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

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.

Fig. 1. CRISPR/Cas9-mediated mutation in DPL.

a Diagram of the target site in the genomic region of DPL. The blue line indicates the PAM (NGG) motif. The blue boxes and black lines indicate exons and introns, respectively. b Targeted mutagenesis in DPL for the dpl-M3, -M4, and -M5 lines. The target sequence is shown in blue letters, for which red dashes indicate deletions, while red letters indicate insertions. c–e Flower buds of MD and a representative line dpl-M3; flower buds in c and d are from plants cultured at 25 °C, and those in e are from plants treated at 12 ± 2 °C (cold stress conditions). Scale bars = 1 cm

Fig. 2. AN4 and DPL expression patterns.

a, b Spatial expression pattern of AN4 and DPL in the opening flower of MD. c Annotation of flower developmental stages in MD; scale bar = 1 cm. d Temporal expression patterns of AN4 and DPL during flower development in MD. SAND was used as the reference gene. Shown are the means ± SDs, n = 3. Asterisks indicate a significant difference: *P < 0.05; **P < 0.01; nonsignificant (NS). e Annotation of different parts of the flower in MD; scale bar = 1 cm

Fig. 3. Alignment of the PhAN4^MD protein sequence with other anthocyanin-related R2R3-MYB transcription factors.

The conserved R2 and R3 domains are indicated by solid lines. Vertical arrows indicate the bHLH-interacting motif ([D/E]Lx2[R/K]x3Lx6Lx3R) in the R3 domain. Red boxes indicate different residues between the AN4^V30 and AN4^MD protein sequences

Fig. 4. Functional analysis of PhAN4^MD.

a Branch of a representative line (AN4OE-4) compared with the MD line, scale bar = 1 cm. b, c Flowers from AN4OE-4 and MD, scale bars = 1 cm. d Transcript levels of early (CHSA, CHSI, F3H, and F3′H) and late (F3′5′H, DFR, ANS, 3RT, 5GT, and GST) anthocyanin biosynthetic genes in AN4OE lines and MD plants. SAND was used as the reference gene. Shown are the means ± SDs, n = 3. Asterisks indicate a significant difference (P < 0.01) from MD according to Student’s t test

Fig. 5. CRISPR/Cas9-mediated mutation in AN4.

a Diagram of the target site in the genomic regions of AN4I and AN4II. The blue line indicates the PAM (NGG) motif. The gray boxes and black lines indicate exons and introns, respectively. b Targeted mutagenesis in AN4I and AN4II of the an4-M1, -M2, and -M6 lines and their corresponding phenotypes. The target sequence is shown in blue letters, for which red dashes indicate deletions, while red letters indicate insertions. c Transcript levels of early (CHSA, CHSI, F3H, and F3′H) and late (F3′5′H, DFR, ANS, 3RT, 5GT, and GST) anthocyanin biosynthetic genes in the an4-M1, -M2, and -M6 lines and MD. SAND was used as the reference gene. Shown are the means ± SDs, n = 3. Asterisks indicate a significant difference (P < 0.01) from MD according to Student’s t test