全 文 ：盐适应过程中香根草内源游离态、结合态、
束缚态多胺含量的变化
周 强 , 於丙军?
( 南京农业大学生命科学学院 , 江苏 南京 210095 )
摘要 : 研究了不同浓度 NaCl 胁迫下 , 香根草 ( Vetiveria zizanioides) 根、叶中的游离态、结合态、束缚态多
胺 (PAs) [包括腐胺 ( Put) , 尸胺 ( Cad) , 亚精胺 ( Spd) 和精胺 ( Spm) ] 含量的变化。在中度盐胁迫
( 100 , 200 mmol L - 1 NaCl ) 9 天时 , 香根草基本能够正常生长 , 但在重度盐胁迫 (300 mmol L - 1 NaCl) 下 , 其
生长受到严重抑制。在上述 3 个不同浓度的 NaCl 胁迫下 , 香根草根、叶中游离态 Put, Cad, Spd, Spm和总
的游离态 PAs含量明显下降 , 在高盐浓度下下降幅更大 ; 结合态 Put, Cad, Spd, Spm和总的结合态 PAs含
量显著上升 , 但在重度盐胁迫下升幅较小或与对照相当 ; 束缚态 Put, Cad和总的束缚态 PAs含量均减少 ,
而束缚态 Spd和 Spm含量在叶中是下降的 , 在根中则增加 , 且在中度盐胁迫下更明显。对根和叶片而言 ,
除游离态 (Spd+ Spm)?Put比值在重度盐胁迫下较对照显著下降外 , 其它游离态、结合态、束缚态和总的
(Spd+ Spm)?Put比值在不同盐胁迫下均上升 , 在中度盐胁迫下更明显。这表明 , 维持多胺总量的稳态和较
高的 (Spd+ Spm)?Put比值是香根草适应中度盐胁迫的一个重要机制。
关键词 : 多胺 ; 盐胁迫 ; (Spd+ Spm )?Put比值 ; 香根草
中图分类号 : Q 945 文献标识码 : A 文章编号 : 0253 - 2700 (2009) 06 - 477 - 09
Changes in Free , Conjugated and Bound Polyamine
Content in Salt Adaptation of Vetiver Grass
( Vetiveria zizanioides, Poaceae)
ZHOU Qiang, YU Bing-Jun
*
( Collegeof Life Sciences, Nanjing Agricultural University, Nanjing 210095 , China)
Abstract: Polyamines (PAs) have been suggested to play roles in plant salt stress adaptation . In this study, alteration of
free, conjugated and bound PAs [ putrescine (Put) , cadaverine (Cad) , spermidine (Spd) and spermine (Spm) ] in roots
and leaves of vetiver grass ( Vetiveria zizanioides) seedlings in responsetosalt stresswere investigated . Under moderate salt
stresses (100 and200 mmol L - 1 NaCl) for 9 days, vetiver grass grewat similar vigor compared with seedlings under normal
growth conditions . However, growth was severely arrested when plants were treated with severe salt stress (300 mmol L - 1
NaCl) . Under three different concentrations of NaCl stress mentioned above, thecontents of free Put, Cad, Spd, Spmand
total free PA substantially decreased in roots and leaves, andmore severe loss of free PAswas observed under higher NaCl
concentration . Conjugated Put, Cad, Spd, Spm, and total conjugated PAs remarkably increased, and theextent of the in-
crease after 300 mmol L - 1 NaCl treatmentwas smaller than thoseafter 100 or 200 mmol L - 1 NaCl treatments . Bound Put,
Cad and total bound PAs decreased in both roots and leaves under salt stress, moreover, bound Spdand Spmdecreased in
leaves while increased in roots in response to salt treatments, and themoreobvious rise was displayed under moderate salt
stresses . With the exception of the significant decreases of free (Spd+ Spm)?Put ratio in roots and leaves after 300 mmol
云 南 植 物 研 究 2009 , 31 (6) : 477～485
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2009.09165
? ?Author for correspondence; E-mail : bjyu@ njau. edu. cn; Tel : 025 - 84399012
Received date: 2009 - 09 - 01 , Accepted date: 2009 - 10 - 22
作者简介 : 周强 ( 1981 - ) 男 , 在读博士研究生 , 主要从事植物逆境生物学研究。
L - 1 NaCl treatment, other ratios of free, conjugated, bound and the total (Spd+ Spm )?Put increased in roots and leaves
of vetiver grass seedlings after different salt treatments, and especially under moderate salt stress . These results indicated
that maintaining homeostasis of total PAs content and high (Spd+ Spm)?Put ratios could be an adaptation mechanism in
vetiver grass to moderate saline environment .
Key words: Polyamines; Salt stress; (Spd+ Spm )?Put ratio; Vetiver grass ( Vetiveria zizanioides)
Salinity is one of the most deleterious abiotic
stresses, which adversely influences plant growth, de-
velopment and crop productivity in the world ( Liu et
al. , 2008) . Salinity can harmthe plant by its osmotic
and ionic effects ( Zhu, 2003) , which disrupts the in-
tegrity of cellular membranes, uptakeof essential nutri-
ents, function of photosynthetic apparatus and many
other physiological and biochemical processes (Demet-
riou et al. , 2007 ) . On the other hand, plants have
evolved mechanisms to cope with salt stress, one of
which is to synthesizesomeorganic substances, such as
polyamines (PAs) , glycine betaine, proline, and sug-
ars (Bartels and Sunkar, 2005 ) .
PAs are small organic cations with aliphatic nitro-
gen and widely observed in different organisms ranging
from bacteria to plants and animals . In plants, PAs are
involved in various physiological and developmental
events such as senescence and stress responses
(Alcázar et al. , 2006 ) . The common PAs arediamine
putrescine ( Put) , cadaverine (Cad) , triamine spermi-
dine (Spd) , and tetramine spermine (Spm) ( Bouche-
reau et al. , 1999) . Plant PAsoccur as free, conjugat-
ed andbound forms, themost common conjugated Put,
Spd, Spm are those that are covalently linked to
hydroxycinnamic acids catalyzed by Put, Spd and Spm
hydroxycinnamoyl transferases, i . e . PHT , SHT and
SpmHT, respectively . The conversion of free PAs to
bound PAs is mostly catalyzed by Transglutaminase
( TGase, EC . 2 . 3 . 2 . 13 ) ( Martin-Tanguy, 2001 ) .
Plant PAs contents were shown to be altered in response
to salt stress (Vasuki and Astrid, 2004) . Application of
exogenous PAs was also reported to improve plant salt
tolerance (Liu et al. , 2006) . Plant PAs aresupposed to
act as free radical scavengers, cellular membrane pro-
tectors andmaintaining cellular ionic balanceduringsalt
stress response ( J iménez-Bremont et al. , 2007) . High
accumulation of free Spd and Spm, or bound PAs were
beneficial for plant to alleviate the salt injury, while
accumulation of Put had adverse effectonplant salt tol-
erance (Zhao et al. , 2003) .
Vetiver grass ( Vetiveria zizanioides) is agramina-
ceous plant native to tropical and subtropical areas . Its
unique morphological , physiological and ecological
characteristics render this plant species capable of
growing in harsh environmental and soil conditions
( e.g . drought, hot, barren, acid, salt and alkali ,
and heavy metal , etc .) . This plant species has been
used for soil and water conservation, rehabilitation of
mines, contaminated soil and saline land, as well as
wastewater treatment . Besides active studies on its bio-
logical traits, current research has been focusedon us-
ing this plant species for conservationof soil andwater,
phytoremediation of heavy metals and other pollutants-
contaminated fields ( Pang et al. , 2003; Lai and
Chen, 2004 ) . Klomjek and Nitisoravut ( 2005 ) ex-
plored the feasibility of using this plant to remove pol-
lutants from saline wastewater . Our former work has
showed that, accumulation of inorganic andorganic os-
molytes invetiver grass seedlings under moderate saline
stress ( less than 200 mmol L - 1 NaCl ) , canplayagood
role in the osmotic adjustment and salt adaptation
(Zhou and Yu, 2009 ) . However, the role of PAs in
salt tolerance response of vetiver grass has not been re-
ported . In this study, we investigated the changes in
free, conjugated and bound PAs in roots and leaves of
vetiver grass under different salt stresses, and the pos-
sible function of PAs in salt adaptation of vetiver grass
was also discussed .
1 Material and methods
1 .1 Plant Materials and Growth Conditions
Vetiver grassseedlingswere obtainedfromhypaethral exper-
imental garden located in thecity of Nanjing, P.R . China . Six-
month-old vetiver grasses with multiple stems were divided into
single seedlings, which were clipped to keep leaf length with
about 0 .3 mand root length with about 0 .15 m . These seedlings
874 云 南 植 物 研 究 31 卷
were fixed in the sheet of foamboard, its roots were dipped into
1?2 Hoagland solution in plastic square boxes, and were cultured
at 28± 2℃ day?22± 2℃ night under light irradiation of 500
mmol? (m2 s) (16 h per day) in the growth chamber .
1 . 2 Salt Treatments
When seedlings resumed growing, they were subjected to
the treatments with following solutions: 1?2 Hoagland solution
(Control ) , 1?2 Hoagland solution + 100 mmol L - 1 NaCl , 1?2
Hoagland solution + 200 mmol L - 1 NaCl , and1?2 Hoagland so-
lution + 300 mmol L - 1 NaCl . All the above solutions were re-
placed by fresh solution in every 2 days andtheir pH wereadjus-
ted to6 .0 . After treatment for 9 days, the vetiver grassseedlings
were sampled and tested .
1 . 3 Determination of Plant Height Growth Rate
Plant height was directly measured using a centimeter ruler
at 0 and 9 days, respectively, and the plant height growth rate
was calculated usingthe followingformula:
Plant height growth rate= (H9 - H0 )?days
H0 and H9 represented the height of seedlings at 0 and 9
days, respectively . The plant height growth ratewas expressed as
means±SE (cm?d) of 6 seedlings .
1 . 4 Extraction and Analysis of PAs
Free, conjugated and bound PAs were extracted and quanti-
fied accordingto Kotzabasis et al. ( 1993) with minor modifica-
tions . 0. 5 g (fr wt) of root or leaf sampleswere homogenized in 5
ml of 5% (v?v) perchloric acid (PCA) water solution and incu-
bated at 4℃ for 1 h . Thehomogenatewas centrifuged at 10 000 g
for 30 min at 4℃ and the supernatant and pellet were collected
separately . The supernatant was used to determine the free and
conjugated PAs, andthe pellet was usedto measure the PCA-in-
soluble bound PAs as follows:
In order to extract the PCA-soluble conjugated PAs, 2 ml of
thesupernatant was mixed with 2 ml of 12 mmol L - 1 HCl and
heated at 110℃ for 18 h in flame-sealed glass ampules . After
acid hydrolysis, HCl was evaporated from the tubes by further
heating at 80℃ and the residue was resuspended in 1 ml of 5%
(v?v) PCA . This solution was used as a source of PCA-soluble
conjugated PAs and free PAs .
For extracting PCA- insoluble bound PAs, the pellet was
rinsed 4 times with 5% (v?v) PCA to removeany trace of solu-
ble PAs and then dissolved by vigorous vortexing in 2 ml of 1
mmol L - 1 NaOH . Themixturewas centrifuged at 10 000 gfor 30
min and the supernatant, including the bound PAs, was hydrol-
ysed under the same conditions as PCA-solubleconjugated PAs .
Free PAs extracted from non-hydrolyzed supernatant, free
plus conjugated PAs extracted from hydrolyzed supernatant and
bound PAs extracted from pellet were derived with benzoyl chlo-
ride as previously described (DiTomaso et al. , 1989 ) . Briefly,
0 . 5 ml of the above supernatant solutionswas mixed with 1 ml of
2 mol L - 1 NaOH . After the addition of 10μl benzoyl chloride,
vortexingfor 20 s, and incubation for 20 min at 37℃ , 2 ml of
ether was added . After centrifugation (1 500 g for 5 min) , 1ml of
theether phase was collected, evaporated to a dry state, and re-
dissolved in 100μl of methanol (HPLC grade) . Benzoyl-poly-
amines ( 10 μl ) were analyzed using a Waters HPLC System
(USA) equipped with an isocratic pump and areverse phase C18
column (Nova-pak, 150×3 .9 mm, particle size 4μm) . Metha-
nol?acetonitrile?H2 O (48∶2∶50) (v?v?v) was used as an isocratic
eluting solvent at a flow rate of 0 .8 ml min- 1 . Polyamine peaks
were detected by a Perkin-Elmer LC-95 absorbance detector at
254 nm . Conjugated polyamine contents were calculated by sub-
tractingthe freepolyamines fromtheacid-solublepolyaminecont-
ents . The fresh weight ( fr wt) of plant sample was transformed
into dry weight ( dry wt) through the sample water content, and
the PAs content was expressed as nmol?g dry wt .
1 . 5 Data Analysis
Each value was expressed as means±SE of three indepen-
dent experiments . The means of the different treatments with
three separate experiments were used to calculate t-values for
statistical analysis . Different letters show significant difference
( P < 0 .05) . All data of theexperiments were analyzed by using
SPSS 13.0 .
2 Results
2 .1 Effect of NaCl Stress on Height Growth Rate
of Vetiver Grass Seedlings
Treatments with 100 , 200 or 300 mmol L - 1 NaCl
for 9 days resulted in significant decrease of plant
height growth rate compared with the control (Fig. 1a) .
However, the plant appearance under moderate salt
stress (100 and 200 mmol L - 1 NaCl ) was as normal as
the control plant did . Severe growth arrest and salt in-
jurywere observed on seedlings treated with severe salt
stress (300 mmol L - 1 NaCl ) (Fig. 1b) . This result in-
dicated that vetiver grass has moderate salt tolerance
capacity .
2 . 2 Effect of NaCl Stress on Free PAs Contents
Put and Spd were the most abundant free PAs in
roots and leaves of vetiver grass seedlings . When com-
pared with the control , the contents of free Put, Cad,
Spd, Spm and total free PAs decreased in both roots
and leaves after 100 , 200 or 300 mmol L - 1 NaCl treat-
ments for 9 days . Treatment with higher concentrations
of NaCl resulted in more severe losses of free PAs
9746 期 ZHOU and YU: Changes in Free, Conjugated and Bound Polyamine Content in Salt Adaptation of . . .
Fig . 1 Effects of NaCl with different concentrations on the plant height growth rate ( a) and plant growth appearance (b) of vetiver
grass seedlings stressed for 9 days . Plant height growth rate was measured and expressed as means±SE ( n= 6 ) .
Different letters show significant difference ( P < 0 .05 )
(Fig. 2 : a-e) . The ratios of free (Spd+ Spm )?Put in-
creased in roots and leaves after 100 or 200 mmol L - 1
NaCl treatments, whereas the significant decreases
wereobserved in roots and leaves after 300 mmol L - 1
NaCl treatment (Fig. 2f) .
2 . 3 Effect of NaCl Stress on Conjugated PAs
Contents
Conjugated Put, Cad, Spd, Spmand total conju-
gated PAs significantly increased in roots and leaves of
vetiver grass seedlings after treated with 100 or 200
mmol L
- 1
NaCl for 9 days . The increase after 200
mmol L - 1 NaCl treatment was greater than that with
100 mmol L - 1 NaCl treatment . Although contents of
conjugated PAs were still higher in seedlings with 300
mmol L - 1 NaCl treatment than in control seedlings, the
extent of the increase after 300 mmol L - 1 NaCl treat-
ment was smaller than those after 100 or 200 mmol L - 1
NaCl treatments (Fig.3 : a-e) . The changes in ratio of
conjugated (Spd+ Spm )?Put in roots and leaves showed
the same trend as above-described . It was noticeable
that the ratios of conjugated ( Spd + Spm )?Put after
100 or 200 mmol L - 1 NaCl treatments weresimilar and
higher than that with 300 mmol L - 1 NaCl treatment
(Fig. 3f) .
2 . 4 Effect of NaCl Stress on Levels of Bound PAs
Bound Put was found to be the most abundant
bound PAs in roots, which accounted for about90% of
the total bound PAs . When comparedwith the control ,
the contents of bound Put, Cad and total bound PAs
decreased in both leaves and roots after salt stress treat-
ments . However, bound Spd and Spmincreased in ro-
ots and decreased in leaves ( Fig. 4 : a-g) . In roots,
the bound ( Spd + Spm )?Put ratios significantly in-
creased after salt stresses, and 200 mmol L - 1 NaCl
treatment resulted in highest increase of the ratios
(Fig. 4h) . The bound ( Spd + Spm )?Put ratios in
leaves increased after 100 or 200 mmol L - 1 NaCl treat-
ments, and declined after 300 mmol L - 1 NaCl treat-
ment, but the alteration on the ratios was not signifi-
cant when compared with the control (Fig. 4i ) .
3 Discussion
Many previous studies have suggested that alter-
ation in polyamine forms and levels plays an important
role in plant abiotic stress response ( Alcázar et al. ,
2006; Bouchereau et al. , 1999 ) . However, there are
some contradictory reports on changes in PAs content
under stresses (Basu et al. , 1988; Yang et al. , 2007) .
This discrepancy might be resulted from plant species,
type and development stage of tissues employed, inten-
sity and duration of stress treatments, and types of
stresses for experiments ( Botella et al. , 2000) . Thus,
the precise role of PAs metabolism in plant stress re-
sponse still remains elusive (Alcázar et al. , 2006) .
In our present study, for the first time, wesimul-
taneously focusedon the changes in contents of endoge-
084 云 南 植 物 研 究 31 卷
nous free, conjugated and bound PAs and their total in
vetivar grass seedlings under salt stress with different
NaCl intensity . Someremarkable changes in contentsof
different formsof PAswereobserved in roots and leaves
of vetiver grass seedlings after 100 , 200 or 300 mmol
L
- 1
NaCl stress for 9 days . The contents of free Put,
Cad, Spd and Spmand their total in roots and leavesof
vetiver grass seedlings significantly decreased in salt-
stressed seedlings than in control seedlings (Fig. 2 : a-
e) . Bound Put, Cad, Spd, Spmand their total also
Fig . 2 Changes in content of free Put ( a) , Cad ( b) , Spd (c) , Spm ( d) , their total ( e) and free ( Spd+ Spm )?Put ratio ( f) in roots
and leaves of vetiver grass seedlings under 100 , 200 , 300 mmol L - 1 NaCl for 9 days, respectively . Polyamine levels are expressed as
means±SE ( n= 3 ) of three independent experiments . Different letters show significant difference ( P < 0 .05)
1846 期 ZHOU and YU: Changes in Free, Conjugated and Bound Polyamine Content in Salt Adaptation of . . .
Fig . 3 Changes in content of conjugated Put (a) , Cad ( b) , Spd ( c) , Spm ( d) , their total ( e) and conjugated ( Spd+ Spm )?Put ratio ( f)
in roots and leaves of vetiver grass seedlings under 100 , 200 , 300 mmol L - 1 NaCl for 9 days, respectively . Polyamine levels are
expressed as means±SE ( n= 3) of three independent experiments . Different letters show significant difference ( P < 0 .05 )
showed similar changes as the above-described with the
exception that bound Spd and Spm in roots was in-
creased ( Fig. 4 : a-g) . The decrease of free PAs was
accompanied with decline of total bound PAs in both
roots and leaves of vetiver grass seedlings under 100 ,
200 and 300 mmol L - 1 NaCl stress (Fig.2 ; Fig. 4 : f,
g) . The contentsof total PAs includingfree, conjugat-
ed and bound forms could be maintained relatively ho-
meostatic in roots and leaves of vetiver grass seedling
under moderate salt stresses ( 100 and 200 mmol L - 1
284 云 南 植 物 研 究 31 卷
NaCl ) , which could be attributed to the increase of
conjugated PAs for compensating the decrease of free
and bound ones . However, under severe stress condi-
tion (300 mmol L - 1 NaCl ) , the total PAs in roots and
leaves significantly declined when compared with the
control (Fig. 5a) .
Fig . 4 Changes in content of bound Put (a for root, b for leaf) , Cad ( c) , Spd ( d) , Spm ( e) , their total (f for root, g for leaf )
and bound ( Spd+ Spm )?Put ratio ( h for root, i for leaf ) in vetiver grass seedlings under 100 , 200 , 300 mmol L - 1 NaCl for
9 days, respectively . Polyamine levels are expressed as means±SE ( n= 3) of three independent experiments .
Different letters show significant difference ( P < 0 .05 )
3846 期 ZHOU and YU: Changes in Free, Conjugated and Bound Polyamine Content in Salt Adaptation of . . .
Fig . 5 Changes in total PAs content ( a) and total ( Spd+ Spm )?Put ratio ( b) in roots and leaves of vetiver grass seedlings
under 100 , 200 , 300 mmol L - 1 NaCl for 9 days, respectively . The values are expressed as means±SE ( n= 3) of
three independent experiments . Different letters show significant difference ( P < 0 .05 )
Although there is little study on the function of
conjugated PAs in plant response to salt stress, it has
been shown that the conjugated PAs are associatedwith
plant resistance to other stresses . For example, the
conjugated PAs were important in enhancing ability of
plant to toleratediseases (Walters, 2003 ) . Piqueras et
al . ( 2002 ) found that high concentration of conjugated
diamines could be correlated with the establishment of
normal growth rate in hyperhydric carnation plants . The
conversionof free PAs to conjugated PAs enhanced the
chilling-toleranceof potato (Mauricio et al. , 1999 ) .
Rodríguez-Kessler et al . (2008) found that conjugated
PAs be related to the mounting of defense mechanisms
of the plant against the infection of the pathogen . The
above mentioned may attribute to their functions, for
example, they could be the preferred substrates for
amineoxidases, stabilize cell membrane and regulateof
the free PA titers and?or in detoxicating phenolic com-
pounds ( Martin-Tanguy, 2001 ) . In our work, it is
very noticeable that, the contents of conjugated Put,
Cad, Spd, Spm and their total increased significantly
in roots and leaves of vetiver grass seedlings under
moderate NaCl stresses (100 or 200 mmol L - 1 NaCl ) ,
while the increase was not significant after severe salt
stress (300 mmol L - 1 NaCl ) when compared with the
control ( Fig. 3 : a-e) . The decrease of free PAs in
vetiver grass seedlings under moderate NaCl stress is
very likely resulted from the conversion or biosynthesis
of conjugated ones . Our data indicate that, moderate
and severe salt stress would cause distinct alternations
on PA forms and levels, leading to either adaptation
and?or salt injury invetiver grass seedlings, and which
can maintain the homeostasis of total PAs during no-
deadly and longer term saline stress . This is also con-
sistent with the different plant growth appearance of
vetiver grass seedlings under moderate and severe salt
stress (Fig. 1 : a, b) .
Under salt stress conditions, salt-tolerant plants
often havehigher (Spd+ Spm )?Put ratio thansalt-sen-
sitive plants, suggesting a protective role of Spd and
Spmin plants against saline environment (Sannazzaro
et al. , 2007) . The increase in the (Spd + Spm )?Put
ratio might beone of the crucial factors for plant stress
tolerance, in partly, for reducing free radicals and al-
leviating lipid peroxidation, as free-radical scavengers
or through the induction of the activities for several an-
tioxidant enzymes such as superoxide dismutase, cata-
lase, and ascorbate peroxidase ( Wen et al. , 2008;
Wang et al. , 2007 ) . In this study, a increase of free
(Spd + Spm )?Put ratios was also observed in salt-
stressed vetiver grass seedlings, although free PAs in
roots and leaves of vetiver grass decreased when com-
pared with the control (Fig. 2 : a-f) . Higher conjugated
and bound (Spd+ Spm )?Put ratios were also found in
vetiver grass seedlings after salt stress treatments
(Fig. 3f; Fig. 4 : h, i ) . Consistently, the ratios of to-
484 云 南 植 物 研 究 31 卷
tal (Spd+ Spm )?Put were also higher in salt-stressed
seedlings than the control plants, especially for the
moderate salt-stressed ones (Fig. 5b) . Thus, mainte-
nance of high (Spd+ Spm )?Put ratio may berelated to
the salt adaptation of vetiver grass .
In conclusion, our results indicate that, maintain-
ing the homeostasis of total PAs by increasing the con-
jugated PAs to compensate the decrease of free and
bound ones, and keeping high (Spd+ Spm )?Put ratios
may jointly contribute to the adaptation of vetiver grass
to moderate saline environment .
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