10 20 30 40 50 60 70 80 90 100 110 120 ACCAAGTTGCCGATGTGCTTATCTGGTCCTGGTGTTTTCTGGGGTAAGAGCTAGCAACCGAATCTTGTATTTCCCACACAGATCCACGGCAACTGTCAGCAACATTGCTGCGAGTACTTG 130 140 150 160 170 180 190 200 210 220 230 240 ATACTGTATCAGTGTATTGCATCTATTGTTGGTGATCACTGTGTAGATCGACAAGGGTCCAAACCGACCACGGCGATAAGCCATGCGTCGTCTGGGACTTTCCCTTCACCACGACACGAG 250 260 270 280 290 300 310 320 330 340 350 360 TCCAAGCTTCGCTGCCCTTGCTTGCAACCAGCGGCCGCCACCGAATGGCACCGTTCACGCGTGCTCCAAAAGCCGCCCACCTCAGTTGGAGCCGGGAAAAGTTGGCAAAAAGCCAATAAA

370 380 390 400 410 420 430 440 450 460 470 480 AAAGACGATGCGCATTGCCCGGATTCCCACTTCGATCCCCCCTCACCTTCGTCTTCCTCTCGACCTCTCCGCTGTCTCTCCGATGGGCTCAGCTTCAGAGCATGATGTCAGGGAGCACCT M R I A R I P T S I P P H L R L P L D L S A V S P M G S A S E H D V R E H L

490 500 510 520 530 540 550 560 570 580 590 600 GCTGGACGTGGACGGCGTCGGGGAAGAAGGGGCGGCGGCGGCGGCGGGGCCGAAGATACGGGTGCGGGGGCTGACGCGGCGGTCGGAGGCGAGAGGCGAGGAGATCCTGCGGGGCGTCGA L D V D G V G E E G A A A A A G P K I R V R G L T R R S E A R G E E I L R G V D

610 620 630 640 650 660 670 680 690 700 710 720 CCTGGACGTGCCGCGCGGGGTGGTCGTCGGCGTCATCGGCCCCAGCGGCAGCGGCAAGTCCACGCTGCTCCGCGCGCTCAACCGCCTCTGGGAGCCCGCGCCGGGCGCCGTGCTCCTCGA L D V P R G V V V G V I G P S G S G K S T L L R A L N R L W E P A P G A V L L D Walker‐A 730 740 750 760 770 780 790 800 810 820 830 840 CGGCGTCGACATctgcggcatcgacgtCCTCGCGCTCCGCCGGAAGGTCGGCATGCTCTTCCAGCTCCCCGCCATGTTCGACGGAACCGTGGCTGACAACGTGCGATACGGGCCACAGCT G V D I C G I D V L A L R R K V G M L F Q L P A M F D G T V A D N V R Y G P Q L als1/star1 muta0on  (dele0on)                                                       Q‐loop  850 860 870 880 890 900 910 920 930 940 950 960 GCAGGGCAAGAAGCTGACGGACGCCGAGGTGCAGAGCCTACTGAGCCTCGCCGACCTGGACCCTGCTCTCTGCTCCAAGCCCGCCTCCGAGCTCTCCGTCGGCCAGGCGCAGCGCGTCGC Q G K K L T D A E V Q S L L S L A D L D P A L C S K P A S E L S V G Q A Q R V A  ABC signature 970 980 990 1000 1010 1020 1030 1040 1050 1060 1070 1080 ATTGGCTCGCACCCTCGCCAACGATCCGGAGGTGCTTCTGCTGGATGAGCCGACGAGCGCGTTGGATCCCATCTCGACGCAGAACATCGAGGAGGCGATCGTGCGGCTGAAGAAGACGAG L A R T L A N D P E V L L L D E P T S A L D P I S T Q N I E E A I V R L K K T R Walker‐B                                        D‐loop 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 GGGGCTCACCACGGTGATGGTGTCGCACAGCGTGAAGCAGATCCAGCGGATCGCCGACCTGGTCTGCCTCCTCGTCGCCGGCGAGGTCGTCGAGGTGCTCCCGCCGTCGGAGCTGTCCGA G L T T V M V S H S V K Q I Q R I A D L V C L L V A G E V V E V L P P S E L S E H‐loop 1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 GGCCAAGCACCCCATGGCACGGCGCTTCCTCGAGCTCAGCTGAAGACTGGCACGACGATCTCCTCCTCCTCTGTATGACGCTTCTTCCGTTTGTGTGATTCGGATGTGTACCTGTAATCT A K H P M A R R F L E L S *

1330 1340 1350 1360 1370 1380 1390 1400 1410 1420 1430 1440 TGAGAAGTTGAGATTATGCCTGTATACTATGTCGGAAATTTTGTGGTGAGATGATCTCAACCCTACTGGCCATAAAATGCATTTTGGTAAATATTCTTTTATCTAAACCCTAGTGGCCTC 1450 1460 1470 1480 1490 1500 1510 1520 1530 CGAGGCTCCGACATTGTATTTTTGCCTGTAATATTGGTACACTACTAAAATGAAATTTGGTACTCCCTCCGTCCCAGATTATAAGGGATTTT

Supplemental Figure 1. STAR1 sequence, conserved motifs and star1 mutation. Yellow letters show star1 mutation position and red ones the motif of ABC transporters.

STAR‐1GFP and 

TMRM stain (mitochondria) 

STAR1‐GFP and 

DsRed‐PTS1 (peroxisome) 

Supplemental Figure 2. STAR1-GFP localization in onion epidermal cells. STAR1-GFP fusion gene was transiently introduced into onion epidermal cells by particle bombardment. Stained with mitochondria specific fluorescence dye, tetramethyl rhodamine methyl ester (TMRM), or co-expression with peroxisome targeted red fluorescence protein, DsRed-PTS1. Bar: 100 µm.

TM1 

TM2  TM3 

TM4  TM5 

TM6 

TM7 Under line: transmembrane domain predicted by TmHMM

STAR2  ALS3 

STAR2  ALS3 

STAR2  ALS3 

STAR2  ALS3 

STAR2  ALS3 

Supplemental Figure 3. Alignment of STAR2 and ALS3. The predicted transmembrane domains are underlined.

A B

Supplemental Figure 4. Expression of STAR1 (A) and STAR2 (B) in different root segments. Seedlings were exposed to a solution containing 50 µM Al for six hours. The root segments were excised at 0-10 mm and 10-20 mm for RNA extraction. The expression levels of STAR1, STAR2 and Actin as an internal control were determined by quantitative RT-PCR. STAR1 and STAR2 expression levels in 10-20 mm without Al were designated as onefold, respectively. Values are means±SD (n=3).

EYFP-C-STAR1 +

EYFP-N-STAR2

EYFP-C-STAR1 +

EYFP-N

EYFP-C +

EYFP-N-STAR2

EYFP-C +

EYFP-N

Supplemental Figure 5. BiFC analysis of interaction between STAR1 and STAR2. Pair of plasmid constructs indicated beneath the figure transiently introduced into protoplasts derived from rice callus by PEG method.

Fluorescence

Transmission

Merged

A B C D

A B

Supplemental Figure 6. Aluminum content in the root segment (A) and cell wall (B). The roots of both wild-type and star1 mutant were exposed to a 0.5 mM CaCl2 solution containing 20 mM Al. After six hours, the roots were excised at 0-1 and 1-2 cm. The cell wall of different root segments were isolated. Data are means±s.d. (n=3).

Supplemental Figure 7. Morin staining of the wild-type (left) and star1 (right) root. Five-day seedlings were exposed to 30 µM Al for 9 h. Root cross sections at 2 mm from tip are shown. Bar = 100 µm.

FeatureNum ProbeUID accessions Description PValueLogRatio

gProcessedSignal

rProcessedSignal average SD average SD

star1 (+Al/-Al) WT (+Al/-Al) 4814 4478 Os09g0518200|AK121725|NA|AK121725 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 89.2 3424.3 33.92 6.06 16.83 1.78 4814 4478 Os09g0518200|AK121725|NA|AK121725 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 89.2 3424.3 33.92 6.06 16.83 1.78

22097 21008 Os01g0734800|AK102415|NA|AK102415 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 49.6 3350.3 61.33 6.12 13.38 0.35 31035 29410 Os01g0597800|AK063325|NA|AK063325 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 24.0 6298.5 304.99 78.83 13.04 2.33 26329 25007 Os04g0206600|AK106302|NA|AK106302 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 86.6 35283.7 334.24 95.23 11.32 6.91

1032 839 Os01g0734800|AK061830|NA|AK061830 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 314.2 7951.5 22.25 3.00 10.31 1.05 29834 28438 Os04g0206500|AK110892|NA|AK110892 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 103.6 12323.3 102.01 22.28 8.76 6.68 27703 26327 Os01g0597800|AK102671|NA|AK102671 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 2584.8 54450.2 18.48 4.80 7.67 1.33 27915 26568 Os02g0206700|CI507529|NA|CI507529 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 81.4 2.9 26.22 2.89 6.77 3.50

5051 4609 Os01g0179600|CI041005|NA|CI041005 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 36.3 12.9 6.82 2.85 5.97 2.11 9864 9300 Os02g0755900|AK104985|NA|AK104985 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 662.9 15865.7 23.35 1.40 5.91 0.28 8823 8276 Os02g0755900|AK073587|NA|AK073587 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 618.7 14921.4 22.87 1.25 5.72 0.25

20256 19270 Os11g0444000|AK105931|NA|AK105931 UDP-glucosyltransferase BX8. 0.0 15.9 33.8 2.39 0.21 4.68 2.73 11981 11411 Os09g0518200|AK064395|NA|AK064395 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 3.6 202.0 43.91 9.66 4.31 2.05 12823 12212 Os05g0527000|CI004090|NA|CI004090 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 30.3 5562.3 178.23 44.15 4.16 0.87 15041 14346 Os05g0526900|CI278387|NA|CI278387 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 15.8 154.5 9.75 2.12 4.11 1.13 35025 33237 Os11g0444000|AK099588|NA|AK099588 UDP-glucosyltransferase BX8. 0.0 20.8 41.1 2.68 0.81 3.79 0.51

39923 37824 Os01g0598000|Os01g0598000|NA|Os01g0598000 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 58.1 2474.7 43.00 8.69 3.17 0.74

6578 6077 Os01g0736100|AK099365|NA|AK099365 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 300.3 106.8 2.62 0.16 2.90 0.35 28181 26753 Os01g0736100|AK103355|NA|AK103355 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 337.4 131.1 2.70 0.24 2.84 0.27 10406 9852 Os07g0674100|AK099614|NA|AK099614 UDP-glucuronic acid decarboxylase. 0.0 41159.8 103316.8 2.40 0.11 2.80 0.06 26745 25477 Os06g0220500|AK063324|NA|AK063324 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 281.0 507.7 1.48 0.30 2.58 0.23 41825 39644 Os06g0289200|AK106250|NA|AK106250 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 139.4 6074.9 38.23 9.28 2.39 1.36

9642 9034 Os07g0510500|AK068586|NA|AK068586 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 1217.3 65.2 19.35 3.73 2.30 0.49

8259 7671 Os03g0358800|Os03g0358800|NA|Os03g0358800 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.8 13.5 14.9 1.25 0.09 2.15 1.50

43102 40747 Os07g0241700|Os07g0241700|NA|Os07g0241700 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 143.0 1287.7 8.87 1.12 2.14 0.10

21145 20161 Os01g0736100|AK064298|NA|AK064298 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 52.9 29.3 2.74 0.62 2.13 0.17 4985 4645 Os02g0755600|AK064105|NA|AK064105 UDP-glucuronosyltransferase. 0.3 4.4 9.6 3.29 2.34 2.11 0.80

13966 13267 Os05g0552700|AK108674|NA|AK108674 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 70.6 39.2 1.83 0.70 2.09 0.29 37406 35487 Os07g0674100|AB183706|NA|AB183706 UDP-glucuronic acid decarboxylase. 0.0 6955.7 13626.6 1.79 0.17 2.08 0.14

785 563 Os06g0289900|CI000741|NA|CI000741 UDP-glucuronosyl/UDP-glucosyltransferase family protein. 0.0 334.5 99.3 3.82 0.39 2.05 0.28

Supplemental Figure 8. Microarray analysis of UDP-glucosyltransferase genes. Wild-type rice (WT; cv. Koshihikari) and star1 mutant were grown in a 0.5 mM CaCl2 for 4 days. Root tip (0-1 cm) from 20-30 plants were sampled after the roots were exposed to a solution containing 0 or 20 µM AlCl3 for 6 h. Microarray was performed according to Agilent Oligo DNA Microarray Hybridization protocols using the Rice Oligo DNA Microarray 44K RAP-DB (G2519F#15241, Agilent Technologies) with three biological replicates. The microarrays were scanned using a DNA micro-array scanner (Agilent Technologies). Signal intensities were detected by Feature Extraction software (Agilent Technologies). Genes annotated with UDP-glucuronosyl/UDP-glucosyltransferase were extracted .

Supplemental Figure 9. RT-PCR of UDP-glucosyltransferase genes. Expression response to Al in the wild-type rice and mutant star1 were detected by RT-PCR. The seedlings were exposed to a 0.5 mM CaCl2 solution containing 0 or 20 µM Al for six hours.

‐      +      ‐      +  Al WT  star1 

      Histone H3

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rha Fuc Ara Xyl Man Gal GlcR

ela

tive

am

ou

nt

of

ne

utr

al su

ga

r

WT -Al

als1 -Al

WT +Al

als1 +Al

WT ‐Al 

als1 ‐Al 

WT +Al 

als1 +Al 

Supplemental Figure 10. Determination of root soluble sugar components. Gel filtration (GF) and gas chromatography (GC) of 80% EtOH soluble root extracts. (A) GF using Superdex Peptide column (ø10-300 mm) and 50 mM ammonium formate buffer (pH 5.3). Arrow show retention time of glucose (control). (B) GC of alditol acetate derivatives of the root extracts. These results suggest that root soluble fractions mainly contain monomeric glucose.

B A

-600

-500

-400

-300

-200

-100

0

100

-110 -100 -90 -80 -70 -60 -50 -40 -30 -20

Cur

rent

(nA

)

Potential (mV)

STAR1+STAR2

STAR1

STAR2

H2O

Supplemental Figure 11. Oocyte two-electrode voltage clamp analysis. Voltage dependent current in UDP-Glc preloaded oocytes expressing both STAR1 and STAR2, STAR1 or STAR2 alone or H2O as control were measured. Means ± SD (n = 12) are shown.

H2O

-1800

-1600

-1400

-1200

-1000

-800

-600

-400

-200

0

200

-110 -100 -90 -80 -70 -60 -50 -40 -30 -20

Cur

rent

(nA

)

Potential (mV)

STAR2 : UDP-Glc

STAR2 : UDP-GlcA

STAR2 : H2O

Supplemental Figure 12. Oocyte two-electrode voltage clamp analysis. Substrate selectivity of STAR2 alone was analyzed using oocyte two-electrode voltage clamp. I/V relationships for STAR2 expressing oocytes were recorded in oocytes preloaded with H2O, UDP-Glc or UDP-GlcA. Means ± SD (n = 12) are shown.

-300

-250

-200

-150

-100

-50

0

50

100

-110 -100 -90 -80 -70 -60 -50 -40 -30 -20

Cur

rent

(nA

)

Potential (mV)

STAR1+STAR2

STAR1+STAR2 with UDP-Glc

Supplemental Figure 13. Oocyte two-electrode voltage clamp analysis. Effect of external UDP-Glc on the voltage dependent current in UDP-Glc preloaded oocytes expressing both STAR1 and STAR2. Means ± SD (n = 12) are shown.

without external UDP‐Glc

with external UDP‐Glc