Background Development ornamental varieties with enhanced floral traits and distinctive characteristics is a central goal in floriculture. Anthocyanins, the principal flavonoid pigments in floral tissues, are synthesized via the well-characterized flavonoid biosynthesis pathway. Chalcone isomerase (CHI) catalyzes the first committed reaction in the flavonoid biosynthetic pathway. Method In this study, we investigated the effects of CHI suppression via RNA interference (RNAi) in three Petunia hybrida cultivars exhibiting distinct petal colors. Leaf discs from three Petunia hybrida lines exhibiting distinct petal colors (blue, pink, and purple) were inoculated with Agrobacterium tumefaciens harboring the recombinant plasmid pBI121 containing the RNAi construct targeting Phchi. Transgenic plants were regenerated on MS medium via microshoots emerging from wounded explant regions and verified by PCR using two primer pairs specific to the CHI silencing vector. Gene expression analysis was conducted for chi and associated pigment biosynthesis genes, including flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), and dihydroflavonol 4-reductase (DFR), comparing transgenic and wild-type plants. Chalcone and naringenin contents were quantified using a NanoDrop ELISA plate reader. Data analysis was performed using SPSS version 16, and mean comparisons were evaluated via T-test at a significance level of α = 0.05. Results Transformation efficiencies for CHI-silenced Petunia lines were 57.89%, 72.0% and 84.0% for the pink, blue, and purple phenotypes, respectively. Phenotypic evaluation revealed not only altered pigment distributions but also novel floral morphologies, such as tetramerous corollas and twisted tubular petal margins, in the CHI-suppressed lines. Quantitative metabolic profiling demonstrated a significant reduction in naringenin levels across all transgenic lines, whereas, chalcone accumulation was significantly elevated in a subset of lines. The expression of CHI gene was markedly reduced in all lines carrying the CHI-RNAi construct; however, transcript levels did not differ significantly between the aberrant floral phenotypes and the control. Transcript levels of key downstream flavonoid biosynthesis pathway genes, including F3H, F3'H, F3'5'H, and DFR were significantly attenuated in CHI-silenced lines compared with wild-type controls. This concerted down‐regulation indicates that suppression of the CHI‐catalyzed step exerts a broader repressive effect on subsequent pathway components. Despite the overall repression of flavonoid-biosynthetic genes in CHI-silenced lines, certain novel phenotypes maintained or even increased expression of specific downstream enzymes. Notably, blue-flowered transformants exhibited F3'H transcript levels comparable to wild-type control, and pink-flowered lines retained F3'5'H expression. These exceptions highlight the nuanced, tissue-specific regulatory responses triggered by CHI suppression. Conclusion Collectively, our results demonstrate that RNAi- mediated silencing of CHI gene is an effective strategy for generating novel pigmentation patterns and floral morphology in Petunia hybrida. Moreover, this work provides critical insights into how targeted manipulation of a single enzymatic step can influence the broader transcriptional network governing anthocyanin biosynthesis.

Downregulation of chalcone isomerase in Petunia hybrida: profound effects on floral morphology and transcriptional remodeling of the flavonoid biosynthetic pathway

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2025年10月30日

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Fatemeh Keykha Akhar,
Abdoreza Bagheri,
Nasrin Moshtaghi,
Abdolkarim Zarei

摘要

Background

Development ornamental varieties with enhanced floral traits and distinctive characteristics is a central goal in floriculture. Anthocyanins, the principal flavonoid pigments in floral tissues, are synthesized via the well-characterized flavonoid biosynthesis pathway. Chalcone isomerase (CHI) catalyzes the first committed reaction in the flavonoid biosynthetic pathway.

Method

In this study, we investigated the effects of CHI suppression via RNA interference (RNAi) in three Petunia hybrida cultivars exhibiting distinct petal colors. Leaf discs from three Petunia hybrida lines exhibiting distinct petal colors (blue, pink, and purple) were inoculated with Agrobacterium tumefaciens harboring the recombinant plasmid pBI121 containing the RNAi construct targeting Phchi. Transgenic plants were regenerated on MS medium via microshoots emerging from wounded explant regions and verified by PCR using two primer pairs specific to the CHI silencing vector. Gene expression analysis was conducted for chi and associated pigment biosynthesis genes, including flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), and dihydroflavonol 4-reductase (DFR), comparing transgenic and wild-type plants. Chalcone and naringenin contents were quantified using a NanoDrop ELISA plate reader. Data analysis was performed using SPSS version 16, and mean comparisons were evaluated via T-test at a significance level of α = 0.05.

Results

Transformation efficiencies for CHI-silenced Petunia lines were 57.89%, 72.0% and 84.0% for the pink, blue, and purple phenotypes, respectively. Phenotypic evaluation revealed not only altered pigment distributions but also novel floral morphologies, such as tetramerous corollas and twisted tubular petal margins, in the CHI-suppressed lines. Quantitative metabolic profiling demonstrated a significant reduction in naringenin levels across all transgenic lines, whereas, chalcone accumulation was significantly elevated in a subset of lines. The expression of CHI gene was markedly reduced in all lines carrying the CHI-RNAi construct; however, transcript levels did not differ significantly between the aberrant floral phenotypes and the control. Transcript levels of key downstream flavonoid biosynthesis pathway genes, including F3H, F3'H, F3'5'H, and DFR were significantly attenuated in CHI-silenced lines compared with wild-type controls. This concerted down‐regulation indicates that suppression of the CHI‐catalyzed step exerts a broader repressive effect on subsequent pathway components. Despite the overall repression of flavonoid-biosynthetic genes in CHI-silenced lines, certain novel phenotypes maintained or even increased expression of specific downstream enzymes. Notably, blue-flowered transformants exhibited F3'H transcript levels comparable to wild-type control, and pink-flowered lines retained F3'5'H expression. These exceptions highlight the nuanced, tissue-specific regulatory responses triggered by CHI suppression.

Conclusion

Collectively, our results demonstrate that RNAi- mediated silencing of CHI gene is an effective strategy for generating novel pigmentation patterns and floral morphology in Petunia hybrida. Moreover, this work provides critical insights into how targeted manipulation of a single enzymatic step can influence the broader transcriptional network governing anthocyanin biosynthesis.