全 文 ：武汉植物学研究 2001, 19 (1) : 31～ 34
J ourna l of W uhan B otan ica l Resea rch
光对水稻非光合组织谷氨酰胺
合成酶同工酶表达的影响
彭　进1　李泽松1　林清华1　张楚富1Ξ
PEN G Shao2B ing2, BENN ET T John2
(1. 武汉大学植物发育生物学教育部重点实验室, 武汉　430072;
2. In terna tiona l R ice R esearch Institu te, P. O. Box 3127, 1271 M akati C ity, Ph ilipp ines)
摘　要: 以前的研究表明, 高等植物叶绿体谷氨酰胺合成酶 (GS2) 受光调节, 但叶片胞液 GS
(GS1)和非光合作用组织中的 GS 很少受光的影响。在本报道中, 笔者运用 GS 活性染色和
W estern b lo tt ing 研究了光对非光合作用组织水稻根 GS 同工酶表达的影响。在阳光的直接
照射下以及在室内不同光照强度下, 可以很清楚地观察到 GSra 和 GSrb 的活性带及其蛋白
质带。但是, 当用尼龙网挡住阳光的直接照射时, GSrb 的活性带和蛋白质带消失; 当阳光被尼
龙网遮挡住后, 其光强度仍然比室内光照强度大得多, 表明光照强度不是影响 GSrb 表达的
主要因素。当分析生长在暗处以及生长在光ö暗转换下的水稻幼苗根 GS 同工酶变化时, 仍然
可以观察到 GSrb 的存在。在所有实验条件下, GSra 都未发生明显变化。这些结果提示, 光对
GSrb 表达的影响可能是由某些光谱相互作用所产生的未知因素造成的。
关键词: 谷氨酰胺合成酶; 同工酶; 光; 水稻; 根
中图分类号: Q 946. 5; Q 947. 8　文献标识码: A 　文章编号: 10002470X (2001) 0120031204
Effect of L ight on Express ion of Glutam ine Syn thetase
Isozym es in Non -photosyn thetic T issues of R ice Plan ts
PEN G J in1, L I Ze2Song1, L IN Q ing2H ua1, ZHAN G Chu2Fu1Ξ ,
PEN G Shao2B ing2, BENN ET T John2
(1. K ey L abora tory of M O E f or P lan t D evelopm en ta l B iology ,W uhan U niversity , W uhan　430072, Ch ina;
2. In terna tiona l R ice R esearch Institu te, P. O. Box 3127, 1271 M akati C ity, Ph ilipp ines)
Abstract: T he p reviou s studies show ed that the exp ression of ch lo rop last GS2
is m edia ted by ligh t bu t cyto so lic GS1 and GS in non2pho to syn thet ic t issues of
h igher p lan ts are lit t le influenced. In the cu rren t repo rt, w e have invest iga ted
that GS isozym es in rice roo ts respond to ligh t u sing GS act ivity sta in ing andΞ Received: 2000206208, A ccep ted: 2000209214.Foundation item s: T he N ational N atu ral Science Foundation of Ch ina (N o. 39870527) ; Cooperative Foundationof In ternational R ice Research Institu te.B iography: Peng J in (1975- ) , m ale, graduate studen t.A u tho r fo r co rrespondence. E2m ail: cfzhang@w hu. edu. cn
W estern b lo t t ing. Bo th GSra and GSrb act ivity and their p ro tein bands w ere
detected at the illum inat ion of sun ligh t and the incandescen t lamp in the con2
t ro lled environm en t cham ber bu t GSrb act ivity and its p ro tein bands w ere no t
ob served under keep ing ou t d irect sun ligh t w ith the nylon net. T he ligh t in ten2
sity after the keep ing ou t sun ligh t w as h igher than that of the incandescen t
lamp. T h is show ed that ligh t in ten sity w as no t a crucia l facto r fo r influencing
GSrb exp ression. T he act ivity and the p ro tein bands of GSrb in rice roo ts w ere
ob served st ill in the ligh tödark tran sit ion and the dark seedlings. GSra act ivity
w as no t rem arkab ly changed at the ligh t t rea tm en t. T he resu lts suggest tha t
the effect of ligh t on the exp ression of GSrb isofo rm in rice roo ts m ay be
ach ieved th rough som e unknow n facto r ( s) resu lted from the in teract ion of
som e spectra.
Key words: Glu tam ine syn thetase; Isozym e; L igh t; R ice; Roo t
In h igher p lan ts, g lu tam ine syn thetase (GS, EC6. 3. 1. 2) is a crit ica l enzym e in n i2
t rogen m etabo lism. GS p resen ts the m u lt ip le isozym es in the differen t t issues and subcel2
lu lar compartm en ts and these isozym es p lay non2overlapp ing ro les in the ammon ium as2
sim ila t ion [1 ]. A cco rd ing to the localiza t ion of the isozym es in the subcellu lar compart2
m en ts, GS isozym es are divided in to cyto so lic fo rm (GS1) and ch lo rop lastöp last id fo rm
(GS2). T he exp ression of GS isozym es is affected by differen t facto rs. L igh t has been
show ed to induce rem arkab ly GS2 level bu t have lit t le effect on the accum u lat ion of GS1
p ro tein and tran scrip t [2 4 ]. In rice roo ts, w e have found tw o GS (GSra and GSrb) iso2
fo rm s[5 ] and they are cyto so lic fo rm s[6 ]. GSra is a con st itu t ive enzym e and GSrb is an in2
ducib le enzym e by ex ternal n it rogen [5 ]. How ever, the info rm at ion fo r GS in non2pho to2
syn thet ic t issues influenced by ligh t is repo rted lit t le. In the cu rren t comm un icat ion, w e
repo rt tha t GS isozym es in rice roo ts are respon sive to ligh t.
1　M a ter ia ls and m ethods
1. 1　Plan t ma ter ia ls and culture
T he germ inated rice (O ry z a sa tiva L. ) YouYou 122 seeds w ere tran sp lan ted to the
nylon net f loa t ing on N H +4 2con ta in ing nu trien t so lu t ion [5 ]. A fter seedlings grew in the
natu ra l ligh t fo r 6 days o r 10 days, a part of the seedlings w ere tran sferred to the condi2
t ion (abou t 280 Λmo lõm - 2õs- 1 at noon) keep ing ou t d irect sun ligh t w ith the nylon net
w ith air tempera tu re abou t 25℃ (n igh t) to 35℃ (day). T he seedlings w ere con t inuou s2
ly cu ltu red fo r 4 days. T he germ inated seeds u sing to the illum inat ion of the incandes2
cen t lamp w ith the differen t ligh t in ten sity w ere grow n in a con tro lled environm en t
cham ber at a 14ö10 h ligh t2dark pho toperiod w ith 30 to 110 Λmo lõm - 2õs- 1 and w ith tem 2
pera tu re 22℃ (n igh t) to 33℃ (day). A part of seedlings grow n fo r 6 days in the ligh t
in ten sity of 110 Λmo lõm - 2õ s- 1 w ere tran sferred to the dark and con t inuou sly cu ltu red
fo r 4 days ( ligh tödark tran sit ion). A t the sam e t im e, a part of the germ inated seeds w ere
grow n in the dark fo r the sam e days.
1. 2　Extraction , a ssay
GS ex tract ion and the act ivity assay w ere carried ou t as described p reviou sly [5 ].
23 武 汉 植 物 学 研 究　　　　　　　　　　　　　　　　　第 19 卷　
W hen crude enzym e w as ex tracted, the ex tract ion buffer of 3 mL w as added to 1 g sam 2
p le.
1. 3　Isoform determ ina tion of GS
A fter nat ive2PA GE, the determ inat ion of GS isofo rm s in the gel w ere carried ou t
w ith act ivity sta in ing and W estern b lo t t ing [5 ] , respect ively. GS1 an t ibody w as u sed to de2
term ine GS isofo rm s. GS1 an t ibody w as p rovided k indly by D r. T. Yam aya, Tohuku U n i2
versity, Japan.
A : A ctivity stain ing; B: W estern b lo t. L anes 1 and 4, the roo ts of 10
and 14 days grow n in the natu re ligh t; lanes 2 and 3, the roo ts of 4
days grow n in the condition keep ing ou t direct sun ligh t w ith the nylon
net after seedlings grew in the natu ral ligh t fo r 6 days and 10 days,
respectively. 50 ΛL of each samp le w as app lied to the slo t of the gel
F ig. 1　Effect of sun l ight and shading with nylon net on the
expression of r ice root GS isoform s
2　Results
2. 1 　 Effect of sun l ight and
shad ing with nylon net on the
expression of r ice root GS
isozym es
T he act ivity sta in ing and
W estern b lo t t ing after nat ive2
PA GE show ed that tw o isozy2
m es of GS, GSra and GSrb,
w ere clearly detected under the
illum inat ion of the natu re ligh t
(F ig. 1: lanes 1 and 4). How 2
ever, w hen the direct illum ina2
t ion of sun ligh t w as kep t ou t
w ith the nylon net, GSrb act ivi2
ty and its p ro tein bands disappeared (F ig. 1: lanes 2 and 3). T he disappeared ex ten t of
GSrb w as affected w ith the in terval overcast and ra in (F ig. 1: lane 3). W e have ob served
the sam e resu lts a t the sim ilar condit ion s w ith O ry z a sa tiva L. cv. IR 72 in the labo ra to ry
A : A ctivity stain ing; B: W estern b lo tt ing. L ane 1, the roo ts grow n in
ligh tödark transit ion; lane 2, the roo ts grow n in the dark; lanes 3 and
4, the roo ts grow n in ligh t in tensity of 30 and 110 Λmo l·m - 2·s- 1,
respectively. 50 ΛL of each samp le w as app lied to the slo t of the gel
F ig. 2　Effect of differen t l ight in ten sity on the expression
of r ice root GS isoform s
of In ternat ional R ice R esearch
In st itu te, Ph ilipp ines. T he
resu lts s uggested that the ex2
p ression of GSrb isofo rm w as
strongly influenced by the spec2
t rum change of the natu ra l ligh t
and w as no t by the difference of
the la t itudes.
2. 2　Effect of l ight in ten sity on
the expression of r ice root GS
isozym es
To analyze w hether the ex2
p ression of GSrb is influenced
by the in ten sity of ligh t, w e de2
term ined the change of GS isozym es in rice roo ts a t the illum inat ion of incandescen t
lamp w ith the differen t ligh t in ten sity. T he bands of GSrb act ivity and its p ro tein at the
differen t ligh t in ten sity t rea tm en t w ere ob served (F ig. 2: lanes 3 and 4) although the
33　第 1 期　　 　 　　　　彭 进等: 光对水稻非光合组织谷氨酰胺合成酶同工酶表达的影响 (英)
amoun t of GSrb of 30 Λmo lõm - 2õs- 1 w as less than that of 110 Λmo lõm - 2õs- 1. T he ligh t
in ten sity from the illum inat ion of the incandescen t lamp w as low er than that of the kee2
p ing ou t d irect sun ligh t w ith the nylon net. T h is indica ted that ligh t in ten sity is no t a
crucia l facto r fo r the appearance of GSrb. It is in terest ing that GSrb act ivity and its p ro2
tein bands cou ld be ob served also w hen rice seedlings grew in ligh tödark tran sit ion o r
dark fo r 4 days (F ig. 2: lanes 1 and 2). T he resu lts show ed that the regu la t ion of ligh t to
GSrb w as no t d irect. T he levels of GSra act ivity and its p ro tein w ere no t rem arkab ly
changed at a ll condit ion s of the ligh t t rea tm en t.
3　D iscussion
T he ligh t2dependen t regu la t ion of the gene exp ression in p lan ts is qu ite comp lica2
ted. T he exp ression of ch lo rop last GS2 in h igher p lan ts including rice w as induced by
ligh t and cyto so lic GS1 in the pho to syn thet ic t issues w as no t affected [2 4 ]. Can tón et
a l. [7 ] repo rted recen t ly that ligh t st rongly st im u la ted GS1 mRNA accum u lat ion in the
co tyledon s of Sco ts p ine seedlings du ring developm en t. Pérez2Garcia et a l. [8 ] repo rted
that the levels of GS1 p ro tein and its t ran scrip t of tom ato leaves w ere increased also due
to the illum inat ion of ligh t under P seud om onas sy ring ae infect ion o r pho sph ino th ricin
trea tm en t and they con sidered that som e ligh t2dependen t facto rs cou ld be invo lved.
How ever, lit t le info rm at ion concern ing GS of non2pho to syn thesis t issues affected by
ligh t w as repo rted. O u r resu lts show that GSrb exp ression is affected indirect ly by ligh t.
To ou r know ledge, the exp ression of GS in non2pho to syn thesis t issues of h igher p lan ts
influenced by ligh t is f irst ob served. How ever, w e do no t know w hat facto r (s) resu lt ing
from keep ing ou t d irect sun ligh t w ith nylon net affect the exp ression of GSrb isofo rm in
rice roo ts. It is po ssib le that unknow n facto r ( s) resu lted from the in teract ion of som e
spectra of sun ligh t g ive p lay to the effect of ligh t on the exp ression of GSrb bu t the un2
know n facto r (s) w ere no t p resen t under the dark.
References:
[ 1 ]　L am H M , Co sch igano K T , O liveira I C, et a l. T he mo lecu lar2genetics of n itrogen assim ilation in to am ino acids
in h igher p lan ts. A nnu R ev P lan t P hy siol P lan t B iol, 1996, 47: 569 593.
[ 2 ]　M cN ally S, H irel B. Glu tam ine syn thetase isofo rm s in h igher p lan ts. P hy siol V eg , 1983, 21: 761 774.
[ 3 ]　T ingey S V , T sai F Y, Edw ards J W , et a l. Ch lo rop last and cyto so lic glu tam ine syn thetase are encoded by homo l2
ogous nuclear genes w h ich are differen tially exp ressed in V ivo. J B iol Chem. 1988, 263: 9 651 9 657.
[ 4 ]　Peterm an T K, Goodm an H M. T he glu tam ine syn thetase gene fam ily of A rabid op sis tha liana: ligh t2regu lation
and differen tial exp ression in leaves roo ts and seeds. M ol Gen Genet, 1991, 230: 145 154.
[ 5 ]　Zhang C F, Peng S B, Peng X X, et a l. Response of glu tam ine syn thetase isofo rm s to n itrogen sources in rice
(O ry z a sa tiva L. ) roo ts. P lan t S ci, 1997, 125: 163 170.
[ 6 ]　L in Q H (林清华) ,L i C G (李常健) , Zhang C F (张楚富) , et a l. Comparative study of imm uno logical p roperties on
glu tam ine syn thetase isozym es from rice p lan ts. A cta B otan ica S in ica , 2000, 42 (5) : 471 475.
[ 7 ]　Cantón F R , SuarezM F, Jo se2E stanyo lM , et a l. Exp ression analysis of a cyto so lic glu tam ine syh thetase gene in
co tyledons of Sco ts p ine seedlings: developm ental, ligh t regu lation and spatial d istribu tion of specific transcrip ts.
P lan t M ol B iol, 1999, 40: 623 634.
[ 8 ]　Pérez2Garcia A , V icen te A , Can tón F R , et a l. L igh t2dependen t changes of tom ato glu tam ine syn thetase in re2
sponse to P eseud om onas sy ring ae infection o r pho sph ino th ricin treatm en t. P hy siol p lan t, 1998, 102: 377 384.
43 武 汉 植 物 学 研 究　　　　　　　　　　　　　　　　　第 19 卷