Metabolism of exogenous auxin by <i>Arabidopsis thaliana</i>: Identification of the conjugate <i>N</i>‐(indol‐3‐ylacetyl)‐glutamine and initiation of a mutant screen

Barratt, Natalie M.; Dong, Wen; Gage, Douglas A.; Magnus, Volker; Town, Christopher D.

doi:10.1034/j.1399-3054.1999.105204.x

Cited by 35 publications

(30 citation statements)

References 38 publications

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“…The generated full scan data served as a database to screen ions characteristic for indoles (Ö stin et al, 1998). Using this procedure, we confirmed the presence of endogenous metabolites previously identified in Arabidopsis (Ö stin et al, 1998;Barratt et al, 1999;Tam et al, 2000) such as IA-Asp, 2-oxoindole-3-acetic acid (OxIAA), and IA-Glu (data not shown), as well as identified two additional conjugates, N- Leu (Fig. 1).…”

Section: Screen For Amide Conjugatessupporting

confidence: 66%

“…Interestingly, this compound is a high-specificity substrate for recently cloned IAR3 hydrolase (Davies et al, 1999). Formation of IA-Leu and IA-Ala was not observed in response to applied IAA (Ö stin et al, 1998;Barratt et al, 1999), which may show that their role in the hormone metabolism is different from the other conjugates. High concentrations of exogenous IAA stimulated the production of IA-Asp and IA-Glu, neither of which is a good substrate for ILR1/IAR3 proteins.…”

Section: Screen For Amide Conjugatesmentioning

confidence: 99%

“…Thus, it can be concluded that if such amide conjugates are present in vegetative tissues, their relative levels must be much lower than the ones we found. Alternatively, their chemical properties could prevent them from being analyzed by GC/MS, as for example, N ␣ -(indole-3-acetyl)-Gln (Barratt et al, 1999) or protein/ peptide-linked IAA molecules (Bialek and Cohen, 1986).…”

Section: Screen For Amide Conjugatesmentioning

confidence: 99%

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Quantitative Analysis of Indole-3-Acetic Acid Metabolites in Arabidopsis

Kowalczyk,

Sandberg

2001

Plant Physiology

184

135

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A general gas chromatography/mass spectrometry (MS)-based screen was performed to identify catabolites and conjugates of indole-3-acetic acid (IAA) during vegetative growth of Arabidopsis. This experiment revealed the existence of two new conjugates: N- and N- . A method for quantitative analysis of IAA metabolites in plant extracts by liquid chromatography-electrospray tandem MS has been developed. The accuracy and precision of the new method are better than 10% for standards close to the detection limit, and are between 6% and 16% for the entire protocol applied to plant extracts. The low detection limits, 0.02 to 0.1 pmol for the different metabolites, made it possible to use as little as 50 to 100 mg of tissue for quantitative analysis. The analysis was performed on different tissues of an Arabidopsis plant at two stages of development, using heavy labeled internal standards of the catabolite 2-oxoindole-3-acetic acid as well as IAA conjugated to amino acids: aspartate, glutamate, Ala, and Leu. Expanding leaves and roots that generally contain high amounts of the free hormone also contained the highest levels of IA-aspartate, IA-glutamate, and 2-oxoindole-3-acetic acid, supporting their role as irreversible catabolic products. The levels of IA-Leu and IA-Ala did not follow the general distribution of IAA. Interestingly, the level of IA-Leu was highest in roots and IA-Ala in the aerial tissues.Indole-3-acetic acid (IAA, auxin) is an important plant hormone controlling a variety of developmental processes. Reliable and sensitive quantification methods for IAA have been developed over the last decade, and a significant amount of information is available on the levels of free hormone in plants (Edlund et al., 1995;Ribnicky et al., 1998;Prinsen et al., 2000). Although the metabolism of IAA is relatively well investigated, the information on the levels and relative importance of its major metabolites is rare. IAA can be catabolized by several pathways including conjugation to sugars and amino acids and non-decarboxylative or decarboxylative oxidation (for review, see Normanly, 1997). Until recently, conjugated IAA species have been quantified indirectly, via hydrolysis to free hormone (Baldi et al., 1989;Bialek and Cohen, 1989). Ester conjugates can be hydrolyzed using mild alkaline conditions at room temperature, and amide conjugates require strong alkaline conditions and at least 100°C for hydrolysis. The latter approach is often called the total IAA method because under such conditions, both types of conjugated IAA are hydrolyzed and are subsequently measured as a sum of free IAA present in the sample before hydrolysis and IAA resulting from hydrolyzed conjugates. This relatively simple method has been favored by many researchers; however, for the biochemical studies on IAA metabolism, it is not specific or selective enough. Furthermore, some IAA-related indolic compounds have been reported to interfere with the assay. Indole-3-acetonitrile (IAN) can be converted to IAA during the strong alkaline hydrolysis, t...

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Section: Screen For Amide Conjugatessupporting

confidence: 66%

Section: Screen For Amide Conjugatesmentioning

confidence: 99%

Section: Screen For Amide Conjugatesmentioning

confidence: 99%

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Quantitative Analysis of Indole-3-Acetic Acid Metabolites in Arabidopsis

Kowalczyk,

Sandberg

2001

Plant Physiology

184

135

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“…5A). The level of IAAsp is usually correlated with the levels of IAA (Ostin et al, 1998;Barratt et al, 1999;Barlier et al, 2000), consistent with the reduced IAA levels in abph1. Thus, the levels of IAA and an IAA conjugate were lower in abph1 embryos, a finding that is consistent with the reduced ZmPIN1a expression in the abph1 SAM.…”

Section: Abph1 Mutant Embryos Have a Reduced Auxin Contentmentioning

confidence: 75%

Studies ofaberrant phyllotaxy1Mutants of Maize Indicate Complex Interactions between Auxin and Cytokinin Signaling in the Shoot Apical Meristem

et al. 2009

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One of the most fascinating aspects of plant morphology is the regular geometric arrangement of leaves and flowers, called phyllotaxy. The shoot apical meristem (SAM) determines these patterns, which vary depending on species and developmental stage. Auxin acts as an instructive signal in leaf initiation, and its transport has been implicated in phyllotaxy regulation in Arabidopsis (Arabidopsis thaliana). Altered phyllotactic patterns are observed in a maize (Zea mays) mutant, aberrant phyllotaxy1 (abph1, also known as abphyl1), and ABPH1 encodes a cytokinin-inducible type A response regulator, suggesting that cytokinin signals are also involved in the mechanism by which phyllotactic patterns are established. Therefore, we investigated the interaction between auxin and cytokinin signaling in phyllotaxy. Treatment of maize shoots with a polar auxin transport inhibitor, 1-naphthylphthalamic acid, strongly reduced ABPH1 expression, suggesting that auxin or its polar transport is required for ABPH1 expression. Immunolocalization of the PINFORMED1 (PIN1) polar auxin transporter revealed that PIN1 expression marks leaf primordia in maize, similarly to Arabidopsis. Interestingly, maize PIN1 expression at the incipient leaf primordium was greatly reduced in abph1 mutants. Consistently, auxin levels were reduced in abph1, and the maize PIN1 homolog was induced not only by auxin but also by cytokinin treatments. Our results indicate distinct roles for ABPH1 as a negative regulator of SAM size and a positive regulator of PIN1 expression. These studies highlight a complex interaction between auxin and cytokinin signaling in the specification of phyllotactic patterns and suggest an alternative model for the generation of altered phyllotactic patterns in abph1 mutants. We propose that reduced auxin levels and PIN1 expression in abph1 mutant SAMs delay leaf initiation, contributing to the enlarged SAM and altered phyllotaxy of these mutants.

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“…Determination of the native substrates for these enzymes will be an important component to defining their role in IAA homeostasis. The IAA conjugates have not been quantified in Arabidopsis, although labeling studies have revealed that IAA-Asp and IAA-Glu are early metabolites that form in response to micromolar concentrations of exogenous IAA (Ö stin et al, 1998;Barratt et al, 1999). Labeling studies with less exogenous IAA (0.5 m) do not yield these conjugates, instead IAA is converted to OxIAA and OxIAA-hexose (Ö stin et al, 1998).…”

mentioning

confidence: 99%

Characterization of Auxin Conjugates in Arabidopsis. Low Steady-State Levels of Indole-3-Acetyl-Aspartate, Indole-3-Acetyl-Glutamate, and Indole-3-Acetyl-Glucose

2000

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Amide-linked indole-3-acetic acid (IAA) conjugates constitute approximately 90% of the IAA pool in the dicot Arabidopsis, whereas ester-linked conjugates and free IAA account for approximately 10% and 1%, respectively when whole seedlings are measured. We show here that IAA-aspartate Asp, IAA-glutamate (Glu), and IAA-glucose (Glc) are present at low levels in Arabidopsis. Nine-day-old wild-type Arabidopsis seedlings yielded 17.4 Ϯ 4.6 ng g Ϫ1 fresh weight IAA-Asp and 3.5 Ϯ 1.6 ng g Ϫ1 fresh weight IAA-Glu, and IAA-Glc was present at 7 to 17 ng g Ϫ1 fresh weight in 12-d-old wild-type seedlings. Total IAA content in 9-d-old Arabidopsis seedlings was 1,200 Ϯ 178 ng g Ϫ1 fresh weight, so these three IAA conjugates together made up only 3% of the conjugate pool throughout the whole plant. We detected less than wild-type levels of IAA-Asp and IAA-Glu (7.8 Ϯ 0.4 ng g Ϫ1 fresh weight and 1.8 Ϯ 0.3 ng g Ϫ1 fresh weight, respectively) in an Arabidopsis mutant that accumulates conjugated IAA. Our results are consistent with IAA-Asp, IAA-Glu, and IAA-Glc being either minor, transient, or specifically localized IAA metabolites under normal growth conditions and bring into question the physiological relevance of IAA-Asp accumulation in response to high concentrations of exogenous IAA.

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Metabolism of exogenous auxin by Arabidopsis thaliana: Identification of the conjugate N‐(indol‐3‐ylacetyl)‐glutamine and initiation of a mutant screen

Cited by 35 publications

References 38 publications

Quantitative Analysis of Indole-3-Acetic Acid Metabolites in Arabidopsis

Quantitative Analysis of Indole-3-Acetic Acid Metabolites in Arabidopsis

Studies ofaberrant phyllotaxy1Mutants of Maize Indicate Complex Interactions between Auxin and Cytokinin Signaling in the Shoot Apical Meristem

Characterization of Auxin Conjugates in Arabidopsis. Low Steady-State Levels of Indole-3-Acetyl-Aspartate, Indole-3-Acetyl-Glutamate, and Indole-3-Acetyl-Glucose

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