Abstract
Key message
FZL is primarily localized to the chloroplast inner envelope and not to the thylakoids, but nevertheless affects the maintenance of thylakoid membranes and photosynthetic protein complexes.
Abstract
The fuzzy-onion-like protein (FZL) is a membrane-bound dynamin-like GTPase located in the chloroplast. We have investigated the chloroplast sub-localization of the endogenous FZL protein and found it to be primarily localized to the inner envelope. Moreover, we observed that mature leaves of fzl mutants start to turn pale, especially in the midvein area of the leaves, 11 days after germination. We therefore assessed their photosynthetic performance as well as the accumulation of thylakoid membrane proteins and complexes after the initial appearance of the phenotype. Interestingly, we could observe a significant decrease in amounts of the cytochrome b6f complex in 20-day-old mutants, which was also reflected in an impaired electron transport rate as well as a more oxidized P700 redox state. Analysis of differences in transcriptome datasets obtained before and after onset of the phenotype, revealed large-scale changes in gene expression after the phenotype became visible. In summary, we propose that FZL, despite its localization in the inner chloroplast envelope has an important role in thylakoid maintenance in mature and aging leaves.
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Acknowledgements
This project was funded by the Deutsche Forschungsgemeinschaft (DFG), TR175, Project B05 to J.S. and SFB1035, Project A04 to S.S. F.S. was financially supported by Rhenac Green Tec AG (Hennef, Germany) for some time of the conducted study. We would like to thank Sabine Grahl for data analysis and help with supervision. Antje von Schaewen and Jörg Meurer are kindly acknowledged for providing antisera. We would further like to thank Chris Carrie and his group for providing isolated mitochondria.
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11103_2018_748_MOESM1_ESM.pdf
Online Resource 1—Phenotype of fzl-2 and fzl-3 mutant plants grown under LED light. a) Immunoblot analysis of fzl-2 and fzl-3 mutant lines using anti-FZL antiserum. Upper panel: The 10% SDS gel was loaded with 15 μg of total protein. Lower panel: Control for equal loading, showing that the antiserum cross-reacts with Rubisco. (c) Phenotypes of fzl-2 and fzl-3 mutants grown in long-day conditions under LED light (200 µM m-2 s-1) show a more pronounced pale green phenotype especially at the mid-rib region. (d) Isolated pea chloroplasts were fractionated into inner envelopes, thylakoid membranes and stroma and samples (equivalent to 15 μg protein) were loaded on a 12 % SDS gel. After electrophoresis and membrane transfer, blots were probed with the anti-FZL antiserum. Antibodies against the inner-envelope protein Tic110 and Tic40, the soluble stromal protein FBPase and the thylakoid membrane protein D1 were used as controls (PDF 300 KB)
11103_2018_748_MOESM2_ESM.pdf
Online Resource 2—Chlorophyll concentration in µg/g fresh weight. Chlorophyll concentration was measured using leaves from 10, 13 and 20-day-old plants grown in LED light, P-value > 0.00001, n > 4. Values correspond to Figure 1 (PDF 590 KB)
11103_2018_748_MOESM3_ESM.pdf
Online Resource 3—Characterization of fzl mutant plants grown under standard light conditions. (a) Phenotype of fzl mutants grown in long-day conditions under 100 µM/s/m2 light. On day 20, fzl plants show a pale green phenotype. (b) Chloroplast size measurement in fzl plants. The diameters of 50 individual chloroplasts were measured using ImageJ software based on confocal micrographs of the chlorophyll autofluorescence of 21-day old leaves. Student’s t-test was performed and P-values were calculated for the 95% confidence interval. P-value <0.00001. (c) Chlorophyll concentration was measured using leaves from 25-day-old plants grown in normal light. In the fzl plants, chlorophyll concentrations were reduced to 70% of WT levels. P-value: 0.004, n=3 (PDF 144 KB)
11103_2018_748_MOESM5_ESM.pdf
Online Resource 5—In silico modeling of FZL with BDLP. In silico structural prediction analysis was performed using Phyre2 web portal. (a) FZL (288-912 aa) structure predicted with 100% confidence interval based on the structure of GDP bound BDLP (PDB Id: 2J68). (b) One on one threading of FZL (288-912 aa) to the GDP bound BDLP protein structure (PDB Id: 2J68). (c) Structure of GDP bound BDLP (PDB Id: 2J68) (PDF 333 KB)
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Patil, M., Seifert, S., Seiler, F. et al. FZL is primarily localized to the inner chloroplast membrane however influences thylakoid maintenance. Plant Mol Biol 97, 421–433 (2018). https://doi.org/10.1007/s11103-018-0748-3
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DOI: https://doi.org/10.1007/s11103-018-0748-3