The rate of synthesis of the endoplasmic reticulum in barley aleurone cells after treatment with gibberellic acid was determined by measurement of [14C1-choline incorporation into acid-insoluble material in a semipurified fraction containing the endoplasmic reticulum. 94% of the 14C incorporated into this fraction is extractable by lipid solvents and only 9% is removed by procedures for nucleic acid extraction. Gibberellic acid increases the rate of synthesis of the endoplasmic reticulum 4-to 8-fold, starting about 4 hr after addition of the hormone (at about the same time as polysome formation). Abscisic acid inhibits this gibberellic acid-enhanced increase in the rate of synthesis of endoplasmic reticulum.Isolated barley aleurone layers respond to exogenous gibberellic acid by synthesizing a-amylase and protease after an 8-to 10-hr lag period (1-3); abscisic acid prevents these characteristic responses to gibberellic acid (4, 5). During the lag period, gibberellic acid causes an increase in the number and proportion of ribosomes that can be isolated as polysomes in the hormone-treated cells, starting 3-4 hr after hormone addition (Evins, manuscript in preparation). The polysomes are, in vivo, probably bound to the endoplasmic reticulum (ER), because electron microscopy studies show extensive development of the rough ER in the aleurone cells of germinating barley (6) as well as changes in the rough ER of isolated barley aleurone layers during both the lag and synthesis phases of a-amylase production (7, 8, and personal communication from E. L. Vigil and M. Ruddat, University of Chicago). However, the quantitative determination of differences in the rate of ER synthesis between hormone-treated and control tissues and the time of the onset of ER synthesis after hormone addition are difficult to establish by electron microscopy. We now report the rate of ER synthesis estimated by the measurement of [14C]choline incorporation into a semipurified ER fraction (method of Nagley and Hallinan, ref. 9). With this method it can be shown that gibberellic acid increases the rate of ER synthesis 4-to 8-fold. This increase starts at about the same time as polysome formation. Abscisic acid inhibits the gibberellic acid-enhanced increase in the rate of ER synthesis within 2 hr after addition. METHODSFor each sample, 10-40 aleurone layers were prepared by the methods of Chrispeels and Varner (1) from barley seeds (Hordeum vulgare L. cv. Himalaya) and incubated for the times specified in 5 ml of 1 mM Na acetate buffer (pH 4.8)-20 mM CaClr-1MM gibberellin A3 on a Dubnoff metabolic shaker at 250C.The method of Nagley and Hallinan (9) was used to measure the rate of synthesis of ER. At the end of the incubation the aleurone layers were transferred for 30 min to a medium containing 5 ACi of [methyl-14C]choline chloride (New England Nuclear Corp., 8.25 Ci/mol) and either 10 mg of neutralized casein acid hydrolysate (per 40 layers) or 1 mM iserine and 1 mM L-methionine (A grade, Calbiochem). Trichloroacetic acid-precipitable m...

The addition of abscisic acid to barley (Hordeurn vulgare L. cv. Himalaya) aleurone layers at the same time as gibberellic acid completely prevents the gibberellin-induced increases in the percentage of polysomes, the formation of polyribosomes, and the synthesis of a-amylase, even when the molar concentration of gibberellic acid is four times greater than the concen- Gibberellic acid and abscisic acid affect both the conversion of monosomes to polysomes and the synthesis of new ribosomes. The gibberellin-stimulated increases in the number of ribosomes and the percentage of polysomes are probably a prerequisite for the hormone induction of enzyme synthesis.

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