Effects of Indole-3-Acetic Acid on the Transcriptional Activities and Stress Tolerance of <i>Bradyrhizobium japonicum</i>

<div><p>A genome-wide transcriptional profile of <i>Bradyrhizobium japonicum</i>, the nitrogen-fixing endosymbiont of the soybean plant, revealed differential expression of approximately 15% of the genome after a 1 mM treatment with the phytohormone indole-3-acetic acid (IAA). A total of 1,323 genes were differentially expressed (619 up-regulated and 704 down-regulated) at a two-fold cut off with <i>q</i> value ≤ 0.05. General stress response genes were induced, such as those involved in response to heat, cold, oxidative, osmotic, and desiccation stresses and in exopolysaccharide (EPS) biosynthesis. This suggests that IAA is effective in activating a generalized stress response in <i>B. japonicum</i>. The transcriptional data were corroborated by the finding that stress tolerance of <i>B. japonicum</i> in cell viability assays was enhanced when pre-treated with 1 mM IAA compared to controls. The IAA treatment also stimulated biofilm formation and EPS production by <i>B. japonicum</i>, especially acidic sugar components in the total EPS. The IAA pre-treatment did not influence the nodulation ability of <i>B. japonicum</i>. The data provide a comprehensive overview of the potential transcriptional responses of the symbiotic bacterium when exposed to the ubiquitous hormone of its plant host.</p> </div

Quantification of the total EPS (A) and acidic carbohydrate content (B).

<p>EPSs were isolated from the supernatant of <i>B. japonicum</i> cultures treated with 1 mM IAA (black bars) or control (grey bars). The differences between the two are statistically significant (<i>P</i> < 0.05). Values are means ± standard errors of the means for three independent experiments with three replications each.</p

Effects of different concentrations of IAA on SBF by <i>B. japonicum</i> in minimal medium.

<p>The amount of biofilm was determined by the crystal violet staining method and calculated as follows: SBF = (B-C)/G, where B is the OD₅₉₅ of the attached biofilm cells, C is the OD₅₉₅ of the stained control wells containing the medium only, and G is the OD₆₀₀ of cells grown in broth. Values are means ± standard errors of the means for three independent experiments with three replications each. An asterisk (*) indicates a significant difference between the treatment and the control (no IAA), with a <i>P</i> < 0.05 using Student’s t-test.</p

Functional classification of the genes differentially expressed after IAA treatment.

<p>The cells harvested from mid-log phase cultures were treated with 1 mM IAA for 3 h. The microarray results were analyzed with a 2.0-fold cut off and <i>q</i> value ≤ 0.05. Black bars represent positive fold-induction values and grey bars represent negative fold-induction values. Functional classifications were derived from <i>B. japonicum</i> genome annotations available through Rhizobase (<a href="http://bacteria.kazusa.or.jp/rhizobase/" target="_blank">http://bacteria.kazusa.or.jp/rhizobase/</a>).</p

Effect of IAA pretreatment on soybean nodulation by <i>B. japonicum</i>.

<p>Panel A, B, and C present the number of nodules, nodule dry weight, and plant dry weight, respectively. For measuring dry weights of nodules and plants, samples were dried at 70 °C for 3 days. An asterisk (*) indicates that the value is statistically different (<i>P</i> = 0.044) from the control value based on Student’s t-test. Values are the mean ± the standard error of the mean for 9 plants.</p

The growth characteristics of <i>B. japonicum</i> in response to IAA treatments.

<p>An arrow indicates the time (t = 44 h; O.D.₆₀₀ ~ 1.0) at which IAA was added to cultures. The symbol legend is as follows: (-●-), control (no IAA); (-○-), 0.25 mM; (--), 0.5 mM; (-Δ-), 1 mM; (-■-), 2 mM; and (-□-), 5 mM. Each point is the mean ± standard error of the mean for three biological replicates.</p

The concentration of IAA in the supernatant plotted as a function of time.

<p>Time on the X-axis corresponds to post-treatment time. The symbol legend is as follows: (-●-), control (no IAA); (--), 0.25 mM; (-Δ-), 0.5 mM; and (-■-), 1 mM. Each point is the mean ± standard error of the mean for two independent experiments, each comprising three replications. No error bar is shown due to too small variances.</p

Schematic overview of the computational and experimental contributions of COMBREX and its users, and the interrelationships of these contributions.

<p>Data and results specific to COMBREX are shown in boxes. External data imported into COMBREX are also shown, with arrows indicating entry points into the cycle. Methodology employed by COMBREX and its users is shown in blue type, as it is used to generate data. Not shown are two critical contributions to COMBREX: genome and cluster data imported from NCBI RefSeq and ProtClustDB, respectively, and NIH funding, which enables the grants that COMBREX issues to experimental laboratories.</p

Definitions of COMBREX functional status symbols and fractions of microbial genes in COMBREX in each status category.

<p>Experimentally characterized proteins are <i>green</i>. (Those in the <i>green</i> set that have been manually curated by the GSDB are also marked with a gold “G.”) Proteins with functional predictions but no experimental evidence are <i>blue</i>. Proteins with no available functional predictions are <i>black</i>.</p