GH3 Auxin-Amido Synthetases Alter the Ratio of Indole-3-Acetic Acid and Phenylacetic Acid in Arabidopsis

Abstract

Auxin is the first discovered plant hormone and is essential for many aspects of plant growth and development. Indole-3-acetic acid (IAA) is the main auxin and plays pivotal roles in intercellular communication through polar auxin transport. Phenylacetic acid (PAA) is another natural auxin that does not show polar movement. Although a wide range of species have been shown to produce PAA, its biosynthesis, inactivation and physiological significance in plants are largely unknown. In this study, we demonstrate that overexpression of the CYP79A2 gene, which is involved in benzylglucosinolate synthesis, remarkably increased the levels of PAA and enhanced lateral root formation in Arabidopsis. This coincided with a significant reduction in the levels of IAA. The results from auxin metabolite quantification suggest that the PAA-dependent induction of GRETCHEN HAGEN 3 (GH3) genes, which encode auxin-amido synthetases, promote the inactivation of IAA. Similarly, an increase in IAA synthesis, via the indole-3-acetaldoxime pathway, significantly reduced the levels of PAA. The same adjustment of IAA and PAA levels was also observed by applying each auxin to wild-type plants. These results show that GH3 auxin-amido synthetases can alter the ratio of IAA and PAA in plant growth and development.

Keywords: Arabidopsis; Auxin; Biosynthesis; Inactivation; Indole-3-acetic acid; Phenylacetic acid.

Fig. 1

Auxin biosynthesis and inactivation pathways and glucosinolate biosynthesis pathways in Arabidopsis. (A) Current IAA and IG pathways. (B) The PAA and BG pathways proposed in this study (dotted square). CYP79B2 and CYP79B3 catalyze the conversion of Trp to IAOx (blue arrow), and CYP79A2 mediates the conversion of Phe to PAOx (red arrow). The dashed arrows represent metabolic pathways in which enzymatic steps are still unknown.

Fig. 2

Phenotypes and auxin metabolite levels of CYP79A2ox plants. (A) Phenotypes of pER8 and CYP79A2ox (79A2ox) plants (10-day-old). Scale bars indicate 1 cm. (B) The number of lateral roots (LR) per the primary root length (cm). Values are mean � SD (n = 50). (C) Auxin metabolite levels in pER8 and 79A2ox plants. Values are mean � SD (n = 4). Differences between pER8 and 79A2ox plants are statistically significant at P < 0.05 (*P < 0.05 and ***P < 0.001, Student's t-test). Note that PAA metabolites are shown in nmol/gFW and IAA metabolites are in pmol/gFW. gFW, gram fresh weight.

Fig. 3

GH3 gene expression levels in CYP79A2ox plants. Expression levels of GH3.1, GH3.2, GH3.3, GH3.5, GH3.6, GH3.9 and GH3.17 genes in pER8 and CYP79A2ox (79A2ox) plants (10-day-old). 18S rRNA gene is used for normalization. Values are mean � SD (n = 4). Differences between pER8 and 79A2ox plants are statistically significant at P < 0.05 (*P < 0.05 and **P < 0.01, Student's t-test).

Fig. 4

Phenotypes and auxin metabolite levels of CYP79B2ox plants. (A) Phenotypes of pER8 and CYP79B2ox (79B2ox) plants (10-day-old). Scale bars indicate 1 cm. (B) The number of lateral roots (LR) per the primary root length (cm). Values are mean � SD (n = 50). (C) Auxin metabolite levels in pER8 and 79B2ox plants. Values are mean � SD (n = 4). Differences between pER8 and 79B2ox plants are statistically significant at P < 0.05 (*P < 0.05 and ***P < 0.001, Student's t-test).

Fig. 5

Auxin metabolite levels in sur2 mutants. (A) Phenotypes of WT and sur2 mutants (8-day-old). Scale bars indicate 5 mm. (B) The levels of IAA, PAA and their conjugates (IAA-Asp, IAA-Glu, PAA-Asp, PAA-Glu) in WT and sur2 mutants (8-day-old). Values are mean � SD (n = 4). Differences between WT and sur2 mutants are statistically significant at P < 0.05 (*P < 0.05, **P < 0.01 and ***P < 0.001, Student's t-test).

Fig. 6

Effect of PAA and IAA treatments on auxin metabolites in Arabidopsis. Twelve-day-old WT seedlings were treated with 20 �M of PAA or 1 �M of IAA for 24 h. (A) Auxin metabolites levels in PAA-treated plants. (B) Auxin metabolite levels in IAA-treated plants. Values are mean � SD (n = 3). Differences between mock and auxin-treated plants are statistically significant at P-value <0.05 (*P < 0.05, **P < 0.01 and ***P < 0.001, Student's t-test).