Supplemental Information

Supplemental Figures

Fig. S1 Phenotypic comparison of mature seeds from different transgenic rice and

their wild type (WT).

A, Appearance comparison of mature seeds from different transgenic rice and their

wild type. B, Seed length (mm) comparison of mature seeds from different transgenic

rice and their wild type. C, Seed width (mm) comparison of mature seeds from

different transgenic rice and their wild type.

Error bars represent statistical differences (SDs) of three biological replicates, and the

different letters indicate statistical significance among transgenic plants and wild-type

at p < 0.05.

Fig. S2 Appearance and free lysine content of dehulled mature seeds from pyramiding

HFL2 line and its parents and wild type (WT).

A, GR-65 and 35S-15 transgenic lines were generated by transformation of GR or 35S

construct into wild-type (WT), respectively; HFL2 pyramid transgenic line was

produced by crossing GR-65 and 35S-15 lines (GR-65/35S-15). B, The free lysine

content in mature seeds of various lines were measured. HFL2-D and HFL2-N were

mature seeds with normal or dark-brown color appearance, respectively, from the

same HFL2 transgenic lines. Bar: 5 mm. Error bars represent SDs of three biological

replicates, and the different letters indicate statistical significance among transgenic

plants and wild-type at p < 0.05.

Fig. S3 Principle component analysis (PCA) of detected metabolomic data in

developing seeds at 10 DAF and leaves.

A, PCA analysis of detected metabolomic data in developing seeds at 10 DAF. B,

PCA analysis of detected metabolomic data in leaves.

Fig. S4 Relative content of metabolites in mature seeds detected by metabolomic

analysis, and the chromatograms for serotonin measurement.

A-D, The relative contents of tryptophan (A), tryptamine (B), serotonin (C) and lysine

(D) in mature seeds. Data were from the metabolomics analysis. E-H, The

chromatograms for serotonin measurement from different samples, including empty

control (E), serotonin standard (F), extracts from mature seeds of wild-type (G) and

HFL1 rice (H), respectively. Error bars represent SDs of three biological replicates,

and the different letters indicate statistical significance among transgenic plants and

wild-type at p < 0.05.

Fig. S5 Dynamic changes of transcriptional expression of TDC and T5H genes

during seed development..

Error bars represent SDs of three biological replicates, and the different letters

indicate statistical significance among transgenic plants and wild-type at p < 0.05.

Fig. S6 Generation and characterization of Ubi::TDC3 transgenic rice.

A, The T-DNA region containing the Ubi::TDC3 construct. (B) Tryptamine and (C)

serotonin contents in calli (n=3). D, PCR confirmation of Ubi::TDC3 insertion in

selected transgenic rice lines. E-G, The relative expression of TDC3 (E, G) and T5H

(F) genes in developing seeds of Ubi::TDC3 transgenic rice. P, Ubi::TDC3 plasmid

DNA. Error bars represent SDs of three biological replicates, and the different letters

indicate statistical significance among transgenic plants and wild-type at p < 0.05.

Fig. S7 Principle Component Analysis (PCA) and ontology (GO) enrichment analyses

of transcriptomes in developing seeds.

A, PCA analysis of transcriptome in developing seeds at 10 DAF. B-C, PCA (B) and

ontology (GO) enrichment (C) analyses of transcriptome in developing seeds at 15

DAF.

Fig. S8 Relative content of metabolites and transcriptional expression levels of genes

associated with jasmonate (JA) metabolism.

A-B, The relative expression of genes encoding gamma-thionin family domain

containing protein (Thionin), defense protein in DEFL family (DEFL), lipoxygenase

(LOX) and β-oxidase (ACX) in developing seeds at 15 DAF. C, The expression of

GDCi genes in developing seeds at 15 DAF. D, The relative content of

N-delta-acetylorithine in mature seeds of different transgenic lines and wild-type

(WT). Error bars represent SDs of three biological replicates, and the different letters

indicate statistical significance among transgenic plants and wild-type at p < 0.05.

Fig. S9 Relative content of indole acetic acid (IAA), salicylate (SA), proline and

homoserine in leaves, developing seeds and mature seeds.

A-B, The relative content of indole acetic acid (IAA) in developing seeds and mature

seeds. C, The fold-change of ICS gene’s expression in developing seeds at 10 DAF

and 15 DAF in HFL1 rice. D-L, The relative content of salicylate (D-F), proline (G-I),

and homoserine (J-L) in leaves, developing seeds and mature seeds. Error bars

represent SDs of three biological replicates, and the different letters indicate statistical

significance among transgenic plants and wild-type at p < 0.05.

Fig. S10 Percentage of dark-brown grain, lysine content in mature seeds, and gene

transcriptional levels in developing seeds at 10 DAF of HFL2 under different

temperature treatments during seed development.

Error bars represent SDs of three biological replicates, and the different letters

indicate statistical significance among transgenic plants and wild-type at p < 0.05.

Fig. S11 Relative expression of TDC3 and T5H genes, and serotonin content in the

seedlings of wild type or under jasmonate treatment.

Error bars represent SDs of three biological replicates, and * indicates statistical

significance between wild-type and its JA treatment at p < 0.05.

Fig. S12 Change of metabolite content within some main metabolic pathways in

mature seeds of HFL1 compared with the WT.

Red font indicates significantly higher in HFL1 mature seeds when compared to the

wild-type (WT), p < 0.05.

Fig. S13 Transcriptional levels of key genes involved in aromatic amino acid

biosynthesis through the shikimic acid pathway.

3DS, 3-dehydroquinate synthase; CS, chorismic acid synthase; AS, anthranilic acid

synthase. Error bars represent SDs of three biological replicates, and the different

letters indicate statistical significance among transgenic plants and wild-type at p <

0.05.

Supplemental Tables

Table S1. List of detected and differential metabolic compounds between transgenic

rice and wild-type (WT) (p < 0.05).

Tissue Plants Total

metabolites

Number of different metabolites*/ WT

Total Up Down

Leaves

WT 232 / / /

GR-14 232 2 1 1

35S-15 232 26 10 16

HFL1 232 21 12 9

Developing

seed at 10 DAF

WT 244 / / /

GR-14 244 1 1 0

35S-15 244 9 3 6

HFL1 244 7 5 2

Mature seeds

WT 223 / / /

GR-14 223 24 13 11

35S-15 223 29 6 23

HFL1 223 41 36 5

HFL1-D 223 46 41 5

HFL1-N 223 36 19 17

GR-65 223 26 10 16

HFL2 223 21 18 3

Note: * means significant difference at p<0.05.

Table S2. Differential metabolites and their fold change in mature seeds among

multiple comparisons within HFL2 and its derived samples.

Biochemical Name

Fold of Change (Ratios of Group Means from Scaled

Imputed Data)

GR-65 /

WT

35S-15 /

WT

HFL2 /

WT

HFL2/

GR-65

HFL2/

35S-15

2-Aminoadipate 1.28 1.88 3.81 2.97 2.03

Lysine 6.51 1.62 130.74 20.09 80.69

Serotonin 2.03 1.51 4.11 2.02 2.72

Tryptophan 0.92 1.14 1.12 1.22 0.99

N6-acetyllysine 1.99 1 9.68 4.87 9.68

Glutamine 1.3 1.06 1.92 1.47 1.82

Arginine 0.9 1.05 2.42 2.7 2.3

Pseudouridine 1.07 0.98 2.05 1.92 2.08

Phenylalanine 0.98 0.88 1.09 1.11 1.24

N-6-trimethyllysine 1.51 1.19 1.96 1.3 1.64

Pipecolate 3.94 1.84 13.2 3.35 7.16

Saccharopine 16.76 1.41 60.18 3.59 42.56

N-delta-acetylornithine 6.75 1.75 26.76 3.96 15.29

gamma-Aminobutyrate 0.81 0.55 1.13 1.39 2.06

Putrescine 2.05 1.93 6.09 2.97 3.16

Ribose 0.86 0.84 0.86 1 1.02

Xylonate 1.14 0.73 1.73 1.51 2.36

Uracil 1.4 0.78 1.71 1.22 2.18

Squalene 1.09 1.08 1.68 1.54 1.56

gamma-Glutamylglutamine 1 1 1.75 1.75 1.75

Citramalate 1.19 3.19 1.9 1.6 0.6

Adenine 0.9 0.71 0.97 1.08 1.36

N-acetylglucosamine 0.5 0.63 0.26 0.52 0.42

Glucosaminate 0.54 0.76 0.7 1.28 0.91

Pantothenate 0.85 0.93 0.81 0.95 0.88

Note: Dark grey shaded cells indicate that the mean values were significantly higher or lower for

that comparison, respectively, as the level of p ≤ 0.05; while light gray shaded cells indicate that at

the level of 0.05 < p < 0.10.

Table S3. Statistical analysis of the number of differentially expressed genes in developing seeds.

Developing seed Comparison p-value threshold Number of differentially

expression genes

Number of

up-regulated genes

Number of

down-regulated genes

10 DAF

GR-14 vs WT 0.05 1203 577 626

35S-15 vs WT 0.05 954 494 460

HFL1 vs WT 0.05 1289 745 544

HFL1 vs GR-14 0.05 1540 734 806

HFL1 vs 35S-15 0.05 745 371 374

15 DAF HFL1 vs WT 0.05 41 30 11

Table S4. The metabolites associated with stress response in mature seeds of HFL1

transgenic rice compared with the wild-type (p < 0.05).

Pathway Biochemical Name KEGG Fold-chan

ge p-value q-value

TCA

Citrate C00158 1.68 0.0269 0.084

Fumarate C00122 1.90 0.0311 0.01325

Malate C00149 2.52 0.002 0.0197

Succinate C00042 1.51 0.01245 0.02052

Glutamate

metabolism

2-Aminobutyrate C02261 1.92 0.0127 0.0593

2-Pyrrolidinone

1.19 0.0757 0.1432

4-Hydroxybutyrate (GHB) C00989 2.36 0.0655 0.1363

arginine C0006 3.70 0.0104 0.054

gamma-Aminobutyrate

(GABA) C00334 3.05 0.0344 0.0949

Glutamine C00064 3.69 0.0151 0.0619

Histidine C00135 4.00 0.001 0.0187

N-acetylglutamate C00624 1.40 0.029 0.0845

N-acetylputrescine C02714 2.51 0.02153 0.02606

Proline C00148 1.63 0.0299 0.0845

Pyroglutamine

1.60 0.0014 0.0187

Stachydrine C10172 2.38 0.0269 0.084

trans-4-Hydroxyproline C00157 1.71 0.0837 0.1551

N-delta-Acetylornithine

27.92 0.0005 0.0141

Polyamines

Agmatine C00179 2.19 0.2061 0.2565

Putrescine C00134 7.36 0.0438 0.1154

Spermidine C00315 2.47 0.3721 0.3293

Glutathione

metabolism 5-Oxoproline C01879 2.02 0.0295 0.0845

Secondary

metabolites

4-Hydroxycinnamate C00811 1.50 0.0761 0.1432

Ferulate C01494 1.41 0.0938 0.1711

Serotonin C00780 14.04 0.0015 0.0187

Amino sugar

and

nucleotide

sugar

Arabinose C00216 1.64 0.0049 0.0366

Arabonate C00878 2.25 0.0226 0.0812

Xylonate C05411 2.00 0.0606 0.1325

Table S5. List of primers used for vector construct and qRT-PCR analyses.

Gene Name Forward Primer(5'→3') Reverse Primer(5'→3') Notes

TDC3 GGATCCACCACCATCTCCACTCCAT GGTACCTATTATTCCACGCACCTCT

TDC1rt GCGAGGGTGAAACCTTCCA GCGAGCCGGTGGAGTCC Byeon et al., 2014

TDC2rt GTGCTGCCTTTAACATTGTTGG CATGTCATTGGACTTTGCTATCTGT Byeon et al., 2014

TDC3rt GACGTCGAGCCCTTCCGC ACCGTCAGCCGCGTGATG Byeon et al., 2014

T5Hq GCGTCCAGAAATCCCCCG GCTCCGTCATCACCCACTCC

TSAα1 CATTCATTCCATTCATCAC GGACAAGACAACTCAGGTAT

TSAα2 CACCAACAGAAAGAATGGAGAAAAT CGAACCCAACAGCCACAGC

TSAβ ATCAACAATGCTGTCGCTCAAGT TGCCCTCACCTCTGCTCCA

GDCi CTCCAAGGGTTCCAGCCAGGTCA CGATGTTGAACCGCCCCGTCTT

GDC CTCCAGAACCGTTGTGTGA TTCCGTTTGTTTTGCCAC

CRO AAAATGGTATTGGCTTGTTC CCTGACTCTGGCTCGTTC

ACX TGGTCCACCTAAGAAGCC CACATCAGCCATAACATCC

3DS CTGTTGGAGGGAAGACCG TCTCGTCCTGAGCAACCAC

CS1 ATTGGGAAGAAACAACATACTG ACTCCACCATCGGCACA

ASI CGAACATACGCCAATCCA CCAGCAAGTGGACGGTTA

ASα1 CATCTTCTCCGACCACCTCACG TCCATAATGCTCCTTGGTATCTTCA

ASβ1 TAGTGGACCCGCAAGAAC CCCAAACAAAGGTGTAGAAG

ASβ2 TTCTGGAACTTGGACCTACC CTCCCTGGATGTGCCTGT