Proteomic Response of Hordeum vulgare cv. Tadmor and Hordeum marinum to Salinity Stress: Similarities and Differences between a Glycophyte and a Halophyte

Abstract

Response to a high salinity treatment of 300 mM NaCl was studied in a cultivated barley Hordeum vulgare Syrian cultivar Tadmor and in a halophytic wild barley H. marinum. Differential salinity tolerance of H. marinum and H. vulgare is underlied by qualitative and quantitative differences in proteins involved in a variety of biological processes. The major aim was to identify proteins underlying differential salinity tolerance between the two barley species. Analyses of plant water content, osmotic potential and accumulation of proline and dehydrin proteins under high salinity revealed a relatively higher water saturation deficit in H. marinum than in H. vulgare while H. vulgare had lower osmotic potential corresponding with high levels of proline and dehydrins. Analysis of proteins soluble upon boiling isolated from control and salt-treated crown tissues revealed similarities as well as differences between H. marinum and H. vulgare. The similar salinity responses of both barley species lie in enhanced levels of stress-protective proteins such as defense-related proteins from late-embryogenesis abundant family, several chaperones from heat shock protein family, and others such as GrpE. However, there have also been found significant differences between H. marinum and H. vulgare salinity response indicating an active stress acclimation in H. marinum while stress damage in H. vulgare. An active acclimation to high salinity in H. marinum is underlined by enhanced levels of several stress-responsive transcription factors from basic leucine zipper and nascent polypeptide-associated complex families. In salt-treated H. marinum, enhanced levels of proteins involved in energy metabolism such as glycolysis, ATP metabolism, and photosynthesis-related proteins indicate an active acclimation to enhanced energy requirements during an establishment of novel plant homeostasis. In contrast, changes at proteome level in salt-treated H. vulgare indicate plant tissue damage as revealed by enhanced levels of proteins involved in proteasome-dependent protein degradation and proteins related to apoptosis. The results of proteomic analysis clearly indicate differential responses to high salinity and provide more profound insight into biological mechanisms underlying salinity response between two barley species with contrasting salinity tolerance.

Keywords: Hordeum marinum; Hordeum vulgare; glycophyte; halophyte; proteome; salinity; stress acclimation; stress damage.

FIGURE 1

Water saturation deficit (WSD; A), proline accumulation (B), osmotic potential (ψ_s; C) and maximum quantum yield of photosystem II photochemistry (F_v/F_m; D) determined in leaf tissues of Hordeum marinum (black columns) and H. vulgare cv. Tadmor (gray columns). Different letters above the columns indicate significant differences between the variants determined by ANOVA, multiple comparisons, Duncan’s multiple range test at 0.05 level.

FIGURE 2

A representative immunoblot (A) and a graph showing the results of densitometric analysis of dehydrin protein relative accumulation (sum of all dehydrin bands detected in a given sample; B). M-H. marinum (black columns), V-H. vulgare cv. Tadmor (gray columns). C, crown; L, leaf; Mk, protein marker All Blue Precision Plus Protein Marker Standard (Bio-Rad), St - internal standard (a mixed sample containing equal amounts of all samples used in the experiment and loaded on each gel). (B) Dehydrin protein relative abundance in the individual samples was expressed as fold changes with respect to the internal standard St. Different letters above the columns indicate significant differences between the variants determined by ANOVA, multiple comparisons, Duncan’s multiple range test at 0.05 level. (C) Proteins transblotted onto nitrocellulose membrane were visualized by staining with Ponceau S (Sigma–Aldrich).

FIGURE 3

Venn diagrams showing the number of identified proteins in all experimental variants studied (A; HVC, Hordeum vulgare, control treatment; HVN, H. vulgare, 300 mM NaCl; HMC, H. marinum, control treatment; HMN, H. marinum, 300 mM NaCl) and Venn diagrams showing identified proteins revealing significant differences in their relative protein abundance between control and salt-treated plants (B) and between H. vulgare and H. marinum genotypes (C). (B,C) the number of proteins showing significant differences between the experimental variants is indicated. Differences were evaluated by unpaired Student’s t-test at 0.05 level.

FIGURE 4

Cluster analysis of 108 proteins identified in at least two experimental variants except for the variants HVC ∩ HMC (21), HVN ∩ HMN (15), HVC ∩ HMN (4), and HVN ∩ HMC (0). HVC, H. vulgare, control treatment; HVN, H. vulgare, 300 mM NaCl; HMC, H. marinum, control treatment; HMN, H. marinum, 300 mM NaCl.

FIGURE 5

Functional classification of identified proteins according to biological process. The upper diagram shows functional classification of all 284 identified proteins in the experiment while the four lower diagrams show functional classification of the identified proteins in the individual experimental variants.

FIGURE 6

A schematic summary of plant response to high salinity (300 mM NaCl) in crown tissues of H. vulgare, a glycophyte, and H. marinum, a halophyte. ↑ means an increased protein relative abundance in salt-treated variant with respect to control while ↓ means a decreased protein relative abundance in salt-treated variant with respect to control. The intensity of the arrows indicates quantitative differences in physiological characteristics and protein levels between H. vulgare and H. marinum. See the abbreviations list and the text for abbreviation explanation.