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In addition to water molecule(H 2 O), plant aquaporins can transports;

中性低分子化合物輸送 Neutral low molecular-weight compounds. In addition to water molecule(H 2 O), plant aquaporins can transports;. Substrate (researcher/paper) CO 2 (Kaldenhoff’s group, Katsuhara’s group) Silica (Ma’s group) Boron (Fujiwara’s group) Glycerol (many works)

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In addition to water molecule(H 2 O), plant aquaporins can transports;

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  1. 中性低分子化合物輸送 Neutral low molecular-weight compounds In addition to water molecule(H2O), plant aquaporins can transports; Substrate (researcher/paper) • CO2 (Kaldenhoff’s group, Katsuhara’s group) • Silica (Ma’s group) • Boron (Fujiwara’s group) • Glycerol (many works) • H2O2(JBC 282:1183) • Ammonia (PMB 54:713) • Arsenite (Ma, Katsuhara)and more…

  2. CO2固定の律速段階　　Limitting steps for CO2 fixation leaf surface with stomata Stomatal conductance 気孔透過性 intercellular space cell walls Internal conductance 葉内透過性 細胞へ（原形質膜で）の透過 an aquaporin in the plasma-membrane of a mesophyll cell cytoplasm chloroplast RuBisCO 炭酸固定酵素の効率 Tissue and cellular structures are not to scale

  3. Aquaporin CO2 • CO2 permeability (Reported) • Oocyte + micro-pH electrode • Transgenic plants + CO2 conductance Ulreich et al. 2003 NtAPQ1 Our group 2004 HvPIP2;1, gas exchange and carbon isotope (C13/C12) ratio PIP cRNA Micropipette Carbonic-anhydrase: CA（enzyme) Micro pH electrode Voltage electrode CO2　→　HCO3-+H+(pH変化検出) CA Need high throughput system →　New yeast system

  4. Screening system to identify aquaporins transporting CO2. Aquaporin genes (Gal-inducebile) Model examination Acetic acid 酢酸添加による細胞内pH低下が GFP蛍光強度の変化で検出できた イネ、オオムギ、シロイヌナズナのPIPsをスクリーニング　 →　CO2透過活性をもつPIPを網羅的に検出

  5. Experimental data 1 sec after addition of NaHCO3 Ｐｏｓｉｔｉｖｅ control (NtAQP1) NaHCO3 Fluorescence 蛍光強度（任意単位）　→ Fluorescence 蛍光強度（任意単位）　→ F ΔF Δt NtAQP1 タバコアクアポリン (CO2透過性が既知のコントロール） HvPIP2;1 オオムギPIPの一つ T228MHvPIP2;1 活性中心の２２８番目のThrをMetに置換した不活性(inactive form)

  6. （CO2透過性アクアポリン遺伝子）／（調べたアクアポリン遺伝子）（CO2透過性アクアポリン遺伝子）／（調べたアクアポリン遺伝子） 　イネ　２／２３　　オオムギ　６／２１　 Future: High photosynthetic activity via improvements of aquaporins

  7. Si permeability (Siは耐病性と収量を向上させる） Lsi1 →positional cloning→ OsNIP2;1

  8. Arsenite (As(OH)3) permeabiliyu DAcr3 yeast assay No As(OH)3 5μM As(OH)3 Aquaporins transporting As(OH)3 pYES2 HvNIP1;1 HvNIP1;2 HvNIP2;1 HvNIP2;2 OsNIP1;1 OsNIP2;1 OsNIP2;2 OsNIP3;1 OsNIP3;2 OsNIP3;3 OsNIP4;1 As(OH）3 Lethal Acr3 Oocytes assay (PNAS 105;9931) LSi1=OsNIP2;1 Lsi6=OsNIP2;2 Growth of yeast ΔACR3 expressing various NIP aquaporins

  9. H2O2 peramiablityH2O2sensitive（Δskn7) yeast assay 0 mM H2O2 0.25 mM H2O2 H2O2 signaling in plants is essential for response to stress defense against pathogens and the regulation of programmed cell death. 0.75 mM H2O2 0.5 mM H2O2 HvPIP2;4 HvPIP2;3 HvPIP2;5 HvTIP2;2 HvPIP2;2 HvPIP2;1 pYES2 (ベクター） →　HvPIP2;5 and HvTIP2;2 probably transport H2O2

  10. アクアポリン研究のアウトプット Possible Applications Two examples Enhancement of growth and stress tolerances H2O, other molecules Improving yield/quality in flowers and fruits Aquaporins Crop improvements via aquaporin engineering (activity, expression regulation…..)

  11. Rice plants overexpressing PIPs OsPIP1;1 ・・・　 salt resistance ↑ seed germination ↑ (PPB 63:151, 2013) OsPIP2;4　・・・ Lpr↑ （Previously descried） HvPIP2;1 Lpr↑　but salt sensitive （PCP 44:1378, 2003） RNAi (Rice) against PIP2s 乾燥ストレス（脱水）耐性強化イネ? PIP expression・・・water uptake Stress-induced RNAi Drought OsNAC6 Down-regulation Tolerance? PIP2 PIP2 乾燥ストレス誘導的に発現抑制 (PIP2をRNAiで抑制） Preventing dehydration Not Yet Performed (まだできていない）

  12. 果実、花卉（Flowers and fruits) 名古屋大学共同研究 Nagoya university 生育旺盛に？ （うまく行かない） Notgood シロイヌナズに導入 P P CaM 35S PIP2 全身で過剰発現 （リン酸化をmimicしたPIP2） 恒常活性型 CaM 35S PIP2 トマトに導入 全身でに発現抑制 （PIP2のアンチセンス鎖） 高糖度で 日持ちの良い果実？ 恒常抑制型 InMYB1 PIP2 PIP2 アサガオに導入 花弁特異的に発現抑制 （PIP2をRNAiで抑制） みずみずしい 花持ちの良い花き？

  13. アサガオInMYB1上流 1 kb In MYB1 gene 「花弁特異的プロモーター」 　 特願2008-264646 　白武勝裕，森本玲奈， 　 飯田滋，星野敦，森田将裕 Little delay? PIP2 Knockdown InMYB1 PIP2 PIP2 花弁特異的に発現抑制 （PIP2をRNAiで抑制） WT

  14. MicroTom Model plant for fruit study CaM 35S PIP2 全身でに発現抑制 （PIP2のアンチセンス鎖） 形質転換体 16系統，41個体 No difference to date

  15. Summary and future of aquaporin research Profiling, physiological analysis Mutants, transgenic Hydraulic conductivity T-DNA Anti-sense／ＲＮＡｉ Overexpression Intracellular trafficking 免疫前血清 Anti-OsPIP2;1 Anti-OsPIP2;5 Functional analyze Expression (mRNA) Molecular engeneering OsPIP1;3(根)OsPIP1;3(根)OsPIP1;1(根) イネの根組織および細胞 Expression (Protein) CO2permeability Si permeability • Water stress tolerant crops • High quality of flowers and fruits • Improvements of plant functions vs Homo-tetramer Hetero-tetramer Structural analysis Mutated aqauporins Interaction (heteromeriozation)

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