in addition to water molecule(h 2 o), plant aquaporins can transports;
DESCRIPTION
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) - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: In addition to water molecule(H 2 O), plant aquaporins can transports;](https://reader035.vdocuments.pub/reader035/viewer/2022070502/568136ef550346895d9e82ae/html5/thumbnails/1.jpg)
In addition to water molecule(HIn addition to water molecule(H22O), O), plant aquaporins can transports; 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…
中性低分子化合物輸送 Neutral low molecular-weight compounds
![Page 2: In addition to water molecule(H 2 O), plant aquaporins can transports;](https://reader035.vdocuments.pub/reader035/viewer/2022070502/568136ef550346895d9e82ae/html5/thumbnails/2.jpg)
Tissue and cellular structures are not to scale
chloroplast
cytoplasm an aquaporin in the plasma-membrane of a mesophyll cell
cell wallsintercellular space
leaf surface with stomata
CO2 固定の律速段階 Limitting steps for CO2 fixation
RuBisCO 炭酸固定酵素の効率
Internal conductance葉内透過性細胞へ(原形質膜で)の透過
Stomatal conductance気孔透過性
![Page 3: In addition to water molecule(H 2 O), plant aquaporins can transports;](https://reader035.vdocuments.pub/reader035/viewer/2022070502/568136ef550346895d9e82ae/html5/thumbnails/3.jpg)
• CO2 permeability
CO2
Aquaporin
• Oocyte + micro-pH electrode
Micropipette PIP cRNA
Carbonic-anhydrase: CA ( enzyme)
Micro pH electrode
Voltage electrode
CO2 → HCO3- + H+ (pH 変化検出 )
Ulreich et al. 2003 NtAPQ1• Transgenic plants + CO2 conductance
Need high throughput system → New yeast system
Our group 2004 HvPIP2;1, gas exchange and carbon isotope (C13/C12) ratio
3
CA
(Reported)
![Page 4: In addition to water molecule(H 2 O), plant aquaporins can transports;](https://reader035.vdocuments.pub/reader035/viewer/2022070502/568136ef550346895d9e82ae/html5/thumbnails/4.jpg)
Screening system to identify aquaporins transporting CO2.
CA - EGFP アクアポリン遺伝子
酵母を用いたCO2 透過性スクリーニング系。ターゲットのアクアポリンがCO2を透過させれば、カーボニックアンヒドラーゼ(CA)が重炭酸イオンを生成して細胞内が酸性化して、pH感受性GFPの発光強度変化で検出できる。 0
1020304050607080
480 500 520 540 560nm
RFU
0 min5 sec30 sec1 min2 min
Acetic acid
Model examination
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
4
Aquaporin genes (Gal-inducebile)
![Page 5: In addition to water molecule(H 2 O), plant aquaporins can transports;](https://reader035.vdocuments.pub/reader035/viewer/2022070502/568136ef550346895d9e82ae/html5/thumbnails/5.jpg)
Experimental data
Time (sec)
450
460
470
480
490
500
CA-EGFPHvPIP2;1T228M HvPIP2;1
0 0.2 0.4 0.6 0.8 1
Positive control (NtAQP1) 1 sec after addition of NaHCO3
Fluo
resc
ence
蛍光
強度
(任
意単
位)
→
ΔFΔt
F
NaHCO3
Fluo
resc
ence
蛍光
強度
(任
意単
位)
→
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)
![Page 6: In addition to water molecule(H 2 O), plant aquaporins can transports;](https://reader035.vdocuments.pub/reader035/viewer/2022070502/568136ef550346895d9e82ae/html5/thumbnails/6.jpg)
genes (ΔF/F)/sec genes (ΔF/F)/sec genes (ΔF/F)/sec genes (ΔF/F)/sec
No PIPs 0.34 ± 0.05 OsPIP2;1 0.43 ± 0.04 HvPIP1;1 0.50 ± 0.06 HvPIP2;1 0.43 ± 0.06
OsPIP2;2 0.37 ± 0.04 HvPIP1;2 0.37 ± 0.07 HvPIP2;2 0.34 ± 0.04
NtAQP1 0.43 ± 0.05 OsPIP2;3 0.33 ± 0.04 HvPIP1;3 0.31 ± 0.08 HvPIP2;3 0.43 ± 0.07
OsPIP2;4 0.34 ± 0.03 HvPIP1;4 0.37 ± 0.02 HvPIP2;4 0.35 ± 0.06
OsPIP1;1 0.33 ± 0.05 OsPIP2;5 0.35 ± 0.03 HvPIP1;5 0.35 ± 0.07 HvPIP2;5 0.31 ± 0.04
OsPIP1;2 0.32 ± 0.03 OsPIP2;6 0.33 ± 0.04
OsPIP1;3 0.36 ± 0.05 OsPIP2;7 0.38 ± 0.05 T228M HvPIP2;1 0.33± 0.03
OsPIP2;8 0.33 ± 0.04
イネ (Os) PIP1;1PIP1;2
PIP1;3
PIP2;1
PIP2;2
PIP2;3
PIP2;4
PIP2;5
PIP2;6
PIP2;7
PIP2;8
TIP1;1
TIP1;2
TIP2;1
TIP2;2
TIP3;1
TIP4;2
TIP5;1
NIP2;1
NIP2;2
NIP2:3
NIP3;2
NIP3;3
CO2 透過性 X X X ○ X X X X X X X X X X ○ X X X X X X X X
オオムギ (Hv) PIP1;1PIP1;2
PIP1;3
PIP1;4
PIP1;5
PIP2;1
PIP2;2
PIP2;3
PIP2;4
PIP2;5
TIP1;1
TIP1;2
TIP2;1
TIP2;2
TIP2;3
TIP3;1
TIP4;1
TIP5;1
NIP1;1
NIP1;2
NIP2;1
CO2 透過性 ○ X X X X ○ X ○ X X ○ X X X X ○ X X ○ X X
( 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
![Page 7: In addition to water molecule(H 2 O), plant aquaporins can transports;](https://reader035.vdocuments.pub/reader035/viewer/2022070502/568136ef550346895d9e82ae/html5/thumbnails/7.jpg)
• Si permeability
(Si は耐病性と収量を向上させる)
Lsi1 →positional cloning→ OsNIP2;1
![Page 8: In addition to water molecule(H 2 O), plant aquaporins can transports;](https://reader035.vdocuments.pub/reader035/viewer/2022070502/568136ef550346895d9e82ae/html5/thumbnails/8.jpg)
pYES2HvNIP1;1HvNIP1;2HvNIP2;1HvNIP2;2OsNIP1;1OsNIP2;1OsNIP2;2OsNIP3;1OsNIP3;2OsNIP3;3OsNIP4;1
No As(OH)3 5μM As(OH)3
Growth of yeast ΔACR3 expressing various NIP aquaporins
As(OH ) 3
Acr3 yeast assay
Aquaporins transporting As(OH)3
Acr3 Lethal
• Arsenite (As(OH)3) permeabiliyu
Oocytes assay (PNAS 105;9931)
LSi1=OsNIP2;1Lsi6=OsNIP2;2
![Page 9: In addition to water molecule(H 2 O), plant aquaporins can transports;](https://reader035.vdocuments.pub/reader035/viewer/2022070502/568136ef550346895d9e82ae/html5/thumbnails/9.jpg)
0 mM H2O2 0.25 mM H2O2
0.5 mM H2O2 0.75 mM H2O2
pYES2( ベクター)
HvPIP2;1
HvPIP2;3
HvPIP2;4
HvPIP2;5
HvTIP2;2HvPIP2;2
→ HvPIP2;5 and HvTIP2;2 probably transport H2O2
• H2O2 peramiablity H2O2 sensitive ( Δskn7) yeast assay
H2O2 signaling in plants is essential for response to stress defense against pathogens and the regulation of programmed cell death.
![Page 10: In addition to water molecule(H 2 O), plant aquaporins can transports;](https://reader035.vdocuments.pub/reader035/viewer/2022070502/568136ef550346895d9e82ae/html5/thumbnails/10.jpg)
H2O, other molecules
アクアポリン研究のアウトプットアクアポリン研究のアウトプットPossible ApplicationsPossible Applications
Improving yield/quality in flowers and fruits
Crop improvements via aquaporin engineering(activity, expression regulation…..)
Two examples
Enhancement of growth and stress tolerances
Aquaporins
![Page 11: In addition to water molecule(H 2 O), plant aquaporins can transports;](https://reader035.vdocuments.pub/reader035/viewer/2022070502/568136ef550346895d9e82ae/html5/thumbnails/11.jpg)
OsNAC6 PIP2
PIP2
乾燥ストレス誘導的に発現抑制(PIP2を RNAi で抑制)乾燥ストレス(脱水)耐性強化イ
ネ ?
OsPIP2;4 ・・・ Lpr ↑( Previously descried )
Rice plants overexpressing PIPs
OsPIP1;1 ・・・ salt resistance ↑ seed germination ↑(PPB 63:151, 2013)
HvPIP2;1 Lpr ↑ but salt sensitive( PCP 44:1378, 2003 ) RNAi (Rice) against PIP2s
PIP expression ・・・ water uptake
Tolerance?
Down-regulation
Drought
Preventing dehydration
Stress-inducedRNAi
Not Yet Performed ( まだできていない)
![Page 12: In addition to water molecule(H 2 O), plant aquaporins can transports;](https://reader035.vdocuments.pub/reader035/viewer/2022070502/568136ef550346895d9e82ae/html5/thumbnails/12.jpg)
シロイヌナズに導入
アサガオに導入
みずみずしい花持ちの良い花き?
高糖度で日持ちの良い果実?
PIP2
InMYB1
花弁特異的に発現抑制( PIP2 を RNAi で抑制)
PIP2
トマトに導入
PIP2CaM 35S
全身で過剰発現(リン酸化を mimic した PIP2 )
P P 生育旺盛に?(うまく行かない) Not good
PIP2全身でに発現抑制
( PIP2 のアンチセンス鎖)
CaM 35S
果実、花卉( Flowers and fruits)
恒常活性型
恒常抑制型
名古屋大学共同研究Nagoya university
![Page 13: In addition to water molecule(H 2 O), plant aquaporins can transports;](https://reader035.vdocuments.pub/reader035/viewer/2022070502/568136ef550346895d9e82ae/html5/thumbnails/13.jpg)
PIP2
InMYB1
花弁特異的に発現抑制( PIP2 を RNAi で抑制)
PIP2
PIP2Knockdown
WT
In MYB1 geneアサガオ InMYB1 上流 1 kb
「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?
![Page 14: In addition to water molecule(H 2 O), plant aquaporins can transports;](https://reader035.vdocuments.pub/reader035/viewer/2022070502/568136ef550346895d9e82ae/html5/thumbnails/14.jpg)
Model plant forfruit study
MicroTom
PIP2
全身でに発現抑制( PIP2 のアンチセンス鎖)
CaM 35S
形質転換体16 系統, 41 個体
No difference to date
![Page 15: In addition to water molecule(H 2 O), plant aquaporins can transports;](https://reader035.vdocuments.pub/reader035/viewer/2022070502/568136ef550346895d9e82ae/html5/thumbnails/15.jpg)
Summary and future of aquaporin researchSummary and future of aquaporin research
T-DNAAnti-sense /RN
Ai
Expression(mRNA)
Homo-tetramer Hetero-tetramer
vs
Hydraulic conductivity
Expression (Protein)
Molecular engeneering
• Water stress tolerant crops• High quality of flowers and fruits• Improvements of plant functions
Overexpression
Functional analyze
Mutants, transgenic
Interaction(heteromeriozation)
Profiling, physiological analysis
イネの根組織および細胞
免疫前血清 Anti-OsPIP2;1 Anti-OsPIP2;5
OsPIP1;3( 根 ) OsPIP1;3( 根 ) OsPIP1;1( 根 )
CO2 permeability Si permeability
Intracellular trafficking
Structural analysisMutated aqauporins
1/16
1/8
1/4
1/2
1
2
4
1 h 2 h 4 h 8 h 12 h 24 h
はるな二条 200 mM NaCl
Rela
tive
ratio
HvPIP1;1
HvPIP1;2
HvPIP1:3
HvPIP1;4
HvPIP1;5
HvPIP2;1
HvPIP2;2
HvPIP2;3
HvPIP2;4
HvPIP2;5