Plant Science 130 (1997) 1 9

Heat- and acid-tolerance of a grass commonly found in

geothermal areas within Yellowstone National Park
Richard G. Stout a,*, Michael L. Summers b, Tulli Kerstetter a,
Timothy R. McDermott b
b

a
Department of Biology, Montana State Uni6ersity, Bozeman, MT 59717, USA
Department ofPlant, Soil and En6ironmental Science, Montana State Uni6ersity, Bozeman, MT 59717, USA

Received 2 May 1997; received in revised form 11 August 1997; accepted 25 August 1997

Abstract
Surveys of geothermally-heated environments in Yellowstone National Park have revealed an exceptionally
heat-resistant grass Dichanthelium lanuginosum. Individuals of this species were able to withstand rhizosphere
temperatures ranging from 40 to 57C. Long-term (July and August, 1996) rhizosphere temperature measurements at
three sites confirmed that geothermal heat maintained high soil temperatures during the night. Plants grown in the lab
from field-collected seed display significantly higher shoot fresh weight when grown at soil temperatures of 35 41C
vs. 2327C. Though there is no difference in root fresh weight of plants grown at these two temperature regimes,
the roots from the warmer soils are significantly shorter and more highly branched compared with plants grown in
the cooler soils. This species also displays acid tolerance both in the field, with rhizosphere pH B 3, measured at
several sites, and when grown in the lab. In response to increased temperature, individual D. lanuginosum plants,
either grown in the lab or collected in the field, expressed a low molecular weight protein that cross-reacted with heat
shock protein antibodies. 1997 Elsevier Science Ireland Ltd.
Keywords: Dichanthelium lanuginosum; Heat-resistance; Heat shock proteins; Geothermal; Yellowstone National
Park; Acid tolerance

1. Introduction
Abbre6iations: DTT, dithiothreitol; HSP, heat shock protein;
MES, 2[N-morpholino]ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; S.E.,
standard error; TTBS, Tween 20 in Tris-buffered saline; YNP,
Yellowstone National Park.
* Corresponding author. Tel.: + 1 406 9944912; fax: + 1
406 9943190; e-mail: ubirs@trex.montana.edu

High temperature stress can severely limit crop

productivity [1,2], and this may be an increasingly
significant problem in the future if global warming becomes a reality [3,4]. One approach to
physiological research in heat tolerance, is to

0168-9452/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved.

PII S 0 1 6 8 - 9 4 5 2 ( 9 7 ) 0 0 2 0 5 - 7

R.G. Stout et al. / Plant Science 130 (1997) 19

identify and study flowering plants adapted to

survive and reproduce in extreme thermal environments. For example, some of the most heattolerant plants have been found in sun-baked
deserts such as Death Valley, CA [5]. Since
geothermally-heated environments are usually
more stable than sun-heated soils, they may be
more likely to select for plants acclimated to the
thermal state, than hot deserts. However, very
little information exists regarding plants adapted
to such geothermal environments [6].
Yellowstone National Park (YNP) contains the
highest number of surface-geothermal features in
the world at a single location [7], and offers an
excellent opportunity to study heat-resistant flowering plants and mosses. A general consensus
appears to be that plants growing at chronic
soil-surface temperatures ] 40C would be considered to be heat-resistant [6,8 10]. In this report, we provide information regarding the
especially heat-resistant grass Dichanthelium
lanuginosum that is commonly found in geothermal environments within YNP.

2. Materials and methods

2.1. Field conditions, temperature and pH

measurements
The surface-geothermal nature of our study
sites usually fell into one or both of the following
broad categories: (a) hot springs and their discharge streams or (b) relatively barren, dry, hot/
steaming ground. The former has moist soils,
typically containing a higher proportion of humus
and with soil pH values more alkaline (within
broad limits) than the latter. Steaming ground
sites can generally be characterized as consisting
primarily of coarse soils of altered rhyolite (volcanic rock, high in silicates) with an acidic pH,
typically B 6, and often occurring on open
ground near steam vents. The precise physical
nature of each thermal site, however, varied somewhat with respect to soil type, pH, and moisture
content.
Soil temperatures were determined using a digital thermocouple thermometer (model 115, Bar-

nant, Barrington, IL) equipped with a 10 cm long

penetration probe (Barnant, c 603-1000). To
monitor and record the degree of diurnal rhizosphere (15 cm below the soil surface) temperature variations, we used data-loggers (Forestry
Suppliers, Jackson, MS) equipped with thermistor
sensor cables. For leaf surface temperature measurements, a surface microprobe (Barnant, c 6231000) was used.
Using a portable digital pH meter equipped
with gel-filled paper-pulp combination pH electrode (c 13-620-299, Fisher Scientific), soil pH
determinations conducted in the field were measured by making a slurry soil and distilled water
(1:2 by volume). Several soil samples, particularly
from the more acidic soils, were brought to the
lab for analysis, including pH verification.

2.2. Plant material, seed germination, growth

conditions
Vascular plant species were determined by comparing field-collected plants with type specimens
in the Montana State University Herbarium with
the assistance of herbarium staff. Plant material
was collected in the field for protein analyses in
the lab. Leaf or whole-plant specimens were immediately wrapped in aluminum foil and frozen in
liquid nitrogen. These specimens were kept in
liquid nitrogen until returned to the lab, where
they were stored at 80C. Seeds from D. lanuginosum were collected at several different study
sites within a 30 mile radius.
To germinate field-collected seeds of D. lanuginosum, they were first scarified using sandpaper
and surface-sterilized in a 0.8% (w/v) solution of
sodium hypochlorite (e.g. 15% (v/v) solution of
Clorox) containing 0.02% (v/v) Triton X-100 for
15 min. The seeds were then rinsed at least ten
times with sterile water and germinated on moistened filter paper in a Petri dish sealed with
parafilm. Alternatively, the seeds were germinated
in a Petri dish on 1% (w/v) agar containing Murashige and Skoog [11] complete medium (Gibco
BRL, Life Technologies, Grand Island, NY) and
1 mM MESKOH, pH 5.7.
When seedlings reached the 23 leaf stage, they
were transferred to vermiculite moistened with tap

R.G. Stout et al. / Plant Science 130 (1997) 19

water and grown at 25 27C under fluorescent

lights (16 h light/8 h dark). Plant trays were
loosely covered with plastic wrap for several
weeks to increase humidity so as to enhance
seedling establishment. When seedlings reached
the 710 leaf stage, they were transferred to 4 in
pots containing sand, vermiculite, or potting soil.
Plants were watered with deionized water three
times per week and with a mineral nutrient solution (Sterns Miracle-Gro, Port Washington, NY)
every 23 weeks. To grow plants at relatively high
soil temperatures, thermostat-controlled heating
mats (Hummert, Earth City, MO) were used. Soil
temperatures were monitored periodically using
the thermocouple thermometer described above.

2.3. Culture conditions under a range of pH

6alues
Seeds were germinated under sterile conditions
as described above. Three plants at the 2 3 leaf
stage were transferred to growth boxes which
consisted of two Magenta plant tissue culture
boxes (GA-7 vessel, Sigma, St. Louis, MO) arranged as a Leonard jar [12]. The bottom box
served as a reservoir for the nutrient solution [13]
which was transferred to the top box via a paper
wick that contained the sand support medium
(industrial quartz c 20/30). The pH of the nutrient solution was adjusted to the desired pH using
sulfuric acid. The upper box was sealed with a
light-transparent membrane that allows gas exchange (Closures, Sun cap, Sigma), and the entire
assembly was autoclaved. Four replicate growth
boxes at each pH were used. After 4 weeks incubation in a 32C growth chamber (16/8 h light/
dark), plants were harvested, dried, and weighed.

2.4. Protein extraction, gel electrophoresis

Plant material used for protein analyses consisted of the following: whole D. lanuginosum
plants collected in the field (during midday when
rhizosphere temperatures were typically the
highest), D. lanuginosum grown to the 7 10 leaf
stage in the lab, and 3-day-old soybean (Glycine
max) seedlings (without cotyledons). The labgrown D. lanuginosum plants and the soybean

seedlings were incubated in 50 ml of 5 mM potassium phosphate buffer, pH 6.0, at either 25 or

45C for 2 h before proteins were extracted.
A total-protein extract from whole plants (except where noted) was obtained as described by
Jinn et al. [14]. Briefly, plant material (0.51.5 g)
was ground using a mortar and pestle at room
temperature (25C) in SDS extraction buffer [50
mM TrisHCl, pH 8.5, 2% (w/v) sodium dodecyl
sulfate (SDS), 1% (w/v) dithiothreitol (DTT) and
1 mM phenylmethylsulfonylfluoride] for 23 min
using 3 ml buffer/g tissue. Grindate was filtered
through cheesecloth and centrifuged at 2000 g
for 5 min at room temperature. The supernatant
was transferred to a 30 ml glass Corex tube, and
to this 5 volumes of 20C acetone was added.
This was sealed with parafilm and stored at
20C overnight. The precipitate was pelleted by
centrifuging at 2000 g for 5 min. The supernatant was poured off, the pellet resuspended in 12
ml of SDS extraction buffer and transferred to
Eppendorf tubes. This cloudy solution was
clarified by incubation in a boiling water bath for
5 min and then centrifugation in a microfuge
(10 000 g) for 2 min. The resulting clear supernatant was used for SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot analysis.
Protein extracts were fractionated on the basis
of molecular weight using SDS-PAGE as described by Laemmli [15]. Gels (8 cm 7 cm 1.5
mm) consisted of a 4% (w/v) acrylamide stacking
gel and a 12.5% (w/v) acrylamide running gel
using a mini-gel apparatus (SE 260 from Hoeffer
Pharmacia Biotech, San Francisco, CA). The
samples (1025 mg protein per sample) were run
for 90120 min at 20 mA (constant current) per
gel. Protein molecular weights were estimated using prestained low-range SDS-PAGE standards
(Bio-Rad Laboratories, Hercules, CA).

2.5. Western blot analysis

Proteins were transferred from the gel to nitrocellulose paper (0.2 mm Hybond-ECL from Amersham Life Sciences, Arlington Heights, IL) as
follows. Immediately after electrophoresis was
completed, gels were soaked in transfer buffer [16]
containing 0.1% (w/v) SDS for 1520 min before

R.G. Stout et al. / Plant Science 130 (1997) 19

electroblotting. Electrotransfer was accomplished

using a semi-dry electrophoretic transfer cell (BioRad Laboratories) according to the instructions
provided. After transfer, the nitrocellulose paper
was stained to detect proteins [17], photographed,
and destained before immunostaining.
The blots were blocked using milk buffer [20
mM TrisHCl, pH 7.6, 0.8% (w/v) sodium chloride, 0.1% (v/v) Tween-20, and 5% non-fat powdered milk] overnight at 5C. The primary
antibody was prepared against one of the soybean
15 18 kDa heat shock proteins (HSPs) as previously described [18] and was diluted 1:2000 in
milk buffer. The blocked blots were incubated for
2 h on a shaker (50 rpm) at room temperature in
this primary antibody solution (0.3 ml solution/
cm2 blot) and then rinsed 3 times in TTBS [20
mM TrisHCl, pH 7.6, 0.8% (w/v) sodium chloride, and 0.1% (v/v) Tween-20]. Blots were incubated in secondary antibody (1:1000 dilution in
milk buffer of donkey anti-rabbit IgG conjugated
to horseradish peroxidase; 0.3 ml solution/cm2
blot) for 1 h at room temperature on a shaker and
then rinsed three times with TTBS. Antibodybinding was visualized by using an enhanced
chemiluminescence system (ECL from Amersham)
following the protocol suggested by the manufacturer.

contributed to the measured temperature. To estimate the geothermal component of the heating,
the rhizosphere temperature of three different individual D. lanuginosum plants at three different
study sites was measured over the course of 2030
days during July and August, 1996. The results
are shown in Fig. 2. This figure indicates that, as
expected, diurnal temperature fluctuations do occur in these geothermally-heated sites. The degree
of this diurnal temperature oscillation varied considerably, however, depending on the site, ranging
from 13 (Fig. 2a) to 6C (Fig. 2b). From the
average low temperatures determined for each site
(see Fig. 2), the contribution of geothermal heat
to the rhizosphere temperatures was estimated to
range from 33 (Fig. 2a) to 47C (Fig. 2b). By
contrast, the low rhizosphere temperature of several grasses growing in nonthermal soils nearby
the geothermal sites ranged from 10 to 14C
during the same period of time (data not shown).
Though of secondary importance to this study,
the leaf surface temperatures of some plants at
most study sites were also measured. These temperatures ranged widely, depending on the following factors: exposure to sunlight, air temperature,
windiness, and proximity to hot water, hot soil or
steam. In general, the maximum leaf temperatures
were observed to range from 38 to 42C.

2.6. Statistical analyses

Differences between treatments with respect to
the effects of soil temperature on shoot and root
growth as well as root length were analyzed by
the Students t-test [19].

3. Results

3.1. Heat tolerance

From surveys of over 20 different geothermal
areas within YNP, D. lanuginosum was found
growing in soils with rhizosphere temperatures
(2 5 cm below the soil surface) ranging from
40 57C (see Fig. 1). Since most of these measurements were taken during midday, it was presumed that both solar- and geothermal-heating

Fig. 1. Rhizosphere temperature range distribution of D.

lanuginosum at different thermal areas within YNP. Rhizosphere temperatures were recorded for heat-resistant individuals (growing at a soil temperature ] 40C at 2 5 cm below
soil surface). Total number of individual plants surveyed=
128.

R.G. Stout et al. / Plant Science 130 (1997) 19

In growth experiments in the lab, these plants

were found to grow at both relatively cool (23
27C) and warm (3541C) soil temperatures as
shown in Table 1. Though they display significantly more shoot fresh weight at the warmer
temperatures (PB 0.001), the plants displayed no
significant difference in root fresh weight (PB
0.05). We have also observed that roots from
plants grown at the warmer soil temperatures are
significantly shorter (PB0.001), are more numerous, and are much more highly branched when
compared with plants grown at the cooler soil
temperatures (see Table 1).

3.2. Heat shock protein expression

Fig. 2. Long-term rhizosphere temperatures of individual D.

lanuginosum plants at three thermal sites. The rhizosphere
temperatures (2 5 cm below the soil surface) were monitored
and recorded :once/h for 2030 days during July and August, 1996. (The arrow in panel c shows when the data-loggers
remote thermistor was somehow dislodged from rhizosphere
of the plant to fall into the adjacent thermal stream. The
readings to the right of the arrow indicate the temperature of
this thermal stream over time).

An important initial step toward elucidating the

physiological nature of D. lanuginosums ability to
grow in thermal soils is to determine whether this
species expresses some proteins in a heat-dependent manner. To investigate this, we used antibodies prepared against one of the soybean
(Glycine max) 1518 kDa heat shock proteins
(HSPs) [18]. These HSPs were selected because
they are expressed in a heat-dependent manner
and because the expression of such small HSPs is
correlated with the development of thermotolerance [14,20]. D. lanuginosum seedlings grown at a
soil temperature of 2527C did not express a
protein recognized by these antibodies (Fig. 3b,
lane C) when incubated at 25C for 2 h in a
buffered sucrose solution. However, in plants
grown and treated in the same way, except that
they were exposed to 45C for 2 h, two low
molecular weight protein bands cross-reacted with
these HSP antibodies in both root and leaf
protein extracts (Fig. 3b, lanes D and E). In
addition, whole-plant protein extracts from fieldcollected D. lanuginosum plants taken from soils
with a temperature of 32C do not display these
bands (Fig. 3b, lane F), whereas cross-reactive
proteins were detected in extracts from D. lanuginosum plants harvested from hot (48C) soils (Fig.
3b, lane G). Similar experiments, using antibodies
against low molecular weight (17.618.1 kDa)
HSPs from Arabidopsis thaliana and pea (Pisum
sati6a), have yielded similar results (data not
shown).

R.G. Stout et al. / Plant Science 130 (1997) 19

Table 1
Effect of soil temperature on growth of Dichanthelium lanuginosum seedlings
Soil temperature Range (C)

2327
2527
3640
3541
a
b

(Experiment
(Experiment
(Experiment
(Experiment

1)a
2)b
1)
2)

Mean fresh weight (g)

Mean root length (cm)

Roots

Leaves

0.3490.06
0.429 0.09
0.389 0.05
0.409 0.04

1.09 9 0.10
1.39 90.13
1.72 90.28
2.27 90.54

15.5 9 0.94
18.6 9 1.52
7.6 9 1.23
7.8 90.87

Experiment 1, data are from 13-week-old plants grown in sand on a light table in the lab (see Section 2). Mean 9S.E., n =12.
Experiment 2, data are from 16-week-old plants grown in vermiculite on a light table in the lab (see Section 2). Mean 9 S.E., n = 9.

3.3. Acid tolerance

Though this species is not restricted to acidic
soils in the surveys conducted for this study,
individual D. lanuginosum plants were repeatedly
observed in very acidic soils (pH B3) within thermal areas of YNP (Fig. 4). This figure shows that
: 20% of the plants had an apparent rhizosphere
pH 53.5. D. lanuginosum plants growing at such
acidic pH values lack apparent morphological
characteristics that distinguish them from plants
of the same species growing in soils with a more
neutral pH. In addition, there was no correlation
between rhizosphere pH and temperature.
D. lanuginosum also tolerates acidic solutions
when grown in sand cultures supplemented with
mineral nutrients (Fig. 5). Grown axenically, these
4-week-old seedlings displayed similar amounts of
both shoot and root growth over a range of pH
values (36.6). Though root growth was restricted
at pH 2 and below, some shoot growth was
evident at pH 2.

4. Discussion
A useful approach to research into the physiology of plant stress tolerance has been to study
plants living in extreme environments [5,21]. Very
little is known about plants adapted to extreme
environments such as geothermal areas [6] which
are characterized by the presence of hot springs,
geysers, and steaming ground [7,8]. Much more is
known about hot springs and their algal vegetation [22] than about the mosses and vascular

plants that grow adjacent to hot springs and on

steaming soils [6]. In surveying such areas within
YNP, we have found that the perennial grass D.
lanuginosum is often found growing in these
geothermally-heated environments. In the survey
conducted for this study, this species, also known
as Panicum thermale or hot springs panic grass,
was the most common heat-resistant vascular
plant found at YNPs thermal features and was
not observed growing independently of geothermally heated soils. D. lanuginosum has apparently
adapted to the stressful conditions imposed by
these extreme environments.
As shown in Figs. 1 and 2, individual D. lanuginosum plants can tolerate extremely high rhizosphere temperatures for extended periods of time.
The rhizosphere temperature fluctuations shown
in Fig. 2 are diurnal in nature. It is important to
note, however, that the low (night) temperatures
in two of the plants (Fig. 2b and 2c) never
dropped below 35C over the course of several
weeks. The ability of some individual D. lanuginosum plants to withstand such long-term soil
temperatures ranks them among the most heattolerant plants known [6,9]. Although other
grasses, such as varieties of pearl millet (Pennisetum glaucum), can survive high soil surface temperatures [23], we have found no reports in the
literature of vascular plants able to tolerate the
chronic high temperatures we report here. Because of the heat-stability of the geothermal soils,
the roots of these plants must be adapted to the
thermal state. D. lanuginosum possesses apparent
HSPs [14,18,20] that protect macromolecules and
organelles during chronic heat stress and, presumably, also some thermostable enzymes.

R.G. Stout et al. / Plant Science 130 (1997) 19

Although the definition of a thermophilic plant

appears somewhat arbitrary in the literature
[9,10,24,25], the ability of D. lanuginosum to proliferate under unusually high-temperatures (e.g.
chronic rhizosphere temperatures above 45C)
clearly places this plant into a novel category as
compared with other vascular plants. Under the
growth conditions used in this study, D. lanuginosum displayed higher shoot fresh weight at

Fig. 4. Rhizosphere pH range distribution of D. lanuginosum

at different thermal areas within YNP. pH values were determined for soil samples collected from within the rhizosphere of
heat-resistant individuals (growing at a soil temperature ]
40C at 2 5 cm below soil surface). Total number of individual plants surveyed = 56.

warm soil temperatures (3541C) than at 23

27C (see Table 1), though the root fresh weight
was not so affected. Although the roots from
plants grown at the cooler soil temperatures were
longer than those grown at the warmer temperatures, the higher-temperature plants had more
numerous short roots. This accounts for the similarity in the root fresh weight between the two
groups of plants. Our observation of shorter,

Fig. 3. Immunoblot of protein extracts from D. lanuginosum

using an antibody against a soybean small heat shock protein.
Proteins were separated by SDS-PAGE, transferred electrophoretically to nitrocellulose paper and stained for protein
(panel a), then probed with an antibody to a class I low
molecular weight heat shock protein from soybean (panel b).
Lane A, molecular weight markers; lane B, extract (32 mg
protein) from heat-treated soybean seedlings as a control; lane
C, whole-plant extract (15 mg protein) from D. lanuginosum
grown at 25C and treated for 2 h at 25C; lane D, shoot
extract (18 mg protein) from 25C-grown D. lanuginosum
treated for 2 h at 45C; lane E, root-extract (10 mg protein)
from same plants used in lane D; lane F, whole-plant extract
(20 mg protein) from D. lanuginosum collected in the field
(rhizosphere temperature = 32C); lane G, whole-plant extract
(22 mg protein) from D. lanuginosum collected in field (rhizosphere temperature = 48C). Blots are representative of results
obtained from several experiments.

Fig. 5. Graph of root and shoot dry-weights of D. lanuginosum

grown in sand cultures under a range of pH conditions. Each
point is the average of four replicate boxes, each containing
three plants. Error bars, where visible, denote the S.E.

R.G. Stout et al. / Plant Science 130 (1997) 19

more highly-branched root systems in the plants

grown at the higher soil temperatures could be the
result of increased ethylene production as a consequence of heat stress on the roots [26]. At the
present time we have no data regarding the effects
of heat on hormone production in these plants.
D. lanuginosums remarkable ability to endure
chronically high root temperature in the field render it worthy of physiological and cellular investigations. Our initial physiological studies are
consistent with the idea that heat resistance (at
least to temperatures above 45C) of this plant is
acquired. This conclusion is primarily based on
the temperature-dependent expression of a low
molecular weight HSP (Fig. 3) observed both in
laboratory-grown and field-collected plants. Some
low molecular weight HSPs can thermostabilize
soluble proteins in vitro [14], probably by acting
as molecular chaperones [20]. The results shown
in Fig. 3 are significant in that they are consistent
with the notion that at least some of the molecular mechanisms responsible for the heat resistance
of D. lanuginosum are inducible and, thus, are
experimentally approachable.
In addition to displaying tolerance to high soil
temperatures, D. lanuginosum can also withstand
extremely acidic soils (Figs. 4 and 5). The failure
of most plants to grow in acidic soils may be due
to a high hydrogen ion concentration, to the
effects of acidic soil pH on nutrient availability,
or the presence of toxic amounts of aluminum or
manganese or both [27,28]. At the present time, it
is not known how D. lanuginosum overcomes
these problems in order to survive in geothermally-heated soils. When grown in nutrient solutions under a range of pH values, this plant
species displayed shoot growth from pH 2.5 to 3.5
and root growth at pH 3.5. These results, when
compared with previous studies [28], suggest that
D. lanuginosum is remarkably acid tolerant.
This report presents data that documents the
occurrence and proliferation of the vascular plant
D. lanuginosum in environments that are extreme
with respect to soil temperature and pH. It is
likely that this interesting species represents a
promising resource for the study of stress physiology in plants.

Acknowledgements
The authors thank C. Seibert and J. Rumely for
assistance in plant identification, C.-Y. Lin for
kindly providing the antibodies to the soybean
heat shock protein, L. Wester for statistical analyses, and M. Johnson for technical assistance. This
work was supported by National Science Foundation grants OSR-9350546 (RGS) and IBN9420798 (TRM), by USDA NRICGP grant
95-37106-2447 (RGS) and by the Montana State
University MONTS program.

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