Auxin catabolism unplugged: Role of IAA oxidation in auxin homeostasis

Stepanova, Anna N.; Alonso, José M.

doi:10.1073/pnas.1613506113

Cited by 33 publications

(22 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similar metabolic analysis of dao1 loss-offunction mutant revealed that the reduction of IAA oxidation in this mutant did not change the levels of free IAA, whereas the IAA conjugates, IAAsp and IAGlu, accumulated at much higher levels in the dao1 mutant than in wild type seedlings [18]. These findings provide evidence to support the idea that DAOand GH3-mediated catabolic pathways influence each other's activities to maintain auxin homeostasis [22]. The functional characterization of GH3, UGT and DAO proteins in planta can be challenging due to large families and functional redundancy.…”

supporting

confidence: 58%

A bacterial assay for rapid screening of IAA catabolic enzymes

Brunoni

Collani

Šimura

et al. 2019

Preprint

View full text Add to dashboard Cite

Background:Plants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3acetyl-L-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. DIOXYGENASE OF AUXIN OXIDATION (DAO) irreversibly oxidizes IAA tooxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways 2 have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on expressing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays. Results:In this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that express the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahighperformance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC-ESI-MS/MS). Experimental procedures were optimized for testing the activity of different classes of IAA-modifying enzymes without the need to purify recombinant protein. Conclusions:This new method represents an alternative to existing in vitro assays. It can be applied to the analysis of IAA metabolites that are produced upon supplementation of substrate to engineered bacterial cultures and can be used for a rapid screening of orthologous candidate genes from non-model species.

show abstract

supporting

confidence: 58%

A bacterial assay for rapid screening of IAA catabolic enzymes

Brunoni

Collani

Šimura

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…Very interestingly, the levels of IAA conjugates, such as IAAsp and IAGlu, were found to be much higher in the dao1 mutant than in wild-type plants and the accumulation of these IAA metabolites was associated with an increase of GH3 gene expression in the dao1 mutant (Porco et al , 2016). These findings demonstrated that DAO1 acts in concert with GH3 genes to maintain optimal auxin levels, and thus the regulation of IAA homeostasis by these auxin inactivation pathways is redundant (Stepanova & Alonso, 2016; Zhang & Peer, 2017). Additionally, differences in enzyme kinetics and expression levels between DAO and GH3 suggested that DAO, having much slower enzyme kinetics compared with GH3 proteins, contributes to maintain constitutively basal auxin levels under normal growth conditions, while GH3 rapidly responds to environmental factors that increase cellular IAA levels (Mellor et al , 2016; Stepanova & Alonso, 2016).…”

Section: Introductionmentioning

confidence: 89%

“…These findings demonstrated that DAO1 acts in concert with GH3 genes to maintain optimal auxin levels, and thus the regulation of IAA homeostasis by these auxin inactivation pathways is redundant (Stepanova & Alonso, 2016; Zhang & Peer, 2017). Additionally, differences in enzyme kinetics and expression levels between DAO and GH3 suggested that DAO, having much slower enzyme kinetics compared with GH3 proteins, contributes to maintain constitutively basal auxin levels under normal growth conditions, while GH3 rapidly responds to environmental factors that increase cellular IAA levels (Mellor et al , 2016; Stepanova & Alonso, 2016). Although the majority of the knowledge about auxin metabolism is based on Arabidopsis thaliana studies, the conjugation and oxidation mechanisms seem to be present also in many other species (Cooke et al , 2002; Ludwig-Müller, 2011; Zhang & Peer, 2017).…”

Section: Introductionmentioning

confidence: 89%

Conifers exhibit a characteristic inactivation of auxin to maintain tissue homeostasis

Brunoni

Collani

Casanova‐Sáez

et al. 2019

Preprint

View full text Add to dashboard Cite

Dynamic regulation of the levels of the natural auxin, indol-3-acetic acid (IAA), is essential to coordinate most of the physiological and developmental processes and responses to environmental changes. Oxidation of IAA is a major pathway to control auxin concentrations in Arabidopsis and, along with IAA conjugation, to respond to perturbation of IAA homeostasis. However, these regulatory mechanisms are still poorly investigated in conifers. To reduce this gap of knowledge, we investigated the different contribution of the IAA inactivation pathways in conifers.• Mass spectrometry-based quantification of IAA metabolites under steady state conditions and after perturbation was investigated to evaluate IAA homeostasis in conifers. Putative Picea abies GH3 genes (PaGH3) were identified by a comprehensive phylogenetic analysis including Arabidopsis and basal land plants. Auxin-inducible PaGH3 genes were identified by expression analysis and their IAA-conjugating activity was explored.• Compared to Arabidopsis, oxidative and conjugative pathways differentially contribute to reduce IAA levels in conifers. We demonstrated that the oxidation pathway plays a marginal role in controlling IAA homeostasis in spruce. On the other hand, an excess of IAA rapidly activates GH3-mediated irreversible conjugation pathways.• Taken together, these data indicate that a diversification of IAA inactivation mechanisms evolved specifically in conifers.

show abstract

“…The first of the mechanisms was evidenced by the appearance of strongly elevated (3-to 8.5-fold) levels of oxIAA hexose (Table 2), the major primary IAA catabolite in Arabidopsis roots 41,42 . This oxidized, inactive form of IAA is generated through the action of two DIOXYGENASE FOR AUXIN OXIDATION (DAO) enzymes (reviewed in 43 ). However, contrary to expectations, over the same time period transcripts encoding DAO1 and DAO2 are reduced 2-to 3-fold [signal log ratio (SLR) of −1.00 to −1.57; Table 4].…”

Section: Continuedmentioning

confidence: 99%

The dynamic response of the Arabidopsis root metabolome to auxin and ethylene is not predicted by changes in the transcriptome

Hildreth

Foley

Muday

et al. 2020

Sci Rep

View full text Add to dashboard Cite

While the effects of phytohormones on plant gene expression have been well characterized, comparatively little is known about how hormones influence metabolite profiles. This study examined the effects of elevated auxin and ethylene on the metabolome of Arabidopsis roots using a highresolution 24 h time course, conducted in parallel to time-matched transcriptomic analyses. Mass spectrometry using orthogonal UPLC separation strategies (reversed phase and HILIC) in both positive and negative ionization modes was used to maximize identification of metabolites with altered levels. The findings show that the root metabolome responds rapidly to hormone stimulus and that compounds belonging to the same class of metabolites exhibit similar changes. The responses were dominated by changes in phenylpropanoid, glucosinolate, and fatty acid metabolism, although the nature and timing of the response was unique for each hormone. These alterations in the metabolome were not directly predicted by the corresponding transcriptome data, suggesting that post-transcriptional events such as changes in enzyme activity and/or transport processes drove the observed changes in the metabolome. These findings underscore the need to better understand the biochemical mechanisms underlying the temporal reconfiguration of plant metabolism, especially in relation to the hormone-metabolome interface and its subsequent physiological and morphological effects.are available in the supplementary materials (Table S1). Data mining for the specific metabolites rutin, IAA and ACC was performed by searching the detected masses of the features in the XCMS positive datasets for masses corresponding to [M + H] + ions and the negative XCMS datasets for masses corresponding to [M − H] − ions. Features were not observed that corresponded to either IAA or ACC, while the details regarding the assignment of rutin are included in Table S1. transcriptome data analysis. Microarray datasets for root samples generated under identical conditions 11,12 were examined for changes in the expression of genes associated with the synthesis, inactivation, or transport of hormone-responsive metabolites, identified from the KEGG database 78 or the recent literature (e.g., in the case of members of the GH, DAO, and PIN families). Results for genes exhibiting a SLR of ≥ 0.5 or ≤ −0.5 in all three biological replicates for at least one time point are presented in Tables 4 and 5, with the data for all examined genes provided in Datafile S5.

show abstract

Auxin catabolism unplugged: Role of IAA oxidation in auxin homeostasis

Cited by 33 publications

References 12 publications

A bacterial assay for rapid screening of IAA catabolic enzymes

A bacterial assay for rapid screening of IAA catabolic enzymes

Conifers exhibit a characteristic inactivation of auxin to maintain tissue homeostasis

The dynamic response of the Arabidopsis root metabolome to auxin and ethylene is not predicted by changes in the transcriptome

Contact Info

Product

Resources

About