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. 2012 Feb;24(2):637-59.
doi: 10.1105/tpc.111.092692. Epub 2012 Feb 3.

Evidence for a role of VIPP1 in the structural organization of the photosynthetic apparatus in Chlamydomonas

André Nordhues  1 , Mark Aurel Schöttler, Ann-Katrin Unger, Stefan Geimer, Stephanie Schönfelder, Stefan Schmollinger, Mark Rütgers, Giovanni Finazzi, Barbara Soppa, Frederik Sommer, Timo Mühlhaus, Thomas Roach, Anja Krieger-Liszkay, Heiko Lokstein, José Luis Crespo, Michael Schroda
Affiliations

Evidence for a role of VIPP1 in the structural organization of the photosynthetic apparatus in Chlamydomonas

André Nordhues et al. Plant Cell. 2012 Feb.

Abstract

The vesicle-inducing protein in plastids (VIPP1) was suggested to play a role in thylakoid membrane formation via membrane vesicles. As this functional assignment is under debate, we investigated the function of VIPP1 in Chlamydomonas reinhardtii. Using immunofluorescence, we localized VIPP1 to distinct spots within the chloroplast. In VIPP1-RNA interference/artificial microRNA cells, we consistently observed aberrant, prolamellar body-like structures at the origin of multiple thylakoid membrane layers, which appear to coincide with the immunofluorescent VIPP1 spots and suggest a defect in thylakoid membrane biogenesis. Accordingly, using quantitative shotgun proteomics, we found that unstressed vipp1 mutant cells accumulate 14 to 20% less photosystems, cytochrome b(6)f complex, and ATP synthase but 30% more light-harvesting complex II than control cells, while complex assembly, thylakoid membrane ultrastructure, and bulk lipid composition appeared unaltered. Photosystems in vipp1 mutants are sensitive to high light, which coincides with a lowered midpoint potential of the Q(A)/Q(A)(-) redox couple and increased thermosensitivity of photosystem II (PSII), suggesting structural defects in PSII. Moreover, swollen thylakoids, despite reduced membrane energization, in vipp1 mutants grown on ammonium suggest defects in the supermolecular organization of thylakoid membrane complexes. Overall, our data suggest a role of VIPP1 in the biogenesis/assembly of thylakoid membrane core complexes, most likely by supplying structural lipids.

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Figures

Figure 1.
Figure 1.
Photobleaching of High Light–Exposed VIPP1-RNAi Strains. (A) VIPP1 protein levels in VIPP1-RNAi strains are strongly reduced. Whole-cell proteins from transformants generated with VIPP1-RNAi construct pMS439 corresponding to 2 μg chlorophyll were separated on a 14% SDS-polyacrylamide gel and analyzed by immunoblotting. CF1β served as loading control. (B) High light sensitivity of VIPP1-RNAi strains. Ten microliters containing 107 cells were spotted onto TAP-NH4 agar plates and photographed directly or after a 4-d incubation at the indicated light intensities. (C) Bleaching of VIPP1-RNAi strains. A control strain and VIPP1-RNAi strain #4 grown in TAP-NH4 medium were exposed to a light intensity of ~1000 μE m−2 s−1 for 10 h. (D) The chlorophyll content rapidly declines in high light–exposed VIPP1-RNAi strains. A control strain (n = 3) and five VIPP1-RNAi strains (#3, #26, #44, #49, and #93) were treated as in (A), and the chlorophyll content was determined. Chlorophyll contents are given relative to the concentrations determined prior to the shift to high light, which were set to 1. Error bars represent se. r.u., relative units. (E) PSII and PSI in VIPP1-RNAi strains are very sensitive to high light. Equal cell densities of a control strain and VIPP1-RNAi strains #3, #14, and #93 were subjected to high light as in (A), and PSII and PSI activities were measured by determining the DCMU-sensitive and -insensitive fractions of the electrochromic shift signal. (F) Subunits of PSII and PSI are rapidly degraded in VIPP1-RNAi strains exposed to high light intensities. Whole-cell proteins from high light–exposed control and VIPP1-RNAi strain #27 grown in TAP-NH4 medium were separated on 14% SDS-polyacrylamide gels and analyzed by immunoblotting.
Figure 2.
Figure 2.
Examination of VIPP1/2 Levels in VIPP1-RNAi and -amiRNA Strains by Immunoblotting. Transformants generated with empty vectors (control strains) or VIPP1-RNAi and VIPP1-amiRNA constructs in the cw15-325 (#12) and cw15-302 (#18) strain backgrounds, respectively, were grown in TAP-NH4 medium. Total protein from the transformants and purified recombinant VIPP1 and VIPP2 at the indicated protein concentrations were separated on 7.5 to 15% SDS-polyacrylamide gels and analyzed by immunoblotting using antisera against VIPP1 and VIPP2. HSP90C served as loading control.
Figure 3.
Figure 3.
Thylakoids in VIPP1-amiRNA Strains Exposed to High Light Intensities Are Extremely Swollen. (A) Electron microscopy image of a cell from the control strain grown at low light intensities. Cells were grown at ~30 μE m−2 s−1 in TAP-NH4 medium. An overview image is shown on the left, and a zoom-in of the region demarcated by the black box is shown on the right. N, nucleus; P, pyrenoid; S, starch. Bars in overview images correspond to 1 μm and those in zoom-ins to 0.2 μm. (B) Electron microscopy image of a cell from the control strain exposed to high light. Cells were grown at ~30 μE m−2 s−1 in TAP-NH4 medium and exposed to ~1000 μE m−2 s−1 for 3.5 h. Images were taken as in (A). (C) Electron microscopy image of a cell from a VIPP1-amiRNA strain grown at low light intensities. VIPP1-amiRNA strain #14 was grown at ~30 μE m−2 s−1 in TAP-NH4 medium. Images were taken as in (A). (D) Electron microscopy image of a cell from a VIPP1-amiRNA strain exposed to high light. VIPP1-amiRNA strain #14 was grown at ~30 μE m−2 s−1 in TAP-NH4 medium and exposed to ~1000 μE m−2 s−1 for 3.5 h. Images were taken as in (A).
Figure 4.
Figure 4.
VIPP1-RNAi Cells Are More Severely Photoinhibited at Very High Light and Replace Photodamaged D1 by de Novo–Synthesized D1 Slower Than Control Cells. Cultures of control and VIPP1-RNAi strain #80 grown in TAP-NH4 medium were split into two parts. One part was supplemented with ethanol-dissolved chloramphenicol (CAP) to a final concentration of 100 μg/mL, while the other received the same volume of pure ethanol. The four cultures were exposed to ~1800 μE m−2 s−1 for 30 min (photoinhibition [PI]) and shifted back to ~30 μE m−2 s−1 (recovery [rec]). Maximum quantum efficiency of PSII during photoinhibition and recovery was measured with a PAM fluorometer as variable fluorescence (FV = FM − F0) normalized to FM. Shown is the average of two independent experiments; error bars represent se.
Figure 5.
Figure 5.
High Light Sensitivity Is Alleviated but Not Abolished in VIPP1-RNAi Strains Grown on Nitrate. (A) PSII in VIPP1-RNAi strains is less high light sensitive in cells grown on nitrate compared with ammonium. Control (Con) and VIPP1-RNAi strains #12 and #27 were grown in TAP-NO3 or TAP-NH4 medium. Cells were exposed to ~1000 μE m−2 s−1 and maximum quantum efficiency of PSII was measured with a PAM fluorometer as described in Figure 4. Shown is the average of two independent experiments. Error bars represent se. (B) PSII in VIPP1-RNAi strains is less sensitive to photoinhibition in cells grown on nitrate compared with ammonium. Control and VIPP1-RNAi strains #5, #27, and #41 were grown in TAP-NO3 or TAP-NH4 medium. Cells were exposed to ~1800 μE m−2 s−1 for 1 h (photoinhibition [PI]) and shifted back to ~30 μE m−2 s−1 (recovery [rec]). Maximum quantum efficiency of PSII was measured with a PAM fluorometer as described in Figure 4. Shown is the average of four independent experiments. Error bars represent se. (C) Subunits of PSII and PSI are less prone to degradation in high light–exposed VIPP1-RNAi strains grown on nitrate. Whole-cell proteins from nitrate-grown control and VIPP1-RNAi strain #27 exposed to ~1000 μE m−2 s−1 for 7 h were separated on 14% SDS-polyacrylamide gels and analyzed by immunoblotting. For comparison, whole-cell proteins from VIPP1-RNAi strain #27 grown on ammonium and exposed to high light for 3 h was loaded next to the other samples. (D) RNA gel blot analysis of high light–exposed control and VIPP1-RNAi strains. Control and VIPP1-RNAi strain #12 were grown in TAP-NO3 or TAP-NH4 medium. Cells were exposed to ~1000 μE m−2 s−1 for 5 h, and RNA was extracted from samples taken at the indicated time points and subjected to RNA gel blot analysis. CBLP2 served as loading control.
Figure 6.
Figure 6.
VIPP1-RNAi Strains Are Affected in Some Photosynthesis Parameters. (A) Thylakoid membrane energization. Control (Con) and VIPP1-RNAi strains #2-7, #2-19, #3-30, #4-19, and #4-49 were grown in TAP-NO3 or TAP-NH4 medium and kept in low light (LL) of ~30 μE m−2 s−1 or exposed to high light (HL) of ~1000 μE m−2 s−1 for 3 h. Maximum thylakoid membrane energization was then determined by measuring the ECS in saturating light. Shown is the average of eight and six measurements on the control strain, and of 12 and 10 measurements on VIPP1-RNAi strains grown on NO3 and NH4, respectively. Error bars represent se. Chl, chlorophyll. (B) ATP synthase activity. The activity of the ATP synthase was inferred from the decay kinetic of the ECS signal during a short dark interval using the same cells as described in (A). (C) Maximum quantum efficiency of PSII. FV/FM was measured after 5 min of far-red illumination, followed by 10 min of dark adaptation of the cell suspension using control and VIPP1-RNAi strains #4, #9, #40, #53, #72, #111, and #129 grown in low light. Shown is the average of four and two measurements on the control strain, and of 13 and 10 measurements on VIPP1-RNAi strains. Error bars represent se. (D) Chlorophyll a/b ratios. Chlorophyll was extracted with 80% acetone from the same cells as described in (C), and the chlorophyll concentration was determined spectrophotometrically. Asterisks indicate the significance of the difference to untreated controls grown on nitrate and ammonium as nitrogen source, respectively (t test, P value ≤ 0.05). (E) Cytochrome f reduction kinetics. Control and VIPP1-RNAi strains #4, #9, and #72 were grown in TAP-NH4 medium. Cytochrome f reduction was initiated by switching off saturating red light at time point zero. The fully reduced state of cytochrome f in the dark was normalized to zero and the fully oxidized state to one. Kinetics were recorded four times for each strain. Averages for the control and all three VIPP1-RNAi strains are shown. (F) Cytochrome f oxidation kinetics. Control and VIPP1-RNAi strains #27, #29, and #30 were grown in TAP-NH4 medium. Cytochrome f oxidation was initiated by switching on a saturating red light pulse at time point zero. Six replicates of the control strain and two each for the VIPP1-RNAi strains were recorded and averages plotted. Again, the fully reduced state of cytochrome f in the dark was normalized to zero and the fully oxidized state to one. (G) Fluorescence induction kinetics. Control and VIPP1-RNAi strains #27, #29, und #30 were grown in TAP-NH4 medium. Dark-adapted cells trapped in state 1 were supplemented with DCMU, and fluorescence was induced by switching on the light. Six replicates of the control strain and two each for the VIPP1-RNAi strains were recorded and averages plotted. The F0 values were normalized to zero and the FM values to one.
Figure 7.
Figure 7.
PSII of VIPP1-RNAi Strains Is Highly Susceptible to Heat Stress. (A) PSII maximum quantum efficiency of control and VIPP1-RNAi strains exposed to heat stress. Control and VIPP1-RNAi strains #5, #20, #27, and #41 were grown in TAP-NO3 or TAP-NH4 medium, and cells were exposed to 40°C at ~5 μE m−2 s−1. FV/FM was measured over time with a PAM fluorometer as described in Figure 4. Shown is the average of five and seven independent experiments for control and VIPP1-RNAi strains, respectively. Error bars represent se. (B) PSII subunits are only mildly affected by heat stress. Whole-cell proteins were extracted from control and VIPP1-RNAi strain #27 grown and heat-stressed as described in (A). Whole-cell proteins were separated on 14% SDS-polyacrylamide gels and analyzed by immunoblotting. (C) RNA gel blot analysis of heat-stressed control and VIPP1-RNAi strains. Control and VIPP1-RNAi strain #20 were grown and heat stressed as described in (A). RNA was extracted from samples taken at the indicated time points and subjected to RNA gel blot analysis. CBLP2 served as loading control.
Figure 8.
Figure 8.
Levels of Thylakoid Membrane Core Complexes Are Slightly Lower in VIPP1-RNAi Cells Than in Control Cells. (A) Ratios of thylakoid membrane core complexes in VIPP1-RNAi cells relative to control cells. A control strain and VIPP1-RNAi strain #111 were metabolically labeled using 15NO3 and 14NO3, respectively, as nitrogen source and maintained at ~30 μE m−2 s−1 and 25°C for 1 h (Cont light), exposed to photoinhibitory light of ~2000 μE m−2 s−1 for 1 h at 25°C, or heat-shocked at 40°C for 1 h at ~5 μE m−2 s−1. After mixing control and VIPP1-RNAi cells from each treatment at a 1:1 ratio, proteins in membrane-enriched fractions were separated by SDS-PAGE and digested tryptically in gel. Peptides were eluted, desalted, and analyzed by liquid chromatography-MS/MS. Peptide identification and quantification was performed as described previously (Mühlhaus et al., 2011). Quantification values for single core complex subunits (SU) were computed from quantified peptides, and the average ratio of light (VIPP1-RNAi) to heavy (control) subunits was calculated for the different core complexes. Error bars represent se, and asterisks indicate the significance of the difference of the ratio from one (assuming equal variance; t test, P value ≤ 0.05). (B) Ratios of respiratory chain core complexes in VIPP1-RNAi relative to control cells. Ratios of respiratory chain core complexes were determined as described in (A).
Figure 9.
Figure 9.
Immunofluorescence Microscopy Detects VIPP1 in Distinct Punctae and as Diffuse Material in the Chloroplast. Control cells from the cw15-325 background were grown at ~30 μE m−2 s−1 in TAP-NH4 medium and fixed and processed for immunofluorescence (IF) microscopy as described in Methods. The signal recognized by the affinity-purified anti-VIPP1 antibody is shown in green. Triangles indicate potential rod-like extensions. Bars = 5 μm.
Figure 10.
Figure 10.
VIPP1-RNAi/amiRNA Strains Harbor Aberrant Structures at the Origin of Thylakoid Membranes. (A) Electron microscopy image of a cell from VIPP1-amiRNA strain #18. Cells were grown at ~30 μE m−2 s−1 in TAP-NH4 medium. An overview image is shown on the left, and zoom-ins of the regions demarcated by black boxes are shown on the right. Triangles indicate regions at the origin of multiple thylakoid membrane ramifications. CV, contractile vacuole; N, nucleus; P, pyrenoid; S, starch. Bars in overview images = 1 μm, those in zoom-ins = 0.2 μm. (B) Electron microscopy image of a cell from the control strain. Cells were grown at ~30 μE m−2 s−1 in TAP-NH4 medium. Symbols are as in (A). (C) Electron microscopy image of a cell from VIPP1-RNAi strain #27. Cells were grown at ~30 μE m−2 s−1 in TAP-NO3 medium. Abbreviations are as in (A). (D) Electron microscopy image of a cell from VIPP1-RNAi strain #27. Cells were grown at ~30 μE m−2 s−1 in TAP-NH4 medium. Abbreviations are as in (A).
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