全 文 ：第27卷 第3期 作　物　学　报 V ol. 27, N o. 3
2001 年5月 A CTA A GRONOM ICA S IN ICA M ay, 2001
Role of Key Enzymes for Photosyn thesis in the D iurna l Changes of Pho-
tosyn thetic Ra te in R iceΞ
J IAN G D e2A n　LU Q ing　W EN G X iao2Yan　ZH EN G B ing2Song　X I H ai2Fu
(College of lif e S cience, Z hej iang U niversity , H uaj iach i, H angzhou 310029, China)
Abstract　　T he diurnal variations of net pho tosyn thetic rate (Pn) , activities of R ubisco and R u2
bisco activase (RCA ) asw ell as RCA and A T P con ten ts in flag leaves of two cultivarsw ere m ea2
sured to exp lo re the possible effect of R ubisco activase in diurnal changes of Pn and rubisco activa2
tion. T he results show ed that Pn , in itial R ubisco and RCA activities arose the typ ical pattern
w ith two peak s in the morn ing and afternoon, respectively, and a m idday dep ression at noon.
How ever, RCA and A T P con ten tsw ere m arkedly low in the morn ing than in the afternoon. R e2
gression analysis indicated that Pn w as very sign ifican tly co rrelative to in itial R ubisco activity dur2
ing the day, but on ly co rrelated w ith RCA activity in the morn ing upon Pn increasing and in the
afternoon upon Pn decreasing. It suggested that the m idday dep ression of Pn w as invo lved in los2
ing of the in itial R ubisco activity. RCA activity co rrelated w ith A T P con ten t on ly in early morn2
ing w hen A T P con ten t w as low er, but no t at noon and in late afternoon w hen A T P con ten t w as
h igher. But RCA con ten t increased slow ly w ith increasing A T P con ten t in early morn ing and did
fast at noon and in the afternoon, imp lying that in v ivo A T P m igh t be invo lved in no t on ly acti2
vation but also syn thesis of RCA oröand p ro tect the enzym e from degradation.
Key words　　R ice, Pho tosyn thetic rate, R ubisco, R ubisco activase, D iurnal change
R ubisco is a key enzym e fo r pho tosyn thesis and pho to resp iration. T he purified R ubisco re2
quires activation by h igh concen trations of CO 2 and M g2+ at h igh pH to attain its m ax im al activi2
ty. Its activation in v ivo at physio logical concen trations of CO 2 in ch lo rop last m ay be con tro lled
by: 1) ligh t2induced changes in strom alM g2+ and H + concen trations, 2) R ubisco activase[ 1～ 4 ].
T here are som e reports on the diurnal changes of R ubisco activities in soybean [ 5, 6 ] , sp inach and
sugarbeet[ 7 ]. W e have also repo rted that R ubisco activities and percen tage of activation w ere
m arkedly low in early morn ing and late afternoon [ 8, 9 ], and at that tim e Pn w as sign ifican tly posi2
tively related w ith the in itial R ubisco carboxylation activity in rice p lan t[ 9 ]. How ever, it is still
unknow n if the diurnal changes of Pn and R ubisco activity are regulated in v ivo by R ubisco acti2
vase (RCA ). T he diurnal variations of Pn , R ubisco and RCA activities, as w ell RCA and A T P
con ten ts in rice flag leaves w ere m easured in th is experim en t to investigate the in terrelation be2
tw een them and the ro le of RCA in v ivo.
1　M a ter ia ls andM ethods
1. 1　Plan t culture
R ice (O ry za sativa subsp. ind ica cvs. Zhenong966 and Zhenong 952) seedlings at a leafΞ This research w as supported by the N ational N atural Science Foundation (No. 39570432)
Received on: 1999212216, A ccep ted on: 2000204226

stage of 3～ 4 w ere transp lan ted on M ay 7, 1998, and m anaged as the p revious paper[ 10 ]. T he
experim en t w as conducted in the H uajiach i Campus, Zhejiang U niversity, H uangzhuo.
1. 2　M ea surem en t of photosyn thetic ra te (Pn)
M easurem en t of Pn outdoor w ith an infrared CO 2 analyzer (M odel 305) w as done on flag
leaves attached to the m ain stem s at the m ilk stage on July 7 and 8, 1998. Bo th daysw ere a fine
day. To illustrate clearly, the data in F igures show ed the results on July 7 because bo th days had
the sam e pattern.
1. 3　A ssay for activ ities of Rubisco carboxyla tion and Rubisco activa se
T he assay w as conducted w ith N aH 14CO 3 fo llow ing the m ethods of Robinson et al. [ 12 ] and
L an et al. [ 13 ] A fter Pn w asm easured, 0. 1g of the leaf w as ground in a mortar and pestle w ith liq2
uid N 2 p lus a little quartz sand and 1% inso luble PV P (po lyvinylpo lypyrro lidone) to pow der, and
then homogen ized w ith 1. 9 mL of the coo led ex traction buffer con tain ing BT P (B is2T ris2
p ropane) 10 mmolöL (pH 7. 0) , ED TA 1 mmolöL , M gC l25 mmolöL , A T P 0. 4 mmolöL , D T T
15 mmolöL , phenylm ethysulfonyl fluo ride 1 mmolöL , benzam idine 2 mmolöL , leupep tin 0. 01
mmolöL , 0. 1% Β2m ercap thano l. T he homogenate w as cen trifuged at 15000×g fo r 10 m in. T he
supernatan t as a crude ex tract of R ubisco and R ubisco activase w as imm ediately used to assay ac2
tivities of the enzym es.
T he m easurem en t fo r in itial R ubisco carboxylation activity began as soon as 50 ΛL of the ex2
tract w as m ixed w ith 450 ΛL reaction m edium finally consisting of H EPES2KOH 50 mmolöL
(pH 8. 0) , ED TA 1 mmolöL , M gC l2 20 mmolöL , D T T 2. 5 mmolöL , N aHCO 3 (con tain ing
1. 665×106 Bq of N aH 14CO 3 fo r each samp le) 10 mmolöL , R uBP 0. 6 mmolöL. T he reaction
w as term inated w ith 100 ΛL of HC l 2 molöL after reaction 30s. T he in itial R ubisco carboxylation
activity w as calculated on basis of 14C2fixed.
T he m easurem en t fo r the to tal R ubisco activity w as conducted by fo llow ing p rocedure: that
first, 50 ΛL of the enzym e ex tract w as activated in 75 ΛL of the activation m edium (H EPES 50
mmolöL (pH 8. 0) , ED TA 0. 67 mmolöL , M gC l233 mmolöL and N aHCO 310 mmolöL ) at 30℃
fo r 10 m in, and second, the activated enzym e w as m ixed w ith reaction m edium (H EPES2KOH
50 mmolöL (pH 8. 0) , ED TA 1 mmolöL , M gC l2 20 mmolöL , D T T 2. 5 mmolöL , N aHCO 3
[N aHCO 3 (con tain ing 1. 665×106Bq of N aH 14CO 3 fo r each samp le) 10 mmolöL ], final vo lum e
w as 450 ΛL and then 50 ΛL of R uBP (0. 6 mmolöL ) w as added to the m ix ture. A fter reaction
30s, the reaction w as term inated w ith 100 ΛL of HC l (2 molöL ). T he to tal R ubisco carboxyla2
tion activity w as calculated on basis of 14C2fixed.
T he m easurem en t of RCA activity w as based on the sam e p rogram as the m easurem en t of
R ubisco to tal activity w ith the addition of A T P 5 mmolöL , phosphocreatine 5 mmolöL , 5 un its
of phosphocreatine k inase and 50 Λg of R ubisco inactivated w ith R uBP 0. 4 mmolöL. RCA activi2
ty w as calculated from difference betw een th is activity and the to tal R ubisco carboxylation activi2
ty [ 12 ].
1. 4　M ea surem en ts of RCA and ATP con ten ts
0. 5 m l of the enzym e ex tract w as p recip itated finally w ith 35% saturated (N H 4) 2 SO 4 and
203　　　　　　　　　　　　　　　　　作　　物 　　学　　报　　　　　　　　 　　　　　　　　27卷

cen trifugation 15000×g fo r 10 m in. T he deposit con tain ing RCA w as disso lved again w ith 25 ΛL
of the ex tract m edium. T he quan tification of RCA w as done w ith the single radial imm unodiffu2
sion m ethods[ 14 ] using the rabbit serum an tibody, w h ich w as obtained w ith m ethod described by
T an et al. [ 15 ] , and RCA from rice leaf as the standard acco rding to our p revious paper[ 16 ]. A T P
w as ex tracted w ith bo iling T ris2HC l 0. 02 molöL (pH 7. 5) and its con ten t w as determ ined w ith
bio lum inescence assay [ 11 ].
F ig. 1　　D iurnal changes in net pho tosyn thetic rate (Pn) , avtivities of
in itial R ubisco ( IRA ) and R ubisco activase (RCA ) , con ten ts of R ubisco
activase and A T P in the rice flag leaves of two cultivars on July 7,
1998 A , cv. Zhenong 952; B , cv. Zhenong 966
2　Results
2. 1　D iurna l var ia-
tion s in photosyn-
thetic ra te (Pn ) and
activ ities of Rubisco
carboxyla tion and
Rubisco activa se
O n basis of leaf
area the diurnal
changes of Pn and
activities of in itial
R ubisco carboxyla2
tion and R ubisco ac2
tivase show n in F ig.
1, A 1 and B 1, ap2
peared the typ ical
pattern including
two peak s and a
m idday dep ression in bo th cultivars. T he first peak of Pn occurred around 8 o′clock in bo th culti2
vars and the second peak did at 14～ 16 o′clock. T he peak in the morn ing w as h igher than that in
the afternoon. In itial R ubisco carboxylation activity also had two peak s w ith the m idday dep res2
sion. How ever, the pattern w as a little differen t betw een two cultivars, that kep t h igher activity
during 8～ 12 o′clock and w as h ighest activity at 15 o′clock in Zhenoong 966 (F ig. 1, B1) , but
h ighest peak at 8 o′clock w ith deep m idday dep ression in Zhenong 952 (F ig. 1A 1). T he diurnal
change pattern of RCA activity w as sim ilar w ith that of Pn in Zhenong 952 (F ig. 1,A 1) but no t
in Zhenong 966 in w h ich RCA activity rose a little at 12: 00 (F ig. 1,B1).
2. 2　D iurna l changes in con ten ts of Rubisco activa se and ATP
A s illustrated in F ig. 1, A 2 and B2, RCA con ten ts on the basis of leaf area had an increase
w ith tim e p roceeding. It w as relatively low er befo re 10: 00 but rather h igher after 12: 00. N o de2
p ression at noon w as seen in bo th cultivars. A T P con ten t on the basis of leaf area also rose as day
tim e p rogressed, it increased faster w ith increasing ligh t in tensity befo re 8: 00 and then kep t
rather stable h igher levels in bo th cultivars (F ig. 1, A 2 and B 2).
3033期　　　J IAN G D e2A n et al. : Ro le of Key Enzym es fo r Pho tosyn thesis in the D iurnal⋯⋯　　　

2. 3　Rela tion sh ips between the enzym es activ ities and Pn
T he relationsh ip s betw een the activities of two key enzym es and Pn w ere dep icted in F ig. 2.
Pn w as linearly sign ifican tly co rrelative to bo th in itial R ubisco carboxylation activity and RCA ac2
tivity in the morn ing w h ile Pn increased during 5: 40～ 8: 00, befo re the m idday dep ression (F ig.
2, A 1 and B 1). O n ly the in itial R ubisco carboxylation activity had linearly sign ifican t co rrelation
w ith Pn during 8: 30～ 14: 00 in the m idday dep ression of Pn , indicating that the dep ression of Pn
m igh t be m ain ly caused by low er in itial R ubisco carboxylation activity but no t by low er RCA ac2
tivity (F ig. 2,A 2 and B 2). T here w ere curvilinear co rrelation betw een Pn and the activities of in i2
tial R ubisco carboxylation and RCA in late afternoon (14: 30～ 19: 00) (F ig. 2, A 3 and B3) , im 2
p lying that bo th enzym es affected Pn partly w ith low ering of ligh t in tensity.
F ig. 2　　R elationsh ip s betw een net pho tosyn thetic rate and activities of in itial R ubisco
carboxylation (A 1- 3) o r R ubisco activase (B 1- 3)
1, 2 and 3 show data gathered, during 5: 40～ 8: 00, 8: 30～ 14: 00 and 14: 30～ 19: 00, respectively.
　　To determ ine w hether the diurnal change of in itial R ubisco activity w as resulted from regu2
lation by RCA activity, regression analysis w as perfo rm ed in differen t tim e2befo re, during and
after the m idday dep ression, respectively. T he result show n in F ig. 3 elucidated linearly positive
co rrelation betw een in itial R ubisco carboxylation activity and RCA activity on ly in the morn ing
upon Pn increase (F ig. 3,A 1) and in late afternoon upon Pn decrease (F ig. 3, C1). N o sign ifican t
co rrelation betw een in itial R ubisco carboxylation activity and RCA activity at noon suggested that
low er in itial R ubisco carboxylation activity w as no t caused by low er RCA activity in th is case.
403　　　　　　　　　　　　　　　　　作　　物 　　学　　报　　　　　　　　 　　　　　　　　27卷

F ig. 3　　R elationsh ip s betw een in itial R ubisco activities and R ubisco activase activities
(A 1, B 1, C1) , R ubisco activase activities and A T P con ten ts(A 2, B2, C2)
A , B and C indicate data gathered during 5: 40～ 8: 00, and 8: 30～ 14: 00 and 14: 30～ 19: 00, respectively.
F ig. 4　　R elationsh ip betw een RCA con ten ts and A T P con ten ts
in the rice flag leaves during a day A , cv. Zhenong
952; B , cv. Zhenong 966
2. 4　Rela tion sh ip be-
tween RCA and ATP
　D ependen t of A T P,
in v itro, RCA catalyzes
activiation of R ubisco,
but it is still unknow n
w hether RCA activity
w as determ ined by leaf
A T P con ten t in v ivo.
F ig. 3, A 2, B2 and C2
show ed that a linearly
positive co rrelation be2
tw een RCA activities and A T P con ten ts in the leaf w as found on ly befo re 8 o′clock (F ig. 3 A 2).
R egression analysis betw een RCA con ten ts and A T P con ten ts show ed positively curvilinear co rre2
lation (F ig. 4). N evertheless, RCA con ten ts ro se slow ly w hen A T P con ten ts w ere below 90
nmol m - 2 and the farm er did fast w hen the latter over 90 nmol m - 2 in bo th cultivars.
5033期　　　J IAN G D e2A n et al. : Ro le of Key Enzym es fo r Pho tosyn thesis in the D iurnal⋯⋯　　　

3　D iscussion
3. 1　Role of the photosyn thetic key enzym es in d iurna l changes of Pn
Several repo rts[ 9～ 10, 17 ] had show ed that m idday dep ression of Pn w as caused by low ering of
C i and closing of stom ata. How ever, low Pn in early morn ing and late afternoon w as determ ined
by bo th in ternal and environm en tal facto rs, w h ich w ere low in itial R ubisco activity [ 9 ] and pho to2
syn thetic pho ton flux [ 10 ]. T he m idday dep ression of bo th Pn and R ubisco carboxylation activity
occurred in a fine day. T here w as a closely positive co rrelation betw een Pn and R ubisco activity
during the daytim e (F ig. 2, A 1～ 3) , suggesting that m idday dep ression of Pn invo lve in the re2
duction of R ubisco activity. Fukayam a et al. [ 18 ] repo rted a positive co rrelation betw een R ubisco
activase con ten t and oxygen evo luation during leaf developm en t, and M artinez et al. [ 19 ] found
that a h igh2yield m aize cultivar had h igher R ubisco activase con ten t. How ever, no available data
on diurnal changes of RCA activity w as repo rted. O ur results show ed that the m idday dep ression
also appeared in R ubisco activase activity in bo th cultivars. A sign ifican tly positive co rrelation be2
tw een RCA activities and Pn (F ig. 2 B 1～ 3) and betw een RCA activities and R ubisco carboxyla2
tion activities (F ig. 3, A 1 and C1) show ed on ly in the morn ing and late afternoon but no t during
the m idday dep ression (F ig. 3, B1). T hese results imp lied that RCA directly participated in to ac2
tiviation of R ubisco on ly in the condition, w here Pn increased o r decreased w ith sharp increase o r
decrease in pho tosyn theic pho ton flux. But the low RCA activity seem ed no t a m ain facto r fo r the
m idday dep ression of Pn and in itial R ubisco activity at noon. O ther unknow n facto r ( s) m igh t
cause in itial R ubisco activity to low er in sp ite of low RCA activity at that tim e.
3. 2　Rea son for d iurna l var ia tion in Rubisco activa se
T he obvious diurnal change in R ubisco activity did no t result from the change in R ubisco
con ten t as repo rted in barley [ 20 ] and in our p revious paper[ 9 ]. Sugar phosphates p roduced in
ch lo rop lasts can bind R ubisco very tigh tly and m ake it inactivate[ 2～ 4, 21 ]. T he inactivated R ubisco
needed activation by R ubisco activase depend on A T P [ 12, 15, 22 ]. T herefo re, A T P levels in the
leaves m igh t con tro l the diurnal changes of R ubisco activase activities. O ur result show ed that on2
ly in the morn ing upon Pn increasing RCA activity w as co rrelative w ith A T P con ten t (F ig. 4) ,
although nearly every repo rt show ed that A T P w as necessary to keep RCA activity in v it2
ro
[ 2～ 4, 15, 16, 22 ]
. T hat A T P con ten t in flag leaf w as h igher in the m idday and late afternoon could
no t exp lain the result that RCA activity depends on A T P in vivo. Perhap s an ano ther unknow n
facto r regulates the m idday dep ression of RCA activity. R ubisco activase con ten t w as positively
curvilearly co rrelated to A T P con ten t (F ig. 4) and the purified tobaco R ubisco activase could be
decomposed by itself [ 15 ] , suggesting that A T P m igh t also participate in syn thesis of R ubisco acti2
vase p ro tein o röand p ro tect it from decomposition. T herefo re, w e th ink that A T P con ten t in leaf
also invo lve two functions, one fo r activiation of RCA w hen RCA con ten t is low er in the morn ing
and the o ther fo r exp ression of RCA.
603　　　　　　　　　　　　　　　　　作　　物 　　学　　报　　　　　　　　 　　　　　　　　27卷

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水稻光合关键酶类在光合日变化中的作用
蒋德安　陆　庆　翁晓燕　郑炳松　奚海福
(浙江大学生命科学学院　杭州华家池 310029)
提　要　研究了2个水稻品种的剑叶净光合速率 (Pn)、R ubisco 初始活力和R ubisco 活化酶活力、R ubis2
co 活化酶含量和A T P 含量的日变化, 以明确在体内R ubisco 活化酶是否对光合作用和R ubisco 活力的
日变化起调节作用。结果表明, 2个品种剑叶的 Pn、R ubisco 初始活力和R ubisco 活化酶活力在一天内
变化较大, 三者的日变化模式基本一致, 即具有两个峰和一个低谷。R ubisco 活化酶和A T P 的含量随
时间推移而上升, 下午明显高于上午。回归分析显示, 无论在“午休”前、后或“午休”期间, Pn 极显著
正相关与R ubisco 初始活力。这表明R ubisco 初始活力对光合日变化有很大的影响。R ubisco 活化酶活
力只有在“午休”前和“午休”后才分别与 Pn 和R ubisco 初始活力有显著正相关, “午休”期间无显著相
关。说明光合“午休”涉及R ubisco 初始活力的下降, 但“午休”前的光合上升和“午休”过后的光合再下
降与 R ubisco 和R ubisco 活化酶的活化和钝化有关。R ubisco 活化酶活力与A T P 含量只有在午休前才
有显著直线正相关, 但全天中其含量与A T P 含量有显著指数正相关, 表明体内超过一定范围的A T P
浓度可能参与R ubisco 活化酶的合成或防止其降解。
关键词　水稻; 光合速率; 日变化; R ubisco; R ubisco 活化酶, A T P
7033期　　　J IAN G D e2A n et al. : Ro le of Key Enzym es fo r Pho tosyn thesis in the D iurnal⋯⋯