Three classes of hemoglobins are required for optimal vegetative and reproductive growth of Lotus japonicus. Genetic and biochemical characterization of the atypical LjGlb2-1.

Legumes express two major types of hemoglobins: symbiotic (leghemoglobins) and non-symbiotic (phytoglobins). The latter are categorized into three classes according to phylogeny and biochemistry. Using knock-out mutants, we show that all three phytoglobin classes are required for optimal vegetative and reproductive development of Lotus japonicus. Furthermore, the mutants of two class 1 phytoglobins showed different phenotypes: Ljglb1-1 was smaller and had more pods, whereas Ljglb1-2 had no vegetative phenotype and produced fewer pods. Non-nodulated plants lacking LjGlb2-1 showed delayed growth and alterations in the leaf metabolome linked to amino acid processing, fermentative and respiratory pathways, and hormonal balance. The mutant leaves accumulated salicylic acid and contained less methyl jasmonic acid, suggesting cross-talk between LjGlb2-1 and the signaling pathways of both hormones. We conclude that LjGlb2-1 is not a leghemoglobin but an unusual class 2 phytoglobin, based on the expression of LjGlb2-1 in leaves, the alterations of flowering and fruiting of nodulated Ljglb2-1 plants, the developmental and biochemical phenotypes of the mutant fed on ammonium nitrate, and the heme coordination and reactivity of the protein toward nitric oxide. For comparison, we have characterized also a close relative of LjGlb2-1 in Medicago truncatula, MtLb3, and conclude that this is an atypical leghemoglobin.