モデル植物のSTOP1転写因子を用いたアルミニウム及び低pHストレスの機能生物学的解析

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Title

モデル植物のSTOP1転写因子を用いたアルミニウム及び低

_{pHストレスの機能生物学的解析( 本文(Fulltext) )}

Author(s)

大山, 慶直

Report No.(Doctoral

Degree)

博士(農学) 甲第628号

Issue Date

2014-03-13

Type

博士論文

Version

ETD

URL

http://hdl.handle.net/20.500.12099/49108

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

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STOP1

pH

2013

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STOP1

pH

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2

3

1 Al

pH

9

2 STOP1

44

3

60

4

67

71

(5)

3 2007

GMO

30% 4 ha

(Baligar and Ahlrichs, 1998)

( ,

(6)

4 (Al) Al pH Al pH pH 4.5 Al3+ pH5.5 Al Al pH Al3+ 50 Al Al Al Al ( 2003) Al Al (Yamamoto et al,.2010) Al Al Al

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5 Al Al Al Al Al Al pH Al pH 5.5 Al Al 20 % pH Al Al Ca Al (Internal Tolerance) Al

(Exclusion Mechanism) (Taylor,

1991) ( )

Al

ABC ALS3 (Aluminum Sensitive 3) ( ) (Larsen et al., 1997, 2005)

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6

(Huang et al., 2009) STAR2 AtALS3

Al

( ) Al (Pellet et al.,

1995)

( Columbia-0) (AtALMT1;

Al-activated malate tranporter 1) Al

(Hoekenga et al., 2006) ALMT1

Al (distal part of

the transition zone: DTZ) Al

(Kobayashi et al., 2007) Al ( ) Al (Delhaize et al., 1993) ALMT ( ) Al pH Al (Kinraide TB, 1998,2003 Koyama et al., 2001) STOP1

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7

pH Al

AtALMT1 ( ) STOP1 (Sensitive TO

Proton rhizotoxicity 1)

(Iuchi et al., 2007) STOP1

pH Al

STOP1 Al

(Yamaji et al., 2009) AtSTOP1 Al

pH STOP1 ( ) pH

( cv. xanthi)

( )

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8

NtSTOP1 ( )

-RNAi(RNA interference; RNA )

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9

1 Al

pH

1-1

ORF (Open reading flame; ) 798

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10

(Figure 1) Al

STOP1 pH Al

STOP1

( ) (Figure 2) ALMT1

(Kobayashi et al., 2007) STOP1

(Kobayashi et al., 2013) STOP1

Al pH (Iuchi et al., 2007) C2H2 ( Cysteine histidine ) STOP1 C4 (Vallee et al., 1991) (Krishna et al., 2003) C2H2 STOP1

(Englbrecht et al., 2004) STOP2

CaMV35S ( 35S

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11

(Kobayashi et al., 2013) CaMV35S

ALMT1

Al (Kobayashi et

al., 2013) STOP2 STOP1

STOP1

ART1 (Al resistance transcription factor 1 : Os12g0170400)

(Yamaji et al., 2009)

STOP1 ART1

AACT1(Al-activated citrate transporter 1 : Os10g0206800) Al AACT1 AACT1 STAR1 2 TaSTOP1 (Garcia-Oliveira et al., 2013) STOP1 STOP1 Al

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12

degenarate-PCR RACE (rapid amplification of cDNA ends)

RNAi

(NtSTOP1-KD) NtSTOP1

Al NtSTOP1

Al

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13

2-2

total-RNA 1/50 MGRL (Fujiwara et al., 1992) ( ) (NaH2PO4 Na2HPO4 ) (Table. 1) MGRL (Ca(NO3)2) (CaCl2) (NaNO3) 4 4 / 12 h / 12 h 22 pH5.5 2 10 80 total-RNA

RNA (Suzuki et al., 2004)

100 mM Tris-HCl 10 mM EDTA 5 %

2-2 % LDS 0.6 M NaCl 0.4 M 100 mM

Tris-HCl 25 mM EDTA 1.4 M NaCl 2 %(w/v) CTAB ( cetyl trimethyl ammonium bromide)

600 µl 5

15000 rpm 5

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14 rpm 4 10 3 (35 % GTC 0.2 M pH4.0 ) / (24 : 1) 14,000 rpm 4 10 2 66 % GTC (guanidine thiocyanate)

RNA RNase RNase

0.6 15 16,000 rpm 20 75 % 150 µl 16,000 rpm 20 DNase RNase-free 42.5 µl RNase-free 250 2

DEPC Diethylpyrocarbonate RNase

DNase

RNA DNA

DNase RNase-free

42.5 µl RNA ×10 DNase 5 µl RNase (20 U) 0.5

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15 µl 150 µl / (24 : 1) 150 µl 13,000 rpm 5 3 M (NaOAc) 30 µl 300 µl 10 16,000 rpm 4 20 RNA 75 % 150 µl 16,000 rpm 20 20 µl RNase free 80 RNA RNA DNase total-RNA 150 µl 8 M 50 µl 16,000 rpm 30 75 % 150 µl 16,000 rpm 5 RNase-free 50 µl 3 M NaOAc 30 µl 300 µl 10 16,000 rpm 20 RNA 75 % 150 µl 16,000 rpm 20 15 µl RNase-free

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16 DNase RNA NanoDrop 1000 (LMS ) RNA 1 µg 100 ng / µl 260 nm (A260) 280 nm (A280) A260 /A280 1.9 A260 230 nm (A230) A260/A230 2.2 RNA 1 µg 10 µM 1 µl RNase-free 17.5 µl 70 10 RNA 2 10 mM dNTP 1 µl ×5 RTbuffer 5 µl RNase-inhibitor 0.5 µl RTase 1 µl (Table. 2) 42 60 70 15 cDNA 4 U RNaseH 37 20 30

3’RACE oligo dT AP 5’RACE RNase-free Rv1

(GSP) PCR

oligo dT 5’RACE

RTase RNase-free

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17

C (dC-tailing)

5’ RACE

Fw

Fw PCR

RNaseH cDNA WizardSV Gel and PCR

Clean-Up System (promega ) 50 µl nucleotide-free

cDNA 10 µl ×5 TdT buffer 5 µl 0.1 % BSA (bovine serum

albumin) 2.5 µl 2 mM dCTP 2.5 µl 24 µl 94 2 2 1 µl TdT ( ) 37 10 65 10 30 PCR 20 3 4 3 64 (degeneration) STOP

degenerate- (Table 3) degenarate-PCR

degenerate-Table 3. degenarate-PCR RACE 1stPCR

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18 PCR 1stPCR 100 2ndPCR 3 PCR ( ) Tm PCR (Figure 3) 1stPCR 10 µl 2ndPCR 50 µl 10 µl ×10 Ex taq buffer 1 µl 2 mM dNTP 0.8 µl 5 µM Fw Rv 1 µl DNA 1 µl 5.15 µl

Ex taq Takara 0.05 µl PCR (Table. 4)

5’RACE 1st PCR oligo dG AAP 2ndPCR AUAP

3’RACE 2 oligodT PCR DNA PCR DNA DNA 100 µl 3 M NaOAc 10 µl 100 % 200 µl 15 16,000 rpm 4 20 70 % 150 µl 16,000 rpm 4 20 10 µl DNA

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19

( )

1.5 ml DNA2 µl 1 µl DNA Ligation

Kit ver2.1 (Takara ) solution 3 µl 16 6

PCR Ex taq TdT PCR 3’ pT7Blue-Tvector (Takara : Figure 4) 1.5 ml 50 µl (E.coli JM109 ) 30 42 45 2 SOC 250 µl 1 37 X-Gal

(5-bromo-4-chloro-3-indolyl- -D-galactoside: ) IPTG

(isopropylthio- -D-galactoside: ) 100 ng/µl

(ampicillin ; Amp: ) LB

37

LB 10 µl 1 µl

DNA PCR

pT7Blue-Tvector (Figure 5) T7 for PT7

(5’-AGCTCTAATACGACTCACTATAGG-3’) M13M4

(5’-GTTTTCCCAGTCACGAC-3’) PCR PCR

94 / 20 sec 58 / 20 sec 72 / 60 sec (3’ RACE) 90 sec (5’ RACE)

3’ 5’ 4

LB 2 µl 37

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20 )

DNA Big Dye Terminater ver3.1 (Applied

Biosystems ) PCR DNA ABI PRISM

3100 (Applied Biosystems ) Finch TV ver4.1

cDNA GenBank

cDNA PCR DNA

Prime STAR Max (Takara ) PCR

RNAi RNAi

21 22

bp SiRNA (short interfering RNA) RNA

RNAi SiRNA

RNAi ( )

NtSTOP1 pBI121

Xba Sma ( TOYOBO )

RNAi

(CICDH: cytosolic iso-citrate dehydrogenase; At1g65930) 1

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21 (Figure 6)

mRNA UTR (un-translational region)

ATG 0 -87 225

312 PCR 5’

PCR taq Prime STAR Max

Sma CICDH

BamH

DNA pBI121

DNA Ligation Kit ver2.1 (Takara )

Xba Sac PCR

-CICDH pBI121

RNAi

( ) 100 ng / µl

( ) LB 1 37

QIAprep Spin Miniprep Kit

-KD

NtSTOP1-KD

( , LBA4404 )

(Horsch et al., 1985) (

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22

22 / 12 / 12

KD

KD Actin ( )

PCR (semi-quantitive PCR) (Figure

6B) PCR SYBR Nucleic Acid Gel Stain

(invitrogen ) AE-6932GXES-US ( ) pH RNA 4 4 KD 15 KD 3 1/50 (4 µM Cd2+_{) Al}3+ ₃ _{(0.4 µM La}3+₎ (200 µM Mn2+₎ _AlCl₃_{(4 µM Al}3+₎ pH5.0 1/50 dose Al pH5.0 2 4 µM (AlCl3) 1/50 pH AlCl3 pH 5.2 5.0 4.7 HCl 1/50

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23 pH

2 7

pH5.5 AlCl3

1/50

(Pico Scopeman KENIS Tokyo Japan) (MC-300 KENIS) 5 pH 5 Al NtSTOP1-KD 1 % pH5.5 150 ml 1/50 2 6 2 ml 1 % 20 M AlCl3 pH 5.0 24 Al AlCl3 1 ml Hammp

NADH / NAD+ _{(Hammp et al., 1984)}

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24

20 M AlCl3 pH 5.0 24 Al

degenearte-PCR

RACE MATE ALS

Al GenBank degenarate (Table 3) degenerate-PCR RACE PCR (RT-real time PCR) Al KD 3

cDNA Power SYBR

Green PCR master mix

PCR ABI PRISM 7000 (Applied

Biosystems ) Primer3 ver0.4.0

(http://frodo.wi.mit.edu/primer3/) (Table

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25

1-3

STOP1 ( Ex taq ) ( ) CLUSTALW (http://www.genome.jp/tools/clustalw/) NtSTOP1 514 cDNA 1761bp

NCBI BLAST AtSTOP1

cDNA 76 % 54 % (NtSTOP1 aa.266-385) 90 % ( ) STOP1 cDNA (XM_004250324.1) 88 % (XP_004250372) 86 % 99 % NtSTOP1 EMBL-EBI (InterPro: protein sequence analysis & classification http://www.ebi.ac.uk/interpro/)

AtSTOP1 C2H2 ( C2H2

) WoLF PSORT

(http://wolfpsort.org/)

14 12

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26 NtSTOP1-KD Al pH NtSTOP1 Al pH Cd2+ _Mn2+ _La3+ KD (Figure 7) 4 Al -KD Al KD 2 Al KD (Figure 8) NtSTOP1 Al pH pH5.5 pH KD Al (Figure 8 & 9) Figure 8 pH5.0 6 M Al 50 % 12 M Al 80 % pH pH4.75 50 % pH4.0 90 % pH pH6.0 pH5.5 Al Al

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27 2 2 Al KD Al 10 (Figure 10, left) Al KD (Figure 10, right) Al GenBank Table 3 degenerate-PCR RACE ; GenBank AB811784, ;

GenBank AB811783 GenBank

PCR 1 KD Al KD Al 100 (Figure 11A) KD NtSTOP1

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28

Al

KD NtSTOP1

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29 Figure 1

Growth response of compared with that of STOP1-KO and AtALMT1-KO with Al and H+ rhizotoxicities. In STOP1-KO, distracted gene plant, root growth is greatly inhibited in low-pH, such as pH4.7, conditions and as ALMT1-KO, distracted gene plant, could not elongate their roots. Therefore, STOP1 is the necessary factor to gain Al and low pH stress tolerance. (Iuchi et al., 2007)

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30 Figure 2

Schematic representation of the overall domain structure and mutation. The four zinc-fingers (ZF1–ZF4) and the position of the mutation are also indicated. Asterisks indicate conserved motif of zinc-fingers. STOP1 could not to link with Zn2+_{because of substitution of 266th amino acid residue His to Tyr.}

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34 94 B 72 30sec 30sec 20sec 4 4 72 2min N cycle

3step-stepdawn-PCR for degenarate PCR and RACE

94 A ( 0.5 /cycle) 72 30sec 30sec 20sec 10 cycle 94 2min Figure 3

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35 Figure 4

The whole genomic map of pT7Blue vector. The black arrow’s part contains

multiple-cloning-site (MCS). This vector was used for transformation to E.coli. This image can be downloaded from Novagen’s website.

(http://www.merckmillipore.jp/life-science-research/vector-table-novagen-pt7blue-ve ctor-table/japanese/c_HICb.s1Ozp8AAAEhEV0Ldcaf)

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36 Figure 5

Sequences of T7 for pT7 primer and M13M4 primer and possession of lacZ region in pT7Blue vector. Both primers were used with colony-PCR for confirming presence of insert DNA fragment, and sequencing. The distance between T7 for pT7 and M13M4 is about 144bp.

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38

A

B

Figure 6

Transgenic tobacco plants with suppressed by RNAi inhibition. These were obtained by -mediated transformation using a vector carrying an RNAi construct. Partial cDNA of sandwiched the first intron of NADP-ICDH of , and the gene construct was driven by the

Cauliflower mosaic virus 35S promoter (A). Suppression of was analyzed in T3 seed progeny of three independent transgenic lines ( -KD1, 2, and 3) by RT-PCR using as the internal control (B).

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39 Figure 7

Tolerance of KD1 to rhizotoxic metals was compared with that of WT. Means and SD of relative root length (+toxicants/control) are shown ( = 5). Tolerance to toxic metals was tested by adding the following compounds to control (pH 5.0, no toxicant) solution: 4 µM CdCl2, 0.4 µM LaCl3, 200 µM MnSO4, or 4.0 µM

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40 Figure 8

Al- and H+_{-tolerance of transgenic tobacco plants with} _{suppressed by}

RNAi inhibition. Transgenic tobacco was obtained by -mediated transformation using a vector carrying an RNAi construct. Seedlings of T3 seed progenies of NtSTOP1-KD and wild-type (WT) were grown hydroponically

for 1 week in Al- +

toxicity (pH 5.2, 5.0, and 4.7). Means and SD of relative values (% of control; pH 5.0, 0Al for Al, pH 5.5 for pH from five seedlings are shown.

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41 Figure 9

Root growth of tobacco seedlings in hydroponic culture at pHs 5.5 and 5.0. Wild type and NtSTOP1-KD lines were grown in hydroponic solutions at pHs 5.5. and 5.0 for 1 week. Root length at pH 5.5 was used as control for evaluating H+_{tolerance, while}

that at pH 5.0 was used as control for evaluating Al-tolerance. Asterisks indicate significant difference as compared with WT ( -test, <0.05)

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42 Figure 10

Aluminum accumulation in root tip and organic acid excretion in response to Al. Citrate and malate excretion from roots of WT and -KD lines in control (pH 5.0, Al 0) and Al-cont

mean values in control solution (± SD). Black bars, mean values in Al solutions ( = 3). Asterisks indicate significant difference between complemented line and WT in either conditions respectively. ( -test, <0.05)

*

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43

A

B

Figure 11

Transcript levels of genes orthologous to those associated with Al tolerance in in lines. Seedlings were incubated in Al-toxic (pH 5.0, 20 t samples were used for transcript analyses. Transcript levels of citrate-transporting MATE,

(homologous to ) (A), and (homologous to ) (B), were compared between KD lines and wild-type (WT). Transcript levels were relatively quantified by real-time PCR using as the internal control, and then fold-change (relative to WT control) values were calculated. Means and SD of three replications are shown.

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44

2 STOP1

2-1

1 STOP1 NtSTOP1 Al ALMT1 MATE (orthologous) Al STOP1 STOP1 Al Al 2007 MATE

(Liu et al., 2009) MATE

ALS3 (Larsen et al., 1997 and 2005) AtSTOP1

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45 STOP1 Al pH pH pH pH K+ (Walker D.J. et al., 1998) pH

H+_-ATPase _ATPase _(Yan

et al., 1998) pH (Bose et al., 2010) pH H+ ₍ ) (1~2 ) pH pH Ca2+ (Koyama et al., 2001 ) (Kinraide et al., 2003) Ca2+ _(Shomer

et al., 2006 Kinraide and Wang, 2010)

pH STOP1

pH

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46 STOP1 pH (Kobayashi et al., 2013) pH pH pH STOP1 1 NtSTOP1 pH AtSTOP1 NtSTOP1 pH NtSTOP1

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47

2-2

total-RNA 1

total-RNA

RNA (Suzuki et al., 2004) 1

DNase RNA 1 DNase 1 DNase RNA 1 C (dC-tailing) 5’ RACE 1 1

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48 DNA 1 ( ) 1 1 (2848bp) (626bp) Sfi

CDS Prime STAR Max (Takara )

pBE2113 Sfi 5’- GGCCNNNN^NGGCC-3’ 50 4

(DH- )

100 ng/µl LB

1 37 (Figure 12)

1 QIAprep Spin Miniprep Kit

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49

( ) GV3101

Floral dip (Clough and Bent, 1998)

T0 T2 T2 AtSTOP1 STOP1 (Col-0) AtSTOP1 ( ) Col-0 3 (BC3F5) pH 5.5 pH5.0 pH Al pH5.5 Al 2 total-RNA 4 4 30 Al pH5.5 2 µM AlCl3 1/50 pH AlCl3 pH 4.7 1/50

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50 pH

2 5

(Pico Scopeman KENIS ) (MC-300 KENIS ) 5 5 PCR Al 10 µM AlCl3 Al RNA 3 1 total-RNA PCR Primer3 ver0.4.0 UBQ1 ( : At3g52590) Al

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51

2-3

1 CLUSTALW

Prime STAR Max

PCR cDNA

Genbank

LjSTOP1 510 PnSTOP1 509

PpSTOP1 588 NCBI AtSTOP1

BLAST STOP1

PnSTOP1 62 % CsSTOP1 59 % PpSTOP1 57 % LjSTOP1

55 % (AtSTOP1 aa.246-385)

91 % 91 % 74 %

90 %

STOP1 AtSTOP1 C2H2

(Figure 13) WoLF PSORT

14 LjSTOP1 12 PpSTOP1 2 CsSTOP1 13 PnSTOP1 13 AtSTOP1 14 13 AtSTOP1 STOP1 STOP1

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52 STOP1 (Figure 14) STOP1 STOP1 pH Col-0 20 % 30 % 80 % ( ) 30 % 50 % (Figure 15B) 1 NtSTOP1 -KD NtSTOP1 30 % Al Col-0

PpSTOP1 (Figure 15A)

PpSTOP1 Col-0

Al

3 Al

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53 ALMT1 Al STOP1 1 pH STOP1 AtSTOP1 Al

STOP1 AtSTOP1 ALMT1 Al

Al

STOP1

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54 Figure 12

Indicating figure of the cassettes for transgenic STOP compliment in pBE2113 promoter fragment was connected STOP1 orthologue to regulate their transcription, as called cassette. Then, the STOP1 orthologue cassettes were transformed to pBE2113 for making STOP compliment lines by the T-DNA method.

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55 Figure 13

Deduced amino acid alignment of STOP1-like proteins from various plant species. Strictly conserved amino acids are highlighted with black, while residues belonging to conserved amino acid groups are highlighted in gray. Alignment was carried out using a WEB program ClustalW (http://www.genome.jp/tools/clustalw/).

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56 Figure 14

Phylogram of STOP1-1 like proteins in various plant species. Distance indicator shows relatedness of proteins. Bold font indicates that function of protein has been supported by experiments using mutants [ ,

(GenBank ID, NM_103160); , (NM_001072803)], RNAi

suppression [ , (AB811781)], gene knock-out

[ , (AB811779)], and/or complementation

assays [ (tea), (AB811780); (black poplar),

(AB811779); (AB811782)]. Putative orthologs

are shown in regular font: (AC232513.1);

(XM_002891054.1); (XM_003556158), (XM_002270160),

(BT117929), (TC13125),

(AK320912), (Jcr4S27000.20), (NM_001051470),

(HM122494), (XM_003564671),

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57

A

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58 Figure 15

Relative root length (% of that in WT) of complemented lines is shown in Al-toxic (A, pH 5.5, 1 , -EDTA) and low-pH stress (B, pH 4.7) conditions. Complemented lines carried or like proteins [from tobacco ( ), black poplar

( ), tea ( ), ( ), or

( )]. Values are means ± SE ( = 3). Asterisks indicate significant difference between complemented lines and ( -test, <0.05).

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59 Figure 16

Recovery of transcriptions of suppressed genes in -mutant (Sawaki et al., 2009) was analyzed in complemented lines after exposure to

Al-5.0 for 24 h) conditions. Transcript levels of (

), ALS3 ( ),

(

), were quantified by real-time PCR. Mean values ± SE ( = 3) are shown. Asterisks indicate significant difference between complemented line and

(MT) ( -test, <0.05).

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60

3

STOP1 Al STOP1 Al (Figure 15A, 16) Al STOP1 STOP1 Al (Figure 16, S1) AtSTOP1 STOP1 (Figure 13) DNA DNA (

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61

) DNA ( ;major groove

) DNA STOP1 STOP1 2 Al Al Al DNA STOP1 Al STOP ART1 STOP1

Os01g0871200 (aa.522) STOP2 Os03g0838800 (aa.385)

AtSTOP1 STOP1 Al

DNA (http://www.kazusa.or.jp/lotus/blast.html) LjSTOP1

BLAST AtSTOP1 LjSTOP1

C2H2

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62 STOP STOP1 2 ( ) STOP Al STOP1 Al STOP1 Al STOP1 Al ( ) Al (Magalhaes et al., 2007) STOP1 Al STOP1 Al Al 3 (Magalhaes et al., 2007) NtSTOP1-KD Al (Figure 10)

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63 Al Al (Delhaize., et al 1993) Al (Yang et al., 2005) Al GUS ALMT1 ALMT1

(Agrawal et al., 2012) ALMT1

Al STOP1 Al

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64 pH STOP1 pH4.7 pH 50 % pH STOP1-KD STOP1 pH ART1 Al (Yamaji et al., 2009) STOP1-KD ( ) STOP1 STOP1 Al pH pH Al pH Al pH Al pH pH pH

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65

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66 Supplemental figure 1

Al inducible malate exudation of WT (Col-0) and complemented lines. Same complemented lines in figure 15 were used for experiment. A method of experiment was implemented in relation to in the 1st chapter. 7d plants were treated at non-Al stress (pH 5.0, -Al) and Al stress (pH 5.0, 10 µM Al), and independently used for each stress condition group for measurement. Values are means ± SE ( = 3). .

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67

4

STOP1 STOP1 500 600 C2H2 90 % AtSTOP1 NtSTOP1 Al ALMT1 MATE NtSTOP1 Al Al pH pH4.7 2 75 % 1 40 % pH pH NtSTOP1 STOP1 pH pH 2 STOP1 STOP Al STOP1 STOP1

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68

STOP1

STOP

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69 STOP1 STOP Al Al STOP1 pH

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70

pH

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71

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