全 文 ：植　物　学　报　 1995, 37( 11): 833～ 841
Acta Bot anica Sinica
阳离子诱导大叶藻叶绿体膜中激发能在
PSⅡ和 PSⅠ之间分配变化的机理*
1高振泮　 2马　红　 1娄清香　 2翟小京
2马桂芝　 2毛大璋　 2李良璧
1(中国科学院海洋研究所 ,青岛 266071)　　　　　 2(中国科学院植物研究所 ,北京 100044)
摘　　要
用 Ca2+和胰酶处理大叶藻 ( Zostera marina )叶绿体膜研究了其类囊体膜多肽成分与
Mg2+ 诱导其 Chl a荧光和类囊体膜表面电荷变化之间的相互关系 ,观察到: 1.在正常的叶绿
体膜中 , Mg2+诱导 PSⅡ荧光强度的增高与其诱导类囊体膜表面电荷密度的降低密切相关 ;
2.用 Ca2+处理这种叶绿体膜 ,除去类囊体膜表面的 32～ 34 kD多肽对 Mg2+ 诱导的上述现象
无影响 ; 3.如果用胰酶消化 Ca2+ 处理过的叶绿体膜 ,进一步除去膜表面的 26 kD多肽 , Mg2+
诱导的这些现象则全部消失。这些实验结果清楚地表明 ,在大叶藻的叶绿体膜中 ,类囊体膜表
面的 26 kD多肽是阳离子诱导这两种相关现象的特异性作用部位。 对阳离子调节激发能在
PSⅡ和 PSⅠ 之间分配的机理进行了讨论。
关键词　叶绿体 ;光系统 ;激发能分配⒇
MECHANISM OF CHANGE IN THE CATION-INDUCED EXCITA-
TION ENERGY DISTRIBUTION BETWEEN PSⅡ AND PSⅠ IN
THE CHLOROPLASTMEMBRANE FROM ZOSTERA MARINA
*
1
Gao Zhen-pan,
2
Ma Hong ,
1
Lou Qing-xiang ,
2
Zhai Xiao-jing ,
2Ma Gui-zhi, 2Mao Da-zhang and 2 Li Liang-bi
1 ( Institute of Oceanology , Academia Sinica , Qingdao 266071)
2( Inst itute of Botany, Academia Sinica, Bei jing 100044)
Abstract
The interrelations between thylakoid polypeptide components and Mg
2+ -induced Chl a
fluorescence and thylakoid surface charge changes w ere investig ated in Zostera marina
chloroplasts treated with Ca
2+
and trypsin. It w as observed that: 1. The increase of Mg
2+
-
induced PSⅡ fluorescence intensi ty w as closely related to the decrease of Mg2+ -induced sur-
⒇　 Receiv ed: 1995-02-21　 Revised: 1995-04-19
　　 Abbreviations: LHCⅡ . Light-h arves ting Chl a /b-protein serving ph otosystemⅡ ; SDS-PAGE. Sodium dodecyl
sulphate-polyacrylamide gel elect rophoresis.
　　We wish to extend ou r herty thanks to Zhang Qun, Feng Li-jie and Lu li-na for thei r technical assi stance.
　* Project supported by the National Natural Science Foundation of China and partly supported by EM SL.
face cha rge density of the thy lakoid membrane in the normal chloroplast; 2. Removal of the
32～ 34 kD polypeptides of the thylakoid surface by Ca2+ ext raction of the chloroplast did no t
af fect the M g2+ -induced phenomena; 3. If the Ca2+ -treated chloroplast w as further digested
by trypsin to remove the 26 kD polypeptide of the membrane surface, the Mg
2+ -induced
phenomena disappeared completely. These results clearly indicated that the 26 kD polypep-
tide of thylakoid surface is the specific acting si te of the cation that induced these two corre-
lated phenomena in the chloroplast from Zostera marina. The mechanism on the regulating
ef fect of the cation on excitation energy distribution betw een PSⅡ and PSⅠ was discussed.
Key words　 Chlo roplast; Photosystem; Exci tation energy distribution
IN TRODUCTION
It is generally agreed that the efficiency of primary photosynthetic processes of ligh t
conversion depends on the dist ribution of the absorbed excitation energ y between PSⅡ and
PSⅠ in terrestrial seed plants. The excitation energy distribution betw een these tw o photo-
sy stems may be regulated by div alent cations[1 ] and monito red by measuring changes in Chl a
fluorescence intensi ty both a t room temperature and liquid N2 temprature
[2 ] . M any re-
searchers
[ 3～ 6]
have demonst rated that the regulating effect of cations on the excitation energ y
distribution is associated w ith LHCⅡ and controlled by the electrostatic property of thy-
lakoid surface. However, the pa rticular molecula r mechanism on the interactions betw een
cation and LHCⅡ is still not w ell explained. The investigations concerning this regulation in
marine plants are deficient y et.
Trypsin has been widely used as a mild chemical modifier in studying the relationship
betw een the st ructure and function of the pro teins on thy lakoid surface[7～ 9 ] . Using t rypsin
as a probe, w e have recently observ ed that the co rrelativ e effect of Mg
2+ -induced Chl a fluo-
rescence and thylakoid surface charge changes is closely related to the 32 kD and 23～ 25 kD
polypeptides on the thylakoid surface in spinach chloroplasts (data unpublished) . Since the
32 kD and 23～ 25 kD polypeptides are the protein components of PSⅡ and LHCⅡ in the
chloroplast, respectiv ely[4, 10 ] and LHCⅡ plays a key role in the cation-induced exci tation en-
ergy dist ribution change
[3, 4 ] , the t rue acting sites of the ca tion which induced f luorescence
and surface charg e changes need to be confirmed. For this purpose, w e ex t racted the chloro-
plast f rom Zostera marina with Ca
2+
to remove 32 kD polypeptide of the thylakoid surface,
and then measured the ef fects of t rypsin on thy lakoid polypeptide components, Mg2+ -in-
duced Chl a f luo rescence and surface charge changes in the Ca
2+ -treated chloroplast.
1　 MATERIALS AND METHODS
1. 1　 Plant materials
Zostera marina was collected f rom Huiquan Wan at Qingdao, Tsing tao , China.
1. 2　 Preparation of chloroplasts and incubation of chloroplasts with Mg2+
According to the methods of Li et al
[9 ] . The normal chloroplast unt reated w ith Mg
2+
was known as CK-chloroplast in the tex t.
1. 3　Extraction of chloroplasts with Ca2+
40 mL of 1 mol /L CaCl2 resoluted in 0. 4 mol /L sucrose-0. 01 mol /L NaCl -0. 05
mol /L t ricine buffer ( pH 7. 8) w as added to the chloroplasts equivalent to about 10 mg Chl
834 植　　物　　学　　报　　　　　　　　　　　　　　 37卷
and ex t racted fo r 90 min on ice in the dark. The mix ture w as centrifug ed at 3000×g fo r 10
min and the precipi tates w ere washed three times wi th the above t ricine buffer ( pH 7. 8) to
remove the residual Ca2+ . The final pallets w ere suspended in the same tricine buffer to the
required concentration for the following assay of t rypsin digestion. The chloroplast was
known as Ca-chloroplast in the tex t.
1. 4　 Digestion of the chloroplasts with trypsin
Ca-chlo roplast w as dig ested w ith 0. 5 g t rypsin /g Chl fo r 5 min on ice in the dark. The
reaction was terminated by adding a 10-fold excess trypsin inhibi tor ( soybean) . The reac-
tion mix ture w as centrifug ed at 3000× g for 10 min, and the precipitates w ere w ashed three
times with the above tricine buffer ( pH 7. 8) to remove the residual trypsin and trypsin in-
hibi tor. The final sediments w ere suspended in the same tricine buf fer to the required con-
centrations for measurements of Chl a fluo rescence intensity and elect ric charge densi ty of
the thylakoid surface. The chlo roplast suspension w as kept in the dark for 30 min at 0℃
before use. This chlo roplast was known as Ca-T-chlo roplast in the tex t.
1. 5　 Measurements of Chl a fluorescence emission spectra at 77 K and electric charge
density of the thylakoid surface, as well as the SDS-PAGE analysis of thylakoid polypep-
tide components
According to the methods of Li et al [11 ] .
1. 6　Measurement of Chl concentration
According to the method of Arnon
[12 ] .
2　 RESU LT S
2. 1　 Effect of trypsin on changes of Mg2+ -induced Chl a fluorescence
Fig. 1 show s the Chl a fluorescence emission spect ra of CK-, Ca-and Ca-T-chloroplast
and the ef fects of Mg
2+
on these spectra. The results indicated that these spectra presented
three peaks at 686 nm, 694 nm and 717 nm, which belong to LHCⅡ , PSⅡ antenna Chl-
protein complex ( Chl aⅡ ) and PSⅠ antenna Chl-protein complex ( Chl aⅠ ) respectiv e-
ly[2 ] . The spect rum characteristics are similar to those of the chloroplasts f rom terrest rial
seed plants, ex cept that the w aveleng th of the f luo rescence emission peak from Chl aⅠ is
shif ted 13 nm to the blue region
[2 ] . The blue shi ft of wavelength may relate to a higher Chl
b content in Zostera marina chlo roplast ( Table 1) .
Table 1　 Chl a /b ratios of the chloropl asts f rom Zostera marina
Chloroplasts Chl a /b ratios
CK-chloroplast 2. 33± 0. 02
Ca-ch loroplast 2. 31± 0. 02
Ca-T-chloroplast 2. 30± 0. 02
On the other hand, f rom the data shown in Fig. 1 w e can also see that incubated CK-
chloroplast w ith 0. 25 mmol /L M gCl2 at 25℃ fo r 2 min significantly increased the f luores-
cence emission intensity a t 686 nm ( F686 ) , 694 nm ( F694 ) and 717 nm ( F717 ) in the spec-
trum ( Fig. 1A) . The Mg
2+
stimulating effects w ere essentially kept unchanged in the emis-
sion spect rum from Ca-chlo roplast, indicating that the pret reatment of the chloroplast w ith
CaCl2 did not af fect the Mg
2+
inductivi ty ( Fig. 1B) . How ever, w hen the Ca
2+
-treated
chloroplast was digested by t rypsin, the Mg
2+
inductivity completely disappeared ( Fig. 1C) .
83511期　 高振泮等: 阳离子诱导大叶藻叶绿体膜中激发能在 PSⅡ和 PSⅠ 之间分配变化的机理
It is generally ag reed tha t in the chloroplasts from terrestrial seed plants the F685 is emit ted
by LHCⅡ , and LHCⅡ plays a key role in the cation-induced excitation energy dist ribution
betw een tw o photosystems
[3, 4 ] . Therefore, on the basis of the purpose for this investig ation
it is possible to use the F686 as an index for the expression of the t rypsin inhibi tory effect on
Mg
2+
-induced fluo rescence changes.
Fig. 1　 Effects of Mg2+ on Ch l a f luorescence emission spect ra of the chloroplast s f rom Zostera marina
A. CK-ch loroplas t; B. Ca-chloroplas t; C. Ca-T-ch loroplas t; —— . - Mg2+ ; - - - .+ Mg2+ ( 0. 25 mmol /L)
Fig. 2 show s the effects of Mg
2+
at different concentrations on the F686 f rom CK-, Ca-
and Ca-T-chloroplast. The results indicated that in the concentration range used in this ex-
periment Mg2+ obviously stimulated the F686 f rom CK-and Ca-chloroplast. The stimulating
ef fect enhanced as Mg
2+
concentration w as increased and achieved the maximum at 2. 5
mmol /L MgCl2 , but the maximum stimulating rates differed f rom each o ther. The percent-
age of the maximum stimulation was about 57% for CK-chloroplast and about 52% fo r Ca-
836 植　　物　　学　　报　　　　　　　　　　　　　　 37卷
chloroplast, i. e. the fo rmer was higher than the lat ter by 5% ( Fig. 2, Curv es 1 and 2) .
The slight dif ference may be due to Ca
2+
-in-
duced conformational change of the chloro-
plast membrane in the ex t raction process
with CaCl2 . Since the characteristics of
Curve 1 and Curve 2 resemble each other,
and these two chloroplasts have similar Chl
a /b ratio ( Table 1) , it is reasonable to pre-
dict that the o rganization of the change in
cation-induced exci tation energy distribution
is intact ly kept in Ca-chloroplast. In contrast
to this, under the same condi tions the Mg
2+
stimulating ef fects on the F686 completely dis-
appeared in Ca-T-chlo roplast ( Fig. 2, Curve
3) . Because the Chl a /b ratio of the chloro-
plast remains essentially unchanged before
and af ter trypsin digestion ( Table 1) , it i s
likely that t rypsin-induced elimination of
Fig. 2　 Stimulative ef fect s of Mg2+ at dif ferent concent ra-
tions on F686 f rom the chloroplast s of Zostera marina
Curv es 1, 2 and 3 represen t CK-, Ca- and
Ca-T-chloroplast respectiv ely
Mg2+ stimulating ef fect on the F686 only relates to trypsin-induced change of the functional
proteins in the organization of the cation regulating exci tation energ y dist ribution betw een
tw o pho tosystems. Therefore, we have attempted to analyse thylakoid polypeptide compo-
nents of the above three chlo roplasts.
2. 2　 Electrophoretic analysis of trypsin-induced polypeptide component changes of
chloroplast membranes
The separation and identification of thylakoid polypeptide components f rom three types
of chlo roplasts of Zostera marina were perfo rmed wi th SDS-PAGE technique. The experi-
mental results are show n in Fig. 3. Data in the figure show that under the condi tions in this
experiment there w ere 21 and 19 polypeptide bands on the gels of CK- and Ca-chloroplast
membranes respectiv ely, the latter lacked bands 12 and 13 ( Compare Fig. 3A and 3B) ,
whi le there were only 18 polypeptide bands on the gel of Ca-T-chloroplast membrane, lack-
ing bands 12, 13 and 17 ( Compare Fig. 3A and 3C) . The measurements of molecular
weight show ed that these th ree bands represented 34 kD, 32 kD and 26 kD polypeptide re-
spectively. These results are consistent w ith those observed from our previous experiment
using t rypsin digestion of the chloroplast f rom spinach ( data unpublished) .
Since the 32～ 34 kD and 26 kD polypeptides are respectiv ely the main components of
PSⅡ and LHCⅡ in thylakoids of terrestrial seed plants[4, 10 ] , and Ca2+ pret reatment of CK-
chloroplast for removing the 32 kD and 34 kD polypeptides of thylakoid does not remarkably
influence M g
2+ -induced fluorescence changes ( See Fig. 2, Curve 2 and Fig. 3B) , the above
results clearly show that t rypsin-induced elimination of M g
2+
stimulating f luorescence effect
only rela tes to the t rypsin-induced loss of the 26 kD polypeptide of LHCⅡ . In other wo rds,
the 26 kD polypeptide of LHCⅡ is the specific acting si te of Mg2+ which regulates exci tation
energy dist ribution betw een PSⅡ and PSⅠ .
83711期　 高振泮等: 阳离子诱导大叶藻叶绿体膜中激发能在 PSⅡ和 PSⅠ 之间分配变化的机理
Fig. 3　 Densi tometric t racings of th ylakoid polypeptides of Zostera marina f ractionated on SDS-PAGE
A. CK-chloroplas t; B. Ca-chloroplas t; C. Ca-T-ch loroplas t
2. 3　 Effect of trypsin on changes of Mg2+ -induced electric charge density on thylakoid
surf ace
According to a previous report
[6 ]
, the isoelectric point of the chlo roplast membranes in
terrest rial seed plants is pH 4. 3～ 4. 5. At phy siological pH the outer surface of the thy-
lakoid membrane of chloroplasts carries an excess of net negative charg e. So the particles
move tow ard the positiv e pole under the influence of an ex ternal electric field. The elec-
trophoretic v elocity depends on the elect ric charg e density of the thylakoid surface. Barber
[ 6]
suggested that M g
2+
-induced fluo rescence yield change is controlled by the surface electro-
static property of thylakoid membranes. It is necessary, therefore, to determine the effects
of t rypsin digestion on changes of Mg
2+
-induced surface charge of the three chloroplast
membranes f rom Zostera marina.
Fig. 4 and Fig. 5 show the effects of Mg
2+
on the elect rophoretic velosity of CK-, Ca-
and Ca-T-chloroplast. The results indicated that the elect ropho retic mobility of the three
kinds of chloroplast membranes was similar w ithout MgCl2 ( Fig. 4) . Mg
2+
remarkably in-
hibi ted the elect ropho retic mobilities of CK- and Ca-chlo roplast in the presence of MgCl2
( Fig. 4A and 4B) . The inhibitory effect w as increased as Mg
2+
concentration increased in
the range f rom 0 to 2. 5 mmol /L. Both the maximum inhibi tory rate ( about 48% ) and
Mg
2+
concentration required for it ( about 2. 5 mmol /L) w ere qui te similar in these tw o
chloroplast membranes ( Fig. 5, Curves 1 and 2) . In contrast to this, under the same condi-
tions the elect rophoretic mobility of Ca-T-chloroplast w as not o r sligh tly af fected by Mg2+
( Fig. 4C and Fig. 5, Curve 3) . Although the treatment of Mg
2+
at the highest concentra-
tion used in this experiment ( 5 mmol /L ) resulted in a significant decrease of the elec-
trophoretic v elosity in these three chloroplasts ( Fig. 5) , the inhibition w as not related to
Mg2+ -induced fluorescence changes ( See Fig. 2) . It can be regarded as the consequence of
the elect rostatic screening effect of the cation on the net negative charges of the thylakoid
surface[ 6] . Therefore , comparing these experimental results with those of Fig . 2 , w ecan see
838 植　　物　　学　　报　　　　　　　　　　　　　　 37卷
Fig. 4　 Effects of Mg2+ on the elect rophoretic veloci ty of
Zostera marina chloroplasts under an ex ternal elect ric field
A. CK-chloroplast; B. Ca-chloroplast;
C. Ca-T-ch loroplast; □ - Mg2+ ;
□∥ + Mg2+ ( 0. 25 mmol /L)
Fig. 5　 Inhibitory ef fect s of Mg2+ at dif ferent concent ra-
t ions on th e elect roph oretic velosi ty of
Zostera marina chloroplasts
Curv es 1, 2 and 3 represen t CK-, Ca- and
Ca-T-chloroplast respectiv ely
that the increase of Mg
2+ -induced f luorescence intensi ty w as closely related to the decrease
of Mg
2+
-induced surface charg e density of the thylakoid membrane in the chloroplast f rom
Zostera marina, and the elimination of Mg2+ stimulating ef fect on F686 caused by trypsin di-
gestion is associated with the surface charge of the thylakoid, i. e. the cation regulating ef-
fect on excitation energy dist ribution betw een the tw o photosystems is controlled by the sur-
face electrostatic property of the thylakoid membrane.
3　 DISCUSSION
Murata
[1 ]
assumed that the negative correlation between change in M g
2+ -induced PSⅡ
fluorescence intensity and PSⅠ f luorescence intensi ty w as due to the cation-induced in-
hibi tory effect on exci tation energ y transfer f rom PSⅡ to PSⅠ in the chlo roplasts of terres-
trial seed plants. The hypothesis of energy spillover is now widely accepted. Furthermo re, i t
has been suggested that the energy spillover ef fect is associated wi th Mg
2+
-induced st ruc-
tural o r confo rmational changes of thylakoid membranes[2, 13 ] and controlled by the electro-
static property of the membrane surface
[5, 6 ] , w hile LHCⅡ plays a key role in this pro-
cess[3, 4 ] . But the acting sites of the cation on LHCⅡ still remain unclear. The experimental
results in this w ork clearly show that in the normal chloroplast f rom Zostera marina, the in-
crease of Mg
2+
-induced PSⅡ fluo rescence intensi ty is closely related to the decrease of
Mg
2+ -induced surface charge density of the thylakoid membrane. These tw o Mg
2+ -induced
correlative effects are intactly kept in the chloroplast pretreated with Ca
2+
to remove the 32
～ 34 kD polypeptides of the thylakoid surface. And when the dig estion of the Ca2+ -treated
chloroplast w ith trypsin further removes the 26 kD polypeptide of the thy lakoid surface,
these two Mg
2+ -induced correlative ef fects are completely elimina ted ( Compare Fig. 2, Fig.
3 and Fig. 5) . Because the pigment compositions of the chlo roplast remain essentially un-
changed before and af ter the t rypsin dig estion ( Table 1) and the 26 kD polypeptide of the
thylakoid surface is a main component of LHCⅡ [ 4, 10] , these results not only demonst rate
83911期　 高振泮等: 阳离子诱导大叶藻叶绿体膜中激发能在 PSⅡ和 PSⅠ 之间分配变化的机理
that 26 kD polypeptide of LHCⅡ is the specific function site of cation that induced the above
tw o correlativ e effects, but also provide a st rong ev idence fo r Barber s hypothesis[ 6] that
postulated the interactions betw een cation and LHCⅡ , w hich induced elect rostatic property
changes of thylakoid membranes leading to the st ructural or conformational alterations of the
membranes, may be a main reason of the Mg
2+ -induced Chl a fluorescence changes.
How ever, the interaction betw een ca tion and LHCⅡ resulting in electrostatic property
changes of the thylakoid surface can be achiev ed by alternative ways. One is by cation-in-
duced elect rostatic screening and the other is by cation-induced charg e neutralization. Bar-
ber
[6 ]
ag reed to the fo rmer w ay. He suggested that the elect rosta tic screening effect of Mg
2+
on the thylakoid surface is the true reason for the cation-induced Chl a fluorescence changes.
The charge neutralization only induces the thy lakoid restacking w hile there is no associated
large increase in Chl a f luorescence. But the experimental results in this wo rk support the
lat ter w ay. In comparison with Ca-chloroplast , it could be seen that although the outer sur-
face of the thylakoid membranes f rom Ca-T-chloroplast carries a similar amount of net nega-
tiv e charg e, they are not affected by M g
2+
( Fig. 4 and Fig. 5) . Since Ca-T-chlo roplast is on-
ly devoid of the 26 kD polypeptide component of LHCⅡ ( Fig. 3) , these results show that i t
is a true reason for the electrostatic property alteration on the membrane surface that the
cation specifically binds with the carrying elect ric g roups on 26 kD polypeptide of LHCⅡ to
neutrali ze the net negativ e charg e on the thy lakoid surface. Therefo re, based on the results
in this study and the following experimental facts: 1) 26 kD polypeptide of LHCⅡ is located
on the outer surface of thylakoid membranes
[10 ] ; 2) the net negativ e charg es are derived
from the carboxyl g roups associated wi th acidic amino acid residues of the proteins on the
surface of thylakoid membranes
[6 ]
and 3) the carboxyl group is the cation-sensi tiv e binding
site[14 ] ; our conclusion is that the speci fic binding of Mg2+ w ith the carboxyl g roup of LHC
Ⅱ 26 kD polypeptide of thylakoid surface, w hich neutrali zes net negativ e charg e of the
membrane surface to result in the st ructural or conformational alterations, may be the main
reason for the cation-induced changes in excita tion energy distribution betw een the two pho-
tosystems.
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84111期　 高振泮等: 阳离子诱导大叶藻叶绿体膜中激发能在 PSⅡ和 PSⅠ 之间分配变化的机理