全 文 ：广 西 植 物 Guihaia 28(5)：675— 680 2008年 9月
Effects of long-lasting brassinosteroid TS303
DONG Deng-Feng1，2．LI Yang-Rui¨ ，HANG Li—Geng ，
LIANG He2，HUANG Jing-Hua2
(1．K v L口6 Df Crop Genetic Improvement and Biotechnology，Nanning 530007，China；
2．CDz e 0f Agric“h“Fe，Guangxi University，Nanrfing 530004，China)
Abstract：The effects and the action mechanism of long lasting brassinosteroid(coded as TS303)and propyl dihydro—
iasmonate(PDJ)on peanut resistance to chiling were studied，The enhancement of chilling tolerance was indicated by
the reduction of malondialdehyde and electro1yte leakage．Chilling stress decreased superoxide dismutase(SOD)and
catalase(CAT)activities and relative water content while it increased the peroxidase(POD)activity and soluble saccha—
ride and proline c。ntents．TS303，PDJ and their mixture TNZ ameliorated the ir~ury caused by chilling stress through
Dreven g decreases in CAT and SOD activities and relative water content by enhancing the increases m Prol ne and
so1uble saccharide contents．TS303 exhibited better effect than PDJ on preventing decreases in SOD and CAT activi—
ties,meanwhile，PDJ exhibited better effect than TS303 on enhancing the increases in soluble saccharide and p 。l “。
contents，and it might be the difference in action mechanism that resulted in the additive or synergistic protec Ve e±iec
on cell membrane system．
Key words：brassinosteroid；chiling；peanut(Arachis hypogaea)；propyl dihydrojasmonate
CLC Number：Q565．2，Q945 Document Code：A Article 1)：1o00—3142(2008)05_0675—06
Bra【ssinosteroids can protect plants from injuring
induced by biotic and abiotic stresses(Zhou et a1．，
2002)．However，nature Brassinosteroids usualy ex—
hibit a short一1ived(2—3 d)or sporadic effect when used
in field，Attempts to prolong their effect have been
made and one chemically modified brassinosteroid
(TS303)，which display long-lasting effect has been re—
Dorted(Sasse，1997)．TS303，whose two active hydrox—
yl groups are combined by ethyl groups，has no physio—
logical effect itself． However，it can be hydrolyzed to
aclive plant BR showing long term effect from the 5 th
daY after it’s used(Takstsuto et a1．，1996)．
Jasmonic acid(JA)and its esters(e．g．Methyl Jas—
monate，MeJA)are linolenic acid derived cyclopen—
tanone-based compounds of wide distribution in plants,
whieh Dlay an important role in plant defense．They
can activate genes involving in pathogen and insect re—
sistance(Creelman et a1．，1997)as well as those enco—
ding osmotins(Xu et a1．，1994)which are correlation to
abiotic stress．Jasmonates usually exhibited better
physiological effects than abscisic acid but used at two
orders of magnitude lower concentration，so it would be
a Dotential substitute for the expensive abscisic acid
(Miyamoto et a1．，1997)．Propyl dihydrojasmonate
(PDJ)，a kind of synthetic jasmonate whose C9一CIO vi—
nyl bond is saturated by hydrogen and carboxyl group
F0undati0n item：Supported by Postdoctora1 Research Foundation of Guangxi Academy of Agriculture Sciences(GNBH52275)；the
Natura1 Science Foundation of Colege of Agriculture，Guangxi University(X061013)
Biography：DONG Deng-Feng(1971．)，Male，Born in Jinshan County of Hubei Province，Philosophy Doc or and a。soc p oiesso ·
Researching on plant stress physiology and molecular biology．
Author f0r corre~,；pondence，E-roail：lyr@gxaas．net
676 广 西 植 物 28卷
esterifled by n-propyl，has stronger penetrability and
steadier effect than other jasmonates．
Peanut，originated in South America，is susceptible
to chiling inj uries when exposed to nonfreezing tem—
peratures in the range of 0-15℃ ．In subtropical tern-
perature and high altitude areas，such as western-
southern of China，the frequent occurrences of cold
snaps in spring cause damage to peanut． Therefore
chilling stress appears to be a major limiting factor for
spring—peanut growth and agronomic productivity．In
this study，peanut seeds were pretreated with modified
chemical regulators TS303，PDJ and their mixture，the
resulting seedlings were subsequently stressed with
chilling，and then some resistance-related indices were
investigated at different stressed days with the aim to
substantiate the ameliorating effects and to further ex—
plore the mechanism．
1 Materials and methods
Peanut(Arachis hypogaea L．“Guihua 1 7”)seeds
were selected and surface sterilized with 30 H2 O2 for
5 min and washed thoroughly with de-ionized water，
soaked in distilled H2 O(as contro1)，0．1 mg／L TS303
(lama corporation，Japan)，1 mg／L PDJ(Zeon corpo—
ration，Japan)and TNZ(0．1 mg／L TS303+1 mg／L
PDJ)for 12 h，respectively，sown into plastic pots con—
taining vermiculite in a growth chamber with a light／
dark temperature of 28／20 ℃ ，800／~mol·rn- ·s-
light 10 h／dark 14 h cycle，7O一85 9／6 relative humidity，
watered with one-fifth strength Hoagland solution(pH
5．5-6．O)every 3 d．The temperature was switched to
6 ℃ for low temperature stress for 9 d after the
plants’second compound leaves were fully expanded
(12一day-old)，while other cultivation conditions were
similar to those before stress．Peanut 1eaves of 0 d，3
d，6 d and 9 d under chilling stress were harvested to
determine the following indices．
Electrolyte leakage was measured as described by
Lutts et a1．(1996)．
Samples for determination of contents of protein
and MDA，activities of SOD，POD，CAT and APX were
prepared by freezing 0．5 g of leaves in liquid nitrogen
to prevent proteolytic activity，followed by grinding
with 5 mL extraction buffer(O．1 mol／L phosphate
buffer，pH 7．5，containing 0．5 mmol／L EDTA，10
mg／mL PVP and 1 mmol／L ascorbic acid)in a chilled
pestle and mortar，the homogenate was centrifuged at
13 000× g，4 ℃ for 2O mi nutes，the supernatant was
used to analyze．
MDA content was determi ned according to Zhao
et a1．(1994)．1 mL of the supernatant was added to 3
mL of 5 mg／mL thiobarbituric acid(TBA)in 0．2 g／mL
trichloroacetic acid(TCA)．The mixture was heated at
100℃ for 20 mi n in a sealed tube and then cooled in
an ice bath．After eentrifugation at 5 000 × g for 10
mi n，the absorbance of the supernatant was recorded at
450，532 and 600 nm with a spectrometer．The concen—
tration of MDA was calculated by the folowi ng formu—
la：C(uM)=6．45(A532一A600)一0．56A450．
Protein content was determined according to Brad—
ford(1976)using bovine serum albumin as a standard．
SOD activity was determined according to Gianno—
potitis&Ries(1977)：one unit of SOD activity was de—
fined as the amount of enzyme inhibiting photochemical
reduction of NBT by 50 per mi nute．
Ascorbate peroxidase(APX)was assayed by re—
cording the decrease in absorbance due to ascorbic acid
at 290 nm(Nakano& Asada，1981)．Enzyme activity
was computed by referring to a standard curve of
known concentrations of AsA．and one unit of APX ac—
tivity was defined as the amount of enzym e catalyzing
the consumption of 1／~mol AsA per minute．
Catalase(CAT)activity was assayed by measuring
the rate of decomposition of H2 02 at 240 nm in a reac—
tion mixture as described by Chance Maehly(1955)．
Enzym e activity was computed by referring to a stand—
ard curve of known concentrations of hydrogen perox—
ide，and one unit of CAT activity was defined as the a—
mount of enzym e catalyzing the conversion of 1／xmol
H2 02 into water per minute．
POD activity was determined by monitoring the
increase in absorbance at 470 nm as guaiacol was oxi—
dised，according to the method of Chance& Maehly
(1955)．
Relative water content(RWC)was estimated gray—
5期 董登峰等：长效油菜素内酯 TS303和二氢茉莉酸丙酯增强花生抗寒能力 677
imetrically according to the method of Fenlg et a1．
(2003)．Leaves fresh weight(FW)，turgid weight(TW)
and dry weight(DW)were measured and RWC was com—
puted as RWC( ) (FW-DW)／(TW-DW)x 100．
Proline was extracted with boiling 30 mg／mL aque—
OUS sulfosalicylic acid and determined by acid ninhydrin
reagent as described by Bates et a1．(1973)．
Soluble saccharide was extracted with 8O 0A ethano1
and estimated by anthrone reagent using glucose as stand—
ard，according to the method of Yemm & Willis(1954)．
Experiments described here were performed with
four replicates；all parameters taken for the experiments
processed by analysis of variance(ANOVA)and the means
were compared by Duncan’s Multiply Range Test(DM—
RT)at the 5 significance(P<0．05)limits in SAS．
0 3 6 9
Treatm ent duration(d)
Fig．1 Efects of TS303，PDJ and TNZ on malondialdehyde
content(A)and electrolyte leakage(B)in
peanut Ieaves under chilling stress
Data represent the mean士 SE from four replicate experiments．
Treatments labeled common 1owercases within same treatment
day show no significant diference(P< 0．05)．The same below．
2 Results
2．1 Effects of TS303，PDJ and TNZ on malondialdehyde
content and electrolyte leakage in pean ut leaves under
chilling stress
Under low-temperature exposure，MDA contents
in leaves were dramaticaly increased． Meanwhile
TS303 significantly decreased MDA accumulation
caused by chilling．PDJ alone did not show significant
effect on decreasing MDA contents，and it even signifi—
cantly increased(by 48．42 9／6)before chilling stress．
Similar results had been reported in peanut(Kumari et
nZ．，2006)and Scenedesmus incrassatulus(Fedina &
Benderliev，2000)treated with methyl jasmonate，an an～
alog of PI)J．However，when PDJ was used in combi—
nation with TS303．a significant decreased of MDA
content was demonstrated throughout the chilling peri—
od(Fig．1：A)．
Electrolyte leakage was increased both rapidly and
stably with increasing days of stress．TS303，PDJ and
TNZ slowed down the leaking resulted by chilling．
The peanut leaves treated with TS303，PDJ and TNZ
leaked 32．29 ，22．82 and 61．17 9／6 less than the
control，respectively，on the 6 th day of chilling；and
35．58％，24．61 and 65．44 less，respectively，on
the 9th day of chiling(Fig．1：B)．
2．2 Effects of I 03。PDJ and TNZ on activities of su-
peroxide dismutase，ascorbate peroxidase。catalase an d
peroxidase in peanut leaves under chilling stress
SOD。( T and APX activities were found to de—
crease gradually with chiling stress．TS303 treated，a—
lone or in combination with PDJ，both significantly
slowed down the decreasing in SOD and CAT activities
induced by chilling，and they even induced increasing in
SOD activity in the first three days．PDJ did not show
a significant effect of slowing down on the decreasing
in SOD and CAT activities when was used alone．
TS303，PDJ and TNZ did not show significant effects
on APX activity(Fig．2：A，B，O ．
POD activity in the control peanut leaf was rapid—
ly increased in the first three days，while decreased in
the following days。 However，POD activities in the
chemicals treated peanut leaves were increased slowly
but consistently during the stress period． Generally，
POD activities of TS303一treated peanut leaves were
higher than those of PDJ—treated on the corresponding
stress days(Fig．2：D)．
2．3 Effects of TS303。PDJ and TNZ on water，soluble
saccharide and prolinecontents in peanut leaves under
chiling stress
During the chilling stress，relative water content
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678 广 西 植 物 28卷
(RWC)of peanut leaf was decreased，while soluble sac—
charide and proline contents were increased gradually．
Increasing in the relative water content as wel1 as solu—
ble saccharide and proline were showed in TS303 and
PDJ treatments used alone or in combination．These
effects magnified with increasing days of stress．On the
9th day of chilling stress，TS303，PDJ and TNZ was
found to increase relative water content by 4．72 ，
1．
1．0
0．9
O．日
0．7
0．6
0．5
O．4
0．3
0．2
0 1
0
12．o9％ and 17．94 ，respectively；soluble saccharide
content by 27．85％，51．33 and 57．63 ，respective—
ly；and proline content by 17．29 9／6，38．15 9，6 and
59．O5 9／6，respectively(Fig．3：A，B，C)．Among treat—
ments，TNZ exhibited the best effect．TS303 interacted
with PDJ as additive，and even synergistic manner in
some cases(e．g．action on RWC and proline content in
the 9th day of chiling stress)．
O 3 6 9 0 3 6 9
Fig．2 Effects of TS303，PDJ and TNZ on activities of superomde dismutase(A)，ascorbate
peroxidase(B)，catalase(C)and peroxidase(D)in peanut leaves under chiling stress
3 Discussion
Damage caused by chilling stress is，at least in
part，due to membrane lipid peroxidation(Lu &
Huang，2004)．MDA is one of the main products of
plant lipid peroxidation． The increasing of electrolyte
leakage was considered to be a symptom of stress in—
duced membrane damage and deterioration，and proven
to be sensitive and accurate marker and thus usefu1 for
assessing the chilling damage(Simon，1 974)．TS303
and PDJ could significantly decrease electrolyte leakage
and MDA accumulation resulted by chilling stress，in—
dicating an enhancement on resistance of peanuts to
chilling stress．
Plants are severely affected by abiotic and biotic
stresses partly because the production and quenching of
reactive oxygen species(ROS)in plant cells can not be
maintained in a balanced state(Bowler，1992)．ROS
such as superoxide radical，hydrogen peroxide and hy—
droxyl radical can seriously disrupt normal metabolism
through oxidative damage of lipids，proteins and nucleic
acids(Imlay& Linn，1988；Jiang et a1．，2002)．Plants
have evolved specific protective mechanisms，involving
in antioxidant molecules and antioxidative enzymes
such as SOD，CAT ，POD and APX so as to defend
themselves against oxidants(Jiang 8L Zhang，2002)．
SOD catalyses the conversion of the superoxide anion
to H2。2．CAT，APX and a variety of general PODs
(Chang et a1．，1984)catalyze the breakdown of H2 O2
in different organeles．Therefore，this enzyme system
cooperatively eliminates the damaging effects of toxic
oxygen species．In the present study，chilling weakened
the activities of SOD，CAT and APX，but increased to—
tal POD activity．The increasing of total POD activity
might be a compensative result due to CAT decreasing
and useful in the defense mechanism of plants against
H2 o2．As a mater of fact，the increasing in total POD
(ub]ojdbE n)套^{l3∞×d《
9 8 7 6 5 4 3 2 1
0 O O 0 0 0 0 O O O
0 9 8 7 6 5 4 3 2 ， 0
一熹学JdL．6E n)备 B∞o。c
9 8 7 6 5 4 3 2 ， 0
一c!。菩 I6E n) 》{l撼8∞ 一I_9口 暑．-l n)^l若3B上vu
5期 董登峰等：长效油菜素内酯 TS303和二氢茉莉酸丙酯增强花生抗寒能力 679
卯
∞
善
’_一 ∞
詈 ∞
3l】
∞
10
O
O 3 6 9
Tm~rnenl duration(d)
Fig．3 Effects of TS 303，PDJ and TNZ on water(A)，
soluble saccharide(B)and proline(C)contents in
peanut leaves under chilling stress
activity seemed to be a common response to various
oxidative stress factors(Inshida a1．，1985；Kumari et
a1．，2006)．Ts303 and TNZ inhibited the decreases in
activities of SOD and CAT ，and enhanced the increases
in activity of POD during the whole chilling period．
Present results suggested that TS303 and TNZ induced
stress tolerance in plants may be caused，at least in part，
by increasing antioxidant activities，which in turn reduc—
ing stress-related oxidative dama ge to cell membranes．
It is wel established that the RWC reflects the water
status and is related to the growth and plant chilling re—
sistance(Feng et a1．，2003)，IB303，PDJ and TNZ all
slowed down the decreasing in water and postponed the
onset of tissue desiccation under the chilling stress．High—
er content of soluble saccharide and proline had been sug—
gested as important factors conferring chiling tolerance
(Uemura& Steponkus，1998；Hores et a1．，1988)．Accu—
mulation of saccharide and proline might be an adaptive
change to maintain water content． Besides osmoregula—
tion，the cryoprotective action of soluble saccharide and
proline might also be involved in the stabilization of mem-
brane system as well as provision of a store of carbon，ni—
trogen and energy，and act as precursors to other protective
compounds(Uemura& Steponkus，1998；fang＆ Kao，
1999)．PD and TNZ significantly enhanced the incre—
ments of soluble saccharide and proline induced by chiling
throughout the stress period，and TS303 did the same on
the 6th and／or 9th days．
Collectively，TS303 and PDJ could ameliorate the
injury caused by chilling stress by preventing the de—
crease in CAT and SOD activities and relative water con—
tent by enhancing the increment in proline and soluble
saccharide contents．TS303 exhibited better effect than
PDJ on preventing decreases in antioxidative enzymes ac—
tivities，meanwhile PDJ exhibited better effect than
TS303 on enhancing the increases in soluble saccharide
and proline contents，and it might be the difference in ac—
tion mechanism that resulted in the additive or synergis—
tic protective effect on eel membrane system．
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鸵岳l∞{B阱救∞蔼 l已 ∞艏∞ ∞箍∞俘帕l‘，o
善 詈 (％)】u8§ 垂 蓑 意 歪 茗蓉u 33e羔嗣lIos ju墨|0u ‘lI2
68O 广 西 植 物 28卷
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长效油菜素内酯 TS303和二氢茉莉酸
丙酯增强花生抗寒能力
董登峰 ，2，李杨瑞l*，江立庚 ，梁 和2，黄京华
(1．广西作物遗传改良和生物技术重点实验室，南宁 530007；2．广西大学 农学院，南宁 530004)
摘 要：长效油菜素内酯TS303和二氢茉莉酸丙酯(PDJ)浸种能增强花生对低温的忍耐能力，二者显著降低低
温诱导的丙二醛含量和电解质渗漏率。低温降低超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性以及相对含
水量，但增加过氧化物酶(POD)活性以及可溶性糖和脯氨酸含量。TS3O3和PDJ以及它们的混合物 TNZ都能
延缓低温伤害引起的s0D和 CAT活性下降，并能通过增加可溶性糖和脯氨酸含量来提高相对含水量。TS303
在延缓SOD和CAT活性降低方面效果比PDJ好，但 PDJ在增加可溶性糖和脯氨酸含量方面效果比TS303强，
由于TS303和PDJ作用机理不同，二者混合使用表现出加成或协同效应。
关键词：油菜素内酯；二氢茉莉酸丙酯；花生；低温
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