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 relationship
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 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 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 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
prior to being placed at 30°C under continuous light at 115 mmolm
− 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 Bqml
35
S 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 TrisGlycine 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 mlml; Triton X – 100,
0.005 mlml; 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
