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
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 com- pared to that for ‘Chinese Spring’. Etiolated ‘Chi-
nese Spring’ and DT1BS leaf segments were placed in continuous light for 20 h at temperatures rang-
ing from 10 to 45°C and the level of chlorophyll accumulation determined by the SPAD chloro-
phyll meter. The results showed that the optimum temperature
for chlorophyll
accumulation in
DT1BS was 30°C. This temperature is identical to
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 Spring’ CS and
DT1BS were grown at 30°C under 16 h8 h lightdark 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
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-incu- bated 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.
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
