全 文 ：V o l. 29, N o. 3
pp. 349—352　M ay, 2003
作　物　学　报
A CTA A GRONOM ICA S IN ICA
第 29 卷 第 3 期
2003 年 5 月　349—352 页
D iurna l Changes of Phosphoenolpyruva te Carboxyla se Activ ity in L eaves of
F ield-grown M a ize In troduced in to Tibetan Pla teau
Ξ
YAN G J ia2D ing
(Cold & A rid R eg ions E nv ironm en ta l and E ng ineering R esearch Institu te, T he Ch inese A cad emy of S ciences, L anz hou , Guansu 730000, Ch ina)
Abstract　 In the day course of pho to syn thesis of m aize in troduced in to T ibetan P lateau, during its jo in t ing
stage, the pho sphoeno lpyruvate carboxylase (PEPC) act ivity in pho to syn thet ic leaves undulated mo re gen t ly
and w as alw ays h igher than the net pho to syn thet ic rate (P n) at every t im e po in t. By studying the variat ion of
difference betw een P n and PEPC act ivity th roughout the day, the influence of environm ental facto rs (e. g.
ligh t in tensity and am bien t temperatu re) and stom atal status on pho to syn thesis w as analysed.
Key words　M aize; T ibetan P lateau; Pho sphoeno lpyruvate carboxylase
引种到青藏高原大田的玉米叶片中磷酸烯醇式丙酮酸羧化酶活性的日变
化
杨甲定
(中国科学院寒区旱区环境与工程研究所, 甘肃兰州 730000)
摘　要　引种到青藏高原大田的玉米, 其拔节期的全天光合进程中, 叶片中磷酸烯醇式丙酮酸羧化酶 (PEPC) 活性总是
大于相应时间点的净光合速率 (P n) , 且全天变化幅度较 P n 缓和。通过研究 PEPC 活性和 P n 之间差异的全天变化, 分析
了环境因子 (如光强、气温)和气孔状态对光合作用的影响。
关键词　玉米; 青藏高原; 磷酸烯醇式丙酮酸羧化酶
中图分类号: Q 945111　　　文献标识码: A
　　D efin it ion of the lim it ing facto rs in the pho to2
syn thet ic p rocess is a p rerequ isite to genet ica lly in2
creasing the pho to syn thet ic ra te of crop species[ 1 ].
A nd it is a lso favo rab le to increase crop yield agro2
nom ica lly becau se pho to syn thet ic ra te is often the
p rim ary lim ita t ion fo r h igh yield under field condi2
t ion s[ 2, 3 ]. So Giffo rd et a l. p ropo sed tha t im p rove2
m en t in the m ax im um rate of leaf pho to syn thesis
m ay becom e essen t ia l to fu rther increase in yield
po ten t ia l[ 4 ].
U nder field environm en ta l condit ion s, pho to2
syn thesis of leaves is a lw ays the resu lt of com p lex
diverse facto rs and react ion s, such as CO 2 concen2
t ra t ion ( am b ien t and o r in tercellu la r ) , am b ien t
tem pera tu re, ligh t in ten sity, the act ivity of car2
boxylase and the capacity of electron tran spo rt etc.
Fu rtherm o re, P n (net pho to syn thet ic ra te) is ind i2
rect ly associa ted w ith leaf w ater po ten t ia l, leaf age
and availab le so il nu trien ts as w ell[ 5 ]. So in o rder
to im p rove crop m anagem en t, it w ou ld be u sefu l to
understand the facto rs tha t lim it pho to syn thesis in
the field condit ion s.
It is w ell estab lished tha t leaf K ranz anatom y
and PEPC con tribu te to h igh pho to syn thet ic ab ilityΞFoundation item: State Key Basic Research and D evelopm ent P lan (‘973’, G2000048704).
Biography: 杨甲定 (19712) , 男, 甘肃甘谷人, 硕士。从事植物逆境生理与植物基因工程研究。Co rresponding autho r. Yang J ia2ding (E2m ail:
yangjiading@ sina. com ).
Received (收稿日期) : 2002204223, A ccep ted (接受日期) : 20022092011

in C 4 p lan ts, includ ing m aize [ 6 ]. H igh act ivity of
PEPC (pho sphoeno lpyruvate carboxylase ) is an
im po rtan t necessary facto r to m ain ta in h igh pho to2
syn thet ic ra te in C 4 p lan ts besides o ther eco log ica l
o röand physio log ica l facto rs. By analyzing the d if2
ferences betw een PEPC act ivity and pho to syn thet ic
ra te over the day, it is feasib le to study w h ich
facto r ( s ) shou ld be respon sib le fo r the d iu rna l
changes of pho to syn thesis in field2grow n m aize
w h ich w as in troduced from low eleva t ion p la in to
the T ibetan P la teau.
1　M a ter ia ls and M ethods
111　Plan t ma ter ia l and growing cond ition s; Ga s
exchange m ea surem en ts
T hese item s w ere exact ly sam e as w hat w ere
repo rted in ou r fo rm er paper [ 7 ].
112　A ssay of enzym e activ ity
11211　Ex tract ion of enzym e
T he upper parts of th ree leaves on w h ich gas
exchange m easu rem en ts w ere m ade at every t im e
po in t w ere imm edia tely excised and frozen in liqu id
N 21
PEPC (EC41111131) w as ex tracted by a m od2
if ica t ion of the m ethod described by H ague and
Sim s[ 8 ]. F rozen leaf m ateria l (5g) w as ground in
10mL ice2co ld ex tract ion so lu t ion [ T ris2H 2SO 4
(011m o löL , pH 810) , con ta in ing 5% glycero l (vö
v) , 1% PV P (w öv) , D T T ( 7mm o löL ) , ED TA
( 1mm o löL ) ] in a co ld g lass m o rta r. T he ho2
m ogenate w as filtered th rough fou r layers of
cheeseclo th and cen trifuged at 20000 × g fo r
15m in, and the superna tan t w as m easu red fo r en2
zym e act ivity as soon as po ssib le. A ll p rocedu res
w ere carried ou t a t 4℃.
11212　Enzym e act ivity assay
T he act ivity of PEPC w as assayed by m easu r2
ing the decrease in A 340nm a t 25℃ u sing a W FZ8002
D 3A dig ita l spectropho tom eter (Beijing, Ch ina ).
A cco rd ing to som e researchers[ 9, 10, 11 ] , the assay
system con ta ined ( in Λm o l) : T ris2HC l, pH 810
(100) , PEP (sodium salt, 115) , NADH (disod ium
salt, 0117) , N aHCO 3 (20) , M gC l2 (20) , 20 un its
of m ala te dehydrogenase, an app rop ria te vo lum e of
enzym e ex tract ion above and dist illed w ater in a to2
ta l vo lum e of 3mL. T he un it of PEPC act ivity w as
ca lcu la ted as Λm o l CO 2·m - 2· s- 1, based on the
est im ate tha t 1g fresh w eigh t of frozen leaf
m eteria l w as equ iva len t to 215 × 10- 3 m 2 of leaf
area.
2　Results and d iscuss ion
211　D iurna l trends of Pn and PEPC activ ity
A s show n in F ig11, P n of m aize leaves in2
creased from 2145 to 35183 ( Λm o l CO 2 ·m - 2 ·
s
- 1) over the period from 8: 00 to 13: 00; then fluc2
tua ted sligh t ly from 13: 00 to 19: 001 A fter 19:
00, P n declined m arkedly. T he m ax im um P n
(39103 Λm o l CO 2·m - 2·s- 1) occu rred a t 15: 001
T hese ind ica ted obviou sly tha t the P n vs t im e cu rve
had a sing le peak [ 7 ]. W h ile the act ivity of PEPC in2
creased from 26158 to 46152 (Λm o l CO 2·m - 2·
s
- 1) from 8: 00 to 13: 001 T he m ax im um act ivity
of PEPC (46195 Λm o l CO 2·m - 2· s- 1 ) a lso oc2
cu rred a t 15: 001 T hen PEPC act ivity leveled off
and fell g radually from 15: 00 to 21: 001
A pparen t ly, the d iu rna l varia t ion of PEPC act ivity
exh ib ited a sim ila r bu t gen t le t rend com pared to
tha t of leaf P n.
F ig11　D iurnal changes of P n and PEPC activity. Every po in t
stands fo r the m ean of 15 (fo r P n ) o r 3 ( fo r PEPC activity)
values from th ree leaves. V ertical bars indicate the standard
erro rs. ( P n, net pho to synthetic rate; PEPC, pho s
phoeno lpyruvate carboxylase. )
Com paring the trends of PEPC act ivity and P n
(F ig11) , it w as in terest ing ly found tha t f luctua2
t ion of PEPC act ivity w as clearly less than tha t of
P n , p robab ly m ean ing tha t P n shou ld be associa ted
053 　　　作　　物　　学　　报 29 卷　

w ith and m o re sen sit ive to m o re endogenou s and
exogenou s facto rs than PEPC. Fu rtherm o re,
PEPC act ivity a lw ays exceeded P n th roughou t the
day, as w as sim ila rly repo rted in ribu lo se 1, 52b is2
pho sphate carboxylase study [ 12 ]. It is know n tha t
the sub stra te fo r PEPC is HCO -3 , ra ther than CO 2
becau se PEPC does no t con ta in b io t in [ 13 ]. A nd P n
w as m easu red a t a ir level of CO 2, w h ile PEPC ac2
t ivity w as m easu red a t sa tu ra t ing HCO -3 w ho se
concen tra t ion w as grea ter than 1000 m o löL. T hu s
m easu red PEPC act ivity in fact ind ica ted the po ten2
t ia l capacity w h ich pho to syn thet ic leaves cou ld
reach under certa in op t im al condit ion s theo ret ica l2
ly. F rom th is view po in t, w e cou ld p ropo se tha t P n
of in troduced m aize leaves st ill have som e space to
be increased.
212　D ifferences between PEPC activ ity and Pn
W e cou ld reasonab ly suppo se tha t the d iffer2
ence betw een P n and PEPC act ivity w ou ld be con2
stan t if the influence of each facto r associa ted w ith
pho to syn thesis w ou ld no t vary th roughou t the
day. In fact, the d ifference betw een P n and PEPC
act ivity exh ib ited som e varia t ion s over the day as
show n in F ig121
F ig12　T he difference values betw een the m eans of P n and of
PEPC activity th roughout the day. ( P n, net pho to synthetic
rate; PEPC, pho sphoeno lpyruvate carboxylase. )
A s show n in F ig12, nearly con stan t d iffer2
ences fo rm 8: 00 to 9: 00 m ean tha t the increase of
PEPC act ivity shou ld be ch iefly respon sib le fo r
tha t of P n , w h ile o ther facto rs be secondary. It has
been repo rted tha t pho to syn thet ic PEPC act ivity
w as sub ject to reversib le ligh t act iva t ion by p ro tein
pho spho ryla t ion in m aize leaves[ 11, 14, 15 ]. So it
shou ld be reasonab ly concluded tha t increasing
PA R (F ig13) w as the u lt im ate im petu s of increas2
F ig13　D iurnal variat ions of pho to synthetically active radiance
( PA R ) , am bien t temperature (TA ) and in tercellu lar CO 2 con2
cen tration (C i). (T hese data w ere cited from the paper of Yang
et a l. [7 ])
ing P n in th is period.
Fo rm 9: 00 to 15: 00, due to tha t declin ing C i
w as disadvan tageou s to P n (F ig13) , the increasing
of P n (F ig11) and gradual decrease of d ifferences
(F ig12) suggested tha t facto rs o ther than PEPC
act ivity shou ld p lay m o re im po rtan t parts to in2
crease P n and the m o st po ssib le ones w ere PA R and
am b ien t tem pera tu re. E specia lly a t 15: 00 w hen C i
w as at its m in im um , the P n exh ib ited its m ax i2
m um. T h is m ean s tha t the o ther facto rs excep t C i
m u st be in an op t im al situa t ion fo r leaf pho to syn2
thesis. T herefo re, w e suggested tha t supp lying
CO 2 fert ilizer of certa in con ten t to m aize comm un i2
ty shou ld be efficien t to p rom o te P n fu rther.
A s show n in F ig11, 2 and 3, P n declined gen t ly
and the d ifferences gradually becam e larger desp ite
the h igh level of PEPC act ivity and increasing C i
from 15: 00 to 19: 001 Besides decreasing of PA R
and am b ien t tem pera tu re, the accum u la t ion of
pho to syn thet ic p roduct ( e. g. so lub le
carbohydra te ) w ou ld a lso con tribu te to th is
t rend [ 16 ] , though there ex isted d ifferen t
view s[ 17, 18 ].
A fter 19: 00, acco rd ing to the criterion sug2
gested by Farquhar and Sharkey [ 19 ] , the stom ata l
facto r w as no t the reason fo r P n decreasing becau se
of increasing of C i. Con sidering the h igh PEPC ac2
t ivity, the facto r w h ich shou ld be respon sib le fo r
decreasing of P n w as the sharp decreasing PA R
(F ig13) , becau se enzym e react ion s of CO 2 assim i2
la t ion requ ire assim ila to ry pow er ( A T P and
153　3 期 Yang J ia2D ing: D iu rnal Changes of Pho sphoeno lpyruvate Carboxylase A ctivity ⋯

NAD PH ) from electron tran spo rt, w h ich in tu rn
depends on ab so rbed irrad iance [ 5 ].
Acknowledgm en ts
W e thank the Cen ter L abo ra to ry of T ibetan
A cadem y of A gricu ltu ra l Sciences fo r p rovid ing
m any experim en ta l facilit ies.
References
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253 　　　作　　物　　学　　报 29 卷