rice triosephosphate isomerase gene 5' sequence directs in ...460 xu et al. plant physiol. v31....

Plant Physiol. (1994) 106: 459-467

Rice Triosephosphate Isomerase Gene 5' Sequence Directs @-Glucuronidase Activity in Transgenic Tobacco but

Requires an Intron for Expression in Rice'

Yong Xu, Hong Yu, and Timothy C. Hall*

Institute of Developmental and Molecular Biology, Texas A&M University, College Station, Texas 77843-31 55

In rice (Oryza sativa L.), cytosolic triosephosphate isomerase (TPI) i s encoded by a single gene. TPI catalyzes a vital step in glycolysis, and RNA blots showed that the tpi gene is expressed in all vegetative tissues (root, culm, and leaves) and in rice suspension cells. No effect of light on expression was detected, but submergence of rice seedlings resulted in elevated levels of TPI mRNA in roots and culms. The 2767-bp 5' upstream sequence of the tpigene was fused translationally with the @-glucuronidase (gusA) gene, and the resulting construct, TPI-GUS, was found to express constitutive, high levels of GUS activity in transgenic tobacco (Nicotiana tabacum) plants. However, the same construct yielded no GUS activity in stably transformed rice plants, and RNA blots showed that no GUS mRNA could be detected even though stable integra- tion of functional copies of the construct was confirmed by South- ern blot and genomic polymerase chain reaction analyses. Tran- sient assays using particle bombardment yielded high levels of GUS expression from the TPI-GUS construct in tobacco leaves, but essentially no expression in rice, barley, or maize leaves. When the first intron of the tpi gene was included in the construct (TPI-intl- GUS), transient GUS activity was routinely obtained in rice leaves, revealing that the first intron of the rice tpi gene i s crucial for i ts expression in rice. TPI-intl -GUS also directed transient GUS expression in maize and barley leaves, but little or no activity was obtained from this construct in tobacco, tomato, or soybean leaves. These results with the rice tpi promoter are in accordance with mounting evidence that differences in gene expression exist between monocots and dicots.

TPI catalyzes the essential isomerization reaction between dihydroxyacetone phosphate and ~-glyceraldehyde-S-P in the glycolytic pathway. In addition to glycolysis, this reaction links almost all the triosephosphate-involving metabolic pathways in plants, such as gluconeogenesis, fatty acid bio- synthesis, the pentosephosphate pathway, and photosynthetic carbon dioxide fixation (Miemyk, 1990). Therefore, TPI represents a vital, or housekeeping, enzyme in both prokar- yotes and eukaryotes and its coding sequence is highly conserved among phylogenetically distant species (Swinkels et al., 1986). In spite of this great importance of cytosolic TPI in cell metabolism, our previous work has established that it

This work was supported by grants from the Rockefeller Foun- dation and the Texas Advanced Technology Program; Y.X. was supported by a predoctoral fellowship from the Rockefeller Foundation.

* Corresponding author; fax 1-409-862-4098. 459

is encoded as a single copy gene in the rice (Oryza sativa L.) genome (Xu and Hall, 1993; Xu et al., 1993b).

In contrast to the situation for facultative genes, few studies exist conceming molecular mechanisms of housekeeping gene regulation in higher plants. Given the important functions of TPI in various plant metabolic networks, we felt that it represented an excellent candidate for such studies, and we now report the expression of GUS directed by the TPI 5' regulatory sequence in stably transformed tobacco (Nicotiana tabacum) and rice plants. Additionally, we examined the effect of two environmental factors, light and anaerobiosis, on the expression of the tpi gene.

Our studies also permitted an examination of the role of introns in gene expression and a comparison of tpi gene promoter activity in monocots and dicots. Whereas introns

nents for expression of many animal genes (Buchman and Berg, 1988; Jonsson et al., 1992), few examples exist in dicots for their contribution to gene expression. However, introns have been shown to be important in the expression of several monocot genes. Examples include maize alcohol dehydrogenase 1 (Adhl) (Callis et al., 1987; Mascarenhas et al., 1990; Luehrsen and Walbot, 1991), maize shrunken 1 (Vasil et al., 1989; Maas et al., 1991), rice actin 1 (McElroy et al., 1990), and oat phytochrome (Bruce et al., 1989). Interestingly, the studies reported here reveal that the first TPI intron is not required for GUS expression by the TPI 5' sequence in dicots (tobacco, tomato, and soybean) but is essential for expression in monocots (rice, barley, and maize), further suggesting that fundamental differences in regulation of gene expression exist between dicots and monocots.

have been shown to be important, sometimes vital, compo-

MATERIALS AND METHODS

Plant Materials

Rice (Oryza sativa L. cv Gulfmont and cv Taipei 309) and tobacco (Nicotiana tabacum cv Xanthi) plants were grown essentially as described previously (Battraw and Hall, 1990; Bustos et al., 1991). For wild-type plants, root, culm (base of leaf sheaths and mesocotyl), and leaf tissues were harvested from 1- or 2-week-old seedlings. For transgenic plants, roots

Abbreviations: GUS, 8-glucuronidase; MUG, 4-methylumbelli- feryl-8-o-glucuronide; nt, nucleotide; ocs, octopine synthase gene; TPI, triosephosphate isomerase; X-gluc, 5-bromo-4-chloro-3-indolyl glucuronide.

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460 Xu et al. Plant Physiol. V31. 106, 1994

and leaves were harvested either from Magenta boxes or from 1-month-old plants grown in the greenhouse. Tissues were frozen in liquid nitrogen and stored at -8OOC until use. For transient assays, mature leaves were harvested from the various plant species and put on 1% agar plates immediately before bombardment.

For submergence treatment, 2-week-old rice seedlings were completely submerged for 20 h in 10 mM Tris-HC1 (pH 7.0) (Springer et al., 1986). Root, culm, and leaf tissues were harvested, frozen in liquid nitrogen, and stored at -8OOC until use. For light treatment, rice seeds were germinated in the dark for 2 weeks. Half of the etiolated rice seedlings were immediately harvested, and the other half were exposed to light for 2 h before harvesting. Seedlings grown under normal conditions were harvested at the same time as control tissues.

Construction of Chimeric Genes

The 2767-bp sequence upstream from the AUG start codon of the t p i gene was translationally fused to a fragment containing GUS coding and ocs 3' terminator sequences isolated from plasmid Kiwi 101 (Janssen and Gardner, 1989). The resulting construct, TPI-GUS (Fig. lB), was used for transient assays and for rice transformation. TPI-GUS was cloned into the binary vector pBIN19 (Bevan, 1984) for tobacco transformation.

A PCR fragment containing 90 bp of the TPI 5' region and the complete first exon (39 bp), the first intron (860 bp), and the first 9 bp of the second exon of the t p i gene was used to replace the region between the two NcoI sites in TPI-GUS to make the TPI-intl-GUS construct (Fig. 1B). The short TPI exon sequences flanking the intron were retained in the construct because of the likely requirement for a correct environment for intron splicing. Both constructs (TPI-GUS and TPI-intl -GUS) contain identical sequences upstream of their translation start codons. The difference between the two constructs is within the open reading frame: TPI-GUS starts with the first AUG of GUS, whereas TPI-intl-GUS begins with the first AUG of TPI and fuses 16 amino acid residues of TPI to the initiation Met of the GUS protein.

Tobacco and Rice Transformation

Tobacco seeds were surface sterilized and germinated on Murashige and Skoog medium (Murashige and Skoog, 1962) without phytohormones. Leaf disc transformations were carried out according to Horsch et al. (1985). Regeneration of transformed tobacco plants was camed out as described by Bustos et al. (1991). Rice transformation was camed out as described by Battraw and Hall (1990) using electroporation of rice protoplasts.

RNA and DNA Isolation and Blot Hybridization

Total RNA was isolated (Sambrook et al., 1989) from vegetative tissues (roots, culms, and leaves) of 2-week-old plants, rice suspension calli, and leaves of 1-month-old transgenic rice and tobacco plants. RNA gel-blot hybridizations were performed as previously described (Xu and Hall, 1993). All the blots were stripped and reprobed with an 18s rDNA

El E2E3 E4 E5 E6E7E8E9 5' (2767 bP) I Rice Cytosolic TPI Genomic Structure +

3 3 z z // I, ~~ 4 9 I I k b I

i$ TF'I5' /intron l \ GUS OCS 3'

TPI-intl -GUS El E2 (9 bp)

Figure 1. Restriction maps of the tpi gene and derivative constructs. A, Structure of rice cytosolic TPI cDNA (1051 bp; C,enBank accession N o . M87064) and the corresponding genomic clone (6246 bp; GenBank accession No. L04967). Black boxes repre!,ent TPI coding regions. Restriction sites are indicated (the EcoRl sites at each end are derived from added linkers). 6, Structures of thl. TPI-GUS and TPI-intl-GUS constructs used for expression in plant tissues. In TPI- GUS, 2767 bp 5' of the TPI coding region was translationally fused at the AUG codon with the GUS coding sequence; as indicated, this fusion retains t h e Ncol site at t h e AUG initiation codon of the GUS coding region. In TPI-intl-GUS, the first intron (860 bp) of the rice tp/ gene together with the first exon (El , 39 bp) and 9 bp of t h e second exon (E2) were inserted between the TPI 5' flanking sequence and the GUS coding sequence; there is no Ncol site at the AUG initiation codon. T h e ocs gene polyadenyLition sequence (ocs 3') was included at the 3' end of both chimeric gene constructs.

probe (Eckenrode et al., 1985) to verify relatively equal loading of the total RNA in each lane.

Total DNA was isolated from ground transgenic rice leaves by a modified Cetrimide (hexadecyltrimethylammonium bromide, sigma) procedure of Taylor and Powell (1982). DNA gel-blot analysis was performed as previously described (Xu and Hall, 1993) using a fragment derived from the GUS coding region as a probe.

PCR Analysis

DNA isolated from leaves (3-5 cm in length) of putative transgenic plants was used for PCR (Edwards et al., 1991). For the initial screening of transgenic plants, primers GSl and GS2 (see "Results") were used. Thirty-five cycles of PCR (1 min each at 92OC, 55OC, and 72OC) were carried out using rice total DNA (approximately 55 ng) as temphte, Taq DNA polymerase, and standard PCR buffer (Perkin -Elmer). Total DNA, isolated by the Cetrimide procedure clf Taylor and Powell (1982), was used for amplification of furctional copies of the integrated construct. Primers GSl and 1 3 3 (see "Re-



Rice Triosephosphate Isomerase Gene Expression 461

suits") were used. Total DNA was first digested with Kpnland approximately 100 ng of digested DNA was used forPCR. The initial PCR cycle (3 min at 92°C, 4 min at 55°C,and 5 min at 72°C) was followed by 35 additional cycles (1.5min at 92°C, 2 min at 55°C, and 4 min at 72°C).

Microprojectile Bombardment

Transient plant transformations were accomplished bybombarding (Sanford, 1990) mature leaves from variousplant species with the TPI-GUS or TPI-intl-GUS construct.Bombardment was carried out using a PDS-1000/He ParticleDelivery System (Bio-Rad), following the protocols providedby the manufacturer. Leaf tissues were flattened on 1% agarto cover about 9 cm2 total area in the center of a Petri plate.CsCl-purified plasmid DNA was coated onto the 1.6-/im goldparticles (approximately 1 mg of particles coated with 1 jtgof plasmid DNA for each shot). Plasmid Kiwi 101 (Janssenand Gardner, 1989), which contains a dual promoter drivingthe gusA reporter gene, was used in each bombardment as apositive control. Dicot (tobacco, soybean, and tomato) leaftissues were placed at level 4 and a helium pressure of 900psi was used; monocot (rice, barley, and maize) leaf tissueswere also placed at level 4, but a helium pressure of 1800 psiwas used. After bombardment, samples were incubated inthe dark at 25°C for 24 h before staining for GUS activity.The leaf tissues were cleared with 95% ethanol to facilitatelocation of blue spots resulting from GUS activity. Thenumber of blue spots per shot was determined under themicroscope.

GUS Enzyme Assays

GUS activity was determined by measuring the rate ofhydrolysis of the substrate MUG (Sigma) to form the fluores-cent product 4-methylumbelliferone, as described by Jeffer-son (1987). Protein concentrations were estimated using thecolorimetric assay of Bradford (1976). Histochemical localiza-tion of GUS activity was performed by incubating tissueswith the substrate X-gluc (Research Organics, Cleveland,OH), as described by Jefferson et al. (1987).

RESULTS

Ubiquitous tpi Gene Expression Is Not Regulated byLight but Can Be Induced in Rice Seedlings underSubmergence Treatment

Previously, we reported the isolation of a rice cDNA cloneand the corresponding genomic sequence encoding cytosolicTPI, a vital, housekeeping enzyme (Xu and Hall, 1993; Xu etal., 1993b). Whereas at least nine tpi genes may exist in maize(Marchionni and Gilbert, 1986), there is only one in rice.DNA sequencing analysis revealed that the tpi gene containseight introns within the coding region (Fig. 1A) whose posi-tions are conserved between rice and maize tpi genes. Theexpression pattern of the rice tpi gene was studied by RNAblots using the TPI cDNA coding region as a probe (Fig. 2).Under normal growing conditions, tpi gene expression wasfound in all vegetative tissues (roots, culms, and leaves) inrice plants and in rice suspension cells (Fig. 2A). Higher levels

i • •III IBS rRNA

B Submergence-treated Seedlings Non-treated Seedlings

J

TPI

ADH

ISSrRNA

Dark-grown Seedlings Light-grown Seedlings

TH

ISSrRNA

Figure 2. RNA gel-blot analysis of rice cytosolic tpi gene expression.A, Total RNA (5 ^g) isolated from 2-week-old rice root, culm, andleaf tissues and from suspension calli was separated on a 1.0%formaldehyde agarose gel, electrophoretically transferred to a nylonmembrane, and hybridized with a "P-labeled rice TPI cDNA probe(Xu and Hall, 1993). B, Total RNA was isolated from 2-week-oldrice seedlings grown under normal conditions and from seedlingsunder submergence for 20 h. The same blot was sequentiallyprobed with rice cytosolic TPI cDNA and a maize Adhl partialcDNA (Dennis et al., 1984). C, Total RNA samples were preparedfrom 2-week-old light-grown roots, culms, and leaves, and fromdark-grown seedlings (DS) and dark-grown seedlings with 2 h oflight treatment (DS+L). In all the experiments, the same blot wasstripped and reprobed with an 18S rDNA probe to verify relativelyequal loading of RNA in each lane.

of TPI steady-state mRNA (approximately 1100 nt) werefound in roots and calli than in leaves and culms.

Anaerobic stress is known to induce the first and lastenzymes of the glycolytic pathway at the mRNA level (Den-nis et al., 1987). For certain intermediate glycolytic enzymeslike enolase, small increases in activity were observed inmaize (Bailey-Serres et al., 1988); however, there was littleeffect of anaerobiosis on the abundance of enolase mRNA(Van Der Straeten et al., 1991). We found that, after 20 h ofsubmergence, a marked increase in TPI message occurred inroots and culms in rice seedlings (Fig. 2B), but no increase ofTPI mRNA in leaf tissues was discerned. The same blot was



462 Xu et al. Plant Physiol. Vol. 106, 1994

stripped and hybridized using a partial maize Adhl cDNAclone (Dennis et al., 1984). Adhl message (approximately2000 nt) was found to be strongly induced (at least 10-fold)in submergence-treated roots and culms (Fig. 2B). As withTPI expression, we did not observe an increase in Adhlmessage in submergence-treated rice leaves. Instead, a de-crease in Adhl mRNA in submergence-treated leaves wasevident (Fig. 2B).

When rice plants were grown in the dark, the tpi gene wasexpressed at a level similar to that for plants grown in thelight (Fig. 2C). When etiolated rice seedlings were exposed tolight for 2 h, there was no dramatic change in the steady-state TPI mRNA (Fig. 2C). This result suggested that rice TPIexpression is not induced by light, which is consistent withthe fact that the gene encodes the cytosolic form of TPI ratherthan the plastid form.

The 5' Upstream Sequence of Rice Cytosolic tpi GeneDirects Constitutive gusA Reporter Gene Expression inTransgenic Tobacco

To identify promoter elements regulating expression of therice tpi gene, a chimeric construct was made that contained2767 bp of the tpi gene 5' flanking sequence fused transla-tionally to the GUS gene (gusA) (Jefferson et al., 1987) andan ocs synthase 3' terminator sequence (Fig. IB: TPI-GUS).The TPI-GUS construct was used for T-DNA-mediated to-bacco transformation by the leaf disc technique (Horsch etal., 1985). Seventeen transformed plants, confirmed to ex-press GUS by histochemical staining (Jefferson, 1987), wereself-fertilized. Derivative homozygous R, plants shown tocontain a single-copy transgene by Southern analysis (datanot shown) were subjected to further analysis. The distribu-tion of GUS activity in transgenic tobacco was consistentwith the ubiquitous expression pattern of the tpi gene in rice,GUS activity being detected by MUG assays in all tissuestested (data not shown). Histochemical analysis showed rel-atively high levels of GUS activity in metabolically activetissues, as illustrated in Figure 3 for a representative plant.Regions of high activity included the root tip meristematicregion (Fig. 3A), the lateral root initials (Fig. 3B), the axillarybud initials (Fig. 3C), and young leaves (Fig. 3D).

The 5' Upstream Sequence of Rice Cytosolic tpi Gene Failsto Direct GUS Expression in Transgenic Rice Tissues

To characterize TPI promoter activity in transgenic riceplants, the TPI-GUS construct was introduced into rice (cvTaipei 309) by electroporation-mediated protoplast transfor-mation (Battraw and Hall, 1990). Surprisingly, of all 12transgenic plant lines identified as positive for the TPI-GUSconstruct by PCR analysis (Fig. 4B), none showed GUSactivity using either histochemical staining or fluorescentMUG assay (data not shown). RNA blot analysis was per-formed to determine if GUS message was present in thesetransgenic rice plants. As shown in Figure 5, GUS messageof the correct size (approximately 1900 nt) was detected onlyin TPI-GUS-transformed tobacco plants but not in transgenicrice plants. Southern blot analysis indicated that multiplecopies of the chimeric gene were present in the PCR positive

Figure 3. Histochemical localization of GUS activity in tobaccoplants transformed with the TPI-GUS construct. Tobacco seedlingsof homozygous R, plants were used for histochemical staining(Jefferson et al., 1987) to determine spatial patterns of GUS expres-sion directed by the rice cytosolic TPI 5' regulatory sequence. A,Primary root. B, Primary root with lateral root initials. C, Axillarybud. D, Young leaf.

transgenic plants, and three independent transformants (Fig.4C, plants Rl, R13, and R20; R9 and R14 are sibling plantsof R13) were shown to contain at least one intact copy of theTPI-GUS construct (consisting of about 1.5 kb of the tpi 5'region and complete gusA and ocs 3' regions).

To explore the reason why the TPI 5' flanking sequencethat directed a high level of GUS activity in transgenictobacco plants did not give any detectable GUS expressionin transgenic rice plants, transient expression analyses wereundertaken using both tobacco leaves and rice leaves. Plas-mid pKiwi 101 (Janssen and Gardner, 1989), which containsa hybrid mannopine synthase-cauliflower mosaic virus 35Spromoter driving the GUS coding sequence, was used tooptimize the bombardment conditions for both tobacco (Fig.




TPI5'TPI-GUS

PCR fragment expected (350 bp)PCR fragment expected (2.85 kb)GUS probe (1.8kb)

I Southern fragment expected (4.7 kb)

• M l 2 3 4 5 6 7 8 9 1011121314151617181920

-350bp

C h Rl R9 R13 RM R17 R20R20MW.T.

4.7 kb—»• ——

Figure 4. Molecular analysis of rice plants transformed with TPI-GUS. A, Probes and expected gene fragments. PCR primers areshown by arrows and expected PCR fragments are shown as shadedbars. Primers CS1 and CS2 were used for initial screening and theexpected PCR product (350 bp) spans the junction of the TPI 5'sequence and the gusA coding region. Primers CS1 and CS3 wereused for genomic PCR amplification from transgenic rice genomicDNA. The CUS probe (1.8 kb) and the expected fragment (4.7 kb)in Southern analysis are shown in black bars. B, Ethidium bromide-stained gel showing PCR analysis of rice plants. A positive (+) control(using the TPI-CUS plasmid as template), a negative (-) control (noDNA template), molecular markers (M) (BRL 1-kb ladder), and theexpected PCR product (350 bp) are indicated. C, Southern blotanalysis of rice plants transformed with TPI-GUS. Blots were pre-pared using SamHI/Xbol-digested DNA isolated from six transgenicplants (Rl, R9, R13, R14, R17, R20) and a nontransformed (W.T.)rice plant. R20(U) is undigested DNA from plant R20. The blot washybridized with the 1.8-kb GUS probe and a one-copy plasmidreconstruction (1x) was included. D, Ethidium bromide-stained gelshowing the 2.85-kb PCR product amplified from genomic DNA oftransgenic rice plants R13 and R20.

6A) and rice leaves (Fig. 6B). Bombardment of tobacco leaveswith the TPI-GUS construct reproducibly yielded many bluespots (about 80-100/shot; Fig. 6C), consistent with the resultsfrom stably transformed tobacco (Fig. 3). Surprisingly, incontrast, no expression was observed from the TPI-GUSconstruct on rice leaves in five of seven separate bombard-ment experiments (Fig. 6D). In two experiments, a total ofthree blue spots were found on 10 bombarded leaf sections(each approximately 2.5 cm long X 0.5 cm wide). Thesecomparative transient assays show that, whereas the 5' flank-ing sequence of the rice tpi gene is sufficient to direct reportergene expression in tobacco tissues, it is inefficient at orincapable of driving gusA gene expression in rice.

Conclusive evidence that the sequence inserted into therice genome was functional was obtained using DNA frag-ments amplified directly by PCR from transgenic rice plantsR13 and R20 in bombardment experiments. The genomicPCR was performed using two primers (Fig. 4A: GS1 andGS3) to amplify a DNA fragment containing about 350 bpof the TPI 5' flanking sequence, the complete gusA codingregion, and 650 bp of the ocs 3' region (Fig. 4A). Theamplified sequences (Fig. 4D) yielded as many blue spots inbombarded tobacco leaves as did the TPI-GUS construct (datanot shown, but similar to that of Fig. 6C). These resultsconfirm that at least one copy of a DNA segment capable ofexpressing GUS activity in tobacco was integrated in eachtransgenic rice plant tested. They also exclude the possibilitythat the sequence context of the TPI 5' leader-GUS codingregion boundary did not permit translational initiation.

Inclusion of the First Intron of the Rice tpi Gene AllowsGUS Expression in Transient Assays in Monocots butDebilitates GUS Expression in Dicots

The lack of transient and stable expression of GUS in riceleaves from TPI-GUS implied that an additional sequenceelement from the tpi gene is essential for its expression inrice. Because introns of many animal and monocot plantgenes have been shown to dramatically influence geneexpression, we decided to determine if the first inrron of thetpi gene could assist in driving expression in rice. To do this,construct TPI-intl-GUS was made by inserting TPI intron 1(860 bp), together with exonl (39 bp) and the first 9 bp ofexon2, in front of the GUS coding region in the TPI-GUSconstruct (Fig. IB: TPI-intl-GUS).

Comparative bombardment transient assays using_IPI-GUS and TPI-intl-GUS constructs were performed on leavesof several plant species. As illustrated by a semi-quantitativeblue-spot count assay (Fig. 7), dramatically different levels ofGUS expression were observed for the two constructs de-pending on whether monocot or dicot leaves were bom-barded. In three monocot plants (rice, maize, and barley),TPI-intl-GUS routinely gave GUS expression, whereas TPI-GUS did not (Fig. 6, D and F; Fig. 7). The reverse results

GUS-»-

18s rRNA

Tl wt Rl R9 R13 R14 R17 R20

- 4••••••tf

Figure 5. RNA gel-blot analysis of GUS expression in transgenicplants. Each lane contained 5 jtg of total RNA isolated from leaftissues of tobacco (T1) and six rice plants (Rl, R9, R13, R14, R17,R20) transformed with the TPI-GUS construct, and an untrans-formed wild-type rice (wt). The blot was subsequently hybridizedwith the 1.8-kb GUS probe (see Fig. 4A) and an 18S rDNA probe.The expected size of the GUS message (approximately 1900 nt) isindicated by an arrow.



464 Plant Physiol. Vol. 106, 1994

Figure 6. Transient expression of TPI promoter activity in tobacco (A, C, and E) and rice (B, D, and F) leaves. Histochemicalanalysis of CUS activity was carried out by X-gluc staining 24 h after microprojectile bombardment. Plasmid pKiwi 101was used as a positive control (A and B). In the same experiment, TPI-CUS and TPI-int-GUS constructs were used tobombard tobacco (C and E) or rice leaves (D and F). Bombardment without DNA or with a promoterless plasmid did notgive any blue spots (not shown).

were seen in three dicot plants (tobacco, soybean, and to-mato), where high levels of GUS expression were observedwith TPI-GUS, but the GUS expression was dramaticallyreduced with TPI-intl-GUS (Fig. 6, C and E; Fig. 7). Theresults from these transient assays strongly suggest that thefirst intron of the tpi gene is very important for gene expres-sion from the TPI promoter in monocot tissues, whereas theTPI 5' upstream sequence itself is sufficient to direct geneexpression in dicot plants and inclusion of the first TPI introndebilitates its expression in dicots.

DISCUSSION

Regulation of Cytosolic tpi Gene Expression in Rice

Genes encoding TPI have been cloned in very few plantspecies (Marchionni and Gilbert, 1986; Sato et al., 1990; Xuet al., 1993b) and, to the best of our knowledge, no reporton plant tpi gene regulation has appeared. Our results indi-

cate that the expression of rice cytosolic TPI is ubiquitous,with higher expression levels in roots and calli than in culmsand leaves (Fig. 2A). Since TPI is a vital, or housekeeping,gene, the nontissue-specific expression pattern was expected.However, we also observed some variation in the levelof TPI message in individual tissues (data not shown), indi-cating that tpi gene expression may be modulated duringdevelopment.

Anaerobic conditions have been shown to induce severalgenes of the glycolytic pathway (Dennis et al., 1987). How-ever, there is a significant difference in the level of inductionin that the first and the last enzymes of the glycolytic pathwayare strongly induced by anaerobiosis, whereas the interme-diate glycolytic enzymes are generally induced to a fairly lowlevel, or not at all (Kelley and Freeling, 1984; Bailey-Serres etal., 1988). We observed a clear increase in the steady-statelevel of TPI mRNA in rice seedlings after 20 h of submergencetreatment; however, the level of induction was much less




100

90

B 80

% 70

2 5 0

2 30

- i 60

m 5 4 0

5 20

1 0

” - . Rice Barley Maize Tobacm soybean Tomato

Figure 7. Histogram of transient GUS expression in leaf tissues by particle bombardment using TPI-GUS and TPI-intl -GUS. X-gluc staining was performed 20 h after bombardment and the tissues were treated with 95% ethanol to allow visualization of the blue spots. Values represent the mean of three independent shooting experiments for each construct.

than that of the Adhl gene (Fig. 2C). In general, our results agree with the hypothesis that intermediate steps in the glycolytic pathway may be less responsive to anaerobiosis than are the initial and final parts of the pathway. In our experiments, we also observed organ-specific induction: the increase in TPI mRNA level under submergence conditions occurred in root and culm tissues but not in leaves. We found that tissue-specific message levels for Adhl were similar to those of TPI, i.e. Adhl was not induced in leaves, which differs from a previous report by Xie and Wu (1989) showing anaerobic induction of rice Adhl expression in both roots and leaves.

Light is essential for normal plant growth and development, not only as a source of energy but also as a stimulus that regulates diverse developmental and metabolic processes. Numerous studies on light-responsive plant genes have shown that rapid increase in the transcript levels from these genes occurs in etiolated seedlings and dark-adapted plants in response to light (Gilmartin et al., 1990). In green tissues, TPI is involved not only in glycolysis but also in photosynthetic carbon fixation. As is true for other photosynthetic proteins, chloroplast-specific TPI has been shown to be light inducible (Nelson et al., 1984). Thus, it is very likely that the gene encoding the plastid form TPI is regulated by light. The result by RNA blot analysis (Fig. 2, B and C), that the rice cytosolic tpi gene is not light-inducible but is induced by submergence, supports our belief that the chloroplast tp i gene is highly diverged from cytosolic TPI in rice (Xu and Hall, 1993).

TPI 5‘ Regulatory Sequences Function as Expected in Transgenic Tobacco

The expression of GUS activities from the 5’ flanking sequence of the tp i gene in leaves, roots, and stems of transgenic tobacco plants transformed by the TPI-GUS construct (Fig. 3) is consistent with the ubiquitous expression of rice cytosolic TPI observed in wild-type rice plants and suspension calli (Fig. 2A). Such expression is expected for a gene encoding a vital, housekeeping enzyme, and our findings of widespread spatial distribution of TPI activity parallel those of Shih and Goodman (1988) for the GapC gene, which

encodes cytosolic glyceraldehyde-3-P dehydrogenase. TPI and GapC are both vital enzymes in glycolysis. Shih and Goodman (1988) proposed that the metabolic state of the cells is a factor regulating expression of the GapC gene in tobacco leaves, and our data showing high expression in metabolically active tissues (Fig. 3) suggest that rice tpi gene expression may also be linked to the metabolic state of plant cells. This concept requires further analysis of t p i gene expression, e.g. by using different SUC concentrations in the growth medium, as was undertaken by Shih and Goodman (1988) in their studies on the GapC gene.

lack of Expression from TPI-GUS in Transgenic Rice May Reflect the Absence of an Essential cis-Acting Element

Our data indicate that the lack of GUS activity in transgenic rice plants is probably due to an intrinsic insufficiency of the TPI-GUS construct. We first eliminated the possibility that the gene had been inactivated by sequence alterations or rearrangements during production of the transgenic plants. A 2.85-kb TPI-GUS fragment was PCR amplified from the rice genome and subsequently shown to express GUS when used to bombard tobacco leaves (Fig. 4). Another potential explanation for the lack of GUS expression in rice stably transformed with TPI-GUS might be co-suppression (Jorgen- sen, 1990). However, methylation and other presumed causes for co-suppression are unlikely to operate in transient assays, and our results indicated that TPI-GUS gave essentially no GUS expression in bombarded rice (Fig. 6D), maize, or barley leaves (Fig. 7). Although the possibility of methylation of the construct in stably transformed rice plants cannot be completely ruled out, our transient assay results strongly suggested that the absence of an essential cis-acting element in TPI-GUS is a more plausible explanation for the lack of GUS expression. This notion is further supported by the transient results using the intron-containing construct (TPI-intl -GUS), which routinely gave GUS expression in bombarded rice (Fig. 6F), barley, or maize leaves (Fig. 7).

In most studies of intron enhancement of gene expression in plants, introns were inserted in the 5’ untranslated region in such a way that the mature mRNA produced after splicing contains additional sequences upstream of the translation initiation codon when compared to the intronless construct. In our studies, the two constructs (TPI-GUS and TPI-intl- GUS) being compared contained identical 5’ regulatory sequences up to the AUG translational start codons. The only difference between the two constructs is that TPI-GUS starts with the AUG of GUS and TPI-intl-GUS begins with the first AUG of TPI and fuses 16 amino acid residues of TPI to the initiation Met of the GUS protein (see “Materials and Methods”). Since the GUS coding sequence is so widely used in transgenic plants, there is little reason to think that the use of this translational start sequence can be the reason for lack of expression of the TPI-GUS construct in rice. Although the sequences immediately downstream of the AUG initiation site may have some differential impact on translational efficiency of the two constructs, we think that TPI intron 1 contributes significantly to the functionality of the TPI promoter in monocots and to the dramatically different GUS



466 Xu et al. Plant Physiol. Vcd. 106, 1994

expression pattems between monocot and dicot plants (Fig. 7).

Introns Play an Essential Role in the Expression of TPI and Other Genes in Monocot Plants

The spatial regulation of several dicot genes is unchanged when they are expressed as transgenes in monocots (e.g. tomato rbcS [Kyozuka et al., 19931 and potato Pin2 [Xu et al., 1993a]), and some monocot genes function normally in transgenic dicots (e.g. wheat Cab [Lamppa et al., 19851 and rice chitinase [Zhu et al., 19931). However, a number of studies have revealed differences in gene regulation between dicots and monocots. For example, neither wheat rbcS nor maize Adhl genes are expressed in transgenic tobacco under their own 5' regulatory sequences (Keith and Chua, 1986; Ellis et al., 1987), and differences in transcription factors and rec- ognition of promoter sequences have been suggested (Last et al., 1991).

It is intriguing that the intronless rice TPI 5' sequence functions well as a promoter in transgenic tobacco and two other dicot tissues but not in its own genetic background (or in maize or barley, the two other monocots tested) in the absence of an intron. It appears likely that the first intron of TPI is vital to expression of the t p i gene in monocot tissues but not in dicots. A similar finding has been reported for the maize shrunken 1 gene of maize, where intron 1 enhances chimeric gene expression in rice and maize protoplasts approximately 100-fold but inhibits reporter gene expression in tobacco protoplasts (Maas et al., 1991). Additionally, maize Adhl intron 1 dramatically increases foreign gene expression in maize and rice protoplasts (Callis et al., 1989; Luehrsen and Walbot, 1991) but does not increase gene expression in tobacco (Last et al., 1991). The dramatic reduction of GUS expression in dicots is likely to result from improper or inefficient splicing of the monocot introns in dicot species, causing premature translational termination due to in-frame stop codons within the intron sequence. This would be consistent with the notion that the requirements for splicing are different between monocot and dicot plants (Goodall and Filipowicz, 1991).

Enhancement of gene expression by introns can occur at the transcriptional level as a result of the intron bearing a transcriptional enhancer element (Oshima et al., 1990; Schultz et al., 1991) and can also occur at the posttranscrip- tional level through splicing-dependent mechanisms that lead to efficient polyadenylation, increased mRNA stability, or increased mRNA export to the cytoplasm (Buchman and Berg, 1988; Huang and Gorman, 1990). All plant introns studied so far that enhance gene expression appear to function only within the transcriptional unit in an orientation-dependent manner and result in increased steady-state levels of mRNA (Callis et al., 1987; Luehrsen and Walbot, 1991; Tanaka et al., 1991). It has been proposed (Callis et al., 1987; Luehrsen and Walbot, 1991) that the primary mechanism of intron- mediated stimulation of expression in plants is not sequence specific, but rather depends on the splicing process, and that critical factors include both the structure and the position of the intron. The basis for the requirement of the TPI intron for expression in rice remains to be determined. As we have

shown, TPI is a single-copy gene, and chimlxic reporter constructs using the TPI promoter can be exp1,essed stably and transiently in both monocot and dicot tissues. These attributes make the tpi gene a very favorable example for studying the differential role of introns in gene expression in the two major classes of higher plants. Among the questions to be fcicused on in the near future is whether the first intron of TPI Ifunctions as a transcriptional enhancer 01' if processes involved in its splicing are critical for the observed intron- mediated regulation of expression seen in rice.

ACKNOWLEDGMENTS

We thank Dr. Wallace G. Buchholz for discussion and advice, Mr. Jon Seay for help with rice tissue culture, and Ms. Ddxa Begum for assistance in tobacco transformation.

Received May 5, 1994; accepted June 30, 1994. Copyright Clearance Center: 0032-0889/94/106/045S/09.

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