A Family of Auxin-Conjugate Hydrolases That Contributes to Free Indole-3-Acetic Acid Levels during Arabidopsis Germination

Abstract

Auxins are hormones important for numerous processes throughout plant growth and development. Plants use several mechanisms to regulate levels of the auxin indole-3-acetic acid (IAA), including the formation and hydrolysis of amide-linked conjugates that act as storage or inactivation forms of the hormone. Certain members of an Arabidopsis amidohydrolase family hydrolyze these conjugates to free IAA in vitro. We examined amidohydrolase gene expression using northern and promoterb-glucuronidase analyses and found overlapping but distinct patterns of expression. To examine the in vivo importance of auxin-conjugate hydrolysis, we generated a triple hydrolase mutant, ilr1 iar3 ill2, which is deficient in three of these hydrolases. We compared root and hypocotyl growth of the single, double, and triple hydrolase mutants on IAA-Ala, IAA-Leu, and IAAPhe. The hydrolase mutant phenotypic profiles on different conjugates reveal the in vivo activities and relative importance of ILR1, IAR3, and ILL2 in IAA-conjugate hydrolysis. In addition to defective responses to exogenous conjugates, ilr1 iar3 ill2 roots are slightly less responsive to exogenous IAA. The triple mutant also has a shorter hypocotyl and fewer lateral roots than wild type on unsupplemented medium. As suggested by the mutant phenotypes, ilr1 iar3 ill2 imbibed seeds and seedlings have lower IAA levels than wild type and accumulate IAA-Ala and IAA-Leu, conjugates that are substrates of the absent hydrolases. These results indicate that amidohydrolases contribute free IAA to the auxin pool during germination in Arabidopsis.Although auxins are essential regulators of many aspects of plant growth and development, our understanding of how levels of this hormone are controlled remains incomplete. One component of auxin homeostasis is conjugation of the auxin indole-3-acetic acid (IAA) to different moieties, including esterification to sugars and amide linkage to amino acids and peptides. IAA-Leu, IAA-Ala, IAA-Asp, IAA-Glu, and IAA-Glc have been identified in Arabidopsis seedlings (Tam et al., 2000;Kowalczyk and Sandberg, 2001). Several IAA-peptide conjugates have been identified in bean seeds (Bialek and Cohen, 1986;Walz et al., 2002) and Arabidopsis (Walz et al., 2002); in fact, IAApeptide conjugates are the major IAA conjugates in Arabidopsis seeds Park and Cohen, 2003). Different IAA conjugates apparently have specific functions in plants, such as storage, transport, or inactivation of IAA (Cohen and Bandurski, 1982;Bartel et al., 2001). In general, endogenous IAA conjugates that are biologically active and hydrolyzed in plants may function as auxin storage forms, whereas conjugates inactive in bioassays may have roles in IAA degradation Ljung et al., 2002).Several Arabidopsis screens have uncovered IAAamino acid conjugate-resistant mutants that help to delineate conjugate functions (Bartel and Fink, 1995;Campanella et al., 1996;Davies et al., 1999;Lasswell et al., 2000;Magidin et al., 2003;LeClere et al., 2004). Two of these mutants, ilr1 and iar3, are defective...

Conversion of Endogenous Indole-3-Butyric Acid to Indole-3-Acetic Acid Drives Cell Expansion in Arabidopsis Seedlings

“…Hypocotyl elongation and cotyledon cell expansion also depend on auxin. The triple hydrolase mutant, which has decreased free IAA levels in whole seedlings (Rampey et al, 2004), displays reduced hypocotyl lengths when grown in the light at 22°C but not at 28°C ( Fig. 6A; Rampey et al, 2004), suggesting that conjugates provide some of auxin that drives hypocotyl elongation at normal growth temperatures.…”

“…The triple hydrolase mutant, which has decreased free IAA levels in whole seedlings (Rampey et al, 2004), displays reduced hypocotyl lengths when grown in the light at 22°C but not at 28°C ( Fig. 6A; Rampey et al, 2004), suggesting that conjugates provide some of auxin that drives hypocotyl elongation at normal growth temperatures. When we examined ibr1 ibr3 ibr10 for a similar phenotype, we found that 8-d-old ibr1 ibr3 ibr10 seedlings displayed wild-type hypocotyl lengths at both temperatures (Fig.…”

“…We examined root hairs in a triple mutant defective in three IAA-amino acid conjugate hydrolases (Rampey et al, 2004): ILR1, an IAA-Leu and IAA-Phe hydrolase (Bartel and Fink, 1995); IAR3, an IAA-Ala Figure 2. ibr mutants display root hair elongation defects.…”

“…Mutation of Arabidopsis (Arabidopsis thaliana) genes encoding IAA-amino acid hydrolases, including ILR1, IAR3, and ILL2, reduces plant sensitivity to the applied IAA-amino acid conjugates that are substrates of these enzymes, including IAA-Leu, IAA-Phe, and IAA-Ala (Bartel and Fink, 1995;Davies et al, 1999;LeClere et al, 2002;Rampey et al, 2004), which are present in Arabidopsis (Tam et al, 2000;Kowalczyk and Sandberg, 2001;Kai et al, 2007).…”

Modulating plant hormones by enzyme action

“…14 In contrast, formation of IAA-aspartate and IAA-glutamate marks auxin for subsequent metabolic degradation and conjugation of IAA with either alanine or leucine maintains a ready pool for formation of the active free acid form of auxin. 15,16 Thus, metabolism of IAA and JA provides a simple means of modulating plant hormone receptor occupancy and controlling gene responses. Some GH3 family of proteins catalyze the conjugations of various amino acids to plant hormones, including JA and IAA; however, Characterization of multiple group II GH3 genes in Arabidopsis also demonstrates critical roles in plant growth and development primarily through the conjugation of IAA to amino acids.…”