Application of Long-Chained Auxin Conjugates Influenced Auxin Metabolism and Transcriptome Response in Brassica rapa L. ssp. pekinensis.

Auxin amino acid conjugates are considered to be storage forms of auxins. Previous research has shown that indole-3-acetyl-L-alanine (IAA-Ala), indole-3-propionyl-L-alanine (IPA-Ala) and indole-3-butyryl-L-alanine (IBA-Ala) affect the root growth of Brassica rapa seedlings. To elucidate the potential mechanism of action of the conjugates, we treated B. rapa seedlings with 0.01 mM IAA-, IPA- and IBA-Ala and investigated their effects on the auxin metabolome and transcriptome. IBA-Ala and IPA-Ala caused a significant inhibition of root growth and a decrease in free IAA compared to the control and IAA-Ala treatments. The identification of free auxins IBA and IPA after feeding experiments with IBA-Ala and IPA-Ala, respectively, confirms their hydrolysis in vivo and indicates active auxins responsible for a stronger inhibition of root growth. IBA-Ala caused the induction of most DEGs (807) compared to IPA-Ala (417) and IAA-Ala (371). All treatments caused similar trends in transcription profile changes when compared to control treatments. The majority of auxin-related DEGs were found after IBA-Ala treatment, followed by IPA-Ala and IAA-Ala, which is consistent with the apparent root morphology. In addition to most YUC genes, which showed a tendency to be downregulated, transcripts of auxin-related DEGs that were identified (UGT74E2, GH3.2, SAUR, IAA2, etc.) were more highly expressed after all treatments. Our results are consistent with the hypothesis that the hydrolysis of conjugates and the release of free auxins are responsible for the effects of conjugate treatments. In conclusion, free auxins released by the hydrolysis of all auxin conjugates applied affect gene regulation, auxin homeostasis and ultimately root growth inhibition.

Conifers exhibit a characteristic inactivation of auxin to maintain tissue homeostasis.

Dynamic regulation of the concentration of the natural auxin (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 angiosperms and, along with IAA conjugation, to respond to perturbation of IAA homeostasis. However, these regulatory mechanisms remain poorly investigated in conifers. To reduce this knowledge gap, we investigated the different contributions of the IAA inactivation pathways in conifers. MS-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 based on a comprehensive phylogenetic analysis including angiosperms 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 concentrations in conifers. We demonstrated that the oxidation pathway plays a marginal role in controlling IAA homeostasis in spruce. By contrast, 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.

Indole-3-acetylaspartate and indole-3-acetylglutamate, the IAA-amide conjugates in the diploid strawberry achene, are hydrolyzed in growing seedlings.

MAIN CONCLUSION: Indole-3-acetylaspartate and indole-3-acetylglutamate are the stored auxin amino acid conjugates of the achene of the diploid strawberry and serve as sources of auxin during seedling growth. The edible part of the strawberry, a pseudocarp, has long been known to enlarge in response to auxin produced by the developing achenes, the botanical true fruit. Auxin homeostasis involves a complex interaction between biosynthesis, conjugate formation and hydrolysis, catabolism and transport. Strawberry tissues are capable of synthesizing auxin conjugates, and transcriptome data support the expression of genes involved in IAA conjugate formation and hydrolysis throughout embryo development and subsequent seedling growth. Using a highly sensitive and selective mass spectrometric method, we identified all the low molecular weight indole-auxin amino acid conjugates in achenes of F. vesca as consisting of indole-3-acetylaspartate (IAasp) and indole-3-acetylglutamate (IAglu). In contrast to what has been proposed to occur in Arabidopsis, we determined that IAasp and IAglu are hydrolyzed by seedlings to provide a source of free IAA for growth.

Auxin biosynthesis and storage forms.

The plant hormone auxin drives plant growth and morphogenesis. The levels and distribution of the active auxin indole-3-acetic acid (IAA) are tightly controlled through synthesis, inactivation, and transport. Many auxin precursors and modified auxin forms, used to regulate auxin homeostasis, have been identified; however, very little is known about the integration of multiple auxin biosynthesis and inactivation pathways. This review discusses the many ways auxin levels are regulated through biosynthesis, storage forms, and inactivation, and the potential roles modified auxins play in regulating the bioactive pool of auxin to affect plant growth and development.

Metabolic profiles of 2-oxindole-3-acetyl-amino acid conjugates differ in various plant species.

Auxins are a group of phytohormones that play a key role in plant growth and development, mainly presented by the major member of the family - indole-3-acetic acid (IAA). The levels of free IAA are regulated, in addition to de novo biosynthesis, by irreversible oxidative catabolism and reversible conjugation with sugars and amino acids. These conjugates, which serve as inactive storage forms of auxin and/or degradation intermediates, can also be oxidized to form 2-oxindole-3-acetyl-1-O-ß-d-glucose (oxIAA-glc) and oxIAA-amino acids (oxIAA-AAs). Until now, only oxIAA conjugates with aspartate and glutamate have been identified in plants. However, detailed information on the endogenous levels of these and other putative oxIAA-amino acid conjugates in various plant species and their spatial distribution is still not well understood but is finally getting more attention. Herein, we identified and characterized two novel naturally occurring auxin metabolites in plants, namely oxIAA-leucine (oxIAA-Leu) and oxIAA-phenylalanine (oxIAA-Phe). Subsequently, a new liquid chromatography-tandem mass spectrometry method was developed for the determination of a wide range of IAA metabolites. Using this methodology, the quantitative determination of IAA metabolites including newly characterized oxIAA conjugates in roots, shoots and cotyledons of four selected plant models - Arabidopsis thaliana, pea (Pisum sativum L.), wheat (Triticum aestivum L.) and maize (Zea mays L.) was performed to compare auxin metabolite profiles. The distribution of various groups of auxin metabolites differed notably among the studied species as well as their sections. For example, oxIAA-AA conjugates were the major metabolites found in pea, while oxIAA-glc dominated in Arabidopsis. We further compared IAA metabolite levels in plants harvested at different growth stages to monitor the dynamics of IAA metabolite profiles during early seedling development. In general, our results show a great diversity of auxin inactivation pathways among angiosperm plants. We believe that our findings will greatly contribute to a better understanding of IAA homeostasis.

Auxin conjugated to fluorescent dyes--a tool for the analysis of auxin transport pathways.

Auxin is a small molecule involved in most processes related to plant growth and development. Its effect usually depends on the distribution in tissues and the formation of concentration gradients. Until now there has been no tool for the direct tracking of auxin transport at the cellular and tissue level; therefore the majority of studies have been based on various indirect methods. However, due to their various restrictions, relatively little is known about the relationship between various pathways of auxin transport and specific developmental processes. We present a new research tool: fluorescently labelled auxin in the form of a conjugate with two different fluorescent tracers, FITC and RITC, which allows direct observation of auxin transport in plant tissues. Chemical analysis and biological tests have shown that our conjugates have auxin-like biological activity and transport; therefore they can be used in all experimental systems as an alternative to IAA. In addition, the conjugates are a universal tool that can be applied in studies of all plant groups and species. The conjugation procedure presented in this paper can be adapted to other fluorescent dyes, which are constantly being improved. In our opinion, the conjugates greatly expand the possibilities of research concerning the role of auxin and its transport in different developmental processes in plants.

Compensatory mutations in predicted metal transporters modulate auxin conjugate responsiveness in Arabidopsis.

Levels of the phytohormone indole-3-acetic acid (IAA) can be altered by the formation and hydrolysis of IAA conjugates. The isolation and characterization of Arabidopsis thaliana mutants with reduced IAA-conjugate sensitivity and wild-type IAA responses is advancing the understanding of auxin homeostasis by uncovering the factors needed for conjugate metabolism. For example, the discovery that the IAA-Ala-resistant mutant iar1 is defective in a protein in the ZIP family of metal transporters uncovered a link between metal homeostasis and IAA-conjugate sensitivity. To uncover additional factors impacting auxin conjugate metabolism, we conducted a genetic modifier screen and isolated extragenic mutations that restored IAA-amino acid conjugate sensitivity to the iar1 mutant. One of these suppressor mutants is defective in a putative cation diffusion facilitator, MTP5 (At3g12100; formerly known as MTPc2). Loss of MTP5 function restored IAA conjugate sensitivity to iar1 but not to mutants defective in IAA-amino acid conjugate amidohydrolases. Our results are consistent with a model in which MTP5 and IAR1 transport metals in an antagonistic fashion to regulate metal homeostasis within the subcellular compartment in which the IAA-conjugate amidohydrolases reside, and support previous suggestions that the ion composition in this compartment influences hydrolase activity.