Tissue‐specific profiling of the Arabidopsis thaliana auxin metabolome

Abstract

SUMMARYThe plant hormone auxin is believed to influence almost every aspect of plant growth and development. Auxin transport, biosynthesis and degradation combine to form gradients of the hormone that influence a range of key developmental and environmental response processes. There is abundant genetic evidence for the existence of multiple pathways for auxin biosynthesis and degradation. The complexity of these pathways makes it difficult to obtain a clear picture of the relative importance of specific metabolic pathways during development. We have developed a sensitive mass spectrometry-based method to simultaneously profile the majority of known auxin precursors and conjugates/catabolites in small amounts of Arabidopsis tissue. The method includes a new derivatization technique for quantification of the most labile of the auxin precursors. We validated the method by profiling the auxin metabolome in root and shoot tissues from various Arabidopsis thaliana ecotypes and auxin over-producing mutant lines. Substantial differences were shown in metabolite patterns between the lines and tissues. We also found differences of several orders of magnitude in the abundance of auxin metabolites, potentially indicating the relative importance of these compounds in the maintenance of auxin levels and activity. The method that we have established will enable researchers to obtain a better understanding of the dynamics of auxin metabolism and activity during plant growth and development.

Regulation of Auxin Homeostasis and Gradients in Arabidopsis Roots through the Formation of the Indole-3-Acetic Acid Catabolite 2-Oxindole-3-Acetic Acid

“…Additional IAA metabolites have been identified in Arabidopsis, such as IAA-Glc, IAA-Val, IAA-Phe, 6-OH-IAA-Val, and 6-OH-IAA-Phe (Kai et al, 2007), IAA-Trp (Staswick 2009), and IAA-Ala and IAA-Leu (Kowalczyk and Sandberg, 2001;Rampey et al, 2004). These and other studies (Tam et al, 2000;Kubeš et al, 2012;Novák et al, 2012;Peer et al, 2013) clearly show that oxIAA is a major IAA degradation product in Arabidopsis tissues and that IAA amino acid conjugates are normally present in much lower quantities. and oxIAA (open triangles) was monitored after feeding 7-DAG wild-type Arabidopsis Col-0 seedlings with liquid medium containing 10 mM of the labeled precursor for 3, 6, 12, and 24 h. Enrichment of the IAA and oxIAA pools is expressed as the isotope ratio, the ratio of labeled to unlabeled compound, after correction for natural isotope abundances.…”

“…It is known that auxin can be conjugated to amino acids and sugars to either storage forms or to irreversible degradation products, and several genes involved in IAA conjugation and deconjugation have been identified (reviewed in Ludwig-Müller, 2011;Korasick et al, 2013). Data from our laboratory and others have demonstrated that oxidation of IAA to 2-oxindole-3-acetic acid (oxIAA) is another important pathway for IAA degradation in plant species such as Arabidopsis (Östin et al, 1998;Kowalczyk and Sandberg 2001;Kai et al, 2007;Novák et al, 2012;Peer et al, 2013), maize (Zea mays; Reinecke and Bandurski, 1983), and Pinus sylvestris (Ernstsen et al, 1987). These findings have mainly been based on the use of radioactive and labeled precursor feeding and/or mass spectrometry identification and quantification of the main IAA degradation products.…”

“…Some of these conjugates may act as reversible storage forms of IAA that can later be hydrolyzed to produce free IAA. The most abundant AA conjugates in Arabidopsis seedlings are normally IAA-Asp and IAA-Glu (Östin et al, 1998;Tam et al, 2000;Kowalczyk and Sandberg, 2001;Kai et al, 2007;Novák et al, 2012). pathways in plants, making it difficult to study the specific regulation and function of IAA catabolism in planta.…”

cis-Cinnamic Acid Is a Novel, Natural Auxin Efflux Inhibitor That Promotes Lateral Root Formation

“…Extraction and purification of auxin and its metabolites was done as described previously with minor modifications (Novák et al, 2012). Frozen samples were homogenized using a MixerMill (Retsch GmbH) and extracted in 1 mL 50 mM sodium phosphate buffer (pH 7.0) containing antioxidant (1% sodium diethyldithiocarbamate) and a cocktail of deuterium and 13 C 6 -labeled internal standards of IAA and its metabolites.…”

“…After sample application, the column was washed with 2 mL 5% methanol and then eluted with 2 mL 80% methanol. Eluates were evaporated to dryness and dissolved in 20 mL of mobile phase prior to mass analysis using a 1290 Infinity LC system and 6460 Triple Quad LC-MS system (Agilent Technologies; Novák et al, 2012).…”