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環境応答生理学 2015
Physiology of Environmental Responses
(484208)
Class 6 (7-July, 2015)
江崎文一、且原真木(柴坂三根夫)
Ezaki, Katsuhara (Shibasaka)
植物のアクアポリン
(Plant aquaporins)
Aquaporin =
MIP (membrane intrinsic protein)
PIP(plasm-membrane…)
(原形質膜型)
TIP(tonoplast….)
(液胞膜型)
NIP(Nodulin26-like…)
SIP(small …) ER signaling?
XIP(x …)
シロイヌナズでは35個のMIP
(35 Major Intrinsic Protein in Arabidopsis)
(ヒトでは13個(human)、微生物は1から2個(bacteria))
XIPs are found in some plants (tomato,
cotton, moss) but functions are not yet
known
Why many in plants?
細胞の水透過性をきめる
細 Determining cell water permeability
胞
外
(
土
壌
)
水
環
境
水透過性高い:細胞質の体積を維持
High permeability: maintain cytoplasm
(Tyerman et al. J Exp Bot 50:1055, 1999)
液胞 Vacuole
•Wet
核
•Dry
•Salt stress
(variable)
nucleus
(細胞体積の90%以上)
(More than 90% volume)
細胞質(cytoplasm)
Plasma-membrane
Cell wall
Number of MIP (aquaporin) genes in the genomes (at 2014)
rice (Oryza sativa)
Arabidopsis thaliana
maize (Zea mays)
poplar (Populus trichocarpa)
soybean (Glycin max)
cotton (Gossypium hirsutum)
tomato (Solanum lycopersicum)
potato (Solanum tuberosum)
39
35
36
55
66
48
47
41
Redundancy, Different localization,
Developmental expression
Different function (substrate or stress response)
Seed maturation (formation and dehydration)
TIP3;1 and 3;2
(old name a- and b-TIP)
Germination (root emergency)
TIP1;1 and others
(old name g-TIP)
(root elongation)
TIP2;1(old name d-TIP)
PIP2;1 and other PIPs
PIPs in stomata (孔辺細胞のPIP)
Aquaporins in tomato
AtPIP2;2, 2;3, 2;5, 2;8
(in future)
Artificial regulation of stomatal movements
and fruit development via aquaporins
若い果実で多く発現するもの
(Young fruits)
Organ- and developmental-dependent expression analysis
Data from RiceXPro
OsPIP2;1 aquaporin
Generally expressed
OsTIP3;1 aquaporin
Seed specific
Different expression in tissue
How to detect the in tissue distribution?
Indirect immunofluorescense (間接蛍光抗体法)
Alexa 647 (red) conjugated
anti-rat IgG goat antibody
Anti-HvPIP1s rat antibody
(IgG)
(common among HvPIP1s)
HvPIP1s
Alexa 488 (green) conjugated
anti-rabbit IgG goat antibody
Anti-HvPIP2;1 (or 2;2) rabbit antibody (IgG)
(specific to each molecular species)
HvPIP2s
(明視野)
内皮
(抗PIP1s抗体)
根毛
中心柱
皮層
後生木部
表皮
(抗PIP2;2抗体)
(Overlay)
(PCP 52:663)
環境応答性 (Response to the environments)
Rice PIPs
OsPIP2;1
10
OsPIP2;5
8
6
4
2
0
-11
8
0
2
4
6
12
OsPIP2;1
10
OsPIP2;5
8
6
4
Submergence (冠水)
TIP expression ↑
Cell elongation
Node elongation (節間伸長)
2
0
2.0
-11
8
0
2
4
6
light , but evaporation-dependence
実は蒸散要求(湿度)に応答
Relative protein amount
12
(x 106 copies / 1μg total RNA)
Humid (RH=90%)
mRNA amounts
(x 106 copies / 1μg total RNA)
mRNA amounts
Dry (RH=40~50%)
冠水適応に関与するOsTIPsの同定
(浮イネ Deepwater rice)
OsTIP1:1
1.5
1.0
0.5
0
2.0
OsTIP2;2
1.5
1.0
0.5
0
N H A N H A
東北農業研究センター
1 day
3 days
Submergence
A:all 全冠水
H:Half 半冠水
N:non 無冠水
名古屋大
植物アクアポリンの多様な発現調節と活性調節
Various expressional and activity regulation in plant aquaporins
Tissue, organ, and developmental specificity
phosphorylation
= activation = opening
Aquaporin activity depends:
protein amount (expression)
gating (phosphorylation, pH, Ca,….)
heteromerlization
intracellular trafficking
ナシ果実の肥大 (pear fruit ripping)
Water
初期 early stage
Cell division
中期~後期 mid to late stages
Cell elongation with water absorption
TIP aquaporin・・・expression ↑
PIP aquaporin・・・constant expression
activation with phosphorylation
脱水耐性・・・・水透過性の制御(抑制)+浸透調節
Dehydration tolerance・・・Lpr regulation + osmotic regulation
Lpr depends on PIP regulation
<multi reactions>
• Gating via phosphorylation
(minutes)
• Internalization
(1-2 hours)
• Degradation and expression
(> several hours)
Kjellbom et al.
4:308-314(1999)
Standard sol.
Early Effects of Salinity on
GFP-LTP
Water Transport in
Arabidopsis Roots.
Molecular and Cellular
Features of Aquaporin
PIP1;1-GFP
Expression. Boursiac et al.
Plant Physiology (2005)
139:790
PIP2;1-GFP
Stimulus-induced downregulation of root
water transport involves reactive oxygen
species-activated cell signalling and plasma
membrane intrinsic protein internalization.
Boursiac et al. The Plant Journal (2008) 56,
207–218
100 mM NaCl
45 min
120 min
原形質膜マーカー
タンパク
Trafficking of Plant Plasma
Membrane Aquaporins: Multiple
Regulation Levels and Complex
Sorting Signals
Chevalier and Chaumont (2015)
PCP 58 819
チューリップの開花
Opening tulip flower (Azad)
• 低温 → 高温で開花
Low temp→high temp, then opening
• 花の基部の細胞が吸水
Flower cells (lower part) uptake water
• PIPの発現は恒常的
PIPs express constantly
• TgPIP2;2がリン酸化で高温で活性化
TgPIP2;2 activation by phosphorylation under high temp
貧栄養(Low minerals)
(Carvajal)
根のアクアポリン発現量が減少
(aquaporin expression ↓)
→ 根の吸水量減少 (root water permeability↓)
→ 地上部の成長抑制 (shoot growth↓)
葉の就眠運動 (leaf movements)
(Moshelion)
朝(day):アクアポリンの活性上昇 amount/ activity↑
→ 細胞の吸水増加(water influx) → 葉が開く
leaf open
夜(night):アクアポリンの活性低下 amount/ activity↓
→ 細胞が脱水 (deydration) → 葉が閉じる
leaf close
Substrates (輸送基質):
water
Glycerol
B(OH)3
Si(OH)4
As(OH)3
NH3
Lactate
CO2
H2O2
Low molecular weight, neutral
光合成の3つの律速段階とCO2透過性アクアポリンの関係
(photosynthesis and CO2-permeablr aquaporins)
大気
Air
CO 固定の律速段階
2
気孔の透過
Stomatal conductance
Stomata
気孔
CO2
細胞間隙
Intercellular space
炭酸固定酵素の効率
Cell
wall
葉肉細胞 mesophyll cells
原形質膜 Plasmamembrane
細胞質
アクアポリン Mesophyll condctance:
葉緑体
CO2 permeability via AQP
Chloroplast
RuBisCO activity
細胞壁
CO2 transport activity
using oocyte
External CO2/H2CO3濃度
PIP cRNA
Oocyte(卵母細胞)
CA
Micropipette
Carbonic-anhydrase
(CA)
CA
Micro pH electrode
(pH電極)
Aquaporin
Voltage
electrode
CO2
CA
-
CO2 → HCO3 +
H+
(pH変化検出)
(PCP 55: 251, 2014)
Salinity stress
Drought/osmotic stress
Ionic stress
Osmotic stress
(K+ deficiency/excess Na+
influx)
Aquaporin
Na+ toxicity
Inhibitions of:
photosynthesis
protein synthesis
enzyme activity
<Signal transduction>
Dehydration
Inhibitions of:
water uptake
cell elongation
leaf development
(Cell death)
Ion homeostasis
Na+ extrusion/compartmentation/
K+ reabsorption
Osmotic adjustment
Accumulations of ions/solutes/organic
compounds
Recovery/Adaptation
A schematic summary of the stresses that plants suffer and resultant
responses of plants to detrimental effects for survival under high salinity.
適合溶質(compatible solute)
→
• 浸透圧を上げる (osmotic)
• シャペロン活性 (chaperon)
• スカベンジャー活性 (scavenger)
細胞質に蓄積される特殊な有機物質
(special organics in the cytosol)
ベタイン(betain):本来はアミノ酸のアミノ基
に3つのメチル基が付いた化合物の総称。
H3N+-
→ (CH3)3N+-
もっとも存在量の多いグリシンベタイン(=
トリメチルグリシン)を、単にベタインと呼ぶ
ことも多い。
タバコ葉の例 (プロリン蓄積)
Proline accumulation in tobacco
グリシンベタインの合成系
塩ストレス誘導性
Salt-stress inducible
左: 150 mM NaCl
右: 150 mM NaCl + 5mM betaine aldelyde (external effective)
塩ストレス環境とイオン輸送系
その1(理論的側面と分子機構)
Salt stress and ion transport #1
(theory and molecular aspect)
細胞の構造と無機イオン輸送の関係
(Inorganic) Ion transport and cell structure
Nuc
Vacuole
Exclude
Isolation
Ion
Selection
ATP
Ion(low concentration)
Accumulation
Tolerant/sensitive determinants and transport system
• Tolerant?Sensitive?
Elements
(toxic materials)
標的
Target
Detoxification
Compartmentation
Cell/Individuals
Tolerant mechanisms: compartmentation, no absorption,
exclusion, to be insensitive
•Sensitive(感受性): Originally no tolerant mechanism
Inactivation of tolerant mechanism
→ Disturbance in metabolism and others
(Injory mechanism)
輸送の基本 ・・・ 濃度勾配(濃度差)
Movement ・・・ Gradient (Concentration)
「電気化学ポテンシャル」(electrochemical potential)とは
「濃度差と電位差の両方を考慮したもの」
(including both gradient of concentration and electric field)
+
+
+
+
+
+
- - -(minus charge)
---
+
+
+
+
+
+
+
+
+
10倍の濃度差と、59mVの電位差がつりあう
Balance between 10-hold concentration gradient and 59 mV
電気化学ポテンシャル(の正式)
Electrochemical potential (formula)
標準状態の化学ポテンシャル (standard chemical potential) μ*
気体定数 (Gas Constant) R 絶対温度 (Abs. Temp.) T 活量 (activity) a
粒子のイオン価 (charge No.) z ファラデー定数 (Faraday Const.) F
電位 (electric potential) φ
平衡電位 (Reversal (Equivalent) potential) Em or Erev
ネルンストの式: Nernst equation
膜内外で一価(下図の場合K+)が平衡しているときは・・・
 [K  ]in 
 RT  [M ]in 
  59 log   (mV )
Em 
ln
zF  [M ]out 
 [K ]out 
外側基準
(outer is zero standard)
K+ K + K+ K + K+
K+ K+ K+ K+ K+
K+
(In case of K+)
ポンプ (Pump)
エネルギーを直接使って、電気化学ポテンシャ
ル勾配に逆らって、物質を輸送する
(Against electrochemical gradient, using energy)
H+
H+ H+
H+
H+
ATP
ADP + Pi
H+
Example: H+ -ATPase
輸送速度は毎秒102個
(102 molecules per sec)
トランスポーター (Transporter)
ある物質X(たいていH+)の電気化学ポテン
シャル勾配を利用して、目的の物質Yを濃度勾
配に逆らって輸送する (Using electrochemical
gradient of X (H+in most case), transporting Y
against Y’s electrochemical gradient)
Na+
H+
H+
Na+
Na+
H+
+
H
+
Na
H+
H+
Na+
Na+
Na+H+
Example: Na+-H+ -antiporter
輸送速度は毎秒103個
(103 molecules per sec)
チャネル (channel)
一部のトランスポーター (Some Transporter)
目的の物質を、電気化学ポテンシャル勾配
濃度勾配にしたがって(促進的に)輸送する
(Transport X according to its electrochemical
gradient) +
K+
K+
- - - -
K
K+
K+
glucose
glucose
- - - -
K+
Example: K+ -channel
輸送速度は毎秒108個以上
(108 molecules per sec)
glucose
Example: Glucose transporter
輸送速度は毎秒103個
(103 molecules per sec)
細胞膜電位 (静止電位)
Membrane potential (Resting potential)
⋍ K+の平衡電位
(動物も植物も)
Em of K+
細胞膜電位 = 非起電性成分
Membrane potential
non-electrogenic ones
(細胞内の非移動性負電荷)
動物と植物は
ここが違う!
Plant unique
intracellular immobile negative charges
+
起電性成分(起電性ポンプ)
electrogenic ones (electrogenic pump)
Resting potential of living cells
Animal cells
Plant cells
Na+-K+-ATPase (electrogenic pumps) H+-ATPase
2K
+
H+
3Na
+
out
in
ATP
ADP+Pi
-70 ~ -90 mV
ATP
ADP+Pi
-100 ~ -200 mV
Exclusion of Na+(How to low [Na+]cyt)
Animal
High[Na+]outが基本
Plant
[Na+]out -freeが基本
Na+-K+-ATPase
2K+
3Na+
H+
out
in
No Na+-K+-ATPase except sea algae
(海産藻類は例外)
ATP
Na+
ADP+Pi
For exclusion of [Na+]cyt ・・・ Na+/H+ antiporter
二次輸送システムの基本的な違い
X
Animal
Plant
Using Na+ gradient
Using H+ gradient
Na+
symport antiport
(co-trasnport)
Y
X
H+
Y
Ion channel study using the electrophysiology
Ion flux was detected
as electric current
(cell recording)
従来の電気生理
(classical electrophysiology)
パッチクランプ
(Patch Clamp)
1991年
ノーベル賞
(Single channel recording)
Example of ATP-dependent cation channel
open
ion+
close
Erev
(逆転電位)
↑外向き電流
outward current
ion+
↓内向き電流
inward current
Outward current ・・・
+ ion efflux (外向きの流れ)
または
- ion influx(内向きの流れ)
Erev tells flux ions and selectivility
 [ K  ]in 
 RT  [M ]in 
  59 log   (mV )
(1) Erev 
ln
zF  [M ]out 
 [ K ]out 
 RT  Pk[ K  ]in  PNa[ Na ]in  PCl[Cl  ]out 

(2) Erev 
ln



F
 Pk[ K ]out  PNa[ Na ]out  PCl[Cl ]in 
 [ K  ]in  a[ Na ]in 

(3) Erev  59 log 

 [K ]out  a[ Na ]out 
PNa
a
, PCl  0
Pk
(1)イオンMの平衡電位(ネルンストの式)と、MがK+の場合
(2)3つの主要イオンで電位が決まる場合のGoldmanの式
(3)Cl-の透過性が低い場合、Na+とK+の選択性αが求められる
If Cl- permeabiliy (PCl) is low, selectivity between Na+ and K+ is calculated.
Erev
(逆転電位)
PNa/PK (α)= 0.28
(淡水産車軸藻の場合)
Na+はK+のチャネルを
通って細胞に入る
原形質膜
K+ out in
[K+]in = 100, [K+]out = 0.1,
[Na+]in = 0, [Na+]out = 100
(cyt side is “in”)
Na+
Ca2+
ATP
耐塩性の高い車軸藻(汽水産)の場合、αはほとんど
0、すなわちNa+はK+のチャネル通れず、細胞に入ら
ない (In blackish characae, α ≒0)
カリウムチャネルの分子構造
Molecular structure of K channel
放線菌のKcsAチャネル
Actinomycetes Kcs
(現代科学2004年1月
号より)
By Mackinonn
2003年ノーベル賞
K+ out in
Na+
Na-K co-transporter
Arabidopsis HKT
(Science 270:1660, 1995)
Na/K 共輸送
symport
「植物の膜輸送システム」
(秀潤社)より
Na-K co-transporter Arabidopsis HKT (Science 270:1660, 1995)
K+ out in
Na+
Arabidopsis HKT
X
300 mM NaCl入り培地
Na+透過に関与するのは
A240とL247
別のNa輸送システム(SOS系)
Salt stress sensitivity test/bending assay
inversion
5-day-old plant
without salt stress
wild type
Sensitive
Mutant
(SOS mutant)
by Dr. Zhu
salt stress for several days
SOS1-mutant
K+
Na+
KAT
AKT
HKT
・・・
SOS3 SOS2
Na+
X
NHE
Na+
H+
SOS1
(Na/H antiporter)
SOS2: CIPK24
SOS3: CBL4
AtHKT1とSOS1の植物体内で
の生理的役割