A Plant Gene Encoding One-Heme and Two-Heme Hemoglobins With Extreme Reactivities Toward Diatomic Gases and Nitrite.

Irene Villar,Jesús I Martínez,Brandon Reeder,Manuel Becana,Stefania Abbruzzetti,Carmen Pérez-Rontomé,Michael T Wilson,Raul Huertas,Estíbaliz Larrainzar,Giulietta Smulevich,Lisa Milazzo,Michael Udvardi,Maria C Rubio

doi:10.3389/fpls.2020.600336

Abstract

In plants, symbiotic hemoglobins act as carriers and buffers of O2 in nodules, whereas nonsymbiotic hemoglobins or phytoglobins (Glbs) are ubiquitous in tissues and may perform multiple, but still poorly defined, functions related to O2 and/or nitric oxide (NO). Here, we have identified a Glb gene of the model legume Medicago truncatula with unique properties. The gene, designated MtGlb1-2, generates four alternative splice forms encoding Glbs with one or two heme domains and 215–351 amino acid residues. This is more than double the size of any hemoglobin from plants or other organisms described so far. A combination of molecular, cellular, biochemical, and biophysical methods was used to characterize these novel proteins. RNA-sequencing showed that the four splice variants are expressed in plant tissues. MtGlb1-2 is transcriptionally activated by hypoxia and its expression is further enhanced by an NO source. The gene is preferentially expressed in the meristems and vascular bundles of roots and nodules. Two of the proteins, bearing one or two hemes, were characterized using mutants in the distal histidines of the hemes. The Glbs are extremely reactive toward the physiological ligands O2, NO, and nitrite. They show very high O2 affinities, NO dioxygenase activity (in the presence of O2), and nitrite reductase (NiR) activity (in the absence of O2) compared with the hemoglobins from vertebrates and other plants. We propose that these Glbs act as either NO scavengers or NO producers depending on the O2 tension in the plant tissue, being involved in the fast and fine tuning of NO concentration in the cytosol in response to sudden changes in O2 availability.

Highlights

Hemoglobins are ubiquitously present in the archaea, bacteria, and eukaryotes (Vinogradov et al, 2005)
We show that the gene is transcriptionally activated by hypoxia and nitric oxide (NO) in roots, and its expression localized in the meristems and vascular bundles of roots and nodules
The expression of MtGlb1-2 is induced by low O2 and further enhanced in the presence of NO3− or GSNO (Figure 2) and the gene promoter is active in root and nodule tissues (Figure 3)

Summary

Introduction

Hemoglobins are ubiquitously present in the archaea, bacteria, and eukaryotes (Vinogradov et al, 2005). They typically comprise a heme prosthetic group and a polypeptide of 6–8 α-helices. Binding of O2 occurs exclusively with the Fe2+ of the heme, whereas NO can bind Fe2+ with high affinity and Fe3+ with low affinity. Hemoglobins can be classified structurally according to the axial coordination of the heme Fe. Pentacoordinate hemoglobins have a His residue at the fifth position (proximal) leaving the sixth position (distal) to be occupied by water or gaseous ligands, whereas hexacoordinate hemoglobins have a second His, or much less frequently Lys, Gln, or Tyr, at the sixth position (Becana et al, 2020)

Methods

Results

Conclusion