全 文 ：书圆锥南芥适应高山环境极端高温的系统性耐热能力
*
唐 婷1，2，郑国伟1
＊＊，李唯奇1＊＊
(1 中国科学院昆明植物研究所中国西南野生生物种质资源库，云南 昆明 650201;2 中国科学院大学，北京 100049)
摘要:高山生态系统的主要气候特征是温度变化幅度较大，目前研究主要集中于高山植物的抗冻机制，而
很少关注其对极端高温 (高于 45 ℃)的适应性。本研究发现高山物种圆锥南芥跟拟南芥比，具有更强的
基础耐热性和获得性耐热性。通过叶绿素荧光检测发现在极端高温下，圆锥南芥具有更稳定的光系统 II
和更有效的能量耗散机制来维持更高水平的光合效率。通过电导率和丙二醛含量的检测发现圆锥南芥的膜
伤害更小，膜流动性与脂肪酸链的长度和不饱和度紧密相关，圆锥南芥脂肪酸具有更低的 16 ∶ 3 含量，更
长的碳链，不饱和度没有明显的变化，这些可能有助于维持膜的稳定。另外，更高表达量的 HSP101 和
HSP70可能为圆锥南芥提供了更好的保护作用。以上结果表明，圆锥南芥能利用生理生化活动的调整来适
应高山环境中的极端高温，这种耐热策略与低地耐热植物相似，因此圆锥南芥具有系统性耐热能力，可以
作为研究植物耐热分子机制的模式物种。
关键词:高山植物;圆锥南芥;耐热能力;光系统;脂肪酸;热激蛋白
中图分类号:Q 945 文献标识码:A 文章编号:2095－0845(2014)06－683－15
Adaptation to Extremely High Temperature in an Alpine Environment:
Systemic Thermotolerance in Arabis paniculata
TANG Ting1，2，ZHENG Guo-Wei1＊＊，LI Wei-Qi1＊＊
(1 Germplasm Bank of Wild Species in Southwest China，Kunming Institute of Botany，Chinese Academy of Sciences，
Kunming 650201，China;2 University of Chinese Academy of Sciences，Beijing 100049，China)
Abstract:Alpine ecosystems are characterised by frequent fluctuations between high and low temperatures． The re-
sistance of alpine plants to low temperatures has received considerable attention，but little is known about their adap-
tation to extremely high temperatures (＞45 ℃)． In this study，the alpine species Arabis paniculata was shown to
display superior basal thermotolerance and acquired thermotolerance than its relative Arabidopsis thaliana． Our chlo-
rophyll fluorescence data suggest that under heat shock conditions，A. paniculata has a thermostable photosystem II
(PSII)and that efficient non-photochemical quenching maintains a high level of photosynthetic efficiency． Assays of
ion leakage and malondialdehyde (MDA)content revealed that membrane damage caused by high temperatures was
less severe in A. paniculata than in A. thaliana． The degree of unsaturation and fatty acid chain length was closely cor-
related with membrane fluidity． Compared with A. thaliana，A. paniculata had a lower 16 ∶ 3(roughanic acid)content，
longer fatty acid chain length and no major alterations in the level of unsaturation of membrane fatty acids;this might
enable the maintenance of stable membrane fluidity． Furthermore，more extensive accumulation of heat shock proteins
(HSPs)，such as HSP101 and HSP70 in A. paniculata compared with A. thaliana，might correlate with better protec-
tion against high temperature in A. paniculata than in A. thaliana． Our findings suggest that the alpine plant
A. paniculata uses all of these physiological and biochemical adjustments to adapt to high temperature，and that similar
植 物 分 类 与 资 源 学 报 2014，36 (6):683～697
Plant Diversity and Ｒesources DOI:10．7677 /ynzwyj201414047
＊
＊＊
Funding:NSFC (31300251)and XiBuZhiGuang Project
Author for correspondence;E-mail:weiqili@mail. kib. ac. cn;gwzheng@mail. kib. ac. cn
Ｒeceived date:2014－03－18，Accepted date:2014－06－11
作者简介:唐 婷 (1987－)女，博士，主要从事植物逆境分子生理学研究。E-mail:tangtinghnkjdx@163. com
to lowland tropical species，A. paniculata exhibits systemic thermotolerance． Accordingly，A. paniculata might be a
useful model plant to study the molecular and physiological mechanisms that contribute to thermotolerance in plants．
Key words:Alpine plants;Arabis paniculata;Thermotolerance;Photosystem;Fatty acids;Heat shock proteins
High temperature is a critical environmental
factor that limits the geographic distributions of wild
species． Plants have evolved multiple strategies to a-
dapt to this type of stress． All of these strategies can
be classified into three levels:1)morpho-anatomical
and phenological responses;2) physiological re-
sponses，such as appropriate modification of photo-
synthesis，decreases in the unsaturation of mem-
brane lipids，and the accumulation of secondary me-
tabolites;and 3)molecular responses，such as the
accumulation of stress proteins and antioxidants
(Wahid et al．，2007)． The particular strategies a-
dopted depend on the species itself and its environ-
ment． For examples，whereas some cultivars of tropi-
cal crop chickpea have heat-escape mechanisms as-
sociated with early phenology and leaf reflectance，
others have heat-tolerance mechanisms that involve
increases in membrane stability and the accumula-
tion of HSPs (Devasirvatham et al．，2012)． Loise-
leuria procumbens，a typical alpine plant，adjusts
the thermotolerance of its PSII complex for a 4. 8 ℃
diurnal variability in its capacity to adapt to the fluc-
tuation in extreme temperatures (Braun et al．，
2002)． Plants can also adopt unusual approaches to
adapt to heat stress． Saussurea medusa and Solms-
Laubachia linearifolia change their overall composi-
tion rather than the level of unsaturation of mem-
brane lipids in response to increases and decreases
in temperature in an alpine environment (Zheng et
al．，2011)．
Alpine environments are associated with a range
of abiotic stresses，such as low and high tempera-
tures，extreme radiation，drought，and nutrient dep-
rivation． Low temperature is generally assumed to be
the main factor that limits the growth and develop-
ment of alpine plants，and the adaptation of alpine
plants to low temperatures is well documented (Kr-
ner，2003;Marquez et al．，2006;Martin et al．，
2010;Yamori et al．，2005)． On the other hand，
high temperature is another factor that might con-
strain the growth of alpine plants． Alpine plants with
small stature usually grow close to the soil surface
and are surrounded by bare soil，which can reduce
plants’exposure to cooling winds． Meanwhile，the
location of plants on a slope that favors the optimal
interception of solar radiation might also promote in-
creases in the ambient temperature they experience
(Salisbury and Spomer，1964;Krner and Cochrane，
1983;Krner and Larcher，1988)． These factors
can cause alpine plants to suffer from heat damage in
microhabitats． For example， the temperature was
found to reach as high as 80 ℃ in south-facing bare
and raw humus (Turner，1958)． The day ambient
temperature can reach 40 ℃ in the alpine scree on
the Hengduan Mountains (Zheng et al．，2011)．
Therefore，it seems likely that alpine plants have de-
veloped strategies to tolerate heat stress，considering
of course that the challenges posed by long-term ex-
posure to extreme cold exceed those posed by rela-
tively transient exposure to high temperatures (Krn-
er，2003)． Only a few reports describe the heat tol-
erance of alpine plants． Some interesting and impor-
tant questions that remain concern the strategies that
alpine plants have adopted to tolerate high tempera-
ture and whether these strategies are same as those
adopted by lowland tropical or desert plants．
Compared with most physiological processes，
photosynthesis is extremely sensitive to heat stress，
with complete inhibition of PSII possible before other
types of cell injury take place (Berry and Bjorkman，
1980)． Chlorophyll fluorescence is a good indicator
of changes in PSII under stress (Maxwell and John-
son，2000;Ｒohácˇek，2002)． Heat stress can de-
crease the maximum quantum yield of PSII (Fv /Fm)
(Petkova and Stefanov，2009)，with Fm providing
clues to understand plants’ stress responses and
486 植 物 分 类 与 资 源 学 报 第 36卷
changes in Fm reflecting the efficiency of heat dissi-
pation (NPQ)(Maxwell and Johnson，2000)． Plants
increase NPQ significantly during heat stress to dissi-
pate excess energy and thus prevent them from suf-
fering heat damage (Haldimann and Feller，2004)．
Membranes are also sensitive to high tempera-
tures (Wahid et al．，2007)． Heat stress causes
membrane damage by affecting lipid hyperfluidity
(Havaux，1998)． Alterations in lipid fluidity could
induce changes in the permeability of membranes，
membrane lipid peroxidation，and even affect the
composition of membrane fatty acids (Meriga et al．，
2004;Sairam et al．，2000;Wahid et al．，2007)．
Ｒeduced levels of fatty acid unsaturation are associ-
ated with increased tolerance of heat stress (Muraka-
mi et al．，2000)． Most reports of alpine plants de-
scribe their cold tolerance;in general，the mem-
branes of alpine plants are more fluid than their low-
land counterparts as a result of a special phospholi-
pid structure formed during the long period of cold
acclimation (Beck，1994)． However，the possibility
that the composition and saturation of membrane li-
pids of alpine plants might change after exposure to
high temperature remains to be investigated．
The induction of HSPs is very important for
plant resistance to heat stress． The majority of the
most abundant HSP family members， such as
HSP101 and HSP70，play important roles in resol-
ving the problems caused by protein misfolding and
aggregation (Queitsch et al．，2000)． HSP101 and
HSP70 are conserved and have similar functions in
most organisms， ranging from eubacteria to eu-
karyotes (Boorstein et al．，1994;Queitsch et al．，
2000)． HSP101 is required for both acquired ther-
motolerance and basal thermotolerance in germina-
ting maize kernels (Nieto-Sotelo et al．，2002)．
HSP101 was also shown to improve the induction of
HSA32 in order to prolong the duration of heat accli-
mation in Arabidopsis (Wu et al．，2013)． The in-
duction of HSP70 was also shown to improve the
thermotolerance of cells and tissues (Schffl et al．，
1999)． Loss of HSP70 renders Arabidopsis plants sen-
sitive to heat treatment (Latijnhouwers et al．，2010)．
The current classification of Arabis and Arabi-
dopsis indicates that these two genera are highly pa-
raphyletic． Arabis paniculata (or Arabis alpine ht-
tp:/ /www. efloras. org / florataxon. aspx?flora _ id =
2＆taxon_id = 200009229) ，a member of the sister
group closest to the model plant Arabidopsis thaliana
(Koch et al．，1999) ，is a typical perennial alpine
plant，which can adapt to the pace of repeated cy-
cles between vegetative and reproductive develop-
ment with the changing seasons (Bohlenius et al．，
2006)． Its flowering response to winter temperature
is age-dependent (Bergonzi et al．，2013)． The ma-
ximum daytime temperature of the environments in
which A. paniculata is indigenous exceeds 32 ℃ dur-
ing most seasons (Zheng et al．，2011)． In the pres-
ent study，we used A. paniculata as a example to in-
vestigate the heat tolerance of alpine plants． We
characterized the thermotolerance of the species by
comparing the responses of PSII，membrane injury，
unsaturation of fatty acids，and changes in the abun-
dances of HSP proteins between A. paniculata and
A. thaliana． Our findings indicated that A. paniculata
exhibited systemic thermotolerance．
1 Materials and methods
1. 1 Plant materials and heat treatment
Arabis alpina has a wide distribution，covering
all European mountain systems，the Canary Islands，
North Africa，the high mountains of East Africa and
Ethiopia，the Arabian Peninsula and mountain ran-
ges of Central Asia in Iran and Iraq (Koch et al．，
2006)． A． paniculata is synonymous with A． alpina，
which grows in Yulong Snow Mountain in Lijiang，
Yunnan Province，China． Seeds of A. paniculata
were collected from Yulong Snow Mountain． Seeds of
A. paniculata and A. thaliana (Columbia ecotype)
were sown and plants were grown side-by-side under
all of the conditions tested． For the plate-grown
plants，surface-sterilised seeds were sown on solidi-
fied Murashige and Skoog medium that contained 1%
sucrose． The plates with seeds were put in the dark
5866期 TANG Ting et al．:Adaptation to Extremely High Temperature in an Alpine Environment:Systemic …
at 4 ℃ for 5 d and transferred to a growth chamber at
22 ℃ under 120 μmol m－2 s－1 light for 9 d． Seedlings
of two species at this age showed identical rates of
growth characteristics and size，and were used to as-
say both basal and acquired thermotolerance． Plates
with seedlings were exposed to 45 ℃ for 3 h in the
dark (Li et al．，2011)，which was 100% lethal for
A. thaliana，and then left at room temperature with
low light (22 ℃，4 μmol m－2 s－1)for recovery (Gao
et al．，1998)． For the soil-grown plants，seedlings
grown in soil at 22 ℃ under 120 μmol m－2 s－1 light
for about 4 or 6 weeks had similarly sized rosettes in
the two species． Exposure to 50 ℃ or 52 ℃ for 2 h
caused 100% lethality in A. thaliana plants of this
age． For the acquired thermotolerance test，the seed-
lings were treated at 37 ℃ for 2 h，followed by a 1 h
exposure to 22 ℃，which is a minor alteration of a
previously reported treatment (Larkindale et al．，
2005)． Thereafter，the effects of more severe heat
treatments were assessed． Each experiment was per-
formed at least three times．
1. 2 Measurement of chlorophyll fluorescence
Measurement of chlorophyll fluorescence before
and after heat treatments was performed using an IM-
AGING-PAM chlorophyll fluorometer and the Ima-
ging Win software application (Walz;Effeltrich，
Germany)，as described previously (Woo et al．，
2008)． A dark-light induction curve was generated
to assess the capacity to adapt to different conditions
of light and darkness． After 20 min of dark adapta-
tion，plants were subjected to a saturating pulse
(＞1 800 μmol photons m－2 s－1)，and the minimal
fluorescence (F0)，the maximal fluorescence (Fm)
and Fv /Fm ratio were then determined． False-colour
images of the Fv /Fm parameter that were captured u-
sing the Imaging Win software are shown (Woo et
al．，2008)． This was followed by a 40-second expo-
sure to darkness，and then 6 minutes of actinic illu-
mination (111 μmol photons m－2 s－1)，with satura-
ting flashes delivered at 20-second intervals． The ac-
tinic intensity simulated growth conditions． The actu-
al quantum yield of PSII photochemistry［Y(II) ］，
the yield of non-regulated energy dissipation ［Y
(NO) ］，the yield of regulated energy dissipation［Y
(NPQ) ］and an estimate of the fraction of PSII cen-
ters that are open (qL)were obtained after allowing
adaptation to the light conditions for 6 minutes prior
to taking the final measurements．
1. 3 Detection of ion leakage
Ion leakage from leaves was measured by slight
modification of a previously reported method (Welti
et al．，2002)． Leaves were harvested both after heat
treatment (50 ℃ for 2 h)and after a 3 d-recovery
period at 22 ℃ followed the heat treatment． Deionised
water was added，and conductance of the water was
measured after gentle rocking at 22 ℃ for 3 h． The i-
onic strength of the water was measured after the so-
lution had been boiled in a water bath at 100 ℃ for
30 minutes and cooled to 22 ℃ ． Leaked ions were
calculated as the percentage of the initial conductivi-
ty relative to the final conductivity．
1. 4 Detection of lipid peroxidation
We used the content of MDA as an indicator of
the lipid peroxidation in leaves． Levels of MDA were
measured by minor modification of a previously de-
scribed method (Mishra and Singhal，1992)． Leav-
es were sampled both after 2 h of exposure to 52 ℃
and after a 2 d-recovery period at 22 ℃ following the
heat treatment． Leaf samples were ground under li-
quid nitrogen，homogenised in 5 mL of 10% trichlo-
roacetic acid (TCA)and centrifuged at 6 000 r·min－1
for 10 minutes． After mixing 1 mL of the supernatant
with 1 mL of thiobarbituric acid solution (0. 6% in
10% TCA)，the mixture was incubated at 95 ℃ for
20 minutes，quickly cooled in an ice bath，and then
centrifuged at 6 000 r·min－1 for 10 minutes． The ab-
sorbance of the supernatant was measured at 450 nm，
532 nm，and 600 nm． The concentration of MDA was
calculated as follows:6. 45× (A532－A600)－0. 56×
A450．
1. 5 Fatty acid analysis
Fatty acid analysis was performed as described
previously，with minor modifications (Miquel and
Browse，1992)． Initially，500 mg of leaves was ground
686 植 物 分 类 与 资 源 学 报 第 36卷
under liquid nitrogen， transferred to a centrifuge
tube with 5 mL of chloroform /methanol / formic acid
(10 ∶ 10 ∶ 1，by volume)，and stored overnight at
－20 ℃ ． After centrifugation at 6 000 r·min－1 for 10
minutes，the supernatant was transferred to a new
glass tube and the precipitate was re-extracted using
2. 2 mL of chloroform /methanol /water (5 ∶ 5 ∶ 1，by
volume)． The two extractions were combined and the
combination was supplemented with 3 mL of 0. 2
mol·L－1 H3 PO4 /1 mol·L
－1 KCl before vortexing．
Subsequently，the supernatant was transferred to a
new glass tube，the chloroform phase was dried with
N2 and the remainder was dissolved in 0. 25 mL of
chloroform． We then added 0. 25 mL of toluene
(with 50 μg of triheptadecanoin)and 1 mL of 2. 5%
H2SO4 in methanol，keeping the sample at 80 ℃ in
a water bath for 90 min． The sample was then extrac-
ted three times with 1. 5 mL of 0. 9% NaCl and 1 mL
of hexane． The organic phases from each extraction
were combined and dried under N2，and then dis-
solved in 0. 4 mL of hexane for analysis by gas chro-
matography．
1. 6 Protein extraction and immunoblotting of
HSPs
Total protein was isolated using a previously de-
scribed procedure (Fan et al．，1997)． Seedlings
were ground in homogenization buffer using a pestle
(50 mmol·L－1 Tris-HCl，pH 7. 5;10 mmol·L－1
KCl，1 mmol·L－1 EDTA，0. 5 mmol·L－1 phenylm-
ethylsulfonyl fluoride，and 2 mmol·L－1 dithiothrei-
tol)in precooled mortar that was kept on ice． The ho-
mogenate was centrifuged for 10 min at 7 000 r·min－1
at 4 ℃ ． Protein contents in the supernatants were
determined using a dye-binding assay with Coomass-
ie Brilliant Blue at 595 nm． The same amount of su-
pernatant protein from each sample was separated by
sodium dodecyl sulfate polyacrylamide gel electro-
phoresis analysis and then transferred onto polyvinyl-
idene difluoride filters． The filters were first probed
with HSP101- or HSP70-specific antibodies，and
then incubated with a secondary antibody conjugated
to alkaline phosphatase． HSPs were visualised by
staining the blot for phosphatase activity． Given the
high conservation of HSP sequences and the consist-
ent sizes of HSPs，the HSP profiles were comparable
across these two related species． Each measurement
was performed independently at least three times．
1. 7 Data processing
The data were subjected to one-way analysis of
variance (ANOVA)analysis with SPSS 19. 0． Sta-
tistical significance was tested by Fisher’s least sig-
nificant difference (LSD)method． The double-bond
index (DBI)was calculated using the following for-
mula:DBI = (∑［N × mol% fatty acid］)/100，
where N is the number of double bonds in each fatty
acid molecule (Zheng et al．，2011)．
2 Ｒesults
2. 1 A. paniculata showed strong basal and ac-
quired thermotolerance under high-temperature
conditions
We compared the thermotolerance of the alpine
plant A. paniculata with that of its relative，the mod-
el plant A. thaliana，after heat treatment at 52 ℃ for
2 h． We found that whereas seedlings of A. panicula-
ta withered slightly after heat shock，recovered after
3 d，and grew normally after 10 d，A. thaliana with-
ered substantially after heat shock and could not re-
cover after 10 d (Fig. 1A)． In order to investigate
the photosynthetic activities under heat stress，we
compared the Fv /Fm ratios of the two species． As
shown in Fig. 1B，Fv /Fm of both plants decreased
dramatically after heat shock，and decreased to al-
most zero in A. thaliana． Whereas the Fv /Fm could
recover to the level of the control after heat-treated
A. paniculata plants were transferred to 22 ℃，a
similar recovery of Fv /Fm was not observed in A. th-
aliana (Fig. 1B)． Moreover，we found that A. panic-
ulata was more tolerant than A. thaliana after a 2 h
exposure to 52 ℃ after a 2 h pretreatment at 37 ℃
(Fig. 1C)． The Fv /Fm ratio of A. paniculata was also
higher than that of A. thaliana during the period of
exposure to heat (Fig. 1D)． The results indicated
that A. paniculata was markedly superior to A. thaliana
7866期 TANG Ting et al．:Adaptation to Extremely High Temperature in an Alpine Environment:Systemic …
Fig. 1 Phenotypes and chlorophyll fluorescence imaging of soil-cultured plants following heat treatment with and without acclimation
A． Photographs taken of six-week-old seedlings of A. paniculata (upper line)and A. thaliana (lower line)before heat treatment (Con-
trol)，after heat shock (HS，52 ℃ for 2 h) ，and recovery for 3 d (Ｒ3 d)and 10 d (Ｒ10 d) ;B． False-colour images of the Fv /Fm pa-
rameter after direct heat treatment． Images represent the maximum quantum yield of PSII (Fv /Fm);C． Photographs taken of seedlings of
A. paniculata (upper line)and A. thaliana (lower line)before heat treatment (Control) ，after heat shock with acclimation (HS，37 ℃
for 2 h，then 52 ℃ for 2 h) ，and recovery for 3 d (Ｒ3 d)and 10 d (Ｒ10 d) ;D． False-colour images of the Fv /Fm parameter after accli-
mation to heat treatment． Images represent the maximum quantum yield of PSII (Fv /Fm);E． The lethal temperature of A. paniculata after
heat treatment． Six-week-old seedlings were left at 54 ℃，58 ℃，and 62 ℃ for 2 h in the dark． The photographs were taken after 12 d of
recovery at room temperature (22 ℃)with dim illumination． There were three seedlings for each treatment per species． Each measurement
was repeated independently at least three times
886 植 物 分 类 与 资 源 学 报 第 36卷
in terms of both its basal and acquired levels of ther-
motolerance． As shown in Fig. 1A and Fig. 1E，the
temperatures that kill A. thaliana and A. paniculata
(lethal temperatures)were 52 ℃ and 62 ℃，respe-
ctively． The 10 ℃-difference in the thermotolerant
limits of the two species indicates that，compared
with A. thaliana，A. paniculata is better adapted to
the high-temperature extremes occasionally encoun-
tered in alpine environments．
2. 2 Photochemical activities in A. paniculata
exceeded those in A. thaliana under heat shock
After 3 h of heat shock at 45 ℃，the Fv /Fm of
A. paniculata decreased to about half of that of the
control，whereas in A. thaliana it decreased to al-
most zero after heat treatment (Fig. 2A)． Following
the same heat treatment，F0 increased slightly and
Fm decreased in both species;the decline of Fm in
A. thaliana exceeded that in A. paniculata (Fig. 2B
and 2C)． The decrease in Fv /Fm resulted from a
slight increase in F0 and a dramatic decrease in Fm ．
After heat treatment，qL and Y(II)both decreased
dramatically to almost zero in both plant species
(Fig. 3A and 3B)． Whereas Y(NPQ) increased
more than two-fold in A. paniculata， it decreased
from 0. 22 to almost zero in A. thaliana (Fig. 3C)．
In contrast with no obvious increase in Y(NO)in
A. paniculata，this parameter increased approximate-
ly three-fold in A. thaliana (Fig. 3D)． Given that Y
(NO)approaches 1 in A. thaliana，heat treatment
might cause a nearly complete breakdown in photo-
chemistry． The increases in Fv /Fm and Y(II) in
A. paniculata after heat treatment suggested that the
photochemical efficiency of PSII is higher in A. pan-
iculata than in A. thaliana． Moreover，the higher Y
(NPQ)value indicated more effective mechanism
for dissipating excess heat energy in A. paniculata
than in A. thaliana，and the lower Y(NO)value re-
flected less serious heat damage in A. paniculata
compared with A. thaliana．
Whereas the Fv /Fm ratio for A. paniculata in-
creased from 0. 41 to 0. 62 after 3 d of recovery，Fv /
Fm in A. thaliana failed to recover after heat shock
(Fig. 2A)，and the plants were expected to die soon
thereafter． In A. paniculata，the recovery of Fv /Fm
was attributed primarily to a decrease in F0 and an
increase in Fm ． By contrast，in A. thaliana，F0 also
decreased and Fm decreased dramatically (Fig. 2B
and 2C)． After 3 d of recovery，qL and Y(II)in
A. paniculata increased from almost zero to values
comparable to those observed for the unstressed con-
trol;nonetheless，the same two parameters remained
at almost zero in A. thaliana (Fig. 3A and 3B)． The
Y(NPQ)of A. paniculata declined to the level of the
control after 1 d of recovery，and did not change too
much after 3 d of recovery;however，in A. thaliana，
the level of Y(NPQ)increased a little after 1 d of
recovery and remained at a low level，which was ap-
proximately one-third of that of the control after 3 d of
recovery (Fig. 3C)． Whereas Y(NO)in A. paniculata
could also recover to the control level，it remained at
Fig. 2 Effects of high temperature on the maximal efficiency of PSII photochemistry (Fv /Fm，A)，the minimal fluorescence
yield (F0，B)，and the maximal fluorescence yield (Fm，C)in A. paniculata and A. thaliana
Dashed lines (with circles)and solid lines (with squares)represent A. paniculata and A. thaliana，respectively． The 9 d-old seedlings
grown on agar-based medium were treated at 45 ℃ for 3 h，and then allowed to recover (22 ℃，4 μmol m－2 s－1)for 3 d． There were
three replicates for each plate，and the experiment was repeated at least three times． Values are mean ± S. D，n= 8
9866期 TANG Ting et al．:Adaptation to Extremely High Temperature in an Alpine Environment:Systemic …
Fig. 3 Effects of high temperature on parameters related to PSII chlorophyll fluorescence in A. paniculata and A. thaliana
Dashed lines (with circles)and solid lines (with squares)represent A. paniculata and A. thaliana，respectively． The 9 d-old seedlings
grown on agar-based medium were treated at 45 ℃ for 3 h，and then allowed to recover (22 ℃，4 μmol m－2 s－1)for 3 d． There
were three replicates for each plate，and the experiment was repeated at least three times． Values are mean ± S. D，n= 8
a level more than twice that of the control in
A. thaliana (Fig. 3D)． These results indicated that，
compared with A. thaliana，A. paniculata possessed
a more stable photosystem that was not easily affect-
ed by heat stress． Moreover，the more efficient heat
dissipation and rapid repair strategies used by
A. paniculata enabled it to withstand exposure to a
high temperature．
2. 3 After heat stress，the level of membrane
damage in A. paniculata was less severe than that
observed in A. thaliana
High temperatures can disturb membrane sys-
tems． To determine the direct injury caused by heat
stress in cellular membrane structures，we investiga-
ted levels of ion leakage in leaves． As shown in
Fig. 4，ionic leakage of leaves increased more than
two-fold after exposure to 50 ℃ for 2 h;there was no
difference in this regard between A. paniculata and
A. thaliana． However，after 3 d of recovery，the level
of ion leakage in leaves of A. paniculata decreased to
the level observed in the control，whereas more than
90% of the ions leaked through the membrane in
A. thaliana． These results indicated that slight injury
occurred in both plants after heat shock，but that
A. paniculata could soon repair its membranes;in
contrast，membrane damage became progressively
more severe in A. thaliana during the recovery stage．
In order to investigate the damage that resulted
from membrane lipid peroxidation caused indirectly
by heat stress，we determined the MDA content be-
fore and after heat treatment (Fig. 5)． The MDA
contents of both were higher after heat treatment and
subsequent recovery than before heat treatment． Af-
ter heat treatment，the MDA levels of A. paniculata
and A. thaliana increased by 98% and 118%，re-
spectively to final concentrations of 4. 64－9. 20 μmol
g－1 and 5. 11－11. 14 μmol g－1，respectively;during
the period of recovery，the respective figures were
195% and 328% (4. 64－13. 68 μmol g－1 and 5. 11－
21. 88 μmol g－1，respectively)． In addition，after 2 d
096 植 物 分 类 与 资 源 学 报 第 36卷
of recovery，the MDA level of A. thaliana was signi-
ficantly higher than that of A. paniculata． These re-
sults indicated that heat shock had similar effects on
the levels of indirect membrane damage in both
plants，but that it was more severe in A. thaliana
during the subsequent recovery period．
2. 4 The unique composition of fatty acids in
A. paniculata before and after heat treatment
might contribute to its high level of thermotoler-
ance
Given that the fluidity of cellular membranes is
mainly affected by the composition of their constitu-
ent fatty acids，we measured the fatty acids in the
membranes of A. paniculata and A. thaliana before
and after heat treatment (Table 1)． Before heat treat-
ment，the levels of most fatty acids in A. thaliana
differed from those in A. paniculata，except for 16 ∶ 0
(palmitic acid)． In particular，the levels of 16 ∶ 1
(palmitoleic acid)and 16 ∶ 3 (roughanic acid)were
20-and two-fold more in A. thaliana than in A. pan-
iculata，respectively． The content of 18 ∶ 3 (linolenic
acid)of A. thaliana was 9% less than that of A. pan-
iculata． The total relative content of C-16 fatty acids
(expressed as a percentage of all fatty acids) in
A. paniculata was 22. 44%，which was 8. 02% less
than that in A. thaliana． By contrast，the level of C-
18 fatty acids was 8. 02% more in A. paniculata than
in A. thaliana;this indicated that the average length
of fatty-acid carbon chains was longer in A. panicula-
ta than in A. thaliana． Moreover，the DBI of A. pan-
iculata was higher than that of A. thaliana． These re-
sults indicated species-specific differences in the
basal composition of membrane fatty acids at 22 ℃ ．
After heat treatment，the trend with which fatty
acids changed in A. paniculata also differed from that
in A. thaliana (Table 1)． For example，the relative
content of 16 ∶ 3 was constant at the control level and
then declined slightly in A. paniculata． By contrast，
it declined continually from 10. 75% to 10. 18% af-
ter heat treatment，and then to 6. 71% after 3 d of
recovery in A. thaliana． In addition， the relative
content of 16 ∶ 0 of A. paniculata decreased slightly
after heat treatment，and then remained stable after
3 d of recovery． However，in A. thaliana，the con-
tent of 16 ∶ 0 decreased from 16. 86% to 14. 95% af-
ter heat treatment and then increased to 22. 73% af-
ter 3 d of recovery． Whereas the DBI of A. paniculata
remained unchanged during the entire treatment
Fig. 4 Effects of high temperature on ion leakage in A. paniculata and
A. thaliana． Four-week-old soil-cultured plants were heat treated
(“Heat shock”)at 50 ℃ for 2 h．“Ｒecovery”means the seedlings re-
mained at room temperature for 3 d after heat treatment． Different low-
er-case letters indicate that the values are significantly different (P＜
0. 05)． The values were subjected to one-way analysis of variance
(ANOVA)to determine statistical significance． There were five repli-
cates for each test，and the experiment was repeated at least three
times． Values are mean ± S. D，n= 5
Fig. 5 Effects of high temperature on the contents of MDA in A. pan-
iculata and A. thaliana． Measurements were taken after direct heat treat-
ment(“Heat shock”)with six-week-old seedlings in soil culture (52 ℃
for 2 h)．“Ｒecovery”means that the seedlings remained at room tempera-
ture for 2 d after heat treatment． Different lower-case letters indicate that
the values are significantly different(P＜0. 05)． The values were subjected
to one-way analysis of variance (ANOVA)to determine statistical signifi-
cance． There were five replicates for each test，and the experiment was re-
peated at least three times． Values are mean ± S. D，n=5
1966期 TANG Ting et al．:Adaptation to Extremely High Temperature in an Alpine Environment:Systemic …
Table 1 Fatty acid composition of total lipids from leaves of A. paniculata and A. thaliana in the control group，and those subjected to heat
acclimation at 37 ℃ for 2 h (HA)，heat shock at 52 ℃ for 2 h (HS) ，and post-heat recovery for 1 d and 3 d (Ｒ1 and Ｒ3，respectively)．
Values in the same row with different letters are significantly different (P＜0. 05)． An asterisk means that the value in A. paniculata
is different from that in A. thaliana in the same treatment． Values are means ± standard deviation (n= 5)
Fatty acid
(mol%)
Plant species
Treatment
Control HA HS Ｒ1 Ｒ3
16 ∶ 0
A. paniculata 16. 75 ± 0. 65ab 17. 73 ± 0. 83a 16. 57 ± 0. 44b* 17. 61 ± 1. 06ab 17. 66 ± 1. 05ab*
A. thaliana 16. 86 ± 1. 27b 16. 75 ± 1. 19b 14. 95 ± 0. 39c 17. 11 ± 0. 36b 22. 73 ± 0. 76a
16 ∶ 1
A. paniculata 0. 12 ± 0. 05a* 0. 11 ± 0. 04a* 0. 11 ± 0. 04a* 0. 13 ± 0. 03a* 0. 11 ± 0. 02a*
A. thaliana 2. 44 ± 0. 13b 2. 40 ± 0. 28b 2. 74 ± 0. 18a 2. 61 ± 0. 12ab 2. 58 ± 0. 13ab
16 ∶ 2
A. paniculata 0. 79 ± 0. 23a* 0. 70 ± 0. 07ab* 0. 70 ± 0. 06ab* 0. 53 ± 0. 09c 0. 62 ± 0. 05bc*
A. thaliana 0. 41 ± 0. 05ab 0. 45 ± 0. 06a 0. 44 ± 0. 03ab 0. 42 ± 0. 07ab 0. 38 ± 0. 04b
16 ∶ 3
A. paniculata 4. 79 ± 0. 93a* 4. 79 ± 0. 18a* 4. 34 ± 0. 29ab* 3. 98 ± 0. 28bc* 3. 71 ± 0. 13c*
A. thaliana 10. 75 ± 0. 33a 10. 24 ± 0. 42b 10. 18 ± 0. 39b 8. 30 ± 0. 31c 6. 71 ± 0. 39d
Sum of 16Cs
A. paniculata 22. 44 ± 0. 67ab* 23. 33 ± 0. 92a* 21. 73 ± 0. 35b* 22. 25 ± 1. 10ab* 22. 09 ± 1. 18b*
A. thaliana 30. 46 ± 1. 22b 29. 84 ± 0. 97b 28. 32 ± 0. 37c 28. 45 ± 0. 40c 32. 4 ± 0. 93a
18 ∶ 0
A. paniculata 4. 54 ± 0. 45b* 6. 53 ± 1. 23a* 5. 37 ± 0. 67ab 5. 78 ± 0. 67ab* 5. 39 ± 1. 52ab*
A. thaliana 7. 47 ± 0. 51b 7. 83 ± 0. 88b 5. 88 ± 0. 73c 7. 32 ± 0. 76b 9. 99 ± 0. 99a
18 ∶ 2
A. paniculata 15. 02 ± 1. 07c* 14. 97 ± 0. 59c* 16. 40 ± 0. 44ab* 15. 56 ± 0. 93bc* 16. 68 ± 0. 55a*
A. thaliana 13. 40 ± 0. 60b 13. 24 ± 0. 30b 14. 69 ± 0. 84a 13. 53 ± 0. 39b 13. 89 ± 0. 55b
18 ∶ 3
A. paniculata 57. 99 ± 1. 62a* 55. 17 ± 1. 72b* 56. 50 ± 1. 00ab* 56. 42 ± 1. 21ab* 55. 84 ± 1. 86b*
A. thaliana 48. 67 ± 1. 99b 49. 09 ± 1. 52b 51. 12 ± 1. 19a 50. 70 ± 0. 72a 43. 73 ± 1. 06c
Sum of 18Cs
A. paniculata 77. 56 ± 0. 67ab* 76. 67 ± 0. 92b* 78. 27 ± 0. 35a* 77. 75 ± 1. 10ab* 77. 91 ± 1. 18a*
A. thaliana 69. 54 ± 1. 22b 70. 16 ± 0. 97b 71. 68 ± 0. 37a 71. 55 ± 0. 40a 67. 60 ± 0. 93c
DBI
A. paniculata 2. 20 ± 0. 04a* 2. 11 ± 0. 06b 2. 17 ± 0. 03ab 2. 14 ± 0. 04b* 2. 13 ± 0. 06b*
A. thaliana 2. 08 ± 0. 05b 2. 08 ± 0. 06b 2. 17 ± 0. 03a 2. 08 ± 0. 03b 1. 82 ± 0. 04c
process，it increased from 2. 08 to 2. 17 after heat
treatment，and it decreased to 1. 82 after 3 d of re-
covery in A. thaliana． These results indicated that
whereas the composition and level of unsaturation of
membrane fatty acids remained relatively stable in
A. paniculata regardless of the imposition of heat
shock， it changed dramatically when A. thaliana
plants were given a heat-shock treatment，and again
when they were allowed to recover from exposure to
the elevated temperature．
2. 5 Upon heat stress，heat shock proteins accu-
mulated more rapidly in A. paniculata than in
A. thaliana
Most of the main members of the HSP family
accumulate in plants subjected to heat treatments． To
investigate the role of HSPs in the thermotolerance of
A. paniculata，we tested the content of HSP101 and
HSP70 of A. paniculata and A. thaliana following
their exposure to 45 ℃ for different periods of time．
As shown in Fig. 6，HSP101 could be induced in
both species after 30 min of heat treatment;the con-
tent of HSP101 increased as the duration of heat
treatment increased，and peaked after 4 h and 2. 5 h
of heat treatment in A. paniculata and A. thaliana，
respectively． Subsequently，the level of HSP101 de-
clined as the duration of heat treatment increased
further． The level of HSP101 induced by heat treat-
ment was higher in A. paniculata than in A. thaliana．
In addition，HSP70 was constitutively expressed in
both plants and the levels of the protein were higher
in A. paniculata than in A. thaliana． The content of
HSP70 of A. paniculata changed little after different
durations of heat treatment． However，in A. thaliana，
the level of HSP70 declined by about 60% after 30
min of treatment compared with that in the control，and
it disappeared completely after 5 h of heat treatment．
296 植 物 分 类 与 资 源 学 报 第 36卷
Fig. 6 Effects of high temperature on the expression of HSP101 and HSP70 in A. paniculata (upper lane)and A. thaliana (lower
lane)． Total proteins from 60 9 d-old seedlings grown on agar-based medium were treated at 45 ℃ for different durations in the dark
were electrophoretically separated on SDS-PAGE gels，transferred to filters，and reacted with antibodies against HSP101 and HSP70．
The lanes of HSPs in Figure 6 were visualised by staining the blot for phosphatase activity
The finding that the accumulation of HSPs occurred
more rapidly and reached a higher level in A. panic-
ulata than in A. thaliana suggested that they might
play better protective roles by conferring resistance
to heat stress．
3 Discussion
Plants that are indigenous to alpine ecosystems
have evolved a series of cold-tolerance strategies that
enable their adaptation to long periods of low temper-
ature． However，alpine environments frequently en-
counter temporary periods of high temperature，
which resemble the heat shock process commonly
applied in a laboratory setting． The present study re-
vealed at least some of the physiological and bio-
chemical strategies that enable the alpine plant
A. paniculata to adapt to high temperatures． We
found that both the basal thermotolerance and ac-
quired thermotolerance of A. paniculata were remark-
ably superior to those of its relative A. thaliana． Un-
der high-temperature conditions，A. paniculata dis-
played appropriate adjustments in terms of its photo-
synthetic system，maintenance of membrane stabili-
ty，different changes in the composition of its mem-
brane fatty acids to those observed in A. thaliana，as
well as faster and more extensive accumulation of
HSPs than was observed in A. thaliana． These re-
sults suggest that A. paniculata uses these four strate-
gies to adapt to the high temperatures found in alpine
environments． The same four strategies are correlated
closely with the tolerance of lowland plants to heat
shock (Sharkey and Zhang，2010)． Thermal stabili-
ty of PSII is enhanced by the stability of thylakoid
membranes，which is in turn influenced by lipid
composition;accumulation of HSP protects PSII and
thylakoid and plasma membranes from high tempera-
ture-induced damages．
One of the main factors that affect thermotoler-
ance is photosynthetic activity (Wahid et al．，2007)．
For instance，Fm decreased after heat shock and
then increased rapidly in heat-tolerant tropical beans
(Petkova et al．，2007)． In our experiment，the de-
crease in Fv /Fm resulted mainly from the decline of
Fm in two plants subjected to heat shock． At high
temperatures，a decline in Fm probably indicated
changes in the structures of PSII (Mishra and Sin-
ghal，1992)． The continual decline of Fm suggested
that structural damage of PSII was too severe to ena-
ble recovery in A. thaliana． However，this decline of
Fm and its subsequent increase during the recovery
stage in A. paniculata showed a strong capacity for
photosynthetic repair in this species． Therefore，like
3966期 TANG Ting et al．:Adaptation to Extremely High Temperature in an Alpine Environment:Systemic …
certain tropical plants，A. paniculata retains a high
level of photosynthetic efficiency under heat shock
conditions by ensuring the structural integrity and
function of PSII． The high thermal stability of PSII
in A. paniculata might also result from its high thyla-
koid membrane stability and /or the substantial accu-
mulation of HSPs，because PSII thermal stability is
enhanced by the stability of thylakoid membranes
and the accumulation of HSP protects PSII from
damage induced by high temperatures (Barua et al．，
2003)．
Plants have also evolved the ability to dissipate
excess light energy as heat in order to protect photo-
synthetic organs and to deal with the excess light ab-
sorbed in PSII through NPQ (Demmig-Adams et
al．，1996;Hendrickson et al．，2004;Kramer et
al．，2004)． Many tropical plants，including grape-
vine and the heat-tolerant species Wedelia trilobata，
maintain relatively high photochemical activity by in-
ducing a rapid increase in NPQ after heat treatment
(Luo et al．，2011;Song et al．，2010)． It is possi-
ble that the change of photochemical efficiency in
A. paniculata might be closely associated with the
dissipation of excess light energy as heat at high tem-
peratures． Y(NPQ)increased dramatically after the
imposition of heat shock and then declined during
the recovery process;meanwhile，Y(II)showed the
opposite trend in A. paniculata (Fig. 3)． These find-
ings suggest that the strong capacity for energy dissi-
pation in A. paniculata might contribute to both the
heat tolerance of PSII in this species and the effi-
cient use of heat energy after exposure to a high tem-
perature． However，the continual and synchronous
declines in Y(NPQ)and Y(II)in A. thaliana sub-
jected to heat shock suggest that A. thaliana has lost
this ability． In alpine ecosystems，the maximal level
of solar radiation is intense，and this can produce
extremely high temperatures in certain alpine micro-
climates (Krner，2003)． A. paniculata might cope
with these short-term and extreme increases in tem-
peratures by efficient dissipation of the heat．
Analyses of ion leakage in the present study re-
vealed that the membrane damage caused by heat
shock was reversible in A. paniculata but irreversible
in A. thaliana (Fig. 4 and Fig. 5)． The maintenance
of the integrity and fluidity of biological membranes
in A. paniculata could be attributed to three features
of its membrane lipid composition． One is the differ-
ent basal composition of membrane fatty acids． The
resistance of PSII of plants to short-term heat stress
was previously shown to be negatively correlated with
the content of 16 ∶ 3 fatty acids (Ｒoutaboul et al．，
2012)． The present study indicated that heat treat-
ment of A. paniculata had no obvious effect on the
very low level of 16 ∶ 3 fatty acids observed prior to
heat treatment． This might improve the stability of
the photosynthetic thylakoid membranes and eventu-
ally increase thermotolerance by reducing the level of
lipid unsaturation of thylakoids． The longer fatty acid
chains found in A. paniculata compared with those in
A. thaliana might also account for the superior heat
tolerance of the former． Longer fatty acid chains
might promote adaptation to high temperatures by
making the membrane environment less fluid and
more gel-like (Chintalapati et al．，2004)． The third
factor that potentially accounts for the greater ther-
motolerance of A. paniculata relative to A. thaliana is
the absence of obvious alterations in the degree of
unsaturation of fatty acids in the former． The reduced
level of membrane damage in A. paniculata might be
attributed to the accumulation of higher levels of
HSPs，because HSPs protect membrane integrity
and mediate membrane fluidity (Hong et al．，2003;
Tsvetkova et al．，2002)．
The accumulation of HSPs is positively correla-
ted with the thermotolerance of plants (Al-Whaibi，
2011)． For example，the desert plant Prosopis chil-
ensis might adapt to its harsh natural environment by
rapid accumulation of HSPs on a daily basis whenev-
er higher temperatures are experienced (Ortiz et al．，
1995)． In the present study，the induction of HSP101
was rapid，and the observation that levels of HSPs
remained elevated for a longer time in A. paniculata
than in A. thaliana is consistent with the general
496 植 物 分 类 与 资 源 学 报 第 36卷
consensus that HSPs protect against damage caused
by heat stress． It is well documented that HSP70
might play an important role in signalling the synthe-
sis of other HSPs as a signal transducer (Sorger，
1991)． The present study demonstrated that a rapid
decline in levels of HSP70 and HSP101 occurred af-
ter 5 h of exposure to 45 ℃ in A. paniculata，and
that the same synchronous trend to decrease levels of
HSP70 and HSP101 was also apparent in A. thaliana．
This relationship suggests that HSP101 and HSP70
might cooperate to play protective roles in many
physiological processes． It should be noted that plant
HSPs are often induced by both high and low tem-
peratures (Parsell and Lindquist，1993)． Given that
extreme temperatures are ubiquitous in alpine envi-
ronments，it seems that like desert plants，alpine
plants use the accumulation of HSPs to sense stress
conditions in a timely manner．
A. paniculata exhibited substantial heat toler-
ance through the advantages conferred by adaptations
at the levels of photosynthesis，membrane stability，
and the accumulation of HSPs． The strategies adopt-
ed by A. paniculata to cope with heat stress provide
an example of a thermotolerance mechanism used by
alpine plants． Compared with lowland tropical or
desert plants，the strategies that alpine plants use to
tolerate high temperatures might be systemic and u-
niversal，apart from some differences in the response
to high temperature between high and low attitude
plants． However，the molecular mechanisms or exact
signalling pathways that alpine plants use to with-
stand high temperatures are unclear． Therefore，it is
of vital significance to study the molecular mecha-
nisms of thermotolerance by exploring the genetic re-
sources of alpine plants． Investigation of the relatives
of A. thaliana with a special capacity to tolerate stress
seems especially promising in this regard (Bressan et
al．，2001)． A. paniculata possesses many character-
istics that make it an excellent choice as a model
system． These include its small stature，easy cultiva-
tion，a short life cycle，close relatedness to an ex-
tensively studied model plant，and outstanding ther-
motolerance． Accordingly，we recommend that A. pan-
iculata be adopted as a model plant to study the mo-
lecular genetic regulation of thermotolerance in plants．
Acknowledgements:The authors would like to thank Kun
Xu，Zhikun Wu，and Weiwei Liu for providing seeds of
A. paniculata． The authors also thank Yanxia Jia for help with
chlorophyll fluorescence assays and Wei Huang for critical
advice．
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7966期 TANG Ting et al．:Adaptation to Extremely High Temperature in an Alpine Environment:Systemic …