全 文 :Received 2002-08-30 , Accepted 2002-12-31.
Project supported by NSFC(39970077).
*Corresponding author(E-mail:wangyong_nkdx@eyou.com).
Significant Role of Cytokinins in Maintaining the Life of Fronds in
Spirodela polyrrhiza
LI Wei-Min , LIU Qing-Dai , XIONG Yan , WANG Shu-Fang , WANG Ning-Ning , WANG Yong*
(College of Life Sciences , Nankai University , Tianjin 300071)
Abstract:Spirodela polyrrhiza P143 half-fronds without
roots and pockets were cultivated on liquid medium under
long-day conditions with or without 6-benzyladenine(6-
BA).After 4 d of cultivation on a medium without 6-BA
the half-fronds began to lose their chlorophyll and de-
crease in photosynthetic activity , and on the 12th d of
cultivation these half-fronds were almost dead.But when
cultivated on the medium to which 6-BA was added half-
fronds increased significantly their dry weight , chlorophyll
and soluble protein contents , capacity of Hill reaction and
the levels of the ribulose-1 , 5-bisphosphate carboxylase/
oxygenase small subunit and the rbcS mRNA encoding it.
The 6-BA-treated half-fronds had a mean longevity of 80
d , almost the same as that of the intact fronds.There-
fore , it is proposed that cytokinins are necessary for main-
taining the activities of duckweed fronds.
Key words:cytokinin;Spirodela polyrrhiza;senescence;longevity
Excised leaves or leaf discs have been used exten-
sively to study the effect of cytokinins on leaf senescence
(Richmond and Lang 1957 , Mothes et al.1959 , Thi-
mann and Laloraya 1960 , Parthier and Wollgiehn 1961 ,
Osborne 1962 , Sugiura et al.1962 , Gunning and
Barkley 1963 , Srivastava and Ware 1964 , Von Abrams
and Pratt 1968 , Tavares and Kende 1970).In these ex-
periments the senescence process was usually delayed by
cytokinin treatment , but not prevented.For example , the
green color or protein level of detached leaf parts has been
reported to decrease after cytokinin treatment , only at a
less significant rate than the control not treated with cy-
tokinin(Richmond and Lang 1957 ,Sugiura et al.1962 ,
Gunning and Barkley 1963 , Srivastava and Ware 1964 ,
Von Abrams and Pratt 1968).There were also reports
that senescence of the leaves of a few species was not de-
layed by the application of cytokinins , but was delayed by
the addition of other hormones (Noodén and Leopold
1978).It has also been reported that in attached leaves
of lemon (Ilan and Goren 1979), white lupine (Davey
andVan Staden 1978)and Sitka spruce (Lorenzi et al.
1975)the endogenous cytokinin activity remained high
during senescence.It seems , therefore , that the shortage
of cytokinin in leaves is not the only or most significant
factor that initiates the senescence process.Cytokinins
may play a significant role in maintaining the living state
of leaves , and leaves themselves have normally enough
endogenous cytokinin.Only when leaves are in serious
shortage of endogenous cytokinins , the senescence process
will be initiated.
Duckweeds are small water plants floating on or be-
low the surface of lakes and other standing waters.They
are among the simplest flowering plants , consisting of a
leaf-like structure called frond , with or without roots.At
the base of each frond there are two meristematic pockets ,
one on each side of the frond midvein.On the ventral
surface of the pockets meristematic cells are located , from
which daughter fronds and roots originate.Daughter
fronds will separate from their parents after maturation and
new daughter fronds will be produced.Thus , within a
short period , a large population can be produced from a
single mother frond through vegetative budding (Landolt
1986).Because of their smallness , rapid vegetative mul-
tiplication and growth on liquid medium duckweeds have
often been used as one of the model plants to study plant
physiological problems , when aseptically cultivated like
microorganisms within laboratory under controlled condi-
tions (Landolt and Kandeler 1987).Duckweeds have
been shown to take up cytokinins easily through the lower
surface of the frond from the surrounding medium (Lan-
dolt and Kandeler 1987).Therefore , we hoped that ex-
cised half-fronds could be a better system for investigating
the role of cytokinins played in the activity , senescence
and longevity of the leaf or leaf-like structures.
215Journal of Plant Physiology and Molecular Biology 2003 , 29(3):215-220
1 Materials and Methods
1.1 Aseptic culture of duckweed plants and half-
fronds
Spirodela polyrrhiza strain P143 plants were grown
aseptically on a modified DATKO medium in f lasks as de-
scribed previously (Wang and Kandeler 1994).As short-
day plants they were cultivated under long-day conditions
(16 h light and 8 h dark)with a light intensity of about
45μmol m-2 s-1 , to prevent flowering.The temperature
was 22-26℃during the light phase and 18-22℃dur-
ing the dark phase.Plants were transferred to fresh medi-
um every 7 d.
Uniformly mature fronds were cut into two parts and
the half-fronds without roots and pockets were grown for
investigation.These half-fronds were divided into two
groups , one of which was cultivated on the medium with
6-BA and the other on the medium without 6-BA.Experi-
mental materials were collected after 4 , 8 and 12 d.
1.2 Determination of dry weight , chlorophyll and
soluble protein contents
For dry weight determination , plant materials were
harvested , dried at 85℃ and weighed every 0.5 h to con-
stant weight.Chlorophyll was extracted from these sam-
ples with 96% ethanol.After centrifugation , the super-
natant was used to determine chlorophyll content accord-
ing to the method of Wintermans and De Mots (1965).
Soluble proteins were extracted from fresh tissue of half-
fronds with Tris-glycine 10 mmol/L , PMSF (phenyl-
methyl sulfonyl floride)1 mmol/L and 1% β-mercap-
toethanol , and quantified spectrophotometrically after be-
ing stained with Coomassie Brilliant Blue G250(Bradford
1976).Bovine serum albumin (BSA)was used as the
standard.
1.3 Hill reaction
Chloroplasts were isolated by using the following
procedure.Half-frond tissues were collected and ground
with a mortar and pestle in pre-cooled extraction buffer
containing Tris-HCl(pH 7.5)0.02 mol/L , sucrose 0.4
mol/L and KCl 0.03 mol/L.The mortar was kept on ice
during extraction.The extract was then centrifuged at 500
×g for 1 min.The supernatant was collected and cen-
trifuged at 3 000×g for 2min to pellet the chloroplasts.
The chloroplasts were subsequently suspended in the ex-
traction buffer.
Hill reaction activity was measured in terms of pho-
toreduction of 2 ,6-dichlorophenol indophenol(DCPIP)as
described by Holt and French(1948).The reaction mix-
ture contained 3 mL extraction buffer , 1.5 mL 0.003%
DCPIP dissolved in Tris-HCl 0.02 mol/L , and 0.5 mL
chloroplast suspension.The absorbance change of 600 nm
was recorded after 5 min of exposure to light with a inten-
sity of about 335 μmol m-2s-1.
1.4 SDS-PAGE
Standard operation procedure of sodium dodecylsul-
phate polyacrylamide gel electrophoresis (SDS-PAGE)
was carried out(Laemmli 1970).The stacking gel was
5% and the resolving gel was 12.5%.Protein bands
were visualized by staining with Coomassie Brilliant Blue
R250.
1.5 RNA blot analysis
Total RNA was extracted using the acid guanidinium
thiocyanate method as described before (Wang et al.
1999)and quantified spectrophotometrically.For North-
ern analysis , RNA samples were fractionated elec-
trophoretically in a 1.2% formaldehyde-agarose gel ,
transferred onto Nylon membrane (Hybond XL Amer-
sham)by capillary blotting in 10×SSC and hybridized
with [ 32P] -labeled cDNA probe generated by the random
primer method (Promega).Hybridization was done at
65℃ for 24 h in 1%BSA , EDTA 1 mmol/L , Na2HPO4
(pH 7.2)0.5 mol/L.The membrane was washed twice
with a solution of 1%BSA , EDTA 1 mmol/L , Na2HPO4
(pH 7.2)40 mmol/L and 5%SDS at 65℃ for 5 min
each , 3 times with a solution of EDTA 1 mmol/L ,
Na2HPO4(pH 7.2)40mmol/L and 1%SDS at 65℃ for
5 min each.The blot was then exposed to X-ray film at
-20℃with two intensifying screens for 2 d.
2 Results
2.1 Effect of 6-BA on the senescence and longevity
of half-fronds
We first investigated the concentration range of 6-BA
effective in preventing senescence and in keeping the bio-
logical activity of half-fronds.The beginning of and total
loss of green colorwere used respectively as the beginning
of senescence and death of half-fronds.As shown in Fig.
1 , both the effect of 6-BA on senescence and that on
216 植物生理与分子生物学学报 29卷
longevity were found to be concentration-dependent , in
the range tested from 10
-8
to 10
-5
mol/L.6-BA at a
concentration higher than 10-6 mol/L postponed the
senescence of half-fronds for more than 40 d and pro-
longed the longevity for more than 80 d.Therefore , 6-BA
10
-6
mol/L was used in the following experiments.
Fig.1 Effect of 6-BA concentration on the senescence and
longevity of half-fronds
Half-fronds without roots and pockets were cultivated on a medium
with different concentrations of 6-BA.The lengths of vertical lines
with caps represent the standard deviation(n=30).
2.2 Changes in dry weight , chlorophyll and soluble
protein contents
To investigate changes in dry weight and chlorophyll
content , two separate experiments were performed and
each treatment comprised 4 replicates of 100 half-fronds.
Changes in dry weight observed are shown in Fig.
2A.The dry weight of half-fronds increased significantly
within 8 d of cultivation on the medium either with or
without 6-BA.This is accounted for by the photosynthetic
products that accumulated , but did not get transported out
of the half-fronds.Then the half-fronds not treated with
6-BA stopped increasing in dry weight , but those treated
with 6-BA increased their dry weight further , and on the
12th d of cultivation the increase was about 200% of the
original value , which was the result of increased thickness
we have observed.
Changes in chlorophyll content are shown in Fig.
2B.The chlorophyll content in half-fronds cultivated on
the medium without 6-BA decreased gradually and
reached half of the original value after 8 d of cultivation.
In contrast , the chlorophyll content of half-fronds culti-
vated on the medium with 6-BA increased gradually and
Fig.2 Changes in dry weight(A), chlorophyll(B) and soluble
protein(C)content
Half-fronds were cultivated on a medium with or without 6-BA 10-6
mol/ L and harvested on day 0 , 4 , 8 and 12 , respectively.The
lengths of vertical lines with caps represent standard deviations (n
=8).
reached more than twice the original level after 8 d of cul-
tivation.This result agrees with the dark green color ob-
served for these half-fronds.
For soluble protein analysis , two separate experi-
ments were performed.The average from two experiments
is shown in Fig.2C.The soluble protein content in half-
fronds cultivated on medium without 6-BA changed little
during 12 d of cultivation , but those cultivated on the
mediumwith 6-BA increased their protein content by more
than 150%.This indicated that the 6-BA-treatment had
increased the biological activity of half-fronds.
2.3 Changes in the protein level of Rubisco small
subunit
The soluble proteins were separated by SDSPAGE
2173期 李伟敏等:细胞分裂素在维持紫萍叶状体生命中的重要作用(英文)
and stained with Coomassie Brilliant Blue to see if the
protein level of the small subunit(SSU)of ribulose-1 ,5-
bisphosphate carboxylase/oxygenase (Rubisco), the key
enzyme in CO2 fixation , changed during cultivation.
Fig.3 showed that 6-BA treatment of the half-fronds
caused a significant increase in the level of SSU , which
might imply an increase in photosynthetic activity.
Fig.3 Comparison of levels of Rubisco small subunit(SSU)sepa-
rated by SDS-PAGE and stained with Coomassie Brilliant Blue
An aliquot of extracted proteins representing the same number of
half-fronds was loaded in each lane.M:Molecular weight marker;
Lane 0:Before treatment;Lanes 1 , 3 and 5:Without 6-BA;Lanes
2 , 4 and 6:With 6-BA 10-6 mol/L.Half-fronds were cultivated
for 4 d(lanes 1 and 2), 8 d(lanes 3 and 4)and 12 d(lanes 5 and
6)before harvesting.
2.4 Changes in Hill reaction activity
During cultivation of half-fronds on the medium
without 6-BA the capacity of Hill reaction in chloroplasts
decreased.After 4 d of cultivation it dropped to about
60% and after 12 d to only about 10% of the original
value , which indicated dramatic loss of photosynthetic ac-
tivity (Fig.4).On the other hand , chloroplasts from
half-frond tissues kept on medium to which 6-BA 10-6
mol/L was added had higher photosynthetic activity.Af-
ter 12 d of cultivation , the capacity of Hill reaction in
these half-fronds was more than 2 times that before 6-BA-
treatment(Fig.4).
Fig.4 Changes in Hill reaction
Data are presented as percentage of the original capacity , which is
taken 100.
2.5 Changes in rbcS gene expression
The mRNA levels of the rbcS gene(s), encoding the
small subunit of Rubisco , were assayed to detect changes
in gene expression.Total RNA was extracted , separated
on an agarose gel and , after blotting to a nylon-mem-
brane , was probed with a cDNA fragment corresponding to
an rbcS gene of P143 plants cloned previously (Liu et
al.2002).
As shown in Fig.5 , within the first 8 d of cultiva-
tion , the rbcS mRNA level was higher in half-frond tis-
sues kept on medium either with or without 6-BA than in
those directly used for RNA extraction after cutting (lane
0).After 12 d of cultivation , however , it was almost un-
detectable in half-frond tissues grown on the medium
without 6-BA , but that cultivated on the medium with 6-
BA had still a very high rbcS mRNA level.
Fig.5 Expression of rbcS gene in half-frond tissues cultivated on
medium with(lanes 2 , 4 and 6)or without(lanes 1 , 3 and 5)6-
BA 10-6mol/ L
Lane 0:Before treatment;Lanes 1 and 2:Cultivated for 4 d;Lanes
3 and 4:Cultivated for 8 d;Lanes 5 and 6:Cultivated for 12 d.
An aliquot of 10μg of total RNA was loaded in each lane.A:Au-
toradiograph of the membrane probed with the32P-labeled rbcS cDNA
fragment.B:Photograph of the gel stained with ethidium bromide
used as a loading control.
3 Discussion
Cytokinins are a class of phytohormones.They are
known to be involved in diverse processes of plant growth
and development , such as cell division , shoot initiation ,
leaf and root differentiation , chloroplast biogenesis and
senescence(Davies 1995 , Mok andMok 2001).Howev-
er , in our knowledge , there has not been any report that
cytokinins are needed in maintaining the photosynthetic
activity or the living state of leaves.
Duckweeds are water plants and absorb nutrients
mainly with their lower surface of fronds (Landolt and
Kandeler 1987).Therefore , parts of mature fronds can be
cultivated on medium and under suitable growth condi-
tions to investigate the longevity of a frond and the influ-
ence of substances by adding them into the medium.Cut-
ting off roots of P143 strain plants did not affect the
218 植物生理与分子生物学学报 29卷
longevity of fronds(data not shown).Half-fronds without
roots and pockets did not initiate senescence within two
months when they were cultivated in the darkness(results
will be reported in another paper).However , they began
to senesce after being cultivated on the cytokinin-free
medium and under long-day conditions for 4 d (Fig.1).
Here the light intensity was only about 45μmol m-2s-1 ,
which was not extensive enough to cause any damage or
senescence of the intact plants.If 6-BA or another kind
of cytokinins(data not shown)was included in the medi-
um the half-fronds increased their biological activities ,
such as the photosynthesis , chlorophyll biosynthesis , pro-
tein synthesis , and the expression of the rbcS gene(Figs.
2-5).The longevity of these half-fronds was as long as
that of the intact plants (more than 80 d at 10-6 mol/L
6-BA).These results demonstrate that cytokinins play a
significant role inmaintaining the biological activity of the
fronds.When fronds are in a serious shortage of cy-
tokinins the senescence process of fronds will be initiated.
Half-fronds died eventually though they were culti-
vated on a medium containing 6-BA and refreshed every 7
d , and under suitable long-day and temperature condi-
tions(data not shown).Cultivating intact plants on medi-
um with 6-BA prolonged their longevity but did not pre-
vent their senescence(data not shown).These data show
that there must be another endogenous factor or factors
that initiate leaf(frond)senescence when the endogenous
cytokinins are still not in serious shortage.Therefore ,
shortage of endogenous cytokinins should not be associat-
ed with all cases of leaf senescence , such as lemon(Ilan
andGoren 1979), white lupine (Davey and Van Staden
1978)and Sitka spruce (Lorenzi et al.1975).
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细胞分裂素在维持紫萍叶状体生命中的重要作用
李伟敏 刘清岱 熊 延 王淑芳 王宁宁 王 勇*
(南开大学生命科学学院 , 天津 300071)
摘要:将无根和囊的紫萍(Spirodela polyrrhiza)P143
品系半叶状体分别放在含有和不含 6-苄基嘌呤的
培养液上于长日照条件下培养。在不含 6-BA的培
养液上培养4 d以后 ,半叶状体开始失绿 ,光合能力
降低;第 12天时 ,半叶状体已接近死亡 。但是 ,培养
在含有6-BA的培养液上的半叶状体干重 、叶绿素和
可溶性蛋白质含量 、希尔反应活力 、核酮糖-1 , 5-二
磷酸羧化酶/加氧酶的小亚基和其编码基因 rbcS
mRNA水平显著增加 。这些 6-BA 处理过的半叶状
体平均寿命可达 80 d ,几乎等同于完整植物体上叶
状体的寿命 ,因此提出细胞分裂素对于紫萍叶状体
生物学功能的维持是必需的。
关键词:细胞分裂素;紫萍;衰老;寿命
中图分类号:Q945 , Q946
*通讯作者(E-mail:wangyong_nkdx@eyou.com)。
220 植物生理与分子生物学学报 , Journal of Plant Physiology and Molecular Biology 2003 , 29(3):215-220