SULTR3;5 - STOP1 転写制御による植物の酸性土壌耐性機構に関する研究

Figure 9. Schematic representation of regulating genes by STOP1 in relation to low pH and Al tolerance. Major genes identified as downregulated in the stop1 mutant are shown with possible functions in low pH and Al stress tolerance. Except for AKT1, other genes were downregulated in the stop1 mutant. AtMATE was reported by Liu et al. (2009). Underlined genes were greatly down-regulated in the stop1 mutant with both low-pH and Al treatments (Table 3).

Al defense system

GDH

Tonoplast

Cytosol

GAD

ME

Malate

Al³⁺

Malate

AtALMT1 ALS3 Low pH defense

systems

AtTDT

K⁺

2-47

AtALMT1-KOࡸALS3-KOࡣపpHẘᛶ࡟ឤཷᛶ࡛ࡣ࡞࠸ࠋࡑࢀࡺ࠼ࠊstop1 ኚ␗య࡛㌿෗ᢚไࡉࢀࡓࡇࢀ௨እࡢ㑇ఏᏊࡀࠊపpHẘᛶࡢ⪏ᛶࢩࢫࢸ࣒࡜ࡋ࡚ᶵ

⬟ࡋ࡚࠸ࡿ࠿ࡶࡋࢀ࡞࠸ࠋDNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ (e.g. stop1 ኚ␗య࡜㔝⏕ᆺ ࡢẚ㍑࣐࢖ࢡࣟ࢔ࣞ࢖)ࡢḟ࡟⾜࠺༙ᐃ㔞 RT-PCR ࡣࠊSTOP1-KO ࡸ┦⿵⤌᥮య ( i.e. CaMV 35S:: STOP1ᑟධstop1ኚ␗య)࡟࠾࠸࡚ࠊ࢖࢜ࣥᜏᖖᛶࡸpHㄪ⠇ࡢ

㑇ఏᏊࡀSTOP1࡟ࡼࡗ࡚ඹㄪ⠇ࡉࢀ࡚࠸ࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓ (Fig. 9)ࠋ౛࠼ࡤࠊ

ᾮ⬊⭷ࢪ࢝ࣝ࣎࢟ࢩࢺࣛࣥࢫ࣏࣮ࢱ࣮࡛࠶ࡿ AtTDT (ࣜࣥࢦ㓟ࢺࣛࣥࢫ࣏࣮ࢱ࣮

ࡢ୍✀)ࡣࠊstop1 ኚ␗య࡛㌿෗ᢚไࡉࢀࡓ㑇ఏᏊࡢ 1 ࡘ࡜ࡋ࡚㑅ࡤࢀࡓࠋࡇࡢ㑇

ఏᏊࡢT-DNAᤄධኚ␗యࡣ⣽⬊㉁ࡢpHᜏᖖᛶࡀᶵ⬟ࡋ࡞ࡃ࡞ࡗࡓࡓࡵࠊࢩࣟ࢖

ࢾࢼࢬࢼࡢpHㄪ⠇ࡢ㑇ఏᏊ࡜ࡋ࡚ሗ࿌ࡉࢀࡓ (Hurth et al., 2005)ࠋࡇࡢ㑇ఏᏊ ࡣstop1ኚ␗యࡢపpHฎ⌮࡛኱ࡁࡃ㌿෗ᢚไࡉࢀ࡚ࡣ࠸࡞࠿ࡗࡓࡀ (FC = 0.75)ࠊ ࡑࡢపpH㉸ឤཷᛶࡢ୍㒊ࡣㄝ࡛᫂ࡁࡿࠋ௚ࡢ᳜≀✀ࡢపpH⪏ᛶ࡟㔜せ࡞㑇ఏᏊ ࡢ࠸ࡃࡘ࠿ࡶࠊstop1 ኚ␗య࡛㌿෗ᢚไࡉࢀ࡚࠸ࡓࠋ౛࠼ࡤࠊᵝࠎ࡞⏕≀✀࡛ K⁺ ࡢ㍺㏦ࡸࡑࡢᜏᖖᛶࡣ୺࡞ pH ㄪ⠇ᶵᵓ࡜ࡋ࡚▱ࡽࢀ࡚࠸ࡿ (Zhang and Kone, 2002)ࠋ୺せ࡞K⁺ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࡢAKT1 (Lee et al., 2007)ࢆㄪ⠇ࡍࡿ࢟ࢼ࣮ࢮ

ࢆࢥ࣮ࢻࡍࡿCIPK23ࡢ㌿෗ᢚไࡣࠊAKT1άᛶࡢῶᑡࢆᘬࡁ㉳ࡇࡍࡇ࡜࡛stop1 ኚ␗యࡢ pH ㄪ⠇࡟ᙳ㡪ࢆ୚࠼࡚࠸ࡿ࠿ࡶࡋࢀ࡞࠸ࠋSO4ࡸ NO3࡟㛵ࡍࡿ௚ࡢࢺ

ࣛࣥࢫ࣏࣮ࢱ࣮ࡶࠊ᳜≀ࡢpHㄪ⠇࡟ᐤ୚ࡋ࡚࠸ࡿ࠿ࡶࡋࢀ࡞࠸ࠋࢺ࢘ࣔࣟࢥࢩ࡛

ࡣࠊపpHᇵᆅࡢ᰿ࡢ⏕⫱ࡀࠊK2SO4ࡢῧຍ࡛ᨵၿࡉࢀࡿ (Yan et al., 1992)ࠋࡑ ࡢ࠺࠼ࠊ 㧗⃰ᗘ ࡢ K⁺ࡣ Al ฎ⌮ࡋ ࡓࢥ ࣒ࢠࡢ⣽ ⬊ෆࡢ 㓟ᛶ ໬ࢆ㜵࠸ ࡛࠸ࡿ

(Lindberg and Strid, 1997)ࠋ୍᪉ࠊNO3ࡢ㏣ຍࡣࠊ㓟⣲Ḟஈୗࡢࢺ࢘ࣔࣟࢥࢩࡢ

࢔ࢩࢻ࣮ࢩࢫࢆ⦆࿴ࡋ࡚࠸ࡿ(Libourel et al., 2006)ࠋࡲ࡜ࡵࡿ࡜ࠊࡇࢀࡽࡢࢹ࣮ࢱ ࡣࠊ࢖࢜ࣥᜏᖖᛶࡸ࢖࢜ࣥ㍺㏦࡟㛵ࢃࡿ㑇ఏᏊࡢ㌿෗ᢚไࡀࠊstop1 ኚ␗యࡢ H⁺ ឤཷᛶࢆᘬࡁ㉳ࡇࡍࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࠋࡇࡢྍ⬟ᛶࡣࠊࢩࣟ࢖ࢾࢼࢬࢼࡢ K⁺ࢺ

ࣛࣥࢫ࣏࣮ࢱ࣮ࡢ᭱㏆ࡢ◊✲࡛❧ドࡉࢀ࡚࠸ࡿࠋcation-proton ࢔ࣥࢳ࣏࣮ࢱ࣮࡛

࠶ࡿCHX13ࡢ㐣๫Ⓨ⌧ࡣࠊK⁺ไ㝈᮲௳࡟࠾࠸࡚ࠊపpH (pH 4.3 and 5.6; Zhao et

al., 2008)࡛ࡢ⏕⫱ࡣࢃࡎ࠿࡟ᨵၿࡋࡓࡇ࡜࠿ࡽࠊࢩࣟ࢖ࢾࢼࢬࢼࡢ K⁺ᜏᖖᛶࡣ

H⁺ឤཷᛶ࡟㛵㐃ࡀ࠶ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋ

STOP1ࡢᶵ⬟㞀ᐖࡣࠊpHㄪ⠇ࡢ௦ㅰࣃࢫ࢙࢘࢖࡜ࡋࡓࠊ᳜≀⣽⬊ෆࡢpHᜏ

ᖖᛶࣉࣟࢭࢫ࡜ண᝿ࡉࢀࡿ࣓࢝ࢽࢬ࣒࡟ࡶᙳ㡪ࢆ୚࠼࡚࠸ࡿࠋpH stat⤒㊰ࡢ୍㒊 ࡣࠊࣜࣥࢦ㓟㓝⣲ (malic enzyme)ࡸࣆࣝࣅࣥ㓟࢝ࣝ࣎࢟ࢩ࣮ࣛࢮࠊ࢔ࣝࢥ࣮ࣝࢹ

ࣄࢻࣟࢤࢼ࣮ࢮࠊࡲࡓࡣங㓟⬺Ỉ⣲㓝⣲ (lactate dehydrogenase)࡟ࡼࡗ࡚ᵓᡂࡉ

ࢀࠊH⁺ࢆᾘ㈝࡛ࡁࡿ (Roberts et al., 1992; Sakano, 1998)ࠋ௚ࡢpH stat⤒㊰࡛ࠊ GABA shuntࡣGDHࠊGADࠊGABA-T࡛ᵓᡂࡉࢀࠊ⣽⬊㉁ࡢH⁺ㄪ⠇࡟㛵୚ࡋ࡚

࠸ࡿ (Bown and Shelp, 1997)ࠋGABA shunt⤒㊰ࡣࠊ⣽⬊㉁ࡢ࢔ࢩࢻ࣮ࢩࢫࢆᘬ ࡁ㉳ࡇࡍప㓟⣲≧ែࡸ௚ࡢࢫࢺࣞࢫ࡟ࡼࡗ࡚㧗ࡲࡿ (Crawford et al., 1994)ࠋ௒ᅇ ࡢ⤖ᯝࡣࠊࡇࢀࡽࡢpHㄪ⠇ࣃࢫ࢙࢘࢖ࡀstop1ኚ␗య࡛ᢚไࡉࢀ࡚࠸ࡿࡇ࡜ࢆ♧

ࡋ࡚࠸ࡿ (Fig. 7)ࠋࡇࢀࡽࡢ⤒㊰ࡢ୺࡞࢔࢖ࢯࣇ࢛࣮࣒࡛࠶ࡿ GDH1ࠊGDH2ࠊ GAD1ࠊGABA-TࠊME1ࠊME2ࡣstop1ኚ␗య࡛㌿෗ᢚไࡉࢀ࡚࠸ࡓ (Table 3; Fig.

7)ࠋGAD࢔࢖ࢯࣇ࢛࣮࣒ࡢ1ࡘ࡛࠶ࡿGAD4ࡣࠊstop1ኚ␗య࡛㌿෗ಁ㐍ࡉࢀ࡚

࠸ࡓࡀ (Table 4)ࠊGAD4 Ⓨ⌧࡟ࡼࡿ㈉⊩ࡣࠊGAD1 ࡼࡾࡶᑠࡉࡃࠊࢺ࣮ࢱࣝࡢ GADάᛶࡢホ౯ࡀ᭱㏆ࡢ◊✲࡛⾜ࢃࢀ࡚࠸ࡿ (Miyashita and Good, 2008)ࠋ୍᪉

࡛ࠊ௚ࡢ㌿෗ᢚไࡉࢀࡓ㑇ఏᏊࡢⓎ⌧ࣞ࣋ࣝࡣࠊ㔝⏕ᆺ࡜stop1ኚ␗య࡛࠿࡞ࡾఝ

࡚࠸ࡓࡇ࡜࠿ࡽࠊࡇࢀࡽࡢ⤒㊰ࡣstop1ኚ␗య࡛ᢚไไᚚࡉࢀ࡚࠸ࡿࡇ࡜ࢆ♧ࡋ࡚

࠸ࡿࠋࡇࢀࡣstop1ኚ␗యࡢH⁺ឤཷᛶ࡟ᙳ㡪ࢆ୚࠼࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿࠋ⯆࿡῝

࠸ࡇ࡜࡟ࠊࡇࢀࡽࡢ㑇ఏᏊࡢ࣮࢜ࢯࣟࢢࡣࠊ௚ࡢ⏕≀✀࡛㓟ᛶ⎔ቃࡢ㐺ᛂ࡟㔜せ࡛

࠶ࡿ࡜ࡋ࡚ྠᐃࡉࢀ࡚࠸ࡿ (Castanie-Cornet et al., 1999)ࠋ኱⭠⳦࡛ࡣࠊGADࡣ పpHᇵᆅ࡛ࡢ⏕ࡁṧࡾ࡟ᚲ㡲࡞ࡶࡢ࡛࠶ࡿ (Yohannes et al., 2004)ࠋstop1ኚ␗

య࡛㌿෗ᢚไࡉࢀࡓࡇࢀࡽࡢ㑇ఏᏊࡣࠊࢩࣟ࢖ࢾࢼࢬࢼࡢపpH⪏ᛶ࣓࢝ࢽࢬ࣒࡟

㛵୚ࡋ࡚࠸ࡿ࠿ࡶࡋࢀ࡞࠸ࠋ

STOP1࡟ࡼࡗ࡚ㄪ⠇ࡉࢀࡓ௚ࡢ㑇ఏᏊࡣࠊ㐣ཤ࡟H⁺⪏ᛶ㑇ఏᏊ࡜ࡋ࡚࡛ࡣ࡞

ࡃࠊ௚ࡢ⏕≀Ꮫⓗ⌧㇟࡟ࡼࡗ࡚ྠᐃࡉࢀࡓ࡜ࡋ࡚ࡶࠊపpH⪏ᛶࡢᶵ⬟ࢆᣢࡗ࡚࠸

ࡿྍ⬟ᛶࡀ࠶ࡿࠋ౛࠼ࡤࠊstop1 ኚ␗య࡛㌿෗ᢚไࡉࢀࡓ㑇ఏᏊࡢ 1 ࡘ࡛࠶ࡿ

PGIP1 ࡣࠊ⑓ཎయឤᰁࡢ㜵ᚚࢩࢫࢸ࣒࡟㛵ࢃࡿࢱࣥࣃࢡ㉁࡜ࡋ࡚ࡼࡃ▱ࡽࢀ࡚࠸

ࡿࠋRudrappaࡽࡣࠊⴥ࡬ࡢ⑓ཎ⳦࡟ࡼࡿឤᰁࡀAtALMT1ࡢⓎ⌧ࢆㄏᑟࡋࠊ᰿ᅪ

ࡢ᭷⏝⳦ࢆᙉ໬ࡋࡓࡇ࡜ࢆሗ࿌ࡋࡓ (Rudrappa et al., 2008)ࠋࡇࢀࡣࠊSTOP1ࡀ

⏕≀ⓗࠊ㠀⏕≀ⓗ᭷ᐖᅉᏊࡢ୧᪉ࡢ㜵ᚚࢩࢫࢸ࣒࡟ᒓࡍࡿከ㠃ᛶ㑇ఏᏊ࡛࠶ࡿࡇ࡜

ࢆ♧ࡋ࡚࠸ࡿࡇ࡜࠿ࡽࠊSTOP1ࡣPGIPⓎ⌧ࢆㄪ⠇ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿࠋ୍᪉

࡛ࠊPGIP1ࡶ᰿ࡢ⣽⬊ቨ࡟࠾ࡅࡿᶵ⬟࡟ࡼࡾࠊH⁺⪏ᛶࡢ₯ᅾⓗ࡞ᙺ๭ࢆᣢࡗ࡚࠸

ࡿࠋ࣌ࢡࢳࣥ㡿ᇦࡢ࣏ࣜ࢞ࣛࢡࢶࣟࣥ㓟 (polygalacturonic acid)࡟⤖ྜࡍࡿࡇࡢࢱ

ࣥࣃࢡ㉁ࡣࠊ‘‘egg box’’࡜࠸ࢃࢀࡿᵓ㐀≀ࢆᵓ⠏ࡍࡿ (Spadoni et al., 2006)ࠋࡇࡢ ᵓ㐀≀ࡣࠊඹ᭷⤖ྜ࡟ࡼࡿ࣌ࢡࢳࣥ㙐ࡢ⤖ྜ࡟ᚲ㡲࡛࠶ࡾ (O’Neill et al., 2004)ࠊ పpH ᮲௳࡟ࡼࡿࡑࡢᙅయ໬ࡣࠊࢩࣟ࢖ࢾࢼࢬࢼ᰿ࡢ H⁺ẘᛶࡢ୍ࡘ࡜ࡉࢀ࡚࠸ࡿ

(Koyama et al., 2001)ࠋH⁺⪏ᛶ࡟࠾ࡅࡿPGIP1ࡢᙺ๭ࡣᮍࡔ᫂ࡽ࠿࡟࡞ࡗ࡚࠸࡞

࠸ࡀࠊ㓝ẕ࡟࠾࠸࡚⣽⬊ቨࡢᏳᐃࢆಖࡘࠊ㢮ఝࡢᶵ⬟ࢆࡶࡘࢱࣥࣃࢡ㉁ࡀపpH⪏ ᛶ࣓࢝ࢽࢬ࣒ࡢ1ࡘ࡜ࡋ࡚ྠᐃࡉࢀ࡚࠸ࡿࠋ࣐ࣥࣀ࣮ࢫ㒊ศࢆ㍺㏦ࡍࡿࢱࣥࣃࢡ㉁

࡛࠶ࡿCWP2 (for cell wall 2) ࡣࠊ㓝ẕ࡟࠾࠸࡚పpH᮲௳࡛⣽⬊ቨࢆᏳᐃࡉࡏࡿ

ࡇ࡜ࡀ࡛ࡁࡿࡓࡵࠊH⁺⪏ᛶ࡟ᚲ㡲࡛࠶ࡿ (Skrzypek et al., 1997)ࠋ⯆࿡῝࠸ࡇ࡜

࡟ࠊPGIP1 ࡣ୰ᛶ᮲௳࡛࣌ࢡࢳࣥ࡞࡝ࡢᇶ㉁ࢆᏳᐃࡉࡏࡿࡓࡵࠊ⑓ཎయࡢ࣏ࣜ࢞

ࣛࢡࢶࣟࢼ࣮ࢮ (polygalacturonase)ࢆ㜼ᐖࡍࡿ୍᪉࡛ࠊ࣌ࢡࢳࣥࡢྜᡂࡣప pH ᮲௳࡛ቑຍࡍࡿ(Spadoni et al., 2006)ࠋຍ࠼࡚ࠊPGIP1ࡢ࣍ࣔࣟࢢ࡛࠶ࡿPGIP2

ࡶstop1ኚ␗య࡛᭷ព࡟㌿෗ᢚไࡉࢀ࡚࠸ࡓࡀࠊ࣐࢖ࢡࣟ࢔ࣞ࢖ࡢࢫ࢟ࣕࣥࢹ࣮ࢱ

ࡢࢡ࢜ࣜࢸ࢕࣮ࢥࣥࢺ࣮ࣟࣝࡢẁ㝵࡛᤼㝖ࡉࢀ࡚࠸ࡓ (Table 6)ࠋࢩࣟ࢖ࢾࢼࢬࢼ

᰿ࡢH⁺⪏ᛶ࡟㛵ࡍࡿࡇࢀࡽPGIP ࢱࣥࣃࢡ㉁ࡢ㈉⊩࡟ࡘ࠸࡚ࠊࡉࡽ࡞ࡿ◊✲࡟ࡼ

ࡾࠊホ౯ࡍࡿࡇ࡜ࡀᚲせ࡛࠶ࡿࠋ඲యⓗ࡟ࠊstop1ኚ␗య࡛㌿෗ᢚไࡉࢀ࡚࠸ࡓ㑇 ఏᏊࡣࠊ᳜≀ࡸ௚ࡢ⏕≀✀ࡢపpH⪏ᛶࡸpHᜏᖖᛶ࡟㛵ࡍࡿ㑇ఏᏊ࡜ࡋ࡚Ỵᐃࡉ

ࢀࡓࠊᵝࠎ࡞㑇ఏᏊ࡛ᵓᡂࡉࢀ࡚࠸ࡿࠋ᳜≀ࡢ◊✲࡟࠾ࡅࡿᐇ㦂ⓗ࡞ドᣐࡣ㝈ᐃⓗ

࡛࠶ࡿࡀࠊ࢖࢜ࣥ㍺㏦࡜Ⅳ⣲ࠊ❅⣲ (CN) ௦ㅰࡢኚື࡟ࡼࡿ㐃ᦠࡣࠊపpH⎔ቃ࡟

㐺ᛂࡍࡿࡓࡵ࡛࠶ࡾࠊ௚ࡢ⏕≀✀࡛ࡶඹ㏻࡛࠶ࡿࠋ౛࠼ࡤࠊ㨶㢮ࡢపpH㐺ᛂࡣ࢝

࣒ࣜ࢘ࠊࢼࢺ࣒ࣜ࢘ࠊỈ⣲㍺㏦ࡸ CN ௦ㅰࡢኚ໬࡛࠶ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜ࡉࢀ

(Hirata et al., 2003)ࠊࡉࡽ࡟pH 0௜㏆࡛⏕ᜥࡍࡿࣂࢡࢸࣜ࢔ࡣࠊ௚ࡢ୰ᛶ௜㏆࡛

⏕ᜥࡍࡿࣂࢡࢸࣜ࢔࡟ẚ࡭ࠊ࢖࢜ࣥࢺࣛࣥࢫ࣏࣮ࢱ࣮ࡸCN௦ㅰࡢ㓝⣲ࡢࢥࣆ࣮ᩘ

ࡀከ࠿ࡗࡓ (Fütterer et al., 2004)ࠋSTOP1ไᚚ㑇ఏᏊࡢࡉࡽ࡞ࡿ◊✲࡛ࠊ㧗➼᳜

≀ࡢ」㞧࡞pH⪏ᛶࢩࢫࢸ࣒ࡀ᫂ࡽ࠿࡟࡞ࡿ࠿ࡶࡋࢀ࡞࠸ࠋ

ไᚚࢱࣥࣃࢡ㉁ࡢ୍⩌ (e.g. protein kinasesࠊtranscriptional regulators)ࡣࠊ

stop1ኚ␗య࡛㌿෗ᢚไࡉࢀࡓࠋࡑࡇ࡟ࡣࢩࣟ࢖ࢾࢼࢬࢼSTOP1ࡢ2ࡘ࠶ࡿ࣍ࣔ

ࣟࢢࡢ࠺ࡕࡢ1ࡘࡀྵࡲࢀ࡚࠾ࡾࠊࡇࢀࢆSTOP2࡜ྡ௜ࡅࡓࠋSTOP2ࡣSTOP1

࡜㠀ᖖ࡟ࡼࡃఝࡓ Cys-2-Hys-2 zinc finger domain ࢆᣢࡗ࡚࠸ࡓ(Iuchi et al., 2007)ࠋSTOP2 ࡢ ᶵ ⬟ ࡣ ࡲ ࡔ ᫂ ࡽ ࠿ ࡟ ࡞ ࡗ ࡚ ࠸ ࡞ ࠸ ࡀ ࠊ ௚ ࡢ zinc finger transcription factor࡜ྠᵝࠊSTOP1࡜࡜ࡶ࡟࠸ࡃࡘ࠿ࡢ㑇ఏᏊࡢ㌿෗ࢆඹไᚚࡋ

࡚࠸ࡿ࠿ࡶࡋࢀ࡞࠸ࠋ౛࠼ࡤࠊzinc finger transcription factor proteinࡢDOF1

(At1g51700)ࡣࠊࡑࡢ࣍ࣔࣟࢢࡢDOF2࡜ඹྠ࡛ࠊ᰿࡛࢔࣑ࣀ㓟࡜Ⅳ໬Ỉ⣲ࡢ௦ㅰ

ࡢ㓝⣲ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊ (e.g. PEP and PPDK; Yanagisawa et al., 2004)ࡢ㌿෗

ࢆไᚚࡋ࡚࠸ࡿ (Yanagisawa, 2000)ࠋSTOP2ࡢᙺ๭ࡢỴᐃࡣࠊSTOP1࡟ࡼࡗ࡚

ㄪ⠇ࡉࢀࡿ㑇ఏᏊࡢ㌿෗ࣃࢫ࢙࢘࢖ࢆ⌮ゎࡍࡿ࠺࠼࡛㔜せ࡞ㄢ㢟࡛࠶ࡿࠋ୍᪉࡛ࠊ

H⁺࡟ࡼࡿstop1ኚ␗యࡢ㑇ఏᏊ㌿෗ไᚚࡣAlฎ⌮࡛ࡶ㉳ࡇࡗࡓࠋࡇࢀࡣࠊ୧⪅ࡢ

ࢫࢺࣞࢫࡀྠࡌࢩࢢࢼࣝࢆㄏᑟࡍࡿࡇ࡜࡛ࠊ㑇ఏᏊⓎ⌧ࢆᘬࡁ㉳ࡇࡍࡇ࡜ࢆ♧ࡋ࡚

࠸ࡿࠋH⁺ࡸAl࡟ࡼࡿ⣽⬊㉁ࡢ㓟ᛶ໬ࡢㄏᑟ࡛ࠊpHࢆឤ▱ࡍࡿGFPࢆ⏝࠸ࡓࢩࣟ

࢖ࢾࢼࢬࢼ࡛☜ㄆࡉࢀࡓࠋAl ฎ⌮ࡣࠊప pH ฎ⌮ୗ࡛ࡢࢩࣟ࢖ࢾࢼࢬࢼ᰿ࡢ⣽⬊

㉁࡟㏣ຍⓗ࡞㓟ᛶ໬ࢆࡶࡓࡽࡍ(Moseyko and Feldman, 2001)ࠋAl࡟ࡼࡿ⣽⬊⭷

H⁺-ATPaseࡢ㜼ᐖࡣࠊ࠸ࡘࡃ࠿ࡢ᳜≀✀࡛ሗ࿌ࡉࢀ࡚࠸ࡿࡀ(Ahn et al., 2001)ࠊ ࡑࢀࡽࡶ⣽⬊㉁ࡢ㓟ᛶ໬࡟ࡼࡿࡶࡢ࠿ࡶࡋࢀ࡞࠸ࠋࡉࡽ࡞ࡿ◊✲࡛ࠊAl ฎ⌮࡟࠾

ࡅࡿࠊSTOP1࡟ࡼࡗ࡚ไᚚࡉࢀࡿ㑇ఏᏊࡢάᛶ໬ࣉࣟࢭࢫ࡟㛵ࡍࡿศᏊ࣓࢝ࢽࢬ

࣒ࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜ࡀ࡛ࡁࡿࠋ

stop1 ኚ␗యࡣࠊAtALMT1-KO ࡟ẚ࡭ࠊ㓟ᛶᅵተ࡟࠾ࡅࡿ⏕⫱ࡀⰋࡃ࡞࠸ࠋ

୍᪉ࠊAl ⪏ᛶࣞ࣋ࣝࢆỈ⪔᱂ᇵ࡛ ᐃࡋࡓ࡜ࡇࢁࠊࡑࡢ⤖ᯝࡣ㠀ᖖ࡟ࡼࡃఝ࡚࠸

ࡓ (Fig. 4b, 4c; Figure 6A in Iuchi et al., 2007)ࠋࡇࢀࡣࠊH⁺ࡢឤཷᛶࡀࠊ㓟ᛶᅵ ተ࡟࠾ࡅࡿstop1ኚ␗యࡢ⏕⫱࡟ᙳ㡪ࢆ୚࠼࡚࠸ࡿࡇ࡜ࢆ♧ࡍࠋࡇࡢࡇ࡜ࡣࠊࢩࣟ

࢖ࢾࢼࢬࢼࡢရ✀ࢆ⏝࠸ࡓ㐣ཤࡢ⏕⫱ᐇ㦂࡟ࡼࡗ࡚ࡶᨭᣢࡉࢀࡿ࠿ࡶࡋࢀ࡞࠸ࠋࢩ

ࣟ࢖ࢾࢼࢬࢼࡢྛရ✀ࢆྠࡌ㓟ᛶᅵተ࡛⏕⫱ࡉࡏࡿ࡜ࠊAl ⪏ᛶ⛬ᗘࡀఝ࡚࠸ࡿሙ

ྜࠊࡑࡢ⏕⫱ࡣH⁺⪏ᛶ࡜┦㛵ࡀ࠶ࡿ(Ikka et al., 2007)ࠋࡇࢀࡣH⁺⪏ᛶࡀࠊ㓟ᛶ ᅵተ࡟࠾ࡅࡿࢩࣟ࢖ࢾࢼࢬࢼࡢ⏕⫱ࢆỴᐃࡍࡿࡢ࡟㔜せ࡞せᅉࡢ 1 ࡘ࡜࡞ࡗ࡚࠸

ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋSTOP1࡟ࡼࡗ࡚ไᚚࡉࢀࡿH⁺⪏ᛶࡢ㑇ఏᏊ (Fig. 9) ࡢࡉ

ࡽ࡞ࡿ◊✲ࡣࠊ௚✀ࡢస≀ࡢ H⁺⪏ᛶࢆࠊ㑇ఏᏊ⤌᥮࠼ࡸẚ㍑ࢤࣀ࣒ࡢ࣐࣮࣮࢝ࢭ

ࣞࢡࢩࣙࣥ࡟ࡼࡗ࡚ᨵⰋࡍࡿࡇ࡜࡟㈉⊩ࡍࡿ࠿ࡶࡋࢀ࡞࠸(Iuchi et al., 2007)ࠋ

➨

2

❶ ࣮ࣘ࢝ࣜࡢ

Al

⪏ᛶ㑇ఏᏊ࡜

AtSTOP1

࣍ࣔࣟࢢ㑇ఏ Ꮚ࡜ࡢ㛵㐃ᛶ

2-1

⥴ゝ

࣮ࣘ࢝ࣜࡣᶞᮌࡢ୰࡛ࡶᡂ㛗ࡢ㏿࠸✀ࡢ1ࡘ࡛࠶ࡾࠊࣃࣝࣉ⏕⏘ࡢ୺ཎᩱ࡜࡞

ࡿ (e.g., Greaves et al. 1997; Campinhos 1999)ࠋ࣮ࣘ࢝ࣜࡢ᳜ᯘᆅࡔࡅ࡛ୡ⏺୰

࡛1700୓ha௨ୖࡢࣉࣛࣥࢸ࣮ࢩࣙࣥࡀ✌ാࡋ࡚࠸ࡿ (FAO 2001)ࠋ࣮ࣘ࢝ࣜࡣࠊ ࡑࡢᆅᇦࡢ⮬↛⎔ቃ᮲௳࡟㐺ᛂࡋࡓရ✀ࡀ⏕⫱ࡋ࡚࠸ࡿࡀࠊ⧄⥔㛗ࡀ㛗࠸ࠊ࠶ࡿ࠸

ࡣࣃࣝࣉ⏕⏘ᛶࡀ㧗࠸࡜࠸࠺ࡼ࠺࡞ࠊᕤᴗⓗ࡟㐺ࡋࡓ≉ᛶࢆᣢࡗ࡚࠸ࡿ࡜ࡋ࡚㑅ᢤ ࡉࢀࡓ࢚࣮ࣜࢺࢡ࣮ࣟࣥࡣࠊ௚ࡢ⏕⫱ᆅᇦ࡛ᡂ㛗ࡀᝏࡃ࡞ࡿࡇ࡜ࡀ࠶ࡿࠋ࣐࣮࣮࢝

㑅ᢤࡸ㑇ఏᏊᑟධ࡟ࡼࡿศᏊ⫱✀ࡣࠊࣉࣛࣥࢸ࣮ࢩࣙࣥࡢ⎔ቃࢫࢺࣞࢫ࡟ᑐࡍࡿ᢬

ᢠᛶࢆ୚࠼ࡿྜ⌮ⓗ࡞ᡭἲࡢ1ࡘ࡛࠶ࡿ (Raymond and Apiolaza 2004)ࠋᐇ㝿ࠊ ศᏊ࣐࣮࣮࢝ࡢ☜❧ࡢࡓࡵࡢࢤࣀ࣒㓄ิࡀᅜ㝿ඹྠ஦ᴗ࡜ࡋ࡚㐍⾜ࡋ࡚࠾ࡾ

(DOE Joint Genome Institute and the Eucalyptus Genome Network, EUCAGEN)ࠊ㑇ఏᏊᙧ㉁㌿᥮ࡢ᪉ἲࡣ࠸ࡃࡘ࠿ࡢ✀࡛☜❧ࡉࢀ࡚࠸ࡿ (Ito et al.

1996)ࠋ࠶ࡿ≉ᐃࡢᙧ㉁ࢆㄪ⠇ࡍࡿ㑇ఏᏊࡢྠᐃࡣ㔜せ࡛࠶ࡾࠊຠᯝⓗ࡞⫱✀ᡓ␎

ࢆᐇ⾜ࡍࡿࡓࡵࡢṧࡉࢀࡓၥ㢟Ⅼ࡛࠶ࡿࠋ

ሷࢫࢺࣞࢫࡸ㓟ᛶࢫࢺࣞࢫࡢࡼ࠺࡞ᅵተࢫࢺࣞࢫࡣࠊ࣮ࣘ࢝ࣜࡢࣉࣛࣥࢸ࣮ࢩ

ࣙࣥ࡟࠾ࡅࡿᡂ㛗ࡢపୗࡢ୺せ࡞ཎᅉ࡛࠶ࡿ (White et al. 2009; Feikema and

Baker 2011)ࠋ㓟ᛶᅵተࡣள⇕ᖏᆅᇦ࡟ᗈࡃศᕸࡋ࡚࠸ࡿࡓࡵࠊAlࢫࢺࣞࢫࡢࡼ࠺

࡞㓟ᛶᅵተࢫࢺࣞࢫ⪏ᛶ࡟㔜せ࡞㑇ఏᏊࡢྠᐃࡣࠊ⫱✀࡟࠾ࡅࡿศᏊ࣐࣮࣮࢝ࡢ㐍 Ṍ࡟㔜せ࡛࠶ࡿࠋUDP-glucoseࢆ฼⏝ࡍࡿ⪏ᛶࡸ (Fukuda et al. 2007)ࠊ⣽⬊⭷⾲

㠃ࡢప㈇㟁Ⲵࡢࡼ࠺࡞ (Wagatsuma et al. 1995)ࠊᵝࠎ࡞⪏ᛶ࣓࢝ࢽࢬ࣒ࡀከ✀ࡢ

᳜≀࡛Ⓨぢࡉࢀ࡚ࡁࡓࡀࠊ᭷ᶵ㓟ᨺฟࡣከࡃࡢ᳜≀✀࡟ඹ㏻ࡢAl⪏ᛶ࣓࢝ࢽࢬ࣒

࡜ࡋ࡚ㄆ▱ࡉࢀ࡚ࡁࡓ (Ma et al. 2001; Kochian et al. 2004)ࠋ᰿࠿ࡽࡢ᭷ᶵ㓟ᨺ ฟࡣࠊ᰿➃ࡢAlឤཷᛶ⤌⧊ࢆࡑࡢẘᛶ࠿ࡽಖㆤࡋ࡚࠸ࡿ (Delhaize et al. 1993)ࠋ

ࣜࣥ㓟࡟ࡼࡿAlẘᛶࡢ୙άᛶ໬ࡣࠊࢯࣂ (buckwheat)࡛ㄆࡵࡽࢀࡓࡀ (Zheng et

al. 2005)ࠊ᭷ᶵ㓟ࠊࡍ࡞ࢃࡕࣜࣥࢦ㓟ࠊࢡ࢚ࣥ㓟ࠊࢩࣗ࢘㓟ࡢ᰿࠿ࡽࡢᨺฟࡣࢯࣂ

ࡢࡶࡗ࡜ࡶ㔜せ࡞Al⪏ᛶ࣓࢝ࢽࢬ࣒࡛࠶ࡿ࠿ࡶࡋࢀ࡞࠸࡜ࡉࢀࡓ (e.g. Ma et al.

1998; Zheng et al. 1998)ࠋ࠸ࡃࡘ࠿ࡢ◊✲࡛ࠊ᭷ᶵ㓟௦ㅰࡢᨵኚࡣAl⪏ᛶࡢ᭷ᶵ 㓟ᨺฟࡢቑᙉ࡟ᙺ❧ࡘ࡜ࡉࢀ (e.g., canola, Anoop et al. 2003; alfalfa, Tesfaye et al. 2001)ࠊ ࡉ ࡽ ࡟ ⣽ ⬊ ⭷ ࡢ ㍺㏦ ⬟ ຊ ࡶ ᭷ ᶵ 㓟 ᨺ ฟ ࡢ ಁ 㐍せ ᅉ ࡜ ሗ ࿌ ࡉ ࢀ ࡓ (Furukawa et al. 2007; Magalhaes et al. 2007)ࠋࡇࢀ࡟ఝࡓ࣓࢝ࢽࢬ࣒࡜ࡋ࡚ࠊ

࣍࣡࢖ࢺ࣮ࣝࣆࣥ (white lupin)ࡸ (Neumann et al. 1999; Kihara et al. 2003)ࠊ ࢽࣥࢪࣥ⣽⬊ࡢኚ␗య࡛ࡣ (Takita et al. 1999; Ohno et al. 2003)ࠊࣜࣥ㓟Ḟஈ࡟

ࡼࡗ࡚᭷ᶵ㓟ᨺฟࡀ㧗ࡵࡽࢀࡿࡇ࡜ࡀุ᫂ࡋࡓࠋࡇࢀࡽࡢࣉࣟࢭࢫ࡟㛵㐃ࡍࡿ㔜せ

࡞㑇ఏᏊࡣࠊ࣐࣮࣮࢝㑅ᢤࡢ㝿࡟㓟ᛶᅵተ⪏ᛶࢆ㧗ࡵࡿศᏊ࣐࣮࣮࢝ೃ⿵࡜࡞ࡿྍ

⬟ᛶࡀ࠶ࡿࠋ

Alᛂ⟅ᛶࣜࣥࢦ㓟ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࠊALMT1 (Aluminum activated malate transporter 1) ࡣࠊࢥ࣒ࢠ࡛ྠᐃࡉࢀ (Sasaki et al. 2004)ࠊࡑࡢᚋࢩࣟ࢖ࢾࢼࢬ ࢼ࡛ࡶྠᐃࡉࢀࡓ (Hoekenga et al. 2006)ࠋ୍᪉ࠊALMT1࡜␗࡞ࡾࠊmultidrug and toxic compound extrusion (MATE) ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࣇ࢓࣑࣮ࣜ࡟ᒓࡍࡿࢡ

࢚ ࣥ 㓟 ࢺ ࣛ ࣥ ࢫ ࣏ ࣮ ࢱ ࣮ ࡣ ࠊ ᵝ ࠎ ࡞ ᳜ ≀ ✀ ࡛ ༢ 㞳 ࡉ ࢀ ࡚ ࠸ ࡿ (Rogers and Guerinot 2002)ࠋࢡ࢚ࣥ㓟ࢆ㍺㏦ࡍࡿMATEࡢ࠸ࡃࡘ࠿ࡣࠊAlᛂ⟅ᛶ࡛࠶ࡾࠊࣔ

ࣟࢥࢩ (sorghum)ࡸ (Magalhaes et al. 2007; Maron et al. 2010)ࠊ࣒࢜࢜ࢠ (barley) ࡛ ࡣ (HvAACT1: Hordeum vulgare aluminum-activated citrate transporter 1, Furukawa et al. 2007)ࠊ Al⪏ᛶ࡟㛵୚ࡍࡿ࡜≉ᐃࡉࢀࡓࠋ㑇ఏᏊ

ゎᯒ࡛ࡣࠊ࠸ࡃࡘ࠿ࡢရ✀ࡢ᭷ᶵ㓟ᨺฟ࡟ࡼࡿAl⪏ᛶࡢ㐪࠸ࡀࠊ᭷ᶵ㓟ࢺࣛࣥࢫ

࣏࣮ࢱ࣮ࡢ㌿෗ࣞ࣋ࣝࡢ㐪࠸ (Sasaki et al. 2006; Furukawa et al. 2007)࡛࠶ࡾࠊ MATE ࢱࣥࣃࢡ㉁ࡢ᭷ᶵ㓟㍺㏦⬟ຊ (Raman et al. 2008)࡟ࡼࡗ࡚ㄝ࡛᫂ࡁࡿࡇ

࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋࡇࢀ࡟ຍ࠼ࠊࢩࣟ࢖ࢾࢼࢬࢼ࡛༢㞳ࡉࢀࡓAtSTOP1ࡣ㌿෗ᅉ Ꮚ࡜ࡋ࡚ാࡁࠊࡇࡢୗὶ࡟ࡣAl⪏ᛶࠊపpH ⪏ᛶ࡟ᐤ୚ࡍࡿ࡜⪃࠼ࡽࢀࡿ㑇ఏᏊ ࡀከࡃྵࡲࢀ࡚࠸ࡓࠋࡇࢀࡣࠊMATEࢺࣛࣥࢫ࣏࣮ࢱ࣮ࡸࠊSTOP1ࡢୗὶ࡟࠶ࡿ

㑇ఏᏊࡀࠊ㓟ᛶᅵተ⪏ᛶࢆྥୖࡉࡏࡿ࣐࣮࣮࢝㑅ᢤࡢࡓࡵࡢྜ⌮ⓗ࡞ࢱ࣮ࢤࢵࢺ࡜

࡞ࡿࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࠋ

ࡇࡢ❶࡛ࡣࠊ࣮ࣘ࢝ࣜࡢ᰿࠿ࡽࡢ᭷ᶵ㓟ᨺฟࡸࠊࢡ࣮ࣟࢽࣥࢢ࡟ࡼࡿࢡ࢚ࣥ㓟

㍺㏦ࡢMATE ࢺࣛࣥࢫ࣏࣮ࢱ࣮࣍ࣔࣟࢢࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡢ◊✲࡟ࡘ࠸࡚ሗ࿌

ࡍࡿࠋࡲࡓࠊ࣮ࣘ࢝ࣜࡢSTOP1࣍ࣔࣟࢢ㑇ఏᏊࢆ༢㞳ࡋࠊࡑࢀࡀAlࠊపpH⪏ᛶ

࡟ാ࠸࡚࠸ࡿࡢ࠿ࠊࡲࡓࠊ㌿෗ᅉᏊ࡜ࡋ࡚ാࡁࠊ᪤▱ࡢMATEࢆࡣࡌࡵ࡜ࡍࡿAl

⪏ᛶ㑇ఏᏊࢆไᚚࡋ࡚࠸ࡿࡢ࠿ㄪ࡭ࡓ⤖ᯝࢆሗ࿌ࡍࡿࠋ

22-2

ᐇ㦂ᮦᩱཬࡧᐇ㦂᪉ἲ

᳜≀ᮦᩱ࠾ࡼࡧ⳦య⣔⤫

ࣘ ࣮ ࢝ ࣜ ࢝ ࣐ ࣝ ࢻ ࣞ ࣥ ࢩ ࢫ (Eucalyptus camaldulensis var. obtuse, Location: EMU CREEK PETFORD)ࡣAustralian Tree Seed Center࠿ࡽྲྀᚓࡋ ࡓࠋࡲࡓࠊ࣮ࣘ࢝ࣜGUT5ࡣࠊࢢࣛࣥࢹ࢕ࢫ (Eucalyptus grandis) ࡜࣮ࣘࣟࣇ࢕ࣛ

(E. urophylla) ࢆ஺㓄ࡋ࡚㑅ᢤࡉࢀࡓࢡ࣮ࣟࣥရ✀࡛࠶ࡿࠋࡇࡢࢡ࣮ࣟࣥࡣin vitro

࡛⤌⧊ᇵ㣴ࠊ⥅௦ࡉࢀ࡚࠸ࡿ (Kawazu et al., 1996)ࠋࢩࣟ࢖ࢾࢼࢬࢼࡣ㔝⏕ᆺ࡜ࡋ

࡚Colombia-0 (Col-0)ࢆࠊT-DNAᤄධኚ␗య࡜ࡋ࡚ALS3-KO (SALK_061074)ࢆ౑⏝

ࡋࡓࠋ௚࡟ࠊࢱࣂࢥBY-2 (Nicotiana tabacum, Bright-Yellow 2)ࠊ࣮ࣘ࢝ࣜẟ≧᰿

ࡢㄏᑟࡢࡓࡵࠊRi ࣉࣛࢫ࣑ࢻࢆᣢࡘ hyper-virulent Agrobacterium rhizogenes strain ATCC15834 (American type culture collection, VA, USA)ࢆ౑⏝ࡋࡓࠋࢩࣟ

࢖ࢾࢼࢬࢼࡢ⤌᥮యసᡂ࡟ࡣࠊAgrobacterium tumefaciens GV3101ࢆ౑⏝ࡋࡓࠋ

ࣘ

࣮࣐ࣘ࢝ࣜ࢝ࣝࢻࣞࣥࢩࢫᐇ⏕ࡢ⏕⫱᮲௳ཬࡧࢫࢺࣞࢫฎ⌮᮲௳

࣮ࣘ࢝ࣜ✀Ꮚࢆḟளሷ⣲㓟࡛✀Ꮚ⁛⳦ࡋ (1% available chlorine; 10Υ࡛ 20 ศ)ࠊ๓ᇵ㣴ࡣࢩࣟ࢖ࢾࢼࢬࢼ࡛in vitro⏕⫱ࡋࡓKobayashi et al. (2007)ࡢ᪉ἲࢆ

ࡶ࡜࡟ࠊᑡࡋࡢኚ᭦ࢆࡋࡓ࠺࠼࡛ࠊ↓⳦ⓗ࡞᮲௳࡛⾜ࡗࡓࠋ4ࡘࡢࢼ࢖ࣟࣥࡢ࣓ࢵ

ࢩࣗࢩ࣮ࢺ(1.3 cm square of 50 mesh per inch; 20 seedlings per sheet)ࢆࠊࣉࣛ

ࢫࢳࢵࢡࡢ࣓ࢵࢩࣗࢩ࣮ࢺ (5 cm square)࡟⨨ࡁࠊ150 mlࡢ๓ᇵ㣴⁐ᾮࡢධࡗࡓ

ࣉ ࣛ ࢫ ࢳ ࢵ ࢡ ࣏ ࢵ ࢺ ࡟ ᾋ ࠿ ࡭ ࠊ ᧛ ✀ ࡋ ࡓ ࠋ ๓ ᇵ 㣴 ⁐ ᾮ ࡢ ⤌ ᡂ ࡣ ࠊ ᨵ ኚ Hoagland-Arnon⁐ᾮ (0.4 mM Ca(NO3)2, 0.1 mM NH4H2PO4, 0.2 mM MgSO4, 40 M KCl, 5.4 M EDTA-Fe, 1 M MnCl2, 4.6 M H3BO3, 0.076 M ZnSO4, 0.032 M CuSO4, 0.001 M (NH4)6Mo7O24)࡟ࠊ1 %࡜࡞ࡿࡼ࠺sucroseࢆຍ࠼ࠊ pHࢆ5.6࡟ㄪᩚࡋࡓࡶࡢ࡛࠶ࡿࠋࡇࡢ࣏ࢵࢺࡣ23Υࠊ16᫬㛫᫂ᮇ (20 mol E m^-2 s^-1) ࡛⥔ᣢࡋࡓࠋ

๓ᇵ㣴6᪥ᚋࠊᗂ᳜≀యࢆᨭᣢࡋ࡚࠸ࡿ࣓ࢵࢩࣗࢩ࣮ࢺࢆࢥࣥࢺ࣮ࣟࣝ⁐ᾮࡢ ධࡗࡓ᪂ࡋ࠸ࣉࣛࢫࢳࢵࢡ࣏ࢵࢺ࡟⛣ࡋ᭰࠼ࠊ1᫬㛫๓ฎ⌮ࢆ⾜ࡗࡓࠋࢥࣥࢺ࣮ࣟ

ࣝ⁐ᾮࡣࠊ๓ᇵ㣴⁐ᾮ࠿ࡽࣜࣥ㓟ࢆ㝖ࡁࠊpHࢆ4.8 ࡟ㄪᩚࡋࡓࡶࡢ࡛࠶ࡿࠋ๓ฎ

⌮ࡋࡓᗂ᳜≀యࡣࠊ⥆ࡅ࡚ࢫࢺࣞࢫฎ⌮⁐ᾮࡢ࣏ࢵࢺ࡟⛣ࡋ᭰࠼ࡓࠋRNA ᢳฟࡢ ࡓࡵࡢࢫࢺࣞࢫฎ⌮⁐ᾮࡣࠊࢥࣥࢺ࣮ࣟࣝࠊCu (ࢥࣥࢺ࣮ࣟࣝ⁐ᾮ㸩1 M CuSO4, pH 4.8)ࠊNaCl (ࢥࣥࢺ࣮ࣟࣝ⁐ᾮ㸩30 mM NaCl, pH 4.8)ࠊAl (ࢥࣥࢺ࣮ࣟࣝ⁐ᾮ 㸩50 M AlCl3, pH 4.6)ࠊపpH (ࢥࣥࢺ࣮ࣟࣝ⁐ᾮ, pH 4.0)࡛࠶ࡿࠋ᰿࠿ࡽᨺฟࡉ

ࢀࡓ᭷ᶵ㓟ࢆᅇ཰ࡍࡿࡓࡵࠊ๓ฎ⌮ࡋࡓᗂ᳜≀యࡢ࣓ࢵࢩࣗࢩ࣮ࢺࡣ3 mlࡢࢫࢺ

ࣞࢫฎ⌮⁐ᾮࡲࡓࡣࢥࣥࢺ࣮ࣟࣝ⁐ᾮࡢධࡗࡓ6࢙࢘ࣝࣉ࣮ࣞࢺ࡟⛣ࡋ᭰࠼ࠊฎ⌮

ࢆ⾜ࡗࡓࠋࢱࣥࣃࢡ㉁ࡢࣜࣥ㓟໬ࠊ⬺ࣜࣥ㓟໬ࡢ㜼ᐖ๣ࡢຠᯝࢆㄪ࡭ࡿࡓࡵࠊ࢝ࣜ

ࢡࣜࣥ (calyculin)ࠊK-252aࠊࢫࢱ࢘ࣟࢫ࣏ࣜࣥ (staurosporin)ࠊࢩࢡࣟࢫ࣏ࣜࣥ

(cyclosporine)ࢆ⤊⃰ᗘ5 M࡟࡞ࡿࡼ࠺ຍ࠼࡚ฎ⌮ࡋࡓࠋࡍ࡭࡚ࡢฎ⌮୰ࡣࠊ25Υࠊ 㐃⥆᫂ᮇ (20 mol E m^-2 s^-1) ࡟⥔ᣢࡋࡓࠋ

ࣘ

࣮ࣘ࢝ࣜGUT5ࡢ⏕⫱᮲௳ཬࡧࢫࢺࣞࢫฎ⌮᮲௳

↓⳦ⓗ࡟⏕⫱ࡋࡓGUT5ࢆࠊ4࠿ࡽ5ᯛࡢⴥ࡜2 cmࡢ᰿ࢆṧࡋ࡚ษ᩿ࡋࠊ1%

ࡢࢫࢡ࣮ࣟࢫࢆຍ࠼ࡓ1/5⃰ᗘᨵኚHoagland-Arnon⁐ᾮ (pH 5.6) 150 mlࡢධࡗ ࡓࣉࣛࢫࢳࢵࢡࡢ࣏ࢵࢺ࡟ࠊࣉࣛࢫࢳࢵࢡ࣓ࢵࢩࣗࡢࣇ࣮ࣟࢺୖ࡛⏕⫱ࡋࡓࠋ ᗘ ᮲௳ࡣ23Υࠊ↷ᗘ20 E m⁻² s⁻¹ࠊ16᫬㛫᫂ᮇ࡛࠶ࡿࠋࡇࢀࡽࡣRNAᢳฟࡸࠊࢡ࢚

ࣥ㓟ᨺฟ ᐃ࡟౑⏝ࡋࡓࠋ⤌᥮GUT5ẟ≧᰿ࡣࠊ1% ࢫࢡ࣮ࣟࢫࢆຍ࠼ࠊࣜࣥ㓟ࢆ

㝖࠸ࡓ1/4 ⃰ᗘࡢB5 ᐮኳ (1%) ᇵᆅ (pH 4.6) ࢆࢥࣥࢺ࣮ࣟࣝ࡜ࡋࠊAl ฎ⌮༊ࡣ 50 M AlCl3 (pH 4.6)ࠊపpHฎ⌮༊ࡣpH 4.0࡟ㄪᩚࡋࡓᇵᆅࢆ౑⏝ࡋࠊ3᪥㛫ฎ⌮

ࡋࡓࠋ ᗘ᮲௳ࡣ22Υࠊ↷ᗘ20 E m⁻² s⁻¹ࠊ16᫬㛫᫂ᮇ࡛࠶ࡿࠋࢩࣟ࢖ࢾࢼࢬࢼ ࡣỈ⪔᱂ᇵ࡛➨1❶࡜ྠࡌ᮲௳࡛⾜ࡗࡓࠋࢥࣥࢺ࣮ࣟࣝࡣ1/50⃰ᗘࡢMGRLᇵᆅ

࡟200 M ࡟࡞ࡿࡼ࠺CaCl2 ࢆຍ࠼ࠊࣜࣥ㓟ࢆ㝖ࡁࠊpH 5.0࡟ㄪᩚࡋࡓࡶࡢ࡛࠶ࡿࠋ

Alฎ⌮༊ࡣࢥࣥࢺ࣮ࣟࣝ⁐ᾮ࡟2 M࡟࡞ࡿࡼ࠺Alࢆῧຍࡋࡓࠋᗂ᳜≀యࡣ25Υࠊ

↷ᗘ35 E m⁻² s⁻¹ࠊ12᫬㛫᫂ᮇ࡛7᪥㛫᱂ᇵࡋࡓࠋ⁐ᾮࡣ2᪥ࡈ࡜࡟᭦᪂ࡋࡓࠋ

RNAᢳฟࠊ㏫㌿෗ࠊDNAࢩ࣮ࢡ࢚ࣥࢫࠊ㓄ิゎᯒ

Total RNAࡣSuzukiࡽࡢ᪉ἲ࡟ࡼࡗ࡚ᢳฟࡋࡓ (Suzuki et al., 2004)ࠋTotal RNAࡣTranscriptor High Fidelity cDNA synthesis kit (Roche Applied Science, Tokyo, Japan)࡟ࡼࡗ࡚㏫㌿෗ࡋࡓࠋ㑇ఏᏊ㓄ิࡣ ABI PRISM 3130xl DNA

sequencer࡜ABI BigDye terminator system (ver3.1)ࢆ౑⏝ࡋ࡚ゎᯒࡋࡓࠋ㓄ิ

ゎ ᯒ ࠊ ࢔ ࣑ ࣀ 㓟 ࢔ ࣛ ࢖ ࣥ ࣓ ࣥ ࢺ ࠊ ⣔ ⤫ ᶞ ࡣ CLUSTALW (http://www.ebi.ac.uk/Tools/msa/clustalw2/)࡛⾜࠸ࠊ⭷ࢱࣥࣃࢡࡢ㈏㏻㡿ᇦண ࡣ HMMTOP (http://www.enzim.hu/hmmtop/)࡛⾜ࡗࡓࠋ

ࢡ

ࢡ࢚ࣥ㓟㍺㏦MATEࣇ࢓࣑࣮ࣜ㑇ఏᏊࡢ࣮ࣘ࢝ࣜ࣍ࣔࣟࢢࡢ༢㞳

ࢡ࢚ࣥ㓟㍺㏦MATEࡢcDNA᩿∦ࡣࠊdegenerate primerࢆ⏝࠸࡚Alฎ⌮ࡋ ࡓ᰿࠿ࡽᢳฟࡋࡓcDNAࢆ㗪ᆺ࡟nested PCR࡟ࡼࡗ࡚ᚓࡓࠋdegenerate primer ࡣࠊࢩࣟ࢖ࢾࢼࢬࢼ (At3g08040)ࠊ࢖ࢿ (Os03g11734.1)ࠊLupinus albus (Q3T7F5) ࡢࢡ࢚ࣥ㓟㍺㏦ MATE ࡢಖᏑ㡿ᇦࢆࡶ࡜࡟タィࡋࡓࠋ1st PCR ࡣࠊforward, 5’-GCIGCIGAYCCIYTIGCI, reverse, 5’-RCARAAIGTIACIGCIACIACࠊ2nd PCR forward, 5’-GAYACIGCITTYATHGGI, reverse, 5’-RTCYTTRAAICCICKRAA࡛

࠶ࡿࠋቑᖜ⏘≀ࢆࢧࣈࢡ࣮ࣟࢽࣥࢢࡋࠊABI PRISM 3130xl DNA sequencer࡜ABI BigDye terminator system (ver 3.1) ࡛㓄ิࢆỴᐃࡋࡓࠋࡑࡢᚋࠊSMART RACE cDNA Amplification Kit (Takara-bio, Ohtsu, Japan)࡛ྛ㑇ఏᏊ30࠿ࡽ50ࡢ᩿∦ࡀᚓࡽ

ࢀࠊ㓄ิࢆỴᐃࡋࠊ඲㛗ࢆᚓࡓࠋ༢㞳ࡋࡓࢡ࣮ࣟࣥࡢ㓄ิࡣGenbankࡢࢹ࣮ࢱ࣋

࣮ ࢫ ࡟ Ⓩ 㘓 ࡋ ࡓ (Accession numbers: EcMATE1, AB725912; EcMATE2, AB725913; EcMATE3, AB725914; EcMATE4, AB725915)ࠋGUT5 ࡢ ALS3ࠊ MATE1ࠊSTOP1ࡣOhyamaࡽ (Ohyama et al., 2013)ࡢ᪉ἲ࡛༢㞳ࡋࡓࠋ᏶඲㛗cDNA ࡣKiharaࡽ (Kihara et al., 2003) ࡢ᪉ἲ࡛3ʹࠊ5ʹ RACE (rapid amplification of cDNA ends) ἲ࡟ࡼࡾ༢㞳ࡋࡓࠋ᰿࠿ࡽtotal RNAࢆᢳฟࡋࠊTranscriptor High Fidelity cDNA synthesis kit (Roche Applied Science, Tokyo, Japan)ࢆ⏝࠸࡚oligo dTࣉࣛ࢖࣐࣮࡛㏫

㌿෗ࡋࡓ⏘≀ࢆ degenerate PCR ࡜ RACE ࡟౑⏝ࡋࡓࠋ㓄ิࡣ ABI PRISM 3130xl

DNA sequencer࡛Ỵᐃࡋࡓࠋ࢔࣑ࣀ㓟㓄ิࡢ࢔ࣛ࢖࣓ࣥࣥࢺ࡜⣔⤫ᶞࡣGENETYX

software version 11.01 (Genetyx, Tokyo, Japan) ࡜ ClustalW (http://www.ddbj.nig.ac.jp/index-e.html)࡛ స ᡂ ࡋ ࠊ ⭷ ㈏㏻㡿 ᇦ ண ࡣ HMMTOP (http://www.enzim.hu/hmmtop/) ࢆ ౑ ⏝ ࡋ ࡓ ࠋ ࡇ ࢀ ࡽ ࡢ ࢜ ࣮ ࢯ ࣟ ࢢ 㑇 ఏ Ꮚ 㓄 ิ ࡣ GenBank ࡟Ⓩ㘓ࡋࡓࠋAccession number ࡣ AB826006 (EguSTOP1)ࠊAB826007 (EguMATE1)ࠊAB826008 (EguALS3)࡛࠶ࡿࠋ

㑇

㑇ఏᏊࡢ㌿෗㔞ゎᯒ

ྛ㑇ఏᏊࡢ㌿෗ࣞ࣋ࣝࡣࠊLightCycler 480 SYBR Green I Master kit (Roche)

࡜ࣜ࢔ࣝࢱ࢖࣒PCR (LightCycler 480 SYBR Green I Master kit (Roche))࡛ゎᯒ ࡋࡓࠋ౑⏝ࡋࡓࣉࣛ࢖࣐࣮ࡣ௨ୗࡢ࡜࠾ࡾ࡛࠶ࡿࠋ࡞࠾ࠊෆ㒊ᶆ‽࡜ࡋActinࢆ౑

⏝ࡋ࡚࠸ࡿࠋ

EcMATE1: forward 5’-AGTCTCCCTTATCAGCATTGCTTCA,

reverse 5’-TAAACGTTGTGGAAGAAGTCCTTCTCTAAT, EcMATE2: forward 5’-ATGCCAGAGGACAGTGTTCAGCATCT,

reverse 5’-TGCAGTGTCAATTAGGGAAGCAACAGGATC, EcMATE3: forward 5’-GCGTTGAATCTTTCTTGATTTTG,

reverse 5’-CAGTCTCCCCACTTCAAGAATTA, EcMATE4: forward: 5’-CACAGGCGGCTTTGCTGCAA,

reverse 5’-AGGCTGATAACTATTGGCGCTG, EcActin: forward 5’-GTTGCACCTCCTGAGAGAAAGT,

reverse 5’-TAGCTCACCAACAAAGACCTTGC, EguSTOP1: forward 5ʹ-GTTTAATGAGTTCACGCGATCTGCT,

reverse 5ʹ- CACTAAAATCAGGCCACCTCGTTC, EguALS3: forward 5ʹ- AACCCTAGCACAACTCGAATCC,

reverse 5ʹ- CTCAACATGGTGCAAGAAAATG, EguMATE1: forward 5ʹ- AGTCTCCCTTATCAGCATTGCTTCA,

reverse 5ʹ- TAAACGTTGTGGAAGAAGTCCTTCTCTAAT, EguActin: forward: 5ʹ- GTTGCACCTCCTGAGAGAAAGT,

reverse 5ʹ- TAGCTCACCAACAAAGACCTTGC.

࣋

࣋ࢡࢱ࣮ᵓ⠏࠾ࡼࡧ࢔ࢢࣟࣂࢡࢸ࣒ࣜ࢘࡟ࡼࡿᙧ㉁㌿᥮

EcMATE1-4ࢆMini-Ti࡟᥋⥆ࡋࡓࣉࣛࢫ࣑ࢻ࣋ࢡࢱ࣮ࡣࠊ᳜≀య࡬ࡢᙧ㉁㌿

᥮ ࡟ ᵓ ᡂ ࡉ ࢀ ࡓ Gateway ࣋ ࢡ ࢱ ࣮ ࡛ ࠶ ࡿ pGWA2 ࢆ ౑ ⏝ ࡋ ࡚ ᵓ ⠏ ࡋ ࡓ (Nakagawa et al. 2009)ࠋpGWA2ࡣpBI121ࢆࡶ࡜࡟ࠊcauliflower mosaic virus 35Sࣉ࣮ࣟࣔࢱ࣮࡜Agrobacterium nopaline synthase terminator (NOS-T)ࡢ㛫

࡟㑇ఏᏊࢆᑟධࡍࡿGatewayࢡ࣮ࣟࢽࣥࢢࢧ࢖ࢺࢆᣢࡗ࡚࠸ࡿࠋࡇࡢ࢝ࢭࢵࢺࡣࠊ

T-DNA 㡿ᇦ࡟࠶ࡾࠊࡑࡢ㞄࡟ࡣࣁ࢖ࢢ࣐ࣟ࢖ࢩࣥ⪏ᛶ࡜࢝ࢼ࣐࢖ࢩࣥ⪏ᛶࡢ࢝ࢭ

ࢵࢺࡀ࠶ࡿࠋEcMATE1-4ࡣ Gatewayࢡࣟࢼ࣮ࢮࡢㄆ㆑㓄ิࢆ᥋⥆ࡋࡓࣉࣛ࢖࣐

࣮࡛PCRቑᖜࡋࠊT-DNAࡢGatewayࢡ࣮ࣟࢽࣥࢢࢧ࢖ࢺ࡟ᑟධࡋࡓࠋࡇࢀ࡟ࡼ

ࡾࠊẟ≧᰿࡛MATEࢆ㐣๫Ⓨ⌧ࡉࡏࡿpGWA2-EcMATE1-4 ࢆసᡂࡋࡓࠋࡍ࡭࡚

ࡢࢡ࣮ࣟࢽࣥࢢ࣋ࢡࢱ࣮ࡣࠊGateway system (Invitrogen)ࡢᶆ‽ࣉࣟࢺࢥࣝ࡟ᚑ

࠸ࠊTOPOࢡ࣮ࣟࢽࣥࢢࡢࡓࡵࡢPCRࣉࣛ࢖࣐࣮ࡣ௨ୗࡢ࡜࠾ࡾ࡛࠶ࡿࠋ EcMATE1: forward 5’-CACCATGGCCGAGGACTCTGATGTTCGTG,

reverse 5’-TCATAAACGTTGTGGAAGAAGTCCTTCTCTAAT, EcMATE2: forward 5’-CACCATGCCAGAGGACAGTGTTCAGCATCT,

reverse 5’-TTATGAAGCCTGTGGTGTACGTTGACCC, EcMATE3: forward 5’-CACCATGCCTCTGTCTATGTTCTTCAAGGA,

reverse 5’-TTAGTTATTTAGAAATCCCCAAGGTCCCATGCC,

EcMATE4: forward: 5’-CACCATGGAGCCTCTGGAGGGTTCG, reverse 5’-TCAGCCCCAGAGAAAATTCCAAGGACCC.

pGWA2-EcMATE1 ࡣ Diazࡢ᪉ἲ࡟ᚑ࠸ (Diaz et al. 1989)ࠊAgrobacterium

rhizogenes ATCC15834࡟࢚ࣞࢡࢺ࣏࣮ࣟࣞࢩ࡛ࣙࣥᑟධࡋࠊ⤌᥮ẟ≧᰿ࢆㄏᑟࡍ

ࡿࡓࡵࠊ↓⳦ⓗ࡟⏕⫱ࡉࡏࡓࢱࣂࢥBY-2ࡢⴥ࡟ឤᰁࡉࡏࡓࠋឤᰁࡉࡏࡓࢱࣂࢥⴥ (1 cm square)ࡣࠊ1 % (w/v) galactose ࡜acetosyringone (20 g/ml) ࢆྵࢇࡔ1/4

⃰ᗘࡢ B5 ᐮኳᇵᆅ (1%) ࡛ 2 ᪥㛫ᇵ㣴ࡋࠊ6.25 mg/l meropenem (Sumitomo pharmaceutical Co., Japan, Tokyo) ࡛ὙίࡋࡓࠋὙίࡋࡓⴥࡣࠊࡉࡽ࡟sucrose (1 % w/v)ࠊ meropenem (6.25 mg/l)࡜ 1 ppmࡢnaphthylacetic acid (NAA)ࢆྵ

ࢇࡔ1/4⃰ᗘࡢB5ᐮኳᇵᆅ (1%) ࡛ᇵ㣴ࡋࡓࠋᇵᆅࡣẟ≧᰿ࡀㄏᑟࡉࢀࡿࡲ࡛2 㐌㛫ࡈ࡜࡟᭦᪂ࡋࡓࠋㄏᑟࡉࢀࡓ⤌᥮ẟ≧᰿ࡣmeropenem ࡢ௦ࢃࡾ࡟50 g/ml of hygromycinࢆྵࢇࡔྠࡌᐮኳᇵᆅ࡛㑅ᢤࡋࡓࠋᇵ㣴ࡣ22Υࠊ16᫬㛫᫂ᮇ (20

mol E m^-2 s^-1) ࡛⥔ᣢࡋࡓࠋ

EguSTOP1-RNAi (RNA interference) (pGWB80-EguSTOP1-RNAi)ࡢ࣋ࢡࢱ࣮సᡂ

ࡶྠᵝ࡟⾜ࡗࡓࠋEguSTOP1 ࡢ㓄ิ 250 bp ࢆ GATEWAY binary vector pGWB80 (Nakagawa et al. 2009)࡟ ᑟ ධ ࡋ ࡓ ࠋpGWB80 ࡣ ࢩ ࣟ ࢖ ࢾ ࢼ ࢬ ࢼ isocitrate

dehydrogenase (At1g65930)ࡢ࢖ࣥࢺࣟࣥࡢ୧➃࡟GATEWAY࡟ࡼࡾ┦⿵ࠊ㏫┦⿵࡟

࡞ࡿࡼ࠺᩿∦ࢆᑟධࡍࡿࡇ࡜࡛ RNAi ࢆᵓ⠏࡛ࡁࡿࡼ࠺࡟ࡋࡓ࣋ࢡࢱ࣮࡛࠶ࡿࠋ pGWB80-EguSTOP1-RNAi ࡣ ୖ グ ࡜ ྠ ᵝ ࡟ ࢚ ࣞ ࢡ ࢺ ࣟ ࣏ ࣞ ࣮ ࢩ ࣙ ࣥ ࡛ Agrobacterium rhizogenes ATCC15834࡟ᑟධࡋࠊGUT5ࡢⴥ࡟ឤᰁࡉࡏࡿࡇ࡜࡛⤌᥮

ẟ≧᰿ࢆᚓࡓࠋࡲࡓࠊpBI121 (with a GUS cassette in the T-DNA)ࢆྠᵝ࡟⤌ࡳ᥮࠼ࡓ ẟ≧᰿ࢆࢥࣥࢺ࣮ࣟࣝ࡜ࡋࡓࠋ

ࢩࣟ࢖ࢾࢼࢬࢼ┦⿵ヨ㦂ࡢ࣋ࢡࢱ࣮ࡣࠊEguALS3࡜AtALS3 (At2g37330)ࡢࣉࣟ

࣮ࣔࢱ࣮ (ATGࡢୖὶ⣙ -700 bp)ࠊ3’➃㡿ᇦ (stopࢥࢻࣥ࠿ࡽ400 bp)ࢆ᥋⥆ࡍࡿࡓ

ࡵ overlap extension PCR (Horton et al., 1989) ࢆ⾜ࡗ࡚సᡂࡋࡓࠋࡇࡢ DNA ࡣ pBIG-HYGࡢT-DNA㡿ᇦࡢGUSࡢ㒊ศ࡟ᑟධࡋࠊAgrobacterium tumefaciens strain GV3101࡛ࢩࣟ࢖ࢾࢼࢬࢼALS3-KOࣛ࢖ࣥ࡟floral dipἲ (Clough et al., 1998)࡛ឤ ᰁࡉࡏࡓࠋT2✀Ꮚࢆ⏕⫱ヨ㦂ࠊ㌿෗ゎᯒ࡟⏝࠸ࡓࠋ

࢔

࢔ࢢࣟࣂࢡࢸ࣒ࣜ࢘ࢆ⏝࠸ࡓ୍㐣ⓗⓎ⌧࡟ࡼࡿ EcMATE1ࠊEguSTOP1 ࡢ⣽⬊ෆ ᒁᅾ

EcMATE1࠾ࡼࡧEguSTOP1ࡢ⣽⬊ෆᒁᅾࡣSparkes (2006)ࡢ᪉ἲࢆࡶ࡜࡟ࠊ ࢱ ࣂ ࢥ ᰿ ࡢ ࢔ ࢢ ࣟ ࣂ ࢡ ࢸ ࣜ ࢘ ࣒ ࢆ ౑ ⏝ ࡋ ࡓ ୍ 㐣 ⓗ Ⓨ ⌧ ࡛ ゎ ᯒ ࡋ ࡓ ࠋgreen fluorescent protein (sGFP; see Sawaki et al. 2009)ࢆࢥ࣮ࢻࡋࡓcDNAࢆྛ㑇ఏ ᏊࡢCᮎ➃࡟PCR࡛᥋⥆ࡋࡓࠋࡑࢀࡒࢀࡢ㑇ఏᏊࡣࠊಶู࡟PCRቑᖜࡋࡓࡀࠊ ࡑࡢ࡜ࡁ࡟౑⏝ࡋࡓࣉࣛ࢖࣐࣮ࢆ௨ୗ࡟♧ࡍࠋ

EcMATE1: forward 5’-TTAACCCGGGATGGCCGAGGACTCTGATGT-3’,

reverse 5’-TCGCCCTTGCTCACCATTAAACGTTGTGGAAGAAGT-3’

sGFP: forward 5’-ACTTCTTCCACAACGTTTAATGGTGAGCAAGGGCGA-3’

reverse 5’-CGAAGAGCTCTTACTTGTACAGCTCGTCCA-3’

EcMATE1-sGFPࠊEguSTOP1-sGFPࡣEcMATE1ࡲࡓࡣEguSTOP1ࡢforward

࡜sGFP ࡢreverse ࣉࣛ࢖࣐࣮࡛PCR ࡟ࡼࡗ࡚⤖ྜࡋࡓࠋࡇࡢ DNAࡣ pBI121 ࡢ -glucuronidase ࡜ ⨨ ࡁ ᥮ ࠼ ࠊhypervirulence Agrobacterium tumefaciens EHA101 ࡟ᑟධࡋࡓࠋᐮኳᇵᆅ (1 % (w/v) sucrose ࢆྵࡴ 1/2 ⃰ᗘ MS ᇵᆅ (Murashige and Skoog 1962) containing)࡛ࢱࣂࢥ᰿ࢆ4㐌㛫⏕⫱ࡋࠊ⵨␃Ỉ࡛Ὑ ίࡋࡓࠋࡑࡢᚋࠊ150 MࡢacetosyringoneࢆྵࡴAgrobacterium buffer (pH 5.6 of 10 mM MES/KOH, 10 mM of MgCl2, OD660 = 0.2)࡟ᾐࡋࡓࠋࡇࡢᗂ᳜≀యࢆ‵

ࡽࡏࡓࣇ࢕ࣝࢱ࣮࣮࣌ࣃ࣮ୖ࡛2᪥㛫⥔ᣢࡋࠊ᰿⣽⬊ࡢGFPࢆ⺯ග㢧ᚤ㙾 (Axio,

Zeiss-Japan, Tokyo)࡛ほᐹࡋࡓࠋ

᰿᰿࠿ࡽᨺฟࡉࢀࡓࣜࣥࢦ㓟࡜ࢡ࢚ࣥ㓟ࡢᐃ㔞

GUT5ࡢࢡ࢚ࣥ㓟ᨺฟᐇ㦂࡛ࡣࠊ㡰໬3㐌㛫ࡢ᳜≀యࢆ౑⏝ࡋࡓࠋ

⁐ᾮ୰ࡢ ࢡ࢚ࣥ㓟 ࡜ ࣜࣥࢦ㓟 ࡣࠊKihara (2003)ࡢ᪉ἲ࡛ࠊNAD⁺/NADH cycling method (Hampp et al. 1984)࡟ࡼࡿ㓝⣲཯ᛂ࡛ ᐃࡋࡓࠋࣜࣥࢦ㓟࡜ࢡ࢚

ࣥ㓟ࡣ໬Ꮫ㔞ⓗ࡟malate dehydrogenaseࡸcitrate lyase࡟ࡼࡗ࡚NADH࡟ኚ᥮

ࡉࢀࠊࡑࡢᚋNADHࢆcycling method࡛ᐃ㔞໬ࡋࡓࠋ

⤌᥮ࢱࣂࢥẟ≧᰿ࡢ᭷ᶵ㓟ᨺฟ

⤌᥮ࢱࣂࢥẟ≧᰿ࡣࠊ2 % sucrose ࢆྵࡴ1/4⃰ᗘMS solution (pH 5.6)࡛1 㐌㛫๓ᇵ㣴ࡋࡓࠋษࡾྲྀࡽࢀࡓ᰿➃ (3 cm; 100 root tips)ࡣࠊ30 mlࡢࢥࣥࢺ࣮ࣟ

ࣝ⁐ᾮ (1 % (w/v) sucroseࢆྵࡴ1/20⃰ᗘMS solution (-Pi), pH 4.8)ࡢධࡗࡓ100 mlࣇࣛࢫࢥ୰࡛2᫬㛫๓ฎ⌮ࢆ⾜ࡗࡓࠋ๓ฎ⌮ᚋࠊ᰿ࡣ30 ml ࡢࢥࣥࢺ࣮ࣟࣝ⁐

ᾮࡲࡓࡣAlࢫࢺࣞࢫ⁐ᾮ (๓ฎ⌮⁐ᾮ + 25 M AlCl3)࡟⛣ࡋ᭰࠼ࡓࠋࡇࡢᇵᆅࡣࠊ 24᫬㛫࡛᭦᪂ࡋࠊ48᫬㛫ࡲ࡛ฎ⌮ࡋࡓࠋࡍ࡭࡚ࡢࣇࣛࢫࢥࡣᬯᡤࠊ25Υ࡛ᅇ㌿ᇵ 㣴 (50 rpm)ࡋࡓࠋ24᫬㛫ฎ⌮ࡋࡓᇵᆅࡣ᭷ᶵ㓟ゎᯒ࡟౑⏝ࡋࡓࠋ⤌᥮ẟ≧᰿ࡢᡂ 㛗ࡣ᭷ᶵ㓟ᨺฟヨ㦂࡟౑⏝ࡋࡓࢥࣥࢺ࣮ࣟࣝ࡜Alࢫࢺࣞࢫ⁐ᾮ࡛3᪥㛫ࡢ᰿ఙ㛗

࡟ࡼࡗ࡚ヨ㦂ࡋࡓࠋ

⤫ィゎᯒ

ࡍ࡭࡚ࡢᐇ㦂ࡣᑡ࡞ࡃ࡜ࡶ2ᅇ཯᚟࡛⾜ࢃࢀࠊ෌⌧ᛶࢆ☜ㄆࡋ࡚࠸ࡿࠋ⤫ィゎ ᯒࡣ࣐࢖ࢡࣟࢯࣇࢺࡢ࢚ࢡࢭࣝࢆ౑⏝ࡋࠊstudentࡢt-testࢆ⾜ࡗࡓࠋ

22-3

ᐇ㦂⤖ᯝ

࣮ࣘ࢝ࣜࡢ᭷ᶵ㓟ᨺฟ

ᵝࠎ࡞᳜≀ࡣ Al ࡟ᛂ⟅ࡋ࡚᭷ᶵ㓟ࢆᨺฟࡋ࡚࠸ࡿࡀࠊᨺฟࡍࡿ᭷ᶵ㓟ࡢ✀㢮

ࡸㄏᑟ᫬㛫࡟㛵ࡍࡿᨺฟࣃࢱ࣮ࣥࡢ㐪࠸ࡀ࠶ࡿࠋணഛᐇ㦂࡛ࠊ࣮࣐ࣘ࢝ࣜ࢝ࣝࢻࣞ

ࣥࢩࢫࡢ᰿࠿ࡽᨺฟࡉࢀࡿ୺࡞᭷ᶵ㓟ࢆ ᐃࡋࡓ⤖ᯝࠊࣜࣥࢦ㓟࡜ࢡ࢚ࣥ㓟ࡢ୧᪉

ࢆ᳨ฟࡋࡓࡀࠊࢩࣗ࢘㓟ࡣ᳨ฟࡉࢀ࡞࠿ࡗࡓࠋᚑࡗ࡚ࠊᇵᆅ୰ࡢࣜࣥࢦ㓟࡜ࢡ࢚ࣥ

㓟ࢆᐃ㔞ࡍࡿࡇ࡜࡟ࡋࡓࠋ

ࣜࣥࢦ㓟࡜ࢡ࢚ࣥ㓟ࡢ࡝ࡕࡽࡀ᰿࠿ࡽᨺฟࡉࢀࡿ୺࡞᭷ᶵ㓟࠿ㄪ࡭ࡿࡓࡵ࡟ࠊ

᧛✀1㐌㛫ࡢ࣮࣐ࣘ࢝ࣜ࢝ࣝࢻࣞࣥࢩࢫᗂ᳜≀య (᰿㛗5-10 mm)ࢆAl⁐ᾮ࡛ฎ⌮

ࡋࡓࠋࢡ࢚ࣥ㓟ᨺฟࡣࠊAl⃰ᗘࡀ25 M௨ୖ࡛ቑຍࡍࡿࡀࠊ୍᪉࡛ࣜࣥࢦ㓟ᨺฟ ࡣAl࡟ࡼࡿᛂ⟅ࡣ࡞࠿ࡗࡓ (Fig. 10a)ࠋAl⁐ᾮ୰ࡢࢡ࢚ࣥ㓟ᨺฟࡣࡑࡢ᫬㛫࡟౫ Ꮡࡋࡓ (Fig. 10b)ࠋᨺฟ㔞ࢆ3᫬㛫ࡈ࡜࡟ㄪ࡭ࡓ࡜ࡇࢁࠊᑡ࡞ࡃ࡜ࡶ12᫬㛫ࡲ࡛

ቑຍࡋࡓࠋࡇࢀࡽࡢ⤖ᯝࡣࠊ࣮࣐ࣘ࢝ࣜ࢝ࣝࢻࣞࣥࢩࢫࡀAlㄏᑟᛶࢡ࢚ࣥ㓟ᨺฟ ࡢ⬟ຊࢆᣢࡗ࡚࠸ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋ

ࡉࡽ࡞ࡿ᰿࠿ࡽࡢࢡ࢚ࣥ㓟࡜ࣜࣥࢦ㓟ᨺฟࢆẚ㍑ࡍࡿࡓࡵࠊ௚ࡢࢫࢺࣞࢫ࡛ฎ

⌮ࡋࡓ᫬ࡢࠊ୧⪅ࡢ᭷ᶵ㓟ᨺฟ㔞ࢆㄪ࡭ࡓࠋࡑࢀࡒࢀࡢฎ⌮⃰ᗘࡣࠊ50 M Alࠊ పpH (pH4.0)ࠊ1.0 M Cuࠊ30 mM NaCl ࡛ࠊࢥࣥࢺ࣮ࣟࣝ⁐ᾮࡢ᫬࡜ẚ㍑ࡋ࡚

50%ࡢ᰿ఙ㛗㜼ᐖࢆ♧ࡍ⃰ᗘ࡛࠶ࡿ (Fig. 10c)ࠋࡍ࡭࡚ࡢฎ⌮࡟࠾࠸࡚ࠊࣜࣥࢦ㓟 ࡢᨺฟࣞ࣋ࣝࡣఝࡓࡼ࠺࡞ࡶࡢ࡛࠶ࡗࡓࡀࠊࢡ࢚ࣥ㓟ᨺฟࡣAlࡢࡳ࡟࠾࠸᳨࡚ฟ

ࡉࢀࡓ (Fig. 10d)ࠋࡇࢀࡽࡢ⤖ᯝࡣࢡ࢚ࣥ㓟ࡀAlࢫࢺࣞࢫ࡟ᑐࡋ࡚≉␗ⓗ࡟ᨺฟ

ࡉࢀࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋ

Figure 10. Citrate and malate release profile in Eucalyptus camaldulensis. a Response to Al concentration. b Time-course response to Al (50 M) stress. c Growth of roots with various stressors (50 M AlCl3, low pH (pH 4.0), 1.0 M CuSO4 and 30 mM NaCl) for 3 days. d Citrate release with various stressors (identical to conditions used for c). Each solution was adjusted to pH 4.6 except the low pH treatment. Each experiment was replicated three times and mean ± SD are shown.

PCRἲࢆ฼⏝ࡋࡓEcMATEࡢ༢㞳

ࡍ࡛࡟ሗ࿌ࡉࢀࡓࢡ࢚ࣥ㓟㍺㏦ MATE 㑇ఏᏊࡢ࢔࣑ࣀ㓟㓄ิࡢಖᏑ㡿ᇦ࠿ࡽ

సᡂࡋࡓdegenerate primerࢆ౑⏝ࡋࡓ࡜ࡇࢁࠊEcMATEࣇ࢓࣑࣮ࣜ㑇ఏᏊࢆࢥ

࣮ࢻࡍࡿcDNA᩿∦ࡀ⣙40ಶᚓࡽࢀࡓࠋࡇࡢ᩿∦࠿ࡽసᡂࡉࢀࡓࣉࣛ࢖࣐࣮ࢆ⏝

࠸࡚3’RACEࠊ5’RACEࢆ⾜࠸ࠊ4 ࡘࡢ␗࡞ࡿ EcMATE ࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ

㑇ఏᏊࢆ༢㞳ࡋࡓࠋࡇࢀࡽࡢcDNAࢡ࣮ࣟࣥࡣdegenerate primerࡢ఩⨨࡟ಖᏑ ࡉࢀࡓ࢔࣑ࣀ㓟㓄ิࢆᣢࡗ࡚࠾ࡾࠊ502࠿ࡽ579ࡢ࢔࣑ࣀ㓟ࢆࢥ࣮ࢻࡋ࡚࠸ࡿࢱࣥ

ࣃࢡ㉁࡛࠶ࡾ (Table 7)ࠊࡇࢀࡽࡢ㑇ఏᏊࡣ5’࡜3’ࡢUTR㡿ᇦࢆᣢࡗ࡚࠸ࡓࠋ

EcMATEࡢ࢔࣑ࣀ㓟㓄ิࡣ௚ࡢ᳜≀✀ࡢࢡ࢚ࣥ㓟㍺㏦MATEࡢ඾ᆺⓗ࡞≉ᚩ

ࢆᣢࡗ࡚࠸ࡓ (Fig. 11a)ࠋࡍ࡭࡚ࡢ EcMATEࢱࣥࣃࢡ㉁ࡣ 12 ࡢ⭷㈏㏻㡿ᇦࢆᣢ ࡗ࡚࠾ࡾࠊ௚ࡢࢡ࢚ࣥ㓟㍺㏦MATE ࢱࣥࣃࢡ㉁࡛ࡶྠᐃࡉࢀ࡚࠸ࡿࠋ⣔⤫ᶞ࡟ࡼ

ࡿゎᯒ࡛ࡣࠊEcMATE1࡜௚ࡢ2ࡘࡢࢱࣥࣃࢡ㉁ࡀྠࡌࢢ࣮ࣝࣉ࡟ධࡗࡓࡀࠊ୍᪉

࡛ࠊEcMATE3࡜EcMATE4ࡣูࡢࢢ࣮ࣝࣉ࡟ศ㢮ࡉࢀࡓ (Fig. 11b)ࠋࡇࢀࡽࡢࡍ

࡭࡚ࡢࢱࣥࣃࢡ㉁ࡣࠊ༢Ꮚⴥ᳜≀ࡢ࣍ࣔࣟࢢࡼࡾࡶࠊࢩࣟ࢖ࢾࢼࢬࢼࠊ࣍࣡࢖ࢺࣝ

࣮ࣆࣥࡢࡼ࠺࡞཮Ꮚⴥ᳜≀࡟㏆࠿ࡗࡓࠋAtFRD3 (Arabidopsis thaliana ferric redictase defective 3)ࡣ㕲ྲྀᚓ࡟㛵ಀࡍࡿࢡ࢚ࣥ㓟ࢺࣛࣥࢫ࣏࣮ࢱ࣮࡛࠶ࡿࡀࠊ

EcMATE1ࠊEcMATE2࡟᭱ࡶ㏆ࡃࠊ୍᪉࡛ࠊAtMATEࡣࢩࣟ࢖ࢾࢼࢬࢼࡢAlᛂ

⟅ࢡ࢚ࣥ㓟㍺㏦MATE࡛࠶ࡿࡀࠊEcMATE4࡟᭱ࡶ㏆࠿ࡗࡓࠋ

᰿ẘᛶฎ⌮࡟ᛂ⟅ࡍࡿEcMATE㑇ఏᏊࡢ㌿෗ࣉࣟࣇ࢓࢖ࣝ

EcMATE 㑇ఏᏊࡢⓎ⌧ࢆㄪ࡭ࡿࡓࡵࠊᵝࠎ࡞ࢫࢺࣞࢫฎ⌮࡟࠾ࡅࡿ᰿࡜ᆅୖ

㒊ࡢ㌿෗ࣞ࣋ࣝࢆㄪ࡭ࡓࠋEcMATE4ࡢ㌿෗ࡣ᰿࡜ᆅୖ㒊ࡢ୧᪉࡛㌿෗ࡉࢀ࡚࠸ࡓ ࡀࠊ୍᪉࡛ EcMATE1ࠊ2ࠊ3 ࡣᵝࠎ࡞ࢫࢺࣞࢫ࡛ᆅୖ㒊ࡼࡾࡶ᰿࡛㧗࠸㌿෗㔞࡛

࠶ ࡗ ࡓ ( F i g . 1 2 )ࠋ ᰿ ࡛ 㧗㌿ ෗ ࡍ ࡿ E c M A T E ࣍ ࣔ ࣟ ࢢ ࡢ ࠺ ࡕ ࡛ ࠊ

Table 7. 䚷Basal information of MATE family genes in E. camaldulensis.

gene name ORF (bp) amino acid (AA) transmembrane helices

EcMATE1 1740 579 12

EcMATE2 1641 546 12

EcMATE3 1509 502 12

EcMATE4 1608 534 12

The transmembrane helices were predicted by the HMMTOP program.

Figure 11. Amino acid alignment (a) and phylogenetic tree (b) of citrate-transporting MATE proteins from Eucalyptus and other plant species.

EcMATE1–4, Eucalyptus camaldulensis; AtMATE (At1g51340) and AtFRD3 (At3g08040), Arabidopsis thaliana; OsFRDL1 (Os03g0216700), Oryza sativa;

HvAACT1 (BAF75822), Hordeum vulgare; SbMATE (ABS89149), Sorghum bicolor; LaMATE (AAW30733), Lupinus albus; ZmMATE1 (FJ015156.1), Zea maize. TM1–12 in a indicates transmembrane regions predicted by HMMTOP, and the box indicates the highly conserved region. The scale bar in b indicates amino acid substitutions per site. The actual value depends on the branch lengths in the tree.

FFigure 12. Comparison of expression level of EcMATE genes in the shoots with various rhizotoxic treatment. Amplicons for EcMATE1-4 genes in semi-quantitative RT-PCR were analyzed by gel electrophoresis. The Actin gene was used as an internal control.

EcMATE1ࡢ㌿෗ࡣ50 %ᡂ㛗㜼ᐖࢆ㉳ࡇࡍࢫࢺࣞࢫฎ⌮ࡢ୰࡛Al࡜పpH࡟ᛂ⟅

ࡋࡓ (Fig. 10c)ࠋEcMATE1ࡢ㌿෗ࡣAl࡜పpH࡟ࡼࡗ࡚ㄏᑟࡉࢀࠊNaCl (30 mM)

࡜㖡 (1.0 M)࡛ࡣㄏᑟࡉࢀ࡞࠿ࡗࡓ (Fig. 13a)ࠋEcMATE1 ࡢ᭱ࡶ㧗࠸ fold change (i.e., Al/control) ࡣࠊAlฎ⌮4᫬㛫ᚋ࡛6.4ࡔࡗࡓࡀࠊࡇࡢ࡜ࡁࢡ࢚ࣥ㓟 ᨺฟࡀጞࡲࡗࡓ (Figs. 10b, 13b)ࠋEcMATE1ࡢ㌿෗ࡣAl࡜పpH࡛ࢫࢺࣞࢫᙉᗘ

࡟ẚ౛ࡋ࡚ㄏᑟࡉࢀ࡚࠸ࡓ (Fig. 13c, d)ࠋຍ࠼࡚ࠊEcMATE1ࡢ㌿෗ࡣ᰿ඖࡼࡾࡶࠊ

᭷ᶵ㓟ࡀᨺฟࡉࢀࡿ᰿➃࡛㧗࠿ࡗࡓ(Figs. 13e, 14)ࠋࡇࢀࡽࡢ⤖ᯝࡣࠊEcMATE1 ࡣ࣮࣐ࣘ࢝ࣜ࢝ࣝࢻࣞࣥࢩࢫ࡛Alㄏᑟࢡ࢚ࣥ㓟ᨺฟ࡟㔜せ࡞ᙺ๭ࢆࡋ࡚࠸ࡿࡇ࡜

ࢆ♧ࡋ࡚࠸ࡿࠋ

EcMATE1ࡢ⣽⬊ෆᒁᅾ

EcMATE1ࡢ⣽⬊ෆᒁᅾࢆྍど໬ࡍࡿࡓࡵࠊ࢔ࢢࣟࣂࢡࢸ࣒ࣜ࢘ࢆ౑⏝ࡋࡓ୍

㐣ⓗⓎ⌧࡟ࡼࡾ EcMATE1:sGFP ࡢᒁᅾࢆゎᯒࡋࡓࠋ⣽⬊㉁ෆ sGFP ࡀࢱࣂࢥ᰿

࡟ᑟධࡉࢀࡓ᫬ࠊ⣽⬊ࡢ඲యࡀ⺯ග࡛᫂ࡿࡃගࡗ࡚࠸ࡓ (Fig. 15a, c)ࠋ୍᪉ࠊ EcMATE1:sGFPࡢⓎ⌧ࡣࠊ⣽⬊ࡢ➃࡛⥳ࡢ⺯ගࡀከࡃほᐹࡉࢀࡓ (Fig. 15b, d)ࠋ

ࡇࢀࡣEcMATE1ࢱࣥࣃࢡ㉁ࡀ⣽⬊⭷࡟ᒁᅾࡋ࡚࠸ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋ

ࢱࣂࢥẟ≧᰿࡛ࡢEcMATE1ࡢⓎ⌧

EcMATE1 ࡢ Al ᛂ⟅ࢡ࢚ࣥ㓟ᨺฟᶵ⬟ࢆㄪ࡭ࡿࡓࡵࠊRi ࣉࣛࢫ࣑ࢻ࡜ࠊ

EcMATE࡜Km ⪏ᛶ㑇ఏᏊࢆT-DNA 㡿ᇦ࡟⤌ࡳ㎸ࢇࡔTi ࣉࣛࢫ࣑ࢻࢆసᡂࡋࠊ

h y p e r- v i r u l e n A . r h i z o g e n e s ࢆ ౑ ࠸ ⤌ ᥮ ࢱ ࣂ ࢥ ẟ ≧ ᰿ ࢆ స ᡂ ࡋ ࡓ ࠋ C a M V 3 5 s : : E c M AT E 1 ࢆ ⤌ ࡳ ᥮ ࠼ ࡓ ࢱ ࣂ ࢥ ẟ ≧ ᰿ ࡣ ࠊA l ᇵ ᆅ ࡛ G U S

( -glucuronidase) ࢆ⤌ࡳ᥮࠼ࡓࢥࣥࢺ࣮ࣟࣝ࡜ẚ࡭ࠊࢡ࢚ࣥ㓟ࢆࡼࡾከࡃᨺฟࡋ

ࡓ ࠋE c M A T E 1 ⤌ ᥮ య ࡣ A l ࡢ ྵ ࡲ ࡞ ࠸ ᇵ ᆅ ࡛ ࡢ ࢡ ࢚ ࣥ 㓟 ᨺ ฟ ࡀ

FFigure 13. MATE gene expression analysis of E. camaldulensis roots treated with ion stress. a Expression of MATE1-4 to various stressors for 24 h (see conditions in Fig. 10d), b Timecourse analysis of EcMATE1 expression treated with 50 M Al by real-time PCR. Dose response analysis of Al (c) and low pH (d).

e Relative expression of EcMATE1 in root tip (0-5 mm) and mature part of roots (>5 mm) with or without 50 M Al for 24 h. Expression level was quantified by realtime PCR. Means of three replicates and the error bar ± SD are indicated.

Figure 14. Position of the organic acid efflux in the roots was visualized by the method of Takahashi et al. (1999, Simple visual detection of Al-tolerance level in plant root by decoloring the filter paper stained by hematoxylin : Study using the root of carrot seedling. Jap J Soil Sci Plant Nutr, in Japanese 70:554̺557).

Briefly, primary roots of seedlings were placed on a filter paper, which was pre-stained in 0.2% hematoxiline and 5 M of AlCl3 at pH 4.5 for 1 hour and supported on a grass plate. After 12 hours of incubation, distained position of the hematoxiline-stained-paper was photographed.

Figure 15. Subcellular localization of EcMATE1::sGFP fusion protein in tobacco root cells. Genes for fusion protein of EcMATE1::sGFP (a, b) and cytosol-localizing sGFP (c, d) were introduced to tobacco by Agrobacterium transformation then visualized by a fluorescent microscope at 3 days after infection (a, c). Bright field images (b, d) are also shown. Bar indicates 50 m.

ቑຍࡋ࡞࠿ࡗࡓ (Fig. 16a)ࠋຍ࠼࡚ࠊEcMATE1⤌᥮ࢱࣂࢥẟ≧᰿ࡢᡂ㛗ࡣࠊGUS

⤌᥮ࢥࣥࢺ࣮ࣟࣝࡼࡾࡶⰋࡃ࡞ࡗࡓ (Fig. 16b)ࠋ௚ࡢ࣍ࣔࣟࢢ࡛ࡣࠊEcMATE3ࡣ Alᛂ⟅ࢡ࢚ࣥ㓟ᨺฟࢆ♧ࡋࡓࡀ (Fig. 17)ࠊ୍᪉࡛Al࡟ࡼࡿ㌿෗ㄏᑟࡣ࡞࠿ࡗࡓ (Fig. 13a)ࠋࡇࢀࡽࡢ⤖ᯝࡣࠊEcMATE1ࡣ࣮࣐ࣘ࢝ࣜ࢝ࣝࢻࣞࣥࢩࢫ࡟࠾ࡅࡿAl ᛂ⟅ࢡ࢚ࣥ㓟㍺㏦MATEࢆࢥ࣮ࢻࡋ࡚࠸ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋ

pprotein kinase࡜phosphataseࡢ㜼ᐖ๣࡟ᑐࡍࡿEcMATE1㌿෗ᛂ⟅

࣮ࣘ࢝ࣜࡢࢡ࢚ࣥ㓟ᨺฟࡀࢱࣥࣃࢡ㉁ࡢࣜࣥ㓟໬ࠊ⬺ࣜࣥ㓟໬ࡢࣉࣟࢭࢫ࡟ࡼ

ࡗ࡚ㄪ⠇ࡉࢀ࡚࠸ࡿ࠿࡝࠺࠿ࢆㄪ࡭ࡿࡓࡵࠊAlᛂ⟅ࢡ࢚ࣥ㓟ᨺฟࡀᵝࠎ࡞protein kinase inhibitorࡸphosphatase inhibitorࡢᏑᅾ࡛࡝ࡢࡼ࠺࡟ኚ໬ࡍࡿࡢ࠿ㄪ࡭

ࡓ (Fig. 18)ࠋAl ᛂ ⟅ ࢡ ࢚ ࣥ 㓟 ᨺ ฟ ࡣ protein kinase inhibitor (K-252aࠊ staurosporine)࡜phosphatase inhibitor (calyculin Aࠊcyclosporine A)ࡢ୧᪉࡟ᙳ 㡪ࢆཷࡅࡿࠋCyclosporin Aࡣᨺฟࢆ40㸣㜼ᐖࡋࠊ୍᪉࡛௚ࡢ㜼ᐖ๣ࡣᨺฟࢆ70㸣 ௨ୖ㜼ᐖࡋࡓ (Fig. 18a)ࠋK-252aࠊstaurosporineࠊcalyculinࡢ㜼ᐖ๣ࡣEcMATE1 ࡢ㌿෗ࢆ㜼ᐖࡋࡓࡀ (Fig. 18b)ࠊEcMATE2ࡢ㌿෗ࡣኚ໬ࡋ࡞࠿ࡗࡓ (Fig. 19)ࠋ ࡇࢀࡽࡢ⤖ᯝࡣࠊEcMATE1ࢆ㏻ࡌࡓAlᛂ⟅ࢡ࢚ࣥ㓟ᨺฟࡀࠊࡑࡢㄏᑟࣉࣟࢭࢫ

࡟࠶ࡿࢱࣥࣃࢡ㉁ࡢࣜࣥ㓟໬ࠊ⬺ࣜࣥ㓟໬࡟㛵ಀࡋ࡚࠸ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋ

࣮ࣘ࢝ࣜSTOP1┦ྠ㑇ఏᏊࡢ༢㞳

AtSTOP1 㑇ఏᏊࡢ࣮࢜ࢯࣟࢢࡣ degenerate PCRࠊRACE ࡟ࡼࡗ࡚࣮ࣘ࢝ࣜ

GUT5࠿ࡽ༢㞳ࡉࢀࡓࠋ1959 bpࡢ༢୍ࢡ࣮ࣟࣥࡀ༢㞳ࡉࢀࠊ217 bpࡢ3’➃࡜158 bp

ࡢ 5’➃ࠊORF ࡣ 528 ࡢ࢔࣑ࣀ㓟࡛ᵓᡂࡉࢀ࡚࠸ࡓ (Fig. 20)ࠋ࢔࣑ࣀ㓟㓄ิ࡟ࡣ

zinc-finger domainࡢ㓄ิࡀྵࡲࢀࠊ௚ࡢ᳜≀✀ࡢSTOP1 (AtSTOP1, ART1, NtSTOP1, AtSTOP2, PpSTOP1) ࡜㧗࠸┦ྠᛶࢆ♧ࡋࡓ(zinc-finger domain ࡢ㓄ิ࡛

Figure 16. Citrate release and growth of transgenic tobacco hairy roots carrying EcMATE1. a Citrate release at 24 h in Al (25 M at pH 4.8) and control (pH 4.8) media. b Relative root elongation (25 M Al at pH 4.8-0 Al control at pH 4.8).

GUS transgenic lines were used as a control experiment. Mean ± SD are shown (n = 3). Asterisks indicate significant difference compared with each control (GUS) line by student t test (P<0.05)

Figure 17. Citrate and malate excretion from the transgenic tobacco hairy roots carrying EcMATE1-4. Citrate (a) and malate (b) excretion at 24 h in Al (25 M at pH 4.8) and control (pH 4.8) media are shown (Means ± SD, n=3). GUS transgenic lines were used as a control experiment. Asterisks indicate significant difference compared with each control (GUS) line by student t-test (P < 0.05).

Figure 18. Effect of protein kinase and phosphatase inhibitors on citrate release and EcMATE1 expression. a Citrate release to Al solution (50 M, pH 4.6) from E. camaldulensis roots in the presence of protein kinase inhibitors (K-252a and staurosporin A) and protein phosphatase inhibitors (calyculin A and cyclosporin A) or the absence of protein phosphorylation/dephosphorylation inhibitors. b Transcripts of EcMATE1 in roots treated with Al solution (50 M, pH 4.6) alone or supplemented with calyculin A or K-252a. Citrate released into the medium and the transcript levels of EcMATE1 were analyzed after 24-h treatment. Each experiment was replicated three times and mean ± SD are shown. Calyculin A and K-252a greatly repressed Al responsive citrate release from the roots

FFigure 19. Expression level of actin and EcMATE2, a constitutively expressing homologue, under Al treatment in the presence or absence of various kind of inhibitors for protein phosphorylation and de phosphorylation (see detail in Fig.

17 legend).

FFigure 20. Deduced amino acid sequence of EguSTOP1 and orthologous genes in other plant species. Letters highlighted with black are strictly conserved among orthologues and those with gray belong to the same amino acid group.

C2H2 domain 1

EguSTOP1 0 --- 0 AtSTOP1 0 --- 0 OsART1 0 --- 0 Zea mays 0 --- 0 NtSTOP1 0 --- 0 PpSTOP1 1 MQGGNQQPAPSPTNDSLSTLAFLHHQLTQQFSTSNLPNTPLLSSSLGFSMSGNAQDQSHTHQNIQQQQIQ 70 AtSTOP2 0 --- 0 EguSTOP1 1 ---MDSKMDLEERLRAETWGKPSSVNDLPQRVPPD 32 AtSTOP1 1 ---MET 3 OsART1 0 --- 0 Zea mays 1 ---MEGRMTSLEATMKVS 15 NtSTOP1 1 ---MDPDDSLSEDPWTKPSSSGNELLKIVPS 28 PpSTOP1 71 SMISGGGSRGHAQVGSRSGESPGGLFQHLQHGNRDSNLDQHQFYNETTNQLFSENVLNQASLVEQRVRSI 140 AtSTOP2 0 --- 0 EguSTOP1 33 RQTPFPNFASHKNLQKREDQDPSISNYGMRIEPSFSEFNRPSECQPPLPSNPISQDRGVQMNDILQLGKT 102 AtSTOP1 4 EDDLCNTNWGSSSSKSREPGSSDCGNSTFAGFTSQQKWEDASILDYEMGVEPGLQESIQANVDFLQGVRA 73 OsART1 1 ---MDRDQMTNTMRDQAANLTSMNPLFYPFMADDALLGMAPPPPQQLLPSVSIQH 52 Zea mays 16 SLMASSMSRNADPDQQTLRPNSVEQFYFPRPGQSLPGIPPFFGPPSSSLYLPNDNEAKFGNQFESNPSQN 85 NtSTOP1 29 DNHSFTNFNLH--AQKWEG--SSYLDQQTRIEQQFSGF---TQPKHTYQMDQ-HGNQMNENHDSTST 87 PpSTOP1 141 TEHLSMLQDRIQQLQALVPLISQLSHFQNEGNVLAQQQVASAAVVSITSQLAMVAVDLLLQSGVNTGTQP 210 AtSTOP2 1 ---MHIHMMNRDEHIAKKVEGSI 20 EguSTOP1 103 QEWDPKAMLNNLSFLEQKIHQLQELVHAIVGRRGPVLGRPDELVAQQQQLITADLTSIIIQLISTAGSLL 172 AtSTOP1 74 QAWDPRTMLSNLSFMEQKIHQLQDLVHLLVGRGGQLQGRQDELAAQQQQLITTDLTSIIIQLISTAGSLL 143 OsART1 53 MDWSPDTMLDNLTFIEEKIRQVKDVIRSMAGRRASSSSAATP----EQQLVNADLTCLIVQLISTAGSLL 118 Zea mays 86 TDWDPQAIVSNLTFLEQKIKQVKDIVQSMSNRENQVAGGSSELAAKQH-LVTADLTSIIIQLISTAGSLL 154 NtSTOP1 88 KDWDPSNLLNNLSFLEQKIHQLQELVHLIVGRRGQTGLQGNDLIVQQQQLITADLTSIIVQLVSTAGSLL 157 PpSTOP1 211 NHSKEMHLSQLLQNAANPNFSQQNAPRHDLRAPHMSHWMEKLLGGSFAASEGVNAAITSGNGGIRFSGGG 280 AtSTOP2 21 SSFSGETSTSSKQIYVNPVTTTGTKSMEDDDVSLSLLYNLSTLHEKVHQIQSLVSFYMVSTNNINQSSGS 90 EguSTOP1 173 PSVKNS--LSSASTPPIRQLGQLGGILNNSGSGIGLDSNLVLPSQ--GGSKVPDQSNQVDPMDQSAID-- 236 AtSTOP1 144 PSVKHN--MSTAPGPF---TGQPGSAVFPYVREAN---NVASQSQ--NNNNCGAREFDLPKPVLVDEREG 203 OsART1 119 PSLKNSSFLSRTTPPPAAAAGAAQAVSLAAGESSSSARNNETNREDEEEQMGSPDYDELFKVWTNGGAMD 188 Zea mays 155 PSMKNP--LLSSNPAV-RQLGNTLGSPMGLGMNANQRPSVDSKTDIPDTGKTSDYDELMNSLNPTQDERD 221 NtSTOP1 158 PTMKHS--LSSVSHAA-SQLVQFGGVTVPSATCTN-GGGLPCNDG--GVTKVEDQSNHVDQLRDCGIEQS 221 PpSTOP1 281 GNFPVLIDDVENSGQQSLSGAQQVGGNGSAGSMIHYPMSNMIGKDFGSILDGKDRKPSDVLGSSSREGDS 350 AtSTOP2 91 TSLAVANIGSLVQEIITAASSMLYTCQQLQIGSNNNNNDIDNDQTVDAMVLEFSRQETDPGHDFVQESTN 160 EguSTOP1 237 -NL---EDHESKDDEDGDEGENLPPGSFEILQLEKEEILAPHTHFCTICGKGFKRDA 289 AtSTOP1 204 HVV---EEHEMKDEDDVEEGENLPPGSYEILQLEKEEILAPHTHFCTICGKGFKRDA 257 OsART1 189 ECV---GAAGDEQDARENPAAAAEEE----KYEVLQLEEDEILAPHTHFCGICGKGFKRDA 242 Zea mays 222 EMIKCPNPCDGEGSELTPMEDHDVKESDDGGEGENLPPGSYVVLQLEKEEILAPHTHFCLICGKGFKRDA 291 NtSTOP1 222 HAV---DGHESKDEDEAEEEENLPPGSYEILQLEKEEILAPHTHFCTICGKGFKRDA 275 PpSTOP1 351 GANEFISVELRGVNAGLEDLDNDSRVDDEGSDSDNISPGSFDLVEMDATEILAEHTHFCEICGKGFKRDA 420 AtSTOP2 161 LFGVQERGQISFPDQNLDWYNTETINPKKDKHRSKPSSGSYDILELDVADLLAKYTHYCQICGKGFKRDA 230 EguSTOP1 290 NLRMHMRGHGDEYKTPAALAKPHKE--AGSEMMLI-KRYSCPYAGCKRNKDHKKFQPLKTILCVKNHYKR 356 AtSTOP1 258 NLRMHMRGHGDEYKTAAALAKPNKESVPGSEPMLI-KRYSCPFLGCKRNKEHKKFQPLKTILCVKNHYKR 326 OsART1 243 NLRMHMRGHGDEYKSAAALAKPPPPPEGEEQPPQPERRYSCPHAGCKRNRMHASFQPLKTILCVKNHYKR 312 Zea mays 292 NLRMHMRGHGDEYKTPAALAKPTKD--SGADHAPV-TRYSCPFVGCKRNKEHKKFQPLKTILCVKNHYKR 358 NtSTOP1 276 NLRMHMRGHGDEYKTPAALAKPHKE--PSSEPTLI-KRYSCPNVGCKRNKEHKKFQPLKTILCVKNHYKR 342 PpSTOP1 421 NLRMHMRGHGDVYKTAAALARPDRG--TQIPTSNASRRYSCPYVGCKRNKKHRKFQPLKTLLCVKNHYRR 488 AtSTOP2 231 NLRMHMRAHGDEYKTREALISPTSQ-DKKGGYSLKKHYYSCPQHGCRWNQRHEKFQPLKSVICAKNHYKR 299 EguSTOP1 357 THCDKSYTCSRCNTKKFSVIADLKTHEKHCGKDKWLCSCGTTFSRKDKLFGHITLFQGHTPAIPFDENKG 426 AtSTOP1 327 THCDKSFTCSRCHTKKFSVIADLKTHEKHCGKNKWLCSCGTTFSRKDKLFGHIALFQGHTPAIPLEETKP 396 OsART1 313 SHCEKRHVCGRCGAKRFSVMADLKTHEKHCGRDRWLCSCGTSFSRKDKLFAHVALFQGHAPALPPPPPPP 382 Zea mays 359 SHCDKSYTCSRCNTKKFSVIADLKTHEKHCGRDKWLCSCGTTFSRKDKLFGHVALFQGHTPALPMEDVKV 428 NtSTOP1 343 THCEKAYTCSRCNIKKFSVIADLKTHEKHCGKDKWLCSCGTTFSRKDKLFGHIALFQGHTPAVPLDETKG 412 PpSTOP1 489 SHCPKVLNCQKCSTKKFSVVADLKTHEKHCGREKWLCSCGTTFSRKDKLVGHIGLFVGHAPAMPLHDMEG 558 AtSTOP2 300 SHCPKMYMCRRCSVKHFSVLSDLRTHEKHCGDIKWVCSCGTKFSRKDKLMSHVSLFLGHVPAHGSSKPPT 369 EguSTOP1 427 GLSLQGEHNEDTNKVGNVSF-SFGSSTPSSGGVQNIMEDVKGNVDDPTSFFSPLSFEASNFGGFNEFTRS 495 AtSTOP1 397 SASTSTQRGSSEGGNNNQGMVGFNLGSASNANQETTQPGMTDGRICFEESFSPMNFDTCNFGGFHEFPRL 466 OsART1 383 TSGRRRHKQEEPEFTWGGGGGNEFLDVKGIAGVGSGSGGGDEFFSAGSFGAMDFGFGQLDASLAMLLPSE 452 Zea mays 429 SEASEQPQDSEPMNEMARSN-VYSFPCSSSDGISNLD---MKMADDVRGYFSPLNFDPC-FGALDDFTRP 493 NtSTOP1 413 SAGTSDRGQTSEVTMKARQE-DFKVNASHGNEFQDPR-DIKSAADDPGSYFSPLNFDTSNLNGFQEFPRP 480 PpSTOP1 559 GVGSNLIVDHHQSPAEAFGLGSKSPMAFWE--- 588 AtSTOP2 370 ITLK--- 373

EguSTOP1 496 AFDDSEGAFSFLLQASCNYPQKNGGQSSSNNLE- 528

AtSTOP1 467 MFDDSESSFQMLIANACGFSPRNVGESVSDTSL- 499

OsART1 453 QFAGDHQEENGDK--- 465

Zea mays 494 GFDISENPFSFLPSGSCSYGQQNGDS--- 519

NtSTOP1 481 PFDESDSSFSFLLSGSCEYPPHKAAKYMSFTELE 514

PpSTOP1 588 --- 588

AtSTOP2 373 --- 373

C2H2 domain 1

C2H2 domain 2

C2H2 domain 3 C2H2 domain 4

72㹼94%ࡢ┦ྠᛶࠊFig. 21a, 21b)ࠋࡇࡢ࣮ࣘ࢝ࣜSTOP1ࢱࣥࣃࢡ㉁ (௨ୗࠊ

EguSTOP1) ࡣࠊ⣽⬊㉁࡟ᒁᅾࡋ࡚࠸ࡿsGFP࡜඲ࡃ␗࡞ࡿᒁᅾࢆ♧ࡋࠊEguSTOP1

ࡣ୍㐣ⓗⓎ⌧ゎᯒ࡟ࡼࡾ᰾࡛⺯ගࡀほᐹࡉࢀࡓ (Fig. 22)ࠋࡇࡢ⤖ᯝ࠿ࡽࠊEucSTOP1 ࡣಖᏑࡉࢀࡓzinc-finger domainࢆᣢࡕࠊ௚ࡢ᳜≀✀ࡢSTOP1࡜ඹ㏻ࡋ࡚࠸ࡿࡇ࡜

ࡀ♧ࡉࢀࡓࠋ

Ohyamaࡽࡣࢩࣟ࢖ࢾࢼࢬࢼࡢstop1ኚ␗య࡟௚✀ࡢSTOP1㢮ఝࢱࣥࣃࢡ㉁ࢆ

┦⿵ࡋࡓ࡜ࡇࢁࠊH⁺ឤཷᛶࡀᅇ᚟ࡋࡓ (Ohyama et al., 2013)ࠋྠࡌᐇ㦂᮲௳࡛ࠊ

AtSTOP1ࣉ࣮ࣟࣔࢱ࣮࡟EguSTOP1 ࡢ᏶඲㛗cDNA ࢆ᥋⥆ࡋࡓ࣋ࢡࢱ࣮ࢆࢩࣟ࢖

ࢾࢼࢬࢼࡢstop1ኚ␗య࡟ᑟධࡋࡓ┦⿵⤌᥮యࢆసᡂࡋࡓ࡜ࡇࢁࠊpH 5.5࡛ࡣstop1 ኚ␗య࡜5⣔⤫ࡢ┦⿵⤌᥮యࡣ㔝⏕ᆺࡢCol-0࡜ྠࡌ⏕⫱ࢆ♧ࡋࡓࠋ୍᪉ࠊpH 4.7

࡛ࡣࠊstop1ኚ␗యࡣపpH㉸ឤཷᛶࡢࡓࡵ᰿ࡢᡂ㛗ࡣ࡞ࡃࠊ┦⿵⤌᥮యࡣstop1ኚ

␗యࡢపpH㉸ឤཷᛶࡢ୍㒊ࢆᅇ᚟ࡋࠊ᰿ࡢᡂ㛗ࡣ㔝⏕ᆺࡢ⣙50㸣ࡢ⏕⫱࡛࠶ࡗࡓ

(Fig. 23)ࠋࡇࢀࡣEguSTOP1ࡀࠊstop1ኚ␗య࡟ࡼࡗ࡚ᢚไࡉࢀࡓࢩࣟ࢖ࢾࢼࢬࢼࡢ

࠸ࡃࡘ࠿ࡢ H⁺⪏ᛶ㑇ఏᏊࢆάᛶ໬ࡉࡏࡿࡇ࡜ࢆ♧ࡋࠊEguSTOP1 ࡢᶵ⬟ࡣ㌿෗ᅉ Ꮚ࡛࠶ࡿ࡜ホ౯ࡋࡓࠋ

RNAi࡟ࡼࡿ࣮ࣘ࢝ࣜSTOP1ࣀࢵࢡࢲ࢘ࣥẟ≧᰿ࡢゎᯒ

GUT5 ࡣ Agrobacterium rhizogenes ࡟ࡼࡿ⤌᥮ẟ≧᰿ࢆసᡂ࡛ࡁࡿࠋ EguSTOP1ࡢAl³⁺࡜H⁺ㄪ⠇ࢆホ౯ࡍࡿࡓࡵࠊEguSTOP1 ࡢRNAiࢥࣥࢫࢺࣛࢡ ࢺ (Fig. 24a)ࢆᑟධࡋࡓ⤌᥮ẟ≧᰿ࡢ⏕⫱ᛂ⟅ࢆࠊEguSTOP1 RNAiࡢධࡗ࡚࠸

࡞࠸ࣛ࢖ࣥࢆࢥࣥࢺ࣮ࣟࣝ࡜ࡋ࡚ẚ㍑ࡋࡓࠋEguSTOP1ࡢRNAi ࣀࢵࢡࢲ࢘ࣥࣛ

࢖ࣥ (EguSTOP1-KD; knock-down)ࡢ EguSTOP1 ㌿෗㔞ࡣࢥࣥࢺ࣮ࣟࣝࡢ 10%

࡛࠶ࡗࡓ (Fig. 24b)ࠋEguSTOP1-KDࡣపpHᇵᆅ (pH4.0)࡛⏕⫱ࡋࡓ᫬ࠊ㔝⏕ᆺ

࡟ ẚ ࡭ ࠊ 4 0 % ࡢ ⏕ ⫱ 㜼 ᐖ ࢆ ཷ ࡅ ࡓ ( F i g . 2 4 c ) ࠋ

Figure 21. Amino acid alignment and phylogenetic tree of EguSTOP1 and orthologous genes in other plant species. Predicted C2H2 zinc finger domains (a) and phylogenetic tree of overall proteins (b) were shown. Strictly conserved amino acids are highlighted with black, while the amino acids belong to the same amino acid group were shown with gray (a). The overall similarity of proteins was analyzed with Clustal W, and then visualized in the phylogenetic tree (b). The bar indicates nucleotide substitutions per site. The orthologous genes are AtSTOP1 (TAIR: At1g34370), AtSTOP2 (TAIR: At5g22890), PpSTOP1 (GenBank: AB811779), NtSTOP1 (GenBank: AB811781), OsART1 (GenBank: AB379846), Brachypodium distachyon (GenBank: XP_003564719), Glycine max (GenBank: XP_003556206), Malus x domestica (GenBank:

ADL36633), Populus trichocarpa (GenBank: XP_002327330), Vitis vinifera (GenBank: XP_002270196), Zea mays (GenBank: NP_001149728).

B A

CTICGKGFKRDANLRMHMRGHC CTICGKGFKRDANLRMHMRGHC CQICGKGFKRDANLRMHMRAHC CEICGKGFKRDANLRMHMRGHC CTICGKGFKRDANLRMHMRGHC CGICGKGFKRDANLRMHMRGHC

CPYAGCKRNKDHKKFQPLKTILCVKNHYKRTHC CPFLGCKRNKEHKKFQPLKTILCVKNHYKRTHC CPQHGCRWNQRHEKFQPLKSVICAKNHYKRSHC CPYVGCKRNKKHRKFQPLKTLLCVKNHYRRSHC CPNVGCKRNKEHKKFQPLKTILCVKNHYKRTHC CPHAGCKRNRMHASFQPLKTILCVKNHYKRSHC

CSRCNTKKFSVIADLKTHEKH CSRCHTKKFSVIADLKTHEKH CRRCSVKHFSVLSDLRTHEKH CQKCSTKKFSVVADLKTHEKH CSRCNIKKFSVIADLKTHEKH CGRCGAKRFSVMADLKTHEKH

CSCGTTFSRKDKLFGHITLFQGH CSCGTTFSRKDKLFGHIALFQGH CSCGTKFSRKDKLMSHVSLFLGH CSCGTTFSRKDKLVGHIGLFVGH CSCGTTFSRKDKLFGHIALFQGH CSCGTSFSRKDKLFAHVALFQGH EguSTOP1

AtSTOP1 AtSTOP2 PpSTOP1 NtSTOP1 OsART1

EguSTOP1 AtSTOP1 AtSTOP2 PpSTOP1 NtSTOP1 OsART1

* * * * * * * *

278 246 219 409 264 231

365 335 308 497 351 321

C2H2 domain 1

327 297 270 459 313 283

393 363 336 525 379 349

C2H2 domain 2

C2H2 domain 3 C2H2 domain 4

Populus trichocarpa Vitis vinifera

Malus x domestica EguSTOP1

Glycine max

AtSTOP1 NtSTOP1

Brachypodium distachyon Zea mays

AtSTOP2

OsART1

PpSTOP1

0.1

a

b

Figure 22. Fluorescence microscopy image of EguSTOP1::sGFP by Agrobacterium-mediated transient assay in tobacco leaf cells. Fluorescent images (left) and bright field images (right) of EguSTOP1::sGFP and sGFP are shown. The bar indicates 50 μm.

Figure 23. Root growth inhibition of Arabidopsis stop1 mutants expressing AtSTOP1 promoter-driven EguSTOP1 in Al and low pH stress treatments. Seedlings were grown in Al-toxic (pH5.5, Al 2 μM), low pH-toxic (pH 4.7) and control solutions for 5 days. Mean ± SD relative root lengths (% to Control) are shown (n = 5). Different letters indicate significant difference from Col-0 (Tukey’s test, P < 0.05).

0 10 20 30 40 50 60 70 0 20 40 60 80 100

120 Al

Low pH

Re la tive root length (% to control)

a

b b b b b b

AtSTOP1pro::EguSTOP1 in stop1

d a

bc b

bc bc

c

Figure 24. Vector construct for RNAi suppression of EguSTOP1 (a), suppression of EguSTOP1 transcript levels (b) and growth assay of transgenic hairy roots in low pH and Al treatments (c). Control transgenic hairy roots were not carrying EguSTOP1 (white bar) and in EguSTOP1-KD plants (black bar) the STOP1-orthologue was suppressed. Transcript levels were compared after exposure to Al toxic solution. Means and SE are shown (n = 3).

(b) For each line, three independent transgenic hairy roots were grown for 3 days on control (pH 5.6), low pH (4.0) or Al (pH 4.0, Al 100 M) medium. Means and SE are shown (n = 3). An asterisk indicates significant difference from control (student’s t-test, P

< 0.05).

0.0 0.3 0.6 0.9 1.2 1.5

0mM Al 25Al pH5.6 pH4.0

Root elongation (cm / 3d) control RNAi

0 PM Al 25 PM Al 0

0.1 0.2 0.3

control control RNAi control RNAi pH4.6 100 uM Al,

pH4.6

pH4.0 Relative Expression (STOP1 / actin) PM

* *

A

B

C

CaMV35S

EguSTOP1 OP1 ST Egu

NOS-T

NPTII HPT

pGWB80-EguSTOP1

*

a

b

c

ຍ࠼࡚ࠊAlࢫࢺࣞࢫฎ⌮ (25 M Al)࡛ࡢ⏕⫱ࡣࢥࣥࢺ࣮ࣟࣝࡼࡾࡶ㔜࠸㜼ᐖࢆཷ

ࡅࡓࠋࢥࣥࢺ࣮ࣟࣝࡣ0 M Al࡟ẚ࡭50 %ࡢ㜼ᐖࢆཷࡅࡓࡀࠊEguSTOP1-KDࡢ

᰿㛗ࡣ15 %࡟࡞ࡗ࡚࠸ࡓ (Fig. 24c)ࠋࡇࢀࡣEguSTOP1ࡀẟ≧᰿ࡢAl࡜పpH

⪏ᛶࡢㄪ⠇ࡋ࡚࠸ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋ

EguSTOP1ࣀࢵࢡࢲ࢘ࣥẟ≧᰿ࡢAl⪏ᛶ㑇ఏᏊ࣮࢜ࢯࣟࢢࡢ㌿෗ኚື

ඛ㏙ࡢ࡜࠾ࡾࠊ࣮࣐ࣘ࢝ࣜ࢝ࣝࢻࣞࣥࢩࢫ࡟࠾࠸࡚Alᛂ⟅ࢡ࢚ࣥ㓟ᨺฟࡀAl

⪏ᛶ࣓࢝ࢽࢬ࣒࡟ྵࡲࢀ࡚࠾ࡾࠊAlㄏᑟ࡛Ⓨ⌧ࡍࡿEcMATE1 ࡟ࡼࡗ࡚ᨺฟࡀ㉳

ࡇࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡲࡓࠊbacterial-type ABC transporter ࢆࢥ࣮ࢻࡍࡿ

ALS3 / STAR2 (sensitive to Al rhizotoxicity 2)ࡣAtSTOP1 / ART1㌿෗ᅉᏊ࡟ࡼ

ࡗ࡚ㄪ⠇ࡉࢀࡿ㑇ఏᏊ࡜ࡋ࡚≉ᐃࡉࢀࡓ (Yamaji et al., 2009)ࠋࡇࡢ⤖ᯝ࠿ࡽࠊࡇ

ࢀࡽ2ࡘࡢ㑇ఏᏊࡀEguSTOP1-KD࡛㌿෗ᢚไࡉࢀࡿ࠿ㄪ࡭ࡓࠋ

Al ᛂ⟅ࡢ MATE transporter ࡸ ALS3 ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡣࠊGUT5 ࠿ࡽ

degenerate PCRࠊRACE࡟ࡼࡗ࡚༢㞳ࡋࡓࠋ࣮ࣘ࢝ࣜGUT5࠿ࡽ4ಶࡢࢡ࢚ࣥ㓟

㍺㏦MATEࢆ༢㞳ࡋࡓࠋࡑࡢ1ࡘ (EguMATE1)ࡣࠊ582ࡢ࢔࣑ࣀ㓟ࢆࢥ࣮ࢻࡋࠊ

࣮࣐ࣘ࢝ࣜ࢝ࣝࢻࣞࣥࢩࢫࡢEcMATE1࡜96㸣ࡢ┦ྠᛶ࡛࠶ࡗࡓࠋࡇࢀࡽࡢ࣮ࣘ

࢝ࣜMATEࡣࠊ᪤▱ࡢࢡ࢚ࣥ㓟㍺㏦MATE ࡟ࡼࡿ⣔⤫ᶞ࡛AtFRD3ࡸLaMATE

࡜ྠࡌࢢ࣮ࣝࣉ࡟ᒓࡋ࡚࠸ࡓ(Fig. 25a)ࠋࢩࣟ࢖ࢾࢼࢬࢼ AtALS3 ࡢ࣮ࣘ࢝ࣜ

GUT5࣍ࣔࣟࢢ(௨ୗࠊEguALS3)ࡣࠊAtALS3 (78%)࡜STAR2 (rice ALS3; 71%)

ࢆࡶ࡜࡟༢㞳ࡋࡓࠋEguALS3ࡣࠊ285ࡢ࢔࣑ࣀ㓟࡛ᵓᡂࡉࢀࠊ௚᳜≀✀࡜ඹ㏻ࡢ 7ࡘࡢ⭷㈏㏻ࢻ࣓࢖ࣥࢆᣢࡗ࡚࠸ࡓ (Fig. 25b)ࠋEguALS3ࡢAl⪏ᛶ࡬ࡢ㈉⊩ࢆホ ౯ࡍࡿࡓࡵࠊࢩࣟ࢖ࢾࢼࢬࢼࡢ T-DNA ᤄධࣀࢵࢡ࢔࢘ࢺࣛ࢖ࣥ (ALS3-KO)ࡢ┦

⿵ヨ㦂ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊEguALS3 ࡣ ALS3-KO ࡢ Al ឤཷᛶࢆ๻ⓗ࡟ᅇ᚟ࡉ ࡏ (Fig. 26)ࠊ࣮ࣘ࢝ࣜࡢAl⪏ᛶࡢᶵ⬟ࢆᣢࡗ࡚࠸ࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋ

Figure 25. Comparison of the deduced amino acid sequences of orthologous genes for citrate transporting MATE and ALS3/STAR2 transporters. Phylogenetic tree analysis of EguMATE1 and previously identified citrate transporting MATE proteins in various plants (a). The orthologous genes are EcMATE1 (GenBank: AB725912), EcMATE2 (GenBank:

AB725913), EcMATE3 (GenBank: AB725914), EcMATE4 (GenBank: AB725915), AtMATE (TAIR: At1g51340), AtFRD3 (TAIR: At3g08040), OsFRDL1 (RAP-DB:

Os03g0216700), HvAACT1 (GenBank: BAF75822), SbMATE (GenBank: ABS89149), LaMATE (GenBank: AAW30733) and ZmMATE1 (GenBank: FJ015156.1). The bar indicates nucleotide substitutions per site. Amino acid alignment of AtALS3 (TAIR:

At2g37330), STAR2 (rice homologue of ALS3; GenBank: AB379845) and EguALS3 (b).

Black boxed residues are identical and gray boxes indicate similar amino acids. Putative transmembrane domains are indicated with bars above the letters.

A

B

EguALS3 1 -MDIVSGAATGLGLEWDWVFDVEHLGWMVEFLKGMVKPAAALAVVFMAAALSYTQRLGLEREMVYAISRA 69 AtALS3 1 ---MDLKWDDFFN--DYEWLIVFLKGMVKPAAALVVVLLAVILSYSQNLSLEGEMIYSVSRS 57 OsSTAR2 1 MMASMAALLQRLLVVVNQVDPGAPGFWR-EFLVGMLKPVAATAVVAMAVALSFTQRLGLEGEMLYAMARA 69 EguALS3 70 FLQLSIIGFVLQFIFNRENSFWIILAYLFMVSVAGYTAGQRAKHVPRGKYIAGASILAGTAVTMFLLVLL 139 AtALS3 58 FLQLSVIGFVLQFIFNQENSGWIILAYLFMVSVAGYTAGQRARHVPRGKYVAGLSILAGTSITMFLLVLL 127 OsSTAR2 70 FLQLSVIGFVLQFIFTQKSAAWILLAYLFMVTVAGYTAGQRARHVPRGKHIAAVSILAGTSVTMALLVAL 139 EguALS3 140 NVFPFTPRYIIPVAGMMVGNAMTVTGVTMKRLRDDIRTQMNLVETALALGATPRQATHQQVKRALVIALS 209 AtALS3 128 NVFPFTPRYMIPIAGMLVGNAMTVTGVTMKQLRDDIKMQLNLVETALALGATPRQATLQQVKRALVISLS 197 OsSTAR2 140 RVFPFTPRYIIPVAGMMVGNAMTVTGVTMKKLREDVGMQRGVVETALALGATPRQATARQVRRSLVIALS 209 EguALS3 210 PVIDNAKTVGLISLPGAMTGLIMGGASPIEAIQLQIVVMNMLIGASTVSSIMSTYLCWPGFFTKAYQLES 279 AtALS3 198 PVLDSCKTVGLISLPGAMTGMIMGGASPLEAIQLQIVVMNMMVGAATVSSITSTYLCWPSFFTKAYQLQT 267 OsSTAR2 210 PVIDNAKTVGLIALPGAMTGLIMGGASPLEAIQLQIVVMNMLMGASTVSSILSTYLCWPAFFTGAFQLND 279

EguALS3 280 KVFSSE 285

AtALS3 268 HVFSSD 273

OsSTAR2 280 AVFAAD 285

TM1

TM2 TM3 TM4

TM5

TM6 TM7

EguMATE1 EcMATE1

EcMATE2 AtMATE1

EcMATE4

EcMATE3 ZmMATE1

AtFRD3

LaMATE

SbMATE

OsFRDL1 HvAACT1

0.1

a

b

Figure 26. In planta complementation assay of EguALS3 in a T-DNA insertion mutant of Arabidopsis thaliana. Vector construct for in planta complemented assay (a). The complemented lines were obtained by introducing EguALS3 into the T-DNA insertion mutant of ALS3 in Arabidopsis. All lines were grown in solution in the presence or absence of Al (pH 5.0, Al 2 μM), and then relative root length was calculated (b). Means and SE (n

= 5) were shown. Expression of EguALS3 in the complemented lines was compared by RT-PCR.

a

b

Figure 27. Transcripts levels of EguMATE1 and EguALS3 in control transgenic and EguSTOP1-suppressed hairy roots. For each line, three independent transgenic hairy roots were grown for 3 days on control (pH 5.6), low pH (pH 4.0) and Al (pH 4.0, Al 100 μM) medium. Means and SE are shown (n = 3). Transcript levels for EguMATE1 and EguALS3 were quantified by real-time PCR in the control and EguSTOP1-KD hairy roots. Means and SE of three replications are shown. Different letters indicate significant difference (Tukey’s test, P < 0.05).

0.0 0.2 0.4 0.6 0.8 1.0

1 2 3

0 0.01 0.02 0.03 0.04 0.05 0.06

control control RNAi pH4.6 100uM Al, pH4.6

EguALS3 EguMATE1

Relativ e Ex pression (gene / actin)

a a

b

a a

b

100PM Al, pH4.6

EguMATE1࡜EguALS3ࡣAl࡟ࡼࡗ࡚ࢥࣥࢺ࣮ࣟࣝࣛ࢖࡛ࣥ㌿෗㔞ࡀ㧗ࡃ࡞ࡾࠊ

EguSTOP1-KD࡛ࡣ㌿෗㔞ࡣᢚไࡉࢀ࡚࠸ࡓ (Fig. 27)ࠋࡇࢀࡣ࣮ࣘ࢝ࣜࡢ୺࡞

Al⪏ᛶ㑇ఏᏊࡀࠊEguSTOP1࡟ࡼࡗ࡚ㄪ⠇ࡉࢀ࡚࠸ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋ

22-4

⪃ᐹ

ከࡃࡢ᳜≀✀ࡣࠊ᰿࠿ࡽ᭷ᶵ㓟ࢆᨺฟࡍࡿࡇ࡜࡛᰿➃ࡢ Al ឤཷᛶ⣽⬊ࢆಖㆤ ࡍࡿ (Ma et al. 2001; Kochian et al. 2004)ࠋࡇࡢᡓ␎ࡣࠊ༢Ꮚⴥ࡜཮Ꮚⴥࡢ୧᪉ ࡢⲡᮏ᳜≀࡛ඹ㏻࡛࠶ࡿࠋࡇࡢ◊✲࡛ࡣࠊ࣮ࣘ࢝ࣜࡀAl࡟ᛂ⟅ࡋ࡚ࢡ࢚ࣥ㓟ࢆᨺ ฟࡍࡿࡇ࡜ࢆぢฟࡋࡓ (Fig. 10a)ࠋࡇࢀࡣᮌᮏ᳜≀ࡢ᰿࠿ࡽࡢ᭷ᶵ㓟ᨺฟࡶࠊAl

⪏ᛶࡢᙺ๭ࢆᣢࡘࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋ࣮ࣘ࢝ࣜࡣAlࢫࢺࣞࢫࣇ࣮ࣜࡢ≧ἣ࡛᰿ᅪ

࡟ࣜࣥࢦ㓟࡜ࢡ࢚ࣥ㓟ࢆᨺฟࡋ࡚࠸ࡿࡀࠊAl ẘᛶ࡛ࢡ࢚ࣥ㓟ᨺฟࡢࡳࡀቑຍࡋࡓ

(Fig. 10d)ࠋࡇࢀࡣࠊ᳜≀ࡢࢡ࢚ࣥ㓟ᨺฟMATEࢱࣥࣃࢡ㉁࡜ࡋ࡚▱ࡽࢀࡿ࣍ࣔࣟ

ࢢ࡟㏆࠸EcMATEࢺࣛࣥࢫ࣏࣮ࢱ࣮ࡢAl㌿෗ㄏᑟ࡟ࡼࡗ࡚ㄝ࡛᫂ࡁࡿࠋ

MATEࡣ 12ࡢ⭷㈏㏻㡿ᇦࢆᣢࡗ࡚࠾ࡾࠊ1 ࡘࡢಖᏑ㡿ᇦࡣࢡ࢚ࣥ㓟ᨺฟ⬟ຊ

࡜⤖ࡧ௜ࡅࡽࢀ࡚࠸ࡿ (Yang et al. 2011)ࠋ࣮࣐ࣘ࢝ࣜ࢝ࣝࢻࣞࣥࢩࢫ࠿ࡽ4ࡘࡢ MATE ࣍ࣔࣟࢢࢆ༢㞳ࡋࠊࡍ࡭࡚ࡢ㑇ఏᏊࡣࢡ࢚ࣥ㓟㍺㏦ MATE ࡟ඹ㏻ࡢ≉ᚩࠊ ࡍ࡞ࢃࡕ 12 ࡢ⭷㈏㏻㡿ᇦ࡜᪤▱ࡢࢡ࢚ࣥ㓟㍺㏦ MATE ࡢಖᏑ㡿ᇦࢆᣢࡗ࡚࠸ࡓ (Fig. 11a)ࠋMATE࣍ࣔࣟࢢࡢ㛫࡛ࠊEcMATE1ࡣࠊ၏୍Alฎ⌮࡛㌿෗ㄏᑟࡉࢀࠊ Al࡜పpHࡢ≉␗ᛶࡣAlᛂ⟅᭷ᶵ㓟ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࡢⓎ⌧ࣃࢱ࣮ࣥ࡟ఝ࡚࠸ࡓ (Fig. 13)ࠋ౛࠼ࡤࠊࢩࣟ࢖ࢾࢼࢬࢼ࡛Alᛂ⟅ࣜࣥࢦ㓟ᨺฟࢆㄪ⠇ࡍࡿAtALMT1 ࡣࠊࢫࢺࣞࢫ≉␗ᛶࢆㄪ࡭ࡿᐇ㦂࡛Al࡟ࡼࡗ࡚ㄏᑟࡉࢀ࡚࠸ࡓ (Kobayashi et al.

2007)ࠋࡲࡓࠊࢩࣟ࢖ࢾࢼࢬࢼࡢࢡ࢚ࣥ㓟㍺㏦MATE (AtMATE) ࡣࠊAl⪏ᛶ࡬ࡢ

㈉⊩ࡀAtALMT1࡯࡝࡛ࡣ࡞࠸ࡀࠊఝࡓAlㄏᑟࣃࢱ࣮ࣥࢆ♧ࡋࡓ (Liu et al. 2009)ࠋ

Agrobacterium rhizogenesis ࡟ࡼࡿࢱࣂࢥẟ≧᰿࡛ࡢEcMATE1 Ⓨ⌧࡛ࠊAl ᛂ⟅ࢡ࢚ࣥ㓟ᨺฟࡀቑຍࡋࡓ (Fig. 16)ࠋࡇࢀࡣEcMATE1ࡢࢡ࢚ࣥ㓟㍺㏦ࡀࢱࣂ

ࢥࡢ⣽⬊࡛ᶵ⬟ࡍࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋࡇࡢ⤖ᯝࡣࠊࢩࣟ࢖ࢾࢼࢬࢼ⤌᥮య࡛

SbMATEࢆⓎ⌧ࡋࠊࢡ࢚ࣥ㓟ᨺฟࢆቑຍࡉࡏࡿࡇ࡜࡛Al⪏ᛶࢆ♧ࡋࡓ⤖ᯝ࡜ఝ࡚

࠸ࡿ (Magalhaes et al. 2007)ࠋࡇࢀࡽMATEࢱࣥࣃࢡ㉁ࡢⓎ⌧࡟ࡼࡿࢡ࢚ࣥ㓟㍺

㏦ࡣࠊAl࡟ࡼࡿάᛶ໬࡛ㄝ࡛᫂ࡁࡿࠋ༸ẕ⣽⬊ࡢ◊✲࡛ࡣࠊMATEࢱࣥࣃࢡ㉁ࡀ Al ࡜ ┤ ᥋ ┦ ஫ స ⏝ ࡍ ࡿ ࡇ ࡜ ࡟ ࡼ ࡗ ࡚ ά ᛶ ໬ ࡉ ࢀ ࡿ ྍ ⬟ ᛶ ࢆ ♧ ࡋ ࡓ (e.g., Magalhaes et al. 2007)ࠋࡇࡢAlάᛶ໬ࡢࣉࣟࢭࢫ࡟࠾ࡅࡿࠊࢱࣥࣃࢡ㉁ࡢࣜࣥ㓟

໬ࡣࠊࢩࣟ࢖ࢾࢼࢬࢼࡢALMT1-typeࣜࣥࢦ㓟ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࡢAlᛂ⟅ࣜࣥࢦ 㓟ᨺฟᐇ㦂ࢆࡶ࡜࡟⾜ࡗࡓ (Kobayashi et al. 2007)ࠋ㢮ఝࡢࢱࣥࣃࢡ㉁ࡢࣜࣥ㓟

໬ࣉࣟࢭࢫࡀࠊ࣮ࣘ࢝ࣜࡢ EcMATE1 ࢆάᛶ໬ࡋࡓྍ⬟ᛶࡀ࠶ࡾࠊᐇ㝿ࠊࢱࣥࣃ

ࢡ㉁ࡢࣜࣥ㓟໬ࠊ⬺ࣜࣥ㓟໬ࡢ㜼ᐖ๣ῧຍࡣࠊAl ᛂ⟅ࢡ࢚ࣥ㓟ᨺฟ࡟ᙳ㡪ࢆཬࡰ

ࡋࡓࠋ

᭷ᶵ㓟ᨺฟࡣࠊᵝࠎ࡞᳜≀࡛ࢫࢺࣞࢫ⪏ᛶ࡟ᙺ๭ࢆᯝࡓࡋ࡚࠸ࡿࠋ౛࠼ࡤ Al

⪏ᛶࡸྍ⤥ែࣜࣥ㓟 (Neumann et al. 1999)ࠊ㕲⋓ᚓ (Miethke and Marahiel 2007)ࠊ᰿ᅪ⑓ཎ⳦࡟ᑐࡍࡿ᢬ᢠᛶ (e.g., Rudrappa et al. 2008)࡞࡝࡛࠶ࡿࠋศᏊ

⏕≀Ꮫ◊✲࡟ࡼࡾࠊ᭷ᶵ㓟ᨺฟࡢࢺࣛࣥࢫ࣏࣮ࢱ࣮࡜ࡑࡢㄪ⠇ࡀࠊ᭷ᶵ㓟ࡢ✀㢮࡜

ࢫࢺࣞࢫᛂ⟅ࡢỴᐃ࡟㔜せ࡞せ⣲࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋ㏆ᖺࠊ࣒࢜࢜ࢠࡢAlᛂ

⟅ HvAACT1 ࢡ࢚ࣥ㓟ᨺฟࡢ࣓࢝ࢽࢬ࣒ࡣࠊࣉ࣮ࣟࣔࢱ࣮ࡢ㐍໬ࡀࡁࡗ࠿ࡅ࡟࡞

ࡗࡓ࡜ᥦ᱌ࡉࢀࡓࠋHvAACT1ࡢࣉ࣮ࣟࣔࢱ࣮ゎᯒ࡛ࠊ㕲⋓ᚓࡢࡓࡵࡢࢡ࢚ࣥ㓟ᨺ ฟMATEࡢࣉ࣮ࣟࣔࢱ࣮ࡢኚ໬ࡀࠊ⤖ᯝ࡜ࡋ࡚Alẘᛶ࠿ࡽ᰿➃ࢆಖㆤࡍࡿࡓࡵࡢ 㑇ఏᏊࡢⓎ⌧ࣃࢱ࣮ࣥࡶኚ࠼ࡓࡇ࡜ࢆ♧ࡋ࡚࠸ࡿ (Fujii et al. 2012)ࠋEcMATEⓎ

⌧ࡣࠊ᰿ඖࡼࡾࡶ᰿➃࡛ᑡࡋ㧗ࡃ࡞ࡾ (Fig. 13e)ࠊ⭷ᒁᅾ࡛࠶ࡗࡓࡀ(Fig. 15)ࠊࡇ

ࢀࡣHvAACT1࡜ఝ࡚࠸ࡓࠋEcMATE1࡜Al⪏ᛶࡢ㑇ఏⓗ㛵㐃ᛶࡣᮍࡔ᫂ࡽ࠿࡟

࡞ࡗ࡚࠸࡞࠸ࡀࠊEcMATE1ࡢࡉࡽ࡞ࡿ◊✲ࡣࠊᮌᮏ᳜≀✀࡟࠾ࡅࡿAlᛂ⟅ࢡ࢚

ࣥ㓟ᨺฟࡢ㐍໬ࡢ⌮ゎ࡜࠸࠺ព࿡࡛⯆࿡῝࠸ࠋ

௨๓ሗ࿌ࡉࢀࡓࡼ࠺࡟ࠊSTOP1┦ྠ㑇ఏᏊࡣᵝࠎ࡞᳜≀✀࡛Al⪏ᛶ㑇ఏᏊࢆ

ㄪ⠇ࡋ࡚࠸ࡿࠋࢩࣟ࢖ࢾࢼࢬࢼ࡛stop1ኚ␗యࡣAtALMT1 (Iuchi, et al., 2007)

࡜ ALS3 (Sawaki, et al., 2009)ࡢ㌿෗㔞ࢆᢚไࡋࠊ୍᪉࡛ࠊ࢖ࢿࡢ ART1 (rice STOP1-like protein) (Yamaji, et al., 2009)ࢆษ᩿ࡋࡓኚ␗య࡛ࡣࠊSTAR2 (the rice orthologue of ALS3)ࢆ㌿෗ᢚไࡋࡓࠋຍ࠼࡚ࠊ㏫㑇ఏᏛࢆ౑ࡗࡓࠊࢱࣂࢥ࡛

ࡢ STOP1-like protein (NtSTOP1)㑇ఏᏊࡢ RNAi ࣀࢵࢡࢲ࡛࢘ࣥࡣࠊNtMATE

࡜NtALS3ࡢ㌿෗㔞ࡀᢚไࡉࢀࠊAlឤཷᛶ࡜࡞ࡗࡓ (Ohyama, et al., 2013)ࠋ௨

ୖࡢ⤖ᯝ࠿ࡽࠊSTOP1-like proteinࡢ㏫㑇ఏᏛࡣࠊ௚ࡢ᳜≀✀࡛Al⪏ᛶ㑇ఏᏊࢆ

Ỵᐃࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋᮇᚅ㏻ࡾࠊEguSTOP1ࡢRNAi ࣀࢵ

ࢡࢲ࢘ࣥࡣࠊࢡ࢚ࣥ㓟ᨺฟMATE࡜ALS3ࡢ࣮࢜ࢯࣟࢢ㑇ఏᏊࡢ㌿෗㔞ࢆᢚไࡋࠊ

ྠ᫬࡟Al ⪏ᛶ⛬ᗘࡶῶᑡࡋࡓ (Fig.27)ࠋࡇࢀࡣࠊSTOP1-like proteins ࡢRNAi

㌿෗ᢚไࡀࠊ௚ࡢ᳜≀✀࡛ࡶAl⪏ᛶ㑇ఏᏊࡢ≉ᐃ࡟᭷⏝࡞࢔ࣉ࣮ࣟࢳ࡛࠶ࡿࡇ࡜

ࢆ♧ࡋ࡚࠸ࡿࠋ

ඛ㏙ࡢ࡜࠾ࡾࠊEucalyptus camaludrensisࡣAl࡟ᛂ⟅ࡋ࡚ࢡ࢚ࣥ㓟ࢆᨺฟࡋࠊ ࢡ࢚ࣥ㓟ᨺฟMATE (EcMATE1)ࡢ㌿෗ࣞ࣋ࣝ࡟ࡼࡗ࡚ㄪ⠇ࡉࢀ࡚࠸ࡿࠋGUT5ࡶ

ࡲࡓAl࡟ࡼࡗ࡚EguMATEࡢ㌿෗㔞ࡀୖࡀࡾࠊ᰿࠿ࡽ᭷ᶵ㓟ࢆᨺฟࡍࡿࠋࡇࢀࡣࠊ

Alㄏᑟࢡ࢚ࣥ㓟ᨺฟࡀ࠸ࡃࡘ࠿ࡢ࣮ࣘ࢝ࣜ✀࡛Al⪏ᛶ࣓࢝ࢽࢬ࣒ࡢ1ࡘ࡜࡞ࡗ࡚

࠸ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋEguSTOP1-KD ẟ≧᰿࡟࠾࠸࡚ࠊEcMATE1 ㌿෗㔞ࡣ ALS3-like protein (EguALS3)࡜࡜ࡶ࡟ᢚไࡉࢀ࡚࠸ࡓ (Fig. 27)ࠋࡇࢀࡣࠊ⤌᥮ẟ

≧᰿࡟࠾࠸࡚Alឤཷᛶࡀ㧗ࡲࡿࡇ࡜࡛ㄝ࡛᫂ࡁࡿྍ⬟ᛶࡀ࠶ࡿࠋࡇࢀࡽࡢ▱ぢࡣࠊ STOP1-like protein࡜Al⪏ᛶ㑇ఏᏊࡢ୧㑇ఏᏊ࡟ࡼࡿㄪ⠇ࡀࠊ࣮࡛ࣘ࢝ࣜࡶಖᏑ ࡉࢀ࡚࠸ࡿࡇ࡜ࢆ♧ࡋࡓࠋMATE ࡸ ALS3 ࡢ㌿෗ᢚไࡣࢱࣂࢥ࡛ࡶྠᵝ࡟ほᐹࡉ

ࢀ (Ohyama, et al., 2013)ࠊࢩࣟ࢖ࢾࢼࢬࢼ ((Sawaki, et al., 2009); ALS3, (Liu, et al., 2009); MATE)ࠊ࢖ࢿ (rice ALS3-like protein, (Yamaji, et al., 2009))࡛ࡶሗ࿌

ࡉࢀ࡚࠸ࡿࠋࡇࢀࡣSTOP1-like proteins࡜ࡑࢀࡀไᚚࡍࡿ㑇ఏᏊ⩌ࡀከࡃࡢ᳜≀

✀࡛Ꮡᅾࡍࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋຍ࠼࡚ࠊࢩࣟ࢖ࢾࢼࢬࢼࡢEguSTOP1┦⿵⤌᥮

య࡛ࡣࠊH⁺ឤཷᛶࡣ㒊ศⓗ࡟ᅇ᚟ࡋ࡚࠸ࡿࡇ࡜࠿ࡽࠊࢩࣟ࢖ࢾࢼࢬࢼࡢH⁺⪏ᛶࢆ

ᢸ࠺࠸ࡃࡘ࠿ࡢ㑇ఏᏊࡢ㌿෗㔞ࡀࠊ࣮ࣘ࢝ࣜࡢSTOP1࡟ࡼࡗ࡚άᛶ໬ࡉࢀࡓࡇ࡜

ࢆ♧ࡋ࡚࠸ࡿࠋࡇࢀࡣࠊไᚚࡍࡿ㑇ఏᏊࡢࢱ࣮ࢤࢵࢺ࡜࡞ࡿcis-࢚࣓ࣞࣥࢺࡀࢩࣟ

࢖ࢾࢼࢬࢼ࡜࣮࡛ࣘ࢝ࣜಖᏑࡉࢀࠊࡉࡽ࡟ಖᏑࡉࢀࡓSTOP1-like proteinsࡢᵓ㐀

࡟ࡼࡗ࡚┦஫స⏝࡛ࡁࡓ⤖ᯝ࡛࠶ࡿ࡜♧၀ࡉࢀࡓࠋSTOP1-like protein ࡟ࡼࡿ㌿

෗ㄪ⠇ࡢࡉࡽ࡞ࡿ◊✲ࡀࠊࡇࡢྍ⬟ᛶࡢ᳨ド࡟ᚲせ࡛࠶ࡿࠋ

⤖

⤖ㄽ

ᮏ◊✲࡛ࡣࠊSTOP1㌿෗ᅉᏊ࡟ࡼࡿAlࠊపpH⪏ᛶᶵᵓࡢゎ᫂ࢆ┠ⓗ࡜ࡋࡓࠋ ࢩࣟ࢖ࢾࢼࢬࢼSTOP1ࢱࣥࣃࢡ㉁ࡣࠊCys2–His2 zinc finger protein࡜㧗࠸࣍ࣔ

ࣟࢪ࣮ࢆᣢࡗ࡚࠾ࡾࠊAl ⪏ᛶࢆࢥࣥࢺ࣮ࣟࣝࡍࡿ」ᩘࡢ㑇ఏᏊࢆㄪ⠇ࡋ࡚࠸ࡿࠋ ࢩࣟ࢖ࢾࢼࢬࢼSTOP1ࡢୗὶ㑇ఏᏊࢆ࣐࢖ࢡࣟ࢔ࣞ࢖࠿ࡽ㑅ᢤࡍࡿࡇ࡜ࢆ┠ⓗ࡜

ࡋࡓ➨1❶࡛ࡣࠊ㑅ᢤࡋࡓࣜࢫࢺࡢ୰࡟᪤▱ࡢAl⪏ᛶ㑇ఏᏊࠊ࢖࢜ࣥᜏᖖᛶࡸప pHㄪ⠇࡟㛵ࢃࡿ㑇ఏᏊࡀྵࡲࢀ࡚࠸ࡓࠋRT-PCRࡢ⤖ᯝ࠿ࡽࠊ㑅ᢤࡉࢀࡓ㑇ఏᏊ

⩌ࡣ☜࠿࡟࣐࢖ࢡࣟ࢔ࣞ࢖ࡢ⤖ᯝ࡜୍⮴ࡋࠊAlࡲࡓࡣపpHࠊ࠶ࡿ࠸ࡣඹ㏻࡛ാࡃ 㑇ఏᏊࡀྵࡲࢀ࡚࠸ࡿྍ⬟ᛶࡀ㧗ࡲࡗࡓࠋAl ⪏ᛶࡣ」ᩘࡢ㑇ఏᏊࡢ⪏ᛶ⤒㊰࡟ࡼ

ࡗ࡚⥲ྜⓗ࡟⪏ᛶᙧ㉁ࡀᚓࡽࢀࠊࡲࡓపpH⪏ᛶࡣ࣓࢝ࢽࢬ࣒ࡢ▱ぢ⮬యࡀᑡ࡞࠸

ࡓࡵࠊࡇࢀࡽࡢࣜࢫࢺ࠿ࡽ᭦࡞ࡿ Alࠊప pH ⪏ᛶ㑇ఏᏊࡀ≉ᐃࡉࢀࡿࡇ࡜ࡀᮇᚅ ࡉࢀࡿࠋ

➨1❶࡛ࡣࣔࢹ᳜ࣝ≀ࡢࢩࣟ࢖ࢾࢼࢬࢼࢆᮦᩱ࡟◊✲ࢆ⾜ࡗࡓࡀࠊ⏘ᴗ᳜≀࡛

ࡢ◊✲ࡣࠊຠ⋡ⓗ࡛⏕⏘ᛶࡢ㧗࠸ရ✀ࡢ㛤Ⓨ࡟ࡘ࡞ࡀࡿࠋ㓟ᛶᅵተࡀᗈࡃศᕸࡍࡿ

ᮾ༡࢔ࢪ࢔࡛ࣉࣛࣥࢸ࣮ࢩࣙࣥࢆᒎ㛤ࡍࡿ࣮࡛ࣘ࢝ࣜࡣ≉࡟Al࡜పpH⪏ᛶࡢᙉ

໬ࡀ㧗⏕⏘ᛶ࡟ࡘ࡞ࡀࡿࠋEcMATE1ࡢࡼ࠺࡞Al⪏ᛶ㑇ఏᏊࡢ㐣๫Ⓨ⌧ࡣ୍ࡘࡢ

࢔ࣉ࣮ࣟࢳ࡛࠶ࡾࠊ௒ᅇࡢࢣ࣮ࢫ࡛ࡣࠊEcMATE1ࡢ㐣๫Ⓨ⌧࡜᭷ᶵ㓟௦ㅰࡢኚ໬

(e.g., citrate synthase ࡢ 㐣๫ Ⓨ ⌧; ࢭ࢖ࣚ ࢘ ࢔ࣈ ࣛ ࢼࠊAnoop et al. 2003, Nicotiana benthamianaࠊDeng et al. 2009) ࡢࢥࣥࣅࢿ࣮ࢩࣙࣥࡣࠊࢡ࢚ࣥ㓟ᨺ ฟ࡟ࡼࡿຠᯝⓗ࡞Al ⪏ᛶࡢᙉ໬࡟᭷⏝࡛࠶ࡿࠋ࣮ࣘ࢝ࣜࡢ STOP1 ࣍ࣔࣟࢢ࡟ࡼ

ࡿ EcMATE1ࡢㄪ⠇࡟ࡘ࠸࡚ࡣㄪ࡭ࡽࢀ࡚࠸࡞࠸ࡀࠊ➨ 1 ❶࡛᫂ࡽ࠿࡟ࡋࡓࡼ࠺

࡟ࠊAtMATE ࡞࡝ࡢ」ᩘࡢ Al ⪏ᛶ㑇ఏᏊࡢඹไᚚࡣࠊࢩࣟ࢖ࢾࢼࢬࢼ STOP1 (Sawaki et al. 2009)ࠊ࢖ࢿART1 (Yamaji et al. 2009)࡜㛵㐃ࡀ࠶ࡿࠋSTOP1࣍ࣔ

ࣟࢢ࡟ࡼࡗ࡚ㄪ⠇ࡉࢀࡿ㑇ఏᏊࡢ≉ᐃ࡟㛵ࡍࡿࡉࡽ࡞ࡿ◊✲ࡣࠊ࣮ࣘ࢝ࣜࡢAl⪏ ᛶ㑇ఏᏊࡢඹㄪ⠇ࢆ≉ᐃࡍࡿ⯆࿡῝࠸࢔ࣉ࣮ࣟࢳ࡟࡞ࡿ࡜⪃࠼ࡽࢀࠊ࣮ࣘ࢝ࣜࡢ Al⪏ᛶ࡟ຠᯝⓗ࡞ศᏊ⫱✀ࡢ࣐࣮࣮࢝ࡢⓎᒎ࡟฼⏝࡛ࡁࡿࠋ

ᮏ◊✲࡛ࡣࠊ࣮ࣘ࢝ࣜࡢ STOP1 ไᚚࢩࢫࢸ࣒࡟ᒓࡍࡿ2 ࡘࡢ Al ⪏ᛶ㑇ఏᏊ

ࢆ≉ᐃࡋࡓࠋࡇࡢ▱ぢࡣࠊ࣮ࣘ࢝ࣜࡢศᏊ⫱✀࡟᭷⏝࡛࠶ࡿ࠿ࡶࡋࢀ࡞࠸ࠋ౛࠼ࡤࠊ ᶞᮌࡢࣂ࢖࢜ࢸࢡࣀࣟࢪ࣮ࡢ㐍Ṍ (e.g., gene transformation; (Ho, et al., 1998;

Kawazu, et al., 2003))࡟ࡼࡾࠊ࣮ࣘ࢝ࣜࡢ⤌᥮⫱✀ࡢ฼⏝ࡀྍ⬟࡟࡞ࡗࡓ (Kirch, et al., 2011)ࠋᐇ㝿ࠊሷ⪏ᛶ⤌᥮Eucalyptus globulusࡣ⳦ࡢ㓝⣲ࢆ㐣๫Ⓨ⌧ࡉࡏ

ࡿࡇ࡜࡛㛤Ⓨࡉࢀࡓ (Matsunaga, et al., 2012)ࠋ࢔ࢡ࢔࣏ࣜࣥࡢ㐣๫Ⓨ⌧ࡣࠊ࣮ࣘ

࢝ࣜࡢỈ฼⏝ຠ⋡ࡀ㧗ࡲࡗࡓ (Tsuchihira, et al., 2010)ࠋࡇࡇ࡛ࠊ㏆ᖺࡢ࣮ࣘ࢝ࣜ

ࢤࣀ࣑ࢡࢫࡸ SNP ྠᐃࡢ㐍Ṍࡣ(e.g., genome-wide cDNAࠊSNP identification (Novaes, et al., 2008))ࠊᶵ⬟ࢤࣀ࣑ࢡࢫࡸࠊຠ⋡ⓗ࡞⫱✀ࡢᇶ┙ࢆᥦ౪ࡋࡓ (e.g., genomic selection, (Grattapaglia and Kirst, 2008; Grattapaglia and Resende,

2011))ࠋࡇࡢࡼ࠺࡞ᶞᮌࡢࣂ࢖࢜ࢸࢡࣀࣟࢪ࣮࡟ࡼࡿMATEࡸALS3ࡢ㐣๫Ⓨ⌧ࠊ

୧㑇ఏᏊࡢ⪏ᛶຠᯝ࡟ᇶ࡙ࡃ࣐࣮࣮࢝฼⏝㑅ᢤࡣ (e.g., EguMATE ࡸ EguALS3 ࡢ㧗Ⓨ⌧ಶయࡢࢭࣞࢡࢩࣙࣥ)ࠊ࣮ࣘ࢝ࣜAl⪏ᛶࡢྥୖ࡟ຠᯝⓗ࡞᪉ἲ࡛࠶ࡿࠋ

ࡇࢀࡲ࡛ࡢሗ࿌࡛ࡣࠊSTOP1-like proteinࡢᶵ⬟୙඲ࡣࠊ༢Ꮚⴥ᳜≀ (࢖ࢿࡢ ART1ኚ␗య (Yamaji, et al., 2009))ࠊ཮Ꮚⴥ᳜≀ (ࢩࣟ࢖ࢾࢼࢬࢼኚ␗య (Iuchi, et al., 2007); ࢱࣂࢥࣀࢵࢡࢲ࢘ࣥ (Ohyama, et al., 2013))࡛Alឤཷᛶࢆᘬࡁ㉳ࡇ ࡋࡓࠋᮏ◊✲࡛ࡣࠊᗈⴥᶞࡢ࣮ࣘ࢝ࣜSTOP1࣮࢜ࢯࣟࢢ㑇ఏᏊࡢࣀࢵࢡࢲ࢘ࣥ࡟

ࡼࡿ Al⪏ᛶᗘࡢపୗࢆ♧ࡋࡓࠋ㏆ᖺࠊࢩࣟ࢖ࢾࢼࢬࢼ stop1-mutant ࢆ࣍ࢫࢺ࡜

ࡋࡓ௚ࡢᗈⴥᶞ (i.e., tea, black poplar)ࡢSTOP1࣮࢜ࢯࣟࢢ㑇ఏᏊࢆ┦⿵ヨ㦂࡟

ࡼࡾ≉ᐃࡋࡓ (Ohyama, et al., 2013)ࠋࡇࡢࢱࣥࣃࢡࡢάᛶࡀࡲࡔホ౯ࡉࢀ࡚࠸࡞

࠿ࡗࡓ㛫࡟ࠊࢹ࣮ࢱ࣮࣋ࢫ࡟ࡼࡿ᥈⣴࡛㔪ⴥᶞࡀ࣮࢜ࢯࣟࢢࢆᣢࡗ࡚࠸ࡿࡇ࡜ࢆ♧

ࡋࡓࠋࡇࢀࡣSTOP1ไᚚࢩࢫࢸ࣒ (STOP1 ࢱࣥࣃࢡ࡜ࡑࡢไᚚ㑇ఏᏊ)ࡀࠊ㐍໬

࡜ࡋ࡚ྂࡃࠊᶞᮌࢆྵࡴ㝣ୖ⫋≀࡟ᗈࡃඹ㏻ࡋ࡚࠸ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋࢡ࢚ࣥ㓟

㍺㏦ MATE ࡸࠊALS3 ࡢࡼ࠺࡞㌿෗ㄪ⠇ࡉࢀࡓ㑇ఏᏊࡢ࠸ࡃࡘ࠿ࡣࠊᵝࠎ࡞᳜≀

࡛୺࡞Al⪏ᛶ㑇ఏᏊ࡜ࡋ࡚ぢࡘ࠿ࡗ࡚࠸ࡿࠋMATEࡣsorghum (Magalhaes, et al., 2007)ࠊbarley (Furukawa, et al., 2007)ࠊcorn (Tan and Binger, 1986)࡛ぢࡘ࠿ࡾࠊ ࡇࢀࡽࡢࢱࣥࣃࢡ㉁ࡣࠊཎ᰾⏕≀ࢱ࢖ࣉࡢࢺࣛࣥࢫ࣏࣮ࢱ࣮࡜≉ᚩࡀඹ㏻࡛࠶ࡗࡓ (Ryan, et al., 2011)ࠋࡇࢀࡣ㝣ୖ᳜≀ࡢSTOP1ࢩࢫࢸ࣒ࡀ⣽⳦࡜㉳※ࡀྠࡌ࡛࠶

ࡿࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࠋ⏕≀✀㛫ࡢSTOP1ࡢ┦ྠᛶ᳨⣴࡛ࡣࠊ㠀ᖖ࡟ࡼࡃఝࡓ㓟

⪏ᛶ࣓࢝ࢽࢬ࣒ (i.e., transcriptional regulation of some shared genes)ࡀ㓝ẕ (Gregori, et al., 2008)ࡸ့ங㢮 (Gunshin, et al., 1997)࡛ሗ࿌ࡉࢀࡓࡀࠊ⸴㢮࡜௚

ࡢ⏕≀࡛ࡣ┦ྠᛶࡀ↓࠿ࡗࡓࠋࡉࡽ࡞ࡿ㓟⪏ᛶࢩࢫࢸ࣒ࡢ⏕≀✀㛫ࡢẚ㍑◊✲ࡣࠊ ࡑࢀࡒࢀࡢࢩࢫࢸ࣒ࡀ␗࡞ࡿ⏕≀✀࡛⊂❧࡟㐍໬ࡋࡓ࠿࡝࠺࠿ࡢ⟅࠼࡜࡞ࡿ࠿ࡶ

ࡋࢀ࡞࠸ࠋ

せせ⣙

㓟ᛶᅵተ࡛ၥ㢟࡜࡞ࡿAlཬࡧH⁺ࡣ᰿ఙ㛗㜼ᐖࢆᘬࡁ㉳ࡇࡋࠊࣜࣥ㓟ࢆ୙ྍ⤥

ែ࡟ࡍࡿࡓࡵࠊ᳜≀ࡢᡂ㛗ࢆᢚไࡍࡿࠋࡇࢀ࡟ᑐฎࡍࡿࡓࡵࠊ᳜≀ࡣ᰿࠿ࡽࡢ᭷ᶵ 㓟ᨺฟࢆࡣࡌࡵ࡜ࡍࡿᵝࠎ࡞ᑐᛂ⟇ࢆᣢࡗ࡚࠸ࡿࠋࣔࢹ᳜ࣝ≀࡛࠶ࡿࢩࣟ࢖ࢾࢼࢬ ࢼ࡛ࡣ୧ࢫࢺࣞࢫ࡟⪏ᛶࡢᶵ⬟ࢆ୚࠼ࡿ㑇ఏᏊࡀⓎぢࡉࢀࠊSTOP1 (sensitive to proton rhizotoxicity 1)࡜ྡ௜ࡅࡽࢀࡓࠋᮏ◊✲࡛ࡣࠊ⥙⨶ⓗ࡟㌿෗㔞ࢆ ᐃ࡛ࡁ

ࡿ࣐࢖ࢡࣟ࢔ࣞ࢖ࢆ⏝࠸࡚ࢩࣟ࢖ࢾࢼࢬࢼSTOP1ࡀไᚚࡋ࡚࠸ࡿୗὶ㑇ఏᏊࠊࡍ

࡞ࢃࡕAlࠊ࠾ࡼࡧపpH⪏ᛶ࡟ᐇ㝿࡟㛵ࢃࡿ㑇ఏᏊࢆ᥈⣴ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋ ࡉࡽ࡟ࠊᐇ⏝᳜≀࡜ࡋ࡚ࣃࣝࣉ࡟౑⏝ࡉࢀࡿ࣮ࣘ࢝ࣜࡢ᭷ᶵ㓟ᨺฟ࡟ࡼࡿAl⪏ᛶ ᶵᵓࢆㄪ࡭ࡓࠋ

ࢩࣟ࢖ࢾࢼࢬࢼ࡛ࡣࠊAlࢫࢺࣞࢫ࡛ࣜࣥࢦ㓟ࡀᨺฟࡉࢀࡿ࡜࡜ࡶ࡟ࠊࣜࣥࢦ㓟

ྵ㔞ࡀቑຍࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋࡇࡢ᫬ࡢࣜࣥࢦ㓟௦ㅰ࡟㛵ࢃࡿ㑇ఏᏊࡢ㌿෗

ኚືࢆㄪ࡭ࡓ࡜ࡇࢁࠊTCA ࢧ࢖ࢡࣝෆࡢ㑇ఏᏊࡣࡸࡸ㌿෗ࡀᢚ࠼ࡽࢀ࡚࠸ࡿࡢ࡟

ᑐࡋࠊMalic enzymeࠊMalate dehydrogenaseࠊPEP carboxykinase࡞࡝TCAࢧ

࢖ࢡࣝࡢ࿘㎶࡛ാࡃ㑇ఏᏊࡀ㧗࠸㌿෗㔞ࢆ♧ࡋ࡚࠸ࡓࠋࡲࡓࠊAl ࡟ࡼࡿ௦ㅰኚື

ࢆㄪ࡭ࡓ⤖ᯝࠊࢢࣝࢱ࣑ࣥ㓟ࡀῶᑡࡋࠊ࢔ࢫࣃࣛࢠࣥ㓟ࡀቑຍࡋ࡚࠸ࡓࠋ௨ୖ࠿ࡽࠊ Al ฎ⌮࡟ࡼࡿࣜࣥࢦ㓟ቑຍࡣࢭࣜࣥ⤒㊰ࡸࠊࢢࣝࢱ࣑ࣥ㓟࠿ࡽ࢔ࢫࣃࣛࢠࣥ㓟ࢆ

⤒⏤ࡍࡿ⤒㊰࡞࡝ࠊTCA ࢧ࢖ࢡࣝࡢࣂ࢖ࣃࢫ࡜࡞ࡿ⤒㊰ࡀ㔜せ࡞ᙺ๭ࢆࡋ࡚࠸ࡿ

ࡇ࡜ࡀ♧၀ࡉࢀࡓࠋࡉࡽ࡟ࠊSTOP1ࣀࢵࢡ࢔࢘ࢺኚ␗య࡜㔝⏕ᆺࢆẚ㍑ࡋࡓ࣐࢖

ࢡࣟ࢔ࣞ࢖ࡢ⤖ᯝ࠿ࡽࠊAl ⪏ᛶ㑇ఏᏊ࡜ࡋ࡚▱ࡽࢀࡿࣜࣥࢦ㓟ᨺฟࢺࣛࣥࢫ࣏࣮

ࢱ ࣮ ࡢ AtALMT1 ࡸ half type ATP-binding cassette transporter ࡢ ALS3 (Aluminum Sensitive 3) ࡀSTOP1ኚ␗య࡛㌿෗ᢚไࡉࢀ࡚࠸ࡓࠋࡑࡢ࡯࠿࡟ࢺ

ࣛࣥࢫ࣏࣮ࢱ࣮ࡸ࢖࢜ࣥ㍺㏦ࢆㄪ⠇ࡍࡿࢱࣥࣃࢡ㉁ࠊpHㄪ⠇௦ㅰࡢ㑇ఏᏊࡀ㌿෗

ᢚไࡉࢀࡓࡇ࡜࠿ࡽࠊSTOP1ࡀAl࡜H⁺ᛂ⟅ࡢ㌿෗ไᚚࡢࢩࢢࢼࣝఏ㐩࡟㛵ࢃࡿ

ࡇ࡜ࡀ♧၀ࡉࢀࡓࠋపpHฎ⌮࡟࠾ࡅࡿSTOP1ኚ␗యࡢ௦ㅰ≀ࡢゎᯒ࡛ࡶGABA shuntࡸpH statࡢpHㄪ⠇⤒㊰ࡀኚ␗య࡛ῶᑡࡋࡓࠋࡲࡓࠊGFPࢆ⏝࠸ࡓSTOP1 ࡢᒁᅾゎᯒ࡛ࡣࠊSTOP1ࡀ᰾࡟ᒁᅾࡋ࡚࠸ࡓࡇ࡜ࠊࡉࡽ࡟AlࠊH⁺ࡢẘᛶࢆ㜵ᚚࡍ

ࡿᵝࠎ࡞㑇ఏᏊࡢⓎ⌧ㄪ⠇ࢆࡋ࡚࠸ࡿࡇ࡜࠿ࡽࠊSTOP1ࡀ㌿෗ᅉᏊ࡛࠶ࡿྍ⬟ᛶ

ࢆ♧၀ࡋ࡚࠸ࡿࠋ

࣮ࣘ࢝ࣜࡣୡ⏺୰࡛ᕤᴗ⏝࡟᳜ᯘࡉࢀ࡚࠸ࡿࡀࠊ㓟ᛶᅵተࢆࡣࡌࡵ࡜ࡍࡿ୙Ⰻ ᅵተࡢࡓࡵࠊ㧗ᡂ㛗ࠊ㧗཰㔞ࡢಶయ㑅ᢤࠊ⫱✀ࡀㄢ㢟࡜࡞ࡗ࡚࠸ࡿࠋࡇࡢࡇ࡜࠿ࡽࠊ Al ⪏ᛶࢆ௜୚ࡍࡿ᭷ᶵ㓟ᨺฟࡣ㑅ᢤࡢ࣐࣮࣮࢝࡜࡞ࡾᚓࡿࡓࡵࠊ௦⾲ⓗ࡞ရ✀࡛

࠶ࡿ࣮࣐ࣘ࢝ࣜ࢝ࣝࢻࣞࣥࢩࢫࡢ᭷ᶵ㓟ᨺฟ≉ᛶࢆㄪ࡭ࡓࠋ㔜㔠ᒓࠊప pH ࡞࡝ࠊ ᵝࠎ࡞ࢫࢺࣞࢫ࡛᰿ఙ㛗ࡢ㜼ᐖࡀぢࡽࢀࡓࡀࠊ᭷ᶵ㓟ᨺฟࡣAlࡢࡳ࡟ᛂ⟅ࡋ࡚ࢡ

࢚ࣥ㓟ࢆᨺฟࡋࡓࠋḟ࡟ࠊ࣮࡛ࣘ࢝ࣜࡣࢡ࢚ࣥ㓟㍺㏦࡟㛵ࢃࡿࢡ࢚ࣥ㓟ࢺࣛࣥࢫ࣏

࣮ࢱ࣮ࡣᮍ༢㞳࡛࠶ࡗࡓࡓࡵࠊ༢㞳ࡋࡓ࠺࠼࡛ࡑࡢᶵ⬟࡟ࡘ࠸࡚ゎᯒࡋࡓࠋ Multidrug and toxic compound extrusion (MATE) ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࣇ࢓࣑࣮ࣜ

࡟ᒓࡍࡿࢡ࢚ࣥ㓟ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࢆࠊ௚ࡢ᳜≀✀ࡢ㓄ิࢆࡶ࡜࡟ࠊ࣮࣐ࣘ࢝ࣜ࢝

ࣝࢻࣞࣥࢩࢫ࠿ࡽ4 ࡘࡢEcMATE ࣍ࣔࣟࢢࢆ༢㞳ࡋࡓࠋࡇࡢ୰࡛Al ࡟ᛂ⟅ࡋ࡚

㌿෗㔞ࡀቑຍࡋ࡚࠸ࡿ࣍ࣔࣟࢢࡣEcMATE1ࡢࡳ࡛࠶ࡗࡓࠋGFP࡟ࡼࡿᒁᅾゎᯒ

࡛ࡣEcMATE1ࡀ⣽⬊⭷࡟Ꮡᅾࡋࠊࢱࣂࢥẟ≧᰿࡛ࡢEcMATE1 㐣๫Ⓨ⌧య࡛ࡣ

Al ࡟ࡼࡾࢡ࢚ࣥ㓟ᨺฟࡀቑຍࡋࠊ᰿ఙ㛗㜼ᐖࡶᨵၿࡉࢀࡓࠋࡲࡓࠊࢱࣥࣃࢡ㉁ࡢ

ࣜࣥ㓟໬ࠊ⬺ࣜࣥ㓟໬㜼ᐖ๣࡟ࡼࡾࠊ᭷ᶵ㓟ᨺฟ࡜EcMATE1㌿෗㔞ࡣῶᑡࡋࡓࠋ ௨ୖ࠿ࡽࠊEcMATE1ࡣAl⪏ᛶ࡟㈉⊩ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡓࠋ⥆࠸࡚ࠊ࣮ࣘ࢝ࣜ

ࡢSTOP1㑇ఏᏊࢆ༢㞳ࡋࠊ࢔ࢢࣟࣂࢡࢸ࣒ࣜ࢘ࣜࢰࢤࢿࢫ࡟ࡼࡿẟ≧᰿ㄏᑟ࡜࡜

ࡶ࡟RNAi ࡢᑟධ࡟ࡼࡗ࡚ STOP1ࡢ㌿෗ࢆᢚ࠼ࡓ࡜ࡇࢁࠊMATEࡸ ALS3 ࣮࢜

ࢯࣟࢢ㑇ఏᏊࡢ㌿෗ࡣᢚไࡉࢀࡓࠋ࣮ࣘ࢝ࣜSTOP1ࢆࢩࣟ࢖ࢾࢼࢬࢼࡢstop1ኚ

␗య࡟ᑟධࡋࡓ┦⿵ᐇ㦂࡛ࡣࠊపpH⪏ᛶࡀᅇ᚟ࡋࡓࠋࢱࣂࢥⴥ࡬ࡢ GFP⼥ྜࢱ

ࣥࣃࢡࡢᑟධ࡛ࡣࠊSTOP1 ࡣ᰾࡟ᒁᅾࡋ࡚࠸ࡓࠋ௨ୖ࠿ࡽࠊ࣮ࣘ࢝ࣜࡢ STOP1

࡜Al⪏ᛶ㑇ఏᏊࢆྵࡴAl⪏ᛶ㑇ఏᏊࡢࢩ࣮ࣜࢬࢆ≉ᐃࡋࡓࠋࡇࢀࡽࡢ㑇ఏᏊࡣ㓟 ᛶᅵተ⪏ᛶ࡟㛵ࡍࡿศᏊ⫱✀ࡸࢤࣀ࣑ࢵࢡࢭࣞࢡࢩࣙࣥ࡟᭷⏝࡛࠶ࡿྍ⬟ᛶࢆ♧

၀ࡋࡓࠋ

ㅰ ㅰ㎡

௒ᅇࡢ◊✲࡟㛵ࡋࠊ⤌᥮యࡢసᡂ࡜ゎᯒࢆ⾜ࡗ࡚ࡃࡔࡉࡗࡓ⌮໬Ꮫ◊✲ᡤࡢ⠛

ᓮ୍㞝ඛ⏕ࠊ⸨⏣⨾⣖ඛ⏕ࠊ⌮໬Ꮫ◊✲ᡤࣂ࢖࢜ࣜࢯ࣮ࢫࢭࣥࢱ࣮ࡢᑠᯘṇᬛඛ⏕ࠊ

஭ෆ⪷ඛ⏕ࠊ࣋ࢡࢱ࣮ࢆᥦ౪ࡋ࡚ࡃࡔࡉࡗࡓᓥ᰿኱Ꮫࡢ୰ᕝᙉඛ⏕ࠊࡲࡓࠊ࣐࢖ࢡ

ࣟ࢔ࣞ࢖ࠊ௦ㅰ≀ ᐃࢆᢸᙜࡋ࡚ࡃࡔࡉࡗࡓࠊ࠿ࡎࡉDNA◊✲ᡤࡢᰘ⏣኱㍜ඛ⏕ࠊ Ḉ஭ᮃඛ⏕࡟ឤㅰ࠸ࡓࡋࡲࡍࠋ

࣮ࣘ࢝ࣜࡢ◊✲࡟㛵ࡋࠊᣦᑟࡋ࡚ࡃࡔࡉࡗࡓబ⸨ⱱᵝࠊす❑ఙஅᵝࠊᅵᒃᬛோ

ᵝࠊᅵ⫧ᩗᝅᵝࠊఀ⸨୍ᘺᡤ㛗ࠊᐇ㦂ࡢ⿵ຓࢆࡋ࡚ࡃࡔࡉࡗࡓ໭ᮧᬛᵝࢆࡣࡌࡵࠊ

⋤Ꮚ〇⣬᳃ᯘ㈨※◊✲ᡤࡢⓙᵝ࡟ឤㅰ࠸ࡓࡋࡲࡍࠋ࡞࠾ࠊ࣮ࣘ࢝ࣜࡢ◊✲ࡣMETI

ࣉࣟࢪ࢙ࢡࢺࡢ⿵ຓ࡟ࡼࡗ࡚⾜ࢃࢀࡲࡋࡓࠋ

᭱ᚋ࡟ࠊᮏ◊✲ࡢ⥲ྜⓗ࡞ᣦᑟࢆࡋ࡚࠸ࡓࡔࡁࡲࡋࡓࠊᒱ㜧኱Ꮫᛂ⏝⏕≀⛉Ꮫ 㒊ࡢᑠᒣ༤அඛ⏕ࠊᑠᯘభ⌮Ꮚඛ⏕࡟ឤㅰ࠸ࡓࡋࡲࡍࠋ

100

ᘬ

ᘬ⏝ᩥ⊩

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Bouche N, Fromm H (2004) GABA in plants: just a metabolite? Trends Plant Sci 99: 110-115

Bown AW, Shelp BJ (1997) The metabolism and functions of g-aminobutyric acid. Plant Physiol 1115: 1-5

Campinhos Jr E (1999) Sustainable plantations of high-yield shape Eucalyptus trees for production of fiber: the Aracruz case. New Forests 117: 129-143 Castanie-Cornet MP, Penfound TA, Smith D, Elliott JF, Foster JW (1999)

Control of acid resistance in Escherichia coli. J Bacteriol 1181: 3525-3535 Chiu W, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered

GFP as a vital reporter in plants. Curr Biol 66: 325-330

Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743

ドキュメント内 STOP1 転写制御による植物の酸性土壌耐性機構に関する研究 (ページ 48-120)