The separation and identification of plant phospholipids and glycolipids by two-dimensional thin- layer chromatography

Freeze-substitution and Epon embedment were quantitatively evaluated for their effectiveness in retaining water-soluble metabolites in plant tissues. Roughly 99% of the 80% (v/v) ethanol-extractable radioactivity in photosynthetically labeled soybean leaf discs and in petiole fragments containing translocated "C was retained during freeze-substitution in acetone or propylene oxide and embedment in Epon. Substantially more activity was lost from "C-sucrose-infiltrated pith blocks, but most or all of this loss came from the block surface. The procedure was effective for a sucrose concentration as low as 0.004%. Sections floated on water retained most of their UC sucrose, and high resolution autoradiographs could easily be prepared without resorting to dry procedures. Embedded "Csucrose was apparently chemically unreactive, since there was no loss of radioactivity when sections were stained with the periodic acid-Schiff reagent, nor did the embedded sucrose show staining.Histological procedures for the localization of water-soluble compounds in the phloem have followed in large part the experimental approach outlined by Branton and Jacobson (2). This consists essentially of freeze-drying the tissue and, with a recent exception, embedment in paraffin, followed by "dry autoradiography." Fritz and Eschrich (7) have recently vastly improved on this approach by treating freeze-dried tissue with silicone and then embedding in Epon, rather than in paraffin. However, in all cases where sufficient resolution was attained, an irregular distribution of silver grains over the sieve tube lumen was observed (1,7,9,12,15,16).During earlier work with freeze-substitution, it was observed that at least some water-soluble carbohydrates were retained by freeze-substitution and Epon embedment but were lost during aqueous aldehyde fixation (6). While developing this procedure for the embedment of sugars for microscopy, it was also found that the water-soluble contents of cells in freeze-dried preparations were very susceptible to shrinkage (5). This shrinkage might explain the apparent uneven distribution of radioactive water-soluble compounds in the sieve tubes of freeze-dried phloem. With care, this artifact could be virtually eliminated from freeze-substituted tissue, but not from 'This work was supported in part by Grant GB-14719 from the National Science Foundation. freeze-dried tissue. Freeze-substitution, then, seemed to offer the double advantage of providing a much simpler alternative to freeze-drying as well as a more accurate cytological picture of the tissue's water-soluble constituents. Furthermore, embedment of the tissue in Epon greatly improves both the autoradiographic resolution (thinner sections) and the ease of preparing autoradiographs (7, 13). Epon also physically protects the cell structure from the adverse effects of water, thus allowing meaningful cytological observations of the same material.MATERIALS AND METHODS Plant Tissues. Tissue containing "C-labeled metabolites was provided by photosynthetic i...

Section: Resultsmentioning

confidence: 97%

The Retention of Water-soluble Compounds during Freeze-Substitution and Microautoradiography

Fisher

Housley

1972

“…After extraction, polar lipids were separated by one-dimensional TLC (silica gel precoated plates from Merck, Darmstadt). As solvent systems chlo-roform-methanol-acetic acid-water (170:40:30:7, v/v) or chloroform-acetone-methanol-acetic acid-water (50:20:10:10:5, v/v) were used (10). Neutral lipids were separated as described by Mangold (12).…”

mentioning

confidence: 99%

Changes in Chloroplast Membrane Lipids during Adaptation of Barley to Extreme Salinity

Müller¹,

Santarius²

1978

During adaptation of barley (Hordeum vulgare L.) seedlings to extremely high concentrations of sodium chloride in the root space, the content of galactolipids of chloroplast membranes decreased considerably. Alterations in membrane lipids were due to the high concentration of ions rather than to the increase in the water potential. Sodium chloride was accumulated in the leaf cells and affected lipid-synthesizing enzymes such as galactosyl transferase and acylase which are attached to the chloroplast envelope. The return of salt-adapted barley seedlings to a nutrient solution with low salt concentration resulted in a reversal of the observed changes.It is suggested that the decrease in content of galactolipids in biomembranes is one of the factors causing increased salt resistance in barley plants which are adapted to extreme salinity.Many plants are able to adapt to saline environments. While salt resistance is acquired, various morphological, anatomical, and biochemical changes take place within the plant cells (9, 15). It is still unknown which changes are responsible for the increase in salt resistance, and which passively accompany changes in resistance.During the last few years it has turned out that mainly cellular membranes are stress-sensitive sites within plant cells (4). It was found that photophosphorylation and electron transport of isolated thylakoid membranes were rapidly and irreversibly inactivated by high concentrations of inorganic salts (16,18).Plants growing in saline environments either exclude salts from or tolerate them inside their cells. In the latter case, the increase in salt resistance must be due to the protection of the sensitive membranes. This could be achieved either by structural changes of the biomembranes or by the synthesis and accumulation of protective compounds. However, no directly relevant data in this field are available (9). Major building blocks of biomembranes are lipids and proteins. It is assumed that structural changes may be indicated by changes in the lipid and/or protein composition. We wanted to know whether changes in the lipids of cellular membranes accompany and are essential for the development of salt resistance during the adaptation of barley seedlings to extreme salinity. As From leaves of 7-to 14-day-old seedlings, chloroplasts were isolated according to the method B described by Plesnicar and Bendall (14). After filtration, the homogenate was centrifuged for 5 min at 200g and plastids were sedimented from the supernatant by centrifugation for 15 min at 600g. The pellet was resuspended and washed once for 10 min at 2,000g. When thylakoids were needed, isolated chloroplasts were suspended in distilled H20 and freed thylakoids were sedimented for 5 min at 20,000g.Chloroplast envelopes were obtained from isolated chloroplasts according to van Besouw (20). After washing, intact chloroplasts were suspended in 50 mM citrate buffer (pH 5.5) and, after 20 min, the envelopes were stripped off from the swollen chloroplasts by five strokes in a Potter g...

“…Phospholipids are first eluted from silicic acid (Bio-sil HA, 325 mesh, Bio-Rad Laboratories) column according to Vorbeck and Marinetti (18), after removal of neutral lipids with chloroform and galactolipids with acetone, and further fractionated by thin layer chromatography in chloroform-acetone-methanol-acetic acid-water (5:2:1:1: 0.5, v/v) solvents (9). Characterization and identification of each class of components were performed by one-or two-dimensional thin layer chromatography with the aid of specific spray reagents as described earlier (8,17 …”

mentioning

confidence: 99%

Changes in Lipid Composition during Greening of Etiolated Pea Seedlings

Trémolières

Lepage

1971

After 7 days of germination in the dark, the three sections of pea seedlings studied (cotyledons, stems, and young leaves) are rich in linoleic acid; after illumination of the seedlings a very significant increase in linolenic acid is observed in the young leaves section, whereas only small variations are noted in the fatty acid composition of the other sections. The increase in linolenic acid results from the increase in galactolipid content of the young leaves; these already linolenic acid-rich galactolipids are present but onl-in small amounts in the etiolated seedlings (10% of total lipid).Variations in composition of the otlher lipid classes (phospholipids and neutral fats) were also studied. The possibility of fatty acid transport from the cotyledons toward the young leaves during the synthesis of the photosynthetic apparatus is discussed.or clover leaves (17) have shown that during greening the incorporation of acetate-1-'4C into linolenic acid does not clearly indicate how this fatty acid is synthesized as the chloroplast matures. We may assume that linolenic acid is not mainly formed de novo, but from an endogenous precursor already present in the etiolated seedling (perhaps from another fatty acid present in large quantities in the cotyledon). However, the low incorporation of radioactive tracers in the fatty acid portion does not permit us to define further the metabolic pathways of the galactolipid biosynthesis.In this study, we have followed the changes in fatty acid composition of each lipid class during the greening of the etiolated pea seedlings (in which the cotyledons and the young leaves are clearly cut) in order to elucidate the biosynthetic pathways of chloroplast galactolipids as well as the action of light on the formation of these lipids, and to show the relationship between storage lipids and structural lipids during greening. MATERIAL AND METHODSVariations in lipid metabolism as related to greening and formation of the photosynthetic apparatus have been studied mainly in algae, Chllorella and Euglena (5,(12)(13)(14). But it is known that the greening of higher plants differs markedly from that of photosynthetic algae. Whereas algae pass from a heterotrophic metabolism to an autotrophic or semiautotrophic metabolism during greening and this transformation is largely reversible, in higher plants the etiolated seedlings subsist only at the expense of food reserves made by the plant, and greening is essential for its survival and presents an irreversible feature. Moreover, although the lipid compositions of green algae and higher plants have common characteristics such as high levels of galactolipids with polyunsaturated fatty acids and presence of phosphatidylglycerol with trans-3-hexadecenoic acid (7), appreciable differences are encountered between them: (a) in algae, the galactolipid polyunsaturated fatty acids consist of 16 and 18 carbon chain fatty acids, whereas in higher plants, y-linolenic acid alone accounts for 80% of the galactolipid fatty acid (7); (b) For germination...