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 cau...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 IAMH1on chromosomeⅣin Arabidopsis.We generated iamh1 iamh2 double mutants using our CRISPR/Cas9gene editing technology.We showed that disruption of the IAMH 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.展开更多
Indole-3-acetic acid (IAA) a phytohormon of auxin type is synthesized by different ways in plants and some bacteria (Agrobacteria, Pseudomonas and some others). The enzyme indolylacetamide hydrolase transforms indolyl...Indole-3-acetic acid (IAA) a phytohormon of auxin type is synthesized by different ways in plants and some bacteria (Agrobacteria, Pseudomonas and some others). The enzyme indolylacetamide hydrolase transforms indolylacetamide to IAA mainly in bacteria. However, recently published data showed that some plants can also hydrolyze indolylacetamide into IAA. In order to elucidate the role of indolylacetamide as an auxin precursor in plants and bacteria, productive method of determination of the activity of indoleacetamide hydrolase is necessary. The simple, inexpensive and productive method for the measurement of indoleacetamide hydrolase activity was elaborated based on significant difference between IAAM and IAA in color developed with Salkovski’ reagent. The light absorbance increased during conversion of IAAM to IAA by protein extracts from some plant cells and this increase may be used for quantitative estimation of indoleacetamide hydrolase activity. The method is suitable for fast discovery of indoleacetamide hydrolase activity before planning more complicated analyses and for the analysis of many probes at a short time in physiological and biochemical experiments. A detailed protocol for determination of indoleacetamide hydrolase activity by the elaborated method is described.展开更多
文摘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 IAMH1on chromosomeⅣin Arabidopsis.We generated iamh1 iamh2 double mutants using our CRISPR/Cas9gene editing technology.We showed that disruption of the IAMH 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.
文摘Indole-3-acetic acid (IAA) a phytohormon of auxin type is synthesized by different ways in plants and some bacteria (Agrobacteria, Pseudomonas and some others). The enzyme indolylacetamide hydrolase transforms indolylacetamide to IAA mainly in bacteria. However, recently published data showed that some plants can also hydrolyze indolylacetamide into IAA. In order to elucidate the role of indolylacetamide as an auxin precursor in plants and bacteria, productive method of determination of the activity of indoleacetamide hydrolase is necessary. The simple, inexpensive and productive method for the measurement of indoleacetamide hydrolase activity was elaborated based on significant difference between IAAM and IAA in color developed with Salkovski’ reagent. The light absorbance increased during conversion of IAAM to IAA by protein extracts from some plant cells and this increase may be used for quantitative estimation of indoleacetamide hydrolase activity. The method is suitable for fast discovery of indoleacetamide hydrolase activity before planning more complicated analyses and for the analysis of many probes at a short time in physiological and biochemical experiments. A detailed protocol for determination of indoleacetamide hydrolase activity by the elaborated method is described.
