1. Volume 6, Issue 5, Pages 1673-1691 (September 2013)
Arabidopsis Thylakoid Formation 1 Is a Critical Regulator for Dynamics of PSII–LHCII Complexes in Leaf Senescence and Excess Light 
Weihua Huang, Qingbo Chen, Ying Zhu, Fenghong Hu, Lingang Zhang, Zhaoxue Ma, Zuhua He, Jirong Huang 
Molecular Plant 
Volume 6, Issue 5, Pages (September 2013)
DOI: /mp/sst069
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
Physiological Characterization of Pathogen-Infected or Naturally Senescent WT and thf1 Leaves.
(A) Disease symptoms of a representative leaf (upper panel) infected with either virulent strain ES4326 or avirulent strain ES4326 (avrRpm1), and the magnified image of the inoculation site (lower panel) at 3 DPI (day post inoculation). Arrows indicate different phenotypes in Chl degradation between WT and thf1.
(B) Representative leaves from the same position of naturally senescent WT and thf1 plants.
(C) Chl content of the sixth leaf from 3- to 8-week-old WT and thf1 plants. Error bars represent SD (n = 3).
Molecular Plant 2013 6, DOI: ( /mp/sst069)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

3. Figure 2 Characterization of Dark-Induced WT and thf1 Leaves.
(A) Phenotypes (left panel) and Fv/Fm values (right panel) of dark-induced senescent WT and thf1 leaves. The color bar shows Fv/Fm values from 0 to 1. DDI, day of dark incubation.
(B) Chl, Chl a, Chl b content, and Chl a/b ratios of dark-treated leaves. Error bars represent SD (n = 3). fw, fresh weight.
(C) Quantified data of Fv/Fm from the panel of (A). Error bars represent SD (n = 10).
(D) 77 K Chl fluorescence emission spectra of thylakoids extracted from dark-treated leaves of WT and thf1 plants.
Molecular Plant 2013 6, DOI: ( /mp/sst069)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

4. Figure 3 thf1 Is a Cosmetic Stay-Green Mutant.
(A) Ion leakage of WT and thf1 leaves during dark incubation. Error bars represent SD (n = 3).
(B) Expression of THF1 and genes related to senescence or the Chl degradation pathway in WT and thf1 was analyzed by quantitative real-time PCR during dark incubation. Two independent biological replicates were analyzed for all examined genes, and similar results were obtained. In each biological replica, expression of each gene was examined three times. The expression level in WT at 0 DDI was set to 1. Error bars indicate SD (n = 3).
Molecular Plant 2013 6, DOI: ( /mp/sst069)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
Analysis of Photosynthetic Proteins and Complexes during Dark-Induced leaf Senescence.
Samples loaded in each lane were normalized by the fresh weight of leaves. DDI, day of dark incubation.
(A) Immunoblot analysis of LHCII, PSII, and PSI core subunits in WT and thf1 leaves during dark-induced senescence. The asterisk indicates a non-specific band.
(B) BN–PAGE analysis of photosynthetic complexes in thylakoid membranes isolated from WT and thf1 leaves incubated in the dark for 0, 3, and 6 d, respectively. MC, megacomplexes; SC, supercomplexes; M, monomers; D, dimmers; T, trimers.
(C) Immunoblot analysis of D2-contained complexes in WT and thf1 samples at 0, 3, and 6 DDI. The upper and lower images of the film were exposed to the membrane for 2 min and for 30 min, respectively. Black arrows indicate the PSII–LHCII megacomplex. Red arrows indicate the shifted PSII dimer.
Molecular Plant 2013 6, DOI: ( /mp/sst069)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
Ultrastructure of Chloroplasts from WT and thf1 (Green Sector) Leaves Without or With Dark Treatment.
A representative chloroplast is shown on the left, and the magnified structure of the box is shown on the right. Arrows, grana thylakoid; arrowheads, stromal thylakoid; asterisks, plastoglobule. Bars = 500 nm.
Molecular Plant 2013 6, DOI: ( /mp/sst069)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
Genetic Interaction between thf1 and nye1 or nyc1 with Regard to Chl Degradation during Dark-Induced Senescence.
(A) Phenotypes of dark-treated leaves. DDI, day of dark incubation.
(B) Analysis of total Chl, Chl a and Chl b content, and Chl a/b ratio of the same samples as in (A). Data are means ± SD of three independent replicates.
Molecular Plant 2013 6, DOI: ( /mp/sst069)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

8. Figure 7
Genetic Interaction between thf1 and nye1 or nyc1 with regard to PSII Stability during Dark-Induced Senescence.
(A) Fv/Fm values in dark-treated WT, thf1, nye1, nye1 thf1, nyc1, and nyc1 thf1 leaves. Error bars represent SD (n = 10).
(B) Immunoblot analysis of Lhcb1, D1, and D2 proteins during dark incubation. Each lane was normalized by the fresh leaf weight. The level of RbcL stained with Coomassie Brilliant Blue was shown as a loading control.
Molecular Plant 2013 6, DOI: ( /mp/sst069)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

9. Figure 8 THF1 Interacts with Lhcbs In Vitro and In Vivo.
(A) Proteins were immunoblotted with antibodies against Lhcb1 and THF1 after thylakoid membrane complexes were separated by BN– and SDS–PAGE. I, PSII supercomplexes; II, monomeric PSI and dimeric PSII; III, monomeric PSII; IV, CP43 minus PSII; V, trimeric LHCII; VI, monomeric LCHII.
(B) In vitro assay of THF1 interaction with Lhcb1-6. The upper panel showed the CBB-stained gel of the purified GST, GST-Lhcbs, and 6xHis–THF1 separated by SDS–PAGE. The reaction mixture between GST or GST–Lhcbs and 6xHis–THF1 was precipitated with glutathione-agarose beads. The precipitates were probed with anti-THF1 serum (lower panel). Asterisks indicate the purified proteins.
(C) Analysis of the THF1-interacting domain of Lhcb2. Proteins precipitated by glutathione-agorose beads were probed with anti-THF1 antibody (upper panel). The level of purified GST, GST-fused proteins, or 6xHis–GFP input for interaction assay was shown in the lower panel. Asterisks indicate the purified proteins.
(D) pH-dependent interaction between THF1 and Lhcb1-3 in vitro. Protein interaction analysis was conducted at pH 7.0, 7.4, or 8.0, respectively.
(E) BiFC assay of THF1 interaction with Lhcb1 in Arabidopsis protoplasts. Bar = 5 μM.
Molecular Plant 2013 6, DOI: ( /mp/sst069)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

10. Figure 9
The Stay-Green Phenotype of thf1 Leaves in the Dark Is Dependent on Chl b.
(A) Phenotypes of detached leaves from 3-week-old WT, thf1, cao, and thf1 cao plants.
(B) Chl content of non-senescent and 6-d dark-treated leaves. Error bars represent SD (n = 3). fw, fresh weight.
Molecular Plant 2013 6, DOI: ( /mp/sst069)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

11. Figure 10
Analysis of Photosynthetic Complexes under High-Light Stress and a Proposed Model for THF1 Function.
Thylakoid membranes were isolated from detached mature leaves treated without or with high light (600 μmol m–2 s–1) for 6h. Five μg of Chl were loaded in each lane.
(A) BN–PAGE analysis of photosynthetic complexes. MC, megacomplexes; SC, supercomplexes; M, monomers; D, dimmers; T, trimers.
(B) Immunoblot analysis of the blue native gel as shown in (A) using the D2 antibody. The image of the film exposed to the membrane for 1min (upper panel) and for 20min (lower panel). Black arrows indicate the largest PSII–LHCII megacomplex.
(C) A proposed model for the dual role of THF1 in dynamic regulation of PSII–LHCII complexes. THF1 promotes the disassembly of PSII megacomplexes but stabilizes PSII supercomplexes in leaf senescence and high light.
Molecular Plant 2013 6, DOI: ( /mp/sst069)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions