Plant Science 191–192 (2012) 71–81
Contents lists available at SciVerse ScienceDirect
Plant Science
journal homepage: www.elsevier.com/locate/plantsci
Review
The evolution of land plant hemoglobins
Consuelo Vázquez-Limón a , David Hoogewijs b , Serge N. Vinogradov c , Raúl Arredondo-Peter a,∗
a
Laboratorio de Biofísica y Biología Molecular, Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad Autónoma del Estado de Morelos, Av. Universidad
1001, Col. Chamilpa, 62210 Cuernavaca, Morelos, Mexico
b
Institute of Physiology and Zürich Center for Integrative Human Physiology (ZIHP), University of Zürich, Zürich, Switzerland
c
Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, MI 48201, USA
a r t i c l e
i n f o
Article history:
Received 13 March 2012
Received in revised form 24 April 2012
Accepted 25 April 2012
Available online 4 May 2012
Keywords:
Hemoglobin
Non-symbiotic
Leghemoglobin
Truncated
Land plants
Evolution
a b s t r a c t
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 3/3fold 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.
© 2012 Elsevier Ireland Ltd. All rights reserved.
Contents
1.
2.
3.
4.
5.
6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.
What is a globin?—a historical perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.
Diversity of globins in living organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.
Types and distribution of hemoglobins in land plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.4.
Properties and function of land plant hemoglobins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The phylogeny and evolution of land plant hemoglobins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Phylogeny of land plant hemoglobins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Ancient land plant non-symbiotic hemoglobins and the evolution of the non-symbiotic hemoglobin-leghemoglobin lineage . . . . . . . . . .
The origin of land plant hemoglobins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
The putative algal ancestor of land plant non-symbiotic hemoglobins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
The origin and evolution of algal and land plant truncated hemoglobins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rates of evolution of land plant hemoglobins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Going back (>3000 mya) to the (primeval) structural ancestor of 3/3 and 2/2 hemoglobins? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: Hb, hemoglobin; Lb, leghemoglobin; MYA, million of years ago; nsHb, non-symbiotic hemoglobin; nsHb-1, non-symbiotic hemoglobin type 1; nsHb-2,
non-symbiotic hemoglobin type 2; sHb, symbiotic hemoglobin; tHb, truncated (2/2) hemoglobin.
∗ Corresponding author. Tel.: +52 7773297000x3671/3383; fax: +52 7773297040.
E-mail addresses: [email protected], [email protected] (R. Arredondo-Peter).
0168-9452/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.plantsci.2012.04.013
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C. Vázquez-Limón et al. / Plant Science 191–192 (2012) 71–81
1. Introduction
1.1. What is a globin?—a historical perspective
Globins are proteins with a characteristic ␣-helical secondary
structure comprised of helices A−H, known as the myoglobin-fold,
and a heme group ensconced within a hydrophobic cavity formed
by a 3/3 sandwich of helices A, B, C, and E over helices F, G, and
H. Of the two heme-Fe axial sites, the proximal one is coordinated
to a His at position 8 of helix F, while the distal site can coordinate either with a side-chain group of residues located in helix E
or bind small molecule ligands, including O2 , CO, and NO. Historically, the familiar vertebrate O2 -binding hemoglobin, a tetramer
of ␣- and ␤-globins, and myoglobin were among the first proteins
whose sequences and structures were determined over 50 years
ago [1]. At that time, the hemoglobins in metazoans other than vertebrates were investigated mostly in cases where the hemoglobin
presence was visible. These included the larval hemoglobin of
the insect Chironomus [2] and the intracellular hemoglobin of the
annelid Glycera [3]. Comparison of several vertebrate and the invertebrate hemoglobin structures led to the recognition of a highly
conserved tertiary structure, the myoglobin-fold, underpinned by
the conservation of over 30, mostly solvent-inaccessible hydrophobic residues [4], even in cases of <20% identity to vertebrate globins.
The 3/3 ␣-helical myoglobin-fold is not unique: it is shared with
phycocyanins and other proteins [5]. The transport of O2 for aerobic
respiration is thought to be the major function of vertebrate globins
related to their ability to reversibly bind O2 [1]. However, evidence
has accrued over the last two decades indicating that both bacterial and eukaryote globins have enzymatic and sensing functions in
addition to O2 -transport and storage [6].
1.2. Diversity of globins in living organisms
The availability of numerous sequenced genomes over the past
20 years allowed the identification of globins in a wide variety
of organisms, ranging from bacteria to vertebrates. The bacterial globin superfamily encompasses three families/lineages that
belong to two structural classes: the 3/3- and 2/2-fold globins
(Fig. 1) [7,8]. The two globin families/lineages with the 3/3-fold
are the F family comprising the flavohemoglobins and related single domain globins [9], and the S (for sensor) family, encompassing
globin coupled sensors and protoglobins [10,11], and related single domain globins [12]. The third family consists of truncated
myoglobin-fold globins, with the 3/3-fold reduced to a 2/2-fold
due to a shortened or absent helix A and conversion of the F helix
into a loop (Fig. 1) [13–17]. The T family exists in three structurally
distinct subfamilies, T1−T3 [13–17].
Recent genomic information has also greatly extended the
structural and functional diversity of vertebrate globins through
the discovery of novel globins like neuroglobin and cytoglobin
[18,19], which are hexacoordinated [20,21], and perform yet-to-bedetermined functions in nerve and fibroblast-like cells, respectively
[22,23]. Furthermore, the identification of additional globins with
unknown physiological functions and restricted phyletic distributions, globin X in some protostomes and chordates [24], globin Y in
amphibians and monotreme mammals, and globin E the avian eye
globin has added complexity to vertebrate globin gene evolution
[25–29]. Phylogenetic analyses of these vertebrate globins revealed
that erythroid-specific globins have independently evolved O2 transport functions in different lineages [30]. Most recently, a new
metazoan globin lineage was discovered, consisting of large, ca.
1600 residues, chimeric proteins with an N-terminal cysteine protease domain and a central globin domain, named androglobins,
because of their specific expression in testis tissue [31].
All metazoan globins, vertebrate and non-vertebrate, symbiotic
and non-symbiotic plant globins, and many globins in microbial eukaryotes have the 3/3 ␣-helical fold and have sequences
that are homologous to the F family bacterial globins. T family
group 1 and 2 globins occur in microbial eukaryotes (ciliates, stramenopiles, oomycets, opisthokonts, etc.) and in plants [7]. Fungi
are unique in having only flavohemoglobins and S family single
domain globins [32]. We have proposed that eukaryote globins
evolved from the respective bacterial lineage via horizontal gene
transfer resulting from one or both of the accepted endosymbiotic
events responsible for the origin of mitochondria and chloroplasts,
involving an ␣-proteobacterium and a cyanobacterium, respectively [12]. The present status of our knowledge of the three globin
families and their subgroups in bacteria and the relationships
between them and eukaryote globins is shown in Fig. 2. Within this
Fig. 1. Structure of 3/3-folding spermwhale myoglobin and 2/2-folding Chlamydomonas T1 truncated hemoglobin (Brookhaven Protein Data Bank identification number
1MCY and 1DLY, respectively). Helices are indicated with letters A−H. Note the overlapping of helices A, E, and F to helices B, G, and H in the 3/3-folding, and overlapping of
helices B and E to helices G and H in the 2/2-folding.
C. Vázquez-Limón et al. / Plant Science 191–192 (2012) 71–81
73
Fig. 2. Diagrammatic representation of the known chimeric and single domain globins from the three bacterial families and their relationships to eukaryote globins.
GCSs, globin coupled sensors; Cygb, cytoglobin; Fgbs, F family single domain globins; FHbs, flavohemoglobins; Hb, hemoglobin; Mb, myoglobin; Ngb, neuroglobin; Pgbs,
protoglobins; Sgbs, S family single domain globins; T1−T3, T family truncated hemoglobin subfamilies.
Source: Modified from Vinogradov and Moens [6].
framework, all metazoan globins as well as plant hemoglobins are
likely to have emerged from a bacterial F family single domain
globin. This hypothesis has received experimental support from
a recent crystal structure of a globin from the thermophilic bacterium Methylokorus infernorum which was closest to mammalian
neuroglobins, despite only a <20% identity in sequence [33]. Furthermore, it is evident that the F family globins that had one or
more enzymatic functions in the early bacteria, evolved in multicellular eukaryotes with new properties, including reversible
binding of important diatomic ligands, such as O2 , NO, and sulfide, which enabled the evolution of transport and storage functions
[6].
symbioses with the actinobacteria Frankia species [43,44]. Symbiotic hemoglobin (including to leghemoglobins) are apparently
only localized in the nodules of the foregoing N2 -fixing plants
[34,45–48]. In contrast to symbiotic hemoglobins, non-symbiotic
and truncated hemoglobins are widely distributed in land plants
and are localized in tissues from symbiotic and non-symbiotic plant
organs. For example, nshb and thb genes were identified in primitive bryophytes and in evolved monocots and dicots [35,49,50],
and non-symbiotic and truncated hemoglobin transcripts and proteins were detected in embryonic and vegetative plant organs,
such as embryos, coleoptiles and seminal roots, and roots and
leaves, respectively [50–57]. The distribution of the three land plant
hemoglobins is summarized in Fig. 3.
1.3. Types and distribution of hemoglobins in land plants
1.4. Properties and function of land plant hemoglobins
Three types of globins have been identified in land plants: symbiotic hemoglobins, non-symbiotic hemoglobins, and truncated
hemoglobins homologous to bacterial T2 truncated hemoglobins
[34–36]. Non-symbiotic hemoglobins are further classified into
type 1 and type 2 based on O2 -affinity and sequence similarity (see
below) [37,38]. The first plant hemoglobin to be identified was a
leghemoglobin from legume root nodules [39]. The root nodules
are induced by rhizobia, a collective name for an expanding collection of symbioses between plant legumes, of which there are about
18,000 species, and a bacterial partner ␣- and ␤-proteobacteria
[40]. The only known non-legume capable of symbiosis with rhizobia is the small tree Parasponia andersonii (Ulmaceae) [41–43].
The other major group of N2 -fixing symbioses are actinorhizal
plants from four orders also belonging to the rosid clade that form
The best characterized land plant hemoglobins are leghemoglobins and non-symbiotic hemoglobins. Kinetic analysis
revealed that leghemoglobins bind and release O2 with high and
moderate rate constants, respectively [58,59]. An early view of
leghemoglobin function in legume nodules was facilitated O2 diffusion to bacteroids for aerobic respiration [46]. More recent
work has highlighted the absolute requirement of leghemoglobin
for N2 -fixation to occur in nodules [60], supporting the notion
that the 10-fold higher O2 -affinity of leghemoglobin versus myoglobin maintains the low O2 -concentration, necessary to avoid
the inactivation of the O2 -sensitive bacterial Mo-nitrogenase. It
has been reported that NO accumulates during the early stages
of the rhizobia−legume symbiosis and in mature nodules [61].
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C. Vázquez-Limón et al. / Plant Science 191–192 (2012) 71–81
Leghemoglobin binds NO to form nitrosyl-leghemoglobin, thus
leghemoglobins may also function in nodules by binding and modulating levels of NO [62].
In contrast to leghemoglobins, kinetic analysis reveals nonsymbiotic hemoglobins type 1 to have a high O2 -affinity, because
they bind and release O2 with moderate and extremely low rate
constants, respectively [37,38,63,64]. Hence, a variety of functions
other than O2 -transport have been proposed for the non-symbiotic
hemoglobins type 1 [34,65]. Evidence has accumulated over the
last decade indicating that an in vivo function of non-symbiotic
hemoglobins type 1 is the modulation of levels of NO and redox
potentials [66–72]. Thus, these proteins may function in signal
transduction pathways, specifically those involving plant hormones, such as auxins, cytokinins, ethylene, and abscisic acid
that affect a number of physiological processes, including delayed
flowering, seed germination, stomatal closure, and root hair elongation [73]. The rate constants of O2 -binding for non-symbiotic
hemoglobins type 2 are similar to those reported for leghemoglobins and symbiotic hemoglobins. Thus, it is likely that the
in vivo function of the non-symbiotic hemoglobins type 2 is related
to O2 -transport [37]. Arabidopsis non-symbiotic hemoglobin type 2
has been proposed to participate in fatty acid metabolism and in
the accumulation of polyunsaturated fatty acids by facilitating an
O2 -supply in developing seeds [74]. Furthermore, evidence exists
for an involvement of both Arabidopsis non-symbiotic hemoglobins
Brassicaceae
ARATHA nsHb-1/2
BRANAP nsHb-2
EUTHAL nsHb-2
RAPSAT nsHb-1
Caricaceae
CARPAP nsHb-1
tHb
tHb
tHb
Malvaceae
GOSHIR nsHb-1/2
THECAC nsHb-1/2 tHb
Myrtaceae
EUCGRA nsHb-1/2 tHb
Rutaceae
CITCLE nsHb-11 tHbb
CITSIN nsHb-2 tHb
HbCITUNS nsHb-1
ASTSIN
CANLIN
CHAFAS
LOTJAP
LUPLUT
MEDSAT
MEDTRU
GLYMAX
in shoot organogenesis [75]. The inner cavities in non-symbiotic
hemoglobins have been suggested to play a role in determining
the function of these proteins [76]. While less is known about the
land plant truncated hemoglobins, it has been proposed that they
function similarly to bacterial truncated hemoglobins, i.e. by participating in NO metabolism [35].
An interesting possibility is that land plant hemoglobins interact
and function with other proteins. For example, biophysical analyses
(i.e. by UV/vis spectroscopy, tryptophan fluorescence quenching, isothermal titration calorimetry and isoelectric focusing)
showed that soybean ferric leghemoglobin reductase interacts with
and reduces to ferric rice non-symbiotic hemoglobin 1 (Gopalasubramaniam et al., unpublished). Reduced rice non-symbiotic
hemoglobin 1 may bind O2 and NO for dioxygenation of NO, thus
permitting the hemoglobin-based NO-metabolic reactions. Also,
the analysis of the predicted structure of a maize non-symbiotic
hemoglobin showed the existence of a pocket-like region (the N/C
cavity) at the N- and C-terminal ends where interactions with
organic molecules and proteins could be possible. A Lys K94 (which
is located at the EF loop) protrudes into this region suggesting
that K94 may function as a trigger if molecules accommodate
into the N/C cavity. Thus, K94 may sense and transmit signals to
helices E and F, where distal and proximal His are located, respectively. This mechanism could modulate the kinetics and function of
hemoglobins into the plant cell [77].
Fabaceae
PHAVUL
Lb
PISSAT
nsHb-1 sHb PSOTET
Lb
nsHb-1 SESROS
Lb
VIC
VICFAB
Lb
CSAT
VICSAT
nsHb-1 V
VIG
VIGUNG
Lb
tHb
Lb
Lb
nsHb-1
Lb
Lb
Lb
Ulmaceae
PARAND
PARRIG
TREORI
TRETOM
T
TREVIR
Lb
Lb
Lb
Lb
tHb
b
nsHb-1
Salicaceae
POPTRE nsHb-1
tHb
nsHb-1
nsHb-1
POPTRI
nsHb-1
nsHb-1
tHb
nsHb-1
~60 mya
Origin of Lbs
Poaceae
BRADIS
HORVUL
ORYSAT
PANVIR
SETITA
nsHb-1
tHb
nsHb-1
tHb
nsHb-1
tHb
nsHb-1
tHb
nsHb-1
tHb
SORBIC
TRIAES
ZEAMAY
ZEAPAR
Araceae
WOLARR
nsHb-1
tHb
nsHb-1
tHb
nsHb-1
~94 mya
Vitaceae
tHb
VITVIN
nsHb-1
Origin of sHbs
nsHb-1
tHb
Euphorbiaceae
e
RICCOM nsHb-1 tHb
H
MANESC nsHb-1/2 tHb
nsHb-1
Nymphaeceae
EURFER nsHb-1/22
Origin of the
nsHb-1 and nsHb-2
lineages
Pinaceae
ae
PICSIT
nsHb
tHb
Slow evolutionary rate
(stabilizing selection)
~140 mya
Marchantiaceae
e
sHb
MARPOL nsHb
tHb
WGD
~320 mya
Ditrichaceae
CERPUR nsHb
Funariaceae
PHYPAT nsHb
R
Ranunculaceae
AQUCOE nsHb-1
Casuarinacea
Casuarinaceae
Fagaceae
CASG
CASGLA sHb nsHb-1 QUEPET
aceae
Betulaceae
Rosaceae
sacea
ALNFIR nsHb-11
FRAVES
Myricaceae
MALHUP
MYRGAL nsHb-1
MALDOM
Cucurbitaceae
CUCSAT nsHb-1/2 tHb PRUPER
PYRCOM
Datiscaceae
DATGLO tHb
Solanaceae
Asteraceae
ae
CICI
CICINT nsHb-2 SOLLYC nsHb-1/2
Phrymaceae SOLTUB nsHb-1/2
MIMGUT nsHb-1/2 tHb
nsHb-1
nsHb-1
tHb
nsHb-1
nsHb-1 tHb
nsHb-1
tHb
nsHb-1
tHb
tHb
Polygonaceae
RHEAUS nsHb-1
Selaginellaceae
tHb
SELMOE
EL
nsHb
tHb
Fast evolutionary rate
(relaxed selection)
~450 mya
nsHb and tHb
algal ancestors
Fig. 3. Distribution, divergence times, and major events during the evolution of known land plant hemoglobins. Binomial abbreviation for land plants and database accession
number for hemoglobin sequences are indicated in Table 1. Lb, leghemoglobin; nsHb, non-symbiotic hemoglobin; nsHb-1, non-symbiotic hemoglobin type 1; nsHb-2,
non-symbiotic hemoglobin type 2; sHb, symbiotic hemoglobin; tHb, truncated hemoglobin; WGD, whole genome duplication.
C. Vázquez-Limón et al. / Plant Science 191–192 (2012) 71–81
75
Table 1
Binomial abbreviations and database accession numbers for land plant hemoglobins shown in Fig. 3.
Land plant
Abbreviation
Protein
Accession no.
Database
Alnus firma
ALNFIR
nsHb-1
BAE75956.1
GenBank
Aquilegia coerulea
AQUCOE
nsHb-1
tHb
Aquca 019 00153.1
Aquca 001 00509.1
Phytozome
Phytozome
Arabidopsis thaliana
ARATHA
nsHb-1
nsHb-2
tHb
AAB82769.1
AAB82770.1
NP 567901.1
GenBank
GenBank
GenBank
Astragalus sinicus
ASTSIN
Lb
ABB13622.1
GenBank
Brachypodium distachyon
BRADIS
nsHb-1
tHb
XP 003558445.1
XP 003563697
GenBank
GenBank
Brassica napus
BRANAP
nsHb-2
AAK07741.1
GenBank
Canavalia lineata
CANLIN
Lb
AAA18503
GenBank
Carica papaya
CARPAP
nsHb-1
tHb
evm.TU.supercontig 62.94
ACQ91204.1
Superfam
GenBank
Casuarina glauca
CASGLA
nsHb-1
sHb
CAA37898.1
AAA33018.1
GenBank
GenBank
Ceratodon purpureus
CERPUR
nsHb
ABK41124.1
GenBank
Chamaecrista fasciculata
CHAFAS
nsHb-1/sHb
ABR68293
GenBank
Cichorium intybus × Cichorium endivia
CICINT
nsHb-2
CAA07547.1
GenBank
Citrus clementina
CITCLE
nsHb-1
tHb
clementine0.9 024121m
clementine0.9 034092m
Phytozome
Phytozome
Citrus sinensis
CITSIN
nsHb-2
tHb
orange1.1g037487m
orange1.1g030922m
Phytozome
Phytozome
Citrus unshiu
CITUNS
nsHb-1
AAK07675
GenBank
Cucumis sativus
CUCSAT
nsHb-1
nsHb-2
tHb
Cucsa.109820.1
Cucsa.308830.1
Cucsa.161470.2
Phytozome
Phytozome
Phytozome
Datisca glomerata
DATGLO
tHb
CAD33536
GenBank
Eucalyptus grandis
EUCGRA
nsHb-1
nsHb-2
tHb
Eucgr.I01236.1
Eucgr.G02733.1
Eucgr.L03669.1
Phytozome
Phytozome
Phytozome
Euryale ferox
EURFER
nsHb-1
nsHb-2
AAQ22728.1
AAQ22729.1
GenBank
GenBank
Eutrema halophilum
EUTHAL
nsHb 2
tHb
BAJ33934.1
BAJ34404.1
GenBank
GenBank
Fragaria vesca
FRAVES
nsHb-1
tHb
gene19672
gene08771
Superfam
Superfam
Glycine max
GLYMAX
Lb
Lb
Lb
Lb
nsHb-1
tHb
CAA23730.1
CAA23731.1
CAA23732.1
AAA33980.1
AAA97887.1
AAS48191
GenBank
GenBank
GenBank
GenBank
GenBank
GenBank
Gossypium hirsutum
GOSHIR
nsHb-1
nsHb-2
AAX86687.1
AAK21604.1
GenBank
GenBank
Hordeum vulgare
HORVUL
nsHb-1
tHb
AAB70097.1
AAK55410.1
GenBank
GenBank
Lotus japonicus
LOTJAP
Lb
Lb
Lb
nsHb-1
BAB18108.1
BAB18107.1
BAB18106.1
BAE46739.1
GenBank
GenBank
GenBank
GenBank
Lupinus luteus
LUPLUT
Lb
AAC04853.1
GenBank
Malus domestica
MALDOM
nsHb-1
tHb
AAP57676
MDP0000320419
GenBank
Superfam
Malus hupehensis
MALHUP
nsHb-1
ACV41424
GenBank
Manihot esculenta
MANESC
nsHb1
nsHb-2
tHb
cassava4.1 005272m
cassava4.1 018430m
cassava4.1 017779m
Phytozome
Phytozome
Phytozome
Marchantia polymorpha
MARPOL
nsHb
AAK07743.1
GenBank
Medicago sativa
MEDSAT
Lb
AAA32659.1
GenBank
76
C. Vázquez-Limón et al. / Plant Science 191–192 (2012) 71–81
Table 1 (Continued)
Land plant
Abbreviation
Protein
Accession no.
Database
Medicago truncatula
MEDTRU
nsHb-1
AAG29748.1
GenBank
Lb
Lb
tHb
CAA40899.1
CAA40900.1
XP 003603592.1
GenBank
GenBank
GenBank
Mimulus guttatus
MIMGUT
nsHb-1
nsHb-1
nsHb-2
tHb
mgf011036m
mgf016439m
mgf005736m
mgf015565m
Superfam
Superfam
Superfam
Superfam
Myrica gale
MYRGAL
nsHb-1
ABN49927.1
GenBank
Oryza sativa
ORYSAT
nsHb-1
nsHb-1
nsHb-1
nsHb-1
nsHb-1
tHb
AAK72229.1
AAC49881.1
AAK72230.1
AAK72231.1
ABN45744.1
NP 001057972.1
GenBank
GenBank
GenBank
GenBank
GenBank
GenBank
Panicum virgatum
PANVIR
nsHb-1
tHb
Pavirv00001133m
Pavirv00015565m
Phytozome
Phytozome
Parasponia andersonii
PARAND
nsHb-1
AAB86653.1
GenBank
Parasponia rigida
PARRIG
nsHb-1
P68169
GenBank
Phaseolus vulgaris
PHAVUL
Lb
AAA33767.1
GenBank
Physcomitrella patens
PHYPAT
nsHb
tHb
tHb
ABK20873.1
XP 001781680.1
XP 001760820.1
GenBank
GenBank
GenBank
Picea sitchensis
PICSIT
nsHb
tHb
ABR17163
ABK22150
GenBank
GenBank
Pisum sativum
PISSAT
Lb
BAA31156
GenBank
Populus tremula × Populus tremuloides
POPTRE
nsHb-1
tHb
ABM89109.1
ABM89110.1
GenBank
GenBank
Populus trichocarpa
POPTRI
nsHb-1
tHb
XP 002313074.1
XP 002309574.1
GenBank
GenBank
Prunus persica
PRUPER
nsHb-1
tHb
ppa012723m
ppa012268m
Phytozome
Phytozome
Psophocarpus tetragonolobus
PSOTET
Lb
AAC60563.1
GenBank
Pyrus communis
PYRCOM
nsHb-1
AAP57677
GenBank
Quercus petraea
QUEPET
nsHb-1
ABO93466
GenBank
Raphanus sativus
RAPSAT
nsHb-1
AAP37043
GenBank
Rheum australe
RHEAUS
nsHb-1
ACH63214
GenBank
Ricinus communis
RICCOM
nsHb-1
tHb
tHb
tHb
EEF43319.1
XP 002516587.1
XP 002537252.1
XP 002539183.1
GenBank
GenBank
GenBank
GenBank
Selaginella moellendorffii
SELMOE
nsHb
tHb
EFJ10590.1
EFJ07410.1
GenBank
GenBank
Sesbania rostrata
SESROS
Lb
Lb
CAA31859.1
CAA32043.1
GenBank
GenBank
Setaria italica
SETITA
nsHb-1
nsHb-1
tHb
SiPROV021593m|PACid:18193598
Si023403m
SiPROV021323m| PACid:18198243
Superfam
Phytozome
Superfam
Solanum lycopersicum
SOLLYC
nsHb-1
nsHb-2
tHb
tHb
AAK07676.1
AAK07677.1
Solyc08g068090.2.1
Solyc08g068070.2.1
GenBank
GenBank
Superfam
Superfam
Solanum tuberosum
SOLTUB
nsHb-1
nsHb-2
tHb
AAN85431.1
PGSC0003DMP400029554
PGSC0003DMP400025538
GenBank
Superfam
Superfam
Sorghum bicolor
SORBIC
nsHb-1
nsHb-1
tHb
Sb01g042260.1
Sb09g025730.2
EER89990.1
Phytozome
Phytozome
GenBank
Theobroma cacao
THECAC
nsHb-1
nsHb-1
nsHb-2
CGD0027631
CGD0027620
CGD0005903
Superfam
Superfam
Superfam
C. Vázquez-Limón et al. / Plant Science 191–192 (2012) 71–81
77
Table 1 (Continued)
Land plant
Abbreviation
Trema orientalis
TREORI
Trema tomentosa
Accession no.
Database
tHb
CGD0031696
Superfam
nsHb-1
CAB16751.1
GenBank
TRETOM
nsHb-1
CAA68405.1
GenBank
Trema virgata
TREVIR
nsHb-1
CAB63706.1
GenBank
Triticum aestivum
TRIAES
nsHb-1
tHb
AAN85432.1
ACH86231.1
GenBank
GenBank
Vicia faba
VICFAB
Lb
CAA90870.1
GenBank
Vicia sativa
VICSAT
Lb
CAA70431.1
GenBank
Vigna unguiculata
VIGUNG
Lb
Lb
AAA86756.1
AAB65769.1
GenBank
GenBank
Vitis vinifera
VITVIN
nsHb-1
nsHb-1
tHb
CBI32537.3
CBI32538.3
XP 002284484.1
GenBank
GenBank
GenBank
Wolffia arrhiza
WOLARR
nsHb-1
AEQ39061
GenBank
Zea mays ssp. mays
ZEAMAY
nsHb-1
nsHb-1
tHb
AAG01375.1
AAZ98790.1
ACG29525.1
GenBank
GenBank
GenBank
Zea mays ssp. parviglumis
ZEAPAR
nsHb-1
AAG01183.1
GenBank
2. The phylogeny and evolution of land plant hemoglobins
Protein
2.1. Phylogeny of land plant hemoglobins
2.2. Ancient land plant non-symbiotic hemoglobins and the
evolution of the non-symbiotic hemoglobin-leghemoglobin
lineage
In general, the evolution of land plant 3/3 hemoglobins paralleled the major transitions in land plant evolution. There is
at present no agreement on the times at which the transitions
occurred, mostly due to the disagreement of the fossil dates
with the dates provided by molecular phylogenetic analyses. The
molecular phylogenetic analyses tend to push the overall dates to
appreciably earlier times [78–83]. The first relevant transition or
rather series of transitions are the emergence of embryophytes
at 430−450 mya [80], perhaps as early as ∼800 mya [84], followed by the emergence of bryophytes (hornworts, mosses, and
liverworts) over the following 40–50 mya. Thus, the hemoglobins
of Marchantia (liverwort), Physcomitrella patens (moss), and the
spike moss Selaginella (Lycopsid), the oldest living groups of land
plants, are closest to the ancestral embryophyte hemoglobins.
The next major transition comprises the emergence of spermatophytes (seed plants) and the split into gymnosperms (conifers,
cycads, Gnetales, and Ginko) and ancestors of angiosperms (flowering plants): it occurred at about 320 mya, preceded by a whole
genome duplication event [85]. The third transition represents the
diversification of angiosperms at 140−180 mya [82], also preceded
by a whole genome duplication event [85]. Because Euryale ferox
(Nymphaeaceae) has non-symbiotic hemoglobins type 1 and type
2 [86], and because the Nymphaeales (water lilies and relatives)
represent one of the earliest branching angiosperm lineages, it is
tempting to conclude that the latter whole genome duplication
event prior to 150 mya [85,87] represents the origin of the nonsymbiotic hemoglobins type 1 and type 2 from the embryophyte
non-symbiotic hemoglobin. The final major transition is the split
of angiosperms into monocots and dicots at 140−150 mya [78].
It is likely that symbiotic hemoglobins originated thereafter from
a non-symbiotic hemoglobin and spread among nodulating flowering plants, while leghemoglobins evolved only within legumes
at approximately 60 mya (Fig. 3) [43,88,89]. Little is known about
the phylogeny of actinorhizal symbiotic hemoglobins, and it is still
unclear whether or not these proteins evolved with non-legume
angiosperms.
Mosses (Ceratodon purpureus and P. patens) non-symbiotic
hemoglobins are the oldest non-symbiotic hemoglobins characterized so far [90,91], and thus provide insight into the properties
of first land plant non-symbiotic hemoglobins. Specifically, gene
analysis revealed that C. purpureus and P. patens nshb are interrupted by three introns inserted similarly to known land plant nshb,
shb, and lb genes, suggesting that the ancestor globin gene of land
plant nshb, shb, and lb genes also contained the three introns. Furthermore, expression analyses revealed that C. purpureus nshb is
up-regulated by stress conditions that were essential during land
colonization by plants, such as high osmolarity, high and low temperatures, and nutrient deprivation [91,92]. Thus, it is likely that
non-symbiotic hemoglobins played a role during plant adaptation
to the land environment.
Sequence alignment of primitive and evolved non-symbiotic
hemoglobins, symbiotic hemoglobins, and leghemoglobins
revealed that the size of the polypeptide decreased over time
at the N-terminal region, mostly at pre-helix A. A predicted
leader peptidase site was identified in the pre-helix A region of
the C. purpureus and P. patens non-symbiotic hemoglobins [36].
The pre-helix A was suggested to function as a leader peptide
in primitive non-symbiotic hemoglobins [36], similarly to the
Chlamydomonas T1 truncated hemoglobin, which is translocated
from cell cytoplasm to chloroplasts [93]. Thus, a possibility is
that an ancestor to land plant non-symbiotic hemoglobins was
translocated from cytoplasm to cellular organelles, and that nonsymbiotic hemoglobins became cytoplasmic during the evolution
of land plants [36].
Although the crystal structure of moss non-symbiotic
hemoglobins is not known, the structures of C. purpureus and
P. patens non-symbiotic hemoglobins were modeled [91] and
deposited in the Caspur database (http://mi.caspur.it/PMDB/,
identification number PM0074985). The structures were similar
to the experimentally determined crystal structure of land plant
non-symbiotic hemoglobins type 1. Furthermore, the spectroscopic properties of the recombinant C. purpureus and P. patens
78
C. Vázquez-Limón et al. / Plant Science 191–192 (2012) 71–81
non-symbiotic hemoglobins demonstrated them to be hexacoordinated (Vázquez-Limón and Arredondo-Peter, in preparation). The
similarity of the properties of moss non-symbiotic hemoglobins
with those of non-symbiotic hemoglobins type 1 imply that the
first land plant non-symbiotic hemoglobins had high O2 -binding
affinities and were probably not involved in O2 -transport.
A comparison of the predicted structure of moss non-symbiotic
hemoglobins, native rice Hb1 (a non-symbiotic hemoglobin type 1),
and soybean leghemoglobin a revealed the major alterations occurring during the evolution of land plant non-symbiotic hemoglobins
to leghemoglobins [91]. These changes consisted in (i) a hexacoordinate to pentacoordinate transition at the heme-Fe, (ii) a decrease
in the sizes of the CD-loop and the N- and C-terminal regions, and
(iii) generation of a more compact protein structure.
The minimum age of the stem lineage of the N2 -fixing clade
including Rosaceae and Fagales (e.g. Betulaceae, Casuarinaceae,
and Myricaceae) is estimated to be approximately 94 mya [94].
It is reasonable to assume that leghemoglobins evolved from a
non-symbiotic hemoglobin ancestor concomitantly with the emergence of legumes at ca. 60 mya [94,95] and of rhizobial nodulation
providing functional specialization in N2 -fixing nodules [88,89].
The Caesalpinoideae is the oldest subfamily of legumes and contains both nodulating and non-nodulating species. It is thus likely
that the non-symbiotic hemoglobin to leghemoglobin transition
occurred in a caesalpinoid legume. The characterization of a
Chamaecrista fasciculata hemoglobin, a caesalpinoid hemoglobin,
intermediate between non-symbiotic hemoglobins and leghemoglobins, has the same alterations as above [88]. Apparently these
alterations permitted leghemoglobins, and probably other symbiotic hemoglobins with similar structures to evolve O2 -binding
kinetic properties that enabled them to function in maintaining
N2 -fixation in nodules.
3. The origin of land plant hemoglobins
3.1. The putative algal ancestor of land plant non-symbiotic
hemoglobins
The latest estimate of the origin of the last eukaryote common
ancestor is approximately 1200 mya, close to the emergence of bangiophytes (red algae) from the stem lineage of the Archaeplastida
(green algae and land plants) [80]. It is widely accepted that the
paraphyletic groups of algae, the Chlorophyta and the Charophyta,
are the closest relatives to land plants [80,82,96]. Thus, they must
have emerged prior to the origin of embryophytes at 430−450 mya
[79,80]. Several chlorophyte genomes have been sequenced,
including those of the Prasinophyceae algae Micromonas and
Ostreococcus. Although Ostreococcus lucimarinus has no globins,
Ostreococcus tauri, Micromonas pusilla, and Micromonas sp. RCC299
each have a single domain 3/3 globin. Their sequences place them
between bacterial F family single domain globins and bryophyte
and other land plant non-symbiotic hemoglobins [49,97], suggesting that these algal non-symbiotic hemoglobin-like globins share a
bacterial F family single domain globin ancestor with the land plant
non-symbiotic hemoglobins. Unfortunately, a major gap still exists
between evolved green algae (i.e. the direct ancestors of land plants
[96,98]) and bryophyte non-symbiotic hemoglobins, given that the
latest study implicates the Zygnemetales and Coleochaetales as the
two Charophyte sister groups to embryophytes [83] and that no
Charophyte genome sequences are available.
Although the predicted Micromonas and Ostreococcus nonsymbiotic hemoglobin structures have the canonical myoglobinfold and heme-Fe coordination to a proximal His, they show that
the two Micromonas hemoglobins are hexacoordinate, while the
O. tauri hemoglobin is pentacoordinate [97]. This suggests that
both penta- and hexa-coordination existed in algal hemoglobins
and that plant non-symbiotic hemoglobins originated from a hexacoordinated hemoglobin in the ancestor of algae and land plants.
Interestingly, the Micromonas and Ostreococcus nshb genes lack
introns, whereas known land plant nshb genes have three introns.
Genomic information from Charophyte algae should provide information on the intron structure of their hemoglobins and determine
whether the proposal that introns inserted into an algal nshb prior
to the origin of land plant nshb genes [97] is correct or not.
3.2. The origin and evolution of algal and land plant truncated
hemoglobins
Our findings regarding the origin of plant, Chlorophyte, and
Stramenopile T2 truncated hemoglobins were quite different from
those regarding the 3/3 plant hemoglobins. A preliminary analysis
indicated the possibility of horizontal gene transfer from a progenitor bacterium to one of the following bacterial phyla: the Chloroflexi,
Deinococcales, Bacilli, and Actinomycetes [49]. A more recent reexamination corroborated the earlier results and refined them to
show that the six known Chloroflexi T2 truncated hemoglobins were
the closest relatives of the land plant, algal and stramenopile T2
truncated hemoglobins [99]. This finding is not surprising given
that recent studies have demonstrated the horizontal gene transfer of over 50 genes from progenitors of modern Chlamydia to the
ancestral primary photosynthetic eukaryote [84].
The computationally predicted structure of Chlorella T1 truncated hemoglobin and Physcomitrella and Arabidopsis T2 truncated
hemoglobins based on the only known eukaryote truncated
hemoglobin structures, the T1 truncated hemoglobins from
Chlamydomonas and Paramecium (Brookhaven Protein Data Bank
identification number 1DLY and 1DLW, respectively), showed that
the Chlamydomonas and Chlorella T1 truncated hemoglobins were
very similar and that the Physcomitrella and Arabidopsis T2 truncated hemoglobins had the 2/2-fold typical of bacterial T2 truncated
hemoglobins (Fernández and Arredondo-Peter, unpublished).
The number of introns in algal thb genes ranges from 2 to >5
and their positions are variable [49] indicating that introns evolved
under relaxed selection. In contrast, the number and position of
introns in land plant thb genes are conserved with the exception of
a soybean thb gene (Genbank accession number AAS48191), which
has only one intron. The known land plant thbs have three conserved introns, suggesting stabilizing selection during the evolution
of introns in land plant thb genes [49].
4. Rates of evolution of land plant hemoglobins
The evolutionary rates of land plant hemoglobins were estimated from the amino acid substitutions and divergence values
relative to the moss hemoglobins considered to be closest to
the ancestral hemoglobins [100]. The divergence values of nonsymbiotic hemoglobins and leghemoglobins indicate that they did
not evolve at constant rates. Apparently, high variation occurred in
non-symbiotic hemoglobins during the first ∼40 million of years
of land plant evolution, followed by a decreased rate of divergence during the subsequent ∼200 million of years, i.e. during
the trachaeophyte to magnoliophyta (angiosperms) transition. Furthermore, the rate of divergence was higher in non-symbiotic
hemoglobins type 2 and leghemoglobins than in non-symbiotic
hemoglobins type 1, indicating that non-symbiotic hemoglobins
type 2 and leghemoglobins evolved under relaxed selection compared to non-symbiotic hemoglobins type 1. In contrast, land
plant truncated hemoglobins apparently evolved at a rather constant rate. However, the high variability detected between the
Physcomitrella and Selaginella truncated hemoglobins suggests a
C. Vázquez-Limón et al. / Plant Science 191–192 (2012) 71–81
major divergence in land plant truncated hemoglobins during
the bryophyte to trachaeophyte transition [100]. Thus, a general
conclusion is that hemoglobins were highly variable during the colonization of land by plants, but the rate of evolution decreased
prior to the origin of magnoliophyta. However, with the exception of leghemoglobins, which evolved under stabilizing selection
to specifically function in nodules of N2 -fixing legumes (Section
2.2), it is not known whether or not rates of divergence affected
the hemoglobin function (s) during the evolution of land plants.
5. Going back (>3000 mya) to the (primeval) structural
ancestor of 3/3 and 2/2 hemoglobins?
Globins are ancient proteins that originated early in the evolution of life, i.e. more than 3000 mya [7,12]. Prokaryotes contain a
variety of 3/3 and 2/2 hemoglobins as mentioned earlier, and we do
not have at present any clues as to the origins of the two 3/3 (F and
S) families and the 2/2 T family. It appears likely that the two 3/3
families shared a common precursor. Still, we do not know whether
the two structural lineages shared a common ancestor or emerged
separately. The computational analysis of land plant hemoglobin
folding indicates that it proceeds through the formation of folding
modules formed by helices A, B and C, and E, F, G, and H (folding
modules A/C and E/H, respectively) [101]. Modeling of the rice Hb1
(a non-symbiotic hemoglobin type 1) A/C and E/H modules suggests that module E/H overlaps to the Mycobacterium tuberculosis
HbO (a T3 truncated hemoglobin) 2/2-fold. This result implies that
module E/H is an ancient structural motif. Its presence in the common globin ancestor would provide for the emergence of a 3/3-fold
through the addition of module A/C, and for the origin of a 2/2-fold
via the addition of a B/C module.
6. Concluding remarks and future directions
Overall, the outline of plant globin evolution subsequent to land
colonization about 430−450 mya or as early as ∼800 mya [79,80]
appears to be fairly clear. The major events summarized in Fig. 3
include the following. (i) The land plant (embryophyte) 3/3 nonsymbiotic hemoglobins originated from a precursor non-symbiotic
hemoglobin in the ancestor shared with green algae. (ii) The
precursor globin may have descended from a bacterial (Cyanobacteria/Proteobacteria) F family single domain globin as the result of
horizontal gene transfer events accompanying the two accepted
endosymbiotic events to the eukaryote ancestor common to all the
eukaryotes. (iii) The plant 2/2 truncated hemoglobins originated
and vertically evolved from a bacterial 2/2 hemoglobin, probably
as the result of a horizontal gene transfer event from an ancestor of
present day Chloroflexi to either the ancestor of all eukaryotes, or to
the ancestor shared by algae and land plants. (iv) The diversification of angiosperms at 140−180 mya [82], preceded by a whole
genome duplication event [85], is the likely time for the emergence of the non-symbiotic hemoglobins type 1 and type 2 from the
embryophyte 3/3 hemoglobin, as indicated by the presence of both
in E. ferox (Nymphaeaceae) [86], and because the Nymphaeales represent one of the earliest branching angiosperm lineages [87]. (v)
The final major transition is the split of angiosperms into monocots
and dicots at 140−150 mya [78,80]. It is likely that thereafter symbiotic hemoglobins originated from a non-symbiotic hemoglobin and
spread among nodulating flowering plants, while leghemoglobins
evolved only within the Leguminoseae (Fabaceae) soon after their
emergence at about 60 mya [43,88,89,94]. Little is known about
the phylogeny of actinorhizal symbiotic hemoglobins, and it is
still unclear whether or not these proteins evolved with nonlegume angiosperms. (vi) The emergence of symbiotic hemoglobins
(including to leghemoglobins) was accompanied by a transition of
79
the heme-Fe coordination from hexa- to penta-coordination, by
decrease in the lengths of the CD-loop and the N- and C-terminal
regions, and by compaction of the protein structure leading to
decreased mobility of the distal His [88,91]. (vii) Although at least
two major whole genome duplication events were identified in
plant phylogeny [85], the T2 hemoglobins in contrast to the 3/3
hemoglobins do not appear to have undergone any duplication.
This review reveals at least three lacunae in our ability to completely analyze the phylogeny of plant and algal globins. Recent
studies have identified the Charophyte algae as the sister group
to land plants, and more specifically, two of the six orders, the
Zygnemetales and Coleochaetales [82,83]. Undoubtedly, the most
important lacuna is the absence of genomic information about
Charophyte algae. Except for the Picea sitchensis genome, the
absence of genomic information about ferns, ginkos, and cycads
represents the second lacuna. Little is known about the role
and evolution of symbiotic hemoglobins in actinorhizal symbiosis involving the actinobacterium Frankia and representatives of
the orders Cucurbitales, Fagales, and Rosales relative to the leghemoglobins of the legume family. It has been estimated that in
terms of N2 -fixation the contributions of the two types of plantmicrobial symbioses are approximately equal [89]. Hence, the
absence of genomic data about plants with symbiotic hemoglobins
other than leghemoglobins is yet another lacuna that needs to be
filled. Finally, an additional lacuna is the lack of a land plant T2
truncated hemoglobin crystal structure. The sequences of these
proteins are mostly over 160 amino acids, with ∼20 to ∼40 amino
acid extensions at both chain termini, relative to the microbial T2
truncated hemoglobins. Although the predicted structure of the
globin domain from the Physcomitrella and Arabidopsis T2 truncated hemoglobins fits satisfactorily the microbial T2 truncated
hemoglobin structure (Section 3.2), we are ignorant of the folding of
the extensions. Thus, it would be desirable to have both structures
(i.e. the full, globin plus extensions, structure) in order to compare
them and identify the alterations that may have occurred during
the evolution of land plant T2 truncated hemoglobins.
Acknowledgments
Authors are grateful to Gustavo Rodríguez Alonso for providing
Fig. 1. Work in R.A.-P. laboratory has been funded by SEP-PROMEP
(grant no. UAEMor-PTC-01-01/PTC23) and Consejo Nacional de
Ciencia y Tecnología (CoNaCyT grant nos. 25229N and 42873Q),
México. C.V.-L. is a postdoctoral fellow supported by CoNaCyT.
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