Plant glutamyl-tRNA reductases coordinate plant and rhizobial heme biosynthesis in nitrogen-fixing nodules

Heme is biosynthesized in legume root nodules to meet the demand for leghemoglobins (Lbs) and other heme-binding proteins. However, the main source of nodule heme remains unknown. Both the plant host and rhizobia possess a complete heme biosynthetic pathway, differing slightly in the production of 5-aminolevulinic acid (ALA), a key regulatory step catalyzed by glutamyl-tRNA reductase (GluTR) in the plant and by HemA in the rhizobia. Transcriptomic analysis revealed that many plant heme biosynthetic genes, including GluTR2 but not GluTR1, are upregulated in nodules compared to roots, whereas expression of related rhizobial genes, including both HemA1 and HemA2, is generally inhibited under symbiotic conditions compared to free-living conditions. Knockout of Lotus japonicus GluTR2, but not of HemA1 and HemA2, led to a significant decrease (∼50%) in nodule heme content. The stable heterozygous mutant of GluTR1 or transient knockdown of GluTR1 exhibited a ∼20% reduction in nodule heme content. Overexpression of Fluorescent in blue light (FLU), a feedback inhibitor of GluTR activity, caused a much greater reduction in nodule heme content (∼75%) and an increased level of apo-Lb and, in combination with the hemA1 hemA2 mutant, a drastic inhibition of nitrogenase activity (>90%). This study provides genetic evidence supporting a major role of plant GluTRs in coordinating heme biosynthesis between the two symbionts by supplying heme to assemble with cytoplasmic apo-Lbs and by providing ALA for heme synthesis in the bacteroids.