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,TX79415,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
www.elsevier.com/locate/plantsci
1. Introduction
Plants are frequently exposed to elevated soil and air temperatures resulting in a reduction in their growth, development and ultimately produc-tivity. When subjected to a period of sub-lethal elevated temperatures, plants acquire thermotoler-ance which transiently raises the injury threshold and protects them from subsequent, otherwise lethal, high temperatures. This acquisition of ther-motolerance is a complex physiological phe-nomenon which has been shown to involve at least some heat shock proteins (HSPs).
Plants, like all organisms, produce HSPs in re-sponse to various environmental stresses [1 – 3]. At sub-lethal elevated temperatures quantitative in-duction of HSPs occurs with a concomitant
reduc-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 thermotoler-ance [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 ge-netic 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 suc-cessfully 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 Abbre6iations: DT, ditelosomic; HSP, heat shock protein; SDS,
sodium dodecyl sulphate.
* Corresponding author. Tel: +1-806-7495560; fax: + 1-806-7235272.
E-mail address:[email protected] (J. Burke).
latent (constitutive) heat shock factor (HSF) is trimerized, causing it to bind to heat shock ele-ments (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 se-quence motifs and possibly acts on the chromatin to enable access to transcription factors such as HSF and TATA box binding proteins [18]. Multi-ple HSFs have been reported in plants and verte-brates 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 hy-drophobic repeats, but these areas are not well conserved in plants or yeasts. Also in higher eu-karyotes, 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 muta-tions with which a cause and effect relamuta-tionship could be established. We have begun an investiga-tion 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 coin-ciding with an alteration of HSP synthesis.
In this study we used a sensitive chlorophyll accumulation assay [20] to characterize the ac-quired thermotolerance of one of a series of ditelo-somics (DT) (a plant missing one chromosome arm-telocentric) of the hexaploid wheat cultivar ‘Chinese Spring’ [21]. A previous investigation us-ing 2-D gel electrophoresis [23] to analyze the genetic control of HSP synthesis in wheat iden-tified the chromosomal localization of genes con-trolling 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 chro-mosomes 3, 4 and 7 as sites containing HSP controlling loci. However, the study did not ad-dress 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 induc-tion temperatures correlates with acquisiinduc-tion 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 ditelo-somic DT1BS derived from ‘Chinese Spring’ [21] were analyzed and compared in this study. The ditelosomic lines are designated by their home-ologous 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 sup-port, 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
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 fol-lowing 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 ex-cised 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 (approxi-mately 22°C), 34 or 40°C pre-incubation tempera-ture. This labelling procedure enabled the incorporation of label into proteins at a rate inde-pendent 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 water-saturated phenol. The phenol phase was re-ex-tracted 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 centrifuga-tion 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%). Follow-ing resuspension in IEF buffer, insoluble material
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 scintilla-tion 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 electrophore-sis (SDS PAGE) using a 12% SDS polyacrylamide gel following standard protocols [25]. Two-dimen-sional 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 man-ufacturers 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 (Biomax-mr, Kodak) in the presence of a single enhancer screen at −80 °C.
3. Results
3.1. Optimum temperature for chlorophyll accumulation
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 chal-lenge [22]. We subjected ‘Chinese Spring’ and DT1BS to pre-incubation (heat shock) tempera-tures 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 follow-ing 34 and 40°C pre-incubations. ‘Chinese Sprfollow-ing’ (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 condi-tions.
Fig. 1. Temperature allowing optimum chlorophyll accumula-tion. 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 temper-atures. Chlorophyll accumulation was measured by a SPAD chlorophyll meter and measurements are represented as rela-tive 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
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
temperatures. ‘Chinese Spring’ suffered damage from a 50°C treatment even when pre-incubated at 34°C, while a 40°C pre-incubation provided suffi-cient 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 treat-ments, proteins were labelled in vivo (35
S-methion-ine) 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 dif-ferences were evident in band patterns between DT1BS and ‘Chinese Spring’. Several bands evi-dent 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’.
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 exam-ple, 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 tem-perature. A number of abundant proteins of ap-proximately 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
Most of the available evidence indicates that HSPs are involved with acquired thermotolerance, but in plants only HSP101 has been directly impli-cated [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 characteri-zation involved a chlorophyll accumulation assay for which optimal parameters were previously de-termined in a study of Chinese Spring’ wheat [22]. In the present study we show that DT1BS and
Fig. 4. SDS PAGE analysis of 35S-labeled proteins in leaf
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’. Re-ports by Porter et al. [27], Saadalla et al. [28],
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 accumula-tion assay in the leaf segments and is further evidence that DT1BS induces the acquired ther-motolerance system at lower temperatures than the Chinese Spring’ parental line.
Differences at the protein level between ‘Chinese Spring’ and DT1BS in response to elevated tem-perature were examined by 1- and 2-D PAGE. Several differences were evident between ‘Chinese Spring’ and DT1BS protein profiles in 1-dimen-sional PAGE. In particular, a band of approxi-mately 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 temper-ature. 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-di-mensional 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 in-duced 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 indi-cates that DT1BS is capable of HSP induction in response to lower temperatures than ‘Chinese Spring’, in agreement with our phenotypic charac-terization 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 pat-terns 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 signifi-cant levels at all temperatures tested for DT1BS but barely detectable at any temperature in
‘Chi-nese Spring’ seedlings. It is possible that regulation of this, and other proteins, is also partially en-coded 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 chloro-phyll at 40°C compared to ‘Chinese Spring’ seedlings (Fig. 1). It is interesting that putative HSPs are present in DT1BS at 30°C while thermo-tolerance 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 ther-mal challenge. Identification of individual HSPs involved with acquired thermotolerance and ma-nipulation of their levels will aid in answering this question.
In summary, we have identified and character-ized 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 ac-quired thermotolerance correlates closely with the induction of a number of HSPs, and our data suggests that the missing chromosome arm possi-bly encodes at least some regulating factor govern-ing synthesis of certain HSPs. A more detailed investigation of this chromosome arm should re-veal the regulon involved, advancing our knowl-edge of plant acquired thermotolerance. Additionally, greater knowledge of acquired ther-motolerance regulation and the genes regulated should facilitate the manipulation of crops to im-prove their thermotolerance thereby increasing their viability in the face of elevated temperatures.
Acknowledgements
States Department of Agriculture, and does not imply its approval to the exclusion of other prod-ucts that may also be suitable. The cultivar was kindly provided by Dr J. P. Gustafson, USDA-ARS, Columbia, MO.
References
[1] E. Vierling, The roles of heat shock proteins in plants, Annu. Rev. Plant. Physiol. Plant Mol. Biol. 42 (1991) 579 – 620.
[2] M.A. Coca, C. Almoguera, T.L. Thomas, J. Jordano, Differential regulation of small heat shock genes in plants: analysis of a water-stress-inducible and develop-mentally activated sunflower promoter, Plant Mol. Biol. 31 (1996) 863 – 876.
[3] Y. Sanchez, J. Taulien, K.A. Borkovich, S.L. Lindquist, Hsp104 is required for tolerance to many forms of stress, EMBO 11 (1992) 2357 – 2364.
[4] G.C. Li, Z. Werb, Correlation between synthesis of heat shock proteins and development of thermotolerance in Chinese hamster fibroblasts, Proc. Natl. Acad. Sci. 79 (1982) 3218 – 3222.
[5] S.L. Lindquist, E.A. Craig, The heat shock proteins, Ann. Rev. Genetics 22 (1988) 631 – 677.
[6] D.A. Parsell, J. Taulien, S.L. Lindquist, The role of heat shock proteins in thermotolerance, Phil. Trans. R. Soc. 339 (1993) 286.
[7] Y. Sanchez, S.L. Lindquist, Hsp104 required for induced thermotolerance, Science 248 (1990) 1112 – 1114. [8] J.L. Vogel, D.A. Parsell, S.L. Lindquist, Heat-shock
proteins Hsp104 and Hsp70 reactivate mRNA splicing after heat inactivation, Current Biol. 5 (1995) 306 – 317. [9] S.W. Hong, E. Vierling, Mutants ofArabidopsis thaliana
defective in the acquisition of tolerance to high tempera-ture, Proc. Natl. Acad. Sci. 97 (8) (2000) 4392 – 4397. [10] C. Queitsch, S.W. Hong, E. Vierling, S.L. Lindquist,
Heat shock protein 101 plays a crucial role in thermotol-erance in Arabidopsis, Plant Cell 12 (4) (2000) 479 – 492. [11] J.H. Lee, A. Huber, F. Schoffl, Depression of the activ-ity of genetically engineered heat-shock factor causes constitutive synthesis of heat shock proteins and in-creased thermotolerance in transgenic Arabidopsis, Plant J. 8 (1995) 603 – 612.
[12] J.H. Lee, F. Schoffl, An HSP70 antisense gene affects the expression of HSP70/HSC70, the regulation of HSF, and the acquisition of thermotolerance in transgenic
Arabidopsis thaliana, Mol. Gen. Genetics 252 (1996) 11 – 19.
[13] R. Prandl, K. Hinderhofer, G. Eggers-Schumaker, F. Schoffl, HSF3, a new heat shock factor fromArabidopsis thaliana, derepresses the heat shock response and confers thermotolerance when overexpressed in transgenic plants, Mol. Gen. Genetics 258 (1998) 269 – 278.
[14] G. Visioli, E. Maestri, N. Marmiroli, Differential dis-play-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. [15] C.P. Joshi, N.Y. Klueva, K.J. Morrow, H.T. Nguyen, Expression of a unique plastid-localized heat-shock protein is genetically linked to acquired thermotolerance in wheat, Theor. Appl. Genet. 95 (1997) 834 – 841. [16] Y-R.J. Lee, R.T. Nagao, J.L. Key, A soybean 101-kD
heat shock protein complements a yeast HSP104 dele-tion mutadele-tion in acquiring thermotolerance, Plant Cell 6 (1994) 1889 – 1897.
[17] E.C. Schirmer, S.L. Lindquist, E. Vierling, An Ara-bidopsis heat shock protein complements a thermotoler-ance defect in yeast, Plant Cell 6 (12) (1994) 1899 – 1909. [18] F. Schoffl, R. Prandl, A. Reindl, Regulation of the heat-shock response, Plant Physiol. 117 (1998) 1135 – 1141.
[19] A. Ali, S. Bharadwaj, R. O’Carroll, N. Ovsenek, HSP90 interacts with and regulates the activity of heat shock factor 1 in Xenopus Oocytes, Mol. Cell Biol. 18 (1998) 4949 – 4960.
[20] J.J. Burke, Characterization of acquired thermotolerance in soybean seedlings, Plant Physiol. Biochem. 36 (1998) 601 – 607.
[21] E. Sears, L. Sears, The telocentric chromosomes of common wheat. Proceedings of the 5th Wheat Genetics Symposium, New Delhi. (1978) 389 – 407.
[22] P.J. O’Mahony, J.J. Burke, M.J. Oliver, Identification of acquired thermotolerance deficiency within the ditelo-somic series of ‘Chinese Spring’ wheat, Plant Physiol. Biochem. 38 (3) (2000) 243 – 252.
[23] D.R. Porter, H.T. Nguyen, J.J. Burke, Chromosomal location of genes controlling heat shock proteins in hexaploid wheat, Theor. Appl. Genet. 78 (1989) 873 – 878.
[24] J.J. Burke, T.C. Mahan, An electronically controlled eight position thermalplate system, App. Eng. Agri. 9 (1993) 483 – 486.
[25] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680 – 685.
[26] M. Krishnan, H.T. Nguyen, J.J. Burke, Heat shock protein synthesis and thermal tolerance in wheat, Plant Physiol. 90 (1989) 140 – 145.
[27] D.R. Porter, H.T. Nguyen, J.J. Burke, Quantifying ac-quired thermal tolerance in wheat, Crop Sci. 34 (1994) 1686 – 1689.
[28] M.M. Saadalla, J.S. Quick, J.F. Shanahan, Heat toler-ance in winter wheat. II. Membrane thermostability and field performance, Crop Sci. 30 (1990) 1248 – 1251. [29] J.F. Shanahan, I.B. Edwards, J.S. Quick, J.R. Fenwick,
Membrane thermostability and heat tolerance of spring wheat, Crop Sci. 30 (1990) 247 – 251.
[30] R.A. Vierling, H.T. Nguyen, Heat-shock protein genetic expression in diploid wheat genotypes differing in ther-mal tolerance, Crop Sci. 32 (1992) 370 – 377.
