Volume 10, Issue 1, Pages 183-196 (January 2017) The Phytol Phosphorylation Pathway Is Essential for the Biosynthesis of Phylloquinone, which Is Required for Photosystem I Stability in Arabidopsis  Lei Wang, Qingwei Li, Aihong Zhang, Wen Zhou, Rui Jiang, Zhipan Yang, Huixia Yang, Xiaochun Qin, Shunhua Ding, Qingtao Lu, Xiaogang Wen, Congming Lu  Molecular Plant  Volume 10, Issue 1, Pages 183-196 (January 2017) DOI: 10.1016/j.molp.2016.12.006 Copyright © 2017 The Author Terms and Conditions

Figure 1 Spectroscopic Characterization of Wild-Type, vte6-3, and vte6-3 com. (A) P700 redox kinetics in wild-type (WT), vte6-3, and vte6-3 com. P700 redox state was investigated by P700 absorbance at 820 nm induced by far-red light (720 nm). AL, actinic light (80 μmol m−2 s−1); FR, far-red light; SL, saturating light pulse; ox., P700 oxidation; red., P700 reduction; ΔAmax, maximum photo-oxidizable P700. (B) 77K fluorescence emission spectra of thylakoid membranes in WT, vte6-3, and vte6-3 com. After excitation at 436 nm, fluorescence emission signals were acquired and then normalized to the PSII emission maximum at 685 nm. (C) Chlorophyll a fluorescence induction kinetics in WT, vte6-3, and vte6-3 com. AL, actinic light (80 μmol m−2 s−1); SL, saturating light pulse. Molecular Plant 2017 10, 183-196DOI: (10.1016/j.molp.2016.12.006) Copyright © 2017 The Author Terms and Conditions

Figure 2 Phylloquinone and Plastoquinone Contents in Wild-Type and vte6. (A) Phylloquinone contents in wild-type (WT) and vte6 mutants. (B) Plastoquinone contents in WT and vte6 mutants. WT-Col, vte6-3, and vte6-3 com belong to ecotype Columbia-0; WT-Nos, vte6-1, and vte6-2 belong to ecotype Nossen. Data represent means ± SD (n = 3). Significant differences from the wild-type are based on Student's t test (**P < 0.01). Molecular Plant 2017 10, 183-196DOI: (10.1016/j.molp.2016.12.006) Copyright © 2017 The Author Terms and Conditions

Figure 3 PSI Complementary Quantum Yields in Wild-Type and vte6. PSI complementary quantum yields were calculated based on the P700 induction curve in the wild-type (WT) and vte6 mutants. (A and D) Quantum yields of PSI, Y(I). (B and E) Non-photochemical energy dissipation caused by acceptor-side limitation, Y(NA). (C and F) Non-photochemical energy dissipation caused by donor-side limitation, Y(ND). WT-Col and vte6-3 belong to ecotype Columbia-0; WT-Nos, vte6-1 and vte6-2 belong to ecotype Nossen. Data represent means ± SD (n = 3). Molecular Plant 2017 10, 183-196DOI: (10.1016/j.molp.2016.12.006) Copyright © 2017 The Author Terms and Conditions

Figure 4 Analysis of Thylakoid Proteins from Wild-Type, vte6-3, and vte6-3 com. (A) Thylakoid membrane protein immunodetection based on equal loading of total leaf proteins (10 μg). (B) BN–PAGE analysis of thylakoid membrane complexes. Thylakoid membranes (corresponding to 10 μg of chlorophyll) from the leaves of wild-type (WT), vte6-3, and vte6-3 com were solubilized and separated by BN–PAGE. Thylakoid membrane protein complex designations are labeled on the left according to Peng et al. (2008). I(NDH-PSI), NDH-PSI supercomplex; II(PSII SC), PSII supercomplexes; III(PSI + PSII-D), PSI and dimeric PSII, IV (PSII-M), monomeric PSII; V(CP43 free PSII), monomeric PSII without CP43; VI(LHCII-T), trimeric LHCII; (VII)LHCII-M, monomeric LHCII. (C) Two-dimensional (2D) BN/SDS–PAGE separation of thylakoid membrane complexes (10 μg of chlorophyll). After separation in the first dimension in a non-denaturing gel, protein lanes were subjected to a denaturing 2D gel (2D BN/SDS–PAGE) followed by staining with Coomassie brilliant blue. Identities of relevant proteins are indicated by arrows. PSI core subunits PsaA/B and some minor proteins are circled in red. Molecular Plant 2017 10, 183-196DOI: (10.1016/j.molp.2016.12.006) Copyright © 2017 The Author Terms and Conditions

Figure 5 Ultrastructure of Chloroplasts in Wild-Type, vte6-3, and vte6-3 com. (A–F) Electron micrographs of chloroplasts from wild-type (WT) (A), vte6-3 (B), and vte6-3 com (C). Images at high magnification obtained from the regions indicated by a red box in WT (D), vte6-3 (E), and vte6-3 com (F), respectively. GT, granal thylakoids; ST, stromal thylakoids. Bars represent 1 μm for (A) to (C) and 0.2 μm for (D) to (F). Molecular Plant 2017 10, 183-196DOI: (10.1016/j.molp.2016.12.006) Copyright © 2017 The Author Terms and Conditions

Figure 6 Analysis of Thylakoid Protein Complex Assembly in Wild-Type and vte6-3. After pulse-chase labeling with [35S]Met for 20 min in the presence of cycloheximide, leaves were chased for 0, 15, 30, and 60 min with unlabeled Met. Thylakoid protein complexes were isolated and separated by 2D BN/SDS–PAGE on the basis of equal radiation. Labeled proteins were visualized by autoradiography. PSII SC, PSII supercomplexes; PSI + PSII-D, PSI and dimeric PSII; PSII-M, monomeric PSII; CP43-PSII, monomeric PSII without CP43; PSII-RC, PSII reaction center; f.p., free proteins. a, the PSI complex; b, c, and d, the PSI assembly intermediate complexes; e, the PsaA/PsaB heterodimer in PSI (inferred from molecular weight); f, unassembled free proteins of PSI. Molecular Plant 2017 10, 183-196DOI: (10.1016/j.molp.2016.12.006) Copyright © 2017 The Author Terms and Conditions

Figure 7 PSI Stability Analysis in Wild-Type and vte6-3. (A) Maximum level of photo-oxidizable P700 (ΔAmax; normalized to initial values at time zero, t0). Detached leaves from wild-type (WT) and vte6-3 in the presence of lincomycin and cycloheximide were exposed to 80 μmol photons m−2 s−1 for 24 h. Data represent means ± SD (n = 3). Significant difference from the wild-type were based on Student's t test (*P < 0.05; **P < 0.01). (B) Maximum level of photo-oxidizable P700 (ΔAmax; normalized to initial values at time zero, t0). Detached leaves from wild-type (WT) and vte6-3 in the presence of lincomycin and cycloheximide were exposed to 200 μmol photons m−2 s−1 for 24 h. Data represent means ± SD (n = 4). Significant difference from the wild-type were based on Student's t test (*P < 0.05; **P < 0.01). (C) Immunodetection of specific thylakoid membrane proteins. Detached leaves from WT and vte6-3 in the presence of lincomycin and cycloheximide were exposed to 80 μmol photons m−2 s−1 for 24 h. (D) Immunodetection of specific thylakoid membrane proteins. Detached leaves from WT and vte6-3 in the presence of lincomycin and cycloheximide were exposed to 200 μmol photons m−2 s−1 for 24 h. Molecular Plant 2017 10, 183-196DOI: (10.1016/j.molp.2016.12.006) Copyright © 2017 The Author Terms and Conditions

Figure 8 Susceptibility of PSI to High-Light Treatment in Wild-Type and vte6-3. (A) Level of maximum photo-oxidizable P700 (ΔAmax; normalized to initial values at time zero, t0). Detached leaves from wild-type (WT) and vte6-3 were exposed to 900 μmol photons m−2 s−1 for 2 h. Data represent means ± SD (n = 3). Significant difference from the wild-type were based on Student's t test (**P < 0.01). (B) Immunodetection of specific thylakoid membrane proteins. Detached leaves from WT and vte6-3 were exposed to 900 μmol photons m−2 s−1 for 2 h. Molecular Plant 2017 10, 183-196DOI: (10.1016/j.molp.2016.12.006) Copyright © 2017 The Author Terms and Conditions