Table S1. Unigenes putatively involved BCAA metabolism in ...TCHL42225 AMP-binding protein 3.00E -74...

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.