Distinct Characteristics of Indole-3-Acetic Acid and Phenylacetic Acid, Two Common Auxins in Plants methods
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PAA shows biological activity via the TIR1/AFB-mediated pathway
To address the action mechanism of PAA, we performed several auxin activity tests using Arabidopsis. The application of PAA enhanced the formation of lateral roots, although the activity was 10- to 20-fold lower than that of IAA ( A, B). PAA induced the DR5-driven expression of β-glucuronidase (GUS) in the roots of DR5::GUS plants, although the activity was nearly 20-fold lower than that of IAA ( C). This PAA-induced GUS expression was abolished by the co-application of auxinole, a specific inhibitor of TIR1/AFBs ( C) ( ). In addition, we tested the response to PAA in tir1-1 and tir1-1 afb2-3 seedlings using the root elongation assay, and demonstrated that the roots of tir1-1 afb2-3 were resistant to PAA, similarly to IAA ( Supplementary Fig. S1 ). These results indicate the involvement of TIR1/AFBs in the PAA signal transduction pathway. In addition, treatment of the moss P. pat...
PAA shows biological activity via the TIR1/AFB-mediated pathway
To confirm whether PAA functions through the TIR1/AFB-mediated pathway, we analyzed the interaction of PAA and auxin co-receptors using a yeast two-hybrid system ( ). Among six TIR1/AFBs in Arabidopsis, we used TIR1, AFB2 and AFB5 for the interaction analysis in this study ( ). As for Aux/IAAs, eight proteins representing distinct subclades were chosen from 29 Aux/IAAs in Arabidopsis ( ). As shown in A, the interaction of TIR1/AFB2/AFB5 with various Aux/IAAs was promoted in the yeast two-hybrid assay with 100 µM IAA as previously reported ( ). PAA promoted the interaction of relatively limited numbers of TIR1/AFB2/AFB5 and Aux/IAAs in the same concentration used for IAA (100 µM). When the interaction test was carried out with 1 mM PAA, TIR1/AFB2/AFB5-Aux/IAA interaction profiles were similar to those observed in 100 µM IAA ( A). Although the efficiency of auxin u...
IAA and PAA show distinct distribution patterns in maize coleoptiles on auxin transport inhibitor treatment and gravi...
We next studied PAA movement by analyzing the distribution patterns of endogenous auxins in maize coleoptiles. As previously reported, we prepared four segments from etiolated maize coleoptiles by horizontal sectioning and measured auxin levels in each segment ( A-C) ( ). IAA formed concentration gradients from the tip downward in coleoptiles (mock), with the highest level in the tip region, where IAA is most actively produced ( B) ( ). In agreement with this, VT2 and SPI1 genes, which encode co-orthologs of TAA1 and YUC genes, were expressed at a higher level in the tip region ( D) (, ). As shown in C, we found that PAA formed a similar gradient pattern with IAA in coleoptiles (mock), suggesting that both IAA and PAA may be actively produced in the tip region in maize coleoptiles. To investigate the mechanism of PAA movement, we applied the polar auxin transport inhibitor 1- N...
PAA cannot recover the root gravitropism of yucQ mutants
To demonstrate further the distinct characteristics of IAA and PAA, we next investigated the recovery of root phenotypes of yuc3 yuc5 yuc7 yuc8 yuc9 quintuple ( yucQ ) mutants with IAA and PAA (, ). Application of IAA and PAA restored the severe root growth defects of yucQ to wild-type (WT) levels ( A, B). No significant increase in IAA levels was observed in roots of yucQ by treatment with PAA ( C). Similarly, PAA levels were not significantly increased in roots of yucQ by treatment with IAA ( D). When these seedlings were grown horizontally for 24 h, the IAA-restored primary roots of yucQ showed a positive response to gravitropic stimulation ( E). In contrast, no significant response was observed in the PAA-restored primary roots of yucQ ( E). These results suggest that IAA generates concentration gradients, but PAA does not, in response to gravitropic stimulation; thus, these natu...
The GH3 family probably plays a role in PAA metabolism
The constitutive occurrence of PAA-Asp and PAA-Glu in vector control plants ( pER8, D) and a pronounced increase in these PAA-amino acid conjugate levels, as well as IAA-Asp and IAA-Glu levels in β-estradiol-induced YUCox plants ( C, D), suggest that the PAA metabolic pathway involves GH3 genes, which encode enzymes catalyzing the synthesis of IAA-amino acid conjugates ( A) ( ). To investigate this, we generated transgenic plants expressing β-estradiol-inducible GH3.9 ( GH3.9ox ). As previously demonstrated in β-estradiol-inducible GH3.6 ( ), the induction of GH3.9 resulted in root growth defects and loss of gravitropism ( A). The induction of GH3.9 increased the endogenous levels of IAA-Glu by 11-fold ( B). Similarly, PAA-Glu showed a 13-fold increase in GH3.9ox plants ( C). These results indicate the involvement of the GH3 family in PAA metabolism in Arab...
A possible role for the TAA and YUC families in PAA biosynthesis
Because IAA and PAA exist widely in vascular and non-vascular plants, we hypothesized that IAA biosynthetic enzymes are also implicated in PAA synthesis. The YUC family catalyzes a rate-limiting step of the IPA pathway in IAA biosynthesis ( A) ( ). Overexpression of YUC genes promotes IAA production and results in high-auxin phenotypes, such as hypocotyl elongation, primary root growth inhibition and enhanced lateral root growth (, ). To test our hypothesis in vivo, we investigated the metabolic impact of YUC overexpression on PAA biosynthesis using Arabidopsis with β-estradiol-inducible YUC1 ( YUC1ox ), YUC2 ( YUC2ox ) or YUC6 ( YUC6ox ) ( B). Treatment with β-estradiol increased YUC1, YUC2 and YUC6 expression levels by 41-, 142- and 29-fold in YUC1ox, YUC2ox and YUC6ox plants, respectively, compared with those in the vector control plants ( pER8 ) ( Supplementary Fig. S...
A possible role for the TAA and YUC families in PAA biosynthesis
In addition, we examined the enzymatic conversion of PPA to PAA by glutathione S -transferase (GST)-fused YUC2 proteins expressed in E. coli. As shown in E, GST-fused YUC2 proteins converted IPA to IAA, and the reaction was abolished in the absence of NADPH. Similarly, we detected the conversion of PPA to PAA with GST-YUC2 proteins by the decrease of a specific ion for PPA at 3.6 min and the increase of a specific ion for PAA at 4.8 min using LC-ESI-MS/MS ( F). The conversion of PPA to PAA with GST-YUC2 was abolished in the absence of NADPH, similar to the conversion of IPA to IAA ( E, F). From these results, we confirmed that YUC proteins catalyze the conversion of PPA to PAA.
Materials and Methods
The Arabidopsis thaliana ecotype Col-0 was used as the WT plant. Arabidopsis seeds were stratified at 4°C for 2 d in the dark. Seedlings were grown vertically on Murashige and Skoog (MS) medium with 1% sucrose under continuous light conditions at 20-22°C. Oats and barley were grown in soil at 20°C during the day and 15°C at night, with a 16 h photoperiod. For the analysis of auxins in vascular and non-vascular plants, 2-week-old seedlings of Arabidopsis, 7-day-old shoots of oats, 7-day-old shoots of barley, 3-week-old plants of P. patens and 2-week-old plants of M. polymorpha were used. For the analysis of auxins in Arabidopsis tissues, 3-month-old dry seeds, siliques (7 d after flowering), inflorescences, cauline leaves, stems (2 cm from the bottom end) and rosette leaves of 5-week-old plants grown in soil, and roots of 7-day-old seedlings grown on MS agar m...
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To address the action mechanism of PAA, we performed several auxin activity tests using Arabidopsis. The application of PAA enhanced the formation of lateral roots, although the activity was 10- to 20-fold lower than that of IAA ( A, B). PAA induced the DR5-driven expression of β-glucuronidase (GUS) in the roots of DR5::GUS plants, although the activity was nearly 20-fold lower than that of IAA ( C). This PAA-induced GUS expression was abolished by the co-application of auxinole, a specific inhibitor of TIR1/AFBs ( C) ( ). In addition, we tested the response to PAA in tir1-1 and tir1-1 afb2-3 seedlings using the root elongation assay, and demonstrated that the roots of tir1-1 afb2-3 were resistant to PAA, similarly to IAA ( Supplementary Fig. S1 ). These results indicate the involvement of TIR1/AFBs in the PAA signal transduction pathway. In addition, treatment of the moss P. patens with PAA, similarly to 2,4-D, led to typical auxin responses such as the elongation of gametophores ( D) ( ), suggesting that the biological functions of PAA as an auxin are probably evolutionarily conserved in land plants. Fig. 2 Auxin activity of PAA on Arabidopsis and P. patens. (A and B) Enh...
To confirm whether PAA functions through the TIR1/AFB-mediated pathway, we analyzed the interaction of PAA and auxin co-receptors using a yeast two-hybrid system ( ). Among six TIR1/AFBs in Arabidopsis, we used TIR1, AFB2 and AFB5 for the interaction analysis in this study ( ). As for Aux/IAAs, eight proteins representing distinct subclades were chosen from 29 Aux/IAAs in Arabidopsis ( ). As shown in A, the interaction of TIR1/AFB2/AFB5 with various Aux/IAAs was promoted in the yeast two-hybrid assay with 100 µM IAA as previously reported ( ). PAA promoted the interaction of relatively limited numbers of TIR1/AFB2/AFB5 and Aux/IAAs in the same concentration used for IAA (100 µM). When the interaction test was carried out with 1 mM PAA, TIR1/AFB2/AFB5-Aux/IAA interaction profiles were similar to those observed in 100 µM IAA ( A). Although the efficiency of auxin uptake into yeast cells may differ between IAA and PAA, these results indicate that PAA can bind to TIR1/AFBs and Aux/IAAs, and that these auxins may have overlapping roles in plants.
We next studied PAA movement by analyzing the distribution patterns of endogenous auxins in maize coleoptiles. As previously reported, we prepared four segments from etiolated maize coleoptiles by horizontal sectioning and measured auxin levels in each segment ( A-C) ( ). IAA formed concentration gradients from the tip downward in coleoptiles (mock), with the highest level in the tip region, where IAA is most actively produced ( B) ( ). In agreement with this, VT2 and SPI1 genes, which encode co-orthologs of TAA1 and YUC genes, were expressed at a higher level in the tip region ( D) (, ). As shown in C, we found that PAA formed a similar gradient pattern with IAA in coleoptiles (mock), suggesting that both IAA and PAA may be actively produced in the tip region in maize coleoptiles. To investigate the mechanism of PAA movement, we applied the polar auxin transport inhibitor 1- N -naphthylphthalamic acid (NPA) to the tip region of maize coleoptiles ( ) and measured the level of auxins ( B, C). NPA blocked the basipetal transport of IAA and elevated IAA levels by 2.4-fold in the tip region (0-3 mm), and decreased those in the lower regions (from 3-7 mm to 11̵...
To demonstrate further the distinct characteristics of IAA and PAA, we next investigated the recovery of root phenotypes of yuc3 yuc5 yuc7 yuc8 yuc9 quintuple ( yucQ ) mutants with IAA and PAA (, ). Application of IAA and PAA restored the severe root growth defects of yucQ to wild-type (WT) levels ( A, B). No significant increase in IAA levels was observed in roots of yucQ by treatment with PAA ( C). Similarly, PAA levels were not significantly increased in roots of yucQ by treatment with IAA ( D). When these seedlings were grown horizontally for 24 h, the IAA-restored primary roots of yucQ showed a positive response to gravitropic stimulation ( E). In contrast, no significant response was observed in the PAA-restored primary roots of yucQ ( E). These results suggest that IAA generates concentration gradients, but PAA does not, in response to gravitropic stimulation; thus, these natural auxins have distinct transport characteristics. Fig. 6 Recovery of the root growth in yucQ by IAA and PAA. (A and B) Recovery of primary root length in yucQ by auxins. Scale bar = 1 cm. Data are the mean ± SD ( n = 60). The asterisk indicates a significant difference from the WT (Student...
The constitutive occurrence of PAA-Asp and PAA-Glu in vector control plants ( pER8, D) and a pronounced increase in these PAA-amino acid conjugate levels, as well as IAA-Asp and IAA-Glu levels in β-estradiol-induced YUCox plants ( C, D), suggest that the PAA metabolic pathway involves GH3 genes, which encode enzymes catalyzing the synthesis of IAA-amino acid conjugates ( A) ( ). To investigate this, we generated transgenic plants expressing β-estradiol-inducible GH3.9 ( GH3.9ox ). As previously demonstrated in β-estradiol-inducible GH3.6 ( ), the induction of GH3.9 resulted in root growth defects and loss of gravitropism ( A). The induction of GH3.9 increased the endogenous levels of IAA-Glu by 11-fold ( B). Similarly, PAA-Glu showed a 13-fold increase in GH3.9ox plants ( C). These results indicate the involvement of the GH3 family in PAA metabolism in Arabidopsis. Fig. 8 Metabolism of PAA in GH3.9ox plants. (A) Phenotype of GH3.9ox plants with or without β-estradiol treatment. Scale bars = 5 mm. Seedlings were treated with 10 µM β-estradiol for 5 d. (B and C) The levels of IAA metabolites (B) and PAA metabolites (C) increase in GH3.9ox...
Because IAA and PAA exist widely in vascular and non-vascular plants, we hypothesized that IAA biosynthetic enzymes are also implicated in PAA synthesis. The YUC family catalyzes a rate-limiting step of the IPA pathway in IAA biosynthesis ( A) ( ). Overexpression of YUC genes promotes IAA production and results in high-auxin phenotypes, such as hypocotyl elongation, primary root growth inhibition and enhanced lateral root growth (, ). To test our hypothesis in vivo, we investigated the metabolic impact of YUC overexpression on PAA biosynthesis using Arabidopsis with β-estradiol-inducible YUC1 ( YUC1ox ), YUC2 ( YUC2ox ) or YUC6 ( YUC6ox ) ( B). Treatment with β-estradiol increased YUC1, YUC2 and YUC6 expression levels by 41-, 142- and 29-fold in YUC1ox, YUC2ox and YUC6ox plants, respectively, compared with those in the vector control plants ( pER8 ) ( Supplementary Fig. S2 ). As shown in C, the induction of YUC genes greatly increased the levels of the IAA metabolites IAA-aspartate (IAA-Asp) and IAA-glutamate (IAA-Glu) compared with those in pER8 plants, probably due to a rapid inactivation of increased IAA by the GH3 family ( ). We assumed that the levels of the po...
In addition, we examined the enzymatic conversion of PPA to PAA by glutathione S -transferase (GST)-fused YUC2 proteins expressed in E. coli. As shown in E, GST-fused YUC2 proteins converted IPA to IAA, and the reaction was abolished in the absence of NADPH. Similarly, we detected the conversion of PPA to PAA with GST-YUC2 proteins by the decrease of a specific ion for PPA at 3.6 min and the increase of a specific ion for PAA at 4.8 min using LC-ESI-MS/MS ( F). The conversion of PPA to PAA with GST-YUC2 was abolished in the absence of NADPH, similar to the conversion of IPA to IAA ( E, F). From these results, we confirmed that YUC proteins catalyze the conversion of PPA to PAA.
The Arabidopsis thaliana ecotype Col-0 was used as the WT plant. Arabidopsis seeds were stratified at 4°C for 2 d in the dark. Seedlings were grown vertically on Murashige and Skoog (MS) medium with 1% sucrose under continuous light conditions at 20-22°C. Oats and barley were grown in soil at 20°C during the day and 15°C at night, with a 16 h photoperiod. For the analysis of auxins in vascular and non-vascular plants, 2-week-old seedlings of Arabidopsis, 7-day-old shoots of oats, 7-day-old shoots of barley, 3-week-old plants of P. patens and 2-week-old plants of M. polymorpha were used. For the analysis of auxins in Arabidopsis tissues, 3-month-old dry seeds, siliques (7 d after flowering), inflorescences, cauline leaves, stems (2 cm from the bottom end) and rosette leaves of 5-week-old plants grown in soil, and roots of 7-day-old seedlings grown on MS agar media were used. For lateral root formation analysis, WT seedlings were germinated and grown vertically on MS agar medium for 5 d and then on auxin-containing medium for 4 d ( n = 10). The moss P. patens was maintained as protonemata on a plate containing BCDATG medium ( ). To observe their auxin res...
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"text": "To address the action mechanism of PAA, we performed several auxin activity tests using Arabidopsis. The application of PAA enhanced the formation of lateral roots, although the activity was 10- to 20-fold lower than that of IAA ( A, B). PAA induced the DR5-driven expression of β-glucuronidase (GUS) in the roots of DR5::GUS plants, although the activity was nearly 20-fold lower than that of IAA ( C). This PAA-induced GUS expression was abolished by the co-application of auxinole, a specific inhibitor of TIR1/AFBs ( C) ( ). In addition, we tested the response to PAA in tir1-1 and tir1-1 afb2-3 seedlings using the root elongation assay, and demonstrated that the roots of tir1-1 afb2-3 were resistant to PAA, similarly to IAA ( Supplementary Fig. S1 ). These results indicate the involvement of TIR1/AFBs in the PAA signal transduction pathway. In addition, treatment of the moss P. pat..."
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"text": "To confirm whether PAA functions through the TIR1/AFB-mediated pathway, we analyzed the interaction of PAA and auxin co-receptors using a yeast two-hybrid system ( ). Among six TIR1/AFBs in Arabidopsis, we used TIR1, AFB2 and AFB5 for the interaction analysis in this study ( ). As for Aux/IAAs, eight proteins representing distinct subclades were chosen from 29 Aux/IAAs in Arabidopsis ( ). As shown in A, the interaction of TIR1/AFB2/AFB5 with various Aux/IAAs was promoted in the yeast two-hybrid assay with 100 µM IAA as previously reported ( ). PAA promoted the interaction of relatively limited numbers of TIR1/AFB2/AFB5 and Aux/IAAs in the same concentration used for IAA (100 µM). When the interaction test was carried out with 1 mM PAA, TIR1/AFB2/AFB5-Aux/IAA interaction profiles were similar to those observed in 100 µM IAA ( A). Although the efficiency of auxin u..."
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"text": "We next studied PAA movement by analyzing the distribution patterns of endogenous auxins in maize coleoptiles. As previously reported, we prepared four segments from etiolated maize coleoptiles by horizontal sectioning and measured auxin levels in each segment ( A-C) ( ). IAA formed concentration gradients from the tip downward in coleoptiles (mock), with the highest level in the tip region, where IAA is most actively produced ( B) ( ). In agreement with this, VT2 and SPI1 genes, which encode co-orthologs of TAA1 and YUC genes, were expressed at a higher level in the tip region ( D) (, ). As shown in C, we found that PAA formed a similar gradient pattern with IAA in coleoptiles (mock), suggesting that both IAA and PAA may be actively produced in the tip region in maize coleoptiles. To investigate the mechanism of PAA movement, we applied the polar auxin transport inhibitor 1- N..."
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"text": "To demonstrate further the distinct characteristics of IAA and PAA, we next investigated the recovery of root phenotypes of yuc3 yuc5 yuc7 yuc8 yuc9 quintuple ( yucQ ) mutants with IAA and PAA (, ). Application of IAA and PAA restored the severe root growth defects of yucQ to wild-type (WT) levels ( A, B). No significant increase in IAA levels was observed in roots of yucQ by treatment with PAA ( C). Similarly, PAA levels were not significantly increased in roots of yucQ by treatment with IAA ( D). When these seedlings were grown horizontally for 24 h, the IAA-restored primary roots of yucQ showed a positive response to gravitropic stimulation ( E). In contrast, no significant response was observed in the PAA-restored primary roots of yucQ ( E). These results suggest that IAA generates concentration gradients, but PAA does not, in response to gravitropic stimulation; thus, these natu..."
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"name": "The GH3 family probably plays a role in PAA metabolism",
"text": "The constitutive occurrence of PAA-Asp and PAA-Glu in vector control plants ( pER8, D) and a pronounced increase in these PAA-amino acid conjugate levels, as well as IAA-Asp and IAA-Glu levels in β-estradiol-induced YUCox plants ( C, D), suggest that the PAA metabolic pathway involves GH3 genes, which encode enzymes catalyzing the synthesis of IAA-amino acid conjugates ( A) ( ). To investigate this, we generated transgenic plants expressing β-estradiol-inducible GH3.9 ( GH3.9ox ). As previously demonstrated in β-estradiol-inducible GH3.6 ( ), the induction of GH3.9 resulted in root growth defects and loss of gravitropism ( A). The induction of GH3.9 increased the endogenous levels of IAA-Glu by 11-fold ( B). Similarly, PAA-Glu showed a 13-fold increase in GH3.9ox plants ( C). These results indicate the involvement of the GH3 family in PAA metabolism in Arab..."
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"name": "A possible role for the TAA and YUC families in PAA biosynthesis",
"text": "Because IAA and PAA exist widely in vascular and non-vascular plants, we hypothesized that IAA biosynthetic enzymes are also implicated in PAA synthesis. The YUC family catalyzes a rate-limiting step of the IPA pathway in IAA biosynthesis ( A) ( ). Overexpression of YUC genes promotes IAA production and results in high-auxin phenotypes, such as hypocotyl elongation, primary root growth inhibition and enhanced lateral root growth (, ). To test our hypothesis in vivo, we investigated the metabolic impact of YUC overexpression on PAA biosynthesis using Arabidopsis with β-estradiol-inducible YUC1 ( YUC1ox ), YUC2 ( YUC2ox ) or YUC6 ( YUC6ox ) ( B). Treatment with β-estradiol increased YUC1, YUC2 and YUC6 expression levels by 41-, 142- and 29-fold in YUC1ox, YUC2ox and YUC6ox plants, respectively, compared with those in the vector control plants ( pER8 ) ( Supplementary Fig. S..."
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"text": "In addition, we examined the enzymatic conversion of PPA to PAA by glutathione S -transferase (GST)-fused YUC2 proteins expressed in E. coli. As shown in E, GST-fused YUC2 proteins converted IPA to IAA, and the reaction was abolished in the absence of NADPH. Similarly, we detected the conversion of PPA to PAA with GST-YUC2 proteins by the decrease of a specific ion for PPA at 3.6 min and the increase of a specific ion for PAA at 4.8 min using LC-ESI-MS/MS ( F). The conversion of PPA to PAA with GST-YUC2 was abolished in the absence of NADPH, similar to the conversion of IPA to IAA ( E, F). From these results, we confirmed that YUC proteins catalyze the conversion of PPA to PAA."
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"text": "The Arabidopsis thaliana ecotype Col-0 was used as the WT plant. Arabidopsis seeds were stratified at 4°C for 2 d in the dark. Seedlings were grown vertically on Murashige and Skoog (MS) medium with 1% sucrose under continuous light conditions at 20-22°C. Oats and barley were grown in soil at 20°C during the day and 15°C at night, with a 16 h photoperiod. For the analysis of auxins in vascular and non-vascular plants, 2-week-old seedlings of Arabidopsis, 7-day-old shoots of oats, 7-day-old shoots of barley, 3-week-old plants of P. patens and 2-week-old plants of M. polymorpha were used. For the analysis of auxins in Arabidopsis tissues, 3-month-old dry seeds, siliques (7 d after flowering), inflorescences, cauline leaves, stems (2 cm from the bottom end) and rosette leaves of 5-week-old plants grown in soil, and roots of 7-day-old seedlings grown on MS agar m..."
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