Heat Shock Inhibits α-Amylase Synthesis in Barley Aleurone without Inhibiting the Activity of Endoplasmic Reticulum Marker Enzymes

Abstract

The effects of heat shock on the synthesis of a-amylase and on the membranes of the endoplasmic reticulum (ER) of barley (Hordeum vulgare) aleurone were studied. Heat shock, imposed by raising the temperature of incubation from 250C to 400C for 3 hours, inhibits the accumulation of a-amylase and other proteins in the incubation medium of barley aleurone layers treated with gibberellic acid and Ca2 . When ER is isolated from heat-shocked aleurone layers, less newly synthesized a-amylase is found associated with this membrane system. ER membranes, as indicated by the activities of NADH cytochrome c reductase and ATPdependent Ca2+ transport, are not destroyed by heat stress, however. Although heat shock did not reduce the activity of ER membrane marker enzymes, it altered the buoyant density of these membranes. Whereas ER from control tissue showed a peak of marker enzyme activity at 27% to 28% sucrose (1.113-1.120 grams per cubic centimeter), ER from heat-shocked tissue peaked at 30% to 32% sucrose (1.127-1.137 grams per cubic centimeter). The synthesis of a group of proteins designated as heat-shock proteins (HSPs) was stimulated by heat shock. These HSPs were localized to different compartments of the aleurone cell. Several proteins ranging from 15 to 30 kilodaltons were found in the ER and the mitochondrial/plasma membrane fractions of heat-shocked cells, but none of the HSPs accumulated in the incubation medium of heat-shocked aleurone layers.

Effect of Gibberellin and Heat Shock on the Lipid Composition of Endoplasmic Reticulum in Barley Aleurone Layers

“…A notable exception is vanadate-sensitive ATPase activity, a marker enzyme for the plasma membrane, which had a shoulder of activity in the ER microsome fractions. Heat shock decreased the buoyant density of ER microsomes, which is consistent with the observations of Sticher et al (1990).…”

“…Heat-shocked aleurone cells may therefore be able to continue the secretion of proteins as long as secretory proteins enter the endomembrane pathway. Sticher et al (1990) demonstrated that ER-associated Ca2+ transport and Cyt c reductase activities of barley aleurone cells are not diminished during heat shock, indicating that these ER functions are not heat labile. In contrast, heat shock does not affect the synthesis of phytohemagglutinin in heat-shocked soybean cotyledons, but it does stop the posttranslational processing of this protein in the ER (Chrispeels and Greenwood, 1987).…”

“…The suppression of secretory protein synthesis was correlated with the delamination and dissociation of the stacks of ER lamellae and an apparent reduction in amount of ER membranes as viewed by transmission EM (Belanger et al, 1986). Sticher et al (1990) reported that heat shock caused an increase in the apparent buoyant density of ER membranes isolated on continuous SUC gradients and that heat shock suppressed the synthesis of secretory proteins. They noted, however, that heat shock increased the activity of Cyt c reductase 1.2-fold and did not significantly affect the leve1 of ATP-dependent Ca2+ transport activity.…”

“…They noted, however, that heat shock increased the activity of Cyt c reductase 1.2-fold and did not significantly affect the leve1 of ATP-dependent Ca2+ transport activity. From these data concerning marker enzyme activity, they suggested that the principal effect of heat shock was on the organization of ER membranes rather than on the quantity of ER membranes and that the ER reorganized into a "system unable to support the synthesis or transport of secretory proteins" (Sticher et al, 1990). Because the ER lamellae serve as the translation site for secretory protein mRNAs, Plant Physiol.…”

α-Amylase Isoforms are Posttranslationally Modified in the Endomembrane System of the Barley Aleurone Layer

“…iB) and homogenates of whole tissue (Fig. 1 B) Enzyme and Protein Assays a-Amylase was assayed using the standard I2KI procedure (15) and latent IDPase as described previously (25). The tonoplast fraction was identified using the vacuolar membrane marker TP 25 (11) and a nitrate-sensitive ATPase activity (6).…”