AMER. ZOOL., 39:865-876 (1999)
Small Heat Shock Proteins Protect Electron Transport in Chloroplasts and
Mitochondria During Stress1
SCOTT A. HECKATHORN,* 2 - 3 CRAIG A. D O W N S , * AND JAMES S. CoLEMANt
* College of Charleston, Department of Biology, 58 Coming St., Charleston, South Carolina, 29424
tDesert Research Institute, 2215 Raggio Parkway, Reno, Nevada, 89512-1095
SYNOPSIS. Evidence indicates that small heat-shock proteins (Hsps) are involved
in stress tolerance, but the specific cell components or functions that small Hsps
protect or repair are mostly unidentified. We recently showed that the chloroplast
small Hsps of higher plants (1) are produced in response to many environmental
stresses (e.g., heat, oxidative, and high-light stress); and (2) protect (but do not
repair) photosynthetic electron transport in vitro during stress, specifically by interacting with the oxygen-evolving-complex proteins of Photosystem II (PSII) within the thylakoid lumen. However, in vivo evidence of the importance of these Hsps
to photosynthetic stress tolerance is lacking. Here we report positive relationships
between chloroplast small Hsp production and PSII thermotolerance in (1) a heattolerant genotype of Agrostis palustris (bentgrass) and a heat sensitive genotype
which lacks one or more chloroplast small Hsps produced by the tolerant genotype;
(2) ecotypes of Chenopodium album (lambs quarters) from the northern vs. southern U.S. (New York vs. Georgia); and (3) nine Lycopersicon (tomato) cultivars/
species differing in heat tolerance. These in vivo results are consistent with our
previous in vitro observations and indicate that genetic variation in production of
the chloroplast small Hsp is an important determinant of photosynthetic and,
thereby, whole-plant thermotolerance. Recently, we showed that the mitochondrial
small Hsp of plants protects respiratory (specifically Complex I) electron transport
in vitro during heat stress, and here we present evidence for previously unidentified
small Hsps in mitochondria of mammal (rat) cells which also protect Complex I
during heat stress. These results suggest that the mitochondrial small Hsps, like
the small chloroplast Hsps, are general stress proteins that contribute significantly
to cell and organismal stress tolerance.
INTRODUCTION
Small Hsps
Most small Hsps range in size from 1535 kD, are related to the a-crystallin proteins, and share a highly evolutionarily conserved domain referred to as the "heatshock domain" (Vierling, 1991; Arrigo and
Landry, 1994; Boelens and De Jong, 1995;
Caspers et al, 1995; Waters, 1995; Waters
et al, 1996). Virtually all cells examined to
date make one or more small Hsps, which
are often produced only in response to
'From the Symposium on Organismal, Ecological
and Evolutionary Significance of Heat Shock Proteins
and the Heat Shock Response presented at the Annual
Meeting of the Society for Comparative and Integrative Biology, 6-10 January 1999, at Denver, Colorado.
2
3 E-mail: [email protected]
3
stress (Hagemann et al, 1991; Vierling,
1991; Arrigo and Landry, 1994; Boelens
^ D e JonS> l995> Caspers et al, 1995;
Waters, 1995). There is ample evidence that
sma11 Hs s
P PlaY ^ important role in cell
stress
tolerance (e.g., Arrigo and Landry,
*994; Waters et al, 1996). For example, in
vitro
sma11 Hs s
P (including those from
plants) can prevent heat-induced protein aggregation or denaturation and/or help renature heat- or chemically denatured model
enzymes or whole-cell protein extracts
(Jinn et al, 1989; Merck et al, 1993; Lee
et al, 1995, 1997). However, the specific
u
o r
fu n c t i o n s
tha t
small
Hsps protect or repair in vivo are mostly
unidentified,
JQ contrast to most organisms, plants USUally produce more than 20 small Hsps, and
J
New address: Syracuse University, Department of
Biology, 130 College Place, Syracuse, New York,
13244.
c o m p ro n e n t s
sm
\
r
i
_
I
_
J
J
a l l Hsps are often the most abundant and
stress responsive group of Hsps in plants
865
866
S. A. HECKATHORN ETAL.
(Vierling, 1991; Howarth and Ougham,
1993; O'Connell, 1994; Waters et al,
1996). At least five classes of small Hsps
are recognized in plants, two of which contain proteins that localize to the cytosol, and
three of which contain small Hsps that localize to ER, mitochondria, and plastids
(e.g., chloroplasts), respectively. Most of
these small Hsps appear to be nuclear encoded (Vierling, 1991; Waters, 1995; Waters et al, 1996).
Chloroplast small Hsps
Chloroplast-localized small Hsps were
discovered by Kloppstech, Vierling, Restivo, and their co-workers (Kloppstech et al.,
1985; Restivo et al, 1986; Vierling et al,
1986). Early work indicated the existence
of only one chloroplast small Hsp isoform,
but more recent work indicates that more
than one isoform is produced by most species examined to date (e.g., Park et al,
1996; Downs et al, 1999a). Most chloroplast small Hsps are apparently nuclear-encoded (Kloppstech et al, 1985; Vierling et
al, 1986; Heckathorn et al, 1998a), but
there is some evidence to suggest that chloroplast small Hsps can be encoded by the
chloroplast genome (Krishnasamy et al,
1988).
Several early lines of evidence suggested
that the chloroplast small Hsps play a role
in photosynthetic and plant thermotolerance. (1) The chloroplast small Hsp is often
the most abundant and heat responsive of
the plastid Hsps (e.g., Restivo et al, 1986;
Vierling, 1991; Clarke and Critchley,
1994). (2) The chloroplast small Hsp is
highly evolutionarily conserved among
plants and algae (Grimm et al, 1989; Vierling et al, 1989; Waters et al, 1996; Downs
et al, 1998). (3) Phenotypic variation in
production of the chloroplast small Hsp is
correlated with thermotolerance of photosynthesis (specifically Photosytem n, PSII,
the water-oxidizing quinone-reducing protein complex) (Schuster et al, 1988; Stapel
et al, 1993; Clarke and Critchley, 1994;
Heckathorn et al, 1996a). (4) Greater production of the chloroplast small Hsps, both
within (Park et al, 1996; Joshi et al, 1997)
and among species (Downs et al, 1998), is
positively correlated with whole-plant thermotolerance.
Recently, we confirmed that in vitro the
chloroplast small Hsp specifically protects
(but does not repair) PSII function, and
thereby whole-chain electron transport, during heat stress (Heckathorn et al, 1998b;
Downs et al, 1999a). We have also shown
that at least one chloroplast small Hsp localizes to the thylakoid membranes, confirming earlier studies (Kloppstech et al,
1985; Glaczinski and Kloppstech, 1988;
Krishnasamy et al, 1988; Schuster et al,
1988; Grimm et al, 1989; Adamska and
Kloppstech, 1991), and associates with the
Oxygen-Evolving-Complex (OEC) proteins
of PSII (Downs et al, 1999a). Further, we
demonstrated that the chloroplast small Hsp
is produced by leaves in response to a number of other stresses (e.g., drought, highlight, UV-A, and oxidative stress) and protects PSII in vitro during photoinhibitory
high-light and oxidative stress (Downs et
al, 1999&), as suggested earlier (Schuster
et al, 1988; Stapel et al, 1993). Taken together, the evidence indicates that the chloroplast small Hsps are general stress proteins that protect PSII, and consequently,
affect photosynthetic and whole-plant stress
tolerance.
However, in vivo evidence of the importance of genetic variation in the chloroplast
small Hsp to photosynthesis is still lacking.
Further, we have an incomplete understanding of the patterns and importance of natural variation in chloroplast small Hsp production and the extent to which this contributes to variation in organismal thermotolerance and the distribution of species
(Coleman et al, 1995). In this paper, we
examine the relationship between production of chloroplast small Hsps and PSII
thermotolerance in three different taxa. Relationships between natural genetic variation in chloroplast small Hsp production
and thermotolerance would suggest that
variation in production of these Hsps is a
trait on which natural selction can act, and
thus would yield insight into the ecological
and evolutionary importance of chloroplast
small Hsps.
SMALL HSPS IN CHLOROPLASTS AND MITOCHONDRIA
Mitochondrial small Hsps
Mitochondrial small Hsps are closely related to chloroplast small Hsps, exhibiting
high similarity in the C-terminal half of the
proteins, but differing in the N-terminal
halves (Lenne et al., 1995; Waters et al.,
1996; Debel et al, 1997; Lund etal, 1998).
Some mitochondrial small Hsps localize to
the mitochondrial matrix and some to membranes of the mitochondria (Borovskii and
Voinikov, 1993; Lenne and Douce, 1994).
There is evidence to suggest a role for mitochondrial small Hsps in stress tolerance
in plants and fungi (Chou et al., 1989; Plesofsky-Vig and Brambl, 1995). We recently
showed that plant mitochondrial small Hsps
protect respiratory electron transport by
specifically protecting Complex I (NADHubiquinone oxidoreductase) function
(Downs and Heckathorn, 1998). Although
small Hsps localize to mitochondria in
plants and fungi, mitochondria-localized
small Hsps have not yet been identified in
vertebrates (Arrigo and Landry, 1994; Waters et al., 1996). Here we present evidence
for localization of small Hsps to the mitochondria of rat cells and for protection of
Complex I function during heat stress by
these Hsps, as with plant mitochondrial
small Hsps.
METHODS
Plant material
Relationships between chloroplast small
Hsp production and PSII thermotolerance
were examined in three different taxa: (1)
in a heat-tolerant genotype of Agrostis palustris Huds. (creeping bentgrass) and a heat
sensitive genotype which lacks one or more
chloroplast small Hsps produced by the tolerant genotype; (2) in ecotypes of Chenopodium album L. (lambs quarters) from the
cool northern vs. warm southern U.S. (New
York vs. Georgia); and (3) in nine Lycopersicon (tomato) cultivars/species differing in
whole-plant heat tolerance.
In the first experiment, vegetatively propogated clones of heat-sensitive and heat-tolerant genotypes of Agrostis palustris were
obtained from Dr. Dawn Luthe (Mississippi
State University, MS, USA). All plants
originated from a single seed, from which
867
stock callus was propogated (Park et al.,
1996). A thermotolerant callus genotype
was derived following selection for survival
at high temperatures. Both thermotolerant
and non-selected thermosensitive plants
were regenerated from callus. Increased
whole-plant thermotolerance in the selected
genotype was genetically correlated with
the production of 1-2 chloroplast small
Hsps {ca. 25-kD) not produced by the thermosensitive genotype (Park et al., 1996);
both genotypes produce a 27-kD chloroplast Hsp. Adult non-flowering plants were
grown in a controlled environment (25°C
days/20°C nights; 200 u-mol m"2 s'1 photosynthetic photon flux density (PPFD); 14hr photoperiod). A subset of plants of each
genotype were heat stressed by gradually
increasing temperatures from 25°C to 45°C
over an 8-hr period. Photosystem II function {i.e., Fv/Fm, see below) was monitored
before, during, and after the heat stress. The
following day, ¥J¥m was determined in
both pre-heat-stressed and control plants at
both 26 and 41°C (n = 4 plants for each
treatment combination).
In the second experiment, seeds of Chenopodium album were collected from naturally occuring plants near Athens, Georgia
and Syracuse, New York, USA. Plants were
grown from seed in a greenhouse under naturally fluctuating temperatures (15-30°C)
with ca. 12-hr days and 12-hr nights. Natural irradiance was supplemented with 200
(xmol m~2 s"1 PPFD provided by sodiumvapor lamps. When plants were 5 weeks
old, they were transferred to growth chambers for ten days (24°C, 14-hr days and
18°C nights; 400 (xmol m"2 s"1 PPFD).
Some plants of each ecotype were heat
stressed as in Heckathorn et al. (19986).
Leaves from pre-heat-stressed and control
plants of each ecotype were harvested for
determination of chloroplast small Hsp content prior to, and eight hrs after, initiation
of heat stress (n = 6) (as in Heckathorn et
al, 1996a, b; Downs et al. 1998). Briefly,
total leaf proteins were extracted in an
SDS-Tris buffer, fractionated by SDSPAGE, transferred electrophoretically to
PVDF membranes, and probed for the presence of chloroplast small Hsp using polyclonal antiserum specific to the chloroplast
868
S. A. HECKATHORN ETAL.
small Hsps. The content of small Hsp on
immunoblots was determined by scanning
densitometry, using a desktop scanner and
NIH software. Preliminary experiments
were conducted to ensure that the densities
of Hsp on immunoblots were within the linear range of the protein-density relationship. Triplicate lanes of each sample were
run on gels and densitometric values from
these lanes were averaged and then normalized among samples. Also, at eight hrs,
leaves were harvested for chloroplast isolation and photosynthetic electron transport
measurements (n = 3 for each treatment
combination) (see below).
In the third experiment, nine genotypes
of Lycopersicon (tomato) reputedly differing in whole-plant heat tolerance were acquired from the CM. Rick Tomato Genetics
Resource Center (University of CaliforniaDavis, CA, USA): L. esculentum Mill, cultivars Condine Red, Edkawi, Fireball, Gardener, Nagcarlang, Malintka, and Saladette;
L. esculentum v. cerasiforme (Santa Cecilia), from Napo, Ecuador; and L. chilense v.
Huaico Moquegua, from Moquegua, Peru.
Plants of each of the nine genotypes were
grown from seed in a greenhouse as above.
When the plants were 68 days old, they
were transferred to growth chambers (24°C,
14-hr days and 18°C nights; 400 u,mol m"2
s~' PPFD). Following a 36-hr acclimation
period, plants of each genotype were heat
stressed at 42°C for six hrs as in Heckathorn
et al. (19984>); some plants of each genotype remained at control temperatures
throughout. Leaf samples and FJPm data
were collected from each plant before, during, and after heat stress treatments (n = 4
plants for each time point and treatment
combination). Content of chloroplast small
Hsp was determined as described above.
Photosynthetic measurements
Photosynthetic data were collected from
recently expanded leaves from both heatstressed and control plants. The ratio of variable-to-maximum fluorescence of darkadapted leaves (FyFn,), a measure of Photosystem II function, was measured as before {e.g., Heckathorn et al. 1996a, 1991b).
Chloroplast isolation and measurements of
PSII-limited whole-chain electron transport
from PSII-to-PSI was carried out as previously described (Heckathorn et al, 1998b;
Downs et al, 1999a). Photosynthetic electron transport (Pet) and ¥J¥m thermotolerance were determined by calculating the ratio of Pel and Fv/Fm in heat-stressed plantsto-Pel and Fv/Fm in control plants.
Mitochondria! isolation and Complex I
activity
To determine if mammalian mitochondria
have mitochondrial-localized small Hsps
and whether these Hsps protect Complex I
electron transport during heat stress as in
plants, rat PC 12 cells (adrenal pheochromocytoma) were obtained from American
Type Culture Collection (Washington D.C.,
USA) and propagated at 36.5°C and 5%
CO2 in Dulbecco's modified Eagle's medium containing 10% heat-denatured fetal bovine serum, 1% glutamine, and 1% penicillin/streptomycin. Cells were heat stressed
by first ramping the incubator temperature
over one hour from 36.5°C to 38.5°C, maintaining temperature for one hour at 38.5°C,
then ramping the temperature over one hour
from 38.5°C to 41.5°C. The temperature
was then decreased from 41.5°C to 38.5°C
over an hour and maintained at 38°C for 4 6 hr, after which the temperature was decreased from 38°C to 36.5°C over an hour.
At this point, cells were harvested for mitochondrial isolation or whole-cell analysis.
Mitochondria were isolated from nonheat-stressed and heat-stressed cells using a
method modified from Almeida and Medina (1998). Cells were ruptured with a glass/
teflon homogenizer in a mitochondrial isolation buffer (MIB) consisting of 0.3 M sorbitol, 50 mM HEPES/KOH (pH 7.4), 2 mM
EDTA, 2.5 mM DTT, 4 mM L-cysteine, and
20 units of trypsin. The homogenate was
subjected to centrifugation at 1,500 X g for
10 min, after which the pellet was discarded
and the supernatant was centrifuged at
17,000 X g for 15 min. Following this second centrifugation, the supernatant was discarded and the pellet was then resuspended
in MIB. This partially purified preparation
was then subjected to the two differential
centrifugation steps again. The final pellet
was resuspended and layered on a discontinous Percoll step gradient (Downs and
SMALL HSPS IN CHLOROPLASTS AND MITOCHONDRIA
Heckathorn, 1998). The mitochondrial fraction was collected and washed with MIB
lacking trypsin, but containing trypsin inhibitor, and then centrifuged at 10,000 X g
for 10 min. The resulting pellet was resuspended in either a phosphate buffer or a
SDS-PAGE buffer, depending on the assay
(see below). Mitochondria samples to be
spectrophotometrically assayed were frozen
at — 80%C, thawed, and sonicated as in
Downs and Heckathorn (1998), to yield
submitochondrial vesicles (SMVs).
Complete Complex I activity (electron
transport from NADH to a quinone receptor) was measured in SMVs using a modification of the method of Singer (1974), and
followed the oxidation of NADH at 340 nm
using ubiquinone-1 (CoQ,) as the electron
acceptor (Downs and Heckathorn, 1998).
Submitochondrial vesicles assayed at control or heat stress temperatures were incubated with either no protein added, or the
addition of either anti-murine Hsp25 antibody (Ab25) (1:300 v/v) (StressGen, Victoria, BC, Canada, Cat# SPA-801), BSA (0.2
mg/ml), or rabbit IgG (1:300 v/v) (Sigma,
St. Louis, MO, USA). For enzymatic analysis of Compex I activity under heat stress
temperatures, SMVs were incubated at
48°C for 30 min in the presence of 1 mM
KCN (to inhibit electron transport downstream from Complex I) and 0.2 mM
NADH. A concentration of 40 jjLg/|xL of total SMV protein was used for all assays.
To determine if the mitochondrial samples were free of intracellular contamination, mitochondrial samples were solubilized in a buffer containing 1% SDS, 0.05
M Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM
DTT, boiled, and then subjected to SDSPAGE (15.5% polyacrylamide gels). Proteins were subjected to western blotting
(Downs and Heckathorn, 1998; Downs et
al, 1998) and assayed with either (1) antimitochondrial Hsp60 antibody (StressGen,
Victoria, BC, Canada, Cat# SPA-804, SPA805) to ensure that mitochondrial preparations were enriched in intact mitochondria,
(2) anti-aB-crystallin antibody (StressGen,
Victoria, BC, Canada, Cat# SPA-223) to
determine if cytosolic proteins were co-purifying with the mitochondria, or (3) antibody to acetylated Histone H4 (Upstate
869
Biotechnology, Lake Placid, NY, USA,
Cat# 06-761) to ascertain the extent of nuclei contamination. To determine if murine
Hsp25 or a homologue of Hsp25 associated
with the mitochondria, replicate blots were
assayed with antibody against recombinant
murine Hsp25 (same antibody as above).
Replicate gels were assayed by Coomassieblue staining and silver-staining to ensure
equal soluble protein loads per lane. Mitochondrial isolation and western immunoblot
assays were independently replicated four
times.
RESULTS
Photosystem II function, as indicated by
Fv/Fm, decreased at high temperatures in
Agrostis palustris, moreso following a prolonged heat stress (a gradual increase from
25 to 45°C over 8 hr) than following a 30min incubation at 41°C (Fig. 1). Fv/Fm did
not differ between heat-sensitive and heattolerant genotypes in control plants at 26°C
(Fig. 1). Heat-senitive and heat-tolerant genotypes also did not differ when at incubated at 41°C for only 30 min, which is
insufficient time for significant synthesis
and accumulation of Hsps. However, when
plants were pre-heat stressed for eight hrs
and then monitored the next day, allowing
sufficient time for Hsp accumulation both
during and after the heat stress, the heattolerant genotype exhibited significantly
higher ¥J¥m values than the heat-sensitive
genotype at both 26 and 41°C. These results
indicate that PSII sustained irreversible
damage during the prolonged heat stress
and suggest that production of the small
Hsps in the tolerant genotype that are lacking in the sensitive genotype imparted protection to PSII.
Increased photosynthetic thermotolerance was also positively related to chloroplast small Hsp production in ecotypes of
Chenopodium album (Fig. 2). Plants from
the warmer southern U.S. (Georgia) produced more chloroplast small Hsp than did
plants from the cooler northern U.S. (New
York). Thermotolerance of PSII-limited
electron transport was also greater in the
Georgia ecotype compared to the New York
ecotype. Among nine Lycopersicon (tomato) genotypes examined, a highly signifi-
870
S. A. HECKATHORN ETAL.
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The effect of heat stress on F /F , a measure
FIG. 1.
v m
of Photosystem II function, in heat-tolerant and heatsensitive genotypes of Agrostis palustris. F^F^ was
measured on recently expanded dark-adapted leaves
from either unstressed control plants lacking the chloroplast small Hsps or pre-heat-stressed plants containing the small Hsps. The heat-sensitive genotype lacks
the smallest of the three chloroplast small Hsps that
the tolerant genotype produces. Results are means +
1 SE; n = 4. Asterisks indicate significant differences
between genotypes (P < 0.05; ANOVA followed by
/-tests).
cant, positive linear relationship was observed between relative chloroplast small
Hsp content and PSII (Fv/Fm) thermotolerance (Fig. 3).
To determine if small Hsps associate with
the mitochondria of mammal cells, mitochondria from unstressed and heat-stressed
rat PC 12 cells were assayed with a polyclonal antibody to murine recombinant
Hsp25 (Ab25). In mitochondria from heatstressed cells, Ab25 exhibited a cross-reaction with at least four major bands (Fig.
4A). Trypsin treatment of lysed mitochondria indicated that these small Hsps were
susceptible to trypsin degradation (data not
shown).
In replicate blots, anti-aB-crystallin antibody reacted with a single band in whole-
FIG. 2. The effect of heat stress on maximum content
of the chloroplast small heat-shock protein (Hsps) and
photosynthetic thermotolerance (the ratio of PSII-limited whole-chain electron transport rates in heatstressed-to-control plants) in ecotypes of Chenopodium
album from Georgia and New York. For Hsp content,
results are means + 1 SE, n = 6. For photosynthetic
thermotolerance, results are ratios based on mean values of three replicates. Differences between genotypes
in Hsp content were marginally significant (P = 0.09;
f-test).
cell extracts, but did not react with comparable mitochondrial proteins, demonstrating that mitochondria were not contaminated with cytosolic proteins and that
aB-crystallin antibody did not cross-react
with the mitochondrial Hsps (Fig. 4B).
Anti-mitochondrial-Hsp60 antibody reacted
with single bands in both whole-cell and
mitochondrial samples, and mitochondrial
samples contained more Hsp60 than wholecell samples, showing that intact mitochondria were purified (Fig. 4C). Antibody
against Histone H4 reacted with single
871
SMALL HSPS IN CHLOROPLASTS AND MITOCHONDRIA
3.0-
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y = 7.2x - 1.0 i = 0.78 P = .002
•
A) sHsp
•
1.0-
_g eg
2^
e
I
F v /F m thermotolerance
FIG. 3. The relationship between thermotolerance of
Photosystem II {i.e., the ratio of Fv/Fm in heat-stressedto-control plants) and maximum content of the chloroplast small heat-shock protein (Hsp) for nine genotypes of Lycopersicon (n = 4). Least-squares linear
regression analysis was performed for the Hsp-thermotolerance relationship, and the resulting equation is
shown, along with r2 and P values from the associated
correlation analysis and ANOVA.
bands, but only in whole-cell samples, and
not mitochondrial samples, indicating that
there was no nuclei contamination of mitochondrial samples (Fig. 4D).
To determine if the mammalian mitochondrial small Hsps could protect Complex I activity from heat stress, as mitochondrial small Hsps do in plants, we used
the polyclonal anti-Hsp25 antibody as a
non-competitive inhibitor to disrupt the
function of the mitochondrial small Hsps,
as described previously for plant mitochondria and chloroplasts (Downs and Heckathorn, 1998; Heckathorn et al, 19986). The
rate of Complex I activity assayed at 28°C
was higher in non-heat-stressed mitochondria than in pre-heat-stressed mitochondria
(ANOVA; P < 0.0001), indicating that
Complex I was damaged by the cell-culture
heat-stress treatment (Fig. 5). The rate of
Complex I activity when assayed at 48°C
A
WC
hs
B) aB-crystallin
WC WC Mit Mit
con hs con hs
C) Hsp60
we
con
FIG. 4. Mitochondria were isolated from unstressed
and pre-heat-stressed murine {i.e., rat PC 12) cells, subjected to SDS-PAGE (40 (xg per lane), and Western
(immuno) blotting. (A) Western blot assayed with antibody specific to murine Hsp25 (sHsp). (B) Western
blot assayed with an antibody specific to aB-crystallin,
a cytosolic protein. (C) Western blot assayed with antibody specific to mitochondrial Hsp60. (D) Western
blot assayed with an antibody specific to Histone-H4,
a protein found in the nucleus. WC = whole cell; Mit
= mitochondria; con = control; hs = heat stress.
Mit
hs
Mit
con
Mit
con
WC
hs
Mit
hs
D) HistoneH4
we
con
WC
hs
Mit
con
Mit
hs
872
S. A. HECKATHORN
ETAL
400
> <~*
control
at 28°C
control
at 48°C
pre-heat
stress
at 28°C
pre-heat
stress
at 48°C
FIG. 5. The effect of antibodies to recombinant murine Hsp25 on Complex I electron transport from NADH
oxidation to quinone (CoQ,) reduction determined spectrophotometrically at 340 nm. Submitochondrial vesicles
purified from either unstressed (control) or heat-stressed murine PC 12 cells were assayed at either 28°C or 48°C.
Either no protein was added, or BSA (0.2 mg/mL), rabbit IgG (1:300 v/v), or antibody specific to recombinant
murine Hsp25 (Ab25) (1:300 v/v). Results are means +1 SE; n = 6.
was almost 3-fold higher in pre-heatstressed mitochondria than in control mitochondria (P < 0.0001), indicating that acclimation to high temperature occurred in
pre-heat-stressed samples (Fig. 5). This acclimation appeared to be due entirely to the
production of the mitochondrial small Hsps,
because addition of Ab25, which was used
to disrupt mitochondrial small Hsp function, decreased Complex I activity in preheat-stressed mitochondria assayed at 48°C
by about 75% (Tukey's multiple comparison test; P < 0.05), to levels comparable to
controls at 48°C. Addition of IgG or BSA
had no effect upon Complex I activity on
control or pre-heat-stressed samples assayed at either 28°C or 48°C (P > 0.05).
DISCUSSION
Chloroplast small Hsps
Here we report positive relationships between chloroplast small Hsp production and
PSII thermotolerance in (1) a heat-tolerant
genotype of Agrostis palustris (bentgrass)
and a heat sensitive genotype which lacks
one or more chloroplast small Hsps produced by the tolerant genotype; (2) ecotypes of Chenopodium album (lambs quarters) from the cooler northern vs. warmer
southern U.S. (New York vs. Georgia); and
(3) nine Lycopersicon (tomato) cultivars/
species differing in heat tolerance. These
data, when taken together with our previous
results from in vitro studies, indicate that
genetic variation in production of the chloroplast small Hsp may be an important determinant of photosynthetic and, thereby,
whole-plant thermotolerance.
How does the chloroplast small Hsp
work? Evidence from in vitro studies—
Most of the available evidence on the chloroplast small Hsps from our recent work
and past studies from other labs (e.g.,
Kloppstech et al, 1985) is summarized in
a model of the function of the chloroplast
small Hsps depicted in Figure 6. The major
chloroplast small Hsps are known to be nuclear-encoded proteins that are synthesized
SMALL HSPS IN CHLOROPLASTS AND MITOCHONDRIA
873
Chloroplast
Chloroplast During Heat Stress
Outer Envelope
\
Thylakoid
w/ Hsp22
Thylakoid
w/o Hsp22
FIG. 6. A diagramatic representation of our current model of the function of the chloroplast small Hsps. The
nuclear-encoded small heat-shock protein is known to be synthesized in the cytosol (Hsp31) and then imported
into chloroplasts, after which the transit sequence is removed to yield the stromal isoform(s) (Hsp25). The
stromal isoform is presumeably imported into the lumen of the thylakoids, after which a second transit sequence
is removed to yield the thylakoid isoform(s) (Hsp22). Our results indicate that Hsp22 localizes to the thylakoid
lumen where it interacts with Photosystem II (PSII) during stress, particularly the 33-, 23-, and 17-kD proteins
of the oxygen-evolving complex (OEC), protecting PSII function, and thereby, whole-chain electron transport
from heat, oxidative, and photoinhibitory damage.
in the cytosol (e.g., Hsp31, which is ca. 31kD in tomato and lambs quarters) and then
imported into chloroplasts, after which the
transit sequence is removed to yield the
stromal isoform(s) (ca. Hsp25) (Kloppstech
et al, 1985; Vierling et al, 1986; Heckathorn et al, 1998a). In tomato and lambs
quarters, the stromal isoform is presumably
imported into the lumen of the thylakoids,
after which a second transit sequence is removed to yield the thylakoid isoform(s) (ca.
Hsp22). This scenario is however, speculation, and remains to be confirmed. Our previous results indicate that Hsp22 localizes
to the thylakoid lumen where it interacts
with Photosystem II (PSII) during stress,
particularly the 33-, 23-, and 17-kD proteins of the OEC, protecting PSII function,
and thereby, whole-chain electron transport
from heat, oxidative, and photoinhibitory
damage (Heckathorn et al, 1996a, 1997a,
1998ft; Downs et al, 1999a, b). We emphasize that current evidence indicates that
the chloroplast small Hsp exists in vivo
mostly as a homo-oligomer of ca. 200-230
kD (Chen et al, 1994; Suzuki et al., 1998);
thus, the functional form of the chloroplast
small Hsp is likely to be an oligomer, which
is not depicted in Figure 6. We also emphasize that current evidence does not preclude other stress-related functions of the
chloroplast small Hsps, such as protection
of stromal proteins or interaction of the
stromal isoform with the stromal-side of
PSII, or that chloroplast small Hsps fulfill
different functions in different species. For
example, in pea (Pisum sativum L.), a thermosensitive species compared to tomato
and lambs quarters, the chloroplast small
Hsp is localized mostly to the stroma (Chen
et al, 1990; Osteryoung and Vierling,
1994).
874
S. A. HECKATHORN ETAL
This current model of chloroplast small
Hsp function is consistent with our knowledge of how heat stress affects photosynthesis. Photosynthesis is among the most
thermosensitive of plant metabolic processes (Berry and Bjorkman, 1980; Weis and
Berry, 1988). Several specific components
of both photosynthetic electron transport
and the Calvin cycle are relatively thermolabile, compared to other photosynthetic
components, and their thermosensitivity can
limit the efficiency and rate of photosynthesis during heat stress (Berry and Bjorkman, 1980; Weis and Berry, 1988; Pastenes
and Horton; 1996a, b). Within the light reactions, the OEC of PSII is usually the most
heat sensitive of the chloroplast thylakoidmembrane protein complexes involved in
photosynthetic electron transfer and ATP
synthesis and is one of the most thermolabile aspects of photosynthesis in general
(Berry and Bjorkman, 1980; Nash et al.,
1985; Weis and Berry, 1988; Williams and
Gounaris, 1992; Havaux, 1993; Heckathorn
etal., 1997a).
Variation in the production of the chloroplast small Hsp and thermotolerance—
Given the strong evidence indicating a
functional role for the chloroplast small
Hsp in protecting PS II, might inter- and
intra-specific variation in the production of
the chloroplast small Hsp be related to inter- and intra-specific variation in the thermotolerance of photosynthesis? The studies
we discuss here certainly suggest that the
answer to that question is "yes." The small
Hsp that is lacking in the heat-sensitive
Agrostis genotype is the smallest chloroplast small Hsp isoform (Park et al., 1996),
which we have shown localizes to the thylakoid lumen and interacts with the OEC
proteins of PSD (Downs et al, 1999a).
Thus, we would predict a strong reduction
in PS II thermotolerance in the sensitive genotype, simply from this observation. Furthermore, in the case of Lycopersicon genotypes, we were able to explain 78% of
the variation in PSII thermotolerance simply from the relative production of the chloroplast small Hsp. In the case of Chenopodium, individuals taken from a population residing in a hotter habitat (Georgia)
showed increased production of the chlo-
roplast small Hsp and increased photosynthetic thermotolerance vs. plants from a
cooler habitat (NY). The relationship between chloroplast small Hsp and wholeplant thermotolerance/habitat holds for unrelated species as well (Downs etal., 1998).
Mitochondrial small Hsps
We now know that the mitochondrial
small Hsps also protect electron transport
during heat stress in plants (Downs and
Heckathorn, 1998) and certain mammal
cells (shown here). It therefore seems likely
that mitochondrial small Hsps will prove to
be important in protecting mitochondrial
electron transport during other types of
stress as well, and that genetic and phenotypic variation in production of mitochondrial small Hsps will prove to be important
in determining cell and organismal stress
tolerance too. Future research should elucidate what part and function of Complex I
is protected by the mitochondrial small
Hsps.
ACKNOWLEDGMENTS
We thank Dawn Luthe for providing
heat-sensitive and heat-tolerant Agrostis
palustris plants, Paul Preczewski and Samantha Ryan for help with data collection,
Linda Jones for assistance with tissue culture, and the anonymous reviewers for helpful comments on the manuscript. This work
was supported by grants from the National
Science Foundation (to SAH and JSC) and
the Andrew W Mellon Foundation (to
JSC).
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