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The Plant Cell, Vol. 3, 435-443, May 1991 0 1991 American Society of Plant Physiologists

A Parsley 4CL-1 Promoter Fragment Specifies Complex Expression Patterns in Transgenic Tobacco

Karl D. Hauffe," Uta Paszkowski,b Paul Schulze-Lefert,b Klaus Hahlbrock,b Jeffery L. Dangl,bs' and Carl J. Douglasay2

a Department of Botany, University of British Columbia, 3529-6270 University Boulevard, Vancouver, British Columbia V6T 124, Canada

Department of Biochemistry, Max-Planck-lnstitut fijr Züchtungsforschung, Carl von Linne Weg 1 O, D-5000 Koln 30, Germany

The 4CL-1 gene is one of two highly homologous parsley genes encoding 4-coumarate:coenzyme A ligase, a key enzyme of general phenylpropanoid metabolism. Expression of these genes is essential for the biosynthesis of both defense-related and developmentally required phenylpropanoid derivatives. We examined the developmental regulation of the 4CL-1 promoter by analyzing the expression of 4CL-1-8-glucuronidase fusions in transgenic tobacco plants. A 597-base pair 4CL-1 promoter fragment specified histochemically detectable expression in a complex array of vegetative and floral tissues and cell types. The activity of a series of 5' deleted promoter fragments was analyzed in parsley protoplasts and transgenic tobacco plants. Deletions past -210 base pairs led to a drastic decline in 8-glucuronidase activity in protoplasts and loss of tissue-specific expression in transgenic tobacco. These results were put into the context of potential protein-DNA interactions by in vivo footprint analysis of the 4CL-1 promoter in parsley cells. Loss of promoter activity in parsley protoplasts and transgenic tobacco was correlated with the deletion or disruption of the dista1 portion of a large (100-base pair) footprinted region within the first 200 base pairs of the 4CL-1 promoter.

INTRODUCTION

Differentiation of plant cell types often exploits the diversity of the end products of phenylpropanoid metabolism. Lignin and cell wall-bound phenolics are structural components of xylem and other vascular tissues, flavonoid floral pig- ments are attractants for pollinating insects, and a range of flavonoid derivatives serve as either activators or inhibitors of plant-microbe interactions. Synthesis of these compounds, therefore, must be integrated into organ differentiation (Wiermann, 1981). The first and last steps of general phenylpropanoid metabolism are catalyzed by L-phenylalanine ammonia-lyase (PAL) and 4-coumarate:coenzyme A (COA) ligase (4CL), respectively. PAL converts phenylalanine into cinnamic acid, whereas 4CL catalyzes the formation of COA esters of various cinnamate derivatives. These activated derivatives are, in turn, substrates for many branch pathways leading to the accumulation of end products in specialized cell types or in many cell types in response to stress (Hahlbrock and Grisebach, 1979; Hahl- brock et al., 1982).

' Current address: Max-Delbrück-Laboratorium in der Max- Planck-Gesellschaft, Carl von Lime Weg 10, D-5000 Koln 30, Germany.

To whom correspondence should be addressed.

The organization and regulation of phenylpropanoid genes have been areas of active research (see Hahlbrock and Scheel, 1989; Lamb et al., 1989; Dixon and Harrison, 1990; Dixon and Lamb, 1990; Dangl, 1991, for recent reviews). This analysis has focused on their transcriptional control in response to pathogen attack, light stress, or mechanical wounding (Chappell and Hahlbrock, 1984; Cra- mer et al., 1985a, 1985b; Lawton and Lamb, 1987). The hallmark of these responses is the rapid, often cell type- independent accumulation of the respective mRNAs pre- ceding accumulation of protective compounds (Schmelzer et al., 1989). Response of phenylpropanoid genes to environmental triggers is layered atop their normal temporal and spatial expression. Detection of P-glucuronidase (GUS) activity from chimeric promoter-reporter gene fusions allowed detailed definition of cell type-specific expression patterns from PAL and chalcone synthese (CHS) promoters (Bevan et al., 1989; Liang et al., 1989b; Ohl et al., 1990; Schmid et al., 1990; van der Meer et al.,

We have concentrated on the parsley 4CL-1 promoter because 4CL occupies a key position at the branch between general phenylpropanoid metabolism and end prod- uct-specific branches. 4CL is encoded in parsley by only

1990).

436 The Plant Cell

A _ B

Figure 1. Histochemical Localization of GUS Activity in Tobacco Plants Transgenic for 4CL-GUS Construction 99-G1-801, Except asNoted.

Parsley 4CL-1 Expression Patterns 437

two highly homologous genes, which show no evidence of differential regulation (Douglas et al., 1987; Lozoya et al., 1988). This contrasts with the complex organization and differential regulation observed in severa1 species for PAL and CHS genes (Bolwell et al., 1985; van Tunen et al., 1988; Beld et al., 1989; Liang et al., 1989a; Lois et al., 1989). In this paper, we describe the cell type-specific expression specified by the 4CL-1 promoter.

R E SU LTS

Cell Type-Specific Expression from the 4CL-1 Promoter

In situ analysis of 4CL mRNA accumulation in parsley seedlings (Schmelzer et al., 1989) showed that 4CL expression is developmentally regulated. Here, we detailed the ability of the parsley 4CL-1 promoter to direct cell type- specific expression of the GUS reporter gene (Jefferson et al., 1987) in transgenic tobacco. In a separate study (Doug- Ias et al., 1991), we showed that whereas the 4CL-1 gene is regulated by light and elicitor in parsley cells and in transgenic tobacco, 4CL-1 promoter sequences alone are insufficient to mediate regulation by these stimuli.

Histochemical detection of GUS expression from a 4CL-1-GUS fusion containing 597 bp of the 4CL-1 promoter is shown in Figure l . Activity was high in the primary xylem of axillary buds and developing leaf veins (Figure 1A) but undetectable in any other cell types of young stems or leaves. There was no expression in these epidermal cells, or in those of leaves (not shown), where light- induced synthesis of flavonoids was localized (Schmelzer et al., 1988). Tracheary elements were visualized in stained sections (Figure 16) by the autofluorescence of lignin and other cell wall-bound phenolics under UV excitation (Jah- nen and Hahlbrock, 1988). The highest 4CL-1 -GUS activity was found in xylem tissue where tracheary elements were

beginning to differentiate. This activity was not observed in the fully differentiated xylem of a mature leaf petiole (not shown). 4CL-1 -GUS activity was extensive in the secondary xylem of older stems (Figure lC), where GUS activity was restricted to files of ray parenchyma cells lying between highly lignified tracheary elements (Figure 1 D). In emerging lateral roots, GUS expression was limited to subapical cells (Figure 1 E), whereas in elongated roots, expression was observed in vascular tissue, root hairs, and subapical cells (not shown).

Figure 1 also shows that the 4CL-1 promoter was sufficient to drive GUS expression in a variety of floral tissues. In immature flowers, before pollination, this expression was observed in vascular tissue and developing nectaries (Figure 1 F). In postpollination flowers, expression in developing seeds was strong (Figure 1 G), whereas vascular and nectary expression weakened (not shown). Histochemical analysis of embedded cross-sections of developing seeds (Figure 1 H) showed that expression was limited to a single epidermal cell layer. In contrast, 4CL promoter activity in the nectary was observed in all cells (Figure 11). The 4CL-1 promoter was also very active in the mature stigmas (Figure 1 J), where expression was observed only in epidermal cells (Figure 1 K), and expression was also observed in mature pollen grains (not shown). Except for expression in vascular tissue, 4CL-1 -GUS activity in pigmented petal tissue (where flavonoid derivatives accumulate) was limited to epidermal cells (Figure 1 L). To test whether this expression was light dependent, transgenic tobacco plants harboring the 4CL- 1-GUS fusion were placed in complete darkness for 2 weeks after the formation of flower buds (but before emergence of floral tissues from sepals). Flower development continued with the typical etiolation response of elongation, and a lightly pigmented petal resulted (Figure 1M). The 4CL-1 promoter was highly expressed in pigmented petal tissue of both light-grown and dark-adapted plants (Figure 1N). We conclude that expression of the

Figure 1. (continued).

Cells having GUS activity are recognized by indigo dye deposits after staining with X-Gluc. (A) Cross-section through a young stem with an emerging leaf bud. (B) Enlargement of the leaf bud vein in (A) showing the autofluorescence of lignified tracheary elements (arrow) under UV excitation. (C) Cross-section of a mature stem. (D) Autofluorescence under UV excitation of the same section shown in (C). (E) Whole mount of an emerging lateral root. (F) Longitudinal hand section through the receptacle of a flower before pollination. (G) Whole mount of an isolated ovary. (H) Longitudinal section 20 pm thick through the ovary of a flower transgenic for 99-G1-809. (I) Longitudinal section 20 wm thick through the receptacle of a flower, showing the nectary. (J) Stigma whole mount. (K) Longitudinal section 20 pm thick through a stigma. (L) Cross-section 20 pm thick through the pigmented region of a petal. (M) Unstained flowers from light-grown (I) and dark-grown (d) plants. (N) X-Gluc-stained flowers from light-grown (I) and dark-grown (d) plants. n, nectary; o, ovule; r, ray parenchyma cells; t, tracheary elements; v, vascular tissue; ep, epidermal cells. Bars = 50 pm.

438 The Plant Cell

E C G

8!

-210

-180-170

-150

-UO

-130I

-120

r-100

-90

. _ - 80

footprinting analysis of the 4CL-1 promoter in suspension-cultured parsley cells was performed to localize potentialprotein-DNA contacts. Figure 2 shows that several strongin vivo protections from, as well as enhancements of,methylation were apparent over a stretch of around 100nucleotides in comparison to in vitro methylated DNA (-90bp to -190 bp). Figure 3 shows the location of theseresidues in the 4CL-1 promoter sequence. Investigation ofthe region between -200 and -700 failed to reveal anysimilar alterations in methylation patterns (not shown), andtreatment with fungal elicitor resulted in no major changesin putative protein-DNA interactions in this or any part ofthe 4CL-1 promoter to around position -700.

A series of Bal31 exonuclease deletions throughout the4CL-1 promoter were fused to the GUS reporter gene andanalyzed for transient expression in parsley protoplasts.Figure 4 shows that progressive 5' deletion quantitativelydiminished expression in protoplasts but that a major dropoccurred upon truncation from —210 bp to —174 bp (com-pare plasmids 810 and 803, Figure 4). The end point at-174 removed the TATA-distal portion of the in vivofootprinted region. We conclude that a critical c/'s-actingelement is deleted or disrupted within these 36 bp at thedistal end of the footprinted region and that elements distal

Figure 2. In Vivo Footprint Analysis of the 4CL-1 Promoter.

The genomic DNA sequence of G ladders of the noncoding strandof the parsley 4CL-1 gene from -70 bp to -230 bp is presented.The most prominent differences are noted in the pattern of sen-sitivity to in vivo DNA methylation by methyl sulfate in suspension-cultured parsley cells treated 4 hr before methylation with Phyto-phthora megasperma f. sp. glycinea elicitor (lane E) or in untreatedcontrol cells (lane C) relative to G methylation of cloned DNA (laneG). Open arrowheads, G residues hypomethylated in both elicitor-treated and control cells; filled arrowheads, G residues hyperme-thylated in both elicitor-treated and control cells.

4CL-1 promoter in petals is light independent, providedthere is not a very long-lived signal.

Two Hundred Ten Base Pairs of 4CL-1 Promoter IsSufficient for Cell Type-Specific Expression

The data presented above show that the 4CL-1 promoteris able to specify GUS expression in a developmental^complex manner. We defined the c/s-acting elements re-sponsible for quantitative expression of the 4CL-1 pro-moter in parsley and then determined whether any of thesec/s-acting sequences also mediate cell type-specific andtissue-specific expression in transgenic tobacco. In vivo

V V V VfTGTAAATCTCG CCATCACATG CTGCTTCATC TTAGTCAACT TTTTCCCTTC -161A 810 A 803

vv*ATCACCTAAC ACACAATATT TTTCTCACCA ACCCCACTCA TAATTTAATT -111

vwCCCCATTTTA CCCCTAACCA AACCTCATAT ICCGATAAAC TCCCCCTTTA -61

CCAACCCCCA TCCCCTTACC AAACCCTCCA CAT TTGAATTGTT -11

CATCATCTAA CATGTAACAA ACCTCTCTTA CTCATCATCG TTTCAACACC -40

AAAAACACAC ACACAACTAA CATTTTCATT TTCTCATTAT GGGAGATTGTMET

•90

Figure 3. Portion of the 4CL-1 Promoter Showing Features Rel-evant to Its Expression.

The coding strand is depicted with the transcription start site(Douglas et al., 1987) marked by a horizontal arrow above thesequence. The translation initiation codon is indicated by MET.Arrowheads above the nucleotide sequence indicate C residuesthat correspond to those G residues on the noncoding strandshown to have altered sensitivity to in vivo methylation. Openarrows, hypomethylation; filled arrows, hypermethylation. Verticalarrows beneath the sequence show the 5' end points and re-spective names of some promoter deletion derivatives that weretested for transient expression in parsley protoplasts and fortissue-specific expression in transgenic tobacco plants. The pu-tative TATA sequence is boxed. Numbers to the right refer tobase pairs relative to the transcription start site.


4CL promoter GUS repor ter gene

/ - _ = x

-1530 -597

-339 4

-252 - -210 -

-174 - -120 I

-78 I

promoter less

35s

GUS act lv l ty f S.D [pM 4-MU/mg X mlnl PI asm 1 d

99-G 1-800 3133 i 595

80 1 2205 t 399

808 781 i 245

809 739 i 78

810 487 f 184

803 47 i 7

813 41 t 14

812 20 i 4

99-GUS-JD/Kozak 18r 3

pRT99-GUS 1978 t 395

Figure 4. Structure and Transient Expression in Parsley Protoplasts oi 4CL-1 Promoter Fragments Fused to the GUS Gene.

A restriction map of the 4CL-1 promoter region and the GUS gene with the nopaline synthase 3' terminal region is shown at the top. Solid bars represent the promoter sequences fused to the GUS gene in each plasmid construction. The base-pair coordinates at the left indicate the length of the promoter fragments with respect to the 4CL-1 transcription start site. All constructions are in the vector pRT99- GUS-JD (Schulze-Lefert et al., 1989). The 99-GUS-JD/Kozak and pRT99-GUS plasmids shown at the bottom contain, respectively, the GUS gene alone and the cauliflower mosaic virus 35s promoter fused to GUS (Jefferson et al., 1987). The right-hand column shows the specific GUS enzyme activity of each construction. Expression was measured transiently in parsley protoplasts 2 days after DNA transfer. The values are the means (+SD) of 1 O separate experiments. 4-MU, 4-methylumbelliferone.

to -210 bp, not detected by in vivo footprinting, further enhance 4CL-1 transcriptional activity.

These 5' deleted GUS fusions were transferred to tobacco for histochemical analysis of 4CL-1 promoter activity in the tissue and cell types shown in Figure 1. Table 1 summarizes the results obtained from these analyses. Deletion of DNA upstream of -210 bp had no qualitative effect on tissue-specific expression: GUS activity was evident in all tissues and cells in plants transgenic for promoters 210 bp or longer. However, expression was undetectable in any tissues in any plants transgenic for the 120-bp and 78-bp deletions. Expression was partially disrupted in plants transgenic for the 174-bp promoter. No vascular or root expression was evident in any of these plants, but weak cell-specific GUS expression was clearly evident in certain floral organs of three plants expressing relatively high levels of GUS activity (determined fluoro- metrically). Thus, the 36 bp between -210 bp and -174 bp appears to be required both for efficient basal expression in parsley tissue culture cells and for part of the complex pattern of tissue-specific expression in transgenic tobacco. Sequences downstream of -1 74 bp, which by

themselves specified very low levels of expression in parsley cells, appear to be sufficient for some aspects of the observed tissue-specific expression.

DlSCUSSlON

Our data show that a 21 O-bp promoter fragment from the parsley 4CL-1 gene is sufficient to direct tissue-specific and cell-specific expression of the GUS reporter gene in transgenic tobacco. These observations confirm and ex- tend the compartmentalization of phenylpropanoid gene expression first postulated from enzymatic studies (Rubery and Northcote, 1968; Jones, 1984), defined by in situ hybridization (Schmelzer et al., 1989), and complemented by histochemical analysis of PAL-GUS promoter fusions (Bevan et al., 1989; Liang et al., 1989b; Ohl et al., 1990). We have also characterized in detail expression from the 4CL-1 promoter in floral tissues, where a complex pattern of tissue-specific and cell-specific expression in vascular, nectary, and stigmatic tissue, as well as in developing

440 The Plant Cell

Table 1. Summary of Histochemically Localized GUS Expression in Transgenic Tobacco Plants

Histochemically Detectable Expression

5’ End Point GUS Positive/ Ovule/ Root Construction (bP) Total Plants” Vascularb Nectary Stigma Pollen Peta1 Seed Coat Tip

99-G1-801 -597 618 + + + + + + + 99-G1-808 -339 415 + + + + + + + 99-G1-809 -252 517 + + + + + + + 99-G1-810 -21 o 6/11 + + + + + + + 99-G1-803 -1 74 011 4c - 99-G1-813 -1 20 018 99-G1-812 -78 017

- - - + + + - - - - - - - - - - - - - -

a Individual plants harboring each construction were histochemically screened for vascular expression.

tissue of stems.

stigmata; in two out of five, expression was detected in pollen. None of the five plants had detectable expression in petals or ovules.

Vascular expression, when present, was observed in vascular tissue of leaves, cotyledons, roots, and in primary and secondary vascular

In one out of the five plants in which floral organs were examined, weak but detectable expression was observed in nectaries and

seeds and pollen, was observed. We found that much of the nonvascular expression was limited to epidermal cells. Phenylpropanoid derivatives in floral tissues may serve as insect attractants or microbial repellents in nectaries and may be used as structural components of the developing seed coat. Receptive “wet” stigmata (such as those found in tobacco) have been reported to secrete a variety of phenolic compounds, which may function as protectants against microbial infection or insects, inhibitors or stimu- lators of pollen germination, or as a nutrient source when present as phenolic glycosides (Esau, 1977; Knox, 1984).

We also showed that flavonoid accumulation in petals is probably developmentally triggered rather than light induced. In contrast, phenylpropanoid gene expression in epidermal cell layers of vegetative tissue is primarily light controlled (Schmelzer et al., 1988; Liang et al., 1989b; Douglas et al., 1991). Although our data (Figures 1M and 1N) support the contention that light is not required for 4CL-1 expression in peta1 epidermal cells, we note that the presence in plants of a potential long-lived signal has precedence (Ohl et al., 1989). The reduced pigmentation in etiolated flowers suggests that pigmentation is light enhanced.

We limited the sequences responsible for this myriad of cell type-specific expression modes to 21 O bp upstream of the transcription start site. Furthermore, we showed that a deletion to -1 74 bp abrogates histochemically detectable GUS activity in vascular, root, and some floral tissues. This same deletion also caused a 1 O-fold drop in transient GUS expression in parsley protoplasts. Thus, removal of only the most TATA-dista1 sequences from the broad in vivo footprinted region severely disrupted 4CL-1 expression in both parsley protoplasts and transgenic tobacco, suggesting that at least some cis-acting elements identified by in vivo footprinting are required in common

for tissue-specific expression and expression in undiffer- entiated tissue culture cells. Interestingly, a partially palin- dromic in vivo footprinted sequence, TAGTCAAC (position -178), was disrupted in this deletion and is conserved in PAL promoters (positions -236 in bean gPAL2, Cramer et al., 1989; -258 in PcPAL-1, Lois et al., 1989; -331 in Arabidopsis PAL1, Ohl et al., 1990). We cannot, however, rule out the possibility that this or other element(s) removed in this deletion play a purely quantitative role in 4CL-1 expression and do not by themselves specify cell-specific and tissue-specific expression. Elements downstream of -174 bp are also involved in specifying at least some of the observed 4CL-1 expression modes, for example in certain floral organs. The footprinted sequence CTCAC- CAACCC at position -137 is noteworthy in this regard because it is highly conserved in severa1 PAL and other phenylpropanoid genes (Lois et al., 1989; Ohl et al., 1990). Clearly, more refined mutations of elements within the 21 O-bp promoter are needed to test their roles in specifying cell type-specific expression; these experiments are cur- rently underway.

METHODS

Genomic Sequencing

In vivo footprinting was done according to modifications (Schulze- Lefert et al., 1989) of the original procedure (Church and Gilbert, 1984). Genomic fractions enriched for 4CL-1 or 4CL-2 were prepared as follows: Genomic DNA from dimethyl sulfate-treated parsley cells was digested to completion with BamHl and fraction- ated on sucrose gradients to separate the 4CL-1 and 4CL-2 upstream regions (contained on 3.0-kb and >1 O-kb restriction fragments, respectively). This also resulted in a large enrichment


of the 4CL-1 promoter fragment because greater than 90% of BamHI-digested DNA was larger than 3 kb in size. Genomic fractions were digested with Sau3A to create reference cuts at position +118 of both genes. High specific activity single-stranded probes were generated by priming synthesis over an M13 tem- plate spanning the promoter region.

Plasmid Constructions

Plasmids 99-G1-800 through 99-G1-813 were constructed by cloning 4CL-1 promoter fragments as transcriptional fusions upstream of the GUS gene in pRT99-GUS-JD/Kozak, a derivative of pRT99-GUS-JD (Schulze-Lefert et al., 1989) that contains a con- sensus eukaryotic (“Kozak”) translation start site (Kozak, 1981 ; a gift of R. Jefferson). A 1.5-kb promoter fragment was first isolated by Ba131 deletion of a 2.7-kb BamHl fragment, starting an Sstl site 3’ to the transcription start site. This deletion derivative had a 3‘ end point at position +17 with respect to the transcription start site and was cloned into pRT99-GUS-JD/Kozak (to create 99-G1-800). 5‘ deletion derivatives (99-G1-801 to 99-G1-813) were created by standard procedures using Ba131 and upstream restriction sites.

Tobacco Transformation

4CL-1 -GUS fusions were cloned as EcoRI-Hindlll fragments into BIN19 (Bevan, 1984) and transferred into Agrobacterium tumefaciens by triparental matings with fscherichia coli strains (Ditta et al., 1980). The structure of all BIN19 constructions in Agrobacfer- ium was confirmed using the screening method of Ebert et al. (1987). Tobacco leaf discs were transformed and plants were regenerated by standard methods (Horsch et al., 1985).

Protoplast Preparation and Transformation

Protoplasts were prepared from 5-day-old suspension-cultured parsley cells as described by Dangl et al. (1987), and transformation with 20 pg/106 protoplasts of supercoiled or linearized plasmid DNA was performed as described (Lipphardt et al., 1988; Schulze-Lefert et al., 1989). Cells were kept in constant darkness, and GUS activity was assayed 2 days after transfer using the fluorometric assay (Jefferson, 1987).

Histochemical Localization of GUS Activity

Histochemical localization of GUS activity was performed as described by Jefferson (1 987). Hand sections, tissue for paraffin embedding and sectioning, or whole mounts were taken from 4- month-old greenhouse-grown plants or 3-week-old to 4-week-old axenically grown F1 seedlings. Before staining, tissue was vacuum infiltrated with 0.5% paraformaldehyde, 1 O0 mM sodium phosphate (pH 7.0), 0.4 M sucrose for 1 hr, and then washed twice for 15 min with 100 mM sodium phosphate (pH 7.0). Hand- sectioned tissue or tissue for whole mounts was stained for GUS activity for 3 hr to 16 hr using 5-bromo-4-chloro-3-indole glucu- ronide (X-Gluc, Research Organics, Cleveland, OH or Clontech, Palo Alto, CA) at 0.5 mg/mL in 100 mM sodium phosphate (pH

7.0). After staining, the tissue was cleared in ethanol (25% to 80%) and observed with a Zeiss Axioskope. Autofluorescence was observed using excitation at 365 nm to 425 nm and emission above 450 nm (Zeiss filter set 48771 8).

Thicker tissue sections used for embedding were prefixed on ice as above, washed twice for 15 min with 100 mM sodium phosphate (pH 7.0), and then stained with X-Gluc for 16 hr to 18 hr. The tissue was postfixed in 1% paraformaldehyde, 0.5% glutaraldehyde, 1 O0 mM sodium phosphate (pH 7.0), and washed for 60 min with 100 mM sodium phosphate (pH 7.0), with buffer changes every 10 min. Tissue was dehydrated in a series of aqueous ethanol solutions (v/v) as follows: 25%, do%, and 55% for 20 min each; 70% overnight at 4°C; 8O%, 90%, and 95% for 20 min each at room temperature; 100% twice for 20 min. Subsequently, the tissue was infiltrated with tertiary butanol and paraplast as described by Schmelzer et al. (1989). Embedded tissue was sectioned at 1 O-pm to 20-pm thickness using a standard rotary microtome (Leitz, type 121 2). Subsequent handling of tissue sections (relaxation, binding to microscope slides, solubili- zation of paraffin with xylene) was performed as described by Schmelzer et al. (1 989). Using these methods, endogenous GUS activity, as described in certain floral organs (Plegt and Bino, 1989; Hu et al., 1990), was not observed in any organs examined from untransformed tobacco plants or tobacco plants transgenic for a promoterless GUS gene.

ACKNOWLEDGMENTS

We thank Drs. Sarah Grant, lmre Somssich, and Dierk Scheel for critical reading of the manuscript, Michael Bevan for supplying BIN19, Richard Jefferson for the gift of plasmid DNA before publication, and Tami Chappell and Erika Tabak for help with the manuscript. K.D.H. was the recipient of a Deutsche Akademische Austauschdienst postdoctoral fellowship from the Federal Repub- lic of Germany, P.S.-L. was the recipient of a Fritz Thyssen Foundation predoctoral stipend, and J.L.D. was supported by a postdoctoral fellowship from the National Science Foundation. This work was supported by Natural Science and Engineering Research Council of Canada Operating and Equipment Grants to C.J.D. and by Fonds der Chemischen lndustrie (to K.H.).

Received December 26, 1990; accepted March 5, 1991

REFERENCES

Beld, M., Martin, C., Huits, H., Stuitje, A.R., and Gerats, A.G.M. (1 989). Flavonoid synthesis in Petunia hybrida: Partia1 characterization of dihydroflavonol-4-reductase genes. Plant MOI. Biol.

Bevan, M.D. (1 984). Binary Agrobacterium tumefaciens vectors 13,491-502.

for plant transformation. Nucl. Acids Res. 12, 871 1-8721.

442 The Plant Cell

Bevan, M., Shufflebottom, D., Edwards, K., Jefferson, R., and Schuch, W. (1989). Tissue- and cell-specific activity of a phenylalanine ammonia-lysase promoter in transgenic plants. EMBO

Bolwell, G.P., Bell, J.N., Cramer, C.L., Schuch, W., Lamb, C.J., and Dixon, R.A. (1985). L-Phenylalamine ammonia lyase from Phaseolus vulgaris. Characterization and differential induction of multiple forms. Eur. J. Biochem. 149, 41 1-41 9.

Chappell, J., and Hahlbrock, K. (1 984). Transcription of plant defense genes in response to UV-light or fungal elicitor. Nature

Church, G., and Gilbert, W. (1984). Genomic sequencing. Proc. Natl. Acad. Sci. USA 81, 1991 -1 995.

Cramer, C.L., Bell, J.N., Ryder, T.B., Bailey, J., Schuch, W., Bolwell, G.P., Robbins, M.P., Dixon, R.A., and Lamb, C.J. (1 985a). Co-ordinated synthesis of phytoalexin biosynthetic enzymes in biologically-stressed cells of bean (Phaseoh vulgaris L.). EMBO J. 4, 285-290.

Cramer, C.L., Ryder, T.B., Bell, J.N., and Lamb, C.J. (1985b). Rapid switching of plant gene expression by fungal elicitor. Science 227,1240-1 243.

Cramer, C.L., Edwards, K., Dron, M., Liang, X., Dildine, S.L., Bolwell, G.P., Dixon, R.A., Lamb, C.J., and Schuch, W. (1989). Phenylalanine ammonia-lyase gene organization and structure. Plant MOI. Biol. 12, 367-383.

Dangl, J.L. (1 991). Regulatory elements controlling developmental and stress induced expression of phenylpropanoid genes. In Plant Gene Research, Vol. 8, T. Boller and F. Meins, eds (Vienna: Springer-Verlag), in press.

Dangl, J.L., Hauffe, K.D., Lipphardt, S., Hahlbrock, K., and Scheel, D. (1 987). Parsley protoplasts retain differential re- sponsiveness to U.V. light and fungal elicitor. EMBO J. 6,

Ditta, G., Stanfield, S., Corbin, D., and Helinski, D.R. (1980). Broad range DNA cloning system for gram-negative bacteria: Construction of a clone bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA 77,7347-7351.

Dixon, R.A., and Harrison, M.J. (1990). Activation, structure and organization of genes involved in microbial defense in plants. Adv. Genet. 28,165-234.

Dixon, R.A., and Lamb, C.J. (1990). Molecular communication in interactions between plants and microbial pathogens. Annu. Rev. Plant Physiol. Plant MOI. Biol. 41, 339-367.

Douglas, C.J., Hoffmann, H., Schulz, W., and Hahlbrock, K. (1 987). Structure and elicitor or u.v.-light stimulated expression of two 4-coumarate:CoA ligase genes in parsley. EMBO J. 6,

Douglas, C.J., Hauffe, K.D., Ites-Morales, M.-E., Ellard, M., Paszkowski, U., Hahlbrock, K., and Dangl, J.L. (1991). Exonic sequences are required for elicitor and light activation of a plant defense gene, but promoter sequences are sufficient for tissue specific expression. EM60 J., in press.

Ebert, P.R., Ha, S.B., and An, G. (1987). ldentification of an essential upstream element in the nopaline synthase promoter by stable and transient assays. Proc. Natl. Acad. Sci. USA 84, 5745-5749.

Esau, K. (1977). Anatomy of Seed Plants. (New York: John Wiley & Sons).

J. 8, 1899-1906.

311,76-78.

2551-2556.

1 189-1 195.

Hahlbrock, K., and Grisebach, H. (1979). Enzymatic controls in the biosynthesis of lignin and flavonoids. Ann. Rev. Plant Phys- iol. 30, 105-1 30.

Hahlbrock, K., and Scheel, D. (1989). Physiology and molecular biology of phenylpropanoid metabolism. Annu. Rev. Plant Phys- iol. Plant MOI. Biol. 40, 347-369.

lahlbrock, K., Kreuzaler, F., Ragg, H., Fautz, E., and Kuhn, D.N. (1 982). Regulation of flavonoid and phytoalexin accumulation through mRNA and enzyme induction in cultured plant cells. In Biochemistry of Differentiation and Morphogenesis, L. Jaenicke, ed (Berlin: Springer-Verlag), pp. 34-43.

lorsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholtr, O., Rogers, S.G., and Fraley, R.T. (1985). A simple and general method for transferring genes into plants. Science 227, 1229-1231.

Hu, C., Chee, P., Zhou, J., Miller, P., and O’Brien, W.T. (1990). lntrinsic GUS-like activity. In Progress in Plant Cellular and Molecular Biology, H.J.J. Nijkamp, L.H.W. van der Plas, and J. van Aartrijk, eds (Dordrecht, The Netherlands: Kluwer Aca- demic), pp. 195-200.

Jahnen, W., and Hahlbrock, K. (1988). Cellular localization of nonhost resistance reactions of parsley (Petroselinium crispum) to fungal infection. Planta 173, 197-204.

Jefferson, R.A. (1 987). Assaying chimeric genes in plants: The GUS gene fusion system. Plant MOI. Biol. Rep. 5, 387-405.

Jefferson, R.A., Kavanagh, T., and Bevan, M.W. (1987). GUS fusions: P-Glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901-3907.

Jones, D.H. (1 984). Phenylalanine ammonia lyase: Regulation of its induction, and its role in plant development. Phytochemistry

Knox, R.B. (1 984). Pollen-pistil interactions. In Encyclopedia of Plant Physiology, VoI. 17: Cellular Interactions, L.F. Linskens and J. Heslop-Harrison, eds (Berlin: Springer-Verlag), pp.

Kozak, M. (1981). Possible role of flanking nucleotides in recog- nition of the AUG initiation codes by eucaryotic ribosomes. Nucl. Acids Res. 9,5233-5252.

Lamb, C.J., Lawton, M.A., Dron, M., and Dixon, R.A. (1989). Signals and transduction mechanisms for activation of plant defenses against microbial attack. Cell 56, 21 5-224.

Lawton, M.A., and Lamb, C.J. (1 987). Transcriptional activation of plant defense genes by fungal elicitor, wounding and infection. MOI. Cell. Biol. 7, 335-341.

Liang, X., Dron, M., Cramer, C.L., Dixon, R.A., and Lamb, C.J. (1 989a). Differential regulation of phenylalanine ammonia-lyase genes during plant development and by environmental cues. J. Biol. Chem. 264,14486-14492.

Liang, X., Dron, M., Schmid, J., Dixon, R.A., and Lamb, C.J. (1 989b). Development and environmental regulation of a phenylalanine ammonia-lyase-&glucuronidase gene fusion in transgenic tobacco plants. Proc. Natl. Acad. Sci. USA 86,

Lipphardt, S., Brettschneider, R., Kreuzaler, F., Schell, J., and Dangl, J.L. (1 988). UV-inducible transient expression in parsley protoplasts identifies regulatory cis-elements of a chimeric chalcone synthase gene. EMBO J. 7,4027-4033.

23,1349-1 359.

508-608.

9284-9288.


Lois, R., Dietrich, A., Hahlbrock, K., and Schulz, W. (1989). A phenylalanine ammonia-lyase gene from parsley: Structure, regulation and identification of elicitor and light responsive elements. EM60 J. 8, 1641-1648.

Lozoya, E., Hoffmann, H., Douglas, C.J., Schulz, W., Scheel, D., and Hahlbrock, K. (1988). Primary structures and catalytic properties of isoenzymes encoded by the two 4-coumarate:CoA ligase genes in parsley. Eur. J. Biochem. 176, 661-667.

Ohl, S., Hahlbrock, K., and Schaefer, E. (1989). A stable blue light derived signal modulates UV light induced changes in chalcone synthase gene activity in cultured parsley cells. Planta

Ohl, S., Hedrick, S.A., Chory, J., and Lamb, C.J. (1990). Func- tional properties of the phenylalanine ammonia-lyase promoter from Arabidopsis. Plant Cell 2, 837-848.

Plegt, L., and Bino, R.J. (1 989). P-Glucuronidase activity during development of the male gametophyte from transgenic and non-transgenic plants. MOI. Gen. Genet. 216, 321 -327.

Rubery, P.H., and Northcote, D.H. (1968). Site of phenylalanine ammonia-lyase activity and synthesis of lignin during xylem differentiation. Nature 219, 1230-1243.

Schmelzer, E., Jahnen, W., and Hahlbrock, K. (1988). In situ localization of light-induced chalcone synthase mRNA, chalcone synthase, and flavonoid end products in epidermal cells of parsley leaves. Proc. Natl. Acad. Sci. USA 85, 2989-2993.

177,228-236.

Schmelzer, E., Krüger-Lebus, S., and Hahlbrock, K. (1989). Temporal and spatial patterns of gene expression around sites of attempted funga1 infection in parsley leaves. Plant Cell 1,

Schmid, J., Doerner, P.W., Clouse, S.D., Dixon, R.A., and Lamb, C.J. (1 990). Developmental and environmental regulation of a bean chalcone synthase promoter in transgenic tobacco. Plant Cell2, 61 9-631.

Schulze-Lefert, P., Dangl, J.L., Becker-Andre, M., Hahlbrock, K., and Schulz, W. (1989). lnducible in vivo footprints define sequences necessary for UV-light activation of the parsley chalcone synthase gene. EMBO J. 8,651-656.

van der Meer, I., Spelt, C.E., MOI, J.N.M., and Stuitje, A.R. (1 990). Promoter analysis of the chalcone synthase (chsA) gene of Petunia hybrida: A 67 bp promoter region directs flower- specific expression. Plant MOI. Biol. 15, 95-109.

van Tunen, A.J., Koes, R.E., Spelt, C.E., van der Krol, AR., Stuitje, A.R., and MOI, J.N.M. (1988). Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: Coordinate, light regulated and differential expression of flavonoid genes. EMBO J. 7,1257-1263.

Wiermann, R. (1981). Secondary plant products and cell and tissue differentiation. In The Biochemistry of Plants, Vol. 7, P.K. Stumpf and E.E. Conn, eds (New York: Academic Press), pp.

993-1001.

85-1 16.

a parsley 4cl-1 promoter fragment specifies …labs.bio.unc.edu/dangl/pub/pdf/plant cell-hauffe et...

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