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Title: Subdiffraction-resolution live-cell imaging for visualizing thylakoid membranes

Abstract

The chloroplast is the chlorophyll-containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live-cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three-dimensional structured illumination microscopy (3D-SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D-SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild-type and mutant strains. Using 3D-SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed themore » green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D-SIM. This study demonstrates that 3D-SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.« less

Authors:
ORCiD logo [1];  [2];  [1]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Div.; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology, Howard Hughes Medical Institute
  2. Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology, Howard Hughes Medical Institute
Publication Date:
Research Org.:
Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1560876
Alternate Identifier(s):
OSTI ID: 1462279; OSTI ID: 1559159
Grant/Contract Number:  
SC0018301; AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
The Plant Journal
Additional Journal Information:
Journal Volume: 96; Journal Issue: 1; Related Information: Figure S1. Optical sections of reconstructed three‐dimensional structured illumination microscopy images of the side view of the chloroplast isolated from wild‐type Arabidopsis thaliana leaves.Figure S2. Optical sections of reconstructed three‐dimensional structured illumination microscopy images of the side view of the chloroplast isolated from Arabidopsis thaliana stn7 stn8 mutant leaves.Figure S3. Optical sections of reconstructed three‐dimensional structured illumination microscopy images of the side view of the chloroplast isolated from Arabidopsis thaliana tap38 mutant leaves.Figure S4. Optical sections of reconstructed three‐dimensional structured illumination microscopy images of a wild‐type Chlamydomonas reinhardtii cell.Figure S5. Additional reconstructed three‐dimensional structured illumination microscopy images showing shrinkage of thylakoid membranes at the lobe region of Chlamydomonas reinhardtii chloroplasts under high‐light conditions for 1 day.; Journal ID: ISSN 0960-7412
Publisher:
Society for Experimental Biology
Country of Publication:
United States
Language:
English
Subject:
46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND TECHNOLOGY

Citation Formats

Iwai, Masakazu, Roth, Melissa S., and Niyogi, Krishna K. Subdiffraction-resolution live-cell imaging for visualizing thylakoid membranes. United States: N. p., 2018. Web. doi:10.1111/tpj.14021.
Iwai, Masakazu, Roth, Melissa S., & Niyogi, Krishna K. Subdiffraction-resolution live-cell imaging for visualizing thylakoid membranes. United States. doi:10.1111/tpj.14021.
Iwai, Masakazu, Roth, Melissa S., and Niyogi, Krishna K. Mon . "Subdiffraction-resolution live-cell imaging for visualizing thylakoid membranes". United States. doi:10.1111/tpj.14021. https://www.osti.gov/servlets/purl/1560876.
@article{osti_1560876,
title = {Subdiffraction-resolution live-cell imaging for visualizing thylakoid membranes},
author = {Iwai, Masakazu and Roth, Melissa S. and Niyogi, Krishna K.},
abstractNote = {The chloroplast is the chlorophyll-containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live-cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three-dimensional structured illumination microscopy (3D-SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D-SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild-type and mutant strains. Using 3D-SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D-SIM. This study demonstrates that 3D-SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.},
doi = {10.1111/tpj.14021},
journal = {The Plant Journal},
number = 1,
volume = 96,
place = {United States},
year = {2018},
month = {7}
}

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Figures / Tables:

Figure 1 Figure 1: The subdiffraction-resolution live-cell imaging analysis of the grana size of A. thaliana chloroplasts. Chl fluorescence from A. thaliana chloroplasts in the mesophyll tissue observed and analyzed by 3D-SIM. As compared with the reconstructed widefield image (a), reconstructed 3D-SIM image (b) of chloroplasts in the WT showed distinct roundmore » shape structures, indicating the individual grana. The reconstructed 3D-SIM images of chloroplasts in (b) the WT control, (c) the stn7 stn8 mutant, (d) the tap38 mutant, and the WT acclimated to (e) far-red (FR) light, and (f) blue light (BL) conditions were analyzed to measure the differences in the grana size. Scale bars, 5 μm. FWHM of grana from (g) the WT control (n = 300 from 8 chloroplasts), (h) the stn7 stn8 mutant (n = 124 from 8 chloroplasts), (i) the tap38 mutant (n = 251 from 8 chloroplasts), and the WT chloroplasts acclimated to (j) FR light (n = 149 from 8 chloroplasts) or (k) BL (n = 172 from 8 chloroplasts). Inset numbers indicate mean FWHM ± SD of all chloroplasts, and data represent means ± SD for each chloroplast.« less

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    Works referencing / citing this record:

    Regulation of Oxygenic Photosynthesis during Trophic Transitions in the Green Alga Chromochloris zofingiensis
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