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Identification of acquired thermotolerance deficiency within the ditelosomic series of `Chinese Spring' wheat§
Plant Physiol. Biochem., 2000, 38 (3), 243−252 / USDA-ARS © 2000 Published by Éditions scientifiques et médicales Elsevier SAS. All rights reserved S098194280000735X/FLA
Identification of acquired thermotolerance deficiency within the ditelosomic series of ‘Chinese Spring’ wheat§ Patrick O’Mahony, John J. Burke*, Melvin J. Oliver Plant Stress and Germplasm Development Unit, USDA-ARS, 3810 4th Street, Lubbock TX 79415, USA
* Author to whom correspondence should be addressed (fax +1 806 723 5272; email [email protected]) (Received March 22, 1999; accepted October 28, 1999) Abstract — The relative contribution of individual heat shock proteins to acquired thermotolerance was evaluated through analysis of chromosomal deletions in a ditelosomic series of the hexaploid wheat (Triticum aestivum L.) cultivar ‘Chinese Spring’. This study describes the identification of a line within this ditelosomic series that exhibited a reduced level of acquired thermotolerance. Changes in the temperature sensitivity of chlorophyll accumulation were used as an indicator of acquired thermotolerance. The temperature providing maximum chlorophyll accumulation was 30 °C in leaves under continuous light. A 30-min challenge temperature of 48 °C prior to the light exposure was shown to inhibit subsequent chlorophyll accumulation. Preincubation at 40 °C for 4 h before the 30-min 48 °C challenge triggered the acquired thermotolerance system of the plant resulting in chlorophyll accumulation upon exposure to light. Evaluation of the ditelosomic series revealed reductions in acquired thermotolerance levels in the DT7DS line relative to controls. Two-dimensional SDS polyacrylamide gel analysis was used to identify reduction in the level of two low molecular mass heat shock proteins in DT7DS. USDA-ARS © 2000 Published by Éditions scientifiques et médicales Elsevier SAS Acquired thermotolerance deficiency / ditelosomic / heat shock / Triticum aestivum CELTEC, cellular thermoelectric controller / DAP, days after planting / HSP, heat shock protein
1. INTRODUCTION Stress as a result of elevated temperatures is a common threat to plant survival and crop yields worldwide. Elevated temperatures have physiological [2, 4, 5, 10] and morphological [20] effects on plants, many of which result in the retardation of growth and development. However, when plants are exposed to elevated non-lethal temperatures, they acquire thermotolerance which can transiently raise their injury threshold and protect them from subsequent, otherwise lethal, temperatures. Plants produce heat shock proteins (HSPs) in response to various environmental stresses [9, 25, 28]. At sublethal elevated temperatures, quantitative induc§
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tion of HSPs occurs with a concomitant reduction in the synthesis of most other proteins. This reorganization of metabolic priorities coincides with the acquisition of thermotolerance [17, 18, 28]. Significant evidence is available from yeast studies which link HSP induction to the acquisition of thermotolerance [21, 24, 25, 28, 30]. To date, however, only indirect evidence exists to suggest that HSP induction is associated with acquired thermotolerance in plants. In Arabidopsis, a correlation has been observed between modulated heat shock protein synthesis and heat shock factor activity and expression with levels of thermotolerance [14, 15, 23, 29]. Studies in thermosusceptible and thermotolerant recombinant inbred lines of wheat detected a genetic relationship between expression of a plastid localized HSP26 and acquired thermotolerance [13]. In addition, other studies have demonstrated that an acquired thermotolerance deficient yeast that carries a mutated HSP104 gene can be successfully complemented by a plant HSP 101 genes from soybean [16] and Arabidopsis [26].
Plant Physiol. Biochem., 0981-9428/00/3/ USDA-ARS © 2000 Published by E´ditions scientifiques et médicales Elsevier SAS. All rights reserved
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Obtaining direct evidence to link HSP with acquired thermotolerance in higher plants has been hampered by a lack of mutants with which a cause and effect relationship could be established. This shortfall may be circumvented to a degree by analysis of heat shock responses in aneuploid genetic stocks where specific chromosomal deletions result in a lack of, or reduction in, acquired thermotolerance coincident with an alteration of HSP synthesis. In this study, we used a sensitive chlorophyll accumulation assay [7] to identify acquired thermotolerance deficiencies within the ditelosomic (a plant with one chromosome missing an arm-telocentric) series of the hexaploid wheat cultivar ‘Chinese Spring’ [27]. A previous investigation using 2D gel electrophoresis [22] to analyze the genetic control of HSP synthesis in wheat identified the chromosomal localization of a number of low molecular mass HSPs. Variations in relative HSP levels suggested that the homoeologous DT lines 3, 4 and 7 contain the majority of the controlling genes indicating chromosomes 3, 4 and 7 as sites containing HSP controlling loci. However, this study did not address the possible functional relationship between specific HSP changes and levels of acquired thermotolerance. In this study of ditelosomics, we report the use of a similar strategy to that of Porter et al. [22] to detect two HSPs which may be involved in acquired thermotolerance, and to localize them to a specific chromosome arm.
2. RESULTS In order to accurately identify acquired thermotolerance deficiencies using the chlorophyll inhibition assay [6], it is necessary to determine several pertinent temperature parameters in the parental line. These parameters include the optimum temperatures for (a) chlorophyll accumulation, (b) minimum challenge temperature resulting in the maximum decrease in chlorophyll accumulation and (c) preincubation temperature yielding maximum chlorophyll accumulation following a subsequent challenge temperature.
2.1. Optimum temperature for chlorophyll accumulation A range of temperatures from 10 to 45 °C were used to find the temperature that allowed for the maximum accumulation of chlorophyll in excised leaf tissue. The temperature sensitivity of chlorophyll accumulation in Plant Physiol. Biochem.
Figure 1. Determination of optimum temperature for chlorophyll accumulation in leaf segments of Chinese Spring wheat exposed to continuous light for 20 h. Error bars represent standard error.
Chinese Spring wheat is shown in figure 1. Chlorophyll levels increased with temperature from 10 to 30 °C and then declined to minimal levels at 45 °C. A temperature of 30 °C was chosen for use in subsequent experiments to ensure maximum chlorophyll accumulation.
2.2. Determination of optimum challenge temperature The temperature-induced inhibition of chlorophyll accumulation subsequent to a high temperature challenge was determined by exposing etiolated leaf segments to temperatures between 44 and 56 °C for 30 min prior to the 30 °C/20 h light treatment (figure 2). A 35 and 72 % reduction in chlorophyll accumulation was observed subsequent to a 44 and 46 °C challenge, respectively. A 30-min challenge at 48 °C, or above, prior to the 30 °C light treatment resulted in more than a 95 % inhibition of chlorophyll accumulation. A 48 °C challenge temperature was used in subsequent experiments to inhibit chlorophyll accumulation.
2.3. Preincubation temperature yielding maximum protection The acquisition of thermotolerance was evaluated by preincubating wheat leaf segments at temperatures from 34 to 46 °C for 4 h prior to a 30-min/48 °C challenge (figure 3). The inability to accumulate chlorophyll upon exposure to continuous light because of a prior 30-min/48 °C challenge was quantified as the amount of chlorophyll accumulated as a percentage of
Acquired thermotolerance within Chinese Spring wheat
Figure 2. Determination of suitable challenge temperature for maximum inhibition of chlorophyll production in leaf segments of Chinese Spring wheat. Error bars represent standard error.
the untreated control (incubated at 30 °C throughout). An increase in the preincubation temperature from 34 to 40 °C resulted in more protection from the effects of the challenge temperature as measured by a greater degree of chlorophyll accumulation. A 75 % reduction in chlorophyll accumulation was observed with a 36 °C preincubation, a 53 % reduction occurred at 38 °C, and the least reduction in chlorophyll accumulation (48 %) was seen with a 40 °C preincubation. This decrease in the reduction of chlorophyll accumulation represents thermotolerance acquired during preincubation at 40 °C. Preincubation at temperatures above 40 °C provided less protection to where little or no chlorophyll accumulation was observed with 44 or 46 °C preincubations (figure 3). The findings to this point revealed that maximum chlorophyll accumulation occurs at 30 °C upon continuous light exposure; that 30-min challenges at 48 °C or above prevent subsequent chlorophyll accumulation; and that preincubation at 40 °C for 4 h prior to a 48 °C challenge delivered the maximum level of protection.
2.4. Identification of acquired thermotolerance mutants Having defined the parameters of our screening system, a selection of ditelosomics (DTs) of Chinese Spring wheat were examined for a reduction in the level of acquired thermotolerance. Each viable DT line was preincubated at 40 °C for 4 h, challenged at 48 °C for 30 min and chlorophyll accumulation ability assessed following a 30 °C/20 h light treatment. Table I presents the results of our initial screen of these lines.
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Figure 3. Evaluation of a range of preincubation temperatures inducing maximum thermal protection in leaf segments of Chinese Spring wheat. Error bars represent standard error.
This was a preliminary screen only as some seeds were available in limited quantity and others had germination problems. However, based on observed altered levels of acquired thermotolerance, nine of the 34 lines were selected and subjected to a more rigorous statistical analysis following the production of seed from the original stocks. These lines were DT1BS, DT3DL, DT3BL, DT4DS, DT4DL, DT5AL, DT6DL, DT6BS and DT7DS. Lack of sufficient seed quantity and poor germination rates for DT5BL prohibited similar analysis. In addition to the DT7DS line, DT7AS and DT7BS also showed a reduction in acquired thermotolerance levels but in both cases the results were difficult to reproduce in latter seed batches derived from the original parent lines (data not shown). The nine lines identified were subjected to a more extensive version of the primary screen (for statistical purposes between four and eight samples were examined). Figure 4 presents a bar graph representation of the results that we obtained from this screen of the nine selected lines. The Chinese Spring control (labeled WT for wild-type) accumulated approximately 50 % of the chlorophyll observed in nonchallenged Chinese Spring seedlings. The vertical line in this figure serves as a reference point for comparison of the DTs with the Chinese Spring parent. Seven of the nine lines evaluated accumulated chlorophyll to levels close to that of the WT control. Four of the seven lines DT3DL, DT4DS, DT4DL and DT6BS were statistically the same as the WT seedlings, with the three remaining lines DT3BL, DT5AL and DT6DL having chlorophyll levels slightly above or below the WT seedlings. Two lines exhibited chlorophyll levels well removed from those of the WT sample. The vol. 38 (3) 2000
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Table I. Relative acquired thermotolerance levels within the ditelosomic series of Chinese Spirng wheat relative to Chinese Spring controls. Acquired thermotolerance levels similar to control levels are indicated with a (+) sign; those lines with increased thermotolerance over controls are indicated with either a (++, or +++) sign; those lines having acquired thermotolerance levels below controls are indicated with a (–) sign; and those that were not determined are indicated with ND. Ditelosomic line
Thermotolerance level
Ditelosomic line
Thermotolerance level
1AL 1AS 1BS 1DL 1DS 2AS 2BL 2DL 2DS 3AL 3BL 3DL 3AS 3BS 3DS 4BS 4DS
+ + ++ ND ND ND ND + + + ++ + ND ND ND + +
4AL 4DL 5AL 5BL 5DL 6AS 6BS 6DS 6AL 6BL 6DL 7AS 7BS 7DS 7AL 7DL 7DL
+ + + ++ + + +++ + ND ND + – – – + + +
DT1BS line consistently had greater chlorophyll levels than the WT showing only 10–20 % injury following preincubation and challenge and a more detailed characterization of this line is currently underway. The DT7DS line, however, could only accumulate approximately 7 % of the chlorophyll that the wild-type line accumulates. Thus it appears that the DT7DS line is not capable of establishing a significant level of acquired thermotolerance following a 40 °C for 4 h preincubation that would protect the metabolic activities associated with chlorophyll accumulation in the light from a high temperature challenge. No other physiological abnormality was observed in whole plant studies where the DT7DS line germinated and grew at similar rates to the wild-type (data not shown).
Figure 4. Identification of the levels of acquired thermotolerance in several ditelosomics of Chinese Spring wheat. Individual bars represent the level of chlorophyll accumulation as percentage of control values following a preincubation to induce acquired thermotolerance. Error bars represent standard error.
to 25 °C, remaining high until 30 °C and then declining sharply, reaching minimal levels at 45 °C. This temperature sensitivity profile is almost identical to that for the wild-type Chinese Spring wheat (figures 1, 5). Thus the reduction in chlorophyll accumulation for DT7DS seen in figure 4 is not the result of a change in temperature sensitivity of chlorophyll accumulation in this line. These data also demonstrate that 30 °C can be used for optimum chlorophyll accumulation in the DT7DS line which enables the direct comparisons to wild-type following heat treatments. The greening rates of wild-type and DT7DS wheat were compared by incubating leaf segments for 20 h at
2.5. Characterization of the acquired thermotolerance mutant DT7DS To ensure that this result was due to an altered acquired thermotolerance pathway in the DT7DS line and not a general altering of the temperature sensitivity of the plant, a temperature optimum curve for chlorophyll accumulation in this line was generated. The results of this analysis are presented in figure 5. Chlorophyll levels increased with temperature from 10 Plant Physiol. Biochem.
Figure 5. Determination of optimum temperature for chlorophyll accumulation in leaf segments of the ditelosomic DT7DS. Error bars represent standard error.
Acquired thermotolerance within Chinese Spring wheat
Figure 6. Comparative greening rates (A) of Chinese Spring and the ditelosomic DT7DS wheat leaf segments exposed to light at 30 °C, and comparison of challenge temperatures inhibiting chlorophyll accumulation (B) in control Chinese Spring wheat (●) and DT7DS (·). Error bars represent standard error.
30 °C in light. Chlorophyll accumulated in the DT7DS at rates similar to that of wild-type (figure 6), indicating that any differences observed in the preincubated and challenged DT7DS tissues did not arise from an inherent difference in chlorophyll accumulation rate compared to control Chinese Spring seedlings. The challenge response curve for chlorophyll accumulation in DT7DS seedlings was also compared with the challenge response curve of control Chinese Spring seedlings (figure 6). Similar temperature sensitivities were observed between the DT7DS and control Chinese Spring seedlings when they were directly challenged without preincubation to induce acquired thermotolerance. These results show that the DT7DS line does not have unique temperature characteristics that distinguish it from control seedlings as might be the case in temperature sensitive plants. Finally, the temperature response curve for acquisition of thermotolerance was evaluated by incubating DT7DS leaf segments at 34, 36, 38, 40, 42, 44 and 46 °C for 4 h prior to the 30-min/48 °C challenge (figure 7). Following a 20-h treatment in light at 30 °C, chlorophyll accumulation was evaluated. As for wildtype, preincubation at 40 °C resulted in the greatest retention of the ability of the tissue to accumulate chlorophyll after a high temperature challenge. Nevertheless, as seen in figure 4, the absolute level of acquired thermotolerance observed in the DT7DS seedlings resulted in significantly less accumulation of chlorophyll than the Chinese Spring seedlings. It is worth noting that variations of even 1° in temperature at or near the sensitivity threshold for these plants can alter the final levels of accumulation of chlorophyll. Therefore, the data for DT7DS is presented relative to control data and not as absolute values.
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Figure 7. Comparison of the range of preincubation temperatures providing protection in DT7DS (●) and Chinese Spring wheat (·) from injury by a subsequent thermal challenge. Error bars represent standard error.
2.6. Two-dimensional gel electrophoresis analysis of Chinese Spring wild-type and DT7DS Once it had been established that the DT7DS line was deficient in the ability to acquire thermotolerance compared to the wild-type Chinese Spring wheat, we were interested in determining if this phenotype could be linked to a concomitant change in the synthesis of heat shock proteins. To accomplish this, leaf segments from control Chinese Spring seedlings and DT7DS seedlings were treated in the dark at 22 and 40 °C for 4 h in the presence of [35S]-labeled amino acid mix. Labeled proteins were separated by 2D gel electrophoresis and detected by fluorography. Protein patterns for wild-type and DT7DS treated at 22 °C were qualitatively identical, and only minor comparative quantitative differences could be determined (figure 8). The protein patterns from each line were also essentially identical following a 4-h/40 °C preincubation (heat shock) treatment, with two notable exceptions. The synthesis of two low-molecular mass heat shock proteins was consistently reduced or absent in the heat shocked DT7DS line, as evidenced by the absence of these proteins in the DT7DS-40 °C fluorograph presented in the lower right panel of figure 8. These proteins have molecular masses of 31 and 32 kDa, arrowed and labeled HSP31 and HSP32 respectively in the magnified insert for the CS-40 °C fluorograph of figure 8, and have dissimilar pI values of approximately 5.0 and 5.5, respectively. These proteins are considered heat shock proteins as neither are present in the protein pattern from the control treated at 22 °C. The analysis was repeated in four independent experiments, and while other quantitative and qualitative vol. 38 (3) 2000
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Figure 8. Autoradiographs of 2D gel electrophoresis separation of radiolabeled proteins from Chinese Spring and DT7DS leaf segments at room temperature (22 °C) and heat shocked at 40 °C for 4 h. Error bars represent standard error.
spot differences were noted between the patterns from the replicate experiments, the only consistent and reproducible change was that seen for the synthesis of the 31- and 32-kDa proteins.
3. DISCUSSION This study utilized the chlorophyll accumulation assay [6] to assess the level of acquired thermotolerance in wild-type and a ditelosomic series of Chinese Spring wheat. Acquired thermotolerance is defined as the ability to retain viability when subjected to lethal Plant Physiol. Biochem.
elevated temperatures following a sublethal elevated temperature treatment (acquisition treatment). Burke [6] pointed out that in order to use this sensitive viability assay (since lack of greening results in tissue death), it is first necessary to establish the optimal temperature parameters for all aspects of the assay (optimal chlorophyll accumulation, optimal acquisition preincubation temperature and challenge temperature) for each plant species under investigation. Since this had not been accomplished for Chinese Spring wheat, the first part of this study was aimed at establishing the assay parameters. The results demonstrate that for Chinese Spring wheat, the optimal temperature for chlorophyll accumulation in the light
Acquired thermotolerance within Chinese Spring wheat
is 30 °C (figure 1), the preincubation temperature for optimal acquisition of thermotolerance is 40 °C (figure 3), and the most useful challenge temperature that results in maximum inhibition of chlorophyll accumulation is 48 °C (figure 2). The assay conditions determined from these data were then utilized to assess the level of thermotolerance in a series of ditelosomic lines that are a set of aneuploids that each carry a distinct and specific deletion of an individual chromosome arm. In this way, we could assign a chromosomal location to genes that control the ability of a plant, in this case Chinese Spring wheat, to acquire thermotolerance. This information is crucial to our attempts to map and isolate thermotolerance genes. The data we have presented in this report clearly demonstrate that genes important for the acquisition of thermotolerance reside in the chromosome arm that is deficient within the ditelosomic line DT7DS, the short arm of chromosome 7 of the D genome (DT7DS). This is not meant to suggest that all genes involved in acquired thermotolerance reside in this chromosomal arm, only that one or more genes that can be revealed using this strategy do. The observations that the level of acquired thermotolerance is elevated in the DT1BS line and somewhat reduced in the DT3BL and DT6DL lines suggest the presence of several more thermotolerance related genes. That the gene(s) we have demonstrated reside on DT7DS are involved in acquired thermotolerance and not some other temperature related trait (e.g. the temperature sensitivity of chlorophyll accumulation) can be shown from the confirmatory experiments of our analysis (figures 5, 6, 7). A role for heat shock proteins (HSPs) in the phenomenon of acquired thermotolerance has been established [18, 28]. Most of the information to date on acquired thermotolerance in eukaryotes has derived from studies in yeast. A similar role for HSPs in plants has been shown indirectly [14, 15, 29] and inferred from studies using plant HSPs to complement a yeast HSP-mutant deficient in acquired thermotolerance [16, 26]. In higher plants, however, direct correlation between acquired thermotolerance and HSP induction has not been described. Heat shock proteins are generally categorized as high molecular mass HSPs (65–110 kDa), or low molecular mass sHSPs (15–30 kDa). Plants produce both categories of HSP in response to sublethal temperatures [19] but the majority, up to thirty, are sHSPs located in the cytosol, organelles and endoplasmic reticulum [11, 12, 28]. Though the precise function of sHSPs is enigmatic, evidence is accumulating for a
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role as molecular chaperones [3]. In this study, the majority of HSPs synthesized in response to heat shock were in the low molecular mass range and coincided with a down regulation of many other proteins. In this investigation, we demonstrate that the reduction of acquired thermotolerance in the ditelosomic wheat cultivar DT7DS can be correlated with the reduction of two low molecular mass HSPs (31 and 32 kDa). These data point to a possible direct link between acquired thermotolerance and the production of HSPs. That such a strong reduction of acquired thermotolerance can be linked to the lack of only two low molecular mass HSPs is somewhat surprising as the general consensus is that many HSPs play a role in this complex trait. However, as it is likely that the interrelationships among the various roles that the HSPs could play in acquired thermotolerance are closely linked, the lack of only one or two proteins could cause a severe reduction in the level of protection afforded by the 4-h pretreatment at 40 °C. Although our evidence is strong, we have not shown a direct cause and effect relationship between HSP synthesis and acquired thermotolerance but have established a basis from which more direct genetic approaches can be launched.
4. METHODS 4.1. Plant material Hexaploid wheat (Triticum aestivum L, 2n = 6 x = 42) cultivar ‘Chinese Spring’ and a number of the ditelosomic (DT) series derived from Chinese Spring [27] (kindly provided by Dr J.P. Gustafson, USDA-ARS, Columbia, MO) were analyzed in this study. Seeds were germinated and seedlings grown between two layers of water saturated germination paper support surrounded by a layer of wax paper in a glass beaker in the dark at 27 °C. Because the metabolic rate varies from the axis to the tip of monocot leaves, specific leaf sections were evaluated in this study. In each treatment three 2-cm leaf segments 1 cm from the leaf tip of three separate leaves were excised and placed on 1 % agarose in a 35 × 10 mm diameter tissue culture dish (Corning). A specific portion of the leaves were used in the analyses in order to compensate for the fact that the metabolic rate varies from the axis to the tip of monocot leaves. The 2-cm section 1 cm from the leaf tip was determined to be the area of the leaf that exhibited maximum chlorophyll accumulation during greening (data not presented). All viable vol. 38 (3) 2000
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ditelosomic lines were screened initially for a deficiency in acquired thermotolerance from these lines. A group of nine lines were selected for further study. The lines are designated by their homoeologous group (1–7), genome A, B or D and the chromosome arm length (L = long, S = short).
4.2. Temperature and light treatments Unless otherwise stated, temperature treatments were achieved using an electronically controlled eight position thermal plate system [8]. Thermal plates were covered with 3MM water-saturated filter paper on which the culture dishes containing the leaf segments were placed. The thermal plates were covered with Glad Wrap (which is gas permeable) to prevent the 3MM paper from drying out and reducing temperature transfer from the plates to the culture dish. Leaf segments were treated at the specified temperature prior to being placed under continuous light at 115 µmol⋅m–2⋅s–1 (two Philips F40/AGRO AGROLITE fluorescent bulbs and two 75 W incandescent bulbs) for 20 h. 4.3. Determination of the temperature for maximum chlorophyll accumulation The temperature that supported development of maximum chlorophyll levels was identified before evaluating the high temperature stress characteristics of the wheat leaves. Tissue culture dishes containing etiolated leaf segments were exposed to continuous illumination at 10, 15, 20, 25, 30, 35, 40 or 45 °C for 20 h to identify the optimum temperature for chlorophyll accumulation. Chlorophyll accumulation was determined following the light treatment by the procedure of Arnon [1]. 4.4. Challenge temperature determination The challenge temperature, a 30-min temperature exposure that prevents subsequent chlorophyll accumulation, was determined by placing segments of etiolated wheat leaves in culture dishes containing 1 % agarose and incubating them in the dark for 30 min at 30 (control), 44, 46, 48, 50, 52, 54 or 56 °C. Following the 30-min temperature challenge, the leaf segments were given a 20-h light treatment at the optimal temperature for chlorophyll accumulation, and the chlorophyll content of leaf segments was determined as described above. The temperature exposure that inhibited chlorophyll accumulating by more than 90 % of the control chlorophyll levels was chosen as the challenge temperature for subsequent germplasm evaluation. Plant Physiol. Biochem.
4.5. Preincubation temperature determination Burke [6, 7] demonstrated that a 4-h preincubation at an elevated, sublethal temperature could reduce the level of injury experienced by plants subsequently exposed to a formerly lethal challenge temperature. To determine the appropriate preincubation temperature for ‘Chinese Spring’ wheat, etiolated leaf segments were preincubated for 4 h at 30 (control), 34, 36, 38, 40, 42, 44 or 46 °C. Following the preincubation period, the leaf segments, with the exception of the 30 °C control, were exposed to the predetermined 30-min challenge temperature. The control and challenged leaf segments were placed at 30 °C for 20 h under continuous light and subsequent chlorophyll accumulation evaluated as described above.
4.6. Screening for acquired thermotolerance deficiencies Seedlings of all viable ditelosomic lines (34 of 42 available lines, the remainder carry embryonic lethal chromosome arrangements) were screened initially for acquired thermotolerance deficiency by the following procedure. Seeds were germinated and grown in the dark at 27 °C for 5 d or until leaves were approximately 5 to 10 cm in length. Leaf segments were incubated at the identified preincubation temperature for 4 h then challenged at the challenge temperature for 30 min. The leaf segments were then transferred to continuous light at 30 °C after which chlorophyll content was determined as described above. This was a preliminary study and in some cases only two samples were studied due to insufficient seed quantity or germination problems. Subsequently nine lines were chosen for further analysis based on apparent altered acquired thermotolerance along with seed availability and acceptable germination rates.
4.7. In vivo labeling and protein isolation Proteins were labeled in vivo by allowing excised leaf segments (3 cm) to stand for 4 h in water containing 1.85⋅107 Bq⋅mL–1 [35S]-trans label (ICN) at either room temperature as control (approximately 22 °C) or at the preincubation temperature identified above. This labeling procedure enabled the incorporation of label into proteins at a rate independent from uptake rates (data not presented). Following treatments, leaf segments were washed in distilled water to remove excess radioactivity, the apical 1 cm removed and the remaining 2 cm pulverized in Tris/Glycine extraction buffer (Tris base, 0.1 M, pH 8.4; Glycine, 0.1 M). Cell debris was removed by centrifugation at 14 000 × g for
Acquired thermotolerance within Chinese Spring wheat
10 min. Proteins were extracted from the supernatant with an equal volume of water-saturated phenol. The phenol phase was re-extracted with 0.5 volumes of extraction buffer, and proteins were precipitated overnight at –20 °C by addition of 2.5 volumes of 0.1 M ammonium acetate in methanol. After recovery by centrifugation, the protein pellet was washed once in 0.1 M ammonium acetate in methanol, air dried and resuspended in IEF buffer (urea, 9 M; DTT, 0.65 M; 3-10 Pharmalyte, 0.02 mL⋅mL–1; Triton X-100, 0.005 mL⋅mL–1; bromophenol blue, 0.001 %). Following resuspension in IEF buffer, insoluble material was removed by centrifugation at 14 000 × g for 2 min, the supernatant moved to a new tube and stored at –20 °C. The quantity of labeled protein in each sample was determined by liquid scintillation analysis using a Packard Tri-Carb 1500 liquid scintillation counter.
4.8. Two-dimensional gel electrophoresis Two-dimensional separation of radiolabeled proteins was achieved using the Immobiline DryStrip Kit and ExcelGel SDS on the Multiphor II electrophoresis system (Pharmacia). Procedures followed the manufacturers instructions with some modifications. Acetic acid was used instead of Pharmalyte 3-10 in the rehydration solution for IEF dry strips. Approximately 200 000 cpm of each sample were loaded on each gel. The SDS-PAGE gel after the final protein separation step was treated with fixer (10 % acetic acid and 30 % methanol) for 30 min and fluor (55 % acetic acid, 15 % ethanol, 30 % xylene and 0.8 % 2,5-diphenyl oxazole) for 1 h. The gel was then washed for 2× 2-min washes in distilled water, covered with wet cellulose acetate and dried on to the cellulose acetate membrane for 2 h at 45 °C. Labeled proteins were detected by fluorography by exposure to X-Ray film (BIOMAX-MR, Kodak) in the presence of a single enhancer screen at –80 °C.
Acknowledgments The authors wish to thank Jacob Sanchez for his excellent technical assistance throughout this study. This study supported in part by grant No. 96-351003168 from the NRI Competitive Grants Program/ USDA.
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[15] Lee J.H., Huber A., Schoffl F., Depression of the activity of genetically engineered heat-shock factor causes constitutive synthesis of heat shock proteins and increased thermotolerance in transgenic Arabidopsis, Plant J. 8 (1995) 603–612. [16] Lee Y.R.J., Nagao R.T., Key J.L., A soybean 101-kD heat shock protein complements a yeast HSP104 deletion mutation in acquiring thermotolerance, Plant Cell 6 (1994) 1889–1897. [17] Li G.C., Werb Z., Correlation between synthesis of heat shock proteins and development of thermotolerance in Chinese hamster fibroblasts, Proc. Natl. Acad. Sci. USA 79 (1982) 3218–3222. [18] Lindquist S.L., Craig E.A., The heat shock proteins, Annu. Rev. Genet. 22 (1988) 631–677. [19] Mansfield M.A., Key J.L., Synthesis of low molecular weight heat shock proteins in plants, Plant Physiol. 84 (1987) 1007–1017. [20] Otto B., Ohad I., Kloppstech K., Temperature treatments of dark-grown pea seedlings cause an accelerated greening in the light at different levels of gene expression, Plant Mol. Biol. 8 (1992) 887–896. [21] Parsell D.A., Taulien J., Lindquist S.L., The role of heat shock proteins in thermotolerance, Phil. Trans. R. Soc. 339 (1993) 279–286. [22] Porter D.R., Nguyen H.T., Burke J.J., Chromosomal location of genes controlling heat shock proteins in hexaploid wheat, Theor. Appl. Genet. 78 (1989) 873–878.
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[23] Prandl R., Hinderhofer K., Eggers-Schumaker G., Schoffl F., HSF3, a new heat shock factor from Arabidopsis thaliana, derepresses the heat shock response and confers thermotolerance when overexpressed in transgenic plants, Mol. Gen. Genet. 258 (1998) 269–278. [24] Sanchez Y., Lindquist S.L., HSP104 required for induced thermotolerance, Science 248 (1990) 1112–1114. [25] Sanchez Y., Taulien J., Borkovich K.A., Lindquist S.L., Hsp104 is required for tolerance to many forms of stress, EMBO J. 11 (1992) 2357–2364. [26] Schirmer E.C., Lindquist S.L., Vierling E., An Arabidopsis heat shock protein complements a thermotolerance defect in yeast, Plant Cell 6 (1994) 1899–1909. [27] Sears E., Sears L., The telocentric chromosomes of comon wheat, Proc. 5th Wheat Genetics Symposium, New Delhi, 1978, pp. 389–407. [28] Vierling E., The roles of heat shock proteins in plants, Annu. Rev. Plant. Physiol. Plant Mol. Biol. 42 (1991) 579–620. [29] Visioli G., Maestri E., Marmiroli N., Differential display-mediated isolation of a genomic sequence for a putative mitochondrial LMW HSP specifically expressed in condition of induced thermotolerance in Arabidopsis thaliana (L.) Heynh, Plant Mol. Biol. 34 (1997) 517–527. [30] Vogel J.L., Parsell D.A., Lindquist S.L., Heat-shock proteins Hsp104 and Hsp70 reactivate mRNA splicing after heat inactivation, Current Biol. 5 (1995) 306–317.
Identification of acquired thermotolerance deficiency within the ditelosomic series of ‘Chinese Spring’ wheat§ Patrick O’Mahony, John J. Burke*, Melvin J. Oliver Plant Stress and Germplasm Development Unit, USDA-ARS, 3810 4th Street, Lubbock TX 79415, USA
* Author to whom correspondence should be addressed (fax +1 806 723 5272; email [email protected]) (Received March 22, 1999; accepted October 28, 1999) Abstract — The relative contribution of individual heat shock proteins to acquired thermotolerance was evaluated through analysis of chromosomal deletions in a ditelosomic series of the hexaploid wheat (Triticum aestivum L.) cultivar ‘Chinese Spring’. This study describes the identification of a line within this ditelosomic series that exhibited a reduced level of acquired thermotolerance. Changes in the temperature sensitivity of chlorophyll accumulation were used as an indicator of acquired thermotolerance. The temperature providing maximum chlorophyll accumulation was 30 °C in leaves under continuous light. A 30-min challenge temperature of 48 °C prior to the light exposure was shown to inhibit subsequent chlorophyll accumulation. Preincubation at 40 °C for 4 h before the 30-min 48 °C challenge triggered the acquired thermotolerance system of the plant resulting in chlorophyll accumulation upon exposure to light. Evaluation of the ditelosomic series revealed reductions in acquired thermotolerance levels in the DT7DS line relative to controls. Two-dimensional SDS polyacrylamide gel analysis was used to identify reduction in the level of two low molecular mass heat shock proteins in DT7DS. USDA-ARS © 2000 Published by Éditions scientifiques et médicales Elsevier SAS Acquired thermotolerance deficiency / ditelosomic / heat shock / Triticum aestivum CELTEC, cellular thermoelectric controller / DAP, days after planting / HSP, heat shock protein
1. INTRODUCTION Stress as a result of elevated temperatures is a common threat to plant survival and crop yields worldwide. Elevated temperatures have physiological [2, 4, 5, 10] and morphological [20] effects on plants, many of which result in the retardation of growth and development. However, when plants are exposed to elevated non-lethal temperatures, they acquire thermotolerance which can transiently raise their injury threshold and protect them from subsequent, otherwise lethal, temperatures. Plants produce heat shock proteins (HSPs) in response to various environmental stresses [9, 25, 28]. At sublethal elevated temperatures, quantitative induc§
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tion of HSPs occurs with a concomitant reduction in the synthesis of most other proteins. This reorganization of metabolic priorities coincides with the acquisition of thermotolerance [17, 18, 28]. Significant evidence is available from yeast studies which link HSP induction to the acquisition of thermotolerance [21, 24, 25, 28, 30]. To date, however, only indirect evidence exists to suggest that HSP induction is associated with acquired thermotolerance in plants. In Arabidopsis, a correlation has been observed between modulated heat shock protein synthesis and heat shock factor activity and expression with levels of thermotolerance [14, 15, 23, 29]. Studies in thermosusceptible and thermotolerant recombinant inbred lines of wheat detected a genetic relationship between expression of a plastid localized HSP26 and acquired thermotolerance [13]. In addition, other studies have demonstrated that an acquired thermotolerance deficient yeast that carries a mutated HSP104 gene can be successfully complemented by a plant HSP 101 genes from soybean [16] and Arabidopsis [26].
Plant Physiol. Biochem., 0981-9428/00/3/ USDA-ARS © 2000 Published by E´ditions scientifiques et médicales Elsevier SAS. All rights reserved
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Obtaining direct evidence to link HSP with acquired thermotolerance in higher plants has been hampered by a lack of mutants with which a cause and effect relationship could be established. This shortfall may be circumvented to a degree by analysis of heat shock responses in aneuploid genetic stocks where specific chromosomal deletions result in a lack of, or reduction in, acquired thermotolerance coincident with an alteration of HSP synthesis. In this study, we used a sensitive chlorophyll accumulation assay [7] to identify acquired thermotolerance deficiencies within the ditelosomic (a plant with one chromosome missing an arm-telocentric) series of the hexaploid wheat cultivar ‘Chinese Spring’ [27]. A previous investigation using 2D gel electrophoresis [22] to analyze the genetic control of HSP synthesis in wheat identified the chromosomal localization of a number of low molecular mass HSPs. Variations in relative HSP levels suggested that the homoeologous DT lines 3, 4 and 7 contain the majority of the controlling genes indicating chromosomes 3, 4 and 7 as sites containing HSP controlling loci. However, this study did not address the possible functional relationship between specific HSP changes and levels of acquired thermotolerance. In this study of ditelosomics, we report the use of a similar strategy to that of Porter et al. [22] to detect two HSPs which may be involved in acquired thermotolerance, and to localize them to a specific chromosome arm.
2. RESULTS In order to accurately identify acquired thermotolerance deficiencies using the chlorophyll inhibition assay [6], it is necessary to determine several pertinent temperature parameters in the parental line. These parameters include the optimum temperatures for (a) chlorophyll accumulation, (b) minimum challenge temperature resulting in the maximum decrease in chlorophyll accumulation and (c) preincubation temperature yielding maximum chlorophyll accumulation following a subsequent challenge temperature.
2.1. Optimum temperature for chlorophyll accumulation A range of temperatures from 10 to 45 °C were used to find the temperature that allowed for the maximum accumulation of chlorophyll in excised leaf tissue. The temperature sensitivity of chlorophyll accumulation in Plant Physiol. Biochem.
Figure 1. Determination of optimum temperature for chlorophyll accumulation in leaf segments of Chinese Spring wheat exposed to continuous light for 20 h. Error bars represent standard error.
Chinese Spring wheat is shown in figure 1. Chlorophyll levels increased with temperature from 10 to 30 °C and then declined to minimal levels at 45 °C. A temperature of 30 °C was chosen for use in subsequent experiments to ensure maximum chlorophyll accumulation.
2.2. Determination of optimum challenge temperature The temperature-induced inhibition of chlorophyll accumulation subsequent to a high temperature challenge was determined by exposing etiolated leaf segments to temperatures between 44 and 56 °C for 30 min prior to the 30 °C/20 h light treatment (figure 2). A 35 and 72 % reduction in chlorophyll accumulation was observed subsequent to a 44 and 46 °C challenge, respectively. A 30-min challenge at 48 °C, or above, prior to the 30 °C light treatment resulted in more than a 95 % inhibition of chlorophyll accumulation. A 48 °C challenge temperature was used in subsequent experiments to inhibit chlorophyll accumulation.
2.3. Preincubation temperature yielding maximum protection The acquisition of thermotolerance was evaluated by preincubating wheat leaf segments at temperatures from 34 to 46 °C for 4 h prior to a 30-min/48 °C challenge (figure 3). The inability to accumulate chlorophyll upon exposure to continuous light because of a prior 30-min/48 °C challenge was quantified as the amount of chlorophyll accumulated as a percentage of
Acquired thermotolerance within Chinese Spring wheat
Figure 2. Determination of suitable challenge temperature for maximum inhibition of chlorophyll production in leaf segments of Chinese Spring wheat. Error bars represent standard error.
the untreated control (incubated at 30 °C throughout). An increase in the preincubation temperature from 34 to 40 °C resulted in more protection from the effects of the challenge temperature as measured by a greater degree of chlorophyll accumulation. A 75 % reduction in chlorophyll accumulation was observed with a 36 °C preincubation, a 53 % reduction occurred at 38 °C, and the least reduction in chlorophyll accumulation (48 %) was seen with a 40 °C preincubation. This decrease in the reduction of chlorophyll accumulation represents thermotolerance acquired during preincubation at 40 °C. Preincubation at temperatures above 40 °C provided less protection to where little or no chlorophyll accumulation was observed with 44 or 46 °C preincubations (figure 3). The findings to this point revealed that maximum chlorophyll accumulation occurs at 30 °C upon continuous light exposure; that 30-min challenges at 48 °C or above prevent subsequent chlorophyll accumulation; and that preincubation at 40 °C for 4 h prior to a 48 °C challenge delivered the maximum level of protection.
2.4. Identification of acquired thermotolerance mutants Having defined the parameters of our screening system, a selection of ditelosomics (DTs) of Chinese Spring wheat were examined for a reduction in the level of acquired thermotolerance. Each viable DT line was preincubated at 40 °C for 4 h, challenged at 48 °C for 30 min and chlorophyll accumulation ability assessed following a 30 °C/20 h light treatment. Table I presents the results of our initial screen of these lines.
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Figure 3. Evaluation of a range of preincubation temperatures inducing maximum thermal protection in leaf segments of Chinese Spring wheat. Error bars represent standard error.
This was a preliminary screen only as some seeds were available in limited quantity and others had germination problems. However, based on observed altered levels of acquired thermotolerance, nine of the 34 lines were selected and subjected to a more rigorous statistical analysis following the production of seed from the original stocks. These lines were DT1BS, DT3DL, DT3BL, DT4DS, DT4DL, DT5AL, DT6DL, DT6BS and DT7DS. Lack of sufficient seed quantity and poor germination rates for DT5BL prohibited similar analysis. In addition to the DT7DS line, DT7AS and DT7BS also showed a reduction in acquired thermotolerance levels but in both cases the results were difficult to reproduce in latter seed batches derived from the original parent lines (data not shown). The nine lines identified were subjected to a more extensive version of the primary screen (for statistical purposes between four and eight samples were examined). Figure 4 presents a bar graph representation of the results that we obtained from this screen of the nine selected lines. The Chinese Spring control (labeled WT for wild-type) accumulated approximately 50 % of the chlorophyll observed in nonchallenged Chinese Spring seedlings. The vertical line in this figure serves as a reference point for comparison of the DTs with the Chinese Spring parent. Seven of the nine lines evaluated accumulated chlorophyll to levels close to that of the WT control. Four of the seven lines DT3DL, DT4DS, DT4DL and DT6BS were statistically the same as the WT seedlings, with the three remaining lines DT3BL, DT5AL and DT6DL having chlorophyll levels slightly above or below the WT seedlings. Two lines exhibited chlorophyll levels well removed from those of the WT sample. The vol. 38 (3) 2000
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Table I. Relative acquired thermotolerance levels within the ditelosomic series of Chinese Spirng wheat relative to Chinese Spring controls. Acquired thermotolerance levels similar to control levels are indicated with a (+) sign; those lines with increased thermotolerance over controls are indicated with either a (++, or +++) sign; those lines having acquired thermotolerance levels below controls are indicated with a (–) sign; and those that were not determined are indicated with ND. Ditelosomic line
Thermotolerance level
Ditelosomic line
Thermotolerance level
1AL 1AS 1BS 1DL 1DS 2AS 2BL 2DL 2DS 3AL 3BL 3DL 3AS 3BS 3DS 4BS 4DS
+ + ++ ND ND ND ND + + + ++ + ND ND ND + +
4AL 4DL 5AL 5BL 5DL 6AS 6BS 6DS 6AL 6BL 6DL 7AS 7BS 7DS 7AL 7DL 7DL
+ + + ++ + + +++ + ND ND + – – – + + +
DT1BS line consistently had greater chlorophyll levels than the WT showing only 10–20 % injury following preincubation and challenge and a more detailed characterization of this line is currently underway. The DT7DS line, however, could only accumulate approximately 7 % of the chlorophyll that the wild-type line accumulates. Thus it appears that the DT7DS line is not capable of establishing a significant level of acquired thermotolerance following a 40 °C for 4 h preincubation that would protect the metabolic activities associated with chlorophyll accumulation in the light from a high temperature challenge. No other physiological abnormality was observed in whole plant studies where the DT7DS line germinated and grew at similar rates to the wild-type (data not shown).
Figure 4. Identification of the levels of acquired thermotolerance in several ditelosomics of Chinese Spring wheat. Individual bars represent the level of chlorophyll accumulation as percentage of control values following a preincubation to induce acquired thermotolerance. Error bars represent standard error.
to 25 °C, remaining high until 30 °C and then declining sharply, reaching minimal levels at 45 °C. This temperature sensitivity profile is almost identical to that for the wild-type Chinese Spring wheat (figures 1, 5). Thus the reduction in chlorophyll accumulation for DT7DS seen in figure 4 is not the result of a change in temperature sensitivity of chlorophyll accumulation in this line. These data also demonstrate that 30 °C can be used for optimum chlorophyll accumulation in the DT7DS line which enables the direct comparisons to wild-type following heat treatments. The greening rates of wild-type and DT7DS wheat were compared by incubating leaf segments for 20 h at
2.5. Characterization of the acquired thermotolerance mutant DT7DS To ensure that this result was due to an altered acquired thermotolerance pathway in the DT7DS line and not a general altering of the temperature sensitivity of the plant, a temperature optimum curve for chlorophyll accumulation in this line was generated. The results of this analysis are presented in figure 5. Chlorophyll levels increased with temperature from 10 Plant Physiol. Biochem.
Figure 5. Determination of optimum temperature for chlorophyll accumulation in leaf segments of the ditelosomic DT7DS. Error bars represent standard error.
Acquired thermotolerance within Chinese Spring wheat
Figure 6. Comparative greening rates (A) of Chinese Spring and the ditelosomic DT7DS wheat leaf segments exposed to light at 30 °C, and comparison of challenge temperatures inhibiting chlorophyll accumulation (B) in control Chinese Spring wheat (●) and DT7DS (·). Error bars represent standard error.
30 °C in light. Chlorophyll accumulated in the DT7DS at rates similar to that of wild-type (figure 6), indicating that any differences observed in the preincubated and challenged DT7DS tissues did not arise from an inherent difference in chlorophyll accumulation rate compared to control Chinese Spring seedlings. The challenge response curve for chlorophyll accumulation in DT7DS seedlings was also compared with the challenge response curve of control Chinese Spring seedlings (figure 6). Similar temperature sensitivities were observed between the DT7DS and control Chinese Spring seedlings when they were directly challenged without preincubation to induce acquired thermotolerance. These results show that the DT7DS line does not have unique temperature characteristics that distinguish it from control seedlings as might be the case in temperature sensitive plants. Finally, the temperature response curve for acquisition of thermotolerance was evaluated by incubating DT7DS leaf segments at 34, 36, 38, 40, 42, 44 and 46 °C for 4 h prior to the 30-min/48 °C challenge (figure 7). Following a 20-h treatment in light at 30 °C, chlorophyll accumulation was evaluated. As for wildtype, preincubation at 40 °C resulted in the greatest retention of the ability of the tissue to accumulate chlorophyll after a high temperature challenge. Nevertheless, as seen in figure 4, the absolute level of acquired thermotolerance observed in the DT7DS seedlings resulted in significantly less accumulation of chlorophyll than the Chinese Spring seedlings. It is worth noting that variations of even 1° in temperature at or near the sensitivity threshold for these plants can alter the final levels of accumulation of chlorophyll. Therefore, the data for DT7DS is presented relative to control data and not as absolute values.
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Figure 7. Comparison of the range of preincubation temperatures providing protection in DT7DS (●) and Chinese Spring wheat (·) from injury by a subsequent thermal challenge. Error bars represent standard error.
2.6. Two-dimensional gel electrophoresis analysis of Chinese Spring wild-type and DT7DS Once it had been established that the DT7DS line was deficient in the ability to acquire thermotolerance compared to the wild-type Chinese Spring wheat, we were interested in determining if this phenotype could be linked to a concomitant change in the synthesis of heat shock proteins. To accomplish this, leaf segments from control Chinese Spring seedlings and DT7DS seedlings were treated in the dark at 22 and 40 °C for 4 h in the presence of [35S]-labeled amino acid mix. Labeled proteins were separated by 2D gel electrophoresis and detected by fluorography. Protein patterns for wild-type and DT7DS treated at 22 °C were qualitatively identical, and only minor comparative quantitative differences could be determined (figure 8). The protein patterns from each line were also essentially identical following a 4-h/40 °C preincubation (heat shock) treatment, with two notable exceptions. The synthesis of two low-molecular mass heat shock proteins was consistently reduced or absent in the heat shocked DT7DS line, as evidenced by the absence of these proteins in the DT7DS-40 °C fluorograph presented in the lower right panel of figure 8. These proteins have molecular masses of 31 and 32 kDa, arrowed and labeled HSP31 and HSP32 respectively in the magnified insert for the CS-40 °C fluorograph of figure 8, and have dissimilar pI values of approximately 5.0 and 5.5, respectively. These proteins are considered heat shock proteins as neither are present in the protein pattern from the control treated at 22 °C. The analysis was repeated in four independent experiments, and while other quantitative and qualitative vol. 38 (3) 2000
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Figure 8. Autoradiographs of 2D gel electrophoresis separation of radiolabeled proteins from Chinese Spring and DT7DS leaf segments at room temperature (22 °C) and heat shocked at 40 °C for 4 h. Error bars represent standard error.
spot differences were noted between the patterns from the replicate experiments, the only consistent and reproducible change was that seen for the synthesis of the 31- and 32-kDa proteins.
3. DISCUSSION This study utilized the chlorophyll accumulation assay [6] to assess the level of acquired thermotolerance in wild-type and a ditelosomic series of Chinese Spring wheat. Acquired thermotolerance is defined as the ability to retain viability when subjected to lethal Plant Physiol. Biochem.
elevated temperatures following a sublethal elevated temperature treatment (acquisition treatment). Burke [6] pointed out that in order to use this sensitive viability assay (since lack of greening results in tissue death), it is first necessary to establish the optimal temperature parameters for all aspects of the assay (optimal chlorophyll accumulation, optimal acquisition preincubation temperature and challenge temperature) for each plant species under investigation. Since this had not been accomplished for Chinese Spring wheat, the first part of this study was aimed at establishing the assay parameters. The results demonstrate that for Chinese Spring wheat, the optimal temperature for chlorophyll accumulation in the light
Acquired thermotolerance within Chinese Spring wheat
is 30 °C (figure 1), the preincubation temperature for optimal acquisition of thermotolerance is 40 °C (figure 3), and the most useful challenge temperature that results in maximum inhibition of chlorophyll accumulation is 48 °C (figure 2). The assay conditions determined from these data were then utilized to assess the level of thermotolerance in a series of ditelosomic lines that are a set of aneuploids that each carry a distinct and specific deletion of an individual chromosome arm. In this way, we could assign a chromosomal location to genes that control the ability of a plant, in this case Chinese Spring wheat, to acquire thermotolerance. This information is crucial to our attempts to map and isolate thermotolerance genes. The data we have presented in this report clearly demonstrate that genes important for the acquisition of thermotolerance reside in the chromosome arm that is deficient within the ditelosomic line DT7DS, the short arm of chromosome 7 of the D genome (DT7DS). This is not meant to suggest that all genes involved in acquired thermotolerance reside in this chromosomal arm, only that one or more genes that can be revealed using this strategy do. The observations that the level of acquired thermotolerance is elevated in the DT1BS line and somewhat reduced in the DT3BL and DT6DL lines suggest the presence of several more thermotolerance related genes. That the gene(s) we have demonstrated reside on DT7DS are involved in acquired thermotolerance and not some other temperature related trait (e.g. the temperature sensitivity of chlorophyll accumulation) can be shown from the confirmatory experiments of our analysis (figures 5, 6, 7). A role for heat shock proteins (HSPs) in the phenomenon of acquired thermotolerance has been established [18, 28]. Most of the information to date on acquired thermotolerance in eukaryotes has derived from studies in yeast. A similar role for HSPs in plants has been shown indirectly [14, 15, 29] and inferred from studies using plant HSPs to complement a yeast HSP-mutant deficient in acquired thermotolerance [16, 26]. In higher plants, however, direct correlation between acquired thermotolerance and HSP induction has not been described. Heat shock proteins are generally categorized as high molecular mass HSPs (65–110 kDa), or low molecular mass sHSPs (15–30 kDa). Plants produce both categories of HSP in response to sublethal temperatures [19] but the majority, up to thirty, are sHSPs located in the cytosol, organelles and endoplasmic reticulum [11, 12, 28]. Though the precise function of sHSPs is enigmatic, evidence is accumulating for a
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role as molecular chaperones [3]. In this study, the majority of HSPs synthesized in response to heat shock were in the low molecular mass range and coincided with a down regulation of many other proteins. In this investigation, we demonstrate that the reduction of acquired thermotolerance in the ditelosomic wheat cultivar DT7DS can be correlated with the reduction of two low molecular mass HSPs (31 and 32 kDa). These data point to a possible direct link between acquired thermotolerance and the production of HSPs. That such a strong reduction of acquired thermotolerance can be linked to the lack of only two low molecular mass HSPs is somewhat surprising as the general consensus is that many HSPs play a role in this complex trait. However, as it is likely that the interrelationships among the various roles that the HSPs could play in acquired thermotolerance are closely linked, the lack of only one or two proteins could cause a severe reduction in the level of protection afforded by the 4-h pretreatment at 40 °C. Although our evidence is strong, we have not shown a direct cause and effect relationship between HSP synthesis and acquired thermotolerance but have established a basis from which more direct genetic approaches can be launched.
4. METHODS 4.1. Plant material Hexaploid wheat (Triticum aestivum L, 2n = 6 x = 42) cultivar ‘Chinese Spring’ and a number of the ditelosomic (DT) series derived from Chinese Spring [27] (kindly provided by Dr J.P. Gustafson, USDA-ARS, Columbia, MO) were analyzed in this study. Seeds were germinated and seedlings grown between two layers of water saturated germination paper support surrounded by a layer of wax paper in a glass beaker in the dark at 27 °C. Because the metabolic rate varies from the axis to the tip of monocot leaves, specific leaf sections were evaluated in this study. In each treatment three 2-cm leaf segments 1 cm from the leaf tip of three separate leaves were excised and placed on 1 % agarose in a 35 × 10 mm diameter tissue culture dish (Corning). A specific portion of the leaves were used in the analyses in order to compensate for the fact that the metabolic rate varies from the axis to the tip of monocot leaves. The 2-cm section 1 cm from the leaf tip was determined to be the area of the leaf that exhibited maximum chlorophyll accumulation during greening (data not presented). All viable vol. 38 (3) 2000
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ditelosomic lines were screened initially for a deficiency in acquired thermotolerance from these lines. A group of nine lines were selected for further study. The lines are designated by their homoeologous group (1–7), genome A, B or D and the chromosome arm length (L = long, S = short).
4.2. Temperature and light treatments Unless otherwise stated, temperature treatments were achieved using an electronically controlled eight position thermal plate system [8]. Thermal plates were covered with 3MM water-saturated filter paper on which the culture dishes containing the leaf segments were placed. The thermal plates were covered with Glad Wrap (which is gas permeable) to prevent the 3MM paper from drying out and reducing temperature transfer from the plates to the culture dish. Leaf segments were treated at the specified temperature prior to being placed under continuous light at 115 µmol⋅m–2⋅s–1 (two Philips F40/AGRO AGROLITE fluorescent bulbs and two 75 W incandescent bulbs) for 20 h. 4.3. Determination of the temperature for maximum chlorophyll accumulation The temperature that supported development of maximum chlorophyll levels was identified before evaluating the high temperature stress characteristics of the wheat leaves. Tissue culture dishes containing etiolated leaf segments were exposed to continuous illumination at 10, 15, 20, 25, 30, 35, 40 or 45 °C for 20 h to identify the optimum temperature for chlorophyll accumulation. Chlorophyll accumulation was determined following the light treatment by the procedure of Arnon [1]. 4.4. Challenge temperature determination The challenge temperature, a 30-min temperature exposure that prevents subsequent chlorophyll accumulation, was determined by placing segments of etiolated wheat leaves in culture dishes containing 1 % agarose and incubating them in the dark for 30 min at 30 (control), 44, 46, 48, 50, 52, 54 or 56 °C. Following the 30-min temperature challenge, the leaf segments were given a 20-h light treatment at the optimal temperature for chlorophyll accumulation, and the chlorophyll content of leaf segments was determined as described above. The temperature exposure that inhibited chlorophyll accumulating by more than 90 % of the control chlorophyll levels was chosen as the challenge temperature for subsequent germplasm evaluation. Plant Physiol. Biochem.
4.5. Preincubation temperature determination Burke [6, 7] demonstrated that a 4-h preincubation at an elevated, sublethal temperature could reduce the level of injury experienced by plants subsequently exposed to a formerly lethal challenge temperature. To determine the appropriate preincubation temperature for ‘Chinese Spring’ wheat, etiolated leaf segments were preincubated for 4 h at 30 (control), 34, 36, 38, 40, 42, 44 or 46 °C. Following the preincubation period, the leaf segments, with the exception of the 30 °C control, were exposed to the predetermined 30-min challenge temperature. The control and challenged leaf segments were placed at 30 °C for 20 h under continuous light and subsequent chlorophyll accumulation evaluated as described above.
4.6. Screening for acquired thermotolerance deficiencies Seedlings of all viable ditelosomic lines (34 of 42 available lines, the remainder carry embryonic lethal chromosome arrangements) were screened initially for acquired thermotolerance deficiency by the following procedure. Seeds were germinated and grown in the dark at 27 °C for 5 d or until leaves were approximately 5 to 10 cm in length. Leaf segments were incubated at the identified preincubation temperature for 4 h then challenged at the challenge temperature for 30 min. The leaf segments were then transferred to continuous light at 30 °C after which chlorophyll content was determined as described above. This was a preliminary study and in some cases only two samples were studied due to insufficient seed quantity or germination problems. Subsequently nine lines were chosen for further analysis based on apparent altered acquired thermotolerance along with seed availability and acceptable germination rates.
4.7. In vivo labeling and protein isolation Proteins were labeled in vivo by allowing excised leaf segments (3 cm) to stand for 4 h in water containing 1.85⋅107 Bq⋅mL–1 [35S]-trans label (ICN) at either room temperature as control (approximately 22 °C) or at the preincubation temperature identified above. This labeling procedure enabled the incorporation of label into proteins at a rate independent from uptake rates (data not presented). Following treatments, leaf segments were washed in distilled water to remove excess radioactivity, the apical 1 cm removed and the remaining 2 cm pulverized in Tris/Glycine extraction buffer (Tris base, 0.1 M, pH 8.4; Glycine, 0.1 M). Cell debris was removed by centrifugation at 14 000 × g for
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10 min. Proteins were extracted from the supernatant with an equal volume of water-saturated phenol. The phenol phase was re-extracted with 0.5 volumes of extraction buffer, and proteins were precipitated overnight at –20 °C by addition of 2.5 volumes of 0.1 M ammonium acetate in methanol. After recovery by centrifugation, the protein pellet was washed once in 0.1 M ammonium acetate in methanol, air dried and resuspended in IEF buffer (urea, 9 M; DTT, 0.65 M; 3-10 Pharmalyte, 0.02 mL⋅mL–1; Triton X-100, 0.005 mL⋅mL–1; bromophenol blue, 0.001 %). Following resuspension in IEF buffer, insoluble material was removed by centrifugation at 14 000 × g for 2 min, the supernatant moved to a new tube and stored at –20 °C. The quantity of labeled protein in each sample was determined by liquid scintillation analysis using a Packard Tri-Carb 1500 liquid scintillation counter.
4.8. Two-dimensional gel electrophoresis Two-dimensional separation of radiolabeled proteins was achieved using the Immobiline DryStrip Kit and ExcelGel SDS on the Multiphor II electrophoresis system (Pharmacia). Procedures followed the manufacturers instructions with some modifications. Acetic acid was used instead of Pharmalyte 3-10 in the rehydration solution for IEF dry strips. Approximately 200 000 cpm of each sample were loaded on each gel. The SDS-PAGE gel after the final protein separation step was treated with fixer (10 % acetic acid and 30 % methanol) for 30 min and fluor (55 % acetic acid, 15 % ethanol, 30 % xylene and 0.8 % 2,5-diphenyl oxazole) for 1 h. The gel was then washed for 2× 2-min washes in distilled water, covered with wet cellulose acetate and dried on to the cellulose acetate membrane for 2 h at 45 °C. Labeled proteins were detected by fluorography by exposure to X-Ray film (BIOMAX-MR, Kodak) in the presence of a single enhancer screen at –80 °C.
Acknowledgments The authors wish to thank Jacob Sanchez for his excellent technical assistance throughout this study. This study supported in part by grant No. 96-351003168 from the NRI Competitive Grants Program/ USDA.
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