1. Volume 7, Issue 2, Pages 336-355 (February 2014)
Salinity Stress in Roots of Contrasting Barley Genotypes Reveals Time-Distinct and Genotype-Specific Patterns for Defined Proteins 
Katja Witzel, Andrea Matros, Marc Strickert, Stephanie Kaspar, Manuela Peukert, Karl H. Mühling, Andreas Börner, Hans-Peter Mock 
Molecular Plant 
Volume 7, Issue 2, Pages (February 2014)
DOI: /mp/sst063
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 Schematic of the Experimental Set-Up and Sampling Regime.
(A) Salinity stress was imposed on 6-day-old barley seedlings and NaCl concentration was increased in a step-wise manner until the desired salinity range was reached. The harvesting time points for ion content measurement and proteomic analysis are indicated by black boxes.
(B) Overview of the 2-DE analysis of root tissue.
Molecular Plant 2014 7, DOI: ( /mp/sst063)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
Development of Barley Seedlings cvs. Steptoe and Morex Grown in the Presence of NaCl.
(A) Assembled representative images of plants.
(B) The fresh weight (mg) of the second and third leaf after 10 d of treatment. Both leaves have developed during the salt-stress treatment. Values represent means ± SE of n = 20 plants, from one representative experiment. Asterisks indicate statistical differences between the respective control and treatment at P ≤
Molecular Plant 2014 7, DOI: ( /mp/sst063)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
Effect of NaCl Concentration and Exposure Time on the Leaf Content of Na+ (A), K+ (B), and Ca2+ (C).
Plants were harvested after 1, 4, 7, and 10 d of exposure to salinity. Data represent means and standard errors of three independent biological replicates. Asterisks indicate statistical differences between both genotypes at P ≤ 0.05.
Molecular Plant 2014 7, DOI: ( /mp/sst063)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
Representative 2-DE Gel from a cv. Morex Root, Indicating the Positions of Spots Identified by Mass Spectrometry.
(A) Positions of spots present only in cv. Steptoe profiles were estimated based on the surrounding spot patterns.
(B) Spot 198 (marked in (A)) was more abundant during salinity treatment in the salt-tolerant cv. Morex as compared to the salt sensitive cv. Steptoe. The differential expression of spot 198 was verified using immunoblotting (see Figure 7). Sample loading, separation, and protein visualization were as described in the ‘Methods’ section.
Molecular Plant 2014 7, DOI: ( /mp/sst063)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
Hierarchical Clustering Analysis and Functional Classification of Protein Expression Patterns.
(A) Columns represent 2-DE gels prepared from root samples of cvs. Steptoe and Morex plants grown in 0mM (control), 100mM, and 150mM NaCl for 1, 4, 7, or 10 d. Rows show means of normalized spot values from three biological and three technical replicates (n = 9), using color coding based on a relative scale (–3.0 to +3.0). Protein spots showing similar expression patterns have been grouped, and spot numbers are given for each row. The five distinguishable clusters are separated by white bars.
Molecular Plant 2014 7, DOI: ( /mp/sst063)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

7. Figure 6 Differential Protein Expression induced by Salinity Stress.
(A, C) Intersection of squares show relations in protein expression changes between time points and treatments obtained from the rank products test for each cultivar. Crossing points refer to the experimental sample (e.g. day one at 100 mM NaCl, located on the top left). Intersections with other conditions are labeled by letters A through Q. The two numbers appearing within an intersection area refer to the extent of the protein expression change: the number closest to the crossing point (proximal number) is the number of proteins up-regulated, while the distal one indicates the number of down-regulated proteins.
(B, D) Protein expression changes at selected intersections. Normalized spot values were extracted from SameSpots software. Black bars represent control and gray bars the salinity stress treatment.
Molecular Plant 2014 7, DOI: ( /mp/sst063)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

8. Figure 7
Heat Map Visualization of Protein Abundance Using 2-DE and Immunoblotting for Selected Salinity Responsive and Genotype-Specific Proteins.
Blots were probed with the HMGB2 antibody from Arabidopsis (Launholt et al., 2006), a Catharanthus roseus HDS antibody (Oudin et al., 2007), and the Arabidopsis antibody raised against flavodoxine-like quinone reductase FQR1 (Laskowski et al., 2002). Rows display the abundance of the protein spot on 2-DE gels as derived from normalized spot volumes (▲) (spots 85 and 120 for HMGB2, spot 137 for HDS, spot 198 for oxidoquinone reductase) and the signal intensity of the Western blot band determined by densitometric measurement (●) using color coding based on a relative scale (–3 to +3).
Molecular Plant 2014 7, DOI: ( /mp/sst063)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

9. Figure 8
Content of Endogenous Free and Total Salicylic Acid (SA), of Jasmonic Acid, and of Abscisic Acid in Leaves and Roots of Both Cultivars under Control (0mM NaCl) and Salinity Stress (150 mM NaCl) Conditions.
Samples were analyzed from three independent cultivations in three technical replicates each. Mean values from the three independent cultivations are displayed. Statistically significant values were determined by Student’s t-test and are marked with * for p < 0.05, with ** for p < 0.01, and with *** for p <
Molecular Plant 2014 7, DOI: ( /mp/sst063)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

10. Figure 9
Schematic Representation of Salt-Tolerance Mechanisms in Barley cv. Morex.
Molecular Plant 2014 7, DOI: ( /mp/sst063)
Copyright © 2014 The Authors. All rights reserved. Terms and Conditions