Theses Doctoral

PhD student:

Irene Villar Rúa


Manuel Becana, Mª Carmen Rubio

Date of presentation:


Faculty and University:

Facultad de Ciencias, Universidad de Zaragoza

Qualification grade:

Sobresaliente cum laude


Structure and function of atypical hemoglobins from model legumes


In this thesis, we have characterized atypical leghemoglobins (Lbs) and nonsymbiotic hemoglobins (Glbs) of the model legumes Medicago truncatula and Lotus japonicus.

The first aim was to identify and characterize a class 1 Glb of M. truncatula, designated as MtGlb1-2 (Medtr4g068870). We found that the MtGlb1-2 gene is expressed in the meristems and vascular bundles of roots and nodules. This gene gives rise to four alternative spliced forms encoding proteins with one or two heme domains. To our knowledge, this is a unique case in plants. Using a combination of UV-visible, laser flash photolysis, resonance Raman and electron paramagnetic resonance spectroscopies, we characterized two of those proteins: MtGlb1-2.1 with two hemes and MtGlb1-2.4 with a single heme. To investigate heme function, we generated mutants of the two proteins in which the distal histidines were replaced by leucines. Our study showed that MtGlb1-2.1 and MtGlb1-2.4 display extreme reactivity toward physiological ligands such as molecular oxygen, nitric oxide (NO) and nitrite, as well as with the model ligand carbon monoxide. These unusual features led us to propose that MtGlb1-2 may act by either scavenging or producing NO, depending on the oxygen availability in the plant cells.

The second aim was to characterize MtLb3 (Medtr1g090810) of M. truncatula and LjGlb2-1 (Lj5g3v1699110) of L. japonicus. The LjGlb2-1 protein was annotated in the databases as Lb, but our results indicate that it is an anomalous Lb or a class 2 Glb. Our biochemical analyses showed that both LjGlb2-1 and MtLb3 have unusual properties in their interaction with NO and in their nitrite reductase (reduction of nitrite to NO in microaerobiosis) and NO dioxygenase (oxygenation of NO to nitrate in aerobiosis) activities. These are in between those described for Lbs and Glbs. Also, we phenotyped knockout mutant plants for each hemoglobin gene under symbiotic and nonsymbiotic conditions. This phenotyping indicated that MtLb3 may be irrelevant for plant growth, at least under normal conditions, whereas LjGlb2‑1 is important for plant growth and nodulation and is involved in the flowering and fruiting of nodulated plants.