GmDOF3.1-GmCPX Module Regulates Nodulation and Nitrogen Fixation Abilities in Soybean

Abstract

Soybean nodule nitrogen fixation is very important, which can provide a large amount of nitrogen supply for its own growth and that of other crops, but the mechanism is largely unclear. In the present study, a coproporphyrinogen oxidase gene, GmCPX, was identified to facilitate soybean nodulation and nitrogen-fixation under the regulation of transcription factor GmDOF3.1. GmCPX was predominantly expressed in nodule which was highly induced after the inoculation of rhizobium. Overexpression of GmCPX significantly increased nodule numbers, fresh weights, nitrogenase activities, total nitrogen and ammonium nitrogen contents in transgenic soybeans, and also significantly increased the infection cell numbers and areas, heme contents and haemoglobin contents, while significantly decreased the ROS contents. Oppositely, the decreased corresponding characteristics and increased ROS contents were observed in RNAi transgenic soybeans and EMS mutant. The allelic variation analysis of GmCPX in 547 resequencing accessions found that three upstream SNPs were associated with the related traits of nodulation and nitrogen fixation, which further induced a copy number variation of recognition element for transcription factor GmDOF3.1. Further analyses found that GmDOF3.1 negatively regulated the expression of GmCPX, and the opposite phenomena to GmCPX transgenic lines were found in GmDOF3.1 overexpression and RNAi transgenic soybeans. Moreover, the genetic variation analysis of GmDOF3.1 in resequencing soybeans demonstrated its negative regulation for nodulation and nitrogen fixation. Thus, the regulation of GmDOF3.1-GmCPX module to soybean nodulation and nitrogen fixation was fully verified via forward- and reverse-genetics strategies and could be applied in the genetic improvements of corresponding characteristics in soybean.