Elucidation of the Function of Dihydrochalcones in Apple

Dihydrochalcones (DHCs) are specialised metabolites with a limited natural distribution, found in significant amounts in Malus x domestica Borkh. (cultivated apple) and wild Malus species. Among them, M. x domestica accumulates significant amounts of phloridzin, whilst trilobatin and sieboldin are abundant in some wild relatives. DHCs have demonstrated a wide range of bioactive properties in biomedical models. Some DHCs have also been reported to act as flavour enhancers. Phloridzin may act as an anti-diabetic compound by blocking sodium-linked glucose transport and renal reabsorption of glucose in kidneys. Despite the protective effects reported in mammal models, little is known about how these metabolites are biosynthesised and what is their function in planta, where it has been hypothesised a role for phloridzin in plant growth. The biosynthetic pathway leading to DHC formation has been proposed in apple, and some steps have been characterised recently. DHC pathway diverts from the main phenylpropanoid pathway most probably from 4-coumaroyl-CoA by the action of a yet unknown reductase that would produce 4-dihydrocoumaroyl-CoA. Then, chalcone synthase (CHS) catalyses its condensation to form phloretin. Phloretin can be directly glycosylated at position 2′- or 4′ by the previously characterised 2′- and 4′-O-UDP-glycosyltransferases PGT1 and PGT2, to produce phloridzin or trilobatin, respectively. However, sieboldin has been postulated to derive from hydroxylation in position 3 of phloretin before been glycosylated, and the key responsible enzyme producing 3-hydroxyphloretin has not been yet discovered. The main aim of this PhD proposal was to provide a better understanding of the physiological functions of DHCs in apple, as well as to contribute to the elucidation of the biosynthetic pathway as the molecular basis for future genetic engineering in apple. Towards this aim, functional characterisation was conducted in MdPGT1 knockdown apple lines by RNAi silencing and CRISPR/Cas9 genome editing to assess the physiological effect of targeting a key biosynthetic gene involved in phloridzin biosynthesis. In addition, molecular, transcriptomic and metabolomic analyses were integrated to evaluate candidate genes accounting for 3-hydroxylase activity involved in DHC biosynthesis in wild Malus species accumulating sieboldin. Moreover, a de novo transcriptome assembly was carried out in an intergeneric hybrid between M. x domestica and Pyrus communis L. known to accumulate intermediate levels of DHCs compared to apple, in order to identify additional genes potentially involved in DHC pathway. We compared the physiological effect of reducing phloridzin through PGT1 knockdown by RNAi silencing and CRISPR/Cas9 genome editing. Knockdown lines exhibited characteristic impairment of plant growth and leaf morphology as reported in literature, whereas genome edited lines exhibited normal growth despite reduced foliar phloridzin. Bioactive brassinosteroids and gibberellins were found to be key players involved in the contrasting effects on growth observed following phloridzin reduction. Moreover, a cytochrome P450 from wild M. toringo (K. Koch) Carriere syn. sieboldii Rehder, and M. micromalus Makino was identified as dihydrochalcone 3-hydroxylase (DHCH), proving to produce 3-hydroxyphloretin and sieboldin in yeast. Different DHCH allele isoforms found in domesticated apple and M. toringo and M. micromalus correlated with sieboldin accumulation in a Malus germplasm collection. Finally, the assembled de novo transcriptome of the intergeneric apple/pear hybrid integrated to functional annotation and metabolomic analysis resulted in the identification of genes potentially involved in DHC biosynthesis, providing the basis for future biochemical characterisation. Altogether these results contribute to get insight into the roles of DHCs in apple and to illustrate how CRISPR/Cas9 genome editing can be applied to dissect the contribution of genes involved in phloridzin biosynthesis in apple. Furthermore, the present PhD thesis contributes to the state-of-the-art by elucidating key missing steps in the biosynthesis of DHCs, which could be relevant for future establishment of genetic engineered lines that contribute to assess physiological effects of altering DHCs content, as well as to establish heterologous expression systems to produce de novo DHCs.