Volume 10, Issue 1, Pages 143-154 (January 2017) Dynamic Changes of IsiA-Containing Complexes during Long-Term Iron Deficiency in Synechocystis sp. PCC 6803  Fei Ma, Xin Zhang, Xi Zhu, Tianpei Li, Jiao Zhan, Hui Chen, Chenliu He, Qiang Wang  Molecular Plant  Volume 10, Issue 1, Pages 143-154 (January 2017) DOI: 10.1016/j.molp.2016.10.009 Copyright © 2017 The Author Terms and Conditions

Figure 1 Induction of IsiA in Synechocystis sp. PCC 6803 under Iron Deficiency. Cells were harvested after 1–6 days of iron deficiency treatment. (A) Immunoblot analysis was performed using an antibody specific to IsiA (upper panel). Equal loading was confirmed by Coomassie brilliant blue (CB) staining (lower panel). (B) Densitometric quantitation of IsiA levels was determined using ImageJ and calculated as a relative value at day 1, which was set to 1 for easy comparison. All data are shown as the means ± SD (n = 5) in all figures and tables. Molecular Plant 2017 10, 143-154DOI: (10.1016/j.molp.2016.10.009) Copyright © 2017 The Author Terms and Conditions

Figure 2 77 K Fluorescence Emission Spectra and Immunoblot Analysis of Thylakoid Membranes Isolated from Synechocystis sp. PCC 6803 Subjected to Long-Term Iron Deficiency. Thylakoid membranes were isolated from cells exposed to iron deficiency for 1–15 days. (A) 77 K fluorescence emission spectra of the thylakoid membranes isolated from cells under long-term iron deficiency with an excitation wavelength at 430 nm. Samples were set to 15 μg mL−1 Chl a. The spectra were normalized to the fluorescence intensity at 780 nm. (B) The relative fluorescence intensity of F687 to F696 during long-term iron deficiency. (C) Thylakoid membranes (0.5 μg mL−1 Chl a) isolated from cells under long-term iron deficiency were separated by SDS–PAGE and stained with Coomassie brilliant blue, and immunoblotting was performed using antibodies specific to IsiA, PsbA, and PsaD. To increase the chance of detecting low levels of IsiA in the first 3 days, the amount of loaded sample was increased to 6 μg mL−1 Chl a, as indicated by the arrow. (D) Densitometric quantitation of IsiA, PsbA, and PsaD protein levels by immunoblot analysis of thylakoid membranes. The densitometric quantitation of protein levels was performed using ImageJ, and the value at day 6, when the induced IsiA was saturated, was set to 1 for easy comparison. Molecular Plant 2017 10, 143-154DOI: (10.1016/j.molp.2016.10.009) Copyright © 2017 The Author Terms and Conditions

Figure 3 Sucrose Gradient Fractionation of Thylakoid Protein Complexes and Immunoblot Analysis of the Fractions Isolated from Synechocystis sp. PCC 6803 under Long-Term Iron Deficiency. Thylakoid membranes were isolated from cells after 1–15 days of iron deficiency. (A) Thylakoid protein complexes were separated by step sucrose gradient ultracentrifugation. Fractions are as indicated. (B) Immunoblot analysis of sucrose gradient fractions using antibodies specific to IsiA, PsbA, and PsaD. (C–E) Densitometric quantitation of IsiA (C), PsaD (D), and PsbA (E) protein levels in immunoblot analysis. The densitometric quantitation of protein levels was performed using ImageJ, and the value at day 6, at which point F4 appeared, was set to 1 for easy comparison. Molecular Plant 2017 10, 143-154DOI: (10.1016/j.molp.2016.10.009) Copyright © 2017 The Author Terms and Conditions

Figure 4 IsiA Distribution on Sucrose Gradient Fractions Isolated from Synechocystis sp. PCC 6803 after 15 Days of Iron Deficiency Treatment. (A) Serial sampling and immunoblot analysis were performed in the sucrose gradient fractions after 15 days of iron deficiency treatment. Fractions are as indicated, and the numbers (1–6) above the blot indicate serial samples taken from F3 to F4 and used for further determination of the IsiA/PsaD ratio in panel (B). (B) The relative abundance of IsiA to PsaD in serial samples (1–6) of F3 and F4. (C and D) Immunoblot analysis of the F2 fraction in the HT-3 mutant cells (in which CP47 is tagged with 6× His) or isiA-his mutant cells (in which IsiA is tagged with 6× His) before (F2) or after (F2-elution) immobilized metal affinity chromatography (IMAC). (E) Immunoblot analysis of the F3 and F4 fractions before (F3 or F4) or after (F3-elution or F4-elution) IMAC. (F) The relative abundance of IsiA to PsaD in (E). Molecular Plant 2017 10, 143-154DOI: (10.1016/j.molp.2016.10.009) Copyright © 2017 The Author Terms and Conditions

Figure 5 77 K Fluorescence Emission Spectra of Sucrose Gradient Fractions Isolated from Synechocystis sp. PCC 6803 under Long-Term Iron Deficiency. Thylakoid protein complexes were separated by step sucrose gradient ultracentrifugation and the resulting fractions, i.e., F2 (A), F3 (B), and F4 (C), were sampled and set to 15 μg mL−1 Chl a. The excitation was performed at 430 nm and the spectra were normalized to the fluorescence intensity at 780 nm. Molecular Plant 2017 10, 143-154DOI: (10.1016/j.molp.2016.10.009) Copyright © 2017 The Author Terms and Conditions

Figure 6 77 K Fluorescence Emission Spectra of Sucrose Gradient Fractions Treated with Dodecyl Maltoside. Thylakoid protein complexes isolated from Synechocystis sp. PCC 6803 under long-term iron deficiency were separated by step sucrose gradient ultracentrifugation. F3 at day 3 (A) and day 15 (B) and F4 at day 15 (C) were collected and treated with 0.3% DM. Excitation was performed at 430 nm, and the samples were set to 15 μg mL−1 Chl a. The spectra were normalized to the fluorescence intensity at 780 nm. Molecular Plant 2017 10, 143-154DOI: (10.1016/j.molp.2016.10.009) Copyright © 2017 The Author Terms and Conditions

Figure 7 Sucrose Gradient Fractionation and 77 K Fluorescence Emission Spectra Analysis of DM-Treated F4. F4 isolated by sucrose gradient ultracentrifugation after 15 days of iron deficiency treatment were further solubilized with 0.3% DM and re-fractionated by step sucrose gradient ultracentrifugation (A). The resulting two fractions, 15d-F4 0.3% DM-up and 15d-F4 0.3% DM-down were sampled and set to 15 μg mL−1 Chl a for analysis of 77 K fluorescence emission spectra (B). Excitation was performed at 430 nm, and the spectra were normalized to the fluorescence intensity at 780 nm. Molecular Plant 2017 10, 143-154DOI: (10.1016/j.molp.2016.10.009) Copyright © 2017 The Author Terms and Conditions

Figure 8 Effect of the Deletion of PsaL on the Formation of IsiA-PS I HF/LF-Supercomplexes. (A) Induction of IsiA in psaL mutants under iron deficiency. (B) Sucrose gradient ultracentrifugation of the thylakoid protein complexes. Fractions are as indicated. (C) Immunoblot analysis of the sucrose gradient fractions using antibodies specific to IsiA. Molecular Plant 2017 10, 143-154DOI: (10.1016/j.molp.2016.10.009) Copyright © 2017 The Author Terms and Conditions