多面性を持つストレス耐性遺伝子AtALMT1の発現制御に関する研究

Title

多面性を持つストレス耐性遺伝子AtALMT1の発現制御に関

_{する研究( 本文(Fulltext) )}

Author(s)

小林, 安文

Report No.(Doctoral

Degree)

博士(農学) 甲第612号

Issue Date

2013-09-10

Type

博士論文

Version

ETD

URL

http://hdl.handle.net/20.500.12099/47823

ከ㠃ᛶࢆᣢࡘࢫࢺࣞࢫ⪏ᛶ㑇ఏᏊ

AtALMT1

ࡢ

Ⓨ⌧ไᚚ࡟㛵ࡍࡿ◊✲

2 0 1 3 ᖺ

ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ㎰Ꮫ◊✲⛉

⏕≀㈨※⛉Ꮫ

(ᒱ 㜧 ኱ Ꮫ)

ᑠ ᯘ Ᏻ ᩥ

ከ㠃ᛶࢆᣢࡘࢫࢺࣞࢫ⪏ᛶ㑇ఏᏊ

AtALMT1

ࡢ

Ⓨ⌧ไᚚ࡟㛵ࡍࡿ◊✲

┠ḟ

ᗎㄽ

1 㡫

➨

1 ❶

⣽⬊⭷⾲㠃࡛ࡢ㟼㟁ⓗࣔࢹࣝ࡟ᇶ࡙ࡃ

Al

_{, H}

_{ẘᛶࡢศᏊ⏕⌮Ꮫⓗゎᯒ}

_{6 㡫}

➨

2 ❶

ྛ✀ࢩࢢࢼࣝᅉᏊ࡟ᛂ⟅ࡍࡿ

AtALMT1

ࡢⓎ⌧ไᚚᶵᵓ

38 㡫

⥲ྜ⪃ᐹ

66 㡫

⤖ㄽ

72 㡫

ㅰ㎡

73 㡫

ᘬ⏝ᩥ⊩

74 㡫

ᗎㄽ

2005 ᖺ࡟ 65 ൨ேࢆ㉸࠼ࡓୡ⏺ேཱྀࡣ㸪ᅜ㝿㐃ྜ࡟ࡼࡿ᥎ᐃ࡛㸪2050 ᖺ࡟ࡣ 96 ൨ே㸪

᭱኱ࡢண ್࡛ࡣ109 ൨ே࡟㐩ࡍࡿ࡜ண᝿ࡉࢀ࡚࠸ࡿ (World Population Prospects The

2012 division; http://esa.un.org/wpp/index.htm). 1950 ᖺ௨㝆㸪໬Ꮫ⫧ᩱࡸ㎰⸆౑⏝㔞㸪✐ ≀స௜㠃✚ࡢቑຍཬࡧ㎰ᴗᢏ⾡ࡢⓎ㐩࡟ࡼࡾ✐≀⏕⏘㔞ࡣቑ⏘ࡉࢀ࡚ࡁࡓ㸬ࡋ࠿ࡋ࡞ࡀࡽ㸪 ᅜ㐃ୡ⏺㣗⣊ィ⏬ (WFP)࡟ࡼࡿሗ࿌࡛ࡣ㸪ቑຍࡍࡿேཱྀ࡟㣗⣊⏕⏘ࡀ㏣࠸ࡘ࠿࡞࠸ࡓࡵ㸪 8 ൨ 5000 ୓ேࡀᰤ㣴୙Ⰻࡸ㣚㣹࡟㝗ࡗ࡚࠸ࡿ㸬≉࡟㸪῝้࡜࡞ࡗ࡚࠸ࡿࡢࡀ㛤Ⓨ㏵ୖᅜ࡛ ࠶ࡾ㸪㣚㣹ேཱྀࡣ1 ᖺ࡟ 400 ୓ேࡢ࣮࣌ࢫ࡛ቑຍࡋ⥆ࡅ࡚࠸ࡿ㸬ࡇࢀࡽࡢ㛤Ⓨ㏵ୖᅜ࡛ࡣ㸪 ㎰ᴗศ㔝࡟࠾ࡅࡿ⛉Ꮫᢏ⾡ࡀ༑ศ࡟ᾐ㏱ࡋ࡚࠾ࡽࡎ㸪ࡇࡢ៏ᛶⓗ࡞㣚㣹≧ែࡀᝏ໬ࡍࡿഴ ྥ࡟࠶ࡿ㸬1996 ᖺ࡟㛤ദࡉࢀࡓୡ⏺㣗⣊ࢧ࣑ࢵࢺ࡟࠾࠸࡚ 2015 ᖺࡲ࡛࡟ᰤ㣴୙㊊ேཱྀࡢ ༙ῶࢆ┠ᶆ࡜ࡍࡿࡇ࡜ࡀ᥇ᢥࡉࢀࡓࡀ㸪ࡑࡢࡓࡵ࡟ࡣ㣚㣹ேཱྀࢆẖᖺ2600 ୓ேῶࡽࡉ࡞ࡅ ࢀࡤ࡞ࡽ࡞࠸㸬⌧ᐇ࡟㸪100 ൨ேࡢேཱྀࢆ㣴࠺ࡓࡵ࡟ࡣᑡ࡞ࡃ࡜ࡶ 40 ൨ ha ࡢ⪔ᆅࡀᚲせ ࡜ࡉࢀࡿࡀ㸪⌧ᅾࡢ⪔ᆅ㠃✚ࡣ࠾ࡼࡑ15 ൨ ha ࡛࠶ࡿ㸬ࡋ࠿ࡋ࡞ࡀࡽ㸪₯ᅾⓗྍ⪔ᆅࡢከ ࡃࡀၥ㢟ᅵተ࡛࠶ࡿࡓࡵ⪔ᆅ㠃✚ࡢቑຍഴྥࡣㄆࡵࡽࢀ࡞࠸㸬ᆅ⌫ୖࡢ㝣ᆅ㠃✚ (ịᗋ࡛そ ࢃࢀࡓᅵᆅࢆ㝖ࡃ)ࡣ, ࠾ࡼࡑ 130 ൨ ha ࡛࠶ࡿࡀ㸪ၥ㢟ᅵተ࡜ࡉࢀࡿ㓟ᛶᅵተࡀ⣙ 30% (3950 ୓ ha)ࢆ༨ࡵࡿ㸬⌧ᅾ㸪㓟ᛶᅵተ㠃✚ࡢ࠺ࡕ 67%ࡀ᳃ᯘᆅᖏ㸪18%ࡀࢧࣂࣥࢼ࡟ࡼࡾ ༨ࡵࡽࢀ㸪4.5㸣 (179 ୓ ha)ࡔࡅࡀ✐≀⏕⏘࡟฼⏝ࡉࢀ࡚࠸ࡿ㸬୍᪉࡛㸪20 ୡ⣖ᚋ༙࠿ࡽ ຍ㏿ࡋࡓ᳃ᯘఆ᥇ཬࡧࡑࢀ࡟క࠺ࣉࣛࣥࢸ࣮ࢩࣙࣥ࡟ࡼࡾᑡ࡞ࡃ࡜ࡶ250 ୓ ha ࡢ⇕ᖏ㞵ᯘ ࡀᾘኻࡍࡿ࡜࡜ࡶ࡟㸪ࡉࡽ࡞ࡿ㓟ᛶᅵተࡢຎ໬ࢆᘬࡁ㉳ࡇࡋࡓࡲࡲᨺ⨨ࡉࢀ࡚࠸ࡿ㸬㓟ᛶ ᅵተ࠿ࡽ⏕ࡌࡿᖺ㛫⏕⏘≀ࡢ౯್ࡣ⣙ 1,290 ൨ࢻࣝ࡟┦ᙜࡍࡿ࡜ࡉࢀ㸪ୡ⏺⤒῭࡟୚࠼ࡿ

ᙳ㡪࡟ࡘ࠸࡚ࡶィࡾ▱ࢀ࡞࠸ (von Uexküll and Mutert, 1995)㸬ᚑࡗ࡚, ࡇࡢ㓟ᛶᅵተࢆ⪔ ᆅ࡜ࡋ࡚᭷ຠ࡟฼⏝ࡍࡿࡇ࡜ࡀ㸪㣗⣊ၥ㢟ࡢゎỴ࡟኱ࡁࡃ㈉⊩ࡍࡿ࡜ࡉࢀ࡚࠸ࡿ㸬

㓟ᛶᅵተࡢᨵⰋ࡟ࡣ, ᅵተ㓟ᗘࢆ▹ṇࡍࡿࡓࡵࡢ▼⅊㉁⫧ᩱࡢ᪋⫧ࡀ᭷ຠ࡛࠶ࡿ. ࡲࡓ, 㓟ᛶᅵተࡢ✀㢮ࡢ1 ࡘ࡛࠶ࡿ andisol ࡛ࡣ, ࣜࣥ㓟▼⭯ࡸ▼⭯ࡢ᪋⫧ࡶຠᯝⓗ࡛࠶ࡿࡇ࡜ࡀ ▱ࡽࢀ࡚࠸ࡿ (Meriño-Gergichevich et al., 2010)㸬ࡋ࠿ࡋ࡞ࡀࡽ, ࡇࢀࡽ⫧ᩱ᪋⫧࡟క࠺཰ 㔞ࡢቑຍ࡟㛵ࡋ࡚, ࡝ࡢࡼ࠺࡟ࢫࢺࣞࢫࡀ㍍ῶࡉࢀ࡚࠸ࡿ࠿ࢆศᏊ࡛ࣞ࣋ࣝ᫂ࡽ࠿࡟ࡋࡓ ◊✲ࡣ࡯࡜ࢇ࡝⾜ࢃࢀ࡚࠸࡞࠸. ࡇࢀࡽࢆ⌮ゎࡍࡿࡇ࡜࡛, ከᵝ࡞㓟ᛶᅵተ࡟ᑐࡋ࡚, ᅵተ ࡢ㓟ᗘ▹ṇ࠾ࡼࡧ㣴ศࣂࣛࣥࢫࢆ⪃៖ࡋ, ࡑࡢᅵተ࡟㐺ࡋࡓ᪋⫧ࡀྍ⬟࡜࡞ࡿ࡜⪃࠼ࡽࢀ ࡿ. ࡲࡓ, ࡇࢀࡽ⫧ᩱࡢ᪋⫧ࢆຠᯝⓗ࡟⾜࠺ࡇ࡜ࡀ࡛ࡁࢀࡤ, 㛤Ⓨ㏵ୖᅜ࡟࡜ࡗ࡚ࡢ⤒῭ⓗ ࡞㈇ᢸࡀゎᾘࡉࢀࡿ㸬 ୍᪉㸪᳜≀࡟ࡣ⎔ቃࢫࢺࣞࢫ࡟ᑐࡍࡿ₯ᅾⓗ࡞⪏ᛶไᚚᶵᵓࢆഛ࠼ࡿ㸬ࡇࢀࡽ⪏ᛶᶵᵓ ࡣ㸪ࡲࡎ㸪᳜≀ࡀ⣽⬊࡛ࣞ࣋ࣝࢫࢺࣞࢫࢆㄆ㆑ࡋ㸪ࢩࢢࢼࣝఏ㐩⤒㊰ࢆ௓ࡋ࡚᝟ሗࢆ୰⥅ ࡍࡿࡇ࡜࡟ጞࡲࡿ㸬ࡇࡢࢩࢢࢼࣝࡣ㸪⏕⌮ⓗኚ໬ (౛࠼ࡤ㸪ẼᏍࡢ㛤㛢)ཬࡧ㑇ఏᏊⓎ⌧ࢆ ㄪ⠇ࡋ㸪ศᏊ㸪⣽⬊࡛ࣞ࣋ࣝࡢ㐣⛬ࢆᨵኚࡍࡿࡇ࡜࡛⪏ᛶࢆ⋓ᚓࡍࡿ࡜ࡉࢀࡿ (Knight and Knight, 2001)㸬᱂ᇵస≀ཬࡧ✐≀ࡢᨵⰋ࡛⏕ࡌࡓᙧែኚ໬࡟ࡣ㸪㌿෗ไᚚᅉᏊࡢኚ␗ࡸⓎ ⌧㔞ࡢኚ໬㸪ㄪ⠇ࢱࣥࣃࢡ㉁ࡢⓎ⌧㔞ࡢኚ໬ࡀ㛵ಀࡋ࡚࠸ࡿ (Riechmann, 2002)㸬ࢫࢺࣞ ࢫ⪏ᛶᶵᵓࢆศᏊ࡛ࣞ࣋ࣝ᫂ࡽ࠿࡟ࡋ㸪ศᏊ⫱✀࡟ࡼࡗ࡚⪏ᛶ᳜≀ࢆ⋓ᚓࡍࡿᡭἲࡣ㸪ୡ ⏺ⓗ࡟ぢ࡚ࡶ┒ࢇ࡟ྲྀࡾ⤌ࡲࢀ࡚࠾ࡾ㸪ྍ⪔ᆅࡢᣑ኱ࡸ⏕⏘ᛶࡢྥୖ࡟ࡼࡾ㣗⣊ၥ㢟ࡢゎ Ỵ࡟㈉⊩ࡍࡿࡇ࡜ࡀᮇᚅ࡛ࡁࡿ㸬⌧ᅾ, ⎔ቃࢫࢺࣞࢫ࡟ᑐࡍࡿ⪏ᛶᶵᵓࡢ௜୚ࡸ཰㔞ࡢቑຍ ࢆຠ⋡ⓗ, ຠᯝⓗ࡟ศᏊ⫱✀࡟ࡼࡾ⾜࠺ࡇ࡜ࡀồࡵࡽࢀ࡚࠸ࡿ. ࢖ࢿ࡟࠾࠸࡚, ᵝࠎ࡞㑇ఏ Ꮫⓗᡭἲࢆ⏝࠸࡚, ࡼࡾගྜᡂຠ⋡ࡢ㧗࠸ C4ᆺගྜᡂ⤒㊰ࢆᑟධࡍࡿヨࡳࡀ࡞ࡉࢀ࡚࠸ࡿ. C4ᆺගྜᡂ⤒㊰࡟ྵࡲࢀࡿ㓝⣲ࢱࣥࣃࢡࡸ௦ㅰ⏘≀ࡢ㍺㏦యࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࢭࢵࢺࢆ ⣽⬊≉␗ⓗ࡞Ⓨ⌧ࢆ♧ࡍࣉ࣮ࣟࣔࢱ࣮ࢆ⏝࠸࡚Ⓨ⌧ࡉࡏࡿࡔࡅ࡛࡞ࡃ, C3ᆺ࡜C4ᆺ࡛ࡣⴥ ࡢ⤌⧊ᵓ㐀ࡀ኱ࡁࡃ␗࡞ࡿࡓࡵ, ⤌⧊ᵓ㐀ࡢ㠃࡛ࡢᨵኚࡶᚲせ࡛࠶ࡿ࡜ࡉࢀ࡚࠸ࡿ (von Caemmerer et al., 2012).

㓟ᛶᅵተ࡟Ꮡᅾࡍࡿࢫࢺࣞࢫ࡟ࡣ㸪ྍ⁐໬࡟క࠺࢔࣑ࣝࢽ࣒࢘ (Al)㸪࣐ࣥ࢞ࣥ(Mn)࢖࢜ ࣥ⃰ᗘࡢቑຍཬࡧ㐣๫࡟Ꮡᅾࡍࡿࣉࣟࢺࣥ (H+_{)࡟ࡼࡿ୺࡟᰿ࡢఙ㛗㜼ᐖ㸪ᚲ㡲ᰤ㣴ඖ⣲࡛} ࠶ࡿ࣐ࢢࢿࢩ࣒࢘ (Mg)㸪࢝ࣝࢩ࣒࢘ (Ca)Ḟஈ㸪ྍ⁐ᛶపୗ࡟క࠺ࣜࣥ (P)㸪ࣔࣜࣈࢹࣥ (Mo)ࡢḞஈࡀᣲࡆࡽࢀࡿ (Marschner, 1995)㸬㓟ᛶᅵተ࡛ࡢ⏕⫱㜼ᐖࡣ, ୺࡟ Al ࢫࢺࣞࢫ ࡟ࡼࡿ࡜ࡉࢀࡿ. ࡋ࠿ࡋ࡞ࡀࡽ, 㓟ᛶᅵተ࡟༊ศࡉࢀࡿ᭷ᶵ㉁ᅵተ (histosol)࡛ࡣ㸪ᅵተ⁐ ᾮpH ࡀ 4 ௨ୗ࡜࡞ࡾ㸪⁐ᾮ⤌ᡂ࡟࠾࠸࡚ 3 ౯ Al ࢖࢜ࣥ (Al3+_{)ࡼࡾࡶ H}+_{ࡢ๭ྜࡀ㧗ࡃ࡞}

ࡾ, ୺࡟ H+࡟ࡼࡿ㞀ᐖࡀ⏕ࡌ࡚࠸ࡿ࡜ࡉࢀࡿ (Kidd and Proctor, 2001)㸬H+_{ࡑࡢࡶࡢ࡟ࡼ}

ࡿపpH ࢫࢺࣞࢫࡣ㸪⪏ᛶᶵᵓ࡟ࡘ࠸࡚ࡢヲ⣽࡞◊✲ࡀ࡞ࡉࢀ࡚࠸࡞࠸㸬histosol ࡣ㸪࠾ࡼ ࡑ200 ୓ ha ࡟ࡶཬࡪࡇ࡜࡟ຍ࠼㸪ࡑࡢ௚ࡢ㓟ᛶᅵተ࡟࠾࠸࡚ࡶ Al ࢫࢺࣞࢫࡔࡅ࡛࡞ࡃప pH ࢫࢺࣞࢫࡀᏑᅾࡋ࡚࠸ࡿ࡜ࡉࢀࡿ. ᚑࡗ࡚, Al, H+ẘᛶཬࡧࡑࢀࡽࡢ⪏ᛶᶵᵓࢆゎ᫂ࡀ ᚲせ࡜ࡉࢀࡿ. ⌧ᅾ, ᵝࠎ࡞᳜≀✀࡟ᑐࡍࡿࣜࢯ࣮ࢫࡢᩚഛࡀ┒ࢇ࡟⾜ࢃࢀ࡚࠸ࡿ. ࣔࢹ᳜ࣝ≀࡜ࡋ࡚ ࢤࣀ࣒᝟ሗ➼ࡢࣜࢯ࣮ࢫᩚഛࡀඛ⾜ࡋ࡚࠸ࡿࢩࣟ࢖ࢾࢼࢬࢼ࡛ࡣ, 㑇ఏᏛⓗ࡞ゎᯒࡀ, 㓟 ᛶᅵተࢫࢺࣞࢫࡢ⪏ᛶศᏊᶵᵓࡢゎ᫂࡟㈉⊩ࡋ࡚࠸ࡿ. ࢩࣟ࢖ࢾࢼࢬࢼ Al-activated malate transporter (AtALMT1)ࢆ௓ࡋ࡚᰿ᅪ࡟ᨺฟࡉࢀࡿࣜࣥࢦ㓟࡜ Al ࡜ࡢ࣮࢟ࣞࢺ໬

࡟ࡼࡿAl ẘᛶࡢ↓ẘ໬ࡀ⪏ᛶ࡟㔜せ࡛࠶ࡿ (Hoekenga et al., 2006). ࡲࡓ, ࢩࣟ࢖ࢾࢼࢬ

ࢼ࡛ࡢࢡ࢚ࣥ㓟ᨺฟ࡟㛵୚ࡍࡿ࡜ࡉࢀࡿMultidrug and toxin efflux family (AtMATE)ࡶ

ࡲࡓAl ẘᛶࡢ㍍ῶ࡟ᚲせ࡛࠶ࡿ࡜ࡉࢀࡓ (Liu et al., 2009). ⣽⳦ᆺࡢ ABC ࢺࣛࣥࢫ࣏࣮

ࢱ࣮ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊAluminum sensitive 3 (ALS3) ཬࡧSensitive to Al rhizotoxicity

1 (AtSTAR1) ࡢኚ␗ࡣ, Al ឤཷᛶࢆᘬࡁ㉳ࡇࡋࡓ. ALS3 ࡣᑟ⟶࡬ࡢ Al ᤼ฟ࡟㛵୚ࡍࡿࡇ ࡜ࡀ♧၀ࡉࢀ࡚࠸ࡿ (Larsen et al., 1997; Larsen et al., 2005). ୍᪉, AtSTAR1 ࡟ࡘ࠸࡚ ࡣ, UDP-ࢢࣝࢥ࣮ࢫ (UDP-Glc)ࡢ㍺㏦άᛶࢆᣢࡕ, ⣽⬊እ࡟㍺㏦ࡉࢀࡓ UDP-Glc ཬࡧࡑ ࡢㄏᑟయࡀ, ⣽⬊ቨࡢ Al ⤖ྜ㒊఩ࢆ㐽ⶸࡍࡿࡼ࠺࡟⣽⬊ቨ⤌ᡂࢆᨵኚࡍࡿ࡜⪃࠼ࡽࢀ࡚࠸

(sensitive to proton rhizotoxiity 1)ࡀึࡵ࡚༢㞳ࡉࢀࡓࡀ, STOP1㑇ఏᏊࡢኚ␗ࡣ㓟ࡔࡅ

࡛࡞ࡃAl ࢫࢺࣞࢫ࡟ᑐࡋ࡚ࡶឤཷᛶࢆ♧ࡋࡓ (Iuchi et al., 2007). ࡇࡢ Al ㉸ឤཷᛶࡣ, Al

ฎ⌮࡟ࡼࡿ AtALMT1 ࡢⓎ⌧ㄏᑟཬࡧࡑࢀ࡟௜㝶ࡋࡓࣜࣥࢦ㓟ᨺฟࡀᾘኻࡋ࡚࠸ࡿࡇ࡜ࡀ

ཎᅉ࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀࡓ. ຍ࠼࡚, STOP1 ࡢ᰾ᒁᅾᛶཬࡧࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ゎᯒ࡟ ࡼࡾ, Al ཬࡧప pH ⪏ᛶ࡟㛵୚ࡍࡿ」ᩘࡢ㑇ఏᏊࡢⓎ⌧ࡀ, ㌿෗ไᚚᅉᏊ࡜ࡋ࡚ᶵ⬟ࡍࡿ STOP1 ࢆ௓ࡋ࡚ไᚚࡉࢀ࡚࠸ࡿࡇ࡜ࢆሗ࿌ࡋࡓ (Sawaki et al., 2009).

᳜≀ࡢ⏕⫱࡟ᙳ㡪ࡍࡿ⎔ቃኚ໬( ᗘ, CO2 ⃰ᗘ, ஝⇱ཬࡧሷศ➼)࡟ᛂ⟅ࡍࡿ㑇ఏᏊࡣ, 」ᩘࡢࢫࢺࣞࢫ࡟ᛂ⟅ࡍࡿࡶࡢࡀከࡃᏑᅾࡍࡿ (Ahuja et al., 2010). ౛࠼ࡤ, Glycine-rich RNA-binding protein 7 (GRP7)ࡣ, ሷ, ஝⇱ཬࡧప ࢫࢺࣞࢫ࡟ࡼࡗ࡚Ꮝ㎶⣽⬊࡛ㄏᑟࡉ ࢀࡿ (Kim et al., 2008). ẼᏍ㛤㛢ࡢไᚚࡣ, ࡑࢀࡽࡢࢫࢺࣞࢫ࡟ᑐࡍࡿ⪏ᛶ⋓ᚓ࡟୰ᚰⓗ ࡞ᙺ๭ࢆᯝࡓࡋ࡚࠾ࡾ, GRP7 ࡢⓎ⌧ᛂ⟅ᛶࡣ⎔ቃ࡟㐺ᛂࡍࡿࡓࡵ࡟㒔ྜࡀࡼ࠸࡜⪃࠼ࡽࢀ ࡿ. ࡉࡽ࡟, 㠀⏕≀ⓗࢫࢺࣞࢫ࡜⏕≀ⓗࢫࢺࣞࢫᛂ⟅࡟ࡘ࠸࡚, ࢩࢢࢼࣝఏ㐩⤒㊰ࡢࢡࣟࢫ ࢺ࣮ࢡࡀ㏆ᖺ, ὀ┠ࡉࢀ࡚࠸ࡿ (Fujita et al., 2006). AtALMT1Ⓨ⌧ᛂ⟅࡟㛵ࡋ࡚, Al ࡟ࡼࡿㄏᑟ࡯࡝࡛ࡣ࡞࠸ࡀ, ప pH ࡟ࡼࡾ᭷ព࡟ㄏᑟࡉ ࢀࡿࡇ࡜ࡀ☜ㄆࡉࢀ࡚࠸ࡿ (Kobayashi et al., 2007). ࡉࡽ࡟, ᳜≀⑓ཎ⳦ཬࡧච␿ᛂ⟅ࢆ ᘬࡁ㉳ࡇࡍ࢚ࣜࢩࢱ࣮࡟ࡼࡗ࡚ㄏᑟࡉࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀࡓ (Rudrappa et al., 2008;

Lakshmanan et al., 2012). AtALMT1ࡣSTOP1 ࢆ௓ࡋ࡚Ⓨ⌧ࡀไᚚࡉࢀ࡚࠸ࡿࡀ, ࡑࢀ௨

እࡢⓎ⌧ไᚚ࡟㛵ࢃࡿᅉᏊࡸ඲యⓗ࡞ࢩࢢࢼࣝఏ㐩⤒㊰ࡣ᫂ࡽ࠿࡜࡞ࡗ࡚࠾ࡽࡎ, ࡇࢀࡽ 」ᩘࡢᛂ⟅ᛶࡢㄝ᫂࡟⮳ࡗ࡚࠸࡞࠸. ࡲࡓ, ᵝࠎ࡞⎔ቃᅉᏊࡣ, ᳜≀ࡢෆᅾⓗ࡞ࣉࣟࢢ࣒ࣛ ࢆάᛶ໬ࡍࡿࡓࡵࡢࢩࢢࢼࣝ࡜ࡋ࡚᳜≀࡟ཷᐜࡉࢀࡿ (McCarty and Chory, 2000). 㓟ᛶ ᅵተ㞀ᐖ࡟ᑐࡋ࡚, ẚ㍑ⓗ࡟◊✲ࡀ㐍ᒎࡋ࡚࠸ࡿ Al 㞀ᐖ࡛ࡉ࠼, ࢩࢢࢼࣝࡢཷᐜ࡟㛵ࡍࡿ ▱ぢࡣஈࡋ࠸.

ᮏ◊✲࡛ࡣ, 」ᩘࡢࢩࢢࢼࣝᅉᏊ࡟ᛂ⟅ࡍࡿ࡜ࡉࢀࡿAtALMT1࡟ࡘ࠸࡚, Ⓨ⌧ไᚚᶵᵓ

ᵓࢆ᳨ドࡍࡿ. ຍ࠼࡚, 㓟ᛶᅵተ࡛ࡢ⏕⫱㜼ᐖࢆᘬࡁ㉳ࡇࡍ Al3+_{, H}+_{ẘᛶ࡟ࡘ࠸࡚, ࢫࢺࣞ} ࢫ⁐ᾮ࡛ࡢ࢖࢜ࣥ✀ࡢ᥎ᐃཬࡧ࢖࢜ࣥ✀࡜⣽⬊⭷⾲㠃࡜ࡢ㟼㟁ⓗ࡞┦஫స⏝ࢆ⪃៖ࡋࡓศ Ꮚ⏕⌮Ꮫⓗゎᯒࡀ, ⪏ᛶ㑇ఏᏊࡢⓎ⌧ไᚚ࡟᪂ࡓ࡞▱ぢࢆࡶࡓࡽࡍࡇ࡜ࢆ♧ࡍ.

➨

1 ❶

⣽⬊⭷⾲㠃࡛ࡢ㟼㟁ⓗࣔࢹࣝ࡟ᇶ࡙ࡃ

Al

_{, H}

_{ẘᛶࡢศᏊ}

⏕⌮Ꮫⓗゎᯒ

ᗎㄽ

2009). ⣽⬊⭷ୖ࡟ࡣ, 㞀ᐖࡢࢱ࣮ࢤࢵࢺ࡜࡞ࡿᵓᡂᡂศࡸ Al3+_{ࢆཷᐜࡍࡿࣞࢭࣉࢱ࣮ࢱࣥ} ࣃࢡ㉁ࡀᏑᅾࡍࡿ࡜ࡉࢀࡿࡀ, ࡑࢀࡽ࡜ Al3+_{࡜ࡢ㛵ಀ࡟ࡘ࠸࡚ࡢヲ⣽࡞ゎᯒࡣ⾜ࢃࢀ࡚࠸} ࡞࠸. ࡜ࡇࢁ࡛, ⁐ᾮ୰ࡢ Al ࢖࢜ࣥ✀ࡣ, ⁐ᾮ pH ཬࡧඹᏑࡍࡿ࢔ࢽ࢜ࣥ࡟ࡼࡾ࢖࢜ࣥ✀ࡢ๭ ྜࡀ」㞧࡟ኚ໬ࡍࡿ. ᙉ࠸ẘᛶࢆ♧ࡍ࡜ࡉࢀࡿ Al3+_{ࡣ, pH ࡀ 4 ௜㏆࡛ 90㸣௨ୖࢆ༨ࡵࡿࡀ,} pH ࡢୖ᪼࡟క࠸, AlOH2+_{, Al(OH)2+ࡢ๭ྜࡀቑຍࡍࡿ. ࡇࢀࡽỈ㓟໬࢔࣑ࣝࢽ࣒࢘࢖࢜ࣥ} ✀ࡣ୙Ᏻᐃ࡛࠶ࡾ, 㔜ྜ࡟ࡼࡾ Al13ࡢᙧᡂࡸAl(OH)3ࡢỿẊࢆ⏕ࡌࡿ (Kinraide, 1991). ࢥ ࣒ࢠ࡟࠾࠸࡚, ࢖࢜ࣥ✀ࢆ⪃៖ࡋࡓỈ⪔ヨ㦂⣔࡛ࡢẘᛶ࢖࢜ࣥࡢゎᯒࡼࡾ, Al3+_ࡸH+_ẘᛶཬ ࡧCa2+_{࡟ࡼࡿ㍍ῶຠᯝࡣ, Speciation-Gouy-Chapman-Stern (SGCS)ࣔࢹ࡛ࣝィ⟬ࡉࢀࡿ} ⣽⬊⭷⾲㠃 (PM)࡛ࡢ࢖࢜ࣥάືᗘ ({Ion}PM)ࢆ⏝࠸ࡿࡇ࡜࡛ㄝ᫂ࡉࢀ࡚࠸ࡿ (Kinraide and Wang, 2010). ࢥ࣒ࢠ࡛ࡣ, ᵝࠎ࡞⁐ᾮ᮲௳࡛ࡢ᰿㛗ࢹ࣮ࢱࡀ, SGCS ࡛ࡢࣔࢹࣜࣥࢢ ࡼࡾ᥎ᐃࡉࢀࡓ{Al3+_{}PM, {H}+_{}PM, {Ca}2+_{}PM࡛⾲ࡉࢀࡿ㠀⥺ᙧᅇᖐᘧࢆ⏝࠸࡚㧗┦㛵࡛ᅇᖐ࡛} ࡁ, ⏝࠸ࡽࢀࡿᘧ࠿ࡽẘᛶཬࡧ㍍ῶᶵᵓࡀᥦၐࡉࢀ࡚࠸ࡿ (Kinraide, 2003; Kinraide et

Ǎ0$O&O3ࢆྵࡴ200 Ǎ0&D&O2⁐ᾮ࡛ࡣ, {Al3+}PM, {Al3+}bulkࡣࡑࢀࡒࢀ, 9.80, Ǎ0

࡛࠶ࡿࡀ, pH 4.0 ࡟ㄪᩚࡋࡓሙྜ, {Al3+_{}PM, {Al}3+_{}bulkࡣࡑࢀࡒࢀ, 1.59, Ǎ0 ࡜ィ⟬ࡉ}

ࢀࡿ. ⣽⬊⭷࡟Ꮡᅾࡍࡿࣜࣥ⬡㉁ࡣ, ᙅ㓟ᛶഃ࡟ゎ㞳ᐃᩘࢆᣢࡕ, pH ࡀ 4.5 ௨ୖ࡛㈇㟁Ⲵࣜ

࢞ࣥࢻ࡜࡞ࡿ. pH 4.0 ࡜ẚ㍑ࡋ࡚ pH 5.0 ࡛ࡢ{Al3+_{}bulk ࡣప࠸ࡀ, {Al}3+_{}PMࡀࡼࡾ኱ࡁ࡞್}

ࢆ♧ࡍࡇ࡜ࡣ, ࡇࡢ㈇㟁Ⲵࣜ࢞ࣥࢻࡢゎ㞳࡟౫Ꮡࡋ࡚, ⣽⬊⭷⾲㠃㟁఩ࡀ pH 4.0 ࡛ࡣ-12.9

mV ࡛࠶ࡿࡢ࡟ᑐࡋ࡚ pH 5.0 ࡛ࡣ-35.5 mV ࡜㈇㟁఩ࡀ኱ࡁ࠸ࡓࡵ, ࢝ࢳ࡛࢜ࣥ࠶ࡿ Al3+

ࡀPM ࡟㞟✚ࡉࢀࡿࡇ࡜࡟ࡼࡿ. ࡇࡢ㟼㟁ⓗࣔࢹࣝࡼࡾ, Ca ࡢ㍍ῶᶵᵓ࡟ࡘ࠸࡚, ㈇㟁Ⲵࡀ

୰ ࿴ ࡉ ࢀ ࡿ ࡇ ࡜ ࡟ ࡼ ࡿ ẘ ᛶ ࢖ ࢜ ࣥ ࡢ ⨨ ᥮ (Mechanism I) ཬ ࡧ PM ࡢ Ca2+_{ࡢ ᅇ ᚟}

(Mechanism II)ࡢ PM ࡛⏕ࡌࡿᶵᵓ࡜ Mechanism III ࡟ศ㢮ࡉࢀࡿ PM ࡜┤᥋㛵ಀࡋ࡞࠸ Ca ࡢ⏕⌮ⓗᛂ⟅࡟ࡼࡿᶵᵓࢆᥦၐࡋ࡚࠸ࡿ (Kinraide, 2003; Kinraide et al., 2004). ⁐ᾮ ୰࠾ࡼࡧ⣽⬊⭷⾲㠃࡛ࡢ࢖࢜ࣥ✀ࢆ᥎ᐃࡍࡿࡇ࡜࡛, ࢩࣟ࢖ࢾࢼࢬࢼ࡟࠾࠸࡚ࡶྠᵝࡢࢫ ࢺࣞࢫࡢホ౯ࢆ⾜࠺ࡇ࡜ࡀ࡛ࡁࡿࡀ, ࡇࡢࣔࢹࣜࣥࢢࡣ, ẘᛶ࢖࢜ࣥ࡟ᑐࡍࡿ᰿㛗㜼ᐖࡔ ࡅࢆᣦᶆ࡜ࡋ࡚㆟ㄽࡉࢀ࡚࠸ࡿࡓࡵ, ศᏊ࡛ࣞ࣋ࣝࡢ㞀ᐖᶵᵓࡸ⪏ᛶᶵᵓࢆゎᯒࡍࡿࡓࡵ ࡟ࡣ, ࡉࡽ࡞ࡿ᳨ドࡀᚲせ࡛࠶ࡿ. ࡇࢀࡽࣔࢹࣝࢆ⪃៖ࡋࡓゎᯒࡣ, ࡇࢀࡲ࡛࡟ࢩࣟ࢖ࢾࢼࢬࢼ࡛ࡣ⾜ࢃࢀ࡚࠸࡞࠸. ࢩࣟ ࢖ࢾࢼࢬࢼࢆ⏝࠸ࡓỈ⪔ヨ㦂⣔࡛ࡢ◊✲ࢆẚ㍑ࡍࡿ࡜, Al ⪏ᛶࡢ QTL ゎᯒ࡟⏝࠸ࡽࢀࡓẚ ㍑ⓗ㧗࠸pH ࡟タᐃࡉࢀࡓప࢖࢜ࣥᙉᗘࡢᇵᆅ࡛ Al ⃰ᗘࡀప࠸᮲௳ Ǎ0$O&O3, pH 5.0) ࡜pH 4 ௜㏆࡟タᐃࡉࢀࡓ㧗࢖࢜ࣥᙉᗘࡢᇵᆅ࡛ Al ⃰ᗘࡀ㧗࠸᮲௳ ǍM, pH 4.2) ࡛ࡢ ᰿ 㛗 㜼 ᐖ ࡀ, 㠀ᖖ࡟ఝࡓ㑇ఏ ⓗ࡞ຠᯝ࡟ࡼ ࡗ࡚ࡶࡓ ࡽࡉࢀ࡚࠸ࡓ (Kobayashi and Koyama, 2002; Hoekenga et al., 2003). Ca ⃰ᗘ࡟ࡘ࠸࡚, ᚋ⪅ࡢ⁐ᾮ⣔࡛ࡣ࠾ࡼࡑ 3 mM

࡛࠶ࡿࡢ࡟ᑐࡋ࡚, ๓⪅ࡢ⁐ᾮ⣔ ࡛ࡣ Ǎ0 ࡜࠿࡞ࡾపࡃㄪ〇ࡉࢀ࡚࠾ࡾ, {Ca2+_}PM࡜㛵

㐃ࡋࡓ{Al3+_{}PMࢆ⏝࠸ࡿࡇ࡜࡛ㄝ᫂ࡍࡿࡇ࡜ࡀ࡛ࡁࡿ࡜⪃࠼ࡽࢀࡿ.}

ࢩࣟ࢖ࢾࢼࢬࢼ࡛ࡣAl3+_ࡸH+_{⪏ᛶ࡟㈉⊩ࡍࡿ㑇ఏᏊࡢT-DNA ᤄධ࡟ࡼࡾᶵ⬟ࡢ◚ቯࡉ}

ኚ␗ࡀ⏕ࡌࡓ㑇ఏᏊࡢᶵ⬟࡟౫Ꮡࡋ࡚, ẚ㍑ⓗ㧗࠸⁐ᾮ pH ᮲௳ ࡢప Al ⃰ᗘ࡛ࡢឤཷᛶ⛬ ᗘࡀ␗࡞ࡗ࡚࠸ࡓ (Sawaki et al., 2009). AtALMT1 ཬࡧ ALS3 ࡣࡑࢀࡒࢀ, ᰿ᅪࡢ Al ࢆ᤼

㝖ࡍࡿࡓࡵࡢAl ㄏᑟᆺࡢࣜࣥࢦ㓟ᨺฟ, ⣽⬊ෆ࡛ࡢ Al 㝸㞳࡟㛵୚ࡋ࡚࠾ࡾ, AtALMT1-KO,

ALS3-KO ࡜ࡶ࡟, Al ㉸ឤཷᛶ⾲⌧ᆺࢆ♧ࡍ. ࡲࡓ, STOP1 ࡣAtALMT1ཬࡧALS3ࢆྵࡴ

Al ⪏ᛶ㑇ఏᏊ࡜ H+_{⪏ᛶ࡟㛵୚ࡍࡿ࡜ࡉࢀࡿ㑇ఏᏊࡢⓎ⌧ࢆไᚚࡍࡿ㌿෗ไᚚᅉᏊ࡛࠶ࡾ,}

STOP1-KO ࡣ Al ཬࡧ H+_{࡟㉸ឤཷᛶࢆ♧ࡋࡓ (Iuchi et al., 2007; Sawaki et al., 2009). ࡇ}

ࢀࡽឤཷᛶࢆ♧ࡍKO ࡢẚ㍑ⓗ㧗࠸⁐ᾮ pH ᮲௳ࡢప Al ⃰ᗘ࡛ࡢ㜼ᐖࡣ, ୖ㏙ࡋࡓ SGCS ࡛ࡢࣔࢹࣜࣥࢢࡼࡾ, PM ࡛ࡢẘᛶ࢖࢜ࣥ࡜㛵㐃ࡀ࠶ࡾ, ࡑࡢẘᛶࢆホ౯ࡍࡿࡇ࡜࡟ᙺ❧ࡘ ࡜ᮇᚅ࡛ࡁࡿ. 㑇ఏᏊⓎ⌧ࡢࡼ࠺࡞⣽⬊ෆ࡛ࡢศᏊࣞ࣋ࣝࡢᛂ⟅ࡶࡲࡓ㟼㟁ⓗࣔࢹࣝ࡟ᇶ ࡙ࡃ PM ࡛ࡢ Al3+_{ࡢㄏᘬࢆᨭᣢࡍࡿ࡜ண᝿ࡉࢀࡿ. ᚑࡗ࡚, ࢩࣟ࢖ࢾࢼࢬࢼ࡟ࡼࡿゎᯒࡣ,} SGCS ࡢࣔࢹࣜࣥࢢࡢ᳨ド࡟㐺ࡋ࡚࠾ࡾ, Ca2+_{㍍ῶࡢ㛵㐃ࡋࡓAl}3+ _{ཬࡧ H}+_{ẘᛶࡢࡉࡽ࡞ࡿ} ⌮ゎ࡟ࡘ࡞ࡀࡿ࡜⪃࠼ࡽࢀࡿ. {Al3+_{}PMࢆ᥎ᐃࡍࡿࡓࡵ࡟, ⁐ᾮ୰࡛ࡢ Al ࢖࢜ࣥ✀ཬࡧඹᏑࡍࡿ࢖࢜ࣥ✀ࢆṇ☜࡟᥎ᐃ} ࡍ ࡿ ᚲ せ ࡀ ࠶ ࡿ. SGCS ࣉࣟࢢ࣒ࣛࡣ, ༢⣧࡞⁐ᾮ࡛ࡢ࢖࢜ࣥ✀᥎ᐃ࡟㐺ࡋ࡚࠸ࡿ (Kinraide and Wang, 2010). ᮏ❶࡛ࡣ, ࢩࣟ࢖ࢾࢼࢬࢼࡢ⏕⫱࡟㐺ࡋࡓᚲ㡲ᰤ㣴⣲ࢆྵࡴ Ỉ⪔ᇵᆅ࡟㛵ࡋ࡚, ᇵᆅࡸᅵተ⁐ᾮࡢ࢖࢜ࣥ✀᥎ᐃࣉࣟࢢ࣒ࣛ࡜ࡋ࡚ᗈࡃ⏝࠸ࡽࢀ࡚࠸ࡿ GEOCHEM-EZ (Famoso et al., 2010; Shaff et al., 2010)࡜ SGCS ࢆ⤌ࡳྜࢃࡏ࡚࢖࢜ࣥ✀

ࡢ᥎ᐃ࡜άືᗘࡢ⟬ฟࢆ⾜࠸, ࢩࣟ࢖ࢾࢼࢬࢼኚ␗ᰴཬࡧ㔝⏕ᰴ⣔⤫࡛ࡢ Al3+_{, H}+_ẘᛶ࡜

Ca2+_{ࡢ㍍ῶຠᯝࢆPM ࡛ࡢ࢖࢜ࣥάືᗘ࡟ࡼࡾㄝ᫂ࡍࡿ. ࡉࡽ࡟, Al}3+_{ᛂ⟅ᛶࢆ♧ࡍ࣐࣮࢝}

࣮㑇ఏᏊࡢⓎ⌧ᛂ⟅ࡀ{Al3+_{}PM࡜㛵㐃ࡋ࡚࠸ࡿࡇ࡜ࢆ♧ࡍ. ࡲࡓ, ⣽⬊⭷⬡㉁ࡢࣜࣥ⬡㉁࠿}

ࡽ⢾⬡㉁࡬ࡢྜᡂ⤒㊰࡟ྵࡲࢀࡿ㓝⣲phosphatidate phosphohydrolase (PAH) 1 ཬࡧ 2 ࢆ

ࢥ࣮ࢻࡍࡿ㑇ఏᏊ࡟ኚ␗ࡀ⏕ࡌ, ࣜࣥ⬡㉁㔞ࡀ㔝⏕ᰴ࡜␗࡞ࡿ஧㔜ኚ␗ᰴ pah1pah2 ࡛ࡢ

Al3+_{άືᗘ࡟ᑐࡍࡿᛂ⟅ࢆゎᯒࡋࡓ. ࡇࢀࡽࡢ᳨ドࡼࡾ, Al ẘᛶ࡟ࡼࡿ㞀ᐖᶵᵓࡸ⪏ᛶᶵᵓ}

ᐇ㦂ᮦᩱཬࡧᐇ㦂᪉ἲ

౪

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ࢩࣟ࢖ࢾࢼࢬࢼ㔝⏕ᰴColumbia-0 (Col-0; JA58)✀Ꮚࡣ, RIKEN BioResource Center

(RIKEN BRC)ࡼࡾධᡭࡋࡓ. SALK ࢩࣟ࢖ࢾࢼࢬࢼ T-DNA ᤄධ KO ᰴ, AtALMT1-KO

(SALK_009629), ALS3-KO (SALK_061074) ཬ ࡧ STOP1-KO (SALK_114108) ࡣ

Arabidopsis Biological Resource Center (ABRC)ࡼࡾධᡭࡋࡓ. pah1pah2஧㔜ኚ␗ᰴࡣ,

ᮾிᕤᴗ኱Ꮫࡢ኱⏣༤அ༤ኈࡼࡾᥦ౪ࡉࢀࡓ✀Ꮚࢆゎᯒ࡟⏝࠸ࡓ.

Ỉ⪔᱂ᇵ

Ỉ⪔ヨ㦂࡟ࡼࡿ᰿㛗 ᐃࡣ, Kobayashi ࡽ (2007)ࡢࣇ࣮ࣟࢺᘧỈ⪔᱂ᇵἲ࡟ࡼࡾ⾜ࢃࢀ

ࡓ. ᇶᮏᇵᆅ࡟ࡣ, 1/50 ᙉᗘࡢ MGRL ᇵᆅ (Fujiwara et al., 1992)ࡼࡾ Na·PO4ࢆ㝖ࡁ,

Ca(NO3)2ࢆ CaCl2  Ǎ0࡜ NaNO3  Ǎ0࡟ኚ᭦ࡋࡓᇵᆅࢆ⏝࠸ࡓ. Na2EDTA ࡜

FeSO4ࡀࡑࢀࡒࢀǍ0, Ǎ0 ࡜࡞ࡿࡼ࠺࡟ྵࡲࢀ࡚࠸ࡿࡇࡢ 1/50 ᙉᗘࡢ MGRL

ᇵᆅࢆHigh-EDTA ࡜ࡋࡓ (Table 1) . ୍᪉, FeSO4 Ǎ0ࢆࡑࡢࡲࡲ࡟, Na2EDTA ࢆ

 Ǎ0 ࡟ኚ᭦ࡋࡓᇵᆅࢆ Low-EDTA ࡜ࡋࡓ. Al3+_{ཬࡧ H}+_{ẘᛶࡢࢫࢺࣞࢫฎ⌮⁐ᾮࡣ,} ྛᇶᮏᇵᆅࢆ⏝࠸࡚10 mM AlCl3ࢆ⤊⃰ᗘࡀ0 ࠿ࡽ Ǎ0 ࡜࡞ࡿ⠊ᅖ࡛ῧຍࡋ, ⁐ᾮ pH ࢆ 4.5, 4.7, 5.0, 5.2, 5.5 ࡟ 1N HCl ཬࡧ 1N NaOH ࢆ⏝࠸࡚ㄪᩚࡋࡓ. ࡲࡓ, Ca2+_{࡟ࡼࡿẘᛶ࢖} ࢜ࣥࢫࢺࣞࢫࡢ㍍ῶࡣ, ୍ᐃࡢ AlCl3⃰ᗘࡲࡓࡣpH ࡟ᑐࡋ࡚, CaCl2⤊⃰ᗘࡀ0 ࠿ࡽ 5 mM ࡜࡞ࡿ⠊ᅖ࡟ㄪ〇ࡋࡓฎ⌮⁐ᾮࢆ⏝࠸࡚ヨ㦂ࢆ⾜ࡗࡓ. ࣐ࢢࢿࢩ࣒࢘࢖࢜ࣥ(Mg2+_)࡟ࡼࡿ ྠᵝࡢ㍍ῶస⏝࡟ࡘ࠸࡚ࡣ, CaCl2⃰ᗘ࡜ MgCl2⃰ᗘࢆ⤌ࡳྜࢃࡏࡓฎ⌮⁐ᾮࢆㄪ〇ࡋࡓ.

ᑦ, ฎ⌮⁐ᾮࡣ 2 ᪥ẖ࡟஺᥮ࡋࡓ. ᳜≀యࡣ, ᫂ᮇ 12 ᫬㛫 (photosynthetic photon flux GHQVLW\33)'ǍPROP-2 sec-1)/ᬯᮇ 12 ᫬㛫ࡢ࿘ᮇ (24±2Υ)࡛ 7 ᪥㛫᱂ᇵࡋ, ᚤᑠィ  ࣘࢽࢵࢺ (MC-300, KENIS)࡟ࡼࡾ᰿㛗ࢆ ᐃࡋࡓ. ྛࢩࣟ࢖ࢾࢼࢬࢼ㔝⏕ᰴཬࡧኚ␗ᰴ

Table 1. Nutrient composition of the modified

MGRL medium

Element

Concentration

MgSO

ȝM

MnSO

ȝM

FeSO

ȝM

ZnSO

20 nM

CuSO

20 nM

KNO

ȝM

H

BO

ȝM

(NH

)

MO

O

0.48 nM

CoCl

2.6 nM

Na

EDTA

ȝM

CaCl

ȝM

NaNO

ȝM

ࡣ 15 ಶయ᱂ᇵࡋ, ୖ఩ 10 ಶయࡢ᰿㛗ࡢᖹᆒ್ࢆゎᯒ࡟⏝࠸ࡓ. Ⓨ⌧㔞ゎᯒ࡟ࡘ࠸࡚ࡣ, Col-0 (⣙ 100 ಶయ)ࢆ Na·PO4 ࢆྵࡴ 1/50 MGRL ⁐ᾮ (High-EDTA, pH 5.6)࡛ 10 ᪥㛫᱂

ᇵࡋࡓ. ᰿㛗 ᐃ࡛⏝࠸ࡓ Low-EDTA ࢆᇶᮏ⁐ᾮ࡜ࡋࡓ Al3+_{ࢫࢺࣞࢫཬࡧ Ca}2+_ࡲࡓࡣ Mg2+_{᮲௳ࢆฎ⌮⁐ᾮ࡜ࡋ࡚⏝࠸ࡓ.} ᅵ⪔ヨ㦂ࡣ, ᮾ໭኱ᏛᕝΏᐇ㦂㎰ሙ (ᐑᇛ┴⋢㐀㒆㬆Ꮚ⏫)ࡼࡾ᥇ྲྀࡋࡓከ㔞ࡢ஺᥮ᛶ࢔ ࣑ࣝࢽ࣒࢘ࢆྵࡴ඾ᆺⓗ࡞㠀࢔ࣟࣇ࢙ࣥᆺ㯮࣎ࢡᅵተࢆヨ㦂ᅵተ࡜ࡋ࡚⏝࠸࡚⾜ࡗࡓ. ᅵ ተࡣ㸪᪥㝜࡛㢼஝ࡋࡓᚋ㸪ࡩࡿ࠸ (┠ᖜ:2 mm)࡟࠿ࡅ㸪࠶ࡽ࠿ࡌࡵࡼࡃᨩᢾࡋࡓ㸬ᰤ㣴ሷ ࡜ࡋ࡚㸪ᅵተ100 g ࠶ࡓࡾ KCl 0.048 g㸪MgSO4 0.036 g㸪(NH4)2SO4 0.132 g ୪ࡧ࡟ NaH2PO4 0.25 g ࢆຍ࠼ࡓ. Ca ⫧ᩱ࡟ࡼࡿ⏕⫱ࢆホ౯ࡍࡿࡓࡵ࡟ᰤ㣴ሷࢆྵࡴᅵተ࡟ᑐࡋ࡚ ᅵተ100 g ࠶ࡓࡾ CaCO3 0.15㸪0.40 g ཬࡧ CaSO4 0.20 g ࢆ⤌ࡳྜࢃࡏࡓฎ⌮༊ࢆㄪ〇ࡋ ࡓ. ᰤ㣴ሷࢆ㥆ᰁࡲࡏࡿࡓࡵ 1 㐌㛫⛬ᗘᨺ⨨ࡋ㸪ᅵተ pH ࡢ ᐃࢆ⾜ࡗࡓ㸬ࡲࡎ㸪ᅵተ࡜⵨ ␃Ỉࡀ1:2.5 ࡢẚ࡟࡞ࡿࡼ࠺࡟ΰྜࡋ㸪30 ศ㛫ᨩᢾࡋࡓ㸬1 ᫬㛫௨ୖ㟼⨨ࡋࡓᚋ㸪෌ᗘᨩ ᢾࡋ㸪࢞ࣛࢫ㟁ᴟpH ࣓࣮ࢱ࣮ࢆ⏝࠸࡚ ᐃࡋࡓ㸬ᰤ㣴ሷࢆ༑ศ࡟㥆ᰁࡲࡏࡓᅵተࡣ㸪⫱ⱑ ⏝ࣉࣛࢫࢳࢵࢡ࣏ࢵࢺ[2.5×2.5×4.0 (cm)]࡟⣙ 10 g ࡎࡘศὀࡋ㸪࢖࢜ࣥ஺᥮Ỉ࡛‵ࡽࡏࡓ㸬 5 ᪥㛫㸪4Υ࡟࡚ప 㺃྾Ỉฎ⌮ࡋࡓ✀Ꮚࢆ 1 ฎ⌮༊࡟ࡘࡁ 25 ⢏ࡎࡘ᧛✀ࡋ, Ỉ⪔ヨ㦂࡜ྠ ᵝࡢ᱂ᇵ᮲௳࡛4 㐌㛫⏕⫱ࡉࡏࡓ㸬 ࢖ ࢖࢜ࣥάᗘᗘ࡟ᑐࡍࡿ᰿㛗ࢹ࣮ࢱࡢ㠀⥺ᙧᅇᖐゎᯒ

ྛฎ⌮⁐ᾮࡣGEOCHEM-EZ (Shaff et al., 2010)ࢆ⏝࠸࡚ᇵᆅ୰࡛ࡢ࢖࢜ࣥ✀ࢆ᥎ᐃࡋ

ࡓ. GEOCHEM-EZ ࡟ࡼࡾ᥎ᐃࡉࢀࡓ࢖࢜ࣥ⃰ᗘ (Al3+_{, Ca}2+_{, Mg}2+_{, Na}+_{, K}+_ཬࡧH+_{) ࢆ}

SGCS ࣉࣟࢢ࣒ࣛ (Kinraide and Wang, 2010)࡟ᤄධࡋ, {Ion}PMཬࡧ{Ion}bulk ࢆィ⟬ࡋࡓ.

Ỉ⪔ヨ㦂ࡼࡾᚓࡽࢀࡓCol-0, ALS3-KO, AtALMT1-KO ཬࡧSTOP1-KO ࡢ᰿㛗ࢹ࣮ࢱࡣ,

ࢥ࣒ࢠ࡛ᥦၐࡉࢀࡓᅇᖐᘧࢆ⏝࠸࡚MYSTAT12 (SYSTAT, Evanston, IL, USA)࡟ࡼࡾ㠀⥺

ࡓಀᩘࡢ᭷ពᛶࡣ, ࡑࢀࡒࢀࡢಀᩘ࡟ᑐࡍࡿ 95㸣ಙ㢗༊㛫ࡀ 0 ࢆྵࡴ࠿࡝࠺࠿ุ࡛ᐃࡋࡓ.

R

RNA ᢳฟཬࡧ㑇ఏᏊⓎ⌧㔞ゎᯒ

Total RNA ࡀ Suzuki ࡽࡢ RNA ᢳฟ᪉ἲࢆ⏝࠸࡚ᢳฟࡉࢀࡓ (Suzuki et al., 2003)㸬1.5 ml ࢳ࣮ࣗࣈ࡟ධࢀࡓ᰿㒊ࡣ, ࣁࣥࢻࢻࣜࣝ࡜ 1.5 ml ࢳ࣮ࣗࣈ⏝࣌ࣞࢵࢺ࣑࢟ࢧ࣮࡛⁐ゎࡋ

࡞࠸ࡼ࠺࡟ᾮయ❅⣲ࢆ⏝࠸࡚᏶඲࡟☻○ࡋࡓ㸬ࡇࢀ࡟ᢳฟbuffer [100 mM Tris-HCl (pH

9.5), 10 mM EDTA (pH 8.0), 5% (v/v) 2-mercaptoethanol, 2% (w/v) Lithium dodecyl sulfate, 0.6 M NaCl, 0.4 M Na-citrate]ࢆ 600 ǍO ຍ࠼㸪ᡭ࡛ᬮࡵ࡚⼥ゎࡋࡘࡘᠱ⃮ࡋࡓ㸬 15,000 rpm㸪 ᐊ ࡛ 5 ศ㛫㐲ᚰศ㞳ࡋࡓᚋ㸪ୖΎࢆูࡢ 1.5 ml ࢳ࣮ࣗࣈ࡟⛣ࡋ㸪ୖΎ࡟

ᑐࡋ࡚1 vol.ࡢ chloroform/isoamylalcohol (24:1)ࢆຍ࠼࡚⃭ࡋࡃ᧠ᢾࡋࡓ㸬ḟ࡟㸪 15,000

rpm㸪4Υ࡛ 5 ศ㛫㐲ᚰศ㞳ࡋࡓᚋ㸪Ỉᒙࢆูࡢ 1.5 ml ࢳ࣮ࣗࣈ࡟⛣ࡋ㸪0.7 vol.ࡢ 3.5% (w/v) guanidine thiocyanate, 200 mM NaOAc (pH 4.0)ࢆỈ㣬࿴ࣇ࢙ࣀ࣮࡛࣓ࣝࢫ࢔ࢵࣉࡋࡓ⁐ ᾮ ࢆຍ࠼㸪⃭ࡋࡃ᧠ᢾࡋࡓ㸬ࡇࢀࢆᐊ ࡛ 3 ศ㛫ᨺ⨨ࡋ㸪ຍ࠼ࡓࣇ࢙ࣀ࣮ࣝ⁐ᾮࡢ 0.5 vol.

ࡢchloroform/isoamylalcohol ࢆຍ࠼㸪෌ᗘ㸪ᡭ࡛⃭ࡋࡃᨩᢾࡋࡓ㸬14,000 rpm㸪4Υ࡛ 5

ศ㛫㐲ᚰศ㞳ࡋ㸪ỈᒙࢆRNase free ࡢ 1.5 ml ࢳ࣮ࣗࣈ࡟⛣ࡋࡓ㸬௨㝆ࡢ᧯సࡣ, RNase free

ࡢ ࣐ ࢖ ࢡ ࣟ ࣆ ࣌ ࢵ ࢺ ⏝ ࢳ ࢵ ࣉ, ࢳ࣮ࣗࣈཬࡧヨ⸆ࢆ⏝࠸ࡓ㸬ࡇࡢỈᒙ࡟ 0.6 vol.ࡢ

isopropanol ࢆຍ࠼㸪ᐊ ࡛ 10 ศ㛫ᨺ⨨ࡋࡓ㸬ࡇࢀࢆ 16,000 rpm㸪4Υ࡛ 15 ศ㛫㐲ᚰศ 㞳ࡋ㸪ୖΎࢆྲྀࡾ㝖࠸ࡓ㸬ࡉࡽ࡟㸪75% EtOH ࢆຍ࠼㸪16,000 rpm㸪4Υ,࡛ 5 ศ㛫㐲ᚰศ 㞳ࡋ࡚࣌ࣞࢵࢺࢆὙίࡋ㸪ୖΎࢆྲྀࡾ㝖࠸ࡓ㸬ࡇࡢ࣌ࣞࢵࢺࢆ㍍ࡃ㢼஝ࡋࡓᚋ㸪ǍO ࡢ

RNase free H2O ࡟⁐ゎࡋࡓ㸬RNA ⁐ᾮ ǍO ࡟཯ᛂᾮ >ð'1DVH,%XIIHUǍO8

DNase (RNase-free) (Takara Bio Inc., Shiga, Japan), 20 U RNase inhibitor (Toyobo,

Osaka, Japan)]ࢆຍ࠼㸪37Υ࡛ 30 ศ㛫཯ᛂࡉࡏࡓ㸬ࡇࢀ࡟ ǍO51DVHIUHH+2O㸪ǍO

Ỉ㣬࿴ࣇ࢙ࣀ࣮ࣝ㸪Ǎl chloroform/isoamylalcohol (24:1)ࢆຍ࠼࡚⃭ࡋࡃᨩᢾࡋࡓ㸬ࡇ

࡟⛣ࡋࡓ㸬ǍO ࡢ 3 M NaOAc ࡜ 1 vol.ࡢ isopropanol ࢆຍ࠼࡚⃭ࡋࡃΰྜࡋ㸪ᐊ ࡛ 10 ศ㛫㟼⨨ࡋࡓ㸬15,000 rpm㸪ᐊ ࡛ 15 ศ㛫㐲ᚰศ㞳ࡋ࡚ୖΎࢆᤞ࡚ࡓᚋ㸪ỿẊ࡟ 75㸣 EtOH ࢆ  ǍO ຍ࠼㸪15,000 rpm㸪4Υ࡛ 5 ศ㛫㐲ᚰࡋ࡚ୖΎࢆᤞ࡚ࡓ㸬ࡇࡢ࣌ࣞࢵࢺࢆ

10ǍO ࡢ RNase free H2O ࡟⁐ゎࡋࡓ㸬RNA ⁐ᾮࡢ⃰ᗘ ᐃཬࡧ㉁ࡣ㸪ගᗘィ NanoVue

Plus (GE Healthcare, Tokyo, Japan)ࢆ⏝࠸࡚ A260ࡢ್ࡼࡾRNA ⃰ᗘࢆỴᐃࡋ, A260/A280

ཬࡧA260/A230࡛ࢱࣥࣃࢡ㉁ཬࡧከ⢾ࡢΰྜࢆࡑࢀࡒࢀ☜ㄆࡋࡓ㸬ణࡋ㸪ྛ྾ගᗘࡣணࡵA310

(୙⁐ᛶࡢΰධ≀ࡢᣦᶆ್)ࢆᘬ࠸ࡓ್ࢆ⏝࠸ࡓ㸬ǍJ ┦ᙜࡢ RNA ⁐ᾮ࡟ RNase free H2O

ࢆຍ࠼࡚ǍO ࡟ࡋ㸪70Υ࡛ 10 ศ㛫཯ᛂࡉࡏࡓᚋ㸪ị෭ࡋࡓ㸬ࡇࡢΰྜᾮ࡟㏫㌿෗཯ᛂᾮ [5

ð57EXIIHUǍOP0G173ǍO51DVHLQKLELWRUǍO57DVHǍO (Toyobo), oligo dT(18) SULPHUǍO]ࢆຍ࠼࡚ ǍO ࡟ࡋ㸪TaKaRa PCR Thermal Cycler Dice (Takara Bio Inc.)ࢆ⏝࠸࡚㏫㌿෗཯ᛂ (95Υ/15 sec, 62Υ/30 sec)ࢆ 35 ࢧ࢖ࢡࣝ⾜ࡗࡓ㸬ࡇࢀ࡟ 1/4 TE

ࢆ280 ǍO ຍ࠼࡚ cDNA ⁐ᾮ࡜ࡋࡓ㸬࡞࠾㸪㏫㌿෗཯ᛂ࡟⏝࠸ࡓ oligo dT(18) primer ࡟ࡣ㸪

[5’-TTTTTTTTTTTTTTTTTTv(A/C/G)-3’] ࢆ⏝࠸ࡓ㸬ᐃ㔞ⓗࣜ࢔ࣝࢱ࢖࣒ PCR ࡣ, Table 2

࡟グࡋࡓࣉࣛ࢖࣐࣮ᑐ࡜SYBR Premix Ex Taq II (Takara Bio Inc.)ࢆ⏝࠸࡚, ゎᯒࢯࣇࢺ

࢙࢘࢔ࢆ௜ᒓࡋࡓThermal Cycler Dice Real Time System II (Takara Bio Inc.)࡟ࡼࡾ᳨ฟ

ཬࡧゎᯒࢆ⾜ࡗࡓ. ཯ᛂࡣ, ྛฎ⌮ 3 ࢧࣥࣉ࡛ࣝ⾜࠸, ┠ⓗ㑇ఏᏊࡢ㌿෗㔞ࡣUBQ1㌿෗㔞

࡛ṇつ໬ ࢆ⾜ࡗࡓ.

P PI ᰁⰍ

ᇶᮏᇵᆅ (pH 5.5)࡛ 5 ᪥㛫᱂ᇵࡋࡓ㔝⏕ᰴ (Col-0)ཬࡧSTOP1-KO ᗂ᳜≀యࢆ Ǎ0

ࡲࡓࡣǍ0&D&O2ࢆྵࡴపpH (pH 4.7)ࢫࢺࣞࢫ⁐ᾮ࡟ 90 ศ㛫ᾐₕࡋࡓ. ฎ⌮ᚋ, ᗂ᳜

≀య᰿㒊ࡣ, SURSLGLXP LRGLGH  ǍJPO࡛ 15 ⛊㛫ᰁⰍࡋ, ⺯ග㢧ᚤ㙾 (IMT-2-21-RFL, Olympus)ࢆ⏝࠸࡚ほᐹࡋ, ࢹࢪࢱ࣓ࣝ࢝ࣛࣘࢽࢵࢺ 30'& ǂ2/-1, Olympus)࡟ࡼࡾ⏬ ീࢆ᧜ᙳࡋࡓ.

Table 2. Sequences of primers used for semi-quantitative RT-PCR.

Name Sequence (5'-)

At1G73220 _RF _{TGGAAGGAACAGCCACAAGA}CAGTGGAGAATGGGAATGGA At3G03910 _RF _{GATTCGTTGATAGAAAATTTGAGAA}TTCTTGATACCCCCCCATG At5G19890 F AGAAACTCGCGATCCCAAAC

R CCAGGCCCTCCACTCAATAC At3G28510 F AATACGTCCCTGCCCATTTC R TCGTAGGCTTGGCTTCTCTTC At5G13320 _RF TGCCCTAACAACACCGAAAG_{GAACCCGTCCTCACAACCTC} At2G41380 _RF CTGTTGATCGGAAGAGTTGCA_{CAGGAATGCTGGAACGACAA} At5G05340 _RF CTCTTCAACGGCGGCTCTAC_{CAAACCTTGCGGATTTCACC} At5G57560 F ATGATTGGGCAACGAGAGGT

R CCCTGTTTCGAGGCATTAGG At1G74710 _RF CTGCTCTGCATCCAACTCCA_{ATTCACTCTCCTCGCCACCA} At3G17790 (PAP17) _RF CGAACGGTGAGCTTCAGAGA_{ACTGATTGAAGGAGCCACGA} At2G38940 (PHT1;4) _RF TGGACCCAATGCTACAACCT_{CGAGTTCCTGACCCCAATC} AT3G52590 (internal std.) F TCGTAAGTACAATCAGGATAAGATG

IICP-MS ࡟ࡼࡿ᰿㒊 Al ྵ㔞 ᐃ

Col-0 ཬࡧ pah1pah2 ࡣ, P ࢆ㝖࠸ࡓ Al ฎ⌮⁐ᾮ (2 ǍM AlCl3, pH 5.0)࡛ 7 ᪥㛫⏕⫱ࡉࡏ, ࢖

࢜ࣥ஺᥮Ỉ࡛Ὑίࡋࡓ᰿㒊ࢆᅇ཰ࡋࡓ. ᰿㒊ࡣ, 2 ml ⃰ HNO3 (Electronic chemical grade;

Kanto Chemical, Tokyo, Japan)࡜ 0.5 ml 㧗⣧ᗘ H2O2 (semiconductor grade; Santoku Chemical,

Tokyo, Japan)࡛ศゎฎ⌮ࢆ⾜࠸, 㓟ศゎࢩࢫࢸ࣒ DigiPREP (SCP Science, Quebec, Canada)ࢆ

⏝࠸࡚஝⇱ࡉࡏࡓ. ࢧࣥࣉࣝࡣ㉸⣧Ỉ࡛ 2%࡟ᕼ㔘ࡋࡓ HNO3࡛⁐ゎࡉࡏࡓ. Al ྵ㔞ࡣ, ㄏᑟ

⤖ྜࣉࣛࢬ࣐㉁㔞ศᯒィ (ICP-MS; ELAN DRC-e; Perkin Elmer, Waltham, MA)࡟࡚ࣉࣟࢺࢥ

ࣝ࡟ᚑࡗ࡚ ᐃࡋࡓ. ᑦ, ᳜≀య 150 ಶయࢆ 1 ࢭࢵࢺ࡜ࡋ, 3 ࢭࢵࢺ ᐃࢆ⾜ࡗࡓ.

ࣔࣜࣥᰁⰍ

pah1pah2ཬࡧࡑࡢCol-0 㔝⏕ᰴࣂࢵࢡࢢࣛ࢘ࣥࢻࡣ, ୖグࡢ Na·PO4ࢆྵࡴ1/50 MGRL

ᇵᆅ࡛5 ᪥㛫᱂ᇵࡋࡓᚋ, Na·PO4ࢆྵࡲ࡞࠸1/50 MGRL ᇵᆅ࡟⛣ࡋ࡚ࡉࡽ࡟ 5 ᪥㛫᱂ᇵ ࡋࡓ. ྛᗂ᳜≀యࡣ, Ǎ0 $O&O3ࢆྵࡴAl ฎ⌮⁐ᾮ࡛ 24 ᫬㛫ฎ⌮ࢆ⾜ࡗࡓ. ࣔࣜࣥᰁⰍ ࡣ, EtOH ࡟⁐ゎࡋ࡚ 10 mM ࡟ㄪ〇ࡋࡓࣔࣜࣥ⁐ᾮࢆ 10 mM MES ⦆⾪ᾮ࡛ᕼ㔘ࡋ, 100 Ǎ0 ࡜ࡋࡓࣔࣜࣥ⁐ᾮࢆ⏝࠸࡚⾜ࡗࡓ. Al ฎ⌮ࢆ⾜ࡗࡓᗂ᳜≀యࢆ⣧Ỉ࡛Ὑίࡋ, ࣔࣜࣥ⁐ ᾮ࡟࡚15 ศ㛫ᰁⰍࢆ⾜ࡗࡓ. ᰁⰍᚋ, వศ࡞ࣔࣜࣥࢆྲྀࡾ㝖ࡃࡓࡵ࡟ 10 mM MES ⦆⾪ᾮ ࡛ᗂ᳜≀యࢆ10 ศ㛫ᾐₕࡋ, ⺯ග㢧ᚤ㙾 (BX51, Olympus)ࢆ⏝࠸࡚ほᐹࡋࡓ.

⤖ᯝ

୰࡛Al ẘᛶ࡟ᙉ࠸⪏ᛶࢆ♧ࡍ⣔⤫ࡢ㸯ࡘ࡛࠶ࡿ (Figs. 1 and 18). Col-0 ࡟⏤᮶ࡍࡿ Al ㉸ឤ

ཷᛶኚ␗ᰴAtALMT1-KO, ALS3-KO ୪ࡧ࡟ Al ཬࡧ㓟㉸ឤཷᛶኚ␗ᰴSTOP1-KO ࡣ pH

5-5.5 ࡛ Ǎ0 ࡲ࡛ࡢ Al ᮲௳࡛᰿㛗ࡀ㜼ᐖࡉࢀࡿࡇ࡜ࢆ♧ࡋࡓ (Fig. 1). ࡇࡢࡇ࡜ࡣ, ࡇࢀ ࡽࡢኚ␗ᰴࡀpH 5 ௨ୖࡢ᮲௳࡛ࡢ᰿ẘᛶ࢖࢜ࣥࡢẘᛶࢆホ౯࡛ࡁ, ᚋ㏙ࡍࡿ Ca ࡟ᑐࡍࡿ ẘᛶࡢ㍍ῶᶵᵓ࡟ࡘ࠸࡚ࡶ༑ศ࡞ࢹ࣮ࢱࢆ⋓ᚓࡍࡿࡇ࡜࡛ࢩ࣑࣮ࣗࣞࢺ࡛ࡁࡿ࡜⪃࠼ࡽࢀ ࡿ. SGCS ࡢࣔࢹࣜࣥࢢࡣ, ༢⣧⁐ᾮ࡛ࡢ࢖࢜ࣥ✀᥎ᐃ࡟㐺ࡋ࡚࠸ࡿࡀ, ࢩࣟ࢖ࢾࢼࢬࢼࡢ⏕ ⫱࡟⏝࠸ࡓᰤ㣴⁐ᾮ࡛ࡢAl ẘᛶࢆࢩ࣑࣮ࣗࣞࢺࡍࡿࡓࡵ, ࡼࡾṇ☜࡞࢖࢜ࣥ✀᥎ᐃࡀ㔜せ ࡛࠶ࡿࡇ࡜ࢆ♧ࡋࡓ (Fig. 2). ⤖ᯝ࡜ࡋ࡚, ⥙⨶ⓗ࡞⁐ᾮ୰ࡢ࢖࢜ࣥ✀᥎ᐃࡀྍ⬟࡞ GEOCHEM-EZ ࡼࡾᚓࡽࢀࡿ್ࢆ฼⏝ࡋ࡚, SGCS ࡟ࡼࡿ⣽⬊⭷⾲㠃࡛ࡢ࢖࢜ࣥ✀᥎ᐃࢆ

⾜࠺ࡇ࡜࡜ࡋࡓ. Figure 1 ࡣ, Al ẘᛶࡢᣦᶆ࡜ࡉࢀࡿ AlCl3⃰ᗘ, {Al3+}bulkཬࡧ{Al3+}PM࡟ᑐ

ࡍࡿ᰿㛗㜼ᐖࢆ♧ࡋࡓ. AlCl3⃰ᗘ࡟ᑐࡍࡿ᰿㛗㜼ᐖ࡛♧ࡉࢀࡓࡼ࠺࡟, ㉸ឤཷᛶኚ␗ᰴࡢ

᰿㛗㜼ᐖࡣ, EDTA ⃰ᗘ࡟ࡼࡾᙳ㡪ࡉࢀࡓ (Fig. 1; left pannels). EDTA ࢆྵࡴᰤ㣴ᇵᆅ࡟

ࡘ࠸࡚GEOCHEM-EZ ࡟ࡼࡾ᥎ᐃࡉࢀࡓ{Al3+_{}bulk࡟ᑐࡋ࡚᰿㛗ࢆࣉࣟࢵࢺࡍࡿ࡜, EDTA}

⃰ᗘ࡟ࡼࡿᕪࡣᾘኻࡋࡓ (Fig. 1; center pannels). ࡇࢀࡣ, ⁐ᾮ୰࡛ࡢ⇕ຊᏛⓗ࡞཯ᛂࡼ

ࡾEDTA ࡀ Al3+_{࡜⤖ྜࡋ, ⁐ᾮ୰ࡢ Al}3+_{ࢆῶᑡࡉࡏࡿࡇ࡜࡟ࡼࡿ. ඲࡚ࡢ㉸ឤཷᛶኚ␗ᰴ}

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Figure 1. Aluminum inhibition of root growth of Arabidopsis seedlings in hydroponic

culture with different concentrations of EDTA. Seedlings were grown for 7 d in basal media containing different concentrations of AlCl3 (0, 0.5, 1, or 2 ȝ0) at pH 5.0 or 5.5. Root

lengths were plotted as a function of AlCl3concentration (left-hand panels), Al3+activity in

bulk-phase media (middle panels), and Al3+ _{activity at the PM surface (right-hand panels)}

were calculated using GEOCHEM-EZ and SGCS. Values are means + SE (n = 10) (SE bars are not shown if they overlap the symbols). Curves were fitted according to the nonlinear regression model Root Length = a + bexp(c[AlCl3] or {Al3+}bulkor {Al3+}PM). Note the high

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Figure 2. Aluminum inhibition of root growth of Arabidopsis seedlings in

hydroponic culture with different concentrations of EDTA. Root lengths shown in Fig. 1 were replotted as a function of Al3+_{activity in bulk-phase media (left-hand}

panels) and Al3+ _{activity at the PM surface (right-hand panels) calculated using}

ࢀࡓ (Fig. 1; center pannels). ࡇࡢ pH 5.5 ࡛ࡢ㜼ᐖࡣ, ᗎㄽ࡛㏙࡭ࡓࡼ࠺࡟{Al3+_}bulk࡟ᑐ

ࡋ࡚, ࡼࡾ኱ࡁ࡞{Al3+_{}PMࢆ⏕ࡌࡿࡇ࡜࡟ࡼࡿ࡜᥎ᐃࡉࢀࡿ. AtALMT1-KO ࡜ALS3-KO ࡢ}

᰿㛗ࢆSGCS ࡼࡾ᥎ᐃࡉࢀࡓ{Al3+_{}PM࡟ᑐࡋ࡚ࣉࣟࢵࢺࡍࡿ࡜, pH 5.0 ཬࡧ pH 5.5 ࡛ࡢࢹ}

࣮ࢱࢭࢵࢺ࡟ᑐࡍࡿᅇᖐ᭤⥺ࡣ࡯ࡰ1 ࡘ࡟᥋ࡋࡓ (Fig. 1; right pannels). ࡇࡢࡇ࡜ࡣ, ༢

⣧⁐ᾮ࡟࠾ࡅࡿࢥ࣒ࢠࡢゎᯒ࡜ྠᵝ࡟, {Al3+_{}bulkࡼࡾࡶ{Al}3+_{}PMࡀ, ᰤ㣴⁐ᾮ࡟࠾ࡅࡿࢩࣟ}

࢖ ࢾ ࢼ ࢬ ࢼࡢ Al ẘᛶࢆỴᐃࡍࡿせᅉ࡛࠶ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿ. 㓟㉸ឤཷᛶ࡛࠶ࡿ

STOP1-KO ࡢ᰿㛗㜼ᐖࡣ, ௚ࡢឤཷᛶኚ␗ᰴ࡜᫂ࡽ࠿࡟␗࡞ࡗ࡚࠾ࡾ, pH 5 ࠿ࡽ 5.5 ࡢ⠊

ᅖ࡟࠾࠸࡚ H+_{ẘᛶࡀほᐹࡉࢀࡓ (Fig. 1; bottom pannels). ௨ୖࡢ⤖ᯝࡼࡾ, SGCS ࡢ}

GEOCHEM-EZ ࡜⤌ࡳྜࢃࡏࡓࣔࢹࣜࣥࢢࡣ, Al3+_{, H}+_ẘᛶࡢCa2+_{࡟ࡼࡿ㍍ῶࡢホ౯࡟㐺}

ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿ.

A

Al ㉸ឤཷᛶኚ␗ᰴࡢ{Al3+_{}PMẘᛶࡢCa}2+_㍍ῶ

{Al3+_{}PMẘᛶࡢCa ࡟ࡼࡿ㍍ῶࢆホ౯ࡍࡿࡓࡵ,} AtALMT1_{-KO ࡜} ALS3_{-KO ࢆ, ␗࡞ࡿ}

CaCl2⃰ᗘࡢAl ฎ⌮⁐ᾮ (pH 5.0 ཬࡧ 5.5 ࡢ 1 ࡲࡓࡣ Ǎ0$O&O ࢆྵࡴ⁐ᾮ)࡛᱂ᇵࡋࡓ.

୧ኚ␗ᰴ࡛ࡣ, ࡇࡢ Al ฎ⌮࡟ࡼࡾ᰿㛗ࡀ㜼ᐖࡉࢀࡿࡀ, Ca ࡢቑຍ࡟క࠸᰿㛗㔞ࡣቑຍࡋࡓ

(Fig. 3). pH 5.5 ࡢ Ǎ0$O ฎ⌮⁐ᾮ࡛, CaCl2ࡢ0.2 mM ࠿ࡽ 5.0 mM ࡬ࡢቑຍࡣ, {Al3+}PM

ࢆǍ0 ࠿ࡽ Ǎ0 ࡟ኚ໬ࡉࡏࡿࡢ࡟ᑐࡋ࡚, {Al3+_{}bulkࡣǍ0 ࠿ࡽ Ǎ0 ࡜࡞}

ࡾ, Ca2+_ࡣ{Al3+_{}PMࢆ኱ࡁࡃపୗࡉࡏࡿࡀ, {Al}3+_{}bulkࡣ࡯࡜ࢇ࡝ኚ໬ࡋ࡞࠸. ࡇࢀࡽࡢ᮲௳ୗ}

࡛ࡢ᰿㛗್ࡣ, {Al3+_{}PM࡟ᑐࡋ࡚㧗࠸┦㛵ࢆ♧ࡋࡓࡀ, {Al}3+_{}bulk࡛ࡣ┦㛵ࡣㄆࡵࡽࢀ࡞࠿ࡗ}

ࡓ (Fig. 3). ᚑࡗ࡚, ࡇࡢࡼ࠺࡞ pH > 5 ࡢ᮲௳࡛ࡢ Al ẘᛶ࡟ࡘ࠸࡚, Ca2+_ࡀ୺࡟{Al3+_}PMࢆ

ῶᑡࡉࡏࡿࡇ࡜࡛ẘᛶࡢ㍍ῶࡀ⏕ࡌࡿࡇ࡜ࢆ♧ࡋ, Figure 1 ࡛ࡢ⤖ᯝ࡜ྠᵝ࡟, {Al3+_}PMࡀ

Al ẘᛶࢆỴᐃࡍࡿせᅉ࡛࠶ࡿࡇ࡜ࢆᨭᣢࡍࡿ. ࡲࡓ, ࢥ࣒ࢠ࡛ᥦၐࡉࢀࡓ Ca2+_{㍍ῶᶵᵓࡢ}

୰ ࡛, {Al3+_{}PM ࡢ 㟼 㟁 ⓗ ⨨ ᥮ (Mechanism I), ⏕ ⫱ ࡟ ༑ ศ ࡞ PM ࡛ ࡢ Ca}2+_{ࡢ ᅇ ᚟}

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Figure 3. Calcium alleviation of Al toxicity in ALS3-KO and AtALMT1-KO under weakly

acidic conditions. Seedlings were grown in Al-toxic solutions containing various levels of CaCl2, AlCl3, EDTA, and pH as shown. Circle size represents CaCl2concentration. Values

are means + SE (n = 10) (SE bars are not shown if they overlap the symbols). Open arrows and closed arrows indicate points at which 2 mM and 5 mM CaCl2(in 1PM AlCl3at pH 5.5)

induced 50–70% root growth inhibition or nearly no inhibition, respectively. The drawn curves were fitted to the nonlinear regression model Root Length = a + bexp(c{Al3+_}

{{Al3+_{}PM࡟ᑐࡍࡿAl ㄏᑟ㑇ఏᏊࡢⓎ⌧ᛂ⟅} Al ẘᛶ࡟ࡘ࠸࡚, {Al3+_{}PMࡀ㔜せ࡞せᅉ࡛࠶ࡾ, {Ca}2+_{}PM࡟ࡼࡗ࡚㍍ῶࡉࢀࡿࡇ࡜ࢆ♧ࡋ} ࡓ. ࡋ࠿ࡋ࡞ࡀࡽ, ࡇࢀࡣࢥ࣒ࢠ࡛ࡢゎᯒ࡜ྠᵝ࡟, ᰿㛗ࢆᣦᶆ࡜ࡋ࡚ホ౯ࡉࢀࡓ⤖ᯝ࡛࠶ ࡿ. {Ca2+_{}PM࡜㛵㐃ࡍࡿ{Al}3+_{}PM࡟ᑐࡍࡿᛂ⟅ࡢࡉࡽ࡞ࡿホ౯ࢆࡍࡿࡓࡵ࡟, PM ࡛ࡢ Al}3+ 㞟✚ࢆホ౯ࡍࡿࡓࡵࡢAl ࣂ࢖࣐࣮࣮࢜࢝࡜ࡋ࡚, Al 㞀ᐖ࡜㛵㐃ࡍࡿ Al ㄏᑟ㑇ఏᏊࡢⓎ⌧ࢆ ᐃ㔞ࡋࡓ. Al ㄏᑟ㑇ఏᏊࡣ, ࣐࢖ࢡࣟ࢔ࣞ࢖ࢹ࣮ࢱࡢẚ㍑ゎᯒࡼࡾ,  Ǎ0 $O ᮲௳ (Sawaki et al., 2009)࡜ẚ㍑ࡋ࡚ࡼࡾ῝้࡞ Al ᮲௳ Ǎ0) (Zhao et al., 2009)࡛Ⓨ⌧ࡀㄏ

ᑟࡉࢀࡓ㑇ఏᏊࡀ㑅ࡤࢀࡓ (Table 3 and 4). ㉸ឤཷᛶኚ␗ᰴ࡛ẘᛶ࡜࡞ࡿ{Al3+_{}PMࡣ, ࡑࡢ}

࡛ࣞ࣋ࣝ㜼ᐖࢆ⏕ࡌ࡞࠸Col-0 ࡛, Al ࣂ࢖࣐࣮࣮࢜࢝㑇ఏᏊࡢⓎ⌧ࢆㄏᑟࡍࡿࡇ࡜ࡀண᝿ࡉ

ࢀࡿ. Col-0 ࡀ pH 5.5 ࡢ Ǎ0$O ฎ⌮⁐ᾮ࡛ 6 ᫬㛫ฎ⌮ࡉࢀࡿ࡜, {Al3+_{}PMࡀ5.08 Ǎ0 ࡜}

࡞ࡿ0.5 mM CaCl2࡛Al ㄏᑟ㑇ఏᏊࡢ᫂ࡽ࠿࡞ㄏᑟࡀㄆࡵࡽࢀࡓ (Fig. 4A). Figure 3 ࡛♧

ࡉࢀࡓࡼ࠺࡟, ࡇࡢ᮲௳࡛ࡣ Al ㉸ឤཷᛶኚ␗ᰴࡢ᰿㛗ࡣ᏶඲࡟㜼ᐖࡉࢀࡓ. ኚ␗ᰴ࡛ࡢ㜼 ᐖࡀ᏶඲࡟㍍ῶࡉࢀࡓCa2+_{ࣞ࣋ࣝ࡜࡞ࡿ5 mM CaCl2࡛ࡣ, ࡑࢀࡽ㑇ఏᏊࡢⓎ⌧㔞ࡣ, Al} ࢆྵࡲ࡞࠸0.5 mM CaCl2࡛ࡢⓎ⌧㔞࡜ྠ⛬ᗘ࡛࠶ࡗࡓ. ᚑࡗ࡚, Al ㄏᑟ㑇ఏᏊࡢⓎ⌧ࡀ, Ca2+_{࡟ࡼࡗ࡚ㄪ⠇ࡉࢀࡿ{Al}3+_{}PM ࡟ᑐࡍࡿ㉸ឤཷᛶኚ␗ᰴࡢ᰿㛗ᛂ⟅࡜㛵㐃ࡋ࡚࠸ࡿࡇ࡜} ࢆ᫂ࡽ࠿࡟ࡋࡓ. ࡲࡓ, SGCS ࡟࠾࠸࡚, Mg2+_ࡣCa2+_{࡜➼ࡋ࠸{Al}3+_{}PMࡢపୗࢆᘬࡁ㉳ࡇࡍ.} ゎᯒ࡟⏝࠸ࡓ࡯࡜ࢇ࡝ࡢ㑇ఏᏊࡣ, 0.5 mM Ca ࡜ 4.5 mM Mg ࡜ࢆ⤌ࡳྜࢃࡏࡓ⁐ᾮ࡛ࡢⓎ ⌧㔞ࡀ5 mM Ca ࡛ࡢⓎ⌧㔞࡜ྠ⛬ᗘ࡛࠶ࡗࡓ (Fig. 4A). ࡇࢀࡽࡢ⤖ᯝࡣ, SGCS ࡛ࡢࣔࢹ ࣜࣥࢢ࡟ᇶ࡙ࡃ{Al3+_{}PMࡢẘᛶࡀ⣽⬊ෆᛂ⟅ࢆ⏝࠸᳨࡚ドࡉࢀࡓࡇ࡜ࢆ♧ࡍ.}

{Al3+_{}PM࡟ᑐࡍࡿAl ⪏ᛶ㑇ఏᏊAtALMT1ཬࡧALS3ࡢⓎ⌧ᛂ⟅}

Al ࣂ࢖࣐࣮࣮࢜࢝࡜ࡋ࡚⏝࠸ࡓ Al ㄏᑟ㑇ఏᏊࡢⓎ⌧ᛂ⟅ゎᯒࡣ, {Al3+_{}PMࡀ⣽⬊ෆᛂ⟅}

Ta bl e 3 . Lis t o f A l r es po ns iv e g en es e xp re ss in g a g re ate r in du ctio n in th e WT tr ea te d w ith 2 5 ȝ0 A l th an in th e WT tr ea te d w ith 1 0 ȝ0 Al . AGI c od e Lo g2 Fo ld C ha ng e (t re at m en t/c on tr ol ) Lo g2 Fo ld Ch an ge (25A l/10A l) D es cr ip tio n 10A l a P-va lu e 25A l b P-va lu e A t3g 28510 2. 2 0. 04 4. 3 0. 02 2. 0 A A A -t ype A T Pa se f am ily pr ot ei n A t1g 74710 2. 1 0. 03 3. 9 0. 02 1. 8 is oc hor is m at e s yn th as e 1 ( IC S1) A t1g 08430 4. 6 0. 03 6. 4 0. 02 1. 7 A L M T 1/ A T A L M T 1 ( A L-A C TI V A TED M A LA TE TR A N SP O R T ER 1 ); m ala te tr an sm em br an e t ran sp or te r/ A t2g 41380 2. 8 0. 04 4. 3 0. 02 1. 5 em br yo-ab un da nt pr ot ei n-re la te d A t5g 57560 2. 4 0. 03 3. 9 0. 03 1. 5 T C H 4 ( T O U C H 4) ; hydr ol as e, a ct in g on g lyc os yl bo nd s / xyl og lu ca n: xy lo gl uc os yl tr an sf er as e A t3g 54150 1. 6 0. 03 3. 1 0. 02 1. 5 em br yo-ab un da nt pr ot ei n-re la te d A t5g 05340 2. 9 0. 04 4. 3 0. 02 1. 4 pe roxi da se , pu ta tiv e A t1g 52130 1. 9 0. 04 3. 2 0. 04 1. 3 ja ca lin le ctin fa m ily p ro te in A t1g 66180 1. 8 0. 03 3. 2 0. 04 1. 3 as pa rt yl pr ot ea se f am ily pr ot ei n A t5g 13320 2. 2 0. 05 3. 4 0. 02 1. 2 PB S3 ( A V R PP H B S U SC E PT IB L E 3) A t3g 47480 2. 7 0. 03 3. 8 0. 03 1. 1 ca lc ium -b in di ng E F ha nd f am ily pr ot ei n A t1g 17170 2. 0 0. 03 3. 0 0. 02 1. 0 AT GS T U 24 (A R A B IDO PS IS T HAL IANA GL UT A T HI O N E S -T R ANS FE R A SE (C L A SS T A U ) 24) ; g lut at hi on e t ra ns fe ra se A t5g 39670 2. 1 0. 04 3. 2 0. 02 1. 0 ca lc ium -b in di ng E F ha nd f am ily pr ot ei n O nly th e lis te d g en es s ho w ed th at fo ld c ha ng es w er e m or e th an tw ic e a nd p -v alu es w er e le ss th an 0 .0 5 b oth in 1 0A l a nd 2 5A l a nd th e f old in du ctio n in 25 A l w as m or e th an tw ic e g re ate r th an th at in 1 0A l. a C ont ro l( -A l a t pH 5. 0 f or 24 h) ve rs us A l ( 10 ȝ0 at pH 5. 0 f or 24 h) in th e W T us ed in S aw ak ie t al ., 2009. b C on tr ol (-A l a t pH 5. 0 f or 24 h) ve rs us A l ( 25 ȝ0 at pH 5. 0 f or 24 h) in th e W T us ed in Z ha o e t a l., 20 09 .

Ta bl e 4 . Lis t o f g en es u niq ue ly in du ce d b y A l b y c om pa ra tiv e m ic ro ar ra y in Zh ao e t a l. ( 20 09 ) f or w hic h in du ctio n in th e WT tr ea te d w ith 25 ȝ0 Al w as g re ate r th an in th e WT tr ea te d w ith 1 0 ȝ0 Al . AGI c od e Lo g2 Fo ld C ha ng e (T re at m en t/C on tr ol ) Lo g2 Fo ld Ch an ge (25A l/10A l) D es cr ip tio n 10A l a 25A l b A t1g 73220 2. 1 5. 3 3. 2 AT OC T 1 ( A R A B IDOP SI S T HAL IANA OR GAN IC C A T ION /C AR N IT INE TR A N SP O R TER 1) ; c ar bo hy dr at e tr an sm em br an e tr an sp or te r/ c ar nitin e tr an sp or te r/ tr an spor te r A t5g 19890 1. 1 3. 5 2. 5 pe roxi da se , pu ta tiv e A t1g 08430 4. 6 6. 4 1. 7 A L M T 1/ A T A L M T 1 ( A L-A C TI V A TED M A LA TE TR A N SP O R TER 1 ); m ala te tr an sm em br an e t ran sp or te r/ A t3g 03910 0. 6 1. 8 1. 3 gl ut am at e de hy dr og en as e, pu ta tive O nly th e lis te d g en es s ho w ed th at th e f old in du ctio n in 2 5A l w as m or e th an tw ic e g re ate r th an th at in 1 0A l a nd th e p -v alu e w as le ss th an 0 .0 5. a C ont ro l( -A l a t pH 5. 0 f or 24h ) ve rs us A l ( 10 ȝ0 at pH 5. 0 f or 24h ) i n t he W T us ed in S aw ak ie t a l., 20 09 . b C ont ro l( -A l a t pH 5. 0 f or 24h ) ve rs us A l (25 ȝ0 at pH 5. 0 f or 24h ) i n t he W T us ed in Z ha o e t a l., 20 09 .









0

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0.5

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At1g73220

At5g13320

At3g28510

At5g05340

At5g57560

At1g74710

At2g41380

At3g03910

At5g19890

$

Figure 4. Expression levels of Al biomarker genes in Al-toxic solutions with different levels

of CaCl2and MgCl2. Roots of wild-type Col-0 seedlings, grown for 10 d in control solutions,

were incubated for 6 h in 1 PM Al at pH 5.5. A) Al-responsive genes that are sharply upregulated by Al stress in a dose-dependent manner (i.e., greater in 25PM than in 10 PM) and are highly upregulated in Al compared with other stressors in agreement with the above selection criterion (Supplemental Tables S1 and S2). Transcripts were determined by semiquantitative RT-PCR. UBQ1 transcript was used as the internal control. B) Meansr SE of expression levels of AtALMT1 and ALS3 relative to those in plants in zero Al (n = 3). Different letters indicate significant difference (Tukey’s test, P < 0.05).

ᚚࡉࢀࡿࡇ࡜ࡀ༑ศண᝿࡛ࡁࡿ. ࡑࡇ࡛, AtALMT1࡜ALS3ࡢⓎ⌧㔞ࢆᐃ㔞ࡍࡿࡇ࡜࡟ࡼ

ࡾ, ࡇࢀࢆ᳨ドࡋࡓ (Fig. 4B).  Ǎ0 $O ࡛ 6 ᫬㛫ฎ⌮ࡉࢀࡓ Col-0 ᰿㒊࡛ࡢ AtALMT1,

ALS3ࡣ, 0.5 mM Ca ࢆྵࡴࢥࣥࢺ࣮ࣟࣝ⁐ᾮ࡛ࡢⓎ⌧㔞࡜ẚ㍑ࡋ࡚ࡑࢀࡒࢀ 4.2, 2.5 ಸㄏ

ᑟࡉࢀࡓ. ࡑࢀࡒࢀࡢⓎ⌧ㄏᑟࡣ, {Al3+_{}PMẘᛶࡀ{Ca}2+_{}PM࡟ࡼࡗ࡚㍍ῶࡉࢀࡿ᮲௳࡛ᢚไ}

ࡉࢀࡓ. {Al3+_{}PM ࡀ 1.27 Ǎ0 ࡜࡞ࡿ 2.0 mM Ca ࢆྵࡴ⁐ᾮ࡛ࡣ,} AtALMT1_{-KO ཬࡧ}

ALS3-KO ࡣ, ࡑࢀࡒࢀ 70㸣ཬࡧ 50㸣ࡢ㜼ᐖࢆ♧ࡋࡓ (Fig. 3). ࡇࡢ᮲௳࡛ࡢ AtALMT1,

ALS3ࡢㄏᑟࡣࡑࢀࡒࢀ, 3.4, 1.9 ಸ࡛࠶ࡗࡓ. ࡇࢀࡽ⪏ᛶ㑇ఏᏊࡀ, ࡑࢀࡒࢀࡢ KO ᰴ࡛㜼

ᐖࡀ⏕ࡌጞࡵࡿ⃰ᗘ࡛ࡢ{Al3+_{}PM ࡟ࡼࡾ, ㄏᑟࡀᘬࡁ㉳ࡇࡉࢀࡿࡇ࡜ࢆ♧၀ࡋࡓ. ኚ␗ᰴ}

ࡢ᰿㛗㜼ᐖࡀㄆࡵࡽࢀ࡞࠿ࡗࡓ5 mM Ca ࡢ Al ฎ⌮⁐ᾮ࡛ࡣ, AtALMT1ࡣ, ࢥࣥࢺ࣮ࣟࣝ

⁐ᾮ࡛ࡢⓎ⌧㔞࡟ᑐࡍࡿㄏᑟࡀ2.4 ಸ࡜⥔ᣢࡉࢀ࡚࠸ࡓࡢ࡟ᑐࡋ࡚, ALS3ࡣࢥࣥࢺ࣮ࣟࣝ

⁐ᾮ࡛ࡢⓎ⌧㔞࡜᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ (Fig. 4). ࡇࢀࡣ, AtALMT1 ࡀࣜࣥࢦ㓟ᨺ

ฟ࡟ࡼࡗ࡚᰿➃࡛ࡢᛴ㏿࡞Al ᤼㝖ࢆ⾜࠺ࡇ࡜࡟ᑐࡋ࡚ (Hoekenga et al., 2006), ALS3 ࡣ

྾཰ࡉࢀࡓAl ࢆ㝸㞳ࡍࡿ㛗ᮇⓗ࡞ Al ⪏ᛶ࡟ᚲせ࡜ࡉࢀ (Larsen et al., 1997; Larsen et al.,

2005), ࡇࢀࡽᶵ⬟ᛶࡢ㐪࠸ࡀᙳ㡪ࡍࡿ࡜⪃࠼ࡽࢀࡿ. ࡇࡢ⤖ᯝࡣ, Al ⪏ᛶ㑇ఏᏊࡀ㔝⏕ᰴ

࡛㜼ᐖࢆ⏕ࡌጞࡵࡿࡼࡾࡶ࠿࡞ࡾప࠸{Al3+_{}PM࡟ࡼࡗ࡚ㄏᑟࡉࢀࡿࡇ࡜ࢆᙉࡃ♧၀ࡍࡿ.}

A

AtALMT1-KO, ALS3-KO, STOP1-KO ࡟ࡼࡿ Al ཬࡧ H+_{ẘᛶ࡟ᑐࡍࡿCa ㍍ῶᶵᵓࡢࢩ࣑} ࣮ࣗࣞࢩࣙࣥ SGCS ࡛ࡢࣔࢹࣜࣥࢢࢆ⏝࠸࡚, ࢥ࣒ࢠ࡛ࡣ, PM ࡛ࡢ Al3+_ཬࡧH+_{ẘᛶ࡟ᑐࡍࡿCa ㍍ῶ} ࡀ3 ࡘࡢᶵᵓ࡛ホ౯ࡉࢀࡓ (Kinraide et al., 2004). ⋓ᚓࡉࢀࡓࢩࣟ࢖ࢾࢼࢬࢼ᰿㛗ࢹ࣮ࢱ ࡟ࡘ࠸࡚, SGCS ࡛ࡢࣔࢹࣜࣥࢢ࡜ࢥ࣒ࢠ࡛ᥦၐࡉࢀࡓᅇᖐᘧࢆ⏝࠸ࡓ㠀⥺ᙧᅇᖐศᯒ࡟ࡼ ࡿゎᯒࢆ⾜࠺ࡇ࡜࡛, ࢩࣟ࢖ࢾࢼࢬࢼ࡛ࡢ Al3+_ཬࡧH+_{࡟ᑐࡍࡿCa ㍍ῶᶵᵓཬࡧኚ␗ᰴ࡛} ࡢCa ㍍ῶ࡟ᑐࡍࡿ㑇ఏⓗᙧ㉁ࡢ㐪࠸ࢆ♧ࡍࡇ࡜࡟ᙺ❧ࡘ࡜⪃࠼ࡽࢀࡿ. ᘧ1 ࡣ, ẘᛶ࢖࢜ࣥ࡜㍍ῶຠᯝࢆ♧ࡍ࢖࢜ࣥ (Ca2+_{)࡜ࡢ┦஫స⏝࡟ࡼࡿ᰿㛗 (}RL_)ᛂ⟅ࢆ

⾲ࡍᅇᖐᘧ࡛࠶ࡾ, 㠀⥺ᙧᅇᖐศᯒࡼࡾᚓࡽࢀࡿᘧ୰ࡢಀᩘ್࡟ࡼࡾ㍍ῶᶵᵓࡀ᥎ᐃࡉࢀ

ࡿ. ᑦ, ᘧ 1 ࡛ࡢಀᩘa, bࡣ, ࢥ࣒ࢠ࡛ࡢᅇᖐศᯒ࡟࠾࠸࡚ࡑࢀࡒࢀ, ฎ⌮๓ࡢ᰿㛗್, 㜼

ᐖࢆ⏕ࡌ࡞࠸᮲௳࡛ࡢ᰿㛗ࡢ᭱኱್࡜ࡋ࡚ಀᩘ್ࡀ᥎ᐃࡉࢀࡿ. ゎᯒ࡟⏝࠸ࡿ᰿㛗ࢹ࣮ࢱ

ࡣ, Ⓨⱆᚋ࠿ࡽࡢ್࡛࠶ࡿࡓࡵa ࢆ 0 ࡜ࡋ࡚ᅇᖐศᯒࢆ⾜࠸, b࡟ࡘ࠸࡚ࡣ24.3 ࡜᥎ᐃࡉࢀ

ࡓࡇ࡜࠿ࡽᘧ1 ࡣᘧ 2 ࡛⾲ࡉࢀࡓ.

RL = a + bexp[c1(1 + c2/10000{Ca2+}PM){Al3+}PM+ d1/100(1 + d2/10000{Ca2+}PM){H+}PM]

(ᘧ 1)

RL = 24.3exp[c1(1 + c2/10000{Ca2+}PM){Al3+}PM+ d1/100(1 + d2/10000{Ca2+}PM){H+}PM]

(ᘧ 2)

ྛኚ␗యࡢ᰿㛗ࢹ࣮ࢱࡣ, {Ion}PM࡟ᑐࡋ࡚ࣉࣟࢵࢺࡍࡿ࡜ከḟඖⓗ࡞ᣦᩘ㛵ᩘᛂ⟅ࢆ♧

ࡋࡓ (Fig. 5). ᚑࡗ࡚ᘧ 1 ࢆ㐺⏝࡛ࡁࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿ. ಀᩘ c1, d1ࡢ㈇್ࡣࡑࢀࡒࢀ,

{Al3+_{}PM, {H}+_{}PM࡟ᑐࡍࡿ᰿㛗ࢆᣦᩘ㛵ᩘⓗ࡟ῶᑡࡉࡏࡿ. STOP1-KO ࡛ࡢd1ࡢ㈇್ࡀ኱ࡁ}

࠸ࡇ࡜ࡣ, ẚ㍑ⓗ㧗࠸ pH ᮲௳(pH > 5)࡛ H+_{ឤཷᛶ࡜࡞ࡿࡇ࡜ࢆ⾲ࡋ࡚࠸ࡿ (Table 5). ಀ} ᩘc2, d2ࡢ㈇್ࡣ, c1(1 + c2/10000{Ca2+}PM) ࡲࡓࡣd1(1 + d2/10000{Ca2+}PM)࡛⾲ࡉࢀࡿࡼ ࠺࡟, c1, d1ࡢ㈇್ࢆᑠࡉࡃࡍࡿࡓࡵ, {Ca2+}PM࡟ࡼࡿ㍍ῶࡢ⛬ᗘࢆ♧ࡍࡇ࡜࡜࡞ࡿ. ᅇᖐศ ᯒ࡟⏝࠸ࡓ᰿㛗ࢹ࣮ࢱࡣ, ⣽⬊⭷⾲㠃࡛ࡢ Ca Ḟஈ࡟ࡼࡿ᰿㛗㜼ᐖࡢᙳ㡪ࢆ㑊ࡅࡿࡓࡵ, {Ca2+_{}PMࡀ 1 mM ࡼࡾᑠࡉ࠸᮲௳࡛ࡢ᰿㛗್ࢆྲྀࡾ㝖࠸࡚࠾ࡾ, ᘧ 2 ࡢ c2, d2࡟ࡼࡗ࡚} mechanism III ࡢຠᯝࡀホ౯ࡉࢀࡿ. ಀᩘ c2 ࡣ, ඲࡚ࡢኚ␗ᰴ࡟࠾࠸࡚᭷ព࡛࡞ࡃ,

mechanism III ࡀ{Al3+_{}PMẘᛶࡢ㍍ῶ࡟㈉⊩ࡋ࡚࠸࡞࠿ࡗࡓ. ୍᪉࡛, ಀᩘd2ࡣ᭷ព࡛࠶ࡾ,}

mechanism III ࡟ࡼࡗ࡚{H+_{}PMẘᛶࢆ㍍ῶࡍࡿ࡜ࢩ࣑࣮ࣗࣞࢺࡉࢀࡓ. STOP1-KO ࡢd2ࡢ}

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STOP1-KO

Figure 5. Three-dimensional scatter plots of root lengths of AL3S-KO, AtALMT1-KO, and

STOP1-KO in various Al3+_{and H}+ _{rhizotoxic solutions containing various concentration of}

CaCl2. Root length was plotted against computed {Al3+}PM and {H+}PM combinations at

different {Ca2+_} PM.

Table 5. Summary of statistics from regression analysis according to Equation 2 Genotype r2 _{n c} 1 c2 d1 d2 ALS3-KO 0.814 99 -0.158 ns -1.737 -0.928 AtALMT1-KO 0.901 99 -0.307 ns -1.136 -0.635 STOP1-KO 0.882 99 -0.203 ns -3.002 -0.381

၀ࡉࢀࡿ.

SSTOP1-KO ࡢ H+_{ẘᛶ࡟ࡼࡿ᰿➃㞀ᐖࡢCa}2+_㍍ῶ

H+_{ࡣ, ప Ca ⃰ᗘࡢ⁐ᾮ࡛ࢩࣟ࢖ࢾࢼࢬࢼ᰿➃ࡢ㠀ྍ㏫ⓗ࡞㞀ᐖࢆᘬࡁ㉳ࡇࡍ. ࡲࡓ, ࡇ}

ࡢ㞀ᐖࡣ, Ca2+ _{(ࡲࡓࡣ࣍࢘㓟)ࡀ⣽⬊ቨࡢ࣌ࢡࢳࣥࢆᏳᐃ໬ࡍࡿࡇ࡜࡟ࡼࡗ࡚㍍ῶࡍࡿ࡜}

ࡉࢀࡿ (Koyama et al., 2001). ࡇࡢ⣽⬊ቨࡢᏳᐃ໬࡟ࡼࡿ㍍ῶࡀ, STOP1-KO ࡛ࡢ

mechanism III ࡟ࡼࡿ Ca ㍍ῶ࡟㛵ಀࡍࡿ࡜⪃࠼ࡽࢀࡿ. ࡑࡇ࡛, STOP1-KO ࡜ Col-0 ࡛ࡢ

᰿➃ࡢ㞀ᐖࢆẚ㍑ࡋࡓ. STOP1-KO, Col-0 ࡜ࡶ࡟, pH 4.7 ࡢ Ǎ0&D&O2ࡢ༢⣧⁐ᾮ࡛

90 ศ㛫ฎ⌮ࡉࢀࡓ᰿➃ࡣ, ⣽⬊Ṛ࡟క࠸⣽⬊⭷ࢆ㏻㐣ࡍࡿ propidium iodide (PI)ࡢ⺯ගࡀ ほᐹࡉࢀࡓ (Fig. 6). ࡇࡢ Ca ⃰ᗘ᮲௳࡟࠾࠸࡚, ࡯࡜ࢇ࡝ࡢ᰿➃࡛㞀ᐖࢆ⏕ࡌࡓࡀ,

STOP1-KO ᰿➃ࡣ, Col-0 ࡜ẚ㍑ࡋ࡚㞀ᐖࡢ⛬ᗘࡀ኱ࡁ࠿ࡗࡓ. ຍ࠼࡚, Ca ⃰ᗘࢆ Ǎ0

࡜ࡋࡓ⁐ᾮ࡛ྠᵝࡢฎ⌮ࢆ⾜࠸, 㞀ᐖࡢ㍍ῶࢆ☜ㄆࡋࡓ. STOP1-KO ࡛ࡢ㞀ᐖࡢ㍍ῶຠᯝ

ࡣ, Col-0 ࡼࡾࡶ᫂ࡽ࠿࡟ᑠࡉ࠿ࡗࡓ (Fig. 6). ࡇࡢ⤖ᯝࡼࡾ, STOP1-KO ࡣ, Ca2+_ࡢ⣽⬊ቨ

ࡢᏳᐃ໬ࢆྵࡴmechanism III ࡢ㍍ῶᶵᵓ࡟Ḟᦆࡀ⏕ࡌ࡚࠸ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿ. ⣽⬊⭷⾲㠃ࡢ㈇㟁Ⲵ㧗ᐦᗘኚ␗ᰴࡢAl3+_ឤཷᛶ ࡇࢀࡲ࡛ࡢ⤖ᯝࡣ, Al ẘᛶࡢホ౯࡟ SGCS ࡢࣔࢹࣜࣥࢢ࡟ᇶ࡙ࡃゎᯒࡀ᭷ຠ࡞ᡭἲ࡛࠶ ࡿࡇ࡜ࢆᣦ♧ࡋ࡚࠸ࡿ. ࡇࡢࣔࢹࣝ࡟╔┠ࡍࡿࡇ࡜࡛, ᪂つࡢ Al ឤཷᛶ࡜࡞ࡿ㑇ఏᙧ㉁ࡢ ༢㞳, ྠᐃࡀᮇᚅ࡛ࡁࡿ. SGCS ࡛ࡢࣔࢹࣜࣥࢢࡼࡾ, PM ࡛ࡢ㈇㟁Ⲵᐦᗘࡢቑຍࡣ, Al3+_࡟ ឤཷᛶ࡜࡞ࡿࡇ࡜ࡀண᝿ࡉࢀ, ⣽⬊⭷ᵓᡂᡂศ࡟㛵ࡍࡿ◊✲࡛ᐇ㦂ⓗ࡟ᨭᣢࡉࢀ࡚࠸ࡿ (Wagatsuma and Akiba, 1989; Yermiyahu et al., 1997; Khan et al., 2009). ࡉࡽ࡞ࡿ᳨ド

ࢆ⾜࠺ࡓࡵ, ㈇㟁Ⲵᐦᗘࡢ㧗࠸ኚ␗ᰴ࡜㔝⏕ᰴ࡜ࡢ Al ⪏ᛶࡢẚ㍑ࢆ⾜ࡗࡓ. pah1pah2஧㔜

ኚ␗ᰴࡣ, PAH άᛶࢆḞᦆࡋ࡚࠾ࡾ, P Ḟஈ᮲௳ୗ࡛ PM ࡛ࡢ୺࡞㈇㟁Ⲵࣜ࢞ࣥࢻ࡛࠶ࡿࣜ ࣥ⬡㉁ࡀ⥔ᣢࡉࢀࡿ (Nakamura et al., 2009). ୍᪉࡛, Col-0 ࡣ, P Ḟஈ≧ែ࡛ P ࢆ౪⤥ࡍ





&D&O

_

˩0





&D&O

_

˩0

&RO

6723

.2

Figure 6 Ca2+ _{alleviation of H}+ _{damage in root tips of WT Col-0 and STOP1-KO.}

Seedlings grown for 5 d in a control solution (pH 5.5) were soaked in a solution containing 200 or 400 ȝ0 CaCl2(pH 4.7) for 90 min and then stained with propidium

ࡿࡓࡵ࡟ࣜࣥ⬡㉁ࢆ㟁Ⲵࡢ࡞࠸⢾⬡㉁࡟ኚ᥮ࡍࡿ. ᚑࡗ࡚, pah1pah2 ࡣ, P Ḟஈ≧ែ࡛

Col-0 ࡼࡾࡶ PM ㈇㟁Ⲵᐦᗘࡀ㧗ࡃ Al3+_{ࢆࡼࡾ㞟✚ࡍࡿࡓࡵAl}3+_{࡟ឤཷᛶࢆ♧ࡍ࡜⪃࠼ࡽࢀ}

ࡿ.

P ⃰ᗘࡢ␗࡞ࡿ⁐ᾮ࡟ࡘ࠸࡚, ࡑࢀࡒࢀ GEOCEM-EZ ࡛{Al3+_{}bulkࡀྠ⛬ᗘ࡜࡞ࡿࡼ࠺࡟}

ㄪ〇ࡋࡓ⁐ᾮ࡛ pah1pah2 ࡜ Col-0 ࢆ⏕⫱ࡉࡏࡓ. P ࢆ㝖࠸ࡓᇵᆅ࡛ࡢ᰿㛗ヨ㦂ࡼࡾ,

pah1pah2ࡣCol-0 ࡼࡾࡶ Al3+_{࡟ឤཷᛶࢆ♧ࡋࡓ (Fig. 7A). ୍᪉, pah1pah2࡜Col-0 ࡢ㈇}

㟁Ⲵᐦᗘཬࡧ{Al3+_{}PMࡀ➼ࡋࡃ࡞ࡿ⏕⫱࡟༑ศ࡞P ࢆྵࡴᇵᆅ (Ǎ0Na·PO4)࡛ࡣ, Al ⪏}

ᛶ࡟᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ (Fig. 7A). P ࡢ᭷↓࡟క࠺᳜≀యࡢ P ᰤ㣴≧ែࡢ㐪࠸ࡣ P Ḟஈㄏᑟ㑇ఏᏊࡢⓎ⌧㔞࡟ࡼࡗ࡚♧ࡋࡓ (Fig. 7B). ࡉࡽ࡟, ㄏᑟ⤖ྜࣉࣛࢬ࣐㉁㔞ศᯒ

ィ (ICP-MS)࡟ࡼࡾ ᐃࡉࢀࡓ P Ḟஈ≧ែࡢpah1pah2᰿㒊ࡢAl 㞟✚㔞ࡣ, Col-0 ࡜ẚ㍑ࡋ

࡚᭷ព࡟㧗࠿ࡗࡓ (Fig. 7C). ࡲࡓ, P Ḟஈ≧ែࡢᗂ᳜≀యࢆ Al ฎ⌮⁐ᾮ࡛ 24 ᫬㛫ฎ⌮ࢆ⾜

࠸, ࣔࣜࣥᰁⰍࢆ⏝࠸࡚᰿➃࡛ࡢ Al ࢆ᳨ฟࡋࡓ࡜ࡇࢁ, pah1pah2ࡣ, Col-0 ࡼࡾࡶᙉ࠸⺯ග

ࢆ࿊ࡋࡓ. ࡇࡢ pah1pah2 ࡢ᰿➃࡛ࡢ Al 㞟✚ࡣ, ศ⿣ཬࡧᡂ㛗㡿ᇦ࡛ࡢ⺯ගࢆ࿊ࡍࡿ

AtALMT1-KO ࡢ㞟✚࡜᫂ࡽ࠿࡟␗࡞ࡗ࡚࠸ࡓ (Fig. 7D). pah1pah2ࡣ, AtALMT1Ⓨ⌧࡟

౫ᏑࡍࡿAl ᤼㝖ᶵᵓࡣṇᖖ࡛࠶ࡿࡓࡵࡑࡢ㒊఩࡛ࡢ㞟✚ࡣᙅࡃ, ࡑࡢᶵᵓ࡟ᙉࡃ౫Ꮡࡋ࡚

࠸࡞࠸௚ࡢ㒊఩࡛ࡢAl 㞟✚ࡀㄆࡵࡽࢀࡓ. ࡇࢀࡽࡢ⤖ᯝࡣ, ண᝿ࡉࢀࡓ pah1pah2ࡢ⾲⌧

ᆺࢆ༑ศ࡟ㄝ࡛᫂ࡁࡿࡶࡢ࡛࠶ࡿ.

ᅵተ࡛ࡢ⏕⫱ヨ㦂ࡼࡾ, ẚ㍑ⓗ㧗࠸ pH ࡟ㄪᩚࡋࡓ㓟ᛶᅵተ ࡟࠾࠸࡚, pah1pah2ࡣAl3+

࡟ឤཷᛶࢆ♧ࡋ, ୰࿴ຠᯝࡢప࠸ CaSO4ࡢ᪋⫧ࡣ, ࡑࡢ⏕⫱ࢆᨵၿࡋࡓ (Fig. 7E). ࡇࡢᅵ

ተ࡛ࡢ⏕⫱ࡣ, ⁐ᾮ⣔࡛☜ㄆࡉࢀࡓẚ㍑ⓗ㧗࠸ pH ࡛ࡢ{Al3+_{}PM࡟ࡼࡿẘᛶ࡜Ca}2+_࡟ࡼࡿ㍍

ῶࢆᨭᣢࡋ࡚࠸ࡿ. ௨ୖࡢ SGCS ࡢࣔࢹࣜࣥࢢ࡟ᇶ࡙ࡃࢩ࣑࣮ࣗࣞࢩࣙࣥཬࡧ㈇㟁Ⲵᐦᗘ

ࡀ㧗࠸ࡇ࡜ࡀAl3+_{ឤཷᛶࢆ㧗ࡵࡿࡇ࡜ࢆ♧ࡋࡓศᏊ⏕⌮Ꮫⓗヨ㦂ࡣ, {Al}3+_}PMࡀAl3+_ẘᛶࡢ

0 20 40 60 80 100 0.54 1.08 0 50 100 0.54 1.08 Re la tiv e ro o t le n g th (% o f – A l) g /100 g soil CaCO3 0.40 0.15 0.15 CaSO4 0.0 0.0 0.20 pH (H2O) 5.5 5.1 5.0

A

B

E

SDKSDK

D

C

R

oot

ti

p

*

*

Col-0 pah1_pah2

*

Figure 7. Al resistance of Col-0 and pah1pah2 double mutant in Al-toxic solution in the

presence or absence of phosphate. (A) Seedlings were grown for 7 d in Al-toxic solutions ({Al3+_}

bulk, 0.54 or 1.08PM) in zero (total Al added, 2 or 4 PM) or 35 PM Pi (total Al added,

5 and 9.7 PM) at pH 5.0. Means of relative root length r SE are shown (n = 10). Asterisks indicate the significance of difference from Col-0 by Student’s t-test (P < 0.05). (B) Expression of Pi starvation-responsive genes in roots of Col-0 grown for 7 d in solution in the presence or absence of phosphate, as used in root growth experiments. Transcripts were determined by semiquantitative RT-PCR. UBQ1 transcript was used as the internal control. (C) Al content of roots of Col-0 and pah1pah2 with Al stress treatment. Seedlings were grown for 10 d in Al toxic solutions ({Al3+_}

bulkof 0.54PM, total added Al 2 PM) in zero Pi

at pH 5.0. Al concentration in the roots was quantified using ICP-MS. Meansr SE (n = 4) are shown. Asterisks indicate the significance of difference from Col-0 by Student’s t-test (P < 0.05). (D) Morin staining of Col-0 and pah1pah2 after incubation in Al-toxic solution for 24 h. Bar = 20 ȝP. (E) Seedlings of Col-0 and pah1pah2 were grown for 4 weeks on acidic andosol fertilized with CaCO₃, and a combination of CaCO₃and CaSO₄. Soil pH was determined. White bar = 1 cm.

⪃ᐹ

Ǎ0$O&O3ࢆྵࡴǍ0&D&O2⁐ᾮࡢྛpH ࡛ࡢ⣽⬊⭷⾲㠃㟁఩ (ǙPM), {Ion}bulkཬ

ࡧ{Ion}PMࢆ⏝࠸࡚, ᨵࡵ࡚㟼㟁ⓗࣔࢹࣝ࡟ࡘ࠸࡚ㄝ᫂ࡍࡿ (Fig. 8). ⁐ᾮ pH ࡢୖ᪼࡟క࠸,

ǙPMࡀ኱ࡁࡃ࡞ࡾ, ㄏᘬࡉࢀࡿ Al3+ࡀቑຍࡍࡿࡓࡵ, {Al3+}bulkࡢῶᑡ࡟ࡶ㛵ࢃࡽࡎ, {Al3+}PM

ࡣ⥔ᣢࡉࢀࡿ. ྠᵝ࡟, {Ca2+_{}PMࡶpH 4.0 ࡛ࡢ Ǎ0 ࡟ᑐࡋ࡚ pH 5.6 ࡛ࡣ Ǎ0 ࡜}

⣙ 15 ಸ࡟ቑຍࡍࡿ. ࡇࡢ{Ca2+_{}PM ࡢቑຍࡣ{Al}3+_{}PM࡜➇ྜࡍࡿࡓࡵ, pH 5 ࢆ㉸࠼ࡿ࡜}

{Al3+_{}PMࡀῶ⾶ࡍࡿ. ࢥ࣒ࢠ࡛ࡣ, ᵝࠎ࡞᮲௳ࡢỈ⪔⁐ᾮ⣔࡛ࡢ᰿ఙ㛗ࢹ࣮ࢱࢆࡶ࡜࡟,}

{Al3+_{}PM, {H}+_{}PM, {Ca}2+_{}PM ࡟ ᑐ ࡍ ࡿ ᰿ ఙ 㛗 ᛂ ⟅ ࡀ ࢩ ࣑ ࣗ ࣞ ࣮ ࢺ ࡉ ࢀ ࡚ ࠸ ࡿ (Fig. 8)}

(Kinraide et al., 2004; Kinraide and Wang, 2010). ౛࠼ࡤ, ୖグࡢ᮲௳࡟ࡘ࠸࡚, pH4.4 ௨

ୗ࡛ࡣ, H+_{࡟ࡼࡿ㜼ᐖࡀ⏕ࡌࡿࡀ, ǙPMࡢୖ᪼࡟ࡼࡾ{Al}3+_{}PMࡣపୗࡋ࡚࠾ࡾ, Al}3+_࡟ࡼࡿ㜼 ᐖࡣ㍍ῶࡉࢀࡿ. pH ࡢୖ᪼࡟క࠸, ǙPMࡣ኱ࡁࡃ࡞ࡾ, {Al3+}PMࡢቑຍ࡟ࡼࡿ㜼ᐖࡀ㢧ⴭ࡟ ⾲ࢃࢀࡿࡀ, pH ࡀ 5 ࢆ㉸࠼ࡿ࡜, ୖ㏙ࡋࡓࡼ࠺࡟{Ca2+_{}PMࡀ኱ࡁࡃቑຍࡍࡿࡓࡵ, Al}3+_࡟ࡼ ࡿ㜼ᐖࡀ㍍ῶࡉࢀࡿ. ࡇࡢࡼ࠺࡟, ᰿ఙ㛗㜼ᐖࡔࡅ࡛ࡣ࠶ࡿࡀ, PM ࡛ࡢάືᗘࢆ⏝࠸࡚᫂ ☜࡟ㄝ࡛᫂ࡁࡿࡇ࡜࠿ࡽ, ࡑࢀࡽࢆ㓟ᛶᅵተ⪏ᛶࡢศᏊᶵᵓࡢゎᯒ࡟ᛂ⏝࡛ࡁࡿ࡜⪃࠼ࡽ ࢀࡿ. ᮏ◊✲࡛ࡣ, ࢩࣟ࢖ࢾࢼࢬࢼࢆ⏝࠸࡚ pH ࡀ 5 ௨ୖࡢᰤ㣴⁐ᾮ࡛ࡢ Al3+_࡜H+_{ࡢẘᛶࡢࢩ} ࣑࣮ࣗࣞࢺࢆ⾜ࡗࡓ. ゎᯒ࡟⏝࠸ࡓឤཷᛶኚ␗ᰴࡣ, pH 5 ௨ୖ࡛ Al3+_ࡲࡓࡣH+_{࡟ᑐࡋ࡚ឤ} ཷᛶࢆ♧ࡋࡓ. GEOCHEM-EZ ࡜ࡢ⤌ࡳྜࢃࡏ࡟ࡼࡿ SGCS ࡛ࡢࣔࢹࣜࣥࢢ࡟ࡼࡗ࡚, 」 㞧࡞ᰤ㣴⁐ᾮ⣔࡛ࡢࢩࣟ࢖ࢾࢼࢬࢼࡢ᰿ఙ㛗ࡀ {Al3+_{}PM࡛ࢩ࣑࣮ࣗࣞࢺ࡛ࡁࡿࡇ࡜ࢆ᫂ࡽ}

࠿࡟ࡋࡓ. Al ឤཷᛶኚ␗ᰴ࡛ࡢ Al ᰿㛗㜼ᐖࡣ, {Al3+_{}bulkࡼࡾࡶ {Al}3+_{}PM࡜㧗࠸┦㛵ࢆ♧ࡍ}

ࡇ࡜࡟ຍ࠼࡚, Al ㄏᑟࢆ♧ࡍ㑇ఏᏊࡢⓎ⌧ࣃࢱ࣮ࣥ࡟ࡘ࠸࡚ࡶ PM ࡛ࡢ Al3+_{ࡢㄏᘬ࡜㛵㐃ࡍ}

ࡿࡇ࡜ࢆ♧ࡋࡓ. ࡉࡽ࡟, {Ca2+_{}PM ࡢቑຍ࡟క࠺{Al}3+_{}PM ࡢῶᑡࡣ, ᰿ఙ㛗㜼ᐖࡢ㍍ῶ࡜୍}

























    























    











{A

l

}

(ȝ0

)

{A

l

}

(ȝ0

)

ȥ

(mV

)









{A

l

}

(ȝ0

)

{C

a

}

(ȝ0

)

{H

}

(ȝ0

)

RRE

RRE

pH

A

B

C

























    

Figure 8. Computed wheat seedling responses to pH adjustment of a growth medium

composed of 200 ȝ0 CaCl2 and 0.5 ȝ0 AlCl3. Published models (Kinraide et al., 2004;

Kinraide and Wang, 2010) were used to compute solution species, PM surface electrical potentials (ȥPM), PM-surface ion activities ({Al3+}PM, {Ca2+}PM, {H+}PM), and growth

responses. Total relative root elongation (on a scale of 0 to 100) was computed as 100 times the product of partial relative root elongations (on a scale of 0 to 1) in response to individual ions (RRETotal= 100(RRE{Ca2+_}

PM㽢RRE{H+}PM㽢RRE{Al3+}PM). The H+-induced reduction in

the negativity of ȥPMreduced the attraction of cations. {Ca2+}bulkwas nearly constant at 178

+ 1 ȝ0, but at lower pH values {Ca2+_}

PMbecame marginally insufficient. Despite the decline

in {Al3+_}

bulk as pH increased, the increasing negativity of ȥPM caused an increase in

{Al3+_}

PMuntil it reached a maximum at pH 5.0.

࡙࠸࡚Ca ㍍ῶ࡜㛵㐃ࡋࡓ Al3+_ཬࡧH+_{ẘᛶࢆㄝ࡛᫂ࡁࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿ.} ࣔࢹࣜࣥࢢࢆ⏝࠸ࡓゎᯒࡣ, {Ca2+_{}PM࡛⾲ࡉࢀࡿCa ㍍ῶᶵᵓࡀ, ẘᛶ࢖࢜ࣥཬࡧ㑇ఏᏊ} ᆺ࡛␗࡞ࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓ. Al ẘᛶࡣ, Ca2+_࡟ࡼࡿAl3+_{ࡢ㟼㟁ⓗ⨨᥮࡜⣽⬊⭷࡟࠾࠸࡚} ᚲせ࡜ࡉࢀࡿCa2+_{㔞ࡢᅇ᚟ (Mechanism I ཬࡧ II)࡟ࡼࡾ㍍ῶࡉࢀࡓࡀ, I ࡜ II ௨እࡢᶵ⬟} ᮍ▱࡞ᶵᵓ (Mechanism III)࡟ࡼࡿ㍍ῶࡣ, ឤཷᛶኚ␗ᰴ࡛ࡢࢩ࣑࣮ࣗࣞࢩࣙࣥ࡟࠾࠸࡚ ㄆࡵࡽࢀ࡞࠿ࡗࡓ (Table 5). ୍᪉, H+_{ẘᛶ࡛ࡣ, MechanismIII ࡟ࡼࡿ㍍ῶࡀ⏕ࡌࡿ࡜ࡉࢀ,}

STOP1-KO ࡣ ௚ ࡢ ኚ ␗ᰴ ࡜ ẚ ㍑ ࡋ ࡚ ࡑ ࡢ ຠ ᯝࡀ ᑠ ࡉ ࠸ ࡇ ࡜ ࡀ ♧ ࡉ ࢀࡓ (Table 5).

STOP1-KO ࡛ࡣ MechanismIII ࡟ᐤ୚ࡍࡿᶵᵓࡀḞᦆࡋ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀࡿ. ⣽⬊ቨ

ᵓᡂᡂศ࡛࠶ࡿ࣒ࣛࣀ࢞ࣛࢡࢶࣟࢼࣥII (RGII) (O'Neill et al., 2004)࡜࣏ࣜ࢞ࣛࢡࢶࣟࣥ

㓟 (PGA) (Carpita and Gibeaut, 1993)ࡢ Ca2+_{࡟ ࡼ ࡿ Ᏻ ᐃ ໬ ࡣ H}+_{ẘ ᛶ ࢆ ㍍ ῶ ࡍ ࡿ}

(Koyama et al., 2001). stop1 ኚ␗ᰴ࡛ࡣ PGA ࡢᏳᐃ໬࡟㛵୚ࡍࡿ polygalacturonase

inhibitor protein 1 (PGIP1) (Spadoni et al., 2006)ཬࡧ࣌ࢡࢳࣥࡢᯫᶫᵓ㐀࡟ᚲ㡲࡞࣍࢘⣲

ࡢ౪⤥࡟㛵ࢃࡿ borate transporter 2 (BOR2)ࡢⓎ⌧ࡀ㔝⏕ᰴ࡜ẚ㍑ࡋ࡚ᢚไࡉࢀ࡚࠸ࡓ

(Sawaki et al., 2009). ࡇࡢࡇ࡜ࡣ, STOP1-KO ࡟࠾࠸࡚ప pH ࢫࢺࣞࢫ࡟ࡼࡿ᰿➃㞀ᐖࢆ

పῶࡉࡏࡿ⣽⬊ቨࡢᏳᐃ໬⬟ຊࡀపୗࡋ࡚࠾ࡾ, ࡇࡢᏳᐃ໬ࡀ MechanismIII ࡟ྵࡲࢀࡿ ࡜ࢆ♧၀ࡍࡿ. ࡲࡓ, ྛኚ␗ᰴࡢឤཷᛶせᅉࡀ␗࡞ࡿࡇ࡜ࢆࣔࢹࣜࣥࢢࢆ⏝࠸ࡓࢩ࣑ࣗࣞ ࣮ࢩ࡛ࣙࣥ᥎ᐃࡋࡓ. ᚑࡗ࡚, ྠᵝࡢゎᯒ࡟ࡼࡗ࡚ Al3+_{࠾ࡼࡧ H}+_{ឤཷᛶࡢ␗࡞ࡿᙧ㉁ࢆᣢ} ࡘಶయ࡟ࡘ࠸࡚, ศᏊ⏕⌮Ꮫⓗ࡛ࣞ࣋ࣝ≉ᚩࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜ࡀᮇᚅ࡛ࡁࡿ. ෆᅾᛶࡢ᭷ᶵ࢝ࢳ࢜ࣥࡀAl ẘᛶࡢ㍍ῶ࡟࠾࠸࡚ Ca ࡜㢮ఝࡋࡓᶵ⬟ࢆᣢࡘࡇ࡜ࡀ♧၀ࡉ ࢀ࡚࠸ࡿ. ࢩࣟ࢖ࢾࢼࢬࢼ࡟࠾࠸࡚, ᰿ࡢ Al ᤼㝖⬟࡟␗ᖖࢆ♧ࡍኚ␗యࡀ༢㞳ࡉࢀ, ᰾ᒁ ᅾᛶࡢࣜ࣎ࢯ࣮࣒⏕ྜᡂᅉᏊࢆࢥ࣮ࢻࡍࡿ࡜᥎ᐃࡉࢀࡿ㑇ఏᏊ࡟ኚ␗ࡀ⏕ࡌ࡚࠸ࡿࡇ࡜ࡀ ᫂ࡽ࠿࡟ࡉࢀࡓ (Nezames et al., 2012). ࡇࡢ Al ឤཷᛶኚ␗యࡣ, ࣏ࣜ࢔࣑ࣥࡀῶᑡࡋ࡚ ࠾ࡾ, ࢝ࢳ࢜ࣥᛶ࣏ࣜ࢔࣑࡛ࣥ࠶ࡿࢫ࣑࣌ࣝࢪࣥࡢῧຍ࡟ࡼࡾ㔝⏕ᰴ࡜ྠ⛬ᗘࡢ⪏ᛶࢆ♧ ࡋࡓ. ࡇࡢ⤖ᯝࡣ, ⣽⬊ቨ࡟ᨺฟࡉࢀࡓ࢝ࢳ࢜ࣥᛶ࣏ࣜ࢔࣑ࣥࡀ, Ca2+_࡟ࡼࡿ{Al3+_}PMࡢῶ