GmEXPB2, a Cell Wall β-Expansin, Affects Soybean Nodulation through Modifying Root Architecture and Promoting Nodule Formation and Development

Abstract

Nodulation is an essential process for biological nitrogen (N2) fixation in legumes, but its regulation remains poorly understood. Here, a β-expansin gene, GmEXPB2, was found to be critical for soybean (Glycine max) nodulation. GmEXPB2 was preferentially expressed at the early stage of nodule development. β-Glucuronidase staining further showed that GmEXPB2 was mainly localized to the nodule vascular trace and nodule vascular bundles, as well as nodule cortical and parenchyma cells, suggesting that GmEXPB2 might be involved in cell wall modification and extension during nodule formation and development. Overexpression of GmEXPB2 dramatically modified soybean root architecture, increasing the size and number of cortical cells in the root meristematic and elongation zones and expanding root hair density and size of the root hair zone. Confocal microscopy with green fluorescent protein-labeled rhizobium USDA110 cells showed that the infection events were significantly enhanced in the GmEXPB2-overexpressing lines. Moreover, nodule primordium development was earlier in overexpressing lines compared with wild-type plants. Thereby, overexpression of GmEXPB2 in either transgenic soybean hairy roots or whole plants resulted in increased nodule number, nodule mass, and nitrogenase activity and thus elevated plant N and phosphorus content as well as biomass. In contrast, suppression of GmEXPB2 in soybean transgenic composite plants led to smaller infected cells and thus reduced number of big nodules, nodule mass, and nitrogenase activity, thereby inhibiting soybean growth. Taken together, we conclude that GmEXPB2 critically affects soybean nodulation through modifying root architecture and promoting nodule formation and development and subsequently impacts biological N2 fixation and growth of soybean.

Figure 1.

Expression pattern and histochemical localization of GmEXPB2 in soybean. A, GmEXPB2 expression in various plant tissues, including root, nodule, stem, leaf, flower, pod, and seed. B, GmEXPB2 expression in noninoculated roots (N-root) and inoculated roots at 4 dai with rhizobia (4d-root) and nodules at 7, 14, 21, 30, and 40 dai. The relative expression value was calculated by the ratio of the expression value of GmEXPB2 to that of soybean housekeeping gene TefS1 (accession no. X56856). Each bar represents the mean of four biological replicates with se. C, Histochemical detection of the GUS expression when fused to the GmEXPB2 promoter in roots and nodules of soybean transgenic composite plants. I to VIII and XII, Histochemical localization analysis of GmEXPB2 in the longitudinal sections (I–IV) and cross sections (V–VIII and XII) of transgenic soybean nodules at different developmental stages. IX to XI, Expression of the CK vector with CaMV35S promoter in longitudinal section (IX) and cross sections (X and XI) of nodules. Soybean transgenic composite plants harboring ProGmEXPB2::GUS were grown in low-N nutrient solution for 4 d (I and V), 7 d (II and VI), 14 d (III and VII), 21 d (XII), and 30 d (IV and VIII), while CaMV35S::GUS expression analyzed in transgenic nodules at 14 (IX and X) and 21 (XI) d after rhizobia inoculation was used as CK. NVB, Nodule vascular bundle; Nc, nodule cortex. Bars = 20 µm (A and E), 50 µm (B, C, F, G, I, and J), and 100 µm (all other images).

Figure 2.

Effects of OX and knockdown (RNAi) lines of GmEXPB2 on nodulation of soybean transgenic composite plants under LP or HP conditions. A, Growth performance of nodules. B, Nodule number. C, Nodule dry weight. D, Average nodule size. E, Nitrogenase activity. Soybean transgenic composite plants inoculated with rhizobia were grown in sand culture under LP (10 µm KH₂PO₄) or HP (500 µm KH₂PO₄) conditions for 30 d. Nodules were classified into two groups according to their diameter (D): big (≥2 mm) and small (<2 mm) nodule groups. CK refers to transgenic nodules harboring empty vector. Each bar represents the mean of four biological replicates with se. Asterisks represent significant differences between OX or RNAi lines and CK plants for the same trait at the same P value in Student’s t tests (*, 0.01 < P ≤ 0.05; **, 0.001 < P ≤ 0.01; and ***, P ≤ 0.001). ns, Not significant at 0.05 value.

Figure 3.

Toluidine blue-stained nodule cross sections of OX and knockdown lines of GmEXPB2. Soybean transgenic composite plants inoculated with rhizobia were grown in sand culture under LP (10 µm KH₂PO₄, A–F) or HP (500 µm KH₂PO₄, G–L) conditions for 30 d. D to F was amplified image for A to C and J to L was amplified image for G to I showing the infection zone. A, D, G, and J, GmEXPB2-overexpressing soybean nodules. C, F, I, and L, GmEXPB2-suppressing soybean nodules. B, E, H, and K, Transgenic soybean nodules carrying empty vector. Bars = 200 μm (A–F) and 100 μm (G–L).

Figure 4.

Effects of overexpressing GmEXPB2 on root architecture of soybean whole transgenic lines. A, Root hair growth of 2-d-old soybean seedlings. The red box represents the amplifying region of root hair zone for OX or wild-type (WT) soybean plants, respectively. B, Primary root length including root hair and nonroot hair zone of 2- and 3-d-old seedlings. C, Cross-sectional images of soybean root hairs. D, Root hair number per whole cross section. E, Percentage of root hairs to epidermal cells per section. OX refers to soybean whole transgenic lines overexpressing GmEXPB2. Each bar was the mean of 10 biological replicates with se. Asterisks represent significant differences between OX and wild-type plants for different parameters at the same growth period in Student’s t tests (*, 0.01 < P ≤ 0.05; **, 0.001 < P ≤ 0.01; and ***, P ≤ 0.001). Bars = 100 μm.

Figure 5.

Effects of overexpressing GmEXPB2 on root cell division and elongation of soybean whole transgenic lines. A, Root apex. The distance between black arrows in each root represents the elongation zone, and the red arrow pointed the demarcation between meristematic and elongation zone of root apex. B, Sizes of the meristematic and elongation zones. C, Number of cortex cells in the meristematic and elongation zones. D, Average length of cortex cells in the meristematic and elongation zones. Wild-type (WT) and three transgenic soybean lines overexpressing GmEXPB2 (OX1–OX3) were grown on paper culture system for 2 d. Each bar was the mean of 10 biological replicates with se. Asterisks represent significant differences between OX and wild-type lines for the same trait in Student’s t tests (*, 0.01 < P ≤ 0.05; **, 0.001 < P ≤ 0.01; and ***, P ≤ 0.001). ns, Not significant at 0.05 value. Bars = 100 μm.

Figure 6.

Confocal microscopy of root hairs of soybean seedlings inoculated with rhizobia USDA110 carrying GFP. A to F, Root hairs in the preinfection period at 2 dai. G to L, Infected root hairs formed ITs at 3 dai. M to R, IT growth and reaching to the cortical root cells at 4 dai. A to C, G to I, and M to O, Wild-type (WT) plants. D to F, J to L, and P to R, Whole transgenic soybean lines overexpressing GmEXPB2. S, Green fluorescent area per image represents bacteria adhesion to root hairs. T, IT number per 2-mm length of roots. Each bar represents the mean of 12 biological replicates with se. Asterisks represent significant differences between OX and wild-type CK lines for the same trait in Student’s t tests (*, 0.01 < P ≤ 0.05; and ***, P ≤ 0.001). Bars = 100 μm (A–F) and 50 μm (all other images).

Figure 7.

Effects of overexpressing GmEXPB2 (OX) on nodulation of soybean whole transgenic lines. A, Photographs showing soybean nodules of 25-d-old seedlings under both LP and HP conditions. B, Nodule number per plant. C, Nodule dry weight per plant. D, Nitrogenase activity. OX1 to OX3 refer to three independent soybean whole transgenic lines overexpressing GmEXPB2. WT1 and WT2 refer to wild-type soybean plants. LP and HP were 5 and 500 µm KH₂PO₄, respectively. Each bar represents the mean of four biological replicates with se. Asterisks represent significant differences between OX and wild-type lines for the same trait at the same P value in Student’s t tests (*, 0.01 < P ≤ 0.05; **, 0.001 < P ≤ 0.01; and ***, P ≤ 0.001). ns, Not significant at 0.05 value.

Figure 8.

Plant growth and N and P content as affected by overexpressing GmEXPB2 (OX) in soybean whole plant transgenic lines. A, Photographs showing soybean growth performance. B, Dry weight. C, N content. D, P content. OX1 to OX3 refer to three independent soybean transgenic lines overexpressing GmEXPB2. LP and HP were 5 and 500 µm KH₂PO₄, respectively, which were applied for 25 dai. Each bar represents the mean of four biological replicates with se. Asterisks represent significant differences between OX and wild-type (WT) plants for the same trait at the same P value in Student’s t tests (*, 0.01 < P ≤ 0.05; **, 0.001 < P ≤ 0.01; and ***, P ≤ 0.001).

Figure 9.

Schematic comparison of GmEXPB2 expression involved in soybean nodulation, including indirect pathway through root architecture modification and direct pathway through promotion of nodule formation and development between wild-type (WT) and overexpressing GmEXPB2 (OX) soybean plants. Compared with the wild type, OX plants modified root architecture with a longer root hair zone and denser root hairs, which might increase the rhizobium infection events. Overexpressing GmEXPB2 might also directly facilitate the growth of nodule primordia; thereby, OX of GmEXPB2 in either soybean hairy roots or whole transgenic plants resulted in increased nodule number, nodule mass, and thus elevated plant N content as well as biomass.