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(American Journal of Botany. 2002;89:401-409.)
© 2002 Botanical Society of America, Inc.

Physiology and Biochemistry

Slow heating of barley aleurone layers to heat-shock temperature preserves heat-shock-sensitive cellular properties1

Mark K. Johnston, Paul A. S. Benson, Tracy M. Rodgers and Mark R. Brodl2

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

Received for publication June 12, 2001. Accepted for publication September 20, 2001.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
In barley (Hordeum vulgare L. cv. Himalaya) aleurone layers, heat shock causes the selective suppression of {alpha}-amylase synthesis by destabilizing this secretory protein's mRNA. The lamellar stacks of the endoplasmic reticulum (ER), which serve as the site of {alpha}-amylase mRNA translation, are dissociated by heat shock, suggesting that heat-shock-induced changes in ER may be important in selectively targeting {alpha}-amylase mRNAs for destabilization. We have found that samples maintained at heat-shock temperature (40°C) for 18 h recover the ability to synthesize {alpha}-amylase and that the ER membranes in these samples contain membrane phospholipids with enhanced levels of fatty acid saturation. This present study investigated whether gradual warming to 40°C over 3–6 h (ramping) would preserve {alpha}-amylase synthesis by permitting ER membrane phospholipid retailoring during the gradual temperature increase. Analyses by sodium dodecyl-sulfate polyacrylamide gel electrophoresis revealed that {alpha}-amylase synthesis was markedly increased in ramped samples. Furthermore, northern hybridization analyses and transmission electron microscopy showed that these samples had increased {alpha}-amylase mRNA levels and stacks of ER lamellae, respectively. Gas chromatographic analyses of ER membrane phospholipids indicated that the fatty acids of ramped samples were more saturated than their heat-shocked counterparts. These data indicate that heat-induced increases in aleurone ER membrane phospholipid fatty acid saturation may be important in maintaining secretory protein expression at normally nonpermissive heat-shock temperatures.

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
 
One typical response of organisms to environmental stress is altering their pattern of gene expression. In general, most stress responses involve the induction of a suite of stress proteins that function to reestablish normal conditions following stress-induced cellular damage and/or to secure a state that will protect the organism from subsequent stress exposure. In addition, many stress responses involve the suppression of the synthesis of at least a subset of the proteins normally synthesized (Cherry, 1994 ). Mechanisms for the induction of stress proteins have been intensively investigated over the past two decades, but less attention has been paid to mechanisms that are involved in the suppression of proteins synthesized at normal temperatures during stress.

The heat-shock response is one of the best characterized stress responses. In plants, heat shock induces the synthesis of a set of high- and low-molecular-mass heat-shock proteins (HSPs), many of which have been shown to have chaperone-like functions that are thought to mediate the refolding of proteins denatured by heat (Vierling, 1991 ; Nover, 1994 ). In addition, the HSPs are correlated to the establishment of thermotolerance, permitting the organism to survive exposure to subsequent heat stress that might otherwise prove lethal (Nover, 1994 ). In soybean, pea, and tomato, heat shock not only induces HSP synthesis, it also suppresses the synthesis of all normal-temperature proteins (Nagao et al., 1986 ). This is accomplished at the translational level. The mRNAs encoding normal-temperature proteins persist in the cytoplasm, but there is a bias established that permits the translation of only HSP mRNAs during heat shock (Nagao et al., 1986 ; Nover, Scharf, and Neumann, 1989 ). At the other end of the spectrum, normal-temperature protein synthesis in maize continues uninterrupted, along with the synthesis of HSPs (Cooper and Ho, 1987 ).

In the aleurone layer of gibberellic acid (GA3)-treated barley grains, the suppression of normal-temperature protein synthesis during heat shock is selectively targeted for a particular class of proteins. Heat shock induces the synthesis of HSPs, and while the synthesis of nonsecretory protein continues, the synthesis of secretory proteins is selectively and rapidly suppressed (Belanger, Brodl, and Ho, 1986 ; Brodl and Ho, 1991 ). The proteins secreted by barley aleurone layers are hydrolytic enzymes, such as {alpha}-amylase, protease, and nucleases, and they are important to the mobilization of the endosperm reserves that sustain the heterotrophic growth of the embryo until it can photosynthesize on its own. The embryo is the source of gibberellins, which serve as a signal to the aleurone layer that the embryo is ready to germinate. The heat shock-induced suppression of secretory protein synthesis in aleurone layers is correlated with the destabilization of their otherwise stable mRNAs (Brodl and Ho, 1991 ). In the case of the secreted hydrolase {alpha}-amylase, the half-life of its mRNA is reduced from 100 h to ~30 min (Brodl and Ho, 1991 ). Heat shock also causes a dissociation of the stacks of ribosome-rich endoplasmic reticulum (ER) lamellae; only small fragments of ribosome-poor ER remain following a 3-h heat shock (Belanger, Brodl, and Ho, 1986 ). The fact that secretory proteins are translated by ER-bound ribosomes has led us to hypothesize that heat-shock-induced changes in ER membranes affect the normal translation or translocation or both of secretory protein mRNAs, targeting them for degradation (Belanger, Brodl, and Ho, 1986 ; Brodl and Ho, 1991 ).

The translation of mRNAs encoding secretory proteins begins in the cytoplasm on "free" ribosomes. Secretory protein mRNAs encode an amino terminal signal sequence that, once translated, is bound by a signal recognition particle (SRP), which simultaneously binds to the ribosome (Gilmore, 1993 ). This binding causes a transient arrest of protein synthesis. Translation resumes after the SRP-ribosome-mRNA-nascent polypeptide complex docks at an SRP-binding protein that is an integral membrane protein of the ER. With the hydrolysis of guanosine 5'-tri-phosphate (GTP), the SRP is released, translation resumes, and the growing polypeptide chain is cotranslationally translocated through an aqueous channel (Crowley et al., 1994 ).

The involvement of integral membrane proteins in this process of translation and translocation provides a potential site for alterations in the ER membrane physical state to affect the expression of secretory protein mRNAs. Temperature changes influence membrane fluidity, which can affect integral membrane protein function. For example, alterations in SRP-binding protein activity could inhibit the release of SRP, prolonging a translational arrest that might make secretory protein mRNAs prone to destabilization. Stalled translation has been demonstrated in yeast to lead to mRNA instability (Parker and Jacobson, 1990 ; Capronigro, Muhlrad, and Parker, 1993 ).

Plants tailor their membrane lipid composition to maintain a certain degree of viscosity, and they retailor that composition during times of temperature stress. This response is called homeoviscous adaptation. Such adaptation is another potential means for establishing thermotolerance (Pearcy, 1978 ; Raison, Roberts, and Berry, 1982 ). We have documented heat-shock-induced changes in ER lipid composition during heat shock. Heat-shocked barley aleurone layers increase the synthesis of phosphatidyl choline (PC) and have a higher degree of fatty acid saturation in ER membrane phospholipids and in PC isolated from total lipid extracts (Grindstaff, Fielding, and Brodl, 1996 ). This increase in fatty acid saturation is maintained during extended exposure to heat-shock temperature, when, after 18 h at 40°C, aleurone layers recover the synthesis and secretion of {alpha}-amylase, and {alpha}-amylase mRNA levels recover to ~75% of non-heat-shocked controls (Campbell, Fielding, and Brodl, 1997 ). These changes in ER membrane biochemistry would increase membrane viscosity at high temperature, working toward the maintenance of integral membrane protein function. In the present paper, we report on the results of experiments examining whether aleurone layers gradually brought to 40°C over a 3 to 6-h period (heat stress vs. heat shock) sustain the expression of {alpha}-amylase and whether any such expression is correlated with changes in ER membrane phospholipid composition. Hypothetically, these periods of heat stress could permit the membrane retailoring necessary to preserve integral membrane protein function at heat-shock temperature.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant material
The embryos of barley grains (Hordeum vulgare L. cv. Himalaya, obtained from the 1988 harvest of the Agronomy and Soils Club, Washington State University, Pullman, Washington, USA) were removed using a razor blade. The resulting "half-seeds" were surface sterilized and imbibed to prepare isolated aleurone layers according to the procedure of Belanger, Brodl, and Ho (1986) . Aleurone layers were incubated 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 (20 mmol/L sodium succinate, pH 5.0, 20 mmol/L CaCl2). 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, samples were prepared and analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as described by Grindstaff, Fielding, and Brodl (1996) . Equal amounts of total protein were electrophoresed for each sample.

For secreted proteins, 20 aleurone layers per sample were incubated in sterile 50-mL flasks with 4 mL of incubation buffer (above). At appropriate times, the incubation medium was collected and {alpha}-amylase activity was assayed according to the procedure of Jones and Varner (1967) . Total protein in each sample was assayed using the dye-binding method of Bradford (1976) . The densities of protein bands on fluorographs were measured using NIH Image (National Institutes of Health, Bethesda, Maryland, USA).

RNA isolations and northern blot analysis
One hundred aleurone layers per sample were incubated in sterile 250-mL flasks in 20 mL of incubation buffer (above). RNA was isolated using a guanidine-HCl method (Chirgwin et al., 1979 ) as modified by Belanger, Brodl, and Ho (1986) . Northern hybridizations were performed as described by Campbell, Fielding, and Brodl (1997) . The densities of the bands on the resulting fluorographs were measured using NIH Image.

Electron microscopy
Tissue samples ~0.5 mm2 were chemically fixed, dehydrated, infiltrated, and embedded according to the procedure of Campbell, Fielding, and Brodl (1997) . Sections ~60 nm thick were cut on a diamond knife, stained with 1.5% aqueous uranyl acetate and Reynold's lead citrate (Hayat, 1989 ), and viewed in a JEOL JEM 100SK (JEOL, Tokyo, Japan) transmission electron microscope at original magnifications of 5000–19 000x.

In vivo labeling, extraction, and analysis of phospholipids
Twenty aleurone layers per sample were incubated in 4 mL 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, obtained from New England Nuclear) and homogenized with acid-washed silica sand in a mortar and pestle. Lipids were extracted by chloroform-methanol, isolated by thin-layer chromatography, and quantified by liquid scintillation counting as described by Grindstaff, Fielding, and Brodl (1996) .

Cell fractionation
Two hundred aleurone layers per sample were incubated in sterile 250-mL flasks in 20 mL incubation buffer. Samples were homogenized and then fractionated on continuous sucrose gradients, and organelle marker enzyme activity was assayed as described by Grindstaff, Fielding, and Brodl (1996) .

Fatty acid isolation and analysis
Phospholipids were extracted from microsomal preparations (as outlined for cell homogenates) and analyzed by gas chromatography (GC) (columns of 10% SP-2330 on 100/120 Supelcoport; Supelco, Bellefonte, Pennsylvania, USA) according to Grindstaff, Fielding, and Brodl (1996) . The identity of each peak was determined by comigration with fatty acid standards (Sigma, St. Louis, Missouri, USA). Butylated hydroxytoluene was added, and samples were stored under nitrogen gas to minimize oxidation. Samples of phosphatidylcholine (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
 
Expression of {alpha}-amylase continues at 40°C when samples are heat stressed rather than heat shocked
Heat stress (rather than heat shock) was applied to samples in one of two "ramping" profiles. The "fast ramp" constituted an increase of 2.5°C every 30 min from the "normal" temperature of 25°C to the heat-shock temperature of 40°C. Once at 40°C, samples were maintained at that temperature for 60, 120, and 240 min ("hold" periods). The "slow ramp" was identical, except that the interval between temperature increases was 60 min rather than 30 min (Fig. 1).



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  		Fig. 1. Sample manipulations for fast and slow ramp and ramp and hold incubations. Samples were incubated in the presence of 1 µmol/L GA3 at 25°C for 16 h (fast) or 13 h (slow) prior to ramping. For the fast ramp, samples were shifted to shaking water baths that were 2.5°C warmer every 0.5 h. For the slow ramp, samples were shifted to shaking water baths that were 2.5°C warmer every 1 h. After reaching 40°C for the appropriate interval, samples were also held for 1.0, 2.0, or 3.0 h at 40°C (ramp and hold). Labeling took place during the last 0.5 h for samples labeled with [35S]methionine or the last 1.0 h for samples labeled with [3H]glycerol

 
To determine the effect of heat stress on protein synthesis in barley aleurone layers, SDS-PAGE analysis of [35S]-methionine-labeled proteins was performed. In these studies, samples were labeled during the last 30 min of incubation, and samples were taken at each temperature step of the ramp. Heat shock (an abrupt shift to 40°C) induces the synthesis of HSPs and suppresses the synthesis of {alpha}-amylase (Belanger, Brodl, and Ho, 1986 ; Fig. 2A, lanes 1 and 3). However, if samples were brought to heat shock temperature along the fast ramp (Fig. 2A), the synthesis of {alpha}-amylase was much more vigorous (over fivefold greater than heat-shocked controls), and the synthesis of HSPs was not so prominently induced. At the 40°C step, there was a notable reduction in the synthesis of {alpha}-amylase, but digitization measurements revealed that it was still over threefold greater than the level of {alpha}-amylase synthesis observed in heat-shocked samples. This same trend was apparent in samples brought to heat-shock temperature along the slow ramp (Fig. 3A). At each step of the ramp, the synthesis of {alpha}-amylase was approximately at control levels, even at 40°C. As was the case with the fast ramp, the synthesis of HSPs was not nearly as vigorous as in the heat-shocked sample.



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  		Fig. 2. Protein synthesis during fast ramp (A) and fast ramp and hold at heat-shock temperature (B). Aleurone layers were incubated in the absence (–GA) or presence (+GA) of 1 µmol/L GA3 for 19 h at 25°C. In samples exposed to heat shock (HS), the final 3 h of the 19-h incubation period was at 40°C. Ramped samples were incubated as described in Fig. 1 . The ramped samples (A) were labeled, then homogenized when the sample reached the indicated ramp temperature. The ramp and hold samples (B) were labeled, then homogenized when the hold period reached the indicated hours at 40°C following ramping. Equal amounts of total protein were electrophoresed into 11% SDS-containing polyacrylamide gels. The gel was dried and visualized fluorographically. The numbers under MW indicate the positions and sizes of molecular mass standards in kilodaltons. Arrow indicates the position of HSP 70. Arrowhead marks the position of {alpha}-amylase. FR = fast ramp; SR = slow ramp

 


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  		Fig. 3. Protein synthesis during slow ramp (A) and slow ramp and hold at heat-shock temperature (B). Aleurone layers were incubated in the absence (–GA) or presence (+GA) of 1 µmol/L GA3 for 19 h at 25°C. Samples were incubated as described in Fig. 2 , except the ramp rate was 2.5°C/60 min. Equal amounts of total protein were electrophoresed into 11% SDS-containing polyacrylamide gels. The gel was dried and visualized fluorographically. The numbers under MW indicate the positions and sizes of molecular mass standards in kilodaltons. Arrow indicates the position of HSP 70. Arrowhead marks the position of {alpha}-amylase

 
Although the synthesis of {alpha}-amylase continued during the ramping periods, the amount of time spent at 40°C was brief compared to the 3 h at 40°C typical of our heat-shock procedure. For this reason, the synthesis of proteins in barley aleurone layers ramped to 40°C and held at that temperature for extended periods of time was examined. In the first 1 h of the fast ramp hold period, the level of {alpha}-amylase synthesis declined; however, a further hour at 40°C resulted in the reversal of this decline in synthesis (Fig. 2B, lanes 8–10). At the 3-h hold period, {alpha}-amylase was synthesized at levels approximating non-heat-shocked controls and nearly 12-fold greater than the level of synthesis in heat-shocked samples (Fig. 2B). This reduction in {alpha}-amylase synthesis after 1 h at 40°C was reproducible in four independent experiments. The synthesis of HSPs was strongly induced during the entire hold period following fast ramp.

Aleurone layers exposed to slow ramp heat stress treatment also continued the synthesis of {alpha}-amylase at 40°C at levels comparable to non-heat-shocked samples and at levels over 14-fold greater than heat-shocked controls (Fig. 3B). Unlike the fast-ramped samples, however, there was no decline in {alpha}-amylase synthesis at any time during the hold period. Furthermore, the synthesis of HSPs was not as vigorous as in the fast-ramped samples and tapered off after the samples were held at 40°C for 1 h (Fig. 3B, lanes 8–10). Both the sustained synthesis of {alpha}-amylase and the tapering off of HSP synthesis were reproducible in four independent experiments.

To determine whether heat-stressed aleurone layers continued the secretion of {alpha}-amylase, the activity of {alpha}-amylase in the incubation medium was assayed during the ramp and hold periods. An abrupt heat shock reduced the activity of {alpha}-amylase in the incubation medium 19-fold (from 5.6 to 0.3 {Delta}A260·min–1·mL–1) (Fig. 4). In fast-ramped, heat-stressed samples activity was reduced 2.5-fold during the ramp period and 3-fold during the hold period (from 5.6 to lows of 2.3 and 1.9 {Delta}A260·min–1·mL–1, respectively). In slow-ramped, heat-stressed samples activity was reduced 1.9-fold during the ramp period and 2-fold during the hold period (from 5.6 to lows of 2.9 and 2.8 {Delta}A260·min–1·mL–1, respectively).



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  		Fig. 4. The {alpha}-amylase activity in the incubation medium of samples heat stressed by fast and slow ramps with and without hold periods. Samples were incubated as described in Fig. 1 . At the end of each ramp period or at the end of the designated hold period, the incubation medium was collected and replaced with fresh medium. The samples of incubation medium were assayed for {alpha}-amylase activity. The experiment was repeated three times. Differences in activity between independent samples were <7%

 
Because heat shock suppresses {alpha}-amylase synthesis by destabilizing its mRNA (Belanger, Brodl, and Ho, 1986 ; Brodl and Ho, 1991 ), it was predicted that heat-stressed barley aleurone layers should have higher levels of {alpha}-amylase mRNA than heat-shocked samples. The {alpha}-amylase mRNA levels in samples heat stressed under fast and slow ramp conditions and during subsequent hold periods were examined by northern hybridization analyses (Fig. 5). Incubation with GA3 induces the expression of two distinct isoforms of {alpha}-amylase, an isoform with an isoelectric point of pH 4.5 (detected by clone E) and an isoform with an isoelectric point of pH 5.8 (detected by pM/C) (Nolan, Lin, and Ho, 1986 ). Aleurone layers subjected to heat stress by the fast ramp procedure had an average (both isozymes) of 15% of the amount of the {alpha}-amylase mRNA in nonstressed control samples, but they had an average of 12-fold more {alpha}-amylase mRNA than heat-shocked control samples. A similar trend was observed in aleurone layers subjected to heat stress by the slow ramp procedure, where {alpha}-amylase mRNA levels were an average of 17% of nonstressed control samples but an average of 15-fold greater than heat-shocked control samples.



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  		Fig. 5. The {alpha}-Amylase mRNA levels in samples heat stressed by fast and slow ramps with and without hold periods. RNA was extracted from barley aleurone layers incubated as described in Fig. 1 . An equal amount of total RNA from each sample was electrophoresed into formaldehyde-containing 1.5% agarose gels. The RNA was capillary blotted onto hybridization membranes and probed with nick-translated cDNA clones for the low (clone E) and high (pM/C) pI isozymes of {alpha}-amylase. Total RNA yields for each sample were approximately equivalent. The bands shown represent the only radioactivity detectable on the two fluorograms

 
Transmission electron microscopy was used to examine the effect of heat stress on ER ultrastructure. Aleurone layers incubated at 25°C contained organized stacks of rough ER lamellae (Fig. 6A). Heat shock caused these stacked ER lamellae to dissociate and fragment, and ribosome density decreased sharply (Fig. 6B) (Belanger, Brodl, and Ho, 1986 ). When aleurone layers were subjected to either fast or slow ramp heat stress plus a 3-h hold period (rather than heat shock) the stacks of ER lamellae remained intact (Fig. 6C and D). In addition, though the density of ribosomes along the ER diminished from 46.0 ± 1.7 ribosomes/micron of ER in nonstressed samples to 37.3 ± 4.7 in fast ramp and hold samples and to 40.1 ± 3.8 in slow ramp and hold samples, ribosome density was significantly higher in the heat-stressed samples than the 12.2 ± 4.6 ribosomes/micron observed in heat-shocked samples.



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  		Fig. 6. Ultrastructure of aleurone cells exposed to fast ramp and hold and slow ramp and hold heat stress. (A) Electron micrograph of barley aleurone cells incubated in the presence of 1 µmol/L GA3 for 19 h at 25°C. (B) Electron micrograph of barley aleurone cells incubated in 1 µmol/L GA3 for 16 h at 25°C then shifted to 40°C for 3 h. (C) Electron micrograph of barley aleurone cells incubated according to the fast ramp and hold protocol in Fig. 1 . (D) Electron micrograph of barley aleurone cells incubated according to the slow ramp and hold protocol in Fig. 1 . Figure abbreviations: ag = aleurone grain; arrowheads = ER fragments; brace = ER lamellae; p = plastid; s = spherosome. Bar (upper right) in each micrograph = 1.0 µm

 
Heat stress induces biochemical changes in ER phospholipids
Heat shock increases PC synthesis and increases the level of fatty acid saturation in PC-associated and ER membrane phospholipids (Grindstaff, Fielding, and Brodl, 1996 ). It has been hypothesized that these changes may be important to the formation of heat-tolerant ER membranes (Grindstaff, Fielding, and Brodl, 1996 ). In addition, we have found that following 18 continuous hours at 40°C, {alpha}-amylase synthesis recovers and the ER membranes reform with increased levels of saturation in the fatty acids of their phospholipids (Campbell, Fielding, and Brodl, 1997 ). If such changes are important to adaptation to high temperature, it is likely that ramping aleurone layers to heat-shock temperature induces similar changes to help secure the normal secretory protein expression observed during the fast and slow ramp heat stresses. Aleurone layers exposed to both the fast and slow ramp heat stress procedures were labeled with [14C]-glycerol, and total lipid extracts from whole cells were analyzed by thin layer chromatography (TLC). The radioactivity in five major classes of phospholipids was measured by liquid scintillation counting. Heat stress by either ramp procedure increased the incorporation of [14C]-glycerol into PC from 56.61% to ~69% (70.90% for fast ramp and 67.45% for slow ramp), as compared to 75.51% in heat-shocked samples (Table 1). The levels of [14C]-glycerol incorporated into the other phospholipids decreased in proportion to one another; none of the other individual phospholipids changed significantly from its control sample. When fast-ramped aleurone layers were held at 40°C for 2 h, the level of [14C]-glycerol incorporated into PC was maintained at 69.36%. When slow-ramped aleurone layers were held at 40°C for 2 h, the level of [14C]-glycerol incorporated into PC decreased from 67.45% to 63.11%, but a t test of arcsine-transformed percentage data showed that the difference between these two samples is of marginal significance (P < 0.001).


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  		Table 1. Incorporation of [14C]glycerol into phospholipids of aleurone layers incubated with GA3 during heat shock and heat stress. Twenty aleurone layers per sample were incubated in the absence (–GA) or presence (+GA) of GA3 at 25°C for 13 h (slow and slow and hold) or 16 h (all other samples). Samples were then ramped from 25° to 40°C at temperature increases of 2.5°C/30 min (fast) or 2.5°C/60 min (slow) or shifted abruptly to 40°C for 3 h (+GA/HS). The –GA and +GA control samples were incubated for a total of 19 h at 25°C. Ramped samples were homogenized immediately following ramping; hold samples were maintained at 40°C for 2 h following ramping, then homogenized. All samples were labeled with [14C]glycerol for 1 h immediately prior to homogenization. One hundred microliters of lipids extracted from the cell homogenates (300 µL total) were separated by thin layer chromatography and quantified by liquid scintillation counting. Values represent the percentage of total [14C]glycerol incorporation into the five listed phospholipid classes. Results are given as the mean ± SE of six independent experiments, two replicates each. Means ± SE of heat-shocked samples that differ from their corresponding control group are indicated by *P < 0.005, **P < 0.01 (t test). cmpm = counts per minute (radioactivity); PA = phosphatidic acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PI = phosphatidylinositol; PS = phosphatidylserine

 
To determine the temperature at which the increase in [14C]-glycerol incorporation into PC occurs, aleurone layers were slow ramped to 40°C, and samples were taken at every 2.5°C increment (every hour) in the ramp. Significant increases in the incorporation of [14C]-glycerol into PC were observed beginning at 30°C (Table 2). The incorporation of [14C]-glycerol into PC continued to increase until it peaked at 72% of total incorporation at 37.5°C. At 40°C, the incorporation of [14C]-glycerol into PC declined to 64.38% of total incorporation. The difference between this sample and the 37.5°C sample was significant, according to a t test of the arcsine-transformed percentage data (P < 0.05).


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  		Table 2. Incorporation of [14C]glycerol into phospholipids of aleurone layers incubated with GA3 during slow-ramp heat stress. Twenty aleurone layers per sample were incubated in the presence of GA3 at 25°C for 13 h. Samples were then exposed to heat stress via the "slow ramp." The temperatures in the table below represent the final temperature in the ramping procedure to which the layers were exposed prior to homogenization. The samples were labeled during that final hour with [14C]glycerol. One hundred microliters of lipids extracted from the cell homogenates (300 µL total) were separated by thin layer chromatography and quantified by liquid scintillation counting. Values represent the percentage of total [14C]glycerol incorporation into the five listed phospholipid classes. Results are given as the mean ± SE of three independent experiments, two replicates each. Means ± SE of heat-shocked samples that differ from their corresponding control group are indicated by *P < 0.005, **P < 0.01 (t test). cpm = counts per minute (radioactivity); PA = phosphatidic acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PI = phosphatidylinositol; PS = phosphatidylserine

 
Data from TLC analyses of [14C]-glycerol-labeled phospholipids indicated that heat stress increased the synthesis of PC. It was not clear from these studies, however, whether heat stress affected the saturation of fatty acids in membrane phospholipids. To investigate fatty acid saturation, GC analyses of fatty acids hydrolyzed from the phospholipids isolated in total lipid extracts of ER membranes purified on continuous sucrose gradients were performed. Fractions from the continuous sucrose gradients were assayed for catalase, cytochrome c oxidase, cytochrome c reductase, IDPase, and vanadate-sensitive ATPase activities. Although a small shoulder of vanadate-sensitive ATPase activity was present in fractions containing maximal amounts of cytochrome c reductase activity, these fractions were free of contaminating marker enzyme activities from other organelles (data not shown). In peak ER-containing fractions, heat shock increased the ratio of saturated to unsaturated fatty acids from 0.56 to 0.88 (Table 3). When aleurone layers were ramped to 40°C under fast ramp conditions, the ratio of saturated to unsaturated fatty acids increased to 1.09. When held at 40°C following the fast ramp, the aleurone layers maintained a saturated to unsaturated fatty acid ratio of 0.96. Samples heat stressed by the slow ramp protocol also increased the level of fatty acid saturation in ramped and ramped-hold samples to saturated to unsaturated fatty acid ratios of 1.11 and 1.02, respectively. In heat-shocked and heat-stressed samples, the change in the ratio of saturated to unsaturated fatty acids was the result of decreases in 18-C unsaturated fatty acids and increases in myristic, palmitic, and stearic acids. In the heat-stressed samples there were greater increases in myristic and palmitic acids than in the heat-shocked samples, while levels of 18:0 fatty acids were similar in the heat-stressed and heat-shocked samples.


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  		Table 3. Fatty acid composition of ER membranes from barley aleurone layers ramped to heat-shock temperature. Aleurone layers were incubated in 1 µmol/L GA3 for either 19 h (+GA) at 25°C, 16 h at 25°C then 3 h at 40°C (+GA/HS), or ramped to 40°C then either homogenized or maintained at 40°C for 4 h following ramping (hold). Samples were ramped from 25° to 40°C at temperature increases of 2.5°C/30 min (fast) or 2.5°C/60 min (slow). Lysates from chopped samples were layered onto sucrose step gradients. The turbid fraction at the 20%/50% interface was then layered on a continuous 20–50% sucrose gradient. Total lipid extractions were performed on fractions containing peak levels of cytochrome c reductase activity. Fatty acids in the samples were hydrolyzed from phospholipids, methylated, and analyzed by gas chromatography. Results are given as percentage of moles (mol%) and are averages from three independent experiments, two replicates each. Errors of analysis were estimated to be within 5% of individual values

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Collectively, the data presented here indicate that gradual warming of barley aleurone layers to heat-shock temperatures permits the continued expression of {alpha}-amylase when an abrupt change to heat-shock temperature would otherwise rapidly inhibit {alpha}-amylase expression. This expression was characterized by a substantial increase in the ability to synthesize {alpha}-amylase at heat-shock temperature, by increased levels of {alpha}-amylase secretion during heat shock, by increased levels of {alpha}-amylase mRNA over heat-shocked samples, and by the presence of stacked ER lamellae in samples ramped and held at heat-shock temperatures. During this period of gradual warming to heat-shock temperatures, there were increases in PC synthesis, and the phospholipids from ER membranes isolated from these heat-stressed samples had enhanced levels of fatty acid saturation.

The properties that we have documented in heat-stressed barley aleurone layers characterize a classic example of acquired thermotolerance, a condition where normally deleterious temperatures can be tolerated by the organism. Typically thermotolerance is established by a brief, sublethal heat shock, followed by a recovery period and then lethal heat shock (Vierling, 1991 ; Parsell and Lindquist, 1993 ). However, as has been seen in this study and others (Hong and Vierling, 2000 ), thermotolerance can also be established by slow heating to heat-shock temperatures. In contrast to most of the previous studies on the establishment of thermotolerance, we have examined discrete cellular functions and structures rather than the overall survival of the organism. From such a perspective it is easier to appreciate the physiological concerns an organism must address at the cellular level in order meet the challenge of surviving lethal heat shock.

Our results indicate that there were differences between the fast and slow ramps with regard to {alpha}-amylase synthesis. In samples ramped to heat shock via the slow ramp, there was no apparent suppression of {alpha}-amylase synthesis when the samples were brought to and maintained at 40°C; however, in samples ramped to heat shock via the fast ramp, the synthesis of {alpha}-amylase was reduced following 1 h at 40°C. Although protein synthesis was suppressed at this time, it recovered to nearly control levels by 2 h at 40°C. This is in sharp contrast to the 18 h at 40°C required for the recovery of {alpha}-amylase synthesis in barley aleurone layers abruptly exposed to heat shock (Campbell, Fielding, and Brodl, 1997 ). From the [14C]glycerol labeling data, it became apparent that the aleurone layers increased the level of phospholipid synthesis after they were raised to 30°C, and this increase in phospholipid synthesis continued for at least the next 4 h as the temperature increased by slow ramp to 40°C. From the GC data of the fatty acid composition of ER phospholipids, this increase in lipid synthesis appeared to result in an increase in the degree of fatty acid saturation and a shortening of the chain length toward 14- and 16-carbon fatty acids. These differences in {alpha}-amylase synthesis at 40°C in slow-ramped, fast-ramped, and heat-shocked samples indicate that there is a period for retailoring that may require more than the 3-h fast ramp period for completion. Therefore, the rapidity of the onset of the heat stress appears to be important, and the ability of a plant to acclimate to high temperature stress in the field may be correlated to the speed at which it or its critical tissues can retailor membrane biochemistry.

We also observed differences between the fast and slow ramps with regard to HSP synthesis. In slow-ramped samples there was markedly less HSP synthesis during the hold period than in the fast-ramped samples. Western blot analysis of HSP 70 levels indicated that the accumulation of HSP 70 in the slow-ramped sample was greater than in the fast-ramped sample following 2 h at 40°C (data not shown). This reduced HSP synthesis during more gradual temperature ramps is also observed in Chinese hamster ovary cells (Herman et al., 1981 ; Anderson et al., 1988 ) and pea seedlings (Chen et al., 1990 ). This may suggest that part of the reason that fast-ramped samples synthesized less {alpha}-amylase 1 h into the hold period could be because there were not yet sufficient levels of HSPs present for providing thermoprotection to normal-temperature protein synthesis. Many of the HSPs have been shown to be chaperones, and it has been proposed that they have an important role in renaturing heat-denatured proteins (Vierling, 1991 ; Waters, Lee, and Vierling, 1996 ). Yet we have previously shown that HSPs alone are not sufficient to establish thermotolerance or to provide thermoprotection to the synthesis of secretory proteins in heat-shocked barley aleurone cells (Lanciloti, Cwik, and Brodl, 1996 ). In the present paper, "slow ramp and hold" aleurone layers have reduced levels of [14C]-glycerol incorporation into PC as compared to their fast-ramped counterparts, indicating that heat-induced incorporation of new phospholipids (as a component of heat adaptation) is closer to completion. In light of these observations and the data on heat shock and ER membrane biochemistry cited above, we propose that in the case of barley aleurone layers (and perhaps other secretory tissues) HSPs are necessary but not alone sufficient for providing thermoprotection or thermotolerance; the cells must also address issues of membrane viscosity (which cannot apparently be resolved by HSPs) to acquire a fully thermoprotected or thermotolerant state.

Given the role of membrane fluidity in the modulation of the function of integral membrane proteins, it appears that during the ramping period aleurone cells made adjustments to the degree of fatty acid saturation in ER membrane phospholipids, presumably to maintain an optimal degree of viscosity for the proper function of integral membrane proteins important to the translation and translocation of proteins at the ER. The principle of homeoviscous adaptation (Mazliak, 1989 ; Quinn, Joo, and Vigh, 1989 ) states that cells exposed to temperatures that are higher than normal promote fatty acid saturation in membrane phospholipids, thereby decreasing their fluidity at high temperature. Conversely, chilling temperatures will increase the degree of fatty acid unsaturation in their membrane phospholipids, thereby increasing their fluidity to maintain an appropriate level of membrane fluidity. This principle can be demonstrated by fusing protoplasts with liposomes derived from cold-acclimated plants or from mono- and diunsaturated species of PC; the resulting protoplasts are freezing tolerant (Steponkus et al., 1988 ). Reciprocally, Arabidopsis thaliana mutants with lesions in key fatty acid desaturases exhibit enhanced thermostability in chloroplast functions (Hugly et al., 1989 ; Kunst, Browse, and Somerville, 1989 ). The nature of the changes in fatty acid saturation reported here is consistent with the principle of homeoviscous adaptation. Furthermore, the magnitude of the changes in membrane phospholipid saturation observed here is comparable to the magnitude of changes seen in other species where heat stress-induced loss of chloroplastic function was reestablished by membrane modification (Pearcy, 1978 ; Raison, Roberts, and Berry, 1982 ; Hugly et al., 1989 ; Kunst, Browse, and Somerville, 1989 ).

In barley aleurone layers, there is a direct correlation between increased membrane saturation, secretory protein synthesis, and secretory protein mRNA stability (Grindstaff, Fielding, and Brodl, 1996 ; Campbell, Fielding, and Brodl, 1997 ). We have recently shown that heat shock prevents the release of SRPs from the ER during heat shock in barley aleurone layers, potentially keeping secretory protein mRNAs in a protracted state of translational arrest and a target for mRNA turnover machinery (Chu, Brodl, and Belanger, 1997 ). As a ribosome translates a secretory protein mRNA, an SRP binds to the emerging nascent polypeptide chain, causing translational arrest until the ribosome can bind to an SRP receptor on the ER (Rapoport, Jungnickle, and Kutay, 1996 ). With a rapid heat shock, alterations in membrane fluidity could affect SRP receptor function before compensating changes in membrane fluidity could be made. The heat-stressed aleurone layers, on the other hand, have sufficient time to alter the biochemistry of their ER membranes to maintain the proper function of these integral membrane proteins at heat shock temperature. It will be informative to determine the effect of fast and slow ramp heat stress on the release of SRPs from the ER, for if alterations in integral membrane components are important, then the modifications in lipid saturation during heat shock should have a positive effect on the release of SRP at heat-shock temperatures.


   FOOTNOTES
 
1 The authors thank Dr. Bill Geer (Biology Department, Knox College) for assistance with lipid analysis and Dr. John Rogers (Department of Biochemistry, Washington State University, Pullman, WA) for the gift of {alpha}-amylase cDNA clones. 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. B. Back

2 Author for reprint requests. Present address: Trinity University, Department of Biology, 715 Stadium Drive, San Antonio, Texas 78212, USA (fax: 309-341-7718; mbrodl{at}trinity.edu ) Back


   LITERATURE CITED
TOP
 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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