全 文 ：第 26卷 第 4期
2005年 10月 　　　　　　　　　　
华南农业大学学报
Journa l o f South China Agricultural University
　　 　　　　　　　　Vo.l 26, No. 4
Oc.t 2005
　R eceived date:2005 - 02 - 25　　　　B iography:CHONG Yun-xiao(1974 -), fem ale, Ph. D. ;E-m a il:cyx04@ scau. edu. cn
Foundation item:M ajo r Sta te Basic Research Deve lopm ent P rogram of Ch ina (G1999045711)
Effect of ammonia on the ultrastructure
of duckweed species
CHONG Yun-xiao1, 2 , HU Hong-y ing2 , Q IAN Y i2
(1 Co llege of Re sources and Env ironment, South Ch ina Agric. Univ. , Guang zhou 510642, Ch ina;
2 Env ironm en ta l S im ulation and Pollu tion Contro l S ta teKey Joint Lab, Depar tm en t o f
Env ironm ental Science and Eng inee ring, Tsinghua Unive rsity, Be ijing 10084, Ch ina)
Abstrac t:The change s of u ltrastructu re unde r scanning elec tron m icro scopy and transm ission e lectron m icroscopy we re de-
scribed in two duckw eed spec ie s o f Lenma aequinoctia lis and Spirodela polyrrihza cu ltiva ted in amm onia medium in present
study, contrasted by the ones g row ing unde r nitra te m ed ium. The result show ed tha t outstanding ce ll wa ll in lowe r epide rm is
becam e crink ly and disappeared partly in fronds under ammonia medium. The stom ata on the uppe r epide rm is opened much
w ider farm ore than ones under n itratem edium. The ch lo roplastm em brane lim iting became blurred and disappeared partly in
fronds unde r amm on ia m ed ium. The whole chlorop la st o f fronds unde r amm on iam edium presented the trending o f disassem-
b ly. And the in te rna lm em brane ne tw ork including g rana lame llae and strom a lam e llae break o f.f In contrast to one s under ni-
trate medium , m ore starch g ra in appeared am ongst strom a. Based on the re su lts of observa tion, the tox icitym echanism o f un-
ionized amm onia (NH
3
) and ammon ium ion (NH +
4
) to duckweed w ere respec tive ly discussed.
K ey words:duckw eeds;ammonia;u ltrastructure; tox icity
CLC num ber:Q942. 1　　　　　Document code:A　　　　Article ID:1001 - 411X (2005) 04 - 0077 - 04
氨对紫背浮萍和稀脉浮萍超微结构的影响
种云霄 1, 2 , 胡洪营 2 , 钱　易 2
(1华南农业大学 资源与环境学院 , 广东 广州 510642;
2清华大学 环境科学与工程系 环境模拟与污染控制国家重点实验室 , 北京 100084)
摘要:选择紫背浮萍和稀脉浮萍 , 利用 SEM和 TEM 分别对在对照液硝酸盐氮培养液和含有较高氨氮培养液中生长
个体的超微结构进行了观察和分析. 结果表明 , 与在硝酸盐氮培养液中生长的个体相比 , 在氨氮培养液中生长的 2
种浮萍上表皮的气孔张开程度明显地增大 ,下表皮细胞突起的细胞壁消失 , 细胞发生明显的变形;生长在含有氨氮
培养液中的 2种浮萍个体的叶绿体内膜结构均受到严重的损伤 ,叶绿体中淀粉粒明显增多 , 整个叶绿体处于解体状
态 , 而生长于硝酸盐氮培养液中浮萍的叶绿体则完好无损.
关键词:浮萍;氨;超微结构;毒性
　　Duckw eeds species, name ly Lemnaceae species,
are a group of free-floating aqua tic plan ts. They have
lea f-like body, called frond. The frond could take up
nutrient such as n itrogen and phosphorus from w ate r.
To contro l eutrophication w ith low cost, it is a prefe rred
cho ice to convert these nu trients into p lant protein by
duckw eeds up-tak ing[ 1 -5] . Duckw eed species are suit-
ab le to be poultry and fish feed
[ 6, 7]
.
Ammonia, la rgely existing in domesticw astew ate r,
is main nitrogen resource to duckw eed species. How ev-
er, ammon ia is toxic to aqua tic organism and cou ld in-
h ib it the g row th at high concen tration. In addition , due
to their sma ll size and ease o f grow th , duckw eed species
w ere o ften used as expe rimenta lm aterial to testing the
aquatic p lant toxicity of ammonia in w ater
[ 8 - 10]
. Bu t the
past investiga tions o r testing mo re paid mo re a ttentions
to the relationsh ip between ammonia leve l and visua l
symp tom such as frond number change, co lo r, roo t
change e tc.
N itrate is ano ther nitrogen resource for duckw eed
species and generally it has no toxicity except extreme ly
high leve.l Using the fronds g row ing unde r n itratemedi-
um as comparison, the damage of ammon ia to ultra-
structure of tw o duckw eed species fronds we re described
here. The objective is to m ake clear the toxic mecha-
nism o f ammonia to duckw eed species, furthe rmore, the
mechanism to aqua tic p lan.t
1　Mater ials and m ethods
1. 1　Collection and acc lmi ation of duckweed spec ies
Spirodela polyrrihza (Linn. ) Sch le id and Lemna
aequ inoctialisWelw itsch are respective ly common spe-
cies o f two main genera ofLemna and Spirodela in Lem-
naceae. And they w ere often used in w astew ater treat-
men .t Thema terialsw ere co llec ted from aw etland near-
by D ianchi Lake ( located in Kunm ing C ity, Yunnan
Prov ince, China) and acclimated to the artificia l bal-
ance media suitab le to culture o f duckw eed
[ 11]
.
1. 2　Cultivation of duckweed spec ies
The fronds of S. polyrrihza and L. aequinoctia lis
were cultiva ted firstly to adapt the cond itions w ith ni-
trate or ammonia as only nitrogen resource. So the ni-
trogen in the artificia l ba lance med ium was adjusted to
tw o conditions:ρ(NH +4 -N)=20mg /L andρ(NO -3 -N)
=20 mg /L by replacing NH4NO3 w ith NH 4C l and
N aNO 3 respec tive ly. The pH o fmedium was adjusted to
7.0±0.3. Tw en ty hea lthy fronds o f each species w ere
placed into them edia. The cu ltivation w as pe rformed in
plastic containe r w ith 200 mL medium. In order to
make frond adap t the grow th condition fu lly, the culti-
va tion lasted 24 day s. A t the end of the cultiva tion, 5
fronds o f in each condition w ere taken out random ly fo r
the observation of ultrastruc ture. The experimen ts w ere
conducted in laborato ry cond ition w ith fluorescent lamps
of light intensity o f 2 000 - 3 000 lx (16 h-light, 8 h-
dark). The tempe ra tu re w as 26 -30 ℃.
1. 3　Treatment of samples for SEM＆ TEM
The fronds w ere fixed in 4% (w) g lutara ldehyde
and postfixed in 1% (w ) osm ium te trox ide in
0.1mo l /L phosphate buffer w ith pH 7. 1. Dehydration
w as accomplished in a g raded acetone series lastly.
Some samples w ere taken out for the observation of
ch lo roplast o f parenchymatic tissue by transm ission e lec-
tion m icroscopy (TEM). The samples we re infiltrated
and embedded in Epon (Spurr). Thin sectionsw e re cu t
w ith a diamond knife, stained w ith Reyno ld′s lead cit-
ra te and exam ined w ith aH itachi-600 TEM at 80 kV at
las.t O the r samp les we re taken out for the obse rvation of
the epiderm is o f upper and low e r sides by scanning e-
lec tron m icroscopy (SEM). The whole frond w as cu t
in to segments firstly, fixed in g lu taraldehyde, dehyd ra-
ted in acetone, critical-point dried, sputtered w ith go ld
and exam ined w ith aH itachi-505 SEM a t las.t
2　Results
2. 1　Changes of ultrastructure in lower epiderm is
of frond
　　The low er epide rm is of fronds grow ing unde r am-
mon iamedium looked obv iously diffe rent from ones un-
der nitrate medium. For fronds unde r ammonia medi-
um , the outstand ing w all betw een cells became very
crink ly in S. polyrrihza (F ig. 1c) and even d isappeared
partly in L. aequinoctia lis (Fig. 1d), wh ich made the
ce ll take on blurred array. The w all be tw een ce lls stand
ou t on the low er epide rm is o f frond unde r n itrate medi-
um , which separated the ce lls and made the ce ll ar-
range orderly (Fig. 1a and F ig. 1b). It seemed tha t am-
mon ia in themedium could direc tly cause the damage to
the w all o f lowe r ep iderm is ce l.l
2. 2　Changes of ultrastructure in upper epiderm is
of frond
　　Like the cond itions of low er epiderm is, the uppe r
epiderm is of fronds under ammonia medium looked ob-
viously diffe rent from tho se unde r n itrate medium. The
most change w as the behavio r of stom ata. They kep t
open sligh tly in frond under nitra te med ium (Fig. 2a
and Fig. 2b), but opened much bigge r under ammonia
medium (Fig. 2c and Fig. 2d). The epiderm is ce lls
su rround ing stomata a lso we re a little d iffe ren.t They
w ere more expanding unde r ammonia med ium , no t like
one under nitra te medium. It seemed that ammonia
could affect themob ility of stomata of duckw eed species
fronds.
78 　　 华　南　农　业　大　学　学　报　　　 第 26卷　
F ig. 1　The SEM pho tog raph of the low er ep iderm is
F ig. 2　The SEM pho tog raph of the uppe r epide rm is
2. 3　Changes of ultrastructure of the chloroplast
The ch lo roplasts of fronds unde r nitra te medium
kep t comp lete structu re (Fig. 3a and F ig. 3b). The
membrane of the ch loroplastw as very c lear. The lame l-
lae fo rm ing grana piled up densely togethe r. The stroma
lamellae connecting the g rana kept continuous and
formed intact stroma lame llae netwo rk. The dense stro-
ma con tained a starch g ra in and several g lobu l.i On the
contrary, the ch lo roplast of fronds under ammon ia w as
destroyed evidently (Fig. 3c and Fig. 3d). The mem-
b rane became blurred and disappeared partly, even dis-
appeared to tally in S. polyrrihza (Fig. 3c). The g rana
lamellae w as not be adp ressed densely any mo re, bu t
arrayed loose. The strom a lamellae connec ting g rana
w as cut off, which made stroma lame llae ne tw ork b reak
of.f M ore starch grain appeared among st stroma. The
ch lo roplast of fronds under ammonia tended to be disas-
semb ly. A ll these changes presen ted mo re obvious fo r
S. polyrrihza (Fig. 3c).
　　 P:p las tog lobu li S: s tarch grain SL:s trom a lam ellae G:grana M e:
m emb rane of ch lorop las t
F ig. 3　The SEM photograph o f the chlorop last of the fronds
3　D iscussion
It was observed that the size o f fronds under ammo-
nia med ium was sma lle r than ones under nitrate medi-
um. This shou ld have re lationship w ith the dam age of
ammonia to the ce ll w all, which possib ly caused by the
NH
+
4 . B ritto et a l
[ 12]
has proposed tha tNH
+
4 cou ld re-
79　第 4期 　　　　　种云霄等:氨对紫背浮萍和稀脉浮萍超微结构的影响 　　　
duce the content o f some essentia l cations such as the
calcium , magnesium in plan t issue by rep lacemen.t
These ca tions, especially calcium had very importan t
ro le to keep the strength of p lant cellw al.l Possibly it is
NH
+
4 that resulted in the changes o f low er epide rm is ce ll
w a ll under ammonia medium.
Fo r the higher plan t, the behavio r of stoma ta is
ma in ly contro lled by the osmo tic pressure of stomata l
guard ce l.l M any factors could affec t the osmotic pres-
su re o f stom atal gua rd ce ll such as po tassium , abscisic
acide etc. Bu t there is se ldom repo rt that ammonia
could cause the change o f the o smo tic pressure. Fur-
thermore, Seve ri e t al[ 13] founded tha t the stoma ta in
fu ll grown fronds ofLemna spec ies a lw ays kept open and
the guard ce ll had lost the function, bu t for Spirodela
species this func tion still exists. From the observation
resu lt o f this study, bo th stoma ta of S. polyrrihza and
L. aequinoctialis opened w ide r under ammonia medium
than nitrate medium. We didn’ t consider tha t the sto-
ma ta behav ior w as caused by the effect of ammonia to
the osmo tic pressure. W e tend to conc lude tha t the
crimpy shape of othe r epiderm is ce lls, especially those
of low er epiderm is draw ou t the stomata ind irectly,
which w as caused by the toxicity ofNH
+
4 indirec tly.
W ho le chlorop last of p lant is w raped by double lay-
ermembranes. The inside one is stroma lame llae and
grana lamellae, which is composed of fo lding sing le lay-
erm embrane. Based on the resu lt of this study, the
membrane structure o f two duckw eed species unde r am-
mon ia medium was damaged evidently. The unionzed
ammonia (NH 3) could disturb ce ll membrane when it
traverses large ly. Obv iously, un ionzed ammonia (NH3)
resu lted in the damage o f the chlorop last because itw as
ex istent in ammoniamed ium of this study. The level of
NH3 was ve ry low , about 0.15 mg /L according the e-
quilibrium be tw een un-ionized ammonia (NH3 ) and
ammonium ion (NH+4 )[ 9] . But it resu lted in g rea tdam-
age to the ch lo roplast o f duckw eeds. This con firmed tha t
the tox icmechanism of ammon ia to duckw eed should be
mo re attributed to NH3 instead o fNH
+
4 .
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【责任编辑　周志红】
80 　　 华　南　农　业　大　学　学　报　　　 第 26卷