Kopecna Supplementary Material PP - Plant physiology · The samples were homogenized, extracted in cold water with 25% ammonia (ratio 4:1), and purified by solid-phase extraction

Kopečná et al. - Structure and function of nucleoside hydrolases from Physcomitrella and maize catalyzing hydrolysis of purine, pyrimidine and cytokinin ribosides

1

Supplementary material

Supplementary Methods Cloning of gene replacement vectors for generation of Physcomitrella knockout mutants

A 5´-fragment of 1057 bp and a 3´-fragment of 1014 bp were amplified from genomic DNA using the primer pairs 389/390 and 393/394, respectively (Table S3), to construct a PpNRH1 knockout vector. The obtained PCR fragments were further amplified using the primer pairs 391/392 and 395/396 to introduce restriction sites MluI/SpeI (at the 5'end) and BsiWI/XbaI (at the 3'end). The fragments were further digested with above restriction enzymes and ligated into the pBNRF vector (Schaefer et al., 2010, DNA Repair 9: 526–533), which finally resulted in the vector pBNR_PpNRH1-KO conferring G418 resistance. The PpNHR2 knockout vector was prepared using a 5´- (1127 bp) and 3´-fragments (1045 bp) amplified with primer pairs 407/408 and 411/412, respectively. Both fragments were further amplified using the primer pairs 409/410 and 413/414 to introduce restriction sites HindIII/XhoI and BamHI/MluI, respectively. The fragments were further ligated into the pBNRF vector leading to the final vector pBNR_PpNRH2-KO conferring resistance towards hygromycin. The PpNHR3 knockout vector was prepared using a 5´- (1082 bp) and 3´-fragments (1005 bp) amplified with primer pairs 415/416 and 419/420, respectively. Both fragments were further amplified using the primer pairs 417/418 and 421/422 to introduce restriction sites SphI/XhoI and EagI/MluI, respectively. The fragments were further ligated into the pBNRF vector leading to the final vector pBZR_PpNRH3-KO conferring resistance towards zeocin. For the transformation, the respective transformation cassette was liberated from the vector by double digest using XbaI and MluI (pBNR-PpNRH1-KO), HindIII and MluI (pBHR_PpNHR2-KO and pBZR_PpRH3-KO).

Quantification of purine/pyrimidine bases and ribosides

The samples were homogenized, extracted in cold water with 25% ammonia (ratio 4:1), and purified by solid-phase extraction with the addition of the following stable-labeled internal standards: 100 pmol of [13C5]adenine, [13C5]adenosine, [15N4]hypoxanthine, [15N2]xanthosine, and [15N4]inosine (Cambridge Isotope Laboratories, Andover, MA, USA). All samples were loaded onto mixed-mode anion exchange sorbent Oasis MAX cartridges (Waters, Milford, MA, USA) prewashed with methanol and 5% ammonia, washed with 5% ammonia and 2% tetrabutylammonium hydroxide in methanol, and eluted with methanol acidified with 2% formic acid. The eluates were dried and dissolved in 20 µl of mobile phase prior to mass analysis using an Acquity UPLC system and a triple quadrupole mass spectrometer Xevo TQ MS (Waters MS Technologies, Manchester, UK). Each sample was injected onto a Pinnacle DB IBD column (100 x 2.1 mm, 1.9 μm; Restek, Bellefonte, PA, USA) and eluted with a linear gradient (0-4 min, 3/97% A/B; 4-5 min, 3/97-50/50% A/B; flow rate, 0.25 ml min-1; column temperature, 35 °C), where A was methanol and B was 0.1% formic acid in water. Quantification was obtained by monitoring the precursor ([M+H]+) and the appropriate product ions. MRM transitions were selected according to the described methods (Farrow and Emery, 2012, Plant Methods 8:42) and optimized as follows: adenine/adenosine 136>119/268>136, guanine/guanosine 152>135/284>152, hypoxanthine/inosine 137>119/269>137, uracil/uridine 111>42/243>111, and xanthine/xanthosine 153>110/285>153. The capillary voltage was set to 1.0 kV, source/desolvation gas temperatures were 120/550 °C, cone/desolvation gas flow rates were 70/550 l/h, and argon was used as collision gas with a flow 0.21 ml min-1. MS/MS conditions were ranged in 16-28 V (cone voltage) and 8-18 eV (collision energy). The limits of detection (signal to noise ratio 1:3) were close to 50 fmol for all purines/pyrimidines. The linear range was established to be 0.1–100 pmol per injection with a correlation coefficient of 0.995–0.999. Chromatograms were analyzed using the MassLynx V4.1 software (Waters), and the compounds were quantified by standard isotope dilution analysis.


2

Table SI. Data collection and refinement statistics of plant NRHs. ZmNRH3 apoenzyme PpNRH1 apoenzyme

Space group P212121 P212121 Asymmetric unit 1 dimer 4 dimers

Unit cell (Å) a 76.4 125.9 b 85.8 126.1 c 87.4 253.6

== (°) 90.0 90.0 Resolution (Å) 47.8-2.49(2.65-2.49) 44.7-3.35 (3.55-3.35)

Observed reflections 146580 (23116) 284354 (44562) Unique reflections 19953 (2953) 58554 (9170) Completeness (%) 98.6 (91.8) 99.5 (98)

I/ (I) 10.73 (1.77) 12.37 (2.46) Rsym

(%)a 11.9 (98.4) 11.8 (69.2) Rcryst

(%)b 20.5 20.5 Rfree

(%)c 24.1 21.4 RMSD

bond lengths (Å)b 0.008 0.008 bond angles (°) 1.11 1.15

Solvent molecules B average value (Å2)

Dimer AB/Ca2+ 70/60.3 72.2/66.4 Dimer CD/Ca2+ -- 80.2/63.5 Dimer EF/Ca2+ -- 97.6/79.3 Dimer GH/Ca2+ -- 138.2/116.2

Solvent

57 (39 waters)

53.2 (16 waters)

a Rsym = ƩhklƩiIi(hkl) -<I(hkl)> / ƩhklƩIi (hkl), where Ii(hkl) is the i th observed amplitude of reflection hkl and <I(hkl)> is the mean amplitude for all observations i of reflection hkl. b Rcryst = ƩFobs – Fcalc / ƩFobs c 5 % of the data were set aside for free R-factor calculation. Values for the highest resolution shell are in parentheses


3

Table SII. Identities (in %) of ZmNRH3 and PpNRH1 with other NRHs from maize and Physcomitrella patens and from other species. Amino acid identity was calculated by Lalign (Huang & Miller, 1991, Adv. Appl. Math. 12: 337–357).

ZmNRH3 PpNRH1

ZmNRH3 -- 48.9 ZmNRH2a 74.2 50.2 ZmNRH2b 75.8 48.9 ZmNRH1a 50.6 60.7 ZmNRH1b 50.6 61.0 PpNRH1 48.9 -- PpNRH2 59.6 52.0 PpNRH3 57.6 52.1 AtNRH1 68.2 51.9 AtNRH2 51.9 60.1 L. major 35.4 33.3 C. fasciculata 35.7 32.0 S. pombe 38.7 36.6 T. vivax 26.2 29.3 E.coli YbeK 35.6 34.2 E.coli YeiK 34.7 30.2


4

Table SIII. Primer pairs used for the cloning of NRHs and for the site-directed mutagenesis of PpNRH1. FP stands for forward primer, RP for reverse primer, Ta for annealing temperature used.

Enzyme Primer pair Ta [°C]

PpNRH1 FP: 5´-ATAGGATCCGGCGGTGCCTGAGAA-3´ RP: 5´-ATAGGTACCCTATGGGGCTGTAAGTC-3´

61.0

PpNRH2 FP: 5´-CAGGAATTCGATGGTGGGAGAGGAGACGAT -3´ RP: 5´-ATACTCGAGTCAAGGGCTGCACAGGAGTTT -3´

60.0

PpNRH3 FP: 5´-AAGGAGCTCGATGGACGTGAAAGCTGCTGGC -3´ RP: 5´-CTGCTCGAGTCATTTCAGTCGAGAGCTGCAAAG -3´

66.9

D25A FP: 5´-P-CTCCCGGCATTGATGAT-3´ RP: 5´-P-CGGTGTCGATGATGACTTTC-3´

50.0

H99A FP: 5´-P–CGGCGCCGATGGGCTCGGCA-3´ RP: 5´-P-GCCACAAAATCAGCGATCCGTTCCTTC-3´

63.2

Y241A FP: 5´-P-GCCGCCACGTATCATCGTGAAG-3´ RP: 5´-P-GAAATGGGATGCTGCATACAAGT-3´

60.0

Y244A FP: 5´-P-GCTCATCGTGAAGCGTATGACATAGATGC-3´ RP: 5´-P-GCTCATCGTGAAGCGTATGACATAGATGC-3´

59.0

H245A FP: 5´-CGCCACGTATGCTCGTGAAGCGTATGACATAGATGC-3´ RP: 5´-GCATCTATGTCATACGCTTCACGAGCATACGTGGCG-3´

62.5

E247A FP: 5´-P-CAGCGTATGACATAGATGCTATTTACCTGC-3´ RP: 5´-P-CACGATGATACGTGGCGTAGA-3´

58.0

E247K FP: 5´-P-AAAGCGTATGACATAGATGCTATTTACC-3´ RP: 5´-P-ACGATGATACGTGGCGTAGA-3´

54.3

Y249A FP: 5´-P-GCTGACATAGATGCTATTTACCTGC-3´ RP: 5´-P-CGCTTCACGATGATACG-3´

58.0

D250A FP: 5´-P-CCATAGATGCTATTTACCTGCATGAC-3´ RP: 5´-P-CATACGCTTCACGATGATACG-3´

54.8

D252A FP: 5´-P-CTGCTATTTACCTGCATGACCCTG-3´ RP: 5´-P-CTATGTCATACGCTTCACGA-3´

59.0

Y255A FP: 5´-P-GCCCTGCATGACCCTGCAAC-3´ RP: 5´-P-AATAGCATCTATGTCATACGC-3´

60.0

ZmNRH1a FP: 5´-CGCGAATTCTGCGGCGGTCGAGGGGACG-3´ RP: 5´-ATCCTCGAGTTAGTCATCAGTCATCAACC-3´

64.0

ZmNRH1b FP: 5´-CGTGAGCTCTGCGGCGGTCGAGGGGACGAA-3´ RP: 5´-ATACTCGAGTTAGTCATCGGTCATCAGCCT-3´

62.0

ZmNRH2a FP: 5´-CTAGAGCTCGGAGCGGGACGGCCAGCAGAC-3´ RP: 5´-CTCCTCGAGTCATGGTGCCATGAGCAGC-3´

66.0

ZmNRH2b FP: 5´-ATCGAGCTCGATGGGGCAGGACGGCCAGCAGAT-3´ RP: 5´-AGACTCGAGTCATGGTGCCATGAGCAGCTTC-3´

65.0

ZmNRH3 FP: 5´-CGAGAATTCCAAGATCATCATTGACACG-3´ RP: 5´-ATACATATGTCACGGCTTGGTGACGAGCTCC-3´

61.0

D8A of ZmNRH3 FP: 5´-TCCCGGCATCGATGACAGCGTG-3´ RP: 5´-GCCGTGTCAATGATGATCTTGGAATTCGGA-3´

62.6


5

Table SIV. Primers used for generation of gene replacement vectors and genetic analysis of P. patens knockout mutants.

Primer No. Sequence Tm

[°C]

389 5´-CGCAGACCTTCGTCAGACTC-3´ 57.6 390 5´-GGCTGAGCATGTCACTTGTGTAAGA-3´ 59.0 391 5´-CCTTACGCGTGACGGGGACGAAT-3´ 63.3 392 5´-CCCTGACTAGTGTAAGAGGAATCTTCCGT-3´ 60.5 393 5´-GCCCTGAGTCCTGTAATCTT-3´ 54.0 394 5´-CATAACTCTGACGCATGGAATGA-3´ 55.0 395 5´-CCTACGTACGAATGCTGTGGCAATTTGA-3´ 60.9 396 5´-CCCTTCTAGAGTGTGAGGCCCTT-3´ 60.2 407 5´- CCTGCACTTAGTCTTCTCAGGAT-3´ 56.1 408 5´-GAACATGACGGTACTCTGCATCT-3´ 56.7 409 5´- CCCTTCAAAGCTTACCACCTGCTGCTA-3´ 62.4 410 5´-CCCTTGACTCGAGCAATACAGAACGGAA-3´ 61,7 411 5´-CAGAACCGTAGGTTGAATGGTGTTT-3´ 57.5 412 5´-CGAATTGCGCTGGAGGGATAA-3´ 57.6 413 5´-CCCTGGATCCGTAGGTTGAATGGT-3´ 60.5 414 5´-CCTTACGCGTTGGCAGCAAAGA-3´ 60.5


6

Figure S1. NRH gene models for Physcomitrella patens, Zea mays and Arabidopsis thaliana based on cloned cDNAs. Physcomitrella gene models were constructed using cloned cDNA sequences (this work, GenBank accession numbers JQ649322, JX861385-9) and gene models from the Physcomitrella genome (Pp1s357_22V6.1, Pp1s140_172V6.1, Pp1s5_276V6.1). Maize gene models were constructed using cloned cDNAs (this work, HQ825159, HQ825160, HQ825161, JQ594984 and HQ825162) and gene models from the maize genome (GRMZM2G029845, GRMZM2G134149, GRMZM2G085960, GRMZM2G015344 and GRMZM2G104999). Arabidopsis gene models were taken from the TAIR database (At2g36310 and At1g05620).

85 116 60 186 105 97 108 136 109280322389226330189333691

112 116 60 183 105 97 108 136 1092162541532032044593301453

94 116 60 183 105 97 108 2492211971184373381425

AtNRH1 97 116 60 183 105 97 108 136 109848191898013695275

AtNRH2 52 116 60 186 105 97 108 136 10912114019411794237107263

ZmNRH1a 61 116 60 186 105 97 108 136 112460128499266102254117117

Exon 1 Exon 9Exon 8Exon 7Exon 6Exon 5Exon 4Exon 3Exon 2

94 116 60 183 141 97 1082492211611184373381425

116 60 183 105 97 108 2492211971184373381425

PpNRH1

PpNRH2

PpNRH3

PpNRH3_v2

PpNRH3_v3

PpNRH3_v4 116 60 183 141 97 108 3712492211611184373381425

ZmNRH1b 58 116 60 186 105 97 108 136 1122411275819499277115121

ZmNRH2a 64 116 60 183 105 97 108 136 1092219979808936877928

ZmNRH2b 64 116 60 183 105 97 108 136 109219100808090446821314

ZmNRH3 34 116 60 183 105 97 108 136 1099710410310391460951437

94

94

136235

118

136235

118

371


7

Figure S2. Influence of pH and temperature on catalytic activity of recombinant PpNRH1. Assays were performed at 25°C using inosine (400 µM) as a substrate. Conversion at different pH values was measured by reverse phase HPLC. Error bars represent standard deviation (n=3). Conversion at different temperatures was measured at pH 7. Error bars represent standard deviation (n=3).

6 7 8 9 100

20

40

60

80

100

Rel

ativ

e a

ctiv

ity

(%

)

pH

Citrate/phosphate Glycine/NaOH

16 24 32 40 48 56

Temperature (°C)


8

Figure S3. Confirmation of cytokinin riboside conversion by ZmNRH3, ZmNRH2b and PpNRH1. Conversion of cytokinin ribosides was analyzed by HPLC-UV on a BetaMax neutral column (150 x 2.1 mm, 5 µM particle size, Thermo) using 20 mM amonium formate, pH 4.0 (A) and methanol (B) at flow rate 0.3 ml min-1 and monitored at 268 nm. iPR and iP were separated isocratically (A/B = 50/50), tZR and tZ were separated isocratically (A/B = 30/70). The figure shows the reaction performed in the presence of ZmNRH2b. Retention time was first determined for cytokinin riboside and base, namely iPR with iP and tZR with tZ. Then the reaction mixture was analyzed before enzyme addition and after 1 hour incubation. The protein was removed by ultracentrifugation using a 10 kDa filters.

3 4 5

0.0

0.4

0.8

1.2

1.6

2 3 4 5

0.0

0.4

0.8

1.2

1.6iP iPR

Rel

ativ

e in

tens

ity (

%)

Time (min)

standards unreacted iPR reaction mixture

after 1h

tZRtZ

Time (min)

standards unreacted tZR reaction mixture

after 1h


9

Figure S4. Gel permeation chromatography of PpNRH1 and ZmNRH3. Gel permeation chromatography was performed on Superdex 200 HR 10/30 column (GE Healthcare) connected to a BioLogic Duo Flow liquid chromatograph (Bio-Rad). The column was equilibrated and run with 50 mM potassium phosphate buffer, pH 7.0, containing 150 mM NaCl at a flow rate of 0.7 ml min-1. The molecular mass values of both enzymes were evaluated after calibration of the column with protein standards from Gel filtration markers kit (Sigma-Aldrich), which corresponded to thyroglobulin, apoferritin, β-amylase, alcoholdehydrogenase, albumin and carbonic anhydrase (left panel). The calibration curve is shown in right panel. The elution profile of both NRHs with calculated molecular mass values of 81 and 76 kDa are shown in blue and red color.

10 12 14 16 18 20 22-0.03

0.00

0.03

0.06

0.09

0.12

0.15

10 12 14 16 18 204.2

4.5

4.8

5.1

5.4

5.7

6.0

~81 kDa~76 kDa

Abs

orba

nce

at 2

80 n

m

Elution volume (ml)

PpNRH1 ZmNRH3

200 kDa150 kDa

66 kDa29 kDa

669 kDa

443 kDa

y = -0.215x + 8.569

R2 = 0.991

log

(M

ole

cula

r w

eig

ht)

Elution volume (ml)


10

Figure S5. Structural comparison of plant NRH (ZmNRH3, this work) with NRH from E. coli (YeiK, PDB 3B9X). (A) Superposition of ZmNRH3 monomer (pink) with YeiK monomer (grey). The RMSD is 1.55 Å over 294 C residues and the sequence similarity is 34.0 %. The inosine substrate is shown in yellow. The large structural difference between the two structures corresponds to the β10-β11 loop due to their different oligomerization state and interface formation. (B) Substrate binding sites of YeiK (grey) and ZmNRH3 (pink). Amino acid residues are labeled. H-bonds between the enzyme and the docked inosine molecule (in violet) in ZmNRH3 and the calcium ion coordination are shown as black dashed lines. Residue W226 was kept flexible. H-bond interactions between YeiK and the purine ring of inosine molecule (in yellow, PDB 3B9X) are shown as orange dashed lines.


11

Figure S6. Production of PpNRH1 protein variants. (A) Bio-Safe Coomassie Blue-stained SDS-PAGE gels of partially purified wild-type PpNRH1 and mutated forms (10 µL were loaded in each lane). The red arrow indicates the migration of the produced enzyme (~ 38 kDa). (B) Far-UV CD spectra of purified WT and mutated PpNRH1 proteins (0.5 mg mL-1) in 20 mM Tris-HCl buffer, pH 8.0. Each far-UV CD spectrum is the average of three scans in order to obtain the optimal signal to noise ratio. After background subtraction and data smoothing, each CD signal was converted to mean residual ellipticity []´. A B

200 210 220 230 240 250 260

-14

-12

-10

-8

-6

-4

-2

0

2

[]´

x1

0-3 (

deg

cm

2 dm

ol-1

)

Wavelength (nm)

WT D25A H99A Y241A Y244A H245A E247A Y249A D250A D252A Y255A


12

Figure S7. Delayed bud development within 4 weeks after aerial inoculation with protonema suspension (without addition of cytokinin). T-test of mutant values vs WT was performed, *** - p value < 0.001, ** - p value < 0.01. Assay reveals delay in early bud development only (after longer growth periods the differences in number of buds between mutants and wild type diminishes). Protonema was grown on Knop medium and examined microscopically by counting number of buds per view field (0.82 x 0.82 cm).

WT

d|NRH1#

29

d|NRH1#

28

d|NRH2#

13

d|NRH2#

56

d|NRH3#

7

d|NRH3#

23

0

10

20

30

40

50

60

**

******

***

***

Num

ber

of b

uds

(0.8

2 x

0.82

cm

)

***


13

Figure S8. Alignment of plant NHR sequences. NRH sequences were obtained from the NCBI, TGI, Phytozome or TIGR databases. NRH proteins from S. pombe, E. coli, L. major, C. fasciculata, T. vivax, P. patens, Z. mays, A. thaliana, G. max, M. truncatula, N. tabacum, O. sativa, P. trichocarpa, S. moellendorffii, S. lycopersicum, T. aestivum and V. vinifera were used in the alignment. 10 20 30 40 50 60 70 80 90 100 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| T.vivax -----------------------------------MAKNVVLDHDGNLDDFVAMVLLASNTEKVRLIGALCTDADCFVENGFNVTGKIMCLMHNNMNLPL ZmNRH1A ----------------------------MAAVEGTTKKKVIIDTDPGIDDAMA-IFVALRSPELEVLGLTTTFGNVHTALA-TRNALHLLEAVGRTDIPV ZmNRH1B -----------------------------MAAVEGTKKKVIIDTDPGIDDAMA-IFLALRSPELEVLGLTTTFGNVHTALA-TRNALHLLEAVGRTDIPV Triti1 ------------------------------MAAVTKKKKVIIDTDPGIDDAMA-IFVALRSPELEVVGLTTIFGNVYTALA-TRNALHLLETAGRTDIPV Oryza1 --------------------------------MTTTKKKLVIDTDPGIDDAMA-IFVALRSPEVELLGLTTIFGNVYTTLA-TRNALHLLEAVGRTDIPV AtNRH2 -------------------------------MAIGDRKKIIIDTDPGIDDAMA-IFVALNSPEVDVIGLTTIFGNVYTTLA-TRNALHLLEVAGRTDIPV Tomato1 --------------------------------MATEPKKIIIDTDPGIDDAMA-IFVALESPEVEVIGLTTIYGNVYTTLA-TRNALHLLDIAGRTDIPV Nicoti1 --------------------------------MATEPKKIIIDTDPGIDDAMA-IFVALQSPEIKVIGLTTIYGNVYTSLA-TRNALHLLDVAGRTDIPV Poplar1 --------------------------------MAAEPKKIIIDTDPGIDDAMA-IFLALRSPEVEVIGLTTIYGNVYTTLA-TRNALHLLEVAGRTDIPV Vitis1 ---------------------------------MAEPKKIIIDSDPGIDDAMA-IFVALQSPEVDVIGLTTIYGNVYTTLA-TRNALHLLEIAGRTDIPV Selagi1 ---------------------------MAPASAHVIQKKVIIDTDPGIDDAMA-ILLAFQSPELDVIGLTTTFGNVSTSMA-TQNALHLCELAGREDIPV PpNRH1 --------------------MAVPENVVGTIKQSSPPKKVIIDTDPGIDDAMA-IFFALKSPELDVIALTTIYGNVRTPTA-TVNALHLLEFAGREDIPV Selagi2 --------------------MPERCDDEGIAFGRSRKTKIIIDADPGIDAAMA-IIMALRSPEVEVIGITTIFGSAHTPDV-TRNALHLLEVMGSTHVPV PpNRH2 -----------MVGEETMDRSADLMAEYVEVSKNPARKRVIIDTDPGIDDMMA-IFMAFEAPGIEVIGLTTIFGNVDIDLA-TKNALHLCEMTGHPEIPV PpNRH3 -----------------MDVKAAGSVAQDQFSKHAGRRKVIIDTDPGIDDMMA-ILMAFQAPEIEVIGLTTIFGNVNTDLA-TINALHLCEMAGHPEIPV Oryza3 -----------------MDLQEAAMEARNGHRIPPTEEKVIIDTDPGIDDSVA-IMMAFEAPGVKVVGLTTIFGNCTTSHA-TRNALILCDRAGRPEVPV Tritic3 -------------------------------------MKLIIDTDPGIDDSVA-IMMAFQAPGVEVLGLTTIFGNCTTAYA-TRNALILCEKAGRPDVPV ZmNRH3 -------------------------------------MKIIIDTDPGIDDSVA-ILMAFQMPGVQVLGLTTIFGNCTTEHA-TRNALILCEKASHLEVPV ZmNRH2A ---------------------------MERDGQQTRRDKLIIDTDPGIDDSMA-ILMAFRAHTLEIIGLTTIFGNVDTEGA-TCNALLLCERAGHPEVPV ZmNRH2B ---------------------------MGQDGQQIRRDKLIIDTDPGIDDSMT-ILMAFRAPSVEIIGLTTIFGNVDTKGA-TRNALLLCERAGCPEVPV Tritic2 ----------------------------MGEDGQIRRDRVIIDTDPGIDDSMT-ILMAFGEPSVEIIGLTTIFGNVTTEYA-TRNALLLCERAGHPEVPV Oryza2 ----------------------------MGSNEQIHRDKLIIDTDPGIDDSMT-ILMAFRAPTVEIIGLTTIFGNTTTKNA-TQNALLLCERAGHPEVPV AtNRH1 ----------------MDCGMENCNGGISNGDVLGKHEKLIIDTDPGIDDSMA-ILMAFQTPELEILGLTTVFGNVSTQDA-TRNALLLCEIAGFPDVPV Glymax1 ----------------------MASLFNNANGVLGKSEKLIIDTDPGIDDSMA-IFMAFQSPDVEVLGLTTIFGNTTTEVS-TRNALLLCEIAGRENIPV Medic1 ------------------MNGLVNNGNGGVKAVLDKAEKLIIDTDPGIDDSMA-ILMAFHCPEVEVIGLTTVFGNAQTEDA-TRNALLLCEIAGRQNVPV Tomato2 ------------------MSICDGDLVDSNNSFAKQREKIIIDTDPGIDDSMT-ILMAFQTPEVEIIGLTTIFGNVTTKDA-TRNALLLCEAAGYPDVPV Nicoti2 -------------MSNCDGDLVDGFVTTNNYSFAKQREKIIIDTDPGIDDSMA-ILMAFQTPEVEIIGLTTIFGNVTTKDA-TRNALLLCETAGYPDVPV Poplar2 MDGVLGSETESHVIMDGTVNIFHGERDGVLAGSTAKPEKLIIDTDPGIDDTMA-ILMAFQSPELEVLGLTTIFGNVSTEDA-TRNALLLCEIAGRPDVPV Vitis2 ----------MECVMLSSHGGLCDASYDVVSNSPVQPDKVIIDTDPGIDDSMA-ILMAFQTPELEILGLTTVFGNVTTKDA-TRNALLLCEIAGRPDVPV S.pombe -------------------------------------MKIIIDTDPGQDDAIT-ALLAIASPEIELLGVTTVAGNVPVSMT-TRNALQMLDLAGRPDIPV YbeK ------------------------MSQRNTQQGATMALPILLDCDPGHDDAIA-IVLALASPELDVKAITSSAGNQTPEKT-LRNVLRMLTLLNRTDIPV YeiK ----------------------------------MEKRKIILDCDPGHDDAIA-IMMAAKHPAIDLLGITIVAGNQTLDKT-LINGLNVCQKLE-INVPV L.major -----------------------------------MPRKIILDCDPGIDDAVA-IFLAHGNPEIELLAITTVVGNQSLEKV-TQNARLVADVAGIVGVPV C.fasci -----------------------------------MAKKIILDCDPGLDDAVA-ILLAHGNPEIELLAITTVVGNQTLAKV-TRNAQLVADIAGITGVPI 110 120 130 140 150 160 170 180 190 200 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| T.vivax FPIGKSAATAV-NPFPKEWRCLAKNMDDMPILNIPENVELWDKIKAENEKYEGQQLLADLVMNS-EEKVTICVTGPLSNVAWCIDKYGEKFTSKVEECVI ZmNRH1A AEGSHVTIKKATKLRIASFVHGSDGLGNQDFPP-P-ATKPVDQSAAA--------FLVEQANLY-PGQVTIVALGPLTNLALAVEL-DPSFPKKIGQIII ZmNRH1B AEGSHVTIKKATKLRIASFVHGSDGLGNQDFPP-P-ATKPVDQSAAA--------FLVEQANLY-PGQVSVVALGPLTNLALAVEL-DPAFPEKIGQIII Triti1 AEGSHVTIKKATKLRIASFVHGSDGLGNQNFPP-P-AGKAVDQSAAA--------FLVEQANLY-PGQVTVVALGPLTNLALAVEL-DPSFPKKIGQIII Oryza1 AEGSHVTIKKATKLRIASFVHGSDGLGNQNFPP-P-TGKPLDQSAAA--------FLVEQANLY-PGQVTVVALGPLTNLALAIEL-DPSFPKKIGQIVI AtNRH2 AEGTHKTFLNDTKLRIADFVHGKDGLGNQNFPP-P-KGKPIEKSGPE--------FLVEQAKLC-PGEITVVALGPLTNLALAVQL-DPEFSKNVGQIVL Tomato1 AEGSHVTITKGTKLRIADFVHGTDGLGNQNFPA-P-NGKPIEQNAAD--------FLVQQASLY-PGKITVVALGPLTNIALAIQS-DPDFVKNIGQIVV Nicoti1 AEGSHVTITKGTKLRIADFVHGTDGLGNQNFPA-P-NGKPIDQNAAE--------FLIQQASLY-PGKVTVVALGPLTNIALAIQS-DPAFVKNIRQIVV Poplar1 AEGSHVTITKGTKLRIADFVHGADGLGNQNFDP-P-KGKPVEQSAAA--------FLVEQAKLH-PGKVTVVALGPLTNIALAIEL-DPEFCKNIGQIVL Vitis1 AEGSHVTITKGTKLRIADFVHGADGLGNQNFPP-S-AGKPIEQSAAA--------FLIEQAKLY-PGKVTVVALGPLTNIALAIEL-DPGFSKNIGQIVL Selagi1 AQGLHKSLKGDTKEHAVDFIHGKDGLGNTNPPA-P-KGKPIDMTAPE--------FFISKVKEF-PGEVTIIALGPLTNLGKAVEM-DPSFAKLVGEIVI PpNRH1 SEGFRTSLRGELKERIADFVHGADGLGNTYPTL-S-DRKPIDTFAPD--------YLIQKVNEF-PGEITIVALGPLTNLAAAVEC-DPTFAKKVGQIII Selagi2 AEGNIKPMAGG-VPYIQDFANGLDGLGNIATAD-P-ATKKSAMSACD--------FLVDTVSKY-PGEITIVALGPLTNLSQAFQK-EECVVKLVARVVV PpNRH2 AEGPSEPLKRV-KPRIAYFVHGSDGLGNTFQAN-P-KGQKSSKSAAD--------FLLEKVAEF-PGEVTVVALGPLTNIALAIQK-DPNFVKNIGQLVV PpNRH3 AEGPSEPLKRV-KPRIAYFEHGSDGLGETYQAK-P-NFQKLSKDAAD--------FLIENVTEF-PGEVTVVGLGPLTNLALAIQK-DSNFAKNVGQLVV Oryza3 AEGSAEPLKGG-KPHVADFVHGSDGLGNTSFPD-PTTTNKVEQSAAE--------FLVDKVSES-PGEISVLALGPLTNIALAMKK-DSSFASKVKRIVV Tritic3 AEGSAEPLKGG-KPQVADFVHGSDGLGNVSVPE-P-TTKKAEQTAAE--------FLVDKVSQF-PGEVSVLALGPLTNLALAIKM-DPSFVSKVKKIVV ZmNRH3 AEGSHEPLKGG-KPHVADFVHGPDGLGNVDLPD-P-TIKKVEESATD--------FLVDKVSRF-PGEVSVLALGPLTNIALAIKK-DPSFVKNVKKIVV ZmNRH2A AEGSHEPLKGG-KPRIADFVHGSDGIGNLFLPA-P-SAKKVEESAAD--------FMVNKVSEF-PGEVSVLALGPLTNVALAIKR-DPSFASKVNKIVV ZmNRH2B AEGSHEPLKGG-KPRVADFVHGSDGIGNLFLPA-P-SAKKVEESAAD--------FLINKVSEF-PGEVSVLALGPLTNVALAIKR-DPSFASKVKKIVV Tritic2 AEGSPEPLKGG-EPRVADFVHGSDGLGNLSLPA-P-TTKKVEESAAE--------FMVNKVSQF-PGEISVLALGPLTNVALAIKR-DSSFASKVKKIVV Oryza2 AEGSAEPLKGG-EPRVADFVHGSDGLGNLFLPA-P-TSKKVDENAAE--------FMVNKVSQF-PGEVSILALGPLTNVALAIKR-DPSFASKVKKIVV AtNRH1 AEGSSEPLKGG-IPRVADFVHGKNGLGDVSLPP-P-SRKKSEKSAAE--------FLDEKVEEY-PGEVTILALGPLTNLALAIKR-DSSFASKVKKIVI Glymax1 AQGSPEPLKGG-TPRVADFVHGKDGLGNTFLPP-P-KGEKIEKSASE--------FLVEKVSEY-PGEVSVLALGPLTNVALAIKR-DSAFASKVKRIVI Medic1 AEGSTEPLKGG-RPRVADFVHGKDGLGNLFLPD-P-KTNKIDKSASE--------FLVEKVSES-PGEVTVLALGPLTNIALAIKR-DSSFASKVKRIVV Tomato2 AEGSPEPLKGG-EPRVADFVHGSDGLGNLFLPS-P-NSKKIDKSASE--------FLVEKVSEY-PGEVSILALGPLTNLALAVKR-DSTFASKVKRVVV Nicoti2 AEGSPEPLKRG-EPRVADFVHGSDGLGNLFLPC-P-NSKKNDNSASE--------FLVEKVSEY-PGEVSILALGPLTNLALAVKR-DSTFASKVKRVVV Poplar2 AEGSPEPLKGG-IPTVPDFIHGSDGLGNTFLSP-P-KAKKIGKSASE--------FLLDKVSEY-PGEVSILALGPLTNLALAIKR-DSSFASKVKRIVV Vitis2 AEGSSGPLKGG-EPRVADFIHGSDGLGNIFLPQ-P-KAKKIEKNAAE--------FLVDKVSEY-PGEVSILALGPLTNVALAIKR-DSSFASKVKKVVV S.pombe YAGSNKPLLRA--PITATHVHGASGFEGAVLPP-P-SRKENEGHAVD--------FIIDTLRNNEPGTITICTIGPLTNIALALNK-APEVIQRAKQIVM YbeK ASGAVKPLMR--NLIIADNVHGESGLDGPALPE-P-TFAPQNCTAVE--------LMAKTLRES-EEPVTIVSTGPQTNVALLLNS-HPELHSKIARIVI YeiK YAGMPQPIMR--QQIVADNIHGDTGLDGPVFEP-L-TRQAESTHAVK--------YIIDTLMAS-DGDITLVPVGPLSNIAVAMRM-QPAILPKIREIVL L.major AAGCTKPLVRG--VRNASHIHGETGMGNVSYPP-EFKTKLDGRHAVQ--------LIIDLIMSHEPKTITLVPTGGLTNIAMAVRL-EPRIVDRVKEVVL C.fasci AAGCDKPLVR--KIMTAGHIHGESGMGTVAYPA-EFKNKVDERHAVN--------LIIDLVMSHEPKTITLVPTGGLTNIAMAARL-EPRIVDRVKEVVL


14

210 220 230 240 250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| T.vivax MGGAVDVRGNVFLPSTDGTAEWNIYWDPASAKTVFGCPGLRRIMFSLDSTNTVPVRSPYVQRFGEQTNFLLSILVGTMWAMCTHCELLRDG--------D ZmNRH1A LGGAYSVNGNV-----NPAAEANIFGDPDAADIVFTC-GADILAVGLNVTHQVVLTDADREKLEQCDSKYARYL-------CKIMGIYFDYHKDA-YFIK ZmNRH1B LGGAYSVNGNV-----NPAAEANIFGDPDAADIVFTC-GADILAVGLNVTHQVVLTDADREKLEQCDSKYARYL-------CKLMGVYFDYHRDA-YFIK Triti1 LGGAYSVNGNV-----NPAAEANIFGDPDAADIVFTC-GADILAIGINITHQVLLSDVDRKKLEQSDSKYARYL-------SNILGYYYDYHMDA-YSIT Oryza1 LGGAYSVNGNV-----NPAAEANIFGDPDAADIVFTS-GADILAVGINITHQVVLSDADREKLEQSDSKYARYL-------SKILGLYYDYHKDA-YFIK AtNRH2 LGGAFAVNGNV-----NPASEANIFGDPEAADIVFTC-GADIIAVGINVTHQVIMTADDKDKLASSKGKLAQYL-------CKILDVYYDYHLTA-YEIK Tomato1 LGGAFSVNGNV-----NPAAEANIFGDPDAADIVFTS-GADVLAVGINVTHQVVLTDFDRDELAKSNGKYAKYL-------DKILGVYFGYHHEA-YSTK Nicoti1 LGGSFAVNGNV-----NPAAEANIFGDPDAADIVFTS-GADVLAVGINVTHQVVLTDSDRNELAKSNGKFAKYL-------DKILDVYFGYHHDA-YSTK Poplar1 LGGAFSVNGNV-----NPAAEANIFGDPDAADIVFTC-GADILAVGINVTHQVVLTDAERDKLIQSNGKFAQYL-------CKILEVYFSYHQEA-YSMR Vitis1 LGGAFAVNGNV-----NPAAEANIFGDPEAADIVFTS-GADILAVGINVTHQVVLTDADREKLAQSNGKFAQYL-------CKILEVYFSYHRDA-YNTK Selagi1 LGGAFAVNGNV-----NPAAEANVLGDPLAADIVLTC-GANTKIIGINITHQVTMTGPQIEELRRSDSKYGRYL-------CDVIMCYLNYHRDY-YNID PpNRH1 LGGAFQVNGNV-----NPAAEANIYGDPEAADIIFTC-GADILVVGINITHQVYWTGKDLEDLGRSDSKFGKYL-------YAASHFYATYHREA-YDID Selagi2 LGGSFFSSGNA-----NPASEANVICDPEAADMVFSS-GARITVIGINVSTQVVLDGRDLSKLSDSSTESKRSL-------HEMCKFYREWHKKS-DLFD PpNRH2 LGGAFNASGNV-----NPAAEANIFGDPEAADLVFTS-GMDTLVIGISLTTQVIFNDQDLSEIRDSGGKYGKYI-------YDCCRFYHDFHLES-DHFD PpNRH3 LGGSFNASGNV-----NPAAEANLFGDPEAADIVFTS-GMNTLAIGIDLTTQVIFNEDDLCEIRDSGGDYGKYI-------YDCCRFYHDWHLSS-DHLD Oryza3 LGGAFFAAGNA-----TPSAEANIHSDPEAADIVFTS-GADIYVVGLNITTQVYFTDKDMLELRNSKGKHAQFL-------CDICKFYRDWHVHS-YGVD Tritic3 LGGAFFAAGNA-----TPSAEANIHSDPEAADMVFTS-GADIAVVGLNITTQVSFTEADLSELRRSGGKQAQFL-------CDICKFYSDWHLHS-YGDP ZmNRH3 LGGAFFAAGNA-----TPSAEANIHSDPEAADMVFTS-GADIYVVGLNITTQVSFTDKDLLELRNSQGKYAQFL-------CDVCKFYLDWHTES-YGAP ZmNRH2A LGGAFFVAGNV-----NPAAEANILGDPEAADIVFTS-GADVIVVGINITTQVCLTDEGLLELRNSKGKHAPFL-------SETCKFYRDWHVKS-DGFH ZmNRH2B LGGAFFAAGNV-----NPAAEANIHGDPEAADIVFTS-GADIVVVGINITTQVCLTDEDLLELRNSKGKHAAFL-------YEMCKFYRDWHAKS-DGFH Tritic2 LGGAFFAAGNV-----NPAAEANIYGDPDAADVVFTS-GADIDVVGINITTQCCFTDEDLLELKNSKGVHTQFL-------CDMCKFYRDWHEKS-DGFQ Oryza2 LGGAFFAAGNV-----SPAAEANIYGDPEAADIVFTS-GADVDVVGINITTQVCFTDEDLLELRNSKGKHAQFL-------CDMCQFYRDWHAES-DGFH AtNRH1 LGGAFFSLGNV-----NPAAEANIYGDPEAADVVFTS-GADITVVGINITTQLKLSDDDLLELGNCKGKHSKLI-------SDMCKFYRDWHVKS-DGVY Glymax1 LGGAFFALGNV-----NPAAEANIHGDPEAADIVFTS-GADVVVVGINITTQVQFTDADLLDLKESKGKYAPFL-------SDICKFYRDFHVKS-DRVH Medic1 LGGAFFALGNV-----NPAAEANIYGDPEAADVVFTS-GADVVVVGINITTQVQLTDADLLELKESKGKHASLL-------SDMCKFYRDWHVKS-DGVY Tomato2 LGGSFFALGNV-----NPAAEANIYGDPEAADVVFTS-GANIDVVGINITTQVKLKDADLEELKQSKGKYAKFV-------CDMCKFYRDWHVKS-DGVY Nicoti2 LGGSFFALGNV-----NPAAEANIYGDPEAADIVFTS-GANIDIVGINITTQVKLTDADLEELRQSEGKHAKIL-------GNMCKFYRDWHVKS-DGVY Poplar2 LGGAFFALGNV-----NPAAEANIHGDPEAADLVFTS-GANIVVVGINITTQVKFTDGDLLELRQSKGKYAKIL-------SDMCKFYRDWHVKS-DGVY Vitis2 LGGAFFALGNV-----NPAAEANIYGDPEAADVVFTS-GANIVVVGINITTQIKFTDADLHQLRHSEGRYAQFI-------SDICKFYRDWHVKS-DGVY S.pombe MAGAFSEVGNI-----TPAAEFNIYVDPHAAQMVLSS-GIPIVMMPLDITHQLHTSAKRIARMEALPNRVGPVV-------AAWLRMEKAYEAKK-YGTD YbeK MGGAMG-LGNW-----TPAAEFNIYVDPEAAEIVFQS-GIPVVMAGLDVTHKAQIHVEDTERFRAIGNPVSTIV-------AELLDFFLEYHKDEKWGFV YeiK MGGAYG-TGNF-----TPSAEFNIFADPEAARVVFTS-GVPLVMMGLDLTNQTVCTPDVIARMERAGGPAGELF-------SDIMNFTLKTQFEN-YGLA L.major MGGGYH-TGNA-----SPVAEFNVFIDPEAAHIVFNE-SWNVTMVGLDLTHQALATPAVQKRVREVGTKPAAFM-------LQILDFYTKVYEKE-HDTY C.fasci MGGGYH-EGNA-----TSVAEFNIIIDPEAAHIVFNE-SWQVTMVGLDLTHQALATPPILQRVKEVDTNPARFM-------LEIMDYYTKIYQSN-RYMA 310 320 330 340 350 360 370 380 390 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| T.vivax GYYAWDALTAAYVVDQKVANVDPVPIDVVVDKQPNEGATV----------RTDA-ENYPL-TFVA--RNPEAEFFLDMLLRSARAC---- ZmNRH1A GVYLHDPTTVIAAVNPSLLTYTEGVVRVQTVG-ITKGLTVFDNTKKRYGEITAW-SGMPT-VKVA--VTVDAPAVVELMMQRLMTDD--- ZmNRH1B GAYLHDPTTVIAAVNPSLLTYTEGVVRVQTVG-ITKGLTVFDNTKKRYGEITAW-SGMPT-VKVA--VTVDAPAVVELIVQRLMTDD--- Triti1 GVYLHDPTTLLAAVDPSLMTYTEGVVRVQTDG-ITKGLTVFDNTKKTYGEITAW-CGKPT-VKVA--VTVDAPAVVELIMQRLLTDD--- Oryza1 GVYLHDPATLIAAVDPSLMTYTEGVVRVQTDG-ITKGLTVFDTTKKRYGEITAW-TGKPT-VKVA--VTVDAPAVVEMIMQRLTTDD--- AtNRH2 GVYLHDPATILAAFLPSLFTYTEGVARVQTSG-ITRGLTLLYNNLKRFEEANEW-SDKPT-VKVA--VTVDAPAVVKLIMDRLMES---- Tomato1 GVYLHDPTALLAAVDPSLITYTEGVVRVQTNG-ITRGLTLFYNKQKRFGEVSEW-SDKPS-VKVA--VTVDAPRVLKLVMERLINS---- Nicoti1 GVYLHDPTALLAAVDPSLITYTEGAVRVQTSG-ITRGLTLFYNKQKRFAEVTEW-CDKPS-VKVA--VTVDATKVLKLVMERLINS---- Poplar1 GVYLHDPTALLAAVNPSLLTYTEGAVRVQTTG-ITRGLTLLYDKQKRFGEVTEW-TDKPT-VKVA--VTVDAPTVVKLLMERLMES---- Vitis1 GVYLHDPTTLLAAVNPSLITYTEGVVRVQTSG-ITRGLTLFYNKQKRFAEVTEW-TDKPT-VKVA--VTVDAPAVVKLVMDRLIDS---- Selagi1 AIYLHDPATVIAAIDPSLFTYAEGVVRVQQEG-ICRGLTLFNNTRKKWAVPTAW-CNKPP-VKVA--VTVDADKLVVLLKERLMAS---- PpNRH1 AIYLHDPATMVAAVDPSLMTYATGAVRVQKDG-ICKGLTLFNNSNKVWHDPTDW-CGIPP-VKVA--VTVDRERVASLLKERLTAP---- Selagi2 GIYVHGPTCLAALIAPSYFSYKSGALRVETLG-VCRGYTLFDMGLKKWNGLNPW-VGRPH-VDVA--WTVDAPKVLELVLERLIHTDT-- PpNRH2 GIFLHDPTCMAALLDPSLFTYRKGAVRVETEG-LCVGYTLLDMGLKNWVGKNHW-TGVPL-IDVG--MTVDADGVRSLVKKLLCSP---- PpNRH3 GIFLHDPTCMAALLDPTLFTFKNGAVRVETEG-PCLGHTILGMGLKNWVKETSW-TGVPP-IDVA--ITVDADGVRNLVKKLLCSSRLK- Oryza3 ALFLHDPVSFTALVHPEYFTFKKGVVRVETQG-ICKGHTSMDMGLKKWNSDNPW-TGYSP-ISVA--WTVDVPKVLAYAKELLFNAQ--- Tritic3 AVFLHDPVSLAALVRPELFTFRKGVVRVETQG-ICRGHTSMDMGLKKFKSENAW-SGYTP-ISVA--WTVDKAKVVAFVKELLKN----- ZmNRH3 VIFLHDPVSFAALVRPELFTFKKGVVRVETQG-ICVGHTSMDMLLKKWNSENPW-TGYSP-ISVA--WTVDVPKVVAFVKELVTKP---- ZmNRH2A GIFLHDPVSFTAVLHPEYFTFKKGVVRVETQG-ICTGHTLMDQGLKKWNSENPW-SGYKP-ISVA--WTVDVPKVIGFVKKLLMAP---- ZmNRH2B GIFLHDPVSFTAVLHPEYFTFKKGVVRVETQG-ICTGHTLMDQGLKKWNSENPW-SGYKP-ISVA--WTVDVPKVISFIKKLLMAP---- Tritic2 GIFLHDPVSFTALVHPEYFTFKKGVVRVETQG-ICTGHTLMDHGLKKWNSENPW-SGYAP-ISVA--WTVDVPKVLAYVKKLLMAP---- Oryza2 GIFLHDPVSFTALVHPEYFTFKKGVVRVETQG-ICTGHTLMDQGLKKWNSENPW-SGYKP-ISVA--WTVDVPNVLAFVKELLMAP---- AtNRH1 GVYLHDPVSFVAVVRPDLFTYKKGVVRVETQG-ICVGHTLMDQGLKRWNGSNPW-VGYSP-ISVA--WTVDVEGVLEYVKAKLMKP---- Glymax1 GIFLHDPVSFVAVVRPDLFTYKKGVVRVETQG-ICVGHTLLDQGLKKWNTSNPW-TGYSP-VAVA--WTVNVKGVLDYIRELLMKP---- Medic1 GIFLHDPVSFVAVVRPDLFTYKKGVVRVETQG-LCVGHTLLDQGLKRWNSSNPW-TGYSP-ISVA--WTVNVEGVLSYIKELLMKP---- Tomato2 GIFLHDPVSFAALVWPELFTFKKGVVRVETQG-ICVGHTLMDQGLKKWNTSNPW-TDYSP-VSVA--WTVDVDEVLDYIKKTLMKP---- Nicoti2 GIFLHDPVSFVALVRPELFTFKKGVVRVETQG-ICVGHTLLDQGLKKWNTSNPW-TDYSP-VSVA--WTVDVDEVLNYIKKMLMKS---- Poplar2 GIFLHDPVSFVALVRPDLFTYKNGVVRVETQG-ICAGHTLMDQGLKRWNSSNPW-SGFSP-VAVA--WTVTVDEVLNYVRQLLMK----- Vitis2 GIFLHDPVSFAALVRPDLFTYKEGVVRVETQG-ICLGHTLMDQGLKIWNSSNPW-TGYSP-VSVA--WTVDVDGVLNYIKKLLMKL---- S.pombe GGPLHDPNTVMWLLRPDIYSGRKVNVQIETQSELTMGMSVVD-----W-----WQVGLLP-ANVTFLRTVDDDEFYEVLIERLGRLP--- YbeK GAPLHDPCTIAWLLKPELFTTVERWVGVETQGKYTQGMTVVD-----YYYLTG---NKPN-ATVM--VDVDRQGFVDLLADRLKFYA--- YeiK GGPVHDATCIGYLINPDGIKTQEMYVEVDVNSGPCYGRTVCDELGV---------LGKPANTKVG--ITIDTDWFWGLVEECVRGYIKTH L.major GK-VHDPCAVAYVIDPTVMTTERVPVDIELNGALTTGMTVVD-------FRYPR-PKNCR-TQVA--VKLDFDKFWCLVIDALERIGDPQ C.fasci AAAVHDPCAVAYVIDPSVMTTERVPVDIELTGKLTLGMTVAD-------FRNPR-PEHCH-TQVA--VKLDFEKFWGLVLDALERIGDPQ

Kopecna Supplementary Material PP - Plant physiology · The samples were homogenized, extracted in cold water with 25% ammonia (ratio 4:1), and purified by solid-phase extraction

Documents