The Pattern and Control of Phospholipid Metabolism in Wheat Aleurone Tissue

Exposure of isolated aleurone tissue from the wheat (Triticam aetirum) variety Kite which contains the Rht2 allele, to low temperature (5°C) for 20 h prior to addition of exogenous GA3, resulted in significant changes in the content of lipids, especially phospholipids. Sigificat low temperature-induced changes in both the head group and acyl contents of two phospholipids, phosphatidylcholine and phosphatidylethanolamine, were detected. More importantly, these changes displayed a very close temporal relationship with the low temperature-induced increase in GA3 sensitivity. Further, this relationship was paralleled by a highly significant correlation between the changes in the phospholipids and the changes in a-amylase production. These results underline the possibility that the GA3 receptor sites are membranbased lipids.Aleurone tissue of cereal seeds, when exposed to GA3, synthesizes and secretes hydrolytic enzymes, including a-amylase, into the starchy endosperm (5). Deembryonated seed or isolated aleurone layers thus provide convenient experimental systems for the study of GA3-induced a-amylase production. In the preceding paper, low temperature was shown to increase the GA3 sensitivity of otherwise GA3-insensitive aleurone tissue of several wheat varieties. More cubation of the isolated aleurone layers were as described in the preceding paper. Where dry seeds were involved, the aleurone layers were dissected from deembryonated seeds after boiling them for 5 min.Extraction and Analysis of Phospholipids. At the end of the preincubation period, the aleurone layers were immediately frozen in liquid N2 and freeze-dried and lipids were extracted using hot, water-saturated butan-l-ol (28). The total lipid extract was subjected to partition chromatography on Sephadex G-25 to yield a purified total lipid fraction, which was further fractionated by adsorption chromatography on silicic acid column. This fractionated the total lipids into neutral lipids, glycolipids, and phospholipids. These methods have been described in detail previously (1, 28).Phospholipids were separated into individual classes by TLC as described earlier (13) and quantified by GLC according to Douglas and Paleg (3). All experiments were replicated three times, and data were subjected to analysis of variance as well as multivariate analysis of variance.

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Low Temperature-Induced GA₃ Sensitivity of Wheat

Singh

Paleg²

1984

“…Within 4 h, increased incorporation of radiolabeled choline into a microsomal fraction (10) and of radiolabeled orthophosphate into phospholipids (23) is observed. On the other hand, radiolabeled acetate and glycerol are not incorporated into phospholipids to any great extent in aleurone cells (14, 23,32,35 are predominantly used for new phospholipid synthesis (35).…”

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confidence: 99%

Does Gibberellic Acid Induce the Transfer of Lipase from Protein Bodies to Lipid Bodies in Barley Aleurone Cells?

Fernandez¹,

Staehelin²

1987

We have examined the effect of gibberellic acid (GA3) on the distribution of the enzyme responsible for mobilizig storage triacyglycerol in aleurone cells of HordeEm Pulgare L. cv Himalaya. Using cellular fractionation techniques, we find that, in cells that have not been exposed to hormone, neutral lipase activity is principally associated with a pellet containing the membranes of protein bodies. If the cells are exposed to GA3 for at least 1 hour, the majority of the lipase activity becomes associated with the lipid body fraction. The nature of the in vivo association between lipid bodies and protein bodies was examined using ultrarapid freezing followed by freeze-fracture electron microscopy. Our Few investigators have looked at the processes that supply the phospholipid precursors and energy in aleurone layers, i.e. the reactions involved in the hydrolysis of triacylglycerols. Enzymes involved in the release of energy from fatty acids via the glyoxylate cycle appear within 1 to 2 d ofgermination in whole wheat seeds and can be induced by GA3 in embryo-less seeds (9). Lipases that are involved in triacylglycerol hydrolysis also appear in whole wheat seeds after 1 d of germination but cannot be induced by GA3 in embryo-less seeds (30). Jelsema et al. (20) have shown, however, that isolated wheat aleurone layers that have not been exposed to GA3 do contain acid lipase activity. In addition, their observations present an interesting enigma. Although triacylglycerols, the apparent substrate, are found within lipid bodies, the acid lipase activity is located principally in a pellet containing protein bodies. When the cells are exposed to GA3, the lipase activity disappears from the pellet (20).We have reexamined the distribution of lipase and GA3-induced changes in that distribution more closely using cellular fractionation techniques and electron microscopy. Our biochemical data indicate that lipase is associated with protein bodies as well as with lipid bodies. The distribution oflipase between these two organelles appears to be controlled by GA3. Since our structural data indicate that lipid bodies can bind to the surface ofprotein bodies and that the monolayer that surrounds the lipid body becomes continuous with the outer leaflet of the bilayer that surrounds the protein body, we propose that lipase may be transferred between the two organelles by lateral diffusion. A preliminary report of our biochemical findings appeared in Fernandez and Staehelin (13).MATERIALS AND METHODS Plant Materials. Aleurone layers of Hordeum vulgare L. cv Himalaya (1979 harvest, Washington State University, Pullman, WA) were prepared from surface-sterilized, embryo-less half seeds (7) imbibed for 4 d in 50 mm L-arginine (18). Layers were incubated on a rotary shaker at 26°C in 2 mm sodium-acetate buffer (pH 4.8) containing 20 mM CaCl2, 7.5 mg/ml chloramphenicol, and either 10 uM GA3 (Sigma Chemical Co.) or H20.Isolation of lipid bodies. Lipid bodies were isolated using a modification ofa procedure developed originally fo...

“…Extraction and purification of phospholipids were carried out as described by Varty and Laidman (35).…”

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confidence: 99%

Effects of Abscisic Acid and Ethylene on the Gibberellic Acid-Induced Synthesis of α-Amylase by Isolated Wheat Aleurone Layers

Varty¹,

Arreguı́n²,

Gómez³

et al. 1983

Gibberellic acid-induced a-amylase synthesis in wheat aleurone layers (Triticum aestivum L. var Potam S-70) escaped from transcriptional control 30 h after addition of the hormone, as evidenced by the tissue's loss of susceptibility to cordycepin. Abscisic acid inhibited the accumulation of a-amylase activity when added to the tissue during this cordycepin-insensitive phase of enzyme induction. a-Amylase synthesis was not restored by the addition of cordycepin, indicating that the response to abscisic acid was not dependent upon the continuous synthesis of a short lived RNA. When ethylene was added simultaneously or some time after abscisic acid, the accumulation of a-amylase activity was sustained or quickly restored. The loss of susceptibility to cordycepin was completely prevented when aleurone layers were incubated with a combination of gibberellic and abscisic acids from the start of the induction period. This effect of abscisic acid was not reversed by ethylene. On the basis of these observations, it is suggested that abscisic acid inhibits both the transcription and translation of a-amylase mRNA, and that only the latter site of action is susceptible to reversal by ethylene.The rate of incorporation of Imethyl-'4Clcholine into phospholipids was also inhibited by abscisic acid. Ethylene reversed this effect. The effects of abscisic acid and ethylene on phospholipid synthesis were not dependent upon the presence of gibberellic acid. No direct relationship was found between the control of a-amylase synthesis and membrane formation by abscisic acid and ethylene.