Synthesis and Degradation of the Phytohormone Indole-3-Acetic Acid by the Versatile Bacterium Paraburkholderia xenovorans LB400 and Its Growth Promotion of Nicotiana tabacum Plant

Abstract

Plant growth-promoting bacteria (PGPB) play a role in stimulating plant growth through mechanisms such as the synthesis of the phytohormone indole-3-acetic acid (IAA). The aims of this study were the characterization of IAA synthesis and degradation by the model aromatic-degrading bacterium Paraburkholderia xenovorans LB400, and its growth promotion of the Nicotiana tabacum plant. Strain LB400 was able to synthesize IAA (measured by HPLC) during growth in the presence of tryptophan and at least one additional carbon source; synthesis of anthranilic acid was also observed. RT-PCR analysis indicates that under these conditions, strain LB400 expressed the ipdC gene, which encodes indole-3-pyruvate decarboxylase, suggesting that IAA biosynthesis proceeds through the indole-3-pyruvate pathway. In addition, strain LB400 degraded IAA and grew on IAA as a sole carbon and energy source. Strain LB400 expressed the iacC and catA genes, which encode the α subunit of the aromatic-ring-hydroxylating dioxygenase in the IAA catabolic pathway and the catechol 1,2-dioxygenase, respectively, which may suggest a peripheral IAA pathway leading to the central catechol pathway. Notably, P. xenovorans LB400 promoted the growth of tobacco seedlings, increasing the number and the length of the roots. In conclusion, this study indicates that the versatile bacterium P. xenovorans LB400 is a PGPB.

Keywords: IAA degradation; IAA synthesis; Paraburkholderia xenovorans LB400; indole-3-acetic acid (IAA); plant growth-promoting bacteria (PGPB).

Figure 1

Organization of genes in the IPyA and IAM IAA anabolic pathways in P. xenovorans LB400 and other bacteria. (a) Organization of the ipdC and iad1 genes of the indole-3-pyruvate (IPyA) pathway. (b) Organization of iaaH and iaaM genes of the indole-3-acetamide (IAM) pathway. Gene sizes and intergenic regions are accurately represented to scale. C2 denotes the minor chromosome, and MP represents the megaplasmid.

Figure 2

Growth and indole-3-acetic acid (IAA)/anthranilic acid (AA) biosynthesis by P. xenovorans LB400. (a) Growth in the presence of tryptophan. Cells were cultivated in different media (M9 or LB) with glucose (30 mM) and/or tryptophan (10 mM). (b) IAA biosynthesis. (c) AA biosynthesis. Each value represents an average ± SD of three independent experiments. IAA/AA production were not detected in M9 medium with tryptophan or glucose as sole carbon and energy sources. Significant differences for each time point (24 h, 48 h, 72 h) between the conditions of IAA/AA biosynthesis were analyzed by one-way ANOVA followed by the LSD Fisher test. Means with different letters indicate significant differences (p ≤ 0.05).

Figure 3

Expression of the ipdC and iaaH genes in P. xenovorans LB400 grown with different carbon sources. (a) RT-PCR (15, 20, 25, and 30 amplification cycles) of ipdC gene after 24 h growth. (b) RT-PCR (25 and 30 amplification cycles) of ipdC gene after 48 h growth. (c) RT-PCR (30 amplification cycles) of iaaH gene after 48 h growth. RNA samples were purified from LB400 cells harvested at 24 and 48 h. The 16S rRNA gene was selected as a reference gene. MM, molecular markers; 1, cells grown in M9 medium supplemented with glucose (30 mM); 2, cells grown in M9 medium supplemented with glucose (30 mM) and tryptophan (10 mM); 3, cells grown in LB medium supplemented with tryptophan (10 mM).

Figure 4

Proposed P. xenovorans LB400 biosynthetic pathways for the indole-3-acetic acid (IAA) and anthranilic acid (AA). Strain LB400 probably employs the IPyA pathway for IAA synthesis. The key substrates, metabolites, and products include anthranilic acid (AA), tryptophan (Trp), indole-3-pyruvic acid (IPyA), indole-3-acetaldehyde (IAAld), and indole-3-acetic acid (IAA). The enzymes involved in the pathway are an aminotransferase, indole-3-pyruvate decarboxylase (IpdC), and indole-3-acetaldehyde dehydrogenase (Iad1). Additionally, enzymes related to the metabolism of tryptophan to AA are tryptophan 2,3-dioxygenase (KynA), kynurenine formamidase (KynB), and kynureninase (KynU). Enzymes involved in the conversion of chorismate via AA to tryptophan are anthranilate synthase components I and II (TrpE and TrpD), indole-3-glycerolphosphate synthase/N(-5-phosphoribosyl) anthranilate isomerase (TrpC), and tryptophan synthase subunits β and α (TrpB and TrpA). Compounds and genes measured in this study are highlighted in red. The synthesis of the compounds IAA and AA (determined by HPLC) and the expression of the ipdC gene that encodes the enzyme indole-3-pyruvate decarboxylase (determined by RT-PCR). The genes encoding enzymes not highlighted are also present in the genome of strain LB400. The gene locus of the genes corresponding to strain LB400 are shown in grey.

Figure 5

Indole-3-acetic acid (IAA) aerobic degradation pathway and gene clusters involved in IAA catabolism in P. xenovorans LB400, P. phytofirmans PsJN, and Ps. putida 1290. (a) IAA degradation pathway wherein the iac genes encoding the catabolic enzymes are indicated. The IAA aerobic degradation pathway was adapted from Laird et al. [18]. (b) The iac and cat genes involved in peripheral IAA pathway and central catechol pathway. The iac genes (grey) encode enzymes involved in the conversion of IAA to catechol, which is further metabolized by enzymes encoded by the cat genes (black). Gene sizes and intergenic regions are accurately represented to scale. C1 and C2 denote the major and minor chromosomes, respectively.

Figure 6

P. xenovorans LB400 growth on IAA and other carbon sources and IAA degradation. (a) LB400 growth on IAA (0.5, 1.5, and 3.0 mM). (b) Degradation of IAA (0.5, 1.5, and 3.0 mM). (c) LB400 growth on IAA (0.5 mM), IAA (0.5 mM) + glucose (5.0 mM), and glucose (5.0 mM). (d) Comparison of IAA (0.5 mM) degradation in co-culture with or without glucose (5.0 mM). Each value is a mean ± SD of three independent trials. Significant differences of the last three points of (d) were analyzed by one-way ANOVA followed by the LSD Fisher test. Means with different letters indicate significant differences (p ≤ 0.05).

Figure 7

Degradation of IAA by P. xenovorans LB400 resting cells. This assay was performed with concentrated LB400 cells (turbidity 600 nm = 8.0) incubated in phosphate buffer (5.0 mM, pH 7.0) with IAA (0.5 mM). Each value is a mean ± SD of three independent assays. Significant differences were analyzed by one-way ANOVA followed by the LSD Fisher test. Means with different letters indicate significant differences (p ≤ 0.05).

Figure 8

Expression of the iacC and catA genes in P. xenovorans LB400 cells incubated with different carbon sources. The expression was measured by RT-PCR. (a) Expression of the iacC gene. (b) Expression of the catA gene. (c) Expression of the 16S rRNA gene (reference gene). MM, molecular markers (UltraRanger 1 kb DNA ladder); 1, negative control; resting cells with 2, IAA (1 mM); 3, IAA (1 mM) + glucose (5 mM); 4, glucose (5 mM); 5, cells grown on salicylate (5 mM) + glucose (5 mM).

Figure 9

Growth promotion of P. xenovorans LB400 on Nicotiana tabacum seedlings. (a) Representative N. tabacum seedlings post-treatment with strain LB400 and water, respectively. (b) Effects of strain LB400 on the number of roots in N. tabacum. (c) Effects of strain LB400 on root and stem length in N. tabacum. Each value is a mean ± SD of 12 biologically independent replicates. Significant differences were analyzed by one-way ANOVA followed by the LSD Fisher test. Means with different letters indicate significant differences (p ≤ 0.05).