Silencing MDPGT1 by conventional trensgenesis and CRISPR/CAS9 genome editing provides new insights into the of Phloridzin in apple development

Dihydrochalcones (DHCs) constitute a class of metabolites derived from the phenylpropanoid pathway, which have been shown to exert benecial properties both in human health and plants. Among DHCs, phloridzin is the most abundant polyphenolic compound in appe (Malus x domestica), resulting from the action of a key phloretin-specic UDP-2'O-glycosyltransferase (MdPGT1). In order to better understand the role of DHCs in apple, this work aimed to study the eect of silencing MdPGT1 by traditional transgenesis and precise CRISPR/Cas9-mediated genome editing. Then, metabolic, transcriptomic and hormonal analyses were performed in knockdown lines. RNAi-silenced lines exhibited characteristic impairment of plant growth and leaf morphology, whereas edited lines exhibited a normal leaf phenotype. Nevertheless, both approaches led to a signicant reduction of ploridzin in knockdown lines. To delve into the processes that may account for these changes, an RNA-Seq analysis identied 5,004 dierentially expressed genes. These genes were involved in phenylpropanoid and avonoid pathways, as well as photosynthesis, redox metabolism and hormone signal transduction. Moreover, RT-qPCR expression analyses showed a reduction of transcripts involved in the metabolism of indole-3-acetic acid (IAA), salicylic acid (SA) and jasmonic acid (JA). To further characterise the metabolic eects of silencing MdPGT1, knockdown lines were subjected to a phytohormone proling. JA derivatives showed contrasting trends in RNAi and CRISPR/Cas9 lines, whilst IAA and SA were consistently down- and upregulated, respectively. Altogether, these results suggest that phloridzin regulates a range of integrated processes, including stress related hormones and secondary metabolism, ultimately impacting plant development. Silencing MdPGT1 by conventional silencing and CRISPR/Cas9 genome editing also provides insights into the common and distinctive features underlying specic eects of phloridzin on apple development.