Enhancement of the flower longevity of petunia by CRISPR/Cas9-mediated targeted editing of ethylene biosynthesis genes

Abstract

The transcriptional activation of genes that encode the ethylene biosynthesis enzyme 1-aminocyclopropane-1-carboxylate oxidase (PhACO3 and PhACO4) during petunia flower senescence has been reported. However, no studies have elaborately investigated their specific roles in ethylene production and flower longevity using genetic manipulation. Hence, we used the CRISPR/Cas9 system to edit the genes (PhACO3 and/or PhACO4) involved in ethylene production and flower longevity in petunia cv. Mirage Rose. The use of the CRISPR/Cas9 system with a sgRNA, which was designed from exon 2 of PhACO3, allows for the specific editing of the genes PhACO3 and/or PhACO4 with high mutation frequency, consequently producing different types of zygotes. The PhACO3 and PhACO4-edited lines 8 and 9 showed remarkably reduced ethylene production (approximately 2.8- to 3.0-fold in corollas and 1.5-fold in pistils) during flowering and extended flower longevity (approximately 9.5 d), while the PhACO3-edited bi-allelic and PhACO4-edited homozygous T0 mutant lines (14 and 23) showed enhanced flower longevity (approximately 8.0 d) compared with 6.0 d for the WT line. This was associated with reduction of PhACO4 protein levels in PhACO4-edited lines, which was confirmed using Western blot analysis and Image J software. Moreover, there was no undesirable editing effect on the PhACO1 gene. The transmission of the edited alleles to the T1 generation was also observed, and ethylene production and flower longevity were identical to those of the T0 mutant lines. Taken together, this study demonstrated not only the single and combined role of PhACO3 and PhACO4 in ethylene production in petunia flowers but also reports improvements in flower longevity by editing of the aforementioned genes using the CRISPR/Cas9 system. Therefore, our study can pave the way for the editing of homologous genes in other ornamental plants using the CRISPR/Cas9 system with a common sgRNA, thus allowing for a time- and cost-effective approach to advancing plant biology and the floricultural industry.