| dc.description.abstracteng | Jasmonoyl-isoleucine (JA-Ile) is a phytohormone that orchestrates responses to wounding, feeding insects or necrotrophic pathogens in plants. JA-Ile-metabolism has been studied intensively (Wasternack, 2015) and most of the enzymes of the JA-Ile-synthesis as well as of its catabolism have been described (Koo, 2018; Wasternack, 2015). However, the enzyme(s) responsible to specifically glycosylate 12-hydroxy-JA (12-OH-JA) are still elusive although the metabolite 12-O-glucosyl-JA (12-O-Glc-JA) has been described in plants (Koo, 2018; Miersch et alia (et al.), 2008; Seto et al., 2009). 12-O-Glc-JA triggers tuber formation in potato (Yoshihara et al., 2014) and induces leaf-closing in Mimosa (Nakamura et al., 2011). Furthermore, the glycoside might have individual functions as storage- or transport-compound (Miersch et al., 2008).
Therefore, this work investigated the glycosylation of 12-OH-JA. Four UDP-dependent glycosyltransfereases (UGTs) (UGT76E1, UGT76E2, UGT76E11, and UGT76E12) were identified as wound-induced and JA-related and sequence-analyses identified the catalytic motifs in all of them. UGT76E1, UGT76E2, UGT76E11, and UGT76E12 were heterologously expressed and purified from bacterial cultures. The UGT enzymes were characterized biochemically by different activity assays in conditions as equal as possible to determine their individual substrate tolerances and specificities. In addition, a new approach was established which exposes the enzymes to their native substrate to challenge their selectivity. Through this approach, it was possible to investigate purified proteins as close as possible to their natural environment. Finally, the kinetic parameters of the UGTs were recorded with their preferred substrates in a continuous assay. Altogether, UGT76E1 and UGT76E2 were characterized as 12-OH-JA-UGTs. UGT76E11 showed activity towards oxylipins and an unknown compound with the calculated sum formula C11H18O3. UGT76E12 showed high activity towards an artificial hydroxy-FA, and three oxylipins as well as to 12-OH-JA with minor extent.
This is the first report of UGTs that specifically glycosylate oxylipins. So far, glycosylated oxylipins have been found in L. paucicostata (Kai et al., 2010) but not in A. thaliana. However, glycosylation may increase the solubility of the FA-derived compounds in the plant cell and may be involved in oxylipin regulation or even signaling (confer Wasternack & Feussner, 2018).
To investigate the physiological role of the candidate UGTs, a CRISPR/Cas9 approach was designed. A ugt765e1 mutant plant line was originally identified by screening for an altered metabolite profile of 12-OH-JA, 12-OH-JA sulfate, and 12-O-Glc-JA. Due to potential redundancy of the candidates forming 12-O-Glc-JA, higher order mutants will be necessary to investigate the consequences of a 12-O-Glc-JA-depletion in vivo. | de |