全 文 ：Physiology and Ultrastructure of Azolla imbricata
as Affected by Hg2+ and Cd2+ Toxicity
SHI Guo_Xin＊ , XUQin_Song , XIE Kai_Bin , XU Nan , ZHANG Xiao_Lan ,
ZENG Xiao_Min , ZHOU Hong_Wei , ZHU Lei
(School of Life Sciences , Nanjing Normal University , Nanjing 210097 , China)
Abstract:　The toxic effects of different gradient concentrations of Hg2+ and Cd2+ on chlorophyll content ,
chlorophyll a/b value , photosynthetic O2 evolution , respiration rate , anti_oxidase system (superoxide dismu-
tase (SOD), catalase(CAT), peroxidase(POD))and ultrastructure of the cells of Azolla imbricata(Roxb.)
Nakai were studied.The results showed that with Hg2+and Cd2+ increase , chlorophyll content and chlorophyll
a/b value , photosynthetic O2 evolution decreased drastically;respiration rate peaked at 2 mg/L pollutant and
declined afterwards.The activities of SOD , CAT and POD increased first and decreased afterwards except the
activity of POD , which decreased with the increasing of Cd2+ concentration.Ultrastructural observation showed
that the extent of ultrastructural damage was much more serious with higher pollutant concentration and longer
time of stress.This resulted in swelling of chloroplast , disruption and disappearance of chloroplast membrane
and disintegration of chloroplasts;swelling of cristae of mitochondria , deformation and vacuolization of
mitochondria;condensation of chromatin in nucleus , dispersion of nucleolus and disruption of nuclear mem-
brane.The experimental results showed:(1)Hg2+ and Cd2+pollution not only destroyed physiological activi-
ties , but also caused irreversible damage to its ultrastructure , thus leading the cells to death;(2)With in-
crease in the stress of Hg2+ and Cd2+ , ultrastructural damage was related to the changes of plant physiology;(3)The toxic symptoms of plant showed an evident correlation between dose and effect;(4)The toxicity of
Cd2+on A.imbricata is heavier than that of Hg2+ under the same treatment time and concentration.The
lethal concentration of Hg2+ to A.imbricata ranged from 3.5 to 4 mg/L , and that of Cd2+ ranged from 3 to
3.5mg/L.The damage of cell ultrastructure on Anabaena azollae Strasburger was observed.The results indi-
cated that tolerance of Azolla imbricata for Hg2+ and Cd2+was higher than that of A.imbricata.
Key words:　Azolla imbricata;Hg2+;Cd2+;physiology;ultrastructure
　　Azolla imbricata (Roxb.)Nakai is a ferny hy-
drophyte plant , which is widely distributed in Asia , Eu-
rope , America and Oceania.In China , its individuals are
mainly living in the South and the Yangtze River basin
district.A.imbricata has been utilized as a green ma-
nure in the growth of rice for its good adaptability , fast
breeding speed and high amount of azotification.Efforts
have been taken to study its growth conditions , as well as
commensalisms and azotification mechanism of Anabaena
azollae Strasburger(Shi et al , 1981;Calvert and Peters ,
1981;Sun et al , 1984;Calvert et al , 1985).
In the past decade , heavy metal pollution resulted
from atmospheric sedimentation , geological leakage , un-
treated industrial effluents , and waste discharge has re-
ceived increasing attention (Sugiyama , 1994;Lorenz et
al , 1997;di Toppi et al , 1998).It was well document-
ed that aquatic heavy metal pollution gave a negative im-
pact on the normal growth of hydrophytes (Shi et al ,
2000;Zhou et al , 2001).The study on the physiological
and biochemical effects and the change of ultrastructure of
plants suffering from heavy metal pollution will be helpful
for further investigating poisoning mechanism of heavy
metals.
In the present study , A.imbricata was cultivated in
Hg2+_ and Cd2+_polluted water , which had obvious toxic
action on the plants.The chlorophyll contents , photosyn-
thetic O2 evolution , respiration rate , activities of superox-
ide dismutase (SOD), peroxidase (POD)and catalase(CAT), together with the changes of cellular structures
were studied , leading to increasing understanding of reac-
tions and tolerance of hydrophytes to the heavy metal
stresses.The basic characteristics and regularity of Hg2+
and Cd
2+
effects on physiological and biochemical func-
tions and cell structures were also investigated.
1　Materials and Methods
1.1　Materials
Azolla imbricata (Roxb.)Nakai was collected from
the culture pool in Dongshan town , Suzhou , China , and
was cultivated in hydrophytes culture pool.Similar fronds
were selected on June! 2001 , and then kept in eleven
glass aquariums.After being cultivated in distilled water
Received:2002-08-11　Accepted:2002-09-28
Supported by the National Natural Science Foundation of China(39770046), Key Program for S cience and Technology of Educational Ministry of China(01043)
and the Natural Science Foundation of the Educational Committee in Jiangsu Province.
＊Author for correspondence.Tel:+86(0)25 5891571;E_mail:.
植　物　学　报　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Acta Botanica Sinica　2003 , 45(4):437-444 http://www.chineseplantscience.com
for 2 d , five aquariums were added with 2 , 4 , 6 , 8 and
10 mg/L HgCl2 , respectively , while other five received
the same gradient concentrations of CdCl2.The remaining
aquarium was used as a control(0).All aquariums were
put into Forma_3744 totally enclosed illumination culture
instrument at 24 ℃.The illumination procedure consisted
of 12 h light(70 μmol·m-2·s-1)and 12 h dark , alter-
nately.Samples were analyzed under the transmission
electron microscope (TEM) from the second day and
physiological and biochemical indexes were measured after
5_day culture.
1.2　Methods
1.2.1 　Measurement of chlorophyll content 　The
chlorophyll content was determined according to Liu et al(1999)with 754 spectrophotometer.
1.2.2　Measurement of photosynthetic O2 evolution
and respiration rate　The photosynthetic O2 evolution
and respiration rate were measured according to Qin et al(1999)by oxygen electrode method.
1.2.3　Measurement of enzyme activity　The sample
was put in a pre_cooled mortar , in which 0.05 mol/L
phosphoric acid buffer was added.After a grinding in ice_
bath , solid phase was separated centrifugally at 10 000
r/min for 20 min and the supernatant was analyzed.The
activities of SOD and CAT were measured using a reagent
kit purchased from Nanjing Jiancheng Bioengineering In-
stitute of Jiangsu Province , China.The POD activity was
determined by using the guaiacol method (Maehly ,
1955).Each experiment was repeated three times.
1.2.4　Observation of leaf cell ultrastructure　The
last 3rd leaves from the top on the plant in every glass
aquarium were pre_fixed in 2.5% glutaraldehyde and
post_fixed in 2% osmium tetroxide (OsO4), then dehy-
drated in an ethanol series and embedded in Epon 812.
The embedded samples were sectioned(70 nm)with a di-
amond knife (LKB Products).The ultrathin section was
stained with uranyl acetate and lead citrate.Hitachi 600_
A_2 transmission electronmicroscope(TEM)was used for
section observation and photography.Both Azolla imbrica-
ta and Anabaena azollae samples were prepared with
above method.
2　Results
2.1　Physiological and biochemical changes
2.1.1 　Chlorophyll content and chlorophyll a/b
value　Both chlorophyll content and chlorophyll value in
Azolla imbricata decreased with the increase of the Hg2+
and Cd2+ gradient concentrations (Figs.1 , 2).Results
indicated that the chlorophyll contents fell by 11.18%
and 6.14%, respectively , when the concentration of
Hg
2+
and Cd
2+
was 2 mg/L.In low_concentration treat-
ments(<2mg/L), the chlorophyll contents in the fronds
treated with Hg
2+
declined more drastically than those
treated with Cd2+.However , when the concentration of
both Hg2+ and Cd2+ rose up to 10mg/L , the chlorophyll
contents decreased by 49.48%and 71.73% as compared
with control , respectively.This indicates that under the
condition of high_concentration treatment , the toxicity of
Cd2+was more serious than that of Hg2+.The results of
statistical analysis indicated that the changes of the above
indexes both had reached significant levels(r(Hg2+)=
-0.958 7 , r(Cd2+)=-0.985 0).
The chlorophyll a/b values dropped down syn-
chronously with the augmentation of Hg2+ and Cd2+ con-
centrations , and the differences reached significant levels
as well (r (Hg2+)= -0.997 2 , r (Cd2+)=-0.995 5).
2.1.2　Photosynthetic O2 evolution　Figure 3 illus-
trates that the augmentation of the concentrations of Hg2+
and Cd2+ gradually reduced the photosynthetic O2 evolu-
tion.When the frond was treated with 2 mg/L Hg2+ and
Cd2+ , its photosynthetic O2 evolution declined by
14.59% and 21.75%, respectively.Treated with 10
mg/L Hg2+ and Cd2+ , the POE diminished significantly
by 54.02%(r (Hg2+)=-0.982 2)and 47.36%(r
(Cd2+)=-0.952 2), respectively.
2.1.3　Respiration rate　When the frond was treated
with 2 mg/L Hg2+ , its respiration rate ascended by
4.22% in regard to the control value.It decreased by
10%when treated with 4mg/L Hg2+.When treated with
10 mg/L Hg2+ , it decreased by 76.19%(r (Hg2+)=-0.941 7).When the frond was treated with 2 mg/L
Cd2+ , its respiration rate enhanced by 33.06%.When
Fig.1.　Effects of Hg2+and Cd2+ stress on chlorophyll content.
Fig.2.　Effects of Hg2+ and Cd2+ stress on chlorophyll a/ b value.
438　 植物学报　Acta Botanica Sinica　Vol.45　No.4　2003
Fig.3.　Effects of Hg2+ and Cd2+ stress on photosynthesis oxygen
evolution rate.
Fig.4.　Effects of Hg2+ and Cd2+ stress on respiration rate.
Fig.5.　Effects of Hg2+ and Cd2+ stress on SOD activity.
NU , nitrite unit.
the treatment concentration increased up to 6 mg/L , the
respiration rate began to decline as compared to the con-
trol.When the concentration was 10 mg/L , it reduced by
83.33%(r (Cd2+)=-0.823 3).
2.1.4　SOD activity　It could be seen from Fig.5 that
the activity of SOD augmented sharply with the rise of
Hg2+ treatment concentration.When the frond was treated
with 8 mg/L Hg2+ , it increased by 110.5%.As the
concentration continued to rise , the activity began to go
down , and at the concentration of 10 mg/L , the SOD ac-
tivity was affected by only 98.59% (r (Hg2+)=-0.830 1).The opposite trend was observed with the
application of Cd2+.As a matter of fact , when the frond
was treated with 2 mg/L Cd2+ , the activity of SOD
reached its peak , increasing up to 46.43%.When the
concentration increased to more than 4 mg/L , it declined
rapidly , and at 10 mg/L , it decreased by 34.58%(r
(Cd2+)=-0.640 8).
2.1.5　POD activity　Two different tendencies of POD
activity could be found with the rise of Hg2+ concentra-
tion.When the frond was treated with low concentration
Hg
2+ , the activity of POD increased significantly.At 4
mg/L , it reached its peak value , and rose by 39.62%
compared with control(Fig.6).Then , it began to de-
cline , and at 10 mg/L , it reduced by 56.67% (r
(Hg2+)=-0.755 8).It could be seen clearly that POD
activity dropped rapidly when treated with low concentra-
tion of Cd2+.At 2 mg/L , it declined by 51% and at 6
mg/L , decreased by 91.36%.Even the treatment con-
centration increased further , the activity remained approx-
imately unchanged.At 10 mg/L , it decreased by
92.18%(r(Cd2+)=-0.893 6).
2.1.6　CAT activity　When the frond was treated with
2 mg/L Hg2+ or Cd2+ , its activity of CAT increased a
little , while treated with 4 mg/L Hg2+ or Cd2+ , it
reached the peak , and augmented by 37.01% and
63.1%, respectively (Fig.7).With the further rise of
concentration , it declined.At 10 mg/L , for Hg2+ , it
decreased by 64.04%(r (Hg2+)=-0.552 2), for
Cd2+ , it fell by 88.23%(r (Cd2+)=-0.606 0).
Fig.6.　Effects of Hg2+ and Cd2+ stress on peroxidase (POD)
activity.
Fig.7.　 Effects of Hg2+ and Cd2+ stress on catalase (CAT)
activity.
SHI Guo_Xin et al:Physiology and Ultrastructure of Azolla imbricata as affected by Hg2+ and Cd2+Toxicity 439　
2.2　Ultrastructural changes
2.2.1　Chloroplast　The normal chloroplast in the leaf
of A.imbricata appears to be oval , with a clear inner
structure.Grana layer is arranged orderly.The complete
membrane system is composed of grana thylakoid and stro-
ma layer(Fig.8).In the leaf cell of fronds treated with 2
mg/L Cd2+ for 5 d , it could be clearly observed that the
chloroplast seemed to be global.The electron density of
440　 植物学报　Acta Botanica Sinica　Vol.45　No.4　2003
the chloroplast stroma decreased , and a part of grana was
vacuole shape.In the cell of fronds treated with 6 mg/L
Cd
2+ , the inner structure of chloroplast was disorganized ,
and breakage occurred on the outer membrane of some
chloroplasts(Fig.9).Treated with the same concentra-
tion of Hg2+ , only the swelling of thylakoid grana and
layer , together with the formation of global chloroplast
could be observed.After 5 d of growing , the outer mem-
brane was not broken.The dose of 8 mg/L Cd2+ for 6 d
damaged most outer membrane of chloroplast , which dis-
appeared , and the swollen grana and stroma were dis-
persed into the cytostromata(Fig.10).When treated with
10 mg/L Hg2+ for 6 d , damaged thylakoid grana and lay-
ers could be observed (Fig.11).
2.2.2　Nucleus　The nucleus in the control leaf cell
appears to be oval , with a clear nuclear envelope.There
is a distinct boundary between nucleolus and caryoplasms ,
and chromatin is well distributed in caryoplasms(Fig.12).The nuclear envelope was still maintained for
5 d after 2 mg/L Cd2+ application.Although the mixing
of caryo_plasms and nucleolus was found in the boundary
of nucleolus , the nucleolus remained intact (Fig.13).
When treated with 6 mg/L Cd2+ for 5 d , most cell nucle-
olus disappeared , and chromatin agglomerated into a
lumpish matter with fairly high electron density (Fig.
14).With 10mg/L Cd2+ few cell nuclear envelope broke
and nucleolus disintegrated after 6 d.Other organelles
were mostly damaged , however many vacuous structures
existed(Fig.15).When treated with 10 mg/L Cd2+ for 8
d , only rudimental membranes and flocculation matters
with high electron density could be observed(Fig.16).
2.2.3 　Mitochondria 　Mitochondria are in a large
quantity and well distributed in the leaf cells of A.imbri-
cata , appearing to be round , oval and oblong , with a dis-
tinct tubular cristae.When treated with 2 mg/L Cd2+ for
4 d , it could be observed that the electron density of some
region decrease and some cristae swell a little.However ,
the outer membrane was still entire (Fig.17).Some mi-
tochondria cristae decreased in number(Fig.19)and few
formed to be vacuolar in one side (Fig.20).When treat-
ed with 8 mg/L Cd2+ for 6 d , cristae have been damaged
or appeared dim under TEM(Fig.10).Further , the out-
er membranes of some mitochondria broke down , or the
whole mitochondria disappeared.When treated with 2
mg/L Hg2+ for 4 d , it could be observed that mitochon-
dria swollen from one side.They looked vacuolar.Then
the whole vacuolar mitochondria were also found (Fig.
18).
2.2.4　Leaf cavity hair of A.imbricata　The multi_
cell hair(Sun et al , 1984)is formed by the division of
epidermic cells in the leaf cavity.Some mitochondria(Figs.21 ,22)and chloroplast are found in ingrowing cell
wall of the hair.It is well recognized that hair has impor-
tant physiologic function in the material_exchange process
of the fern_alga commensal (Peter et al , 1978;Neu-
muller and Bergman , 1981).When treated with 2 mg/L
Hg2+ and Cd2+ for 4 d , leaf cavity hair began to shed
and disappeared.When treated with 8 mg/L Cd2+ for 6
d , no hair could be observed in the leaf cavity (Fig.23).
2.2.5　Anabaena azollae cell　Anabaena azollae is a
kind of Cyanophyceae , belonging to the prokaryotes.The
alga cells are filled with compressed membrane_like pho-
tosynthesis layer , thylakoid , and fragmentary polyhedron(Fig.24).Although no ingrowing of cell wall existed on
the leaf cavity surface , A.azollae cells are often dis-
tributed over the leaf cavity because of the large quantity
of mitochondria.This alga is a single_cell stringy struc-
ture.However , its tolerance to Hg2+ and Cd2+ was high-
er than that of A.imbricata leaf cell.When treated with
8 mg/L Cd2+ for 4 d , the alga cell was still complete and
only decreased electron density could be observed under
TEM.In addition , the distribution of thylakoid was still
in order.When treated with 10 mg/L Cd2+ for 8 d , the
thylakoid of alga cell was damaged(Fig.25), and few al-
ga walls were damaged.Thylakoid and polyhedron fell in
the leaf cavity (Fig.26).
→
Figs.8-26.　8.Ultrastructure of control leaf cell , showing chloroplast , ×20 000.9.Leaf cells of Azolla imbricata treated with 6 mg/ L
Cd2+ for 5 d , showing round chloroplast , ×15 000.10.Leaf cells of A.imbricata treated with 8 mg/ L Cd2+ for 6 d , showing round disrupt-
ed chloroplast and mitochondria, ×12 000.11.Leaf cells of A.imbricata treated with 10 mg/ L Hg2+ for 6 d , showing disruption of chloro-
plast , ×15 000.12.Ultrastructure of control leaf cells , showing nucleus and nucleolus , ×7 000.13.Leaf cells of A.imbricata treated
with 2 mg/L Cd2+ for 5 d , showing nucleus and nucleolus , ×7 000.14.Leaf cells of A.imbricata treated with 6 mg/L Cd2+ for 5 d , show-
ing nucleus , ×11 000.15.Leaf cells of A.imbricata treated with 10 mg/ L Cd2+ for 6 d , showing nucleus , ×7 000.16.Leaf cells of A.
imbricata treated with 10 mg/L Cd2+ for 8 d , showing disrupted cell contents , ×7 000.17.Leaf cells of A.imbricata treated with 2 mg/ L
Cd2+ for 4 d , showing mitochondria , ×30 000.18.Leaf cells of A.imbricata treated with 2 mg/L Hg2+ for 4 d , showing mitochondria ,
×30 000.19.Leaf cells of A.imbricata treated with 2 mg/L Cd2+for 5 d , showing mitochondria , ×30 000.20.Leaf cells of A.imbrica-
ta treated with 2 mg/L Cd2+ for 5 d , showing mitochondria , ×30 000.21.Showing a leaf cavity hair and symbiotic algae , ×1 000.22.
Showing ingrowing cell wall of cell wall of hair , ×10 000.23.Leaf treated with 8 mg/ L Cd2+ for 6 d , showing symbiotic algae.×450.24.
Showing symbiotic algae and epidermal cell of leaf cavity.×8 000.25.Leaf treated with 10 mg/ L Cd2+ for 8 d , showing broken thylakoid
and carboxysome in cell of symbiotic algae , ×10 000.26.Leaf treated with 10 mg/ L Cd2+ for 8 d , showing disintegration of symbiotic algae
cell , ×12 000.
Abbreviations:A , algae;CP , chloroplast;Cs , carboxysome;CW , cell wall;H , hair;LC , leaf cavity;Mi , mitochondria;N , nucleus;Nu ,
nucleolus;Th , thylakoid.
SHI Guo_Xin et al:Physiology and Ultrastructure of Azolla imbricata as affected by Hg2+ and Cd2+Toxicity 441　
　　The structure of A.imbricata cultivated in Hg2+
and Cd2+ gradient concentrations changed significantly.
When treated with 4 mg/L Cd2+ for 4 d , the fronds began
to shrink and etiolate gradually.The old leaf rotted and
the center leaf etiolated after 10 d of culture , and all
leaves died after 20 d.When treated with the same con-
centration Hg2+ for 20 d , about 20% leaves still survived
and all died after 35 d.Compared with the above results ,
the control grew normally with the exception of the weak
etiolation of the old leaf.
3　Discussion
Chlorophyll is a major pigment participating in pho-
tosynthesis process , whose content and a/b value are an
indication of the activity of photosynthesis.It was com-
monly believed that leaves etiolated due to heavy metals
poisoning (Sun et al , 1985;Shi et al , 2000).In the
present experiment , the etiolation under different gradient
concentrations was related to corresponding decrease of
chlorophyll content and a/b value.Researches by Stobart
et al (1985)indicated that the decrease of chlorophyll
content was caused by two reasons.One was that pro-
tochlorophllide reductase was inhibited by heavy metals;
the other was that the synthesis of aminolaevulinic acid
had been affected.The authors concluded that effects of
both substances on chlorophyll were to influence its pro-
duction , and the gradually decrease of chlorophyll in ma-
ture leaves mainly resulted from the destruction of the in-
ner_membrane structure caused by heavy metals poisoning(Sun et al , 1985;Shi et al , 2000).We found that
when treated with 6 mg/L Cd2+ for 4 d , the inner struc-
ture of chloroplast was disorder(Fig.9).It is well docu-
mented that the completeness and order were necessary for
normal and efficient light_energy transformation in chloro-
plast(Li et al , 1987).With the increase of concentra-
tion , the structure of chloroplast was damaged(Fig.10).
This was another important reason explaining the decrease
of chlorophyll content.Results showed that the damage of
chloroplast matched with the decrease of chlorophyll con-
tent , a/b value and photosynthetic O2 evolution.
Resistance to heavy metals and other adversities ex-
isted in plants.From the stressed respiration rate of Hg2+
and Cd2+ , it could be clearly seen that when treated with
2 mg/L Cd2+ or Hg2+ , the respiration rate reached its
peak , increasing by 33.06% and 4.22%, respectively.
However , a large amount of ATP was consumed in the
process of anti_stress(Marschner , 1995).Ultrastructural
observation demonstrated that when treated with 2 mg/L
Cd2+ or Hg2+ for 4 d , mitochondria cristae had been
partly damaged (Figs.17 , 18).The change of inner
structure of mitochondria led to the deduction of adhesion
area in the soluble matrix of the enzymes required in the
tricarboxylic acid cycle.Thus , cellular respiration weak-
ened , and the aerobic glycometabolism was blocked(Xu
et al , 2001).Therefore , the phenomenon that the respi-
ration rate peaked transitorily and declined rapidly in the
stress of Cd2+ and Hg2+was associated with the extent of
mitochondria damage.
The unbalance between the production and the elimi-
nation of the active oxygen , caused by heavy metals , led
to the accumulation of free radicals in the fronds , which
were harmful to the plants(Cilina et al , 1994;Wong ,
1997).The cell membrane structure was further dam-
aged , causing the confusion of metabolism(Wong , 1997;
Luo et al , 1998).Superoxide anion(O-·2 )is a kind of
active oxygen free radicals.We had ever observed that af-
ter treatment with Cd
2+
or Hg
2+ , the content of O-·2 in
the fronds of Nymphaea tetragona and Brasenia schreberi
increased significantly , and the accumulation rate in-
creased with the increase of Cd2+ or Hg2+ concentration(Lu et al , 1999;Xu et al , 2000).Hydroxide radical
(·OH)formed by O-·2 in secondary reaction could destroy
biomacromolecules and membrane lipid (Rosen and
Rauckman , 1984).An efficient active oxygen scavenging
system was composed of SOD , CAT and POD in the
fronds(Chis , 1992).With the conjunct effects of SOD
and CAT , potentially harmful O-·2 and H2O2 were trans-
formed into harmless H2O and O2 , and the production of
·OH decreased(Scandalions , 1993).O-·2 was dismutat-
ed to H2O2 andO2 by SOD , andH2O2 could be eliminated
by CAT and POD efficiently.The results showed that
antioxidant enzyme system in A.imbricata fronds was se-
riously destroyed with Cd2+ and Hg2+ treatment.Al-
though a resistance peak usually occurred under low_con-
centration treatment , this was mainly caused by the in-
duction of active oxygen free radical , leading to a tran-
sient and irritable increase (Yan et al , 1996;Li et al ,
1999).With the increase of the concentration , enzyme
activity quickly declined.This was one of the key reasons
for the apolexis and death of plants in the stress of Cd
2+
and Hg
2+.Moreover , the change of ultrastructure , espe-
cially the degradation of nucleus , was an important sign of
cell death.
Although in the present research , Anabaena azollae
was not separated from the A.imbricata frond , the re-
sults obtained here were consistent with the study on other
hydrophytes(Lu et al ,1999;Li et al , 1999;Xu et al ,
2000).
The results herein further confirmed the conclusion
drawn in our previous publication (Marschner , 1995):
the harm of heavy metals to plants was expressed in the
membrane and non_membrane structure , the physiological
activities and biochemical reaction of plant cells , instead
of one enzyme or one cell organelle.The difference was
produced just because of the difference in tolerance.Ac-
cording to our experimental results , A.imbricata was
sensitive to Hg2+ and Cd2+.The lethal concentration of
Hg2+and Cd2+ was 3.5 -4.0 mg/L and 3.0-3.5
mg/L , respectively.When treated with 2 mg/L Hg2+and
Cd2+ , leaf cavity hairs began to shed , and the impor-
tance of hair in the materials exchange had been con-
firmed (Peter et al , 1978;Neumuller and Bergman ,
1981).Therefore , when A.imbricata was polluted by
442　 植物学报　Acta Botanica Sinica　Vol.45　No.4　2003
Hg2+ and Cd2+ low_concentration , its symbiosis , materi-
al_exchange and azotification were seriously affected.
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(Managing editor:WANG Wei)
SHI Guo_Xin et al:Physiology and Ultrastructure of Azolla imbricata as affected by Hg2+ and Cd2+Toxicity 443　
Hg2+和 Cd2+胁迫对满江红生理和细胞超微结构的影响
施国新＊　徐勤松　解凯彬　徐　楠　张小兰　曾晓敏　周红卫　朱　蕾
(南京师范大学生命科学学院 , 南京 210097)
摘要:　研究了在梯度浓度 Hg2+和 Cd2+胁迫下 , 满江红(Azolla imbricata (Roxb.)Nakai)的叶绿素含量 、叶绿素 a/b
比值 、光合放氧速率 、呼吸速率 、抗氧化酶系(超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD))和细胞
超微结构受Hg2+和 Cd2+的毒害影响。结果显示:随着胁迫程度的增大 , 叶绿素含量 、叶绿素 a/ b 比值 、光合放氧速
率明显下降 ,呼吸速率均在 2 mg/ L浓度下达到峰值 ,尔后下降;SOD、CAT、POD的活性均出现不同程度的应激性升
高(除 POD在 Cd2+处理时下降), 尔后下降。电镜观察发现 ,随着污染物浓度的增加和胁迫时间的延长 , 叶绿体出
现膨大 、破损和解体;线粒体嵴突膨胀和线粒体变形及空泡化;核染色质凝集 , 核仁消失 , 核膜破裂。实验结果表
明:Hg2+和 Cd2+污染不仅损害植物的生理活性 , 而且也破坏细胞的超微结构 , 最终导致植物死亡;随着 Hg2+和
Cd2+胁迫的增大 ,细胞超微结构的损伤程度和植物的生理变化是同步的;植物受毒害的程度表现出明显的剂量效
应关系;在同一处理时间和浓度下 , Cd2+对满江红的毒性大于 Hg2+。Hg2+对满江红的致死浓度为 3.5 ～ 4.0 mg/L ,
Cd
2+为 3.0 ～ 3.5 mg/ L。对满江红鱼腥藻(Anabaena azollae Strasburger)细胞的超微结构变化观察表明 , 满江红鱼腥藻
对Hg2+和 Cd2+的耐受性明显高于满江红。
关键词:　满江红;Hg2+;Cd2+;生理;超微结构
中图分类号:Q945　　　文献标识码:A　　　文章编号:0577-7496(2003)04-0437-08
收稿日期:2001-10-08　接收日期:2002-09-28
基金项目:国家自然科学基金(39770046);教育部科学技术研究重点项目(01043);江苏省教育厅自然科学基金。
＊通讯作者。 Tel:025_5891571;E_mail:。
(责任编辑:王　葳)
444　 植物学报　Acta Botanica Sinica　Vol.45　No.4　2003