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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

A fast and efficient protocol is presented for the isolation of plastoglobule lipid droplets associated with various photosynthetic organisms. The successful preparation of isolated plastoglobules is a crucial first step that precedes detailed molecular investigations such as proteomic and lipidomic analyses.

Abstract

Plastoglobule lipid droplets are a dynamic sub-compartment of plant chloroplasts and cyanobacteria. Found ubiquitously among photosynthetic species, they are believed to serve a central role in the adaptation and remodeling of the thylakoid membrane under rapidly changing environmental conditions. The capacity to isolate plastoglobules of high purity has greatly facilitated their study through proteomic, lipidomic, and other methodologies. With plastoglobules of high purity and yield, it is possible to investigate their lipid and protein composition, enzymatic activity, and protein topology, among other possible molecular characteristics. This article presents a rapid and effective protocol for the isolation of plastoglobules from chloroplasts of plant leaf tissue and presents methodological variations for the isolation of plastoglobules and related lipid droplet structures from maize leaves, the desiccated leaf tissue of the resurrection plant, Eragrostis nindensis, and the cyanobacterium, Synechocystis sp. PCC 6803. Isolation relies on the low density of these lipid-rich particles, which facilitates their purification by sucrose density flotation. This methodology will prove valuable in the study of plastoglobules from diverse species.

Introduction

The current understanding of plastoglobule composition and function has emerged through detailed proteomic and lipidomic studies1,2,3,4,5. Such studies have been greatly aided by a rapid and effective method of isolation that relies on their very low density for efficient separation using sucrose gradients. Initial methods of plastoglobule isolation were achieved from species such as the beech tree (Fagus sylvatica), scotch broom (Sarothamnus scoparius), onion (Allium cepa), spina....

Protocol

1. Crude plastoglobule isolation

  1. Crude plastoglobule extraction from un-stressed maize leaf tissue
    1. Acquire six healthy maize seedlings approximately 3 weeks old and nearly at the V5 growth stage, weighing approximately 120 g.
    2. Clip off all the leaves at the base of the stem, rapidly dunk them in an ice bath, and transport to the cold room.
    3. Working under a green safety lamp, remove the maize leaves from the ice bath and snip them into smaller pieces (around 5 c.......

Representative Results

Upon completion of step 1 of the protocol, one should be able to readily see a considerable amount of plastoglobule/lipid droplet material floating on (or near) the top layer of the sucrose cushion (Figure 1B-C). Other fractions could also be collected at this stage. For example, the thylakoids will be pelleted and can be re-suspended with medium R 0.2 for subsequent analyses. After subsequent centrifugation, purified plastoglobules will be obtained at or ne.......

Discussion

To minimize physiological/biochemical changes to the material and protect certain photo- and thermo-labile prenyl-lipid pigments that are a rich component of plastoglobules, it is critical to perform the isolation at 4 °C and protected from light. As indicated above, the initial steps are performed in the cold room under a safety lamp using a green-emitting light bulb. The subsequent steps performed in the laboratory are under dimmed lights and use ice or refrigerated centrifugation. For similar reasons, the inclusi.......

Acknowledgements

Research in the Lundquist lab group is supported by grants from the NSF (MCB-2034631) and USDA (MICL08607) to P.K.L. The authors thank Dr. Carrie Hiser (MSU) for support in the development of the cyanobacterial plastoglobule isolation method.

....

Materials

NameCompanyCatalog NumberComments
AEBSFMilipore SigmaP7626
Antipain.2HClBachemH-1765.0050BA
AprotininMilipore SigmaA6106
AscorbateBDHBDH9242
BestatinSigma AldrichB8385
Beta-Glycerophosphate. 2Na5H2OEMD Millipore35675
Bovine Serum AlbuminProliant Biological68700
ChymostatinSigma AldrichC7268
Eragrostis nindensisN/AN/A
E-64Milipore SigmaE3132
French Pressure cell (model FA-079)SLM/AmincoN/A
HEPESSigma AldrichH3375
LeupeptinSigma AldrichL2884
Magnesium ChlorideSigma AldrichM8266
Multitron shaking incubatorInfors HTN/A
Phospho-ramidon.2 NaSigma AldrichR7385
Potassium HydroxideFisher ChemicalsM16050
Reduced CysteineMP Biochemicals101444
Sodium FluorideSigma AldrichS7920
Sodium Ortho-vanadateSigma Aldrich450243
Sodium Pyrophosphate · 10H2OSigma Aldrich3850
SorbitolSigma AldrichS3889
SucroseSigma AldrichS9378
Sylvania 15 W fluorescent Gro-Lux tube light bulb, 18"WalmartN/A
Synechocystis sp. PCC 6803N/AN/A
Optima MAX-TL UltracentrifugeBeckman CoulterA95761
Waring Blender (1.2 L)VWR58977-227Commercial blender
Zea maysN/AN/A

References

  1. Lundquist, P. K., Shivaiah, K. K., Espinoza-Corral, R. Lipid droplets throughout the evolutionary tree. Progress in Lipid Research. 78, 101029 (2020).
  2. Lundquist, P. K., et al.

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PlastoglobuleLipid DropletIsolationPlant Leaf TissueCyanobacteriaThylakoidSucrose GradientUltracentrifugationSonicationMaizeLeaf FiltrateCentrifugationResuspensionBiochemical Analysis

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