Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria

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 studies^1,2,3,4,5. 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), spinach (Spinacia oleracea), pansy (Viola tricolor), pepper (Capsicum annuum), and pea (Pisum sativum)^{6,7,8,9,10,11,12,13}. An updated method to isolate chloroplast plastoglobules in a more efficient and better yielding manner was later presented by Ytterberg et al.^3,14. While initially employed for the study of the plastoglobules of Arabidopsis thaliana leaf chloroplasts, we have successfully employed this updated method for the healthy leaf tissue of other plant species, both monocot and dicot, including maize (Zea mays), tomato (Solanum lycopersicum), lovegrass (Eragrostis nindensis), purple false brome (Brachypodium distachyon), and wild tobacco (Nicotiana benthamiana; unpublished results). Furthermore, the isolation method has been successfully adapted to the plastoglobules of cyanobacteria, including Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120¹⁵, and the desiccated leaf tissue of the resurrection plant, E. nindensis.

Chloroplast plastoglobules of healthy leaf tissue are physically connected to the thylakoid membranes¹⁶. Despite this physical continuity, the two chloroplast sub-compartments maintain distinct lipid and protein compositions, although the regulated exchange of lipid and protein between the two compartments has been proposed^2,4,17,18,19. In fact, an interesting hemifusion model has recently been proposed for the trafficking of neutral lipids between chloroplasts and cytosol¹⁹. Due to the physical continuity of plastoglobules and thylakoids, the isolation method with healthy leaf tissue begins with the collection of a pelleted crude thylakoid preparation, which is subsequently sonicated to separate the plastoglobules from the thylakoids, which is in contrast to methods used for isolating cytosolic lipid droplets²⁰. Ultracentrifugation on a sucrose cushion then floats the low-density plastoglobules up through the sucrose, effectively separating them from the thylakoids, nuclei, and other high-density material. In contrast, plastoglobules in cyanobacteria, as well as those of desiccated leaf tissue, evidently exist in vivo in a free-floating form. Hence, their isolation involves directly floating on a sucrose gradient. This article demonstrates the isolation method from healthy leaf tissue and further demonstrates two variations that can be used to isolate plastoglobules from desiccated leaf tissue or cyanobacterial cultures, greatly expanding the physiological breadth and evolutionary context in which plastoglobules can be studied.

Isolated plastoglobules can subsequently be used for any number of downstream analyses to investigate molecular characteristics. We have used the isolated plastoglobules from A. thaliana leaf tissue for extensive proteomic and lipidomic analysis under differing environmental conditions or genotypes, demonstrating the selective modification of protein and lipid composition in adaptation to stress^2,4,21,22. In addition, in vitro kinase assays that demonstrate trans-phosphorylation activity associated with isolated plastoglobules have been performed²², the oligomeric states of protein components has been investigated using native gel electrophoresis ²¹, and protease-shaving assays have been performed²³.

The primary benefit of this method is the relative speed of the procedure. In our experience, the protocols outlined below can be fully completed within approximately 4 h. An alternate method to isolate plastoglobules from leaf tissue has been described, which allows the simultaneous isolation of other chloroplast sub-compartments²⁴. This alternative method offers some clear advantages when quantitative comparison to the other chloroplast sub-compartments is necessary or desired. However, this alternative method is also more tedious and will provide a significantly lower yield of isolated plastoglobules from comparable quantities of leaf tissue. When a focused study of plastoglobules is the aim, the methodology outlined here is the optimal choice. Nonetheless, total leaf and crude thylakoid aliquots can be collected during the sample preparation, and it is highly recommended to do so, to have reference samples for subsequent comparison.

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 cm x 5 cm) using scissors.
   4. Gently but thoroughly grind half of the clipped leaf tissue in a commercial blender in 350 mL of grinding buffer (Table 1). Start-stop the blender 5x-6x to ensure that all the leaves are cut. Do not use higher than level 7 on the blender.
   5. Filter the homogenate through one layer of 25 µm nylon cloth on a large funnel into four 250 mL centrifuge bottles. Then, repeat step 1.1.4 with the second half of the clipped leaf tissue.
   6. Evenly divide the filtrate between the four bottles and centrifuge for 6 min at 1,840 x g at 4 °C. Remove an aliquot of the leaf filtrate and set it aside prior to centrifugation to be stored as a representative total leaf sample.
   7. Pour off the resulting supernatant and gently resuspend the pellet in 12 mL of medium R 0.2 containing 0.2 M sucrose (Table 1) by swirling and resuspending with gentle movements of the brush. After resuspending each pellet, carry the suspension over to the next bottle and repeat the resuspension. Pool the suspensions into one bottle.
   8. Distribute the pooled suspension between six 3 mL ultracentrifuge tubes, reaching a maximum volume of 2.5 mL in each tube.
   9. Sonicate each tube 4x, for 10 s each time, using a tip sonicator at an amplitude of 100%. Be careful to keep the sonicator horn submerged and away from the liquid surface to prevent frothing.
   10. Slowly move the sonicator horn up and down within the suspension during each round. Alternate between the four tubes, returning each tube to an ice bucket after each sonication to allow the sample to cool.
   11. Centrifuge the sonicated crude thylakoid suspension at 150,000 x g for 30 min at 4 °C. Harvest the resulting floating pad of crude plastoglobules from the surface of the sucrose cushion (readily seen as a yellow, oily pad) using a syringe and 22 G needle by skimming the surface of the cushion with the opening of the needle, recovering approximately 500 µL from each tube. Deposit into a 2.0 mL tube.
   12. Collect aliquots of the crude thylakoid prior to and after the sonication and release of plastoglobules. Continue to step 2.1. Alternatively, store the crude plastoglobules at −80 °C and purify at a later time.
2. Crude plastoglobule extraction from desiccated E. nindensis leaf tissue
   1. Acquire a pot (comprising of three individual plants per pot) of fully desiccated, 8-9-week-old E. nindensis (nearly 40 g of tissue).
   2. Clip off all the leaf tissue from the base of the plant, just above the soil, using scissors and immediately dunk the tissue in an ice bath. Transport the tissue to the cold room.
   3. Working under a green safety lamp, snip the E. nindensis leaves into smaller pieces (around 5 cm x 5 cm) using scissors.
   4. Gently but thoroughly grind the leaves with 100 mL of grinding buffer (Table 1) in a commercial blender. Start-stop the blender several times to ensure that all leaves are being cut. Do not use higher than level 7 on the blender.
   5. Filter the homogenate through one layer of 25 µm nylon cloth on a large funnel into a 250 mL conical flask. Remove an aliquot of the leaf filtrate and set it aside prior to centrifugation to be stored as a representative total leaf sample.
   6. Add the appropriate amount of sucrose to bring to a final concentration of 0.5 M and distribute the solution into 250 mL centrifuge tubes.
      NOTE: A higher concentration of sucrose compared to the Z. mays preparation is used to ensure flotation of the cytosolic lipid droplets prior to the subsequent sonication/release of the thylakoid-bound plastoglobules.
   7. Centrifuge the tubes at 45,000 x g for 30 min at 4 °C. A mixture of plastoglobules and cytosolic lipid droplets will be readily seen as a yellow, oily pad on or near the surface of the sucrose cushion (Figure 1B). Collect the floating material using a syringe with a 22 G needle by skimming the surface of the cushion with the opening of the needle, and deposit into a tube.
   8. Discard the remaining supernatant after collecting the free-floating plastoglobule/lipid droplet mixture.
   9. To isolate the plastoglobules connected to residual thylakoid, resuspend the pellet in 12 mL of medium R 0.2 by swirling and resuspending with gentle movements of the brush. Pool the suspensions into one bottle. Continue to step 1.1.8.
3. Crude plastoglobule extraction from cyanobacteria
   1. Grow a 50 mL culture of Synechocystis sp. PCC 6803 to the stationary phase (about 7-10 days) and adjust the cell density to an OD₇₅₀ of 2.0 using a spectrophotometer. For culture conditions, use BG-11 media and grow in an incubator at a light intensity of 150 µmol photons/m²/s, 2% CO₂, 32 °C, and with continuous shaking at 150 rpm.
   2. To remove the polysaccharides, wash the cells by centrifuging the 50 mL culture at 6,000 x g for 45 min at 4 °C and subsequently removing the supernatant. Continue by washing the cells twice in 50 mL of buffer A (Table 1). Remove an aliquot of the cell homogenate and set it aside prior to centrifugation to be stored as a representative total cell sample
   3. Resuspend the washed pellet in 25 mL of Buffer A and break the cells using a French pressure cell at 1,100 psi, repeating the process 3x (put the sample on ice between each cycle to avoid protein denaturation) until the lysed color changes from green to red-blue-green under white light. Use a pre-cooled cell and perform this step in the cold room.
   4. Distribute the resulting homogenate between eight 3 mL ultracentrifuge tubes filled to a maximum of 2.5 mL in each tube and then carefully overlay with 400 µL of medium R, producing a step gradient.
      NOTE: Refer to Yang, et al. and Kelekar, et al. for detailed descriptions of sucrose gradient preparation^25,26.
   5. Carefully balance the tubes by adding additional medium R, as necessary, and centrifuge for 30 min at 150,000 x g at 4 °C. The thylakoid and other heavier organelles (including any polyhydroxyalkanoate bodies) will pellet, while plastoglobules will be readily seen as a yellow, oily pad on or near the top of the sucrose gradient (Figure 1C).
   6. Harvest the resulting floating pad of crude plastoglobules with a syringe and 22G needle and deposit into a 2 mL tube. Scrape plastoglobules off the side of the ultracentrifuge tube wall with the needle tip if necessary.
   7. Continue to step 2.2. Alternatively, store crude plastoglobules at -80 °C and purify later.

2. Harvesting pure plastoglobules

1. Plant tissue processing
   1. Produce sucrose gradients in 2.5 mL ultracentrifuge tubes by first layering with 500 µL of crude plastoglobules from step 1.1 or step 1.2 mixed with 500 µL of medium R 0.7, to bring to a total volume of 1 mL, then overlay with 400 µL of medium R 0.2, followed by overlaying with 400 µL of medium R.
      NOTE: Refer to Yang et al. and Kelekar et al. for detailed descriptions of sucrose gradient preparation^25,26.
   2. Carefully balance the tubes by adding excess medium R to the top layer, as necessary. Then centrifuge at 150,000 x g for 1.5 h at 4 °C.
   3. Harvest the resulting floating pad of pure plastoglobules (Figure 1A) with a syringe and 22G needle and deposit into a 2 mL tube. Scrape plastoglobules off the top of the centrifuge tube wall with the needle tip if necessary.
   4. Aliquot the pure plastoglobules and flash freeze in liquid nitrogen. Store directly at -80 °C or lyophilize to a dry powder.
2. Cyanobacteria processing
   1. Produce a sucrose gradient in four 2.5 mL ultracentrifuge tubes layered first with 500 µL of the crude plastoglobules from step 1.3 mixed with 750 µL of medium R 0.7, then overlay with 750 µL of medium R 0.2.
      NOTE: Refer to Yang et al. and Kelekar et al. for detailed descriptions of sucrose gradient preparation^25,26.
   2. Centrifuge the sucrose gradient at 150,000 x g for 90 min at 4 °C. Collect the pure plastoglobules (Figure 1C) with a syringe and 22 G needle from the top phase of the sucrose gradient and transfer to a 1.5 mL tube.
   3. Aliquot the pure plastoglobules and flash-freeze in liquid nitrogen. Store directly at −80 °C or lyophilize to a dry powder.

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...

Materials

Name	Company	Catalog Number	Comments
AEBSF	Milipore Sigma	P7626
Antipain.2HCl	Bachem	H-1765.0050BA
Aprotinin	Milipore Sigma	A6106
Ascorbate	BDH	BDH9242
Bestatin	Sigma Aldrich	B8385
Beta-Glycerophosphate. 2Na5H2O	EMD Millipore	35675
Bovine Serum Albumin	Proliant Biological	68700
Chymostatin	Sigma Aldrich	C7268
Eragrostis nindensis	N/A	N/A
E-64	Milipore Sigma	E3132
French Pressure cell (model FA-079)	SLM/Aminco	N/A
HEPES	Sigma Aldrich	H3375
Leupeptin	Sigma Aldrich	L2884
Magnesium Chloride	Sigma Aldrich	M8266
Multitron shaking incubator	Infors HT	N/A
Phospho-ramidon.2 Na	Sigma Aldrich	R7385
Potassium Hydroxide	Fisher Chemicals	M16050
Reduced Cysteine	MP Biochemicals	101444
Sodium Fluoride	Sigma Aldrich	S7920
Sodium Ortho-vanadate	Sigma Aldrich	450243
Sodium Pyrophosphate · 10H2O	Sigma Aldrich	3850
Sorbitol	Sigma Aldrich	S3889
Sucrose	Sigma Aldrich	S9378
Sylvania 15 W fluorescent Gro-Lux tube light bulb, 18"	Walmart	N/A
Synechocystis sp. PCC 6803	N/A	N/A
Optima MAX-TL Ultracentrifuge	Beckman Coulter	A95761
Waring Blender (1.2 L)	VWR	58977-227	Commercial blender
Zea mays	N/A	N/A