Amino acid conjugation of oxIAA is a secondary metabolic regulation involved in auxin homeostasis

Dynamic regulation of the concentration of the natural auxin indole-3-acetic acid (IAA) is essential to coordinate most physiological and developmental processes and responses to environmental changes (reviewed in Friml, 2022). Auxin inactivation plays a crucial role in auxin homeostasis and metabolism. The primary enzymes involved in auxin metabolism have been known for some time. Members of the acyl acid amide synthetases belonging to the GRETCHEN HAGEN 3 (GH3) and the amidohydrolase IAA-LEUCINE RESISTANT 1 (ILR1) and ILR1-like (ILL) families catalyze the conjugation of IAA to amino acids and hydrolysis of the IAA-amino acid conjugates, respectively (LeClere et al., 2002; Staswick et al., 2005). The DIOXIGENASE FOR AUXIN OXIDATION (DAO) enzymes were shown to catalyze the oxidation of IAA to form oxIAA (Porco et al., 2016; Zhang et al., 2016). Albeit these IAA- inactivating enzymes appeared to participate in different catabolic routes, very recently, it was reported that GH3, ILR1, and DAO are part of a single linear pathway rather than two distinct ways (Hayashi et al., 2021). According to this model, IAA is mainly inactivated by GH3 enzymes, DAO functions as an oxidase of IAA-amino acid conjugates to produce oxIAA-amino acid conjugates downstream of GH3, and oxIAA is produced from oxIAA-amino acid hydrolysis by ILR1. Therefore, DAO and ILR1 enzymes appeared to play a role in this pathway that differs from that assigned initially. Although GH3 enzymes are known to possess catalytic promiscuity accepting various substrates and amino acids, a possible additional role of GH3s in auxin inactivation was not investigated (Staswick et al., 2005; Westfall et al., 2012). Besides, while the new model proposed by Hayashi et al. (2021) was described to occur in angiosperms, whether it operates in nonflowering species remains unknown (Ross & G elinas- Marion, 2021). GRETCHEN HAGEN 3 proteins are highly conserved all over the plant kingdom, whereas DAO and DAO-like enzymes have specifically evolved with angiosperms (Okrent & Wildermurth, 2011; Brunoni et al., 2020; Kaneko et al., 2020; Takehara et al., 2020). Here, we report the evidence of oxIAA-amino acid conjugation being catalyzed by the group of IAA-conjugating enzymes belonging to the Group II of GH3s. Our work suggests that the contribution of this pathway to auxin homeostasis is species-dependent.