Abstract
The steady-state levels of Ca2+ within the endoplasmic reticulum (ER) and the transport of 45Ca2+ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca2+-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca2+ level in the ER was measured using the Ca2+-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca2+ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 μM. Transport of 45Ca2+ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA3) and Ca2+. 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 45Ca2+ 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 45Ca2+ transport was into the ER. The sensitivity of 45Ca2+ transport to inhibitors and the Km of 45Ca2+ uptake for ATP and Ca2+ transport in the microsomal fraction of barley aleurone cells. The rate of 45Ca2+ transport is stimulated several-fold by treatment with GA3. This effect of GA3 is mediated principally by an effect on the activity of the Ca2+ transporter rather than on the amount of ER.
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Abbreviations
- CCR:
-
cytochrome-c reductase
- DCCD:
-
dicyclohexylcarbodiimide
- EGTA:
-
ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- ER:
-
endoplasmic reticulum
- FCCP:
-
carbonylcyanide p-trifluoromethoxyphenyl hydrazone
- GA3 :
-
gibberellic acid
- IDPase:
-
inosine diphosphatase
- Mon:
-
monensin
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Bush, D.S., Biswas, A.K. & Jones, R.L. Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels. Planta 178, 411–420 (1989). https://doi.org/10.1007/BF00391870
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DOI: https://doi.org/10.1007/BF00391870