Molecular Cloning and Characterization of a Moss (Ceratodon purpureus)
Nonsymbiotic Hemoglobin Provides Insight into the Early Evolution of Plant
Nonsymbiotic Hemoglobins
Verónica Garrocho-Villegas and Raúl Arredondo-Peter
Laboratorio de Biofı́sica y Biologı́a Molecular, Facultad de Ciencias, Universidad Autónoma del Estado de Morelos, Cuernavaca,
Morelos, México
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 O2
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.
Introduction
Nonsymbiotic hemoglobins (nsHbs) are plant proteins
that reversibly bind O2 and other gaseous ligands. These
proteins have been detected in seeds, roots, stems, leaves,
and flowers from plants growing in normal conditions
(Taylor et al. 1994; Andersson et al. 1996; Lira-Ruan
et al. 2001; Ross et al. 2004); however, levels of nsHbs
increase when plants are subjected to stress conditions,
such as microaerobiosis and anoxia (Taylor et al. 1994;
Trevaskis et al. 1997; Lira-Ruan et al. 2001). Based on
O2 affinity and sequence similarity nsHbs are classified into
class 1 and class 2 (nsHbs-1 and nsHbs-2, respectively)
(Trevaskis et al. 1997). The O2 affinity of nsHbs-1 and
nsHbs-2 is very high and high, respectively (ArredondoPeter et al. 1997; Duff et al. 1997; Trevaskis et al.
1997). The very high O2 affinity of nsHbs-1 results from
an extremely low O2 dissociation rate constant, which suggests that in vivo these proteins do not release O2 after oxygenation and therefore have functions other than O2
transport (Arredondo-Peter et al. 1998). Recent evidence
showed that a possible function of nsHbs-1 is to modulate
the levels of NO and redox potentials (Igamberdiev et al.
2005; Hebelstrup et al. 2007). The O2 dissociation rate constant of nsHbs-2 is higher than that of nsHbs-1, which suggests that an in vivo function for nsHbs-2 could be O2
transport (Trevaskis et al. 1997).
The nsHbs have been identified in bryophyte, gymnosperm, and angiosperm species and are therefore widespread in land plants (Garrocho-Villegas et al. 2007).
Gene and protein sequence comparison suggests that
nsHbs-1 and nsHbs-2 evolved from a common ancestor;
however, it is still unclear if an nsHb-1–like or nsHb-2–like
protein was the ancestor to plant nsHbs. Full bryophyte
cDNA sequences coding for moss (Physcomitrella patens
Key words: Ceratodon, evolution, gene expression, globin,
molecular modeling, nonsymbiotic.
E-mail: [email protected].
Mol. Biol. Evol. 25(7):1482–1487. 2008
doi:10.1093/molbev/msn096
Advance Access publication April 16, 2008
Ó The Author 2008. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
and Ceratodon purpureus) nsHbs and a partial gene sequence coding for a liverwort (Marchanthia polymorpha)
nsHb were deposited in the GenBank database (with accession numbers AF218049, AF309562, and AY026341, respectively). The phenetic analysis of plant Hb sequences,
including those from moss nsHbs, showed that bryophyte
nsHbs are ancestral but intermediate to nsHbs-1 and nsHbs2 (Garrocho-Villegas et al. 2007; Gopalasubramaniam,
Kovacs, et al. 2008). Thus, the apparent oldest land plant
nsHbs are bryophyte nsHbs. However, despite limited reports (Arredondo-Peter et al. 2000; Trent et al. 2001), bryophyte nsHbs remain uncharacterized and thus very little is
known about the properties of primeval plant nsHbs. Here,
we report the cloning and molecular characterization of
a moss (C. purpureus) nsHb, including an analysis of gene
expression in plant organs and modeling of the protein tertiary structure.
Materials and Methods
Plant Material and Growing Conditions
Ceratodon purpureus protonemas were kindly provided by Dr Ralf Reski (Albert-Ludwigs-Universität,
Freiburg, Germany) as an axenic culture. Protonemas were
cultured in Knop medium (Reski and Abel 1985) and
grown in a plant growing chamber (Biotronette Plant
Growth Chamber, Lab-Line Instruments, Melrose Park,
IL) at 25 °C with 16/8-h light/dark periods. Ceratodon purpureus gametophytes were obtained after culturing protonemas for 8 weeks. Normal and stressed (see below)
plants were harvested, immediately frozen in liquid N2,
and stored at 70 °C until used.
The (nshb) gene expression in plants subjected to
stress conditions was evaluated in 2-week-old protonemas
by duplicate. Osmotic-, salt-, and nitrate-stress conditions
were generated by adding 10% sucrose (Umeda et al.
1994), 250 mM NaCl (Umeda et al. 1994), and 85 mM
Ca(NO3)2 (Ohwaki et al. 2005) to the growth medium, respectively. Cold-stress conditions were generated by incubating protonemas at 4 °C for 24 h (Takahashi et al. 1994).
Characterization of a Moss Hemoglobin
Dark-stress conditions were generated by incubating the
protonemas in darkness for 24 h inside a plant growth
chamber (Lira-Ruan et al. 2001). Flooding-stress conditions were generated by fully covering the protonemas with
distilled water for 24 h (Taylor et al. 1994; Lira-Ruan et al.
2001). Heat-shock stress conditions were generated by incubating the protonemas at 42 °C for 2 h (Higo K and Higo
H 1993). Control protonemas (i.e., those growing under
normal conditions) were cultivated for 2 weeks, and control
and stressed protonemas were harvested at the same time.
Total DNA and Poly(Aþ) RNA Isolation
Total DNA was isolated from ;100 mg of C. purpureus
protonemas using the cetyltrimethylammoniun bromide
(CTAB) method (Crose and Amorese 1978). Poly(Aþ)
RNA was isolated from plant tissues using the QuickPrep Micro
mRNA Purification kit (Amersham Biosciences, Little Chalfont Buckinghamshire, United Kingdom) following the
manufacturer’s instructions. Total DNA and poly(Aþ)
RNA were quantitated by spectrophotometry assuming
1 A260 5 50 or 40 lg/ml for DNA or RNA, respectively
(Ausubel et al. 1995).
Polymerase Chain Reaction Amplification, Gene
Cloning, and DNA Sequencing
Primers were designed for polymerase chain reaction
(PCR) to amplify the C. purpureus nshb gene using sequences at the start and stop codons of the C. purpureus nsHb
cDNA (GenBank accession number AF309562). The
primer sequences (degenerated for the NcoI and EcoRI restriction sites [underlined]) were (sense) primer Ceratodon
nsHb (CerHb)/ATG-NcoI: 5#-CCATGGCACCACCGACAGTCG-3# and (antisense) primer CerHb/TAA-EcoRI:
5#-GAATTCTTACTGAGCAGCAGCCCTC-3#. Total C.
purpureus DNA (;1 lg) was used as a template for
PCR amplification. PCR components and concentrations
were 1 lM of each sense and antisense primer, 400 lM
of each deoxynucleotide triphosphate, and 1 U of Taq
DNA polymerase (Invitrogen, Carlsbad, CA) in 1X PCR
buffer containing 4 mM MgCl2 in a final volume of
25 ll. PCR amplification was carried out for 35 cycles
at 55 °C for annealing using a thermalcycler (Minicycler,
MJ Research, Watertown, MA). PCR products were detected in a 1.2% agarose gel after staining with ethidium
bromide, isolated from the gel using the GeneClean kit
(Q-BIOgene, Carlsbad, CA), and cloned into the pCR2.1
cloning vector (Invitrogen) following the manufacturer’s
instructions. Insert DNA was fully sequenced in both orientations at the Molecular Biology Facility of the Cell
Physiology Institute of the National Autonomous University of México.
Southern Blot Analysis
Southern blot analysis of the total C. purpureus DNA
was done as described by Arredondo-Peter et al. (1997) using the C. purpureus nshb gene as a probe. Membranes
were prehybridized and hybridized at 55 °C and washed
1483
at high stringency (60 °C) in 2 standard saline citrate
(SSC)/0.1% sodium dodecyl sulfate (SDS) twice for
5 min each and 0.5 SSC/0.1% SDS twice for 15 min each.
Gene Expression Analysis by Reverse Transcriptase–
Polymerase Chain Reaction
Expression of the C. purpureus nshb gene was examined in protonemas and gametophytes (4 and 10 weeks old,
respectively) and in protonemas grown under stress conditions (see above) by reverse transcriptase–polymerase chain
reaction (RT-PCR) using the GeneAmp RNA PCR kit
(Applied Biosystems, Branchburg, NJ) as described by
Arredondo-Peter et al. (1997). Both reverse transcription
and PCR amplification were performed using the CerHb/
ATG-NcoI and CerHb/TAA-EcoRI primers and 100 ng
of template poly(Aþ) RNA. Amplification conditions were
identical to those used for the amplification of the C. purpureus nshb gene (see above). After gel electrophoresis,
PCR fragments were stained with ethidium bromide, and
the gel was photodocumented, scanned, and analyzed using
the ImageJ 1.36b software (http://rsb.info.nih.gov/ij/). The
relative abundance of the CerHb transcripts in control and
differentially treated protonemas was calculated from the
intensity of each band using as reference the intensity detected in nsHb amplicon observed in protonemas grown under normal conditions.
In Silico Analysis and Molecular Modeling
Multiple sequence alignment and cluster analysis of
the C. purpureus nsHb and selected plant Hbs were performed using the Neighbor-Joining method of the ClustalX
program (Thompson et al. 1997). The tertiary structure of
the C. purpureus nsHb was predicted by homology modeling using the crystal structure of the rice Hb1 (Hargrove
et al. 2000) (PDB ID 1D8U) as a template as described by
Gopalasubramaniam, Garrocho-Villegas, et al. (2008).
Results and Discussion
Cloning of a C. purpureus nshb Gene
The nsHbs evolved alongside land plants after bryophyte-like organisms colonized land about 450 MYA.
Thus, it is likely that these proteins played roles in plant
adaptation to the terrestrial environment (Ross et al.
2002; Garrocho-Villegas et al. 2007). To characterize
a bryophyte nsHb, we designed PCR primers from the
C. purpureus Hb cDNA (GenBank accession number
AF309562) and used them to amplify fragments of 540
and 950 bp in length from template C. purpureus DNA
(supplementary fig. S1, Supplementary Material online).
These PCR fragments were cloned and sequenced. Blast
analysis (Altschul et al. 1990) with sequences from the
GenBank database revealed that sequence of the 540-bp
fragment had no similarity to plant and nonplant Hbs
and that it corresponded to a nonspecific amplification.
However, the coding sequence of the 950-bp fragment
was identical to that from the C. purpureus Hb cDNA, thus
1484 Garrocho-Villegas and Arredondo-Peter
showing that it corresponded to the C. purpureus hb (cerhb)
gene. The CerHb cDNA and gene sequences were compared to identify exon and intron sequences. The cerhb
gene has 4 exons interrupted by 3 introns (supplementary
fig. S2, Supplementary Material online), which are located
at identical positions as all the known plant nshbs and leghemoglobin (lbs). This observation indicates that the ancestral nshb gene was interrupted by 3 introns located in
identical positions as those in modern nshb and lb genes.
Characterization of the CerHb Protein Sequence
The cerhb gene codes for a predicted protein of 177
amino acid residues in length (supplementary fig. S2, Supplementary Material online) with a calculated molecular
mass of 19,535 Da. Sequence analysis showed that CerHb
contains distal (H86) and proximal (H121) His and Phe B10
(F52), which are highly conserved in plant Hbs. However,
in Ceratodon and Physcomitrella nsHbs (see below), the
CD1 position is occupied by Tyr (Y66) instead of Phe,
which is conserved in evolved nsHbs (supplementary fig.
S3, Supplementary Material online). This observation
shows that Tyr in ancestral nsHbs occupied the CD1 position and was replaced by Phe during the evolution of nsHbs.
Arredondo-Peter et al. (2000) identified and partially
characterized an nsHb sequence from the moss P. patens.
Multiple sequence alignment showed that moss (C. purpureus and P. patens) nsHbs are the longest known plant Hbs
and that the size of plant nsHbs decreased over time, mostly
due to a loss of amino acid residues at the N-terminal (prehelix A) region (supplementary fig. S3, Supplementary
Material online) (Ross et al. 2002). Also, sequence alignment revealed that the C. purpureus and P. patens nsHbs
are 80% identical and that the highest sequence variability
occurs at the N-termini regions (i.e., within the first
35 amino acid residues). The identity value of moss nsHbs
is similar to that observed between evolved nsHbs. For
example, the identity value between rice and maize nsHb1s
is approximately 80%. This observation suggests that
nsHbs evolved at approximately the same rate and that
major changes during plant nsHbs evolution occurred at
the N-terminal region (see below).
Identification of the cerhb Gene Copy Number
The number of nshb gene copies varies with each
species in higher plants. For example, single and multiple
nshb gene copies exist in barley (Taylor et al. 1994) and rice
(Arredondo-Peter et al. 1997; Lira-Ruan et al. 2002), respectively. Also, the search for homologs to the globin fold
in the SUPERFAMILY database (Gough et al. 2001) identified several Hb sequences in the P. patens genome (data
not shown). When total C. purpureus DNA was subjected
to Southern blotting with the cerhb probe, 2 hybridizing
fragments of 4 and 6–7 kb in length were detected in
the DNA digested with EcoRI (fig. 1). This observation
suggests that at least 2 copies of the nshb gene exist
in the C. purpureus genome. The existence of cDNAs
coding for CerHb indicates that cerhb is functional in
C. purpureus.
FIG. 1.—Southern blot analysis of the Ceratodon purpureus total
DNA using the cerhb gene as a probe. Hybridization signals were
detected by colorimetry as described by Arredondo-Peter et al. (1997).
Panel (a) C. purpureus total DNA digested with HindIII, EcoRI, and
BamHI restriction enzymes. Panel (b) hybridization signals after DNA
transfer to a nylon (Hybond Nþ, Amersham Biosciences) membrane.
Molecular size markers are shown in base pairs.
Expression of the cerhb Gene in C. purpureus Grown
under Normal and Stress Conditions
The nsHb transcripts and proteins have been reported
to exist in several organs from plants growing under normal
and stress conditions (Taylor et al. 1994; Andersson et al.
1996; Arredondo-Peter et al. 1997; Trevaskis et al. 1997;
Lira-Ruan et al. 2001; Ross et al. 2001). We used RTPCR and specific primers for CerHb to evaluate the cerhb
gene expression in C. purpureus gametophytes and protonemas growing under normal and stress conditions. We selected stress conditions that others have evaluated for nshb
gene expression in diverse higher plants; also, these stress
conditions have been postulated to be primary plant adaptations during land colonization (i.e., osmoregulation,
osmoprotection, temperature tolerance, and nutrient availability) (Rensing et al. 2008). We amplified transcripts of
the expected molecular size (531 bp) from the C. purpureus
gametophytes and protonemas grown under normal and
stress conditions. Figure 2a shows that the cerhb gene expresses in C. purpureus organs. Analysis of the relative
abundance for the CerHb transcripts showed that cerhb expression is higher in gametophytes than in protonemas.
Also, the cerhb gene over- and underexpresses in protonemas grown in sucrose-, heat-shock-, cold- and nitrate-, and
salt-, dark- and flooding-stress conditions, respectively
(fig. 2b). These observations suggest that higher levels of
CerHb could be required for differentiating gametophytes
and that CerHb functions during the plant response to
osmotic (sucrose), temperature (heat and cold), and high levels of nitrate stresses but not in salt, dark, and flooding
stresses.
Characterization of a Moss Hemoglobin
1485
FIG. 2.—Ceratodon purpureus nshb gene expression in C.
purpureus protonemas and gametophytes growing under stress and
normal conditions, respectively. Panel (a) CerHb transcripts (531 bp in
length) amplified by RT-PCR. Panel (b) relative abundance of the
CerHb transcripts; histogram was constructed using the densitometric
values from the gel shown in panel (a). Data are representative of 2
replicates. Ga, gametophytes; C1 and C2, control protonemas 1 and 2,
respectively; Su, sucrose-stress condition; He, heat-shock stress
condition; Co, cold-stress condition; Sa, salt-stress condition; Da,
dark-stress condition; Fl, flooding-stress condition; and NO3, nitratestress condition. C1 and C2 were used as controls for He, Co, Sa and
Da, and Fl and NO3, respectively. For experimental details, see the
Materials and Methods.
Predicted Tertiary Structure of CerHb
Nothing is known about the tertiary structure of bryophyte nsHbs. Understanding the structural properties of
nsHbs is important to clarifying the role these proteins play
in plant organs. In silico methods exist to predict with high
reliability the tertiary structure of proteins from template
structures (Sáenz-Rivera et al. 2004; Gopalasubramaniam,
Garrocho-Villegas, et al. 2008). Predicting a structure
yields insights into potential evolutionary pathways for
nsHbs. Because CerHb and rice Hb1 are approximately
50% identical, we predicted the tertiary structure of CerHb
using rice Hb1 as a template. Figure 3a shows that the predicted CerHb folds into the globin fold and that its structure
is quite similar to that of native rice Hb1, including the positions of helices E and F, where distal and proximal His are
located, respectively. However, the structures differ in that
the N- and C-termini regions are longer in CerHb than in
rice Hb1, the CD-loop folds differently, and an (2 turns)
a-helix exists within the prehelix A region of predicted
CerHb, which may facilitate protein translocation. Ross
et al. (2002) detected leader peptidase-like sites in moss
nsHbs and suggested that the ancestor to plant nsHbs
was translocated to cellular organelles. The existence of
an a-helix at the prehelix A region in predicted CerHb
(fig. 3a) is consistent with the possibility that CerHb
(and other bryophyte nsHbs) may function inside cell organelles. If this is correct, it is possible that nsHbs became
cytoplasmic during evolution (Ross et al. 2002). The examination of the amino acid residues that are essential for ligands binding showed that the positions of the proximal
and distal His, Phe B10, Tyr/Phe CD1, and Tyr 158 are
quite similar in predicted CerHb and native rice Hb1
(fig. 3b). These observations suggest that heme–Fe in
FIG. 3.—Panel (a) overlay of predicted CerHb (gray) and native
rice Hb1 (black) tertiary structures. Helices (including to prehelix A) are
indicated with letters A–H. Arrow shows the predicted (2 turns) a-helix
within the CerHb prehelix A. The predicted structure of CerHb is
deposited in the Protein Model Database (http://mi.caspur.it/PMDB/)
under the ID number PM0075013. Panel (b) overlay of selected amino
acids in the predicted CerHb (gray) and native rice Hb1 (black) heme
pocket. For experimental details, see the Materials and Methods.
CerHb is hexacoordinate and that the kinetic properties
of CerHb and rice Hb1 are similar. Thus, it is likely that,
because of a low O2 dissociation rate constant, the O2 affinity of CerHb is rather high, and was, as well, in ancestral
nsHb.
The Structural Evolution of CerHb and Plant nsHbs
Comparative analysis of the CerHb, rice Hb1, and soybean leghemoglobin a (Lba) tertiary structures (i.e., primitive to evolved plant Hbs) reveals major evolutionary
changes that probably occurred during plant Hbs evolution
(fig. 4). These changes include 1) a hexacoordinate to pentacoordinate transition at the heme prosthetic group (Hoy
et al. 2007; Gopalasubramaniam, Kovacs, et al. 2008), 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. Some of these structural changes have been described for the nsHb to Lb transition and for the specialization of a symbiotic function for Lb (Gopalasubramaniam,
Kovacs, et al. 2008). Thus, the pattern of structural evolution for nsHbs has apparently been conserved for 450 Myr,
which allowed nsHbs to specialize and help plants adapt to
and diversify within the land environment.
1486 Garrocho-Villegas and Arredondo-Peter
FIG. 4.—Comparison of the predicted CerHb and native rice Hb1 and soybean Lba tertiary structures and surface charge distribution. Blue and red
colors represent positively and negatively charged amino acid residues, respectively. Coordinates for the rice Hb1 and soybean Lba structures were
obtained from Brookhaven Protein Database with the ID numbers 1D8U and 1BIN, respectively. The arrow above the figure illustrates the postulated
transition from primitive to evolved plant Hbs.
Supplementary Material
Supplementary figures S1–S3 are available at Molecular Biology and Evolutiononline (http://www.mbe.
oxfordjournals.org/).
Acknowledgments
The authors wish to express their gratitude to Dr Ralf
Reski (Albert-Ludwigs-Universität, Freiburg, Germany) for
kindly providing the C. purpureus protonemas, to an anonymous reviewer for useful suggestions to improve the contents of this article, and to Miss Gillian Klucas for
corrections made to improve the English language. This
work was funded by Consejo Nacional de Ciencia y Tecnologı́a (CoNaCyT project no. 42873Q), México. V.G.-V. is
a postdoctoral fellow financed by CoNaCyT (IdAP 9272).
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Geoffrey McFadden, Associate Editor
Accepted April 7, 2008