Heat shock inhibits release of the signal recognition particle from the endoplasmic reticulum in barley aleurone layers.

When barley (Hordeum vulgare) aleurone layers are subjected to heat shock there is a selective degradation of the normally stable mRNAs encoding secreted proteins. Messages for nonsecreted proteins are not degraded. The synthesis of heat shock proteins is not required for this selective message degradation. Our hypothesis explaining this phenomenon is that a component of the early steps in the synthesis of secreted proteins is damaged by heat shock, resulting in a selective halt in translation on secretory mRNAs, which may in turn lead to degradation of those messages. The first committed step in the synthesis of secreted proteins is the binding of the nascent signal sequence to the signal recognition particle. We have obtained cDNA clones and antibodies for the barley 54-kDa subunit of the signal recognition particle. In cell fractionation experiments, more signal recognition particle was bound to the endoplasmic reticulum membranes and less was in the free particle fraction following a heat shock. The results suggest that heat shock inhibits the release of the signal recognition particle from the endoplasmic reticulum. This would, in turn, inhibit the resumption of translation and may be the underlying cause of the secretory message degradation.

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

The heat-shock responses of barley (Hordeum vulgare L. cv Hi- malaya) aleurone layers incubated with or without gibberellic acid (GA3) were compared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that heat shock blocked the synthesis and secretion of secretory proteins from GA3-treated layers but not untreated layers. This suppression of secretory protein synthesis has been correlated with changes in endoplasmic reticulum (ER) membranes (F.C. Belanger, M. R. Brodl, T.-h.D. Ho [1986] Proc Natl Acad Sci USA 83: 1354-1358; L. Sticher, A.K. Biswas, D.S. Bush, R.L. Jones [1990] Plant Physiol 92: 506-513). Our secretion data suggested that the ER membranes of aleurone layers incubated without GA3 may be more heat shock tolerant. To investigate this, the lipid profiles of membrane extracts in aleurone layers labeled with [14C]glycerol were examined. Heat shock markedly increased [14C]glycerol incorporation into phosphatidylcholine (PC), and gas chromatography revealed an increase in the amount of saturated fatty acids associated with thin layer chromatography-purified PC in GA3-treated layers. In contrast, aleurone layers incubated without GA3 at normal temperature contained PC-associated fatty acids with a greater degree of saturation than GA3-treated layers. Heat shock modestly increased the degree of fatty acid saturation in untreated aleurone layers. This same trend was noted in fatty acids isolated from ER membranes purified by continuous sucrose density centrifugation. We propose that increased fatty acid saturation may help sustain ER membrane function in heat-shocked aleurone layers incubated in the absence of GA3.

Heat Shock Causes Selective Destabilization of Secretory Protein mRNAs in Barley Aleurone Cells.

The aleurone layer of GA(3)-stimulated barley (Hordeum vulgare L., cv Himalaya) grains is normally devoted to the synthesis and secretion of hydrolytic enzymes. Heat shock, however, suppresses the synthesis of the main hydrolytic enzyme, alpha-amylase, by destabilizing its otherwise highly stable mRNA (FC Belanger, MR Brodl, T-hD Ho [1986] Proc Natl Acad Sci USA 83: 1354-1358). In this paper we document that heat shock causes the suppression of the synthesis of some normal cellular proteins, while the synthesis of other normal cellular proteins is unaffected by heat shock. There are two major isozymic forms of alpha-amylase encoded by distinct mRNAs. The mRNA levels for both isozymic forms and the mRNA levels of two other secretory proteins, a protease and an endochitinase, were markedly reduced during heat shock. However, the levels of actin and beta-tubulin mRNAs, both nonsecretory proteins, were not diminished during heat shock. In addition, the levels of three other mRNA species detected by a set of unidentified cDNA clones (the sequence of one shows that it lacks a signal sequence) remained unchanged during heat shock. These data indicate that there are two classes of normal cellular protein mRNAs with regard to the effect of heat shock upon their persistence in the cell, and suggest that the distinction between them is whether or not they encode secretory proteins.

Heat shock proteins are not required for the degradation of alpha-amylase mRNA and the delamellation of endoplasmic reticulum in heat-stressed barley aleurone cells.

When barley (Hordeum vulgare) aleurone layers are heat shocked, the synthesis and secretion of alpha-amylase and other secretory proteins is arrested and the synthesis of heat shock proteins (hsps) is induced. alpha-Amylase mRNA, normally a very stable mRNA, is actively degraded during heat shock. In addition, endoplasmic reticulum (ER) is delamellated during heat shock, possibly causing the destabilization of the mRNA for the secreted alpha-amylase. To ascertain whether or not hsps play any role in the destabilization of alpha-amylase mRNA or in the delamellation process of ER, heat shocked cells were treated with the transcription inhibitor cordycepin, which effectively inhibits the synthesis of hsps yet does not affect alpha-amylase synthesis after this enzyme has been fully induced by gibberellic acid (12 hours). In the absence of hsp expression, heat shock still causes the destabilization of alpha-amylase mRNA and the delamellation of ER. Alternatively, the synthesis of hsps may be induced in the absence of temperature increase by incubating cells in the presence of arsenite. Arsenite-induced expression of some hsps in the absence of increased temperature does not result in the destabilization of alpha-amylase mRNA or in the delamellation of ER. If cordycepin or cycloheximide are used to inhibit hsp synthesis during heat shock, the tissue recovers from heat shock with normal recovery kinetics. Although hsps have been implicated in the establishment of thermotolerance, our observations indicate that hsps do not play a role in the other heat shock-induced changes observable in aleurone cells. Furthermore, if the synthesis of hsp mRNA is inhibited during heat shock (by cordycepin) hsp mRNAs are synthesized later, during recovery, indicating that there is a stable inducer of hsp synthesis in aleurone tissues.

Heat shock causes destabilization of specific mRNAs and destruction of endoplasmic reticulum in barley aleurone cells.

In response to a phytohormone, gibberellic acid, the aleurone layers of barley seeds synthesize and secrete alpha-amylases, which are coded by a set of stable mRNAs. When aleurone layers are subjected to heat shock treatment, the synthesis of alpha-amylase is suppressed while heat shock proteins are induced. The suppression of alpha-amylase synthesis is not the result of translational control as reported in several other systems. Rather, the sequences of alpha-amylase mRNA are rapidly degraded during heat shock as shown by in vitro translation and dot blot hybridization with a cDNA probe. Upon recovery from heat shock, the tissue resumes the synthesis of alpha-amylase in 2-4 hr. However, in the presence of a transcription inhibitor, cordycepin, the resumption of synthesis of alpha-amylase does not take place, indicating that new transcription of alpha-amylase genes is necessary for this recovery process. The degradation of alpha-amylase mRNAs correlates with the rapid destruction of endoplasmic reticulum as observed by electron microscopy, a phenomenon that has not been reported previously as a heat shock response. Since alpha-amylase mRNA is associated with the endoplasmic reticulum via membrane-bound polyribosomes, we suggest that the destruction of the endoplasmic reticulum during heat shock causes the destabilization and the eventual degradation of alpha-amylase mRNA.