IAMH1 (INDOLE-3-ACETAMIDE HYDROLASE 1, AT4G37550) is a soluble amidohydrolase of the acetamidase/formamidase (FmdA_AmdA) family, encoded in tandem with its 90%-identical paralog IAMH2 (AT4G37560) on chromosome 4. IAMH1-like genes are present across the green lineage, from Chlamydomonas and Physcomitrella to flowering plants. IAMH1 was identified in a forward-genetic screen for mutants insensitive to indole-3-acetamide (IAM) but with normal auxin responses. Loss-of-function alleles resist exogenous IAM, and complementation, including with a functional IAMH1-GFP fusion, restores IAM sensitivity. Together with IAMH2, IAMH1 is required to convert exogenous or bacterial iaaM-derived IAM into the auxin indole-3-acetic acid (IAA), and iamh1 iamh2 double mutants suppress iaaM-driven auxin overproduction. The double mutants develop normally and keep wild-type IAA levels, so the IAM route seems to contribute little to bulk auxin under standard conditions. IAMH1 is broadly expressed in seedlings and flowers; unlike IAMH2 its expression is not light-responsive, although it is upregulated in iamh2 mutants under low light. An IAMH1-GFP fusion is excluded from the nucleus, and a leaf proteome detects the protein in the cytosolic fraction, with additional high-throughput detections in vacuolar and membrane fractions that remain unresolved.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000325 plant-type vacuole | HDA PMID:15539469 The vegetative vacuole proteome of Arabidopsis thaliana reve... | UNDECIDED | Summary: IAMH1 was detected in a multi-method proteomic inventory of isolated vegetative vacuoles from Arabidopsis leaves. Reason: This is a high-throughput organelle proteomics detection, and the per-protein identification data were not examined, so no judgement is made. It differs from the cytosol HDA row, which is accepted on the same tier of evidence, because the cytosol detection has orthogonal corroboration (non-nuclear IAMH1-GFP, a soluble enzyme with cytosolic substrate and product) and a vacuolar location has none. For context, the paper acknowledges contamination of the vacuole preparations, excluding about 15% of hits as potential chloroplast or mitochondrial contaminants. The quotes below describe the datasets, not the per-protein identification of IAMH1. Supporting Evidence: PMID:15539469 The number of proteins identified by each of the methods is shown in Figure 2 (these groups of proteins do not include 72 [15% of total] potential chloroplast or mitochondrial contaminants, see below). PMID:25293756 Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. |
| GO:0005829 cytosol | HDA PMID:25293756 A proteomic strategy for global analysis of plant protein co... | ACCEPT | Summary: IAMH1 was detected in a size-exclusion chromatography proteomic profiling of the crude cytosolic fraction of Arabidopsis leaves. Reason: The per-protein proteomic identification was not examined (the quote below describes the dataset, not IAMH1 specifically), so the ACCEPT rests on orthogonal corroboration. A cytosolic location fits the soluble amidohydrolase and its functional IAMH1-GFP fusion, which is clearly excluded from the nucleus, and the substrate IAM and product IAA are cytosolic metabolites. Supporting Evidence: PMID:25293756 Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. PMID:32327358 Expression of IAMH1-GFP fusion driven by IAMH1 promoter showed that IAMH1 was clearly not expressed in the nucleus |
| GO:0005886 plasma membrane | HDA PMID:28887381 Global Analysis of Membrane-associated Protein Oligomerizati... | UNDECIDED | Summary: IAMH1 was detected in a protein-correlation-profiling study of membrane-associated protein complexes. Reason: This is a high-throughput membrane-fraction proteomics detection, and the per-protein data were not examined, so no judgement is made. It differs from the cytosol HDA row, which is accepted on the same tier of evidence, because the cytosol detection has orthogonal corroboration (non-nuclear IAMH1-GFP, a soluble enzyme with cytosolic substrate and product) and a plasma-membrane location has none. For context, UniProt records no predicted transmembrane segment for IAMH1, and that study showed that many soluble proteins partition between cytosolic and membrane-associated pools. Supporting Evidence: PMID:28887381 Over 150 proteins had a complicated localization pattern, and were clearly partitioned between cytosolic and membrane-associated pools. |
| GO:0009507 chloroplast | ISM GO_REF:0000122 | MARK AS OVER ANNOTATED | Summary: The AtSubP sequence-based predictor assigns IAMH1 to the chloroplast. Reason: This is a single computational prediction with no experimental support. None of the proteomic datasets that detect IAMH1 (cytosol, vacuole, membrane fractions) places it in plastids. The IAMH1-GFP study reports only nuclear exclusion. Not removed outright because plastid targeting has not been tested experimentally. Supporting Evidence: PMID:25293756 Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. PMID:32327358 Expression of IAMH1-GFP fusion driven by IAMH1 promoter showed that IAMH1 was clearly not expressed in the nucleus |
| GO:0016811 hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the acetamidase/formamidase (FmdA_AmdA, IPR004304) family signature. Reason: The term is correct but general. Genetic evidence identifies indole-3-acetamide as the physiological linear-amide substrate. iamh1 alleles were isolated as IAM-insensitive, and complementation restores IAM sensitivity. The iamh1 iamh2 double mutant fails to convert exogenous or iaaM-derived IAM into IAA. GO:0043864 indoleacetamide hydrolase activity is a descendant of this term (is_a amidase activity). No in vitro assay of purified IAMH1 has been reported, so the replacement rests on genetic and metabolite evidence plus family membership. Proposed replacements: indoleacetamide hydrolase activity Supporting Evidence: PMID:32327358 Among the ORFs in the mapping interval, At4g37550 and At4g37560 encode putative Acetamidase/Formamidases, which potentially have the hydrolase activities that can break an amide bond. PMID:32327358 the IAMH1 genomic fragment fully restored the IAM sensitivity of the iamh1–1 mutant. PMID:32327358 Such an observation further supports the hypothesis that IAMH1 is involved in converting IAM to IAA in Arabidopsis. |
| GO:0009684 indoleacetic acid biosynthetic process | IMP PMID:32327358 Two homologous INDOLE-3-ACETAMIDE (IAM) HYDROLASE genes are ... | NEW | Summary: IAMH1, redundantly with IAMH2, is required to convert IAM into IAA in planta. Disrupting both genes abolishes iaaM-driven auxin overproduction and most of the IAM-induced rise in IAA and IAA conjugates. Reason: IAMH1 is proposed to catalyse the hydrolytic step of the IAM branch of IAA biosynthesis (IAM to IAA), so it performs a step of the process. The IMP code rests on the iamh1 single mutants: both iamh1-1 and the T-DNA allele iamh1-2 are IAM-resistant, and the IAMH1 genomic fragment restores IAM sensitivity. The iamh1 iamh2 double-mutant data (suppression of iaaM-driven auxin overproduction and of the IAM-induced rise in IAA conjugates) would be IGI with IAMH2. Comparator check (QuickGO, taxon 3702): AMI1, which catalyses the same IAM to IAA step, carries GO:0009684 by IDA. Caveats: the in vivo evidence concerns IAM supplied exogenously or made by the bacterial iaaM transgene. Steady-state IAA and development are normal in the double mutant, so this route contributes little to endogenous auxin under standard conditions. Moya-Cuevas et al. (PMID:41362017) further propose that IAM acts as a signalling molecule in its own right, inducing ABA biosynthesis, and describe the IAM pathway as contributing to IAA only under specific conditions; X. Gao et al. (PMID:42844995) cite that work as questioning whether the pathway contributes to auxin biosynthesis under standard conditions. Neither paper disputes that IAMH1 converts IAM to IAA, so the annotation is kept, but its physiological weight is limited. Supporting Evidence: PMID:32327358 Neither iamh1–1 nor iamh1–2 showed obvious developmental defects under normal growth conditions, despite that both mutant alleles were resistant to IAM. PMID:32327358 the IAMH1 genomic fragment fully restored the IAM sensitivity of the iamh1–1 mutant. PMID:32327358 Disruption of the IAMH genes also suppressed the developmental abnormalities caused by overexpression of the iaaM gene, suggesting that the IAMHs are the main enzymes in converting IAM into IAA in Arabidopsis. PMID:32327358 In contrast, IAM treatment in iamh1 iamh2 double mutants did not induce a substantial increase in IAA-Asp and IAA-Glu conjugates |
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Download this section (compressed HTML)Q: Does purified IAMH1 hydrolyse IAM in vitro, with what kinetics, and does it retain the formamidase or acetamidase activity of its bacterial and fungal family members?
Q: The iamh1 single mutant was isolated as IAM-insensitive even though IAMH2 is 90% identical, and conversely only IAMH2 is light-responsive and required for low-light hypocotyl elongation. What determines the non-redundant contribution of each paralog (expression level, tissue pattern or catalytic efficiency)?
Q: Does the residual IAM-to-IAA conversion in iamh1 iamh2 double mutants depend on AMI1 or other amidase-signature enzymes?
Experiment: Purify recombinant IAMH1 and IAMH2 and compare kinetic parameters with IAM, formamide, acetamide and phenylacetamide.
Experiment: Generate iamh1 iamh2 ami1 triple mutants and test residual IAM sensitivity and IAM-derived IAA formation.
Experiment: Determine the subcellular localization of IAMH1-GFP at higher resolution, with organelle markers, to test the chloroplast prediction and the vacuolar and membrane proteomic detections.
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