Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone, indole-3-acetic acid

Abstract

Acylamidohydrolases from higher plants have not been characterized or cloned so far. AtPsMll is the first member of this enzyme family from a higher plant and was identified in the genome of Arabidopsis thaliana based on sequence homology with the catalyticdomain sequence of bacterial acylamidohydrolases, particularly those that exhibit indole-3-acetamide amidohydrolase activity. AtAMll polypeptide and mRNA are present in leaf tissues, as shown by immunoblotting and RT-PCR, respectively. AtAMll was expressed from its cDNA in enzymatically active form and exhibits substrate specificity for indole-3-acetamide, but also some activity against L-asparagine. The recombinant enzyme was characterized further. The results show that higher plants have acylamidohydrolases with properties similar to the enzymes of certain plant-associated bacteria such as Agrobacterium-, Pseudomonasand Rhodococcus-spQCÍQS, in which these enzymes serve to synthesize the plant growth hormone, indole-3-acetic acid, utilized by the bacteria to colonize their host plants. As Índole-3-acetamide is a native metabolite in Arabidopsis thaliana, it can no longer be ruled out that one pathway for the biosynthesis of indole-3-acetic acid involves indole-3-acetamide-hydrolysis by AtPsMll.

Transcription Analysis of Arabidopsis Membrane Transporters and Hormone Pathways during Developmental and Induced Leaf Senescence

“…8A) is observed in NS and DET, but not DIS. Downstream of Trp, the Arabidopsis amidohydrolase AtAMI1 converts in vitro indole-3-acetamide to IAA (Pollmann et al, 2003). As AtAMI1 transcription decreases during NS, DIS, and DET (Supplemental Fig.…”

Expression ofAMIDASE1(AMI1) is suppressed during the first two days after germination

“…17 This might be of particular importance in so far as such a HAP factor (HAP5c, At1g08970) is located directly upstream of the AMI1 gene. 6 Among other things, HAP factors are known to be involved in the regulation of flowering and to bind to CCAAT box motifs in the promoter region of their target genes. 18 Two such CCAAT boxes can be found in the AMI1 promoter, 266 and 462 bp upstream of the start codon, respectively.…”

“…But perhaps more importantly, AMI1 expression is suppressed at developmental stages or in tissues where LEC1 and HAP5c expression identified AMI1 from thale cress, which was the first IAM hydrolase known from plants. 6 Since that time, several studies were conducted to examine the properties of the enzyme, including intracellular localization studies, tissue specific expression analyses, and the analysis of the molecular mode of action of AMI1. 10,11 In order to enable in-depth studies on the regulation of the AMI1 gene expression, we generated an AMI1 promoter reporter gene construct (pAMI1::GUS) by fusing the complete intergenic region between the upstream located gene (At1g08970) and the predicted third exon of AMI1 to the uidA (GUS) gene and re-entered it into Arabidopsis.…”

“…4,5 One of this biosynthetic routes proceeds via indole-3-acetamide (IAM), which is hydrolyzed to IAA by specific IAM amidohydrolases, e.g., AMIDASE1 (AMI1) from Arabidopsis. 6 However, it is yet not entirely clear how IAM is produced in higher plants. In order for plant pathogens to re-program the cellular metabolism of their hosts for nutrient production, they synthesize IAA by an IAM-dependent two-step pathway.…”

Subcellular localization and tissue specific expression of amidase 1 from Arabidopsis thaliana

“…However, recently, the occurrence of IAM as an endogenous compound has been proven in leaves of sterile-grown Arabidopsis thaliana plants (Pollmann et al 2002). Proof for IAM as a substrate of enzymatic conversión in this tissue carne from the cloning of a specific IAM-hydrolase (AMIl) from the same species and the demonstration of AMIl expression in leaves using RT-PCR (Pollmann et al 2003). Thus, a pathway leading from L-Trp via IAM to IAA, hitherto known only from bacteria such as Agrobacterium tumefaciens (Weiler and Schróder 1987), must be considered in higher plants.…”