全 文 ：园 　艺 　学 　报 　2007, 34 (3) : 549 - 554
Acta Horticulturae Sinica
Rece ived: 2007 - 01 - 16; Accepted: 2007 - 05 - 21
Supported by National Natural Science Foundation of China (30571285, 30671452) , and by the Specialized Research Fund for the Doctoral
Program of H igher Education (20050434009)3 Author for correspondence ( E2mail: hqyang@ sdau1edu1cn)
Study on Root M itochondr ia l Character istics and Root Cell
Dea th of M a lus hupehensis Rehd. under W a ter Stress
MA Huai2yu1 , X IAO J ing1 , and YANG Hong2qiang1, 23
(1 College of Horticulture, Shandong A gricu ltura l U niversity, Taipian, Shandong 271018, China; 2 S tate Key Labora tory of C rop
B iology, Taipian, Shandong 271018, China)
Abstract: The changes of root m itochondrial membrane potential (Δψm) , H2 O2 concen2
tration and the activity of caspase23 /7, the key enzyme of p rogrammed cell death were studied
under 20% 2PEG6000 treatment. The results showed that root m itochondrialΔψm declined
slowly in the first six days of PEG treatment, then declined rap idly. The content of m itochon2
drial H2 O2 increased slowly in the first six days, then accumulated speedy inside of m itochon2
dria. TUNEL assay was carried out and root paraffin slices disp layed distinct positive reaction
spots, which were more and more with the p rolonging treatment. The activity of caspase23 /7 in
root kep t at low level from the first day to the sixth day, and then rose sharp ly on the ninth and
twelfth day of PEG treatment. These indicate that 20% PEG6000 can induce p rogrammed cell
death in M alus hupehensis Rehd. roots at a certain treating2phase.
Key words: M alus hupehensis Rehd. ; Root; W ater stress; M itochondria; Programmed
cell death; Caspase
水分胁迫下湖北海棠根系线粒体及细胞死亡特性研究
马怀宇 1 , 肖　静 1 , 杨洪强 1, 23
(1 山东农业大学园艺学院 , 山东泰安 271018; 2 作物生物学国家重点实验室 , 山东泰安 271018)
摘 　要 : 用 20% PEG6000处理湖北海棠 (M alus hupehensis Rehd. ) 根系 , 研究了水分胁迫条件下根系
线粒体膜电位 (Δψm)、线粒体 H2O2含量、细胞程序性死亡关键酶类 caspase23 /7活性的变化。结果显示 ,
在 20% PEG6000处理前 6 d, 根系线粒体Δψm缓慢降低 , 第 6天后急剧下降。线粒体 H2O2含量在处理的第
1～6天里缓慢升高 , 第 6天后则快速上升。TUNEL原位检测显示 , PEG6000处理能够使根系切片上出现清
晰的阳性反应斑点 , 且随着处理时间的延长阳性反应斑点增多 ; 处理第 3天和第 6天 , 根系中类 caspase23 /
7活性较低 , 而处理第 9天和第 12天时类 caspase23 /7活性成倍升高 , 这表明 20% PEG6000能够诱导湖北海
棠根系细胞程序性死亡。
关键词 : 湖北海棠 ; 根系 ; 水分胁迫 ; 线粒体 ; 细胞程序性死亡 ; Caspase
中图分类号 : S 661 　　文献标识码 : A　　文章编号 : 05132353X (2007) 0320549206
One of the earliest detectable reactions of a cell upon abiotic stress, such as soil drought, is the formation
of superoxide and subsequent accumulation of H2 O2. H2 O2 can be a signaling molecule that triggers p ro2
grammed cell death ( James et al. , 2003 ). M itochondria are the major source of reactive oxygen species
Acta Horticulturae Sinica Vol. 34
(ROS) in p lant root cells and may lead to alteration of the cellular reduction2oxidation ( redox) potential (L iu
et al. , 2002). M itochondria is believed to p lay an important role as a cellular trigger in the induction of PCD
(Halestrap et al. , 2000; Reed & Green, 2002).
A s we know, soil drought could accelerate suberization p rocess of app le roots which is made of exanimate
cells. So it was hypothesized that there m ight be close relationship among H2 O2 , m itochondria and app le root
cell death. M alus hupenensis Rehd. is a special app le stock germp lasm resource in China, and it has a high a2
bility of apom ixis and little diversity among seedling offsp ring. Hence, M alus hupenensis Rehd. is a good ex2
perimental material for researchers and can be widely used in app le physiological research ( Yang & J ie,
1997). PEG6000 was emp loyed to simulate soil drought in this experiment to investigate the characteristic
changes of m itochondria and root cell death. The aim is to establish a foundation for discovery of the relation2
ship among m itochondria, ROS, cell death and root suberization in app le roots.
1　M a ter ia ls and M ethods
111　Plan t ma ter ia l
The roots used in this study were taken from app le seedlings (M a lus hupenensis Rehd. ) , which were
grown in culture pans ( sand∶soil∶sawdust = 1∶1∶1).
112　Trea tm en t
To induce water stress, seedlings with sim ilar growth vigour were p laced in black p lastic bucket contai2
ning 1 /2 comp lete nutrient solution supp lemented with 20% PEG6000 when seedlings had at least seven eu2
phylla. Control p lants were cultured as above without PEG6000. In order to keep roots cultured in nutrient so2
lution under well2aerated condition, all buckets were installed with aerating2pump s. Each index was deter2
m ined with three rep lications.
113　Root m itochondr ia isola tion
Isolation of m itochondria from app le white roots for H2 O2 content and trans2membrane potential measure2
ment was performed according to Yang et al. (2004) , with slight modifications. (1) W hite roots from seed2
ling p lants treated with 20% PEG6000 were ground to mush in mortar m ixed with separate buffer (014 mol/L
glucose, 0105 mol/L Tris2HCl, 0101 mol/L EDTA, 011% BSA, 0105% β2mercap toethanol, pH 714) for 3
m inutes; (2) Centrifuge at 10 m in at 1 000 g, 4℃; (3) D iscard the deposit, centrifuge at 10 m in at 10 000
g, 4℃; (4) D iscard the supernatant, wash the deposit with separate buffer for two times. And then store the
supernatant at 0℃. The operation of root m itochondria isolation should be finished in 1 hour, and the isolated
m itochondria should be used up in 2 hours.
114　M itochondr ia l tran s2m em brane poten tia l m ea surem en t
Measurement of m itochondrial trans2membrane potential was made according to B raidot et al. ( 1998) ,
with slight modifications. M itochondrial deposit was suspended in analysis buffer ( 0125 mol/L sucrose, 2
mmol/L Hepes, 015 mmol/L KH2 PO4 , 412 mmol/L sodium succinic acid, pH 714). Subsequently suspen2
sion was stained with 1μg/mL Rh123 for 30 m inutes in the dark at 25℃, and then washed with analysis buff2
er for 3 times. Protein concentration of suspension was adjusted to 013 mg/mL. Fluorescence intensity of each
examp le was registered 3 times at 52second intervals by using spectrofluorimeter (λex 505 nm, λem 534 nm ).
The fluorescence intensity changes were p resented as average of 3 registered values (Unit: Relative Fluores2
cence Units, RFU s).
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115　M itochondr ia l H2 O2 rela tive con ten t m ea surem en t
Measurement of m itochondrial H2 O2 content is according to Zhou et al. ( 2004) , with slight modifica2
tions. Resuspended m itochondrial deposit with resp iration buffer (220 mmol/L glucose, 70 mmol/L sucrose, 2
mmol/L Hepes, 215 mmol/L KH2 PO4 , 215 mmol/L MgCl2 , 015 mmol/L EDTA, 011% BSA, pH 714) , and
m ixed with 6 mmol/L succinic acid, 10μmol/L lum inal and 10μg/mL HRP. The suspension was measured by
super2weak2lum inescence instrument. Hydrogen peroxide content was p resented as the fluorescence intensity.
The fluorescence intensity of each examp le was registered over 50 s at 37℃ for 3 times. And the fluorescence
intensity changes of examp les were p resented as average of 3 values (Unit: Relative L ight Units, RLU s).
116　D NA fragm en ta tion detection
For whole2mount TUNEL ( term inal deoxynucleotidyl transferase2mediated dUTP nick end labelling) ,
samp les from the 20% PEG treated app le roots were fixed in formal dehyde2acetic acid2ethanol ( FAA) for 24
h at 4℃ and then dehydrated through 75% , 95% , and 2 ×100% ethanol ( v /v in ddH2 O) at room tempera2
ture for 1 h each step. App le root paraffin2cut sections were made in normal way. Subsequently slides were
subjected to TUNEL assay according to kit introduction p rovided by Promega, and then slideswere viewed with
a visible2light m icroscope.
117　Ca spa se23 /72like protea se activ ity a ssay
Proteins were extracted from app le white roots by grinding in mortar with potassium phosphate buffer (pH
712) on ice. Enzyme activity was assayed according to kit of Caspase2GloTM 23 /7 activity assay supp lied by
Promega (Unit: Relative L ight Units, RLU s).
2　Results and Ana lysis
211　Change of m itochondr ia l H2 O2 rela tive con ten t in apple roots
M itochondrial electric leak was the main source of H2 O2 in p lant root cells. H2 O2 is the most stable ROS
and it can rap idly diffuse across cell membranes. A t high concentrations, it is toxic and can trigger p ro2
grammed cell death (James et al. , 2003). Lum inal, as a lipophilic p robe, can across m itochondrial mem2
brane freely and be oxidized by H2 O2 at the existence of exogenous HRP. Hence, with steady concentration of
exogenous HRP, lum inescence intensity of lum inal was directly correlated with the content of H2O2 in the in2
ner m itochondria.
A t the initial stages of 20% PEG2treating, the content of H2 O2 in root m itochondria kep t a low level on
day 3 and day 6, and then got a severe increase on day 9 and day 12 whereas the 20% PEG2untreated control
p lant roots were still at a low level of H2 O2 content in the treatment ( Fig11).
212　Changes of m itochondr ia l m em brane poten tia l in apple roots
It was shown that the m itochondrial permeability transition and change in m itochondrial trans2membrane
potential (△ψm) were the first signals of PCD in most analyzed cases of apop tosis in animal cells (Halestrap et
al. , 2000). Rh123 could flow into m itochondrial matrix along the trans2membrane potentialwith special alterna2
tive character. Fluorescence intensity of Rh123 in m itochondrial matrix depends upon the existence of △ψm, it
meant that the higher △ψm the higher fluorescence intensity was registered. Fig. 2 demonstrated that the △ψm
of 20% PEG treated roots was to keep reduction in the treating days. 20% PEG caused a slow decrease in m ito2
chondrial △ψm of app le root in one to six days of treatment and a severe decrease on day 9 and day 12; How2
ever, compared to those treated p lant roots, △ψm of untreated p lant roots was still at a higher level.
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Acta Horticulturae Sinica Vol. 34
F ig. 1　M itochondr ia l H2O 2 rela tive con ten t changes
in 20% PEG trea ted apple roots
F ig. 2　M itochondr ia l m em brane poten tia l changes
in 20% PEG trea ted apple roots
213　D NA fragm en ta tion detection by TUNEL in apple roots
One of the specific features of PCD is the cleavage of DNA at internucleosomal sites by DNA endonucleas2
es. In order to detectDNA fragmentation in situ, fragmented DNA was end2labelled app lying the TUNEL ( ter2
m inal deoxynucleotidyl transferase2mediated dUTP nick end labelling) method. Treated app le roots were fixed
and subjected to TUNEL. The dark TUNEL2positive reactions were found in both treated and untreated root
cells. However the p icture of 20% PEG treated root cells for 9 days or 12 days showed that root cells contained
multip le TUNEL2positive which were more than that of untreated control or treated roots for 6 days ( Fig. 3).
It was suggested that there were more p rogrammed death cells in 20% PEG treated roots for 9 or 12 days.
F ig. 3　TUNEL a ssay of apple root slices after 20% PEG trea ting ( 125 ×)
a. Control; b. 6 days of treatment; c. 9 days of treatment; d. 12 days of treatment.
214　Ca spa se23 /72like protea se activ ity a ssay in apple roots
Caspase is the key enzyme of p rogrammed cell death in animal. The activity of caspase2like p roteases had
been detected in soybean ( Florence et al. , 2003). To investigate whether or not the 20% PEG2induced app le
root cell death is associated with the activation of caspase2like p roteases, the caspase23 /7 activity was assessed
by fluorometric determ ination of free fluorescentAMC generated by p roteolytic cleavage from the nonfluorescent
conjugated substrate DEVD2AMC. The amount of free fluorescent AMC generated is p roportional to the content
of activated caspase23 /7 in the root2extracted suspension. Table 1 suggested that the 20% PEG2induced PCD
in app le root cellswas accompanied by distinct activation of caspase23 /7. The activity of caspase23 /72like p ro2
tease can be exam ined in both untreated and treated roots whereas was very low in the untreated roots. Two
phases of changed caspase23 /7 p roteases activities were observed: The first phase was at a low level had un2
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No. 3 MA Huai2yu et al. : Study on RootM itochondrial Characteristics and Root Cell Death of ⋯
consp icuous difference with untreated control app le roots on day 3 and day 6; The second phase was markedly
elevated on day 9 and day 12.
Table 1　Ca spa se23 /7 activ ity of apple roots after 20% PEG trea ting (RLU s/mg Pro. )
Treatment 0 days 3 days 6 days 9 days 12 days
Control 46169 ±1162 47198 ±11993 50164 ±21677 60141 ±41778 61109 ±41391
PEG 46169 ±1162 48165 ±11995 51179 ±11475 207169 ±31940 382117 ±51835
　　Note: The values in the table are the mean ±SD of 3 repeats.
3　D iscussion s
Since the idea of PCD was introduced into botanical field, it has been found that PCD is involved in the
p rocess of cell death that occurs during development and in response to environmental triggers in a wide variety
of different biological system. In p lants, PCD is believed to be essential for development and survival. Study
on the mechanism of PCD operation showed that m itochondria and H2O2 p lay a key intermediary role in the in2
ducing course of PCD. Neither morphological changes of m itochondria nor degradation of m itochondrial DNA
was observed in early stage of apop tosis. It is impossible to induce apop tosis in isolated nuclei without m ito2
chondria or m itochondria extracts. On the other hand, cells without nuclei are capable to initiate mechanism
responsible for PCD (Halsetrap et al. , 2002). It is well known that p lants utilize detoxifying and scavenging
enzymes as well as non2enzymatic antioxidants to keep a balance between ROS p roduction and elim ination.
However, the uncontrolled p roduction of ROS can cause extensive damage to p lant cells, the rap id induction of
ROS under stress conditions ( i1e. , the oxidative burst) initially results in the p roduction of O2 -· , which was
oxygenated by SOD to the H2 O2 and induce physiological and chem ical changes in cells, such as DNA degra2
dation, p rotein denaturation, membrane permeability augment and lip ids peroxidation, and metabolic disor2
der, etc (Johnson et al. , 2003). It had been p roved that a certain concentration of H2O2 could induce PCD
of tobacco (Reed & Green, 2002). M itochondrial resp iration state 4 supp lying electrons for the course of oxy2
genating O2 into ROS is regulated by △ψm , in a word, the descent of △ψm would enhance m itochondrial res2
p iration intensity ( Ichas & Mazat, 1998). So the phenomenon we had observed in this study indicated that
app le root △ψm descent was a stimulator of inducing H2 O2 accumulation in m itochondria when app le roots
were subjected to 20% PEG stress. And L iu et al. (1999) had shown the evidence that ROS from resp iration
chain had a negative2feedback effect on △ψm inversely, indicating there was inter2enhancing2ship between
△ψm descent and H2 O2 accumulation. W e also obtained sim ilar result in this study that △ψm and H2 O2 con2
tent in m itochondria had the parallel changing curve, hence, it is hypothesized that the negative2feedback am2
p lification cycle between △ψm and H2 O2 accumulation in m itochondria is the magnifying app roach of cell2
death2inducing stimuli in cells.
The observations of p rogrammed cell death in animal cells strongly suggest that the existence of a series of
special executors2caspases regulate this p rocess, in which all diverse signaling pathways ultimately lead to the
activation of caspases, a fam ily of cysteine p roteases that cleave substrate p rotein after an aspartic acid (A sp)
residue. Since it has been reported that activation of caspase2like p roteases following PCD stimuli involved in
many p lant system s undergoing PCD ( Florence et al. , 2003) , we had exam ined the caspase232like activity in
app le roots subjected to 20% PEG stress. Caspase232like activity was readily detectable in app le roots using
Ac2DEVD2AMC, a fluorogenic substrate specific for caspase23 of animal cells. In addition, the change curve
of caspase232like activity in app le roots was the same as the results of TUNEL exam ination. A ll these facts in2
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Acta Horticulturae Sinica Vol. 34
dicated that app le roots p rogrammed cell death was induced at a certain stage when subjected to the 20% PEG
stress.
The change of △ψm and H2 O2 content could be divided into two phases in the 20% PEG treatments:
(1) Slow changing phase, in which TUNEL2positive reactions and caspase232like activity were at comparative2
ly low level, suggested that there were few root cells operated p rogrammed death; (2) Severe changing phase,
there were more TUNEL2positive reactions in cells and caspase232like activity severely raised, too, which sug2
gested that more root cells were operating p rogrammed death. The results obtained in this experiment indicated
that the change of △ψm and H2 O2 content had close relationship with p rogrammed cell death in root cells
when subjected to water stress. And it was hypothesized that only when H2 O2 accumulation amounted to a cer2
tain valvular value or △ψm descended to a certain level that p rogrammed cell death could be started up , dem2
onstrating that the important function of m itochondria in initiating root cell death p rogram.
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