CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation

Abstract

Summary Legume nodules contain high concentrations of leghemoglobins (Lbs) encoded by several genes. The reason for this multiplicity is unknown. CRISPR/Cas9 technology was used to generate stable mutants of the three Lbs of Lotus japonicus. The phenotypes were characterized at the physiological, biochemical and molecular levels. Nodules of the triple mutants were examined by electron microscopy and subjected to RNA‐sequencing (RNA‐seq) analysis. Complementation studies revealed that Lbs function synergistically to maintain optimal N2 fixation. The nodules of the triple mutants overproduced superoxide radicals and hydrogen peroxide, which was probably linked to activation of NADPH oxidases and changes in superoxide dismutase isoforms expression. The mutant nodules showed major ultrastructural alterations, including vacuolization, accumulation of poly‐β‐hydroxybutyrate and disruption of mitochondria. RNA‐seq of c. 20 000 genes revealed significant changes in expression of carbon and nitrogen metabolism genes, transcription factors, and proteinases. Lb‐deficient nodules had c. 30–50‐fold less heme but similar transcript levels of heme biosynthetic genes, suggesting a post‐translational regulatory mechanism of heme synthesis. We conclude that Lbs act additively in nodules and that the lack of Lbs results in early nodule senescence. Our observations also provide insight into the reprogramming of the gene expression network associated with Lb deficiency, probably as a result of uncontrolled intracellular free O2 concentration.

Celebrating 20 Years of Genetic Discoveries in Legume Nodulation and Symbiotic Nitrogen Fixation

“…Leghemoglobins are essential for buffering oxygen in nodules at low levels while transporting it rapidly to rhizobia. RNAi and CRISPR/Cas9 of LjLB1, LjLB2, and LjLB3 resulted in nodules with elevated oxygen concentrations but reduced ATP:ADP ratios and defective SNF (Ott et al, 2005;Wang et al, 2019). Importantly, nif genes (nifH) were not induced in these nodules (Ott et al, 2009).…”

Plant hemoglobins: a journey from unicellular green algae to vascular plants

“…The mutant nodules showed an increase in the internal free O 2 concentration and alterations in the expression of many genes, including bacterial nif and fix (Ott et al , ). Subsequent work using CRISPR/Cas9 to obtain single, double and triple ( lb123 ) mutants concluded that the three Lbs act additively to maintain optimal N 2 fixation (Wang et al , ). This study also provided RNA‐sequencing information of c. 20 000 genes of the lb123 nodules.…”

“…This study also provided RNA‐sequencing information of c. 20 000 genes of the lb123 nodules. These Lb‐free nodules showed cell vacuolization, accumulation of poly‐β‐hydroxybutyrate, disruption of mitochondria and enhanced concentrations of superoxide and peroxide (Wang et al , ).…”

Molecular Basis of Root Nodule Symbiosis between Bradyrhizobium and ‘Crack-Entry’ Legume Groundnut (Arachis hypogaea L.)

“…With the advent of newer technologies like genome editing, manipulating genome structures has become easier. Genome editing tools like CRISPR/Cas9 were employed to knockout leghemoglobin (Lbs) genes in L. japonicus, and uncovered their synergistic roles in symbiotic nitrogen fixation [128]. This study concluded that Lbs acts additively in nodules and the lack of Lbs results in early nodule senescence.…”

“…This study concluded that Lbs acts additively in nodules and the lack of Lbs results in early nodule senescence. It also provided insight into the reprogramming of the gene expression network in the absence of Lbs, probably as a result of an uncontrolled free O 2 concentration [128]. Various genomics approaches and biotechnology efforts in this direction will help us to learn more about the molecular basis of nodulation at the genomic/proteomic/metabolomic level, which would effectively translate into major gains in research and contribute to more sustainable agriculture (Figure 4).…”