Regulation of α-amylase-encoding gene expression in germinating seeds and cultured cells of rice

Gene, 122 (1992) 247-253 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

247

0378-1119/92/$05.00

06832

Regulation of a-amylase-encoding cultured cells of rice (Recombinant

DNA;

gibberellic

acid treatment;

Su-May Yu a,*, Wen-Shyong Ray Wud a Institute of Molecular Biology, Academia

gene expression in germinating

aleurone;

enhancer;

Tzou a,b, Wan-Sheng

DNA mobility-shift

Lo a, Yen-Hong

seeds and

assay)

Kuo a,c, Hung-Tu

Leeb and

Sinica, Nankang, Taipei, Taiwan, Republic of China, 11529; b Institute of Life Science, National Tsing-Hua University,

Hsinchu, Taiwan, Republic of China, 30043; ‘Department

of Agronomy,

National Taiwan University, Taipei, Taiwan, Republic of China, 10764; d Section of

Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853. USA. Tel. (607) 255-5710: Fax (607) 255-2428 Received

by J.L. Slightom:

19 June 1992; Accepted:

2 August

1992; Received

at publishers:

7 September

1992

SUMMARY

Four a-amylase-encoding cDNA (&olmy-C) clones were isolated from a cDNA library derived from poly(A)+RNA of gibberellic acid (GA,)-treated rice aleurone layers. Nucleotide sequence analysis indicates that the four cDNAs were derived from different aAmy genes. Expression of the individual aAmy gene in germinating seeds and cultured suspension cells of rice was studied using gene-specific probes. In germinating seeds, expression of the &my genes is positively regulated by GA, in a temporally coordinated but quantitatively distinct manner. In cultured suspension cells, in contrast, expression of the aAmy genes is negatively and differentially regulated by sugars present in the medium. In addition, one strong and one weak carbohydrate-starvation-responsive &my genes have been identified. Interactions between the promoter region (HS501) of a rice aAmy gene and GA,-inducible DNA binding proteins in rice aleurone cells were also studied. A DNA mobility-shift assay showed that the aleurone proteins interact with two specific DNA fragments within HS501. One fragment is located between nt -131 to -170 and contains two imperfect directly repeated pyrimidine elements and a putative GA,-response element. The other fragment is located between nt -92 to - 130 that contains a putative enhancer sequence. The interactions between aleurone proteins and these two fragments are sequence-specific and GA-responsive.

INTRODUCTION

During germination of a cereal grain, the embryo synthesizes gibberellins which diffuse to the aleurone cells and

Correspondence to: Dr. S.-M. Yu, Institute

of Molecular

demia Sinica, Nankang, Taipei, Taiwan, Republic Tel. (886)2-789-9209; Fax (886)2-782-6085. Abbreviations:

aAmy,

r-amylase;

&my-C, cDNA clone of c&q; GARE, GA-response element;

Biology,

of China,

c&my, gene (DNA)

Aca-

11529.

encoding

aAmy;

bp, base pair(s); GA,, gibberellic acid; kb, kilobase or 1000 bp; nt, nucle-

otide(s); OSamy, Oryza sativa sc-amylase gene; RAmy, rice cr-amylase gene; OS, Ovyza saliva; SDS, sodium dodecyl sulfate.

act as a signal to activate the synthesis and secretion of aAmy and other hydrolases. These enzymes digest the starch stored in the endosperm and provide sugars for the growth of young seedlings. Little is known of the molecular mechanisms which connect GA with the activation of OrAmy gene expression. The effects of GA on aAmy gene transcription were demonstrated from runoff transcription experiments using nuclei from barley (Jacobsen and Beach, 1985) and oat (Zwar and Hooley, 1986) aleurone protoplasts and from transient expression experiments using c&my promoter-reporter gene constructs in aleurone protoplasts (Huttly and Baulcombe, 1989; Salmenkallio et al., 1990; Jacobsen and Close, 1991; Skriver et al., 1991). The transient expression system has also been applied to iden-

248 tify a conserved GARE in the promoter regions of two barley aAmy genes (Lanahan et al., 1992; Skriver et al., 1991). Using gel retardation assays, putative transcription factors which bind to the 5’ noncoding region of a low p1 rice Wzmy gene were identified (Ou-Lee et al., 1988; Yu et al., 1990). The results suggest that activation of aAmy synthesis by GA may be mediated by the interaction of protein factors with specific sequences in the promoter regions of the &my genes. Previously, we reported that in addition to the hormonal regulation in germinating seeds, the expression of &my genes in rice is also subject to metabolic repression in cultured cells by the availability of carbohydrate nutrients (Yu et al., 1991). To understand how GA and sugars regulate dmy gene expression in rice, it is important to identify &my cDNA clones representing different aAmy genes. These clones, in turns, would be used to isolate their corresponding genomic clones. We cloned four c&my cDNAs, and their gene-specific probes were used to study the expression of cdmy genes in germinating seeds and cultured cells of rice. In addition, to understand better the interactions between the promoter region of a rice dmy gene and the GA-inducible aleurone proteins present in the rice seeds, DNA mobility-shift assays were performed to identify trans-acting proteins and c&acting sequences required for dmygene expression. Two specific DNAregions which interact with the GA-dependent protein factors were identified.

1

a?imyC-C *my.9-C aAmy7-c _Yl(j_C

60

CCCGGGA~~ccTn;cLyjcTGAGGGAGAc GGXNXATGG * ***r***C****G*C*****CAAG**C*****G*htAC******* *******c**G*~G**G***G*cGc****~*G*~*****~*~**~****~****~ l******C**G*c****G*****cGT*+rA*G*A***********G

uAmyC-C LLWIYB-c aAmy7-c aAmylO-c

dmy6-C aAmys-c aAmy7-C aAnlylO-c 181

****G*******CC**‘PAf*G******m

a~my6-C a~my8-C aAnly7-c @amylO-c

G*GAGGA*CAG

2

TCCGA*****c&*m*AATn;TCCA**m* l-c-rGA*C**TG**cG*GAmccA*AAA*

*T*CCTc*GT~ *T*ccTc*GT~ “Scar” “PWI”

G*GAGGA***G

aiimyb-C a~my8-C aAmy7-c aAmy10-c

aAmy8-C aAmy7-C

Fig. 1. The nt sequences

programs

ofthe 3’ regions of four rice aAmy cDNA clones.

was performed

nique. The nt sequence AND DISCUSSION

*cT*T*GcGATc*AGT*G**

a~myb-C WAmys-c aAmy7-C aAmy10-c

DNA sequencing RESULTS

240

l-mxa3AGAAaA-GGAmAm rnGA~CTGCAYGGGC_CcArnA

a_amyb-C a~my8-C aAmy7-c aAmy10-c

from the Sequence

Computer

Group,

with the dideoxy chain-termination

analysis

and comparisons

Analysis

University

Software

of Wisconsin,

tech-

were carried out using Package

Version

of the Genetics

5.0, June 1987. Se-

(a) Cloning and characterization of the rice cDNA The rice cDNA library was screened with the c&my gene OSamy-c (Kim and Wu, 1992) as the probe. Four of the &my cDNA clones showing different restriction patterns were chosen for subcloning into the plasmid vector pBluescript. The resultant clones were designated as &my&C, olAmy7-C, dmy8-C, and dmylO-C with insert sizes of 0.6, 1.0, 1.4, and 1.5 kb, respectively. The 3’-end regions of these cDNA clones were further subcloned and sequenced (Fig. 1). The sequenced 3’ regions of aAmyC, dmy7-C, and c&my&C are identical to those of the reported rice dmygenes RAmy3B (SutliEet al., 1991), RAmylA (Huang et al., 1990a), and RAmy3E (Huang et al., 1990b), respectively. The genomic DNA corresponding to aAmylO-C has not yet been reported.

quences

(b) Construction of the rice aAmy gene-specific probes Comparison of nt sequences of the 3’ untranslated regions shows very low identity (23-27x) among the four c&my cDNA clones (Fig. l), except dmy7-C and WlmylOC which showed 69% identity. Restriction sites were selected for separation of the nonhomologous (gene-specific)

library in l,gtll using Amersham’s cDNA synthesis and cloning systems. The cDNA library consisted of approximately 2 x 10’ independent recombinant clones. Approx. 2 x lo4 plaques were screened, using the 32P-

sequence

are aligned, identity.

star. The translation

and gaps (dash lines) are introduced

Identical

to maximize

nt among the four clones are indicated

stop codons

and polyadenylation

by a

signals are under-

lined. The 5’ boundaries of the gene-specific regions are indicated arrowheads and the restriction enzymes used for DNA truncation indicated

below their corresponding

sites. The nt sequence

by are

is numbered

from the first nt ofthe sequenced regions. Accession number for rAmylO-C in GenBank, EMBL, and DDBJ is M81143. Methods: conditions for preparation of aleurone RNA, construction of the cDNA library, and screening for xAmy cDNA clones were as follows. Rice (Ovyzae sativacv. Labelle)

seeds were surface

min, washed

Na,hypochloride

for 20

extensively with sterile distilled H,O, and incubated

in sterile

10 pM GA,/20

sterilized

mM CaC1,/20

in 2.5%

mM Na.succinate

for different lengths of

time. The germinating embryos were cut off and the aleurone layers were peeled off the endosperm. The collected aleurone layers were immediately frozen in liquid N, and stored at -70°C until use. Total RNA was isolated from the frozen aleurone layers according to the method of Belanger et al. (1986). Poly(A)‘RNA (1 pg) was purified with HYBOND-mAP affinity paper (Amersham). Poly(A) + RNA was used to construct a cDNA

labeled 1.5.kb fragment of the rice genomic clone, OSamy-c (Kim and Wu, 1992), as the probe. The cDNA clones in lgtll were cleaved with EcoRI and subcloned into the EcoRI site of pBluescript and maintained in E. coli strain XLl-B

(Stratagene,

La Jolla, CA).

249

&my

&my

Chly

afTmy

6-C 7-C 8-C ,&Cos::y6-C 7-C 8-C ,0-C"%my6-C 7-C 8-C IO-C"?'6-C

Clone Probe

_____

aRmy6-G-3

Panel

1

2

7-C B-C ,CI-C~~-?~~-C7-C 8-C ,0-c""-","" aRmy8-G-3

aRmy7-G-3'

’

dtmy

aRmylO-G-3’

’

4

3

5

tlarker (kb) 3,02,01.61,00,5-

Fig. 2. Southern OSarny-c

blot analysis

was digested

demonstrating

specificity

with BamHI+EcoRI,

of the same gel as shown in panel 1 were blotted h. After hybridization, vectors

the membranes

were also hybridized

of pBluescript for preparation the restriction

were washed

(the 3-kb bands)

between the T3 promoter of 32P-labeled gene-specific enzymes

indicated

of the ClAmy gene-specific

then electrophoresed to GeneScreen

membranes,

probes.

the antisense

and hybridized

RNA probes

site where the cDNAs

scripts of sizes 210, 112, 119, and 50 nt, representing (Amersham, SP-6 tested) was used to label the probe.

contained

were inserted.

of the four truncated

&my6-C-3’,

clAmy7-C-3’,

regions from the homologous regions of these four cDNA clones and for the preparation of antisense RNA probes. The restriction enzymes used and the nt sequences of.the gene-specific regions are given in Fig. 1. The gene-specific sequences corresponding to each of the four cDNAs were designated as c&my&C-3’, uAmy7-C-3’, dmy8-C-3’, and uAmy1 O-C-3’. Appropriate regions were selected for aAmylO-C-3’ which had little identity with cxAmy7-C-3’. Cross-hybridizations were then performed to determine the gene-specificity, and the results showed that each probe only hybridizes to its respective parental cDNA (Fig. 2). None of these gene-specific probes hybridized to OSamyc, which was originally used as the probe to screen the cDNA library. The results demonstrated that the four genespecific probes axe able to discriminate different aAmy genes. Similar gene-specific probes for these genes were not available from other reports. (c) Expression of aAmy genes in germinating seeds of rice To determine whether the expression of different members of the t&my gene family are regulated in the same manner during seed germination, gene-specific probes were used to study the expression of individual dmy genes in GAS-treated germinating seeds. The accumulation of &my mRNA in aleurones as a function of time after GA, addition was determined by RNA blot analysis (Fig. 3A). A probe made from OSamy-c containing the coding region of a rice cdmy gene was supposed to hybridize to mRNAs of most, if not all, dmy genes. The &my mRNA was barely

cDNAs

were digested

bromide.

with the 32P-labeled

M Na,.citrate

gene-specific

oIAmy&C-3’,

probes

and

at 42°C for 12

of the multiple

are shown at the left margin.

were truncated

cDNAs

with EcoRI,

(Panels 2-5) Four replicates

pH 7.6) and 0.1% SDS at 55°C for 40 min. The

a stretch of 62-bp sequences

M, markers

probes were as follows. The four “Imy cDNAs

in Fig. 1. In vitro transcription

(Panel 1) The &my

gel, and stained with ethidium

in 0.1 x SSC (0.15 M NaC1/0.015

because

and EcoRI

on 1% agarose

at the 5’ ends of the gene-specific

with T3 RNA polymerase and ClAmylO-C-3’,

cloning

Methods:

regions using

yields antisense-strand

respectively.

Finally,

sites

conditions tran-

[x-~‘P]UTP

detectable at day 1, rapidly accumulated and reached their maximal levels at day 4, then rapidly turned over between days 4 and 5. A rice actin cDNA clone, pcRAc1.3 (McElroy et al., 1990), whose expression was not appreciably affected by GA was used as an internal control. The level of mRNA shown in Fig. 3A was quantified by measuring the signal intensity of the autoradiogram using a densitometer. The relative mRNA accumulation of each dmy gene on each day was determined by comparison of the mRNA levels with their peak level at day 4 (Table I). The mRNA of each aAmy gene accumulated at a similar rate, except for that of rxAmy8-C which almost reached the peak level by day 3. However, the mRNAs of ctAmy6-C and dmy8-C were turned over at higher (twofold) rates than those of cdmy7-C and dmylO-C . At day 5, the mRNA levels of cxAmy7-C and dmylO-C were reduced by half; in contrast, those of aAmy&C and dmy8-C were reduced to one-fourth of their highest levels. After this time, all the mRNA levels decreased at similar low rates. The results show that expression of the four cdmy genes in germinating seeds are temporally coordinated but quantitatively distinct. (d) Expression of aAmy genes in cultured suspension cells of rice We have already shown that the expression of &my genes in cultured suspension cells of rice is induced by the deprivation of carbohydrate nutrient (Yu et al., 1991). In that report, OSamy-c was used as a probe to study the

250

Days after Germination

(A)

TABLE

I

Relative accumulation

of &my

tected by &my gene-specific

123456

Probe

Probes

mRNA

in germinating

Days after germination

a

1

2

3

4

OSamy-c

0

25

13

sulmy6-C-3’

0

23

13

&4my7-C-3’ &4my8-C-3’

0 0

26 31

&4mylO-C-3’

0

23

5

6

100

61

41

100

27

26

12

100

98 64

100

50 27

48 23

100

41

44

osamy-c aAmy6-C-3’ aAmy7-G3’

a Level of dmq)mRNA

aAmySG3’

autoradiograms pcRAcl.3.

aAmylO-C-3’

mRNA

HK350 pcRAcl.3

(B) Probe

Size

Days in Culture

1500

bp

cAmy6-C-3’

210

bp

dmy7-C3’

112

bp

orAmy8G3

119

bp

OSam y-c

dmylO-C3’

poscx-3’

50 bp

174

bp

Fig. 3. Accumulation of &my mRNA in germinating seeds and cultured suspension cells of rice. (Panel A) Time course of accumulation of &4my mRNA in GA,-treated rice seeds. (Panel B) Relative mRNA levels of the &my genes in the cultured suspension cells of rice during later growth stages. Rice seeds were germinated in 10 nM GA, for different lengths of time. The germinating embryos were cut off, and the total aleurone RNA was purified from the embryoless half seeds, according to the method of Belanger et al. (1986). Rice suspension cells were cultured as described previously (Yu et al., 1991). RNA was purified from cells grown in the sucrose-containing medium for 8, 10, 12, and 14 days. Total RNA (5 ng) was applied to each lane. The RNA dot blot analysis was performed according to the method of Thomas (1983). The 1.5.kb OrAmy DNA in-

was determined

by densitometric

shown in Fig. 3A and corrected

The relative mRNA

then determined

by dividing

rice seeds as de-

probes

accumulation

the &my

scanning

with the mRNA for each &my

mRNA

of the level of

gene was

level of each day by the

level (peak level) of day 4.

expression of the entire c&my gene family in suspension cells. Here, gene-specific probes were used to determine the expression pattern of different dmygenes. We have shown that the sugars (analyzed by the anthrone reaction) in the sucrose-containing medium were depleted to almost undetectable levels by day 12. A concomitant increase in &my mRNA was observed on day 12 (Yu et al., 1991). Therefore, RNAs purified from cells grown in the sucrosecontaining medium for 8, 10, 12, and 14 days were used for the RNA blot analysis (Fig. 3B). A cDNA clone randomly chosen from the same cDNA library whose expression was not affected by sugar depletion, pOScx, was used as an internal control. The level of mRNA shown in Fig. 3B was also quantified, and the relative mRNA accumulation of each &my gene every day was determined by comparison of mRNA levels with their basal level at day 8 (Table II). Expression of dmy7-C and olAmy8-C was induced 6- and 37-fold, respectively, on day 12 and continued to increase at day 14. Expression of cxAmylO-C was induced later, with a fivefold increase at day 14. Expression of dmyb-C also increased fourfold on day 12; however, it decreased to basal level by day 14. Expression of another aAmy gene, xAmy3-C, increased fivefold after sugar starvation (S.-M.Y., unpublished result). Therefore, among the five dmy genes examined so far, dmy8-C is the most abundantly expressed gene after sugar depletion. In addition, it is worthwhile noting that &my??-C is one of the major genes whose tran-

sert of O&my-c BamHI+EcoRI, labeled with

(Kim and Wu, 1992) was excised from pBluescript by gel-purified as described in Maniatis et al. (1982) and

[ a-32P]dCTP

using the random

primer method (Feinberg

and

Vogelstein, 1983). The gene-specific probes corresponding to each of the four rice wimy cDNAs were prepared and labeled as described in Fig. 2. The size of the mRNA

detected

by all of the probes

is 1.6 kb.

251 TABLE

II

binding

Relative accumulation at later growth

of &my mRNA in cultured

stages as detected

suspension

by cckny gene-specific

cells of rice

probes

Days in culture a

Probes

8

10

12

14

OSamy-c

1.0

3.8

39.5

38.8

&4my6-C-3’

1.0

1.3

4.1

1.2

GCAmy7-C-3’

1.0

1.8

6.2

9.8

c&nys-C-3’

1.0

2.2

37.0

44.5

CYAmylO-C-3’

1.0

1.3

1.2

5.0

could be detected

between

the protein

extract and

fragment A (Fig. 4B, lane 2). Comparison of nt sequences among fragments A, B, and C reveals that they share some similarity (Fig. 4C). It is not clear whether the weak binding of fragment A to the proteins was due to low affinity or nonspecific binding. Nevertheless, the results indicate that there are strong protein binding sites within fragments B and C.

scripts constitute the 40-fold increase of total ovlmy transcripts as detected with a probe of OSamy-c. The results show that expression of the four aAmy genes in response to carbohydrate starvation in the cultured cells is temporally and quantitatively regulated.

(f) GA-dependent and sequence-specific protein factors which hind to HS501 We carried out another protein/DNA binding assay to determine whether or not the DNA-binding protein is GAinducible. Proteins were extracted from the aleurone tissues of de-embryoed seeds which had been treated with or without GA, for three days. Only the GA-treated aleurone extract gave rise to three complexes using fragment B (Fig. 5, lane 4) or C (data not shown) as probes. The aleurone extract did not bind to fragment A (data not shown). No interaction was detected between fragment B and the aleurone extract from an untreated sample (Fig. 5, lane 2). The results indicate that the aleurone proteins which bind to fragments B and C are GA-dependent.

(e) Specific regions of the promoter of a rice aAmy gene interact with protein factors in the GA-treated aleurone layers HS501 is a DNA fragment which is located at the 5’ end promoter region of a rice aAmy gene, OSamy-b (Ou-Lee et al., 1988) and its nt sequence has been presented (Yu et al., 1990). The nt sequence of HS501 was later found to be identical to that of RAmy3C, which encodes a complete rice cdmy isozyme (Sutliff et al., 1991). The nt sequence of HS501 includes 260 bp of the 5’ noncoding region and 270 bp in the first and a part of the second exon. HK350 is a 3’-end-deleted derivative of HS501 and contains the entire 5’ noncoding region (260 bp) and the first exon regions (90 bp) of HS501. RNA blot analysis showed that the c&my mRNA of aleurone cells detected by probing with HK350 was also increased after GA, treatment (Fig. 3A). We have shown that the 5’ end of HS501 is important for a stable protein-DNA complex formation (Ou-Lee et al., 1988; Yu et al., 1990). To localize the protein binding sites in HS501 more precisely, we synthesized three consecutive double-stranded 40-bp oligonucleotides, designated as A, B, and C, which are located at the 5’ end of HS501 (Fig. 4A). Proteins were extracted from the aleurone tissues of GA,-treated germinating seeds, and interactions between aleurone proteins and the synthetic DNA fragments were detected by the gel retardation assay (Fig. 4B). Interaction of the extract with fragments B and C resulted in the formation of complexes B 1, B2, and B3 (Fig. 4B, lanes 4 and 6) even though the Bl band is very weak. Practically no

(g) Conclusions (I) The availability of gene-specific probes corresponding to each of the four aAmy cDNAs has enabled us to examine the abundance of mRNA encoding specific c&my isozymes. Expression of the individual c&my gene was found to be coordinately regulated, and their mRNAs accumulated at similar rates and levels in the aleurone layer of germinating rice seeds. However, differences in the turnover rates of the mRNA of different &my genes indicate a possible differential regulation of the expression of different aAmy genes in germinating seeds. The four &my genes expressed in germinating seeds were expressed constitutively at low levels in cultured cells when sugars were still present in the medium. Expression of three of the four dmy genes was induced after sugars were depleted from the medium, and only dmy6-C displays a different expression pattern from the other three genes. Our preliminary experiments have shown that the turnover of c&my mRNA in germinating seeds and cultured cells involves both transcriptional and posttranscriptional controls (G.S. and S.M.Y., unpublished observation). More detailed investigations are now underway to improve the understanding of the regulatory mechanisms of aAmy gene expression. (2) It is not known whether the GA- and sugar-regulated expression of the same &my genes operate through the same or a different molecular mechanism. Because expression of c&my??-C was GA-regulated in germinating seeds, and it is one of the major metabolite-regulated genes in cultured suspension cells, it would be a good model gene

h Level of “Imy mRNA was determined by densitometric scanning of the autoradiograms shown in Fig. 3B and corrected with the mRNA level of pOScx-3’.

The relative

mRNA

accumulation

then determined by dividing the ovlnzy mRNA mRNA level (basal level) of day 8.

for each c&xy gene was level of each day by the

252 (A)

-209 I

B

-170 !

A

-130 I

-

c

-90 1

-26 I I TATA

0

-GA

+I

+GA

I o_) mRNA

(8)

DNA Probe

A ---

Protein Extract Lane

- + 123456

B

C

+ -

+

Fragment -209 A

-170

TGGAGCCCACAACGCTATCCAAGGCTTTATCTAACTTCCT -169

-130 ******x*

B

~TTGG~TT~T~~TGAGCGCAACTC -129

C

-90

GTCGCCGTGCCGTTGCGTTTCTCGTTAGGAGCAACTGAAC ---__-----_

Fig. 4. Binding of aleurone protein to the 5’-specific DNA fragments elements

corresponding

and a GARE

with fragments boric acid/l

to the sequence

-like element.

DNA fragments

Open box indicates

the position

A, B, and C. The labeled DNA was electrophoresed

mM EDTA.

B2, and B3 indicate

of a rice Amy gene. (A) Fragments

at the 5’ end of HS501.

Filled box indicates of the 1 I-bp putative

positions

of the three protein-DNA

complexes.

enhancer-like

on a 6.8% polyacrylamide

The gel was run for 2.5 h at 12 V/cm. Symbols

+ and - indicate

F indicates

the position

A, B, and C were three consecutive

the position

of two imperfect, element.

gel. The running

reactions

layer extract

Fig. 5. Binding

and DNA mobility-shift

of the GA-inducible

aleurone

(gel-retardation) proteins

de-embryoed rice seeds after 3 days of imbibition to Fig. 4. Symbols + and - refer to the presence complexes.

F indicates

position

assay have been described

to the specific

DNA

fragment

(B) Interaction protein

extract,

of HS501.

proteins

respectively.

of the free DNA probe. (C) The nt sequences

previously

pyrimidine

of aleurone

buffer was 45 mM Tris base/45

with or without

A, B, and C. Numbers indicate positions of the three fragments relative to the transcription start point. Underlining elements. Stars indicate the position of the GARB-like element. Broken line indicates position of the enhancer-like aleurone

40-bp synthetic

directly repeated

mM Bl,

of fragments

indicates positions of the pyrimidine element. Methods for preparation of

(Yu et al., 1990). + GA and

-GA:

protein

extracts

prepared

from

with or without GA,, respectively. The labeled DNA was electrophoresed as described in the legend or absence of protein extract, respectively. Bl, B2, and B3 indicate positions of the three protein-DNA

of the free DNA probe.

for such studies. Molecular mechanisms underlying the two different modes of regulation and interactions between them will be the focus for further studies. (3) Aleurone tissues contain proteins that interact with fragments B and C of HS501 only in the presence of GA. Fragment C contains an ll-bp fragment (5’-GTTGCGTTTCT) from nt - 108 to - 118 which is similar to the animal core enhancer [ GTGGzzl(T)G] (Gillies et al., 1983; Weiher et al., 1983). Fragment B contains two pyrimidine elements (CCTCFTTT) from nt - 145 to - 152 and nt -157 to -164 which are similar to the consensus sequences ($CTTTTF) found in several &my genes of rice,

wheat, barley, and other GA-inducible genes such as j?-glucanase, carboxypeptidase, and aleurain (Huang et al., 1990a). Promoter deletion analysis demonstrated that sequences encompassing two ofthe three pyrimidine elements in the promoter region of a wheat c&my gene, dmy2/54, are required for high level expression and GA, regulation of this gene (Huttly and Baulcombe, 1989). Mutation of the pyrimidine element in the promoter region of a barley drily gene, AmJj32b, significantly decreases both the absolute level of expression and the effect of GA on expression (Lanahan et al., 1992). In addition, the sequence immediately 3’ to the second pyrimidine element in fragment B of

253

HS501, reading 5’-TAAATGAG from nt -138 to -145, shows partial identity with the putative GARE element 5’-TAACAZAZ (Huang et al., 1990a; Lanahan et al., 1992), which is shown to mediate the hormonal regulation of &my gene expression (Skriver et al., 1991; Lanahan et al., 1992). Further studies involving footprinting experiments and in vivo functional assays for the protein/DNA interacting sites in the promoter region will be necessary for determining whether the GA-responsive proteins, the pyrimidine elements, and the putative GARE element repre-

Huttly,

sent the trans- and &-regulatory elements responsible for GA stimulation of the rice aAmy gene expression in germinating seeds.

Lanahan,

A.K. and Baulcombe,

lated by gibberellin

oat aleurone

is regu-

protoplasts.

EMBO

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