Transcript No Slide Title - stress

Arabidopsis Aquaporins
AtTIPI
Slide 1
AtTIPK AtTIPD
AtTIPC AtTIP2C
AtTIPH
AtTIP2G
AtTIPG
AtMIPH
AtMIPK
AtTMP2c
AtMIPL
AtPIP2a
PIP
TIP
Clustal-X/TreeView
AtTIPA
AtTIPB
GLPG F E. coli
AtMIPG
AtPIP3
AtMIPI
AtPIP1a
AtPIP1b
AtMIPF
AtTMPB
AtNLM6
AtMIPM
AtNLM14
BoMIP
AtNLM3
AtNLM1
AtNLM2
AtNLM12
AtNLM10
AtNLM9
AtNLM4
AtNLM13
NLM-B
Quigley F (Arizona)
AtNLM5
0.1
AtNLM11
NLM-A
Slide 2
Quigley F (Arizona)
Location of AQP Genes on Chromosomes
10
5
Mb
TMPB
TIPB
20
NLM14
30
TIPL
TIPA
NLM4
(15)
Ch-I
MIPI
Ch-II
NLM8
TIPK
NLM7
NLM3
MipH
PIP1b
(4)
rDNA
TIPg MIPL TMP2c
NLM13
NLM6
TIPD
TIP2G
TIPI
Ch-III
MIPF
Ch-IV
(14)
TIPH
NLM5
TIPC
NLM1 NLM2
MIPM
PIP3
MIPK
PIP1a
(3)
NLM12
Ch-V
PIP2a NLM11
NLM9
NLM10
TIP2c
MIPG
(12)
* - NLM7 and TipL encode for 3 transmembrane domains.
Loci identified by the same color scheme encode closely related genes.
- duplicated chromosome portions with AQPs.
- position of centromers (Mb from end).
Slide 3
Exon-Intron Structure of Arabidopsis AQP Genes
NLM-B
++
#
NLM-A
H1
TIP
PIP
H2
LB
H3
H4
H5
H6
NPA
NPA
*
LE
+
**
+
Arrows show the position of introns within aquaporin sub-classes
H1-6 are transmembrane segments.
LB, LE: Loops B and E with conserved N-P-A motif.
*Intron absent from TIPc, TIP2c, TIPg, TIP2g and TIPh.
+Intron
++Intron
**Intron absent from TIPh.
sliding in MIPg (appears where intron 2 in TIPs is found).
absent from NLM3 & NLM5.
#Intron absent from NLM14.
Quigley F (Arizona)
Slide 4
Structure of the putative Pseudogene TIPL*
NLM-B
++
#
NLM-A
H1
TIP
PIP
H2
H3
H5
H6
NPA
NPA
*
**
TIPL
TIP sub-family
H4
H4
ATG
H5
H6
STOP
H3 missing, truncated AQP with 2 TM, no EST reported.
*We had considered NLM14 a Pseudogene before, but closer inspection indicates that is an authentic gene.
Quigley F (Arizona)
Slide 5
Structure of the putative Pseudogene NLM7*
NLM-B
++
#
NLM-A
H1
TIP
PIP
NLM7
H2
H3
H4
H5
H6
NPA
NPA
*
**
ATG
Attila
Tn-like sequence
fragment
H3
H4
H5
H6
frame shift, homology continues
NLM-A sub-family
Protein start after H3; frame-shift (sequence error?)
Would lead to short peptide, but sequence homology
Continues (5’ and 3’ including the C-terminus;
no ESTs reported
*We had considered NLM14 a Pseudogene before, but closer inspection indicates that is an authentic gene.
Quigley F (Arizona)
Slide 6
Structure of the putative Pseudogene NLM8*
NLM-B
++
#
NLM-A
H1
TIP
PIP
H2
H3
H4
H5
H6
NPA
NPA
*
**
STOP
NLM8
H1
H2
H3
H5
H6
Different reading frame
ATG
NLM-A sub-family
Predicted longer N-terminal sequence;
TM H3,4, and 5 are missing;
Homology exists but a different reading frame is used
Protein with longer C-terminal end.
No ESTs reported.
*We had considered NLM14 a Pseudogene before, but closer inspection indicates that is an authentic gene.
(Computer prediction missed Exon 1 - but close inspection indicates its presence - no ESTs reported.
Quigley F (Arizona)
Slide 7
AtTIP2g is close to a Mutator-like Element
ORF
mudrA
ORF
at3g26530
AtTIP2g
ORF
1Kb
9 bp target site duplication TTAAAAAAA
Chromosome 3 BAC, MFE16;
MULE-23 (Mutator-like element, group 23*, 12,268 bp)
The region 5’ of the ATG to the insertion site of MULE-23 is 178 bp in length.
* Zhihui Yu, Stephen I. Wright and Thomas E.Bureau, Genetics 156: 2019-2031 (2000)
Quigley F (Arizona)
Slide 8
Mesembryanthemum AQPs are found in different Locations
membrane localization
TO
PM
MIP-A
*
41kD
*
MIP-B
32 kD
#
MIP-C
MIP-F
26 kD
33 kD
To - tonoplast; PM - plasma membrane.
Antibodies raised against peptides.
MIP-A, B, C align with PIPs.
MIP-F aligns with TIPs.
*dimer
Even in the unstressed
state some PIPs are
found in more than
one membrane.
Analysis by continuous
sucrose gradient
fractionation confirmed
this fact (using antibodies
to markers of PM, To,
mitochondria, chloroplasts,
nuclei and the
endoplasmic reticulum
Under stress conditions
(drought, salinity, ABA)
the redistribution is
additionally affected.
Barkla et al., 1999
Kirch et al., 2000
Vera-Estrella et al., 2001
and unpublished data.
forms (~41 kD)
product
#degradation
Barkla, B, Vera-Estrella, R. & Pantoja, O. (UNAM, Mexico)
Slide 9
Expression of GFP
under control of the
Ice plant promoter MipB
in Arabidopsis.
For components of the vector
see
Grebenok et al., 1997.
The promoter is not active
in the primary root meristem
But in the elongation zone.
MipB is active,
as in the ice plant,
in lateral root meristems
in Arabidopsis.
Shuhua Yuan, MS thesis, (Arizona) 2001
Slide 10
We have been using the stopped flow photometer to determine the osmotic permeability of the
tonoplast from the halophyte Mesembryanthemum crystallinum, as well as changes induced by
salt and drought stress. The figure below shows original recordings of the changes in light scattering
resulting from the imposition of a 100 mOsmol osmotic gradient (hyperosmotic) or under iso-osmotic
conditions (iso-osmotic) in tonoplast vesicles from M. crystallinum leaves. The rate constant (kos)
was obtained by fitting a single exponential to the curve, from where we have determined a Pos
0.6
hyperosmotic
(100 mOsmol)
Light Intensity (au)
0.5
0.4
0.3
0.2
0.1
Analysis of
tonoplast vesicles
using stopped-flow
photometric
determination of
light-scattering.
iso-osmotic
0.0
-0.1
0.0
0.2
0.4
0.6
0.8
1.0
Time (s)
of 1205 µm s -1, a value twice that of tobacco cell cultures (Maurel et al., 1997) and 14-times higher than
that for wheat roots (Niemietz and Tyerman, 1997). From similar experiments carried out at different
temperatures, we have calculated a value for the activation energy for water movement across the tonoplast
vesicles of M. crystallinum of 8.97 kJ/mol -1 K -1, a smaller value than that reported for the tonoplast of wheat
Roots (23.32 kJ mol -1 K -1) and tobacco cell cultures (10.47 kJ mol-1 K -1).
Vera-Estrella, R. & Pantoja, O. (UNAM, Mexico)
Slide 11
Conductance of a Na+/K+-Transporter in Xenopus Oocytes
1 Na+
1 Na+; 0.3 K+
0.3 K+
50 nA
water injected oocyte
2.5 min
1 Na+
1 Na+; 0.3 K+
0.3 K+
50 nA
cRNA injected oocyte
5 min
Carlos Muñoz-Garay and Omar Pantoja (IBT, UNAM, Cuernavaca, Mexico)
Slide 12
Internal Concentration as a Fraction of External
Influx and Efflux of Deuterium Tracer to Follow Water Movement in Zea Maize
Normal Tissue
Fit of Normal Tissue
Stressed Tissue
Fit of Stressed Tissue
The flux of deuterated
water into and out of
corn roots, +/- 150 mM NaCl,
was measured by NMR.
_____
1.2
1
0.8
0.6
0.4
0.2
0
0
500
1000
1500
Time (sec)
Exodermal
Layer
2000
2500
3000
The analysis, using models
for N-layers (N-= 1, 2, etc.),
indicated a major influence
of the endodermis and
trans-cellular flux of water
through all cells of the cortex
as the most likely route for
water movement.
______
In stress tissues water flux is reduced
most likely by reduced amounts of AQPs
See: Rosenberg et al. (2000)
ASPP Annual Meeting, abstract #454
Endodermal
Layer
Rosenberg, J & Shachar-Hill, Y (New Mexico State)
Slide 13
Rice AQPs - Salt Shock
Water Channel ESTs - Time Course in Rice
OC104E01 - WCP-I
Decline & recovery
of transcripts
during NaCl stress
Water channels
(1 PIP; 3 TIP)
OC03G03 - WCP-II
OC104A02 - WCP-IV
3.0
OC03F03 - WCP-III
2.0
1.0
Complete recovery after 7d (+)
+1.6-fold
0.0
PIP-AQP
-1.6-fold
-1.0
-2.0
6 hours
24 hours
1 week
-3.0
10.5
Decline
after
15 min (
11.5
12.5
13.5
14.5
Log(2) Signal Intensity
)
15 minutes
1 hour
after
3 hours
Kawasaki et al (U. Arizona, 2001)
15.5
NaCl
shock
Arrays can report
strength &
relative strength of
expression/abundance