Fig 1.
Fresh tissue biomass and number of nodules per plant.
The average fresh mass (mg) of shoots, roots, and nodules, the length (mm) of roots and shoots, and the number of nodules on Lotus japonicus in the control (0 µM copper), 300 µM copper, and 450 µM copper treatments added to basal nutrient solution. Each replicate represents the average of multiple plants in a single pot. Plants were harvested at 42 days post germination and 28 days post inoculation. (A) Fresh shoot mass (n = 5). (B) Fresh shoot length (n = 5). (C) Fresh root mass (n = 14). (D) Fresh root length (n = 5). (E) Fresh nodule mass (0 and 300 µM Cu n = 15, 450 µM Cu n = 13). (F) Mean nodule count, showing the number of active (pink) compared to inactive (green-grey) nodules out of that total count (n = 10). Error bars indicate SEM. Letters indicate significant differences between groups (p < 0.05). Nodule mass was transformed prior to analysis to meet test assumptions. Shoot mass, nodule mass, shoot and root lengths, and nodule counts were analyzed via one-way ANOVA followed by a Tukey’s HSD post-hoc test. Root mass was analyzed via Welch’s unequal variances test followed by a Games-Howell post-hoc test.
Fig 2.
Acetylene reduction by nitrogenase and nodule leghemoglobin content.
(A-C) Acetylene reduction assay results, showing the nanomoles of acetylene reduced to ethylene over 30 minutes as a proxy for nitrogenase activity in control (0 µM copper), 300 µM copper and 450 µM copper treatments added to basal nutrient solution. (A) Amount of ethylene produced per whole root mass. (B) Ethylene produced per g of fresh root tissue. (C) Ethylene produced per estimated g of nodule tissue. 0 µM Cu group n = 4, other groups n = 5. (D) Results of the colorimetric pyridine hemochromogen assay showing the concentrations of oxygen-binding leghemoglobin (ng/g of fresh nodule tissue) for control, 300 µM copper, and 450 µM copper treatment groups. 450 µM Cu n = 4, all other groups n = 5). Each replicate represents the average of multiple plants from a single pot. Plants were harvested at 42 days post germination and 28 days post inoculation. Error bars indicate SEM, letters indicate significant differences (p < 0.05), analyzed via a two-way ANOVA followed by a Tukey’s HSD.
Fig 3.
Copper concentrations in shoot, root and nodule tissues.
The concentration of copper (µg/g of dried tissue) taken up into the shoots (green), nodules (pink) and roots (yellow). Each replicate represents the average of multiple plants in a single pot. Plants were harvested at 42 days post germination and 28 days post inoculation. Treatments added to basal nutrient solution are control (0 µM copper), 300 µM copper, and 450 µM copper. Letters indicate significant differences between groups (p < 0.05). Error bars indicate SEM, n = 5 for roots and shoots, n = 4 for nodules. Data were transformed prior to analysis to meet test assumptions and then analyzed with a two-way ANOVA followed by Tukey’s HSD.
Fig 4.
Concentration of malondialdehyde and activity of ROS-scavenging enzyme ascorbate peroxidase in root and nodule tissues.
(A) Thiobarbituric reactive substances assay results showing the µM concentration of the polyunsaturated fatty acid decomposition product malondialdehyde (MDA) in nodules (pink) and roots (yellow) as an estimation of lipid peroxidation occurring in those tissues. (B) Ascorbate peroxidase (APX) activity in U/min/mg in nodule (pink) and root (yellow) tissues. Treatments added to basal nutrient solution are control (0 µM copper), 300 µM copper, and 450 µM copper. APX 450 µM copper nodules n = 4, all other groups n = 5, Each replicate represents the average of multiple plants in a single pot. Plants were harvested at 42 days post germination and 28 days post inoculation. Error bars indicate SEM, letters indicate significant differences (p < 0.05), analyzed via a two-way ANOVA followed by a Tukey’s HSD.