Supplementary Table 1 Proportion of total amino acids in mixed seeds of five T3 transgenic lines or nontransgenic (WT)control plants before and after defatting treatment

Amino acid / Original (%) / Defatted (%)
Transgenic / WT / Transgenic / WT
Gly / 4.4 / 4.4 / 4.4 / 4.5
Ala / 4.4 / 4.4 / 4.4 / 4.4
Val / 4.6 / 4.7 / 4.6 / 4.7
Leu / 7.6 / 7.8 / 7.7 / 7.8
Ile / 4.5 / 4.5 / 4.5 / 4.6
Pro / 5.0 / 5.2 / 5.0 / 5.1
Ser / 5.2 / 5.3 / 5.3 / 5.5
Thr / 4.2 / 4.2 / 4.3 / 4.3
Cys / 1.9 / 1.8 / 2.0 / 1.8
Met / 1.6 / 1.5 / 1.6 / 1.5
Asx / 11.8 / 11.9 / 11.7 / 11.8
Glx / 17.4 / 17.9 / 17.5 / 18.0
Arg / 6.6 / 6.8 / 6.6 / 6.8
Lys / 6.5 / 6.5 / 6.6 / 6.5
His / 2.9 / 2.9 / 2.9 / 2.8
Phe / 5.0 / 5.1 / 4.9 / 5.1
Tyr / 3.5 / 3.5 / 3.3 / 3.3
Trp / 2.9 / 1.4 / 2.8 / 1.5
Total / 100 / 100 / 100 / 100
Total (mg g–1 DW) / 329.0 / 351.2 / 435.4 / 459.9

Trp content is shown in bold.

Supplementary Table 2 Composition (percent by weight) of the three diets for the fish feeding trial

Ingredient / I
(Transgenic) / II
(WT+free Trp) / III
(WT)
Amino acids
Amino acid additive1 / 15.17 / 14.85 / 14.63
Soybean origin / 17.77 / 18.84 / 18.84
Carbohydrate
Dextrin / 20.00 / 20.00 / 20.00
Starch / 10.00 / 10.00 / 10.00
Cellulose / 3.83 / 4.13 / 4.36
Soybean origin / 2.44 / 2.26 / 2.26
Lipid
Fish oil / 5.00 / 5.00 / 5.00
Soybean origin / 0.25 / 0.25 / 0.25
Minerals and vitamins
Mineral mix2 / 5.00 / 5.00 / 5.00
Vitamin mix3 / 1.00 / 1.00 / 1.00
Soybean origin / 3.33 / 2.80 / 2.80
Other
Soybean origin / 16.21 / 15.87 / 15.87

1Amounts of each amino acid are shown in Supplementary Table 3.

2Amounts of each mineral component conform to those described by Ogino et al. (1979).

3Amounts of each vitamin and vitamin-like additive are shown in Supplementary Table 4.

Supplementary Table 3 Amounts (grams per 100 g of diet) of amino acid additives in the three diets for the feeding trial

Amino acid / I
(Transgenic) / II
(WT+free Trp) / III
(WT)
Arg
Additive / 2.07 / 1.98 / 1.98
Soybean origin / 1.15 / 1.24 / 1.24
Total / 3.22 / 3.22 / 3.22
Cys
Additive / 0.81 / 0.82 / 0.82
Soybean origin / 0.34 / 0.33 / 0.33
Total / 1.15 / 1.15 / 1.15
His
Additive / 0.94 / 0.92 / 0.92
Soybean origin / 0.50 / 0.52 / 0.52
Total / 1.44 / 1.44 / 1.44
Ile
Additive / 1.11 / 1.05 / 1.05
Soybean origin / 0.78 / 0.84 / 0.84
Total / 1.89 / 1.89 / 1.89
Leu
Additive / 1.67 / 1.56 / 1.56
Soybean origin / 1.33 / 1.44 / 1.44
Total / 3.00 / 3.00 / 3.00
Met
Additive / 0.68 / 0.68 / 0.68
Soybean origin / 0.28 / 0.28 / 0.28
Total / 0.96 / 0.96 / 0.96
Phe
Additive / 0.85 / 0.94 / 0.94
Soybean origin / 1.50 / 1.41 / 1.41
Total / 2.35 / 2.35 / 2.35
Thr
Additive / 0.92 / 0.88 / 0.88
Soybean origin / 0.75 / 0.79 / 0.79
Total / 1.67 / 1.67 / 1.67
Trp
Additive / 0.00 / 0.22 / 0.00
Soybean origin / 0.49 / 0.27 / 0.27
Total / 0.49 / 0.49 / 0.27
Tyr
Additive / 0.96 / 0.93 / 0.93
Soybean origin / 0.58 / 0.61 / 0.61
Total / 1.54 / 1.54 / 1.54
Val
Additive / 1.76 / 1.70 / 1.70
Soybean origin / 0.80 / 0.86 / 0.86
Total / 2.56 / 2.56 / 2.56
Total amino acids / 24.16 / 24.16 / 23.93

Supplementary Table4 Amounts of each vitamin and vitamin-like additive in the three diets for the feeding trial

Additive / Amount (/kg diet)
Vitamin A / 3750 IU
Vitamin D3 / 3600 IU
Vitamin K / 10 mg
Thiamin (vitamin B1) / 1.5 mg
Riboflavin (vitamin B2) / 6 mg
Pyridoxine (vitamin B6) / 4.5 mg
Pantothenic acid (vitamin B5) / 0.0015 mg
Vitamin B12 / 10 mg
Niacin (vitamin B3) / 15 mg
Biotin / 0.225 mg
Folic acid / 1.5 mg
Inositol / 450 mg
Choline / 1500 mg
Ascorbic acid / 150 mg
Vitamin E / 100 mg

IU: international unit

Supplementary Table 5 Transformation of soybean embryogenic tissue with a gene for a feedback-resistant α subunit of AS (OASA1D)

Transformation vector / No. of plates / Transgenic plants
Somatic embryos / Regenerated plants / Mature plants / Independentlines1 / Lines withincreased Trp2 / Fixed lines3
pgy2OASA1D / 48 / 21 / 228 / 39 / 13 / 10 / 5
p35SOASA1D / 56 / 5 / 43 / 5 / 3 / 3 / 2

1Independent transformation events were confirmed by Southern blot analysis (Fig. 2).

2Transgenic linesthat produced seeds with a free Trp content more than three times that of nontransgenic (wild-type) seeds.

3Transgenic lines showing no segregation for OASA1D.

Supplementary Table 6 AS activity and Trp content of seeds from seven different T2 transgenic lines

Line / Promoter / AS activity1 / Trp content (mg/g DW)2
L-Trp(–) / L-Trp(+) / Inhibition by L-Trp (%) / Free / Protein / Total / Relative total Trp
G13 / Pgy2 / 1.28a / 1.15a / 9.7 / 5.79b / 4.68ab / 10.47b / 2.26
G14 / Pgy2 / 1.00b / 1.00b / 0.0 / 4.20de / 3.95b / 8.14c / 1.75
G19 / Pgy2 / 0.23d / 0.22d / 5.1 / 3.44e / 4.50ab / 7.94c / 1.71
G51 / Pgy2 / 1.26a / 1.22a / 3.3 / 5.20bc / 5.27a / 10.47b / 2.26
G64 / Pgy2 / 0.40c / 0.35c / 11.8 / 8.14a / 4.39ab / 12.54a / 2.70
C25A / P35S / 0.34c / 0.31c / 9.4 / 4.52cd / 3.61b / 8.14c / 1.75
C25B / P35S / 0.33cd / 0.24d / 25.6 / 4.79cd / 4.25ab / 9.04bc / 1.95
Control (wild type) / 0.25d / 0.14d / 43.8 / 0.16f / 4.47ab / 4.64d / 1

All data are means of three independent determinations. Means followed by the same superscript letters do not differ significantly at the 5% level from each otherwithin a column (Tukey-Kramer multiple comparison test).

1AS activity was measured in the absence (–) or presence (+) of 50 µM L-Trp and is represented by pkat (pmol s–1 mg–1 protein).

2Trp content is expressed as milligrams per gram of dry weight (DW).

Supplementary Table 7 LC-ESI-MS peaks whose intensity correlated with that of the Trp peak in seeds of a T3 transgenic line

Peak number / Mass/charge ratio
(m/z) / Retention time
(min) / Assignment1 / Average peak intensity (cps)2 / Correlation coefficient3 / Loading4 for
principal component 1
Wild type / Transgenic
#165 / 323 / 4.97 / 203,831 / 0.87 / 0.02
#235 / 381 / 5.63 / 1,334,604 / 0.90 / 0.13
#321 / 217 / 6.33 / TCCA / 668,816 / 2,376,824 / 0.88 / 0.17
#340 / 565 / 6.40 / 165,354 / 5,345,171 / 0.91 / 0.04
#344 / 433 / 6.56 / 259,234 / 0.79 / 0.20
#378 / 475 / 6.70 / 239,587 / 0.89 / 0.02
#390 / 334 / 6.77 / γ-Glutamyltryptophan / 474,189 / 6,364,486 / 0.88 / 0.60
#501 / 347 / 7.40 / 1,628 / 1,058,822 / 0.88 / 0.01
#529 / 313 / 7.57 / 160,236 / 0.86 / 0.34
#592 / 561 / 7.90 / 135,306 / 0.91 / 0.01
#194 / 205 / 5.20 / Trp / 3,274,201 / 9,796,184

1Assignment was performed by comparison of MS-MS spectra with those of standard compounds.

2Peak intensities (counts per second, or cps) not shown were below the limit of detection.

3Correlation coefficient for comparison of peak intensity with that of Trp.

4Loading factor in PCA analysis (Fig. 5).