Gibberellic-acid-stimulated Ca(2+) accumulation in endoplasmic reticulum of barley aleurone: Ca(2+) transport and steady-state levels.

The steady-state levels of Ca(2+) within the endoplasmic reticulum (ER) and the transport of (45)Ca(2+) into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca(2+)-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca(2+) level in the ER was measured using the Ca(2+)-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca(2+) level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 μM. Transport of (45)Ca(2+) into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA3) and Ca(2+). Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of (45)Ca(2+) occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that (45)Ca(2+) transport was into the ER. The sensitivity of (45)Ca(2+) transport to inhibitors and the Km of (45)Ca(2+) uptake for ATP and Ca(2+) transport in the microsomal fraction of barley aleurone cells. The rate of (45)Ca(2+) transport is stimulated several-fold by treatment with GA3. This effect of GA3 is mediated principally by an effect on the activity of the Ca(2+) transporter rather than on the amount of ER.