Table S1. Unigenes putatively involved BCAA metabolism in the hop glandular trichome EST
database. tBLASTX homolog searches TrichOME database using Arabidopsis genes as reference
(E-value cutoff < e-20).
Biological Processes Unigenes in hops database (EST #)
BCAA synthesis
1. acetohydroxyacid synthase (also known as acetolactate
synthase) catalytic subunits (At3g48560)
TCHL40342 (3 ESTs); EX518328; GD252495; TCHL41924
(3)
2. acetohydroxyacid synthase regulatory subunits
(At2g31810, At5g16290)
No hit
3. ketolacid reductoisomerase (At3g58610) TCHL42097 (32); TCHL42098 (13); GD248597
4. dihydroxyacid dehydratase (At3g23940) TCHL40677 (5)
5. 2-isopropylmalate synthase (At1g18500 and At1g74040) TCHL42263 (4);TCHL41243(2)
6. 3-isopropylmalate isomerase small subunit (At2g43090,
At2g43100 and At3g58990)
TCHL41734 (6);TCHL41733 (6)
7. 3-isopropylmalate isomerase large subunit
(At4g13430)
TCHL42135 (13)
8. 3-isopropylmalate dehydrogenase (At5g14200, At1g31180
and At1g80560)
TCHL42472 (2); GD249374
9. Threonine dehydratase/deaminase (At3g10050) GD246647
10. Branched-chain amino acid aminotransferase
(At1g10060, At1g10070, At3g49680, At5g65780 and
At1g50110 )
TCHL41709 (9); TCHL41708 (10); TCHL42249 (4);
GD249346
BCAA degradation
1. Branched-chain amino acid aminotransferase (At1g10060,
At1g10070, At3g49680, At5g65780 and At1g50110)
TCHL41709 (9); TCHL41708 (10); TCHL42249 (4);
GD249346
2. branched-chain keto acid dehydrogenase (BCKDH) E1 α
subunit (At1g21400)
TCHL41197 (3); GD252349; GD252293
3. BCKDH E1 β subunit (At3g13450) TCHL41910 (2); GD244031; GD250050
4. BCKDH E2 (At3g06850) TCHL41824 (5); TCHL42019 (2)
5. BCKDH E3 (At3g17240) TCHL40490 (2); GD248119
6. Isovaleryl-CoA-dehydrogenase (At3g45300) TCHL41225 (2); GD253310
7. Methylcrotonyl-CoA carboxylase α subunit (At1g03090) No hit.
8. Methylcrotonyl-CoA carboxylase β subunit (At4g34030) ES654853
9. Enoyl-CoA Hydratase (At3g60510 and At4g31810) GD252787, GD248114, GD248998, GD242840, TCHL40319
(4), TCHL41763 (7), GD247219, EX520912
10. HMG-CoA lyase (At2g26800) ES653388, GD252825, TCHL40912 (2)
Others
1. acyl-CoA thioesterase 9 contigs,15 singlatons (total 60 ESTs);
TCHL41706 (19, HlTE1); TCHL41407 (6, HlTE2);
TCHL40305 (5, HlTE3); TCHL40472/TCHL42469 (4,
HlTE4)
Table S2. Candidate HlCCL genes from the hop glandular trichome EST database.
Unigenes Functional Annotation E-value EST number Corresponding hop CCL
TCHL42480 Putative acyl-CoA synthetase 6.00E-39 2 9
TCHL42365 At1g20510 (OPC-8:0 CoA ligase) 0 2 5
× TCHL42294 ATP citrate lyase a-subunit 4.00E-30 3 /
× TCHL42265 Succinyl-CoA ligase alpha 1 subunit 0 4 /
TCHL42225 AMP-binding protein 3.00E-74 4 4
TCHL42032 Acetyl-CoA synthetase 1.00E-69 2 12
TCHL41120 AMP-binding protein 0 2 3
TCHL40924 Putative 4-coumarate:coenzyme A ligase 3.00E-51 2 5
TCHL40901 AMP-binding protein 5.00E-72 2 3
× TCHL40539 Long chain acyl-CoA synthetase 8 0 2 /
TCHL40516 Acyl-activating enzyme 18 0 2 12
TCHL40325 Acyl-activating enzyme 18 1.00E-58 4 12
TCHL40084 Putative amp-binding protein 0 2 2 and 13
TCHL40050 Putative amp-binding protein (At2g17650) 0 60 2 and 13
TCHL40042 Putative amp-binding protein (At2g17650) 5.00E-73 4 2 and 13
TCHL40034 Putative amp-binding protein (At2g17650) 0 56 2 and 13
GD253410 AMP-binding protein 3.00E-84 1 11
× GD253264 Long chain acyl-CoA synthetase 5.00E-48 1 /
GD249671 Putative acyl-CoA synthetase 5.00E-82 1 9
GD249308 4-coumarate--CoA ligase 5.00E-46 1 10
× GD249251 Long chain acyl-CoA synthetase 7 4.00E-86 1 /
GD248985 Acyl-activating enzyme 18 0 1 2 and 13
× GD248695 Long-chain acyl-CoA synthetase 4 2.00E-31 1 /
GD248638 Acyl CoA synthetase 7.00E-81 1 6
GD248394 4-coumarate:CoA ligase 1 6.00E-26 1 1
× GD248376 Putative acyl-CoA synthetase 2.00E-14 1 /
× GD248225 ACS4 4.00E-27 1 /
?? GD247613 Acyl-activating enzyme 13 (Arabidopsis
thaliana ) 1.00E-17 1 8
× GD247568 Putative amp-binding protein (At2g17650) 3.00E-13 1 /
× GD247197 Long chain acyl-CoA synthetase 6 3.00E-90 1 /
× GD246297 Succinyl-CoA ligase beta subunit 3.00E-08 1 /
× EX521530 Acetyl-CoA synthetase 5.00E-22 1 /
EX521472 Putative amp-binding protein (At2g17650) 2.00E-53 1 2 and 13
EX520818 Putative amp-binding protein (At2g17650) 1.00E-33 1 2 and 13
EX520059 4-coumarate--CoA ligase 6.00E-57 1 7
EX517755 AMP-binding protein 6.00E-85 1 3
ES652801 Phenylacetyl-CoA ligase 7.00E-88 1 No full length cDNA obtained
× ES652489 Putative amp-binding protein (At2g17650) 2.00E-22 1 /
??, Firstly removed, and later was included for further analysis in this study.
×, removed from the candidate genes list due to our limited interests.
Table S3. Compounds screened for carboxyl CoA ligase activity in present study.
SCFAs SBCFAs M/LCFAs Dicarboxylic acids Phenylpropionates Aromatic acids
(Group 1) (Group 2) (Group 3) (Group 4) (Group 5) (Group 6)
Acetic acid Isovaleric acid Heptanoic acid Succinic acid Cinnamic acid 2-Hydroxybenzoic acid
Propanoic acid Isobutyric acid Octanoic acid Malonic acid 4-Coumaric acid 4-Hydroxy-3-methoxybenzoic acid
Butanoic acid 2-Methyl butyric acid Nonanoic acid 2-Methylmalonic acid Caffeic acid 4-Hydroxyphenylacetic acid
Pentanoic acid 4-Methylvaleric acid Decanoic acid Tartronic acid Ferulic acid 5-Phenylpentanoic acid
Hexanoic acid 3-Methylvaleric acid Dodecanoic acid Monomethyl-malonate Sinapic acid 3-Phenyllactic acid
2-Methylvaleric acid Tetradecanoic acid Glutaric acid Mandelic acid
Hexadecanoic acid Adipic acid Benzoic acid
Octadecanoic acid
SBCFA: short branched-chain fatty acid;
SCFA: short-chain fatty acid;
M/LCFA: medium/long-chain fatty acid.
Table S4. Primers used in present study (5’ to 3’)
For real-time RT-PCR
CCL1 For: GGGTCGGAGCCGCTATTTT
CCL1 Rev: AACGAACGGTGCGATGGTC
CCL2 For: TGGAAAGTGTTAGGGCCGATGGTG
CCL2 Rev: GAGAAAAATATGAGCCAATGGACACCAATAG
CCL3 For: AAAGCAAAAGCCATTACAAGCC
CCL3 Rev: GTTTCCAATCAAATTCAGGGTCAC
CCL4-1 For: GATCGGAACCGCTGAAAACA
CCL4-1 Rev: TTTCGGTAAGCCCGTAGCC
CCL4-2 For: ATCCTCAACGCCGTCAACC
CCL4-2 Rev: TTCAGACTCGTCCGCCATAAA
CCL5 For: ACCACCAATCCCCTCAACAC
CCL5 Rev: GGTTTGGAATCGGTGATTTGTTT
CCL6 For: TACATCCATTGCCAACATTTTCCC
CCL6 Rev: TCGCCACGAGGCTTGTCAGA
CCL7 For: CCGGGCCTGTGTCAGATT
CCL7 Rev: GCGCAGCCGTGTCACTCT
CCL8 For: GGGCTCAACACAAACTTGCAC
CCL8 Rev: GGTAGGGAGTCCCACAGAAAAA
CCL9 For: TCCGATCGACGAGCTATTTTAGT
CCL9 Rev: CTGTAATCGGGCATGAGTCAAA
CCL10 For: TGAGGGCGGTGGAGATGT
CCL10 Rev: TGACTGGCGGAACCACAA
CCL11 For: AGCCGCCACCGCAAAAT
CCL11 Rev: ACCTCCACGCTGCTCACATT
CCL12 For: GGAGCGTTTAGCACAGCAGC
CCL12 Rev: ACAGAGGGAATAAGCCAACTTCA
CCL13 For: TGGAGAGTGTTAGGGCCGATGGCA
CCL13 Rev: TGAGCCAATGGACACCATTTTAGCTGTACTA
GAPDH For: TCTCCCAGCTCTCAACGGTAA
GAPDH Rev: TGAGACATCGACGGTAGGAACA
TE1 For:TGAGTGAGTATTTCGATGTTCAGCT
TE1 Rev:TTGCGTAGGCAGCGTTGTT
TE2 For: CTCCGGTCAAGTGAGGTATCAAA
TE2 Rev: ACATTCCGAATCCTGTAGCCAT
TE3 For: AAGGGCACAGCAGTTTGGTTAG
TE3 Rev: CCTCGTTGCGAAGAAATTGAA
TE4 For: ATACGGTGAGCCGACAGCAG
TE4 Rev: CTCCCTGAGTGCATAATCAGAAGAC
BCAT1 For: GTCAAGCAAACCGCCATCG
BCAT1 Rev: GAGAAGCATAGGCAAGGAAAGTGTA
BCKDE1-α1 For: GGAGATTGAAACGAGACCCTGTA
BCKDE1-α1 Rev: TTTTCCACCTTCTCAGCCACTT
BCKDE1-β For: AGCCCAAAGCAAGCAAAAGG
BCKDE1-β Rev: GGATAACTGAGCACCCCAACC
BCKDE2 For: TTGGAGGCTATGCCAGAGGTG
BCKDE2 Rev: AAATGGGCGGTAGAGCTTGTC
BCKDE3 For: AATGTTAGCCCACAAGGCAGAG
BCKDE3 Rev: GGGATGAGTGTAAACGACACCAG
IVDH For: TTGGGCAAGAAGGAAAAGGAG
IVDH Rev: GTCGGACATAAGGTAGGACAACATC
VPS For: ACCCAAGTCCAAGATCACCCA
VPS Rev: GATACAGCATCACTCGCTTCACC
For 5’ and 3’-RACE experiments
HlCCL1 3’RACE:TCCAAAGGGTGTGATGTTGACTCACAAAGG
HlCCL1 5’RACE:CGCAGAGAATGACGTCGTTTTGGTGGAAG
HlCCL2 3’RACE: GTGATCCCATCAGCATAAACTACACCTCAGGCACC
HlCCL2 5’RACE: GGTGCCTGAGGTGTAGTTTATGCTGATGGGATCAC
HlCCL3 3’RACE:ACCATCGGTGTCGTCAGTTCGCCTCTG
HlCCL3 5’RACE:CAGAAGCACTCAAAGACATCAGATACGCTCC
HlCCL4 3’RACE:AAAAGACCCAGAAGGAACGGCGAAATCC
HlCCL4 5’RACE:CGATCTCCTTCTCCGTCGGCTTCTTCG
HlCCL5 3’RACE: CCGATTCCAAACCGGTCCTCGCCTTCACGA
HlCCL5 5’RACE: GGCCTCTAGTTCTGCTGGGGCCACCTGATAACC
HlCCL6 3’RACE:TTCTATCATGCCTGCCAAACTGTTCTTCAC
HlCCL6 5’RACE:CGCTGCCCTTGTAGATGCAATAGTTTTCC
HlCCL7 3’RACE: CGATTTGGTCACCTTATCCGGGCCTGTGTCAGA
HlCCL8 5’RACE: CAGCTTCCCCGTAATCTTTGTCTGGTAGGCCCA
HlCCL9 3’RACE: GCTCTCTCTCCTTCTCCCAATAACGAGTCGAGCCT
HlCCL9 5’RACE: TACCTCTGATCCTTCCCTTGGAATTACAGCGC
HlCCL10 3’RACE: GAGGGTGCGTTGAGGGCGGTGGAGATGTACAG
HlCCL10 5’RACE: CTGTACATCTCCACCGCCCTCAACGCACCCTC
HlCCL11 3’RACE:TGTCTCGGCGTCAAGAAGGGTCAAGTCG
HlCCL11 5’RACE:GCTTCTGTGGCTATGAACGACACCTTTGG
HlCCL12 3’RACE:GCACCGCATAAAGCTATTGTCCTCCCTG
HlCCL12 5’RACE:CTGTCATTGCTGCTGTGCTAAACGCTCC
HlCCL13 5’RACE: GCCATCGGCCCTAACACTCTCCATGCTCACCGGG
TE2 3’RACE:TCTACCGCTTATGCCTTTGCAGGCTTGGTG
TE2 5’RACE:GCCATTCTTTGTGGCCTTTGCCTCTGGTC
TE4 3’RACE:GTTTGAGAAATACGGTGAGCCGACAGCAGA
TE4 5’RACE:GCCAGTGCAGGCAGGTCACAAAAGACA
For full length cDNA cloning and recombinant protein expression in E.coli
pENTR/D-TOPO vector
Note: Only HlCCL1, 7 and 9 (N-terminus 6 x His-tag) could be purified by Ni-NTA affinity chromatograpy when subcloned into
Gateway® pDEST-17 vector and expressed in E.coli strain BL21(+).
CCL1ENTR for: CACCATGGAGAACAACAAGCAAGATGATCACC
CCL1ENTR rev: CTAAATGGCTTGTTCAGTAACAAGTTTTGC
CCL2ENTR for: CACCATGGATAACTATAGAAGGCTCCACACTCCGG
CCL2ENTR rev: TCAAGAAAGGCTGCCCATGGCCATTGCTTTTTC
CCL3ENTR for: CACCATGGGTATGGTTGGGAGAGATATAGACG
CCL3ENTR rev: TAAGTTGCTCTTCTTAAGGGCCCCCATC
CCL4ENTR for: CACCATGGAAGATCTGAAGCCGAGACCAGCC
CCL4ENTR rev: CATTCGGCTCCGGCTCGCTCC
CCL5ENTR for: CACCATGGAGAACTCTAGCAAAGCCATAGTCGACG
CCL5ENTR rev: TTAGAGTTTGGAGGTAGCGAGTTTAATGAGATCC
CCL6ENTR for: CACCATGTCGTACACAGTGAAGGTTGAGGAAG
CCL6ENTR rev: TACCCTTGCTTCTTTTGCTTCACTGTAC
CCL7ENTR for: CACCATGGAGAAATCTGGGTATGGAAGAGACGGTGT
CCL7ENTR rev: TCATATGTTGGAGCGAACTTTCTGAATGAGCTC
CCL8ENTR for1: CACCATGCATTTAAACTTAAAGTCCTTGTGCTCTCTC
CCL8ENTR rev: TTAAACGCCCTGTTCAACGGTCAACTTTTTC
CCL9ENTR for: CACCATGGCGAATAATATAACCTTGACTGGTTTGTTG
CCL9ENTR rev: TTAAGCACCAAACTTGGGGACTTTGGCGGTG
CCL10ENTR for: CACCATGGAGAAGTGTTTCAATCCCGAAaCCCAAATC
CCL10ENTR rev: TCATATCTTGGATCGGACTTTCTGAATGAGTTC
CCL11ENTR for: CACCATGGAGGAACTGAAGCCAAGGTTTGC
CCL11ENTR rev: CATCCGAGCCCGACCAACCC
CCL12ENTR for: CACCATGTGTGAGGTGGGAGTAGAGGACTTG
CCL12ENTR rev: AATTTTACTTCGAACCAGCAGTTCATGC
CCL13ENTR for: CACCATGGATAACTATAGAAGGCTCCACACTCCGG
CCL13ENTR rev: GAAAGAGAAAAATATGAGCCAATGGACACCAAT
HlCCLs were subcloned into pEXP5-CT/TOPO vector and expressed in E.coli strain BL21AI
AAE1-PEXP5-FOR: ATGAAAATGGAGGGAACTATCAAATCTCCG
AAE1-PEXP5-REV: TAACTTGCTTCTGCCTTTCTTTGATAAGCTTAC
AAE2-PEXP5-FOR: ATGAGATTCTTGTTAACCAAAAGAGCATTCAGA
AAE2-PEXP5-REV: CAAGCTACCCATTTCATCAGCTTTCTTCCT
CCL2-PEXP5-FOR: ATGGATAACTATAGAAGGCTCCACACTCCGG
CCL2-PEXP5-REV: AGAAAGGCTGCCCATGGCCATTGCTTTTTC
CCL3-PEXP5-FOR: ATGGGTATGGTTGGGAGAGATATAGACGATC
CCL3-PEXP5-REV: TAAGTTGCTCTTCTTAAGGGCCCCCATC
CCL4-PEXP5-FOR-2: ATGGAAGATCTGAAGCCGAGACCAGCC
CCL4-PEXP5-REV-2: CATTCGGCTCCGGCTCGCTCC
CCL6-PEXP5-FOR: ATGTCGTACACAGTGAAGGTTGAGGAAG
CCL6-PEXP5-REV: TACCCTTGCTTCTTTTGCTTCACTGTAC
CCL8-PEXP5-FOR1: ATGCATTTAAACTTAAAGTCCTTGTGCTCTCTC
CCL8-PEXP5-REV: AACGCCCTGTTCAACGGTCAACTTTTTC
CCL10-PEXP5-FOR: ATGGAGAAGTGTTTCAATCCCGAAACCC
CCL10-PEXP5-REV: TATCTTGGATCGGACTTTCTGAATGAGTTC
CCL11-PEXP5-FOR: ATGGAGGAACTGAAGCCAAGGTTTGC
CCL11-PEXP5-REV: CATCCGAGCCCGACCAACCC
CCL12-PEXP5-FOR: ATGTGTGAGGTGGGAGTAGAGGACTTG
CCL12-PEXP5-REV: AATTTTACTTCGAACCAGCAGTTCATGC
CCL13-PEXP5-FOR: ATGGATAACTATAGAAGGCTCCACACTCCGG
CCL13-PEXP5-REV: AGAAAGGCTGCCCATGGCCATTGCTTTTTC
TE1-PEXP5-FOR: ATGTTGCAGACCATCTCATTATTACCAGC
TE1-PEXP5-REV: TCGGGTAAAAAATTTATCAAGTAAAGATGTTATC
TE2-PEXP5-FOR:ATGGATTTGAGCTCTTCCCCTAATCACC
TE2-PEXP5-REV:AATGCAGGCCTCAGCATCCATACG
TE3-PEXP5-FOR: ATGTTGCAGACCTTTTCTCCTTCCTACAAGC
TE3-PEXP5-REV: TGACTCCTCGTTGCGAAGAAATTGAACAAG
TE4-PEXP5-FOR:ATGATGACACCCATTGGAATTAGAATTCG
TE4-PEXP5-REV:GATTTCATTTTCGTATTGGGAGTGTGGG
OM-TE4-PEXP5-FOR: ATGACGCATACCAAGTCATTCTCCACAGAC
OM-TE4-PEXP5-REV:GATTTCATTTTCGTATTGGGAGTGTGGG
HlCCL11 was subcloned into pMAL-C2X vector and expressed in E.coli strain BL21(+)
CCL11pMALfor: TGCTCTAGAATGGAGGAACTGAAGCCAAGGTTTGC (Xba I)
CCL11pMALrev: TGCACTGCAGTTACATCCGAGCCCGACCAACCC (Pst I)
Constructs used in yeast in vivo assays
ESC(LEU)-CCL2-FOR: GCGGCCGCATGGATAACTATAGAAGGCTCCACACTCCGG (Not I)
ESC(LEU)-CCL2-REV: TTAATTAATCAAGAAAGGCTGCCCATGGCCATTGC (Pac I)
ESC(LEU)-CCL4-FOR:GCGGCCGCATGGAAGATCTGAAGCCGAGACCAGCC (Not I)
ESC(LEU)-CCL4-REV:TTAATTAATCACATTCGGCTCCGGCTCGCTCC (Pac I)
ESC(HIS)-VPS for:GGGGGAATTCATGGCGTCCGTAACTGTAGAGCAAATC (EcoR I)
ESC(HIS)-VPS rev:GGGGGCGGCCGCTTAGACGTTTGTGGGCACGCTGT (Not I)
For subcellular localization experiments
HlCL8-1-GFP-FOR: AAGCTTATGCATTTAAACTTAAAGTCCTTGTGCTCTC (Hind III)
HlCL8-1-GFP-REV: GGATCCTTTGCCTGCTTTATGACTGGCTTTC (BamH I)
HlCL2-GFP-FOR: AAGCTTATGGATAACTATAGAAGGCTCCACACTCCG (Hind III)
HlCL2-GFP-REV: GGATCCAGAGAAGGTAGCCACCACATCCCCTG (BamH I)
HICCL4-GFP-FOR: AAGCTTATGGAAGATCTGAAGCCGAGACCAGCC (Hind Ⅲ)
HICCL4-GFP-REV: GGATCCCATTCGGCTCCGGCTCGCTCC (BamH I)
HIVPS-GFP-FOR: GTCGACATGGCGTCCGTAACTGTAGAGC (Sal I)
HIVPS-GFP-REV: GGATCCGACGTTTGTGGGCACGCT (BamH I)
HITE4-GFP-FOR: GTCGAC ATGACACCCATTGGAATTAGAATTC (Sal I)
HITE4-GFP-REV: GGATCCGATTTCATTTTCGTATTGGGAGTG (BamH I)
HITE4-SP-GFP-FOR: GTCGAC ATGATGACACCCATTGGAATTAGAATT (Sal I)
HITE4-SP-GFP-REV: GGATCC TGGATCAAAGAGAAAGACGGTGG (BamH I)
General primers used in this study:
pDEST-17-FOR: GGAGACCACAACGGTTTCCCTC
pMAL-C2X-maLE-FOR: GGTCGTCAGACTGTCGATGAAGCC
T7-Promotor: TAATACGACTCACTATAGGG
GAL10-FOR: GGTGGTAATGCCATGTAATATG
GAL10-REV: GGCAAGGTAGACAAGCCGACAAC
GAL1-FOR: ATTTTCGGTTTGTATTACTTC
GAL1-REV: GTTCTTAATACTAACATAACT
Figure S1. Terpenophenolics profile in different hop tissues measured using HPLC. The HPLC
traces are shown in different scales for convenient visualization.
Figure S2. Amino acid sequence alignment of 13 HlCCLs. The underlined sequence is the
conserved AMP binding motif.
Figure S3. Nucleotide sequence alignment of HlCCL2 and CCL13. The primers designed for
differentiating these two genes are marked in red and blue, respectively.
Figure S4. LC-ESI/MS analyses of enzymatic products of HlCCLs using CoASH and different
substrates. The solid line in the chromatogram represents the enzyme reaction, the dashed line
represents the control reaction using heat-denatured enzyme, and the dotted lines represent the
commercially available standards. Both parent ions of CoASH and CoA conjugated product (m/z)
were selected for scanning.
(a) LC-ESI/MS analysis and mass spectra of enzymatic product of HlCCL1 using free CoA
and coumarate and authentic coumaroyl-CoA.
(b) LC-ESI/MS analysis and mass spectra of enzymatic product of HlCCL2 using free CoA
and 3-methylvaleryl acid.
(c) LC-ESI/MS analysis and mass spectra of enzymatic product of HlCCL2 using free CoA
and 4-methylvaleryl acid.
(d) LC-ESI/MS analysis and mass spectra of enzymatic product of HlCCL3 using free CoA
and propanoic acid and authentic propionyl-CoA.
(e) LC-ESI/MS analysis and mass spectra of enzymatic product of HlCCL4 using free CoA
and 2-methylbutyryl acid.
(f) LC-ESI/MS analysis and mass spectra of enzymatic product of HlCCL8 using free CoA
and malonate and authentic malonyl-CoA. The second panel is the extracted ion
chromatogram using 854.1 (m/z) as target ion only to distinguish the free CoA and malonyl
CoA (both chemicals showed a similar retention time on our LC system).
Figure S5. Tissue-specific expression of genes involved in the BCAA degradation pathway by
qRT-PCR analyses. Transcript levels are expressed relative to those of GAPDH (n = 3; mean ±
SD).
Figure S6. Comparison of the protein sequences of HlTE1 and 3 with homologs from other plant
species. All protein sequences were extracted from Phytozome V8.0 (http://www.phytozome.net/).
Figure S7. Comparison of the protein sequences of HlTE2 and 4 with homologs from other plant
species. All protein sequences were extracted from Phytozome V8.0 (http://www.phytozome.net/).
Among them, HlTE4, At5g48370, At2g30720, Medtr4g128060, Os09g34190, Pp1s387_49 and
Cre01g037350 had a clear signal peptide based on the computational prediction by TargetP and
WolF PSORT. The cleavage site of signal peptide of HlTE4 protein is highlighted with an arrow.
Figure S8. Substrate specificity of recombinant HlTE4.
The CoA esters were used at 450 μM in each assay. Data are expressed as mean ± SD from
triplicate independent assays. N.D., activity not detected. * 2-methylbutyryl-CoA was synthesized
enzymatically using purified HlCCL4. The reaction, including 1 mM 2-methylbutyric acid and
450 μM CoASH, was incubated at room temperature for 2 hours, and 100% CoASH was
incorporated into 2-methylbutyryl-CoA products confirmed spectrophotometrically (412 nm) with
DTNB.