A ditelosomic line of ‘Chinese Spring’ wheat with augmented acquired thermotolerance

Plant Science 158 (2000) 147 – 154 www.elsevier.com/locate/plantsci

A ditelosomic line of ‘Chinese Spring’ wheat with augmented acquired thermotolerance Patrick O’Mahony, John Burke* Plant Stress and Germplasm De6elopment Unit, USDA-ARS, 3810 4th Street, Lubbock, TX 79415, USA Received 18 April 2000; received in revised form 9 June 2000; accepted 9 June 2000

Abstract A study of the ditelosomic series of ‘Chinese Spring’ wheat has yielded a number of lines displaying either an increased or decreased ability to acquire thermotolerance. One such ditelosomic (DT) is termed DT1BS which refers to the missing short arm of chromosome 1 in the B genome. The DT1BS line has the ability to acquire thermotolerance at lower induction temperatures and provide greater protection to the plant against otherwise lethal elevated temperatures. Using a chlorophyll accumulation assay to measure plant health, we show that DT1BS accumulates chlorophyll optimally at the same temperature, and to similar levels as ‘Chinese Spring’. We also show that maximum acquired thermotolerance against a 48°C challenge is induced at 40°C, but significant levels of protection can be obtained at temperatures as low as 34°C in DT1BS or 36°C in ‘Chinese Spring’. Heat-shock protein accumulation is observed in DT1BS at temperatures 4°C lower than the ‘Chinese Spring’ and is correlated with the induction of acquired thermotolerance. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acquired thermotolerance; Heat shock proteins; Chinese Spring wheat; Ditelosomics

1. Introduction Plants are frequently exposed to elevated soil and air temperatures resulting in a reduction in their growth, development and ultimately productivity. When subjected to a period of sub-lethal elevated temperatures, plants acquire thermotolerance which transiently raises the injury threshold and protects them from subsequent, otherwise lethal, high temperatures. This acquisition of thermotolerance is a complex physiological phenomenon which has been shown to involve at least some heat shock proteins (HSPs). Plants, like all organisms, produce HSPs in response to various environmental stresses [1–3]. At sub-lethal elevated temperatures quantitative induction of HSPs occurs with a concomitant reducAbbre6iations: DT, ditelosomic; HSP, heat shock protein; SDS, sodium dodecyl sulphate. * Corresponding author. Tel: +1-806-7495560; fax: +1-8067235272. E-mail address: [email protected] (J. Burke).

tion in the synthesis of many other proteins. This alteration in metabolic priorities coincides with the acquisition of thermotolerance [1,4,5]. Significant evidence is available from yeast studies which link HSP induction to the acquisition of thermotolerance [6–8]. However, to date only HSP101 has been directly linked to acquired thermotolerance in plants [9,10]. In Arabidopsis modulated heat shock protein synthesis as well as heat shock factor activity and expression have been shown to correlate with levels of thermotolerance [11–14]. Studies in thermo-susceptible and thermo-tolerant recombinant inbred lines of wheat detected a genetic relationship between expression of a plastid localized HSP26 and acquired thermotolerance [15]. In addition, other studies have demonstrated that an acquired thermotolerance-deficient yeast that carries a mutated HSP104 gene can be successfully complemented by plant HSP 101 genes from soybean [16] and Arabidopsis [17]. Expression of HSP genes is regulated primarily at the transcriptional level [13]. Upon heat shock

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latent (constitutive) heat shock factor (HSF) is trimerized, causing it to bind to heat shock elements (HSEs) upstream of the HSP gene [18]. Efficient transcription of heat shock genes occurs when 5’ proximal tripartite HSEs bind trimerized HSF. This interaction is enhanced by other sequence motifs and possibly acts on the chromatin to enable access to transcription factors such as HSF and TATA box binding proteins [18]. Multiple HSFs have been reported in plants and vertebrates while for Drosophila and yeast only one has been identified. Control of HSF trimerization and thus transcription of HSP genes in many higher eukaryotes is controlled by C-terminal hydrophobic repeats, but these areas are not well conserved in plants or yeasts. Also in higher eukaryotes, it is proposed that phosphorylation along with feedback control by HSP70 and HSP90 act to repress HSF activity [19]. Obtaining direct evidence to link HSPs with acquired thermotolerance in higher plants has been restricted due to a lack of functional mutations with which a cause and effect relationship could be established. We have begun an investigation of heat shock responses in aneuploid genetic stocks of ‘Chinese Spring’ wheat where specific chromosomal deletions result in a reduction or up-regulation of acquired thermotolerance coinciding with an alteration of HSP synthesis. In this study we used a sensitive chlorophyll accumulation assay [20] to characterize the acquired thermotolerance of one of a series of ditelosomics (DT) (a plant missing one chromosome arm-telocentric) of the hexaploid wheat cultivar ‘Chinese Spring’ [21]. A previous investigation using 2-D gel electrophoresis [23] to analyze the genetic control of HSP synthesis in wheat identified the chromosomal localization of genes controlling a number of low molecular mass HSPs. Variations in relative HSP levels suggested that the homeologous 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, the study did not address the possible functional relationship between specific HSP changes and levels of acquired thermotolerance. Here we characterize the DT1BS line of wheat which had previously been observed to possess greater acquired thermotolerance than ‘Chinese Spring’ [22]. We demonstrate that an up-regula-

tion of HSP synthesis in DT1BS at lower induction temperatures correlates with acquisition of thermotolerance, suggesting that the missing arm may contain at least one form of genetic control for HSP synthesis and acquired thermotolerance in ‘Chinese Spring’ wheat.

2. Materials and methods

2.1. Plant material Hexaploid wheat (Triticum aesti6um L, 2n = 6× =42) cultivar ‘Chinese Spring’ and the ditelosomic DT1BS derived from ‘Chinese Spring’ [21] were analyzed and compared in this study. The ditelosomic lines are designated by their homeologous group (1–7), genome A, B or D and the length of the missing chromosome arm (L, long, S, short). DT1BS was selected for this study based on previous work which suggested that it has augmented acquired thermotolerance [22]. 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 28°C. In each treatment three 2 cm leaf segments, 1 cm from the leaf tip, of three separate 5-day-old leaves were excised and placed on 1% agarose in a 35×10 mm diameter tissue culture dish (Corning). A specific section of the leaves was 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 (data not presented).

2.2. Temperature and light parameters Unless otherwise stated, temperature treatments were achieved using an electronically controlled eight position thermal plate system [24]. Thermal plates were covered with 3MM water-saturated filter paper (Whatman) 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

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prior to being placed at 30°C under continuous light at 115 mmol/m − 2 per s (two Philips F40/ AGRO AGRO LITE fluorescent bulbs and two 75 W incandescent bulbs) for 20 h. Unless otherwise specified, pre-incubation treatments lasted 4 h while challenge treatments were carried out for 30 min at 48°C as previously determined for ‘Chinese Spring’ [22]. Whole plant analysis utilized a 4 h 34 or 40°C pre-incubation in a humidified growth chamber under light conditions. They were then challenged at 50°C for 1 h under light conditions and subsequently allowed to recover at 30°C in the light.

2.3. Chlorophyll determination Relative chlorophyll levels were determined following exposure to continuous light using a SPAD-502 chlorophyll meter (Minolta). At least three tissue samples were used with five readings taken from each sample.

2.4. In 6i6o labelling and protein isolation Proteins were labelled in vivo by allowing excised leaf segments (3 cm) to stand for 4 h in water containing 1.85 × 107 Bq/ml35S trans label (ICN) at either room temperature as control (approximately 22°C), 34 or 40°C pre-incubation temperature. This labelling 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 of leaf tissue 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 000g for 10 min. Proteins were extracted from the supernatant with an equal volume of watersaturated 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; Triton X–100, 0.005 ml/ml; bromophenol blue, 0.001%). Following resuspension in IEF buffer, insoluble material

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was removed by centrifugation at 14 000g for 2 min, the supernatant removed to a new tube and stored at −20°C. The quantity of labelled protein in each sample was determined by liquid scintillation analysis using a Packard Tri Carb 1500 liquid scintillation counter.

2.5. 1 - and 2 -dimensional gel electrophoresis Radiolabeled proteins were separated by one dimensional SDS polyacrylamide gel electrophoresis (SDS PAGE) using a 12% SDS polyacrylamide gel following standard protocols [25]. Two-dimensional separation of radio-labeled 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. Labelled proteins were detected by fluorography by exposure to X-ray film (Biomaxmr, Kodak) in the presence of a single enhancer screen at −80 °C.

3. Results

3.1. Optimum temperature for chlorophyll accumulation The ability of the ditelosomic line DT1BS to accumulate chlorophyll subsequent to a range of temperature treatments was analyzed and compared to that for ‘Chinese Spring’. Etiolated ‘Chinese Spring’ and DT1BS leaf segments were placed in continuous light for 20 h at temperatures ranging from 10 to 45°C and the level of chlorophyll accumulation determined by the SPAD chlorophyll meter. The results showed that the optimum temperature for chlorophyll accumulation in DT1BS was 30°C. This temperature is identical to

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that for ‘Chinese Spring’ (Fig. 1) and the levels of chlorophyll accumulation were equivalent for both at all temperatures except at 40°C. At 40°C chlorophyll accumulation was significantly greater in DT1BS compared to ‘Chinese Spring’.

3.2. Chlorophyll accumulation and the heat shock response Previous work has shown that ‘Chinese Spring’ exposed to sub-lethal elevated temperatures can withstand subsequent, otherwise lethal, 48°C challenge [22]. We subjected ‘Chinese Spring’ and DT1BS to pre-incubation (heat shock) temperatures ranging from 30 to 46°C for 4 h. They were then challenged at 48°C for 30 min and allowed to Fig. 3. Whole plant response to a 50°C – 1h challenge following 34 and 40°C pre-incubations. ‘Chinese Spring’ (CS) and DT1BS were grown at 30°C under 16 h/8 h light/dark cycles for 5 days, subjected to the temperature treatments specified and allowed to recover for 24 h under original growth conditions.

Fig. 1. Temperature allowing optimum chlorophyll accumulation. Etiolated 5-day-old leaf segments (2-cm length, 1 cm from the apex) of ‘Chinese Spring’ (CS) and DT1BS were placed under continuous light for 20 h at the specified temperatures. Chlorophyll accumulation was measured by a SPAD chlorophyll meter and measurements are represented as relative chlorophyll. Error bars represent standard error.

accumulate chlorophyll at 30°C under continuous light for 20 h. The results show a significant difference in ability to acquire thermotolerance between ‘Chinese Spring’ and DT1BS (Fig. 2). DT1BS rapidly acquired thermotolerance above 32°C reaching a peak at 40°C. ‘Chinese Spring’ on the other hand acquired minimal thermotolerance at 34 and 36°C increasing to maximum levels at 38 and 40°C. This demonstrates that DT1BS is more sensitive to temperature elevation and its acquired thermotolerance response is induced at a lower temperature than ‘Chinese Spring’. However, the optimum temperature for inducing this response was 40°C in ‘Chinese Spring’ and DT1BS and at temperatures between 42 and 44°C the ability to induce thermotolerance was diminished in both.

3.3. Whole plant response to ele6ated temperatures

Fig. 2. Optimum temperature for acquiring thermotolerance. Etiolated 5-day-old leaf segments (2-cm length, 1 cm from the apex) of ‘Chinese Spring’ (CS) and DT1BS were pre-incubated at the specified temperatures for 4 h, followed by incubation at 48°C for 30 min. Tissues were then incubated under continuous light for 20 h at 30°C and chlorophyll levels determined as in Fig. 1. Chlorophyll levels are presented as relative chlorophyll and error bars represent standard error.

In order to confirm that the accumulation of chlorophyll following high temperature treatment of leaf segments truly reflected the response of the plant in general, we carried out a number of temperature treatments on whole plants similar to those carried out on the 2 cm leaf segments. The results displayed in Fig. 3 demonstrate that what was observed in 2 cm leaf segments was a valid representation of whole plant response to elevated

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temperatures. ‘Chinese Spring’ suffered damage from a 50°C treatment even when pre-incubated at 34°C, while a 40°C pre-incubation provided sufficient protection. On the other hand, DT1BS only suffered apparent damage when treated at 50°C without pre-incubation, while pre-incubation at 40°C and even 34°C provided ample protection.

3.4. 1 -D and 2 -D PAGE examination of protein profiles during pre-incubation In order to achieve a broad spectrum analysis of protein expression during high temperature treatments, proteins were labelled in vivo (35S-methionine) at 30°C, 34 and 40°C. Protein was extracted from these samples and protein profiles examined by 1-D and 2-D PAGE (Figs. 4 and 5). On analysis by 1-D SDS–PAGE (Fig. 4) several differences were evident in band patterns between DT1BS and ‘Chinese Spring’. Several bands evident in DT1BS at all temperatures were apparent in ‘Chinese Spring’ only at 40°C (indicated by arrows). Some bands present in DT1BS at all temperatures were not evident in ‘Chinese Spring’ at any temperature, e.g. the band marked by (*), representing a constitutive up-regulation of that protein relative to ‘Chinese Spring’.

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In agreement with previous reports, analysis by 2-D PAGE (Fig. 5) showed that many proteins present at 30°C were absent, or present at reduced levels, at 40°C (left hand spot-upper arrow outside each box). Other proteins, presumably HSPs, which were absent from, or at low levels at 30°C, were at increased levels at 40°C. Two of these putative HSPs are identified by the lower arrow outside each magnified inset. Many of the putative HSPs were present in DT1BS tissues even at 30°C while they only began to appear at low levels in ‘Chinese Spring’ tissue treated at 34°C. At 40°C the protein profile of DT1BS was similar to that of ‘Chinese Spring’ with some exceptions. For example, one protein of approximately 60 kDa (right hand spot-upper arrow) was present in DT1BS leaves at 30, 34 and 40°C, but was not strongly represented in ‘Chinese Spring’ tissues at any temperature. A number of abundant proteins of approximately 55 kDa molecular weight (indicated by * in Fig. 5) may be the same proteins evident in SDS–PAGE analysis of in vivo labelled DT1BS tissues but not ‘Chinese Spring’ (Fig. 4). The abundance of these proteins, even under non-stress conditions, and their apparent molecular weight suggests that they may represent the large subunit of rubisco.

4. Discussion

Fig. 4. SDS PAGE analysis of 35S-labeled proteins in leaf tissue incubated for 4 h at the specified temperatures. Arrows indicate some of the differences in protein levels between ‘Chinese Spring’ (CS) and DT1BS.

Most of the available evidence indicates that HSPs are involved with acquired thermotolerance, but in plants only HSP101 has been directly implicated [9,10] while the mechanisms involved remain largely enigmatic. In this study we examined a ditelosomic line of ‘Chinese Spring’ wheat in an effort to identify chromosomal regions encoding genes involved with acquired thermotolerance. To date we have characterized one ditelosomic line DT7DS [22] which has an apparent deficiency in acquired thermotolerance relative to ‘Chinese Spring’ and a concomitant reduction in, or loss of, two putative HSPs. Data presented here describes the characterization of another ditelosomic line (DT1BS) which has an apparent augmentation of acquired thermotolerance. Phenotypic characterization involved a chlorophyll accumulation assay for which optimal parameters were previously determined in a study of Chinese Spring’ wheat [22]. In the present study we show that DT1BS and

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Fig. 5. 2-D PAGE analysis of 35S-labeled proteins of leaf tissues incubated at 30, 34 or 40°C for 4 h. Arrows indicate some proteins down-regulated by elevated temperatures (left hand spot-upper arrow), constitutively expressed proteins found in DT1BS which are barely detectable in ‘Chinese Spring’ (right hand spot-lower arrow), or putative HSPs up-regulated in response to lower temperatures relative to ‘Chinese Spring’ (CS). The asterisk indicates a protein(s) of similar size and expression pattern to the band highlighted by a similar asterisk in Fig. 4.

‘Chinese Spring’ accumulated similar levels of chlorophyll over a range of temperatures, except at 40°C where DT1BS accumulated 5-fold greater chlorophyll levels than ‘Chinese Spring’. We also demonstrate that DT1BS activates its heat shock response at lower temperatures than ‘Chinese Spring’ and thus is more sensitive to elevated temperatures. Identifying genetic differences in the level of acquired thermotolerance in wheat cultivars is not unique to this study. Krishnan et al. [26] used the triphenyltetrazolium chloride viability assay to show greater thermotolerance in the wheat cultivar ’Mustang’ compared to the cultivar ‘Sturdy’. Reports by Porter et al. [27], Saadalla et al. [28],

Shanahan et al. [29] and Vierling and Nguyen [30] also demonstrated genetic diversity for heat tolerance in wheat. What is unique about the findings in this study is not only is there a greater protection at 40°C, but the initiation of acquired thermotolerance in DT1BS occurs at lower temperatures than the control ‘Chinese Spring’. To verify that the effect of elevated temperatures on leaf segments of ‘Chinese Spring’ wheat reflected the response of the whole plant to similar temperature stress, we analyzed the whole plant response to elevated temperatures. ‘Chinese Spring’ leaves were severely damaged by a 50°C challenge even if pre-incubated at 34°C, while a 40°C pre-incubation provided ample protection.

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DT1BS however, appeared to be damaged only by a straight challenge of 50°C while both 34°C and 40°C pre-incubations provided ample protection against a subsequent challenge. This data confirms the results derived from the chlorophyll accumulation assay in the leaf segments and is further evidence that DT1BS induces the acquired thermotolerance system at lower temperatures than the Chinese Spring’ parental line. Differences at the protein level between ‘Chinese Spring’ and DT1BS in response to elevated temperature were examined by 1- and 2-D PAGE. Several differences were evident between ‘Chinese Spring’ and DT1BS protein profiles in 1-dimensional PAGE. In particular, a band of approximately 55 kDa (identified by *) evident in DT1BS incubated at 30°C, 34°C and 40°C which was not apparent in ‘Chinese Spring’ tissues at any temperature. Analysis of proteins by 2-dimensional PAGE (Fig. 5) provided a more detailed profile of modulating protein levels including a cluster of proteins of 55 kDa (indicated by * in Fig. 5). The molecular mass and relative intensities of these proteins coincide with the extra band in the 1-dimensional PAGE analysis in Fig. 4(*) suggesting they may represent the large subunit of rubisco. It is unlikely, however, that the higher levels of these proteins plays a role in the increased acquired thermotolerance of DT1BS since levels appeared to decrease at temperatures above 30°C at which acquired thermotolerance is induced. Numerous other proteins, presumably HSPs, such as the 25 and 26 kDa proteins identified by the lower arrow in the magnified inset were induced by 34°C and 40°C in both ‘Chinese Spring’ and DT1BS (Fig. 5). However, at 30°C these proteins were also present at low levels in DT1BS but not in ‘Chinese Spring’ seedlings. This indicates that DT1BS is capable of HSP induction in response to lower temperatures than ‘Chinese Spring’, in agreement with our phenotypic characterization of DT1BS. This does not represent a preferential induction of all HSPs however, since we found through western analysis that two HSPs, HSP17.6 and HSP101, had similar induction patterns at various temperatures in both ‘Chinese Spring’ and DT1BS (data not shown). Other proteins such as the 60 kDa protein (right hand spot-upper arrow, Fig. 5) were present at significant levels at all temperatures tested for DT1BS but barely detectable at any temperature in ‘Chi-

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nese Spring’ seedlings. It is possible that regulation of this, and other proteins, is also partially encoded by the missing short arm of chromosome 1, thereby allowing higher expression levels. The presence of low levels of putative HSPs in DT1BS at the growth temperature of 30°C may explain its increased ability to accumulate chlorophyll at 40°C compared to ‘Chinese Spring’ seedlings (Fig. 1). It is interesting that putative HSPs are present in DT1BS at 30°C while thermotolerance induction, as measured by chlorophyll accumulation, is observed only at 32°C and above (Fig. 2). It is possible that the HSPs specifically involved with acquired thermotolerance are only induced at 32°C, or that a certain threshold level of HSP(s) is required within the cell in order to provide protection against a certain degree of thermal challenge. Identification of individual HSPs involved with acquired thermotolerance and manipulation of their levels will aid in answering this question. In summary, we have identified and characterized the acquired thermotolerance system of the DT1BS ditelosomic line of ‘Chinese Spring’ wheat which has a demonstrable ability to respond to lower induction temperatures, and to a greater extent than ‘Chinese Spring’ seedlings. This acquired thermotolerance correlates closely with the induction of a number of HSPs, and our data suggests that the missing chromosome arm possibly encodes at least some regulating factor governing synthesis of certain HSPs. A more detailed investigation of this chromosome arm should reveal the regulon involved, advancing our knowledge of plant acquired thermotolerance. Additionally, greater knowledge of acquired thermotolerance regulation and the genes regulated should facilitate the manipulation of crops to improve their thermotolerance thereby increasing their viability in the face of elevated temperatures.

Acknowledgements The authors wish to thank Jacob Sanchez for his excellent technical assistance throughout this study. This study was supported in part by grant no. 96-35100-3168 from the NRI Competitive Grants Program/USDA. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United

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States Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable. The cultivar was kindly provided by Dr J. P. Gustafson, USDAARS, Columbia, MO.

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