W1):W171–W174
Li Q, Huo Q, Wang J, Zhao J, Sun K, He C (2016) Expression of B-class MADS-box genes in response to variations in photoperiod is associated with chasmogamous and cleistogamous flower development in Viola philippica. BMC Plant Biol 16(1):151
Liu G, Lin Q, ** S, Gao C (2022) The CRISPR-Cas toolbox and gene editing technologies. Mol Cell 82(2):333–347
Maher MF, Nasti RA, Vollbrecht M, Starker CG, Clark MD, Voytas DF (2020) Plant gene editing through de novo induction of meristems. Nat Biotechnol 38(1):84
Malecot V, Marcussen T, Munzinger J, Yockteng R, Henry M (2007) On the origin of the sweet-smelling Parma violet cultivars (Violaceae): Wide intraspecific hybridization, sterility, and sexual reproduction. Am J Bot 94(1):29–41
Marcussen T, Jakobsen KS, Danihelka J, Ballard HE, Blaxland K, Brysting AK, Oxelman B (2012) Inferring species networks from gene trees in high-polyploid North American and Hawaiian violets (Viola, Violaceae). Syst Biol 61(1):107–126
Marcussen T, Heier L, Brysting AK, Oxelman B, Jakobsen KS (2015) From gene trees to a dated allopolyploid network: insights from the angiosperm genus Viola (Violaceae). Syst Biol 64(1):84–101
Marcussen T, Ballard HE, Danihelka J, Flores AR, Nicola MV, Watson JM (2022) A revised phylogenetic classification for Viola (Violaceae). Plants 11:2224
One Thousand Plant Transcriptomes I (2019) One thousand plant transcriptomes and the phylogenomics of green plants. Nature 574(7780):679–685
Park S, Yoo KO, Marcussen T, Backlund A, Jacobsson E, Rosengren KJ, Doo I, Goransson U (2017) Cyclotide Evolution: insights from the analyses of their precursor sequences, structures and distribution in violets (Viola). Front Plant Sci https://doi.org/10.3389/fpls.2017.02058
Rizwan K, Khan SA, Ahmad I, Rasool N, Ibrahim M, Zubair M, Jaafar HZE, Manea R (2019) A comprehensive review on chemical and pharmacological potential of viola betonicifolia: a plant with multiple benefits. Molecules 24(17):3138
Saint-Lary L, Roy C, Paris JP, Tournayre P, Berdague JL, Thomas OP, Fernandez X (2014) Volatile compounds of viola odorata absolutes: identification of odorant active markers to distinguish plants originating from France and Egypt. Chem Biodivers 11(6):843–860
Senthil-Kumar M, Mysore KS (2014) Tobacco rattle virus-based virus-induced gene silencing in Nicotiana benthamiana. Nat Protoc 9(7):1549–1562
Slazak B, Kapusta M, Stromstedt AA, Slomka A, Krychowiak M, Shariatgorji M, Andren PE, Bohdanowicz J, Kuta E, Goransson U (2018) How does the sweet violet (Viola odorata L.) fight pathogens and pests - cyclotides as a comprehensive plant host defense system. Front Plant Sci. https://doi.org/10.3389/fpls.2018.01296
Slazak B, Kaltenbock K, Steffen K, Rogala M, Rodriguez-Rodriguez P, Nilsson A, Shariatgorji R, Andren PE, Goransson U (2021) Cyclotide host-defense tailored for species and environments in violets from the Canary Islands. Sci Rep-Uk. https://doi.org/10.1038/s41598-021-91555-y
Sternberg SH, Redding S, **ek M, Greene EC, Doudna JA (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507(7490):62–67
Sternberger AL, Bowman MJ, Kruse CPS, Childs KL, Ballard HE, Wyatt SE (2019) Transcriptomics identifies modules of differentially expressed genes and novel cyclotides in Viola pubescens. Front Plant Sci 10:156
Trajkovic M, Dragicevic M, Simonovic AD, Subotic AR, Milosevic S, Cingel A, Jevremovic S (2021) Alteration of flower color in Viola cornuta cv “Lutea Splendens” through metabolic engineering of capsanthin/capsorubin synthesis. Horticulturae 7(9):324
Wang J, Bao MZ (2007) Plant regeneration of pansy (Viola wittrockiana) “Caidie” via petiole-derived callus. Sci Hortic-Amsterdam 111(3):266–270
Wu H, Qu X, Dong Z, Luo L, Shao C, Forner J, Lohmann JU, Su M, Xu M, Liu X, Zhu L, Zeng J, Liu S, Tian Z, Zhao Z (2020) WUSCHEL triggers innate antiviral immunity in plant stem cells. Science 370(6513):227–231
Zhu H, Qin SS, Zhang N, Yang DW, Han HR, Wei KH, Li MH (2015) Chemical constituents and biological activities of plants from the Genus Viola. Chem Biodivers 12(12):1777–1808
Acknowledgements
We thank Sang-Gyu Kim for generously providing materials for CRISPR/Cas9 system. This work was supported by the Suh Kyungbae Foundation (SUHF) SUHF-21010053, the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2021R1A2C101007712), Creative-Pioneering Researchers Program through Seoul National University, and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2021R1A5A1032428).
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D.K. and Y.H. initiated and coordinated the research and wrote the manuscript. D.K. conducted in vitro regeneration and genetic transformation of Viola species. J.-Y.P. contributed to plasmid construction, CRISPR/Cas9-mediated genome editing and VIGS. J.W. established and carried out VIGS in Viola. A.M. characterized the conditions for in vitro tissue culture of Viola species. J.Y.B. and S.L. participated in genetic transformation and agroinfiltration.
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Kim, D., Park, JY., Won, J. et al. Construction of Satellite Genetic System for Robust and Versatile Inter-species Gene Function Analyses in Viola. J. Plant Biol. 66, 207–221 (2023). https://doi.org/10.1007/s12374-023-09391-8
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DOI: https://doi.org/10.1007/s12374-023-09391-8