1. Volume 9, Issue 8, Pages 1183-1196 (August 2016)
A Putative Chloroplast-Localized Ca2+/H+ Antiporter CCHA1 Is Involved in Calcium and pH Homeostasis and Required for PSII Function in Arabidopsis 
Chao Wang, Weitao Xu, Honglei Jin, Taijie Zhang, Jianbin Lai, Xuan Zhou, Shengchun Zhang, Shengjie Liu, Xuewu Duan, Hongbin Wang, Changlian Peng, Chengwei Yang 
Molecular Plant 
Volume 9, Issue 8, Pages (August 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1 Molecular and Phenotypic Characterization of ccha1.
(A) Schematic representation of gene structures in ccha1. Untranslated regions (white boxes), exons (black boxes), and introns (bars) are shown. Arrowheads indicate T-DNA insertion sites. PCR primer sequences are shown in Supplemental Table 4.
(B) Phenotypic comparison of 4-week-old wild-type (Col-0), ccha1 mutant, and complemented (Com) seedlings. Bars represent 1 cm. RT–PCR analysis of CCHA1 expression in wild-type, ccha1, and complemented plants. Total RNA was extracted from 5-day-old wild-type, ccha1 mutant, and complemented seedlings, respectively. ACTIN was used as an internal control.
(C) Propidium iodide staining images of epidermal cells of wild-type Col-0, ccha1, and complemented seedlings. Images of palisade mesophyll cells of wild-type Col-0, ccha1, and complemented plants. Bars represent 100 μM.
(D) Growth kinetics of the ccha1 mutant compared with wild-type plants. Values shown are averages ± SE of six replicated experiments.
(E) Chlorophyll contents.
The data are means ± SD. **P < 0.01 and ***P < 0.001 (Student's t-test; compared with the corresponding wild-type control).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

3. Figure 2 Schematic Overview and Subcellular Localization of CCHA1.
(A) Schematic overview of putative domains and motifs in CCHA1 with its amino acid (AA) length. Members of the Uncharacterized Protein Family 0016 family are highly conserved. Solid pink boxes represent two conservative domain, in which two highly conserved internal E-x-G-D-(KR)-(TS) motifs are indicated by the two blue boxes (Motif 1 and Motif 2).
(B) Structure model of Arabidopsis CCHA1. The seven transmembrane spans are depicted.
(C) Localization of CCHA1 to the chloroplast. The fluorescence of CCHA1-YFP specifically coincides with chlorophyll autofluorescence. Bar represents 10 μM.
(D) Electron micrographs of chloroplasts from the ccha1 and wild-type leaves. TEM image of Col-0 (zoom in 8K and 20K) and ccha1 (zoom in 8K and 20K). To quantify the differences between wild-type and ccha1 chloroplasts, 50 chloroplast sections were analyzed from 4-week-old plants representing each genotype. 8K Bar = 1 μM, 20K Bar = 200 nM.
(E) Quantification of the normal chloroplast rate in the wild-type and ccha1 mutant.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

4. Figure 3
Characterization of Chlorophyll Fluorescence and P700 Redox Kinetics of Wild-Type, ccha1 Mutant, and Complemented Plants.
(A) Form 1 to 4-week-old plant (Col-0, ccha1 and Com plants) kinetics of Fv/Fm ratios.
(B and C) Light-response curves of PSII quantum yield and ETR in wild-type and ccha1 mutant plants. The measurements were performed at the following light intensities: 0, 100, 200, 300, 500, 750, and 1000 μmol photons m−2 s−1.
(D) The minimal level of fluorescence (F0) of dark-adapted whole plants with all PSII reaction centers open was determined by switching on a pulsed measuring beam of red light. The Fm level with all PSII reaction centers closed was determined by a saturating pulse in the dark-adapted leaves.
(E) The redox kinetics were investigated by measuring absorbance changes of P700 at 820 nm induced by far-red light (FR; 720 nm). AL, actinic light (120 μmol m−2 s−1).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

5. Figure 4
Analysis of Thylakoid Membrane Protein Accumulation in the Wild-Type, ccha1 Mutant, and Complemented Plants.
(A) BN gel analysis of thylakoid membrane protein complexes. Thylakoid membranes (10 mg of chlorophyll) from 3-week-old wild-type and ccha1 mutant leaves were solubilized with 1% dodecyl-β-D-maltoside and separated by BN gel electrophoresis.
(B) Immunodetection of chloroplast proteins. Thylakoid membrane proteins from the wild-type (Col-0), ccha1 mutant, and rescued plants were separated by 15% SDS–PAGE and electroblotted to PVDF membranes. Samples were loaded on an equal chlorophyll basis. Cytb6/f, cytochrome b6/f complex; ATPase, ATP synthase complex; ATPB, the b subunit of ATP synthase complex. Similar results were obtained with four independent biological replicates.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

6. Figure 5
A Chimeric Version of CCHA1 Partially Suppresses the Ca2+ Sensitivity of the Yeast gdt1Δ Mutant; Measurement of Chloroplast H+-ATPase Activity.
(A) Expression of a truncated version of CCHA1, the Arabidopsis ortholog of Gdt1p, partially suppresses the Ca2+ sensitivity of the yeast gdt1Δ mutant. Wild-type (WT) cells and the gdt1Δ mutant transformed with empty pYES260 plasmid; pYES260-expressing CCHA1 and Gdt1p N-terminal signal peptide fused to the C-terminal of CCHA1 ( AA) (Δ155CCHA1) were precultured in minimal medium without uracil (SD-U) to an OD600 of 0.3, then serial 10-fold dilutions were dropped onto solid SD-U medium supplemented with 900 mM CaCl2 and the plates incubated at 28°C for 4–6 days.
(B) The different mutants were grown in rich liquid medium (YD) to an OD600 of about 0.15, and then adjusted to the same density in liquid YD medium (up) or liquid YD medium supplemented with high concentration of CaCl2 (900 mM, down). OD600 was measured at the indicated times. Error bars represent means ± SEM (n = 3).
(C) Chloroplast H+-ATPase and Ca2+-ATPase activity. ATPase activity in purified chloroplast protein from wild-type, ccha1, and complemented plants. Shown are means ± SD. ∗∗∗P < 0.001, respectively (Student's t-test; compared with the corresponding wild-type control).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

7. Figure 6
The Effects of Ca2+ and Mn2+ on the Phenotype of ccha1 Mutants.
Wild-type, ccha1, and complemented plants are shown. All photographs are representative of >100 plants grown in each condition.
(A) 14-day-old plants grown on half-strength MS medium and supplemented with 50 mM CaCl2, 70 mM CaCl2, 1 mM MnCl2, 2 mM MnCl2. Bar represent 1 cm.
(B) 21-day-old plants were sprayed with water, 50 mM CaCl2, or 15 mM MnCl2. Bars represent 1 cm.
(C) Quantitative analysis of total chlorophyll contents after treatment with CaCl2 and MnCl2. Shown are means ± SD. **P < 0.01 and ***P < (Student's t-test; compared with the corresponding wild-type control).
(D) Representative imaging of [Ca2+]cyt in stomata of the wild-type, ccha1, and Com plants by Fluo-3 staining and confocal assay.
(E) Quantification of the relative Fluo-3 fluorescence intensities indicating [Ca2+]cyt levels in stomata of the wild-type, ccha1, and Com plants. Shown are means ± SD. **P < 0.01 (Student's t-test; compared with the corresponding wild-type control), n = 50.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

8. Figure 7
Effect of pH on ccha1 Mutant Maximal Photochemical Efficiency of PSII (Fv/Fm) and Nonphotochemical Quenching (NPQ).
Arabidopsis seeds were germinated in MS agar medium at different pH levels (4.0, 5.7, and 8.0).
(A) Effects of different pH on the growth of wild-type, ccha1 mutant, and complemented seedlings. The pictures were taken at 7 days after germination. Bar = 0.5 mm.
(B) Effects of different pH on the Fv/Fm from wild-type and ccha1 mutants. The ccha1 mutants showed the characteristic low Fv/Fm ratio under control conditions (±SD, ***P < 0.001 n = 10–20).
(C) NPQ was consistently lower in 3-week-old ccha1 mutants than in wild-type. Actinic light (500 μmol photons m−2 s−1) was switched on at time zero, and plants were left in the dark after 12 min.
(D and E) pH distribution of guard cells was monitored by confocal imaging of the fluorescence from the pH-sensitive dye BCECF-AM (D). Quantification of the relative BCECF-AM fluorescence intensities indicating [pH]cyt levels in stomata (E). Shown are means ± SD. **P < 0.01 (Student's t-test; compared with the corresponding wild-type control), n = 80.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

9. Figure 8 A Putative Model of CCHA1 in the Chloroplast.
CCHA1 targets to the thylakoid membrane. The H+ of the thylakoid lumen drives lumenal Ca2+ and Mn2+ uptake by CCHA1 and release by other potential pathway. An autoinhibited Ca2+-ATPase (ACA1); PPF1, a gibberellin-induced gene controlling flower development, is associated with chloroplasts and thought to encode a putative Ca2+/H+ exchanger. ? represent a suppose Ca2+-ATPase.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions