全 文 ：Cell Cycle Kinetic Analysis in the Cortical Regions of the Lentil
Primary Root During Germination
YU Fu_Gen＊ , PANG Yan_Jun , ZHAO Jian_Chun , WANG Qiang
(Department of Biological Science and Technology , School of Life Sciences , Nanjing University , Nanjing 210093 , China)
Abstract:　Cell cycle kinetic activity in the cortical cells of the lentil(Lens culinaris Medic.cv.Verte du
Puy)primary root during germination was examined both temporally and spatially.Immunohistochemical and
cytological evidence indicated that DNA replication and cell division started in the cortical cells of the lentil
primary root after around 13 and 17 h of imbibition , respectively.The first cells in DNA synthesis and the first
mitotic figures all appeared in the cortical cells about 1 mm from the root_cap junction , but these divided cells
had synthesized their DNA during the maturity of seed instead of during germination.The kinetic pattern of ac-
tivity of the first cell cycle showed that these cells were not activated synchronously , but re_entered the cell cy-
cle in turn depending on their places in the root tip.However , the adjacent cells partially synchronously pro-
ceeded their cell cycle.
Key words:　cell cycle;primary root;germination;immunohistochemistry;Lens culinaris
　　The plant seed germination represents a differentia-
tion process in which the embryo cells undergo changes
from the quiescent state of extremely low metabolism to a
state of fully restored activity , including the activation of
cell cycle.Before germination , the majority of species ex-
amined have a high proportion of 2C state cells(cells ar-
rested in G1 phase)and a low proportion of 4C state cells(cells arrested in G2 phase), or the exclusive 2C state in
quiescent embryos
[ 1-3] .Some species might have a great
number of 4C cells , which Deltour[ 1] attributed to the ex-
perimental uncertainties and side_effects of environmental
conditions.In a study of nuclear DNA replication activity
in seeds of pepper (Capsicum annuum), which has only
2C nuclei in fully matured embryo , Lanteri et al[ 4] noted
an increase of cells with 4C between 48 and 60 h prior to
the radicle protrusion.Similarly , Bino et al[ 2] observed
that 48 h prior to radicle emergence in germinating toma-
to , the 4C/2C ratio rose.At the beginning of maize ger-
mination , 75% of the cells were in G1 phase , but this
population decreased to 25% after 72 h , and most of the
cells entered S phase as a partially synchronised popula-
tion during germination[ 5] .Using 3H_thymidine labelling ,
Baí za et al[ 6] found the presence of distinct cell popula-
tions re_entering the cell cycle synchronously in a stepwise
fashion in embryonic maize tissues during germination.
However , Wyman et al[ 3] found that the majority of cells
in five families of jack pine (Pinus banksiana)embryos
were in 2C states before imbibition.After hydration , an
increase of the 2C_4C nuclei as well as an increase of nu-
clei with a DNA level higher than 4C occurred gradually
and preceded radicle emergence.
In species in which the majority of embryo cells were
arrested in G1 phase , the first mitotic cycle during early
germination was asynchronous[ 1] .The presence of sub_
populations of cells with different cell cycle lengths in the
meristem shows that there is a mechanism of temporal and
spatial regulation of cell proliferation for organising a har-
mony during plant development.Recent researches have
demonstrated that the plant has the same regulation of cell
cycle as the other eukaryotes[ 7-10] .A great many of anal-
yses by flow cytometry have provided numerous results
about temporal cell cycle kinetics with respect to the de-
velopment of plant embryo during germination[ 3 , 5 , 11] .
However , flow cytometry cannot provide the details of the
spatial information of cell cycle because it measures a
mixture of different sub_populations of cells in a plant or-
gan , but knowledge of kinetics of the cell cycle in the root
tip and its regulation both in space and in time is of basic
importance for understanding the growth and development
of the plant tissue
[ 12] .
The purpose of this paper was to study the temporal
and spatial activation and regulation of cell cycles in the
same cell sub_population during the early lentil seed ger-
mination.Iododeoxyuridine(IUdR)labelling and cytolog-
ical observation were used which allowed us to precise a
specific region in sections , and cortical cells were chosen
for this study since they represented the most homogenous
tissue of the root tip.
1　Materials and Methods
Decoated seeds of Lens culinaris Medic.cv.Verte
du Puy were germinated in minicontainers[ 13] .Each mini-
container held 12 seeds.Germination was initiated by in-
jecting 1.8 mL of water into each minicontainer.The
lentil seedlings were grown at 22 ℃ in darkness.
1.1　Percentages of cells in DNA synthesis during
germination
To determine the percentage of cells in DNA synthe-
sis(S phase), IUdR labelling was used[ 14] , and the
Received:2001-12-11　Accepted:2002-04-01
Supported by the Scientific Research Foundation for Outstanding Young Teachers , State Education Ministry and Scientific Research Foundation for Returned Over-
seas Chinese Scholars , State Education Ministry.
＊ Author for correspondence.E_mail:.
植　物　学　报　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　

Acta Botanica Sinica　2002 , 44(7):764-770
seedlings were fixed in chilled 70%ethanol(4 ℃)for 2
h at 1 , 5 , 9 , 13 , 17 , 21 , 25 and 29 h of germination.
For pulse labelling , lentil seedlings were placed for 2 min
in a solution of 2 mmol/L IUdR with 0.05%Tween 20
one hour before fixation.Then , root tips were cut(about
2 mm each)and dehydrated in an ethanol series , embed-
ded in paraffin , cut into 8 μm sections and mounted on
glass slides treated with Vectabond
TM(Vector , Burlin-
gance , CA).
The sections were deparaffinized in xylene , and then
DNA was denatured with 4 mol/L hydrochloric acid for 10
min at room temperature.To identify nuclei labelled with
IUdR , the sections were rinsed three times with Tris_
buffered saline (TBS , 0.05 mol/L , pH 7.6), and incu-
bated for 6 h with monoclonal antibody IU_4(Caltag , San
Francisco , CA)diluted 1∶500 in TBS containing 5%
normal sheep serum.Then , the sections were rinsed three
times with TBS , treated for 2 h with alkaline phosphatase_
conjugated sheep anti_mouse immunoglobulins (Dako ,
Santa Barbara , CA)diluted 1∶50 in TBS , developed with
alkaline phosphatase substrate kit Ⅰ (Vector ,
Burlingame , CA), and lightly counterstained with 5%
Gill s No.1 hematoxylin(Sigma).Each section was de-
hydrated with ethanol and cleared with xylene , and a cov-
erglass was mounted with Canadian Baume.For a control
study , TBS was used in place of the primary antibodies(IU_4).
Nuclei labelled with IUdR(cells in DNA synthesis)
were stained red and unlabelled nuclei were stained light
blue.Percentage of cells in DNA synthesis was calculated
for one axial section of 8 meristems.For each axial sec-
tion , percentage of cells in DNA synthesis were deter-
mined in the cortical regions(0-0.2 , 0.2-0.4 , 0.4-0.6 , 0.6-0.8 , 0.8-1.0 , 1.0-1.2 , 1.2-1.4
and 1.4-1.6 mm from the root cap junction)of the
proximal meristem.
1.2　Mitotic indices during germination
To determine the mitotic indices , the seedlings were
fixed for 24 h by 4%(V/V)formaldehyde in sodium
phosphate buffer(0.1 mol/L , pH 7.4)at 1 , 5 , 9 , 13 ,
17 , 21 , 25 and 29 h of germination.Then , root tips were
cut and rinsed in sodium phosphate buffer and dehydrated
in an ethanol series , then a propylene oxide series and
embedded in Araldite by following the routine treatment
for electron microscopy.Semi_thin sections(1 μm)were
made with an LKB Ultrotome Ⅲ and stained using tolui-
dine blue (1%W/W).Mitotic index was calculated for
one axial section of 8 different meristems.For each axial
section , mitotic indices were determined in the cortical
regions(0-0.2 , 0.2-0.4 , 0.4-0.6 , 0.6-0.8 ,
0.8-1.0 , 1.0-1.2 , 1.2-1.4 and 1.4-1.6 mm in
length from the root cap junction)of the proximal meris-
tem.
2　Results
2.1　DNA synthesis
2.1.1　Changes with increasing germination time　
Figure 1a shows longitudinal axial sections of the lentil
primary root after a short(1 h)IUdR labelling , 29 h after
hydration.The red nuclei were labelled by IUdR.
Changes of DNA synthesis of the cortical cells were ana-
lyzed with increasing germination time.
There were no nuclei labelled by IUdR at the root
tips sampled after 1 h and 5 h of imbibition.Figure 2a
shows the evolution of the changes of DNA synthesis with
increasing germination time in the different cortical re-
gions of the lentil primary root.DNA synthesis of these
cells was not activated synchronously , but in stepwise
fashion , and the cells at 0.8-1.0 mm from the root cap
junction were activated first.After 13 h of germination ,
there were nuclei labelled by IUdR in all regions exam-
ined , and the proportion of cells in S phase at the end(0-0.2 mm and 1.4-1.6mm)was the lowest , and in
the central regions(0.8-1.0 mm and 1.0-1.2 mm)
the highest.During the whole 29 h duration of germina-
tion , therewas only one peak of cells in DNA synthesis in
0-0.2 , 0.2-0.4 , 0.4-0.6 , 0.6-0.8 and 0.8-
1.0 mm.All these peaks represented the first wave of
DNA synthesis and appeared after 17 h of germination.
The studentized residues of these points were above 1.5 ,
so the peaks of these points are statistically significant(Fig.2b).At 21 h , the peaks occurring at 17 h in all
these regions disappeared , although there were more cells
in DNA synthesis in the acropetal regions than in the
basal regions as at 17 h.At 0-0.2 and 0.2-0.4 mm ,
the proportion of cells in DNA synthesis at 25 and 29 h of
germination was less than that at 21 h of germination , but
the opposite was true at 1.0-1.2 and 1.2-1.4 mm.
Therefore , at 25 h there should be cells in the second
wave of DNA synthesis at 1.0-1.2 , 1.2-1.4 and 1.4-1.6 mm.At 1.0-1.2 , 1.2-1.4 and 1.4-1.6
mm , there was only one peak of cells in DNA synthesis in
each region , and the studentized residues of these points
were also above 1.5(Fig.2b).However , these peaks all
appeared at 25 h rather than at 17 h , which was the time
of the occurrence of the peaks in the acropetal regions.
These results show that the majority of cells at 1.0-1.2 ,
1.2-1.4 and 1.4-1.6 mm are not involved in the first
wave of DNA synthesis , but they participate directly in
the coming second wave of DNA synthesis.
At 13 h of germination , the proportion of isolated
cells(labelled cells)was high at the activation of the first
post_embryonic cell cycle in cortical lineage of lentil root
meristem (Fig.1b).This means that the process of seed
maturation and the process of seed germination resulted in
the loss of natural synchrony of sister cells formed by the
last division of the mother cells in embryo.After 29 h of
germination , the proportion of labelled cells in pairs was
high(Fig.1c).This result showed that the sister cells
formed by the first post_embryonic cell division are highly
synchronous in passing the second post_embryonic cell cy-
cle in cortical lineage.
2.1.2　Changes with increasing distances from the
root cap junction 　Figure 2c shows the changes of DNA
synthesis in the cortical cells of the lentil primary root
YU Fu_Gen et al:Cell Cycle Kinetic Analysis in the Cortical Regions of the Lentil Primary Root During Germination 765　
Fig.1.　Longitudinal axial sections of lentil primary roots after 1 h incorporation of IUdR at different periods of imbibition.a.Root tip show-
ing the root cap (RC), the quiescent center(QC), and the meristem with the central cylinder(CC)and the cortex(C).The nuclei colored
in red were labelled by IUdR.The other nuclei were stained in blue by Gill s No.1 hematoxylin.Bar=200 μm.b.Isolated labelled cells(arrows)at 13 h of imbibition.Bar=20μm.c.Pairs of labelled cells(arrows)at 29 h of imbibition.Bar=20μm.d.Detail of the cortical
cells at 1 h of imbibition , showing all nuclei incorporated by IUdR.Bar=20 μm.
766　 植物学报　Acta Botanica Sinica　Vol.44　No.7　2002
with increasing distances from the root_cap junction after
different periods of germination.In all zones , the propor-
tion of cells in DNA synthesis at 13 h was the lowest dur-
ing the whole 29 h period of germination(Fig.2c).Nu-
clei labelled by IUdR in 0.6-0.8 , 0.8-1.0 and 1.0-1.2 mm were more than those in other regions after 13
h germination.At 17 h , more cells were in DNA synthe-
sis at 0.4-0.6 mm.However , at 21 h , more labelling
cells appeared at 0.2-0.4 mm(the studentized residues
of these points approached 1.5 , Fig.2d).At 25 h , the
proportion of cells in DNA synthesis at the basal regions
augmented clearly , and a clear labelling peak appeared at
0.8-1.0 mm (the studentized residue of this point was
above 1.5 , Fig.2d).This phenomenon should be the re-
sult of the second wave of DNA synthesis in the cells of
these regions.At 29 h , the peak retained at 0.8-1.0
mm(the studentized residue was also above 1.5 , Fig.
2d), but the proportion of cells in DNA synthesis in basal
regions declined , as well as at 0-0.2mm.The decrease
of the proportion of cells in DNA synthesis in basal re-
gions was due to cells leaving the second wave of DNA
synthesis , but the decrease at 0-0.2 mm was due to
cells leaving the first wave of DNA synthesis.From this ,
it should be deduced that the peak of the second wave of
DNA synthesis would move from the basal regions to the
acropetal regions with increasing period of germination ,
showing the same tendency as the first wave of DNA syn-
thesis.
2.2　Mitotic index
2.2.1　Changes with increasing germination time　
Figure 3a shows the evolutions of the changes of mitotic
index with increasing germination time in different cortical
regions of lentil primary root.After 17 h of germination ,
there were peaks of cells in division at 0.8-1.0 and 1.0-1.2 mm (the studentized residues of these points were
all above 1.5 , Fig.3b), and the proportion of cells in
DNA synthesis at the same regions was higher than at
other regions after 13 h of germination.However , a 4 h
Fig.2.　a.Changes in the percentages of nuclear DNA synthesis with increasing germination periods in different regions of the primary root of
lentil.b.The studentized residues from a linear regression analysis of the changes in the percentages of nuclear DNA synthesis with increasing
germination periods in different regions of the primary root of lentil.A studentized residue larger than 1.5 is statistically significant , meaning
that a clear peak value appears in the corresponding point in Fig.2a.c.Changes in the percentages of nuclear DNA synthesis in cortical re-
gions(0-1.6 mm from the root cap junction)of the primary root of lentil in different germination periods.d.The studentized residues from
a linear regression analysis of the changes in the percentages of nuclear DNA synthesis in cortical regions(0-1.6 mm from the root cap junc-
tion)of the primary root of lentil in different germination periods.A studentized residue larger than 1.5 is statistically significant , meaning that
a clear peak value appears at the corresponding point in Fig.2c.
YU Fu_Gen et al:Cell Cycle Kinetic Analysis in the Cortical Regions of the Lentil Primary Root During Germination 767　
difference of time was not sufficient for a plant cell cycle
passing from S phase to M phase.Therefore , these divid-
ing cells should have completed their DNA synthesis dur-
ing the formation of embryo , and in 4C state at the begin-
ning of germination.There were peaks of cells in division
at 0-0.2 , 0.2-0.4 , 0.4-0.6 and 0.6-0.8mm af-
ter 25 h of germination(the studentized residues at these
peaks were above 1.5), which corresponded to that of
DNA synthesis in these regions after 17 h of germination ,
showing that the first cell cycle of cells in each region was
partially synchronous.However , all the peaks appeared at
25 h disappeared after 29 h of germination and the peaks
moved to 1.2-1.4 and 1.4-1.6 mm (the studentized
residues were above 1.5 , Fig.3b).The peaks at 1.2-
1.4 and 1.4-1.6 mm should be also the result of the
first wave of DNA synthesis , since cells in these regions
entered the first wave of DNA synthesis later than other
regions.
2.2.2　Changes with increasing distances from the
root cap junction 　Figure 3c shows the changes of
mitotic index in the cortical regions of lentil primary root
with increasing distances from the root cap junction after
different periods of germination.At 17 h , there were di-
viding cells in each region analyzed.However , the mitotic
index of either end of the primary root was much less than
that of the central regions of the primary root.And the
proportion of cells in division was highest at 0.8-1.0
mm after 17 h of germination(at this point the studentized
residue approached 1.5 , Fig.3d).At 21 h , the propor-
tions of cells in M phase in all regions declined and the
peak appeared at 0.4-0.6 mm (the studentized residue
was above 1.5 , Fig.3d).The results above showed that
cells in 4C state scatter around 0.4-0.6 mm , and initi-
ate their cell division in turn depending on their sites in
the primary root.At 25 h , the proportion of cells in divi-
sion in all regions analyzed increased , especially in the
acropetal regions(0-0.2 , 0.2-0.4 , 0.4-0.6 and
0.6-0.8 mm).And the peak was still at 0.4-0.6mm( the studentized residues of these points
were above 1.5 , Fig.3d).At 29 h , the proportion of
Fig.3.　a.Changes in the mitotic index with increasing germination periods in different regions of the primary root of lentil.b.The studen-
tized residues from a linear regression analysis of the changes in the mitotic index with increasing germination periods in different regions of the
primary root of lentil.A studentized residue larger than 1.5 is statistically significant , meaning that a clear peak value appears at the corre-
sponding point in Fig.3a.c.Changes in the mitotic index in the cortical regions(0-1.6 mm from the root cap junction)of the primary root
of lentil in different germination periods.d.The studentized residues from a linear regression analysis of the changes in the mitotic index in the
cortical regions(0-1.6 mm from the root cap junction)of the primary root of lentil in different germination periods.A studentized residue
larger than 1.5 is statistically significant , meaning that a clear peak value appears at the corresponding point in Fig.3c.
768　 植物学报　Acta Botanica Sinica　Vol.44　No.7　2002
cells in division in the acropetal regions(0-0.2 , 0.2-
0.4 and 0.4-0.6 mm)declined clearly , and the peak
occurring at 0.4-0.6 mm after 25 h of germination had
disappeared.However , the proportion of cells in division
augmented;meanwhile the peak of cells in division at 1.2-1.4 mm also appeared.These results showed that the
adjacent cells (in each region analyzed in this experi-
ment)proceed their cell division partially synchronously ,
but cells in different regions initiate their cell division in
turn in space , resembling the initiation of DNA synthesis
of these cells.
3　Discussion
Incorporation of radioactive thymidine into DNA can
be observed early in seed germination.The nature of this
early DNA synthesis has been regarded by some authors as
gene amplification
[ 15 , 16]
or as a mixture of repair and
replicative DNA synthesis
[ 17] .Georgieva et al[ 5] observed
a pronounced increase of G1 nuclei concomitant with an
equal decrease of nuclei in S phase during the initial 4 h
germination of maize seeds.They interpreted this phe-
nomenon as a synchronisation in G1 phase in order to en-
sure the elimination of cells in S orG2 , or to inhibit these
cells from further progression through the cell cycle.Be-
cause they could not detect any mitosis between the initial
S phase and the increase of G1 nuclei , it seemed that this
initial S phase was not similar to the S phase during which
the nuclear DNA content would be duplicated before cell
division.In the 1 h sample during early lentil seed germi-
nation , we observed that the cells in primary roots were
labelled by IUdR.These labelled nuclei were uniformly
stained red , but the nuclei labelled by IUdR 13 h after
imbibition were stained red in varying degrees since these
cells started DNA synthesis in the different period of
time , and contained different quantities of IUdR (Fig.
1d).Therefore , we concluded that the initial integration
of IUdR in nuclei was due to the physical changes of nu-
clei from a dry state to a wet state during imbibition in-
stead of nuclear DNA synthesis , and we did not take into
account this result which was due to an artefact(1 h sam-
ple).
Previous results[ 1] showed that the degree of syn-
chrony of the cell cycle progression was poor in contrast to
the high percentage of G1 phase cells in germinating plant
embryos.Georgieva et al[ 5] observed that during maize
germination most of the cells enter S phase as a partially
synchronised population , and within the initial 60 h of
embryo germination the cells passed through one S phase.
The present study offers the advantage that only a subpop-
ulation of cells is measured.The results above show that
the activation of the cell cycle of cells in different cortical
regions analyzed is not synchronous , and the same is true
for sister cells formed by the last cell division in embryo.
However , for each region analysed in this experiment , we
have found a clear peak of cells in S phase , and a clear
peak of mitoses.From these data , we could conclude that
during early lentil germination the adjacent cortical cells
have traversed partially synchronously the first cell cycle.
Plant cells are surrounded by rigid cell walls.Owing
to this rigid attachment , cell movement and migration as
in animals is not possible for plant cells.Cells are often
arranged in sheets or files , and division must therefore be
coordinated in some way to ensure that neighbouring parts
of the same structure increase in size at the appropriate
rate
[ 10] .Although the activation of the cell cycle in dif-
ferent cortical regions analysed is not synchronous , but
occurs acropetally as a function of time , the mean cell cy-
cle length and the general cell cycle kinetics in the differ-
ent regions can be considered constant.This represents a
harmony for the plant development.Cells in the basal part
of the cortex leave the cycle to enter cell elongation and
full differentiation.The time difference of cell cycle acti-
vation makes cells in the different regions leave cell cycle
in a certain order.Cells in the cortical regions analysed
in this article have undergone rapid cell division.It is
clear that this precise spatial organisation of cell division
ensures the harmony of the development of cortex.
It is known that hormones play a vital role in promot-
ing or inhibiting plant growth , but very little is known
about the mechanism of action of plant_specific mitogenic
signal.In maize germination , it seems that each group of
cells responds to specific signals which appear in the pri-
mary axes sequentially , because the first cell cycle is spe-
cific in timing in different tissues and the mesocotyl meris-
tem is the first to initiate cell division[ 6 , 18] .Cytokinin
could stimulate the increase of DNA synthesis in radicles
of Vaccaria pyramidata during germination[ 15] .This
could indicate that plant hormones participate in the acti-
vation and progression of the first cell cycle during seed
germination.In addition , Tréhin et al[ 19] found that aux-
in and cytokinin were involved in the re_entry of Petunia
protoplasts into the cell cycle.Although in the lentil pri-
mary root each cortical cell maintains an autonomous con-
trol for the activation and progression of its cell cycle , the
coordination of the activation and progression of cell cycle
among different regions exists during the early lentil seed
germination.For example , the cell cycle of cortical cells
is acropetally activated , and the progression of spatial dis-
tribution of cells in nuclear DNA synthesis and mitoses is
similar in different cortical regions.For our next study ,
further focus is probably required on how the cell cycle of
different cells is coordinated and what plays a role in this
coordination.
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兵豆萌发过程中初生根皮层细胞的细胞
周期活动的动态分析
喻富根＊　庞延军　赵剑春　王　强
(南京大学生命科学学院生物科学与技术系 , 南京 210093)
摘要:　观察了兵豆(Lens culinaris Medic.)初生根原皮层组织的细胞周期在其种子萌发过程中时间和空间上的动态
变化。免疫组织化学和细胞学证据表明 ,原皮层细胞分别在种子吸胀大约 13 h 和 17 h开始 DNA复制和细胞分裂。
最早进行 DNA 复制和细胞分裂的细胞位于远基端 1 mm 附近 ,但这些分裂细胞的 DNA复制是在种子成熟过程中完
成的 ,而不是在萌发后。第一个细胞周期的激活样式表明 , 这些细胞并不同步激活 , 而是依次进入细胞周期 ,且进
入的次序与自身在根尖中的相对位置有关。在兵豆初生根原皮层组织中 , 邻近位置上的细胞的细胞周期同步化程
度较高。
关键词:　细胞周期;初生根;萌发;免疫组织化学;兵豆
中图分类号:Q253　　　文献标识码:A　　　文章编号:0577-7496(2002)07-0764-07
收稿日期:2001-12-11　接收日期:2002-04-01
基金项目:教育部优秀年轻教师基金;教育部留学回国人员科研启动基金。
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770　 植物学报　Acta Botanica Sinica　Vol.44　No.7　2002