Plant Science 191–192 (2012) 71–81

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Plant Science

jo urnal homepage: www.elsevier.com/locate/plantsci

Review

The of land plant

a b c a,∗

Consuelo Vázquez-Limón , David Hoogewijs , Serge N. Vinogradov , Raúl Arredondo-Peter

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 and Molecular Biology, Wayne State University, School of Medicine, Detroit, MI 48201, USA

a r t i c l e i n f o a b s t r a c t

Article history: This review discusses the evolution of land plant hemoglobins within the broader context of

Received 13 March 2012

hemoglobins and the three families of bacterial . Most eukaryote hemoglobins, including metazoan

Received in revised form 24 April 2012

globins and the symbiotic and non-symbiotic plant hemoglobins, are homologous to the bacterial 3/3-

Accepted 25 April 2012

fold flavohemoglobins. The remaining plant hemoglobins are homologous to the bacterial 2/2-fold group

Available online 4 May 2012

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.

Keywords:

Although the 3/3 hemoglobins originated in the ancestor of green algae and prior to the emergence

Hemoglobin

Non-symbiotic of embryophytes at about 450 mya, the 2/2 hemoglobins appear to have originated via horizontal gene

Leghemoglobin transfer from a bacterium ancestral to present day Chloroflexi. Unlike the 2/2 hemoglobins, the evolu-

Truncated tion of the 3/3 hemoglobins was accompanied by duplication, diversification, and functional adaptations.

Land plants Duplication of the ancestral plant nshb gene into the nshb-1 and nshb-2 lineages occurred prior to the

−

Evolution monocot dicot divergence at ca. 140 mya. It was followed by the emergence of symbiotic hemoglobins

from a non-symbiotic precursor and further specialization, leading to in

N2-fixing 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 alter-

ation of -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 phylogeny. © 2012 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction ...... 72

1.1. What is a globin?—a historical perspective ...... 72

1.2. Diversity of globins in living ...... 72

1.3. Types and distribution of hemoglobins in land plants ...... 73

1.4. Properties and function of land plant hemoglobins ...... 73

2. The phylogeny and evolution of land plant hemoglobins ...... 77

2.1. Phylogeny of land plant hemoglobins...... 77

2.2. Ancient land plant non-symbiotic hemoglobins and the evolution of the non-symbiotic hemoglobin- lineage ...... 77

3. The origin of land plant hemoglobins ...... 78

3.1. The putative algal ancestor of land plant non-symbiotic hemoglobins ...... 78

3.2. The origin and evolution of algal and land plant truncated hemoglobins ...... 78

4. Rates of evolution of land plant hemoglobins ...... 78

5. Going back (>3000 mya) to the (primeval) structural ancestor of 3/3 and 2/2 hemoglobins? ...... 79

6. Concluding remarks and future directions ...... 79

Acknowledgments ...... 79

References ...... 79

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

72 C. Vázquez-Limón et al. / Plant Science 191–192 (2012) 71–81

1. Introduction are the F family comprising the flavohemoglobins and related sin-

gle domain globins [9], and the S (for sensor) family, encompassing

1.1. What is a globin?—a historical perspective globin coupled sensors and protoglobins [10,11], and related sin-

gle domain globins [12]. The third family consists of truncated

Globins are with a characteristic ␣-helical secondary -fold globins, with the 3/3-fold reduced to a 2/2-fold

structure comprised of helices A−H, known as the myoglobin-fold, due to a shortened or absent helix A and conversion of the F helix

and a heme group ensconced within a hydrophobic cavity formed into a loop (Fig. 1) [13–17]. The T family exists in three structurally

by a 3/3 sandwich of helices A, B, C, and E over helices F, G, and distinct subfamilies, T1−T3 [13–17].

H. Of the two heme-Fe axial sites, the proximal one is coordinated Recent genomic information has also greatly extended the

to a His at position 8 of helix F, while the distal site can coordi- structural and functional diversity of vertebrate globins through

nate either with a side-chain group of residues located in helix E the discovery of novel globins like and

or bind small molecule ligands, including O2, CO, and NO. Histor- [18,19], which are hexacoordinated [20,21], and perform yet-to-be-

ically, the familiar vertebrate O2-binding hemoglobin, a tetramer determined functions in nerve and fibroblast-like cells, respectively

of ␣- and ␤-globins, and myoglobin were among the first proteins [22,23]. Furthermore, the identification of additional globins with

whose sequences and structures were determined over 50 years unknown physiological functions and restricted phyletic distribu-

ago [1]. At that time, the hemoglobins in metazoans other than ver- tions, globin X in some protostomes and chordates [24], globin Y in

tebrates were investigated mostly in cases where the hemoglobin amphibians and monotreme mammals, and globin E the avian eye

presence was visible. These included the larval hemoglobin of globin has added complexity to vertebrate globin gene evolution

the insect Chironomus [2] and the intracellular hemoglobin of the [25–29]. Phylogenetic analyses of these vertebrate globins revealed

Glycera [3]. Comparison of several vertebrate and the inver- that erythroid-specific globins have independently evolved O2-

tebrate hemoglobin structures led to the recognition of a highly transport functions in different lineages [30]. Most recently, a new

conserved tertiary structure, the myoglobin-fold, underpinned by metazoan globin lineage was discovered, consisting of large, ca.

the conservation of over 30, mostly solvent-inaccessible hydropho- 1600 residues, chimeric proteins with an N-terminal cysteine pro-

bic residues [4], even in cases of <20% identity to vertebrate globins. tease domain and a central globin domain, named androglobins,

The 3/3 ␣-helical myoglobin-fold is not unique: it is shared with because of their specific expression in testis [31].

and other proteins [5]. The transport of O2 for aerobic All metazoan globins, vertebrate and non-vertebrate, symbiotic

respiration is thought to be the major function of vertebrate globins and non-symbiotic plant globins, and many globins in micro-

␣

related to their ability to reversibly bind O2 [1]. However, evidence bial have the 3/3 -helical fold and have sequences

has accrued over the last two decades indicating that both bacte- that are homologous to the F family bacterial globins. T family

rial and eukaryote globins have enzymatic and sensing functions in group 1 and 2 globins occur in microbial eukaryotes (ciliates, stra-

addition to O2-transport and storage [6]. menopiles, 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

1.2. Diversity of globins in living organisms

evolved from the respective bacterial lineage via horizontal gene

transfer resulting from one or both of the accepted endosymbiotic

The availability of numerous sequenced genomes over the past

events responsible for the origin of mitochondria and chloroplasts,

20 years allowed the identification of globins in a wide variety

involving an ␣-proteobacterium and a cyanobacterium, respec-

of organisms, ranging from to vertebrates. The bacte-

tively [12]. The present status of our knowledge of the three globin

rial globin superfamily encompasses three families/lineages that

families and their subgroups in bacteria and the relationships

belong to two structural classes: the 3/3- and 2/2-fold globins

between them and eukaryote globins is shown in Fig. 2. Within this

(Fig. 1) [7,8]. The two globin families/lineages with the 3/3-fold

Fig. 1. Structure of 3/3-folding spermwhale myoglobin and 2/2-folding Chlamydomonas T1 truncated hemoglobin (Brookhaven 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 symbioses with the actinobacteria Frankia [43,44]. Sym-

likely to have emerged from a bacterial F family single domain biotic hemoglobin (including to leghemoglobins) are apparently

globin. This hypothesis has received experimental support from only localized in the nodules of the foregoing N2-fixing plants

a recent crystal structure of a globin from the thermophilic bac- [34,45–48]. In contrast to symbiotic hemoglobins, non-symbiotic

terium Methylokorus infernorum which was closest to mammalian and truncated hemoglobins are widely distributed in land plants

, despite only a <20% identity in sequence [33]. Fur- and are localized in tissues from symbiotic and non-symbiotic plant

thermore, it is evident that the F family globins that had one or organs. For example, nshb and thb genes were identified in prim-

more enzymatic functions in the early bacteria, evolved in mul- itive bryophytes and in evolved monocots and dicots [35,49,50],

ticellular eukaryotes with new properties, including reversible and non-symbiotic and truncated hemoglobin transcripts and pro-

binding of important diatomic ligands, such as O2, NO, and sul- teins were detected in embryonic and vegetative plant organs,

fide, which enabled the evolution of transport and storage functions such as embryos, coleoptiles and seminal roots, and roots and

[6]. 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: sym-

biotic hemoglobins, non-symbiotic hemoglobins, and truncated The best characterized land plant hemoglobins are leghe-

hemoglobins homologous to bacterial T2 truncated hemoglobins moglobins and non-symbiotic hemoglobins. Kinetic analysis

[34–36]. Non-symbiotic hemoglobins are further classified into revealed that leghemoglobins bind and release O2 with high and

type 1 and type 2 based on O2-affinity and sequence similarity (see moderate rate constants, respectively [58,59]. An early view of

below) [37,38]. The first plant hemoglobin to be identified was a leghemoglobin function in legume nodules was facilitated O2-

leghemoglobin from legume root nodules [39]. The root nodules to bacteroids for aerobic respiration [46]. More recent

are induced by , a collective name for an expanding collec- work has highlighted the absolute requirement of leghemoglobin

tion of symbioses between plant , of which there are about for N2-fixation to occur in nodules [60], supporting the notion

18,000 species, and a bacterial partner ␣- and ␤-proteobacteria that the 10-fold higher O2-affinity of leghemoglobin versus myo-

[40]. The only known non-legume capable of with rhi- globin maintains the low O2-concentration, necessary to avoid

zobia is the small tree Parasponia andersonii (Ulmaceae) [41–43]. the inactivation of the O2-sensitive bacterial Mo-. It

The other major group of N2-fixing symbioses are actinorhizal has been reported that NO accumulates during the early stages

plants from four orders also belonging to the rosid clade that form of the rhizobia−legume symbiosis and in mature nodules [61].

74 C. Vázquez-Limón et al. / Plant Science 191–192 (2012) 71–81

Leghemoglobin binds NO to form nitrosyl-leghemoglobin, thus in shoot organogenesis [75]. The inner cavities in non-symbiotic

leghemoglobins may also function in nodules by binding and mod- hemoglobins have been suggested to play a role in determining

ulating levels of NO [62]. the function of these proteins [76]. While less is known about the

In contrast to leghemoglobins, kinetic analysis reveals non- land plant truncated hemoglobins, it has been proposed that they

symbiotic hemoglobins type 1 to have a high O2-affinity, because function similarly to bacterial truncated hemoglobins, i.e. by par-

they bind and release O2 with moderate and extremely low rate ticipating in NO metabolism [35].

constants, respectively [37,38,63,64]. Hence, a variety of functions An interesting possibility is that land plant hemoglobins interact

other than O2-transport have been proposed for the non-symbiotic and function with other proteins. For example, biophysical analyses

hemoglobins type 1 [34,65]. Evidence has accumulated over the (i.e. by UV/vis spectroscopy, tryptophan fluorescence quench-

last decade indicating that an in vivo function of non-symbiotic ing, isothermal titration calorimetry and isoelectric focusing)

hemoglobins type 1 is the modulation of levels of NO and redox showed that ferric leghemoglobin reductase interacts with

potentials [66–72]. Thus, these proteins may function in signal and reduces to ferric rice non-symbiotic hemoglobin 1 (Gopala-

transduction pathways, specifically those involving plant hor- subramaniam et al., unpublished). Reduced rice non-symbiotic

mones, such as auxins, cytokinins, ethylene, and abscisic acid hemoglobin 1 may bind O2 and NO for dioxygenation of NO, thus

that affect a number of physiological processes, including delayed permitting the hemoglobin-based NO-metabolic reactions. Also,

flowering, seed germination, stomatal closure, and root hair elon- the analysis of the predicted structure of a maize non-symbiotic

gation [73]. The rate constants of O2-binding for non-symbiotic hemoglobin showed the existence of a pocket-like region (the N/C

hemoglobins type 2 are similar to those reported for leghe- cavity) at the N- and C-terminal ends where interactions with

moglobins and symbiotic hemoglobins. Thus, it is likely that the organic molecules and proteins could be possible. A Lys K94 (which

in vivo function of the non-symbiotic hemoglobins type 2 is related is located at the EF loop) protrudes into this region suggesting

to O2-transport [37]. Arabidopsis non-symbiotic hemoglobin type 2 that K94 may function as a trigger if molecules accommodate

has been proposed to participate in fatty acid metabolism and in into the N/C cavity. Thus, K94 may sense and transmit signals to

the accumulation of polyunsaturated fatty acids by facilitating an helices E and F, where distal and proximal His are located, respec-

O2-supply in developing seeds [74]. Furthermore, evidence exists tively. This mechanism could modulate the kinetics and function of

for an involvement of both Arabidopsis non-symbiotic hemoglobins hemoglobins into the plant [77].

Fabaceae Malvaceae Brassicaceae ASTSIN Lb PHAVUL Lb GOSHIR nsHb-1/2 Lb PISSAT Lb ARATHA nsHb-1/2 tHb CANLIN THECAC nsHb-1/2 tHb CHAFAS nsHb-1 sHb PSOTET Lb BRANAP nsHb-2 Ulmaceae Salicaceae Myrtaceae Lb SESROS Lb nsHb-1 tHb LOTJAP nsHb-1 PARAND POPTRE nsHb-1 EUTHAL nsHb-2 EUCGRA nsHb-1/2 tHb LUPLUT Lb VICFABVIC Lb PARRIG nsHb-1 tHb RAPSAT nsHb-1 Rutaceae MEDSAT Lb nsHb-1 VICSATV CSAT Lb TREORI nsHb-1 POPTRI nsHb-1 Caricaceae CITCLE nsHb-11 tHbb Lb tHb VIGUNGVIG Lb MEDTRU TTRETOM nsHb-1 tHb CARPAP nsHb-1 tHb CITSIN nsHb-2 tHb GLYMAX Lb nsHb-1 tHbb CITUNS nsHb-1Hb- TREVIR nsHb-1 ~60 mya Origin of Lbs Poaceae ~94 mya SORBIC nsHb-1 tHb CasuarinaceaeCasuarinacea Fagaceae BRADIS nsHb-1 tHb TRIAES nsHb-1 tHb Vitaceae CASGLACASG sHb nsHb-1 QUEPET nsHb-1 HORVUL nsHb-1 tHb Origin of sHbs ZEAMAY nsHb-1 tHb VITVIN nsHb-1 Betulaceaeaceae nsHb-1 tHb Rosaceaesacea ORYSAT ZEAPAR nsHb-1 tHb ALNFIR nsHb-11 FRAVES nsHb-1 tHb PANVIR nsHb-1 tHb Araceae Myricaceae nsHb-1 MALHUP nsHb-1 SETITA tHb WOLARR nsHb-1 Euphorbiaceaee MYRGAL nsHb-1 nsHb-1 RICCOM nsHb-1 tHbH Cucurbitaceae MALDOM tHb MANESC nsHb-1/2 tHb CUCSAT nsHb-1/2 tHb PRUPER nsHb-1 tHb Datiscaceae PYRCOM nsHb-1 Nymphaeceae DATGLO tHb EURFER nsHb-1/22

Asteraceaeae Solanaceae Origin of the ~140 mya RanunculaceaeR Pinaceaeae CICINTCICI nsHb-2 SOLLYC nsHb-1/2 tHb nsHb-1 and nsHb-2 AQUCOE nsHb-1 nsHb Phrymaceae tHb PICSIT lineages tHb SOLTUB nsHb-1/2 tHb WGD MIMGUT nsHb-1/2 tHb Slow evolutionary rate (stabilizing selection) RHEAUS nsHb-1 ~320 mya

Ditrichaceae CERPUR nsHb Funariaceae Selaginellaceae PHYPAT nsHb tHb SELMOEEL nsHb tHb

Fast evolutionary rate Marchantiaceaee (relaxed selection) MARPOL nsHbsHb ~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 Aquca 019 00153.1 Phytozome

tHb Aquca 001 00509.1 Phytozome

Arabidopsis thaliana ARATHA nsHb-1 AAB82769.1 GenBank

nsHb-2 AAB82770.1 GenBank

tHb NP 567901.1 GenBank

Astragalus sinicus ASTSIN Lb ABB13622.1 GenBank

Brachypodium distachyon BRADIS nsHb-1 XP 003558445.1 GenBank

tHb XP 003563697 GenBank

Brassica napus BRANAP nsHb-2 AAK07741.1 GenBank

Canavalia lineata CANLIN Lb AAA18503 GenBank

Carica papaya CARPAP nsHb-1 evm.TU.supercontig 62.94 Superfam

tHb ACQ91204.1 GenBank

Casuarina glauca CASGLA nsHb-1 CAA37898.1 GenBank

sHb AAA33018.1 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 clementine0.9 024121m Phytozome

tHb clementine0.9 034092m Phytozome

Citrus sinensis CITSIN nsHb-2 orange1.1g037487m Phytozome

tHb orange1.1g030922m Phytozome

Citrus unshiu CITUNS nsHb-1 AAK07675 GenBank

Cucumis sativus CUCSAT nsHb-1 Cucsa.109820.1 Phytozome

nsHb-2 Cucsa.308830.1 Phytozome

tHb Cucsa.161470.2 Phytozome

Datisca glomerata DATGLO tHb CAD33536 GenBank

Eucalyptus grandis EUCGRA nsHb-1 Eucgr.I01236.1 Phytozome

nsHb-2 Eucgr.G02733.1 Phytozome

tHb Eucgr.L03669.1 Phytozome

Euryale ferox EURFER nsHb-1 AAQ22728.1 GenBank

nsHb-2 AAQ22729.1 GenBank

Eutrema halophilum EUTHAL nsHb 2 BAJ33934.1 GenBank

tHb BAJ34404.1 GenBank

Fragaria vesca FRAVES nsHb-1 gene19672 Superfam

tHb gene08771 Superfam

Glycine max GLYMAX Lb CAA23730.1 GenBank

Lb CAA23731.1 GenBank

Lb CAA23732.1 GenBank

Lb AAA33980.1 GenBank

nsHb-1 AAA97887.1 GenBank

tHb AAS48191 GenBank

Gossypium hirsutum GOSHIR nsHb-1 AAX86687.1 GenBank

nsHb-2 AAK21604.1 GenBank

Hordeum vulgare HORVUL nsHb-1 AAB70097.1 GenBank

tHb AAK55410.1 GenBank

Lotus japonicus LOTJAP Lb BAB18108.1 GenBank

Lb BAB18107.1 GenBank

Lb BAB18106.1 GenBank

nsHb-1 BAE46739.1 GenBank

Lupinus luteus LUPLUT Lb AAC04853.1 GenBank

Malus domestica MALDOM nsHb-1 AAP57676 GenBank

tHb MDP0000320419 Superfam

Malus hupehensis MALHUP nsHb-1 ACV41424 GenBank

Manihot esculenta MANESC nsHb1 cassava4.1 005272m Phytozome

nsHb-2 cassava4.1 018430m Phytozome

tHb cassava4.1 017779m 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

nsHb-1 AAG29748.1 GenBank

Medicago truncatula MEDTRU Lb CAA40899.1 GenBank

Lb CAA40900.1 GenBank

tHb XP 003603592.1 GenBank

Mimulus guttatus MIMGUT nsHb-1 mgf011036m Superfam

nsHb-1 mgf016439m Superfam

nsHb-2 mgf005736m Superfam

tHb mgf015565m Superfam

Myrica gale MYRGAL nsHb-1 ABN49927.1 GenBank

Oryza sativa ORYSAT nsHb-1 AAK72229.1 GenBank

nsHb-1 AAC49881.1 GenBank

nsHb-1 AAK72230.1 GenBank

nsHb-1 AAK72231.1 GenBank

nsHb-1 ABN45744.1 GenBank

tHb NP 001057972.1 GenBank

Panicum virgatum PANVIR nsHb-1 Pavirv00001133m Phytozome

tHb Pavirv00015565m 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 ABK20873.1 GenBank

tHb XP 001781680.1 GenBank

tHb XP 001760820.1 GenBank

Picea sitchensis PICSIT nsHb ABR17163 GenBank

tHb ABK22150 GenBank

Pisum sativum PISSAT Lb BAA31156 GenBank

Populus tremula × Populus tremuloides POPTRE nsHb-1 ABM89109.1 GenBank

tHb ABM89110.1 GenBank

Populus trichocarpa POPTRI nsHb-1 XP 002313074.1 GenBank

tHb XP 002309574.1 GenBank

Prunus persica PRUPER nsHb-1 ppa012723m Phytozome

tHb ppa012268m 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 EEF43319.1 GenBank

tHb XP 002516587.1 GenBank

tHb XP 002537252.1 GenBank

tHb XP 002539183.1 GenBank

Selaginella moellendorffii SELMOE nsHb EFJ10590.1 GenBank

tHb EFJ07410.1 GenBank

Sesbania rostrata SESROS Lb CAA31859.1 GenBank

Lb CAA32043.1 GenBank

Setaria italica SETITA nsHb-1 SiPROV021593m|PACid:18193598 Superfam

nsHb-1 Si023403m Phytozome

tHb SiPROV021323m| PACid:18198243 Superfam

Solanum lycopersicum SOLLYC nsHb-1 AAK07676.1 GenBank

nsHb-2 AAK07677.1 GenBank

tHb Solyc08g068090.2.1 Superfam

tHb Solyc08g068070.2.1 Superfam

Solanum tuberosum SOLTUB nsHb-1 AAN85431.1 GenBank

nsHb-2 PGSC0003DMP400029554 Superfam

tHb PGSC0003DMP400025538 Superfam

Sorghum bicolor SORBIC nsHb-1 Sb01g042260.1 Phytozome

nsHb-1 Sb09g025730.2 Phytozome

tHb EER89990.1 GenBank

Theobroma cacao THECAC nsHb-1 CGD0027631 Superfam

nsHb-1 CGD0027620 Superfam

nsHb-2 CGD0005903 Superfam

C. Vázquez-Limón et al. / Plant Science 191–192 (2012) 71–81 77

Table 1 (Continued)

Land plant Abbreviation Protein Accession no. Database

tHb CGD0031696 Superfam

Trema orientalis TREORI nsHb-1 CAB16751.1 GenBank

Trema tomentosa TRETOM nsHb-1 CAA68405.1 GenBank

Trema virgata TREVIR nsHb-1 CAB63706.1 GenBank

Triticum aestivum TRIAES nsHb-1 AAN85432.1 GenBank

tHb ACH86231.1 GenBank

Vicia faba VICFAB Lb CAA90870.1 GenBank

Vicia sativa VICSAT Lb CAA70431.1 GenBank

Vigna unguiculata VIGUNG Lb AAA86756.1 GenBank

Lb AAB65769.1 GenBank

Vitis vinifera VITVIN nsHb-1 CBI32537.3 GenBank

nsHb-1 CBI32538.3 GenBank

tHb XP 002284484.1 GenBank

Wolffia arrhiza WOLARR nsHb-1 AEQ39061 GenBank

Zea mays ssp. mays ZEAMAY nsHb-1 AAG01375.1 GenBank

nsHb-1 AAZ98790.1 GenBank

tHb ACG29525.1 GenBank

Zea mays ssp. parviglumis ZEAPAR nsHb-1 AAG01183.1 GenBank

2. The phylogeny and evolution of land plant hemoglobins 2.2. Ancient land plant non-symbiotic hemoglobins and the

evolution of the non-symbiotic hemoglobin-leghemoglobin

2.1. Phylogeny of land plant hemoglobins lineage

In general, the evolution of land plant 3/3 hemoglobins par- Mosses (Ceratodon purpureus and P. patens) non-symbiotic

alleled the major transitions in land plant evolution. There is hemoglobins are the oldest non-symbiotic hemoglobins charac-

at present no agreement on the times at which the transitions terized so far [90,91], and thus provide insight into the properties

occurred, mostly due to the disagreement of the fossil dates of first land plant non-symbiotic hemoglobins. Specifically, gene

with the dates provided by molecular phylogenetic analyses. The analysis revealed that C. purpureus and P. patens nshb are inter-

molecular phylogenetic analyses tend to push the overall dates to rupted by three introns inserted similarly to known land plant nshb,

appreciably earlier times [78–83]. The first relevant transition or shb, and lb genes, suggesting that the ancestor globin gene of land

rather series of transitions are the emergence of embryophytes plant nshb, shb, and lb genes also contained the three introns. Fur-

at 430−450 mya [80], perhaps as early as ∼800 mya [84], fol- thermore, expression analyses revealed that C. purpureus nshb is

lowed by the emergence of bryophytes (hornworts, mosses, and up-regulated by stress conditions that were essential during land

liverworts) over the following 40–50 mya. Thus, the hemoglobins colonization by plants, such as high osmolarity, high and low tem-

of Marchantia (liverwort), Physcomitrella patens (moss), and the peratures, and nutrient deprivation [91,92]. Thus, it is likely that

spike moss Selaginella (Lycopsid), the oldest living groups of land non-symbiotic hemoglobins played a role during plant adaptation

plants, are closest to the ancestral embryophyte hemoglobins. to the land environment.

The next major transition comprises the emergence of spermato- Sequence alignment of primitive and evolved non-symbiotic

phytes (seed plants) and the split into gymnosperms (conifers, hemoglobins, symbiotic hemoglobins, and leghemoglobins

cycads, Gnetales, and Ginko) and ancestors of angiosperms (flow- revealed that the size of the polypeptide decreased over time

ering plants): it occurred at about 320 mya, preceded by a whole at the N-terminal region, mostly at pre-helix A. A predicted

genome duplication event [85]. The third transition represents the leader peptidase site was identified in the pre-helix A region of

diversification of angiosperms at 140−180 mya [82], also preceded the C. purpureus and P. patens non-symbiotic hemoglobins [36].

by a whole genome duplication event [85]. Because Euryale ferox The pre-helix A was suggested to function as a leader peptide

(Nymphaeaceae) has non-symbiotic hemoglobins type 1 and type in primitive non-symbiotic hemoglobins [36], similarly to the

2 [86], and because the Nymphaeales (water lilies and relatives) Chlamydomonas T1 truncated hemoglobin, which is translocated

represent one of the earliest branching angiosperm lineages, it is from cell to chloroplasts [93]. Thus, a possibility is

tempting to conclude that the latter whole genome duplication that an ancestor to land plant non-symbiotic hemoglobins was

event prior to 150 mya [85,87] represents the origin of the non- translocated from cytoplasm to cellular organelles, and that non-

symbiotic hemoglobins type 1 and type 2 from the embryophyte symbiotic hemoglobins became cytoplasmic during the evolution

non-symbiotic hemoglobin. The final major transition is the split of land plants [36].

of angiosperms into monocots and dicots at 140−150 mya [78]. Although the crystal structure of moss non-symbiotic

It is likely that symbiotic hemoglobins originated thereafter from hemoglobins is not known, the structures of C. purpureus and

a non-symbiotic hemoglobin and spread among nodulating flow- P. patens non-symbiotic hemoglobins were modeled [91] and

ering plants, while leghemoglobins evolved only within legumes deposited in the Caspur database (http://mi.caspur.it/PMDB/,

at approximately 60 mya (Fig. 3) [43,88,89]. Little is known about identification number PM0074985). The structures were similar

the phylogeny of actinorhizal symbiotic hemoglobins, and it is still to the experimentally determined crystal structure of land plant

unclear whether or not these proteins evolved with non-legume non-symbiotic hemoglobins type 1. Furthermore, the spectro-

angiosperms. scopic 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 hexacoordi- both penta- and hexa-coordination existed in algal hemoglobins

nated (Vázquez-Limón and Arredondo-Peter, in preparation). The and that plant non-symbiotic hemoglobins originated from a hex-

similarity of the properties of moss non-symbiotic hemoglobins acoordinated hemoglobin in the ancestor of algae and land plants.

with those of non-symbiotic hemoglobins type 1 imply that the Interestingly, the Micromonas and Ostreococcus nshb genes lack

first land plant non-symbiotic hemoglobins had high O2-binding introns, whereas known land plant nshb genes have three introns.

affinities and were probably not involved in O2-transport. Genomic information from Charophyte algae should provide infor-

A comparison of the predicted structure of moss non-symbiotic mation on the intron structure of their hemoglobins and determine

hemoglobins, native rice Hb1 (a non-symbiotic hemoglobin type 1), whether the proposal that introns inserted into an algal nshb prior

and soybean leghemoglobin a revealed the major alterations occur- to the origin of land plant nshb genes [97] is correct or not.

ring during the evolution of land plant non-symbiotic hemoglobins

to leghemoglobins [91]. These changes consisted in (i) a hexacoor-

3.2. The origin and evolution of algal and land plant truncated

dinate to pentacoordinate transition at the heme-Fe, (ii) a decrease

hemoglobins

in the sizes of the CD-loop and the N- and C-terminal regions, and

(iii) generation of a more compact protein structure.

Our findings regarding the origin of plant, Chlorophyte, and

The minimum age of the stem lineage of the N2-fixing clade

Stramenopile T2 truncated hemoglobins were quite different from

including Rosaceae and (e.g. , Casuarinaceae,

those regarding the 3/3 plant hemoglobins. A preliminary analysis

and Myricaceae) is estimated to be approximately 94 mya [94].

indicated the possibility of horizontal gene transfer from a progeni-

It is reasonable to assume that leghemoglobins evolved from a

tor bacterium to one of the following bacterial phyla: the Chloroflexi,

non-symbiotic hemoglobin ancestor concomitantly with the emer-

Deinococcales, Bacilli, and Actinomycetes [49]. A more recent reex-

gence of legumes at ca. 60 mya [94,95] and of rhizobial nodulation

amination corroborated the earlier results and refined them to

providing functional specialization in N2-fixing nodules [88,89].

show that the six known Chloroflexi T2 truncated hemoglobins were

The Caesalpinoideae is the oldest subfamily of legumes and con-

the closest relatives of the land plant, algal and stramenopile T2

tains both nodulating and non-nodulating species. It is thus likely

truncated hemoglobins [99]. This finding is not surprising given

that the non-symbiotic hemoglobin to leghemoglobin transition

that recent studies have demonstrated the horizontal gene trans-

occurred in a caesalpinoid legume. The characterization of a

fer of over 50 genes from progenitors of modern Chlamydia to the

Chamaecrista fasciculata hemoglobin, a caesalpinoid hemoglobin,

ancestral primary photosynthetic eukaryote [84].

intermediate between non-symbiotic hemoglobins and leghe-

The computationally predicted structure of Chlorella T1 trun-

moglobins, has the same alterations as above [88]. Apparently these

cated hemoglobin and Physcomitrella and Arabidopsis T2 truncated

alterations permitted leghemoglobins, and probably other sym-

hemoglobins based on the only known eukaryote truncated

biotic hemoglobins with similar structures to evolve O2-binding

hemoglobin structures, the T1 truncated hemoglobins from

kinetic properties that enabled them to function in maintaining

Chlamydomonas and Paramecium (Brookhaven

N2-fixation in nodules.

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 trun-

3. The origin of land plant hemoglobins

cated hemoglobins had the 2/2-fold typical of bacterial T2 truncated

hemoglobins (Fernández and Arredondo-Peter, unpublished).

3.1. The putative algal ancestor of land plant non-symbiotic

The number of introns in algal thb genes ranges from 2 to >5

hemoglobins

and their positions are variable [49] indicating that introns evolved

under relaxed selection. In contrast, the number and position of

The latest estimate of the origin of the last eukaryote common

introns in land plant thb genes are conserved with the exception of

ancestor is approximately 1200 mya, close to the emergence of ban-

a soybean thb gene (Genbank accession number AAS48191), which

giophytes (red algae) from the stem lineage of the Archaeplastida

has only one intron. The known land plant thbs have three con-

(green algae and land plants) [80]. It is widely accepted that the

served introns, suggesting stabilizing selection during the evolution

paraphyletic groups of algae, the Chlorophyta and the Charophyta,

of introns in land plant thb genes [49].

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, 4. Rates of evolution of land plant hemoglobins

including those of the Prasinophyceae algae Micromonas and

Ostreococcus. Although Ostreococcus lucimarinus has no globins, The evolutionary rates of land plant hemoglobins were esti-

Ostreococcus tauri, Micromonas pusilla, and Micromonas sp. RCC299 mated from the substitutions and divergence values

each have a single domain 3/3 globin. Their sequences place them relative to the moss hemoglobins considered to be closest to

between bacterial F family single domain globins and bryophyte the ancestral hemoglobins [100]. The divergence values of non-

and other land plant non-symbiotic hemoglobins [49,97], suggest- symbiotic hemoglobins and leghemoglobins indicate that they did

ing that these algal non-symbiotic hemoglobin-like globins share a not evolve at constant rates. Apparently, high variation occurred in

bacterial F family single domain globin ancestor with the land plant non-symbiotic hemoglobins during the first ∼40 million of years

non-symbiotic hemoglobins. Unfortunately, a major gap still exists of land plant evolution, followed by a decreased rate of diver-

between evolved green algae (i.e. the direct ancestors of land plants gence during the subsequent ∼200 million of years, i.e. during

[96,98]) and bryophyte non-symbiotic hemoglobins, given that the the trachaeophyte to magnoliophyta (angiosperms) transition. Fur-

latest study implicates the Zygnemetales and Coleochaetales as the thermore, the rate of divergence was higher in non-symbiotic

two Charophyte sister groups to embryophytes [83] and that no hemoglobins type 2 and leghemoglobins than in non-symbiotic

Charophyte genome sequences are available. hemoglobins type 1, indicating that non-symbiotic hemoglobins

Although the predicted Micromonas and Ostreococcus non- type 2 and leghemoglobins evolved under relaxed selection com-

symbiotic hemoglobin structures have the canonical myoglobin- pared to non-symbiotic hemoglobins type 1. In contrast, land

fold and heme-Fe coordination to a proximal His, they show that plant truncated hemoglobins apparently evolved at a rather con-

the two Micromonas hemoglobins are hexacoordinate, while the stant rate. However, the high variability detected between the

O. tauri hemoglobin is pentacoordinate [97]. This suggests that Physcomitrella and Selaginella truncated hemoglobins suggests a

C. Vázquez-Limón et al. / Plant Science 191–192 (2012) 71–81 79

major divergence in land plant truncated hemoglobins during the heme-Fe coordination from hexa- to penta-coordination, by

the bryophyte to trachaeophyte transition [100]. Thus, a general decrease in the lengths of the CD-loop and the N- and C-terminal

conclusion is that hemoglobins were highly variable during the col- regions, and by compaction of the protein structure leading to

onization of land by plants, but the rate of evolution decreased decreased mobility of the distal His [88,91]. (vii) Although at least

prior to the origin of magnoliophyta. However, with the excep- two major whole genome duplication events were identified in

tion of leghemoglobins, which evolved under stabilizing selection plant phylogeny [85], the T2 hemoglobins in contrast to the 3/3

to specifically function in nodules of N2-fixing legumes (Section hemoglobins do not appear to have undergone any duplication.

2.2), it is not known whether or not rates of divergence affected This review reveals at least three lacunae in our ability to com-

the hemoglobin function (s) during the evolution of land plants. pletely 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

5. Going back (>3000 mya) to the (primeval) structural

Zygnemetales and Coleochaetales [82,83]. Undoubtedly, the most

ancestor of 3/3 and 2/2 hemoglobins?

important lacuna is the absence of genomic information about

Charophyte algae. Except for the Picea sitchensis genome, the

Globins are ancient proteins that originated early in the evolu-

absence of genomic information about ferns, ginkos, and cycads

tion of life, i.e. more than 3000 mya [7,12]. Prokaryotes contain a

represents the second lacuna. Little is known about the role

variety of 3/3 and 2/2 hemoglobins as mentioned earlier, and we do

and evolution of symbiotic hemoglobins in actinorhizal symbio-

not have at present any clues as to the origins of the two 3/3 (F and

sis involving the actinobacterium Frankia and representatives of

S) families and the 2/2 T family. It appears likely that the two 3/3

the orders Cucurbitales, Fagales, and relative to the leghe-

families shared a common precursor. Still, we do not know whether

moglobins of the legume family. It has been estimated that in

the two structural lineages shared a common ancestor or emerged

terms of N2-fixation the contributions of the two types of plant-

separately. The computational analysis of land plant hemoglobin

microbial symbioses are approximately equal [89]. Hence, the

folding indicates that it proceeds through the formation of folding

absence of genomic data about plants with symbiotic hemoglobins

modules formed by helices A, B and C, and E, F, G, and H (folding

other than leghemoglobins is yet another lacuna that needs to be

modules A/C and E/H, respectively) [101]. Modeling of the rice Hb1

filled. Finally, an additional lacuna is the lack of a land plant T2

(a non-symbiotic hemoglobin type 1) A/C and E/H modules sug-

truncated hemoglobin crystal structure. The sequences of these

gests that module E/H overlaps to the Mycobacterium tuberculosis

proteins are mostly over 160 amino acids, with ∼20 to ∼40 amino

HbO (a T3 truncated hemoglobin) 2/2-fold. This result implies that

acid extensions at both chain termini, relative to the microbial T2

module E/H is an ancient structural motif. Its presence in the com-

truncated hemoglobins. Although the predicted structure of the

mon globin ancestor would provide for the emergence of a 3/3-fold

globin domain from the Physcomitrella and Arabidopsis T2 trun-

through the addition of module A/C, and for the origin of a 2/2-fold

cated hemoglobins fits satisfactorily the microbial T2 truncated

via the addition of a B/C module.

hemoglobin structure (Section 3.2), we are ignorant of the folding of

the extensions. Thus, it would be desirable to have both structures

6. Concluding remarks and future directions (i.e. the full, globin plus extensions, structure) in order to compare

them and identify the alterations that may have occurred during

Overall, the outline of plant globin evolution subsequent to land the evolution of land plant T2 truncated hemoglobins.

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 Acknowledgments

include the following. (i) The land plant (embryophyte) 3/3 non-

symbiotic hemoglobins originated from a precursor non-symbiotic

Authors are grateful to Gustavo Rodríguez Alonso for providing

hemoglobin in the ancestor shared with green algae. (ii) The

Fig. 1. Work in R.A.-P. laboratory has been funded by SEP-PROMEP

precursor globin may have descended from a bacterial (Cyanobac-

(grant no. UAEMor-PTC-01-01/PTC23) and Consejo Nacional de

teria/Proteobacteria) F family single domain globin as the result of

Ciencia y Tecnología (CoNaCyT grant nos. 25229N and 42873Q),

horizontal gene transfer events accompanying the two accepted

México. C.V.-L. is a postdoctoral fellow supported by CoNaCyT.

endosymbiotic events to the eukaryote ancestor common to all the

eukaryotes. (iii) The plant 2/2 truncated hemoglobins originated

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