Phytoglobin: a novel nomenclature for plant globins accepted by the globin community at the 2014 XVIII conference on Oxygen-Binding and Sensing Proteins.

Hemoglobin (Hb) is a heme-containing protein found in the red blood cells of vertebrates. For many years, the only known Hb-like molecule in plants was leghemoglobin (Lb). The discovery that other Hb-like proteins existed in plants led to the term "nonsymbiotic Hbs (nsHbs)" to differentiate them from the Lbs. While this terminology was adequate in the early stages of research on the protein, the complexity of the research in this area necessitates a change in the definition of these proteins to delineate them from red blood cell Hb. At the 2014 XVIII Conference on Oxygen-Binding and Sensing Proteins, the group devoted to the study of heme-containing proteins, this issue was discussed and a consensus was reached on a proposed name change. We propose Phytoglobin (Phytogb) as a logical, descriptive name to describe a heme-containing (Hb-like) protein found in plants. It will be readily recognized by the research community without a prolonged explanation of the origin of the term. The classification system that has been established can essentially remain unchanged substituting Phytogb in place of nsHb. Here, we present a guide to the new nomenclature, with reference to the existing terminology and a phylogenetic scheme, placing the known Phytogbs in the new nomenclature.

A bioinformatics insight to rhizobial globins: gene identification and mapping, polypeptide sequence and phenetic analysis, and protein modeling.

Globins (Glbs) are proteins widely distributed in organisms. Three evolutionary families have been identified in Glbs: the M, S and T Glb families. The M Glbs include flavohemoglobins (fHbs) and single-domain Glbs (SDgbs); the S Glbs include globin-coupled sensors (GCSs), protoglobins and sensor single domain globins, and the T Glbs include truncated Glbs (tHbs). Structurally, the M and S Glbs exhibit 3/3-folding whereas the T Glbs exhibit 2/2-folding. Glbs are widespread in bacteria, including several rhizobial genomes. However, only few rhizobial Glbs have been characterized. Hence, we characterized Glbs from 62 rhizobial genomes using bioinformatics methods such as data mining in databases, sequence alignment, phenogram construction and protein modeling. Also, we analyzed soluble extracts from Bradyrhizobium japonicum USDA38 and USDA58 by (reduced + carbon monoxide (CO) minus reduced) differential spectroscopy. Database searching showed that only fhb, sdgb, gcs and thb genes exist in the rhizobia analyzed in this work. Promoter analysis revealed that apparently several rhizobial glb genes are not regulated by a -10 promoter but might be regulated by -35 and Fnr (fumarate-nitrate reduction regulator)-like promoters. Mapping analysis revealed that rhizobial fhbs and thbs are flanked by a variety of genes whereas several rhizobial sdgbs and gcss are flanked by genes coding for proteins involved in the metabolism of nitrates and nitrites and chemotaxis, respectively. Phenetic analysis showed that rhizobial Glbs segregate into the M, S and T Glb families, while structural analysis showed that predicted rhizobial SDgbs and fHbs and GCSs globin domain and tHbs fold into the 3/3- and 2/2-folding, respectively. Spectra from B. japonicum USDA38 and USDA58 soluble extracts exhibited peaks and troughs characteristic of bacterial and vertebrate Glbs thus indicating that putative Glbs are synthesized in B. japonicum USDA38 and USDA58.

Effect of the synthesis of rice non-symbiotic hemoglobins 1 and 2 in the recombinant Escherichia coli TB1 growth.

Non-symbiotic hemoglobins (nsHbs) are widely distributed in land plants, including rice. These proteins are classified into type 1 (nsHbs-1) and type 2. The O 2-affinity of nsHbs-1 is very high mostly because of an extremely low O 2-dissociation rate constant resulting in that nsHbs-1 apparently do not release O 2 after oxygenation. Thus, it is possible that the in vivo function of nsHbs-1 is other than O 2-transport. Based on the properties of multiple Hbs it was proposed that nsHbs-1 could play diverse roles in rice organs, however the in vivo activity of rice nsHbs-1 has been poorly analyzed. An in vivo analysis for rice nsHbs-1 is essential to elucidate the biological function(s) of these proteins. Rice Hb1 and Hb2 are nsHbs-1 that have been generated in recombinant Es cherichia coli TB1. The rice Hb1 and Hb2 amino acid sequence, tertiary structure and rate and equilibrium constants for the reaction of O 2 are highly similar. Thus, it is possible that rice Hb1 and Hb2 function similarly in vivo. As an initial approach to test this hypothesis we analyzed the effect of the synthesis of rice Hb1 and Hb2 in the recombinant E. coli TB1 growth. Effect of the synthesis of the O 2-carrying soybean leghemoglobin a, cowpea leghemoglobin II and Vitreoscilla Hb in the recombinant E. coli TB1 growth was also analyzed as an O 2-carrier control. Our results showed that synthesis of rice Hb1, rice Hb2, soybean Lb a, cowpea LbII and Vitreoscilla Hb inhibits the recombinant E. coli TB1 growth and that growth inhibition was stronger when recombinant E. coli TB1 synthesized rice Hb2 than when synthesized rice Hb1. These results suggested that rice Hb1 and Hb2 could function differently in vivo.

Rice ( Oryza) hemoglobins.

Hemoglobins (Hbs) corresponding to non-symbiotic (nsHb) and truncated (tHb) Hbs have been identified in rice ( Oryza). This review discusses the major findings from the current studies on rice Hbs. At the molecular level, a family of the nshb genes, consisting of hb1, hb2, hb3, hb4 and hb5, and a single copy of the thb gene exist in Oryza sativa var. indica and O. sativa var. japonica, Hb transcripts coexist in rice organs and Hb polypeptides exist in rice embryonic and vegetative organs and in the cytoplasm of differentiating cells. At the structural level, the crystal structure of rice Hb1 has been elucidated, and the structures of the other rice Hbs have been modeled. Kinetic analysis indicated that rice Hb1 and 2, and possibly rice Hb3 and 4, exhibit a very high affinity for O 2, whereas rice Hb5 and tHb possibly exhibit a low to moderate affinity for O 2. Based on the accumulated information on the properties of rice Hbs and data from the analysis of other plant and non-plant Hbs, it is likely that Hbs play a variety of roles in rice organs, including O 2-transport, O 2-sensing, NO-scavenging and redox-signaling. From an evolutionary perspective, an outline for the evolution of rice Hbs is available. Rice nshb and thb genes vertically evolved through different lineages, rice nsHbs evolved into clade I and clade II lineages and rice nshbs and thbs evolved under the effect of neutral selection. This review also reveals lacunae in our ability to completely understand rice Hbs. Primary lacunae are the absence of experimental information about the precise functions of rice Hbs, the properties of modeled rice Hbs and the cis-elements and trans-acting factors that regulate the expression of rice hb genes, and the partial understanding of the evolution of rice Hbs.

Soybean dihydrolipoamide dehydrogenase (ferric leghemoglobin reductase 2) interacts with and reduces ferric rice non-symbiotic hemoglobin 1.

Ferrous oxygenated hemoglobins (Hb2+O2) autoxidize to ferric Hb3+, but Hb3+ is reduced to Hb2+ by enzymatic and non-enzymatic mechanisms. We characterized the interaction between the soybean ferric leghemoglobin reductase 2 (FLbR2) and ferric rice non-symbiotic Hb1 (Hb13+). Spectroscopic analysis showed that FLbR2 reduces Hb13+. Analysis by tryptophan fluorescence quenching showed that FLbR2 interacts with Hb13+, however the use of ITC and IEF techniques revealed that this interaction is weak. In silico modeling showed that predicted FLbR2 and native Hb13+ interact at the FAD-binding domain of FLbR2 and the CD-loop and helix F of Hb13+.

Variability of non-symbiotic and truncated hemoglobin genes from the genome of cultivated monocots.

Non-symbiotic (nsHb) and truncated (tHb) hemoglobins (Hbs) have been detected in a variety of land plants. The evolution of land plant nsHbs and tHbs at the protein level is well documented; however, little is known about the evolution of genes coding for these proteins. For example, the variability of the land plant nshb and thb genes is not known. Here, we report the variability of the nshb and thb genes from the genome of the cultivated monocots Brachypodium distachyon, Hordeum vulgare (barley), Oryza glaberrima (rice), O. rufipogon (rice), O. sativa (rice) var indica, O. sativa (rice) var japonica, Panicum virgatum (switchgrass), Setaria italica (foxtail millet), Sorghum bicolor (sorghum), Triticum aestivum (wheat), and Zea mays ssp. mays (maize) using sequence comparison and computational methods. Our results revealed that in cultivated monocots variability is higher in nshbs than in thbs, and suggest that major substitution events that occurred during the evolution of the cultivated monocot hbs were A→G and T→C transitions and that these genes evolved under the effect of neutral selection.

The evolution of land plant hemoglobins.

This review discusses the evolution of land plant hemoglobins within the broader context of eukaryote hemoglobins and the three families of bacterial globins. Most eukaryote hemoglobins, including metazoan globins and the symbiotic and non-symbiotic plant hemoglobins, are homologous to the bacterial 3/3-fold flavohemoglobins. The remaining plant hemoglobins are homologous to the bacterial 2/2-fold group 2 hemoglobins. We have proposed that all eukaryote globins were acquired via horizontal gene transfer concomitant with the endosymbiotic events responsible for the origin of mitochondria and chloroplasts. Although the 3/3 hemoglobins originated in the ancestor of green algae and plants prior to the emergence of embryophytes at about 450 mya, the 2/2 hemoglobins appear to have originated via horizontal gene transfer from a bacterium ancestral to present day Chloroflexi. Unlike the 2/2 hemoglobins, the evolution of the 3/3 hemoglobins was accompanied by duplication, diversification, and functional adaptations. Duplication of the ancestral plant nshb gene into the nshb-1 and nshb-2 lineages occurred prior to the monocot-dicot divergence at ca. 140 mya. It was followed by the emergence of symbiotic hemoglobins from a non-symbiotic hemoglobin precursor and further specialization, leading to leghemoglobins in N2-fixing legume nodules concomitant with the origin of nodulation at ca. 60 mya. The transition of non-symbiotic to symbiotic hemoglobins (including to leghemoglobins) was accompanied by the alteration of heme-Fe coordination from hexa- to penta-coordination. Additional genomic information about Charophyte algae, the sister group to land plants, is required for the further clarification of plant globin phylogeny.

Spectroscopic analysis of moss (Ceratodon purpureus and Physcomitrella patens) recombinant non-symbiotic hemoglobins.

Non-symbiotic hemoglobins (nsHbs) are O(2)-binding proteins widely distributed in land plants, including primitive bryophytes. Little is known about the properties of bryophyte nsHbs. Here, we report the spectroscopic characterization of two moss recombinant nsHbs, CerpurnsHb of Ceratodon purpureus and PhypatnsHb of Physcomitrella patens. Spectra showed that the absorption maxima of the ferrous and ferric forms of recombinant CerpurnsHb are located at 418, 531 and 557 nm and 407, 537, 569 (shoulder) and 632 (shoulder) nm, respectively, and of PhypatnsHb are located at 422, 529 and 557 nm and 407, 531, 571 (shoulder) and 647 (shoulder) nm, respectively. These absorption maxima are similar to those of rice Hb1. Also, the absorption maxima of the oxygenated ferrous form of recombinant CerpurnsHb and PhypatnsHb are located at 412, 541 and 575 nm and 414, 541 and 574 nm, respectively, similar to those of oxygenated rice Hb1 and cowpea leghemoglobin II. This evidence indicates that CerpurnsHb and PhypatnsHb are mostly hexacoordinate and that they bind O(2).

What are the origins and phylogeny of plant hemoglobins?

Land plants and algae are now represented by about 40 genomes. Although most are incomplete, putative globins appear to be present in all the ca. 30 land plant genomes and in all except one algal genomes. The globins have either the canonical 3/3 α-helical fold characteristic of vertebrate myoglobin (Mb) or 2/2 α-helical folds, characteristic of bacterial globins with a truncated Mb-fold. In view of the fairly complete picture of the globin superfamily that is now available from analyses of over 1,000 bacterial genomes and >200 other eukaryote genomes, it is now possible to seek answers to the following twin questions: what is the phylogenetic relationship of plant and algal globins to those of other eukaryotes and what is their likely bacterial origin? We summarize below the available results. Molecular phylogenetic analyses indicate that plant and algal 3/3 globins are related to bacterial flavohemoglobins and vertebrate neuroglobins. Furthermore, they also suggest that plant and algal 3/3 and group 1 2/2 Hbs originated from the horizontal gene transfers that accompanied the two generally accepted endosymbioses of a proteobacterium and a cyanobacterium with a eukaryote ancestor. In contrast, the origin of the group 2 2/2 Hbs unexpectedly appears to involve horizontal gene transfer from a bacterium ancestral to Chloroflexi, Deinococcales, Bacillli and Actinomycetes. We present additional results which indicate that the shared ancestry is likely to be with the Chloroflexi alone.

Expression of non-symbiotic hemoglobin 1 and 2 genes in rice (Oryza sativa) embryonic organs.

Rice (Oryza sativa) contains five copies of the non-symbiotic hemoglobin (hb) gene, namely hb1 to hb5. Previous analysis by RT-PCR revealed that rice hb1 expresses in roots and leaves and hb2 expresses in leaves. However, it is not known whether or not hb1 and hb2 express in rice embryonic organs. Here, we report the expression of hb1 and hb2 genes in rice embryonic organs using RT-PCR and specific oligos for Hb1 and Hb2. Our results indicate that hb1 and hb2 genes express in embryonic organs in rice growing under normal conditions. Specifically, hb1 expresses in rice embryos and seminal roots, and hb2 expresses in embryos, coleoptiles and seminal roots. These observations suggest that Hb1 and Hb2 coexist and function in rice embryonic organs.

Expression and localization of a Rhizobium-derived cambialistic superoxide dismutase in pea (Pisum sativum) nodules subjected to oxidative stress.

Two phylogenetically unrelated superoxide dismutase (SOD) families, i.e., CuZnSOD (copper and zinc SOD) and FeMn-CamSOD (iron, manganese, or cambialistic SOD), eliminate superoxide radicals in different locations within the plant cell. CuZnSOD are located within the cytosol and plastids, while the second family of SOD, which are considered to be of bacterial origin, are usually located within organelles, such as mitochondria. We have used the reactive oxygen species-producer methylviologen (MV) to study SOD isozymes in the indeterminate nodules on pea (Pisum sativum). MV caused severe effects on nodule physiology and structure and also resulted in an increase in SOD activity. Purification and N-terminal analysis identified CamSOD from the Rhizobium leguminosarum endosymbiont as one of the most active SOD in response to the oxidative stress. Fractionation of cell extracts and immunogold labeling confirmed that the CamSOD was present in both the bacteroids and the cytosol (including the nuclei, plastids, and mitochondria) of the N-fixing cells, and also within the uninfected cortical and interstitial cells. These findings, together with previous reports of the occurrence of FeSOD in determinate nodules, indicate that FeMnCamSOD have specific functions in legumes, some of which may be related to signaling between plant and bacterial symbionts, but the occurrence of one or more particular isozymes depends upon the nodule type.

Phylogenetic relationships of 3/3 and 2/2 hemoglobins in Archaeplastida genomes to bacterial and other eukaryote hemoglobins.

Land plants and algae form a supergroup, the Archaeplastida, believed to be monophyletic. We report the results of an analysis of the phylogeny of putative globins in the currently available genomes to bacterial and other eukaryote hemoglobins (Hbs). Archaeplastida genomes have 3/3 and 2/2 Hbs, with the land plant genomes having group 2 2/2 Hbs, except for the unexpected occurrence of two group 1 2/2 Hbs in Ricinus communis. Bayesian analysis shows that plant 3/3 Hbs are related to vertebrate neuroglobins and bacterial flavohemoglobins (FHbs). We sought to define the bacterial groups, whose ancestors shared the precursors of Archaeplastida Hbs, via Bayesian and neighbor-joining analyses based on COBALT alignment of representative sets of bacterial 3/3 FHb-like globins and group 1 and 2 2/2 Hbs with the corresponding Archaeplastida Hbs. The results suggest that the Archaeplastida 3/3 and group 1 2/2 Hbs could have originated from the horizontal gene transfers (HGTs) that accompanied the two generally accepted endosymbioses of a proteobacterium and a cyanobacterium with a eukaryote ancestor. In contrast, the origin of the group 2 2/2 Hbs unexpectedly appears to involve HGT from a bacterium ancestral to Chloroflexi, Deinococcales, Bacilli, and Actinomycetes. Furthermore, although intron positions and phases are mostly conserved among the land plant 3/3 and 2/2 globin genes, introns are absent in the algal 3/3 genes and intron positions and phases are highly variable in their 2/2 genes. Thus, introns are irrelevant to globin evolution in Archaeplastida.

Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants.

In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H2O2 system at pH 6.0 and compared it to that from horseradish peroxidase (HRP). Results showed that the affinity of rice Hb1 for H2O2 was 86-times lower than that of HRP (K(m)=23.3 and 0.27 mM, respectively) and that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H2O2 as electron donor was 2838-times lower than that of HRP (k(cat)/K(m)=15.8 and 44,833 mM(-1) min(-1), respectively). Also, results from this work showed that rice Hb1 is not chemically modified and binds CO after incubation with high H2O2 concentration, and that it poorly protects recombinant Escherichia coli from H2O2 stress. These observations indicate that rice Hb1 inefficiently scavenges H2O2 as compared to a typical plant peroxidase, thus indicating that non-symbiotic Hbs are unlikely to function as peroxidases in planta.

Expression and in silico structural analysis of a rice (Oryza sativa) hemoglobin 5.

This work reports the analysis of an additional hemoglobin (hb) gene copy, hb5, in the genome of rice. The amino acid sequence of Hb5 differs from the previously determined rice Hbs 1-4 in missing 11 residues in helix E. Transcripts of hb5 were found to be ubiquitous in rice organs, and hormone- and stress-response promoters exist upstream of the rice hb5 gene. Furthermore, the modeled structure of Hb5 based on the known crystal structure of rice Hb1 is unusual in that the putative distal His is distant from the heme Fe. This observation suggests that Hb5 binds and releases O(2) easily and thus that it functions as an O(2)-carrier or in some aspects of the O(2) metabolism.

Molecular cloning and characterization of a moss (Ceratodon purpureus) nonsymbiotic hemoglobin provides insight into the early evolution of plant nonsymbiotic hemoglobins.

Nonsymbiotic hemoglobins (nsHbs) are widespread in plants including bryophytes. Bryophytes (such as mosses) are among the oldest land plants, thus an analysis of a bryophyte nsHb is of interest from an evolutionary perspective. However, very little is known about bryophyte nsHbs. Here, we report the cloning and characterization of an nshb gene (cerhb) from the moss Ceratodon purpureus. Sequence analysis showed that cerhb is interrupted by 3 introns in identical position as all known plant nshb genes, which suggests that the ancestral nshb gene was interrupted by 3 introns. Expression analysis showed that cerhb expresses in protonemas and gametophytes growing in normal conditions and that it overexpresses in protonemas subjected to osmotic (sucrose), heat-shock, cold-, and nitrate-stress conditions. Also, modeling of the Ceratodon nsHb (CerHb) tertiary structure suggests that CerHb is hexacoordinate and that it binds O(2) with high affinity. Comparative analysis of the predicted CerHb with native rice Hb1 and soybean leghemoglobin a structures revealed that the major evolutionary changes that probably occurred during the evolution of plant Hbs were 1) a hexacoordinate to pentacoordinate transition at the heme prosthetic group, 2) a length decrease at the CD-loop and N- and C-termini regions, and 3) the compaction of the protein into a globular structure.

Use of in silico (computer) methods to predict and analyze the tertiary structure of plant hemoglobins.

Amino acid sequences for more than 60 plant hemoglobins (Hbs) are deposited in databases, but the tertiary structure of only 4 plant Hbs have been reported; thus, the gap between the reported sequences and structures of plant Hbs is large. Elucidating the structure of plant Hbs is essential to fully understanding the function of these proteins in plant cells. Determining the actual protein structure by experimental methods (i.e., by X-ray crystallography) requires considerable protein material and is expensive; thus, this type of work is limited to few laboratories around the world. In silico (computer) methods to predict the tertiary structure of proteins from amino acid sequences have been implemented and are helping reduce the sequence-structure gap. Thus, in silico methods are useful tools for predicting the tertiary structure of several plant Hbs from amino acid sequences deposited in databases. In this chapter, we describe a method for predicting and analyzing the structure of a rice Hb2 from the template structure of native rice Hb1. This method is based on a comparative modeling method that uses programs from the SWISS-MODEL server.

A self-induction method to produce high quantities of recombinant functional flavo-leghemoglobin reductase.

Ferric leghemoglobin reductase (FLbR) is able to reduce ferric leghemoglobin (Lb3+) to ferrous (Lb2+) form. This reaction makes Lb functional in performing its role since only reduced hemoglobins bind O2. FLbR contains FAD as prosthetic group to perform its activity. FLbR-1 and FLbR-2 were isolated from soybean root nodules and it has been postulated that they reduce Lb3+. The existence of Lb2+ is essential for the nitrogen fixation process that occurs in legume nodules; thus, the isolation of FLbR for the study of this enzyme in the nodule physiology is of interest. However, previous methods for the production of recombinant FLbR are inefficient as yields are too low. We describe the production of a recombinant FLbR-2 from Escherichia coli BL21(DE3) by using an overexpression method based on the self-induction of the recombinant E. coli. This expression system is four times more efficient than the previous overexpression method. The quality of recombinant FLbR-2 (based on spectroscopy, SDS-PAGE, IEF, and native PAGE) is comparable to that of the previous expression system. Also, FLbR-2 is purified near to homogeneity in only few steps (in a time scale, the full process takes 3 days). The purification method involves affinity chromatography using a Ni-nitrilotriacetic acid column. Resulting rFLbR-2 showed an intense yellow color, and spectral characterization of rFLbR-2 indicated that rFLbR-2 contains flavin. Pure rFLbR-2 was incubated with soybean Lba and NADH, and time drive rates showed that rFLbR-2 efficiently reduces Lb3+.

Cloning and characterization of a caesalpinoid (Chamaecrista fasciculata) hemoglobin: the structural transition from a nonsymbiotic hemoglobin to a leghemoglobin.

Nonsymbiotic hemoglobins (nsHbs) and leghemoglobins (Lbs) are plant proteins that can reversibly bind O(2) and other ligands. The nsHbs are hexacoordinate and appear to modulate cellular concentrations of NO and maintain energy levels under hypoxic conditions. The Lbs are pentacoordinate and facilitate the diffusion of O(2) to symbiotic bacteroids within legume root nodules. Multiple lines of evidence suggest that all plant Hbs evolved from a common ancestor and that Lbs originated from nsHbs. However, little is known about the structural intermediates that occurred during the evolution of pentacoordinate Lbs from hexacoordinate nsHbs. We have cloned and characterized a Hb (ppHb) from the root nodules of the ancient caesalpinoid legume Chamaecrista fasciculata. Protein sequence, modeling data, and spectral analysis indicated that the properties of ppHb are intermediate between that of nsHb and Lb, suggesting that ppHb resembles a putative ancestral Lb. Predicted structural changes that appear to have occurred during the nsHb to Lb transition were a compaction of the CD-loop and decreased mobility of the distal His inhibiting its ability to coordinate directly with the heme-Fe, leading to a pentacoordinate protein. Other predicted changes include shortening of the N- and C-termini, compaction of the protein into a globular structure, disappearance of positive charges outside the heme pocket and appearance of negative charges in an area located between the N- and C-termini. A major consequence for some of these changes appears to be the decrease in O(2)-affinity of ancestral nsHb, which resulted in the origin of the symbiotic function of Lbs.

Plant hemoglobins: what we know six decades after their discovery.

This review describes contributions to the study of plant hemoglobins (Hbs) from a historical perspective with emphasis on non-symbiotic Hbs (nsHbs). Plant Hbs were first identified in soybean root nodules, are known as leghemoglobins (Lbs) and have been characterized in detail. It is widely accepted that a function of Lbs in nodules is to facilitate the diffusion of O(2) to bacteroids. For many years Hbs could not be identified in plants other than N(2)-fixing legumes, however in the 1980s a Hb was isolated from the nodules of the non-legume dicot plant Parasponia, a hb gene was cloned from the non-nodulating Trema, and Hbs were detected in nodules of actinorhizal plants. Gene expression analysis showed that Trema Hb transcripts exist in non-symbiotic roots. In the 1990s nsHb sequences were also identified in monocot and primitive (bryophyte) plants. In addition to Lbs and nsHbs, Hb sequences that are similar to microbial truncated (2/2) Hbs were also detected in plants. Plant nsHbs have been characterized in detail. These proteins have very high O(2)-affinities because of an extremely low O(2)-dissociation constant. Analysis of rice Hb1 showed that distal His coordinates heme Fe and stabilizes bound O(2); this means that O(2) is not released easily from oxygenated nsHbs. Non-symbiotic hb genes are expressed in specific plant tissues, and overexpress in organs of stressed plants. These observations suggest that nsHbs have functions additional to O(2)-transport, such as to modulate levels of ATP and NO.

A phylogenomic profile of globins.

BACKGROUND: Globins occur in all three kingdoms of life: they can be classified into single-domain globins and chimeric globins. The latter comprise the flavohemoglobins with a C-terminal FAD-binding domain and the gene-regulating globin coupled sensors, with variable C-terminal domains. The single-domain globins encompass sequences related to chimeric globins and "truncated" hemoglobins with a 2-over-2 instead of the canonical 3-over-3 alpha-helical fold. RESULTS: A census of globins in 26 archaeal, 245 bacterial and 49 eukaryote genomes was carried out. Only approximately 25% of archaea have globins, including globin coupled sensors, related single domain globins and 2-over-2 globins. From one to seven globins per genome were found in approximately 65% of the bacterial genomes: the presence and number of globins are positively correlated with genome size. Globins appear to be mostly absent in Bacteroidetes/Chlorobi, Chlamydia, Lactobacillales, Mollicutes, Rickettsiales, Pastorellales and Spirochaetes. Single domain globins occur in metazoans and flavohemoglobins are found in fungi, diplomonads and mycetozoans. Although red algae have single domain globins, including 2-over-2 globins, the green algae and ciliates have only 2-over-2 globins. Plants have symbiotic and nonsymbiotic single domain hemoglobins and 2-over-2 hemoglobins. Over 90% of eukaryotes have globins: the nematode Caenorhabditis has the most putative globins, approximately 33. No globins occur in the parasitic, unicellular eukaryotes such as Encephalitozoon, Entamoeba, Plasmodium and Trypanosoma. CONCLUSION: Although Bacteria have all three types of globins, Archaeado not have flavohemoglobins and Eukaryotes lack globin coupled sensors. Since the hemoglobins in organisms other than animals are enzymes or sensors, it is likely that the evolution of an oxygen transport function accompanied the emergence of multicellular animals.