Recent progress in aquaporin research

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??????
2
Recent progress in aquaporin research

• ???? (expression) ???? (among genes)
• ??/????? (tissue/development specific)
• ????????? (stress response) ?????
  (transgenic)
• ???? (activity) ???? (phosphorylation) ???????
  (intermolecular interaction) ????? (cellular
  trafficking)
• ???????? (phenotype)

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???? Expression
(Normalize to control (0 mM NaCl))
???????PIP??????10??mRNA?
Mild (100 mM) stress ? No changes Severe (200 mM)
stress ? Mostly decrease
var. Haruna-nijyo (salt tolerance middle) 5-day
old seedlings (PCP 52663)
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However, in root hydraulic conductivity (Lpr)
(?????)
Salt tolerant var. K305 Salt sensitive var. I743
(4 hr after salt stress)
Pressure chamber
Sap flow Jv Jv /A Lpr (?p - Y) A total
root surface area ?p applied pressure Y
Yielding pressure
??????PIP?????????PIP ?????????? ????????????
????? (SSPN 5750)
5
33??????????? ?????? (?????)
PIP11 PIP12 PIP13 Related to cold stress PIP21 house keeping (CO2) PIP22 PIP23 PIP24 Sensitive to water stress PIP25 rescue PIP26 internodes panicle neck PIP27 PIP28
TIP11 Related to the flooding TIP12 TIP21 Lateral root TIP22 internodes panicle neck (CO2) (H2O2) TIP31 Putative ABRE in promoter (TIP32) TIP41 TIP42 TIP43 TIP51
NIP11 (NIP12) NIP13 NIP14 NIP21 (Si) (As) (H2O2) NIP22 (Si) (As) NIP31 (B) NIP32 (As) (H2O2) NIP33 (As) (H2O2) NIP41
SIP11 SIP21
???????? (gt105 copies/ µg total RNA)
??, ?????????
????????
?????
?????
?????
??????????????
??????????
RiceXPro http//ricexpro.dna.affrc.go
.jp/ ???? OsMIP family??? ? ?
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???????PIP (?????????????)
?????????????????PIP???
AtPIP22, 23, 25, 28
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????????? (?????????)
RT-PCR
????????????? SiPIP23, 25??
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?????????????(??/???)
Indirect immunofluorescense (???????)
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(???)
(?PIP1s??)
??
??
???
??
????
??
(?PIP22??)
(Overlay)
(PCP 52663)
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(??PIP?????) ???? (????)
AtSIP?????????? ? ER
????? Anti-OsPIP21 Anti-OsPIP25
OsPIP21?OsPIP25???????????(???)???????
???? (??????)
OsPIP13(?) OsPIP13(?) OsPIP11(?)
????anti-Kar2 (Bip)?anti-SIP1s ??anti-(rabbit
IgG) linked with Alexa488
OsPIP11?OsPIP13?????????????ER??????
???? (??????)
?????????????(?????????)????????????????? ????
????????????????
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????????? (Stress response)
?????????OsTIPs??? (??? Deepwater rice) ??
????????????????(??) ????
?? ????
? ? ?
? ? TIP???? ? ????
??????(??)???
????????????
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?????(Temperature)
?? High temperature (??)
??PIP13????????
???????PIP23????????
13
?? (??????)
?????????????????????
????
??? 5ºC 5ºC Shoot ??? 25ºC 5ºC Root
? ????????????????(??)????????
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???????????????? ? ???????? ??????????????????????
????? (???Figleaf Gourd(????????)???)
Days at low temp. Days
at low temp.
?????? ?????? (Western blotting)
Chilling sensitive
Chilling tolerant
????????????????? ??????(?????) ? ???????????? ?
???????????????? ? ?????
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????????
????Phosphprylation???
????????????????? (??)
????(??)??????? Fruit riping
0.8
0.6
??????? Sutaurosporine (kinase inhibitor)
LPr 10-6 ms-1 MPa-1
??????Lpr?????(????)
0.4
0.2
??????????????????????
0
DMSO () 0.1 0.1 0.1 0.1
St (µM) - 0.1 1.0 10.0
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??????
?????????? Flowering
????

• ????????
• ????(????????)???
• ???????????????
• ?????????????????
• (???????????????)

• ?????????

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??????? heteromerization
(First reported in Maize, Plant Cell 16215. 2004)
(?????????????????)
HvPIPs Single injection
HvPIP2s (21 25)
????
HvPIP12 or other HvPIP1s and negative control
????
Co-injection of HvPIP1s and HvPIP2s
(????)
???
Pf????????(????)??????????
RNA?????????
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????????????? ???????? ? N????????
N?
1?
2?
Pf ( x 10-2 cm/s)
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• Co-expression of HvPIP12 and HvPIP24 activated
  each others by two means
• Translocation to the plasmamembrane
• Direct activation

Inactive HvPIP21 T229M can activate PIP21 via
heteromerization
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Inactive HvPIP24 T229M can activate PIP12 via
heteromerization
Inactive HvPIP12 T238M can activate PIP24 via
heteromerization
PIP24T229M ---- Thr 229 is replaced by Met. lt
Loss of water transport activitygt
PIP12T238M ---- Thr 238 is replaced by Met. lt
Loss of water transport activitygt
21
?????? (???/????????)
????????? ????????? (????)
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???????? Internalization/Recycling
Barley Lpr after 100 mM NaCl (Time course)
Salt tolerance low
Salt tolerance middle
Salt tolerance high
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Root hydraulic conductance (Lpr) is regulated by
phosphorylation and possible trafficking of
aquaporins in early phase of salt/osmotic stress
Lpr of Haruna-nijo after salt (100 mM NaCl) or
osmotic (177 mM sorbitol) stress
Isotonic sorbitol induced identical Lpr change
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Root hydraulic conductance (Lpr) is regulated by
phosphorylation and possible trafficking of
aquaporins in early phase of salt/osmotic stress
Lpr of Haruna-nijo after salt (100 mM NaCl) or
osmotic (177 mM sorbitol) stress
Exocytosis inhibitor (brefeldin A BFA )
inhibits temporal recovery of Lpr at 4 h after
stress
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Root hydraulic conductance (Lpr) is regulated by
phosphorylation and possible trafficking of
aquaporins in early phase of salt/osmotic stress
Lpr of Haruna-nijo after salt (100 mM NaCl) or
osmotic (177 mM sorbitol) stress
Proteasome inhibitor (MG132 ) inhibits reduction
of Lpr after stress gt 4 h
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Barley Lpr after 100 mM NaCl ot isotonic
sorbitol/manitol
?? ????
??????? (???????) ??
??????????? (1??) ?????
??????????? (4??) ?????
?????? (4????) ??
????????? (?????4????) ??
??????????????????/????????????????????????
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Model
Salinity (Osmotic) stress
Non-stress
Osmotic stress signal
H2O
PIP aquaporin
OA
(lt4h)
(gt200 mM NaCl)
(gt4h)
Activated
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100 mM NaCl
Early Effects of Salinity on Water Transport in
Arabidopsis Roots. Molecular and
Cellular Features of Aquaporin Expression.
Boursiac et al. Plant Physiology (2005) 139790
Standard sol.
45 min
120 min
???????? ????
GFP-LTP
PIP11-GFP
PIP21-GFP
Stimulus-induced downregulation of root water
transport involves reactive oxygen
species-activated cell signalling and plasma
membrane intrinsic protein internalization. Boursi
ac et al. The Plant Journal (2008) 56, 207218
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Fluorescence recovery after photobleaching
reveals high cycling dynamics of plasma membrane
aquaporins in Arabidopsis roots under salt
stress. Luu et al. Plant Journal (2012)69894
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Selective regulation of maize plasma membrane
aquaporin trafficking and activity by the SNARE
SYP121. Plant Cell (2012)24 3463
CFP or FM4-64
YFP
merge
mYFPZmPIP25 FM4-64
mYFPZmPIP25 mCFPPMA2(PM marker) Co-expression
mYFPZmPIP25 mCFPAtSYP121-Sp2 (??AtSYP121) Co-e
xpression
SNARE Soluble N-ethylmaleimide-sensitive factor
attachment protein receptor
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Nature Cell Biology 14(10), 991-998 (2012)

• Background
• Aquaporin determines Lpr.
• Total root water uptake depends on root
  architecture and amount (including root hair).
• Auxin changes root formation.
• How about relationship between PIPs and Auxin?

Nature 446, 621-622 (2007)
Auxin response factor (ARF) proteins function as
transcription factors controlling
auxin-responsive genes. ARF7 plays a key role
during lateral root formation and emergence.
arf7 loss-of-function mutant ARF7
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Bending induced lateral root formation
Most PIPs decreased after bending.
AtPIPs????
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Bending
PIP amounts water relations
Auxin
Receptor mutant (arf7)
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Lpr-h Root hydraulic conductivity
(root pressure chamber) Lpr-o Root osmotic
conductivity (natural sap exudation)
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GUSPIP21 transcript accumulation mCHERRYPIP
21 protien accumulation mirror image to Auxin
accumulation
IAA reduced PIP21 expression and induce lateral
root formation
Auxin inhibitor increased PIP21 expression and
reduced lateral root formation
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(No Transcript)
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(No Transcript)
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?????
???????
Transgenic rice Expression analysis ltqRT-PCRgt

(WT Nipponbare 24-day old)
Absolute quantification
Internal standarads eEF-1aEukaryotic elongation
factor 1-alpha UBC
Ubiquitin-conjugating enzyme E2

• Mutant lines of OsPIP24
• Overexpression
• Antisense
• T-DNA (POSTECH, Korea)

Acknowledgement The vector for the transgenic
rice plants were kindly provided by Dr. Ichikawa
(NIAS) and Dr. Horiguchi (NIBB).
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Total OsPIP mRNAs in roots and Lpr in OsPIP24
overexpressor
VS. WT
Lpr

OsPIP24
Plt0.05

NOTE Less increases of total OsPIP mRNAs.
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Expressions of OsPIPs in roots of OsPIP24
transgenic lines
? ? ? ? ?
Root specificity
PIP11 PIP12 PIP13 PIP21 PIP22
PIP23 PIP24 PIP25 PIP26 PIP27
PIP28 eEF-1a UBC
x6.4
Plt0.05 Plt0.001
96
OsPIP24
Compensative change against OsPIP24 mRNA.
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Root specificity Root specificity Root specificity ? ? ? ? ?
Changes in roots Wild-type(WT) Osmotic stress /WT Non-stress ? ? ? ? ? ? ? ?
Changes in roots PIP24 T-DNA line Non-stress /WT Non-stress ? ? ? ? ? ? ? ? ? ?
Changes in roots PIP24 Overexpression line(OE) Non-stress /WT Non-stress ? ? ? ? ? ?
Changes in roots PIP24 Overexpression line(OE) Osmotic stress /PIP24 OE Non-stress ? ? ? ? ?
Up-regulation (Plt0.05) Down-regulation(Plt0.05)

• Expression of root specific OsPIPs were
  co-regulated with OsPIP24.

Simultaneous down-regulation under osmotic
stress. Compensative regulation against OsPIP24
in transgenic lines.
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Root spesificity Root spesificity Root spesificity ? ? ? ? ?
Changes in roots Wild-type(WT) Osmotic stress /WT Non-stress ? ? ? ? ? ? ? ?
Changes in roots PIP24 T-DNA line Non-stress /WT Non-stress ? ? ? ? ? ? ? ? ? ?
Changes in roots PIP24 Overexpression line(OE) Non-stress /WT Non-stress ? ? ? ? ? ?
Changes in roots PIP24 Overexpression line(OE) Osmotic stress /PIP24 OE Non-stress ? ? ? ? ?
Up-regulation (Plt0.05) Down-regulation(Plt0.05)

• Expression of root specific OsPIPs were
  co-regulated with OsPIP24.

OsPIP13 may compensate OsPIP24 activity
partially.
Co-expression increased the activity in oocyte
system. (Modified after Matsumoto et al. PCP
50216 (2009))