Gibberellic acid-stimulated α-amylase secretion and phospholipid metabolism in wheat aleurone tissue

1. Turnovers of [14C]glycerol-labelled phospholipids in wheat aleurone tissue have been measured by using a pulse-decay technique. The most metabolically active phospholipids were phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylglycerol. 2. Gibberellic acid action on the tissue led to breakdown of phosphatidylcholine and stimulated turnover of the other phosphatides concomitant with the secretion of alpha-amylase from the tissue. After pulse-labelling of the aleurone tissue with [14C]glycerol, radioactivity was lost from the phospholipids and appeared quantitatively in triacylglycerols, suggesting a stoichiometric metabolism of the former into the latter. Although 1,2-diacylglycerol is an expected intermediate in such a conversion, the patterns of radioactivity in diacylglycerols gave no indication of this. 3. Several aspects of the response of aleurone tissue to gibberellic acid resemble the responses of exocytotic animal tissues to external agonists. In particular, our results and previous reports in the literature suggest that endomembrane flow, exocytosis, phosphatidylinositol turnover and a requirement of Ca2+ for enzyme secretion are common to both plant and animal systems. Although considerable differences also exist between the two, the similarities are sufficient to warrant further consideration that plants and animals might have conserved a similar hormone response-secretion mechanism.

Isolation and characterization of membranes from the cells of maize root tips

A discontinuous sucrose density gradient was used to separate membrane fractions from a homogenate of maize root tips. Endoplasmic reticulum-, Golgi apparatus-, plasma membrane- and mitochondria-rich fractions were identified by their enzymic characteristics and by their appearance under the electron microscope. Maize roots were incubated in vivo with D-[U-14C]glucose, [Me-14C]choline chloride and diazotized [U-3H]sulphanilic acid. The pattern of incorporation of radioactivity into the various membrane fractions was investigated. Analyses of the polypeptide chains of the membrane fractions by SDS-polyacrylamide gel electrophoresis showed that the mitochondria-rich fraction had a different pattern of polypeptides from that of the other membrane fractions. The results are discussed in relation to the hypothesis of endomembrane flow and differentiation.

Incorporation in vivo of [32P]orthophosphate and [Me-3H]choline into rough microsomal, Golgi, and plasma membranes of rat liver

To study membrane biogenesis and to test the validity of the endomembrane flow hypothesis, incorporation of 32P and [Me-3H]choline in vivo into membranes of the rat liver was followed. Rough microsomal, Golgi-rich, and plasma membrane fractions were monitored with marker enzyme assays and shown with morphometric analysis to contain 82% rough microsomes, at least 70% Golgi complexes, and 88% plasma membranes, respectively. Membrane subfractions from the rough microsomal and Golgi-rich fractions were prepared by sonic disruption.At 5 to 30 min after 32P injection, the specific radioactivity of phosphatidylcholine was higher in the rough microsomal membranes than in the Golgi membranes. From 1 to 3 h, the specific activity of phosphatidylcholine in Golgi membranes became higher and reached the maximum at about 3 h. Although the plasma membrane had the lowest specific radioactivity throughout 0.25–3 h, it increased rapidly thereafter to attain the highest specific activity at 5 h. Both rough microsomal and plasma membranes reached their maxima at 5 h.The specific radioactivity of [32P]phosphatidylethanolamine in the three membrane fractions was similar to that of [32P]phosphatidylcholine except from 5 to 30 min, when the specific radioactivity of phosphatidylethanolamine in the Golgi membranes was similar to the rough microsomal membranes.At 15 min to 5 h after [Me-3H]choline injection, more than 90% of the radioactivity in all the membranes was acid-precipitable. The specific radioactivities of the acid-precipitated membranes, expressed as dpm per milligram protein, reached the maximum at 3 h. After [Me-3H]choline injection, the specific radioactivity of phosphatidylcholine separated from the lipid extract of the acid-precipitated membranes (dpm per micromole phosphorus) did not differ significantly in the three membrane fractions. The results indicated rapid incorporation of choline into membrane phosphatidylcholine by the rough endoplasmic reticulum, Golgi, and plasma membranes simultaneously.The data with both 32P and [Me-3H]choline precursors did not support the endomembrane flow hypothesis. The Golgi complexes apparently synthesized phosphatidylethanolamine and incorporated choline into phosphatidylcholine as well as the endoplasmic reticulum. The results are discussed with relevance to current hypotheses on the biogenesis and transfer of membrane phospholipids.