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Dynamic regulation of the concentration of the natural auxin (IAA) is essential to coordinate most of the physiological and developmental processes and responses to environmental changes. Oxidation of IAA is a major pathway to control auxin concentrations in angiosperms and, along with IAA conjugation, to respond to perturbation of IAA homeostasis. However, these regulatory mechanisms remain poorly investigated in conifers. To reduce this knowledge gap, we investigated the different contributions of the IAA inactivation pathways in conifers. MS-based quantification of IAA metabolites under steady-state conditions and after perturbation was investigated to evaluate IAA homeostasis in conifers. Putative Picea abies GH3 genes (PaGH3) were identified based on a comprehensive phylogenetic analysis including angiosperms and basal land plants. Auxin-inducible PaGH3 genes were identified by expression analysis and their IAA-conjugating activity was explored. Compared to Arabidopsis, oxidative and conjugative pathways differentially contribute to reduce IAA concentrations in conifers. We demonstrated that the oxidation pathway plays a marginal role in controlling IAA homeostasis in spruce. By contrast, an excess of IAA rapidly activates GH3-mediated irreversible conjugation pathways. Taken together, these data indicate that a diversification of IAA inactivation mechanisms evolved specifically in conifers.

Dynamic regulation of the concentration of the natural auxin (IAA) is essential to coordinate most of the physiological and developmental processes and responses to environmental changes. Oxidation of IAA is a major pathway to control auxin concentrations in angiosperms and, along with IAA conjugation, to respond to perturbation of IAA homeostasis. However, these regulatory mechanisms remain poorly investigated in conifers. To reduce this knowledge gap, we investigated the different contributions of the IAA inactivation pathways in conifers. MS-based quantification of IAA metabolites under steady-state conditions and after perturbation was investigated to evaluate IAA homeostasis in conifers. Putative Picea abies GH3 genes (PaGH3) were identified based on a comprehensive phylogenetic analysis including angiosperms and basal land plants. Auxin-inducible PaGH3 genes were identified by expression analysis and their IAA-conjugating activity was explored. Compared to Arabidopsis, oxidative and conjugative pathways differentially contribute to reduce IAA concentrations in conifers. We demonstrated that the oxidation pathway plays a marginal role in controlling IAA homeostasis in spruce. By contrast, an excess of IAA rapidly activates GH3-mediated irreversible conjugation pathways. Taken together, these data indicate that a diversification of IAA inactivation mechanisms evolved specifically in conifers.

Dynamic regulation of the concentration of the natural auxin (IAA) is essential to coordinate most of the physiological and developmental processes and responses to environmental changes. Oxidation of IAA is a major pathway to control auxin concentrations in angiosperms and, along with IAA conjugation, to respond to perturbation of IAA homeostasis. However, these regulatory mechanisms remain poorly investigated in conifers. To reduce this knowledge gap, we investigated the different contributions of the IAA inactivation pathways in conifers. MS-based quantification of IAA metabolites under steady-state conditions and after perturbation was investigated to evaluate IAA homeostasis in conifers. Putative Picea abies GH3 genes (PaGH3) were identified based on a comprehensive phylogenetic analysis including angiosperms and basal land plants. Auxin-inducible PaGH3 genes were identified by expression analysis and their IAA-conjugating activity was explored. Compared to Arabidopsis, oxidative and conjugative pathways differentially contribute to reduce IAA concentrations in conifers. We demonstrated that the oxidation pathway plays a marginal role in controlling IAA homeostasis in spruce. By contrast, an excess of IAA rapidly activates GH3-mediated irreversible conjugation pathways. Taken together, these data indicate that a diversification of IAA inactivation mechanisms evolved specifically in conifers.

Dynamic regulation of the concentration of the natural auxin (IAA) is essential to coordinate most of the physiological and developmental processes and responses to environmental changes. Oxidation of IAA is a major pathway to control auxin concentrations in angiosperms and, along with IAA conjugation, to respond to perturbation of IAA homeostasis. However, these regulatory mechanisms remain poorly investigated in conifers. To reduce this knowledge gap, we investigated the different contributions of the IAA inactivation pathways in conifers. MS-based quantification of IAA metabolites under steady-state conditions and after perturbation was investigated to evaluate IAA homeostasis in conifers. Putative Picea abies GH3 genes (PaGH3) were identified based on a comprehensive phylogenetic analysis including angiosperms and basal land plants. Auxin-inducible PaGH3 genes were identified by expression analysis and their IAA-conjugating activity was explored. Compared to Arabidopsis, oxidative and conjugative pathways differentially contribute to reduce IAA concentrations in conifers. We demonstrated that the oxidation pathway plays a marginal role in controlling IAA homeostasis in spruce. By contrast, an excess of IAA rapidly activates GH3-mediated irreversible conjugation pathways. Taken together, these data indicate that a diversification of IAA inactivation mechanisms evolved specifically in conifers.

Dynamic regulation of the concentration of the natural auxin (IAA) is essential to coordinate most of the physiological and developmental processes and responses to environmental changes. Oxidation of IAA is a major pathway to control auxin concentrations in angiosperms and, along with IAA conjugation, to respond to perturbation of IAA homeostasis. However, these regulatory mechanisms remain poorly investigated in conifers. To reduce this knowledge gap, we investigated the different contributions of the IAA inactivation pathways in conifers. MS-based quantification of IAA metabolites under steady-state conditions and after perturbation was investigated to evaluate IAA homeostasis in conifers. Putative Picea abies GH3 genes (PaGH3) were identified based on a comprehensive phylogenetic analysis including angiosperms and basal land plants. Auxin-inducible PaGH3 genes were identified by expression analysis and their IAA-conjugating activity was explored. Compared to Arabidopsis, oxidative and conjugative pathways differentially contribute to reduce IAA concentrations in conifers. We demonstrated that the oxidation pathway plays a marginal role in controlling IAA homeostasis in spruce. By contrast, an excess of IAA rapidly activates GH3-mediated irreversible conjugation pathways. Taken together, these data indicate that a diversification of IAA inactivation mechanisms evolved specifically in conifers.

Dynamic regulation of the concentration of the natural auxin (IAA) is essential to coordinate most of the physiological and developmental processes and responses to environmental changes. Oxidation of IAA is a major pathway to control auxin concentrations in angiosperms and, along with IAA conjugation, to respond to perturbation of IAA homeostasis. However, these regulatory mechanisms remain poorly investigated in conifers. To reduce this knowledge gap, we investigated the different contributions of the IAA inactivation pathways in conifers. MS-based quantification of IAA metabolites under steady-state conditions and after perturbation was investigated to evaluate IAA homeostasis in conifers. Putative Picea abies GH3 genes (PaGH3) were identified based on a comprehensive phylogenetic analysis including angiosperms and basal land plants. Auxin-inducible PaGH3 genes were identified by expression analysis and their IAA-conjugating activity was explored. Compared to Arabidopsis, oxidative and conjugative pathways differentially contribute to reduce IAA concentrations in conifers. We demonstrated that the oxidation pathway plays a marginal role in controlling IAA homeostasis in spruce. By contrast, an excess of IAA rapidly activates GH3-mediated irreversible conjugation pathways. Taken together, these data indicate that a diversification of IAA inactivation mechanisms evolved specifically in conifers.

Background: Plants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-L-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays. Results: In this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC–ESI-MS/MS). Experimental procedures were optimized for testing the activity of differen

Background: Plants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-L-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays. Results: In this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC–ESI-MS/MS). Experimental procedures were optimized for testing the activity of differen

Background: Plants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-L-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays. Results: In this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC–ESI-MS/MS). Experimental procedures were optimized for testing the activity of differen

Background: Plants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-L-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays. Results: In this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC–ESI-MS/MS). Experimental procedures were optimized for testing the activity of differen

Background: Plants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-L-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays. Results: In this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC–ESI-MS/MS). Experimental procedures were optimized for testing the activity of differen