fructose 1,6-bisphosphate aldolase activity in leaves of rice

8/14/2019 Fructose 1,6-Bisphosphate Aldolase Activity in Leaves of Rice

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~ Pergamon PH:S0 0 3 1 - 9 4 2 2 ( 9 7 ) 0 0 4 7 0 - 6Phytochemistry,Vol. 46, No . 8, pp. 1335-1338, 1997 1997 Elsevier Science Ltd. All rights reservedPr in ted in Grea t B r i t a in0031-9422/97 $17.00+0.00

F R U C T O S E 1 ,6 -B I SP H O S P H A T E A L D O L A S E A C T I V I T Y IN L E A V E SO F A R IC E M U T A N T S EL E CT E D F O R E N H A N C E D L Y S IN E

GIDEON W . SCHAEFFER,* FRAN K T. SHARPEa nd RICH A RDC. SICHERUSD A, AR S, P lant Sciences Institute, BeltsviUe, Md . 20 705, U.S.A.

(Received n revised orm 6 May 1997)KeyWordndex--Oryza sativa; G r a m i n e a e ; a l do l a s e ; m u t a n t ; l y si ne .

A b s t r a c t - - U n k n o w n p r o t e in s i s o l at e d f r o m m u t a n t t is su e s o f ri ce (Oryza sativa L . ) r e c o v e r e d f r o m i n h i b i to rs e l e c t i on s w e r e s u b s e q u e n t l y pe p t i de m i c r o s e q u e nc e d . D a t a b a s e s e a r c he s pu t a t i ve l y i de n t i f i e d one pe p t i de a sf r u c t o s e 1 , 6 - b i s phos ph a t e a l do l a s e ( E C 4 . 1. 2. 13 ). T i s s u e s o f m u t a n t r i c e , P I 56 4 7 84 , a nd w i l d t y pe ( c v Ca l r o s e7 6) t is s u e s w e r e e va l u a t e d f o r a l do l a s e a c t i v i t y . T o t a l e n z y m e a c ti v it i es w e r e s l i gh t ly l ow e r i n t h e m u t a n t t h a nt he c on t r o l b u t t h e d i ff e re nc e s w e r e no t s i gn i fi c a n t. A l t h ou gh t he m u t a n t phe no t y pe i s f o r e nha n c e d l y s i ne a ndp r o t e i n , w e a s c r ib e t he s m a l l a l do l a s e d if fe r e nc e s t o phy s i o l og i c a l a d j u s t m e n t s , r a t he r t h a n t o D N A m o d i -f i ca t ions of the a ld olase gene(s ). Ho m olo gie s of r i ce pept ides wi th a ldolase s f ro m a rang e Of species, a s wel l a sr i ce c el l c u l t u r e e x p r e s s e d s e q u e nc e t a g s ( E ST s ) a r e p r e s e n t e d . Som e a m i n o a c i d s s e q u e nc e s a r e h i gh l y c ons e r ve d .T he m u t a n t phe no t y pe e x p r e s s i ng s t r e s s p r o t e i n s i s no t l i k e l y t o b e de f i ne d b y a c ha nge i n r i c e a l do l a s e s . 1997 El sev ie r Sc ience L t d

I N T R O D U C T I O N

Fr u c t o s e - b i s ph os ph a t e a l do l a s e is a w i de ly d i st r i b u t e de n z y m e , a n d f u n c t i o n s i n b o t h t h e c h l o r o p l a s t s a n dc y t o p l a s m o f p l a n ts a n d i n n u m e r o u s a n i m a l t is su e,i nc l u d i ng t he b r a i n , m u s c l e a nd l i v e r . T h i s e nz y m e ,w h i c h ha s a t l e a s t f ou r l o c i i n t h e r i ce ge no m e [ 1] p l a y sa v i t a l r o l e i n c a r b o h y d r a t e m e t a b o l i s m a n d i n t h ep r o d u c t i o n o f t ri o s e p h o s p h a t e s a n d d e r i v a ti v e si m por t a n t i n s i gna l t r a n s du c t i on .M u t a n t s o f ri ce , w i th e n h a n c e d l ys in e in e n d o s p e r mpr o t e i n s [ 2 ] a nd s om e l i ne s w i t h e nha nc e d s e e d p r o -t e in s ha ve a r a nge o f a t t r ib u t e s w h i c h ha ve b e e n p a r -t i a l l y c ha r a c t e r i z e d . W e c onc l u de d f r om pa s t e x pe r i -m e n t s t h a t t h e m u t a n t de s c r i b e d he r e , P I 56 4 7 84 , i scons t i tu t ive for spec i f i c s t re s s - re l a t ed and pos s ib lym e m b r a ne p r o t e i n s s i gn i f i c a n t i n p r o t e i n t r a n s -po r t / e x po r t , i n c l u d i ng f l l , 3 - g l u c a na s e s a nd c h i t i n a s e s[ 3] . A l t e r a t i on s i n p r o t e i n s , p a r t i c u l a r l y m e m b r a n ea nd p r oc e s s i ng p r o t e i n s , m i gh t b e r e f l e c t e d i n t hea c t i v i ty o f a u b i q u i t ou s e nz y m e s u c h a s a l do l a s e . T h i sw o r k w a s b a s e d , i n p a r t , o n t h e o b s e r v a t i o n s t h a te l e c t r opho r e t i c p r o f i l e s de ve l ope d w i t h Coom a s s i eb l u e s how e d s u b t l e q u a n t i t a t i v e d i f f e r e nc e s i n t h eb a n d i n g p a t t e r n s o f m u t a n t s a n d c o n t r o l s se p a r a t e do n b o t h d e n a t u r i n g a n d n o n - d e n a t u r i n g a c r y l a m i d ege ls . P r o t e i n s w e r e e l u te d f r om t he na t i ve ge l s o f th em u t a n t a n d s u b s e q u e n t l y s e p a r a t e d o n S D S - P A G E

* Au thor to wh om correspondence should be addressed.

ge ls , b l o t t e d a nd s e q u e nc e d . M i c r o s e q u e nc i ng da t al e d t o t he i de n t i fi c a t ion o f s e ve r al p r o t e i n s , i n c l u d i ngt he a l do l a s e r e po r t e d he r e .

R E S U L T S A N D D IS C U S S I O NM u t a n t a n d c o n t r o l s e e d l i n g p r o t e i n s s e p a r a t e du n d e r n o n - d e n a t u r i n g c o n d i t i o n s s h o w e d s i m i l a rb a n d i ng pa t t e r n s w i t h s u b t le q u a n t i t a t i v e d i ff e re nc e s .

H o w e v e r , 1 - d im e n s i o n al S D S - P A G E p r o fi le s o f p r o -t e i n s e l u t e d f r om na t i ve ge l s s how e d t he p r e s e nc e o fs u b s t a n t i a l l y g r e a t e r Coom a s s i e po s i t i v e m a t e r i a l a ta b o u t 3 5 k D i n t h e m u t a n t t h a n i n t h e w i l d t y p e .A m i n o a c i d s e q u e n c in g o f t h e p u ri fi e d 3 5 k D b a n d( F i g . 1 ) p r ov i d e s e v i de nc e t ha t t h e p e p t i de i s a n a l do -l as e. F i gu r e 2 i l lu s t ra t e s c om p l e t e ho m o l o gy f o r 21a m i n o a c i d s w i th r i c e c D N A s e q ue n c e s d e ri v e d f r o ma n e x p r e s s i on l i b r a r y o f r ic e s ee d li ng s . I n a d d i t i on t ot h e c o m p l e t e h o m o l o g y o f p r o t e i n s i s o la t e d f r o m r ic et h e re a r e m a n y h i g h l y c o n s e r v e d r e g io n s w h i c h o c c u ra c r o s s a w i de r a ng e o f s pec ie s a nd t is s u e t y pe s i nc l u d -i ng a n i m a l b r a i n a nd l i v e r e x t r a c t s . A m i no a c i d s t ha ta r e u n i q u e f o r p l a n t s a n d h i g h l y c o n s e r v e d i n c l ud e Y( ty ros ine ) a t pos i t ion 3 , K ( lys ine ) a t pos i t ion 9 , andT ( t h r e on i ne ) a t po s i t i on 3 0 a nd G ( g ly c i ne ) a t po s i t i on3 2. T he N t e r m i na l e nd s e e m s va r i a b l e i n p l a n t s y s t e m sb u t m o r e c o n s t a n t i n a n i m a l s y s t e m s . A m i n o a c i d st h a t a r e h i g h l y c o n s e r v e d a c r o s s p l a n t a n d a n i m a ls y s t e m s i nc l u de E ( g l u t a m i c a c i d ) a nd L ( l e u c i ne ) a tpo s i t i on s 6 a nd 7 . A m i no a c i d s A ( a l a n i ne ) a t 1 1 , G

1335


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1336 GIDEON W . SCHAEFFER t al.

kD 12J4 3 - -w h i c h e x p r e s s e s a l te r e d s t r e s s e n z y m e a c t i v i t ie s c o m -p a r e d w i t h th e c o n t r o l i s p r o b a b l y n o t c o n d i t i o n e d b ya m a j o r c h a n g e i n r i c e a l d o l a s e s . H o w e v e r , e n z y m ea c t iv i ti e s d o n o t p r o v i d e i n f o r m a t i o n o n p r o t e i na g g r e g a t i o n a n d p o s t t r a n s l a t i o n a l m o d i f i c a ti o n s s uc ha s p h o s p h o r y l a t i o n a n d g l y c o s y l a t io n w h i c h c o u l dp r o d u c e m o d i f i e d e l e c t r o p h o r e t i c p r o fi l es .

3 0 -- 4 1

OFig 1. SD S-PA GE electrophoret ic prof i le of Coomassie bluestained proteins eluted from non-denatur ing gels of themutan t P I564784 . Ar row m arks the 35 kD ban d which wasmicrosequenced.a t p o s i t i o n s 1 8 a n d 2 0 , a n d t h e t r i p l e t I ( i s o le u c i n e ) , L( l e u c i n e ) a n d A a t p o s i t i o n s 2 1 , 2 2 , 2 3 , r e s p e c t i v e l y ,a r e h i g h l y c o n s e r v e d a c r o s s K i n g d o m s a s a r e L a t 3 5 ,G a t 3 9 a n d E a n d N ( a s p a r a g i n e ) a t p o s i t i o n s 4 1a n d 4 2 r e sp e c t i v e l y . F i n a l l y , E a t p o s i t i o n 4 4 a p p e a r sh i g h l y c o n s e r v e d . T h e s e d a t a s u g g e s t c o d i n g r e g i o n sf o r s e q u e n c e m o d i f i c a t i o n s w h i c h w e r e l i k e l y t o b et o l e r a t e d a n d r e g i o n s in w h i c h c h a n g e s w o u l d p r o b -a b l y a l t e r e n z y m e f u n c t i o n s .

T a b l e 1 i l l u s t ra t e s th e a l d o l a s e a c t i v i t y i n m u t a n ta n d w i l d t y p e e x t r a c t s o f r ic e s e e d li n g s . T h e a l d o l a s ea c t i v i t ie s i n t h i s e x p e r i m e n t w e r e g e n e r a l l y lo w e r i nt h e m u t a n t t h a n t h e c o n t r o l s w h e n e v a l u a t e d o n t h eb a s i s o f c r u d e e x t r a c t s , l e a f a r e a , a n d f r e s h w e i g h t .H o w e v e r , w h e n e x p r e s se d o n a p r o t e i n b a s i s th e s p e c -i f ic e n z y m e a c t i v i t i e s ( /~ m o l m g - 1 p r o t e i n ) o f t h em u t a n t a n d c o n t r o l w e r e s i m i l ar o r h i g h e r i n t h em u t a n t . A n i n i ti a l e x p e r i m e n t d e s i g n e d t o o p t i m i z ec o n d i t i o n s p r o d u c e d s i m i l a r e n z y m e p a t t e rn s . T h ep r o t e i n c o n t e n t s i n c r u d e e x t r a c t s w e r e a l s o l o w e r i nt h e m u t a n t t h a n i n t h e c o n t r o l.

O u r c o n c l u s io n s f r o m t h i s w o r k a r e t h a t a l d o l a s e si s o l a te d f r o m m u t a n t r ic e s e ed l i ng s a p p e a r n o r m a l i nN - t e r m i n a l a m i n o a c i d s e q u e n c e s a n d h a v e s i m i l a re n z y m a t i c p r o p e r t i e s to t h e c o n t r o l e n z y m e s. A f u r th e ri m p l i c a t i o n o f t h i s w o r k i s t h a t t h e m u t a n t p h e n o t y p e

E X P E R IM EN T A L

R i c e , ( O r y z a s a t i v a L . ) se e d s w e r e g e r m i n a t e d i n 1c m d e e p v e r m i c u l i t e / p o t t i n g s o i l m i x i n 1 0 c m c l a yp o t s i n c h a m b e r s w i t h 1 4 h r p h o t o p e r i o d f r o m c o o lw h i t e f l u o r e s c e n t l a m p s a t 9 0 - 1 0 0 # E m - 2 s - 1 . P l a n t sw e r e w a t e r e d d a i l y , c u t a t t h e s o i l l e v e l 1 0 - 1 4 d a y sa f t e r s o w i n g a n d f r o z e n i n l i q u i d N 2 . A b o u t 0 . 2 gf r o ze n p o w d e r w a s t r a n s f e r r e d t o a g r o u n d - g l a s s ti s su eh o m o g e n i z e r a n d e x t r a c t e d a t 0 w i th 4 t o 4 . 5 m l o fe x t r a c t i o n b u f fe r c o n s i s t in g o f 50 m M B i c i n e - N a O H ,p H 8 .1 , 5 m M M g C 12 , 1 m M E D T A , 1 0 % g l y c e r o l ,1 % s o l u b le P V P - 4 0, 0 . 01 % T r i t o n x 1 00 a n d 5 m MD T T . H o m o g e n a t e s w e r e c e n t r i f u g e d a t 27 0 0 0 g f o r1 5 r a i n a t 2 - 4 . S u p e r n a t a n t s w e r e q u i c k l y t r a n s f e r r e dt o 1 .5 m l m i c r o c e n t r i f u g e t u b e s a n d f r o z e n i n l i q u i dN 2 . T h e s p e c t r o p h o t o m e t r i c a s s a y f o r F B P - a l d o l a s e( E C 4 . 1. 2 .1 3 ) a c t i v i t y w a s p e r f o r m e d e s s e n t i a l l y a sd e s c r i b e d i n r e f [4 ]. A s s a y s o l n c o n t a i n e d 5 0 m M T r i s -H C I , p h 7 .5 , 1 m M E D T A , 1 .6 m M f r u c t o s e - l ,6 -b i s p h o s p h a t e , 0 .2 m M N A D H , 10 u n i t s t r i o s e -p h o s -p h a t e i s o m e r a s e , 1 u n i t a l p h a - g l y c e r o p h o s p h a t ed e h y d r o g e n a s e a n d 2 0/~ 1 o f th a w e d l e a f e x t r a c t in at o t a l v o l . o f 1 m l . A s s a y s w e r e p e r f o r m e d a t 2 5 a n dA c h a n g e s m e a s u r e d a t 3 4 0 n m . S o l u b l e p r o t e i n w a sm e a s u r e d a c c o r d i n g t o r e f [5 ] u s i n g B S A a s s t a n d a r da n d f o l lo w i n g v e n d o r ' s s p e c if ic a t io n s .

P r e p s o f p r o t e i n s f o r m i c r o s e q u e n c i n g : S e e d l i n g sg r o u n d i n li q u i d N 2 w e r e e x t r a c t e d x 2 w i t h 5 0 m MT r i s p H 6 .8 , 5 % H O C 2 H s S H , 1 m M p h e -n y l m e t h y l s u l p h o n y l f l u o r i d e ( P M S F ) a n d i n s o l u b leP V P a t 2 - 4 . T h e p r o t e i n s w e r e p p t d w i t h 4 v o l . o fc o l d M e 2 C O a n d p e l l e t e d b y c e n t r i f u g a t i o n a t 1 5 0 0 0g f o r 1 0 r a i n . T h e p e l l e t s w e r e s o l u b i l i z e d w i t h 2 0 m MN a O A c , p H 5 .2 , a n d 5 % g l y c e r o l a n d l a y e r e d o n t on o n - d e n a t u r i n g T r i s / b o r a t e , p H 8 .3 , 5 - 1 6 % p o l y -a c r y l a m i d e g e ls . M a r g i n s o f th e g e l s w e r e s t a i n e d w i t hC o o m a s s i e b lu e a n d 1 - 2 c m s e c ti o n s o f t h e n o n s t a i n e d

Table 1. Fructose-1,6 bisph osph ate aldolase activi ty in crude extracts o f seedlings and expanded (Epd) leaves of an in vitro-der ived mu tant (PI564784) and Calrose 76 (Cal-76) , the source cult ivarLeaf Area Rate Rate RateProtein mg Protein Protein (LA) Fr . wt. #m o lm i n - ~ # m ol m i n - ~ # m o l a i n -Sample (Fr. wt.) -I mg (cm 2) mg sample ~ cm2 g m g -' protein g-~ fr . wt. crn -2 LA

CaI-76 Ep d Le af 12.7 0.213 2.86 13.4 0.225 0.256 3.26 0.055M nt Ep d Le af 10.6 0.157 1.8 11.4 0.17 0.292 3.10 0.046Cal-76 See dling 32.3 0.447 8.73 18.3 0.27 0.389 12.54 0.185M ut Seed ling 26.1 0.383 5.4 15.0 0.22 0.394 10.28 0.151


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kMutant

1 64l (MS$ O64 O46 6 2e 9 5? 648 46 9 510 M11 6612 41 t 614 616 4716 617 1018 1019 9

20

I 2 3 4 6 8 7 8 10 I 11 12 I5 14 15 18 17 18 19 ~ I

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S ~ A " / ~ . . . . . . ~ ' ~ /8 K E " I ~ * Y * " T " ~ * ~ 1K K N I ~ A Y * O T " ~ K ~ I~ s A / ~ - v * - ~ ' ~ /S O K I I ~ L R * V A " ~ K ~ I~ E O K ! I I ~ L R " V T * ~ K ~ IE Q K I / ~ H R * V A " ~ K ~ IP E O K I I ~ Q R " V A S ~ K ~ I

1 842 856 854 846 826 85

Mutmt

7 846 246 8510 7411 861: l 24I $ 2 614 2618 67

21 22 23 24 25 23 27 28 29 30 / 31 32 33 34 35 36 37 38 39 40 I

29

I - '~ M I -6 " ~ E ' - - 8 - 1 N A ~ /

F r u c t o s e 1 ,6 -b i s p h o s p h a t e a l d o l a s e i n r i c e 1 3 37

T i

V G S IT G S IV G ~ ImC:M71 9

o L ;/

I

N V IN V /

ij1 O r y z a e M i v a 1 0 0 % / 2 1 A A c O NA , E S T, R ic e- 10 F l , T b i S P N d ~ a s e "2 " 100%/21 AA cDNA, EST, Rice-11 "3 " 100 %/2 1A A cDNA, EST, R ioe-12 "4 " 100%/21 AA cONA, EST, Rioe-13 "6 " 100%/21 AA ONA, EST, R1e-14 "6 " 9 5 % / 2 1 / ~ c O NA , E ST~ R k ~ - 1 6 "7 A m b ~ tha l l l n l 86% /21 AA eDNA, EST , A ratha ll an l "e Pleura mavum 95%/21AX ~kx ol~ ut , Jeer -0 8pJmioJa ~ 81% /21 AA Chloroplast, precursor 10 ON l i lN ( l am m m m re lnha rd l J l 71% /21 AA Ch lo ro fl lm t11 O r fm ea t l v l 100% 144 AA cDNA, EST "1 2 P l m m m l l v u m 9 5 % / 44 A A C h l o r ~ I~ eO um o r "1 :1 A n d l i d q z ~ tha l lm d 70% 144 AA Suboo ll ul at , c y top lm m k ; "1 4 z ~ m ~ m s ~% / 4 4 AA S u b o e n u W , c y ~ o ~ a s m ~ "16 ~ ~ 91%/44 AA ChkxOl~t, precursor "16 Homo i lap len 50 % n9 AA bra ln , human 17 Idus m u lc t~ 8 50% /39 AA b ra in , m ouse "18 Oryalo laOm xmk:u lue 62 % /39 AA muscle, rabbit "19 Oal lus gal lus 48 % /38 AA l iver, chicken "

41 42 43"E-'-'N'I T

4 4

* (E C 4 . 1 . 2 . 1 3 ) F r u c t o s e - b i s l ~ a ld ola se(D-F r .O me- l ,S- U~te O-g~/oemUe~e-~.L.~a~/~*)F i g 2 . H o m o l o g y o f i so l a t e d / m i c r o s e q u e n c e d p r o t e in s f r o m m u t a n t r i c e s e e d li n g s w i t h F r u c t o s e - l , 6 - b i s p h o s p h a t e a l d o l a sef r o m p l a n t a n d a n i m a l s o ur c e s r e t r ie v e d f r o m G e n b a n k d a t a b a s e s . A m i n o a c i d s fr o m t h e d a t a b a s e c o r r e s p o n d i n g w i t h t h er ic e a m i n o a c i d s a r e d e s i g n a t e d b y * . T h e h i g h l y c o n s e r v e d r e g i o n s a r e i n d i c a t e d b y * a n d s h a d i n g .

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1338 GIDEONW . SCHAEFFER t al.po r t i ons r e p r e se n t ing t he m a j o r p r o t e in b a nds w e r ee lu te d w i th H 2 0 b y sha k ing ove r n igh t . E lu t e d p r o t e in swere re -prec ip i ta ted wi th Me2CO a nd cen t r i fuged . Pel -le ts were re -so lub i l ized in SDS and sepd e lee -t r opho r e t i c a l ly i n 1 2 % SD S P A G E ge l s. Se pd p r o t e in sw e r e b lo t te d i n to P V D F m e m b r a ne s , v isu al ize d w i tha m ido b l a c k in 0 . 5 M H O A c a nd su b j e c t e d t o a m inoac id mic rosequenc ing .Acknowled#ements--The a u tho r s e x p r e s s t he i rapprec ia t ion to T . Heut te for s ign i f icant he lp in them ic r o se q u e nc ing o f t he i so l a t e d p r o t e in s a nd to J .Dud ley fo r technica l a ss istance .

R E F E R E NC E S

1 . N a k a m u r a , H . , Sa toh , W. , H ida k a , S . , K a ga y a ,Y, , E j r i r i , S . and ' Tsu tsum i , K. , Plant MolecularBiology, 1996, 30, 381.2 . Schaeffe r, G . W. , Sharp e , F . T . and D udley , J . T . ,Crop Science, 1994, 34, 1424.3 . Schaeffe r, G . W . and S harpe , F . T . J r . , Theoreticaland Applied Genetics, 1990, 80, 841.4 . K r u g e r , I . a nd Sc hna r r e nb e r ge r , C . , EuropeanJournal o f B iochemistry, 1983, 136, 101.5 . B r a d fo r d , M . M . , Analytical Biochemistry, 1976,72, 248.

fructose 1,6-bisphosphate aldolase activity in leaves of rice

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