GH3 phylogenetic subgroups define divergent routes of auxin inactivation via aspartate and glutamine conjugation.

Auxin conjugation represents a key metabolic mechanism in regulating auxin activity within plant cells. GRETCHEN HAGEN3 (GH3) enzymes conjugate the major naturally occurring auxin indole-3-acetic acid (IAA) with amino acids, thereby contributing to the maintenance of auxin homeostasis. Although the transcription of GH3 genes is auxin regulated, the complexity of this regulation is still not fully understood. Therefore, in this study, we employed β-estradiol-inducible and CRISPR/Cas9-mediated knock-out transgenic tobacco (Nicotiana tabacum) cell lines with modified expression of representative genes from 2 GH3 subgroups with contrasting responses to auxin, NtGH3.1a, and NtGH3.6e. Using IAA metabolite profiling and bacterial enzyme assays, we show that NtGH3.1a preferentially catalyzes the formation of indole-3-acetyl-aspartate (IAA-Asp), while NtGH3.6e facilitates the production of the less-characterized conjugates indole-3-acetyl-glutamine (IAA-Gln) and 2-oxindole-3-acetyl-glutamine (oxIAA-Gln). We further validated these results by testing the Arabidopsis thaliana homologs of both GH3 subgroups, showing that AtGH3.1 favors aspartate, while both AtGH3.5 and AtGH3.6 preferentially utilize glutamine. Finally, subcellular localization analyses using green fluorescent protein (GFP)-tagged NtGH3.1aT and NtGH3.6eT expressed under inducible promoters demonstrated that both enzymes localized to the nucleus and cytoplasm, independently of the presence of auxin. Moreover, we provide evidence of GH3 localization under native promoters, confirming their presence in both compartments. Collectively, our results suggest the evolutionary conservation of amino acid type-preferential IAA conjugation and underscore the functional divergence of GH3 isoforms in IAA metabolism.