Arabidopsis Aquaporins
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Arabidopsis Aquaporins. Slide 1. AtTIPC. AtTIP2C. TIP. AtTIPK. AtTIPH. AtTIPD. AtTIPI. AtTIP2G. AtTIPG. Clustal-X/TreeView. AtMIPH. AtMIPK. AtTIPA. AtTMP2c. AtTIPB. AtMIPL. AtPIP2a. PIP. GLPGF E. coli. AtMIPG. AtPIP3. AtMIPI. AtNLM6. AtPIP1a. AtPIP1b. AtMIPF. AtTMPB.

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Arabidopsis Aquaporins

Slide 1

AtTIPC

AtTIP2C

TIP

AtTIPK

AtTIPH

AtTIPD

AtTIPI

AtTIP2G

AtTIPG

Clustal-X/TreeView

AtMIPH

AtMIPK

AtTIPA

AtTMP2c

AtTIPB

AtMIPL

AtPIP2a

PIP

GLPGF E. coli

AtMIPG

AtPIP3

AtMIPI

AtNLM6

AtPIP1a

AtPIP1b

AtMIPF

AtTMPB

AtMIPM

AtNLM14

BoMIP

AtNLM3

AtNLM1

NLM-A

AtNLM2

AtNLM12

AtNLM10

AtNLM9

AtNLM5

AtNLM4

AtNLM13

NLM-B

0.1

AtNLM11

Quigley F (Arizona)


Quigley F (Arizona)

Slide 2

Location of AQP Genes on Chromosomes

10

20

30

5

Mb

TMPB

TIPB

NLM14

TIPL

TIPA

NLM4

Ch-I

(15)

MIPI

NLM8

TIPK

NLM7

NLM3

MipH

PIP1b

Ch-II

(4)

rDNA

TIPg

MIPL

TMP2c

NLM13

NLM6

TIPD

TIP2G

TIPI

PIP2a

NLM11

Ch-III

(14)

MIPK

PIP1a

MIPF

TIPH

NLM5

TIPC

NLM1

NLM2

MIPM

PIP3

Ch-IV

(3)

NLM12

NLM9

NLM10

TIP2c

MIPG

Ch-V

(12)

- duplicated chromosome portions with AQPs.

* - NLM7 and TipL encode for 3 transmembrane domains.

Loci identified by the same color scheme encode closely related genes.

- position of centromers (Mb from end).


Slide 3

Exon-Intron Structure of Arabidopsis AQP Genes

NLM-B

++

#

NLM-A

LB

LE

TIP

H1

H2

H3

H4

H5

H6

*

+

**

+

PIP

NPA

NPA

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 absent from TIPh.

+Intron sliding in MIPg (appears where intron 2 in TIPs is found).

#Intron absent from NLM14.

++Intron absent from NLM3 & NLM5.

Quigley F (Arizona)


Slide 4

Structure of the putative Pseudogene TIPL*

NLM-B

++

#

NLM-A

TIP

*

**

PIP

H1

H2

H3

H4

H5

H6

NPA

NPA

TIPL

H4

H5

H6

STOP

TIP sub-family

ATG

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*

++

#

H1

H2

H3

H4

H5

H6

*

**

NLM-B

NPA

NPA

ATG

NLM-A

Attila

Tn-like sequence

fragment

H4

H5

H6

H3

NLM7

TIP

frame shift, homology continues

PIP

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*

++

#

H1

H2

H3

H4

H5

H6

*

**

NLM-B

NPA

NPA

STOP

NLM-A

H1

H2

H3

H5

H6

NLM8

Different reading frame

ATG

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.

TIP

PIP

NLM-A sub-family

*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

AtTIP2g

at3g26530

ORF

9 bp target site duplication TTAAAAAAA

Chromosome 3 BAC, MFE16;

MULE-23 (Mutator-like element, group 23*, 12,268 bp)

1Kb

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

MIP-A

41kD

TO

PM

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.

*

*

MIP-B

32 kD

#

MIP-C

26 kD

To - tonoplast; PM - plasma membrane.

Antibodies raised against peptides.

MIP-A, B, C align with PIPs.

MIP-F aligns with TIPs.

MIP-F

33 kD

*dimer forms (~41 kD)

#degradation product

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

Analysis of

tonoplast vesicles

using stopped-flow

photometric

determination of

light-scattering.

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)


1 Na+

1 Na+; 0.3 K+

0.3 K+

50 nA

2.5 min

1 Na+

1 Na+; 0.3 K+

0.3 K+

50 nA

5 min

Slide 11

Conductance of a Na+/K+-Transporter in Xenopus Oocytes

water injected oocyte

cRNA injected oocyte

Carlos Muñoz-Garay and Omar Pantoja (IBT, UNAM, Cuernavaca, Mexico)


Slide 12

The flux of deuterated

water into and out of

corn roots, +/- 150 mM NaCl,

was measured by NMR.

_____

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

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

OC03F03 - WCP-III

3.0

Complete recovery after 7d (+)

2.0

+1.6-fold

1.0

0.0

PIP-AQP

15 minutes

-1.6-fold

-1.0

1 hour

after

NaCl

shock

3 hours

-2.0

6 hours

24 hours

1 week

-3.0

10.5

11.5

12.5

13.5

14.5

15.5

Arrays can report

strength &

relative strength of

expression/abundance

Log(2) Signal Intensity

Decline

after

15 min ( )

Kawasaki et al (U. Arizona, 2001)


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