Regulation of Gene Expression in the Sunflower Embryo - American

Chapter 17

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Regulation of Gene Expression in the Sunflower Embryo C. A. Adams, K. E. Koprivnikar, R. D. Allen, E. A. Cohen, C. L. Nessler, and T. L Thomas Department of Biology, Texas A&M University, College Station, TX 77843 The sunflower embryo is a convenient system for the comparative analysis of gene expression in development. Here we demonstrate the u t i l i t y of this system in the analysis of several genes with different expression patterns and functions. These include the seed storage protein genes of sunflower that encode super-abundant mRNAs and genes that encode less abundant actin, tubulin, and β-lipoxygenase mRNAs. Relatively small changes (-3-fold) in steady state levels of actin, tubulin, and β-lipoxygenase transcripts are observed during embryogenesis. In contrast, very large changes (-100-fold) are observed for seed storage protein mRNAs over the same developmental interval. Compari sons of steady state mRNA levels and relative nuclear RNA synthesis rates of these genes indicate that for the most part mRNA steady state levels are controlled at the level of transcription, although in some cases post-transcriptional control is important. These results are significant in understanding the role of gene regulatory hierarchies in development.

R e g u l a t i o n o f gene e x p r e s s i o n i n development i s a c h i e v e d by a v a r i e t y o f mechanisms i n d i f f e r e n t t i s s u e s and organisms. Control a t t h e l e v e l o f t r a n s c r i p t i o n may be a c c o m p l i s h e d b y t h e p r e s e n c e o r absence o f t i s s u e - s p e c i f i c t r a n s a c t i n g f a c t o r s , by h o l d i n g t h e c h r o m a t i n c o n t a i n i n g t h e gene i n a " s i l e n t " c o n f i g u r a t i o n , o r i n some c a s e s by m e t h y l a t i o n o f t h e gene (reviewed i n I). Postt r a n s c r i p t i o n a l c o n t r o l s may i n v o l v e d i f f e r e n c e s i n t h e r a t e o f p r e mRNA p r o c e s s i n g o r i n t h e s t a b i l i t y o f an mRNA w i t h i n t h e c e l l ( 2 ) , and c o n t r o l o f t h e r a t e a t w h i c h mRNA i s assembled i n t o polysomes (3) ( t r a n s l a t i o n a l c o n t r o l ) . S t u d i e s o f t h e s e c o n t r o l s a r e p l e n t i f u l i n a n i m a l systems (1-3) b u t t h e r e a r e r e l a t i v e l y few p l a n t systems t h a t have been s t u d i e d a s e x t e n s i v e l y . S u n f l o w e r i s an e x c e l l e n t system f o r t h e s t u d y o f gene r e g u l a t i o n d u r i n g embryogenesis. A s i n g l e i n f l o r e s c e n c e p r o v i d e s embryos o f d i f f e r e n t 0097-6156/88/0379-0240$06.00/0 « 1988 American Chemical Society

Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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s t a g e s spanning e a r l y and l a t e d e v e l o p m e n t a l e v e n t s ; f u r t h e r m o r e the s i z e of the s u n f l o w e r i n f l o r e s c e n c e f a c i l i t a t e s the i s o l a t i o n of l a r g e numbers of d e v e l o p m e n t a l l y staged embryos. Thus b i o c h e m i c a l amounts of RNA and p r o t e i n can be i s o l a t e d from n u c l e i f o r t h e s t u d y of t r a n s c r i p t i o n and i s o l a t i o n of t r a n s a c t i n g f a c t o r s . I n a d d i t i o n , a number of genes have been c h a r a c t e r i z e d i n s u n f l o w e r . Two s u n f l o w e r 11S ( h e l i a n t h i n i n ) seed s t o r a g e p r o t e i n (SSP) genes (HaG3 and HaGlO) have been c l o n e d and changes i n e x p r e s s i o n d u r i n g embryogenesis have been d e s c r i b e d ( 4 - 6 ) . Also the s t r u c t u r e of a 2S (albumin) SSP gene (HaG5) from s u n f l o w e r has been determined and shown t o have a s l i g h t l y d i f f e r e n t e x p r e s s i o n p a t t e r n from t h a t o f the h e l i a n t h i n i n s ( 5 - 7 ) . I n t h i s paper we d e s c r i b e the c o n t r o l of gene e x p r e s s i o n i n s u n f l o w e r embryogenesis w i t h r e s p e c t t o HaGlO, HaG5, a c t i n , t u b u l i n , and βl i p o x y g e n a s e (3-LOX) genes. Comparisons o f s t e a d y s t a t e l e v e l s of mRNAs f o r t h e s e genes w i t h r e l a t i v e r a t e s of t r a n s c r i p t i o n a t d i f f e r e n t s t a g e s of development i n d i c a t e t h a t mRNA s t e a d y s t a t e l e v e l s a r e c o n t r o l l e d m a i n l y a t the l e v e l o f t r a n s c r i p t i o n a l t h o u g h i n some cases p o s t - t r a n s c r i p t i o n a l c o n t r o l may be i m p o r t a n t . M a t e r i a l s and Methods Growth of P l a n t s . S u n f l o w e r seeds ( H e l i a n t h u s annuus L. c v . G i a n t grey s t r i p e , N o r t h r u p K i n g Seed Co. M i n n e a p o l i s , MN) were germinated i n s t e r i l e s o i l and grown i n the greenhouse f o r one week b e f o r e t r a n s p l a n t i n g t o the f i e l d . P l a n t s were grown i n the f i e l d d u r i n g the summer and i n growth chambers w i t h 16 h day l e n g t h a t 23°C a t o t h e r times of the y e a r . Embryos were h a r v e s t e d i n l i q u i d N2 and s t o r e d a t -80°C u n t i l needed. RNA I s o l a t i o n . RNA was i s o l a t e d as p r e v i o u s l y d e s c r i b e d (4) except t h a t t i s s u e was homogenized u s i n g a P o l y t r o n (Brinkman I n s t r u m e n t s , Westbury, NY). P o l y A+ RNAs were p u r i f i e d by o l i g o ( d T ) c e l l u l o s e chromatography ( 8 ) . N o r t h e r n B l o t H y b r i d i z a t i o n s . T o t a l o r polyA+RNAs were denatured w i t h d i m e t h y l s u l f o x i d e and g l y o x a l , s i z e f r a c t i o n a t e d on 1% a g a r o s e g e l s , and t r a n s f e r r e d t o n i t r o c e l l u l o s e f i l t e r s . L a b e l e d DNA probes were denatured by b o i l i n g and h y b r i d i z e d t o f i l t e r s f o r 16 h r a t the a p p r o p r i a t e temperature i n 50% formamide, 25 mM phosphate b u f f e r pH 6.8, 5X SET (IX SET - 150 mM N a C l , 20 mM T r i s pH 7.8, 1 mM EDTA), 0.1% sodium d o d e c y l s u l f a t e (SDS), 10% d e x t r a n s u l f a t e , 5X Denhardt's s o l u t i o n (IX Denhardt's = 0.02% f i c o l l , 0.02% BSA, 0.02% p o l y v i n y l p y r r o l i d o n e , 0.02% SDS), 100 ug/ml denatured h e r r i n g t e s t i s DNA, 50 yg/ml p o l y a d e n y l i c a c i d and 10 ug/ml p o l y c y t i d y l i c a c i d . A f t e r h y b r i d i z a t i o n the f i l t e r s were washed f o r l h i n 4X SET wash (4X SET + 0.025M phosphate b u f f e r , 0.2% SDS), 1 h i n 2X SET wash, and 1 h i n IX SET wash a l l a t 50°C. T u b u l i n probed b l o t s were washed i n 50% formamide/ 5X SSC a t 55°C f o r 2h. F i l t e r s were a i r d r i e d and exposed f o r a u t o r a d i o g r a p h y a t -80°C w i t h an i n t e n s i f y i n g screen. R a d i o a c t i v i t y i n each band on the f i l t e r s was measured by e x c i s i n g the band and c o u n t i n g by l i q u i d s c i n t i l l a t i o n o r d e n s i t o metry of a u t o r a d i o g r a p h s u s i n g a Bio-Rad Model 620 v i d e o densitometer.


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Probes f o r HaG5, HaGlO, a c t i n , and β-lipoxygenase c o n s i s t e d o f DNA fragments l a b e l e d w i t h 32p by n i c k t r a n s l a t i o n . HaG5 and HaGlO p r o b e s c o n s i s t e d o f 0.7kb and 1.2kb Eco R I cDNA fragments r e s p e c t i v e l y . The sequences o f each o f t h e s e probes l i e s e n t i r e l y w i t h i n t h e c o d i n g sequences o f t h e genes ( 5 , 6 ) . A c t i n probe was a three k i l o b a s e (kb) soybean genomic c l o n e fragment I s o l a t e d from pSAc3 (9) w h i c h was a g i f t o f D r . R i c h a r d Meagher ( U n i v e r s i t y o f G e o r g i a , A t h e n s , GA). The probe f o r 3 - l i p o x y g e n a s e was a cDNA c l o n e from pea and was k i n d l y p r o v i d e d by D r . Rod Casey (John Innes I n s t i t u t e , N o r w i c h , G r e a t B r i t a i n ) . T u b u l i n probe c o n s i s t e d o f a 1.6 kb t r a n s c r i p t produced from a soybean t u b u l i n genomic c l o n e i n s e r t e d i n t o pSP65 (Promega B i o t e c , Madison, WI) u s i n g Sp6 RNA polymerase according t o the i n s t r u c t i o n s of the manufacturer. This t u b u l i n c o n s t r u c t was k i n d l y p r o v i d e d by Dr. Mark G u i l t i n a n ( 1 0 ) . N u c l e i I s o l a t i o n and Runoff A s s a y s . N u c l e i were i s o l a t e d and p u r i f i e d by t h e p r o c e d u r e o f L u t h e and Quatrano (JUL) e x c e p t t h a t a l l b u f f e r s were a d j u s t e d t o pH 8.6. N u c l e a r r u n o f f r e a c t i o n s were c a r r i e d o u t i n a volume o f 50 μΐ c o n t a i n i n g lOOmM (NH4)2S04, 30mM T r i s HC1 pH 8.6, 7mM M g C l , 2.5mM i n each o f GTP, CTP, ATP, 3mM c r e a t i n e phosphate, 25 ng/ml c r e a t i n e p h o s p h o k i n a s e , 3mM 3m e r c a p t o e t h a n o l , 1 unit/μΐ R N a s i n , and 50 ]iC± P - U T P (New England N u c l e a r , B o s t o n , MA, USA). R e a c t i o n s were s t a r t e d by m i x i n g a l l components, b r i n g i n g t o room temperature and t h e n a d d i n g 10μ1 o f n u c l e i ( 10^ n u c l e i ) . Samples were i n c u b a t e d a t 25°C f o r up t o one h o u r . R e a c t i o n s were stopped by a d d i t i o n o f i c e c o l d 150mM sodium pyrophosphate/3mM UTP w i t h v i g o r o u s m i x i n g and a l l o w e d t o s i t on i c e f o r .10 m i n u t e s . One ml o f i c e c o l d 5% (w/v) TCA was t h e n added t o each sample, v o r t e x e d and i n c u b a t e d on i c e f o r 10 m i n u t e s . TCA p r e c i p i t a b l e c o u n t s were c o l l e c t e d on Whatman GF/C g l a s s f i l t e r s , washed e x t e n s i v e l y w i t h 5% TCA/150mM sodium pyrophosphate, t h e n w i t h e t h a n o l , and f i n a l l y w i t h 2 m l o f d i e t h y l e t h e r . A f t e r a i r d r y i n g , r a d i o a c t i v i t y on t h e f i l t e r s was d e t e r m i n e d by l i q u i d s c i n t i l l a t i o n counting. 2

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I s o l a t i o n o f Heterogeneous N u c l e a r RNA from P - U T P L a b e l e d N u c l e i . Heterogeneous n u c l e a r RNA (hnRNA) was i s o l a t e d by t h e method o f L e e e t a l . (12) w i t h t h e f o l l o w i n g m o d i f i c a t i o n s . P o l y v i n y l s u l f a t e was e l i m i n a t e d from t h e l y s i s b u f f e r , 0.75g o f C s C l was added t o t h e homogenate a f t e r t h e p r o t e i n a s e Κ s t e p , and t h e samples c e n t r i f u g e d o v e r t h e C s C l pad f o r 22 h a t 50,000 rpm a t 15°C i n a TLA 100.3 r o t o r u s i n g a Beckman t a b l e t o p u l t r a c e n t r i f u g e . The r e s u l t i n g RNA p e l l e t was d i s s o l v e d i n 6M g u a n i d i n e hydrochloride/50mM sodium c i t r a t e pH 7.0 and p r e c i p i t a t e d by t h e a d d i t i o n o f 2.5 volumes o f ethanol/-80°C o v e r n i g h t . A f t e r p e l l e t i n g i n an Eppendorf c e n t r i f u g e the RNA was d i s s o l v e d i n 50μ1 o f w a t e r and 75μ1 o f 5M p o t a s s i u m a c e t a t e pH 6.0 was added. The samples were then h e l d a t -20°C o v e r n i g h t and RNA was p e l l e t e d i n an Eppendorf c e n t r i f u g e f o r 30 min. The p e l l e t s were washed w i t h 70% e t h a n o l , d r i e d i n vacuo and redissolved i n hybridization buffer. Dot and S l o t B l o t H y b r i d i z a t i o n s . S l o t b l o t s o f p l a s m i d DNAs c o n t a i n i n g c l o n e s f o r HaG5, HaGlO, a c t i n , t u b u l i n , and 3l i p o x y g e n a s e were made by d e n a t u r i n g t h e DNAs i n 0.5M NaOH a t 100°C


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f o r 10 min f o l l o w e d by a d d i t i o n of an e q u a l volume o f i c e - c o l d n e u t r a l i z a t i o n s o l u t i o n (1M N a C l , 0.3M sodium c i t r a t e , 0.5M T r i s HC1 pH 8.0, 1M HC1) (13) and f i l t e r i n g through n i t r o c e l l u l o s e u s i n g a s l o t b l o t m a n i f o l d from S c h l e i c h e r and S c h u e l l (Keene, NH). Dot b l o t s c o n t a i n i n g t o t a l RNA from s t a g e d embryos were made i n the same manner except t h a t the h e a t i n g i n a l k a l i s t e p was e l i m i n a t e d and a M i n i f o l d ( S c h l e i c h e r and S c h u e l l ) was used. B l o t s were a i r d r i e d and t h e n baked a t 80°C f o r 4 h. B e f o r e use t h e b l o t s were p r e - h y b r i d i z e d i n t h e same manner as N o r t h e r n b l o t s except t h a t f o r s l o t b l o t s p r e - h y b r i d i z a t i o n was done f o r 48 h , 500,000 cpm o f hnRNA from 8-, 13- o r 25-days p o s t - f e r t i l i z a t i o n (DPF) embryo n u c l e i were then added t o each f i l t e r and h y b r i d i z a t i o n c a r r i e d out f o r 72 h a t 40°C. S l o t b l o t s were then washed i n t h e same manner as n o r t h e r n b l o t s and p l a c e d on f i l m f o r 96 h w i t h an i n t e n s i f y i n g screen. Results SSP Genes E x h i b i t Very S i m i l a r P a t t e r n s o f E x p r e s s i o n . F i g u r e s 1 and 2 demonstrate t h a t HaG5 and HaGlO have i d e n t i c a l e x p r e s s i o n p a t t e r n s b o t h q u a l i t a t i v e l y ( F i g u r e 1) and q u a n t i t a t i v e l y ( F i g u r e 2) from 8- through 25-DPF except t h a t HaG5 e x p r e s s i o n i s d e t e c t a b l e by 5 DPF w h i l e HaGlO e x p r e s s i o n i s n o t d e t e c t a b l e u n t i l 7 DPF. B o t h genes r e a c h a peak i n e x p r e s s i o n a t around 13 DPF and then d e c l i n e t o v e r y low l e v e l s by 25 DPF w i t h HalO mRNA b e i n g somewhat more p r e v a l e n t a t 25 DPF than t h a t o f Ha5. Both are undetectable i n d r y seed by RNA g e l b l o t a n a l y s i s ( d a t a n o t shown). S i n c e t h e g e l b l o t method gave more a c c u r a t e q u a n t i t a t i v e d a t a we c o n t i n u e d t o use i t f o r t h e a n a l y s i s of t h e e x p r e s s i o n p a t t e r n o f o t h e r genes of i n t e r e s t . A c t i n , T u b u l i n , and β-Lipoxygenase have S i m i l a r E x p r e s s i o n P a t t e r n s t h a t a r e D i f f e r e n t from t h o s e of SSP Genes. T a b l e I summarizes t h e r e s u l t s of N o r t h e r n g e l b l o t a n a l y s i s of polyA+RNA from embryos a t 8, 13 and 25 DPF w i t h r e s p e c t t o t h e e x p r e s s i o n o f a c t i n , t u b u l i n , and β-LOX. The degree o f e x p r e s s i o n o f r e s p e c t i v e t r a n s c r i p t s a r e r e p o r t e d as p e r c e n t of maximum cpm of probe h y b r i d i z i n g t o t o t a l RNA. Each of t h e s e genes shows t h e same general expression p a t t e r n although a c t i n r a d i o a c t i v i t y d e c l i n e s more r a p i d l y than t h a t o f t u b u l i n , w h i c h i n t u r n d e c l i n e s more r a p i d l y than β-LOX. T a b l e I . E x p r e s s i o n p a t t e r n s f o r a c t i n , t u b u l i n , and β-lipoxygenase genes d u r i n g s u n f l o w e r embryogenesis

Gene Actin Tubulin β-LOX a

Transcript size 1.7 kb 1.6 kb 4.0 kb

Expression l e v e l at i n d i c a t e d DPF 8 13 25 100 43 30 100 60 25 100 65 50

T h e s e numbers a r e t h e average of two s e p a r a t e measurements.


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F i g u r e 1. Dot b l o t a n a l y s i s of HaG5 and HaGlO e x p r e s s i o n . One Ug o f t o t a l RNA from embryos a t t h e i n d i c a t e d day p o s t - f e r t i l i z a t i o n (DPF) was bound t o each dot on n i t r o c e l l u l o s e and t h e dot b l o t s were then h y b r i d i z e d t o t h e a p p r o p r i a t e probe, washed and exposed t o f i l m o v e r n i g h t . "Dry" = mature d r y s e e d , Germ. = g e r m i n a t i n g d r y seed.

F i g u r e 2. N o r t h e r n g e l b l o t of t o t a l RNA from s t a g e d embryos. Numbers i n d i c a t e t h e r e l a t i v e cpm o f probe bound ( e x p r e s s e d as p e r c e n t o f maximum) a t each s t a g e o f development f o r HaG5 and HaGlO.


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P r e v a l e n c e o f S p e c i f i c T r a n s c r i p t s i n Heterogeneous N u c l e a r (hn) RNA I s o l a t e d from 32p-UTP-Labeled N u c l e i . T a b l e I I shows t h e r e s u l t s o f measurement o f t r a n s c r i p t l e v e l s i n hnRNA from d e v e l o p m e n t a l l y s t a g e d n u c l e i l a b e l e d w i t h 32UTP. Both HaG5 and HaGlO n u c l e a r RNA r e a c h a peak a t 13 DPF and d e c l i n e t o v e r y low l e v e l s by 25 DPF. $-LOX t r a n s c r i p t l e v e l s are h i g h e s t i n 8 DPF n u c l e i and d i m i n i s h i n a manner s i m i l a r t o the steady s t a t e RNA l e v e l s ( T a b l e I ) . N e i t h e r a c t i n nor t u b u l i n t r a n s c r i p t s c o u l d be d e t e c t e d above background i n t h e s e e x p e r i m e n t s . V a l u e s shown are p e r c e n t of maximum cpm o f hnRNA from n u c l e i o f d i f f e r e n t DPF embryos t h a t h y b r i d i z e d t o s l o t b l o t s c o n t a i n i n g a t l e a s t a 1 0 0 - f o l d excess o f c l o n e DNA s p e c i f i c t o the gene i n d i c a t e d . Table I I . R e l a t i v e rates of t r a n s c r i p t i o n f o r d i f f e r e n t genes d u r i n g embryogenesis Relative transcription r a t e a t i n d i c a t e d DPFa Gene HaG5 HaGlO 3-LOX a

8 10 10 100

13 100 100 60

25 5 5 10

V a l u e s a t d i f f e r e n t DPF f o r each gene may be compared, comparisons between genes a t s p e c i f i c DPF a r e not v a l i d . Numbers a r e the average o f two d i f f e r e n t e x p e r i m e n t s .

Discussion The genes i n t h i s s t u d y may be d i v i d e d i n t o two groups a c c o r d i n g t o o v e r a l l e x p r e s s i o n p a t t e r n s d e t e r m i n e d by measuring s t e a d y s t a t e mRNA l e v e l s . The SSP genes show a d e f i n i t e peak i n e x p r e s s i o n a t around 13 DPF w h i l e the a c t i n , t u b u l i n , and β-LOX mRNAs appear t o peak e a r l y i n embryo development (at o r b e f o r e 8DPF) and t h e n g r a d u a l l y d e c r e a s e through 25 DPF. These two groups a l s o d i f f e r i n the a b s o l u t e abundance o f t h e i r mRNAs. SSP gene mRNAs were e a s i l y d e t e c t a b l e i n t o t a l RNA whereas the use o f polyA+RNA i s r e q u i r e d f o r the d e t e c t i o n o f a c t i n , t u b u l i n , and β-LOX t r a n s c r i p t s . D i f f e r e n c e s i n the s t r u c t u r e o f t h e s e genes and t h e i r c o r r e l a t i o n t o e x p r e s s i o n p a t t e r n and l e v e l i s c u r r e n t l y under i n v e s t i g a t i o n . The most i n f o r m a t i v e comparison i s t h a t o f s t e a d y s t a t e mRNA l e v e l s t o the r e l a t i v e t r a n s c r i p t i o n r a t e f o r each gene. I n the c a s e o f b o t h SSP genes, the d i f f e r e n c e i n the r e l a t i v e t r a n s c r i p t i o n r a t e a t 8 and 13 DPF i s q u a l i t a t i v e l y s i m i l a r t o the r e l a t i v e s t e a d y s t a t e l e v e l s o f mRNA. However the d i f f e r e n c e i n the l e v e l o f t r a n s c r i p t i o n between 8 DPF and 13 DPF i s l a r g e compared t o t h e d i f f e r e n c e i n s t e a d y s t a t e mRNA l e v e l s between t h e s e p o i n t s . This s u g g e s t s t h a t b o t h t r a n s c r i p t i o n a l and p o s t - t r a n s c r i p t i o n a l controls a r e o p e r a t i v e . The l a t t e r p o s s i b l y i n v o l v e s mRNA s t a b i l i t y w i t h i n the c e l l . I n the case o f the 3-LOX gene the t r a n s c r i p t i o n a l l e v e l s f o l l o w the same p a t t e r n as the steady s t a t e mRNA l e v e l s i n d i c a t i n g a l m o s t e x c l u s i v e l y t r a n s c r i p t i o n a l c o n t r o l o f t h i s gene. For b o t h


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a c t i n and t u b u l i n t h e l e v e l s o f t r a n s c r i p t i o n were u n d e t e c t a b l e i n s i m i l a r e x p e r i m e n t s b u t t h e s e t r a n s c r i p t s were e a s i l y d e t e c t a b l e i n s t e a d y s t a t e mRNA. Thus p o s t - t r a n s c r i p t i o n a l c o n t r o l o f mRNA l e v e l s may be i m p o r t a n t f o r t h e s e genes. The SSP genes s t u d i e d h e r e b e l o n g t o s m a l l gene f a m i l i e s , v a r i o u s members w h i c h w i l l c r o s s - h y b r i d i z e w i t h t h e probes used Ç5,6) . These r e s u l t s thus r e f l e c t c o n t r i b u t i o n s from more than one gene i n each f a m i l y . This i s probably true f o r the a c t i n ( 9 ) , t u b u l i n ( 1 0 ) , and β-LOX (14) genes as w e l l . The r e l a t i v e i m p o r t a n c e of t r a n s c r i p t i o n a l o r p o s t - t r a n s c r i p t i o n a l r e g u l a t i o n o f e x p r e s s i o n may v a r y f o r i n d i v i d u a l genes b u t t h e methods used h e r e do n o t a l l o w us t o d i s t i n g u i s h between d i f f e r e n t gene f a m i l y members. Gene s p e c i f i c probes a r e b e i n g c o n s t r u c t e d f o r some o f t h e s e genes f o r use i n s t u d y i n g d i f f e r e n t i a l r e g u l a t i o n o f members o f t h e same family. Our r e s u l t s suggest t h a t a h i e r a r c h y o f gene r e g u l a t i o n i s o p e r a t i v e d u r i n g h i g h e r p l a n t growth and development and f u r t h e r m o r e t h a t t h e r e l a t i v e importance o f each h i e r a r c h i c a l l e v e l depends on t h e p a r t i c u l a r genes i n v o l v e d . I t may be t h a t d i f f e r e n t l e v e l s of c o n t r o l a r e u t i l i z e d f o r d i f f e r e n t genes because o f s p e c i f i c p h y s i o l o g i c a l consequences. P r e s e n t s t u d i e s a r e f o c u s i n g on t h e r o l e o f t r a n s - a c t i n g f a c t o r s and c i s - a c t i n g elements i n d i f f e r e n t i a l gene e x p r e s s i o n i n s u n f l o w e r . Our u l t i m a t e g o a l i s t o u n d e r s t a n d , i n d e t a i l , t h e mechanisms t h a t c o n t r o l gene e x p r e s s i o n and t h e i r p h y s i o l o g i c a l consequences i n h i g h e r p l a n t s . Literature Cited 1.

Davidson, Ε. H. "Gene Activity in Early Development", 3rd ed.; Academic Press: New York, 1986, pp. 1-44. 2. Cabrera, C. V., Ellison, J. W., Moore, J. G., Britten, R. J., Davidson, Ε. H. J. Mol. Biol. 1982 179, 75-111. 3. Infante, Α. Α., and Heilmann, L. J. Biochemistry 1981 20, 1-8. 4. Allen, R. D., Nessler, C. L., Thomas, T. L. Plant Molec. Biol. 1985 5, 165-173. 5. Cohen, E. A. Masters thesis, Texas A&M University, 1986. 6. Allen, R. D., Cohen, Ε. Α., Vonder Haar, R. Α., Orth, Κ. Α., Ma, D. P., Nessler, C. L., Thomas, T. L. In: Molecular Approaches to Developmental Biology; Firtel, R. Α.; Davidson, Ε. H., Eds.; Alan R. Liss Inc.: New York, 1987; pp. 145-424. 7. Allen, R. D., Vonder Haar, R. Α., Cohen, Ε. Α., Adams, C. Α., Ma, D.-P., Nessler, C. L., Thomas, T. L. Mol. Gen. Genet. 1987 210: 211-218. 8. Aviv, H., Leder, P. Proc. Natl. Acad. Sci. USA 1977 69: 1408-1412. 9. Shah, D. P., Hightower, R. C., Meagher, R. B. Proc. Natl. Acad. Sci. USA 1982 79, 1022-1026. 10. Guiltinan, M. J., Velten, J., Bustos, M. M., Cyr, R. J., Schell, J., Fosket, D. E. Mol. Gen. Genet. 1987 207, 328-334. 11. Luthe, D. S., and Quatrano, R. S. Plant Physiol. 1980 65, 305-308. 12. Lee, J. J., Calzone, F. J., Britten, R. J., Angerer, R. C., Davidson, Ε. H. J. Mol. Biol. 1986 188, 173-183.


17. ADAMS ET AL.


13. Maniatis, T., Fritsch, E. F., Sambrook, J. Molecular Cloning. A Laboratory Manual; Cold Spring Harbor, New York, 1982, p. 331. 14. Start, W. G., Ma, Y., Polacco, J. C., Hildebrand, D. F., Freyer, G. Α., Altschuler, M. Plant Molec. Biol. 1986 7, 11-23. RECEIVED May 25, 1988


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Regulation of Gene Expression in the Sunflower Embryo - American

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