Name: plastoglobule lipid droplet isolation from plant leaf tissue and cyanobacteria
Uploaded: 2022-10-06T14:20:00
Description: Watch this Scientific Journal Video about Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria at JoVE.com

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.

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

<ol>
	<li>Lundquist, P. K., Shivaiah, K. K., Espinoza-Corral, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+throughout+the+evolutionary+tree.">Lipid droplets throughout the evolutionary tree.</a> Progress in Lipid Research. 78, 101029 (2020).</li><li>Lundquist, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+network+of+the+Arabidopsis+plastoglobule+proteome+based+on+quantitative+proteomics+and+genome-wide+coexpression+analysis.">The functional network of the Arabidopsis plastoglobule proteome based on quantitative proteomics and genome-wide coexpression analysis.</a> Plant Physiology. 158 (3), 1172-1192 (2012).</li><li>Ytterberg, A. J., Peltier, J. B., van Wijk, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+profiling+of+plastoglobules+in+chloroplasts+and+chromoplasts.+A+surprising+site+for+differential+accumulation+of+metabolic+enzymes.">Protein profiling of plastoglobules in chloroplasts and chromoplasts. A surprising site for differential accumulation of metabolic enzymes.</a> Plant Physiology. 140 (3), 984-997 (2006).</li><li>Lundquist, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+plastoglobule+kinases+ABC1K1+and+ABC1K3+causes+conditional+degreening,+modified+prenyl-lipids,+and+recruitment+of+the+jasmonic+acid+pathway.">Loss of plastoglobule kinases ABC1K1 and ABC1K3 causes conditional degreening, modified prenyl-lipids, and recruitment of the jasmonic acid pathway.</a> The Plant Cell. 25 (5), 1818-1839 (2013).</li><li>Vidi, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tocopherol+cyclase+(VTE1)+localization+and+vitamin+E+accumulation+in+chloroplast+plastoglobule+lipoprotein+particles.">Tocopherol cyclase (VTE1) localization and vitamin E accumulation in chloroplast plastoglobule lipoprotein particles.</a> Journal of Biological Chemistry. 281 (16), 11225-11234 (2006).</li><li>Lichtenthaler, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastoglobuli+and+the+fine+structure+of+plastids.">Plastoglobuli and the fine structure of plastids.</a> Endeavour. 27 (102), 144-149 (1965).</li><li>Lichtenthaler, H. K., Peveling, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastoglobuli+in+different+types+of+plastids+from+Allium+cepa+L.">Plastoglobuli in different types of plastids from Allium cepa L.</a> Planta. 72 (1), 1-13 (1966).</li><li>Lichtenthaler, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Die+Plastoglobuli+von+Spinat,+ihre+Gr&#246;&#946;e,+Isolierung+und+Lipochinonzusammensetzung.">Die Plastoglobuli von Spinat, ihre Gr&#246;&#946;e, Isolierung und Lipochinonzusammensetzung.</a> Protoplasma. 68 (1-2), 65-77 (1969).</li><li>Lichtenthaler, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastoglobuli+and+lipoquinone+content+of+chloroplasts+from+Cereus+peruvianus+(L)+Mill.">Plastoglobuli and lipoquinone content of chloroplasts from Cereus peruvianus (L) Mill.</a> Planta. 87 (4), 304-310 (1969).</li><li>Simpson, D. J., Baqar, M. R., Lee, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromoplast+ultrastructure+of+Capsicum+carotenoid+mutants+I.+Ultrastructure+and+carotenoid+composition+of+a+new+mutant.">Chromoplast ultrastructure of Capsicum carotenoid mutants I. Ultrastructure and carotenoid composition of a new mutant.</a> Zeitschrift f&#252;r Pflanzenphysiologie. 83 (4), 293-308 (1977).</li><li>Hansmann, P., Sitte, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+and+molecular+structure+of+chromoplast+globules+of+Viola+tricolor.">Composition and molecular structure of chromoplast globules of Viola tricolor.</a> Plant Cell Reports. 1 (3), 111-114 (1982).</li><li>Steinmuller, D., Tevini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+and+function+of+plastoglobuli+:+I.+Isolation+and+purification+from+chloroplasts+and+chromoplasts.">Composition and function of plastoglobuli : I. Isolation and purification from chloroplasts and chromoplasts.</a> Planta. 163 (2), 201-207 (1985).</li><li>Kessler, F., Schnell, D., Blobel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+proteins+associated+with+plastoglobules+isolated+from+pea+(Pisum+sativum+L.)+chloroplasts.">Identification of proteins associated with plastoglobules isolated from pea (Pisum sativum L.) chloroplasts.</a> Planta. 208 (1), 107-113 (1999).</li><li>Grennan, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastoglobule+proteome.">Plastoglobule proteome.</a> Plant Physiology. 147 (2), 443-445 (2008).</li><li>Peramuna, A., Summers, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+and+occurrence+of+lipid+droplets+in+the+cyanobacterium+Nostoc+punctiforme.">Composition and occurrence of lipid droplets in the cyanobacterium Nostoc punctiforme.</a> Archives of Microbiology. 196 (12), 881-890 (2014).</li><li>Austin, J. R., Frost, E., Vidi, P. A., Kessler, F., Staehelin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastoglobules+are+lipoprotein+subcompartments+of+the+chloroplast+that+are+permanently+coupled+to+thylakoid+membranes+and+contain+biosynthetic+enzymes.">Plastoglobules are lipoprotein subcompartments of the chloroplast that are permanently coupled to thylakoid membranes and contain biosynthetic enzymes.</a> The Plant Cell. 18 (7), 1693-1703 (2006).</li><li>Eugeni Piller, L., Abraham, M., Dormann, P., Kessler, F., Besagni, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastid+lipid+droplets+at+the+crossroads+of+prenylquinone+metabolism.">Plastid lipid droplets at the crossroads of prenylquinone metabolism.</a> Journal of Experimental Botany. 63 (4), 1609-1618 (2012).</li><li>Eugeni Piller, L., Glauser, G., Kessler, F., Besagni, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+plastoglobules+in+metabolite+repair+in+the+tocopherol+redox+cycle.">Role of plastoglobules in metabolite repair in the tocopherol redox cycle.</a> Frontiers in Plant Science. 5, 298 (2014).</li><li>Xu, C., Fan, J., Shanklin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+and+functional+connections+between+cytoplasmic+and+chloroplast+triacylglycerol+storage.">Metabolic and functional connections between cytoplasmic and chloroplast triacylglycerol storage.</a> Progress in Lipid Research. 80, 101069 (2020).</li><li>Izquierdo, Y., Fernandez-Santos, R., Cascon, T., Castresana, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplet+isolation+from+Arabidopsis+thaliana+leaves.">Lipid droplet isolation from Arabidopsis thaliana leaves.</a> Bio-Protocols. 10 (24), 3867 (2020).</li><li>Espinoza-Corral, R., Schwenkert, S., Lundquist, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+changes+of+Arabidopsis+thaliana+plastoglobules+facilitate+thylakoid+membrane+remodeling+under+high+light+stress.">Molecular changes of Arabidopsis thaliana plastoglobules facilitate thylakoid membrane remodeling under high light stress.</a> Plant Journal. 106 (6), 1571-1587 (2021).</li><li>Espinoza-Corral, R., Lundquist, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plastoglobule-localized+protein+AtABC1K6+is+a+Mn2+-dependent+kinase+necessary+for+timely+transition+to+reproductive+growth.">The plastoglobule-localized protein AtABC1K6 is a Mn2+-dependent kinase necessary for timely transition to reproductive growth.</a> Journal of Biological Chemistry. 298 (4), 101762 (2022).</li><li>Espinoza-Corral, R., Herrera-Tequia, A., Lundquist, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+topology+and+membrane+interaction+characteristics+of+plastoglobule-localized+AtFBN1a+and+AtLOX2.">Insights into topology and membrane interaction characteristics of plastoglobule-localized AtFBN1a and AtLOX2.</a> Plant Signalling &amp; Behavior. 16 (10), 1945213 (2021).</li><li>Besagni, C., Piller, L. E., Br&#233;h&#233;lin, C., Jarvis, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Plastoglobules+from+Arabidopsis+Plastids+for+Proteomic+Analysis+and+Other+Studies.">Preparation of Plastoglobules from Arabidopsis Plastids for Proteomic Analysis and Other Studies.</a> Chloroplast Research in Arabidopsis. , 223-239 (2011).</li><li>Yang, H., Murphy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+preparation,+sucrose+density+gradients+and+two-phase+separation+fractionation+from+five-day-old+Arabidopsis+seedlings.">Membrane preparation, sucrose density gradients and two-phase separation fractionation from five-day-old Arabidopsis seedlings.</a> Bio-Protocols. 3 (24), 1014 (2022).</li><li>Kelekar, P., Wei, M., Yang, P., Pazour, G. J., King, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Analysis+of+Radial+Spoke+Proteins.">Isolation and Analysis of Radial Spoke Proteins.</a> Cilia: Motors and Regulation. Methods in Cell Biology. 92, 181-196 (2009).</li><li>Chen, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear-encoded+synthesis+of+the+D1+subunit+of+photosystem+II+increases+photosynthetic+efficiency+and+crop+yield.">Nuclear-encoded synthesis of the D1 subunit of photosystem II increases photosynthetic efficiency and crop yield.</a> Nature Plants. 6 (5), 570-580 (2020).</li><li>Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultramicroscopy+reveals+that+senescence+induces+in-situ+and+vacuolar+degradation+of+plastoglobules+in+aging+watermelon+leaves.">Ultramicroscopy reveals that senescence induces in-situ and vacuolar degradation of plastoglobules in aging watermelon leaves.</a> Micron. 80, 135-144 (2016).</li><li>Singh, D. K., Laremore, T. N., Smith, P. B., Maximova, S. N., McNellis, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockdown+of+FIBRILLIN4+gene+expression+in+apple+decreases+plastoglobule+plastoquinone+content.">Knockdown of FIBRILLIN4 gene expression in apple decreases plastoglobule plastoquinone content.</a> PLoS One. 7 (10), 47547 (2012).</li><li>Singh, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FIBRILLIN4+is+required+for+plastoglobule+development+and+stress+resistance+in+apple+and+Arabidopsis.">FIBRILLIN4 is required for plastoglobule development and stress resistance in apple and Arabidopsis.</a> Plant Physiology. 154 (3), 1281-1293 (2010).</li><li>Zheng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gardenia+carotenoid+cleavage+dioxygenase+4a+is+an+efficient+tool+for+biotechnological+production+of+crocins+in+green+and+non-green+plant+tissues.">Gardenia carotenoid cleavage dioxygenase 4a is an efficient tool for biotechnological production of crocins in green and non-green plant tissues.</a> Plant Biotechnology Journal. , (2022).</li><li>Bligh, E. G., Dyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+of+total+lipid+extraction+and+purification.">A rapid method of total lipid extraction and purification.</a> Canadian Journal of Biochemistry &amp; Physiology. 37 (8), 911-917 (1959).</li></ol>

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 &#176;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...

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), 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 proposed2,4,17,18,19. 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 stress2,4,21,22. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AEBSF</td>
			<td>Milipore Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>Antipain.2HCl</td>
			<td>Bachem</td>
			<td>H-1765.0050BA</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Milipore Sigma</td>
			<td>A6106</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbate</td>
			<td>BDH</td>
			<td>BDH9242</td>
			<td></td>
		</tr>
		<tr>
			<td>Bestatin</td>
			<td>Sigma Aldrich</td>
			<td>B8385</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-Glycerophosphate. 2Na5H2O</td>
			<td>EMD Millipore</td>
			<td>35675</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Proliant Biological</td>
			<td>68700</td>
			<td></td>
		</tr>
		<tr>
			<td>Chymostatin</td>
			<td>Sigma Aldrich</td>
			<td>C7268</td>
			<td></td>
		</tr>
		<tr>
			<td>Eragrostis nindensis</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>E-64</td>
			<td>Milipore Sigma</td>
			<td>E3132</td>
			<td></td>
		</tr>
		<tr>
			<td>French Pressure cell (model FA-079)</td>
			<td>SLM/Aminco</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma Aldrich</td>
			<td>L2884</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Multitron shaking incubator</td>
			<td>Infors HT</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-ramidon.2 Na</td>
			<td>Sigma Aldrich</td>
			<td>R7385</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Fisher Chemicals</td>
			<td>M16050</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced Cysteine</td>
			<td>MP Biochemicals</td>
			<td>101444</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Fluoride</td>
			<td>Sigma Aldrich</td>
			<td>S7920</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Ortho-vanadate</td>
			<td>Sigma Aldrich</td>
			<td>450243</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyrophosphate &#183; 10H2O</td>
			<td>Sigma Aldrich</td>
			<td>3850</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorbitol</td>
			<td>Sigma Aldrich</td>
			<td>S3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylvania 15 W fluorescent Gro-Lux tube light bulb, 18&#34;</td>
			<td>Walmart</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Synechocystis sp. PCC 6803</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima MAX-TL Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>A95761</td>
			<td></td>
		</tr>
		<tr>
			<td>Waring Blender (1.2 L)</td>
			<td>VWR</td>
			<td>58977-227</td>
			<td>Commercial blender</td>
		</tr>
		<tr>
			<td>Zea mays</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

plastoglobule lipid droplet isolation from plant leaf tissue and cyanobacteria

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.

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...

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Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria

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.

Plastoglobule lipid droplets are a dynamic sub-compartment of plant chloroplasts and cyanobacteria. Found ubiquitously among photosynthetic species, they ...

The isolation of highly pure plastoglobules is critical to their study. A rapid and effective protocol for their isolation that facilitates their downstream biochemical investigation is presented here. The primary benefit of this method is the relative speed of procedure and its adaptability to numerous plant species and environmental conditions.Particular care must be taken while sonicating thylakoid resuspensions to ensure sufficient force to strip plastoglobules from thylakoids and with subsequent extraction of plastoglobules from sucrose gradient. Demonstrating this procedure will be Dr.Elsinraju Devadasu, a postdoctoral researcher in my laboratory, and Febri Susanto, a PhD student in my laboratory. Begin by acquiring three-weeks-old seedlings of six healthy maize, having V5 growth stage and weighing approximately 120 grams.Clip off all the leaves at the base of the stem and rapidly dunk them in an ice bath before transporting them to the cold room. Working under a green safety lamp, remove the maize leaves from the ice bath and snip them into smaller five-by-five centimeters pieces using scissors. Gently and thoroughly grind half of the clipped leaf tissue in 350 milliliters of the grinding buffer at a level lower than seven on a commercial blender.Start and stop the blender five to six times to ensure that all the leaves are cut. Filter the homogenate through one layer of 25 micron nylon cloth on a large funnel to four 250-milliliter centrifuge bottles. After repeating the blending step with the remaining clipped leaf tissue, evenly divide the filtrate between the four bottles.Remove an aliquot of the leaf filtrate and set it aside to be stored as a representative total leaf sample. Next, centrifuge the filtrate for six minutes at 1, 840 G and four degrees Celsius. Pour off the resulting supernatant and, with gentle swirling movements of the brush, resuspend the pellet in 12 milliliters of medium R 0.2, containing 0.2 molar sucrose.After resuspending the pellet in one bottle, add the suspension to the next bottle and repeat the resuspension to pool the suspensions into one bottle. Distribute the pooled suspension between six three-milliliter ultracentrifuge tubes, reaching a maximum volume of 2.5 milliliters in each tube. Then use the tip sonicator at an amplitude of 100%to sonicate each tube four times for 10 seconds each.Ensure to keep the sonicator horn submerged and away from the liquid surface to prevent frothing. Slowly move the sonicator horn up and down within the suspension during each round. While alternating the four tubes, return each tube to an ice bucket after each sonication to allow the sample to cool.Once done, centrifuge the sonicated crude thylakoid suspension at 150, 000 G for 30 minutes at four degrees Celsius. Skim the cushion surface using a syringe and 22 gauge needle and harvest the resulting yellow and oily floating pad of crude plastoglobules from the sucrose cushion. Recover approximately 500 microliters from each tube and deposit them into a 2.0-milliliter tube.Collect crude thylakoid aliquots before and after the sonication and release of plastoglobules. Once done, either continue the plant tissue processing or store the crude plastoglobules at minus 80 degrees Celsius and purify them later. Grow 50 milliliters of synechocystis species PCC 6802 culture in BG11 media for 7 to 10 days to reach the stationary phase.Using a spectrophotometer, adjust the cell density to an optical density of 2.0 at 750 nanometers. To remove the polysaccharides, centrifuge 50 milliliters of culture and remove the supernatant. Repeat washing of the cells in 50 milliliters of buffer A two times.Resuspend the washed pellet in 25 milliliters of buffer A and break the cells using a French pressure cell at 1, 100 pound square inches. Repeat the process three times in cold conditions until the lysed color changes from green to red, blue, green under white light. Remove the aliquot of the cell homogenate and set it aside to be stored as a representative total cell sample.Distribute the resulting homogenate in eight three-milliliter ultracentrifuge tubes filled to a maximum of 2.5 milliliters in each tube. Overlay this homogenate with 400 microliters of medium R, producing a step gradient. Carefully balance the tubes by adding additional medium R as necessary before centrifuging the tubes.The thylakoid and other heavier organelles, including any polyhydroxyalkanoate bodies, show pellet formation, while plastoglobules form a yellow oily pad on or near the top of the sucrose gradient. Harvest the resulting floating pad of crude plastoglobules with a syringe, as demonstrated earlier, and deposit the harvest into a two-milliliter tube. Scrape plastoglobules off the side of the ultracentrifuge tube wall with a needle tip if necessary.Either continue with cyanobacteria processing or store crude plastoglobules at minus 80 degrees Celsius and purify later. To harvest the pure plastoglobules using the plant tissue processing method, prepare the sucrose gradient described earlier using 500 microliters of crude plastoglobules, 400 microliters of medium R 0.2, and 400 microliters of medium R.After centrifugation, harvest the resulting floating pad of pure plastoglobules into a two-milliliter tube. After aliquoting the pure plastoglobules and flash freezing the aliquots in liquid nitrogen, either store them at minus 80 degrees Celsius or lyophilize them to a dry powder.To harvest pure plastoglobules from cyanobacteria, create a sucrose gradient, as mentioned earlier, using 500 microliters of the crude plastoglobules, 750 microliters of medium R 0.7, and 750 microliters of medium R 0.2. After centrifugation, collect the pure plastoglobules in a 1.5-milliliter tube and aliquot the pure plastoglobules. Once done, flash freeze the pure plastoglobules aliquots in liquid nitrogen and store them directly at minus 80 degrees Celsius or lyophilize to a dry powder.In this method, a significant amount of plastoglobule or lipid droplet material was seen floating on the top layer of the sucrose cushion. After subsequent centrifugation, purified plastoglobules were obtained at or near the surface of the sucrose gradient. After the successful isolation of plastoglobules, immunoblots developed for purity assessment using antibodies raised against arabidopsis thaliana fibrillin 1a and arabidopsis thaliana photosystem II subunit D1 showed that the fibrillin homologues were primarily associated with thylakoids than the plastoglobules.To ensure the efficient and concentrated extraction of the floating pad, place the syringe needle orifice just below the liquid surface of the buffer while completely submerged, then gently pull up. Isolated plastoglobule samples are amenable to subsequent proteomic or lipidomic studies and have been used to demonstrate the changes in the targeted protein-lipid composition under differing environmental conditions. This technique has facilitated new studies of the plastoglobule, including the discovery of associated protein kinase activity and the presence of oligomeric protein complexes through subsequent biochemical investigation.

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