Proteome analysis and pH sensitive ratio imaging: Tools to ...ipnc.ucdavis.edu/powerpoints/R313_0828_1435_Muehling.pdf · Shoot height of faba beans (A), maize cv. Pioneer 3906 (B)

Proteome analysis andProteome analysis andpH sensitive ratio imaging: pH sensitive ratio imaging: Tools to explore the decline Tools to explore the decline in leaf growth under salinityin leaf growth under salinity

K. H. Mühling, B. Pitann, T. Kranz,K. H. Mühling, B. Pitann, T. Kranz,C. M. Geilfus and C. Zörb C. M. Geilfus and C. Zörb

pH

Slide 1

MW

Institute of Plant Nutrition and Soil Science

Slide 1

Plant growth under salinity

Increasing salt concentration (NaCl) in growing medium induces: Ca2+-defficiency symptoms on younger leaves (1st phase) Ca defficiency symptoms on younger leaves (1st phase) Na+-toxicity symptoms on older leaves (2nd phase) shoot growth reduction (1st phase)

control treatment

Slide 2

control 1 mM NaCl

treatment 100 mM NaCl

Physiological mechanisms of growth reductionunder salt stress are yet not fully understood!


Slide 2 under salt stress are yet not fully understood!

1. Physiological traits for 1. Physiological traits for the decline in leaf growth the decline in leaf growth under salinity under salinity Vacuole

Is the Oertli hypothesis still alive? Is the Oertli hypothesis still alive? Apoplast

Cytosol

Slide 3


Slide 3

Na+ flow from soil to leaf

Na+

Leaf[Na+]

Na+

Na+

Slide 4 Root


Slide 4 Root

modified after Epstein, Science (1998)

Na+ toxicity

Oertli hypothesis:Na+ accumulation in the leaf apoplast

Decline in water potential Inhibition of water uptake into

leaf symplastleaf symplast Dehydration of cells and

turgor loss Rolling of leaves

Slide 5

Rolling of leaves


Slide 5

Methodological efforts

Indirect method(in vitro): Isolation of

apoplastic leaf fluids by infiltration-centrifugation method

Detection with ion chromatography

Direct method

1

6

7

4 3 2

5

(in vivo): Ratio imaging

technique Na+-sensitive

Slide 6

3

8

fluorescence indicators


Slide 6

Intra- and Intercellular [Na+]

80M

)5

M)

40

60

r N

a+ (mM

3

4

r N

a+ (m

M

20

40

trac

ellu

lar

1

2

erce

llula

r

00 25 50 75 100

In

0

1

0 25 50 75 100In

te

Slide 7

NaCl treatment (mM) NaCl treatment (mM)

Excluders like maize and wheat do not accumulate Na+ in the leaf apoplast


Slide 7

modified after Mühling & Läuchli (2002)

Shoot height ofShoot height of faba beansfaba beans (A), maize cv.(A), maize cv. Pioneer 3906Pioneer 3906 (B) and(B) and

Growth reduction

Shoot height of Shoot height of faba beansfaba beans (A), maize cv. (A), maize cv. Pioneer 3906Pioneer 3906 (B) and (B) and the maize hybrid the maize hybrid SR3SR3 (C) under increasing salinity(C) under increasing salinity

iti lti i t t lti

ht [c

m]

60

80

100

shoot

60

80

100

30

40

50(A) (B) (C)

sensitive cultivar resistant cultivar

hoot

hei

gh

20

40

60 height [cm20

40

60

10

20

30

salt treatment [mM NaCl]

sh

C 25 50 75 100 125 150 1750

C 25 50 75 100 125 150 175

m]

0C 25 50 75 100 125

0

Slide 8


Slide 8

control 25 mM 50 mM 75 mM 100 mM 125 mM 150 mM 175 mM 125 mM 75 mM 100 mM control 25 mM 50 mM

Kranz et al. (unpublished)

2. pH sensitive ratio 2. pH sensitive ratio imagingimagingimaging imaging

Acid growth theory:g yDecline in apoplastic pH is the major requirement to increase cell wall extensibility, which controls

extension growth (Hager, 1971, 2003).

Slide 9


Slide 9

Pump activity

Plasma membrane Plasma membrane

N +

Plasma membrane

- +

- +K+

Plasma membrane

- +

K+Na+

Na+K+

Na+

Na+

- +

K+

K+

K+

K+

K+

K+

- +

ATPH+ATP

- +

- +

Slide 10

H+H+

H+

***- +

- +***

- +


Slide 10

without salt with salt

Pump activity

Effect of salt treatment in vivo to the hydrolytic acitvity (left side) and the H+ pumping acitvity of the plasmalemma ATPase of maize leaves in vitro

0 00

0.04Mg-ATP

n-1]

0 9

1.2125 mM NaClcontrol

A A49

2

-0.04

0.00

control

125 mM NaCl

activ

ityim

g-1m

in

0.6

0.9

A

-0.08

gramicidine

control

ATPa

se

[µm

ol P

0.3

Slide 11 time (min)0 10 20 30 40 50

-0.12g

0.0


Slide 11 ( )

Zörb et al., JPNSS (2005)

Acid growth theory(Hager et al., 1971, 2003)

What are the potential causes for a reduced growth?

Plasma membrane-ATPase-activity is reducedreduced

CytoplasmpH 7.5

ApoplastpH 5.0

H+

H+

H+ATP

Plasma membrane

- +

- +

- +apoplastic pH is increasing H+

ADP + Pi

- +

- +

Slide 12

no activation of cell wall bound proteins (e.g. expansins)


Slide 12

Pitann, Schubert & Mühling: JPNSS (2009)

Methodological efforts

Direct method (in vivo) Direct method (in vivo) –– Ratio ImagingRatio Imaging

tio 4

90/4

40 n

m0.81.01.21.41.61.82.02.22.4 in vivo

in vitro Regr. Conf. 95%

pH5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0

rat

0.00.20.40.6

Slide 13


Slide 13

pH in the leaf apoplast

3 pH-sensitive fluorescent dyes (FITC, Oregon Green, FTMR) pH-sensitive microelectrodes

5,5

6,0

5,5

6,0

bA A

Salt-sensitive maize Salt-resistant maize

Pioneer 39064,5

5,0

5,5

4,5

5,0

5,5a

3,5

4,0

,

pH

3,5

4,0

,

Slide 14 NaCl Treament [mM]

SR 031 100

3,01 100

3,0


Slide 14 NaCl Treament [mM]

Pitann, Kranz & Mühling: Plant Sci. (2009)

Leaf growth and apoplastic pH

80

Relationship between apoplastic pH and leaf length ofPioneer 3906 ( ) and SR03 ( )

leaf

[cm

]

60

70

80

SR03

h of

7th

l

50

60R2 = 0.003

eaf l

engt

h

30

40

R2 = 0.56

Pioneer 3906

Slide 15 6 0 6 1 6 2 6 3 6 4 6 5

Le

10

20


Slide 15

pH

6,0 6,1 6,2 6,3 6,4 6,5

Kranz et al. (unpublished)

Spatial apoplastic pH gradients

treatment(100 mM NaCl)

control (1 mM NaCl)

Distribution of apoplastic pH in maize leavesDistribution of apoplastic pH in maize leaves

6.76.2stomatal area

( )( )

6.15.7non-stomatal

area

6.0

5.5

Slide 16 control treatment

4.0

5.0


Slide 16 control treatment

Pitann, Kranz & Mühling: Plant Sci. (2009)

pH

3 P t l i3 P t l i

MW

3. Proteome analysis 3. Proteome analysis

under abiotic stress…under abiotic stress

Slide 17


Slide 17

Proteome analysis

Maize leaf: control 100 mM NaCl1) single gel

+ 2) average gel

3) Overlay (zoom)

Slide 18


Slide 18

Proteome analysis

Overlay of spots control and NaCl treatment

2D 3D view

Slide 19


Slide 19

Proteome analysis – Quantification

pH 3 10kDa

220

Control

5050

13.19Down-regulated

Control vs. Saltstress[%]

Modification in protein pattern

20

Treatment

19.41Newly synthesized

19.05Disappeared

9.52Up-regulated

Slide 20KontrolleBehandlung

61.17Total changes


Slide 20

Pitann, Zörb & Mühling: JPNSS (2009)

Proteome analysis - Identification

ät

Protein

Masse / Ladung

Inte

nsitä

Peptide

N- Metabolism: methionine synthase, glutamate ammonium ligase, methionine adenosyl transferase

C Metabolism: rubisco fructose 1 6 bisphosphate aldolase C- Metabolism: rubisco, fructose 1,6 bisphosphate aldolase, glycerin aldehyde-3-phosphatase, ß-glucosidase, chloroplast ATPase…

Biosynthesis/protein modification: DNA poly II Ser/Thr kinase

Slide 21

Biosynthesis/protein modification: DNA poly. II, Ser/Thr kinase, adenosine kinase…


Slide 21

Zörb et al., Plant Sci. (2004)

Proteomics - Expansins

Expansins are known to be acid-activated and have the unique property of cell wall-loosening below pH 5loosening below pH 5

Slide 22


Slide 22

Cosgrove et al. (2000)

Proteome analysis - Expansins

220 kDa220 kDa

pH 3 6,5 10

100 mM NaCl1 mM NaCl

BA

pH 3 6,5 10

50 kDa50 kDa

20 kDa20 kDa

pH 5 6 7

25 kDa 25 kDaC D

pH 5 6 7

7.025gi|14193763ß-Expansin 2[1]

SR03SaltPioneer 3906Salt

pIMW

[kDa]AccessionProtein

SpotNr.

Slide 23 20 kDaß-expansin 2 ß-expansin 2

20 kD


Slide 23 20 kDa 20 kDa

Pitann, Zörb & Mühling: JPNSS (2009)

Acid growth theory(Hager et al., 1971, 2003)

What are the potential causes for a reduced growth?

Plasma membrane-ATPase-activity is reducedreduced

CytoplasmpH 7.5

ApoplastpH 5.0

H+

H+

H+ATP

Plasma membrane

- +

- +

- +apoplastic pH is increasing H+

ADP + Pi

- +

- +

Slide 24

no activation of cell wall boundproteins (e.g. expansins)


Slide 24

Proteome analysis – Subcellular - Apoplast

Expansins are wall-loosening proteins, located within the apoplastof the elongation zone of leaves (Cosgrove, 2000).

kDpH5 9kDa 5 9

90 -

red borderindicates

50 -region of interest for expansin protein

Slide 25 10 -

protein isoforms (~22 kDa)


Slide 25

Shahzad et. al. (unpublished)

10

Post-translational modification: Phospho-Proteomics

Phospho proteom changes (2D GE, Phos Tag stain, fluorescent)

Phos Tag a chelate with unparalleled selectivity for the h h t f t i i dphosphomonoesters of tyrosine, serine and

threonine

phosphorylation under 100 mM NaCl

Slide 26


Slide 26

Zörb et. al. (unpublished)

P t i h d d h t t lt t t t

Phospho-Proteomics

Proteins changed under short-term salt treatmentIEP (pH)

Green: up-regulated: 11

t

p gproteins: (voltage dependent anion channel, sucrose synthase, phosphoglycerate kinase, fructokinase, ….)Blue: down-regulated: 8

t i (14 3 3 lik t i

ecul

arw

eigh proteins: (14-3-3-like protein,

thioredoxine, HSP91,…)

Red: phosphorylated: 7 proteins: (calmodulin, maturase K 40S-riosomal

Mol

e maturase K, 40S riosomal protein S9, …)Black: dephosphorylated: 5 proteins: (glucosyl transferase, fructokinase, triose phospate isomerase, xyloglycane

Slide 27

endotransglycosylase, telomerase elongation inhibitor)

P t


Slide 27


see Poster

Apoplastic proteins

Initial phospho-proteins changed under 1 h short termosmotic treatment in maize leaves (100 mM NaCl)

Phosphorylated: 7 proteins

- calmodulin

- maturase K

- 40S-riosomal protein S9

- ….

Dephosphorylated: 5 proteinsDephosphorylated: 5 proteins

- glucosyl transferase

- triose phosphate isomerase

l l d

Slide 28

-xyloglycane endotransglycosylase (XET)

- telomerase elongation inhibitor


Slide 28 - ….


Cosgrove (1998)

Conclusion

Shoot growth reduction caused by salinity can directly be related to an increase of apoplastic pHincrease of apoplastic pH.

This is due to an inhibition of the plasma membrane H+-ATPase activity in salt-sensitive cultivars.

It was first demonstrated by proteom analysis that the activity of ß-expansins is reduced by 63% under salinity especially in sensitivesalinity especially in sensitive genotypes.

Therefore, the dramatic decrease of the ß-expansin under salt stress may be discussed as a trigger for the Stress is not an exceptional

stress adaptation resistance

Slide 29

be discussed as a trigger for the lower leaf biomass production. circumstance but part of life!

(J. Czichos in Larcher, 1987)


Slide 29

Acknowledgment

Slide 30


Slide 30

Collaborators

Prof. Dr. S. SchubertJustus Liebig UniversityInstitute of Plant NutritionInstitute of Plant Nutrition(PM H+ ATPase)

Prof. Dr. U. SchurrInstitute of Phytosphere

WE LOVE SALINITY !!!

Institute of Phytosphere Research Center Jülich(Digital imaging of leaf growth)

Prof Dr H Felle Prof. Dr. H. FelleJustus Liebig UniversityInstitute of Botany(pH sensitive microelectrodes)

Slide 31


Slide 31

End of lecture

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Slide 32

Documents

Proteome analysis and pH sensitive ratio imaging: Tools to ...ipnc.ucdavis.edu/powerpoints/R313_0828_1435_Muehling.pdf · Shoot height of faba beans (A), maize cv. Pioneer 3906 (B)