Characterization of the heme pocket structure and ligand binding kinetics of non-symbiotic hemoglobins from the model legume lotus japonicus

Plant hemoglobins (Hbs) are found in nodules of legumes and actinorhizal plants but also in non-symbiotic organs of monocots and dicots. Non-symbiotic Hbs (nsHbs) have been classified into two phylogenetic groups. Class 1 nsHbs show an extremely high O2 affinity and are induced by hypoxia and nitric oxide (NO), whereas class 2 nsHbs have moderate O2 affinity and are induced by cold and cytokinins. The functions of nsHbs are still unclear, but some of them rely on the capacity of hemes to bind diatomic ligands and catalyze the NO dioxygenase (NOD) reaction (oxyferrous Hb + NO ? ferric Hb + nitrate). Moreover, NO may nitrosylate Cys residues of proteins. It is therefore important to determine the ligand binding properties of the hemes and the role of Cys residues. Here, we have addressed these issues with the two class 1 nsHbs (LjGlb1-1 and LjGlb1-2) and the single class 2 nsHb (LjGlb2) of Lotus japonicus, which is a model legume used to facilitate the transfer of genetic and biochemical information into crops. We have employed carbon monoxide (CO) as a model ligand and resonance Raman, laser flash photolysis, and stopped-flow spectroscopies to unveil major differences in the heme environments and ligand binding kinetics of the three proteins, which suggest non-redundant functions. In the deoxyferrous state, LjGlb1-1 is partially hexacoordinate, whereas LjGlb1-2 shows complete hexacoordination (behaving like class 2 nsHbs) and LjGlb2 is mostly pentacoordinate (unlike other class 2 nsHbs). LjGlb1-1 binds CO very strongly by stabilizing it through hydrogen bonding, but LjGlb1-2 and LjGlb2 show lower CO stabilization. The changes in CO stabilization would explain the different affinities of the three proteins for gaseous ligands. These affinities are determined by the dissociation rates and follow the order LjGlb1-1 > LjGlb1-2 > LjGlb2. Mutations LjGlb1-1 C78S and LjGlb1-2 C79S caused important alterations in protein dynamics and stability, indicating a structural role of those Cys residues, whereas mutation LjGlb1-1 C8S had a smaller effect. The three proteins and their mutant derivatives exhibited similarly high rates of NO consumption, which were due to NOD activity of the hemes and not to nitrosylation of Cys residues

Characterization of genomic clones and expression analysis of the three types of superoxide dismutases during nodule development in Lotus japonicus

Animal science

Single cell-type transcriptome profiling reveals genes that promote nitrogen fixation in the infected and uninfected cells of legume nodules

2 Pags.- 1 Fig. © 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use,distribution and reproduction in any medium, provided the original work is properly cited.Excessive application of nitrogen fertilizers has inevitably resultedin environmental problems. The symbiotic nitrogen fixation (SNF) that occurs in the root nodules of leguminous plants provides asustainable source of reduced nitrogen in agricultural ecosystems. More than 200 genes have been reported to regulate SNF, including rhizobial infection, nodule organogenesis and senescence (Royet al., 2020). Mature nodules consist mainly of twocell types: infected cells (IC) that contain nitrogen-fixing bac-teroids and uninfected cells (UC) that mediate active metabolismand nutrient transport. Although it is well known that SNFrequires functional specialization, the specific genes responsiblefor transcriptional regulation and carbon/nitrogen metabolismand transport in IC and UC remain largely unexplored.Single-cell transcriptomics has emerged as a powerful tech-nique for investigating spatiotemporal patterns of gene expression.This work was supported by the National Natural Science Foundation of China (31870220, 32000192), the China Post-doctoral Science Foundation (2020M680103), Fundamental Research Funds for the Central Universities 2662020SKPY007 and MCIN/AEI/10.13039/501100011033 (grant PID2020-113985GB-I00)Peer reviewe