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

Akihiro Suzuki,Akiyoshi Tominaga,Hidetoshi Abe,Koichi Futsuki,Masatsugu Hashiguchi,Ken-ichi Kucho,Ryo Akashi,Maki Nagata,Ann M. Hirsch,Mikiko Abe,Susumu Arima,Toshiki Uchiumi

doi:10.1104/pp.109.142638

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.

Highlights

The phytohormone abscisic acid (ABA) is known to be a negative regulator of legume root nodule formation
We isolated a mutant designated enf1 that exhibited decreased sensitivity to exogenous ABA and that was reduced in endogenous ABA concentration
We found that the enf1 mutant produced more root nodules and showed elevated nitrogen fixation activity

Summary

Effect of Endogenous ABA on Nitrogen Fixation Activity

Because enf had a low endogenous ABA concentration, we hypothesized that the decrease in ABA concentration caused the elevation of nitrogen fixation activity To test this hypothesis, we treated wild-type plants at 28 DAI with 20 mM abamine, a specific inhibitor of ABA synthesis (Han et al, 2004). In MG20 roots treated with abamine, the endogenous concentration of ABA decreased to about one-fourth of that of control plants, and at the same time, the acetylene reduction activity per nodule weight was significantly up-regulated (Fig. 7). These results strongly suggest that the decrease in endogenous ABA concentration in enf was responsible for the increased levels of nitrogen fixation activity. Statistical significance is indicated by asterisks (** P , 0.01, by Student’s t test)

Flowering Time

DISCUSSION

Nitrogen fixation activity

Plant Materials and Growth Conditions

Measurement of Dry Weights and Total Nitrogen Contents

Acetylene Reduction Assay

Observation of Sensitivity of Plants to ABA Treatment

Observation of Infection Threads Using Fluorescence Microscopy

ABA and Abamine Treatment

Quantification of NO by Fluorescence Spectrophotometry

Findings

LITERATURE CITED