根圏ミネラルストレスに応答する植物地上部の転写変動に関する研究

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Title

根圏ミネラルストレスに応答する植物地上部の転写変動に

_{関する研究( 本文(Fulltext) )}

Author(s)

澤木, 克亘

Report No.(Doctoral

Degree)

博士(農学) 甲第657号

Issue Date

2016-09-26

Type

博士論文

Version

ETD

URL

http://hdl.handle.net/20.500.12099/55542

※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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᰿ᅪ࣑ࢿࣛࣝࢫࢺࣞࢫ࡟ᛂ⟅ࡍࡿ᳜≀ᆅୖ㒊ࡢ

㌿෗ኚື࡟㛵ࡍࡿ◊✲

2 0 1 6 ᖺ

ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ㎰Ꮫ◊✲⛉

⏕≀㈨※⛉Ꮫ

（岐阜大学）

⃝ᮌඞர

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᰿ᅪ࣑ࢿࣛࣝࢫࢺࣞࢫ࡟ᛂ⟅ࡍࡿ᳜≀ᆅୖ㒊ࡢ

㌿෗ኚື࡟㛵ࡍࡿ◊✲

(4)

┠

┠ḟ

ᗎㄽ

1 㡫

➨1❶ ࣔࢹ᳜ࣝ≀ᆅୖ㒊ࢆ⏝࠸ࡓᰤ㣴Ḟஈᛂ⟅ᛶࡢ

㑇ఏᏊⓎ⌧ࣂ࢖࣐࣮࣮࢜࢝ࡢసᡂ

5 㡫

➨2❶ ࢩࣟ࢖ࢾࢼࢬࢼᆅୖ㒊ࡢ᰿ᅪẘᛶ࢖࢜ࣥᛂ⟅ᛶࡢ

ࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ࣉࣟࣇ࢓࢖ࣜࣥࢢ

43 㡫

➨

3❶

Penicillium phinophilum

᥋✀࡟ࡼࡿ࢖ࢿ࠾ࡼࡧ

ࢩࣟ࢖ࢾࢼࢬࢼࡢ⑓ᐖ᢬ᢠᛶࡢホ౯

77 㡫

⥲ྜ⪃ᐹ

86 㡫

⤖ㄽ

90 㡫

ㅰ㎡

91 㡫

ᘬ⏝ᩥ⊩

92 㡫

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┬␎グྕ

ABA㸸abscisic acid ࢔ࣈࢩࢪࣥ㓟

CTAB㸸Cetyltrimethyl ammonium bromide DNA㸸deoxyribonucleic acid

ET㸸ethylene ࢚ࢳࣞࣥ FC㸸Fold change

GO㸸Gene Ontological

GTC㸸Guanidine Thiocyanate

GWAS㸸Genome Wide Association Study JA㸸jasmonic acid ࢪࣕࢫࣔࣥ㓟

mRNA㸸messenger RNA

NCBI㸸National Center for Biotechnology Information PCR㸸polymerase chain reaction

RNA㸸ribonucleic acid

RT-PCR㸸reverse transcription- polymerase chain reaction

qRT-PCR㸸quantitative reverse transcription-polymerase chain reaction SA㸸salicylic acid ࢧࣜࢳࣝ㓟

TAIR㸸The Arabidopsis Information Resource TE㸸Tris EDTA

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1

ᗎ

ᗎㄽ

᳜≀ࡣᆅ⌫ୖ࡟⣙ 30 ୓✀Ꮡᅾࡋ࡚࠸ࡿ࡜ࡉࢀ࡚࠸ࡿࡀ㸪ࡑࡢ࡞࠿࡛ࢺ࢘ࣔࣟࢥࢩ (Zea

mays) ࡸ࢖ࢿ (Oryza sativa) 㸪ࢥ࣒ࢠ (Triticum aestivum) ࢆࡣࡌࡵ࡜ࡋࡓ✐≀ࡣ㣗ᩱ ※࡜ࡋ࡚ே㛫♫఍ࡢ᰿ᖿࢆᨭ࠼ࡿ᳜≀✀࡛࠶ࡿ㸬᳜≀ࡣ㣗ᩱ㈨※࡜ࡋ࡚ࡢࡳ࡞ࡽࡎ㣫ᩱࡸ ⧄⥔㸪ᘓ⠏㈨ᮦ࡞࡝ࡢᕤᴗཎᮦᩱ࡜ࡋ࡚ከᒱ࡟Ώࡗ࡚฼⏝ࡉࢀ࡚࠾ࡿ㸬ࡑࢀࡒࢀࡢ⏝㏵࡟ 㐺ࡍࡿᙧ㉁ࢆ⋓ᚓࡍࡿࡓࡵ⣖ඖ๓ࡼࡾேⅭ஺㓄࡟ࡼࡗ࡚ከᵝ࡞᳜≀✀ࡢရ✀ᨵⰋࢆ⾜ࡗ࡚ ࡁࡓࡀ㸪஺㓄࡟ࡼࡿရ✀ᨵⰋࡣ┠ⓗᙧ㉁ࢆ⋓ᚓࡍࡿ࡟ࡣ」㞧࠿ࡘ᫬㛫ࡀ࠿࠿ࡿᡭἲ࡛࠶ࡿ㸬㏫㑇ఏᏛⓗゎᯒࡸ㔞ⓗᙧ㉁ᗙ఩ (QTL) ゎᯒ㸪ࢤࣀ࣒࣡࢖ࢻ㛵㐃ゎᯒ (GWAS) ࡞࡝ࡢศᏊ ⏕≀ᏛⓗᡭἲࡢⓎ㐩࡟ࡼࡾᵝࠎ࡞ᙧ㉁࡟㛵୚ࡍࡿ㑇ఏᏊ࠾ࡼࡧ㸪ࡑࡢᶵ⬟ࡢ≉ᐃࡀྍ⬟࡟ ࡞ࡾᙧ㉁㌿᥮ᢏ⾡࡟ࡼࡿ┠ⓗᙧ㉁ࡢᑟධࡀᐜ᫆࡟࡞ࡗࡓ㸬ศᏊ⫱✀ࡢ◊✲ࡣ✲ᴟⓗ࡟ࡣ᳜ ≀ࡢ㑇ఏᏊᶵ⬟ࡢኚ໬࡟ࡼࡿ⏕⌮ᶵᵓࡢᙉ໬࡛࠶ࡿ࡜࠸࠼ࡿ㸬᳜≀ࡢ⏕⌮ᛂ⟅ᶵᵓࢆゎ᫂ ࡋ⎔ቃࢫࢺࣞࢫ᢬ᢠᛶࢆྥୖࡉࡏࡓ᳜≀యࢆసฟࡍࡿࡇ࡜࡛✐≀཰㔞ࡢቑ⏘ࡸ᭷⏝≀㉁ࡢ ⏕⏘㔞ቑຍ࡟⧅ࡀࡿ࡜⪃࠼ࡽࢀ㸪ᐇ㝿࡟⎔ቃࢫࢺࣞࢫ⪏ᛶࢆ⋓ᚓࡋࡓᙧ㉁㌿᥮స≀ࡀసฟ ࡉࢀ࡚࠸ࡿ (Bülowa et al., 1999; Taji et al., 2002)㸬

స≀⏕⏘࡜ࡋ࡚స≀ࡢရ✀ᨵⰋ㸪㎰ᴗࡢᶵᲔ໬㸪໬Ꮫ⫧ᩱࡸ㎰⸆ࡢ㛤Ⓨ࡜࠸ࡗࡓ㎰ᴗᢏ ⾡ࡢ㠉᪂࡟ࡼࡾ㸪㐣ཤ40 ᖺ࠿ࡽ✐≀⏕⏘㔞ࡣ⥳ࡢ㠉࿨࡞࡝ࢆ⤒࡚ 7.4 ಸ࡜࡞ࡾ኱ࡁࡃୖ᪼ ࡋࡓ㸬1960 ᖺ௦௨㝆ࡢ✐≀⏕⏘㔞ࡢఙࡧࡣඛ㐍ᅜࡼࡾࡶⓎᒎ㏵ୖᅜ࡟࠾࠸࡚㢧ⴭ࡛࠶ࡾ 1961 ᖺ࠿ࡽ 2000 ᖺ࡟࠿ࡅ࡚㏵ୖᅜࡢ✐≀⏕⏘ࡣ 3 ಸ࡜࡞ࡾ㸪ࡇࢀࡣேཱྀቑຍ⋡ (2.3 ಸ) ࢆ ୖᅇࡗ࡚࠸ࡿ㸬ୡ⏺ࡢ✐≀ࡢ⪔స㠃✚ࡣ⣙16 ൨ ha㸪∾ⲡᆅࡀ 3.8 ൨ ha ࡛࠶ࡿࡀ㸪FAO ࡟ ࡼࡿ࡜෾ᅵࡸෆ㝣ࡢἙᕝ㸪†἟ࢆ㝖ࡃෆᆅ㠃✚ࡣ⣙130 ൨ ha ࡛㸪ࡇࡢ⣙ 15㸣ࡢ㠃✚࡛ 70 ൨ேࡢ㣗ᩱࢆ⏕⏘ࡋ࡚࠸ࡿࡇ࡜࡟࡞ࡿ (Foley et al., 2011)㸬స≀⏕⏘ࡢ࠺ࡕ 62㸣ࡣே㛫ࡢ 㣗ᩱ࡜ࡋ࡚㸪35㸣ࡣᐙ␆ࡢ㣗ᩱ࡜ࡋ࡚⏕⏘ࡉࢀ࡚࠸ࡿ㸬1985 ᖺ࠿ࡽ 2005 ᖺࡢ㛫࡟⪔సᆅ ࡜∾ⲡᆅࡣ⣙3㸣ࡢ 154 ୓ ha ᣑ኱ࡋ✐≀⏕⏘㔞ࡣ 47㸣ቑຍࡋ࡚࠸ࡿ㸬Ἔ໬స≀ࡣ⏕⏘㠃 ✚ࢆ43㸣㸪⏕⏘㔞ࢆ 57㸣ቑຍࡋ࡚࠸ࡿࡀ㣚㣹ࡣ୍ྥ࡟ῶᑡࡋ࡚࠸࡞࠸㸬2013 ᖺ࡟ୡ⏺ே ཱྀࡣ70 ൨ேࢆ✺◚ࡋ 2050 ᖺ࡟ࡣ⣙ 90 ൨ே࡟࡞ࡿ࡜᥎ ࡉࢀ㸪௒ᚋࡢேཱྀቑຍ࡟కࡗ࡚ୡ

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2

⏺ ⓗ ࡟ 㣗 ᩱ 㟂せ ࡀ 㧗 ࡲ ࡾ 㸪 ⌧ ᅾ ⏕ ⏘ ࡉ ࢀ ࡚ ࠸ ࡿ స ≀ ࡢ 2 ಸ㔞ࡀᚲせ࡜ࡉࢀ࡚࠸ࡿ (Alexandratos, 1999; Cassman, 1999; Federoff and Cohen, 1999; Godfray et al., 2010; Lutz and Samir 2010)㸬ࡲࡓ㏆ᖺࡢ㎰⪔ᆅࡢᣑ኱ࡢࡓࡵ࡟ⲡཎࡢ 70㸣㸪ࢧࣂࣥࢼࡢ 50㸣㸪

ᖏⴠⴥᶞᯘࡢ45㸣㸪⇕ᖏ㞵ᯘࡢ 27㸣ࡀ㛤ᣅࡉࢀ࡚࠾ࡾ㸪ࡇࢀ࡟ࡼࡾ⏕≀ࡢ⏕ᜥᆅࡸከᵝ

ᛶࡸᅵተࡢ≧ែ࡟ⴭࡋ࠸ᝏᙳ㡪ࢆ୚࠼㸪ࡉࡽ࡟୙㐺ษ࡞℺₅ࢆࡣࡌࡵ㸪㐣⪔㉳ࡸ㐣ᨺ∾ࡢ ᮎ㸪ᅵተࡀຎ໬ࡋ⪔సᨺᲠᆅ࡜࡞ࡗ࡚࠸ࡿ (Ramankutty et al., 2008)㸬஦ᐇ㸪ISRIC (International Soil Reference and Information Center) ࡢㄪᰝሗ࿌࡟ࡼࢀࡤ᭱㏆ 20 ᖺ㛫

࡟ே㛫άື࡛20 ൨ ha ࡢᅵተࡀຎ໬ࡋࡓ࡜ࡉࢀ࡚࠾ࡾ㸪⌧ᅾࡢ㎰ᴗࡣ⪔సᆅࡢ 24㸣ࡣ℺₅ ࢆ฼⏝ࡋ࡚㎰స≀ࡢ 34㸣ࢆ⏕⏘ࡋ࡚࠸ࡿࡀ⪔సྍ⬟ᅵተࡢຎ໬࡛స≀ࡢ཰㔞పୗࡀㄢ㢟࡜ ࡞ࡗ࡚࠸ࡿ㸬⮬↛せᅉࡀཎᅉ࡜ࡋ࡚ᅵተຎ໬ࢆᘬࡁ㉳ࡇࡍሙྜࡶ࠶ࡾ㸪ᅵተ౵㣗 (㢼㣗 4.3 ൨ha㸪Ỉ㣗 4.7 ൨ ha)㸪㓟ᛶ㞵ࡢᙳ㡪ࡶྵࡵࡓᅵተࡢ㓟ᛶ໬ࡸሷ㢮໬㸪℺Ỉ㸪ᅵተ࠿ࡽࡢ ⁐⬺ࡸ᭷ᶵ≀ᦆኻࡸ㸪Ẽೃ࠾ࡼࡧᆅ⌮ⓗせᅉ࡟ࡼࡿ஝⇱㸪㧗ప 㸪ẘᛶ㔠ᒓ㸪ప 㸪ᙉග㸪 ᰤ㣴Ḟஈ࡞࡝ࡢ㠀⏕≀ⓗࢫࢺࣞࢫ࡟ࡼࡾ୺せస≀ࡢ⏕⏘ࡸ཰㔞㸪ရ㉁ࢆ㜼ᐖࡋ㸪స≀⏕⏘ ࡢ50㸣ࡀῶᑡࡋ࡚࠸ࡿ࡜ࡉࢀ࡚࠸ࡿ (Hammond-Kosack and Jones, 2000; Wang et al., 2003)㸬ຍ࠼࡚㸪ୡ⏺ࡢ⪔స㠃✚ᆅࡢ 70㸣ࡣ㓟ᛶᅵተࡸ▼⅊㉁ᅵ࢔ࣝ࢝ࣜᅵተ࡜࠸ࡗࡓ୙Ⰻ ᅵተ࡜࿧ࡤࢀࡿ⪔స࡟ࡣ୙ྥࡁ࡞ᅵተࡢᏑᅾࡀၥ㢟࡜࡞ࡗ࡚࠸ࡿ㸬ࡇࡢࡼ࠺࡞ᅵተ࡛㎰ᴗ ࢆ⾜࠺࡟࠶ࡓࡾ໬Ꮫ⫧ᩱࡢ኱㔞ᢞධ࡟ࡼࡿ⎔ቃ㧗㈇ᢸࡢ㎰ᴗࡀ⾜ࢃࢀ࡚࠸ࡿࡀ㸪ᣢ⥆ྍ⬟ ࡞⎔ቃప㈇Ⲵᆺ㎰ᴗ࡬ࡢ㌿᥮ࡀồࡵࡽࢀ࡚࠾ࡾ㸪㎰ᴗ⏕⏘ࢥࢫࢺࡢపୗ࡞ࡽࡧ࡟ᣢ⥆ⓗ㎰ ᴗࡢࡓࡵࡢ㐺ษ࡞᪋⫧ࡣ㈨※⠇⣙ࡢほⅬ࠿ࡽ㔜せ࡜࠸࠼ࡿ㸬᳜≀ࡢಶࠎࡢࢫࢺࣞࢫࡢᛂ⟅ ᶵᵓࡢ⌮ゎ࠿ࡽ⎔ቃࢫࢺࣞࢫ࡬ࡢ⏕⌮ᶵᵓࡢ඲యീࡢゎ᫂ࡣ㣗ᩱၥ㢟ࡢࡳ࡞ࡽࡎ㸪⎔ቃప ㈇Ⲵ㎰ᴗࡢ᥎㐍࡟኱ࡁࡃ㈉⊩ࡋ㸪స≀⏕⏘㔞ࢆ㧗ࡵࡿ㎰ᴗ࡟࠾࠸࡚⏕≀ከᵝᛶࡢ㧗࠸ᆅᇦ ࡸᆅ⌫⎔ቃࡢಖ඲ࢆ⪃៖ࡋࡓᣢ⥆ⓗ㎰ᴗࡢ᪉ἲࡀᚲせ࡛࠶ࡿ㸬௨ୖࡢ஦࠿ࡽ௒ᚋࡢ㎰ᴗ࡟ ࠾࠸࡚ᅵተ୰ࡢẘᛶ࢖࢜ࣥࢫࢺࣞࢫ⪏ᛶࡸᰤ㣴Ḟஈ㐺ᛂ⬟ຊࢆഛ࠼ࡿస≀ࡢసฟࡀồࡵࡽ ࢀ࡚࠸ࡿ㸬 ࣂ࢖࢜࢖ࣥࣇ࢛࣐ࢸ࢕ࢡࢫࡣศᏊ⏕≀ᏛࡢⓎᒎ࡟ࡘ࡞ࡀࡿ᪂ࡋ࠸ᡭἲ࡛࠶ࡾ㸪ࡇࡢᢏ⾡㠉᪂࡟కࡗ࡚⏕యࡢศᏊ඲యࢆ⥙⨶ⓗ࡟ ᐃࡍࡿ࣮࣑࢜ࢡࢫゎᯒᢏ⾡ࡀ┠ぬࡲࡋ࠸Ⓨᒎࢆ

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3 㐙ࡆ࡚࠸ࡿ㸬㏆ᖺࡢᛴ㏿࡞ศᯒᢏ⾡ࡢⓎᒎ࡜࡜ࡶ࡟㑇ఏᏊࡢ⥙⨶ⓗ࡞mRNA ࡢ⥲య (ࢺࣛ ࣥࢫࢡࣜࣉࢺ࣮࣒) ࡸ⣽⬊ෆࢱࣥࣃࢡ㉁⥲య (ࣉࣟࢸ࣮࣒࢜) 㸪⏕యࡢ௦ㅰ≀㉁ࡢ⥲య (࣓ ࢱ࣮࣒࣎ࣟ) ࡞࡝ᵝࠎ࡞✀㢮ࡢ⥙⨶ⓗศᏊ᝟ሗࡢ཰㞟࣭ゎᯒࡀᛴ㏿࡟㐍ᒎࡋ࡚ࡁࡓ㸬ศᏊ⏕ ≀Ꮫᐇ㦂ࡢࣁ࢖ࢫ࣮ࣝࣉࢵࢺ໬࡟ࡼࡗ࡚ከࡃࡢ⏕≀ࡢࢤࣀ࣒ゎㄞࡀ᏶஢ࡍࡿ࡜ඹ࡟㸪኱つ ᶍ࡞࣮࣑࢜ࢡࢫ᝟ሗࡢゎᯒ⎔ቃࡀᩚഛࡉࢀࡘࡘ࠶ࡿ㸬ྛ᳜≀✀ࡢ࣮࣑࢜ࢡࢫ᝟ሗࢆᇶ࡟ࡋ ࡚㔜せ࡞ᙧ㉁ࢆ௚᳜≀✀࡬ᙧ㉁㌿᥮࡛௚ࡢ᳜≀࡟⤌ࡳ᥮࠼ࡿࡇ࡜ࡶྍ⬟࡜࡞ࡗࡓ㸬ࡉࡽ࡟㸪 ࡇࢀࡽࡢ኱つᶍ࣮࣑࢜ࢡࢫ᝟ሗࢆໟᣓⓗ࡟ά⏝ࡍࡿࡇ࡜࡛ᕧ኱࠿ࡘከᵝ࡞⏕࿨⌧㇟ࡢࢹ࣮ ࢱゎᯒࢆྍ⬟࡟ࡍࡿᢏ⾡ࡢ㛤Ⓨ࠾ࡼࡧࡑࢀࡽࡢ᝟ሗࢹ࣮ࢱ࣮࣋ࢫࢆ⤫ྜࡋ㸪⥲యⓗ࠿ࡘ」㞧࡛ືⓗ࡞⏕࿨⌧㇟ࢆ⌮ゎ㸪ᛂ⏝ࡋ㸪⏕࿨యࡢ඲ㇺࢆಠ▔ࡍࡿࡇ࡜࡛⾲⌧ᆺࢆỴᐃࡍࡿศ Ꮚᶵᵓࡸ㔜せᅉᏊࡢྠᐃࡀ㐍ࡵࡽࢀ࡚࠸ࡿ㸬᳜≀ࡣᅛ╔⏕≀࡛⛣ື࡛ࡁ࡞࠸ࡓࡵ⮬↛⎔ቃ ࡛ࡣᵝࠎ࡞ࢫࢺࣞࢫ࡟᫹ࡉࢀ࡚࠾ࡾ㸪㐍໬ࡢ㐣⛬࡟࠾࠸࡚㑇ఏᏊኚ␗ࡀ㉳ᅉ࡜࡞ࡾ⪏ᛶ࡟ 㛵୚ࡍࡿᶵ⬟ࢆ᭷ࡍࡿ㑇ఏᏊࢆ⋓ᚓࡋ㐺ᛂࡋ࡚ࡁࡓ㸬ࣔࢹ᳜ࣝ≀ࡢࢩࣟ࢖ࢾࢼࢬࢼ

(Arabidopsis thaliana) ࡀ 2000 ᖺ࡟ゎㄞࡉࢀ㸪࢖ࢿ㸪࣑ࣖࢥࢢࢧ (Lotus japonicus)㸪ࣄ

࣓ࢶࣜ࢞ࢿࢦࢣ (Physcomitrella patens) ࡞࡝ᵝࠎ࡞⛉ࡢ᳜≀ࢤࣀ࣒ࡀゎㄞࡉࢀ࡚࠸ࡿ㸬ࡑ ࢀ࡟క࠸ࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ࡸ࣓ࢱ࣮࣒࣎ࣟࢆࡣࡌࡵ࡜ࡋࡓᵝࠎ࡞⎔ቃࢫࢺࣞࢫࡸ᳜≀ ࣍ࣝࣔࣥ࡟ࡼࡗ࡚ኚ໬ࡍࡿ࣮࣑࢜ࢡࢫ᝟ሗࡢ㞟✚ࡀጞࡲࡗ࡚࠸ࡿ㸬≉࡟ࢺࣛࣥࢫࢡࣜࣉࢺ ࣮࣒ࡣDNA ࣐࢖ࢡࣟ࢔ࣞ࢖ἲࡀ㛤Ⓨࡉࢀࡓࡇ࡜࡛㸪㑇ఏᏊⓎ⌧ࡢኚ໬ࢆ⥙⨶ⓗ࡟࡜ࡽ࠼ࡿ ᡭẁࡀ࡛ࡁ㸪≉ᐃࡢ᮲௳ୗ࡟࠶ࡿ᳜≀ࡢ㑇ఏᏊࡢ⥲యⓗ࡞ኚືࡢほᐹ㸪≉ᐃࡢ௦ㅰ⤒㊰ࡢ ኚ໬࡜௚ࡢ௦ㅰ⤒㊰ࡢ㛵㐃ᛶ㸪ࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ࢹ࣮ࢱࡢ⵳✚࡟ࡼࡿ኱つᶍ࠿ࡘ⥙⨶ ⓗ࡞ゎᯒἲ࡟ࡼࡾⓎ⌧ไᚚᶵᵓࡢ᥈⣴ࡸඹⓎ⌧㑇ఏᏊࢿࢵࢺ࣮࣡ࢡゎᯒ㸪ࣉ࣮ࣟࣔࢱ࣮ゎ ᯒࡀྍ⬟࡜࡞ࡗࡓ㸬⌧ᅾ㸪኱㔞ࡢࢹ࣮ࢱࡢ⵳✚࠿ࡽ᝟ሗᇶ┙ࡀᩚഛࡉࢀ࡚࠾ࡾ Gene

Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) ࡸ Array Express (http://www.ebi.ac.uk/arrayexpress/) ࡞࡝ࡢࢹ࣮ࢱ࣮࣋ࢫࡀᵓ⠏ࡉ࡚࠸ࡿ㸬ࢺࣛࣥࢫࢡࣜ ࣉࢺ࣮࣒ࢆ⏝࠸ࡓࣉࣟࣇ࢓࢖ࣜࣥࢢࡣ᳜≀ࡢᵝࠎ࡞ࢫࢺࣞࢫ࡟ᛂ⟅ࡍࡿ㑇ఏᏊࢆ≉ᐃࡍࡿ ࡇ࡜࡟㈉⊩ࡋ࡚࠸ࡿ㸬㠀⏕≀ⓗࢫࢺࣞࢫ࡛ࡣ஝⇱ 㸪ᾐ㏱ᅽ㸪ప (Kreps et al.,2002)㸪㧗 (Swindell et al., 2007)㸪ሷ (Chao et al., 2005)㸪ᙉග (Rossel et al., 2002)㸪⣸እ⥺

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4

(Brown et al., 2005)㸪㔠ᒓ (Herbette et al., 2006; Kumari et al., 2008)㸪ᰤ㣴Ḟஈ (Krapp et al., 2011)㸪ෙỈ (Loreti et al., 2005) ࡞࡝ከᒱ࡟Ώࡗ࡚ゎᯒࡉࢀ࡚࠾ࡾ㸪ࡑࢀࡒࢀࡢࢫ ࢺࣞࢫ࡟ᛂ⟅ࡍࡿ㑇ఏᏊࡀ≉ᐃࡉࢀ࡚࠸ࡿ㸬⏕≀ⓗࢫࢺࣞࢫ࡛ࡣ㸪࢘࢖ࣝࢫ (Postnikova and Nemchinov, 2012)㸪ࣂࢡࢸࣜ࢔ (Mark et al., 2005)㸪⣒≧⳦ (Mathinoni et al., 2011)㸪 ᪻⹸ (Kempema et al.,2007; Misra et al., 2010) ࡞࡝㸪ከᵝ࡞⑓ᐖ⹸ࡸ≀⌮ⓗ㞀ᐖ࡛࠶ࡿ 㣗ᐖ࡟ᑐࡍࡿ᳜≀ࡢ⥙⨶ⓗ࡞㑇ఏᏊᛂ⟅ࡢゎᯒ࠾ࡼࡧ㌿෗ไᚚᶵᵓࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸬ࡲ ࡓࢫࢺࣞࢫࢆ」ྜฎ⌮ࡋࡓࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ࢹ࣮ࢱࢆ༢୍ࢫࢺࣞࢫࡢฎ⌮࡜ẚ㍑ࡋࡓ ࡜ࡇࢁ㑇ఏᏊⓎ⌧ࣃࢱ࣮ࣥࡀ␗࡞ࡿᛂ⟅ࢆ♧ࡍࡇ࡜ࡀุ᫂ࡋ࡚࠸ࡿ (Rasmussen et al., 2013)㸬ࡉࡽ࡟✲ᴟⓗ࡞┠ⓗ࡛࠶ࡿᐇ㝿ࡢ㎰ሙ࡛ࡢ᳜≀ࡢ㑇ఏᏊᛂ⟅ࢆᛕ㢌࡟⨨࠸ࡓゎᯒࡶ ጞࡵࡽࢀ࡚࠸ࡿ (Atkinson and Urwin, 2012)㸬ࡇࡢࡼ࠺࡟ࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ࡣከࡃࡢ

◊✲ࡀ㐍ࡵࡽࢀࡘࡘࡶ㸪࠸ࡲࡔ඲ㇺࡀゎ᫂ࡉࢀ࡚࠸࡞࠸ศ㔝࡛࠶ࡿ㸬㏆ᖺ࡛ࡣRNA-seq ࡟ ࡼࡿ㧗ᗘ࠿ࡘṇ☜࡞㑇ఏᏊⓎ⌧ࣞ࣋ࣝࡢᐃ㔞໬ࢆ⾜࠺ࡇ࡜ࡀྍ⬟࡟࡞ࡾ㸪ࡑࡢཎ⌮࠿ࡽ㠀 ࣔࢹࣝ⏕≀ࡢゎᯒࡸ㑇ఏᏊⓎ⌧ࡢไᚚ࡟㛵㐃ࡍࡿmiRNA ࡢ≉ᐃ࡞࡝㸪኱㔞ࡢࢹ࣮ࢱ࠿ࡽⓎ ⌧ไᚚᶵᵓࡢ⌮ゎࡀ㐍ᒎࡋ࡚࠸ࡿ㸬ከᵝ࡞ࢫࢺࣞࢫ✀ࡀ⎔ቃ୰࡟Ꮡᅾࡍࡿ࡞࠿㸪ᅵተ୰ࡢ ẘᛶ࢖࢜ࣥ㞀ᐖࡲࡓࡣ⫧ᩱ୙㊊࡟ࡼࡿᰤ㣴Ḟஈ࡜࠸ࡗࡓ࣑ࢿࣛࣝࢫࢺࣞࢫࡣ㎰ሙ࡛₯ᅾⓗ ࠿ࡘ኱ࡁ࡞ࢫࢺࣞࢫせᅉ࡜࡞ࡾ࠺ࡿ㸬࣑ࢿࣛࣝࢫࢺࣞࢫ࡟ᑐࡍࡿ࣮࣑࢜ࢡࢫ᝟ሗࡢゎᯒ࠿ ࡽᵝࠎ࡞ࢫࢺࣞࢫᛂ⟅ᛶ㑇ఏᏊࡢ≉ᐃ࠾ࡼࡧࢫࢺࣞࢫ⪏ᛶᶵᵓࡢゎ᫂ࡀ㐍ࡵࡽࢀ࡚࠸ࡿࡀ ඲ㇺࡣ᫂ࡽ࠿࡟࡞ࡗ࡚࠸࡞࠸㸬 ᮏ◊✲࡛ࡣ᰿ᅪ㒊ࡢ࣑ࢿࣛࣝࢫࢺࣞࢫ࡟ᑐࡍࡿࢩࣟ࢖ࢾࢼࢬࢼᆅୖ㒊ࡢศᏊ⏕≀Ꮫⓗኚ ໬ࡢືែࢆDNA ࣐࢖ࢡࣟ࢔ࣞ࢖࡟ࡼࡿࢤࣀ࣒࣡࢖ࢻ࡞㑇ఏⓗ᥈⣴࠾ࡼࡧゎᯒࢆ⾜࠸㸪㌿෗ ኚືࡢᕪ␗ࢆ◊✲࠿ࡽศᏊ⏕⌮Ꮫⓗ࡞ࢫࢺࣞࢫᛂ⟅ࢆࣉࣟࣇ࢓࢖ࣜࣥࢢࡍࡿࡇ࡜࡛ࢩࣟ࢖ ࢾࢼࢬࢼᆅୖ㒊ࡢ᰿ᅪ࣑ࢿࣛࣝࢫࢺࣞࢫ࡟ᑐࡍࡿ㌿෗ኚືࡢẚ㍑ゎᯒࢆ⾜ࡗࡓ㸬ࡲࡓ᭷⏝ ᚤ⏕≀࡟ࡼࡿ⑓ᐖ᢬ᢠᛶࡢㄏᑟᶵᵓࡢゎ᫂ࢆ┠ᣦࡋࡓ㸬ࡉࡽ࡟㌿෗཯ᛂࢆᇶ࡟ྛ࣑ࢿࣛࣝ ࢫࢺࣞࢫᛂ⟅ࡢ㑇ఏᏊⓎ⌧ࣂ࢖࣐࣮࣮࢜࢝ࡢసᡂࢆ┠ⓗ࡜ࡋ࡚◊✲ࢆ⾜ࡗࡓ㸬

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5

➨

1 ❶

ᰤ㣴Ḟஈୗࡢࣔࢹ᳜ࣝ≀ࡢᆅୖ㒊ࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ᛂ⟅ࡢゎᯒ

1-1 ⥴ゝ

๓ୡ⣖࠿ࡽࡢ໬Ꮫ㸪ᕤᴗᢏ⾡ࡢⓎᒎ࡟ࡼࡗ࡚㎰ᴗᶵᲔࡢ㛤Ⓨࡸ໬Ꮫ㎰⸆ࡢ〇㐀࡜࠸ࡗࡓ ㎰ᴗᢏ⾡ࡢᨵ㠉ࡀ㐍ࡳ㸪኱つᶍ࠿ࡘᏳᐃⓗ࡞㎰ᴗࡀྍ⬟࡟࡞ࡿ࡟⮳ࡗࡓ㸬ࡑࡢ୍᪉࡛኱㔞 ⏕⏘ᆺ㎰ᴗࡣᅵተ⫧Ỻᗘࢆⴭࡋࡃᦆ⪖ࡉࡏࡿ㸬໬Ꮫ⫧ᩱࡢᢞධࡣᅵተࡢᰤ㣴ศࡢᅇ᚟ࡋస ≀ࡢᏳᐃⓗ࡞཰✭ࢆࡍࡿࡓࡵ࡟୙ྍḞ࡞せ⣲࡛࠶ࡾ㸪≉࡟❅⣲ (N)㸪ࣜࣥ (P)㸪࣒࢝ࣜ࢘ (K) ࡣస≀ࡢ⏕㛗࡟኱ࡁࡃ㛵ࢃࡿ㔜せ࡞ᰤ㣴⣲࡛࠶ࡿ㸬ୡ⏺ࡢ⫧ᩱࡢ౑⏝㔞ࡣᖺࠎቑຍࡋ࡚࠾ ࡾ㸪FAOSTAT (http://faostat.fao.org/site/291/default.aspx) ࡟ࡼࡿ࡜㸪N ※⫧ᩱࡢᾘ㈝㔞 ࡣ1961 ᖺ࡛ࡣ 1160 ୓ࢺ࡛ࣥ࠶ࡗࡓࡢ࡟ᑐࡋ㸪2006 ᖺ࡛ࡣ 1 ൨ 40 ୓ࢺࣥ࡬࡜⣙ 9 ಸ࡟ቑ ຍࡋ࡚࠸ࡿ㸬ࣜࣥ㓟⫧ᩱࡢᾘ㈝㔞ࡣ1961 ᖺ࡛ࡣ 500 ୓ࢺ࡛ࣥ࠶ࡗࡓࡢ࡟ᑐࡋ㸪2006 ᖺ࡛ ࡣ1800 ୓ࢺࣥ࡬࡜⣙ 3.6 ಸ࡟ቑຍࡋ࡚࠸ࡿ㸬ࡇࡢࡼ࠺࡞ᅜ㝿ⓗ࡞⫧ᩱ౑⏝㔞ࡢቑຍࡸ⫧ᩱ ౯᱁ࡢ㧗㦐ࡣୡ⏺ேཱྀቑຍࡸ୰ᅜ㸪࢖ࣥࢻ࡞࡝ࡢ᪂⯆ᅜࡢ㣗⏕άࡢኚ໬࠾ࡼࡧ㈨※኱㔞ᢞ ධᆺ㎰ᴗ࡬ࡢ⛣⾜࡟⏤᮶ࡍࡿࡶࡢ࡜⪃࠼ࡽࢀࡿ㸬ࡲࡓ㏆ᖺࡢ▼Ἔ౯᱁ࡸ⫧ᩱ౯᱁ࡢ㧗㦐࡟ ࡼࡾ㸪኱㔞ࡢ࢚ࢿࣝࢠ࣮ࡸ⫧ᩱᢞධࢆ⾜ࡗ࡚࠸ࡿ኱つᶍ㎰ᴗ࡛㈨※㠃࠾ࡼࡧ⤒῭㠃࡛㎰ᐙ ࡟኱ࡁ࡞㈇ᢸ࡜࡞ࡗ࡚࠸ࡿ㸬 ໬Ꮫ⫧ᩱࡣ㏆௦㎰ᴗ࡟࠾࠸࡚స≀཰㔞ࢆ㧗ࡵࡿ㔜せ࡞せ⣲࡛࠶ࡿࡀ㸪ඛ㏙ࡢࡼ࠺࡟ᅵተ ୰ࡢవ๫⫧ᩱࡣ⎔ቃ୰࡬ὶฟࡋ⎔ቃởᰁࡢ୍ᅉ࡜࡞ࡗ࡚࠸ࡿ (Schachtman et al., 1998; Raun and Johnson, 1999; Strahm and Harrison, 2006)㸬స≀ࡢ⫧ᩱ⋓ᚓ⋡ࡣ㠀ຠ⋡ⓗ࡛㸪

࠾ࡼࡑ30㹼60㸣࡜ぢ✚ࡶࡽࢀ࡚࠾ࡿ㸬ከࡃࡢඛ㐍ᅜࡸ୰ᅜࡢࡼ࠺࡞㎰ᴗ኱ᅜ࡛ࡣ㎰⪔ᆅ࡬

ࡢ໬Ꮫ⫧ᩱࡸ᭷ᶵ㉁㈨ᮦࡢ㐣๫ᢞධ࡟ࡼࡾ㸪ᅵተ୰ࡢ⫧ᩱᡂศࡢ㐣ᗘ࡞⵳✚࡛⎔ቃ୰ࡢ≀

㉁ᚠ⎔ࢆ኱ࡁࡃᨩ஘ࡋ㎰⪔ᆅࡢⲨᗫ㸪ࡦ࠸࡚ࡣ⎔ቃởᰁࡀ㉳ࡁ࡚࠸ࡿ㸬Ṧ࡟N ὶฟࡣ῝้

࡞⎔ቃၥ㢟ࡢཎᅉ࡜࡞ࡾ㸪ᅵተࡸᆅୗỈởᰁ㸪Ἑᕝ㸪†἟ࡢᐩᰤ㣴໬࡟ࡼࡿ㉥₻࡞࡝ࡢỈ ㉁ởᰁࢆᘬࡁ㉳ࡇࡍ㸬᥹ᩓࡋࡓ࢔ࣥࣔࢽ࢔ࡣᗈᇦ࡟ᣑᩓ㸪ὶฟࡋ࡚⮬↛⏕ែ⣔ࡢ≀㉁ᚠ⎔

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6 ࡟ᝏᙳ㡪ࢆ࠾ࡼࡰࡋ❅⣲ᚠ⎔㐣⛬࡛Ⓨ⏕ࡍࡿள㓟໬❅⣲ (N2O) ࡣᆅ⌫ ᬮ໬ࡸ࢜ࢰࣥᒙ ◚ቯࡢせᅉ࡜࡞ࡗ࡚࠸ࡿ㸬ࡲࡓࣜࣥ㖔▼ࡣ෌⏕୙ྍ⬟࡞㈨※࡛࠶ࡿࡓࡵ㸪ᣢ⥆ྍ⬟࡞㎰ᴗ ࢆ᥎㐍ࡍࡿ࡟࠶ࡓࡾ⦆ຠᛶ⫧ᩱࡸ⿕そ⫧ᩱࡢ౑⏝㸪P ࡢᅇ཰᪉ἲࡀồࡵࡽࢀ࡚࠸ࡿ㸬ࡇࢀࡽ ࡢ⎔ቃၥ㢟ࡀ㢧ᅾ໬ࡋࡓ࡞࠿㸪័⾜ࡢ኱㔞㈨※ᢞධᆺࡢ㎰ᴗ᪉ἲࢆぢ┤ࡋᚠ⎔ᆺ㎰ᴗࢩࢫ ࢸ࣒ࡢ┠ᣦࡋࡓᣢ⥆ᆺ㎰ᴗ࡬ࡢ㌿᥮ࡀᚲせ࡛࠶ࡿ㸬㏆ᖺ◊✲࠾ࡼࡧ฼⏝ࡀ㐍ࢇ࡛࠸ࡿ᳜≀ ᕤሙࡸ⢭ᐦ㎰ᴗ࡟ࡼࡿ┤᥋ⓗ࡞᳜≀ࡢᰤ㣴≧ែࡢ᝟ሗ⟶⌮ࡣ௒ᚋࡢᣢ⥆ⓗ㎰ᴗ࡟࠾࠸࡚ᚲ 㡲࡛࠶ࡾ㸪ࣇ࢕࣮ࣝࢻ࡛ࡢ⪔స࡟࠾࠸࡚ࡶ⎔ቃ㈇ᢸࢆ㍍ῶࡋࡘࡘ᳜≀ࡢᰤ㣴≧ែࢆ೺ᖖ࡟ ಖࡘຠ⋡ⓗ࡞᪋⫧᪉ἲࡀᗈࡃồࡵࡽࢀ࡚࠸ࡿ (Morimoto et al., 1996)㸬 ᅵተ୰ࡢᰤ㣴⣲ࡢືែࡣᅵተ୰ࡢ໬Ꮫⓗᛶ㉁࡜ᰤ㣴ඖ⣲ࡢᙧែ࡞࡝ࡢせ⣲ࡀ」㞧࡟⤡ࡳ ྜࡗ࡚࠾ࡾ㸪స≀ࡢᰤ㣴≧ែࡢ⟶⌮ἲࡣᮍࡔ᏶඲࡛ࡣ࡞࠸㸬ᅵተศᯒࡣ㛫᥋ⓗ࡞᳨ᰝ࡛࠶ ࡿࡓࡵ᳜≀ࡢᰤ㣴≧ែࡑࡢࡶࡢࢆ┤᥋᳨ᰝࡋ࡚࠸ࡿࢃࡅ࡛ࡣ࡞ࡃ㸪᳜≀ࡢᰤ㣴≧ែࢆṇ☜ ࡟ ᐃ࡛ࡁ࡞࠸ၥ㢟Ⅼࡀ࠶ࡿ㸬ࣂ࢖࣐࣮࣮࢜࢝ࡣ⏕యᣦᶆ໬ྜ≀࡜ࡋ࡚⑓⌮Ꮫ࠾ࡼࡧ⸆⌮ Ꮫࢆࡣࡌࡵ࡜ࡋ࡚฼⏝ࡉࢀ࡚࠾ࡾ㸪ࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ࡸ࣓ࢱ࣮࣒࣎ࣟ࡞࡝ࡢ࣮࣑࢜ࢡ ࢫゎᯒࡢ⵳✚ࡉࢀࡓࢹ࣮ࢱᇶ┙ࢆ⏝࠸ே㛫ࡢ⾑ᾮࡸᒀ࡞࡝࠿ࡽᣦᶆ≀㉁ࡢྵ㔞ኚ໬ࢆ ᐃ ࡋ⒴ࢆࡣࡌࡵ࡜ࡋࡓᵝࠎ࡞⑌⑓ࡢ᪩ᮇⓎぢ࡟㈉⊩ࡍࡿᢏ⾡࡜ࡋ࡚☜❧ࡉࢀ࡚࠸ࡿ (Pepe et al., 2001)㸬᳜≀࡟⏝࠸ࡽࢀࡿࣂ࢖࣐࣮࣮࢜࢝ࡣ୺࡟ᆅᒙࡢᖺ௦ ᐃࡢุ᩿ᮦᩱ࡜ࡋ࡚฼⏝ ࡉࢀ࡚࠸ࡿᨺᑕᛶⅣ⣲ࡸ௦ㅰ≀㉁㸪ᕧ኱ศᏊ࡞࡝ࢆ⏝࠸ࡓ⎔ቃ࢔ࢭࢫ࣓ࣥࢺ࡟㝈ᐃࡉࢀ࡚ ࠸ࡿ (Ernst and Peterson, 1994; Hughen et al., 2004) 㸬ࣂ࢖࣐࣮࣮࢜࢝ࢆ⏝࠸ࡓస≀ࡢ 㑇ఏᏊኚືࡢ฼⏝ࡋࡓᰤ㣴Ḟஈデ᩿ࡢࣂ࢖࢜࢔ࢵࢭ࢖ࡣ✚ᴟⓗ࡟ヨࡳࡽࢀ࡚ࡣ࠾ࡽࡎ㸪◊ ✲ሗ࿌࡜ࡋ࡚࢖ࢿࡢ⏕㛗ᮇ㛫ࡢุ᩿ (Tarpley et al., 2005) ࡸࢺ࢘ࣔࣟࢥࢩࡢ N ≧ែ (Yang et al., 2011) 㸪ࣄ࣐࣡ࣜ (Helianthus annuus L.) ࡢỈ≧ែ (Marchand et al., 2013) ࢆホ౯ࡍࡿ㑇ఏᏊⓎ⌧ࣂ࢖࣐࣮࣮࢜࢝ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸬

᳜≀ࡢᰤ㣴⟶⌮ࡢほⅬ࠿ࡽ⫧ᩱࡢ฼⏝ຠ⋡ࢆ࠶ࡆࡿ࡟ࡣ᳜≀⮬యࡢᰤ㣴྾཰࠾ࡼࡧ฼⏝ ຠ⋡ࢆྥୖࡉࡏࡿࡇ࡜ࡀᣲࡆࡽࢀࡿ㸬ࡇࢀࡲ࡛ᵝࠎ࡞◊✲࡛ከ✀ࡢ᳜≀࡛ᙧែᏛ㸪⏕⌮Ꮫ ⓗ㸪⏕໬Ꮫⓗᡭἲ࡟ࡼࡿᗈ⠊࡞ゎᯒࡀ⾜ࢃࢀ㸪ᰤ㣴ඖ⣲ࡢᅵተ࠿ࡽࡢ྾཰㸪㍺㏦㸪௦ㅰᶵ ᵓࡀ᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿ (Maathuis 2009)㸬ࡉࡽ࡟㏆ᖺ࡛ࡣศᏊ⏕≀Ꮫⓗ◊✲ࡢⓎᒎ࡟క

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7

࠸㸪ࡑࢀࡽࡢ࣓࢝ࢽࢬ࣒࡟㛵୚ࡍࡿ㑇ఏᏊࡢⓎ⌧ኚືࡸ㌿෗ไᚚᶵᵓ࡟㛵ࡍࡿ◊✲ࡀ⾜ࢃ ࢀ࡚࠾ࡾ㸪N Ḟஈ (Krapp et al., 2011)㸪P Ḟஈ (Hammond et al., 2003)㸪◲㯤 (S) Ḟஈ (Maruyama-Nakashita et al., 2003) ࡞࡝ࡢᵝࠎ࡞ඖ⣲ࡢᰤ㣴Ḟஈฎ⌮࡟ࡼࡿࢺࣛࣥࢫࢡ ࣜࣉࢺ࣮࣒ゎᯒࡀ㐍ࡵࡽࢀ࡚࠸ࡿ (Gojon et al., 2009)㸬ᮏ❶࡛ࡣ⥙⨶ⓗ࡞㑇ఏᏊⓎ⌧ゎᯒ ࢆ⾜࠼ࡿ࣐࢖ࢡࣟ࢔ࣞ࢖ࢆ⏝࠸N㸪P㸪K Ḟஈฎ⌮ࢆ᪋ࡋࡓࢩࣟ࢖ࢾࢼࢬࢼ࠾ࡼࡧ࢖ࢿᆅୖ 㒊ࡢࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ࡢẚ㍑ゎᯒࢆ⾜࠸㸪N㸪P㸪K ≉␗ⓗᛂ⟅㑇ఏᏊࢆ≉ᐃࡋศᏊ⏕ ⌮Ꮫⓗᛂ⟅ࡢࣉࣟࣇ࢓࢖ࣜࣥࢢࢆ┠ᣦࡋࡓ㸬᭦࡟ᰤ㣴⟶⌮ࢶ࣮ࣝ࡜ࡋ࡚㑇ఏᏊⓎ⌧ࣂ࢖࢜ ࣐࣮࣮࢝ࡢ㛤Ⓨࢆ┠ⓗ࡜ࡋࡓ㸬

(13)

8

11-2 ᮦᩱ࠾ࡼࡧ᪉ἲ

౪ヨ᳜≀

ᮏ ᐇ 㦂 ࡛ ࡣ RIKEN BioResource Center ࡼ ࡾ ධ ᡭ ࡋ ࡓ ࢩ ࣟ ࢖ ࢾ ࢼ ࢬ ࢼ 㔝 ⏕ ᰴ Columbia-0 (Col-0; JA58) ࢆ⏝࠸ࡓ㸬࢖ࢿ✀Ꮚࡣᒱ㜧኱Ꮫᛂ⏝⏕≀⛉Ꮫ㒊ᯇ஭◊✲ᐊࡼࡾ ᥦ౪ࡉࢀࡓ࢖ࢿရ✀ࢥࢩࣄ࢝ࣜࢆ⏝࠸ࡓ㸬 ࢩࣟ࢖ࢾࢼࢬࢼᰤ㣴Ḟஈฎ⌮ Ỉ⪔᱂ᇵࡣToda ࡽ (1999) ࡢࣇ࣮ࣟࢺᘧỈ⪔᱂ᇵἲ࡛⾜ࡗࡓ㸬ࢩࣟ࢖ࢾࢼࢬࢼ✀Ꮚࢆ 4Υ࡛ 3 ᪥㛫྾Ỉฎ⌮ࡋࡓ㸬ࡑࡢࡢࡕࢼ࢖࣓ࣟࣥࢵࢩࣗ [34 mesh㸪3038 (mm)] ࢆᣳࢇ ࡔࢫࣛ࢖ࢻ⏝࣐࢘ࣥࢺ[5050 (mm)] (Fuji Firm㸪Japan) ୖ࡟᧛✀ࡋ㸪1cm ゅࡢⓎἻࢫࢳ ࣮ࣟࣝࢆࡘࡅ㸪2%⃰ᗘࡢ MGRL ⁐ᾮ (Table 1) ࡛᫂ᮇ 12 ᫬㛫㸪ගᗘ (photosynthetic photon flux density) 37 μmol m-2_s-1_㸪_{25Υ࡛㸪7 ᪥㛫Ḟஈฎ⌮࡟⏝࠸ࡿᗂ᳜≀యࡣ 14 ᪥㛫㸪}

2 ᪥㛫Ḟஈฎ⌮࡟⏝࠸ࡿᗂ᳜≀యࡣ 19 ᪥㛫᱂ᇵࡋࡓ㸬⁐ᾮࡢ pH ㄪᩚ࡟ࡣ HCl ࡲࡓࡣ NaOH ࢆ౑⏝ࡋ㸪Horiba pH meter F-52 ࠾ࡼࡧప㟁Ẽఏᑟ⋡⏝ pH 㟁ᴟ (Horiba, Kyoto, Japan)

ࢆ⏝࠸ ᐃ㸪pH 5.6 ࡟ㄪᩚࡋ 2 ᪥ࡈ࡜࡟᭦᪂ࡋࡓ㸬ࡑࡢᚋ㸪Ỉ⪔⁐ᾮࢆᰤ㣴ฎ⌮⁐ᾮ (Table 2) ࡟᭰࠼࡚ᗂ᳜≀యࢆ᱂ᇵࡋࡓ㸬࡞࠾ᰤ㣴Ḟஈฎ⌮࡜ࡋ࡚ᰤ㣴⁐ᾮ࠿ࡽྛඖ⣲ࢆῧຍࡋ࡞ ࠸↓ῧຍࡲࡓࡣ1/4 㔞ᰤ㣴ࢆῧຍࡋࡓỈ⪔ᇵᆅࢆసᡂࡋ㸪᳜≀యࢆ᱂ᇵࡋࡓ㸬N Ḟஈ࡛ࡣ NH4NO3࡜NaNO3ࢆ㸪P Ḟஈ࡛ࡣ NaPO4ࢆ㸪K Ḟஈฎ⌮ࡣ KCl ࢆࡑࢀࡒࢀᢤࡁᰤ㣴Ḟஈ ฎ⌮༊࡜ࡋ࡚౑⏝ࡋࡓ㸬ᰤ㣴Ḟஈฎ⌮ᮇ㛫ࡣࡑࢀࡒࢀ14 ᪥㛫⫱࡚ࡓᗂ᳜≀యࡣ 7 ᪥㛫ࡲࡓ ࡣ19 ᪥㛫⫱࡚ࡓᗂ᳜≀యࡣ 2 ᪥㛫↓ῧຍࡲࡓࡣ 1/4 㔞ῧຍࡋࡓฎ⌮ࢆ⾜ࡗࡓ㸬ࡲࡓ 7 ᪥㛫 ᰤ㣴Ḟஈฎ⌮ࢆ᪋ࡋࡓ᳜≀యࢆ᏶඲ᰤ㣴ฎ⌮ᇵᆅ࡟⛣ࡋ3 ᪥㛫⏕⫱ࡋ࡚㏣⫧ฎ⌮༊࡜ࡋ࡚ ᢅࡗࡓ㸬ྛฎ⌮ࢆࡋࡓ᳜≀యࡢᆅୖ㒊ࢆᅇ཰ࡋ㸪-80Υ෭෾ᗜ࡛ಖᏑࡋࡓ㸬 ࢖ࢿᰤ㣴Ḟஈฎ⌮ ࢖ࢿ✀Ꮚࢆ 10㸣ḟளሷ⣲㓟࡛ 10 ศฎ⌮ࡋ㸪࢖࢜ࣥ஺᥮Ỉ࡛ 3 ᅇὙίࡋࡓࡢࡕࢩ࣮ࣕࣞ

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9 ࡟ᧈࡁ࢖࢜ࣥ஺᥮Ỉ࡟ᾐₕฎ⌮ࡋ30Υ࡛ 3 ᪥㛫ദⱆฎ⌮ࢆ⾜ࡗࡓ㸬ࢺࣜ࢝ࣝࢿࢵࢺ H01 [⥙ ┠ࣆࢵࢳ 44 (mm)] (ࢱ࢟ࣟࣥ) ࡟ⓎἻࢫࢳ࣮ࣟࣝࢆࡘࡅ࡚ࣇ࣮ࣟࢺᘧỈ⪔᱂ᇵἲ࡛᱂ᇵ ࡋࡓ㸬ᰤ㣴ฎ⌮⁐ᾮ࡛14 ᪥㛫᱂ᇵࡋ㸪᳜≀యࡢ⏕⫱≧ែࡀ㏆࠸ࢧࣥࣉࣝࢆ㑅ᢤࡋ㸪ࢩࣟ࢖ ࢾࢼࢬࢼࡢᐇ㦂࡜ྠᵝ࡟ᑐ↷༊࡜ࡋ࡚ᰤ㣴ฎ⌮⁐ᾮ࡛㸪ࡉࡽ࡟N㸪P㸪K ᏶඲Ḟஈᇵᆅ࡛ 7 ᪥㛫ฎ⌮ࡋࡓࡢࡕ㸪࢖ࢿᗂ᳜≀యࡢ➨ 2 ⴥࢆࢧࣥࣉࣜࣥࢢࡋࡓ㸬ࡲࡓ㛗ᮇ㛫ᰤ㣴Ḟஈ≧ែ ࡟᫹ࡉࢀࡓ࢖ࢿᆅୖ㒊࡛ࣂ࢖࣐࣮࣮࢜࢝㑇ఏᏊࡢⓎ⌧ኚືࢆ ᐃࡍࡿࡓࡵ㸪7 ᪥㛫᏶඲ᰤ㣴 ᇵᆅ࡛⏕⫱ࡋࡓ࢖ࢿᗂ᳜≀యࢆྛᰤ㣴ඖ⣲1/4 㔞㸪1/10 㔞㸪᏶඲Ḟஈᇵᆅ࡛➨ 5 ⴥࡀⓎ⏕ ࡍࡿࡲ࡛᱂ᇵࡋ㸪➨2 ⴥࢆࢧࣥࣉࣜࣥࢢࡋࡓᚋ㸪౑⏝ࡍࡿࡲ࡛-80Υ࡛ಖᏑࡋࡓ㸬 R RNA ᢳฟ

Total RNA ࢆ Suzuki ࡽࡢ᪉ἲ࡛ᢳฟࡋࡓ (Suzuki et al., 2003)㸬ᅇ཰ࡋࡓ᳜≀యࢧࣥࣉ ࣝࢆ◚○ᶵ (Shake master Neo, BMS) ࢆ⏝࠸࡚෾⤖ࡉࡏࡓࡲࡲ◚○ࡋࡓ㸬ᢳฟ buffer [100 mM Tris-HCl (pH6.8)㸪25 mM EDTA㸪2% (w/v) CTAB㸪1.4 M NaCl㸪5% (v/v) 2-mercaptoethanol] 700 μl ຍ࠼ᠱ⃮ࡋࡓ㸬ࡇࢀࢆ࡛㐲ᚰศ㞳ࡋ (13,000 rpm㸪ᐊ 㸪5 min) 㸪

ୖΎࢆ 1.5 ml ࢳ࣮ࣗࣈ࡟⛣ࡋ࢖࣮ࣥ࢟ࣗ࣋ࢱ࣮࡛ 65Υ㸪5 min ฎ⌮ࡋࡓ㸬ୖΎ࡟

chloroform/isoamylalcohol (24:1) 700 μl ࢆຍ࠼㸪ᡭ࡛⃭ࡋࡃᨩᢾࡋ㸪㐲ᚰศ㞳 (13,000 rpm㸪 4 Υ㸪5 min) ࡋࡓ㸬ୖΎࢆ 1.5 ml ࢳ࣮ࣗࣈ࡟⛣ࡋ㸪ࡇࡢୖΎ࡟ᑐࡋ࡚ 1 vol ࡢࣇ࢙ࣀ࣮ ࣝMix [3.5% (w/v) GTC㸪200 mM NaOAc (pH 4.0) ࢆỈ㣬࿴ࣇ࢙ࣀ࣮࡛࣓ࣝࢫ࢔ࢵࣉ] ࢆ ຍ࠼㸪vortex ࡋ 5 min ᐊ ࡛㟼⨨ࡋࡓᚋ㸪㐲ᚰศ㞳 (14,000 rpm㸪4Υ㸪5 min) ࡋࡓ㸬Ỉ ᒙࢆ1.5 ml ࢳ࣮ࣗࣈ࡟⛣ࡋ㸪ຍ࠼ࡓࣇ࢙ࣀ࣮ࣝ Mix ࡢ 0.5 vol ࡢ CHL/isoamylalcohol ࢆ ຍ࠼ᡭ࡛⃭ࡋࡃᨩᢾࡋࡓᚋ㸪㐲ᚰศ㞳 (14,000 rpm㸪4 Υ㸪5 min) ࡋỈᒙࢆ RNase free ࡢ

ࢳ࣮ࣗࣈ࡟⛣ࡋࡓ㸬࡞࠾ࡇࢀ௨㝆ࡢ᧯సࡣ඲࡚RNase free ࡢࣆ࣌ࢵࢺ࣐ࣥࡢࢳࢵࣉ㸪ࢳࣗ

࣮ࣈ㸪ヨ⸆ࢆ⏝࠸ࡓ㸬ࡇࡢỈᒙ࡟ᑐࡋ࡚0.7 vol ࡢ isopropanol (SIGMA) ࢆຍ࠼ᐊ ࡛ 10

min ᨺ⨨ࡋ㸪㐲ᚰศ㞳 (15,000 rpm㸪4Υ㸪15 min) ࡋ࡚ୖΎࢆྲྀࡾ㝖࠸ࡓ㸬75㸣 ࢚ࢱࣀ ࣮ࣝ 200 μl ࡛࣌ࣞࢵࢺࢆὙίࡋ㸪㐲ᚰศ㞳 (15,000 rpm㸪4Υ㸪5 min) ࡋ࡚ୖΎࢆ㝖ཤࡋ

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10

ྜ࠺㐺ᙜ㔞ࡢRNase free water (SIGMA) ࡟⁐࠿ࡋࡓ㸬

࣐࢖ࢡࣟ࢔ࣞ࢖࡟౪ࡍࡿ RNA ヨᩱࡣΰධࡋ࡚࠸ࡿከ⢾ࢆ㝖ཤࡍࡿࡓࡵ࡟ሷ໬ࣜࢳ࣒࢘

(LiCl) ฎ⌮ࢆ⾜ࡗࡓ㸬30 μl ࡟ㄪᩚࡋࡓ RNA ⁐ᾮ࡟ᑐࡋ 8 M LiCl ࢆ 10 μl ຍ࠼㸪-80 Υ ୍࡛ᬌฎ⌮ࡋࡓ㸬㐲ᚰศ㞳 (15,000 rpm, 4 Υ, 30 min) ࡋ㸪LiCl ⁐ᾮࢆྲྀࡾ㝖ࡁ㸪75 %࢚

ࢱࣀ࣮ࣝࢆ150 μl ຍ࠼㸪㐲ᚰศ㞳 (15,000 rpm, 4 Υ, 5 min) ࢆ⾜ࡗࡓ㸬࢚ࢱࣀ࣮ࣝࢆ㝖

ཤࡋࡓࡢࡕ㸪RNase-free water 50 μl ࢆຍ࠼ࡓ㸬ࡲࡓ 3 M 㓑㓟ࢼࢺ࣒ࣜ࢘ 5 μl㸪isopropanol 50 μl ࢆධࢀ㸪ᐊ ࡛ 10 min 㟼⨨ࡋࡓ㸬㐲ᚰศ㞳 (15,000 rpm, 4 Υ, 15 min) ࡋ㸪75 %࢚

ࢱࣀ࣮ࣝࢆ150 μl ຍ࠼㸪㐲ᚰศ㞳 (15,000 rpm, 4 Υ, 5 min) ࡋࡓ㸬࢚ࢱࣀ࣮ࣝࢆ᏶඲࡟

㝖ཤࡋ㸪㐺ᙜ㔞ࡢRNase-free water ࡟⁐࠿ࡋࡓ㸬

ᐃ㔞ⓗRT-PCR ࡟౪ࡍࡿ RNA ヨᩱࡣࢤࣀ࣒ DNA ࢆ㝖ཤࡍࡿࡓࡵ࡟ DNase ฎ⌮ࢆ⾜ࡗ

ࡓ㸬25 μl ࡟ㄪᩚࡋࡓ RNA ⁐ᾮ࡟ᑐࡋ㸪2.5 μl ࡢ 10DNase I ࣂࢵࣇ࢓࣮ (Takara Bio Inc.)㸪 1 μl ࡢ DNase I (5 U/μl㸪Takara Bio Inc.)㸪0.5 μl ࡢ RNase inhibitor (40 U/μl㸪TOYOBO)㸪 2 μl ࡢ DEPC ฎ⌮Ỉࢆຍ࠼ 37Υ࡛ 60 min ฎ⌮ࡋࡓ㸬ࡇࡢ཯ᛂ⁐ᾮ࡟ RNase free water ࢆ250 μl㸪Ỉ㣬࿴ࣇ࢙ࣀ࣮ࣝ 150 μl㸪chlroform/isoamylalcohol 150 μl ࢆຍ࠼㌿ಽΰ࿴ࡋ ࡓ㸬㐲ᚰศ㞳ࡋ࡚ (ᐊ 㸪15,000 rpm㸪5 min) ୖᒙࢆ᪂ࡋ࠸ࢳ࣮ࣗࣈ࡟⛣ࡋࡓ㸬㞟ࡵࡓỈ

ᒙࡢ1/10 vol ࡢ 3 M 㓑㓟ࢼࢺ࣒ࣜ࢘ (pH 5.2) ࡜Ỉᒙ࡜➼㔞ࡢ isopropanol ࢆຍ࠼࣎ࣝࢸ

ࢵࢡࢫࡋ㸪ᐊ ࡛10 min 㟼⨨ࡋࡓ㸬㐲ᚰศ㞳 (4 Υ㸪15,000 rpm㸪15 min) ࡋ࡚ RNA ỿ

Ẋࢆᅇ཰ࡋࡓ㸬ୖΎࢆ㝖ཤࡋ㸪75 % ࢚ࢱࣀ࣮ࣝ 150 μl ࡛ RNA ỿẊࢆὙίࡋ㸪㐲ᚰศ㞳 (4 Υ㸪16,000 rpm㸪3 min) ࡋࡓࡢࡕ㸪࢚ࢱࣀ࣮ࣝࢆ᏶඲࡟㝖ཤࡋ㸪ࡇࡢ࣌ࣞࢵࢺࢆ 30 μl

ࡢRNase free water ࡟⁐ゎࡋࡓ㸬ࡇࡢ RNA ⁐ᾮࢆ⏝࠸࡚ cDNA ࢆసᡂࡍࡿࡓࡵ㏫㌿෗཯

ᛂࢆ⾜ࡗࡓ㸬1 μg ࡢ RNA ࢆ 1.5 ml ࢚ࢵ࣌ࣥࢻࣝࣇࢳ࣮ࣗࣈ࡟⛣ࡋ㸪཯ᛂ⁐ᾮࡀ 11 μl ࡟

࡞ࡿࡼ࠺RNase free water ࢆຍ࠼ࡓࡢࡕ 70Υ㸪10 min ฎ⌮ࡋࡓᚋ㸪ị෭ࡋࡓ㸬཯ᛂ⁐ᾮ

࡟Oligo dT primer [5’-TTTTTTTTTTTTTTTTTv(A/C/G)-3’] 4 μl㸪10 mM dNTP 2 μl㸪 RNase inhibitor 0.5 μl㸪RTase ࢆຍ࠼㸪TaKaRa PCR Thermal Cycler Dice (Takara Bio Inc.) ࡛ (42Υ/60min㸪99Υ/5 min)࡛཯ᛂࡉࡏࡓ㸬ࡇࡢ཯ᛂ⁐ᾮ࡟ 1/4 TE ࢆ 280 μl ຍ࠼ᐃ

(16)

11

(GE Healthcare, Tokyo, Japan) ࡟ࡼࡗ࡚ィ ࡋࡓ㸬A260ࡢ್࡛RNA ⃰ᗘ㸪A260/A280࡛ࢱ

ࣥࣃࢡ㉁㸪A260/A230࡛ከ⢾ࡢΰྜᗘྜࢆุ᩿ࡋࡓ㸬

࣐

࣐࢖ࢡࣟ࢔ࣞ࢖࠾ࡼࡧࢹ࣮ࢱゎᯒ

࣐ ࢖ ࢡ ࣟ ࢔ ࣞ ࢖ ࢳ ࢵ ࣉ ࡣ Agilent Arabidopsis 4 Oligo Microarray Chip (Agilent Technologies, Tokyo, Japan) ࢆ⏝࠸㸪Cyanine-3 (Cy3) ࡟ࡼࡿ 1 Ⰽἲ࡛⾜࠸㸪Agilent in-situ ࢜ࣜࢦ DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ࣉࣟࢺࢥ࣮ࣝ ver 2.3 ࡟ᚑࡗ࡚⾜ࡗࡓ㸬ᑐ↷༊࠾ࡼࡧྛ

࢖࢜ࣥࢫࢺࣞࢫฎ⌮༊ࡢ⢭〇ࡋࡓ 1 μg RNA ࢆ⏝࠸࡚㸪Cy3 ࡛ࣛ࣋ࣝࡋࡓ CTP (Perkin

Elmer/NEN Life Sciences, Tokyo, Japan) ࢆ⏝࠸࡚ cRNA ࢆྜᡂࡋࡓ㸬ࣁ࢖ࣈࣜࢲ࢖ࢮ࣮

ࢩࣙࣥࢆ⾜ࡗࡓ DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ࢢࣛࢫࢫࣛ࢖ࢻࡣ Agilent Scan Control Software

(Version 6.1) ࢆ⏝࠸࡚ࢫ࢟ࣕࣥࡋ㸪ᮏ◊✲࡛ࡣ෌⌧ᛶࢆ㧗ࡵࡿࡓࡵ㸪3 㐃ࡢ⊂❧ࡋࡓ཯᚟ ࢆ⏝࠸㸪ᚓࡽࢀࡓ࢖࣓࣮ࢪࡢࢩࢢࢼࣝᙉᗘࡣAgilent Feature Extraction software (Version 9.5.1) ࡛ᩘ್໬ࡋࡓ㸬ࡉࡽ࡟ࡇࡢࢹ࣮ࢱࢆ GeneSpring 7.3.1 ࡟ࡼࡗ࡚㑇ఏᏊࡢⓎ⌧㔞ࢆほ ᐹࡋࡓ㸬࣐࢖ࢡࣟ࢔ࣞ࢖ࡢ⏬ീࢹ࣮ࢱࡢࢡ࢜ࣜࢸ࢕ࢳ࢙ࢵࢡ࡜ࡋ࡚㸪flags ࡛ 3 㐃ࡢᐇ㦂⣔ ࡛ࡦ࡜ࡘ࡛ࡶAbsent ࡜ุ᩿ࡉࢀࡓ㑇ఏᏊࢆ᤼㝖ࡋࡓ㸬ྛ࣐࢖ࢡࣟ࢔ࣞ࢖ᐇ㦂⣔࡛Ⓨ⌧ㄏᑟ 㑇ఏᏊࡣ㸪ࡑࢀࡒࢀࡢᰤ㣴Ḟஈฎ⌮ࢆ⾜ࡗࡓྛ࣐࢖ࢡࣟ࢔ࣞ࢖࡛Ⓨ⌧ㄏᑟ㑇ఏᏊୖ఩2.5㸣 ࢆ᥇⏝ࡋ㸪ࢹ࣮ࢱࢭࢵࢺ࡜ࡋ࡚ゎᯒ࡟⏝࠸ࡓ㸬 ᐃ㔞ⓗRT-PCR ゎᯒ

DNase ฎ⌮࠾ࡼࡧ㏫㌿෗཯ᛂࢆ⾜ࡗࡓ cDNA ࢆ SYBR Premix Ex Taq II (Takara Bio Inc.) ࢆ౑⏝ࡋ㸪Thermal Cycle Dice®_{Real Time System II ࠾ࡼࡧ Software Version 4.02}

(Takara Bio Inc.) ࢆ⏝࠸㸪ゎᯒᡭἲࡣ࣐ࢽࣗ࢔ࣝ࡟ᚑ࠸㑇ఏᏊࡢⓎ⌧ゎᯒࢆ⾜ࡗࡓ㸬ゎᯒ

࡟ࡣ3 㐃ࡢ⊂❧ࡋࡓࢧࣥࣉࣝࢆ౑࠸㸪ࣁ࢘ࢫ࣮࢟ࣆࣥࢢ㑇ఏᏊ࡜ࡋ࡚UBQ1ࢆ⏝࠸㸪㌿෗

㔞ࢆṇつ໬ࡋࡓ㸬࡞࠾ᮏゎᯒ࡟౑⏝ࡋࡓࢩࣟ࢖ࢾࢼࢬࢼ࠾ࡼࡧ࢖ࢿ⏝ࡢࣉࣛ࢖࣐࣮㓄ิࡣ ࡑࢀࡒࢀTable 3㸪Table 4 ࡟グ㍕ࡋࡓ㸬

(17)

12 ᅵ ᅵ⪔ヨ㦂 ᅵࡣⶶ⋤ࡼࡾ᥇ྲྀࡋࡓ〓Ⰽ᳃ᯘᅵ (୰ᛶᅵተ) ࢆ⏝࠸ࡓ㸬Toda ࡽ (1999) ࡢᅵ⪔ヨ㦂ࢆ ᇶ࡟㸪ᑐ↷༊ࡢᅵተ࡜ࡋ࡚㸪100g ࡢᅵተ࡟ᑐࡋ࡚ KCl 0.048 g㸪 MgSO4 0.036 g㸪(NH4)2SO4 0.132 g㸪Na2H2PO4 0.132 g㸪CaCO3 0.15 g ࢆஙᲬ࡛☻ࡾ₽ࡋ⢊≧࡟ࡋࡓࡢࡕ㸪ࡇࢀࡽࡢ ᰤ㣴ሷࢆᅵ࡬ῧຍࡋᅵ࡟㥆ᰁࡲࡏࡿࡓࡵ1 㐌㛫ΰྜࡋࡓ㸬ࡲࡓྛᰤ㣴ඖ⣲Ḟஈฎ⌮༊࡜ࡋ ࡚㸪N Ḟஈฎ⌮༊ࡣ (NH4)2SO4㸪P Ḟஈฎ⌮༊ࡣ NaH2PO4 㸪K Ḟஈฎ⌮༊ࡣ KCl ࡢῧຍ 㔞ࢆᑐ↷༊࡜ẚ㍑ࡋ࡚1/4 㔞㸪1/16 㔞㸪↓ῧຍࡢฎ⌮༊ࢆタ⨨ࡋࡓ㸬4Υ࡛ 3 ᪥㛫྾Ỉฎ⌮ ࡋࡓࢩࣟ࢖ࢾࢼࢬࢼ✀Ꮚࢆᅵተ࡟ࣆ࣌ࢵࢺ࣐࡛ࣥྛᰤ㣴ฎ⌮༊࡟30 ⢏᧛✀ࡋ㸪ᅵተ⾲㠃ࡀ ஝⇱ࡋ࡞࠸ࡼ࠺࢖࢜ࣥ஺᥮Ỉࢆࣆ࣌ࢵࢺ࣐࡛ࣥῧຍࡋ࡚28 ᪥㛫⏕⫱ࡋࡓ㸬 ࢖ࢿᆅୖ㒊ࡢN㸪P㸪K Ḟஈࣂ࢖࣐࣮࣮࢜࢝㑇ఏᏊࡢ᥈⣴ ArrayExpress ࡼࡾ Takehisa ࡽ (2015) ࡢ࢖ࢿᆅୖ㒊ࡢᰤ㣴Ḟஈࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ ࢹ࣮ࢱ (http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-66935/) ࢆධᡭࡋゎᯒ ࡟⏝࠸ࡓ㸬Control ẚ࡟ᑐࡋ࡚ 1/4㸪1/16㸪1/64 㔞ࡢྛ୕㐃ࡢᰤ㣴Ḟஈࢹ࣮ࢱࢆp < 0.05㸪

Intensity > 500㸪 FC > 4.0 (up-regulation) ࡲࡓࡣ FC < -4.0 (down-regulation)㸪 ࡛ࣇ࢕ ࣝࢱࣜࣥࢢࢆ⾜࠸㸪㑇ఏᏊࢆ≉ᐃࡋࡓ㸬

(18)

13 Element Concentration NaPO4 35 μM NaNO3 80 μM MgSO4 30 μM CaCl2 200 μM KNO3 60 μM Na2EDTA 1.34 μM FeSO4 172 nM MnSO4 206 nM H3BO3 600 nM ZnSO4 20 nM (NH4)6Mo7O24 0.48 nM CoC12 2.6 nM CuSO4 20 nM Element Concentration NH4NO3 0.5 mM NaNO3 1.0 mM Na-PO4 0.8 mM KCl 0.8 mM CaCl2 0.6 mM MgSO4 0.5 mM Fe-EDTA 0.05 mM H3BO3 50 uM MnSO4 9.0 uM CuSO4 0.3 uM ZnSO4 0.7 uM (NH4)6Mo7O24 0.1 uM

Table 1. Concentrations of nutrients in modified MGRL medium

(19)

14 Name Sequence (5'-) F GGAACACGGTCACTTCTGG R AAGAGCGTTTTCTCCGTTC F AGATGCAGGACGACTCATGG R CTGACTTTGCGGGTTTACC F CTGTGGAGTTGGAGCAGACA R CCAGCCAATTCTGTGATCC F GCCGGTCAACATCATTGTC R GAAAGGTGATGACCGATCC F AGCACTTACTCCACCGCAG R ATGCCGCATATCACAGGAAC F TGTTCCGGGACGAAGTAGTG R GGGCAAAAGCTTCCAGTACC F CCAACAATGCATGGATCAG R CCCGTGTCTTGCACTTTTC F TTCTTCCCTCGAAAGCTCAA R AGGCCCACCAGAGTGTATGA F CCGCTTGACCAGTCTTTAGC R TTTGAACGGTTCGTCTCCTC F GATCGAGGATCCAAATCGTG R TTGTTGGGTCAAAGCACAGA F TCTCTCTCGTGACGCTTTTC R ACCACGAAATCATCTCTCG F ACGGAGAACACGATGTACACAA R ATTGCATGCATGAACCATTG F GCGCTACCACGAAAAAGAAG R AGGTTCAAAACCGTGGTGAG F GCGCTACCACGAAAAAGAAG R AGGTTCAAAACCGTGGTGAG F CAAATTTGGAGGGGTTGATG R ATCATTCCCGTAAGCAACC F CACGCACATCCACTCACA R TGAGCCCCTTCCTCCTTTA UBQ1 F TCGTAAGTACAATCAGGATAAGATG (internal std.) R CACTGAAACAAGAAAAACAAACCCT At2g18660

Table 3. Sequences of primers used for quantitative RT-PCR of the

nutrient deficinecy biomarker in Arabidopsis. At5g15500 At1g73040 At5g16570 At1g80130 At5g23020 At4g11910 At1g03545 At2g14610 At4g23700 At1g14880 At1g21310 At1g51800 At3g38200 At2g26695 At2g44370

(20)

15

Name Sequence (5'-) Sequence (5'-)

Os01g0136100 F CGAGGAACACACAACAAACAAG R TGGTTGCTCTATCTTTCGC Os01g0155800 F CTTTCTGCCCTGCTACACCTAC R CTGCCTGCATGCACTTTA Os02g0276200 F CTGTTTGGTCAGCCATAGG R GATCTTTCCTTGGCAATAGGTG Os02g0503700 F GGGCAATGTCTCTGCTACTA R ATTGTAGCTTCGGGAGGTCA Os02g0719600 F GAGCAGGTGAAGACCAAGTACC R CACACACTGGCACGCTACTTA Os02g0782500 F TCGCCTGAATCATAGCGTAG R GAAACACGAACAGAGCACAAAC Os12g0231000 F GGGAACATTCCCACTATGC R GTAGAAGCGCTTGATGTAGG Os03g0764600 F CTCTGGGCTTCTCAGGAAG R CTGTCTAGCAAGATTCCACGA Os06g0594400 F CACACCAAGCTCAGACAAGA R CTCGTTGTAACCCATGATGC Os04g0679400 F GACTCCAATAACACGCTCATC R GAATGGAATGGAGGTGAGGA Os03g0431200 F CAGAACACGTGCTAAGCAG R GCAGTATACCCATCAACGAAG Os09g0294000 F CAACCTTGTGCATAAAGCTCAG R GAAGCATCGCTCTCAGGTTAC Os12g0503000 F GGCGAGATCATGTATCTTGTG R GGAGTAGTCCAGGTAGGTGTGC Os03g0738600 F CATTGCTGGAGAACATCTACG R CCTCGTAGAGCTTGAGGATGTC Os06g0242000 F GGTGAAACTATTCCAAGAGC R CTCCCACAAAATGTCAGTACC Os12g0548700 F CATCATCAGTTTGATCGTGTCC R TGTCCTTGAGGATCACCTTC Os11g0655900 F CGCGCTCTGGCTCTAGTGTA R ACTTCACCATTACACCACTCG Os03g0699700 F CACACCTGGTGAATTTGACTC R GCAGGTCCTTGATAAGTTGGAC Os08g0150700 F GAAGAAGGGCGTCTACCTC R CCTCCTCAAACCTGGAGAG Os06g0606700 F CGCAGCTAAGGTGTAGTCG R GAACATACTTGGCTGTGGC Os03g0806600 F CCGTTCCAGATGTATGCTG R CATCGTAGTAGCCACCAAATG Os10g0550900 F CATCCCGTACCTCATCAGAC R GTCTTCTGCCACCTCACTTTC Os09g0547500 F GATCATCAACGAGAGGATGC R GCAATAACCAGAGCACGTAC Os10g0552400 F GAGGAAAGAACTGGAAGAAGC R GATACTCTTGCAGCGGCTTA Os07g0258400 F GTAAGGAAACTGGAGGTTGTGG R GAATAGATTGTGCGGCATTAC Os07g0677500 F GCACGTATAAGACGATGGAGTG R GAAACAGAGCAAGGCAAATC Os03g0725200 F GCTGCTGGTCTTCTTGAAC R GAGCGGCAATATCATAGCAAAC Os07g0539900 F GAGTCCCATGATCGACTTC R GCCAAATACAGCATAATCACG Os04g0178300 F CTCCGACTTTACGGATACCAAG R AGTGTTGTACATTGGGGTGAC Os04g0578600 F AGGGACGAGAATCGTGATC R CTTCAGCAAGTGCAGGTAAATG

Os04t0394100 F CTCAAGGCATGTTGAGAGC R CCTTTCGTCAGGAAGTACAC Os11g0658900 F CACACCGAGGTAATAGGGTC R GCAACTTTAGACCTTGGATTGG Os10g0515900 F GAAGACGACGAACAAAGAGACC R GAGCAGAGCTTGATGATCTCG Os10g0513400 F GCACGATTTTCTCCTGTACCTC R CCGGACTGTTGAATTTGTTTC Os01g0525700 F CGTTTGTACATAGAGGCCAACA R CACTTGGTTCATCGTATTTGC

Os05g0505900 F GTCAGTGTTGTTGTCATTGTCG R CTCAATTCTCTCCTCGATCTC Os03g0718100

(house-keeping gene)

Table 4. Sequences of primers used for quantitative RT-PCR of the nutrient deficinecy biomarker in rice.

(21)

16

11-3 ⤖ᯝ

ࢩࣟ࢖ࢾࢼࢬࢼᆅୖ㒊ࡢN㸪P㸪K Ḟஈ≉␗ⓗᛂ⟅㑇ఏᏊࡢ≉ᐃ

ࢩࣟ࢖ࢾࢼࢬࢼ᳜≀యࢆ14 ᪥㛫Ỉ⪔᱂ᇵ⣔࡛⏕⫱ࡋ㸪ྛᰤ㣴Ḟஈฎ⌮ᇵᆅ࡛᱂ᇵࡋࡓᚋ㸪

ࡑࡢ⏕㛗ࢆほᐹࡋࡓ (Fig. 1)㸬ࡇࡢᆅୖ㒊ࢆᅇ཰ࡋ㸪Total RNA ࢆᢳฟࡋ࡚࣐࢖ࢡࣟ࢔ࣞ࢖

࡟౪ࡋࡓ㸬࣐࢖ࢡࣟ࢔ࣞ࢖ࢹ࣮ࢱࡼࡾࣇ࢕ࣝࢱࣜࣥࢢ࡛㑅ᢤࡉࢀࡓ2 ᪥㛫Ḟஈฎ⌮ㄏᑟ㑇 ఏᏊ (N Ḟஈ㸸55 㑇ఏᏊ㸪P Ḟஈ㸸177 㑇ఏᏊ㸪K Ḟஈ㸸126 㑇ఏᏊ) ࠾ࡼࡧ 7 ᪥㛫Ḟஈ ฎ⌮ㄏᑟ㑇ఏᏊ (N Ḟஈ㸸97 㑇ఏᏊ㸪P Ḟஈ㸸119 㑇ఏᏊ㸪K Ḟஈ㸸112 㑇ఏᏊ) ࠿ࡽ㸪ྛ ᰤ㣴Ḟஈ࡟≉␗ⓗᛂ⟅ࢆ♧ࡍ㑇ఏᏊࢆ≉ᐃࡍࡿࡓࡵ࡟2 ᪥㛫ࡲࡓࡣ 7 ᪥㛫ฎ⌮࡛Ⓨ⌧ኚື ࡍࡿ㑇ఏᏊ⩌ࢆ࣋ࣥᅗ࡛ゎᯒࡋࡓ㸬2 ᪥㛫ฎ⌮࡛ࡣ N Ḟஈฎ⌮༊࡛ 51 㑇ఏᏊ㸪P Ḟஈฎ⌮ ༊࡛103 㑇ఏᏊ㸪K Ḟஈฎ⌮༊࡛ࡣ 51 㑇ఏᏊࡀ≉ᐃࡉࢀ (Fig. 2A)㸪7 ᪥㛫ฎ⌮࡛ࡣ N Ḟ ஈฎ⌮༊࡛73 㑇ఏᏊ㸪P Ḟஈฎ⌮༊࡛ 89 㑇ఏᏊ㸪K Ḟஈฎ⌮༊࡛ࡣ 87 㑇ఏᏊࡀ≉ᐃࡉࢀ ࡓ (Fig. 2B)㸬ྛᰤ㣴Ḟஈ≉␗ⓗᛂ⟅࡜ุ᩿ࡉࢀࡓ㑇ఏᏊ⩌ࢆ⏝࠸࣋ࣥᅗ࡟ࡼࡗ࡚ゎᯒࡋࡓ ࡜ࡇࢁ㸪N Ḟஈฎ⌮༊࡛ 15 㑇ఏᏊ㸪P Ḟஈฎ⌮༊࡛ 8 㑇ఏᏊ㸪K Ḟஈฎ⌮༊࡛ 5 㑇ఏᏊ≉ ᐃࡋࡓ (Fig. 2C)㸬N Ḟஈฎ⌮࡛≉ᐃࡉࢀࡓ㑇ఏᏊࡣ N ௦ㅰ࡜ࡋ࡚GLN1;4 (GLUTAMINE

SYNTHETASE 1;4) ࡀྵࡲࢀ࡚࠸ࡓ㸬ࡲࡓ஧ḟ௦ㅰ⏘≀࡟㛵୚ࡍࡿ㑇ఏᏊUF3GTࡸPAP1

(PURPLE ACID PHOSPHATASE 1) ࡀྵࡲࢀ࡚࠸ࡓ㸬P Ḟஈฎ⌮࡛ࢫࢺࣞࢫᮇ㛫࡟ඹ㏻

ࡋ࡚ㄏᑟࡉࢀࡿ㑇ఏᏊ࡜ࡋ࡚IPS2ࡸ⑓ᐖᛂ⟅ᛶ㑇ఏᏊPR1㸪࢝ࢳ࢜ࣥࢺࣛࣥࢫ࣏࣮ࢱ࣮

CHX17 (CATION/H+_{EXCHANGER 17}₎ _{ࡀྵࡲࢀ࡚࠸ࡓ㸬K Ḟஈฎ⌮࡛ࡣ}_At1g51800

ࡸAt2g44370࡜࠸ࡗࡓ⑓ᐖ᢬ᢠࢩࢢࢼࣝ࡟㛵୚ࡍࡿ㑇ఏᏊࡀྵࡲࢀ࡚࠸ࡓ㸬

N Ḟஈฎ⌮࡟ࡼࡿ≉␗ⓗⓎ⌧ኚື㑇ఏᏊ

2 ᪥㛫ࡢ N Ḟஈฎ⌮࡟ࡼࡗ࡚ࢩࣟ࢖ࢾࢼࢬࢼᆅୖ㒊࡛Ⓨ⌧ㄏᑟࡉࢀࡓ N ࢺࣛࣥࢫ࣏࣮ࢱ ࣮ 㑇 ఏ Ꮚ ࡜ ࡋ ࡚ 㸪NRT2:1 (NITRATE TRANSPORTER 2:1) ࡸ UPS1 (UREIDE

PERMEASE 1 (UPS1)㸪MtN21 /EamA-like transporter family㸪N ௦ㅰ㓝⣲㑇ఏᏊ࡜ࡋ࡚

(22)

17

Figure 1. Phenotypes of Arabidopsis seedlings grown in several nutrient content conditions by

hydroponics. Seedlings were grown for 14 or 19 days on control condition (2% MGRL, pH5.6) and transferred each nutrient starvation treatment for 7 or 2 days. Recover treatment was performed transferred to control condition for 3 days after nutrient deficiency treatment for 7 days. Left number represents adding each amount of a nutrient content in solution compared with control.

(23)

18

Figure 2. Venn diagram showing the upregulated genes under nutrient deficiency condition in

Arabidopsis shoots. The comparison was performed using the expression level of (A) 2 days (B) 7 days. The numbers in the intersections represent the extent overlap among three analyses. Genes were selected if the fold change value was in the upper 2.5% of quality-controlled spots in each microarray experiment after 2 days or 7 days treated with N, P or K free solution. (C) Comparison of the upregulated genes affected by the nutrient deficiency treatment in Arabidopsis shoots. Venn diagram showing overlap between genes upregulated for 2 days and 7 days.

(24)

19

5-O-glucosyltransferase ࡞࡝஧ḟ௦ㅰ⏘≀࡟㛵୚ࡍࡿ㑇ఏᏊ⩌ࡀⓎ⌧ୖ᪼ࡋ࡚࠸ࡿࡇ࡜ࡀ

☜ ㄆ ࡉ ࢀ ࡓ 㸬 ௚ ࡟╔ ┠ࡍ ࡿ 㑇 ఏ Ꮚ ࡜ ࡋ࡚ ALLANTOINASE (ALN) ࡸ HXXXD-type

acyl-transferase-like proteinࡀ」ᩘྵࡲࢀ࡚࠸ࡓ㸬7 ᪥㛫ࡢḞஈฎ⌮࡛Ⓨ⌧ㄏᑟࡉࢀࡓ㑇 ఏᏊ⩌࡛ࡣ㸪N ㍺㏦࠾ࡼࡧ௦ㅰ࡟㛵ࢃࡿ㑇ఏᏊ࡜ࡋ࡚ NRT2.5 ࡸGLN1;4 ࡀ≉ᐃࡉࢀࡓ㸬 ࡲࡓ UDP-glucose:flavonoid 3-o-glucosyltransferase ࡞࡝ࡢ஧ḟ௦ㅰ⤒㊰㛵㐃㑇ఏᏊࡀⓎ ⌧ㄏᑟࡉࢀ㸪஧ḟ௦ㅰ⏘≀㛵㐃㑇ఏᏊࡢⓎ⌧ไᚚ࡟㛵ࢃࡿ㌿෗ᅉᏊMYB75ࡀᙉㄏᑟࡉࢀ࡚ ࠸ࡓ㸬 P P Ḟஈฎ⌮࡟ࡼࡿ≉␗ⓗⓎ⌧ኚື㑇ఏᏊ 2 ᪥㛫ࡢ P Ḟஈฎ⌮࡟ࡼࡗ࡚≉␗ⓗⓎ⌧ㄏᑟࡍࡿ㑇ఏᏊ⩌࡟㸪P Ḟஈᛂ⟅㑇ఏᏊ࡜ࡋ࡚௒ ࡲ࡛࡟ሗ࿌ࡉࢀ࡚࠸ࡿ㑇ఏᏊ࡛࠶ࡿPHOSPHATE-RESPONSIVE 1ࡸINDUCED BY PI STARVATION 2 (IPS2) ࡀྵࡲࢀ ࡚࠸ࡓ㸬ࡲࡓ P ࢺ ࣛࣥࢫ࣏࣮ࢱ࣮㑇ఏᏊ࡜ࡋ ࡚ PHOSPHATE TRANSPORTER 1;1 (PHT1;1) ࡀྠᐃࡉࢀࡓ㸬ࡇࡢ㑇ఏᏊ⩌࡟ࡣ⑓ᐖᛂ⟅

ᛶ 㑇 ఏ Ꮚ ࡛ ࠶ ࡿ PATHOGENESIS-RELATED (PR) gene ࡸ DEFENSIN-LIKE PROTEIN 208㸪SAR (systemic acquired resistance) regulator protein NIMIN-1-likeࡀ

ྵࡲࢀ࡚࠸ࡓ㸬άᛶ㓟⣲㝖ཤ⣔㑇ఏᏊ࡜ࡋ࡚ peroxidaseࡸ GSTࡀᙉㄏᑟࡉࢀ࡚࠸ࡿࡇ࡜

ࡀ☜ㄆ࡛ࡁࡓ㸬P Ḟஈ࡛Ⓨ⌧ㄏᑟࡉࢀࡓ㌿෗ᅉᏊࡣ WRKY8㸪WRKY18㸪WRKY51㸪

WRKY60㸪RAP2-9ࡀ᳨ฟࡉࢀࡓ㸬7 ᪥㛫ࡢ P Ḟஈฎ⌮Ⓨ⌧ㄏᑟ㑇ఏᏊ⩌࡟ࡣ P Ḟஈᛂ⟅

㑇ఏᏊ PHOSPHATE STARVATION-INDUCED PROTEIN 2 (PS2 )࠾ࡼࡧPS3ࡸP ࢺࣛ

ࣥࢫ࣏࣮ࢱ࣮㑇ఏᏊPHT1;4ࡸ PHO1;H1ࡀྵࡲࢀ࡚࠸ࡓ㸬ࡲࡓ஧ḟ௦ㅰ⏘≀⏕⏘㓝⣲㑇

ఏᏊPAP14 ࡸ⑓ᐖ᢬ᢠᛶ㛵㐃㑇ఏᏊ PR1 ࡸ PR5㸪࢝࢖ࢿ࣮ࢫ㑇ఏᏊ MAPKKK19ࡀྠ

ᐃࡉࢀࡓ㸬

K Ḟஈฎ⌮࡟ࡼࡿ≉␗ⓗⓎ⌧ኚື㑇ఏᏊ

2 ᪥㛫ࡢ K Ḟஈฎ⌮࡛ࡣ K ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࢆࢥ࣮ࢻࡋ࡚࠸ࡿ㑇ఏᏊࡣ᳨ฟࡉࢀ࡞࠿ࡗ

(25)

20

ࡋ㸪ࢩࢢࢼࣝఏ㐩ࡢᙺ๭ࢆᯝࡓࡍ Ca2+ _{࡟㛵㐃ࡍࡿ㑇ఏᏊ࡜ࡋ࡚} _{calcium-binding protein}

࡛࠶ࡿCML38ࡸCa2+_{-transporting ATPase}_{ࡀྠᐃࡉࢀࡓ㸬ࡲࡓ}_{N ㍺㏦ࢺࣛࣥࢫ࣏࣮ࢱ࣮}

WR3 (nitrate transmembrane transporter) ࡸ N ௦ㅰ࡟㛵୚ࡍࡿ Class I glutamine amidotransferase-like superfamily ࡀ᳨ฟࡉࢀࡓ㸬7 ᪥㛫ࡢḞஈฎ⌮࡛ࡣ㸪K ࢺࣛࣥࢫ࣏

࣮ࢱ࣮㑇ఏᏊ࡛࠶ࡿ HAK5 ࡢⓎ⌧ㄏᑟࡀ☜ㄆ࡛ࡁࡓ㸬ຍ࠼࡚ N ௦ㅰ㓝⣲㑇ఏᏊ࡛࠶ࡿ

GLUTAMATE DECARBOXYLASE 1 (GAD1) ࡸNITRATE REDUCTASE 1 (NIA1) ࡀⓎ

⌧ㄏᑟࡋ࡚࠸ࡓ㸬

ᰤ

ᰤ㣴Ḟஈᛂ⟅㑇ఏᏊࡢGO ゎᯒ

N 㸪 P 㸪 K Ḟ ஈ ࡟ ࡼ ࡿ Ⓨ ⌧ ㄏ ᑟ 㑇 ఏ Ꮚ ࢆ ≉ ᐃ ࡋ ࡓ ࡢ ࡕ 㸪 ࡇ ࡢ 㑇 ఏ Ꮚ ⩌ ࢆ agriGO (http://bioinfo.cau.edu.cn/agriGO/) (Du et al., 2010)ࡢ͆Analysis tool” ࡛͆biological process” ࡟╔┠ࡋ࡚ゎᯒࢆ⾜ࡗࡓ (Fig. 3)㸬N Ḟஈㄏᑟ㑇ఏᏊ⩌࡟ࡣ 2 ᪥㛫ฎ⌮࡛ࡣ ͆secondary metabolic process͇㸪͆response to chemical stimulus͇㸪͆cellular amino acid and derivative metabolic process͇㸪͆response to stress͇ࡀ㸪7 ᪥㛫ฎ⌮࡛ࡣ͆carbohydrate biosynthetic process͇ࡀྠᐃࡉࢀࡓ㸬P Ḟஈㄏᑟ㑇ఏᏊ⩌࡟ࡣ 2 ᪥㛫ฎ⌮࡛ࡣ͆defense response͇㸪͆response to stress͇㸪͆response to stimulus͇㸪͆response to oxidative stress͇㸪 ͆response to bacterium͇㸪͆defense response to fungus͇㸪͆response to other organism͇㸪 ͆response to biotic stimulus͇㸪͆regulation of defense response͇㸪͆regulation of response to stress͇㸪͆response to fungus͇㸪͆multi-organism process͇㸪͆defense response to bacterium͇㸪͆response to chemical stimulus͇㸪͆regulation of response to stimulus͇㸪 ͆response to salicylic acid stimulus͇㸪͆immune response͇㸪͆immune system process͇㸪 ͆innate immune response͇ࡀ㸪7 ᪥㛫ฎ⌮࡛ࡣ͆cellular response to phosphate starvation͇㸪͆response to nutrient levels͇㸪͆response to extracellular stimulus͇㸪 ͆cellular response to starvation͇㸪͆response to starvation͇㸪͆cellular response to nutrient levels͇㸪͆cellular response to extracellular stimulus͇㸪͆response to external

(26)

21 F

Figure 3. GO term enrichment analysis of nutrient deficient-inducible gene group in Arabidopsis shoot using the agriGO tool.

(27)

22

response to nutrient levels͇㸪͆cellular response to extracellular stimulus͇㸪͆response to external stimulus͇㸪“response to stress”㸪“response to stimulus͇㸪͆cellular response to stimulus͇㸪͆cellular response to stress͇㸪͆defense response͇ࡀྠᐃࡉࢀࡓ㸬N Ḟஈㄏᑟ

㑇ఏᏊ⩌࡟ࡣ 2 ᪥㛫ฎ⌮࡛ࡣ᭷ព࡞ GO ᶵ⬟ࡣᏑᅾࡏࡎ㸪7 ᪥㛫ฎ⌮࡛㸪͆response to

stimulus͇㸪͆response to chemical stimulus͇㸪͆response to organic substance͇㸪͆response to stress͇㸪͆response to oxidative stress͇ࡀྠᐃࡉࢀࡓ㸬

ࢩ ࢩࣟ࢖ࢾࢼࢬࢼᰤ㣴Ḟஈࣂ࢖࣐࣮࣮࢜࢝࡟ࡼࡿࢩࣟ࢖ࢾࢼࢬࢼࡢᰤ㣴≧ែࡢホ౯ ௒ᅇࡢᐇ㦂࡛ࡣྛᰤ㣴Ḟஈฎ⌮ࢆ2 ᪥㛫ࡲࡓࡣ 7 ᪥㛫⾜ࡗࡓ᳜≀యࡢࢺࣛࣥࢫࢡࣜࣉࢺ ࣮࣒ࢹ࣮ࢱࢆ⏝࠸ࡓࡀ㸪▷ᮇ࠾ࡼࡧ㛗ᮇฎ⌮࡛ඹ㏻ࡋ࡚ㄏᑟࡉࢀࡿ㑇ఏᏊࡣࢫࢺࣞࢫᮇ㛫 ࡟㛵ࢃࡽࡎⓎ⌧ㄏᑟࡉࢀࡿ㑇ఏᏊ࡛࠶ࡿ࡜࠸࠼ࡿ㸬ࡇࢀࡽࡢ㑇ఏᏊࢆ⏝࠸㸪ࢩࣟ࢖ࢾࢼࢬ ࢼ࡛ᰤ㣴≧ែࢆホ౯ࡍࡿ㑇ఏᏊⓎ⌧ࣂ࢖࣐࣮࣮࢜࢝࡜ࡋ࡚฼⏝࡛ࡁࡿ࠿ᐇ㦂ࢆ⾜ࡗࡓ㸬Ỉ ⪔᱂ᇵ࡛ࢩࣟ࢖ࢾࢼࢬࢼࢆ14 ᪥㛫᱂ᇵࡋࡓࡢࡕᰤ㣴Ḟஈฎ⌮࡜ࡋ࡚ᑐ↷༊࡜ẚ㍑ࡋ࡚ྛᰤ 㣴⣲ࢆ1/4 㔞ῧຍࡋࡓฎ⌮༊࠾ࡼࡧ↓ῧຍฎ⌮༊࡛ 7 ᪥㛫⏕⫱ࡋࡓ㸬ࡇࡢࢩࣟ࢖ࢾࢼࢬࢼᆅ ୖ㒊ࡼࡾcDNA ࢆྜᡂࡋᐃ㔞ⓗ RT-PCR ࡛ࣂ࢖࣐࣮࣮࢜࢝㑇ఏᏊࡢⓎ⌧㔞ࢆ ᐃࡋࡓ (Fig. 4)㸬Ỉ⪔࡟ࡼࡿࢫࢺࣞࢫฎ⌮࡛ࡣ N ࠾ࡼࡧ P Ḟஈฎ⌮ᛂ⟅ࣂ࢖࣐࣮࣮࢜࢝ࡀ㧗࠸෌⌧ᛶࢆ ♧ࡍ࡜࡜ࡶ࡟㏣⫧ฎ⌮࡛Ⓨ⌧㔞ࡣᑐ↷༊࡜ྠ➼࡟ᡠࡗ࡚࠸ࡿࡇ࡜ࡀ☜ㄆ࡛ࡁࡓ㸬୍᪉࡛ K Ḟஈฎ⌮ᛂ⟅ࣂ࢖࣐࣮࣮࢜࢝ࡣK ㏣⫧࡟ࡼࡿᅇ᚟ᛶࡀప࠸ࡲࡓࡣ㐜࠸ࡇ࡜ࡀุ᫂ࡋࡓ㸬 ḟ࡟ᅵ⪔ࢆ⏝࠸ࡓᰤ㣴Ḟஈฎ⌮ୗ࡟࠾ࡅࡿࢩࣟ࢖ࢾࢼࢬࢼᆅୖ㒊ࡢࣂ࢖࣐࣮࣮࢜࢝㑇ఏ ᏊࡢⓎ⌧ኚືࢆ ᐃࡋࡓ㸬ᅵ⪔࡛28 ᪥㛫⏕⫱ࡋࡓ N ࡲࡓࡣ P Ḟஈฎ⌮࡟ࡼࡿ㑇ఏᏊⓎ⌧ ࡣỈ⪔᱂ᇵ࡟ࡼࡗ࡚⏕⫱ࡋࡓࢩࣟ࢖ࢾࢼࢬࢼࡢࢹ࣮ࢱ࡜┦ఝࡍࡿ⤖ᯝ࡜࡞ࡗࡓ (Fig. 6)㸬 ࢖ࢿᆅୖ㒊ࡢᰤ㣴Ḟஈ㑇ఏᏊⓎ⌧ࣂ࢖࣐࣮࣮࢜࢝ࡢ᳨ド ࢩࣟ࢖ࢾࢼࢬࢼ࡛≉ᐃࡋࡓ㌿෗ࣂ࢖࣐࣮࣮࡛࢜࢝ࡣ᳜≀యࡢᰤ㣴≧ែࢆホ౯࡛ࡁࡿࡇ࡜ࡀ ุ᫂ࡋࡓ㸬ḟ࡟ࢩࣟ࢖ࢾࢼࢬࢼࡢ᪉ἲࢆᇶ࡟࢖ࢿ࡛ࣂ࢖࣐࣮࣮࢜࢝㑇ఏᏊࡢ≉ᐃࡀ࡛ࡁࡿ ࠿ࢆヨࡳࡓ㸬ࡲࡎỈ⪔᱂ᇵ⣔ࢆ⏝࠸ࡓᰤ㣴Ḟஈᇵᆅ࡛⏕⫱ࡋࡓ࢖ࢿᆅୖ㒊ࡢ⾲⌧ᆺࢆほᐹ

(28)

23

Figure 4. Expression patterns of specific biomarker genes induced by N, P and K deficiency

condition using qRT-PCR. Seedlings were grown in control condition for 10 days and transferred control (Ctrl) treatment and each element content of (A), (B), (C) quarter or free (D), (E), (F) in (A), (D) -N, (B), (E) -P and (C), (F) -K for 7 days and “Re” indicates resupply treatment for 2 days after stress treatment for 7 days. Date is derived for three biological replications. Relative expression patterns were calculated using levels of each genes transcripts normalized by housekeeping gene,

UBQ1. Means ± SE were calculated from three biological replicates. Different letters indicate

(29)

24

(30)

25

Figure 5. Phenotypes of seedlings grown under nutrient deficiency condition. Seedlings were grown

for 28 days under control and each nutrient starvation treatment on neutral forest brown soil. Left number represents adding each amount of bottom nutrient content in soil compared with control soil.

(31)

26

Figure 6. QuantitativeRT-PCR analysis of gene expression biomarkers under nutrient deficiency

conditions in soil. Plant were grown for 28 days on several nutrition content soil. (A) and (D) N deficnecy, (B), (E) P deficiency (C), (F) K deficiency treatment. (A), (B), (C) quarter content and (D), (E) and (F) free soils. Relative expression patterns were calculated using levels of each genes transcripts normalized by housekeeping gene, UBQ1. Means ± SE were calculated from three biological replicates. Different letters indicate significant differences 5% level by Tukey test. Means ± SE are shown.

(32)

27

(33)

28 ࡋࡓ㸬ྛᰤ㣴Ḟஈ᮲௳࡛⏕⫱ࡋࡓ࢖ࢿ᳜≀యࡣ㸪N Ḟஈฎ⌮༊࡛ 1/4 ᇵᆅ࠾ࡼࡧ 1/10 ᇵᆅ ࡛ࡣ኱ࡁ࡞⏕⫱ᕪࡣぢࡽࢀ࡞࠿ࡗࡓࡀ㸪᏶඲ᇵᆅ࡛᱂ᇵࡋࡓᑐ↷༊࡜ẚ㍑ࡋ࡚ᆅୖ㒊ࡢ⏕ ⫱ࡀ㜼ᐖࡉࢀ࡚࠸ࡓ (Fig. 7)㸬ࡲࡓ P ࠾ࡼࡧ K Ḟஈฎ⌮༊࡛࢖ࢿ᳜≀యࡢ⏕㛗㜼ᐖࢆほᐹ ࡍࡿࡇ࡜ࡣ࡛ࡁ࡞࠿ࡗࡓ୍᪉࡛ྛᰤ㣴ࡢῧຍ㔞ࢆῶᑡࡉࡏࡓᇵᆅ࡛⏕⫱ࡋࡓ࢖ࢿࡣ᏶඲ᇵ ᆅ࡛⏕⫱ࡋࡓ࢖ࢿ࡜ẚ㍑ࡋ࡚⏕㛗ࡢ㜼ᐖࡀ☜ㄆ࡛ࡁࡓࡀ㸪N㸪P㸪K ࡑࢀࡒࢀࡢඖ⣲Ḟஈฎ ⌮࡜ࡢぢศࡅࡀࡘࡁ࡟ࡃ࠸ࡇ࡜ࡀศ࠿ࡗࡓ㸬 ࢖ࢿᆅୖ㒊ࡢᰤ㣴Ḟஈࡢ࣐࢖ࢡࣟ࢔ࣞ࢖ࢹ࣮ࢱࢆゎᯒࡋ㸪Ⓨ⌧ㄏᑟ࠾ࡼࡧᢚไ㑇ఏᏊ࠿ ࡽࣂ࢖࣐࣮࣮࢜࢝㑇ఏᏊࢆ≉ᐃࡋࡓ㸬Ⓨ⌧ㄏᑟࡉࢀࡓ㑇ఏᏊ࡜ࡋ࡚ 1/4 㔞㸪1/16 㔞㸪1/64 㔞ࡢN Ḟஈฎ⌮༊࠿ࡽࡑࢀࡒࢀ 54㸪69㸪88 㑇ఏᏊ㸪P Ḟஈฎ⌮༊࠿ࡽࡑࢀࡒࢀ 234㸪282㸪 249 㑇ఏᏊ㸪K Ḟஈฎ⌮༊࠿ࡽࡑࢀࡒࢀ 22㸪74㸪65 㑇ఏᏊࢆᢳฟࡋࡓ㸬ࡲࡓⓎ⌧ᢚไࡉࢀ ࡓ㑇ఏᏊ࡜ࡋ࡚㸪1/4㸪1/16㸪1/64 㔞ࡢྛ N Ḟஈฎ⌮༊࠿ࡽࡑࢀࡒࢀ 48㸪184㸪198 㑇ఏᏊ㸪 P Ḟஈฎ⌮༊࠿ࡽࡑࢀࡒࢀ 82㸪81㸪77 㑇ఏᏊ㸪K Ḟஈฎ⌮༊࠿ࡽࡑࢀࡒࢀ 13㸪21㸪19 㑇 ఏᏊࡀ≉ᐃࡉࢀࡓ㸬ࡇࢀࡽࡢ㑇ఏᏊ⩌࡛ྛᰤ㣴⃰ᗘ࡟㛵ࢃࡽࡎඹ㏻ࡋ࡚Ⓨ⌧ኚືࡍࡿ㑇ఏ Ꮚࢆ≉ᐃࡍࡿࡓࡵ࣋ࣥᅗ࡟ࡼࡿゎᯒࢆ⾜ࡗࡓ㸬ࡑࡢ⤖ᯝ㸪Ⓨ⌧ㄏᑟ㑇ఏᏊ࡛ࡣN Ḟஈฎ⌮ ࡛30 㑇ఏᏊ㸪P Ḟஈฎ⌮࡛ 167 㑇ఏᏊ㸪K Ḟஈฎ⌮࡛ 13 㑇ఏᏊࡀ≉ᐃࡉࢀࡓ (Fig. 8)㸬 ࡇࢀࡽࡢ㑇ఏᏊ⩌ࡢGO ࢔ࣀࢸ࣮ࢩࣙࣥࡢศ㢮ࢆ agriGO ࡛⾜ࡗࡓ (Fig. 9)㸬N ḞஈⓎ⌧ㄏ

ᑟ㑇ఏᏊ⩌࡛ࡣ͆response to stress͇࡜͆response to stimulus͇ࡀ㸪N ḞஈⓎ⌧ᢚไ㑇ఏ Ꮚ⩌࡟ࡣN ࡟㛵୚ࡋ࡚࠸ࡿ͆cellular nitrogen compound metabolic process͇㸪͆cellular nitrogen compound biosynthetic process͇㸪͆nitrogen compound metabolic process͇㸪 ͆cellular biosynthetic process ͇㸪͆ biosynthetic process ͇㸪͆ metabolic process ͇㸪 ͆nucleobase, nucleoside, nucleotide and nucleic acid metabolic process͇ࡀྵࡲࢀ࡚࠸ࡓ㸬 P ḞஈⓎ⌧ㄏᑟ㑇ఏᏊ⩌࡛ࡣ N ࡟㛵୚ࡍࡿ͆cellular nitrogen compound biosynthetic process͇㸪͆cellular nitrogen compound metabolic process͇㸪Ⰽ⣲ྜᡂ࡟㛵୚ࡍࡿ͆pigment biosynthetic process͇ࡸ͆pigment metabolic process͇ ୍᪉࡛ P ḞஈⓎ⌧ᢚไ㑇ఏᏊ⩌ ࡟ࡶ͆cellular nitrogen compound metabolic process͇ࡀྵࡲࢀ࡚࠸ࡓ㸬

(34)

29

Figure 7. Rice shoot (Koshihikari) growth under nutrient starvation treatments. Plants were grown

by 2% MGRL medium for 14 days and transplanted from various nutrient concentrations on Nutrient medium for 21 days.

(35)

30

Figure 8. Venn diagram summarizing the up- or downregulated genes to nutrient deficiency in rice

shoots. Genes were filtered by FC > 4.0 or < 1/4, Intensity > 500 and p < 0.05 in Array data from Takehisa et al 2015.

(36)

31

(37)

32 ࢖

࢖ࢿᆅୖ㒊࡟࠾ࡅࡿྛᰤ㣴ඖ⣲Ⓨ⌧ㄏᑟ࠾ࡼࡧᢚไ㑇ఏᏊࡢゎᯒ

N ≉␗ⓗ࡟Ⓨ⌧ㄏᑟࡉࢀ࡚࠸ࡿ࡜ุ᩿ࡉࢀࡓ㑇ఏᏊ⩌ࡢᶵ⬟ࢆㄪᰝࡋࡓ࡜ࡇࢁ㸪஝⇱⪏ ᛶ࡟㛵㐃ࡍࡿRESPONSIVE TO ABA GENE 16A (RAB16A) ࡸDehydrin DHN1, HSPࡀ

≉ᐃ࡛ࡁࡓ㸬ࡲࡓN ࢺࣛࣥࢫ࣏࣮ࢱ࣮㑇ఏᏊ࠾ࡼࡧ N ௦ㅰ㛵㐃㓝⣲ࡣࡇࡢ㑇ఏᏊ⩌࡟ࡣᏑ

ᅾࡋ࡞࠿ࡗࡓ㸬P Ḟஈ࡟ࡼࡗ࡚≉␗ⓗ࡟Ⓨ⌧ㄏᑟࡉࢀࡿ㑇ఏᏊ⩌࡛ࡣ㸪P Ḟஈᛂ⟅㑇ఏᏊ Phosphate starvation response regulator-like proteinࡸSimilar to Phi-1 protein㸪⑓ᐖᛂ

⟅ᛶ㑇ఏᏊ Thaumatin ࡸࢪࣕࢫࣔࣥ㓟ㄏᑟ㑇ఏᏊ Os12g0198700㸪യᐖᛂ⟅ᛶ㑇ఏᏊ

WOUND-INDUCED PROTEASE INHIBITOR 1 (WIP1)㸪㔜㔠ᒓ㍺㏦࡟㛵୚ࡍࡿ㑇ఏᏊ

NAS3 (NICOTIANAMINE SYNTHASE 3) ࡀྠᐃࡉࢀࡓ㸬ࡲࡓࡇࡢ㑇ఏᏊ⩌ࡶ N Ḟஈྠ

ᵝ࡟transporter family ࡸ௦ㅰ࡟㛵ࢃࡿ㓝⣲㑇ఏᏊ⩌ࡣ☜ㄆ࡛ࡁ࡞࠿ࡗࡓ㸬K Ḟஈ࡛≉␗

ⓗ࡟Ⓨ⌧ㄏᑟࡉࢀࡿ㑇ఏᏊ⩌ࡣᑡ࡞ࡃ㸪Mn ࢺࣛࣥࢫ࣏࣮ࢱ࣮ OsNRAMP2 ࡸ⑓ᐖᛂ⟅ᛶ

㑇ఏᏊOsPR1bࡸThaumatin-like proteinࡀྵࡲࢀ࡚࠸ࡓ㸬

ḟ࡟㑇ఏᏊⓎ⌧ࡀᢚไࡉࢀࡓ㑇ఏᏊ⩌ࡢᶵ⬟ࢆほᐹࡋࡓ㸬N Ḟஈ࡟ࡼࡗ࡚Ⓨ⌧ᢚไࡉࢀ

࡚࠸ࡿ㑇ఏᏊ⩌࡟ࡣN ௦ㅰ࡟㛵ࢃࡿAsparagine synthetaseࡸ࢔ࢡ࢔࣏ࣜࣥOsTIP2;2ࡀ

᳨ฟࡉࢀࡓ㸬P Ḟஈᢚไ㑇ఏᏊ⩌࡛ࡣ K ࢺࣛࣥࢫ࣏࣮ࢱ࣮OsHAK1ࡸhigh-affinity urea

transporter ࡛࠶ࡿOsDUR3ࡀྵࡲࢀ࡚࠸ࡓ㸬K Ḟஈ࡟ࡼࡿⓎ⌧ᢚไ㑇ఏᏊ⩌࡛ࡣⓎ⌧ㄏ

ᑟ㑇ఏᏊ࡜ྠᵝ࡟⃰ᗘ࡟౫Ꮡࡏࡎඹ㏻ࡋ࡚Ⓨ⌧ᢚไࡍࡿ㑇ఏᏊࡣᑡ࡞ࡃ㸪Ferritin ࡸ

CYP450 family㸪OsVIT2࡛ᵓᡂࡉࢀ࡚࠸ࡓ㸬

ᰤ㣴Ḟஈୗࡢ࢖ࢿᆅୖ㒊ࡢ㑇ఏᏊⓎ⌧ࣂ࢖࣐࣮࣮࢜࢝ࡢホ౯ ࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ゎᯒࢆᇶ࡟ᰤ㣴⃰ᗘ࡟㛵ࢃࡽࡎⴭࡋࡃⓎ⌧ㄏᑟࡲࡓࡣᢚไࡉࢀࡿ 㑇ఏᏊ⩌ࡼࡾⓎ⌧ㄏᑟୖ఩ࡲࡓࡣⓎ⌧ᢚไୗ఩ 6 㑇ఏᏊࢆ㑅ᢤࡋ㸪㑇ఏᏊⓎ⌧ࣂ࢖࣐࣮࢜ ࣮࢝࡜ࡋ࡚௨ୗࡢゎᯒ࡟⏝࠸ࡓ㸬ᵝࠎ࡞ᰤ㣴⃰ᗘ᮲௳ୗ࡛⏕⫱ࡋࡓ࢖ࢿᆅୖ㒊࡛ྛᰤ㣴Ḟ ஈ≧ែ᫬࡟ࣂ࢖࣐࣮࣮࢜࢝㑇ఏᏊࡀ࣐࢖ࢡࣟ࢔ࣞ࢖࡜ྠᵝ࡞㌿෗ኚ໬ࡢഴྥ࡟࠶ࡿ࠿㸪ࡲ ࡓ㏣⫧ฎ⌮࡟ࡼࡗ࡚Ⓨ⌧㔞ࡀᑐ↷༊࡜ྠ⛬ᗘ࡟ᅇ᚟ࡍࡿ࠿ࢆ᳨ドࡍࡿࡓࡵ࡟ᐃ㔞ⓗ RT-PCR ࡟ࡼࡿゎᯒࢆ⾜ࡗࡓ㸬21 ᪥㛫ᰤ㣴ᇵᆅ࡛⏕⫱ࡋࡓࡢࡕ㸵᪥㛫᏶඲Ḟஈᇵᆅ࡛⏕⫱

(38)

33 ࡋࡓ࢖ࢿ᳜≀య࡛ࡣ㸪N ࠾ࡼࡧ P Ḟஈ࡟ࡼࡿⓎ⌧ㄏᑟࡲࡓࡣⓎ⌧ᢚไ㑇ఏᏊࡣࡑࢀࡒࢀ࣐ ࢖ࢡࣟ࢔ࣞ࢖ࢹ࣮ࢱ࡜ྠᵝࡢ㌿෗ᛂ⟅ࢆ♧ࡋ㸪㏣⫧ฎ⌮༊࡛ࡣⓎ⌧㔞ࡀᑐ↷༊࡜ྠ➼࡟ᅇ ᚟ࡋ࡚࠸ࡿࡇ࡜ࡀ☜ㄆ࡛ࡁࡓ (Fig. 10)㸬ḟ࡟㛗ᮇ㛫ᰤ㣴ῧຍ㔞ࢆᑡ࡞ࡃࡋࡓᇵᆅ࡛⏕⫱ࡋ ࡓ࢖ࢿ࡛ࡶ㸪ඛࡢᐇ㦂࡜ྠᵝ࡞⤖ᯝࢆ♧ࡍࡇ࡜ࡀุ᫂ࡋࡓ (Fig. 11)㸬࣐࢖ࢡࣟ࢔ࣞ࢖ࢹ࣮ ࢱࡢᐇ㦂⣔࡜ࡣ␗࡞ࡿ2 ✀ࡢᐇ㦂⣔࡛ N ࠾ࡼࡧ P Ḟஈฎ⌮༊ࡢ࢖ࢿ࡛ࣂ࢖࣐࣮࣮࢜࢝㑇ఏ ᏊࡢⓎ⌧ࡣ㧗࠸≉␗ᛶ࠾ࡼࡧ㧗࠸ឤᗘࢆ♧ࡋ㸪ࡲࡓ㏣⫧ฎ⌮࡟ࡼࡗ࡚Ⓨ⌧㔞ࡣᑐ↷༊࡜ྠ ➼⛬ᗘ࡟ᅇ᚟ࡋ࡚࠸ࡓ㸬

(39)

34 F

Figure 10. Transcript abundances of gene expression biomarkers in response to macronutrient deficiency in rice. Hydroponic culture-grown seedlings for 21 days were exposed to each nutrition condition for 7 days and then extracted RNA from 2rd leaf. qRT-PCR analysis was carried out in triplicate. Expression levels were normalized to the Actin1 transcripts. Relative expression values (stress/control) are expressed. Different letters denote significant differences (Tukey test, P < 0.05).

(40)

35

(41)

36 F

Figure 11. Gene expression of biomarkers are specific for each macronutrient. Biomarker expression values are from qRT-PCR assays of eight biomarker genes. Plants were grown by 1/50 MGRL solution for 1 weeks and were transferred 1/4, 1/10 and free nutrient solution for 7 days. V2 leaf stage then were sampled to extract total RNA. Relative expression patterns were calculated using levels of each genes transcripts normalized by housekeeping gene, Actin1. Means ± SE were calculated from three biological replicates. Different letters indicate significant differences 5% level by Tukey test.

(42)

37 F

(43)

38

11-4 ⪃ᐹ

N㸪P㸪K ࡣ᳜≀࡟᭱ࡶせồࡉࢀࡿඖ⣲࡛࠶ࡾ㸪㎰ᴗ࡛ࡣ⫧ᩱ࡜ࡋ࡚స≀ࡢရ㉁࠾ࡼࡧ཰

✭㔞ࢆᕥྑࡍࡿ㔜せᅉᏊ࡛࠶ࡿ㸬ศᏊ⏕≀Ꮫᢏ⾡ࡢⓎᒎ࡜ඹ࡟㸪ࡇࢀࡽ 3 ඖ⣲࡟㛵ࢃࡿ㌿

෗ᶵᵓࡢゎᯒࡣ㐍ࡵࡽࢀ࡚࠾ࡾᵝࠎ࡞᳜≀✀࡛ࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ゎᯒࡀ㐍ࡵࡽࢀ࡚࠸ ࡿ (N: Jeuffroy et al., 2001; Kraiser et al., 2011, Krap et al., 2013, P: Hammond et al., 2003; Calderon-Vazquez et al., 2008; Hammond et al., 2003; Misson et al., 2005; Wu et al., 2003; Secco et al., 2013)㸬ࡋ࠿ࡋ࡞ࡀࡽ㸪N㸪P㸪K ࡟㝈ࡗ࡚ࡶᆅୖ㒊࡟࠾ࡅࡿ㌿෗ไ ᚚ࡟㛵ࢃࡿ⤫ྜⓗ࡞ゎᯒࡣ⾜ࢃࢀ࡚࠾ࡽࡎ㸪ࡑࡢ࣓࢝ࢽࢬ࣒ࡢ඲ㇺࡣゎ᫂ࡉࢀ࡚࠸࡞࠸㸬 ࡲࡓࢫࢺࣞࢫ⊂⮬ࡢ㌿෗ᛂ⟅࡛࠶ࡿ࠿ࡶ୙࡛᫂࠶ࡾ㸪ᮏ◊✲ࡣྛᰤ㣴ඖ⣲࡟ᑐࡍࡿ⊂⮬ࡲ ࡓࡣᬑ㐢ⓗ࡞᳜≀ࡢ㌿෗ᛂ⟅ࡢ⌮ゎ࡜ゎ᫂࡟㈉⊩ࡍࡿ◊✲࡜ࡋ࡚⾜ࡗࡓ㸬 N Ḟஈ࡟ࡼࡿࢩࣟ࢖ࢾࢼࢬࢼᆅୖ㒊ࡢ㑇ఏᏊⓎ⌧ࡢኚືࡢゎᯒ ࡇࢀࡲ࡛ࡢ◊✲࠿ࡽN Ḟஈฎ⌮࡛ N ࢺࣛࣥࢫ࣏࣮ࢱ࣮㑇ఏᏊࡸ N ௦ㅰ࡟㛵୚ࡍࡿ㓝⣲㑇 ఏᏊࡀⓎ⌧ㄏᑟࡉࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (Krapp et al., 2011)㸬ᮏゎᯒ࡛ࡣ N ḞஈⓎ⌧ ㄏᑟ㑇ఏᏊ⩌࡟N ࢺࣛࣥࢫ࣏࣮ࢱ࣮NRT2.5 ࡸࢢࣝࢱ࣑ࣥࡸ࢔ࣝࢠࢽࣥ࡞࡝ࡢ໬ྜ≀ࢆ㍺㏦ࡍࡿ࢔࣑ࣀ㓟ࢺࣛࣥࢫ࣏࣮ࢱ࣮CAT5㸪N ௦ㅰ㓝⣲ࡢᏑᅾࡀ☜ㄆࡉࢀࡓࡀ㸪᫬㛫ⓗ࡟ඹ㏻ ࡋ࡚Ⓨ⌧ࡋ࡚࠸ࡿ㑇ఏᏊࡣ࡞ࡃ㸪ࢫࢺࣞࢫᮇ㛫ࡀ㛗ᮇ໬ࡍࡿࡇ࡜࡛Ⓨ⌧ㄏᑟࡍࡿ㑇ఏᏊࡀ ኚືࡍࡿࡇ࡜ࡀ♧ࡉࢀࡓ㸬MYB75 ࡣ࢔ࣥࢺࢩ࢔ࢽࣥ⏕ྜᡂ࡟㛵୚ࡍࡿ㌿෗ᅉᏊ࡛࠶ࡾ

(Zhou et al., 2008)㸪MYB62ࡣࣜࣥ㓟Ḟஈ࡟ᛂ⟅ࡍࡿ㌿෗ᅉᏊ࡛࠶ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸

ࡿ (Devaiah et al 2009)㸬⾲⌧ᆺ࠿ࡽ᳜≀య࡟ࡣ࢔ࣥࢺࢩ࢔ࢽࣥࡢ⵳✚ࡀࡳࡽࢀ㸪άᛶ㓟⣲ ࡟ᑐࡍࡿᛂ⟅ࡔ࡜⪃࠼ࡽࢀࡿ㸬GO ゎᯒ࠿ࡽ͆secondary metabolic process” ࡸ “cellular amino acid and derivative metabolic process” ࡞࡝ࡢ஧ḟ௦ㅰ⏘≀࡟㛵୚ࡍࡿᶵ⬟ࡢ㑇ఏ

ᏊࡀⓎ⌧ㄏᑟࡋ࡚࠸ࡿࡇ࡜࠿ࡽN Ḟஈࢫࢺࣞࢫ࡟ᑐࡋ࡚஧ḟ௦ㅰ⏘≀ࡣ㔜せ࡞ᙺ๭ࢆᯝࡓ

(44)

39 P P Ḟஈ࡟ࡼࡿࢩࣟ࢖ࢾࢼࢬࢼᆅୖ㒊ࡢ㑇ఏᏊⓎ⌧ࡢኚືࡢゎᯒ ࣋ࣥᅗゎᯒࡼࡾP ≉␗ⓗⓎ⌧ㄏᑟ㑇ఏᏊ⩌ࢆ≉ᐃࡋࡓࡀ㸪ࡑࡢከࡃࡀ K Ḟஈㄏᑟ㑇ఏᏊ ࡜㧗࠸ぶ࿴ᛶࢆ♧ࡋࡓ (Fig. 2)㸬ࡇࡢ㑇ఏᏊ⩌࡟ࡣ↓ᶵࣜࣥ㓟 (Pi) ㍺㏦య㑇ఏᏊ PHT family ࡀྵࡲࢀ࡚࠾ࡾ㸪᰿ᅪ㒊ࡢ Pi Ḟஈ࡟ᛂ⟅ࡋ࡚ᆅୖ㒊࡛㌿෗ኚືࡀᘬࡁ㉳ࡇࡉࢀ࡚࠸ ࡿࡇ࡜ࡀศ࠿ࡗࡓ㸬2 ᪥㛫࠾ࡼࡧ 7 ᪥㛫ࡢ Pi Ḟஈ࡟ࡼࡿⓎ⌧ㄏᑟ㑇ఏᏊ⩌࡟ࡣPDF1.3࠾ ࡼࡧPDF2.1㸪WRKY18㸪WRKY60ࡀ☜ㄆ࡛ࡁࡓࡀ㸪ࡇࢀࡣከࡃࡢ◊✲࡛ሗ࿌ࡉࢀ࡚࠸ࡿ ࡼ࠺࡟Pi Ḟஈ࡛⑓ᐖᛂ⟅ᛶ㑇ఏᏊࡀⓎ⌧ㄏᑟࡉࢀ࡚࠸ࡿࡇ࡜࠿ࡽ㸪2 ᪥㛫࡜࠸࠺▷ᮇ㛫Ḟ ஈฎ⌮࡛ࡶ᳜≀యᆅୖ㒊ࡣ㎿㏿࡟ᛂ⟅ࡍࡿࡇ࡜ࡀド᫂ࡉࢀࡓ (Xu et al., 2006)㸬ࡉࡽ࡟஧ḟ ௦ㅰ⏘≀⏕⏘࡟㛵୚ࡍࡿྜᡂ㑇ఏᏊࡸ㌿෗ᅉᏊࡀྵࡲࢀ࡚࠸ࡿࡇ࡜࠿ࡽ㸪᳜≀ࡣ P 㣚㣹࡟ ࡼࡿ㓟໬ࢫࢺࣞࢫ࡟ᑐࡋ࡚࢔ࣥࢺࢩ࢔ࢽࣥࢆ⵳✚ࡍࡿࡇ࡜࡛᢬ᢠᛶࢆ⋓ᚓࡋ࡚࠸ࡿ࡜⪃࠼ ࡽࢀࡿ㸬ࡉࡽ࡟⑓ᐖ᢬ᢠᛶ㑇ఏᏊࡢㄏᑟࡀ P Ḟஈ࡛ㄏᑟࡉࢀ࡚࠾ࡾ㸪㛗ᮇ㛫ࡢࢫࢺࣞࢫ࡛ ࡶࡑࡢᛂ⟅ࡀ⥅⥆ࡋ࡚࠸ࡿࡇ࡜ࡀᛂ⟅㑇ఏᏊ࠾ࡼࡧGO ゎᯒ࠿ࡽ᥎ ࡛ࡁࡓ㸬P Ḟஈᙉㄏᑟ 㑇ఏᏊࡀ⏕≀ⓗࢫࢺࣞࢫࡲࡓࡣയᐖࢫࢺࣞࢫ࡛ᛂ⟅ࡍࡿࡇ࡜ࡣ▱ࡽࢀ࡚࠸ࡿࡀ㸪GO ゎᯒ࡟

࠾࠸࡚ࡶ⏕≀ࢫࢺࣞࢫ࡟㛵ࢃࡿᶵ⬟ࡀ᳨ฟࡉࢀࡓ㸬(Uknes et al., 1992; Potter et al., 1993; Chen et al., 2002; Mahalingam et al., 2003)㸬

K Ḟஈ࡟ࡼࡿࢩࣟ࢖ࢾࢼࢬࢼᆅୖ㒊ࡢ㑇ఏᏊⓎ⌧ࡢኚືࡢゎᯒ ᮏゎᯒ࡛ࡣ㸪K ࡟㛵୚ࡍࡿ㑇ఏᏊ࡜ࡋ࡚ K ㍺㏦ࢱࣥࣃࢡ㑇ఏᏊHAK5ࡀྵࡲࢀ࡚࠸ࡓ୍ ᪉࡛㸪௚ࡢK ㍺㏦ࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡋ࡚࠸ࡿ㑇ఏᏊHKT࠾ࡼࡧAKTࡢⓎ⌧ࡣ☜ㄆ࡛ࡁ ࡞࠿ࡗࡓ㸬K Ḟஈ࡟ࡼࡿࢩࣟ࢖ࢾࢼࢬࢼᆅୖ㒊ࡢ㌿෗ኚືࡣ N Ḟஈࡸ P Ḟஈ࡜ẚ㍑ࡋ࡚㌿ ෗ࡀ኱ࡁࡃኚືࡍࡿ㑇ఏᏊࡀᑡ࡞ࡃ㸪2 ᪥㛫࠾ࡼࡧ 7 ᪥㛫࡛ඹ㏻ࡋ࡚Ⓨ⌧ㄏᑟࡲࡓࡣᢚไࡉ ࢀࡿ㑇ఏᏊ⩌ࡣᑡ࡞ࡃ㸪GO ゎᯒ࠿ࡽࡶ㑇ఏᏊᶵ⬟ࡢ᥎ᐹࡣᅔ㞴࡛࠶ࡗࡓ㸬ࡇࢀࡣ௦᭰ඖ⣲ ࡀᏑᅾࡋ࡞࠸N ࡸ P ࡜ࡣ␗࡞ࡾ K+ _{࡟ࡣ௦᭰ඖ⣲࡛࠶ࡿ}_Na+ _{ࡀ࠶ࡾ㸪࠶ࡿ⛬ᗘࡢᶵ⬟ࢆ⿵} ᏶ࡋ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿ㸬≉␗ⓗᛂ⟅ᛶ㑇ఏᏊࡢゎᯒ࡛⣽⬊ቨ࡟㛵ࢃࡿ㑇ఏᏊࡸ

SAUR-like auxin-responsive proteinࡀ᳨ฟࡉࢀࡓࡀ㸪Ⓨ⌧ㄏᑟ㑇ఏᏊ⩌࠾ࡼࡧᢚไ㑇ఏᏊ

(45)

40 ᛶࡀ࠶ࡿ㸬௒ᅇ᳨ฟࡉࢀࡓ㑇ఏᏊ࡛ࡣK ࡟ᑐࡍࡿࢫࢺࣞࢫࡢᛂ⟅ᶵᵓࢆゎ᫂ࡍࡿࡇ࡜ࡣ㞴 ࡋࡃ௒ᚋࡢヲ⣽࡞ゎ᫂ࡀᮇᚅࡉࢀࡿ㸬 ࣔ ࣔࢹ᳜ࣝ≀ࡢᰤ㣴Ḟஈᛂ⟅ᛶࣂ࢖࣐࣮࣮࢜࢝ࡢ≉ᐃ N ࠾ࡼࡧ P ࡣ᳜≀ࡀ᭱ࡶᚲせ࡜ࡍࡿᰤ㣴⣲࡛࠶ࡿ୍᪉࡛㐣๫࡞ᢞධ࡟ࡼࡗ࡚⎔ቃởᰁࡢ ୍ᅉ࡜࡞ࡗ࡚࠸ࡿ㸬ᢞධ㔞࡜᳜≀యࡢᰤ㣴≧ែࡢࢠࣕࢵࣉࡣ኱ࡁࡃ㸪ࣜ࢔ࣝࢱ࢖࣒࡛᳜≀ ࡢᰤ㣴デ᩿ࢆホ౯ࡍࡿࣂ࢖᳨࣐࣮࣮࢜࢝ᰝࡣ┬㈨※ᆺ㎰ᴗ࡟኱ࡁࡃ㈉⊩ࡍࡿࡶࡢࡔ࡜࠸࠼ ࡿ㸬ᮏ◊✲࡛ࡣࣔࢹ᳜ࣝ≀࡛࠶ࡿࢩࣟ࢖ࢾࢼࢬࢼ࠾ࡼࡧ࢖ࢿࢆ⏝࠸ྛᰤ㣴⣲࡟≉␗ⓗᛂ⟅ ࢆ♧ࡍࣂ࢖࣐࣮࣮࢜࢝㑇ఏᏊࡢ≉ᐃᡭἲ࡞ࡽࡧ࡟฼⏝᪉ἲࡢ☜❧ࢆヨࡳࡓ㸬ᗂⱑᮇ࡟࠾ࡅ ࡿྛᰤ㣴ඖ⣲Ḟஈ≧ែୗࡢࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ࢹ࣮ࢱࢆᇶ࡟㑇ఏᏊⓎ⌧ࣂ࢖࣐࣮࣮࢜࢝ ࡢೃ⿵㑇ఏᏊࢆ≉ᐃࡋ㸪Ỉ⪔࠾ࡼࡧᅵ⪔᱂ᇵ࡛⏕⫱ࡋࡓ᳜≀యࢆ⏝࠸࡚ᰤ㣴≧ែࢆホ౯ࡋ ࡓ㸬ᐃ㔞ⓗRT-PCR ゎᯒࡢ⤖ᯝ㸪㑇ఏᏊⓎ⌧ࣂ࢖࣐࣮࣮࢜࢝ࡢⓎ⌧㔞ࡣ࣐࢖ࢡࣟ࢔ࣞ࢖ࡢ ෌⌧ᛶࡀ࠶ࡿࡇ࡜ࢆド᫂ࡋࡓ㸬ࡲࡓ㏣⫧ฎ⌮࡟ࡼࡗ࡚ᑐ↷༊࡜ྠ➼ࡢⓎ⌧㔞ࡲ࡛ᅇ᚟ࡋ࡚ ࠾ࡾ㸪㌿෗ࣂ࢖࣐࣮࣮࡛᳜࢜࢝≀ࡀ೺ᖖ≧ែ࡬ᅇ᚟ࡍࡿࡇ࡜ࡀ☜ㄆ࡛ࡁࡿࡇ࡜ࢆド᫂ࡋࡓ (Fig. 4)㸬ᅵ⪔࡛⏕⫱ࡋࡓࢩࣟ࢖ࢾࢼࢬࢼ᳜≀య࡛ࡣᗄࡘ࠿ࡢࣂ࢖࣐࣮࣮࢜࢝㑇ఏᏊࡀỈ⪔ ᱂ᇵࡋࡓࢩࣟ࢖ࢾࢼࢬࢼࡢࢹ࣮ࢱ࡜␗࡞ࡿ㑇ఏᏊⓎ⌧㔞ࢆ♧ࡋࡓࡀ (Fig. 6)㸪ࡇࢀࡣᅵተ୰ ࡢ↓ᶵᰤ㣴ࡢ⃰ᗘࡣ୙ᆒ୍࡛࠶ࡾ㸪ࡲࡓᅵተ⮬యࡢ໬Ꮫᛶࡶ✀㢮࡟ࡼࡾ኱ࡁࡃ␗࡞ࡗ࡚࠸ ࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡿ୍᪉࡛㸪ከࡃࡢ㑇ఏᏊⓎ⌧ࣂ࢖࣐࣮࣮࢜࢝ࡀᵝࠎ࡞ᅵተࡸᆅᇦ࡟㐺ྜ ࡍࡿỗ⏝ᛶࢆ᭷ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓ㸬 ࢖ࢿࡢࣂ࢖࣐࣮࣮࢜࢝㑇ఏᏊࢆ≉ᐃࡍࡿࡓࡵ࡟㸪ẚ㍑ⓗ㍍ᗘࡢᰤ㣴Ḟஈฎ⌮࡛࠶ࡿ1/4 㔞 ฎ⌮࠿ࡘ㔜ᗘ࡞Ḟஈ≧ែ࡛࠶ࡿ1/64 㔞ฎ⌮᳨࡛▱࡛ࡁࡿⓎ⌧ㄏᑟ࠾ࡼࡧᢚไ㑇ఏᏊࡢ᥈⣴ ࢆ⾜ࡗࡓ㸬࣋ࣥᅗゎᯒࡼࡾ≉ᐃࡋࡓࣂ࢖࣐࣮࣮࢜࢝㑇ఏᏊࢆ⏝࠸㸪ᐃ㔞ⓗ RT-PCR ࡟ࡼࡿ ㌿෗㔞ࡢ ᐃࡋࡓ࡜ࡇࢁ▷ᮇ㛫ฎ⌮ࡢ࢖ࢿ࡛ࡣ㸪࠸ࡎࢀࡢࣂ࢖࣐࣮࣮࢜࢝㑇ఏᏊ࡛ࡶ⁐ᾮ ୰ࡢඖ⣲⃰ᗘ࡟㛵ࢃࡽࡎ㸪≉␗ⓗ࡞Ⓨ⌧ㄏᑟࢆ♧ࡋࡓ (Fig. 10, 11)㸬ࡲࡓࡇࢀࡽࡢࣂ࢖࣐࢜ ࣮࣮࢝㑇ఏᏊࡣ␗࡞ࡿ⏕⫱ᮇ㛫ࡸ᱂ᇵ᮲௳࡛ࡶ㧗࠸ឤᗘ࡛཯ᛂࢆ♧ࡋ㸪᱂ᇵ᮲௳ࡀ␗࡞ࡿ ⎔ቃ࡛ࡶ୍ᐃࡢ⢭ᗘࡀ࠶ࡿࡇ࡜ࢆド᫂ࡋࡓ㸬ࡋ࠿ࡋ௒ᅇࡢᐇ㦂ࡣᰤ㣴ᡂ㛗ᮇࡢ᳜≀య࡛࠶

(46)

41 ࡾ㸪⏕Ṫᡂ㛗ᮇࡢ᳜≀య࡟ᑐᛂ࡛ࡁࡿ࠿ࡣᮍ▱ᩘ࡛࠶ࡿ㸬බඹࢹ࣮ࢱ࣮࣋ࢫ࠿ࡽ⋓ᚓࡋࡓ ࢖ࢿᆅୖ㒊ࡢᰤ㣴Ḟஈ࣐࢖ࢡࣟ࢔ࣞ࢖ࢹ࣮ࢱࡼࡾ㸪ࢩࣟ࢖ࢾࢼࢬࢼ࡜ྠᵝࡢ᪉ἲ࡛≉␗ⓗ ࠿ࡘⴭࡋࡃⓎ⌧ㄏᑟࡲࡓࡣⓎ⌧ᢚไࡉࢀࡿ㑇ఏᏊࢆྠᐃ㸪ά⏝ࡍࡿࡇ࡜࡛㸪ࣂ࢖࣐࣮࢜࢝ ࣮࡛฼⏝࡛ࡁࡿࡇ࡜࠿ࡽ㸪௚ࡢ᳜≀✀࡟࠾࠸࡚ࡶస≀࡬ࡢᛂ⏝ࡢྍ⬟ᛶࡀ㧗ࡲࡿ࡜⪃࠼ࡽ ࢀࡿ㸬㑇ఏ᝟ሗࡀ୙༑ศ࡛࠶ࡿ᳜≀✀࡛ࣂ࢖࣐࣮࣮࢜࢝ೃ⿵㑇ఏᏊࢆ≉ᐃࡍࡿᣦᶆ࡜ࡋ࡚㑇ఏ Ꮚᶵ⬟࡟╔┠ࡋࡓ㸬N ࡜ P ࡛ࡣ㍺㏦ࢱࣥࣃࢡ㉁ࡲࡓࡣ௦ㅰ㓝⣲ࡀ᭷ຊ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓ㸬 ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࡣᵝࠎ࡞᳜≀࡛ࡶᬑ㐢ⓗ࡟Ꮡᅾࡍࡿ࣮࢜ࢯࣟࢢ࡛࠶ࡾ㸪᳜≀✀ࡈ࡜ࡢ cDNA ࣛ࢖ࣈࣛࣜ࠿ࡽࡶ᝟ሗࡀධᡭ࡛ࡁࡿ㸬P ࡟࠾࠸࡚ࡶ࢖ࢿ㸪࢔ࣝࣇ࢓ࣝࣇ࢓ MtPT4

(Harrison et al., 2002)㸪ࢪࣕ࢞࢖ࣔ StPT family㸪ࢺ࣐ࢺLePT family (Nagy et al., 2005) ࡀ༢㞳ྠᐃࡉࢀ࡚࠸ࡿ㸬≉ᐃࡢ᳜≀✀࡬ࡢ࣮࢜ࢯࣟࢢ࡟ᇶ࡙࠸ࡓᛂ⏝ࢆ⪃࠼㸪ࢺࣛࣥࢫ࣏ ࣮ࢱ࣮㑇ఏᏊࡸඖ⣲௦ㅰ㓝⣲㑇ఏᏊࢆ㑇ఏᏊⓎ⌧ࣂ࢖࣐࣮࣮࢜࢝࡜ࡋ࡚᭱㐺࡞ೃ⿵࡛࠶ࡿ ࡜⪃࠼ࡽࢀࡓ㸬௒ᅇ≉ᐃࡋࡓ㑇ఏᏊ࠾ࡼࡧ GO ゎᯒ࡛ྛᰤ㣴ඖ⣲ࡢ㍺㏦ࢱࣥࣃࢡ㉁ࡣᏑᅾ ࡋ࡞࠿ࡗࡓ㸬Yang ࡽ (2011) ࡢሗ࿌࠿ࡽࡶ N ḞஈⓎ⌧ᢚไࣂ࢖࣐࣮࣮࢜࢝࡜ࡋ࡚ྠᐃࡋࡓ 㑇ఏᏊࡢGO annotation ࡣ”transporter”࡟ᒓࡍࡿ㑇ఏᏊࡣᑡ࡞࠿ࡗࡓ㸬ᮏ◊✲࡛ྠᐃࡉࢀ ࡓࢺࣛࣥࢫ࣏࣮ࢱ࣮㑇ఏᏊ࡜ࡋ࡚㸪N Ḟஈ࡛ࡣ NRT family ࡸ URS㸪P Ḟஈ࡛ࡣ PHT family ྠᐃࡉࢀࡓ㸬Ỉ⪔᱂ᇵ࡛ᰤ㣴Ḟஈฎ⌮ࢆ⾜ࡗࡓࢩࣟ࢖ࢾࢼࢬࢼᆅୖ㒊ࡼࡾ≉ᐃࡋࡓ 㑇ఏᏊⓎ⌧ࣂ࢖࣐࣮࣮࢜࢝ࢆᅵ⪔࡛᱂ᇵࡋࡓࢩࣟ࢖ࢾࢼࢬࢼࡢᰤ㣴デ᩿⏝࡟ከࡃࡢࣂ࢖࢜ ࣐࣮࣮࢝ࡀ᳜≀ࡢᰤ㣴≧ែࢆホ౯࡛ࡁࡓࡇ࡜࠿ࡽ㸪⎔ቃせᅉࢆ᤼㝖ࡋࡸࡍ࠸◊✲ᐊࡢᐇ㦂 ⣔࡛᱂ᇵࡋࡓ᳜≀ࡢࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ࢹ࣮ࢱࡣᵝࠎ࡞⎔ቃᅉᏊࡀᏑᅾࡍࡿࣇ࢕࣮ࣝࢻ ሙ࡛ࡶᛂ⏝࡛ࡁࡿྍ⬟ᛶࡀ࠶ࡿࡇ࡜ࢆ♧၀ࡍࡿ⤖ᯝ࡜࡞ࡗࡓ㸬 ᰤ㣴Ḟஈ≧ែୗ࡟࠶ࡿ᳜≀ࡢ㌿෗ኚືࡢ⥲ྜⓗ࠿ࡘ⤫ྜⓗゎᯒࡣᰤ㣴ࢆຠ⋡ⓗ࡟㍺㏦ࡍ ࡿᙧ㉁㌿᥮᳜≀యࡢసฟ࡟㔜せ࡛࠶ࡿ㸬ᮏᐇ㦂࡛ࡣ2 ࡘࡢ᳜≀✀ࢆ⏝࠸࡚ゎᯒࢆ⾜ࡗࡓࡀ㸪 N㸪P㸪K ࡟ඹ㏻ࡋ࡚ᛂ⟅ࡍࡿ㑇ఏᏊࡣ࡯࡜ࢇ࡝ぢࡽࢀ࡞࠿ࡗࡓࡇ࡜࠿ࡽࢩࣟ࢖ࢾࢼࢬࢼ࠾ ࡼࡧ࢖ࢿᆅୖ㒊ࡣḞஈ≧ែୗ࡛ࡢ㌿෗ᛂ⟅ࡣྛᰤ㣴ඖ⣲≉␗ⓗ࡛࠶ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗ ࡓ㸬ࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ࡸ࣓ࢱ࣮࣒࣎ࣟࢆ⏝࠸ࡓࣂ࢖࣐࣮࣮࢜࢝ࢆ᳜≀࡬ࡢ◊✲࡜ࡋ࡚

(47)

42

ᵝࠎ࡞ᡭἲࡀ◊✲ࡉࢀ࡚࠸ࡿ (Phelix and Feltus, 2014)㸬ᮏ◊✲ࡢᐇ㦂᪉ἲ࡛ࢩࣟ࢖ࢾࢼ ࢬࢼࡢ㑇ఏᏊⓎ⌧ࣂ࢖࣐࣮࣮࢜࢝ࡣᐇ㝿ࡢ㎰ሙࢆ᝿ᐃࡋࡓᅵ⪔ヨ㦂࡟࠾࠸࡚ࡶ」ᩘࡢࣂ࢖ ࣐࣮࣮࡛࢜࢝ᰤ㣴Ḟஈ≧ែ࡜≉ᐃ࡛ࡁࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓ㸬ࡲࡓࢩࣟ࢖ࢾࢼࢬࢼྠᵝ㸪 ࢖ࢿᰤ㣴Ḟஈࢫࢺࣞࢫୗ࡟࠶ࡿᗂ᳜≀యࡢ㌿෗ᛂ⟅ࡀ㸪㛗ᮇ⏕㛗᳜≀య࡟ࡶ㐺⏝࡛ࡁࡿࡇ ࡜ࡀ♧ࡉࢀࡓ㸬⌧ᅾ㸪ࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ࢹ࣮ࢱࡀබඹࢹ࣮ࢱ࣮࣋ࢫ࡟ከࡃ㞟✚ࡉࢀ࡚ ࠾ࡾ㸪ࡲࡓḟୡ௦ࢩ࣮ࢣࣥࢧ࣮ࢆ⏝࠸ࡓRNA-seq ࡣࢤࣀ࣒ゎᯒࡉࢀ࡚࠸࡞࠸᳜≀✀࡛ࡶ㌿ ෗ゎᯒࡀྍ⬟࡛࠶ࡾ㸪௒ᚋ㸪ᵝࠎ࡞᳜≀✀ࡢᰤ㣴Ḟஈᛂ⟅ᶵᵓࡢ⌮ゎࡀࡼࡾ㐍ᒎࡍࡿ࡜⪃ ࠼ࡽࢀࡿ㸬⵳✚ࡉࢀࡓ࣮࣑࢜ࢡࢫࢹ࣮ࢱࢆά⏝ࡍࡿࡇ࡜࡛㸪ᰤ㣴฼⏝ຠ⋡ࢆ㧗ࡵࡓᙧ㉁㌿ ᥮యస≀ࡢసฟࡸ㑇ఏᏊⓎ⌧ࣂ࢖࣐࣮࣮࢜࢝ࢆ≉ᐃࡋస≀ࡢᰤ㣴⟶⌮ࢆ⡆౽໬࠿ࡘ᭱㐺໬ ࡍࡿࡇ࡜ࡀ࡛ࡁ㸪㐣ᗘ࡞᪋⫧࡜㸪ࡑࢀ࡟కࡗ࡚Ⓨ⏕ࡍࡿ⎔ቃởᰁࡢ㜵Ṇࡢࡓࡵ┬㈨※ᆺ㎰ ᴗࡢᐇ⌧࡟࠾࠸࡚㔜せ࡞ᙺ๭ࢆᢸ࠺࡜⪃࠼ࡽࢀࡿ㸬

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43

➨

2 ❶

ࢩࣟ࢖ࢾࢼࢬࢼᆅୖ㒊ࡢ᰿ᅪẘᛶ࢖࢜ࣥᛂ⟅ࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒

ࣉࣟࣇ࢓࢖ࣜࣥࢢ

2-1 ⥴ゝ

㏆ᖺࡢᆅ⌫つᶍࡢ⎔ቃኚ໬࡟క࠸⃭ࡋ࠸Ẽೃኚື㸪Ẽ ୖ᪼㸪Ỉ㈨※ࡢᯤῬ࡟ࡼࡿ㎰ᴗ ⎔ቃࡢຎ໬ࡀᅜ㝿ⓗ࡞ၥ㢟࡜࡞ࡗ࡚࠾ࡾ㸪ᐇ㝿࡟ୡ⏺ࡢ୺せ࡞స≀࡛࠶ࡿࢥ࣒ࢠࡸࢺ࢘ࣔ ࣟࢥࢩࡢ⏕⏘㔞ࡣῶᑡࡋ࡚࠸ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (Godfray et al., 2010; Lobell et al., 2011)㸬2016 ᖺ࡟ୡ⏺ேཱྀࡣ 73 ൨ே࡟㐩ࡋ㸪ᑗ᮶ࡢேཱྀቑຍ࡟௜㝶ࡋ࡚῝้໬ࡍࡿ㣗ᩱၥ 㢟ࡀᠱᛕࡉࢀ࡚࠸ࡿ㸬ᗎㄽ࡛㏙࡭ࡓࡀస≀ࡢရ✀ᨵⰋ࡟ࡼࡿ༢఩㠃✚ࡢ཰㔞ቑຍࡣ㝈⏺࡟ ࡁ࡚࠾ࡾ㸪ࡑࡢࡓࡵ㣗ᩱቑ⏘ࡢ᪉ἲ࡜ࡋ࡚᱂ᇵ㠃✚ࡢᣑ኱ࡀゎỴᡭẁ࡜ࡋ࡚ᣲࡆࡽࢀࡿ㸬 ࡋ࠿ࡋ࡞ࡀࡽୡ⏺ࡢᅵተ࡟ࡣ㎰స≀ࢆ᱂ᇵࡍࡿ࡟ࡣ୙㐺ᙜ࡞ᅵተ࡛࠶ࡿ୙ⰋᅵተࡀᗈࡃᏑ ᅾࡋ࡚࠾ࡾ㸪ࡇࢀࡣ⪔సྍ⬟㠃✚ࡢ 67㸣ࢆ༨ࡵ࡚࠸ࡿ࡜ࡉࢀ࡚࠸ࡿ㸬⪔సྍ⬟ᅵተࡢຎ໬ ࡀ㐍ࢇ࡛࠾ࡾ㸪UNEP (ᅜ㐃⎔ቃィ⏬) ࡢㄪᰝ࡛ࡣ⮬↛ⓗࡲࡓࡣேⅭⓗせᅉ࡟ࡼࡾࡇࢀࡲ࡛ 20 ൨ ha ࡟ཬࡪ㠃✚ࡢᅵተຎ໬ࡀ⏕ࡌ࡚࠾ࡾ㸪 ௒ᚋ 20 ᖺ㛫࡟⣙ 1 ൨ 4 ༓୓ ha ࡶࡢ⫧Ỻ࡞ ㎰ᆅࡀᅵተຎ໬࡟ࡼࡗ࡚ኻࢃࢀࡿ࡜ண ࡉࢀ࡚࠸ࡿ㸬 ୙Ⰻᅵተࡣᅵተ୰࡟ᵝࠎ࡞ࢫࢺࣞࢫᅉᏊࢆෆໟࡋ࡚࠸ࡿ㸬㓟ᛶᅵተࡣpH 5.5 ௨ୗࡢᅵተ ࡜ᐃ⩏ࡉࢀ࡚࠸ࡿᅵተ࡛⇕ᖏࡸள⇕ᖏ࡟ᗈࡃᏑᅾࡋ㸪⪔సྍ⬟ᅵተࡢ30㸣ࢆ༨ࡵࡿ࡜ࡉࢀ㸪 ୺࡟ྍ⁐໬ࡋࡓAl ࡀ᳜≀ࡢ⏕⫱ࢆ㜼ᐖࡍࡿ (Foy 1988)㸬▼⅊㉁࢔ࣝ࢝ࣜᛶᅵተࡣ pH 7.0 ௨ୖࡢᅵተ࡛࠶ࡾ୙Ⰻᅵተࡢ50㸣ࢆ༨ࡵ㸪ࡑࡇ࡛ࡣ 3 ౯ࡢỈ୙⁐ែ㕲 (Fe2O3) ࡜࡞ࡾ Fe ྾཰ࢆ㜼ᐖࡋ᳜≀ࡢ⏕㛗ࡀᢚไࡉࢀࡿ㸬ࡲࡓሷ㢮㞟✚ᅵተࡢ㠃✚ࡣ㝣ᆅࡢ6.5㸣࡟┦ᙜࡍࡿ ⣙8 ൨ ha ௨ୖࢆ༨ࡵࡿ࡜ࡉࢀ࡚࠾ࡾ㸪ࡉࡽ࡟㐣๫࡞℺₅࡞࡝࡟ࡼࡗ࡚ Na ࢆࡣࡌࡵ࡜ࡋࡓ ሷ㢮⵳✚ࡲࡓࡣࢯ࣮ࢲ㉁໬ࡀ㐍ࡳ㸪㐣๫࡞ࢼࢺ࣒ࣜ࢘ (Na) ࡸỈࢫࢺࣞࢫ࡟ࡼࡾస≀⏕⏘ ᛶࢆపࡵ࡚࠸ࡿ (FAO, 2005; Frommer et al., 1999; Flowers, 2004)㸬ࡲࡓ୙ⰋᅵተࡣẼೃ

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44 ࡓࡣሷ㢮㞟✚ᅵተ࡜࡞ࡾ㸪ᅵተࡢỈศࡀ⵨Ⓨࡍࡿࡇ࡜࡛ᆅ⾲࡟ሷ㢮ࡀ㞟✚ࡋᅵተ୰ࡢNa ࡸ ࣍࢘⣲ (B) ࡀ᳜≀ࡢ⏕⫱ࢆ㜼ᐖࡍࡿ㸬ே㛫άື࡟ࡼࡿ⎔ቃởᰁࡶ୙Ⰻᅵተࢆసࡾฟࡍせᅉ ࡛࠶ࡾ㸪୙㐺ษ࡞℺₅࡟ࡼࡾᅵተຎ໬ࡀᘬࡁ㉳ࡇࡉࢀሷ㢮㞟✚ᅵተ࡟࡞ࡿࡇ࡜ࡀၥ㢟どࡉ ࢀ࡚࠸ࡿ㸬௚࡟ࡶᕤᴗάື࡟ࡼࡾᕤሙࡸ⢭㘐ᡤ࠿ࡽࡢ᤼↮ࡸᗫỈࡀࡶࡓࡽࡍ㔜㔠ᒓởᰁࡣ ᳜≀ࡢ⏕⫱ࢆⴭࡋࡃ㜼ᐖࡋ㸪ຍ࠼࡚ởᰁࡉࢀࡓᅵተ࡛᱂ᇵࡉࢀࡓస≀ࢆ㣗≀㐃㙐ࢆ௓ࡋ࡚ ே㛫ࡀᦤྲྀࡍࡿࡇ࡜࡛೺ᗣၥ㢟ࢆᘬࡁ㉳ࡇࡋ࡚࠸ࡿ㸬 ቑຍࡍࡿேཱྀࢆ㈥࠺ࡔࡅࡢ㣗ᩱ⏕⏘ࢆ⾜࠺ࡓࡵ࡟୙Ⰻᅵተࡢ㛤ቧࡀᚲ㡲࡛࠶ࡿࡀඛ㏙ࡢ ࡼ࠺࡟୙Ⰻᅵተ࡟ࡣከ✀ࡢࢫࢺࣞࢫᅉᏊࡀᏑᅾࡋ࡚࠾ࡾ⌧ᅾࡢ㎰ᴗ᪉ἲ࡛ࡣᏳᐃⓗ࠿ࡘప ࢥࢫࢺࡢస≀⏕⏘ࡢᐇ⌧ࡣ㞴ࡋ࠸㸬ࡑࡢࡓࡵศᏊ⫱✀ⓗᡭἲ࡟ࡼࡿᙧ㉁㌿᥮ࢆ⏝࠸ࡓࢫࢺ ࣞࢫ⪏ᛶస≀ࡢసฟࡀ⌧ᐇⓗ࡞ゎỴ᪉ἲࡔ࡜࠸࠼ࡿ㸬᳜≀ࡣᅵተ୰࡟Ꮡᅾࡍࡿࢫࢺࣞࢫᅉ Ꮚ࡟ᑐࡋ࡚ከᵝ࡞⪏ᛶᶵᵓࢆഛ࠼࡚࠸ࡿ㸬㏆ᖺ㸪ศᏊ⏕≀Ꮫⓗᡭἲ࡟ࡼࡗ࡚ᅵተࢫࢺࣞࢫ ⪏ᛶᶵᵓࡢゎ᫂ࡀ㐍ᒎࡋ࡚࠾ࡾ㸪ከࡃࡢ᳜≀✀࠿ࡽࢫࢺࣞࢫ⪏ᛶ࡟㛵୚ࡋ࡚࠸ࡿࢱࣥࣃࢡ ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡀ༢㞳≉ᐃࡉࢀ࡚࠸ࡿ㸬ᅵተ୰ࡢ Al3+_{࡟ᑐࡍࡿ᭷ᶵ㓟ᨺฟᶵᵓ࡜ࡋ} ࡚ࢥ࣒ࢠࡸࢩࣟ࢖ࢾࢼࢬࢼ࡛ࡣ Al ᛂ⟅ᛶࣜࣥࢦ㓟ࢺࣛࣥࢫ࣏࣮ࢱ࣮㑇ఏᏊ Al-activated

Malate Transporter (ALMT ) family (Sasaki et al., 2004, Jurandir et al., 2007) ࡸ㸪ࢯࣝ ࣒࢞㸪ࢺ࢘ࣔࣟࢥࢩ࡞࡝࡛ࡣMATE (Multidrug and toxic compound extrusion ) family ࠿ ࡽࢡ࢚ࣥ㓟ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࡀ༢㞳ྠᐃࡉࢀ࡚࠸ࡿ (Magalhaes et al., 2007)㸬࢔ࣝ࢝ࣜᛶ

ᅵተࡸሷ㢮㞟✚ᅵተ࡞࡝࡛ࡣNa ࡀ୺せ࡞ࢫࢺࣞࢫᅉᏊ࡜࡞ࡿࡀ㸪ࡇࢀࡣ Na ࡀ⣽⬊ࡢᾐ㏱

ᅽࢆㄪᩚࡍࡿඖ⣲࡛࠶ࡿ୍᪉࡛㸪㐣๫࡟Ꮡᅾࡍࡿ࡜ᾐ㏱ᅽ࡟ኚㄪࢆࡁࡓࡍẘᛶ࢖࢜ࣥ࡟࡞

ࡿࡇ࡜࡟㉳ᅉࡋ࡚࠸ࡿ㸬᳜≀ࡣ㧗⃰ᗘࡢ Na ࡟ᑐࡋ࡚⣽⬊እ࡬ࡢ᤼ฟ࡜ᾮ⬊࡬ࡢ㝸㞳ࡢ 2

✀㢮ࡢ㐺ᛂ᪉ἲࢆ᭷ࡋ࡚࠸ࡿ㸬AtSOS1 ࡣ Na ࢆ⣽⬊㉁࠿ࡽ⣽⬊እ࡬᤼ฟࡍࡿ Na/H

antiporter ࡜ࡋ࡚ᶵ⬟ࡋ࡚࠸ࡿ (Wu et al., 1996; Shi et al., 2002)㸬ࡲࡓ AtNHX1 ࡣ

AtSOS family ࡜ྠᵝ࡟ Na/H antiporter ࢆࢥ࣮ࢻࡋ࡚࠸ࡿࡀᾮ⬊࡬ࡢ㝸㞳࡜ࡋ࡚ᶵ⬟ࡋ⣽

⬊㉁ࡢNa ࢆᾮ⬊࡬㍺㏦ࡍࡿࡇ࡜࡛⣽⬊ẘᛶࢆᅇ㑊ࡋ࡚࠸ࡿ (Yamaguchi et al., 2005)㸬

Salt Overly Sensitive (SOS) ⤒㊰ࡣሷࢫࢺࣞࢫୗ࡛࢖࣓࢜ࣥ࣍࢜ࢫࢱࢩࢫࡢ⥔ᣢ࡟㈉⊩ࡋ