Soybean metabolites regulated in root hairs in response to the symbiotic bacterium Bradyrhizobium japonicum

Abstract

Nodulation of soybean (Glycine max) root hairs by the nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum is a complex process coordinated by the mutual exchange of diffusible signal molecules. A metabolomic study was performed to identify small molecules produced in roots and root hairs during the rhizobial infection process. Metabolites extracted from roots and root hairs mock inoculated or inoculated with B. japonicum were analyzed by gas chromatography-mass spectrometry and ultraperformance liquid chromatography-quadrupole time of flight-mass spectrometry. These combined approaches identified 2,610 metabolites in root hairs. Of these, 166 were significantly regulated in response to B. japonicum inoculation, including various (iso)flavonoids, amino acids, fatty acids, carboxylic acids, and various carbohydrates. Trehalose was among the most strongly induced metabolites produced following inoculation. Subsequent metabolomic analyses of root hairs inoculated with a B. japonicum mutant defective in the trehalose synthase, trehalose 6-phosphate synthase, and maltooligosyltrehalose synthase genes showed that the trehalose detected in the inoculated root hairs was primarily of bacterial origin. Since trehalose is generally considered an osmoprotectant, these data suggest that B. japonicum likely experiences osmotic stress during the infection process, either on the root hair surface or within the infection thread.

Figure 1.

Distribution of root hair metabolites regulated in response to B. japonicum and identified by GC-MS and UPLC-QTOF-MS into eight functional categories.

Figure 2.

Relative abundance of lactic acid, amino-flavonoid, trehalose, Asp, octadecenoic acid, and an unknown compound in root hairs (RH) and stripped roots (STR) 0, 12, 18, 24, 36, and 48 h after inoculation with the symbiotic bacterium B. japonicum (I) or mock inoculated (NI). Asterisks indicate significantly regulated compounds colonized by B. japonicum. se values are also indicated.

Figure 3.

Average trehalose abundance (bars) determined by GC-MS in root hairs (RH) and stripped roots (STR) inoculated with B. japonicum wild type (WT) or the triple mutant for trehalose synthesis (TM) and in the uninoculated control (UN). The abundance of trehalose for each of the four replicates and each condition is also included (diamonds, squares, triangles, crosses). Asterisks indicate significant differences in trehalose accumulation. se values are also indicated.

Figure 4.

B. japonicum treS, otsA, and treY gene expression levels determined by real-time PCR in root hairs (RH) 12 h after inoculation and in liquid culture (LC). se values are also indicated.