Effect of soybean coumestrol on Bradyrhizobium japonicum nodulation ability, biofilm formation, and transcriptional profile

Abstract

Flavonoids, secondary plant metabolites which mainly have a polyphenolic structure, play an important role in plant-microbe communications for nitrogen-fixing symbiosis. Among 10 polyphenolic compounds isolated from soybean roots in our previous study, coumestrol showed the highest antioxidant activity. In this study, its effect on the soybean nodulation was tested. The soybean symbiont Bradyrhizobium japonicum USDA110 pretreated with 20 μM coumestrol enhanced soybean nodulation by increasing the number of nodules 1.7-fold compared to the control. We also tested the effect of coumestrol on B. japonicum biofilm formation. At a concentration of 2 μM, coumestrol caused a higher degree of biofilm formation than two major soybean isoflavonoids, genistein and daidzein, although no biofilm formation was observed at a concentration of 20 μM each compound. A genome-wide transcriptional analysis was performed to obtain a comprehensive snapshot of the B. japonicum response to coumestrol. When the bacterium was incubated in 20 μM coumestrol for 24 h, a total of 371 genes (139 upregulated and 232 downregulated) were differentially expressed at a 2-fold cutoff with a q value of less than 5%. No common nod gene induction was found in the microarray data. However, quantitative reverse transcription-PCR (qRT-PCR) data showed that incubation for 12 h resulted in a moderate induction (ca. 2-fold) of nodD1 and nodABC, indicating that soybean coumestrol is a weak inducer of common nod genes. In addition, disruption of nfeD (bll4952) affected the soybean nodulation by an approximate 30% reduction in the average number of nodules.

Fig 1

Effect of pretreatment of B. japonicum with different concentrations of coumestrol on soybean nodulation. (A) Numbers of nodules. Values are means ± standard errors of the means from nine replications. The different letters above the bars indicate that the data are significantly different from each other (P < 0.05; by t test using JMP8 statistical software). (B) Distribution of nodules. Positive and negative (−) values on the “Distance from RT (mm)” axis indicate measurements above and below root tips, respectively. The values on the “Number of nodules” axis are the total nodule numbers from nine replications.

Fig 2

Effect of coumestrol, daidzein, and genistein on the biofilm formation ability of B. japonicum. Circles (● and ○), triangles (▲ and △), and squares (■ and □) indicate coumestrol, daidzein, and genistein, respectively. Solid and open symbols are 2 and 20 μM, respectively. All of the open symbols are overlapped because B. japonicum did not form biofilms in 20 μM each compound. To quantify the biofilm formation ability, the specific biofilm-forming ability was normalized by dividing the total biofilm OD₅₉₅ value by the cell growth OD₆₀₀ value. The relative biofilm-forming ability was then calculated by dividing the specific biofilm-forming ability value of each treatment sample by that of the control DMSO. Values are means ± standard errors of the mean for six replications.

Fig 3

Growth of B. japonicum in the presence of coumestrol, daidzein, and genistein. H₂O and DMSO were used as the general and vehicle controls, respectively. Circle symbols indicate controls [solid (●) for H₂O and open (○) for DMSO]. Diamond (♦ and ♢), triangle (▲ and △), and square (■ and □) symbols indicate coumestrol, daidzein, and genistein, respectively. Solid (♦, ▲, and ■) and open (♢, △, and □) symbols indicate 2 and 20 μM, respectively. Values are means ± standard errors of the mean for six replications.

Fig 4

Functional group assignment of genes differentially expressed in the presence of 20 μM coumestrol at a 2-fold cutoff with q value less than 5%. Black and white bars indicate up- and downregulated genes, respectively. Functional classifications were derived from B. japonicum USDA110 genome annotations available through Rhizobase (http://genome.kazusa.or.jp/rhizobase/).

Fig 5

Correlation between microarray and qRT-PCR data for the control versus the 20 μM coumestrol treatment for 15 genes listed in Table S1 in the supplemental material. The genes were selected based on the fold induction and functional categories. Expression differences were log₂ transformed.