全 文 ：满江红鱼腥藻在羽叶满江红大孢子果内
的去向及其超微结构的变化
收稿日期: 1997- 08- 10; 1997- 11- 20修回
郑伟文1　刘利华2　修文琼3
( 1福建省农科院生物技术中心 , 福州 350003;
2福建省农科院中心实验室 ; 3福建省卫生防疫站 )
摘　要　用激光共聚焦扫描图象系统 ( LCSIS)和透射电镜追踪和表征满江红鱼腥藻在发育中的满
江红孢子果内的去向及其超微结构的变化。进入满江红孢子果的鱼腥藻营养细胞连锁体与孢子果
的长轴大体呈平行状态 , 并活跃排列 , 最后形成鱼腥藻营养细胞群体。该群体占据了整个孢子果
果腔 (由囊群盖围合而成 ) 和大孢子囊 (或小孢子囊 ) 周围一切可用的间隙。当果腔开口闭合时 ,
部分营养细胞开始了向厚垣孢子分化的进程。在孢子果发育中期 , 果腔内的营养细胞连锁体都变
成了外部形态类似于厚垣孢子的细胞链。从营养细胞向厚垣孢子的分化经历了细胞质重组和膜系
统重排等一系列超微结构的变化。诸如多角体 ( Carboxy some)、 藻蓝素颗粒、 核糖体和类囊体之
类的细胞内含物在数量、 大小 、 形状和分布等方面随着厚垣孢子本身及其宿主孢子果发育进程而
改变。 文章还讨论了共生藻与自生蓝藻在厚垣孢子分化模式与超微结构特征的异同及其意义。
关键词　满江红 ; 孢子果 ; 鱼腥藻 ; 共生 ; 超微结构
中图分类号　 Q 942
The Fate and Ultrastructural Changes of Anabaena azollae
Within the Debeloping Sporocarps of Azolla
Zheng Weiwen
1 , Liu Lihua
2
and Xiu Wenqiong
3
(1 Biotechnology Center, Fujian Academy of Agricultural Sciences , Fuzhou, P . R . China　 350003;
2 Central laboratory , Fujian Academy of Agricultural Sciences; 3 Fujian Center for Disease Control )
Abstract　 The fa te and ultra structural changes of Anabaena azollae entrapped into the developing
spo ro carps of Azolla pinnata we re fo llowed and charac terized by la ser confocal scanning image sys-
tem ( LCSIS) and t ransmission elec tron micro scopy. Th e entrapped vegeta tiv e Anabaena ho rmogo-
nia tended to run par allelly to the long ax is o f the young spo roca rps and underw ent an ac tiv e divi-
sion, fo rming eventually an Anabaena colony , w hich occupied the entire indusial chamber and all
the space available ar ound the megaspo rang ium or micro spo rangia. Some o f the vegeta tiv e cells ini-
tia ted differ entiation to akinetes w hile the opening of th e chambe r w as clo sed. At an inte rmediate
stag e o f th e spor ocarp development, all the vegeta tiv e cells on th e ho rmogonia g r adually trans-
formed into the akinete-like chains. U ltrast ructurally the pr eakinetes underw ent the cy toplasm
福建农业学报 13( 1): 1～ 8, 1998
Fujian Journal of Agricultural Sciences　　　　　　　　　　　　　　　　　　　　　　　　
r eo rg anization and th e membrane systems rear rangem ent. The inclusions, such as ca rboxy somes,
cyanophycin g ranules, ribosom es and thy lakoids, va ried in number , si ze, and th eir distribution in
the cell along with the differentia tion cour se o f the akinete. The similarities and differ ences in dif-
ferentia tion patte rns and ultrastr uc tur al featur e betw een the endophy tic and fr ee-living cyanobacte-
ria as well a s th eir po ssible significance w ere discussed.
Key Words　 Azolla; Spor ocarp; Anabaena; Symbio sis; Ultr astructur e
Azolla is a g enus of aquatic fer n, which is also only the fern to be symbio tic wi th ni t ro-
gen-fixing cyanobacterium , commonly know as Anabaena azollae. Some o f Azolla species
regula rly have sexua l stage producing mega- and micro-sporocarp, housing the mega- and
micro-spore, respectively. The fertili za tion of egg cells w ithin the megaspo res o ccurs inside
the megaspocarps and the young spo rophy tes initiate thei r development here also ( Lumpkin
and Plucknett , 1980) . This ar rangement is coupled wi th the retention of some Anabaena fi la-
ments, which ini tiate to dif ferentiate into akinetes as they became enclosed in the developing
spo roca rp pai r, leading to the continui ty of the fern-Anabaena symbiosis th rough th e life cy-
cle. The mechanism by which the vegetative cells of Anabaena were par ti tio ned into the de-
veloping sporocarps has been suggested by Calv ert et al . ( 1985) ; Zheng et al . ( 1988,
1994) ; Perkins and Peters ( 1993) . M orpholog y and st ructure of Anabaena cells wi thin the
spo roca rps of Azolla were described ( Moore 1969; Herd et al . 1985; Becking 1987; Braun-
How land and Nierzwicki-Bauer, 1990) . There has been, how ever, a limi ted know ledge about
the fa te and ult rast ructural changes of the vegeta tiv e cells packaged into the young spo ro-
carps although the changes have been simply illust ra ted in a repo rt ( Perkins and Peters,
1993) . As a further contribution tow ard to an understanding of the nature of Azol la-Anabae-
na symbio sis, w e giv e a detailed account of the fate, as observed by using laser confocal scan-
ning image sy stem ( LCSIS) , and changes, as rev ealed by using t ransmission elect ron mi-
croscopy.
1　Materials and Methods
The fronds o f Azolla pinnata examined in this study w ere co llected f rom the pond o f the
campus o f the Univ ersi ty o f Sydney. The spo rula ting f ronds w ere w ashed several times
w ith tap wa ter and dissected carefully wi th fine needle and scalpel under Olympus stereomi-
croscopy. The spo ro carps at v arious developmental phases separated f rom the f ronds w ere
kept, respectively in 1 ml Eppendo rf tubes containing distil led w ater. The specimens fo r LC-
SIS observ ation w ere t ransferred to the tube containing 2% Tween-80 and shook for 2 min in
o rder to precipi tate them into the bot tom of the tube. Th e samples fo r TEM examination
w ere pre-fix ed in 2. 5% g lutaradehyde in PBS ( pH 7. 4) fo r a t least 2 days at 4℃ and washed
three times in PBS again. Then the individual spo roca rps were post-fix ed in 1% osimum
tet roxide in PBS fo r 90 min, washed th ree times in PBS, and dehydrated through an ethano l
2 福建农业学报 第 13卷
series. Finally they w ere incuba ted in propylene oxide and embedded in Epon 812. The sam-
ples were sectioned w ith a diamond or glass kniv es under an LKB Ult ra tome III ult ramicro-
tome, and mounted on copper grids and w ere stained fo r 10 min in urany l aceta te in 50%
ethanol follow ed by 2 min lead ci t rate. The sporocarps w ere examined w ith emphasis on the
symbionts under Bio Rad MRC 600 LCSIS. All sections w ere examined wi th JEOL JEM-
100CX transmission elect ron microscopy at accelerating potential of 80 kv.
2　Results and discussion
2. 1　 LCSIS observations
The mo rphological characteristics of the young sporocarps o f Azolla pinnata were simi-
lar to those observ ed by light microscopy in earlier studies ( Konar and Kapoo r, 1974 ) . At
the early stage ( show n in Fig. 1) the Anabaena were found to be ho rmogonia, w hich w ere
short , roughly st raight , and perhaps mo tile fi laments lacking heterocysts. Th e vegetativ e
ho rmogonia w ere entrapped into a chamber enclo sed by tw o-cell-lay er-thick indusium , w hich
surrounds the enti re megasporangium or microspo rangia. The vegetativ e hormogonia entering
the chamber through the po re at the top o f young spo ro carp tended to run roughly parallel
w ith the long axis o f the spo rocarp and to one another ( Fig. 1) . The entrapped hormogonia
usually consisted o f 10-20 rounded vegetative cells approximately 2. 0-2. 5μm wide and 3. 0-
3. 5μm long ( da ta not show n ) . At this stag e the vegetativ e cells seemed to undergo an activ e
division. The cells fo rmed g radually an Anabaena co lony, occupying eventually the enti re
chamber and all the space avai lable a round th e megaspo rangium or micro sporang ia ( Figs. 1,
2) .
Fig. 1　 A longitudinal section of the young sporocarp at the f irst node of the main stem branches from the stem apex,
showing the Anabaena hormogonia entraped into the chamber of the young sporocarp. a= Anabaena; mas megaspo-
rangium. Bar= 100μm.
Fig. 2　 A longi tudinal section of the developing sporocarp at the third node of the main stem branches, showing the
Anabaena hormogonia in the chamber of the developing sporocarp. mis= micris porangium, Bar= 100μm.
3第 1期 郑伟文等: 满江红鱼腥藻在羽叶满江红大孢子果内的去向及其超微结构的变化
It has been prev iously repo rted that the Anabaena cells actually multiplied w ithin the
spo roca rp cav ity of Azolla , a nd i t occurred, how ever, only af ter the po re of the indusium was
closed ( Becking , 1987; Zheng and Huan 1994 ) . Our data show n tha t enlonga tion and en-
larg ement o f a few vegeta tiv e cel ls o f the ho rmogonia occurred as the po re w as closed, indi-
cating that the vegeta tiv e cel ls morpho logically ini tia ted thei r dif ferentiation into
akinetes. The ex tension of the dif ferentiation occurred f rom one cell to neighbo ring cel ls and
from one hormogonium to ano ther. At an intermediate stag e of the spo roca rp development ,
all the ho rmogonia wi thin the spo rocarps became transformed to the akinete-like chains. The
leng th of the cell increased f rom 3. 5μm to 10- 15μm. According to our observa tions on A-
zolla pinnata , the akinetes only appea red w ithin the chamber of the developing spo ro-
carps. There w as no t any preakinete outside the indusium. This was dif ferent f rom the resul ts
on Azolla mexicana Presl ( Perkins& Peters, 1993 ) in which some of the vegetativ e cells be-
gan to dif ferentiate into akinete-like structure prior to the po re complete closure.
2. 2　 Ultrastructural examination
A typical v egeta tive cell packaged into the forming chamber w as show n in Fig. 3. The
cell w all w as characteristic of g ram negative bacteria wi th peptideg lycan. Betw een the plasma
membrane and tiy lakoids there w ere elect ron-transparent g ranules of gly cogen. Numerous
carboxy somes ( polyheadral bodies) were also apparent. The ul trast ructural characteristics of
the vegetativ e cell seen in the present study w ere similar, to some ex tent , to those repo rted
by Neumuller and Bergman ( 1981) . As the onset of the di fferentia tion o f the vegeta tive cel l
into akinete, the mo rpholo gical changes mentioned above w ere accompanied by several no-
table changes in ul trast ructural characteristics. In the present da ta the cy toplasmic o rganiza-
tion and membrane sy stem arrangement of presumptiv e akinete was suggested to divide to six
stag es as fo llow s : ( 1) The accumula tion of cyanophycin g ranules in cy toplasm w as com-
panied wi th the fo rmation of i rregular elect ron transparent and electron dense intermixed ar-
eas, being simila r to the observ ations on Azol la max icana by Perkins and Peters ( 1993) .
Trace of fibri llar ma terial, which seems to be an envelope precursor secreted f rom cy toplasm
through the cell wall , appea red around the cell ( Fig. 4 ) . ( 2) Thylakoids and ca rboxysomes
disappeared. M ore fibri llar material depo sited surround the cell, and subsequently fo rmed a
discontinues fibrous envelope layer, sepa ra ting f rom the cell w all by an elect ron t ranspa rent
space ( Fig s. 5, 6 ) . ( 3) The cytoplasm w as more electron dense and diso rg aniza tion. The depo-
si tion of the material surrounded the cell wa s ex tensiv e, becoming a continuous and undulate
zone ( Fig. 5, 6 ) . ( 4) Cyanophycin g ranules seemed to fuse each o ther and associate w ith
carboxy somes, and membrane-like st ructures appeared a t the periphery o f the cytoplasm ,
w hich was mostly like to be reappea ring of thy lakoids ( Figs. 5, 7, 9) . Widening o f the elec-
tron t ransparent space and thickening of the peptideg lacan layer in the outer membrane sys-
tem continued ( Fig . 9, 10) . ( 5) The asso ciation o f cyanophycin g ranules leaded to fo rmation
o f a large body ( up to 2μm ) , w hich sometimes occupied nea rly a half o f the volume of cy to-
4 福建农业学报 第 13卷
plasm. The body w as surrounded by an uni t membrane. Thylakoid membranes w ere scat-
tered throughout the cytoplasm, w hich w as accompanied by an increase in ribosome content
( Fig. 6, 8 ) . ( 6) Th e thickness of the peptidegly can reached a maximum level, w hich w as
about twice w ider than that in v egetativ e cell. The pro file of the unifo rm lamella of the enve-
lope lay er was v isible in a ma ture akinete. When akinete became ma ture the envelope consist-
ed the tw o layers wi th di fferent electron densi ties ( Fig. 10 ) .
　　
Fig. 3　 A logitudinal section through the vegetative cel l entrapped into the chamber of the young sporocarp at the
first node of the main stem branches. cs= carbox ysome; t= th ylakoid, Bar= 0. 5μm.
Fig. 4　 A presumptive akinete. Note the dense, disorganized cytoplasm, the accumulation of the cyanophycin granules
in cytoplasm and trace of f ibrillar material ( arrow) around the cell wal l. cy= cyanoph ycin granular, Bar= 0. 5μm.
These stages occur ring at the di fferentia tion process f rom the vegetativ e cell into
akinete indicated that Anabaena inside the developing sporocarps underw ent membrane rear-
rangement ( Perkins and Peters, 1993 ) and cytoplasm reo rg aniza tion. The inclusions, such as
cyanophycine g ranules, carboxy somes, gly cogen g ranules, ribosomes, and thilakoids va ried
in size, shape and number wi th va rious dif ferentiation phases o f bo th Anabaena cel ls and
spo roca rps. How ever, polyphospha te g ranule, lipid body and gas vesicles w ere rarely found
w ithin akinete in our ma terials. An accumulation of la rg e quantities o f cy anophycin g ranules
seemed to be one o f remarkable fea tures, which occured wi thin akinete o f bo th symbio tic
( Perkins and Peters, 1993; Becking , 1987) and free-living cyanobacteria ( Leak and Wilson,
1965; M el ler and Lang, 1968; Clark and Jensen, 1969; Wildman et al . , 1975; Sutherland
et al . , 1979) . In the present study the ma ture akinete show ed membrane systems, to some
ex tent , simi lar to those of f ree-liv ing cyanobacteria. The tw o-layered envelope, fo r ex ample,
5第 1期 郑伟文等: 满江红鱼腥藻在羽叶满江红大孢子果内的去向及其超微结构的变化
　
Fig. 5　 A portion of two developing akinetes showing a cyanophycin granule associated with a carboxysome. en= en-
v olope, Bar= 0. 5μm.
Fig. 6　 A preakinete, showing the cyanophycin granule surrouded byan unit membrance and the deposi tion of fibrous
materials in the granular. Note the thylakoid membranes reappeared within the dence cytoplasm and in negative contrast.
Bar= 0. 5μm.
is common to the mature Anabaena akinete in our material and Nostoc PCC 7524 ( Sutherland
et al . , 1985 ) . How ever, the cell w all of the la ter akinete di ffers f rom that of the fo rmer cel l
in having a g reat ly thickened and less elect ron dense peptideg lycan lay er ( Sutherland et
al . 1979, 1985 ) . The thylakoid a rrangements wi thin advanced akinete appeared very simila r
to that o f v egetative cell described by Numuller and Bergman ( 1981 ) w ho investig ated the
ult rast ructure of Anabaena azollae in Azolla pinnata and was the same as that of blue-g reen
alg ae repo rted by Whit ton ( 1971, tex t-Fig. 1) .
Grilli Caiola and de Vecchi ( 1980 ) suggested there w ere tw o types o f akinetes in Nostoc
isolated f rom cycas co ralloid ro ot. The second type, which contained large and numerous
cyanophycin g ranules, ribo somes, few polyheadral g ranules, represented the mature, quies-
cent akinete, since i t w as able to surv iv e for a long time in unfavorable g row th condi-
tions. Based on the ult rast ructural cha racteristics the akinete of the symbiont found in the
present study is, in a large ex tent, similar to the second type. This w as confi rmed by the fact
that the megaspo roca rps, w hich had been kept for four years at low tempera ture, tog ether
w ith the akinetes could germinated and reestablished a mutualistic symbiosis.
Interestingly , during the spo rulation of Azolla the dif ferentiation and development of
bo th the spo roca rps and the akinetes w ere sychronised. The akinete at the six th stag e men-
tioned above could only be found in a mature megaspo rocarp, w hich f requent ly located at the
6 福建农业学报 第 13卷
　
Fig. 7　 A advanced akinete, showing two carboxysomes associated each other. Bar= 0. 1μm.
Fig. 8　 A nearly mature akinete within the nearly mature megasporocarp at the fifth node of the main stem branch-
es. Note a big cyanophycin body, scattered thylakoids in cytoplasm and the wide electron transparent area between the cell
wal l and the envolope. Bar= 0. 5μm.
Fig. 9- 10　 A comparission of the thickness of peptidoglycan layer in the cel l outer membrane sys tems betweem the
developing ( Fig. 9) and mature ( Fig. 10) akinetes. Note th e envolope ( Fig. 10) consi sted of the tw o layers wi th dif f erent
elect ron densi ties. Bar= 0. 1μm.
six th or seventh branch node o f the main stem. How ever, the facto rs controlling the parallel
development betw een the sporocarps and the akinetes have so far been poo rly under-
sto od. Nevertheless, an a t tempt to know signa l exchange betw een Azolla and Anabaena
should be made besides physical facto rs. This is cur rent ly investiga tion in our lab.
3　Acknowledgments
The first author w ould like to thank Pro f. David Cocain, Dr. Meret V esk fo r al lowing
him to use the faci li ties in the Elect ron Microscope Uni t , the Univ ersity of Sydney , to
Dr. Guy Cox fo r skillful technical assistance, while his stay in Sydney.
7第 1期 郑伟文等: 满江红鱼腥藻在羽叶满江红大孢子果内的去向及其超微结构的变化
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8 福建农业学报 第 13卷