Download Programmed Cell Death during Leaf Senescence in Eucommia

北京大学 政学者论文集（2001 年）
杜仲叶片衰老过程中的细胞程序性死亡
杜仲叶片衰老过程中的细胞程序性死亡
Programmed Cell Death during Leaf Senescence in
Eucommia ulmoides Oliv.*
生命科学学院 植物分子与发育学系 98 级 江枫
Abstract
Leaf tissues from Eucommia ulmoides Oliv. trees were used to study the
mechanism of programmed cell death (PCD) during leaf senescence. Here, we
show that DNA ladders were detected by gel electrophoresis only in senescent
leaves. DNA fragmentation and nuclear DNA condensation were further
confirmed by using the terminal deoxynucleotidyl transferase (TdT)-mediated
dUTP nick end in situ labeling (TUNEL) method. A 20 kDa DNase was detected
only in senescent leaves, which suggested that fragmented DNA would be caused
by this DNase.
Keywords:
DNase―Leaf
senescence―Programmed
cell
death
(PCD) ―Eucommia ulmoides Oliv.
Cell death is a basic biological process that functions in many aspects of animal and
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杜仲叶片衰老过程中的细胞程序性死亡
plant development and in their responses to stress (Greenberg 1996, Wang et al. 1996,
Martins and Earnshaw 1997). Studies on animals have shown that the execution of
programmed cell death (PCD) or apoptosis is controlled by a multistep signaling
pathway (McConkey and Orrenius 1994, Stewart 1994). Like in animals, it is called
as physiological cell death for development and defense (Greenberg 1996), so it may
be that any complex multicellular organism will be found cell death a usuful process
to have in its repertoire. There are many examples on programmed cell death during
plant development, such as senescence of the carpel and petal (Orzáez and Granell
1997a, b), xylogenesis (Mittler and Lam 1995, Fukuda 1996, 1997, Wang and Cui,
1998), aleurone deletion (Wang, M. et al. 1996), the death of the root cap cells (Wang,
H. et al. 1996) and somatic embryogenesis (McCabe et al. 1997).
Leaf senescence is the final stage of leaf development, which has been thought to
be a type of programmed cell death (Noodén and Leopold 1978,Gan and Amasico
1997). The mechanism which controls this process must be genetically regulated by
autonomous (internal) factors such as age, reproductive development and
phytohormone levels, as well as regulated by environmental signals such as stresses,
drought, ozone, nutrient deficiency, pathogen infection, wounding and shading (Gan
and Amasico 1997). At the molecular level, till now, more than 30 leaf senescence
associated genes (SAGs) have been isolated, cloned and characterized. Most of them
encode enzymes that are thought to be involved in cell degeneration and nutrient
mobilization, whereas the identity of some SAGs remains unknown (Basanti et al.
1999). Although so much factors and genes regulated leaf senescence were observed,
it was unclear whether leaf senescence shared any biochemical or genetic pathways
with other types of PCD in plants or animals. Yen and Yang (1998) have reported the
*
Foundation items: Jun Zheng Fudation of Peking University
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杜仲叶片衰老过程中的细胞程序性死亡
detection of DNA fragmentation in naturally senescent leaves from five plant species,
which proved that senescence of the leaves must share some similar pathways
observed in other types of PCD in both plant and animals. In this paper, DNA ladders
and DNA fragmentation were also detected in naturally senescence leaves from
Eucommia ulmoides Oliv. Trees. In addition, we have detected DNase during leaf
senescence, providing important information on the mechanism of leaf senescence.
Materials and Methods
Plant materials
Leaves at different developmental stages, from young (full expansion), green1
(May to Aug.), green2 (Sep. to Nov.), yellow and dry leaves were obtained from
strong and healthy Eucommia ulmoides Oliv. trees which were grown on the campus
of Peking University, Beijing, China.
DNA extraction and analysis
Genomic DNA was isolated from leaf tissues using a CTAB method. Leaf tissues
were ground in liquid N2 immediately after being collected from the plants and then
the frozen samples were homogenized in an extraction buffer that contained 2%
CTAB, 100 mmol/L Tris-HCl, pH 8.0, 20 mmol EDTA, pH 8.0 and 1.4 mmol NaCl,
and the mix incubated at 65℃ for 1 h. After this, Equal volumes chloroform/isoamyl
alcohol (24/1) were added and the tubes inverted 30 min and the samples centrifuged
at 12,000 g for 10 min, repeated twice. The DNA was precipitated by adding 2/3 vol
of isopropyl alcohol at -20℃ for 30 min. DNA samples were digested with DNA-free
RNase for 1 h at 37℃ and the DNA content determined. For DNA analysis, samples
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杜仲叶片衰老过程中的细胞程序性死亡
of 4 μ g DNA were loaded per lane and run on a 1% agarose gel at constant 70 V. The
DNA was visualized by staining with 0.5 μg /ml ethidium bromide.
TUNEL assay
Leaf tissues were cut and fixed in 10% formalin with PBS buffer pH 7.2 overnight.
The samples were dehydrated through a graded series of ethanol and embedded in
paraffin. Sections of 8-10μm thickness were cut using a microtome type 860. After
dewaxing and rehydration, sections were incubated with proteinase K (20μg/ml) for
15 min at 37℃ and rinsed twice with PBS. Then sections were stained for PCD
observation with in situ cell death detection kit, AP (Boehringer, Mannheim,
Germany). Reaction products were dark blue. In positive control, DNase I (grade I,
0.5 mg/ml in 50 mmol/L Tris-HCl, pH 7.5, 1 mg/ml BSA) was added before TUNEL
reaction mixture for 10 min at room temperature to induce DNA strand breaks. In
negative control, terminal transferase was omitted.
Extraction of protein from leaf tissues.
For extraction of the soluble protein, leaves were thoroughly ground in liquid N2 in
a prechilled mortar, and then the powder was suspended in an extraction buffer that
contained 0.5 mol/L Tris-HCl, pH 6.8, 5% 2-mercaptoethanol and 10% glycerol, 2.5%
SDS and a trace of bromophenol blue. After centrifugation at 15,000 g for 15 mins at
4 ℃. The pellets were further ground and washed three times with the same buffer.
All the supermatants were combined together as the extract of soluble protein.
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杜仲叶片衰老过程中的细胞程序性死亡
Detection of molecular species of nucleases using DNA-SDS polyacrylamide gel electrophoresis
Nucleases
with
their
molecular
weights
were
detected
by
DNA-SDS
polyacrylamide gel electrophoresis, as described previously (Sodmergen and S.
Kawano 1991). Separation gel contained 50% acrylamide (Promega), 1.25%
bisacrylamide (Promega), 2 mg/ml salmon sperm DNA (Sigma), 0.4% SDS (Sigma),
1.5 mol/L Tris·HCl, pH 8.8, 0.5% ammonium persulfate, and 0.1% (v/v) TEMED.
Spacer gel contained 12% acrylamide, 0.4% bisacrylamide, 0.4% SDS, 0.5 mol/L
Tris·HCl, pH6.8, 0.5% (w/v) ammonium persulfate, and 0.1% (v/v) TEMED.
Electrophoresis was carried out at constant 20 mA at room temperature. Total time
was about 3-4 hours. After electrophoresis, the protein standard marker was cut off
and stained with 0.25% coomassie blue. The left gels were washed to remove SDS for
1h in the washing buffer (10 mmol/L Tris-HCl,pH 8.5, 0.1 mmol/L EDTA, 0.1
mmol/L EGTA, 0.1% mercaptoethanol and 0.1 mmol/L PMSF).Then shaking in the
washing buffer overnight. The next day, after being washed for 1 h, the gel was
incubated for enzymatic activity in the washing buffer contained 10 mmol/L CaCl2
and 10 mmol/L MgCl2 at 37℃ overnight.
Then the gel was stained with ethidium
bromide for 30 mins. DNase activity was
visualized under UV light.
Results
Detection of
DNA ladder
during leaf
senescence
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The DNA from leaves at different development stages was extracted and analysed
in agarose gels. The results indicate that DNA from young and green1 leaves shows
no degradation (Fig.1, lane1 and lane2). However, as we proceed from green2 to
yellow leaves, bands of DNA degradation can be observed clearer (Fig.1, lane3 and
lane4). The size of the DNA bands corresponds to multiples of around 200bp,
suggesting that they are produced by internucleosomal degradation of the DNA. In the
Fig. 1
dried leaf, the DNA became a smear and the laddering was reduced to become
unvisualized in the agarose gel (Fig.1, lane5).
DNA fragmentation analysis
The DNA fractionation was studied at the cellular level in thin sections of
non-senescent, senescent leaves using the TUNEL reaction. Although the TUNEL
reaction is not exclusive for DNA degradation occurring at the internucleosomal
region, it is a very useful technique to detect in situ those nuclei in which the number
of DNA ends has increased as a consequence of DNA degradation.
In Figure2, the nuclei of mesophyll cells in green2 and yellow leaves were labeled
by TUNEL (Fig.2, C and D) and green2 leaves were labeled stronger than yellow
leaves. By contrast, there were no TUNEL staining in young and green1 leaves, as
shown in Fig.2, A and B, indicating the nuclei of the cells in these leaves remain
intact.
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Fig. 2
Detection of DNase
A 20 kDa DNase
was detected only in
green2
leaves
Fig.3
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(Fig.3,Lane3), which
suggests that fragmented DNA may be caused by this DNase. No DNase was
detected in other developmental stages leaves (Fig.3, Lane1, 2, 4 and 5).
Summary of the experiments (Table 1)
DNA ladders and TUNEL Label were detected only in senescence leaves (green2
and yellow); The degradation of PARP localized around the nuclei and appears before
emergence of DNase; A 20 kDa DNase was detected only in green2 leaves which
began to senescence.
Table 1 Results of the study
Young
Green1
Green2
Yellow
Dry
DNA
ladders
No
No
Yes
Yes
Smear
TUNEL assay
DNase
No
No
Yes
Yes
Not done
No
No
Yes(20kDa)
No
No
Discussion
The relationship between PCD and leaf senescence
Although the leaf senescence has been studied for many years, most of the studies
focussed on the breakdown of the chloroplast and the changing of proteinase activity
(Thomson and Platt-Aloia 1987). It is interesting to note that only a few reports give
DNA damage or alteration at the nuclear levels an important role in leaf senescence
(Yen and Yang 1998). This was possibly because in most cases senescence had been
studied in leaves where the chloroplast was the first organelle showing the effects of
senescence, and the effects on the nuclei occurred rather late (Orzáez and Granell
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1997a). Bleecker and Patterson (1997) has reported that senescence and abscission of
leaves are PCD processes. And Orzáez and Granell (1997a, b) indicated that
senescence of the carpel and petal were also PCD processes. This study indicated that
at the cellular level, senescence of the leaves takes place in a similar manner as that
observed in animals by producing condensed nuclei and yielding fragmented DNA, a
20 kDa DNase, DNA ladders during leaves senescence, and caspase-3-like protease(s)
in early stage of leaf development. These further demonstrated that leaf senescence
was a PCD progress.
And this investigation also found that DNA fragmentation was
detected not only in yellow leaves but also in green2 leaves. This suggested that the
signals triggered natural leaf senescence should occur early, before the leaf began to
show the sign of yellowing in which cells have already entered the third stage of PCD.
Since most genes associated with leaf senescence expressed during the latter phases of
senescence, it is necessary to further identify the genes involved in the initial step of
this process, which should lead to a deeper understanding of its mechanism (Yen and
Yang 1998). But above these suggested that the differentiation of leaf cells should be
last stage of cell differentiation (Cui 1997), the differentiation process of these cells
could be PCD. Once all cells in leaf entered the last stage of PCD, the leaf should
appear the characters of senescence.
Mechanism for PCD in leaf senescence
According to the investigation on animal and medical, PCD comprises of three
stages: initiation, effector and degradation, during which the caspase family plays the
key role. During the third stage of PCD, specific endonucleases attack nuclear DNA in
the internucleosomal linker regions between nucleosomal cores, resulting the
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production of double-stranded, low molecular weight oligonucleosomal DNA
fragments about 180 to 200 bp in size (Cohen 1993). Perez et al. (2000) identified
BFN1, a bifunctional nuclease induced during leaf and stem senescence in
Arabidopsis, which would be an excellent tool with which to study the mechanisms of
the senescence induction. Our experiments also showed that during leaf senescence, a
20 kDa DNase was detected only in green2 leaves (Sep. to Nov.), the stage that DNA
ladders and fragments began to emergence and were strongly detected, which
suggested that fragmented DNA may be caused by this DNase.
In an elegant series of experiments, the groups of Wang and Nagata showed that
the DNA ladder nuclease (now known as caspase-activited DNase, or CAD) pre-exists
in living cells as an inactive complex with an inhibitory subunit, dubbed ICAD
(Nagata 2000). Activation of CAD occurs by means of caspase-3-mediated cleavage
of the inhibitory subunit, resulting in the release and activation of the catalytic subunit
(Liu and Zou et al., 1997; Enari et al., 1998; Sakahira et al., 1998). So PCD signaling
emerged very early during leaf development, perhaps at the stage of budding or earlier.
These suggested that main process of PCD in plants could be the same as in animals,
but there could be different death sign and the direction in which produce from PCD
having gone between plants and animals. The study provides important findings in the
study of the mechanism of PCD in plants.
Acknowledgement
I would like to thank Jun Zheng Foundation for providing me this chance to do
the scientific research, and Cao Jing for sharing of unpublished observations, and my
supervisor Prof. Cui Keming for his instruction on scientific attitude and method and
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his critical reading of this manuscript. I am also grateful to Prof. Sodmergen for
giving his advice on technology, and other teachers and colleagues in our laboratory
for their help.
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作者简介：
江枫，女，1981 年生于福建省福州市，中学就读于福州市第八中学，因学
习成绩优异，被校批准跳级一年，直接参加高三学习。1997 年曾获全国高中数
学联赛一等奖，并获得免试保送北京大学数学学院的机会。但因对生物学有着浓
厚的兴趣，1998 年考入北京大学生命科学学院，就读于植物分子与发育学系。
2000 年获得北京大学学习优秀奖。大学三年学习成绩名列专业第一名，并准备
申请自费留学美国攻读博士学位。
感悟与寄语：
转眼间在北大已经过去了三年，我改掉了自小以来的以学习为中心的生活方
式，明白了生活的灿烂是学习远远不能给予的。既然选择了生物，今后大部分的
时间和精力必然会投入于研究工作中，我很早就期待着能有机会体验一下科学家
的生活。很感谢“君政基金”给予了我一个参与科学研究的机会，尝到了它的酸
甜苦辣，更加对自己的理想——作一名科学家坚定不移。我们实验室的工作是研
究正常发育状态下的树木的细胞程序化死亡，由于树木的生长周期长，与其他的
实验室相比，实验周期长，技术难度大，在国际上也比较少人涉足这个领域，选
择这样的课题，足见导师不急功近利，一心钻研的科学精神。一年时间对于研究
工作是远远不够的，我没有完成发表一篇论文的任务，但是对于研究我有了许多
创新性的见解，这对我在将来的工作是大有裨益的。希望所有向往科学的勇者都
“有志者事竟成”！
指导教师简介：
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北京大学 政学者论文集（2001 年）
崔克明
杜仲叶片衰老过程中的细胞程序性死亡
教授，男，1941 年 2 月生，山东寿光人。1966 年毕业于北京大学
生物学系植物学专业。现任北京大学生命科学学院教授，植物学专业博士生导师，
植物分子及发育生物学系副主任，兼任中国植物学会结构及生殖生物学专业委员
副主任、中国杜仲综合开发协会常务理事、中国林学会杜仲研究会常务副主任委
员、北京植物学会副理事长、国家自然科学基金委员会植物学学科评审组成员、
国家科技奖励评委会委员、北京市学位委员会学科评审组成员和《植物学报》编
委等职。
249