A Novel ABA Insensitive Mutant of Lotus japonicus with a Wilty Phenotype Displays Unaltered Nodulation Regulation  Biswas Bandana , Chan Pick Kuen , Gresshoff Peter M.   Molecular Plant  Volume 2, Issue 3, Pages 487-499 (May 2009) DOI: 10.1093/mp/ssp009 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 Screening for and Isolation of the Beyma ABA Insensitive Mutant. (A) Screening of mutant lines performed in closed tissue culture jars. The figure shows MG-20 (WT) and seven M2 lines (10–20 seeds per M2 line) in a tissue culture jar containing agar medium supplemented with 50 μM ABA. Plants in the jar are 1 week old, at which stage root growth was examined. A total of 3000 M2 lines were screened. Chlorophyll sectors (e.g. visible in line 999) were found on about 1% of M1 plants. (B) MG-20 wild-type root growth in 0 or 50 μM ABA agar medium after 1 week. The black scale bar represents 1.0 cm. (C) Beyma root growth in 0 or 50 μM ABA agar medium after 1 week. Plants were grown in parallel to those shown in Figure 1B. The black scale bar represents 1.0 cm. Molecular Plant 2009 2, 487-499DOI: (10.1093/mp/ssp009) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 Growth Analysis of Beyma. (A) Three-month-old MG-20 plant growing in the absence of exogenously supplied ABA featuring vigorous growth and onset of flowering. (B) Three-month-old Beyma plant grown in parallel to the wild-type shown in Figure 2A. The plant is less vigorous and has smaller leaves (c.f. Figure 2C). Note that flowering has been induced (see upper leaf branch). (C) Leaf size and dehydration response of MG-20 and Beyma immediately after being detached from the plant (top), 1 h after being detached from the plant (bottom). Leaves taken were from 3-month-old plants (as in Figure 2A and 2B). Bejma leaves are extremely stress susceptible. (D) Stomatal opening of leaves of MG-20 (left) and Beyma (right) at 0 and 100 μM ABA. The ABA treatment closes 100% of stomata on MG-20 leaves but Beyma fails to respond. Note that the size of the stomata and the guard cells are equivalent in MG-20 and Beyma. (E) Shoot tip morphology of MG-20 and Beyma. Note that the constitutive stress response of Beyma even in the absence of exogenously supplied ABA, leading to growth reduction. (F) Shoot length measured at different time intervals to record shoot growth difference between MG-20 and Beyma. Data are averages of 15 plants (± standard deviation). Molecular Plant 2009 2, 487-499DOI: (10.1093/mp/ssp009) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 ABA Insensitivity in Beyma. (A) Effect of ABA (in μM) on germination of MG-20 and Beyma. Germination percentage was scored 6 d after placing on filter paper with different concentrations of ABA. (B) MG-20 and Beyma germination at 0 and 100 μM ABA. (C) Effect of ABA concentration (in μM) on nodule number per plant of MG-20 and Beyma grown on plastic growth pouches; scored 4 weeks after inoculation with Mesorhizobium loti NZP2235. Data are averages of 18 plants (± standard deviation). Molecular Plant 2009 2, 487-499DOI: (10.1093/mp/ssp009) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 Difference in Germination Sensitivity between Two Lotus japonicus Ecotypes Gifu and MG-20. (A) Effect of ABA on seed germination of cultivars MG-20 and Gifu. Germination percentage was scored 5 d after placing on filter paper with different concentrations of ABA. (B) MG-20 and Gifu at 0 and 30 μM ABA. Molecular Plant 2009 2, 487-499DOI: (10.1093/mp/ssp009) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 5 Inhibition of Nodulation by ABA Is Local, Not Systemic. (A) Plastic growth pouches, hot-sealed in the middle to achieve liquid separation, were used for split root experiments. Low magnification view is used to illustrate the root structure. MG-20 plants were used for these experiments, with two plants on either side as controls and the plant in the middle with split roots. (B) Close-up showing many nodules on both control and split root plant on the 0 μM ABA (0) side and none on the 50 μM side (50) of the split root plant and only two on the control. Higher magnification view is used to illustrate nodule distribution. (C) Local nodulation inhibition by ABA. Values represents means ± SD (n  =  10). Gifu ecotype is used for comparison. Molecular Plant 2009 2, 487-499DOI: (10.1093/mp/ssp009) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 6 Histology of Nodules Viewed under UV Light Microscope (100× Magnification). (A) MG-20 nodules of plants grown at 0 μM ABA. (B) MG-20 nodules of plants grown at 50 μM ABA. (C) Beyma nodules of plants grown at 0 μM ABA. (D) Beyma nodules of plants grown at 50 μM ABA. Scale bar  =  50 μm. Molecular Plant 2009 2, 487-499DOI: (10.1093/mp/ssp009) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions