Phytoglobin

Phytoglobins (abbreviated as Phytogbs) are ubiquitously distributed in plants as they have been identified in algae and land plants, including primitive bryophytes and evolved monocots and dicots. They can be classified as follows:[3][4]

• 2/2 Phytoglobins (truncated globin family, TrHb2 subfamily)
  • Phytogb3: found in algae and land plants.
• 3/3 Phytoglobins (myoglobin-like family)
  • Phytogb0: the basal type, found in algae, bryophytes and gymnosperms.[4]
  • Phytogb1, 2: only found in angiosperms.
    • Phytogb1: splits into two clades for monocots and dicots respectively. Dicot sub-"clade" is paraphyletic due to the exclusion of phytogb2.[3]
    • Phytogb2: exclusively found in dicots. Includes leghemoglobins (Lbs).[3]

Non-legume symbiotic globins (SymPhytogbs)[4] are scattered among Phytogb1 and Phytogb2.[3] Symbiotic globins generally provide oxygen to symbiotic bacteria that perform nitrogen-fixing. In legumes the bacteria are rhizobia, but in some actinorhizal plants actinomycete Frankia do the job instead.[3]

Phytogbs are coded by genes interrupted by 3 introns (although a 4 introns-containing phytogb gene has been detected in the moss Physcomitrella patens ).[5] The first and third intron of the phytogb genes are localized in the same position as that of the myoglobin genes, which suggests that phytogb and myoglobin genes evolved from a common ancestor more than 600 million years ago.[6] The existence of a second intron in the phytogb genes was predicted by Go using theoretical analysis,[7] which was further verified by cloning and sequencing of soybean lb genes by Marker and collaborators.[8]

Phytogbs are monomeric proteins whose molecular mass ranges from ~17 to ~19 kDa. However, at high (more than 1 mM) concentrations Phytogbs can form dimers. Phytogbs polypeptide chain folds into a particular arrangement of 6 to 7 helices (named with letters A to H) known as the globin fold which forms a hydrophobic pocket where heme is located. Two types of the globin fold have been identified in Phytogbs: the 3/3- and 2/2-folding,[9][10] where helices A, E and F overlap to helices B, G and H and helices B and E overlap to helices G and H, respectively.

Like other globins, heme-Fe in Phytogbs is coordinated at the proximal region by a His amino acid (named as proximal His). The distal region of heme-Fe can be occupied by either a variety of ligands (such as oxygen and nitric oxide) or a distal (frequently a His) amino acid, giving place to penta- or hexa-coordinate Phytogbs, respectively. The Phytogbs2, SymPhytogbs and Lbs are predominantly penta-coordinate whereas Phytogbs1 are predominantly hexa-coordinate and Phytogbs0 and Phytogbs3 are a combination of penta- and hexa-coordinate.[4] Heme-Fe coordination is essential for Phytogb (and other globins) function because it regulates the rate of ligand-binding and –releasing as consequence of the kinetic constants k_on and k_off, respectively. For example, the affinity of soybean Lb and rice Phytogb1 for O₂ (KO₂) is moderate and very high because k_on is 130 and 68 mM⁻¹ s⁻¹, k_off is 5.6 and 0.038 s⁻¹ and KO₂ (i.e. the O₂-affinity resulting from k_on/k_off) is 23 and 1800 mM⁻¹, respectively.[11] This indicates that soybean Lb could function as an O₂-storage or –transport protein and that the function of rice Phytogb1 (and other hexa-coordinate Phytogbs) could be other than O₂-transport because the high affinity of this protein for O₂ results from an extremely low k_off constant.[12]

Like other globins, penta-coordinate Phytogbs reversibly bind and transport O₂. The function of Lbs in nodules was elucidated in 1974 by Wittenberg, Appleby and others.[13] In nodules the concentration of Lbs is very high as they correspond to ~30% of the total soluble proteins. The apparent function of Lbs in nodules is to facilitate the diffusion of O₂ to the respiring bacteroids for nitrogen-fixation. At the same time, Lb contributes to maintain low O₂-levels (~10 nM) to avoid inactivation of the O₂-sensitive nitrogenase that fixes the atmospheric nitrogen.[14]

Furthermore, Phytogbs bind other gaseous ligands, most notably nitric oxide (NO), and exhibit a NO dioxygenase activity.[15] Work by Hill and collaborators during the last ~15 years has shown that levels of endogenous NO varies with the concentration of Phytogbs1 in transgenic maize and alfalfa.[16] Based on these observations, these authors have proposed that a function of oxygenated Phytogbs is to modulate levels of NO via an NO dioxygenase activity and to indirectly regulate a wide variety of cell functions that are modulated by levels of NO. Oxygenated class 1 phytoglobins reacting with NO to produce nitrate represent the main mechanism by which NO is scavenged in plants. The cycle involving nitrate reductase, reduction of nitrite to NO, scavenging NO by phytoglobin was defined as the phytoglobin-NO cycle.[17] Its operation leads to the maintenance of redox and energy status during hypoxia and results in the reduced production of ethanol and lactic acid.[18]

Phytogbs0, 1, 2 and 3 are synthesized at very low concentrations in diverse (embryonic and vegetative) plant organs.[19][20][21] However, concentrations of Phytogbs increase in plants subjected to specific stress conditions, such as flooding[22] and light-limitation.[23] Hence, some Phytogbs have been considered as plant stress-responsive proteins.

Work by Arredondo-Peter, Vinogradov and collaborators has elucidated the major events that could have occurred during the evolution of Phytogbs.[24][25][26] Although Phytogbs evolved from a common ancestor previous to the origin of eukaryotes (i.e. in the eubacteria domain), in plants two evolutionary lineages have been identified for Phytogbs: the (3/3-) Phytogb0, 1 and 2 and SymPhytogb and Lb lineage and the (2/2-) Phytogb3 lineage. Apparently, Phytogbs0 were the ancestors to Phytogbs1 and 2, and SymPhytogbs and Lbs evolved from a Phytogb1 ancestor as an adaptation to the symbiotic nitrogen-fixation. Comparative analysis of moss Phytogb0 with rice Phytogb1 and soybean Lb structure revealed that the major evolutionary changes that probably occurred during the evolution of Phytogbs0, 1 and 2, SymPhytogbs and Lbs were (i) a hexa-coordinate to penta-coordinate transition at the heme group, (ii) a length decrease at the CD-loop and N- and C-termini, and (iii) the compaction of the protein into a globular structure. Furthermore, the structure of the globin domain from Phytogbs3 is highly similar to that of bacterial truncated globins indicating that the 2/2-folding conserved during the evolution of Phytogbs3. However, globin domain from land plant Phytogbs3 is flanked by extra-amino acid sequences that probably originated in the ancestor to land plant Phytogbs3. The functional and evolutionary significance of the extra-amino acid sequences in the Phytogbs3 structure is still not known. 

Phytogbs are potentially useful for a number of biotechnological applications. For example, shortly after the discovery of rice Phytogbs1 an intended application was the use of these proteins as O₂-sensors in electronic devices. Also, over-expression of Phytogbs has been proposed as a strategy to increase crop tolerance to specific stress conditions, such as crop flooding,[27][28] as it couples the early flooding signal ethylene to the low oxygen response of plants.[29][30] Recently, Phytogbs were considered candidates for developing blood substitutes and as additives in veggie burgers.