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Supplementary material
Applied Microbiology and Biotechnology
Successful expression of a novel bacterial gene for pinoresinol reductase and its effect on lignan
biosynthesis in transgenic Arabidopsis thaliana
Masayuki Tamuraa, Yukiko Tsuji
a, Tatsuya Kusunose
b, Atsushi Okazawa
b,i, Naofumi Kamimura
c, Tetsuya
Morid, Ryo Nakabayashi
d, Shojiro Hishiyama
e, Yuki Fukuhara
c, Hirofumi Hara
f, Kanna Sato
a, Toshiya
Muranakab, Kazuki Saito
d,g, Yoshihiro Katayama
h, Masao Fukuda
c, Eiji Masai
c, Shinya Kajita
a,*
aGraduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and
Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan bGraduate School of Engineering, Osaka University, 1 Yamadaoka, Suita, Osaka 565-0871, Japan
cDepartment of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka-cho, Nagaoka,
Niigata 940-2188, Japan dMetabolomics Research Group, RIKEN Center for Sustainable Resource Science, RIKEN, 1-7-22
Tsurumi, Kanagawa 230-0045, Japan eForestry and Forest Products Research Institute, 1 Matsunosato, Tsukuba, Ibaraki 305-8687, Japan
fDepartment of Environmental and Green Technology, Malaysia-Japan International Institute of
Technology, Kuala Lumpur 54100, Malaysia gGraduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba
260-8675, Japan hCollege of Bioresource Sciences, Nihon University, 1866 Mameino, Fujisawa, Kanagawa 252-0880,
Japan iPresent address, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1
Gakuen-cho, Nakaku, Sakai, Osaka 599-8531, Japan
*Corresponding author
Shinya Kajita
Graduate School of Bio-Applications and Systems Engineering, Tokyo University of
Agriculture and Technology, 2-24-16, Nakacho, Koganei, Tokyo 184-8588, Japan
Phone & Fax: +81 42 388 7391
E-mail: [email protected]
Supplementary figures: Figs. S1, S2, S3, S4 and S5.
Supplementary tables: Tables S1, S2, S3 and S4.
pinZ ATGACCAGCCAGGGCCGCATCGTCATCACCGGAGCCTCGGGCCAATATGGACGGCTCGCC 60
mpinZ ATGACAAGTCAAGGTCGTATAGTTATCACAGGAGCTTCGGGACAATATGGAAGGCTTGCT 60
METThrSerGlnGlyArgIleValIleThrGlyAlaSerGlyGlnTyrGlyArgLeuAla 20
pinZ ACGGACCTGCTGATCGCGCAGGGGCTGGCGGACCGGCTCATCCTCATCACCCGCAGCCCC 120
mpinZ ACAGACTTGTTAATCGCACAAGGGCTGGCAGATAGGCTTATCCTTATCACTAGATCTCCA 120
ThrAspLeuLeuIleAlaGlnGlyLeuAlaAspArgLeuIleLeuIleThrArgSerPro 40
pinZ GCCCGCCTCGCCGATCGCGTCGCGCAGGGCTGCACGGTGCGCTATGGCGACTATGACAAG 180
mpinZ GCTCGCCTCGCTGATAGAGTTGCGCAGGGTTGTACAGTAAGATATGGAGACTATGACAAG 180
AlaArgLeuAlaAspArgValAlaGlnGlyCysThrValArgTyrGlyAspTyrAspLys 60
pinZ CCGGAGACGCTGGCCGATGCCGTCCGGGACGCCGAGAAGATGCTGCTCATTTCCGGCACC 240
mpinZ CCAGAGACGTTGGCTGATGCCGTCAGGGATGCTGAGAAGATGTTGCTAATTTCAGGAACT 240
ProGluThrLeuAlaAspAlaValArgAspAlaGluLysMETLeuLeuIleSerGlyThr 80
pinZ CGCGTCGGCGCGCGCGTCGTCCAGCACAAGGCCGCGATCAACGCGGCGGCGGCGGCCGGC 300
mpinZ CGTGTTGGCGCAAGAGTTGTCCAGCACAAAGCCGCAATAAACGCTGCAGCTGCAGCCGGC 300
ArgValGlyAlaArgValValGlnHisLysAlaAlaIleAsnAlaAlaAlaAlaAlaGly 100
pinZ GTCCGCCACATCCTCTACACCAGCTTCATCGGCATCGACGATCCCGCCAATCCCGCCGAG 360
mpinZ GTCAGACACATCCTTTACACCAGCTTTATAGGCATTGACGATCCAGCAAATCCCGCAGAA 360
ValArgHisIleLeuTyrThrSerPheIleGlyIleAspAspProAlaAsnProAlaGlu 120
pinZ GTGCGGCACGACCATATCGAGACCGAGCGGCTCATGCGCGCCTCCGGCATGGCATGGACG 420
mpinZ GTACGGCACGATCATATTGAGACAGAACGGCTAATGCGTGCCTCAGGAATGGCATGGACA 420
ValArgHisAspHisIleGluThrGluArgLeuMETArgAlaSerGlyMETAlaTrpThr 140
pinZ GCGCTGCGGGACGCCCATTATGCCGATGCCATGCTGCTCATGGCGGGCCCGGGGATGATG 480
mpinZ GCGCTGAGGGATGCTCATTATGCAGATGCTATGTTGCTCATGGCAGGTCCAGGGATGATG 480
AlaLeuArgAspAlaHisTyrAlaAspAlaMETLeuLeuMETAlaGlyProGlyMETMET 160
pinZ GCCACCGGCCAATGGGTGAGCAATGCCGGCGACGGGCGCGAGGCGATGGTCTGGCGAGAC 540
mpinZ GCCACCGGCCAATGGGTGTCTAATGCCGGTGACGGGAGAGAAGCTATGGTTTGGCGAGAT 540
AlaThrGlyGlnTrpValSerAsnAlaGlyAspGlyArgGluAlaMETValTrpArgAsp 180
pinZ GATTGCGTGGCCTGCGCGGTCGCCGTGCTCACCACGCCGGGCCATGAGAACAAGGTCTAC 600
mpinZ GATTGTGTGGCTTGTGCGGTTGCCGTGCTCACCACACCTGGTCATGAGAACAAGGTTTAC 600
AspCysValAlaCysAlaValAlaValLeuThrThrProGlyHisGluAsnLysValTyr 200
pinZ AATATCACCGGGCCGGCGCTTCAGACCTTCGACGAGGTCGCCGCGCTGGTGCGCGAGATC 660
mpinZ AATATTACTGGGCCTGCTCTTCAGACTTTCGATGAAGTCGCTGCGTTGGTGAGAGAGATA 660
AsnIleThrGlyProAlaLeuGlnThrPheAspGluValAlaAlaLeuValArgGluIle 220
pinZ ACCGGCCGCCCGCTCGAGCATGTGAAGGTGGGCGATGAAGGCCAATATGCCCTCTTCGAT 720
mpinZ ACCGGAAGACCGCTTGAACATGTAAAGGTGGGTGATGAAGGACAATATGCTCTCTTTGAT 720
ThrGlyArgProLeuGluHisValLysValGlyAspGluGlyGlnTyrAlaLeuPheAsp 240
pinZ GCCATGGGCATTCCGCGCCGCCCGGTGGACGACCAGTATGTGAGGGGCATTCCTTGGAAC 780
mpinZ GCTATGGGAATTCCTCGTCGTCCTGTAGACGATCAGTACGTTAGGGGAATTCCTTGGAAC 780
AlaMETGlyIleProArgArgProValAspAspGlnTyrValArgGlyIleProTrpAsn 260
pinZ AGCGACGACATGGTGACGTTCGGCCGCGCCATTCGCGAGGGTTTCCTGGAGATCTGCACG 840
mpinZ TCTGATGACATGGTGACGTTTGGTAGAGCTATTCGAGAAGGTTTCTTAGAGATCTGCACG 840
SerAspAspMETValThrPheGlyArgAlaIleArgGluGlyPheLeuGluIleCysThr 280
pinZ GACGACGTGGAGAAACTCACCGGTCGCAAGGCGCGCTCCGTGCGCCAGATGATCGAGGAG 900
mpinZ GATGATGTTGAGAAACTTACTGGTCGCAAAGCGAGATCCGTTCGACAGATGATTGAAGAG 900
AspAspValGluLysLeuThrGlyArgLysAlaArgSerValArgGlnMETIleGluGlu 300
pinZ AACCGGGCGATGCTGCAGGCGGCGGCGGACAATGCGGCCCAGCCGGCATGA 951
mpinZ AACAGGGCGATGTTACAAGCTGCAGCTGACAATGCGGCTCAACCGGCATGA 951
AsnArgAlaMETLeuGlnAlaAlaAlaAspAsnAlaAlaGlnProAla*** 316
Fig. S1. Nucleotide sequences of cDNAs for original (pinZ) and codon-optimized pinZ (mpinZ).
Red letters of mpinZ indicate the replaced nucleotides for expected efficient translation of the
transcript in A. thaliana. Deduced amino acid sequence derived from the gene is also indicated in
the third line of each part. An expression cassette for plant transformation had been constructed
with a chemically synthesized cDNA for mpinZ. Complete genome sequence including the
original pinZ and mpinZ has been deposited with accession nos. AP012222 and AB924082,
respectively, in DDBJ, GenBank and EMBL databases.
Fig. S2. Phenotypic observations of the wild-type (first 3 plants from left) and PinZ8 plants. No
significant phenotypic differences were observed between them. White bar = 6 cm
Fig. S3. Analyses by liquid chromatography coupled with a photo-diode array (PDA) and with
both PDA and mass spectrometer (MS) for the compounds, (±)-pinoresinol (A B, and C),
(±)-lariciresinol (D, E and F) and (±)-secoisolariciresinol (G, H and I) and acetosyringone
(internal standard; J, K and L). Chromatograms detected at 280 nm (A, D, G and J) and
negative-ion ESI-MS spectra (C, F I and L) are shown. Absorption spectra of these compounds
measured with photo diode detector (B, E H and K) are also indicated. The retention times of
(±)-pinoresinol, (±)-lariciresinol, (±)-secoisolariciresinol, and acetosyringone (an internal
standard) were 3.1 min, 1.8 min, 1.6 min, and 1.4 min, respectively. Data shown in Fig. 4 was
obtained under the same condition as indicated above.
Fig. S4. Examples of extracted ion chromatogram at m/z 521 obtained by LC-MS/MS analysis
for the detection of expected lariciresinol-glucoside in root extracts from the wild-type (A) and
PinZ8 transgenic (B) roots. Two predominant peaks, both of which give two main product ions
at m/z 329.00 and m/z 359.20, could be detected at retention times of 4.46 min and 4.75 min,
respectively. A quantitative comparison of the peak intensities of these 2 peaks derived from
samples of whole plant, root, leaf, and stem are shown in Fig. S4.
In
ten
sit
yIn
ten
sit
y1×106
1×106
≈≈
4.0 5.0 6.0 7.0 8.0 (min)
5.0 6.0 7.0 8.0 (min)
4.0
Peak 2
Peak 1
Peak 2
Peak 1
A
B
Figure S5. Quantitative comparison of the intensities of the 2 peaks (Peak 1 and Peak 2, Fig. S3)
for the expected compound lariciresinol monoglucoside (Lar-Glc) derived from wild-type (WT)
and PinZ8 transgenic plants without beta-glucosidase treatment. Asterisks represent statistically
significant differences (**, p<0.01, *, p<0.05) between transgenic and wild-type plants by the
Student’s t-test.
0
2000
4000
6000
8000
10000
12000
14000
16000
WT PinZ8 WT PinZ8 WT PinZ8 WT PinZ8
Seedling
2 weeks old
Root
4 weeks old
Leaf
6 weeks old
Shoot
6 weeks old
Peak a
rea
Peak 1
m/z 521
0
10000
20000
30000
40000
50000
60000
WT PinZ8 WT PinZ8 WT PinZ8 WT PinZ8
Seedling
2 weeks old
Root
4 weeks old
Leaf
6 weeks old
Shoot
6 weeks old
Pe
ak a
rea
Peak 2
m/z 512
**
**
*
*
*
**
**
Table S1. Nucleotide sequence of primers used for RT-PCR.
Target
transcript Sense primer Antisense primer
pinz
AtPrR1
AtPrR2
ACTIN2
GCAGCCGGCGTCAGACACAT
TCAAAGAGGCTGGTAACGTAAAG
GAGTTGGAGAAGACCTATGTTTCAG
CAATGAGCTTCGTATTGCTCC
TGCCGGTTGAGCCGCATTGT
ACCCACAACGTAAGTGTAAGGAA
TCACATCCGGATAGAGTTTAGTAGC)
GCATCTGAATCTCTCAGCACC
Table S2. Relative band intensities of RT-PCR products presented in Figs 2B and 2C.
Tissue Gene Wild type PinZ8 a PinZ3
a PinZ6
a PinZ1
a PinZ4
a PinZ5
a PinZ7
a
Root
PinZ 1.00 75.53 37.87 44.89 20.72 9.28 0.24 0.80
AtPrR1 1.00 1.26 1.12 0.67 0.64 1.07 1.17 1.08
AtPrRr2 1.00 3.27 2.07 1.20 0.57 0.51 2.37 2.41
Stem
PinZ 1.00 80.83 49.86 50.27 46.16 24.71 5.04 2.03
AtPrR1 1.00 1.16 0.89 0.76 0.78 0.83 0.81 0.47
AtPrRr2 1.00 1.42 0.95 1.22 1.17 1.26 0.97 0.77 a Each band was scanned and its intensity was obtained by CS analyzer 3.0 (ATTO corporation, Tokyo, Japan). Each value
was normalized with respect to its corresponding actin (ACT2) and subsequent normalized with that of the wild-type
plant.
Table S3. Change in level of lignans and their derivatives in roots characterized by untargeted metabolomic analysis
Assigned compound Retention time
(min) m/z MSI level
a Reference
Fold changeb
PinZ6 plant PinZ8 plant
Lariciresinol 4.953 329.1319 [M-H-CH2O]- 1 Authentic sample 1.60** 1.60**
Lariciresinol analog #1c 3.800 359.1487 [M-H]
- 2 Authentic sample 1.27* 1.06
Lariciresinol analog #2c 4.004 359.149 [M-H]
- 2 Authentic sample 1.05 0.899
Secoisolariciresinol analog 3.760 361.1637 [M-H]- 2 Authentic sample 1.05 5.74**
Syringaresinol 4.595 417.1543 [M-H]- 2 Sim et al. 2013 0.105** 0.105**
Syringaresinol-hexoside 4.347 579.2071 [M-H]- 3 Sim et al. 2013 0.171** 0.0330**
a The level is based on the descriptions developed under Metabolomics Standards Initiative (Sumner et al. 2007).
b Lignan profile as fold changes of the signal intensity in each transgenic line versus the wild-type plant. Data given as
average of 6 independent experiments with 6 biological replicates. Significant differences were analyzed by Tukey
HSD test (**, p < 0.01; *, p < 0.05). c Since there were same compounds assigned more than one, the compounds were numbered sequentially.
Table S4. Change in level of neolignans and their derivatives in roots characterized by untargeted metabolomic analysis
Assigned compound Retention time
(min) m/z MSI level
a
Fold change b
PinZ6
plant
PinZ8
plant
G(8-O-4’)FAc hexoside #1
d 3.302 551.1757 [M-H]
- 2 1.63 1.23
G(8-O-4’)FAc hexoside #2
d 3.397 551.1762 [M-H]
- 2 0.745 1.14
G(8-O-4’)FAc hexoside #3
d 3.476 551.1763 [M-H]
- 2 0.900 0.781
G(8-O-4’)FAc hexoside #4
d 3.548 551.1763 [M-H]
- 2 1.37** 1.36**
G(8-O-4’)FAc hexoside #5
d 3.610 551.1758 [M-H]
- 2 1.34 1.44
G(8-O-4’)FAc hexoside + C12H14O5 #1
d 5.222 789.2598 3 1.19 0.989
G(8-O-4’)FAc hexoside + C12H14O5 #2
d 5.402 789.2600 3 1.14 1.02
G(8-O-4’)FAc hexoside + C12H14O5 #3
d 5.512 789.2598 3 1.24* 0.980
G(8-O-4’)FAc hexoside + C12H14O5 #4
d 5.692 789.2593 3 1.17 0.906
a The level is based on the descriptions developed under Metabolomics Standards Initiative (Sumner et al. 2007).
All assigned compounds were characterized by matching ReSpect MS/MS spectra database (http://spectra.psc.riken.jp/; Sawada et al.
2012). b Neoignan profile as fold changes of the signal intensity in each transgenic line versus the wild-type plant. Data given as average of 6
independent experiments with 6 biological replicates. Significant differences were analyzed by Tukey HSD test (**, p < 0.01; *,
p < 0.05). c G(8-O-4′)FA, Guaiacylglycerol-8-O-4′-feruloyl ether
d Since there were same compounds assigned more than one, the compounds were numbered sequentially.
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