Slide 1. Institute of Plant Nutrition and Soil Science

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1 Proteome analysis and ph sensitive ratio imaging: Tools to explore the decline in leaf growth under salinity K. H. Mühling, B. Pitann, T. Kranz, C. M. Geilfus and C. Zörb ph MW Slide 1

2 Plant growth under salinity Increasing salt concentration (NaCl) in growing medium induces: Ca 2+ -defficiency symptoms on younger leaves (1st phase) Na + -toxicity symptoms on older leaves (2nd phase) shoot growth reduction (1st phase) control 1 mm NaCl treatment 100 mm NaCl Slide 2 Physiological mechanisms of growth reduction under salt stress are yet not fully understood!

3 1. Physiological traits for the decline in leaf growth under salinity Is the Oertli hypothesis still alive? Vacuole Apoplast Cytosol Slide 3

4 Na + flow from soil to leaf Na + Na + [Na + ] Leaf Na + Slide 4 Root modified after Epstein, Science (1998)

5 Na + toxicity Oertli hypothesis: Na + accumulation in the leaf apoplast Decline in water potential Inhibition of water uptake into leaf symplast Dehydration of cells and turgor loss Rolling of leaves Slide 5

6 Methodological efforts Indirect method (in vitro): Isolation of apoplastic leaf fluids by infiltrationcentrifugation method Detection with ion chromatography Direct method (in vivo): Ratio imaging technique Na + -sensitive fluorescence indicators Slide 6

7 Intra- and Intercellular [Na + ] 80 5 In tracellular r Na + (mm M) NaCl treatment (mm) Inte ercellular r Na + (mm M) NaCl treatment (mm) Slide 7 Excluders like maize and wheat do not accumulate Na + in the leaf apoplast modified after Mühling & Läuchli (2002)

8 Growth reduction Shoot height of faba beans (A), maize cv. Pioneer 3906 (B) and the maize hybrid SR3 (C) under increasing salinity sh hoot heigh ht [cm] sensitive cultivar resistant it tcultivar 100 (A) (B) (C) shoot height [cm m] 0 C C C salt treatment [mm NaCl] Slide 8 control 25 mm 50 mm 75 mm 100 mm 125 mm control 25 mm 50 mm 75 mm 100 mm 125 mm 150 mm 175 mm Kranz et al. (unpublished)

9 2. ph sensitive ratio imaging Acid growth theory: Decline in apoplastic ph is the major requirement to increase cell wall extensibility, which controls extension growth (Hager, 1971, 2003). Slide 9

10 Pump activity Plasma membrane Plasma membrane K + K + K + K + K + K + K + Na+ Na + K + N Na+ + Na + ATP *** H + H + H + ATP *** H + Slide 10 without salt with salt

11 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 activity i mg -1 min -1 ] ATPase [µmol P mm NaCl control A A Mg-ATP 125 mm NaCl control gramicidine Slide 11 time (min) Zörb et al., JPNSS (2005)

12 Acid growth theory (Hager et al., 1971, 2003) What are the potential causes for a reduced growth? Plasma membrane-atpase-activity is reduced apoplastic ph is increasing Cytoplasm ph 7.5 Plasma membrane ATP ADP + P i Apoplast ph 5.0 H + H + H + Slide 12 no activation of cell wall bound proteins (e.g. expansins) Pitann, Schubert & Mühling: JPNSS (2009)

13 Methodological efforts Direct method (in vivo) Ratio Imaging tio 490/440 nm rat in vivo in vitro Regr. Conf. 95% ph Slide 13

14 ph in the leaf apoplast 3 ph-sensitive fluorescent dyes (FITC, Oregon Green, FTMR) ph-sensitive microelectrodes 6,0 5,5 Salt-sensitive maize a b Salt-resistant maize A A 6,0 5,5 5,0 4,5 Pioneer ,0 4,5 4,0 4,0 ph 3,5 3,5 3,0 SR ,0 Slide 14 NaCl Treament [mm] Pitann, Kranz & Mühling: Plant Sci. (2009)

15 Leaf growth and apoplastic ph Relationship between apoplastic ph and leaf length of Pioneer 3906 ( ) and SR03 ( ) Le eaf length of 7th leaf [cm] SR03 Pioneer 3906 R 2 = R 2 = Slide ,0 61 6,1 62 6,2 63 6,3 64 6,4 65 6,5 ph Kranz et al. (unpublished)

16 Spatial apoplastic ph gradients Distribution of apoplastic ph in maize leaves stomatal area control (1 mm NaCl) 6.2 treatment (100 mm NaCl) 6.7 non-stomatal area Slide 16 control treatment Pitann, Kranz & Mühling: Plant Sci. (2009)

17 ph MW 3. Proteome analysis under abiotic stress Slide 17

18 Proteome analysis Maize leaf: control 100 mm NaCl 1) single gel + 2) average gel 3) Overlay (zoom) Slide 18

19 Proteome analysis Overlay of spots control and NaCl treatment 2D 3D view Slide 19

20 Proteome analysis Quantification Control kda 220 ph Modification in protein pattern Down-regulated Treatment Up-regulated Disappeared Control vs. Saltstress [%] Newly synthesized Total changes Slide 20 Kontrolle Behandlung Pitann, Zörb & Mühling: JPNSS (2009)

21 Proteome analysis - Identification Protein Peptide Intensitä ät Masse / Ladung N- Metabolism: methionine synthase, glutamate ammonium ligase, methionine adenosyl transferase 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, adenosine kinase Slide 21 Zörb et al., Plant Sci. (2004)

22 Proteomics - Expansins Expansins are known to be acid-activated and have the unique property of cell wall- loosening below ph 5 Slide 22 Cosgrove et al. (2000)

23 Proteome analysis - Expansins 1 mm NaCl 100 mm NaCl 220 kda ph 3 6,5 10 A ph 3 6,5 10 B 220 kda 50 kda 50 kda 20 kda 20 kda Spot Nr. Protein Accession MW [kda] pi Pioneer 3906 Salt SR03 Salt ph ph [1] ß-Expansin 2 25 kda Cgi D kda ß-expansin 2 ß-expansin 2 Slide kda 20 kda Pitann, Zörb & Mühling: JPNSS (2009)

24 Acid growth theory (Hager et al., 1971, 2003) What are the potential causes for a reduced growth? Plasma membrane-atpase-activity is reduced apoplastic ph is increasing Cytoplasm ph 7.5 Plasma membrane ATP ADP + P i Apoplast ph 5.0 H + H + H + Slide 24 no activation of cell wall bound proteins (e.g. expansins)

25 Proteome analysis Subcellular - Apoplast Expansins are wall-loosening proteins, located within the apoplast of the elongation zone of leaves (Cosgrove, 2000). ph kda red border indicates region of interest for expansin protein isoforms (~22 kda) Slide Shahzad et. al. (unpublished)

26 Post-translational modification: Phospho-Proteomics Phospho proteom changes (2D GE, Phos Tag stain, fluorescent) Phos Tag a chelate with unparalleled selectivity for the phosphomonoesters h of tyrosine, serine and threonine phosphorylation under 100 mm NaCl Slide 26 Zörb et. al. (unpublished)

27 Phospho-Proteomics Proteins changed under short-term t salt treatment t t IEP (ph) t Mole ecular weigh Green: up-regulated: 11 proteins: (voltage dependent anion channel, sucrose synthase, phosphoglycerate kinase, fructokinase,.) Blue: down-regulated: 8 proteins: ( like 3 protein, thioredoxine, HSP91, ) Red: phosphorylated: 7 proteins: (calmodulin, maturase K, 40S-riosomal protein S9, ) Black: dephosphorylated: 5 proteins: (glucosyl transferase, fructokinase, triose phospate isomerase, xyloglycane endotransglycosylase, telomerase elongation inhibitor) Slide 27 see Poster Zörb et. al. (unpublished)

28 Apoplastic proteins Initial phospho-proteins changed under 1 h short term osmotic treatment in maize leaves (100 mm NaCl) Phosphorylated: 7 proteins - calmodulin - maturase K - 40S-riosomal protein S9 -. Dephosphorylated: 5 proteins - glucosyl transferase - triose phosphate isomerase Slide 28 -xyloglycane l endo transglycosylase (XET) - telomerase elongation inhibitor -. Cosgrove (1998) Zörb et. al. (unpublished)

29 Conclusion Shoot growth reduction caused by salinity can directly be related to an increase 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 sensitive genotypes. Therefore, the dramatic decrease of the ß-expansin under salt stress may be discussed as a trigger for the lower leaf biomass production. stress adaptation resistance Stress is not an exceptional circumstance but part of life! (J. Czichos in Larcher, 1987) Slide 29

30 Acknowledgment Slide 30

31 Collaborators WE LOVE SALINITY!!! Prof. Dr. S. Schubert Justus Liebig University Institute of Plant Nutrition (PM H + ATPase) Prof. Dr. U. Schurr Institute of Phytosphere Research Center Jülich (Digital imaging of leaf growth) Prof. Dr. H. Felle Justus Liebig University Institute of Botany (ph sensitive microelectrodes) Slide 31

32 End of lecture Slide 32