Dynamic regulation of auxin oxidase and conjugating enzymes AtDAO1 and GH3 modulates auxin homeostasis

Abstract

The hormone auxin is a key regulator of plant growth and development, and great progress has been made understanding auxin transport and signaling. Here, we show that auxin metabolism and homeostasis are also regulated in a complex manner. The principal auxin degradation pathways in Arabidopsis include oxidation by Arabidopsis thaliana gene DIOXYGENASE FOR AUXIN OXIDATION 1/2 (AtDAO1/2) and conjugation by Gretchen Hagen3s (GH3s). Metabolic profiling of dao1-1 root tissues revealed a 50% decrease in the oxidation product 2-oxoindole-3-acetic acid (oxIAA) and increases in the conjugated forms indole-3-acetic acid aspartic acid (IAA-Asp) and indole-3-acetic acid glutamic acid (IAA-Glu) of 438-and 240-fold, respectively, whereas auxin remains close to the WT. By fitting parameter values to a mathematical model of these metabolic pathways, we show that, in addition to reduced oxidation, both auxin biosynthesis and conjugation are increased in dao1-1. Transcripts of AtDAO1 and GH3 genes increase in response to auxin over different timescales and concentration ranges. Including this regulation of AtDAO1 and GH3 in an extended model reveals that auxin oxidation is more important for auxin homoeostasis at lower hormone concentrations, whereas auxin conjugation is most significant at high auxin levels. Finally, embedding our homeostasis model in a multicellular simulation to assess the spatial effect of the dao1-1 mutant shows that auxin increases in outer root tissues in agreement with the dao1-1 mutant root hair phenotype. We conclude that auxin homeostasis is dependent on AtDAO1, acting in concert with GH3, to maintain auxin at optimal levels for plant growth and development.T he plant hormone auxin regulates a myriad of processes in plant growth and development (1). Although significant progress has been made in understanding the molecular basis of auxin transport, perception, and response, the control of auxin metabolism and homeostasis via conjugation and degradation remains less well-studied.Several forms of auxin conjugates have been identified in plants, including ester-linked indole-3-acetic acid (IAA)-sugar conjugates and amide-linked IAA-amino acid conjugates (2). In Arabidopsis thaliana, the Gretchen Hagen3 (GH3) family of auxin-inducible acyl amido synthetases has been shown to convert IAA to IAA-amino acids (3). Most amino acid IAA conjugates are believed to be inactive, and some, such as indole-3-acetic acid aspartic acid (IAA-Asp) and indole-3-acetic acid glutamic acid (IAA-Glu), can also be further metabolized (4-6). The conversion of IAA to indole-3-acetic acid glucose (IAA-glc) is catalyzed by the uridine diphosphate glucosyltransferase UGT84B1 (UGT) (7). The oxidized form of IAA, 2-oxindole-3-acetic acid (oxIAA), has been identified as a major IAA catabolite in Arabidopsis (4, 6, 8) and can be further metabolized by conjugation to glucose (9). oxIAA has been shown to be an irreversible IAA catabolite that has very little biological activity compared with IAA and is not transported via the polar auxin ...

Auxin catabolism unplugged: Role of IAA oxidation in auxin homeostasis

“…In fact, the latter possibility is supported by the observation that the IAA conjugates, IAA-Glu and IAA-Asp, the products of another major route of IAA inactivation, accumulate to much higher levels in the dao1 knockout mutant than in wild-type plants (5-7), whereas a gain-of-function allele of dao1 shows reduced levels of these conjugates (6). Consistent with this observation, an increase in the transcript level of GH3.3, one of the key enzymes in the conjugation of IAA to amino acids, in the dao1 loss-of-function mutant could be observed (5)(6)(7). This idea of the functional redundancy between the DAO-and the GH3-mediated auxin inactivation pathways was further supported by metabolite feeding experiments in which labeled auxin precursors were much more rapidly incorporated into the IAA conjugates, IAA-Asp and IAA-Glu, in the dao1 knockout mutant than in the wild-type controls (6).…”

“…Another possible mechanism that could be behind the large increase in IAA-Asp and IAA-Glu in the dao1 knockout, which was not originally considered, is an increase in the rates of IAA biosynthesis. In fact, when even a minor increase in auxin production was introduced, the mathematical model now predicted large increases in IAA conjugates, resulting in a much better match between the experimentally observed and model-predicted levels of IAA catabolites (7). Consistent with the increase in IAA biosynthesis needed for the mathematical model to accurately reflect the observed changes in auxin catabolites, an increase in the IAA precursor indole-3-pyruvic acid (IPyA) was detected in the dao1 loss-of-function mutant (6,7).…”

“…In fact, when even a minor increase in auxin production was introduced, the mathematical model now predicted large increases in IAA conjugates, resulting in a much better match between the experimentally observed and model-predicted levels of IAA catabolites (7). Consistent with the increase in IAA biosynthesis needed for the mathematical model to accurately reflect the observed changes in auxin catabolites, an increase in the IAA precursor indole-3-pyruvic acid (IPyA) was detected in the dao1 loss-of-function mutant (6,7). Although additional experiments will be required to further demonstrate that the DAO1 disruption indeed results in an increase in the metabolic flux through the IPyA pathway in planta and to determine the exact molecular mechanism responsible for such increase, the computational model was successful at explaining in mathematical terms the interactions between production and two different IAA catabolic pathways.…”

“…With the key genes of the oxIAA pathway at hand and strong experimental evidence for the existence of a compensatory mechanism between the two main IAA catabolic pathways, the question arose as to how these two pathways influence each other's activities. The first clue to answering this question came from the observation by Porco et al (6) that AtDAO1 was auxin inducible, as it has been previously shown for the apple AtDAO1 ortholog, ARRO-1a (11), and for members of the auxin-conjugating enzyme family in Arabidopsis, GH3 (7,12). Thus, one could imagine a simple mechanistic model in which a decrease in the rate of IAA oxidation due to a disruption in DAO1 in Arabidopsis would result in a transient accumulation of IAA.…”

“…Integral to this model was the experimentally observed differential regulation of the two catabolic pathways by IAA. Thus, the GH3-dependent conjugation pathway is rapidly activated by high levels of IAA, whereas the DAO oxidation pathway is much slower to respond to IAA, but can be modulated by even minor changes in the levels of this hormone (6,7,12) (Fig. 1).…”