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(American Journal of Botany. 2003;90:40-48.)
© 2003 Botanical Society of America, Inc.

Physiology and Biochemistry

Heat shock response of warm-incubated barley aleurone layers1

Alisa E. Shaw and Mark R. Brodl2

Department of Biology, Knox College, Galesburg, Illinois 61401 USA

Received for publication May 14, 2002. Accepted for publication July 19, 2002.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Heat shock suppresses secretory protein synthesis in GA3-stimulated barley (Hordeum vulgare cv. Himalaya) aleurone layers by selectively destabilizing their mRNAs and dissociating the stacked rough endoplasmic reticulum (ER) lamellae upon which they are translated. Heat shock also increases phosphatidylcholine (PC) synthesis, and these PC molecules have increased levels of fatty acid saturation. This appears to be adaptive, for aleurone layers maintained at heat shock temperatures for 18 h resynthesize secretory protein mRNAs, rebuild stacked ER lamellae, and resume secretory protein synthesis. In the present study aleurone layers were incubated at warmer than normal pre-heat shock temperatures to determine whether this would favor the formation of heat-resistant ER lamellae that could continue secretory protein synthesis during heat shock. Western blot and SDS-PAGE analyses showed that such treatment did not induce heat shock protein (HSP) synthesis, but it preserved significant secretory protein synthesis during heat shock. Northern hybridizations revealed that levels of mRNAs encoding secretory proteins were several-fold elevated as compared to 25°C preincubated controls, and transmission electron microscopic observations revealed stacked ER lamellae. Thin layer and gas chromatography showed that PC molecules in warm-incubated barley aleurone layers had more fatty acid saturation than did controls. These observations indicate that previous incubation temperature influences both the induction of HSP synthesis and the suppression of normal protein synthesis in the heat shock response. However, we found that it does not affect the temperature at which heat shock becomes lethal.

Key Words: {alpha}-amylase • barley aleurone layer • endoplasmic reticulum • heat shock • heat stress • mRNA stability • phospholipid fatty acid saturation • thermotolerance


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The heat shock response of plants has been intensively investigated for over a decade. In virtually all plants and plant tissues, heat shock induces the synthesis of a set of heat shock proteins (HSPs) (Nover, 1994 ). The induction of HSP gene expression is transcriptionally regulated. Upon exposure to high temperature, monomers and dimers of the heat shock transcription factor form trimers that, in turn, bind to inverted repeats within the heat shock promoter elements (HSEs) of HSP genes, initiating their transcription (Fernandez, O'Brien, and Lis, 1994 ; Morimoto et al., 1994 ). This induction mechanism is highly conserved; constructs containing HSEs from an unrelated organism will drive reporter gene expression in a vigorous heat-dependent manner, even when the organisms are widely evolutionarily divergent (Morimoto et al., 1994 ). Many of the HSPs have chaperone-like functions and are believed to be involved in the refolding of proteins damaged or denatured by heat (Craig, Weissman, and Horwich, 1994 ). Other HSPs appear to be involved in removing cellular components damaged by heat. For example, ubiquitin is an HSP (Bond and Schlesinger, 1986 ), and some HSPs have dual functions as chaperones and as proteases (Spiess, Bell, and Ehrmann, 1999 ). The synthesis of HSPs is correlated with the acquisition of thermotolerance, a state in which plants are protected from subsequent heat shocks that might otherwise prove lethal (Vierling, 1991 ).

In most organisms, the heat shock response also includes the suppression of normal-temperature protein synthesis or at least the suppression of the synthesis of many normal cellular proteins (Brodl et al., 1994 ). The mechanism for the suppression of normal-temperature protein synthesis during heat shock is not as highly conserved as the mechanism for the induction of HSP expression. In yeast, soybean, tomato, pea, and Drosophila, heat shock suppresses the expression of all normal-temperature proteins. Yeast accomplish this at the transcriptional level (Lindquist, 1981 ). The transcription of genes encoding normal-temperature proteins is suppressed, and their cytoplasmic mRNAs have very short half-lives, resulting in a rapid switch to HSP expression (Lindquist, 1981 ). However, soybean (Nagao et al., 1986 ), tomato (Nover, Scharf, and Neumann, 1989 ), pea (Nagao et al., 1986 ), and Drosophila (Storti et al., 1980 ) accomplish this at the translational level. Transcripts encoding normal-temperature proteins remain in the cytoplasm and are not actively translated during heat shock, but they are translationally reactivated during recovery. On the other hand, normal-temperature protein synthesis continues without interruption in heat-shocked maize (Cooper and Ho, 1987 ).

Heat shock in gibberellic acid-induced (GA3) barley aleurone layers causes the selective suppression of secretory protein synthesis (Belanger, Brodl, and Ho, 1986 ). Transcripts encoding secretory proteins are rapidly and selectively destabilized (Brodl and Ho, 1991 ). For example, mRNAs encoding the secretory protein {alpha}-amylase have a normal half-life in excess of 100 h, but this is reduced to approximately 30 min during heat shock (Belanger, Brodl, and Ho, 1986 ; Brodl and Ho, 1991 ). The selective destabilization of secretory protein mRNA during heat shock is correlated with a dissociation and fragmentation of the stacks of endoplasmic reticulum (ER) lamellae that are the hallmark of this secretory cell; only short, single ER fragments with few adhering ribosomes remain following heat shock (Belanger, Brodl, and Ho, 1986 ). Because secretory protein mRNAs are translated by ER-bound ribosomes, we have hypothesized that heat-induced changes in ER membrane structure or biochemistry or both provide a mechanism for selectively targeting secretory protein mRNA for destabilization.

The ER membrane contains integral membrane proteins that are important to the binding of ribosomes, a process that is governed by a protein-RNA complex known as the signal recognition particle (SRP). The ER integral membrane proteins mediate the cotranslational translocation of the secretory protein and the return of SRP to the cytoplasm for recruiting more ribosomes engaged in the translation of secretory protein mRNAs. Heat has been shown to cause damage to membrane-based cellular functions such as photosynthesis (Berry and Björkman, 1980 ), mitochondrial respiration (Lepock et al., 1983 ), and electrolyte partitioning (Burke and Orzech, 1988 ). Temperature shock is believed to cause lipid phase transitions that alter membrane fluidity and normal lipid-protein interaction, which combined impair normal integral membrane protein function (Raison et al., 1980 ; Quinn, 1984 ). In heat-shocked barley aleurone cells, this may impair normal secretory protein translation and translocation, targeting the transcripts and perhaps other components for turnover.

We have documented that in barley aleurone layers, heat shock increases the synthesis of phosphatidyl choline (PC) and that fatty acids from PC and ER membrane-derived phospholipids have increased levels of fatty acid saturation (Grindstaff, Fielding, and Brodl, 1996 ). In addition, when aleurone layers are exposed to extended periods of heat shock temperature (18 h at 40°C), they acclimate to the high temperature as indicated by the resumption of {alpha}-amylase synthesis and the reformation of ER lamellae that have significantly higher levels of fatty acid saturation in membrane phospholipids (Campbell, Fielding, and Brodl, 1997 ). Furthermore when barley aleurone layers are brought to heat shock temperature more gradually (ramped) rather than heat shocked, the synthesis of {alpha}-amylase continues at heat shock temperature (Johnston et al., 2002 ). The ER lamellae in these ramped aleurone layers remain intact, and analysis of ER membrane phospholipids reveals that their fatty acid saturation is significantly increased (Johnston et al., 2002 ). It is hypothesized that the heat-induced "retailoring" of the fatty acid composition of ER membrane phospholipids under these conditions would reduce membrane fluidity and allow the cells to preserve or restore membrane function. This retailoring is called homeoviscous adaptation (Mazliak, 1989 ; Cossins, 1994 ), and the changes in fatty acid saturation in heat shock-adapted barley aleurone layers are similar to changes observed in chloroplast membranes of heat-acclimated plants (Pearcy, 1978 ; Hugly et al., 1989 ). In the present study, we investigated whether incubating barley aleurone layers at warmer temperatures prior to heat shock (30°–34°C rather than the 25°C that is the normal experimental regimen) would increase membrane fatty acid saturation without inducing HSP synthesis and permit increased synthesis of {alpha}-amylase during heat shock.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant material
Embryos and distal tips of barley grains (Hordeum vulgare L. cv Himalaya, obtained from the Agronomy and Soils Club, Washington State University, Pullman, Washington, USA, 1988 harvest) were removed using a razor blade. The resulting "half-seeds" were surface sterilized in a 25% hypochlorite solution and then imbibed asceptically for 4 d on filter paper overlaying vermiculite soaked in 35 mL of incubation buffer (20 mmol/L sodium succinate [pH 5.0], 20 mmol/L CaCl2, 1 µg/mL chloramphenicol) in glass petri dishes (Belanger, Brodl, and Ho, 1986 ). Aleurone layers were dissected from the softened endosperm under aseptic conditions and incubated at the designated temperatures in a shaking water bath at 120 cycles/min.

In vivo labeling and analysis of proteins
Ten aleurone layers per sample were incubated in sterile 25-mL flasks with 2 mL of incubation buffer. Thirty minutes prior to the end of the incubations, the aleurone layers were labeled using 3.7 MBq [35S]-methionine (specific activity >29.6 pBq/mol; obtained from New England Nuclear, Boston, Massachusetts, USA) in 1 mL buffer. At the end of the labeling period, the samples were homogenized and electrophoresed into SDS-containing polyacrylamide gels according to procedures in Grindstaff, Fielding, and Brodl (1996) . Equal amounts of total protein were electrophoresed for each sample. Total protein in each sample was determined using the dye-binding method of Bradford (1976) . The density of protein bands on fluorographs was measured using NIH Image (National Institutes of Health, http://rsb.info.nih.gov/nih-image), according to online instructions.

RNA isolations and northern blot analysis
One hundred aleurone layers per sample were incubated in sterile 250-mL flasks in 20 mL incubation buffer. RNA was isolated, electrophoresed, blotted, and hybridized as described in Campbell, Fielding, and Brodl (1997) . Cloned cDNA for the high pI (pM/C) (Muthukrishnan, Chandra, and Albaugh, 1983 ) and low pI (clone E) (Rogers and Milliman, 1983 ) isozymes of {alpha}-amylase, HSP 70 (Rochester, Winer, and Shah, 1986 ), and soybean actin (Shah and Meager, 1982 ) were nick-translated using a commercial nick-translation kit (Bethesda Research Laboratories, Bethesda, Maryland, USA) in the presence of {alpha}-[32P]-dCTP (specific activity >18.5 Pbq/mol; New England Nuclear). The density of the bands on the resulting fluorographs was measured using NIH Image.

Electron microscopy
Tissue samples of approximately 0.5 mm2 were fixed, postfixed, dehydrated, and embedded according to the procedure of Campbell, Fielding, and Brodl (1997) . Sections approximately 60 nm thick were cut on a diamond knife, stained with 1% methanolic uranyl acetate and Reynold's lead citrate (Hayat, 1989 ), and viewed at original magnifications of 5000–15 000x in a JEOL JEM 100SK (JEOL, Tokyo, Japan) transmission electron microscope.

In vivo labeling, extraction, and analysis of phospholipids
Twenty aleurone layers per sample were incubated in 4 mL of incubation buffer in sterile 50-mL flasks. During the last 1 h of incubation, the samples were labeled with 14.8 KBq [14C]-glycerol (specific activity >0.37 PBq/mmol; New England Nuclear) and homogenized with acid-washed silica sand in a mortar and pestle. Lipids were extracted and separated by thin layer chromatography (TLC) as described by Grindstaff, Fielding, and Brodl (1996) . Phospholipids were identified by comigration with standards (Sigma, St. Louis, Missouri, USA) and visualized using iodine vapor. The resulting lipid-containing spots on the silica gel were scraped from the glass plate and the radioactivity in each spot was assessed using a Beckman LS 6000IC liquid scintillation counter (Beckman Instruments, Palo Alto, California, USA). Total lipid was quantified by the procedure of Bartlett (1959) .

Cell fractionation
Two-hundred aleurone layers per sample were incubated in sterile 250-mL flasks in 20 mL incubation buffer. The cellular contents of gently lysed aleurone layers were fractionated by ultracentrifugation through discontinuous followed by continuous sucrose gradients (Grindstaff, Fielding, and Brodl, 1996 ). The fractions were assayed for marker enzyme activities for ER, Golgi, mitochondria, tonoplast, and plasma membranes according to the procedures of Grindstaff, Fielding, and Brodl (1996) . Sucrose concentrations were measured by refractometry.

Fatty acid isolation and analysis
Phosphatidyl choline reconstituted from TLC plates or phospholipids extracted from ER microsomal fractions were hydrolyzed and methylated according to the procedure of Geer, McKechnie, and Langevin (1986) . The resulting methylated fatty acid esters were analyzed by gas chromatography (GC) (Hewlett-Packard Model 5890 Series II, Palo Alto, California, USA) using a Supelco (Bellefonte, Pennsylvania, USA) 10% SP-2330 on 100/120 Supelcoport chromatographic column. The identity of each peak was determined by comigration with fatty acid standards (Sigma). Butylated hydroxytoluene was added, and samples were stored under nitrogen gas to minimize oxidation. Samples of PC with known fatty acid compositions (Sigma) that were hydrolyzed, methylated, and analyzed by GC indicated that sample oxidation during the extraction and analysis was negligible.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
{alpha}-Amylase expression during heat shock is greater as preheat-shock incubation temperature increases
In our experimental system, the normal incubation temperature for barley aleurone layers prior to heat shock is 25°C. To identify a warmer preincubation temperature that would not be so warm as to induce a heat shock response (initiate HSP synthesis and suppress secretory protein synthesis), the synthesis of proteins in barley aleurone layers was examined in samples incubated at 30°–35°C by SDS-PAGE analyses of proteins labeled with [35S]-methionine (Fig. 1A). In addition, the accumulation of HSPs within these samples was assessed by western hybridization using HSP 70 Ab as a marker for HSP expression (Fig. 1B). When barley aleurone layers that were incubated at 25°C in the presence of GA3 were heat-shocked at 40°C for 3 h, the synthesis of {alpha}-amylase was suppressed, while the synthesis of HSPs was intitiated (Fig. 1A, cf. lanes 1 and 2). Such treatment caused the accumulation of high levels of HSP 70 (Fig. 1B, lane 2). When barley aleurone layers were incubated continuously at temperatures of 30°–35°C for 19 h, the synthesis of {alpha}-amylase persisted at a higher rate, and the synthesis of HSPs was not apparent (Fig. 1A, lanes 3–8). Furthermore, these incubation temperatures did not result in the accumulation of HSP 70, as indicated by western hybridizations (Fig. 1B, lanes 3–8).



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  		Fig. 1. Protein synthesis during continuous exposure to warm incubation temperatures. Aleurone layers were incubated in the absence or presence of 1 µmol/L GA3 for 19 h at the indicated temperature. In lane 2, a 25°C sample was heat shocked by making the final 3 h of the 19-h incubation period 40°C. Samples were labeled with [35S]-methionine for the final 1 h of the incubation period. Equal amounts of total protein were electrophoresed into 11% SDS-containing polyacrylamide gels. The gel was dried and visualized fluorographically (A) or electroblotted onto nitrocellulose and probed with HSP 70 antibodies (B). M and numbers indicate the positions and sizes of molecular mass standards in kilodaltons. Arrow indicates the position of HSP 70; arrowhead indicates the position of {alpha}-amylase

 
To determine whether candidate warm incubation temperatures induce HSP synthesis even if used as a "shock" temperature, experiments were conducted in which samples originally incubated at 25°C for 16 h were abruptly shifted to the candidate warm incubation temperatures for a 3-h warm temperature shock. As before, protein synthesis was monitored by SDS-PAGE analyses of [35S]-methionine-labeled proteins, and HSP 70 accumulation was monitored by western hybridization. In samples shocked at 28°–35°C, the synthesis of {alpha}-amylase continued robustly during the shock period (Fig. 2A, lanes 3–6). These warm temperature shocks did not induce detectable accumulations of HSP 70 (Fig. 2B, lanes 3–6), but SDS-PAGE indicated that a warm temperature shock of 34°C did induce the synthesis of a new protein whose apparent molecular mass was near 70 kD (Fig. 2A, lane 6). However, accumulation of HSP 70 was not detected in western hybridizations using antibodies against HSP 70 (Fig. 2A, lane 6) and HSP 83 (data not shown), except at levels that were not significantly above the background expression in non-heat-shock control lanes. In subsequent experiments, warm incubation temperatures of 30°, 32°, and 34°C were used.



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  		Fig. 2. Protein synthesis during warm incubation temperature "shock." Aleurone layers were incubated in the absence or presence of 1 µmol/L GA3 for 16 h at 25°C then shifted for the final 3 h of incubation to the temperatures indicated above each lane. Samples were labeled with [35S]-methionine for the final 1 h of the incubation period. The fluorograph (A) and western blot (B) were obtained as described in Fig. 1 . M and numbers indicate the positions and sizes of molecular mass standards in kilodaltons. Arrow indicates the position of HSP 70; arrowhead indicates the position of {alpha}-amylase

 
To determine whether warm-incubated barley aleurone layers were more tolerant of heat shock than their 25°C-incubated counterparts, [35S]-methionine-labeled proteins from heat-shocked, warm-incubated aleurone layers were examined by SDS-PAGE. In all cases, irrespective of the preheat shock incubation temperature, exposure to a 3-h heat shock at 40°C markedly reduced {alpha}-amylase synthesis (Fig. 3A, lanes 4–6). However, as the preheat shock incubation temperature increased, the synthesis of {alpha}-amylase was increasingly more robust. Digitization of SDS-PAGE analyses revealed that in heat-shocked 32°C warm-incubated samples {alpha}-amylase synthesis was 1.8-fold greater than in heat-shocked 25°C-incubated controls and was 2.3-fold greater in heat-shocked 34°C warm-incubated samples (Fig. 3B). The opposite was true for the synthesis of HSP 70; as pre-heat shock incubation temperature increased, HSP 70 synthesis decreased by 1.1- and 1.3-fold for 32° and 34°C, respectively (Fig. 3A, lanes 3–6; Fig. 3B).



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  		Fig. 3. Protein synthesis in heat-shocked, warm-incubated barley aleurone layers. Aleurone layers were incubated in the presence of 1 µmol/L GA3 for 16 h at the temperature indicated above the lane then either retained at the indicated temperature (noHS) or shifted to 40°C (HS) for 3 h. Samples were labeled with [35S]-methionine for the final 1 h of the incubation period. Equal amounts of total protein were electrophoresed into an 11% SDS-containing polyacrylamide gel. The gel was dried and visualized fluorographically (A). M and numbers indicate the positions and sizes of molecular mass standards in kilodaltons. Arrow indicates the position of HSP 70; arrowhead indicates the position of {alpha}-amylase. The bands corresponding to HSP 70 and {alpha}-amylase were digitized using NIH Image and their relative levels of expression in pixel counts for each sample in Fig. 3A were graphed (B)

 
The suppression of {alpha}-amylase and other secretory protein synthesis during heat shock results from the selective destabilization of secretory protein mRNA (Belanger, Brodl, and Ho, 1986 ; Brodl and Ho, 1991 ). Therefore, increased levels of {alpha}-amylase synthesis in heat-shocked, warm-incubated barley aleurone layers should be due to an increased abundance of {alpha}-amylase transcripts in these samples relative to the control heat-shock samples. This possibility was investigated by northern hybridization analysis. In all of the warm-incubated samples, the levels of {alpha}-amylase mRNAs were reduced by heat shock to approximately 35% of nonheat-shocked control levels (Fig. 4, cf. lanes 1 and 3–5). In contrast, the level of {alpha}-amylase mRNA in heat-shock control samples was less than 1% of nonheat-shocked control levels and an average of approximately 4% of that in the warm-incubated samples (Fig. 4, cf. lane 2 with lanes 1 and 3–5). The levels of HSP 70 mRNA also varied among the samples. Samples preincubated at 30°C prior to heat shock had HSP 70 transcript levels that were two times greater than samples preincubated at 25° and 32°C prior to heat shock and four times greater than samples preincubated at 34°C prior to heat shock (Fig. 4, cf. lane 3 with lanes 2, 4, and 5).



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  		Fig. 4. The {alpha}-amylase mRNA levels in warm-incubated, heat-shocked barley aleurone layers. RNA was extracted from barley aleurone layers incubated in the presence of 1 µmol/L GA3 for 16 h at the temperature indicated above the lane then either retained at the indicated temperature (noHS) or shifted to 40°C (HS) for 3 h. An equal amount of total RNA from each sample was electrophoresed into agarose gels, capillary blotted onto hybridization membranes, then probed with nick-translated cDNA clones for the low (clone E) and high (pM/C) pI isozymes of {alpha}-amylase and HSP 70. Total RNA yields for each sample were approximately equivalent, and hybridization with an actin clone indicated that actin mRNA was evenly present across all lanes. The bands shown represent the only radioactivity detectable on the individual fluorograms

 
In barley aleurone layers, heat shock causes the delamination and fragmentation of stacked ER lamellae (Belanger, Brodl, and Ho, 1986 ). The continued synthesis of {alpha}-amylase and the enhanced stability of {alpha}-amylase mRNA in warm-incubated samples exposed to heat shock suggested that more of the ER membranes of warm-incubated cells would remain in a stacked configuration during heat shock. To investigate whether this was the case, we examined the ultrastructure of warm-incubated (32°C), heat-shocked barley aleurone cells by transmission electron microscopy. Figure 5 shows that these cells contained stacked ER lamellae comparable in appearance to their counterparts incubated continuously at 25°C. They were not fragmented as was the case with samples incubated at 25°C prior to heat shock. The density of ribosomes along the ER was also higher in warm-incubated, heat-shocked samples than in 25°C heat-shocked controls at 39.9 ± 3.3 ribosomes/µm vs. 11.4 ± 5.2 ribosomes/µm, respectively. Samples incubated continuously at 25°C had 45.4 ± 4.7 ribosomes/µm.



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  		Fig. 5. Ultrastructure of warm-incubated, heat-shocked barley aleurone cells. (A) Electron micrograph of barley aleurone cells incubated in 1 µmol/L GA3 for 19 h at 25°C. (B) Electron micrograph of GA3-treated barley aleurone cell incubated for 16 h at 25°C and then shifted to 40°C for 3 h. (C) As in B except that the preincubation temperature prior to heat shock was 32°C. ag, aleurone grain; arrowheads, ER fragments; bracket, ER lamellae; m, mitochondrion; p, plastid; s, spherosome. A and B are both at 11 000x and C is at 13 000x (final magnifications)

 
Warm incubation induces biochemical changes in ER phospholipids
Earlier studies have shown a consistent and strong correlation between increases in the saturation of ER membrane phospholipids and ER membrane stability at heat shock temperatures (Grindstaff, Fielding, and Brodl, 1996 ; Campbell, Fielding, and Brodl, 1997 ; Johnston et al., 2002 ). The increase in fatty acid saturation appears to be accomplished largely through the synthesis of new PC molecules, because heat shock greatly enhances the incorporation of [14C]-glycerol into PC over other phospholipids (Grindstaff, Fielding, and Brodl, 1996 ; Campbell, Fielding, and Brodl, 1997 ). To determine whether warm incubation or warm incubation followed by heat shock promotes increased PC synthesis, samples were labeled with [14C]-glycerol and lipids were extracted and analyzed by TLC. Warm incubation did not have a significant effect on the incorporation of [14C]-glycerol into the five classes of phospholipids analyzed (Table 1). However, warm-incubated samples that were subsequently heat shocked showed significant increases in the incorporation of [14C]-glycerol into PC. These increases were not as large as those observed in samples incubated at 25°C and then heat shocked. Heat shock also caused a decrease in the incorporation of [14C]-glycerol into phosphatidylinositol and phosphatidylserine in both the warm-incubated and 25°C heat-shocked samples, though the level of the significance of this shift is not statistically as large (P < 0.005, rather than P < 0.01 for PC). There was not a statistically significant difference in the total uptake of [14C]-glycerol between warm- and 25°C-incubated aleurone layers, but the differences in total uptake of [14C]-glycerol between the heat-shocked samples and their respective controls and between the heat-shocked samples themselves were statistically significant.


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  		Table 1. Incorporation of [14C]-glycerol into phospholipids of warm-incubated, heat-shocked barley aleurone layers. Aleurone layers were incubated in the presence of GA3 for 16 h at 25° or 32°C and then maintained for a further 3 h at 25° or 32°C or shifted to 40°C for 3 h (HS) and then homogenized. The final 3 h of each incubation was in the presence of [14C]-glycerol. Lipids were extracted from 100 µL of the cell homogenates, separated by thin layer chromatography (TLC), and quantified by liquid scintillation counting. Values represent the percentage of total [14C]-glycerol incorporation into the five listed phospholipid classes. Results given are the average of three independent experiments, two replicates each. Means ± 1 SE of heat-shocked samples that differ from their corresponding control group are indicated by *P < 0.005, **P < 0.01 (t test)

 
The fatty acids associated with PC were analyzed by GC to determine whether warm incubation or warm incubation followed by heat shock influenced the type or extent of saturation of the fatty acids. Such analyses indicated that warm-incubated samples had a higher saturated to unsaturated fatty acid ratio (0.83) than 25°C-incubated samples (0.61) (Table 2). The warm-incubated samples had increased levels of 18 : 0 fatty acids and decreased levels of 18 : 1, 18 : 2, and 18 : 3 fatty acids over their 25°C-incubated counterparts. When these samples were heat shocked, there was a significant increase in the ratio of saturated to unsaturated fatty acids for the 25°C-incubated sample (from 0.61 to 0.96); however, there was no significant change in this ratio for the warm-incubated sample (from 0.83 to 0.89). In the heat-shocked, 25°C-incubated sample, there were increased levels of 18 : 0 fatty acids, with decreased levels of 18 : 1, 18 : 2, and 18 : 3 fatty acids.


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  		Table 2. Fatty acid composition of TLC-isolated PC from heat-shocked, warm-incubated barley aleurone layers. Aleurone layers were incubated and homogenized as described in Table 1, except that no isotope was used. Total lipids were extracted from each sample and separated by TLC. Spots comigrating with PC were scraped from the TLC plates and reconstituted with chloroform-methanol. Fatty acids were hydrolyzed from the TLC-purified PC, methylated and analyzed by GC. Results are given as mol % and are averages from three independent experiments, two replicates each. Errors of analysis were estimated to be within 4% of individual values. nd = not detected; t = trace

 
Because these changes in fatty acid saturation were determined from PC isolated from whole-tissue extracts, it was important to determine whether there were differences in phospholipid composition in the ER membranes of warm-incubated and warm-incubated, heat-shocked aleurone layers. To accomplish this, lipids were extracted from purified ER membranes and their fatty acid composition was determined. The ER membranes from the warm-incubated samples had significantly higher levels of fatty acid saturation as compared to their 25°C-incubated counterparts (saturated to unsaturated fatty acid ratios of 0.74 vs. 0.48, respectively) (Table 3). When these samples were heat shocked, there were increases in the ratio of saturated to unsaturated fatty acids in both; however, the magnitude of the increase was much greater for the 25°C-incubated samples (from 0.48 to 0.98) than for the warm-incubated samples (from 0.74 to 0.90). As was the case for the total PC extracts, there were increases in the levels of 18 : 0 fatty acids accompanied by decreases in the levels of 18 : 1, 18 : 2, and 18 : 3 fatty acids.


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  		Table 3. Fatty acid composition of microsomal membranes isolated by sucrose density centrifugation from warm-incubated, heat-shocked barley aleurone cells. Aleurone layers were incubated as described in Table 1 and were lysed by chopping instead of homogenization. Microsomal fractions were purified on sucrose step gradients followed by continuous gradient centrifugation. Total lipid extractions were performed on fractions containing peak cytochrome c reductase activities. Fatty acids in the samples were hydrolyzed from phospholipids, methylated, and analyzed by GC. Results are given as mol % and are averages from three independent experiments, two replicates each. Errors of analysis were estimated to be within 4% of individual values. nd = not detected; t = trace

 
Warm incubation does not secure thermotolerance against lethal temperature
Given the difference in the heat shock responses between 25°C and warm-incubated barley aleurone layers, the question arises whether warm incubation enhances the thermotolerance of aleurone layers. When incubated at 25°C, exposure to 47°C is lethal to barley aleurone layers, even if it is preceded by a more mild 40°C heat shock (aleurone layers can recover from <47°C) (Lanciloti, Cwik, and Brodl, 1996 ). We examined whether aleurone layers incubated at 32°C could survive a 47°C challenge by monitoring protein synthesis using SDS-PAGE. As was the case with 25°C-incubated samples, incubation at 32°C prior to lethal heat shock at 47°C did not provide thermotolerance (Fig. 6, lanes 3 and 7). Similarly, incubation at 32°C followed by a heat shock at 40°C to induce HSP synthesis prior to lethal heat shock at 47°C did not provide thermotolerance either (Fig. 6, lanes 4 and 8).



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  		Fig. 6. Protein synthesis in lethally heat-shocked, warm-incubated barley aleurone layers. Aleurone layers were incubated in the presence of 1 µmol/L GA3 for 16 h at either 25 (lanes 1–4) or 32°C (lanes 5–8) as indicated above the lane. Then they were either retained at their respective incubation temperature (for a further 3 h) or given a sublethal heat shock (3 h at 40°C, lanes 2 and 6), a lethal heat shock (20 min at 47°C followed by 6 h at 25°C, lanes 3 and 7), or a sublethal heat shock followed by lethal heat shock (lanes 4 and 8). Samples were labeled with [35S]-methionine for the final 1 h of the incubation period. Equal amounts of total protein were electrophoresed into an 11% SDS-containing polyacrylamide gel. The gel was dried and visualized fluorographically. M and numbers indicate the positions and sizes of molecular mass standards in kilodaltons. Arrow indicates the position of HSP 70; arrowhead indicates the position of {alpha}-amylase

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Our data indicate that when aleurone layers are incubated at temperatures from 25° through 34°C they are increasingly more tolerant of heat shock as the incubation temperature increases. This tolerance was marked by the observations that during heat shock aleurone layers incubated at warmer temperatures showed increased levels of {alpha}-amylase synthesis, greater levels of {alpha}-amylase mRNA, and intact stacks of ER lamellae with greater numbers of attached ribosomes. In contrast, when aleurone layers were incubated at the "normal" 25°C temperature, these cellular features were lost upon heat shock. The tolerance for heat shock temperature in warm-incubated aleurone layers did not, however, secure thermotolerance against lethal heat shock. There appears to be a lethal temperature limit for the aleurone layer that cannot be reset by previous temperature treatments.

Thermoprotection, the ability to continue normal cellular functions during or after otherwise damaging temperature stress, is a property directly correlated with the synthesis of HSPs (Vierling, 1991 ; Parsell and Lindquist, 1994 ). For example, HSP synthesis has been correlated with the maintenance of protein synthesis (Beck and De Maio, 1994 ), intermediate filament organization (Mizzen and Welch, 1988 ), tonoplast and plasma membrane integrity (Mansfield, Lingel, and Key, 1989 ), and ER membrane structure (Chen et al., 1988 ) during heat shock. However, we have shown in this present paper through radiolabeling experiments that HSP synthesis was very minimally induced, if at all, during the warm incubation temperature period or even by warm incubation "shocks." Furthermore, western hybridizations using HSP 70 antibodies did not show accumulation of this HSP during warm temperature incubation, and HSP 70 is normally the most abundant of the barley aleurone HSPs (Belanger, Brodl, and Ho, 1986 ). In a previous study, we showed that heat- or arsenite-induced HSP expression was not sufficient to secure thermoprotection of {alpha}-amylase synthesis, {alpha}-amylase transcript levels, or rough ER lamellae stacks in barley aleurone layers (Lanciloti, Cwik, and Brodl, 1996 ). Rather than HSP expression, thermoprotection in the aleurone layer is more tightly correlated with enhanced levels of fatty acid saturation in ER membrane phospholipids. This is true for warm-incubated aleurone layers, aleurone layers incubated without GA3 (Grindstaff, Fielding, and Brodl, 1996 ), aleurone layers exposed to extended heat shock (Campbell, Fielding, and Brodl, 1997 ), and aleurone layers brought more gradually to heat shock temperatures over 3–6 h (Johnston et al., 2002 ). Nonetheless, because we cannot rule out that HSPs are completely absent, the possibility still exists that HSPs secure thermotolerance even when present in exquisitely low concentrations. We propose, however, that any HSPs would work downstream of the events involved in protecting from the suppression of {alpha}-amylase expression and ER dissociation (e.g., cleaning up integral membrane proteins affected by heat) and that the retailoring of ER membrane phospholipid composition more directly addresses heat-induced damage in the aleurone cell (e.g., by reducing the effects of heat on integral membrane proteins from the start and therefore requiring fewer HSPs).

While warm incubation treatments were able to thermoprotect heat-labile normal cellular processes during sublethal heat shock, they did not enhance aleurone cell survival at lethal temperatures (thermotolerance). Although in most systems the synthesis of HSPs is correlated with thermotolerance, in many systems HSP synthesis does not secure thermotolerance or thermotolerance is established without the synthesis of HSPs (Carper, Duffey, and Garner, 1987 ). For example, even if cycloheximide is used to inhibit protein synthesis during heat shock, thermotolerance is still attained in yeast cells (Hall, 1983 ) and Morris hepatoma cells (Landry and Chrétien, 1983 ), and germinating pollen, which does not make HSPs, becomes thermotolerant following sublethal heat treatments (Altschuler and Mascarenhas, 1982 ). We have shown that HSPs do not secure thermotolerance for 25°C-incubated barley aleurone layers, yet they are tightly correlated with thermotolerance in the germinating embryo of the barley grain (Lanciloti, Cwik, and Brodl, 1996 ). From this observation and the data presented here in Fig. 6, we conclude that for some reason barley aleurone layers are one of the types of tissues that are not able to acquire thermotolerance.

The TLC and GC analyses demonstrated that warm-incubated barley aleurone layers have phospholipids with enhanced levels of fatty acid saturation in their ER membranes. Furthermore, when aleurone layers were heat shocked, membrane phospholipid fatty acid saturation was further enhanced. Because {alpha}-amylase is translated by ER-bound ribosomes, its expression is dependent on the proper function of a series of ER integral membrane proteins used in secretory protein translation and translocation (for review see Rapoport, Jungnickel, and Kutay, 1996 ). The proper function of integral membrane proteins is dependent on proper maintenance of membrane fluidity (Murata and Los, 1997 ). An increase in temperature leads to increased fluidity, which is countered chiefly by increasing the extent of saturated membrane phospholipids (Kates, Pugh, and Ferrante, 1984 ; Cossins, 1994 ). This process of "homeoviscous adaptation" is likely the operating principle behind changes in ER membrane phospholipid biochemistry and the preservation of integral membrane protein-dependent cellular features that we have observed in warm-incubated, heat-shocked barley aleurone layers. The HSPs may be needed to refold or turn over heat-damaged integral membrane proteins, but without the appropriate level of membrane fluidity their normal function would still be inhibited.

Impairment of the function of integral membrane proteins during temperature stress could easily interrupt the translation and/or translocation of secretory proteins, leading in turn to secretory protein mRNA decay and dissocation of stacked ER. The SRP with its associated mRNA-ribosome-nascent polypeptide complex binds to the ER via the SRP receptor (SR-{alpha} and -ß subunits), and the SRP receptor links the translating ribosome to a secretory pore. The nascent polypeptide is inserted through this pore into the ER lumen as it is translated (Rappoport, Jungnickle, and Kutay, 1996 ). These ER integral membrane proteins regulate SRP interactions through GTPase-regulated binding steps; SRP is released following GTP (guanosine 5'-triphosphate) hydrolysis (Connolly and Gilmore, 1993 ; Bacher, Pool, and Dobberstein, 1999 ). We have shown that heat shock inhibits the release of SRP from the ER, potentially protracting the translational arrest that accompanies SRP binding to the ribosome-mRNA-nascent polypeptide complex (Chu, Brodl, and Belanger, 1997 ). Protracted translational arrest has been shown to cause transcript destabilization (Parker and Jacobson, 1990 ; Capronigro, Muhlrad, and Parker, 1993 ). There is mounting evidence that mRNAs encoding secretory proteins are important in organizing ER into flat planes of stacked membranes; as polysomes form they tether SRP receptors and secretory pores into chains along the ER surface, creating a self-assembly system for the planes of ER membranes (Staehelin, 1997 ). We propose that in heat-shocked barley aleurone layers the destabilization of secretory protein mRNAs, which account for a large majority of the cell's transcripts (Varner and Ho, 1976 ), would disassemble the stacked ER. But with homeoviscous adaptation and the preservation of ER integral membrane function, secretory protein mRNA levels and consequently stacked ER membrane configurations are maintained in warm-incubated barley aleurone layers.

The reduced expression of HSPs in the heat shock response of warm-incubated barley aleurone layers may also be linked to the changes in membrane phospholipid fatty acid saturation. As we have found in barley aleurone layers, it appears that changes in fatty acid saturation affect the set point for temperature stress responses in poikilothermic animals (Dietz and Somero, 1992 ), yeast (Carratù et al., 1996 ), and photosynthetic bacteria (Vígh et al., 1993 ; Horváth et al., 1998) as well. In a more direct demonstration of this relationship, Carratù et al. (1996) regulated the {Delta}9 acyl-CoA desaturase gene with constitutive promoters of various strengths. When heat shocked, these yeast mutants synthesized HSPs at levels directly proportional to the degree of fatty acid unsaturation established by the desaturase activity; as the fatty acids of membrane phospholipids became more unsaturated (and more fluid), HSP synthesis increased (Carratù et al., 1996 ). It has been proposed (Murata and Los, 1997 ) that differences in membrane phospholipid fatty acid saturation modulate the activities of integral membrane proteins, such as protein kinase C-activated MAP kinase (Kamada et al., 1995 ) or Ca2+ channels (Monroy and Dhindsa, 1995 ), to generate a signal within the cell indicating the onset of temperature stress. As fatty acid saturation increases to maintain integral membrane protein function, the signaling mechanism for heat stress would be dampened and fewer HSPs would be synthesized.

In summary, the results presented in this paper further strengthen the correlation between increases in fatty acid saturation in ER membrane phospholipids and the continued expression of secretory proteins in barley aleurone layers at the normally nonpermissive heat shock temperature of 40°C. This change is consistent with the principle of homeoviscous adaptation, and in the case of secretory protein synthesis in barley aleurone layers the adaptation may specifically affect integral membrane proteins of the ER that are essential to the translation and translocation of integral membrane proteins. We have hypothesized that homeoviscous adaptation helps to minimize the need for HSPs, because heat-induced malfunction of integral membrane proteins would be reduced following homeoviscous adaptation. In addition, the adapted membranes would potentially influence the heat-sensing signaling cascade, leading to the reduced HSP expression.


   FOOTNOTES
 
1 The authors thank Dr. Bill Geer (Biology Department, Knox College) for assistance with lipid analysis; Dr. John Rogers (Department of Biochemistry, Washington State University, Pullman, WA, USA) for {alpha}-amylase cDNA clones; Dr. Dilip Shah (Monsanto Company, Chesterfield, MO, USA) for HSP 70 and actin clones; Dr. Elizabeth Vierling (Department of Biochemistry, Univerity of Arizona, Tuscon, AZ) for antibodies to HSPs; and Ryan Guynn and Nathaniel Sloan for technical assistance. This work was supported by grants from the National Science Foundation (MCB-9807998) and the U.S. Department of Agriculture (NRICGP-9500996) to M. R. Brodl. Back

2 Author for reprint requests, current address: Department of Biology, Trinity University, San Antonio, Texas 78212 USA (FAX: 210-999-7229; Mark.Brodl{at}trinity.edu ) Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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M. K. Johnston, N. P. Jacob, and M. R. Brodl
Heat Shock-Induced Changes in Lipid and Protein Metabolism in the Endoplasmic Reticulum of Barley Aleurone Layers
Plant Cell Physiol., January 1, 2007; 48(1): 31 - 41.
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