Journal of Thermal Biology 27 (2002) 547–553
Heat-shock protein expression in a perennial grass
commonly associated with active geothermal areas in
western North America
Thamir S. Al-Niemia,b, Richard G. Stouta,b,*
a
Department of Plant Sciences and Plant Pathology, Montana State University, ABS 119, Bozeman, MT 59717, USA
b
Thermal Biology Institute, Montana State University, Bozeman, MT 59717, USA
Received 22 April 2002; accepted 30 May 2002
Abstract
Heat-shock protein (hsp) expression in response to short- (hours) versus long-term (weeks) heat was examined in
Dichanthelium lanuginosum. Expression of cytoplasmic class I small hsps (shsps) and hsp 101 was elicited by 2 h above
401C, along with an increase over basal levels of hsp 70. Elevated levels of heat-induced shsp and hsp 101 persisted for
5–7 days after plants were returned to control temperatures. Protein extracts from roots exposed to 421C for several
weeks displayed increased levels of shsp but decreased hsp 101 levels. This decrease in hsp 101 did not occur in
D. lanuginosum from normothermic environments.
r 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Dichanthelium lanuginosum; Heat-shock proteins; Heat acclimation; Geothermal soils; Yellowstone National Park
1. Introduction
Little is known about the collective cellular mechanisms culminating in the adaptation of plants to longterm (weeks to months) heat stress. The length of time a
plant is subjected to high temperatures is of fundamental
importance with regard to determining the heat-killing
temperature (Levitt, 1980). Most of the information
regarding heat-killing temperatures in plants is, however, based on exposure times ranging from minutes to
hours (Levitt, 1980; Kappen, 1981). In an extensive
review of plant heat shock, Neumann et al. (1989)
comment on the paucity of experimental data on
adaptive cellular changes induced by long-term heat
stress in plants.
*Corresponding author. Department of Plant Sciences and
Plant Pathology, Montana State University, ABS 119, Bozeman, MT 59717, USA. Tel.: +1-406-994-4912; fax: +1-406994-7600.
E-mail address: [email protected] (R.G. Stout).
Using plants adapted to hot desert environments has
provided valuable information regarding plant responses to heat stress, though only a few studies (e.g.,
Drennan and Nobel, 1996) have investigated cellular
processes in plants exposed to long periods (weeks) of
high (X401C) temperatures.
Perhaps this lack of information is not surprising
since few extreme environments, with the exception of
geothermally heated soils, subject plants to continuous
long-term (weeks to months) heat stress. Thus, plants
adapted to geothermally heated environments may be
valuable model plants with regard to studying heat stress
physiology.
We have previously reported data regarding the heat
and acid tolerance of the most common flowering plant
we have observed in several of Yellowstone National
Park’s (YNP) active geothermal areas, namely, the
perennial grass Dichanthelium lanuginosum (Stout et al.,
1997). Populations of D. lanuginosum are commonly
found on hot ground in active geothermal areas not only
in YNP but also in similar environments throughout
0306-4565/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 6 - 4 5 6 5 ( 0 2 ) 0 0 0 2 9 - 3
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T.S. Al-Niemi, R.G. Stout / Journal of Thermal Biology 27 (2002) 547–553
western North America (Brewer, 1868; Hitchcock and
Chase, 1910; Gillette et al., 1961; Malloch, 1978;
Spellenberg, 1975; Pavlik and Enberg, 2001). The roots
of these plants have been shown to tolerate temperatures
above 401C for weeks to months (Stout et al., 1997).
Because of this remarkable ability to tolerate such long
periods of constant heat, the roots of these plants must
possess cellular mechanisms allowing them to acclimate
to long-term heat stress. Although the nature of these
mechanisms is currently unknown, an extensive literature supports the idea that heat-shock proteins (hsps)
are likely candidates.
The cytoplasmic class I small heat-shock proteins
(shsps) have been implicated as components of macromolecular protein chaperone mechanisms underlying
plant thermotolerance (Hernandez and Vierling, 1993;
Park et al., 1996; Waters et al., 1996; Knight and
Ackerly, 2001). Recent evidence (Queitsch et al., 2000)
supports the idea that hsp 101 expression may be
essential for thermotolerance in plants, perhaps by
functioning to reactivate denatured proteins (Glover
and Lindquist, 1998), to promote the translation of
some cellular mRNAs (Wells et al., 1998), or both. The
relative significance of hsps, compared to the expression
of thermally stable proteins, for example, in the
adaptation strategies of organisms to thermally stressful
environments is currently unclear (Coleman et al., 1995;
Knight and Ackerly, 2001).
Although it is clear that hsps are involved in the
acquisition of thermotolerance in plants exposed to
short-term (minutes to hours) heat stress, there is very
little experimental evidence that the same array of hsps
are involved in adaptation of plants to long-term heat
stress. Because of D. lanuginosum’s remarkable heat
tolerance in geothermal soils, we have used it to study
physiological and cellular effects of long-term (weeks)
continuous exposure to high temperatures (>401C).
Here we compare short-term (hours) versus long-term
(weeks) high-temperature exposure on the expression of
several prominent hsps in D. lanuginosum from YNP.
2. Materials and methods
Individuals of D. lanuginosum were collected from
populations located in several geothermal basins within
YNP and in normothermic soils in the vicinity of
College Station, Texas. These plants were vegetatively
propagated in the glasshouse as follows. Individual
plants used in this study were generated by separating
the newly growing plantlets in the crown regions of the
old plants and growing them in a 3.5-in2 plastic pots in a
1:1:1 soil:sand:perlite for 4–6 weeks. Plants were irrigated
5 days a week with 0.01 g/l Miracle-Gro (15-30-15) all
purpose fertilizer (Scott Miracle-Gro products Inc.,
Marysville, OH) and 2 days with tap water.
We have used modified hot-water bath systems as a
means of obtaining heat conditions similar to those
found in the rhizosphere of D. lanuginosum growing on
geothermally heated ground. Heat treatments were
conducted in the glasshouse at 421C (711C) in water
baths filled with 16L 1X Hoagland solution (Hoagland
and Arnon, 1938) and supplied with two lines of
perforated tubing fixed at the bottom of each bath and
connected to air pump for continuous aeration. Shoot
temperature was near glasshouse temperature, 19/261C
night/day. After washing roots gently from soil with tap
water, plant roots were inserted in holes made in a 1-inthick solid insulating foam used as cover for the water
bath. The setup allows roots only to be immersed in the
heated nutrient solution. Care was taken to prevent
shoots from exposure to heat by wrapping thin sponge
pieces around crown area of each plant to fill the gap
around the stem. Eight plants were placed in each water
bath. Plants were grown in the hydroponic system for
additional 3 weeks before heat was turned on. Nutrient
solution was replaced with fresh one every week
throughout the duration of experiments. Nutrient
solution was heated up to the treatment temperature
before each change. Tissue samples were collected from
each plant individually, immediately frozen in liquid
nitrogen and stored at 801C.
Two methods for extraction of total protein from
plant tissue were used. For young seedlings (3–4 weeks
old) used in short-term (hours) heat treatment experiments, total protein extractions were performed essentially as described by Hernandez and Vierling (1993)
with a few modifications. We will refer to this extraction
procedure as the ‘‘PVP method’’. Typically, 100–500 mg
(fresh weight) of plant tissue was ground in liquid
nitrogen using a mortar and pestle. The resulting
powder was immediately homogenized in a protein
extraction buffer (2–3 ml buffer/0.1 g fresh weight of
tissue). The protein extraction buffer consisted of
60 mM tris-HCl (pH 8), 60 mM dithiotreitol (DTT),
2% (w/v) sodium dodecyl sulphate (SDS), 15% (w/v)
sucrose, 5 mM e-amino-Z-caproic acid, 1 mM benzamidine, 0.275% diethyl dithiocarbamic acid and 4%
polyvinylpyrrolidone (insoluble, mol. wt. 240,000). After
grinding, samples were heated to 951C for 5 min, and
insoluble material pelleted by centrifugation for 5 min at
12,000g at room temperature. Five volumes of 201C
acetone was added to the resulting supernatant, with
mixing. Samples were stored at 201C overnight. The
following day, samples were centrifuged for 5 min at
2500g at room temperature to pellet protein precipitate
and were dried briefly at room temperature.
Alternatively, we used a protein extraction procedure
we will refer to as the ‘‘phenol method’’ that was more
effective at extracting total protein from older (weeks to
months old) plant tissue. For this procedure,
600–800 mg of frozen tissues were ground extensively
T.S. Al-Niemi, R.G. Stout / Journal of Thermal Biology 27 (2002) 547–553
under liquid N2 using a prechilled mortar and pestle,
poured immediately on top of 8 ml of 1:1 mixture of
water-saturated phenol and protein extraction buffer
(500 mM Tris-HCl, pH 7.5; 100 mM KCl; 50 mM
Na2EDTA, pH 8.0; 700 mM sucrose, and 2%
2-mercaptoethanol added immediately before use) in a
15 ml Oak Ridge tube and vortexed vigorously for 1 min.
The mixture was then homogenized for 30 s using
Polytron Brinkmann homogenizer model PT10/35
(Brinkmann instrument company, Cantiague road,
Westbury, NY) and shaken vigorously for 10 min at
level 7 on a Evapo-Mix shaker (Buchler instruments,
Fort lee, NJ). The organic phase (upper layer) was
collected by centrifugation at 4000g for 15 min at room
temperature, mixed with equal volume of protein
extraction buffer, shaken again for 10 min and centrifuged. The re-extracted phenol phase was collected,
mixed with 5 volumes of methanol containing 100 mM
ammonium acetate (NH4AC), and placed at 201C
overnight to precipitate protein. Protein pellet was
collected by centrifugation at 1500g at room temperature, washed three times with 10 ml methanol-NH4AC
solution, one time with 5 ml ice-cold acetone, and airdried for 30 min at room temperature.
In both of the above procedures the protein pellets
were resuspended in 1:1 (vol:vol) 2X SDS polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer
(20% glycerol, 4% SDS, 10% DTT, 0.126 mM tris-HCl,
pH 6.8). Protein concentrations were estimated by
spotting small volumes (ca. 1 ml) of resuspended sample
extracts and dilutions of bovine serum albumin (BSA) of
known concentrations onto nitrocellulose paper. After
allowing spots to dry (ca. 5 min at room temperature),
the paper was rehydrated in 100 ml dH2O for ca. 1 min,
then washed in 100 ml 12 mM HCl for ca. 1 min, and,
finally, in 100 ml of a 12 mM HCl solution containing
50 mg copper[II]phthalocyanine 3,4,40 ,400 tetrasulfonic
acid (CPTS) for 5–10 min, which is a modification of a
procedure described by Bickar and Reid (1992). Sample
protein concentration was estimated using the BSA
spots staining intensity as a reference. The procedures
for SDS-PAGE and Western immunoblots were as
previously described (Stout et al., 1997).
Rabbit antisera used for the detection of hsps on
Western blots were as follows. For shsp, antisera against
class I low-molecular weight hsp from Arabidopsis
thaliana (Wehmeyer et al., 1996) and Pisum sativa
(DeRocher et al., 1991) or Oryza sativa (Jinn et al.,
1993) were used. For hsp 70, antiserum against Pisum
sativa hsp 70 (DeRocher and Vierling, 1995) was used.
For hsp 101, antisera against Arabidopsis thaliana
(Queitsch et al., 2000) and Triticum sp. (Wells et al.,
1998) hsp 101 were used. The cross-reactivity of these
antibodies to proteins present in crude protein extracts
from D. lanuginosum and corn seedlings grown in the lab
was tested as follows. Plant specimens (0.5–2 g fresh
549
weight) were incubated in a medium containing 1%
sucrose and 5 mM potassium phosphate buffer, pH 6.0
(Jinn et al., 1989), for 2–4 h at either room temperature
(22–251C) or 401C (heat-treated). Protein extracts were
obtained from these plants and immunoblots were
performed as described above.
3. Results
The expression of small cytoplasmic class I hsps, hsp
70, and hsp 101 in D. lanuginosum in response to shortterm (2 h) heat treatment (451C) was analyzed using
protein blotting with antibodies to these hsps. Constitutive expression was determined in plants maintained at
ca. 21–251C. Only proteins reacting with anti-pea hsp 70
were detected at this temperature in both leaf and root
extracts from D. lanuginosum as well as in pea seedlings
(Fig. 1). Heat-induced expression was determined after
treating whole seedlings for 2 h at 371C (pea and
Arabidopsis) or at 451C (D. lanuginosum). This treatment
strongly induced proteins reacting with the anti-pea and
anti-Arabidopsis small hsps and with the anti-Arabidopsis hsp 101 antibodies in all the extracts tested (Fig. 1).
Also, increased expression over constitutive levels of
proteins reacting with anti-pea hsp 70 in extracts from
both pea and D. lanuginosum resulted in response to this
heat treatment (Fig. 1).
A time course of heat-induced hsp expression
indicates that immunodetectable levels of shsp are
Fig. 1. Hsp expression in glasshouse-grown D. lanuginosum in
response to short-term heat treatment. Total proteins (extracted
using the PVP method) from either 2-week-old Arabidopsis or
Pisum (pea) plants treated for 2 h at 251C (c) or at 371C (hs) or
roots and leaves from 6- to 8-week-old individual D.
lanuginosum plants treated for 2 h at 251C (c) or at 451C (hs)
were electrophoretically separated on SDS-polyacrylamide gels
(10–15 mg protein per lane), and then transferred to nitrocellulose paper. These Western blots were reacted with antibodies
against Arabidopsis hsp 101 and hsp 17.6 and pea hsp 70 and
hsp 18.1.
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T.S. Al-Niemi, R.G. Stout / Journal of Thermal Biology 27 (2002) 547–553
Fig. 2. Time-course of heat-dependent expression of shsp in
glasshouse-grown D. lanuginosum. Total proteins (extracted
using the PVP method) from 6- to 8-week-old whole D.
lanuginosum plants treated at 451C for 30, 60, 90 and 120 min
and for 120 min at 251C (c120) were electrophoretically
separated on SDS-polyacrylamide gels (10–15 mg protein per
lane), transferred to nitrocellulose, and reacted with antibodies
against pea hsp 18.1.
were observed (e.g., Lane C, Fig. 4). Heat-induced hsp
expression was determined using D. lanuginosum plants
grown at an average root temperature of 421C (711C).
This treatment induced proteins in extracts from both
YNP and Texas plants reacting with antibodies to shsp
at all the time points tested, up to 18–20 days of
continuous heat exposure (Fig. 4). In plants derived
from YNP thermal areas, though hsp 101 levels initially
increased in response to the heat treatment, they
typically returned to near, or below, control levels after
5–20 days of heat. In plants derived from Texas
normothermic populations, hsp 101 levels typically
increased in response to heat and remained elevated.
4. Discussion
Fig. 3. Persistence of shsp and hsp 101 during recovery from
heat treatment. D. lanuginosum plants were grown for 17 days
using a waterbath system (see Materials and Methods). They
were initially treated for 3 days at an average rhizosphere
temperature of 42711C and then the heat was turned off
(arrow). After 24 h, waterbath temperature equilibrated with
the glasshouse temperature (22731C). Total proteins (extracted
using the phenol method) from either heat-treated (2 h at 401C)
7-day-old corn seedlings (HC) or D. lanuginosum roots collected
at day 0 (C), 3, 4, 6, 8, 13 and 17 were electrophoretically
separated on SDS-polyacrylamide gels (5 mg protein per lane),
and then transferred to nitrocellulose paper. These Western
blots were reacted with antiserum against class I low molecular
weight hsps from Oryza sativa or against Triticum sp. hsp 101.
present between 30 and 60 min of heat treatment and
peak between 60 and 90 min (Fig. 2).
Elevated levels of shsp and hsp 101 in the roots of
D. lanuginosum induced by a 3-day exposure to 421C
(711C) persist for 5–7 days at control temperatures
(22731C) following the heat treatment (Fig. 3). Hsp 101
levels return to control levels after 8–10 days at control
temperatures after the heat treatment. Small amounts of
immunodetectable shsp persist even after this recovery
time, compared to control roots (no heat treatment) in
which no detectable shsps are present.
The expression of small cytoplasmic class I shsp and
of hsp 101 in roots of D. lanuginosum, propagated from
plants adapted to geothermal areas in YNP and to
normothermic soils in Texas, in response to long-term
(weeks) heat treatments (42711C) of roots was also
analyzed. Constitutive expression was determined using
control D. lanuginosum grown at an average root
temperature of 221C (731C). Under control conditions,
no immunodetectable shsp and low levels of hsp 101
In seeking cellular explanations for the apparent
predominance of D. lanuginosum in the geothermally
heated soils of YNP, the expression of several hsps has
been investigated in plants from populations apparently
adapted to the chronically hot soils of such environments. One possible explanation is that these plants
constitutively express relatively high levels of hsps.
Another is that they strongly express hsps very rapidly
after exposure to high temperatures.
Our data support neither hypothesis. Both in the
constitutive expression of selected hsps and in the heatdependent response of shsp expression, D. lanuginosum
originating from some of YNP’s active geothermal areas
displays responses typical of most plants. Little or no
hsp expression is detectable in plants exposed to normal
temperatures (21–251C), with the exception of hsp 70,
which is constitutively expressed at moderate levels in
most plants tested. Sub-lethal heat shock induced the
expression of all hsps tested, with increased shsp
expression detectable between 30 to 60 min of heat
treatment. Again, these results are typical of most, if not
all, flowering plants of normothermic environments.
Though constitutive or unusually rapid expression of
small cytoplasmic hsps and hsp 101 appears not to be
factors contributing to the apparent adaptation of
D. lanuginosum to geothermal soils, another possible
factor may be the unusual persistence of elevated levels
of hsps once their expression is induced by heat
episodes. This may help to protect these plants from
subsequent extreme periods of heat. Previous evidence
(Chen et al., 1990; DeRocher et al., 1991; Jinn et al.,
1995) has indicated that heat-induced hsps may have a
half-life of 2–4 days after heat shock. Comparable
results for shsps and hsp101 were observed using D.
lanuginosum (Fig. 3), though small levels of shsps were
still detectable after 14 days of recovery from heat
exposure. Though control plants (no heat treatment)
displayed no detectable shsps, the small remnant
amounts of shsps observed here would likely have no
T.S. Al-Niemi, R.G. Stout / Journal of Thermal Biology 27 (2002) 547–553
551
Fig. 4. Hsp expression in glasshouse-grown D. lanuginosum in response to long-term heat treatment. D. lanuginosum plants, derived
from both geothermic (YNP) and normothermic (TEXAS) populations, were grown using a waterbath system at an average
rhizosphere temperature of 22731C (control) or 42711C (heat-treated) for up to 20 days. Total proteins (extracted using the phenol
method) from heat-treated (2 h at 401C) 7-day-old corn seedlings (HC), from control D. lanuginosum roots treated for 10 days (C), and
from heat-treated D. lanuginosum roots collected after 1, 5, 10, 17, and 20 days (YNP) and after 1, 3, 5, 7, 11, and 18 days (TEXAS)
were electrophoretically separated on SDS-polyacrylamide gels (5 mg protein per lane), and then transferred to nitrocellulose paper.
These Western blots were reacted with antiserum against class I low molecular weight hsps from Oryza sativa or against Triticum sp.
hsp 101.
significant effect on root survival of rapid (e.g.,
o30 min) exposure to extreme (lethal or near-lethal)
rhizosphere temperatures.
Our results show no significant differences in hsprelated responses by D. lanuginosum, compared to other
flowering plants, to short-term (hours) heat shock and to
recovery from heat treatment. It is possible, however,
that D. lanuginosum may predominate in geothermally
heated soils in YNP because it possesses unusual cellular
adaptations to long-term (weeks to months) heat
exposure.
Little experimental evidence exists regarding the
cellular effects of continuous long-term exposure of
plants to high (>401C) temperatures. In one of the few
papers dealing with this subject, the roots of three desert
plant species were subjected to long-term (4-week)
exposures to selected temperatures, including temperatures of 401C and higher (Drennan and Nobel, 1996). At
temperatures above 401C, all three species displayed
little or no root elongation due to less cell division and
decreased cell extension.
We have previously reported (Stout et al., 1997) on
the effects of prolonged high soil temperatures (13–16
weeks at 35–411C) on the growth of D. lanuginosum
grown from seed collected from populations in active
geothermal soils within YNP. Though we observed no
significant difference in root fresh weight in the control
versus the heat-treated plants, the roots grown in the
warmer soils were shorter and more highly branched
than the roots grown in cooler soils (23–271C). Clearly,
long-term high rhizosphere temperatures affect
D. lanuginosum root morphology. In characterizing the
effects of such treatments on root physiology, we have
examined the expression of two classes of hsps.
The expression of cytoplasmic class I shsp and hsp 101
was examined in D. lanuginosum grown in the glasshouse
under control (22731C) versus heated (42711C) rhizosphere conditions. These two hsps were chosen because
they have been previously implicated in mechanisms of
plant thermotolerance (see Introduction). Under control
conditions, no proteins reacting to anti-shsp antibodies
were detected throughout the course of the experiments
(typically lasting 3–4 weeks). During these experiments
low levels of immunodetectable hsp 101 proteins were
consistently observed under control conditions. This
finding is in general agreement with the literature
(Queitsch et al., 2000; Young et al., 2001). As expected,
heat treatment induced increased expression of both
shsp and hsp 101 proteins within a day. The levels of
heat-induced shsp remained high throughout the course
of these experiments (up to 20 days). This result
supports the hypothesis that cytoplasmic small hsps
are involved in long-term thermotolerance.
Unexpectedly, the immunodetectable levels of hsp 101
in heat-treated roots of plants derived from
D. lanuginosum adapted to geothermal soils in YNP
decreased after 5–7 days. In some cases, these hsp 101
levels decreased below the low levels detectable in the
control roots (see Fig. 4) or in roots recovering from a
relatively brief (3 days) heat treatment (see Fig. 3).
Repeated attempts to detect hsp 101 in protein extracts
from YNP D. lanuginosum roots exposed to heat for
longer than 10 days (e.g., overloading gels, long film
exposure times) met with failure, though high levels of
shsp were present in these same extracts. In contrast,
under the same conditions, plants derived from
D. lanuginosum collected from normothermic soils in
Texas displayed hsp 101 levels that remained elevated
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T.S. Al-Niemi, R.G. Stout / Journal of Thermal Biology 27 (2002) 547–553
compared to controls throughout the course of these
experiments (up to 18 days). A possible explanation for
this difference is that D. lanuginosum adapted to
geothermic environments of YNP may express proteins,
either other hsps or thermostable isoforms of key
enzymes, in response to long-term (days to weeks) high
temperatures not expressed by normothermic D. lanuginosum. Thus, sustained elevated hsp101 expression in
the geothermic adapted plants may not play a role in
chronic heat tolerance.
5. Summary
In attempting to elucidate cellular mechanisms
involved in the thermotolerance of D. lanuginosum
adapted to geothermally heated soils, we have examined
the expression of several hsps, including cytoplasmic
class I shsp and hsp 101, under a variety of heattreatment conditions. Our results show that, in response
to short-term (hours) heat shock, D. lanuginosum
displays results typical of most vascular plants examined. Since D. lanuginosum can withstand continuous
rhizosphere temperatures >401C for weeks to months in
geothermal soils in YNP, we examined the expression of
shsp and hsp 101 in response to long-term (weeks) heat
(42 711C) treatment of D. lanuginosum roots under
glasshouse conditions. In these long-term heat experiments, we compared individuals derived from
D. lanuginosum populations adapted to geothermic soils
of YNP and to normothermic Texas soils. Although
high levels of shsp were immunodetected throughout the
course of these heat treatments, the levels of hsp 101
unexpectedly decreased after 5–7 days of continuous
heat in the plants derived from YNP geothermic soils, in
contrast to plants derived from populations adapted to
normothermic soils, which displayed sustained elevated
hsp 101 levels. These results support the involvement of
cytoplasmic class I shsp in long-term thermotolerance in
D. lanuginosum adapted to both normothermic and
geothermic soils but do not support similar involvement
of hsp 101 in plants from YNP.
Acknowledgements
We thank Prof. Elizabeth Vierling (Arizona State
University), Prof. Daniel Gallie (UC Riverside) and
Prof. Chu-Yung Lin (National Taiwan University) for
their generous gifts of antibodies. This work was
supported by NASA Grant NAG 5-8807 to the
Montana State University Thermal Biology Institute.
This is journal article 2002-38 from the Montana
Agricultural Experiment Station.
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