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Figure 6. Summary of alkaligrass chloroplast phosphoproteome A. Twenty-eight homologous genes of Na 2 CO 3 -responsive phosphoproteins exhibited diverse expression patterns In order to evaluate the gene expression patterns of the Na 2 CO 3 -responsive phosphoproteins, 28 homologous genes were analyzed through quantitative real-time PCR qRT-PCR analysis with ubiquitin as an internal control Figure S7 , Table S Immunodetection of seven representative Na 2 CO 3 -rsponsive proteins To further evaluate the protein abundances of representative photosynthetic proteins under Na 2 CO 3 treatment, Western blotting was conducted using available antibodies.

Figure 7. Validation of eight alkali-responsive chloroplast proteins with immunoblotting Eight chloroplast proteins from plants under different treatment conditions [0 mM, mM for 24 h 24 HAT , and 24 HAT] were loaded with equal amounts. Figure 8. Discussion Diverse photoprotection mechanisms to counteract Na 2 CO 3 -induced photoinhibition Photosynthesis modulation is critical for plant stress response. Conclusion Although NaCl-responsive mechanisms have been well-studied in various halophytes using proteomics approaches [4] , the Na 2 CO 3 -responsive proteins and corresponding regulatory mechanisms in halophytes were rarely explored.

Materials and methods Plant material treatment and biomass analysis Seeds of alkaligrass [ Puccinellia tenuiflora Turcz. Membrane integrity, osmolytes, and ABA analysis The malondialdehyde content and relative electrolyte leakage were determined using previous methods [68] , [69]. Photosynthesis and chloroplast ultrastructure analysis Photosynthesis and Chl fluorescence parameters were measured using previous methods [25] , [69].

Chloroplast isolation, protein extraction and purity assessment Intact chloroplasts were isolated according to Ni and the colleagues [74] , and the chloroplast protein for iTRAQ and phosphoproteomics analysis was extracted according to Wang and the colleagues [75]. Phosphoproteomic analysis of proteins from chloroplasts and leaves After digestion, the chloroplast peptide samples of control, 24 HAT, and 24 HAT were labeled with stable isotope dimethyl labeling in light, intermediate, and heavy, respectively, according to the method of Boersema and the colleagues [79].

Western blot analysis Western blotting was conducted according to Dai and the colleagues [76]. Competing interests The authors have declared no competing interests.

Notes Handled by Yu Xue. Supplementary material The following are the Supplementary data to this article: Supplementary Figure S1. Supplementary Figure S2. Supplementary Figure S3. Supplementary Figure S4. Supplementary Figure S5. Supplementary Figure S6. Supplementary Figure S7. Supplementary Figure S8: Schematic presentation of Na 2 CO 3 responsive mechanisms in leaves from alkaligrass The Na 2 CO 3 responsive proteins from both leaf and chloroplast proteomes were integrated into subcellular pathways.

Click here to view. Supplementary Figure S9: Workflow of plant treatment and sampling Seeds of Puccinellia tenuiflora were sowed on vermiculite and grown in Hoagland solution in pots.

Supplementary Table S1: Click here to view. Supplementary Table S2: Click here to view. Supplementary Table S3: Click here to view. Supplementary Table S4: Click here to view. Supplementary Table S5: Click here to view.

Supplementary Table S6: Click here to view. Supplementary Table S7: Click here to view. Supplementary Table S8: Click here to view. Supplementary Table S9: Click here to view. Supplementary Table S Click here to view. References 1. Yang C. Osmotic adjustment and ion balance traits of an alkali resistant halophyte Kochia sieversiana during adaptation to salt and alkali conditions. Plant Soil. Comparative effects of salt-stress and alkali-stress on the growth, photosynthesis, solute accumulation, and ion balance of barley plants.

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