The Effect of Chalcone Isomerase Gene Silencing on Pigment Production Pathway in Petunia hybrida with RNAi Technology

Document Type : Research Article


1 Department of Plant Genetic and Production, Faculty of Agriculture, Jahrom University, Jahrom, Iran

2 Professor, Department of Plant Biotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

3 Associated Professor, Department of Plant Biotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

4 Assistant Professor, Department of Plant Genetic and Production, Faculty of Agriculture, Jahrom University, Jahrom, Iran


 Flower color is one of the most significant characteristics in ornamental plant breeding. New varieties of various plants in relation to their flower color have been obtained by monitoring the expression levels of genes involved or regulating the flavonoid and anthocyanin biosynthesis pathway. Flavonoids possess significant and diverse biological functions. They are the major pigments for flowers, fruits, seeds, and leaves. They are natural products that contain a C6-C3-C6 carbon framework and are synthesized by a branched pathway that yields both colored and colorless compounds. The gene encoding chalcone isomerase (CHI) is among the genes and enzymes identified in the flavonoid pathway. This enzyme catalyzes the isomerization of naringenin chalcone into the corresponding flavanone. CHI enzyme belongs to the family of isomerases, specifically the class of intramolecular lyases. Chalcone isomerase has a core 2-layer alpha/beta structure and has attracted much attention recently due to its role in stress response and pigment production. One of the most effective methods of genetic engineering is the reduction of flower pigments by suppression of required enzymes for their biosynthesis. RNA interference (RNAi) has provided the tool for the investigation of genes involved in the production of flower color. Silencing of any gene in the anthocyanin biosynthetic pathway can result in reduced or inhibited anthocyanin production. RNAi technology is an effective gene silencing method and a powerful tool for studying gene function and development of new traits by transformation of viral RNA or hairpin RNA (hpRNA) constructs into plants. The processing of dsRNA into 21-23-nt small interfering RNAs (siRNAs), and the mediators of RNAi, triggers cognate mRNA degradation. The hpRNAi methodology simply requires a transgene construct containing an inversely-repeated sequence of the target gene flanked with a promoter and terminator which effectively function in plants.
Material and Methods
 In this research, with the design and construction of chiRNAi, the transformation of the RNAi construct was carried out of Petunia plants. Potted plants of P. hybrida were grown under standard greenhouse conditions (16-17°C night temperature and 21-24°C day temperature and photoperiod 16/8 (light/dark)). The RNAi construct including the 530 bp cds of the chalcone isomerase (chi) gene and 741 bp of pdk gene as intron between chi sense and antisense were used for transient RNAi-induced silencing. The pBI121-chi530 plasmids were introduced into A. tumefaciens strain LBA4404 by electroporation method. Colonies of A. tumefaciens carrying the desired plasmid were screened by PCR with specific primers for chi gene. RNAi construct co-cultured with petunia’s leave. Samples was kept in dark condition for 3 days and then transferred to branch induction media. Samples were investigated for phenotypical changes and chi gene expression by qRT-PCR.
Results and Discussion
 Transgenic lines showed a reduced number of pigments and a faded flower color. So that, in purple petunia, was shown 5 phenotypical groups. These groups was indicated different levels of chi gene silencing. In pink petunia was seen two groups of phenotypical changes. In these plants, chi-RNAi construct was reduced pigment production and so, these plants had faded colors in petals. Also, the chi gene expression was reduced in all transgenic lines. Generally, the results of this research showed that RNAi can be used as an efficient method for gene silencing. The application of gene silencing can indicate the gene’s function in biosynthesis pathways of various components such as anthocyanins. In addition, the chalcone isomerase gene was identified as one of the effective genes in anthocyanin biosynthesis pathway in Petunia plants that could be involved in the production of color in these plants; hence, chi gene silencing resulted in clear phenotypic alterations in this plant.
 In general the concentration of the target mRNA in a particular tissue could be a factor that influences silencing efficiency. At very low levels of gene expression, small amounts of the silencing target, mRNA, could be completely degraded by the RNA-induced silencing complex (RISC), whereas the presence of higher amounts of the target mRNA may result in incomplete silencing, allowing some residual functional mRNA to be translated into the corresponding protein. This research demonstrated the hpRNA construct has been successfully established for floral tissues of P. hybrida. The hpRNA construct was developed for chi-RNAi silencing of one of the key genes in the anthocyanin biosynthetic pathway in Petunia flowers. The silencing of the chi gene is a prototype for the modification of the anthocyanin biosynthetic pathway in Petunia through gene suppression. This strategy could also be useful for rapid functional analysis of other genes involved in flower development.


Main Subjects

  1. Ahloowalia, B.S., & Maluszynski, M. (2001). Induced mutations - A new paradigm in plant breeding. Euphytica 118, 167–173.

    1. Andersen, Y.M., & Makham, K.R. (2006). Flavonoids: chemistry, biochemistry and applications. CRS Press, Taylor & Francis group, Sound Parkway NW. 1212p.
    2. Chandler, S.F., & Sanchez, C. (2012). Genetic modification: the development of transgenic ornamental plant varieties. Wiley Online Library, 10, 891–903.
    3. Chen, G., Liu, H., Wei, Q., Zhao, H., Liu, J., & Yu, Y. (2017). The acyl-activating enzyme PhAAE13 is an alternative enzymatic source of precursors for anthocyanin biosynthesis in petunia flowers. Journal of Experimental Botany, 68, 457–467.
    4. Delgado-Vargas, F., Jimenez, A.R., & Paredae-Lopez, O. (2000). Natural pigments: Carotenoids, Anthocyanins, and Betalains: characteristics, biosynthesis, processing, and stability. Critical Reviews in Food Science and Nutrition, 40, 173-289.
    5. Dixon, R.A. (2005). Engineering of plant natural product pathways. Current Opinion of Plant Biology, 8, 329–336.
    6. Fujino, N., Tenma, N., Waki, T., Ito, K., Komatsuzaki, Y., & Sugiyama, K. (2018). Physical interactions among flavonoid enzymes in snapdragon and torenia reveal the diversity in the flavonoid metabolon organization of different plant species. The Plant Journal, 94, 372–392.
    7. Fukusaki, E., Kawasaki, K., Kajayama, S., An, C., SuZuki, K., Tanaka, Y., & Kobayashi, A. (2004). Flower color modulations of Torenia hybrida by down regulation of chalcone synthase gene with RNA interference. Journal of Biotechnology, 111, 229-240.
    8. Heilersig, H.J.B., Loonen, A.E.H.M., Bergervoet, M., Wolters, A.M.A., & Visser, R.G.F. (2006). Post-transcriptional gene silencing of GBSSI in potato: effects of size and sequence of the inverted repeats. Plant Molecular Biology, 60, 647-662.
    9. Keykha, F., Bagheri, A., & Moshtaghi, N. (2016a). Analysis of chalcone synthase and chalcone isomerase gene expression in pigment production pathway at different flower colors of Petunia hybrida. Journal of Cell and Molecular Research, 8, 8–14.
    10. Keykha, F., Bagheri, A., Moshtaghi, N., Bahrami, A. R., & Sharifi, A. (2016b). Cellular and Molecular Biology RNAi-induced silencing in floral tissues of Petunia hybrida by agroinfiltration: a rapid assay for chalcone isomerase gene function analysis. Cellular and Molecular Biology, 62, 26–31.
    11. Kim, D.H., & Rossi, J.J. (2008). RNAi mechanisms and applications. Biotechniques, 44, 613-616.
    12. Lin, J.J. (1995). Electrotransformation of Agrobacterium, in Methods in Molecular Biology, 47, Nickoloff, J. A., ed. Humana Press, Totowa, NJ. 171pp.
    13. Nakamura, N., Fukuchi-Mizutani, M., Miyazaki, K., Suzuki, K., & Tanaka, Y. (2006). RNAi suppression of the anthocyanidin synthase gene in Torenia hybrida yields white flowers with high frequency and better stability than antisense and sense suppression. Plant Biotechnology, 23, 13-17.
    14. Nakatsuka, T., Mishiba, K., Abe, Y., Kubota, A., Kakizaki, Y., Yamamura, S., & Nishihara, M. (2008). Flower color modification of gentian plants by RNAi-mediated gene silencing. Plant Biotechnology, 25, 61-68.
    15. Nakatsuka, T., Mishiba, K. I., Kubota, A., Abe, Y., Yamamura, S., Nakamura, N., Tanaka, Y., & Nishihara, M. (2010). Genetic engineering of novel flower color by suppression of anthocyanin modification genes in gentian. Journal of Plant Physiology, 167, 231-237.
    16. Nishihara, M., Nakatsuka, T., & Yamamura, S. (2005). Flavonoid components and flower color change in transgenic tobacco plants by suppression of chalcone isomerase gene. FEBS Letters, 579, 6047-6078.
    17. Nishihara, M., & Nakatsuka, T. (2010). Genetic engineering of novel flower colors in floricultural plants: recent advances via transgenic approaches. Methods in Molecular Biology, 589, 325–347.
    18. Nishihara, M., & Nakatsuka, T. (2011). Genetic engineering of flavonoid pigments to modify flower color in floricultural plants. Biotechnology Letter, 33, 433-441.
    19. Park, E.J & Chen, T.H.H. (2006). Improvement of cold tolerance in horticultural crops. Haworth Food & Agricultural Products, Singapore 69–120.
    20. Potera, C. (2007). Blooming biotech. Nature Biotechnolgy, 25, 963–965.
    21. Tanaka, Y., & Ohmiya, A. (2008). Seeing is believing: engineering anthocyanin and carotenoid biosynthetic pathways. Current Opinion in Biotechnology, 19, 190–197.
    22. Tanaka, Y., Brugliera, F., & Chandler, S. (2009). Recent progress of flower color modification by biotechnology. International Journal of Molecular Sciences, 10, 5350-5369.
    23. Thakur, A. (2003). RNA interference revolution. Electeronic Journal of Biotechnology, 6, 39-49.
    24. To, K.Y., & Wang, C.K. (2006). Molecular breeding of flower color. Floriculture, Ornamental and Biotechnology Volume. Global Science Books, UK. pp, 300-310.
    25. Tsuda, SH., Fukui, Y., Nakamura, N., Katsumoto, Y., Yonekura-Sakakibara, K., Fukuchi-Mizutani, M., Ohira, K., Ueyama, Y., Ohkawa, H., A.Holton, T., Kusumi, T., & Tanaka, Y. (2004). Flower color modification of Petunia hybrida commercial varieties by metabolic engineering. Plant Biotechnology, 21, 377-386.
    26. Voorhuijzen, M.M., Prins, T.W., Belter, A., Bendiek, J., Brünen-Nieweler, C., van Dijk, J.P., Goerlich, O., Kok, E.J., Pickel, B., Scholtens, I.M.J., Stolz, A., & Grohmann, L. (2020). Molecular characterization and event-specific real-time PCR detection of two dissimilar groups of genetically modified Petunia (Petunia x hybrida) sold on the market. Frontiers in Plant Science, 11, 11. eCollection 2020
    27. Wang, Y., Xie, X., Ran, X., Chou, S., Jiao, X., Li, E., Zhang, Q., Meng, X., & Li, B. (2018). Comparative analysis of the polyphenols profiles and the antioxidant and cytotoxicity properties of various blue honeysuckle varieties. Open Chemistry, 16, 637–646.
  • Receive Date: 12 April 2022
  • Revise Date: 03 August 2022
  • Accept Date: 26 September 2022
  • First Publish Date: 26 September 2022