Abstract
Thus far, research on plant hemoglobins (Hbs) has mainly concentrated on symbiotic and non-symbiotic Hbs, and information on truncated Hbs (TrHbs) is scarce. The aim of this study was to examine the origin, structure and localization of the truncated Hb (PttTrHb) of hybrid aspen (Populus tremula L. × tremuloides Michx.), the model system of tree biology. Additionally, we studied the PttTrHb expression in relation to non-symbiotic class1 Hb gene (PttHb1) using RNAi-silenced hybrid aspen lines. Both the phylogenetic analysis and the three-dimensional (3D) model of PttTrHb supported the view that plant TrHbs evolved vertically from a bacterial TrHb. The 3D model suggested that PttTrHb adopts a 2-on-2 sandwich of α-helices and has a Bacillus subtilis -like ligand-binding pocket in which E11Gln and B10Tyr form hydrogen bonds to a ligand. However, due to differences in tunnel cavity and gate residue (E7Ala), it might not show similar ligand-binding kinetics as in Bs-HbO (E7Thr). The immunolocalization showed that PttTrHb protein was present in roots, stems as well as leaves of in vitro -grown hybrid aspens. In mature organs, PttTrHb was predominantly found in the vascular bundles and specifically at the site of lateral root formation, overlapping consistently with areas of nitric oxide (NO) production in plants. Furthermore, the NO donor sodium nitroprusside treatment increased the amount of PttTrHb in stems. The observed PttTrHb localization suggests that PttTrHb plays a role in the NO metabolism.
Highlights
Hemoglobins (Hbs) are ubiquitous in all living organisms
We suggested that in hybrid aspen, PttTrHb may be involved in the modulation of nitric oxide (NO) levels in early reactions involved in root growth, in response to symbiosis with the ectomycorrhizal fungi [16]
The analysis revealed that the amount of PttTrHb increased in cortex and epidermis compared with the plants grown in normal conditions (Fig. 5G)
Summary
Hemoglobins (Hbs) are ubiquitous in all living organisms. It has been suggested that eukaryotes have acquired all their globins via horizontal gene transfer concomitant with the endosymbiotic events that are responsible for the origin of mitochondria and chloroplast [1]. Three kinds of Hbs have been identified: symbiotic (or leghemoglobin), non-symbiotic (divided into groups 1 or 2) and, most recently, truncated Hb (TrHb or GLB3, [2]). Bacterial TrHbs are small hemoproteins with amino acid sequences 20–40 residues shorter than (non)vertebrate Hbs. Because of the deletions throughout the sequence, the three-dimensional (3D) structure of the bacterial TrHb family is a 2-on-2 arrangement of a-helices, compared with the classical 3-on-3 globin fold. The group II TrHbs can be further subdivided into four lineages: Actinobacteria, Proteobacteria, Firmicutes and plants [6,7]. The 3D structures of Mycobacterium tuberculosis HbO (MtHbO, [8]) and Thermobifida fusca HbO (Tf-HbO, [9]) from Actinobacteria, Bacillus subtilis HbO (Bs-HbO, [10]) and Geobacillus stearothermophilus HbO (Gs-HbO, [11]) from Firmicutes, and Agrobacterium tumefaciens HbO (Atu-HbO, [12]) from Proteobacteria have been characterized. No crystal structure for plant group II TrHb has been solved
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