Enhanced nodulation and nitrogen fixation in the abscisic acid low-sensitive mutant enhanced nitrogen fixation1 of Lotus japonicus

Abstract

The phytohormone abscisic acid (ABA) is known to be a negative regulator of legume root nodule formation. By screening Lotus japonicus seedlings for survival on an agar medium containing 70 microM ABA, we obtained mutants that not only showed increased root nodule number but also enhanced nitrogen fixation. The mutant was designated enhanced nitrogen fixation1 (enf1) and was confirmed to be monogenic and incompletely dominant. The low sensitivity to ABA phenotype was thought to result from either a decrease in the concentration of the plant's endogenous ABA or from a disruption in ABA signaling. We determined that the endogenous ABA concentration of enf1 was lower than that of wild-type seedlings, and furthermore, when wild-type plants were treated with abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase, which results in reduced ABA content, the nitrogen fixation activity of abamine-treated plants was elevated to the same levels as enf1. We also determined that production of nitric oxide in enf1 nodules was decreased. We conclude that endogenous ABA concentration not only regulates nodulation but also nitrogen fixation activity by decreasing nitric oxide production in nodules.

Figure 1.

Symbiotic and growth phenotypes of the L. japonicus enf1 mutant. M. loti-inoculated plants were grown in pots containing vermiculite, which was watered with B&D medium. Wild-type MG20 (left) and enf1 (right) L. japonicus plants 28 DAI are shown. Bar = 1 cm.

Figure 2.

Analysis of enf1 symbiotic phenotypes. M. loti-inoculated plants were grown for 28 d in pots containing vermiculite and watered with B&D medium (A–F) or grown on B&D agar-solidified (1.5%, w/v) medium for 12 d (G). The plants on the agar plates were inoculated with M. loti NZP2037 carrying plasmid pHC60 (G). A, Nodule number per plant. B, Nodule weight per plant. C and D, Acetylene reduction activity per plant (C) and per nodule weight (D). E, Number of true leaves per plant. F, Shoot length. G, The number of infection threads (IT) per plant at 12 DAI. Black bars indicate MG20, and gray bars indicate enf1 (A –F). In G, black bars indicate later stages of infection thread formation, whereas white bars refer to the initiation of nodule formation. At least 40 plants (A–F) and 17 to 20 plants (G) were examined in each experiment. Error bars represent se. Statistical significance is indicated by asterisks (* P < 0.05, ** P < 0.01, by Student's t test).

Figure 3.

Nonsymbiotic phenotypes of the enf1 mutant. A to F, Plants were grown for 21 d in vermiculite-filled pots supplied with B&D medium with or without 10 mm KNO₃. G, Plants were grown on B&D agar-solidified (1.5%, w/v) medium. A, MG20 (left) and enf1 (right) grown with no nitrogen. B, MG20 (left) and enf1 (right) grown with 10 mm KNO₃. C and D, Shoot length resulting from growth without KNO₃ (C) or with 10 mm KNO₃ (D). E and F, Number of true leaves per plant in medium without KNO₃ (E) or with 10 mm KNO₃ (F). G, Number of lateral roots per plant at 24 DAS. At least 30 plants were used in each experiment. Error bars indicate se, and statistical significance is indicated by asterisks (* P < 0.05, ** P < 0.01, by Student's t test).

Figure 4.

Effects of 0.5 μm ABA on the growth of enf1. A, MG20 at 17 DAI with M. loti. B, enf1 at 17 DAI with M. loti. Shown are untreated MG20 and enf1 (left) and treated with 0.5 μm ABA (right). C and D, Shoot tips of MG20 (C) and enf1 (D) following 0.5 μm ABA treatment. Bars = 1 cm.

Figure 5.

Endogenous concentration of ABA in enf1. M. loti-inoculated plants were grown for 21 d on vermiculite-filled pots supplied with B&D medium. Endogenous concentrations of ABA in MG20 and enf1 roots (A) and shoots (B) are shown. The data represent averages ± se of six independent experiments derived from eight different plants. Statistical significance is indicated by asterisks (** P < 0.01, by Student's t test).

Figure 6.

Histograms of MG20, enf1, and F2 progeny. Histograms of nodule number per plant (A) and of acetylene reduction activity per plant (B) are shown. Black bars indicate MG20, white bars indicate enf1, and gray bars indicate F2 progeny.

Figure 7.

Effects of ABA concentration on nitrogen fixation activity. M. loti-inoculated plants were grown for 21 d on vermiculite-filled pots supplied with B&D medium. Plant roots at 28 DAI were treated with 0.5 μm ABA, 20 μm abamine, both ABA and abamine, or were untreated (B&D medium control) for 3 d. A, Acetylene reduction activity per nodule weight. B, ABA concentration in root. At least 15 plants were used in acetylene reduction assay. Four different plants were used for measurement of ABA concentration, and three repeats were performed. Error bars indicate the se, and the significance of differences between untreated control and treated values was determined by the two-tailed multiple t test with Bonferroni correction following ANOVA (three comparisons in four groups): * P < 0.05, ** P < 0.01.

Figure 8.

Relative amounts of nitrogen fixation-related gene transcripts in enf1 nodules at 28 DAI. Transcript amounts of nifH, LjLbs, LjHb, Ign1, Sst1, and LjGlu1 were normalized against sigA (used as an internal control for nifH) and ATP synthase (internal control for LjLbs, LjHb, Ign1, Sst1, and LjGlu1) transcripts. The mean value of expression in wild-type plants was set to 1. The data represent averages ± se of three independent experiments of nodules derived from five different plants.

Figure 9.

NO production in nodules. A and B, Quantification of NO produced in nodules of MG20 and enf1 at 21 DAI (A) and 28 DAI (B). C, Quantification of NO in nodules that were treated with abamine. Nodules on the root of 28-d-old plants were treated with 20 μm abamine for 3 d. RFU values per nodule fresh weight at 515 nm, normalized against MG20 plants, are shown. The data represent averages ± se of three independent experiments derived from nodules of six to eight plants. In A and B, statistical significance is indicated by asterisks (** P < 0.01, by Student's t test). In C, the significance of differences among four groups was determined by the two-tailed multiple t test with Bonferroni correction following ANOVA (six comparisons in four groups), and the different letters refer to significant differences at P < 0.01.