Developmental and Hormonal Regulation of Rice a-Amylase ...gene, gusA. The resulting promoter-gusA fusions, pE4/CUS (-232 to +31) and pH4/CUS (-748 to +31), were used separately to

Plant Physiol. (1995) 107: 25-31

Developmental and Hormonal Regulation of Rice a-Amylase( RAmy1A)-gusA Fusion Cenes in

Transgenic Rice Seeds

Kimiko Itoh, Junji Yamaguchi, Ning Huang, Raymond 1. Rodriguez, Takashi Akazawa, and Ko Shimamoto'*

Plantech Research Institute, 1 O00 Kamoshida, Aoba-ku, Yokohama 227, Japan (K.I., K.S.); BioScience Center, Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464-01, Japan (J.Y., T.A.); and Department of

Genetics, University of California, Davis, California 9561 6 (N.H., R.L.R.)

Transgenic seeds of rice (Oryza sativa 1.) were used to investigate temporal, spatial, and hormonal regulation of a rice a-amylase gene, RAmylA. Two overlapping segments of the RAmylA promoter were fused to the coding region of the bacterial reporter gene, gusA. The resulting promoter-gusA fusions, pE4/CUS (-232 to +31) and pH4/CUS (-748 to +31), were used separately to transform rice protoplasts. P-Clucuronidase (CUS) activity was detected in germinated transgenic seeds, although the two constructs showed no significant difference in timing or location of CUS expression. Both constructs first expressed CUS in the scutellar epithelium and then in the aleurone layer. Aleurone expression of CUS activity was strongly induced when embryoless half-seeds were treated with gibberellic acid. CUS expression in the aleurone layer was also suppressed by abscisic acid. These results indicate that the 5 ' regulatory region from -232 to +31 is sufficient for temporal, spatial, and hormonal regulation of RAmylA gene expression.

During cereal seed germination, a-amylase plays an important role in hydrolyzing endospermal starch into metabolizable sugars, which, in turn, supports the growth of young roots and shoots (Akazawa and Hara-Nishimura, 1985; Beck and Ziegler, 1989).

Expression of cereal a-amylase genes involves a complex signal transduction pathway that ultimately controls the transcription of a-amylase genes through the action of hormones (Chrispeels and Varner, 1967; Jacobsen and Beach, 1985) and metabolites (Yu et al., 1991: Karrer and Rodriguez, 1992). The question of how hormones such as GA, and ABA regulate the spatial and temporal expression of a-amylase genes still remains unanswered. Expression studies with isolated aleurone protoplasts provide much of the available information on cis-elements involved in the hormonal regulation of these genes. Deletion studies (Huttly and Baulcombe, 1989; Jacobsen and Close, 1991), site-directed mutagenesis (Gubler and Jacobsen, 1992), and gain-of-function experiments using a minimal promoter

Present address: Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-01, Japan.

* Corresponding author; e-mail [email protected]; fax 81-7437-2-5509.

25

fragment (Skriver et al., 1991) have identified a sequence (TAACAAA) as the GARE. This element is one of three highly conserved sequences previously identified (Huang et al., 1990) in the promoters of severa1 cereal a-amylase genes. Additional evidence that TAACAAA is involved in GA, regulation comes from particle-bombardment studies of intact barley aleurone layers (Lanahan et al., 1992). The presence of a second element, TATCCAC/T motif (box I), or 02S, which acts cooperatively with the GARE for full GA, induction, was identified in the promoters of barley a-amylase genes (Gubler and Jacobsen, 1992; Rogers and Rogers, 1992). Also, the pyrimidine box (CCTTTT), which is one of the three sequences highly conserved in cereal a-amylase genes (Huang et al., 1990), was shown to be involved in the maximal induction of a-amylase genes by GA, in barley (Lanahan et al., 1992) and wheat (Huttly and Baulcombe, 1989).

Proteins that interact in a sequence-specific manner with rice a-amylase promoters have been detected in the extracts of embryoless half-seeds (Ou-Lee et al., 1988; Kim et al., 1992; Yu et al., 1992). These studies indicated that one of the promoter elements that interacts with proteins is the pyrimidine box. Similarly, extracts of oat aleurone cells contain proteins that interact with five distinct sequences within the promoter region of a wheat a-amylase gene, including the pyrimidine box and the GARE (Rushton et al., 1992). More recently, Sutliff et al. (1993) demonstrated that nuclear extracts obtained from barley aleurone layers contained a fraction that exhibited a binding activity when incubated with GA, toward the GARE and TATCCAC/T motif (box I) of a barley low-pI a-amylase gene (Amy32b). The DNase I footprinting experiments using this protein fraction showed that GARE and box I were specifically protected. However, no binding activity toward the pyrimidine box was detected in the barley nuclear extracts used in this study (Sutliff et al., 1993). The observation that different a-amylase genes in a given species are differentially regulated by hormones (Nolan et al., 1987; Chandler and Jacobsen 1991) suggests that the molecular mechanisms underlying this regulation are complex and will require further study.

Abbreviations: GARE, GA,-responsive element; gusA, p- glucuronidase gene; X-glucuronide, 5-bromo-4-chloro-3-indoyl glucuronide.

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26 ltoh et al. Plant Physiol. Vol. 107, 1995

Compared with our understanding of hormonal regulation, less is known about the temporal and spatial regulation of cereal a-amylase gene expression during seed germination. It is well established that the aleurone layer is the major site of a-amylase expression, although significant levels of a-amylase synthesis do occur in the scutellum of germinating seeds (Akazawa and Hara-Nishimura, 1985). In germinating rice seeds a-amylase is first detected in the scutellum of the embryo, and later activity extends to the aleurone layer (Okamoto and Akazawa, 1979; Okamoto et al., 1980). This observation has been confirmed by RNA blot analysis (Karrer et al., 1991) and in situ hybridization (Ranjhan et al., 1992) using gene-specific hybridization conditions. Although these investigations demonstrated temporal and spatial regulation of a-amylase gene expression, molecular mechanisms responsible for such regulation were not elucidated.

The ability to transform rice (Oryza sativa L.) (Shimamoto et al., 1989; Hodges et al., 1991) provided an opportunity to further investigate the temporal and spatial regulation of gene expression in the cereals. Transgenic rice has been used to demonstrate regulated expression of promoter- gusA (GUS) gene fusions in various tissues during development (Kyozuka et al., 1991, 1993; Zhang et al., 1991; Terada et al., 1993). To gain a better understanding of molecular mechanisms controlling rice a-amylase gene expression, we have used two segments of the promoter for the rice a-amylase gene, RAmyZA, to drive the expression of the gusA reporter gene in transgenic rice seeds. Results of analysis on developmental as well as hormonal regulation of the fusion genes during germination of transgenic seeds will be presented.

MATERIALS A N D METHODS

Plasmids

Plasmids were constructed by standard recombinant DNA methods (Ausubel et al., 1989; Sambrook et al., 1989). The promoter for the RAmyZA gene was subcloned as a 2.3-kb EcoRI fragment from the rice genomic DNA clone (hOSg2) into the vector pBluescript M13+KS. The nucle- otide sequence and other characteristics of the gene have been reported elsewhere (Huang et al., 1990). The principal features of these constructs consist of gusA, together with the transcriptional termination sequence of the nopaline synthase gene from pBIlOl (Jefferson, 1987), inserted into the SmaI site of pBluescript (pBSGUS). Promoter-gusA fusion genes were constructed by inserting restriction fragments containing the RAmyZA promoter into pBSGUS. The restriction fragments used to make constructs were the PstI-HindIII fragment (-748 to +31, pH4/GUS) and the PstI-EcoRI fragment (-232 to +31, pE4/GUS). The coordi- nates used to describe these restriction fragments are based on the transcription start point for RAmyZA (Huang et al., 1990).

Rice Transformation

RAmylA/GUS plasmids were co-transformed into rice protoplasts (Oryza sativa L. japonica varieties Nipponbare,

Kinuhikari, and Toride-1) with the hygromycin phospho- transferase gene selection marker by electroporation as previously described (Shimamoto et al., 1989; Kyozuka and Shimamoto, 1991). Hygromycin B-resistant 'calli were screened for GUS activity by incubating porticns of calli with X-glucuronide solution (Jefferson, 19117). GUS- positive calli were further cultured and plants were regenerated from these cultures.

Southern Blot Analysis

Plants obtained from R, seeds that exhibited positive GUS staining were used for Southern blot analysis. For each of the H4/GUS and E4/GUS genes, two independent transgenic lines were used. Total genomic DNA was isolated from mature leaves, digested by restriction enzyme HindIII, and then transferred onto a positively charged nylon membrane (Amersham). The cod ing region of the p s A gene was labeled and amplified with digoxi- genin-11-dUTP by PCR and used for probing the intact RAmyZA/GUS genes. Hybridization and chemilumines- cence signal detection were performed according to the manufacturer's specifications (Boehringer Manrtheim).

CUS Analysis

For histochemical analysis of GUS expression, germinating seeds were hand-cut with a razor and stained with X-glucuronide solution as previously described (Kyozuka et al., 1991; Terada et al., 1993). For quantitative GUS analysis, crude extracts from transgenic rice E eeds were prepared for fluorometric analysis of GUS activity as described previously (Kyozuka et al., 1991; Terada et al., 1993). For developmental studies, RI seeds set on primary transgenic plants were peeled off, sterilized with 1% NaOCl for 10 min, and washed with distilled water. Seeds were germinated in plastic wells containing wai er for 2, 4, 6, and 8 d at 30°C under light. For quantitative measurements of GUS activity during germination, seeds were divided into embryo and endosperm portions. [n the case of embryo, residual endosperm, roots, and shoots were removed before the assay. For each developmental stage, more than 20 seeds were analyzed. For the íinalysis of hormonal regulation of the RAmyZAIGUS genes, transgenic R, seeds were deembryonated and the embryoless seeds were sliced longitudinally into three pieces. Each slice derived from a single seed was treated with acetate buffer (10 mM sodium acetate, pH 5.2), 10P7 M GA, in acetate buffer, or 10p7 M GA, plus 10P5 M ABA in acetate buffer, for 4 d at 30°C in the dark. GlJS activity of each treated slice, which included boíh starchy endosperm and aleurone layers, was determined.

RESULTS

lntroduction of RAmylA/CUS Fusion Cenes into Rice

To examine the expression of the a-amylase gene in germinating transgenic rice seeds, the RAmyZA gene of rice

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a-Amylase Gene Expression in Transgenic Rice 27

was chosen because of its responsiveness to GA (O'Neil etal., 1990) and because it is the most active a-amylase geneexpressed during seed germination (Karrer et al., 1991).Two regions of the RAmylA promoter were fused tothe gusA reporter gene (Fig. 1 A) to produce plasmids pH4/GUS (-748 to +31) and pE4/GUS (-232 to +31). Bothpromoter regions contain three conserved sequences(-214CCTTTT-209/ -147TAACAAA-141, and -130TATCC_

AT~124) found in all GA-responsive cereal a-amylase genesexamined to date (Huang et al., 1990). An additionalpyrimidine box is present in pH4/GUS at position -312.These chimeric RAmylA/GUS genes were introduced intorice by electroporation of protoplasts (Shimamoto et al.,1989) and GUS-positive calli were screened with X-glucu-ronide staining. Transgenic plants were subsequently re-generated from the GUS-positive calli and the presence ofintact RAmylA/GUS genes was confirmed by preliminary

PH4/GUS

gusR \T \- PE4/GUS

B

kb C 1 2 3 4 5

12.0-

5.0-4.0-2.9>

1.6-

probe

kb C 1 ? 3 4 5

12.0-

1.6-

Figure 1. Diagram of RArny!A/CDS fusion genes and Southern blotanalysis of transgenic rice plants. A, Diagram of RAmy1A/G(JS genesused for rice transformation. The positions of conserved sequencesindicated in the diagram are -312, -214, -147, and -130 (relativeto the transcription start site) for the distal pyrimidine box (TCMil),the proximal pyrimidine box (CCTTTT), the TAACAAA box, and theTATCCAT box, respectively. The gusA and terminator regions are notdrawn to scale. The lengths of the complete RAmylA/GUS geneswere 2.9 and 2.4 kb for H4/GUS and E4/GUS, respectively. The term"probe" in A indicates the region of the gusA gene used for theSouthern blot analysis. Restriction sites are H, H/ndlll; E, fcoRI. B,Southern blot analysis of selfed progeny derived from plants trans-formed with the gene H4/GUS. Leaf DNA (2 ^g per lane) digestedwith H/ndlll was used, and the location of the probe is shown in A.Lane C, Untransformed control plants; lanes 1 and 2, two progenyplants of line T21; lanes 3 to 5, three progeny plants of line N35. C,Southern blot analysis of selfed progeny derived from plants trans-formed with the pE4/GUS. Leaf DNA (2 /xg per lane) was probed asdescribed in B. Lane C, Untransformed control plants; lanes 1 to 3,three progeny plants of line K43; lanes 4 and 5, two progeny plantsof line T62.

Southern blot analysis using the GUS coding region as ahybridization probe. In this way, five and six independentfertile transgenic plants were obtained for H4/GUS andE4/GUS genes, respectively. All the transgenic lines re-vealed completely specific patterns of GUS expression,which was first localized in the scutellum of germinatingseeds and later in the aleurone layer (data not shown).

Southern Blot Analysis of Transgenic Rice Plants

To confirm transmission of the H4/GUS and E4/GUSgenes to the offspring, Southern blot analysis was per-formed on progeny (R, generation) derived from selfingtwo primary transformants each carrying H4/GUS(Fig. IB) or E4/GUS (Fig. 1C). The coding region of thegusA gene was used as a probe after digestion of genomicDNA with Hindlll. These plants were grown from seedsthat showed aleurone GUS expression after 6 d of germi-nation. The results indicate that the complete sequences ofthe H4/GUS and E4/GUS chimeric genes, 2.9 and 2.4 kb,respectively, were stably transmitted to the GUS-positiveprogeny (Fig. 1, B and C). In addition to the completecopies of the transgenes, several rearranged copies werealso detected and some were segregated in the progeny(Fig. IB, lanes 3-5, and Fig. 1C). The copy number ofcomplete chimeric genes was estimated at one to three perhaploid genome (data not shown).

Temporal and Spatial Regulation of RAmy1A/GUSExpression during Germination of Transgenic Rice Seeds

To investigate the role of the RAmylA promoter in thetemporal and spatial expression of a-amylase during riceseed germination, histochemical and quantitative GUS as-says on transgenic seeds were performed. Histochemicalanalysis of the H4/GUS chimeric gene showed that GUSactivity could be detected in the scutellar epithelium after2 d of germination and that this activity spread into theadjacent aleurone layer by d 4 (Fig. 2). On d 6, the GUSexpression in the aleurone layer increased to the extent thatit covered all parts of the seed. The blue color seen in theproximal lateral half of 6-d-germinated seeds was due tothe GUS activity in the aleurone layer visible through thehydrolyzed translucent endosperm. The blue staining atthe periphery of the starchy endosperm was probably dueto diffusion of either the blue dye, the product of GUSreaction, or of the GUS protein. To quantify the levels ofGUS activity revealed by histochemical analysis, the GUSactivities of germinating seeds derived from two H4/GUStransgenic lines (N33 and T21) were measured (Fig. 3A).For the analysis we used selfed R, seeds, which containedvariable numbers of transgenes. To overcome problems ofheterogeneity we individually analyzed more than 20seeds for each developmental stage. Scutellar GUS activity(Fig. 3, circles) appeared on d 2, peaked on d 4, and de-creased thereafter. In contrast, a very low level of aleu-rone GUS activity (Fig. 3, squares) was detected on d 2,the activity increased thereafter and peaked on d 6, anddecreased sharply by d 8. These results clearly show thatthe RAmylA/GUS fusion genes are differentially regu-

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28 Itoh et al. Plant Physiol. Vol. 107, 1995

H4/GUS E4/GUSem

2days

4days

Gdays

Figure 2. Histochemical analysis of temporal and spatial regulation of RAmyl/VGUSgene expression during germination oftransgenic rice seeds. Transgenic seeds were germinated in water for 2, 4, and 6 d at 30°C, longitudinally cut in half, andstained with X-glucuronide for 12 h at 37°C. Shoots were removed before staining. The blue color seen in the left side of6-d-germinated seeds was due to GUS activity in the aleurone layer visible through the hydrolyzed translucent endosperm.Starch became hydrolyzed and the proximal half of the endosperm became partially translucent on d 6. Lines used wereT21and K43 for H4/GUS and E4/GUS, respectively, em, Embryo; m, mesocotyl; sc, scutellum; se, scutellar epithelium; en,starchy endosperm; al, aleurone layer.

lated in scutellar and aleurone tissues during rice seedgermination.

Similar experiments were performed on seeds from twolines of rice (K43 and T62) transformed with the E4/GUSgene fusion (Fig. 3B). Histochemical assays revealed pat-terns of expression nearly identical to those observed forthe H4/GUS gene (Fig. 2). Quantitative assays of GUSactivity in the scutellum and the aleurone layers of germi-nated seeds indicated that the E4/GUS gene was first ex-pressed in the scutellum on d 2 and peaked on d 4 (Fig. 3B).A very low level of GUS activity in the aleurone layer wasfirst detected on d 2 and the activity increased on d 4 andpeaked on d 6 (Fig. 3B).

Among the four transgenic lines examined, only T21 hadscutellar activity higher than aleurone activity, although itsoverall developmental profile was identical to that of threeother lines. We do not know what causes this difference.Nevertheless, these results clearly show that the -748 to+31 region in the H4/GUS gene and the -232 to +31promoter in the E4/GUS gene function similarly with re-

spect to the localization of expression and the developmen-tal regulation during seed germination.

In addition to seeds, GUS activities were also detected inthe vascular tissues of mesocotyls (Fig. 2) and youngshoots, but no GUS activities in developed leaves weredetected (data not shown). The mesocotyl and shoot activ-ities were found in both transgenic lines carrying the H4/GUS and E4/GUS genes.

Hormonal Regulation of the RAmylA/GUS Genes in theAleurone Layer of Transgenic Rice Seeds

Next we examined the effects of GA3 and ABA on theexpression of the H4/GUS and E4/GUS genes in seeds.Since we used selfed R, seeds for the analysis, which couldhave variable numbers of fusion genes between differentseeds derived from a single transgenic plant, we cut indi-vidual de-embryonated seeds into three slices and treatedthem with buffer, GA3, or a combination of GA3 and ABA.

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A. H4/GUS

J525-

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$40ui5=35

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Figure 3. Developmental regulation of the RAmy1A/G(JS genes dur-ing germination of transgenic rice seeds. The embryo and the en-dosperm were separated after germination and CUS activity (nmol4-MU min~ ' [half seed]"1) was measured after the number of daysindicated. For each transgenic line more than 20 seeds were indi-vidually examined at each developmental stage. The CUS activityobtained from the endosperm was designated the aleurone GUSactivity (•), since the histochemical study indicated that there wasvirtually no GUS activity in starchy endosperm. Similarly, the GUSactivity found in the embryo was designated the scutellum activity(•). A, The developmental profile of the scutellar and the aleuroneGUS activities of rice seeds transformed with the H4/GUS geneduring germination. For line T21 the aleurone activity (•, left axis)and the scutellar activity (•, right axis) were differentially scaled,since in this line the scutellar activity was higher than the aleuroneactivity. B, The developmental profile of the scutellar and the aleu-rone GUS activities of rice seeds transformed with the E4/GUS geneduring germination.

GUS activity of each slice was measured separately (Fig.4A). The GUS measurements were made on six to eightseeds for each transgenic line, and the mean values wereobtained. The histochemical examination of the seed slicesindicated that those treated with GA3 showed GUS activityin the aleurone layer and that the observed induction of theGUS expression by GA3 in the aleurone layer was sup-pressed by addition of ABA for both chimeric genes (Fig.4B). The blue staining was also detected at the periphery ofstarchy endosperm. This was probably due to diffusion ofeither the blue dye resulting from the GUS reaction or theGUS protein. Results of quantitative GUS analysis demon-strated a significant increase in GUS activity after GA3treatment of both H4/GUS and E4/GUS seeds, althoughabsolute values of GUS activities were highly variable be-

tween lines, presumably due to position effects of trans-genes (Fig. 5). When ABA was added along with GA3, theGUS activity was suppressed (Fig. 5). The degree of GA3induction and ABA suppression was similar for bothH4/GUS- and E4/GUS-derived seeds.

DISCUSSION

We used a homologous transgenic system (i.e. rice plantstransformed with rice promoter/gene fusions) to investi-gate the regulation of a cereal a-amylase gene in planta.Histochemical and quantitative GUS assays of seeds trans-formed with the chimeric genes H4/GUS and E4/GUSrevealed patterns of temporal and spatial regulation for theRAmylA gene, consistent with previous studies using RNAblot analysis and in situ hybridization (Karrer et al., 1991;Ranjhan et al., 1992). These previous analyses demon-strated that RAmylA expression in embryos commencedafter 1 d of germination, became much higher on d 3, butdecreased considerably after 5 d of germination. In con-trast, the aleurone expression was first observed on d 2,increased on d 5, and attained even higher levels on d 6.

Hormone treatment4days

dry seedG A/ABA

B

Buffer GA

GUS Assay

GA+ABA

Figure 4. Effect of hormones on expression of chimeric RAmylA/GUS genes in aleurone layers. A, A single seed assay for hormonalregulation of RAmy1A/GUS expression. A transgenic seed was slicedinto four pieces and the slice containing the embryo was discarded.The other three pieces were treated with buffer, GA3, or GA3 plusABA for 4 d at 30°C. GUS activity of each slice was histochemicallyexamined by X-glucuronide staining or quantified. B, Histochemicallocalization of hormonally regulated expression of the H4/GUS genein transgenic rice seeds. Three seed slices derived from a singletransgenic seed were treated with buffer, GA3, or GA3 plus ABA andstained with X-glucuronide for 12 h at 37°C.

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30 ltoh et al. Plant Physiol. Vol. 107, 1995

h H4IGUS

200 4 I O00

800

600

400

200

O 0 G GIA

B. E4lGUS

0 G GIA

B G GIA

0 G GIA

Figure 5. Quantitative analysis of hormonally regulated expression of the RAmylNGUS gene in the aleurone layer of transgenic rice seeds. The single seed assay (Fig. 4A) was performed with six to eight selfed seeds derived from each transgenic line and the average GUS activities and SE values were obtained. A, Transgenic lines T21 and N33 transformed with the H4/GUS gene. B, Transgenic lines K43 and N51 transformed with the E4/GUS gene. B, Buffer; C, GA,; G/A, CA, and ABA.

RAmylA expression after d 6 was not examined. The two promoter/GUS constructs used in this study expressed GUS activity in the scutellum and aleurone layer of transgenic seeds during germination in a similar fashion. Fur- thermore, they showed a similar pattern of response to GA, and ABA in aleurone layers of embryoless seeds. There- fore, our results demonstrated that the region from -232 to +31 in the RAmylA promoter is sufficient for both developmental and hormonal regulation of the RAmyZA gene in germinated seeds

Within the 232-bp promoter region carried on pE4/GUS are three highly conserved sequence motifs that have been implicated by others as being essential for normal regulation of GA,-responsive a-amylase promoters. Among these three motifs is the sequence TAACAAA, which has been identified as the GARE in the promoters of barley a- amylase genes (Skriver et al., 1991; Gubler and Jacobsen, 1992; Lanahan et al., 1992). The TATCCAC/T motif (boxl) has also been shown to act cooperatively with the GARE for maximal GA induction of a barley a-amylase gene (Gubler and Jacobsen, 1992). However, the role of the pyrimidine box, CCTTTT, in GA,-regulated expression ap- pears to be variable among different a-amylase genes. In a low-pI a-amylase gene of barley, remova1 of this box

caused a reduction in both the absolute level of expression m d the effect of GA, on expression (Lanahan et al., 1992), whereas deletion of this box in a high-pI a-amylase gene of barley did not influence promoter activity (Gubler and Jacobsen, 1992). In a low-pI a-amylase gene of wheat, removal of two pyrimidine boxes resulted in a 5-fold reduction in the level of GA,-regulated expression in tran- isient assays using aleurone protoplasts (Huttly and Baul- combe, 1989). The RAmyZA gene contains two pyrimidine boxes, one located at position -312 and the other at position -214. Our results suggest that one pyrimidine box is sufficient for both hormonal and developmental regulation of the RAmyZA gene. Since the pyrimidine boc does not appear to be as important as the two other highly conserved motifs in GA, induction of a-amylase geries, and no other sequence motifs were conserved among 211 the analyzed cereal a-amylase genes, it is an interestir g specula- tion that the pyrimidine box may play a maior role in tissue-specific expression of a-amylase genes in cereals. Further studies using mutated promoters will k e required to test this possibility.

Transient assays using isolated aleurone pro toplasts or intact aleurone layers have been used extensive ly to iden- tify the regulatory elements involved in hormo na1 regulation of cereal a-amylase genes. Although transient assays using particle bombardment have been useful for elucidat- ing elements important for hormonal regulation of a- amylase genes in the aleurone layer, they have not been applied for developmental analysis of their gene expression. We used a transgenic approach to investigate the tissue-specific and developmental expressic n of the RAmyZA gene during seed germination. The results of this study indicate that the promoter-gusA fusions, which have been extensively used in various transient expression assays, are useful tools for studies in a-amylase gene regulation. Furthermore, our results suggest that the transgenic approach will be useful in deciphering how the various cis-elements in the a-amylase promoter work i ogether or separately to achieve hormonal as well as temporal and spatial expression of a-amylase in the plant. For example, it will be of interest to know which element, he GARE, TATCCAC motif, or the pyrimidine box, plays a major role in developmental regulation of RAmyZA gene cmxpression.

The multigenic nature of cereal a-amylase genes (e.g. Huang et al., 1990) often makes analysis of their gene expression complicated unless gene-specific probes are available. Transgenic rice plants described herc are useful material for the study of transcriptional regulation of a-amylase genes during germination. a-Amylase genes are also regulated by Ca2+ ions (Jones et al., 1993) m d metabolites (Yu et al., 1991; Karrer and Rodriguez, 1992); however, the molecular mechanisms are not knowi. Our material will allow transcriptional regulation of a-amylase genes by these regulators to be further investi@ ated.

Received July 12,1994; accepted September 11, 1994. Copyright Clearance Center: 0032-0889/95/ 107/0025 /07.

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Documents

Developmental and Hormonal Regulation of Rice a-Amylase ...gene, gusA. The resulting promoter-gusA fusions, pE4/CUS (-232 to +31) and pH4/CUS (-748 to +31), were used separately to