Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L.

Abstract

Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten PPO genes (named SmelPPO1-10) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target PPO genes (SmelPPO4, SmelPPO5, and SmelPPO6), which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three PPO genes. The successful editing of the SmelPPO4, SmelPPO5, and SmelPPO6 loci of in vitro regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified in silico proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T1 and T2 progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.

Genome-wide investigation and expression profiling of polyphenol oxidase (PPO) family genes uncover likely functions in organ development and stress responses in populus trichocarpa

“…The expression pattern of PPOs gene in diverse tissues has been depicted in numerous species [17,22,23,34]. Due to the difference in the number of PPOs contained in different species, there is no uniform gene expression profiles of PPOs gene in plants.…”

“…The levle of PPO gene in plants is closely related to stress, and mainly responds to the gnawing of diseases and insects, the invasion of microorganisms, and mechanical injury [11,17]. Through transcriptome dataset analysis, some PPO genes in poplar were significantly induced (PtrPPO 9/13)and inhibited(PtrPPO 11/14/15) by drought.…”

“…A great number of literatures have revealed that the expression of PPO genes in plants is closely related to stress, and mainly responds to the gnawing of diseases and insects, the invasion of microorganisms, and mechanical injury [11,17]. In addition, the PPO family gene can significantly respond to MeJA in Salvia miltiorrhiza and Nicotiana tabacum L [18,19].…”

“…For example, PtrPPO12/13 has a similar expression level in all tissues, while PtrPPO14/15 shows a downward trend under the threat of adversity (Figure 6). Similar expression levels indicate the similar function and structure of tandem repeat genes.The expression pattern of PPOs gene in diverse tissues has been depicted in numerous species[17,22,23,34]. Due to the difference in the number of PPOs contained in different species, there is no uniform gene expression profiles of PPOs gene in plants.…”

Applications and Major Achievements of Genome Editing in Vegetable Crops: A Review

“…Eggplant (Solanum melongena L.) ranks fifth among vegetables in terms of total global production, with 52.3 million tons produced in 2017 (Alam and Salimullah, 2021). Three polyphenol oxidase genes (PPOs; SmelPPO4, SmelPPO5, and SmelPPO6) showing the highest transcript levels in the fruit after cutting were regarded to be associated with enzymatic browning of eggplants, and CRISPR-Cas9-based mutagenesis has been applied to knockout three target PPO genes simultaneously aiming to reduce fruit flesh browning (Maioli et al, 2020).…”