全 文 ：第 25卷　第 3期　　　　　　　　　　　　　植　　　物　　　研　　　究 2005年　7月
Vo .l 25　No. 3　　　　　　　　　　　　BULLETIN OF BOTAN ICAL RESEARCH July, 　2005
Au thor in troduction:Zu Yuangang (1954— ), Ph. D. , Professor, m ajor in p lan t science.
Received date:2005 - 01 - 07
拟南芥叶片细胞包被系统酸性降解的光镜观察
祖元刚　于景华　郭晓瑞　唐中华　张宇亮　孟庆焕
(东北林业大学森林植物生态学教育部重点实验室 , 哈尔滨　150040)
摘　要　观察了拟南芥叶片细胞包括细胞壁和质膜在内的细胞包被系统在酸性条件下酶促降解
的过程 。观察发现 ,处于酸性酶解液中的拟南芥叶片 ,最初细胞壁完整 ,细胞排列有序 ,其后细胞
壁开始部分降解 ,细胞排列逐渐进入无序状态 ,随后细胞壁完全降解 ,去壁的原生质体完全进入游
离状态 ,游离原生质体的质膜也随之降解 ,细胞器溢出后以细胞核为核心积聚 、重组为新的原生质
体 。进一步观察了这一过程中细胞 pH值的改变 ,结果发现 ,酸性酶解过程中细胞倾向于 pH值降
低 ,而细胞器重组产生的新原生质体 pH值向正常水平恢复 。因此 ,酸性环境对拟南芥叶片细胞
包被系统的降解产生重要的影响。
关键词　拟南芥;叶片;细胞包被系统;酸性降解
L ightm icroscopic observation of ac id degradation on cell coat system of
Arabidopsis thaliana leaves
ZU Yuan-G ang　YU Jing-Hua　GUO X iao-Rui　TANG Zhong-Hua　ZHANG Yu-Liang　MENG Q ing-Huan
(Key Laboratory of Fo rest P lant E co lory, M inistry of Educa tion, Northea st Fo restry Unive rsity, H arb in　150040)
Abstract　This articlemain ly focuses on the degradation course of enzyma tic reaction of ce ll coat system
including ce llw all and cytoplasm amembrane o f the Arabidopsis tha liana leaves under acid cond itions.
Under lightm icroscopic obse rvation, the cellw alls of the leaves of A. thaliana which is unde r the acid
condition a re intact and arranged in o rder at first, the reafter the ce llw all beg ins to degrade partly, and
the cells tend to disordered sta te gradually, a fte rw ards the ce llw a lls are to tally deg raded. Protoplasts i-
sola ted by enzyme digestion to tally en ter the free state, and accompanied by the degradation of the cy to-
plasmamembrane of the free protop lasts, hav ing overf low ed, the o rganelle surround the nucleus, and
thus a new pro toplast is reconstituted. W e further observed the change o f pH in the ce ll in this course.
The result indicated that the cell tended to the pH va lue decrease during the course o f the ac id enzym atic
deg rada tion, and the pH o f the pro toplast reconstituted from the o rganelle came back to its no rmal leve .l
In conclusion, the acid env ironmen t had an important e ffec t on the deg rada tion o f the cell coat system of
A. tha liana leaves.
Key words　Arabidopsis thaliana;leaves;cell coat;acid deg rada tion
During the course of the preparation o f the p lant
pro top last, a remarkab le cellular morpho logical cha r-
acteristic is the escape phenomenon of the protop last
from parenchyma cells iso lated by enzymatic solution.
Th is must be accompanied by the deg rada tion o f the
plan t ce llw a lls. But the deta ils in this course need to
be investigated deep ly, especially the inf luence of the
acid enzym atic so lu tion on the protoplast escaped from
the cells also and the change o f the cy top lasm amem-
b rane a fte r the p ro toplasts being free. A s the cellw all
and cytoplasmamembrane a re the ce ll coat system o f
the plan t ce ll, and the system p lay an important ro le
in the course o f the norm al ce ll cyc le. Therefore,
studying on the influence o f acid environment on cell
w a lls and p ro toplast w ill be of scientific sign ificance.
To cla rify the significance, the dynam ic change o f the
ce llw all and the cy toplasma membrane w ere investi-
ga ted continuously o f theA. thaliana leaves under the
acid env ironmen.t
1　Materials andM ethods
1. 1　Preparation for the acid deg rada tionm aterials
Excise the w e ll-deve loped Arabidopsis thaliana
leaves and slice by hand, then observe unde r light
m icroscope o f op tics, se lec t the thin and even sec tions
to do the experimen.t
1. 2　Preparation for the cellw a ll digesting solution
The ce ll w a ll d igesting so lu tion is made up o f
buffer so lu tion and enzyme so lu tion, methodmod ified
slighly. The bu ffe r so lution contains 400 mmo l L -1
M ann ito l, 8 mmo l L -1 ca lcium chloride, 5
mmo l L -1 MES , pH 5. 6 (adjusted w ith KOH).
The enzyme solution con tains 0. 1% BSA , 1% cellu-
lo se and 0. 25% macerozyme[ 1] .
1. 3　The dynam ic m icrocosm ic observa tion o f acid
deg rada tion of cellw all of A. thaliana leaves
The digested mate ria ls w ere put in a m ini-poo l,
and several drops of enzyme solution w ere added, and
covered. Then w e observed the degradation sta te o f
ce ll w alls using Leica DM 4000B upright m icro scope
(400 ×), captured the pic ture w ith Leica DM 320
co ld CCD and N ikon TE-2000E confocalm icroscope.
1. 4　 In te rce llular pH measu remen ts by confoca l laser
scanning m icro scope
W e measured intracellular pH (ph i) in 2′, 7′-
bis ( carboxyethy l)-5 ( 6 ) carboxyfluo resce in
(BCECF-AM )-loaded leaves mon ito red w ith N ikon
confoca l m icro scope. Leaves w e re incuba ted w ith
BCECF-AM (20μmo l L -1) for 30 m in in the dark
and w ashed in 10mmol L - 1 MES-TRIS buffer, pH
6. 2. Leaves we re pu t on slides and their fluorescence
signa ls a t 520 nm and 640 w ere measu red for excita-
tion w avelengths se t at 488 nm. Fluorescence intensi-
ty ra tios(520 /640, FlRs)we re thus calcu lated as in-
dicators o f pH
[ 2]
.
1. 5　Nuc leus dynam ic obse rvation
The DAPI (4′, 6-diam ino-2-pheny lindo le dihy-
droch loride) stock so lu tion is 1mg mL - 1 , and the
DAPIw orking so lution is 10 μg mL - 1 (diluted w ith
distilled w a te r). DAPIw o rking so lution w as added to
the samp le to m ake the fina l concen tration to
1μg mL -1. A fter 10m inutes incubation in the
da rkne ss, the samples we re observed w ith fluorescent
m icroscopy, using an UV filter (excitation 365 nm).
2　Results
2. 1　M icroscop ic features of the ce lls of norma l leav-
es
Be fore the cellw alls o fA. thaliana leaves began
acid degradation, their epiderm is laye rs w ere intact,
the ce lls stood side by side and arranged orderly. The
cells o f the pa lisade tissue of the upper epiderm is of
leafw ere ovate, the cells of the sponge tissue of the
low er epiderm is varied in va rieties, and the number of
the chlorop last in palisade tissuew as less than tha t in
the sponge tissue (P lateⅠ :1).
2. 2　The acid deg rada tion o f the cell w all coa t sys-
tem o f A. tha liana leaves incubated in acid enzyme
so lution
A fte r being incubated in the enzyme so lution fo r
one hour and forty m inu tes, the low er epiderm is of the
leaves began to be acid degradation, but the uppe r
ep iderm is w as in o rde r and clear. The rela tive posi-
tion of the palisade tissues and the sponge tissue w as
unchanged obviously. The mo rpho log ical structure of
the cells of the pa lisade tissue w as changed less,
though the ce lls of the sponge tissue arrayed tigh tly,
for the reason o f its ce llw alls came into acid deg rada-
tion earlie r than those of the palisade tissue, the ce lls
came into disordered sta te (P lateⅠ :2).
A fte r two hours of the acid deg rada tion, there
we re some frac tures in the upper epiderm is. The most
evident is tha t the ce lls o f the pa lisade tissue a ren’ t
distinguished from those of the sponge tissue. A fte r
a ll the ce ll w alls of themesophy ll ce lls deg raded , the
func tion that ce ll w all maintaining the shape of the
328 　　　　　　植　　物　　研　　究　　　　　　　　　　　　　　　　　　25卷
ce ll disappeared. The cells absorbed w a ter, expanded
and the shape changed round from the p rim ary d iffe r-
ent shape. And because of the ex trusion be tw een the
ce lls, some ce lls appeared irregu lar shape(P lateⅠ:
3).
Having been treated two hou rs and forty m inu tes
later, the mesophy ll ce lls we re separate from the up-
per and the low e r ep iderm ises, and the compact ce lls
came to arrange loosely (Pla teⅠ :4). Some p ro to-
plasts w hich ce llw a lls entire ly degraded w ere separate
from the leave s, and the cytoplasma membranes o f
partial pro top lasts w ere furthe r degraded in the acid
enzyme so lution, and the o rganelles escaped from the
pro top lasts (Pla teⅠ :5 ～ 8). It’ s no tab le that the
deg rada tion of the cytoplasmamembrane m ight be ac-
companied by the degradation o f the nuclear mem-
b rane, and some chromosomes escaped from the nu-
cleus(PlateⅠ :7, 8).
2. 3　 The o rgane lle wh ich cy toplasma membranes
w ere degraded reconstitu ted new pro top lasts, ofw hich
the nucleus we re the core
A fte r the cy top lasm membranes we re deg raded,
the o rganelle reconstituted surrounding the nuclear.
The typical cha racteristic is tha t the nuc leus and o ther
organe lle arrayed along the outer fringe cy toplasm
membrane (Pla teⅠ :9 ～ 16). Subsequently, the
broken cy top lasm membrane m ight rew rap these o r-
gane lle, and new protoplast would be formed(Plate
Ⅰ :17, 18). W e also have a lready found this k ind o f
phenomenon in the cou rse o f the leaf explants tissue
cu lture ofCamptotheca acum inata
[ 3]
.
Itw as observed that there a lso appearing the de-
crease of pH du ring the degradation of leaf explants o f
C. acum ina ta unde r the acid cond itions
[ 3]
. The re
w ere the same re sults from the experimen ts of acid
deg rada tion of A. tha liana leaf cell coa.t The pH in
norma l leaves w as 5. 7 ～ 7. 1, w ith the ave rage value
6. 5(Pla teⅠ:19, 20), and that in en tirely deg raded
mesophy ll ce llsw as 5. 2 ～ 5. 7, w ith the ave rage 5. 5,
while pH o f epiderm is ce llsw ithout the deg rada tion o f
ce ll coat system m aintained 7. 1 and that of sligh tly
deg raded epide rm is cells w as 5. 2 ～ 5 (Pla teⅠ :21,
22). The obv ious changes o f pH in protoplasts de no-
vo recombined took place and the pH was 5. 7 ～ 6. 7,
w ith average pH 6. 13 (PlateⅠ :23, 24).
2. 4　The trends of pH varia tion in the course of acid
degradation of cell coat system of A. thaliana leaves
We have a lready found tha t during the cou rse of
the ce llw a ll ac id degradation, the pH of leaf explants
ofC. acum inata in the acid medium decreased. Ac-
co rd ing to the same method, we a lso observed the dy-
nam ic change o f the pH of A. thaliana leaf in the
course o f ce ll coat system acid deg radation and go t the
sim ilar conc lusion: the pH o f A. tha liana leaves
which w ere no t deg raded w as be tw een 5. 7 and 7. 1,
the mean w as 6. 5 (P lateⅠ:19 , 20);the mesophy ll
cells o fw hich the ce ll coa t system w as to tally degrad-
ed, the pH was be tw een 5. 2 and 5. 7 , and the ave r-
agew as 5. 5;the guard cells of epide rm is cou ldn’ t be
degraded, and the pH still kept a round 7. 1 , The epi-
derm is ce ll that faint degrades pH is 5. 2 ～ 5. 6(Pla te
Ⅰ:21 , 22).
The pH o f pro top last from cell organ recomb ina-
tion changes obviously. The pH in different reg ional
is 5. 7 ～ 6. 7, the average pH of pro toplast is 6. 13
(PlateⅠ :23, 24).
3　Discussions
Above men tioned p rocess is sim ilar to the resu lt
from the experimen ts that mesophy ll ce lls changed
from order to disorderw ith part o f plasmam embranes
degraded
[ 3]
. The common characte ristic we re that
they we re all under the acid condition, w ith the spe-
cial enzymes involved in the deg radation o f ce ll
w all
[ 5 ～ 8]
. The acid deg rada tion of ce llw a lls and pro-
top lasts, out-of-orde r o f ce lls[ 5] and consequen tly,
form ing o f themu lti-functional ce ll o r stem ce ll to es-
tab lish the essen tial foundation to diffe rent kinds of
cells
[ 9]
. So w e can make these tw o processes and
acid grow th ca lled acid deg rada tion.
W e have e lucida ted the possib le mechanism s of
escape of plant protoplast from ce ll w alls. It can be
concluded that ac id materia l in ac id enzyme so lu tion,
liquid of prescription make p lant b lade in acid envi-
ronment leading to the fac t plant norma l ce ll the acid
degradation of ce ll w a ll take place at firs.t Appea r
protop last break aw ay from cell w all ente r and sepa-
rate the liquid afterw ards in succession. Then the acid
3293期 祖元刚等:拟南芥叶片细胞包被系统酸性降解的光镜观察
deg rada tion of the membrane the quality happens fu r-
ther, make cell nucleus and cell device is it separate
liqu id to enter. Separa te ce ll device of liqu id regard
ce ll nucleus as cen tre go on ce ll think h ighly o f group
finally , produce the new pro top last of appearancew ith
unan imous shape
[ 10]
. This series changes are sim ilar
to the fo rm ing mechanism s of callus
[ 11]
. The results
of this pape r gave the strong evidence.
In the above process, it is very obv ious that in-
trace llula r pH changes in different stages. Name ly the
norma lmesophy ll cell pH is 6. 5 on average. The in-
trace llula r pH in entire ly deg raded ce lls decreased to
the leve ls low er to 5. 5, wh ile that in de novo com-
b ined protop last rose to 6. 13. These changes indica-
ted that the process o f ce ll deg rada tion w as accompa-
nied w ith the changes o f the ce ll organe lles, the gene
expre ssion, especially intracellular pH decline. The
increases of the new protoplast pH validate the theo ry
of cell recombina tion.
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330 　　　　　　植　　物　　研　　究　　　　　　　　　　　　　　　　　　25卷
P la teⅠ 　P t:Palisade tissue;Sp:Sponge tissue;Ep:Epide rm is;Eu:Upper ep iderm is;E l:Low er epiderm is.
1. Norm al cellw all ofArabidopsis tha liana leaves, w ith epiderm is layer intact, cells arrange neatly, palisade and sponge tissue cells dem arca-
tion line clear;2. A fter acid degradationw ith one hou r 40m in, disp ersion or fractu re in the under ep id erm isw ith the upp er ep iderm is in tact
and clear, the relat ive pos ition of palisad e and sponge tissue cells unchanged, the sponge cellw a llp rior to the palisade to degrad ation and en-
ter d isordered s tate;3. The 2 h acid degradation, th e m esophyll ce lls w all all degrade, unab le to d ifferen t the palisad e tissue cell and the
sponge. M esophy ll cells appearm ainly round, cells arrange neat ly, close and ad joint;4. A fter the 2 h acid degrad at ion, the cells arrange to-
w ards to loose;5. A fter the 2 h acid degradat ion, som e m esophy lls cell whose cellw al l totally degrad ed break away from the leaves b lade
(w h ite arrow) w ith som e ce lls p lasm a mem b rane am ong them break ing and p resen ting irregu larity shape (b lack arrow);6. A fter3 h, allm e-
sophy ll ce lls en ter the free state in the form of protop last;7, 8. E scapes of organel les from escaped p rotop last, the w h ite arrow poin ts to the
chrom osome, and 8 is about p ictu re of f luorescence, red is cholorp las t and b lue is nuclear dyed by DAPI;9～ 16. Recom b ination of d issocia-
ted organel les relying onm em brane and surround ing nu clear. Among th em 10, 12, 14 and 16 are fluorescence p icture and red poin ts to ch lo-
rop last, b lue po in ts to nuclear dyed by DAPI;17, 18. New ly form ed p rotop las t;19, 20. pH ch anges in norma l leaves;21, 22. pH changes
af ter en tirely degraded leaves w ith com parative high pH;23, 24. pH variations in new ly com b ined pro top las.t (all ×400)
3313期 祖元刚等:拟南芥叶片细胞包被系统酸性降解的光镜观察