全 文 ：Received 2002-03-28 , Accepted 2002-08-14.
This work was supported by USDA NRICP grants 99-00647 and
00-00665 to M.-C.Shih.
＊Current address:Monsanto Company , 800 N.Lindbergh Blvd , St.Louis,
MO 63167 , USA.
＊＊Corresponding author(Tel:1-319-335-2071 , Fax:1-319-335-3620 , E-
mail:ming-che-shih@uiowa.edu).
Abbreviations:ADH , alcohol dehydrogenase;DAPI , 4′, 6′-diamidino-2-
phenyindole;GUS , β-glucuronidase;PBS , phosphate buffer saline;PBT ,
PBS plus Tween;PCD , program cell death;RR , Ruthenium Red;
TUNEL , terminal deoxynucleotidyl transferase-mediated dUTP-X nick end
labeling.
Involvement of Calcium in the Anoxia-Signaling Pathway of Arabidopsis
thaliana
Qi Wang
＊　Richard Sjölund　Ming-Che Shih＊＊
(Department of Biological Sciences , Universi ty of Iowa , Iowa City , Iowa 52242, USA)
Abstract:We report here the effects of Ruthenium Red ,
an organellar Ca
2+
flux inhibitor , on the induction of the
expression of ADH gene and the survival of Arabidopsis
plants during anoxia.Our results suggest that Ca2+ is
required for the activation of the ADH gene in an early
stage of anoxia.In addition , our data show that the pres-
ence of Ruthenium Red , which can prolong the survival
time of Arabidopsis under anoxia.We propose that the
anoxia-induced cell death in Arabidopsis may be an active
process , and that this process requires an increase in in-
tracellular Ca
2+ , which can be blocked by Ruthenium
Red.Consistent with this idea , we found that nuclear
condensation and chromosomal fragmentation occurred in
leaf cells of Arabidopsis plants under prolonged anoxia.
From the characteristics of the cell damage in leaves sub-
jected to anoxia , it appears that apoptosis occurs first ,
which is followed by the death of the whole Arabidopsis
plants.
Key words:Arabidopsis thaliana , anoxia , alcohol dehydrogenase ,
calcium signaling
　　Exposure to oxygen deprivation(anoxia)or low oxy-
gen(hypoxia)is a stress commonly encountered by both
plants and animals.To survive prolonged periods of oxy-
gen deficiency , all aerobic organisms have had to evolve
mechanisms for sensing oxygen shortage and to adjust
their cellularmetabolism accordingly.Species across great
evolutionary distances have evolved different anoxic toler-
ance.
Upon transfer from aerobic to hypoxia anoxia condi-
tions , both animal and plant cells switch from aerobic res-
piration to lactic fermentation (Roberts et al.
1984a , b).Continuous lactic fermentation throughout hy-
poxia leads to the acidification of cytoplasm and rapid cell
death in animal tissues.In contrast , after a transient pe-
riod of lactic fermentation , maize root tip cells switch to
alcoholic fermentation and allow glycolysis to continue for
a longer period(Roberts et al.1984a , b).Comparative
studies of cytoplasmic acidosis indicate that cytoplasmic
pH regulation is an important factor in affecting the sur-
vival of plants under hypoxia(Roberts et al.1984a , b;
Xia and Saglio 1992).
Anoxia-treatment of maize seedlings causes the re-
pression of the synthesis of the majority of cellular pro-
teins that exist under normoxia and induces the synthesis
of about 20 anaerobic proteins(ANP)after approximately
90 minutes(Sachs et al.1980).Most of these ANPs are
enzymes involved in glycolysis and fermentation (for re-
view , see Sachs et al.1996).In contrast , more than 60
proteins are synthesized in maize root tip cells under hy-
poxia (Chang et al.2000).Nevertheless , hypoxia-in-
duced proteins include ANPs and other enzymes involved
in primary carbohydrate metabolism (Chang et al.
2000).Transcriptional , post-transcriptional , and transla-
tional controls regulate synthesis of ANPs under low-oxy-
gen stress(Drew 1997 , Fennoy and Bailey-Serres 1995).
Several cis-acting elements and their cognate binding fac-
tors involved in anoxic and hypoxic induction of the ex-
pression of the ADH gene in Arabidopsis and the ADH1
gene in maize have been identified (Ferl and Laughner
1989 , Kyozuka et al.1994 , Dolferus et al.1994 , Ho-
eren et al.1998).
While the transcription of some genes that encode
glycolytic and fermentative enzymes can be induced by
oxygen deficiency , a direct relationship between this in-
duction and cell survival remains to be established.In
fact , hypoxic pretreatment improved the survival of maize
root tip cells during subsequent anoxia , and only a small
amount of ADH enzyme is required for this acclimation
441Journal of Plant Physiology and Molecular Biology　2002 , 28(6):441-448
(Johnson et al.1994).There is no evidence that any of
the glycolytic enzymes is limiting in maize root tip cells
under anoxia or hypoxia (Drew 1997).
There was evidence that Ca
2+
is involved in the sig-
naling pathway leading to the activation of ADH1 in
maize.There was a transient increase in cytosolic Ca2+
concentration early after the flooding of maize roots(Sub-
baiah et al.1994a , b), inhibition of which blocked the
induction of the ADH1 gene(Subbaiah et al.1994a).A
similar anoxia hypoxia-inducible Ca2+ increase was ob-
served in Arabidopsis(Sedbrook et al.1996).Ca2+ sig-
naling is also required for the induction of the Arabidopsis
ADH gene (Chung and Ferl 1999).However , these
studies could not distinguish whether the effect of Ca
2+
was at the transcriptional or posttranscriptional level , be-
cause only the steady-state mRNA levels for the ADH
gene during hypoxia were measured.We report here that
the effect of Ca
2+
on anoxia-signaling pathway leading to
the induction of ADH expression in Arabidopsis is mainly
through transcriptional control.Our data also indicate that
cellular Ca
2+
plays additional roles in the response to an-
oxia in Arabidopsis.
1　Materials and Methods
1.1　Anaerobic treatment of Arabidopsis plants
Construction of a transgenic AG2 line containing the
ADH::GUS transgene has been reported (Conley et al.
1999).Twenty-day-old plants were used for anaerobic
treatment.Arabidopsis seeds were sterilized and germinat-
ed on two layers of filter paper in a petri dish(100 mm×
15 mm)containing 3.5 ml of MS medium at 20℃.After
one week , the seedlings on the filter paper were trans-
ferred onto a plate containing solid MS medium and allow
to grow under a 16-h-light 8-h-dark cycle at 20℃ until
use.For anaerobic treatment , filter paper with plantlets
was submerged in water at 20℃ through which nitrogen
gas(>99.5%purity)was bubbled to remove oxygen.
To determine the survival rate , 3-week-old Arabi-
dopsis plants grown on filters were subjected to anaerobic
treatment as described above.At different time intervals ,
filter paper with planets was transferred onto sterile petri
dishes containing MS medium for observation.Plants that
remained alive after one week were judged to be surviving
anoxia-treatment.
1.2　GUS and ADH enzymatic assays
GUS enzyme activity assays were performed accord-
ing to the procedures described by Jefferson et al.
(1987).Tissues were ground to a fine powder in liquid
nitrogen and resuspended in lysis buffer.Supernatants
from these suspensions were removed to new tubes and
protein concentrations were determined by the Bradford
method(Bradford 1976).Aliquots each containing 25 to
50 μg of protein were combined with 1 mmol L 4-methy-
lumbelliferyl-β-D-glucuronide in 200μl of lysis buffer and
incubated at 37℃.Reactions were stopped by the addit-
ion of 1.8 ml of Na2CO3 0.2 mol L.Fluorescence of the
product formed was quantified using a Model TKO-100
minifluorometer(Hoefer Scientific Instruments , San Fran-
cisco , CA).
ADH enzyme activity assays were performed accord-
ing to the procedures described by Xie and Wu(1989).
Ethanol was used as the substrate and the production of
NADH was measured in a Beckman DU 64 spectropho-
tometer.A unit of ADH enzyme is defined as an increase
in absorbance of 0.01 per minute at 340 nm.
1.3　DAPI staining
Arabidopsis plants subjected to anoxia with variable
lengths of time and control plants were blotted dry and
fixed in 4% paraformaldehyde PBS (1×PBS = NaCl
150 mmol L , KCl 2.5 mmol L , Na2HPO4 5 mmol L and
KH2PO4 2mmol L)at 4℃ for 48 h and washed in double
distilled water twice for 5 min each time.Tissues were
then stained in a solution containing 2μg 100ml of DAPI
for 2 h and examined under a fluorescence microscope.
1.4　TUNEL staining
Tissue samples were fixed in 4%paraformaldehyde
for 4 h to overnight and rinsed twice with PBS(3 chang-
es , 20 min each).Fixed tissues were subsequently treat-
ed with 1% cellulase and 0.2%pectinase in a solution
containing sorbitol 0.4mol L , mannitol 0.4mol L , CaC-
l2
2 mmol L , and Mes 5 mmol L , pH 5.5 for 1 h at
27℃.Tissues were washed with PBS twice for 5 min
each and then permeabilized in 1 ml 0.3%Triton X-100
for 1 h.Permeabilized tissues were washed with PBS
twice for 5 min each time and then treated with proteinase
K 200μg ml in PBT (0.1%Tween 20 in PBS)in a total
volume of 200μl for 15 min at room temperature.After 3
rinses in PBT , 1 ml of 2%H2O2 in MeOH was added
and the sample was incubated for 30 min with 2 changes
of buffer.The tissues were then rinsed three times with
PBT and twice with TdT reaction solution(Tris 30 mmol
L , pH 7.2 , CoCl2 1 mmol L , Na-CaCodylate 30 mg
442 植物生理与分子生物学学报　　　　　　　　　　　　　　　　　　28卷
ml).Terminal transferase reactions were performed in a
reaction volume of 100 μl containing dUTP 15 μmol L ,
fluorescein-dUTP 10 μmol L , 25 units of terminal trans-
ferase at 37℃ for 20 min.The reactions were terminated
by rinsing tissues with PBT 3 times for 20 min each.Tis-
sues were mounted in 50% glycerol PBS and examined in
a Leica DMRBE fluorescence microscope.
2　Results
2.1　Effects of Ruthenium Red on anoxic induction
of the expression of the ADH gene
An organellar Ca
2+
flux inhibitor , Ruthenium Red
(RR), can block anaerobic induction of the ADH gene in
maize seedlings(Subbaiah et al.1994a ,b), suggesting
the involvement of calcium in anaerobic signaling in
maize.We used the AG2 transgenic line , which contains
an ADH promoter and GUS coding region fusion(Conley
et al.1999), to investigate whether RR can similarly af-
fect anaerobic induction of the ADH gene expression in
Arabidopsis.
In 20-day-old AG2 plants , GUS activity increased
within 2 h after the start of the anaerobic treatment , and
showed a 8-fold increase of activity after 8-12 h of treat-
ment(Fig.1).The addition of RR 25μmol L in the be-
ginning of the anaerobic treatment completely abolished
this induction(Fig.1a).We found that the anaerobic in-
duction of the endogenous ADH enzyme activity was also
abolished completely by RR(Fig.1b).These results in-
dicate that Ca
2+
is involved in the anoxic induction of ex-
pression of both the ADH::GUS transgene and the endo-
genous ADH gene.A second set of experiments was per-
formed to determine the effective time of RR addition.In
these experiments , Arabidopsis plants were subjected to
anoxia-treatment in a normal medium without RR.RR
was then added at different times after the initiation of an-
oxia-treatment.12 h after the onset of anoxia-treatment ,
plants were harvested and assayed for GUS activity.Fig-
ure 2 shows that when RR was added during the first 2 h
of the anoxia-treatment , the induction of GUS was abol-
ished.In contrast , additions of RR 3 h or longer after the
onset of anoxia-treatment had no apparent effect on the
induction of GUS. These results suggest
that an intracellular Ca
2+
increase in
Fig.1　Effects of Ruthenium Red on the anoxic induction of the
ADH gene
AG2 plants were subjected to anoxia treatment in the absence(★)
or presence(○)of 25μg ml Ruthenium Red.At different time in-
tervals , plants were harvested and assayed for GUS (a)and ADH
(b)activity.A unit of GUS activity is expressed as pmol 4-methy-
lumbelliferon min -1μg-1 protein.A unit of ADH enzyme is defined
as an increase in absorbance at 340 nm of 0.01 per minute (Xie
and Wu 1989).The data presented are each the average of three
independent experiments.
Fig.2　 Effective time of Ruthenium Red inhibition
Ruthenium Red was added at different times after the initiation of
anoxia treatment of AG2 plants.Plants were harvested 12 h after the
onset of anoxia treatment and assayed for GUS activity.The data pr-
esented are each the average of three independent experiments.Er-
ror bars indicate standard deviations.
the first 2 h of anoxia is involved in the induction of the
expression of the ADH gene.
2.2　Effects of Ruthenium Red on the survival of
Arabidopsis plants under anoxia
To determine the maximal time of survival under an-
oxia , 20-day-old Arabidopsis plants were subjected to
4436期　　　　　　　　　　　　　Qi Wang 等:钙离子参与拟南芥的缺氧信号传导(英文)
anaerobic treatment.At different times after the onset of
anoxia-treatment , some Arabidopsis plants were removed
and returned to normal air for the observation
of the survival rates (see Materials and Methods for
details).There was no visible structural damage to pla-
nts at the time of the removal from anoxia-treatment.
However , in plants treated with more than 18 h of anox-
ia , there was complete chlorosis of leaves and 100% le-
thality 3 d later.In contrast , there was no apparent effect
of anoxia on the survival of plants if the anoxia-treatment
lasted for 12 h(Fig.3).These results indicate that time
that wild-type Arabidopsis could survive under anoxia is
between 12 and 18 h.For comparison , we also examined
the survival times of an adh1-2mutant , which showed no
detectable ADH activity in germinating seedlings.Few of
the seedlings survived 8 h of anoxia-treatment(Fig.3).
Fig.3　Survival of Arabidopsis under anoxia treatment
Arabidopsis plants were subjected to anoxia treatment.At different
times plants were removed and placed under normoxic conditions for
several days to observe necrotic symptoms and lethality.Several
hundred plants were observed each time.The treatments were AG2
under anoxia(★), AG2 under anoxia in the presence of Ruthenium
Red(○), and adh1-2 under anoxia(△).
　　Having observed that the adh1-2 mutant had much
shorter survival time than the wild-type under anoxia , we
expected that the addition of RR would shorten the surviv-
al time of the wild-type Arabidopsis.However , we found
that even in the presence of RR 50 μmol L , under which
anoxic induction of the expression of the ADH gene was
almost completely blocked , Arabidopsis plants could still
survive for more than 16 h under anaerobic condition
(Fig.3).These results suggest that anoxia-induced cell
death in Arabidopsis may be an active process and that
this process requires an increase in intracellular Ca
2+ ,
which is blocked by the presence of RR in the medium.
2.3　Occurrence of programmed cell death in leaf
cells after prolonged anoxia
In animal systems , two pathways of programmed cell
death have been reported(Raff 1998).One pathway in-
volves the occurrence of apoptosis , a form of cell death
that is an active process requiring the signaling through
cellular Ca
2+(Raff 1998).The second pathway does not
involve the occurrence of apoptosis.Numerous reports in-
dicate that hypoxia-induced cell death in animals may in-
volve apoptosis (Rosenbaum et al.1994 , Hill et al.
1995 , Sirimanne and Gluckman 1995).Our data showed
that cell death in Arabidopsis resulting from anoxia is an
active process and requires the involvement of Ca
2+.This
prompted us to investigate whether programmed cell death
would occur after 16 h of anoxia , a time which causes
Arabidopsis plants to die several days later even if they
are transferred to normal air.A series of morphological
and biochemical hallmarks have been used to characterize
apoptosis in animal cells(Raff 1998).We chose to ex-
amine whether nuclear condensation and chromosomal
DNA fragmentation(Tomei and Cope 1991 , 1994), two
hallmarks for the occurrence of apoptotic programmed cell
death , also occur in anoxia-induced cell death in leaves
of Arabidopsis.
To determine whether nuclear condensation has oc-
curred , DAPI staining was used to stain nuclei of leaf and
root cells of Arabidopsis plants subjected to and not sub-
jected to anoxia-treatment.In leaves , there was no de-
tectable difference in size between nuclei from control pla-
nts(Fig.4a)and those from plants subjected to 12 h of
anoxia(Fig.4c).An apparent nuclear condensation in
leaf cells of plants subjected 18 h of anoxia (Fig.4e),
but not in those of the control plants (Fig.4a).There
were no apparent differences in size of nuclei between root
cells from control plants(Fig.4b)and from plants under
12 or 18 h of anoxia(Fig.4d and f).
　　The nuclear TUNEL technique (Gorczyca et al.
1993), in which a terminal transferase and a UTP conju-
gated to a detectable marker are supplied in situ to assay
for an excess of free 3′-hydroxyl DNA ends , was used to
detect chromosomal DNA fragmentation.The results
showed that root cells from a plant subjected to 18 h of
anoxia were TUNEL negative (Fig.5d)when examined
under a fluorescent microscope , but leaf cells from the
same plant gave a positive response
(Fig .5 c ).Both leaf and root cells of the control
444 植物生理与分子生物学学报　　　　　　　　　　　　　　　　　　28卷
Fig.4 　DAPI staining of root and leaf cells under various conditions
The sources of nuclei stained are as follows:(a)leaf cells under normoxic conditions , (b)root cells under normoxic conditions , (c)leaf
cells from plants subjected to 12 h of anoxia , (d)root cells from plants subjected to 12 h of anoxia , (e)leaf cells from plants subjected to
18 h of anoxia and(f)root cells from plants subjected to 18 h of anoxia.All figures are ×135.
plants were TUNEL negative (Fig.5a and b).
3　Discussion
The results presented in Figures 2 and 3 suggest
that intracellular Ca
2+
levels may be involved in the sig-
naling pathways leading to the transcription activation of
the ADH gene in Arabidopsis during anoxia.Our results
also suggest that multiple Ca
2+
signaling steps might have
occurred during anoxia , since we found that the addition
of RR affected both the induction of the expression of the
ADH gene and the survival of Arabidopsis plants during
anoxia.
While the transcription of some glycolytic and fer-
mentative genes can be induced by oxygen deficiency , a
direct relationship between this induction and the cell
survival remains to be established(Drew 1997).In fact ,
4456期　　　　　　　　　　　　　Qi Wang 等:钙离子参与拟南芥的缺氧信号传导(英文)
hypoxic pretreatment improved the survival of root tip
cells during subsequent anoxia in both wild-type and
adh1
-
mutant maize plants.It has been postulated that
only a low amount of ADH enzyme that is transcribed
from a second ADH gene is required for this acclimation
(Johnson et al.1994).In Arabidopsis there is only one
copy of ADH gene.Our data showed that a null adh1-2
mutant has a much shorter survival time under anoxia
(Fig.3), which indicated that the presence of the ADH
enzyme is essential for the survival of Arabidopsis under an-
oxia.
Since we have observed that adh
-
mutants have
much shorter survival time than wild-type under anoxia
(Fig.3), we expected that the addition of RR would
shorten the survival time of Arabidopsis.Surprisingly ,
we found that even in the presence of RR 50 μmol L ,
under which anoxic induction of the ADH gene was com-
pletely blocked , wild-type Arabidopsis plants could still
survive for more than 16 h under anaerobic condition
(Fig.3).These results suggest that anoxia-induced cell
death in leaves of Arabidopsis may be an active process
and that this process requires an increase in intracellular
Ca
2+ , which was blocked by the presence of RR in the
medium.Subbaiah et al.(1994a)reported that the ad-
dition of RR substantially shortened the survival time of
maize seedlings under anoxia.This may be attributed to
the fact that mature Arabidopsis plants , rather than seed-
lings , were used in our experiments.In addition , it was
shown that RR could prevent cell death during aerenchy-
ma formation under hypoxia (He et al.1996).We are
currently investigating the functional role of Ca
2+
in the
anoxia-treated Arabidopsis seedlings.
Our data show that leaf cells , but not root cells ,
display apoptotic symptoms under prolonged anoxia
(Figs.4 and 5), whereas the entire Arabidopsis plant
dies after 18 h of anoxia (Fig.3).We and others have
shown previously that anoxic induction of the expression
of ADH and glycolytic genes mainly occurs in roots and
vascular tissues of Arabidopsis and several other plant
species ( Yang et al. 1993 , Dolferus
et al .1994 , Sachs et al .1996).Therefore , it is
Fig.5　In situ detection of chromosomal DNA fragmentation by the TUNEL method
TUNEL experiments were performed as described in the Materials and Methods.(a)Leaf cells from AG2 plants grown under normoxic condi-
tions.(b)Root cells from AG2 plants grown under normoxic conditions.(c)Leaf cells from AG2 plants subjected to 18 h of anoxia.(d)
Root cells from AG2 plants subjected to 18 h of anoxia.All figures are ×125.
446 植物生理与分子生物学学报　　　　　　　　　　　　　　　　　　28卷
possible that the root system is more tolerant to anoxia
than in the shoot system.In this view the shoot system
dies first under prolonged anoxia , while the roots die
only later.An alternative explanation is that different cell
death pathways are triggered in roots and leaves of Arabi-
dopsis.If this were correct , our finding that root cells
display no apoptotic symptoms under prolonged anoxia
does not necessarily mean that roots are more tolerant to
anoxia than shoots.The latter view is more consistent
with a report by Ellis et al.(1999), in which the root
survival was scored as the ability to initiate new growth
from the anoxia-treated root , while the shoot survival was
scored as the ability to produce new leaves from the an-
oxia-treated shoot meristem.Their data showed that in
Arabidopsis the shoot system is more tolerant to anoxia
than is the root system.However , it has to be pointed
out that the growth conditions used by us were different
from those used by Ellis et al., in which plants were
grown on agar plates.Chung and Ferl(1999)showed
that the induction of the expression of the Arabidopsis
ADH gene in both shoots and roots is conditioned by root
growth environment.
The occurrence of programmed cell death(PCD)in
plants is well documented(He et al.1996 , Ryerson and
Heath 1996 ,Wang et al.1996 , Lam et al.1999).The
PCD can be induced either by pathogens or developmen-
tal signals (Ryerson and Heath 1996 , Wang et al.
1996).A recent report indicated that PCD also occurred
during aerenchyma formation in maize root cells treated
with hypoxia(Arunika et al.2001).However , the se-
quence of events during PCD and the resulting morpho-
logical changes of cells may be different among different
PCD processes.Among PCD systems in plants , patho-
gen-induced hypersensitive response (HR)is the most
well studied (Pennell and Lamb 1997 , Lam et al.
1999).It was proposed that the inhibition of mitochon-
drial electron transport chain and the resulting increases
in cellular reactive oxygen species(ROS)might be re-
sponsible for the occurrence of PCD in the localized le-
sions in HR(Lam et al.1999).Since the TCA cycle
and electron transport chain are known to be inhibited by
oxygen deficiency(Drew 1997), it is conceivable that an
increase in ROS may also trigger PCD in leaf cells under
prolonged anoxic stress (Semenza 1999).We are cur-
rently investigating how anoxia affects the levels of ROS
in both root and leaf cells in Arabidopsis.
Acknowledgement:We thank Hsiao-Ping Peng for assistance in
GUS and ADH enzymatic assay s.
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216
钙离子参与拟南芥的缺氧信号传导
Qi Wang
＊　Richard Sjölund　Ming-Che Shih＊＊
(Department of Biological Sciences , Universi ty of Iowa , Iowa City , Iowa 52242, USA)
摘要:报告了钙流通抑制剂钌红对缺氧条件下拟南
芥中 ADH 基因表达的诱导和植株存活的影响。结
果表明 ,缺氧早期 ADH 基因的激活和表达需要钙离
子 ,钌红处理可以延长缺氧条件下拟南芥植株的存
活期。据此推测:拟南芥中缺氧诱导的细胞死亡是
一个钙离子介导的主动过程 ,钌红通过阻止细胞内
钙离子浓度的增加而抑制这一过程。延长缺氧处
理的时间会导致拟南芥叶片细胞内发生核凝聚
和染色体断裂的现象 ,也进一步验证了这种构想 。
表明缺氧处理引起的叶片细胞损伤直至植株死亡
是一个程序化的过程 。
关键词:拟南芥 , 缺氧处理 , 乙醇脱氢酶 , 钙信号传导
中图分类号:Q945
USDA NRICP基金 99-00647和 00-00665资助。
＊现地址:Monsanto Company , 800 N.Lindbergh Blvd , St.Louis , MO
63167, USA。
＊＊通讯作者(Tel:1-319-335-2071 , Fax:1-319-335-3620 , E-mail:ming-che-
shih@uiowa.edu)。
448 植物生理与分子生物学学报 , Journal of Plant Physiology and Molecular Biology　2002 , 28(6):441-448