Abstract
Polyphenol Oxidases (PPOs) catalyze the conversion of phenolic substrates to quinones, leading to the formation of dark-colored precipitates in fruits and vegetables. This process, known as enzymatic browning, is the cause of undesirable changes in organoleptic properties and the loss of nutritional quality in plant-derived products. In potato (Solanum tubersoum L.), PPOs are encoded by a multi-gene family with different expression patterns. Here, we have studied the application of the CRISPR/Cas9 system to induce mutations in the StPPO2 gene in the tetraploid cultivar Desiree. We hypothesized that the specific editing of this target gene would result in a lower PPO activity in the tuber with the consequent reduction of the enzymatic browning. Ribonucleoprotein complexes (RNPs), formed by two sgRNAs and Cas9 nuclease, were transfected to potato protoplasts. Up to 68% of regenerated plants contained mutations in at least one allele of the target gene, while 24% of edited lines carried mutations in all four alleles. No off-target mutations were identified in other analyzed StPPO genes. Mutations induced in the four alleles of StPPO2 gene, led to lines with a reduction of up to 69% in tuber PPO activity and a reduction of 73% in enzymatic browning, compared to the control. Our results demonstrate that the CRISPR/Cas9 system can be applied to develop potato varieties with reduced enzymatic browning in tubers, by the specific editing of a single member of the StPPO gene family.
Highlights
Polyphenol Oxidases (PPOs; E.C.1.10.3.1, E.C.1.10.3.2, or E.C.1.14.18.1) are copper-containing enzymes, widely distributed among higher plants (Yoruk and Marshall, 2003), that catalyze the oxidation of an extensive range of phenolic compounds to their respective quinones
The CRISPR/Cas9 system was applied in potato to induce mutations in the StPPO2 gene, responsible for most of the PPO activity and enzyme content in tubers (Chi et al, 2014)
We hypothesized that the specific editing of this target gene would result in a lower PPO activity in the tuber and the consequent reduction of the enzymatic browning
Summary
Polyphenol Oxidases (PPOs; E.C.1.10.3.1, E.C.1.10.3.2, or E.C.1.14.18.1) are copper-containing enzymes, widely distributed among higher plants (Yoruk and Marshall, 2003), that catalyze the oxidation of an extensive range of phenolic compounds to their respective quinones. Non-Browning Potatoes Obtained With CRISPR/Cas generated by action of PPOs can undergo self-polymerization or react with amino acids or free radicals in proteins leading to the formation of dark-colored precipitates (Mayer, 2006) This process, known as Enzymatic Browning, is the cause of reduction in quality that alters the color, taste, texture and nutritional value of several fresh and processed fruits and vegetables (Jukanti, 2017). In potato (Solanum tuberosum L.), enzymatic browning is a serious problem for both, producers and the industry, because the tubers can be affected during harvest and post-harvest procedures such as shipping, storage, distribution and blanching (Bachem et al, 1994) This undesired process is controlled in industry by using chemical and/or physical agents (Zhang et al, 2018b). The development of new technologies to control PPOs activity in planta is the most promising and safest approach to avoid undesirable browning compounds in fresh and processed potato derived products
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