DIOXYGENASE FOR AUXIN OXIDATION 1 catalyzes the oxidation of IAA amino acid conjugates

Abstract

Together with auxin transport, auxin metabolism is a key determinant of auxin signalling output by plant cells. Enzymatic machinery involved in auxin metabolism is subject to regulation based on numerous inputs, including the concentration of auxin itself. Therefore, experiments characterizing altered auxin availability and subsequent changes in auxin metabolism could elucidate the function and regulatory role of individual elements in the auxin metabolic machinery. Here, we studied auxin metabolism in auxin-dependent tobacco BY-2 cells. We revealed that the concentration of N-(2-oxindole-3-acetyl)-L-aspartic acid (oxIAA-Asp), the most abundant auxin metabolite produced in the control culture, dramatically decreased in auxin-starved BY-2 cells. Analysis of the transcriptome and proteome in auxin-starved cells uncovered significant downregulation of all tobacco (Nicotiana tabacum) homologs of Arabidopsis (Arabidopsis thaliana) DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), at both transcript and protein levels. Auxin metabolism profiling in BY-2 mutants carrying either siRNA-silenced or CRISPR-Cas9-mutated NtDAO1, as well as in dao1-1 Arabidopsis plants, showed not only the expected lower levels of oxIAA, but also significantly lower abundance of oxIAA-Asp. Finally, ability of DAO1 to oxidize IAA-Asp was confirmed by an enzyme assay in AtDAO1-producing bacterial culture. Our results thus represent direct evidence of DAO1 activity on IAA amino acid conjugates.

The main oxidative inactivation pathway of the plant hormone auxin

“…At a high level of IAA in gh3-sept mutants, the group III GH3 enzymes, such as AtGH3.15 might produce IAA-amino acid conjugates to compensate for an impaired IAA inactivation in gh3-sept mutants, thereby accumulating small amounts of oxIAA. Recent work on IAA metabolite analysis indicated that oxIAA-Asp did not accumulate in DAO1 knockout tobacco BY-2 cultured cells and Arabidopsis dao1-1 mutants, suggesting that DAO1 oxidizes IAA-Asp in planta 28 .…”

Inactivation of the entire Arabidopsis group II GH3s confers tolerance to salinity and water deficit

“…DAOs and GH3s have been suggested to establish parallel and redundant pathways for IAA inactivation, with the GH3 pathway playing a compensatory function, as supported by increased and decreased conjugate concentrations in the DAO1 mutant and overexpressing plants, respectively (Porco et al ., 2016; Zhang et al ., 2016). This model has recently been challenged by the finding that IAA‐Asp is an in vivo substrate for the DAO1 oxidase, which additionally accounts for the formation of oxIAA‐Asp conjugates (Müller et al ., 2021). In a context where DAOs function downstream of group II GH3s, the reduced concentrations of oxIAA and oxIAA‐glc in gh3oct plants found in the present work suggest that a significant fraction of the plant oxIAA pool derives from oxIAA‐aa conjugates.…”

“…Several IAA‐Asp metabolites, including 6‐OH‐IAA‐Asp and oxIAA‐Asp, have been identified in the last few decades (Ostin et al ., 1998; Kai et al ., 2007). Recently, the IAA oxidase DAO1 was found to additionally operate in the GH3 pathway by mediating the formation of oxIAA‐Asp from IAA‐Asp in tobacco and Arabidopsis (Müller et al ., 2021).…”

Role of sugar and auxin crosstalk in plant growth and development

“…Moreover, It has also been proposed that Trp‐independent pathways are major contributors of auxin biosynthesis in maize and Arabidopsis but not in other non‐vascular plants (Normanly et al 1993; Wright et al 1991). Apart from biosynthesis, bioactive auxin levels in the cell are also determined through conjugation and oxidation (Mellor et al 2016; Müller et al 2021; Porco et al 2016). IAA conjugates to sugar through an ester bond, while it is bound to amino acids and peptides through amide‐linkage (Ludwig‐Müller 2011; Woodward & Bartel 2005).…”