Two homologous INDOLE-3-ACETAMIDE (IAM) HYDROLASE genes are required for the auxin effects of IAM in Arabidopsis

a.
Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, 92093-0116, USA
b.
RIKEN Center for Sustainable Resource Science, Kanagawa, 230-0045, Japan
c.
School of Life Sciences, Jilin Normal University, Siping, 136000, China
d.
Key Laboratory of Plant Ecology, Northeast Forestry University, Harbin, 150040, China
e.
State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, Chongqing, 400716, China
f.
Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, Tokyo, 183-8509, Japan
g.
Department of Bioregulation and Biointeraction, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, 183-8509, Japan

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Corresponding author: E-mail address: yundezhao@ucsd.edu (Yunde Zhao)

摘要

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Abstract: Indole-3-acetamide (IAM) is the first confirmed auxin biosynthetic intermediate in some plant pathogenic bacteria. Exogenously applied IAM or production of IAM by overexpressing the bacterial iaaM gene in Arabidopsis causes auxin overproduction phenotypes. However, it is still inconclusive whether plants use IAM as a key precursor for auxin biosynthesis. Herein, we reported the isolation IAM HYDROLASE 1 (IAMH1) gene in Arabidopsis from a forward genetic screen for IAM-insensitive mutants that display normal auxin sensitivities. IAMH1 has a close homolog named IAMH2 that is located right next to IAMH1 on chromosome IV in Arabidopsis. We generated iamh1 iamh2 double mutants using our CRISPR/Cas9 gene editing technology. We showed that disruption of theIAMH genes rendered Arabidopsis plants resistant to IAM treatments and also suppressed the iaaM overexpression phenotypes, suggesting that IAMH1 and IAMH2 are the main enzymes responsible for converting IAM into indole-3-acetic acid (IAA) in Arabidopsis. The iamh double mutants did not display obvious developmental defects, indicating that IAM does not play a major role in auxin biosynthesis under normal growth conditions. Our findings provide a solid foundation for clarifying the roles of IAM in auxin biosynthesis and plant development.
- Auxin /
- Auxin biosynthesis /
- Indole-3-acetamide /
- CRISPR /
- IAMH1 /
- IAMH2

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Fig. 1. Indole-3-acetamide (IAM) is a potential auxin biosynthetic intermediate in plants and IAM treatments affect plant growth and activate the auxin reporter DR5:GUS. A: A proposed Trp-dependent auxin biosynthetic pathway using IAM as the intermediate. This pathway is used by plant pathogenic bacteria such as Agrobacteria, which use the iaaM and iaaH to convert Trp to IAA. The roles of IAM in plant auxin biosynthesis are not clear. B: Five-day-old Arabidopsis seedlings grown on MS media and media containing 20 μM IAA or IAM. Note that IAA inhibits primary root elongation and IAM stimulates hypocotyl growth. C: Activation of DR5:GUS expression by IAA and IAM. Interestingly, IAM mainly activates DR5:GUS expression in aerial tissue, whereas IAA increases DR5:GUS signal in the root.

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Fig. 2. Isolation and cloning of an IAM-resistant mutant (iamh1-1). A: Isolation of an IAM-resistant mutant which did not have elongated hypocotyl and did not display epinastic cotyledons when grown on 20 μM IAM-containing media. B: The expression of DR5:GUS auxin reporter was not induced by IAM treatments in theiamh1-1 mutant. C: The iamh1-1 mutation was identified by map-based positional cloning. The IAMH1 gene is At4g37550. The iamh1-1 mutant harbors a G-to-A mutation in At4g37550 that results in a premature stop codon. D: The iamh1-1 phenotypes were rescued by wild-type (WT) IAMH1 genomic DNA or IAMH1 genomic DNA fused with GFP driven by the IAMH1 promoter.

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Fig. 3. Expression pattern of IAMH1 and subcellular localization of IAMH1. A: The GUS expression patterns of IAMH1:GUS transgenic lines. Note that the reporter has a broad expression pattern. B: Expression of the IAMH1-GFP fusion protein in Arabidopsis roots driven by the IAMH1 promoter. IAMH1 appears to be located in the cytosol.

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Fig. 4. The IAMH2 gene is also involved in converting IAM into IAA. A: IAMH1 has a close homolog, IAMH2. The two genes are tandem repeats located on Chromosome IV. B: A T-DNA insertion in IAMH2 also caused resistance to exogenous IAM. C: Generation of iamh2 alleles in the iamh1-1 background by CRISPR/Cas9 gene editing technology. TGG in red is the PAM site for CRISPR/Cas9. The iamh2-2 allele harbors one T insertion, and the iamh2-3 allele contains a 20-bp deletion.

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Fig. 5. The IAMH genes are major contributors to hydrolysis of IAM in Arabidopsis. A: Disruption of the IAMH genes suppressed the iaaM overexpression phenotypes. Note that overexpression of the bacterial iaaM gene led to long hypocotyls and epinastic cotyledons (left, two independent T₁ plants), which are characteristic auxin overproduction phenotypes. B: Young adult independent T₁ plants of iaaM overexpression lines in the WT (left) andiamh1-1 iamh2-3 (right) backgrounds, respectively. C: WT and double mutant plants contained similar levels of IAA (t-value = −0.31235; P-value = 0.385191). However, IAM treatment caused a dramatic increase of IAA in WT plants, but much less increase in the iamh1-1 iamh2-3 background (t-value = 6.55; P-value = 0.001404). D: IAM treatment caused about 80-fold increase of IAA-Asp conjugate in WT, but only a slight increase in the iamh1-1 iamh2-3 double mutants (t-value = 19.95751; P-value = 0.000019). E: IAM treatment led to an increase in IAA-Glu synthesis in WT plants, but the increase was very small in the iamh1-1 iamh2-3 double mutants (t-value = 13.28318; P-value = 0.000093). ∗ indicates statistically significant.

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