CRISPR/Cas9-mediated targeted mutation reveals a role for AN4 rather than DPL in regulating venation formation in the corolla tube of Petunia hybrida

Bin Zhang,Mingyang Li,Yulong Guo,Renwei Huang,Sha Yang,Xiaojing Xu

doi:10.1038/s41438-021-00555-6

Bin Zhang, Mingyang Li + Show 4 more

Open Access

https://doi.org/10.1038/s41438-021-00555-6

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Journal: Horticulture Research	Publication Date: Jun 1, 2021
Citations: 27	License type: open-access

Affiliation: Southwest University, Guizhou University

Abstract

Venation is a common anthocyanin pattern displayed in flowers that confers important ornamental traits to plants. An anthocyanin-related R2R3-MYB transcription factor, DPL, has been proposed to regulate corolla tube venation in petunia plants. Here, however, we provide evidence redefining the role of DPL in petunia. A CRISPR/Cas9-mediated mutation of DPL resulted in the absence of the vein-associated anthocyanin pattern above the abaxial surface of the flower bud, but not corolla tube venation, thus indicating that DPL did not regulate the formation of corolla tube venation. Alternately, quantitative real-time PCR analysis demonstrated that the spatiotemporal expression pattern of another R2R3-MYB gene, AN4, coincided with the formation of corolla tube venation in petunia. Furthermore, overexpression of AN4 promoted anthocyanin accumulation by increasing the expression of anthocyanin biosynthesis genes. CRISPR/Cas9-mediated mutation of AN4 led to an absence of corolla tube venation, suggesting that this gene in fact determines this key plant trait. Taken together, the results presented here redefine the prime regulator of corolla tube venation, paving the way for further studies on the molecular mechanisms underlying the various venation patterns in petunia.

Highlights

Anthocyanin pigments contribute to the diverse colors and pigmentation patterns of flowers, which are important ornamental characteristics of horticultural plants
This anthocyanin patterning became more obvious in flower buds of Mitchell Diploid’ (MD), when these plants with flower buds were treated at 12 ± 2 °C for 2 weeks, but it was always absent in those of dpl-knockout mutants (Fig. 1e)
Because venation patterning was not affected in dpl mutants, we investigated whether other MYB genes were perhaps involved in regulating petunia venation patterning. qPCR analysis showed that the mRNA abundance of AN4 was much higher in the corolla tube than in the anther or limb, whereas the mRNA abundance of DEEP PURPLE (DPL) was much higher in the limb and corolla tube than in the anther (Fig. 2a)

Summary

Introduction

Anthocyanin pigments contribute to the diverse colors and pigmentation patterns of flowers, which are important ornamental characteristics of horticultural plants. 4-reductase (DFR), anthocyanidin synthase (ANS), and glutathione-S-transferase (GST)[2,3,4,5] Expression of these EBGs is regulated by R2R3-MYB transcription factors[6,7], while the expression of the LBGs is regulated by the ternary complex MYB–bHLH–WD40 consisting of R2R3-MYB, a basic helix-loop-helix (bHLH), and WD40 transcription factors[8,9]. Among these transcription factors, R2R3-MYB genes play major roles in providing the required specificity for LBG expression and determining the spatiotemporal accumulation of anthocyanin in plants[10,11]. In Antirrhinum, the bHLH gene Delila is expressed in its petal epidermis, and Venosa, an R2R3MYB gene, is expressed in cells above the vascular tissues, with venation arising in the overlapping expression domains of the R2R3-MYB and bHLH genes[13]

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