Supplementary material

A single hemoglobin gene in Myrica gale retains both symbiotic and non-symbiotic specificity

Anne B. Heckmann, Kim H. Hebelstrup, Knud Larsen , Nuno M. Micaelo and Erik Ø. Jensen

Protein modelling. A theoretical model of MgHb protein structure was derived using MODELLER (Sali and Blundell, 1993) employing the known X-ray structure of riceHb1 in PDB entry 1D8U (Dordas, et al., 2004) as a template. MgHb and riceHb1 share 64% protein sequence similarity, and the alignment was optimised until a good quality model of the riceHb1 homodimer was achieved. The binding free energy of the two interacting monomers was calculated by Gb = Gdimer – (Gmonomer1 + Gmonomer2), in which Gx = Gel + Gnp + Gstrain – TSsc(Froloff, et al., 1997). The calculations of the terms in the free energy expression was done as described by other authors (Teixeira, et al., 2004). Six systems were simulated: The homodimer X-ray structure of riceHb1 and the two corresponding monomers; the MgHb homodimer and the two corresponding monomers obtained by comparative modelling. The molecular mechanics/dynamics (MM/MD) simulations were performed with the GROMACS package (Berendsen, et al., 1995, Lindahl, et al., 2001) employing the GROMOS force field (Scott, et al., 1999, Van Gunsteren, 1996) in the NVT ensemble. The systems were solvated with the SPC water model (Hermans, et al., 1984). Heat baths (Berendsen, et al., 1984, Van Gunsteren and Berendsen, 1990) were used with separate coupling for the solvent and the solute at 300 K and a coupling constant of 0.1 pico seconds (ps). Non-bonded interactions were treated with the twin-range method (Van Gunsteren and Berendsen, 1990) at 8 and 14 Å updated every 10 steps. Long range interactions were treated with a continuum reaction field using a dielectric constant of 54 (Smith and Vangunsteren, 1994). Each MM/MD simulation was initialised with a first phase of energy minimization in 5000 steps of steepest-descent with all protein heavy atoms restrained with a force constant of 105 kJ/(mol nm), followed by 5000 steps with the main chain atoms restrained and finally, 5000 steps unrestrained. MM/MD runs were started with 50-ps simulations with all protein heavy atoms restrained with a force constant of 105 kJ/(mol nm) with initial velocities taken from a Maxwell-Boltzman distribution at 300K, followed by 50-ps with C atoms restrained. The system was equilibrated in 2 ns and continued for more 3 ns of sampling.

Sequences used for phylogenetic analysis.

Class O: Marchantia sp.: AY026341; Physcomitrella patens: Z98685.

Class I: Oryza sativa U76031, U76028; Zea mays: AF236080; Hordeum vulgare: U94968; Casuarina glauca: X53950; Glycine max: U47143; Medicago sativa: AF172172; Lotus japonicus: AB238220; Parasponia andersonii: U27194; Trema spp.: Y00296, Z99635, AF027215, AJ131349, AJ131350, AJ131351; Citrus unshiu: AY026338; Lycopersicon esculatum: AW035116; Arabidopsis thaliana: U94998.

Class IIa (leghemoglobins): Vicia faba: Z54160, Z54159, Z54158, Z54157; Pisum sativum: AB010831, AB015721, AB015720, AB015719; Medicago sativa: AJ389051, M91077, X54089, M32883, X54085, X54538; Medicago truncatula: X57732, X57733; Sesbania rostrata: X13505, X13815, X13814; Canavalia lineata: U09671; Vigna unguiculata: U33207, U33206; Phaseolus vulgaris: AB004549, K03152; Psophocarpus tetragonolobus: S65139; Glycine max: E01430, E01431, E01432; Lupinus luteus: X77042, X77043. Class IIb: Casurina glauca: X77694, X77695, X77696; Beta vulgaris: BE590299; Lycopersicon esculatum: AY026344; Cichorium intybus: AJ007507, AJ277797; Gossypium hirsutum AY026339; Brassica napus: AY026337; Arabidopsis thaliana: U94999.

Statistical Analysis. The GUS fluorometric assay was based on the investigation of 3-4 transformation lines with 1-4 replicates. In order to test the influence of ACC, constructs and lines were analysed separately. A student’s t-test was used to detect whether the mean GUS expression was significantly different between control (-ACC) and exposed plants (+ACC). Data were log-transformed in order to satisfy assumptions of normality and equal variances. Variances were tested by Bartlett´s test.

Primers:

Hbexon2f (5’-cggaattcatxttygaratxgcxcc-3’), Hbexon2r (5’-cgggatccgcrtgxxxyttxaryttxggrtt-3’) (x = deoxyinosine, y = pyrimidine mix, r = purine mix), Mgpr1 (5’-atgatatggtgccgacgaatt-3’), Mgpr2 (5’-agtagttttacagattcgacctgttct-3’), Myrica-KpnI (5’-cgcggtaccggtttcgattcaag-tttttgaatttgatagta-3’), Myrica-XbaI (5’-cgctctagaaattcgtcggcaccatatcat-3’), Myrica-PstI (5’-cgcctgcagttatgggcagcaaatcgc-3’), AhbIF2 (5-‘tctagacaacaatatgagaagcaact-3’), AhbIR3 (5’-ggtacctctaaatgatttaaagtata-3’), AhbIIF2 (5’-tctagagtaattaaatggctctgtga-3’), AhbIIR3 (5’-ggtacctctttctctctttcttttt-3’), Mg-ubiquitin forw (5’-tcacgttgtcaatggtgtcag-3’) Mg-ubiquitin rew (5’-tcaaggctaagatccaagaca-3’), MgHb forw (5’-ggtggtgaagtcatggacagtaatgaagc-3’), MgHb rew (5’-tgcaaaccttgtcacctcgtagtgctc-3’).

Western blot. Protein extract was separated by SDS/PAGE (15% acrylamid). Approximately 60 g of total root and root nodule protein was used per lane, whereas ~120 g of total leaf protein was used. After electrophoresis the separated proteins were electro-blotted in a transfer buffer (25 mM Tris-HCl, pH 7.5, 0.2 M glycin, 0.1% (w/v) SDS, 20% (v/v) methanol) onto a PVCF membrane (Immobilon-P, Millipore). Blots were developed using the ECL Plus Western Blotting detection reagent (Amershame Biosciences) and detected on X-ray films.

Table S1. Binding free energy of MgHb and riceHb1 homodimers. Free energies were calculated from an average of 20 conformations in the equilibrated part of the MM/MD simulations (3-5 ns).

Model - MgHb
Gcoul / Gsol / Gnp / Gstrain / TSsc
Dimer / -8614.01 / -13472.39 / 335.835 / -1142.782 / -105.013
monomerA / -4239.17 / -6771.38 / 178.955 / -426.598 / -57.5645
monomerB / -4096.1 / -7116.48 / 178.085 / -490.732 / -54.6255
-278.74 / 415.09 / -21.25 / -225.45 / 7.177
∆G = -117.53 kJ/mol (-28.09 kcal/mol)
Xray – riceHb1
Gcoul / Gsol / Gnp / Gstrain / TSsc
Dimer / -8236.78 / -13322.1 / 331.545 / -1317.91 / -91.5755
monomerA / -4093.31 / -6613.3 / 174.826 / -571.824 / -48.322
monomerB / -4010.79 / -6824.18 / 178.458 / -657.891 / -44.194
-132.68 / 115.38 / -21.739 / -88.195 / 0.9405
∆G = -128.17kJ/mol (-30.63 kcal/mol)

Table S2. GUS expression observed in seedlings containing M. gale p2MgHb after 3 days (± S.E.)

M MU/hr/mg protein
Lines / - ACC / + ACC
L2.3 / 13.72 ±3.39 / 35.51 ±11.73
L7.0 / 1.16 ±0.21 / 4.95 ±0.39
L9.2 / 4.45 ±0.78 / 12.98 ±2.85
L11.2 / 8.83 ±0.1 / 13.80 ±2.49
L. Japonicus LjLb2 / 187- / TAAGTTTTTGAAAAGTTTAT-TG-TCTCTTAATAAAACCAATGGCCAGCCA / -138
S. rostrata Srglb3 / 185- / AAA-TTTTTAAAAAGATTAT-TG-TCTCTTAATAATGTCAATGGCCACCC- / -136
G. max Lba / 187- / AAA-TTTTTTAAAAGATCGT-TG-TTTCTTCTTCATCATGCTGATTGACAC / -140
C. glauca symHb1 / 324- / AACTTCAATCCCAAGATGTCCTT-TCTCTTATTGATATTTGAACAACAACA / -274
C. glauca symHb1 / 491- / AACTTCAATCCCAAGATCTCCTT-TCTCTTATTGATATTTGAACAACAACA / -441
C. glauca symHb2 / 326- / AACTTCAATCCCAAGATGTCCTT-TCTCTTATTGATATTCAAACAACAACC / -280
A. thaliana AHb2 / 864- / TGTGTTCACCTCAAGACGCTCGAGTCTCTTGAACAAAAA-GAAAAGGTTTC / -815
A. thaliana AHb1 / 144- / AACTTGCTTATCAAGAA-CTGT---CTCTTCGGTAACACAAAAGGGTCTTT / -98
L. japonicus LjHb1 / 164- / TCAAATCCAAACACGATAATAAA--CTCTTCATTGCCAT-GAAGGGCCAAC / -116
G. max nonsym Hb / 184- / TAAGCCACACAAATGGGAATG-A--CTCCCCATTACAAT-GAAGGGCCAAC / -138
M. gale Hb / 197- / AATGTATACCCAAAGAATCTATG--CTCTTCAAGCCAC--GAAGATTTGAT / -151
C. glauca Hb2 / 209- / GCAATTGACCCAAAGAA-ATG-G--CTTTCGAC-CCAC--GAAGAGCCGGA / -166
P. andersonii Hb / 172- / ATAAAAAACCCAAAGAT-ATG-G--CTCCCCAATACCCT-GAAGAGTTACA / -127
T. tormentosa / 176- / AAAAAAAACCCAAGGAG-ATG-G--CTCTCCAGTACCCT-GAAGAGTTACA / -131

Figure S1. Sequence alignment of promoter regions of symbiotic Hb and nonsymbiotic Hb genes. All sequences are numbered from the translation initiation codon ATG. Motifs resembling the nodulin motifs (AAAGAT and CTCTT) are boxed. In non-symbiotic Hb genes a motif GAGGG is also conserved in the 5’-upstream sequence. Arabidopsis thalianaAHb1 and AHb2(Trevaskis, et al., 1997), Casuarina glauca symbiotic Hb (Jacobsen-Lyon, et al., 1995), C. glauca Hb2 (Christensen, et al., 1991), Glycine maxLba(Hunt, et al., 2002) and G. max non-symbiotic Hb (Andersson, et al., 1996), Lotus japonicus LjLb2 and LjHb1 (Uchiumi, et al., 2002; Shimoda, et al., 2005), Myrica gale (this study), Parasponia andersonii(Landsmann, et al., 1986), Trema tomentosa(Bogusz, et al., 1988).

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