functional properties of a phenylalanine ammonia-lyase ... · and meyerowitz, 1986) with bean pal...

The Plant Cell, Vol. 2, 837-848, September 1990 O 1990 American Society of Plant Physiologists

Functional Properties of a Phenylalanine Ammonia-Lyase Promoter f rom Arabidopsis

Stephan Ohl, Susan A. Hedrick, Joanne Chory, and Christopher J. Lamb’ Plant Biology Laboratory, Salk lnstitute for Biological Studies, 1 O01 O North Torrey Pines Road, La Jolla, California 92037

Phenylalanine ammonia-lyase (PAL) is encoded by a small family of genes in Arabidopsis. We cloned and partially characterized one of these genes, PAL1. The deduced amino acid sequence is highly similar to PAL from bean, parsley, and rice. The promoter contains sequence elements homologous to two putative regulatory elements conserved among severa1 phenylpropanoid genes. The regulation of the PAL1 gene was examined by analysis of P-glucuronidase (GUS) activity in transgenic Arabidopsis containing PAL1-GUS gene fusions. The PAL1 promoter was activated early in seedling development and in adult plants was strongly expressed in the vascular tissues of roots and leaves, but was not active in the root tip or the shoot apical meristem. In flowers, expression was observed in sepals, anthers, and carpels, but not in petals. Transcripts encoded by the endogenous PAL genes and GUS transcripts from the PAL1-GUS gene fusion were induced by wounding, HgCI,-stress, and light. Analysis of the regulatory properties of 5’ deleted promoters showed that the proximal region of the promoter to -290 was sufficient to establish the full tissue-specific pattern of expression and that the proximal region to -540 was responsive to environmental stimuli. Negative and positive elements were located between -1816 and -823 and between -823 and -290, respectively.

INTRODUCTION

Phenylalanine ammonia-lyase (EC 4.3.1 5, PAL) catalyzes the deamination of L-phenylalanine to trans-cinnamic acid, which is the first step in the biosynthesis of a large class of plant natural products based on the phenylpropane skeleton (Dixon et al., 1983; Jones, 1984). Phenylpropa- noids play key roles in plant development and in protection against environmental stresses. Thus, flavonoids are pigments and UV-protectants, and lignin is a major structural polymer in xylem cell walls. lnduction of lignin deposition in peripheral tissues and accumulation of furanocoumarin and isoflavonoid-derived phytoalexins help protect against mechanical damage and potential pathogens. In addition, some phenylpropanoids function as regulatory and signal substances. For example, the Agrobacterium virulence genes and the Rhizobium nodulation genes are induced by compounds such as acetosyringone derived from phenylpropanoid wound metabolites and simple flavonoids such as luteolin, respectively (Downie and Johnston, 1986; Stachel and Zambryski, 1986).

PAL mRNA and enzyme levels are highly regulated during development and in response to environmental cues, correlated with the accumulation of specific phenylpropanoid products (Lamb et al., 1989). PAL is encoded by a family of three to four genes in bean (Cramer et al., 1989), parsley (Lois et al., 1989), and rice (Minami et al.,

’ To whom correspondence should be addressed.

1989), and the transcripts of individual PAL genes show highly different patterns of accumulation (Liang et al., 1989a; Lois et al., 1989). Nuclear run-off transcription experiments have shown that elicitor and UV-light induction of PAL mRNA in cell suspension cultures of bean and parsley reflects transient stimulation of PAL gene transcription (Lawton and Lamb, 1987; Lois et al., 1989). Likewise, analysis of the bean PAL2 promoter in transgenic tobacco and potato plants containing PAL2-P-glucuronidase (GUS) gene fusions has demonstrated tissue- and cell-type-specific PAL transcription during development and in response to wounding and light (Bevan et al., 1989; Liang et al., 1989b). Thus, the bean PAL2 promoter is able to integrate a complex set of developmental and environmental cues into a coherent program of expression attuned to the diverse functions of phenylpropanoid natural products.

To help dissect the signal transduction pathways underlying developmental and environmental regulation of PAL promoters, we are examining PAL regulation in Arabidop- sis and report here on the isolation of an Arabidopsis PAL gene and the functional properties of its promoter. Arabi- dopsis is readily transformable (Valvekens et al., 1988), and, hence, we have been able to examine the properties of this promoter in the homologous system. In addition, a number of hormonal and developmental mutants have been characterized in Arabidopsis that are likely to be involved in signal pathways for PAL regulation (Meyerow-

838 The Plant Cell

itz, 1989). Moreover, Arabidopsis provides an excellentsystem for the creation of transgenic plants containingselectable or screenable marker genes regulated by thePAL promoter, or specific dissected c/s-elements, for usein the identification of novel signal pathway mutants thataffect PAL promoter activity in trans.

As a first step in these studies, we show here that PALis encoded by a small gene family in Arabidopsis. One ofthe genes, designated PAL1, was cloned and partiallycharacterized. The encoded product is highly related toPAL from other species, and the promoter contains puta-tive regulatory elements conserved in the promoters ofseveral phenylpropanoid biosynthetic genes. We demon-strate that PAL1 exhibits a complex pattern of tissue-specific developmental expression and is also induced bywounding, HgCI2-stress, and light. Functional analysis of5' deletions identified regions of the PAL1 promoter in-volved in determining the quantitative level of expression,tissue specificity, and environmental regulation.

RESULTS

Cloning and Characterization of an Arabidopsis PALGene

A 5-kb EcoRI fragment containing an Arabidopsis PALgene (PAL1) was subcloned from a genomic clone isolatedby screening a genomic library from Arabidopsis (Changand Meyerowitz, 1986) with bean PAL cDNA sequences(Edwards et al., 1985).

To determine the complexity of PAL genes in the Ara-bidopsis genome, genomic DNA was digested with EcoRI,Hindlll, BamHI, and Pstl, blotted, and hybridized with alabeled fragment from the Arabidopsis PAL1 gene. Theresult is shown in Figure 1. Under stringent hybridizationand washing conditions, four to five bands of differentintensities were observed, indicating that PAL1 is a mem-ber of a small family of genes.

We sequenced a 3.1-kb fragment that included 1.8 kbof the promoter and 0.7 kb of the coding region, as shownin Figure 2. The deduced amino acid sequence showed75%, 77%, and 67% identity to the corresponding PALsequences from bean (Cramer et al., 1989), parsley (Loiset al., 1989), and rice (Minami et al., 1989), respectively.Sequence comparison indicated that the coding regioncontained a 448-bp intron with AG/GT and AG/AT consen-sus boundaries (Breathnach and Chambon, 1981) at thesame relative position as in the previously characterizedPAL genes from these other species. The transcriptionstart site of PAL1 was mapped by S1 nuclease protectionto a position 129 bp upstream of the putative translationinitiation codon (data not shown).

The promoter and 5'-untranslated region of the PAL1gene exhibited no obvious overall sequence homology to

DCOoLU

T3C E

raCD

I8 . 4 55.694 . 3 23 .68

2 . 3 2

1 .93

1 .371 .26

— 0.70

Figure 1. DNA Gel Blot of Arabidopsis Genomic DNA Probedwith a Fragment from the Coding Region of PAL1.

Five micrograms of DNA per lane were digested with EcoRI,Hindlll, BamHI, or Pstl, separated on a 0.8% agarose gel, blotted,and hybridized with a labeled 190-bp Styl-Hindlll PAL1 fragmentcorresponding to the 5' end of the second exon. The blot waswashed in 0.1 x SSC, 0.1% SDS at 65°C and autoradiographedfor 3 days. Size markers are given in kilobases.

Arabidopsis PAL Promoter 839

- I696 ~ 1636

-1576

-1516

-1456

-1396

-1336

-1276

-1216

-1156

-1096

-1036

-916

-916

-856

-196

-736

-676

-616

-556

-496

-436

-376

-316

-256

-196

-136

-76

-16

1

61

121

181

24 1

3 0 1

361

421

481

541

601

661

721

781

MGGATTTTCCGGTATCCGATTTGAGATTCTCGMGCMTTACCAGTTTCCTCMCMCA l ~ G l y P h ~ S ~ r G l y l l ~ A ~ q P h e C l u t l e L e u G l u A l a I l ~ ~ A ~ ~ A

-1637

-1517

-1517

-1457

-139J

-1337

-127J

-1217

-1151

-1097

-1037

-977

-911

-851

-191

-131

-617

-611

-557

-497

-431

-311

-317

-251

-191

-137

-71

-11

60

120

180

240

300

360

42Q

480

540

600

660

720

780

840

other PAL genes, but a computer-aided nucleotide sequence comparison identified several repeats of two small elements, which are conserved among the promoters of several phenylpropanoid biosynthetic genes and which coincide with inducible in vivo footprints in the parsley PAL promoter (Lois et al., 1989). In the 5’-untranslated leader, a 9-bp element is identical to an element in the bean CHSl5 gene at a similar location, which coincides with an elicitor-inducible DNase I hypersensitive site (Lawton et al., 1990).

Expression of PAL 1-GUS Fusion in Transgenic Arabidopsis

To determine the regulatory properties of the Arabidopsis PALl promoter during seedling development and in response to different environmental stimuli, 1.8 kb of the PALI promoter, subsequently referred to as the “full-length promoter,” was cloned into the binary vector pBll O1 .I (Jefferson et al., 1987) as a transcriptional fusion in front of a promoterless p-glucuronidase (GUS) gene. The result- ing construct, pS0-I, was transformed into Arabidopsis by the root transformation method (Valvekens et ai., 1988), and kanamycin-resistant transgenic plants were analyzed in the T2 generation.

Expression during Early Seedling Development and in Adult Plants

Seeds of transgenic plants transformed with pS0-1 were germinated on medium containing kanamycin and seedlings were stained with 5-bromo-4-chloro-3-indolyl-p-glucuronide (X-Gluc) at various developmental stages, as shown in Figure 3. The strongest GUS staining was found in very young seedlings that had just emerged from the seed coat (48 hr after planting), and staining subsequently declined slowly. GUS staining was found in all tissues of the seedling except the root tip. Staining was most intense in a broad area at the base of the radicle, at the base and the tip of the cotyledons, and associated with the pre- sumptive vascular tissue of the shoot (Figures 3A and 38). Four to 5 days after germination, GUS staining had decreased in most tissues and became progressively re- stricted to the vascular tissue (Figures 3C to 3F). The staining intensity became more variable among independent transformants, but remained higher in the root than the shoot.

To determine the pattern of PALl -GUS expression in adult plants transformed with pS0-1, the TP progeny of

841 GTGMGCT 048

boxed, and sequence elements with similarities to other phenyl-

details). The position of the 448-bp intron is indicated by an arrow (intron sequence not shown), and the deduced amino acid sequence is shown below the nucleotide sequence.

1 yGluAla

Figure 2. Sequence of the P A L ~ Prometer, 5’JJntranslated Re- ProPanoid Promoters are underlined (see Figure 8 and text for gion, and Part of the Coding Region.

The transcription start site is at +1, a putative TATA homology is

840 The Plant Cell


severa1 independent transformants were grown for 3 to 5 weeks on selective medium and stained with X-Gluc to visualize GUS activity. Figure 3 shows that the pattern found in young seedlings was maintained in the adult plant, with stronger staining in roots than in stems or leaves (Figures 3G and 3H). At the tissue level, staining was most pronounced in the vascular tissue (Figures 3L and 3M), but lower levels of GUS activity were occasionally observed in other tissues.

Young flower buds did not stain initially for GUS activity (Figure 30), but staining appeared slowly as the flower buds developed. In a fully developed flower, GUS staining was found in the sepals, in the stamen, and at the tip and base of the carpel, but no staining was detected in petals (Figures 3N and 3P). lntense GUS staining was also observed in pollen grains, and analysis of flower buds at different developmental stages showed that GUS staining in pollen became visible before the pollen developed its yellow pigmentation (data not shown).

Staining intensity could be quite variable even among the progeny of individual transformants and within a plant between different organs. Thus, two leaves of the same size and developmental stage sometimes showed a striking difference in staining intensity. However, the basic qualitative features of high vascular expression, lack of expression in the root tip, and a characteristic pattern of floral expression were always maintained.

lnduction by Wounding, HgCI2, and Light

Three-week-old transgenic plants were wounded, incubated in an HgCI, solution, or transferred to white light after 4-day dark adaptation, and then harvested 6 hr to 12 hr after stimulation. GUS activity was measured using the fluorimetric assay and, to our surprise, no significant induction of extractable GUS activity was observed in these experiments. The results of a typical experiment determining GUS activity 8 hr after wounding or transfer to a HgCI2 solution are shown in Table 1.

Table 1. GUS Activity in Plants Transformed with pS0-1 after Wounding or HgCI2 Treatment

GUS Activity (pmol MU”/min. mg protein)

Control 29.2 k 1.8 Wounded 31.2 * 2.3 HSCIz 27.3 f 1.9

GUS activity was determined from whole plants 8 hr after wounding or transfer to a 100 pM HgCI2 solution. The data are mean values and average deviations from the mean calculated from 14 measurements on individual transformants. a MU, methylumbelliferone.

Because there was some variability in the assay, and a transient environmental induction might not be readily observed against a background of significant GUS activity arising from sustained developmental expression of the gene fusion, we subsequently examined changes in the levels of PAL and GUS transcripts in transgenic plants after wounding, HgCI, treatment, or transfer to white light after 4-day dark adaptation. Total RNA was extracted from whole plants harvested 1 hr to 4 hr after treatment and subjected to RNA gel blot analysis using labeled PALl or GUS probes. The results are shown in Figure 4. Tran- scripts encoded by the endogenous PAL genes were markedly induced by all three stimuli (Figure 4, top panel). Additional experiments measuring mRNA during a longer time course showed that the induction by wounding and HgCI, stress was transient with the highest levels 1 hr to 2 hr after stimulation (data not shown). Light induction resulted in a slower accumulation of PAL transcripts, with a maximum 2 hr to 4 hr after transfer to light.

GUS mRNA was induced by all three stimuli and accumulated with kinetics very similar to those observed for PAL mRNA (Figure 4). In control plants transformed with a cauliflower mosaic virus 35s promoter-GUS gene fusion, the endogenous PAL mRNA was induced by wounding, whereas GUS mRNA remained relatively low (Figure 4, w).

Figure 3. Transgenic Arabidopsis Plants at Different Developmental Stages after Staining with X-Gluc.

(A) to (F) PALl expression in young seedlings at 2 days [(A)], 3 days [(B)], 5 days [(C)], 8 days [(D)], 11 days [(E)], and 15 days [(F)] after planting. (G) Adult plants 25 days after planting. (H) Sector of (G) at higher magnification. (I) and (K) Plants transformed with pS0-4 at 2 days [(I)] and 8 days [(K)] after planting. (L) and (M) Cross-sections from leaves of SO-1 transformants after GUS staining. (L) Cut diagonally through the base of a leaf and the petiole. (M) Cut vertically through the leaf. (N) to (P) Floral organs of plants transformed with pS0-1. (N) Open flower. (O) Young flower bud. (P) Ripening silique. (a) to (S) Control plants transformed with a cauliflower mosaic virus 35s-GUS construct. (a) Young plants. (R) Adult plant, sector. (S) Roots.

842 The Plant Cell

wounding i HgQ2 i i lightC l 2 3 1 2 3 w c l 2 4

PAL

GUS

..I

- 4.4

- 2.4

_ 1.4- 4.4

Figure 4. RNA Gel Blot of PAL mRNA and GUS mRNA in PlantsTransformed with pSO-1.

Whole plants were wounded, transferred to a 100-^M HgCI2solution, or transferred to white light following 4 days of darkadaptation, and harvested 1 hr to 4 hr after treatment. Totalcellular RNA was extracted and fractionated by gel electrophore-sis, blotted, and hybridized to labeled probes homologous to PALand GUS. c, control plants harvested immediately after woundingor transfer to light; w, plants transformed with a cauliflower mosaicvirus 35S promoter-GUS fusion 1 hr after wounding. Size markersare given in kilobases.

Effect of 5' Deletions on Organ-Specific and Tissue-Specific Expression

The effect of 5' promoter deletions on the basal level ofGUS expression was examined in 3-week-old T2 plantstransformed with the constructs pSO-1 to pSO-4 and isshown in Figure 5. Deletion of the promoter from -1832to -816 resulted in a 30% increase in GUS activity extract-able from whole plants. Further deletions of the promoterto —540 and —290 led to stepwise decreases to about30% and 2%, respectively, of the activity of the full-lengthpromoter.

Figure 6 shows the levels of extractable GUS activity inleaves, stems, and roots of adult T2 plants containing thePAL1-GUS gene fusion and consecutive 5' promoter dele-tions (pSO-1 to pSO-4). Progressive deletion of the pro-moter to -290 (pSO-4) did not cause qualitative changesin organ-specific expression. Irrespective of the extent ofthe deletion, the relative expression levels in differentorgans increased in the order leaves < stems < roots.

To investigate the effects of 5' promoter deletions onthe tissue-specific pattern of PAL1 promoter activity, trans-genic plants were histochemically stained for GUS activity.Although the staining intensity in different organs wasfound to be quite variable among the progeny of an indi-vidual transformant, no difference was found in the tissue-specific pattern of GUS expression between plants con-taining the full-length promoter and the various promoterdeletions (Figures 31 and 3K). Thus, plants containing theshortest promoter fragment (pSO-4, -290) still expressed

GUS in the vascular tissue and in the same flower organsas plants transformed with pSO-1 containing the full pro-moter. However, consistent with the markedly reducedoverall GUS activity extractable from the SO-4 plants,GUS staining in the histochemical assay developed muchmore slowly and only after prolonged incubation of tissuespecimens.

Effect of 5' Deletions on mRNA Induction byEnvironmental Stimuli

Transgenic plants SO-1 to SO-4 were wounded, incubatedin an HgCI2 solution, or transferred to light after darkadaptation. Plants were harvested 1 hr to 4 hr after stim-

40000

• 30000 -

" 20000 -

10000-(fl

o

B

pSO 1 pSO 2 pSO 3 pSO 4 pBI101 pBI121

PAL promoter .̂

——l——l——l——l+EJSnaBI CcoRV Mine]] BglH

PAL

PAL promoter

Figure 5. Effect of 5' Promoter Deletions on the Basal Level ofGUS Activity.

(A) GUS activity was determined in whole plants transformed withfour different PAL promoter-GUS fusions (pSO-1 to pSO-4) andtwo control constructs containing the GUS gene without a pro-moter (pBI101) or fused to the cauliflower mosaic virus 35Spromoter (pBI121). GUS activity of the pSO transformants wasdetermined in extracts from 180 to 250 pooled plants from 14 to19 primary transformants per construct.(B) Schematic representation of the chimeric PAL-GUS con-structs. The 5' ends of the PAL promoter in these constructs are-1816 (pSO-1), -832 (pSO-2), -540 (pSO-3), and -290 (pSO-4)relative to the transcription start site.


01 80000 -

5 60000-

- 40000 -

20000-

pS01pSO2pSO3pSO4

o

leaves s tems roots

Figure 6. Organ-Specific GUS Expression in Plants Transformedwith pSO-1 to pSO-4.

GUS activity was determined in leaves, stems, and roots of adultplants that had developed flower buds. The data are mean valuesand average deviations from the mean calculated from sevenindividual transformants per construct.

ulation, and total cellular RNA was isolated and probedwith a fragment homologous to GUS. Figure 7 shows thatthe PAL promoter deleted to -540 (pSO-3) was still induc-ible by all three stimuli. Although deleting the promoterfrom -540 to -290 caused an approximately 15-foldreduction in GUS activity (Figure 5), GUS enzyme activitywas still detectable in SO-4 plants both histochemicallyand by fluorimetric assay. However, plants transformedwith pSO-4 did not accumulate detectable levels of GUSmRNA in either control or induced plants. The data shownin Figure 7 were obtained from the progeny of one individ-ual transformant per construct. However, all primary trans-formants tested in similar experiments (except SO-4plants) were inducible. The data in Figure 7 also show thatthe decrease in GUS activity caused by progressive 5'promoter deletions corresponded closely to the decreasein the respective GUS transcript levels.

DISCUSSION

Isolation and Structure of an Arabidopsis PAL Gene

In this paper we describe the isolation and partial charac-terization of an Arabidopsis PAL gene using a heterolo-gous PAL cDNA probe from bean. A genomic DNA gel blotof DNA digested with four restriction enzymes and probedwith a fragment homologous to a highly conserved part ofthe second exon revealed one to two strong bands andtwo to four weaker bands under stringent hybridizationand washing conditions. The PAL probe was only 190 bpand did not contain an internal site for any of the fourrestriction enzymes used in this experiment. Therefore,the number of bands should reflect the number of PAL-related sequences in the genome. In the experiment shownin Figure 1, genomic DNA was isolated from the Columbia

ecotype, whereas PAL1 was cloned from a library ofLandsberg ecotype DNA. However, using Landsberg DNAin an equivalent experiment resulted in the same patternof bands except for the 8.5-kb band in Hindlll-cut DNA,which was missing in the Landsberg blot (data not shown).The data indicate that the PAL1 gene is a member of asmall family of genes. The prominent band in EcoRI-digested genomic DNA probably corresponds to the 5-kbEcoRI fragment that was initially subcloned from the gen-omic A clone.

Many gene families are smaller in Arabidopsis than inmost other plant species (Meyerowitz, 1987). In contrast,the Arabidopsis PAL gene family is comparable in size tothe PAL gene families in bean, parsley, and rice. Theobservation that the small genome of Arabidopsis containsa PAL gene family of similar complexity to those in specieswith much larger genomes is consistent with the hypoth-esis that individual PAL genes may encode variant prod-ucts with distinct functional specializations. This hypothe-sis was initially proposed to account for the differentialregulation of members of the PAL gene family in bean andparsley (Liang et al., 1989a; Lois et al., 1989) and thedifferent biochemical properties of specific bean PAL en-zyme subunit isoforms (Bolwell et al., 1985).

pSOlwounding HgQ3 light

C l 2 3 1 2 c l 2 4

pSO 2 pBI 121wounding HgCl 2 light w H

C 1 2 3 1 2 C l 2 4 c l 3 1

I

pSO3wounding HgCl2 l ightc 1 2 3 1 2 c 1 2 4

pSO4wounding HgG2 l ightC 1 2 3 1 2 c ! 2 4

pBI 121light

c 1 2 4

rFigure 7. RNA Gel Blot of GUS mRNA from Plants Transformedwith pSO-1 to pSO-4 or pBI121.

Whole plants were wounded, transferred to a 100-AiM HgCI2solution, or transferred to white light following 4 days of darkadaptation, and harvested 1 hr to 4 hr after treatment. Totalcellular RNA was extracted and fractionated by gel electrophore-sis, blotted, and hybridized to labeled probes homologous toGUS. c, control plants harvested immediately after wounding ortransfer to light; w, wounding; H, HgCI2 solution. Size markersare given in kilobases.

844 The Plant Cell

Sequencing 0.7 kb of the putative coding region con- firmed that this Arabidopsis gene encodes PAL. The deduced amino acid sequence showed a high degree of similarity to PAL from bean, parsley, and rice, with most differences representing conservative changes. The amino terminus is quite divergent in both length and sequence. As expected, the sequences from the three dicotyledonous plants are more closely related to each other than to the rice PAL sequence.

Developmental and Tissue-Specific Regulation

To facilitate analysis of the tissue-specific developmental regulation and stress induction of the PALl gene and to obtain some information on the functional organization of its promoter, we generated four PALl promoter-GUS transcriptional fusions and analyzed these constructs in transgenic Arabidopsis. As expected, considerable variation was found in the absolute levels of expression in different transformants, probably due to positional effects from insertion of the transgene at different sites in the genome. This problem was overcome by analyzing severa1 independent transformants in each case. Moreover, qualitative properties such as tissue-specific patterns of expression did not vary between independent transformants containing the same construct.

The regulation of a reporter gene by a heterologous promoter has been shown in many systems, including the bean PAL2-GUS gene fusion in transgenic tobacco, to be an accurate reflection of the intrinsic regulatory properties of the promoter (Benfey and Chua, 1989; Bevan et al., 1989; Liang et a[., 198%; Schmid et al., 1990). The Arabidopsis PALl promoter was very active during the early stages of seedling development. GUS staining was found in all tissues except the root tip and was most pronounced at the base of the radicle. The cotyledons accumulate high levels of anthocyanins at this stage; we propose that during the 1 st day or 2nd day after emerging from the seed coat the seedling is vulnerable to UV light and pathogens, and, thus, PAL is induced in the shoot for rapid accumulation of pigments and in the root possibly for defense against microbial attack. In adult plants, PAL was expressed in the vascular tissue of roots, shoots, and leaves. This observation is consistent with the vascular expression of the bean PALP-GUS gene fusion in transgenic tobacco and potato (Bevan et al., 1989; Liang et al., 1989b) and supports the hypothesis that PAL expression associated with the initiation of lignin synthesis is an early marker for xylem differentiation during vascular development.

In Arabidopsis flowers, the PALl -GUS gene fusion was expressed in sepals, anthers, and the carpel but not in the white unpigmented petals. After the flower bud opened and the stigma became susceptible to fertilization, GUS staining developed at the base of the stigma. The func-

tional significance of PALl expression in this tissue is not known, but a possible explanation would be that during outgrowth of the pollen tube the pollen specifically interacts with a defined region of the stigma. A second zone of PALl -GUS expression of unknown function developed at the base of the ripening silique in the abscission zone of sepals and petals. The strong expression found in pollen correlates with pigment biosynthesis. Staining pollen from different developmental stages showed that the onset of PALl expression slightly preceded the accumulation of the yellow pollen pigment, which in petunia was identified as the phenylpropanoid tetrahydroxychalcone (de Vlaming and Kho, 1976).

The PALl promoter apparently confers a different pattern of expression in Arabidopsis flowers than the bean PAL2 promoter in tobacco flowers (Liang et al., 1989b). Thus, PALP expression, measured fluorimetrically, was high in petals but was very low in sepals. Another striking difference in tissue-specific expression is that the ,bean PAL2 promoter confers strong expression in tobacco root tips and shoot apical meristems (Liang et al., 1989b), whereas the Arabidopsis PALl promoter is silent in these tissues. The differences in floral expression may reflect fundamental differences in the underlying signal pathways between Arabidopsis and tobacco, rather than functional differences between the Arabidopsis PALl and bean PAL2 promoters, because the bean CHSl5 promoter, which is strongly expressed in bean and transgenic tobacco flowers, is not active in petals of transgenic Arabidopsis (J. Kooter, unpublished observation). However, the differences between bean PAL2 and Arabidopsis PALl expression in apical meristems may reflect intrinsic properties of these promoters underlying the differential regulation of members of the PAL gene family.

lnduction by Wounding, HgCI2, and Light

PAL is induced in many plant species by various stress- related stimuli including wounding, heavy-metal stress, and light. In bean and parsley, this induction has been shown to be due to transcriptional activation of the respective genes (Lawton and Lamb, 1987; Lois et al., 1989). Sur- prisingly, we were unable to detect a significant increase in GUS activity in Arabidopsis plants transformed with the PALl -GUS gene fusion after various treatments that are known to induce PAL in other systems. However, meas- urement of PAL and GUS mRNA in transgenic plants showed that both endogenous PAL genes and the gene fusion were rapidly induced in response to wounding, HgCI, stress, and light. The coordinate induction of the gene fusion and the endogenous PAL genes indicated that the PALl promoter contained the regulatory elements necessary for environmental induction.’ The RNA gel blot data (Figure 4) showed that environmental induction of PAL and GUS transcripts was transient, and, hence, in-


creases in GUS activity from transient induction of GUS mRNA might well not be detected against the basal GUS activity accumulated from continuing developmental expression of the gene fusion. The rapid induction of PAL transcripts by wounding and HgCI, stress is consistent with the rapid induction of PAL transcripts by treatment of Arabidopsis cell suspension cultures with the microbial elicitor 0l-l,4-endopolygalacturonic acid lyase (Davis and Ausubel, 1989).

Effect of 5’ Deletions on Promoter Activity

To gain some information on the functional organization of the PALl promoter, we compared the expression of three different 5’-deleted promoter-GUS fusions with the full- length promoter construct and determined whether specific regulatory properties were changed by the deletions. Deleting the promoter from -1816 to -832 gave a 30% increase in GUS activity, suggesting that a negative cis- element might be located in this region. Although some variation in GUS activity was found among individual plants transformed with the same construct, the values in this experiment were obtained from a large number of primary transformants. Negative regulatory elements severa1 hundred nucleotides upstream from the transcription start site have been found in a number of plant promoters including the bean CHS15 promoter (Dron et al., 1988; J. Kooter, unpublished results) and a tobacco chlorophyll a/b-binding protein promoter (Castresana et al., 1988), and may be a common feature.

By deleting the promoter from -832 to -540 and further to -290, we observed stepwise reductions in promoter activity, suggesting that at least two positive cis-elements were located in this region. Although both GUS activity and mRNA levels decreased upon deleting from -832 to -540, inducibility by wounding, stress, and light, as measured by the ratio of induced to basal level, was not signif- icantly altered. Therefore, we conclude that elements re- quired for PAL induction by exogenous stimuli are located downstream of position -540. Although the proximal 290 nucleotides of the promoter still retained all aspects of tissue-specific regulation, wound, stress, and light inducibility could not be demonstrated for this construct, possibly because of its very low overall level of expression. Thus, at present we do not know whether this construct has lost inducibility or whether induced GUS mRNA levels were still below the detection limit.

Comparison of the Arabidopsis PALl promoter with promoters from other phenylpropanoid genes identified sequence elements homologous to two “boxes” conserved among many phenylpropanoid genes (Cramer et al., 1989; Lois et al., 1989) that had been shown to display elicitor- inducible and light-inducible footprints in vivo (Lois et al., 1989). The sequence comparison in Figure 8 shows that the PALl promoter contains three elements (box 1) ho-

mologous to the proximal element from the parsley PAL promoter and two elements (box 2) homologous to the dista1 element. The sequence comparison includes homologous elements from the bean PAL2 promoter. In addition, we found a 9-bp AC-rich element in the 5’-untranslated region of PALl (box 3) that is also present in the bean CHSl5 gene at a very similar location and coincides with an elicitor-inducible hypersensitive site in this gene (Lawton et al., 1990). The -290 promoter fragment still contains one homolog of each of the boxes 1, 2, and 3. Our data do not prove that these elements are involved in regulation of the Arabidopsis PALl promoter but are consistent with this hypothesis, and further functional analysis of the promoter will be necessary to establish their regulatory functions.

The isolation of the Arabidopsis PALl gene and characterization of the functional properties of its promoter open up two new approaches for dissection of the underlying signal pathways in addition to the direct characterization of cis-elements and cognate transcription factors. First, the effects of well-characterized Arabidopsis mutants on PALl promoter activity can be analyzed. For example, auxin is thought to be a major factor determining the spatial organization of the vascular system (Sachs, 1986), and, thus, expression of PALl in the initial stages of xylem differentiation may be affected in the axr l mutant, which

boX 1 j iars ley PAI. -119

b e m PAL t 1 1 3

bean PAI. -89 A.t. PAL - 6 4

A . t . PAL -359 A.t. PAL -435

consensus

aAtaCaC acgcgc acacCaC cAaaCaC sAaatsC cAtcaca

~CT$ACCT~C$

box 2

parsiey PRL -208 AATCcaACGgTTgagATTAaTc bean PAI. -151 AATCcttCGcTTgtCATTAtTt h . t . PAL -169 cATCtaACGgTcatCATTAaTt A . t , ~ ~ ~ - 1 6 3 1 AtgaagACGcaTtgCccaAtTa

c%CtwC= consensus

b o x 3 AACCAACAA A . t . PAL t22 bem CHS t23

Figure 8. Conserved Sequence Elements in the Promoters of Severa1 Phenylpropanoid Biosynthetic Genes.

Matches to the consensus sequences noted by Lois et al. (1989) (boxed areas) and matches of at least 4/6 (box 1) or 3/4 (box 2) are shown in capital letters. The positions of the sequences are given relative to the transcription start sites (see text for details). The corresponding bean sequences are from the PAL2 gene (Cramer et al., 1989).

846 The Plant Cell

is insensitive to auxin (Estelle and Somerville, 1987). Like- wise, detl affects many aspects of photomorphogenesis, including accumulation of flavonoid pigments, and, hence, may modulate PALl expression, as is the case with severa1 other light-regulated genes (Chory et al., 1989). Finally, the ethylene-insensitive mutant ein (Bleecker et al., 1988) may impact stress activation of the PALl promoter. Analysis of PALl promoter activity in these genetic backgrounds will help delineate the terminal stages of the specific signal pathways disrupted by these mutations. In addition, transgenic Arabidopsis containing marker genes fused to the PALl promoter could be used to screen for nove1 mutants with defects in the signal pathways involved in PAL regulation. This approach is currently being tested with transgenic plants containing either the Arabidopsis alcohol de- hydrogenase gene or bacterial genes for resistance to hygromycin and streptomycin under the control of the full- length PALl promoter and, in principle, could be extended to synthetic promoters containing specific cis-elements dissected from PAL1.

DNA and RNA Blots

DNA isolation and DNA and RNA transfer and hybridization were performed according to standard protocols (Ausubel et al., 1987). RNA was isolated according to Chomczynski and Sacchi (1 987) except that the plant material was homogenized with a Brinkmann homogenizer (Brinkmann, Westbury, NY) for 30 sec at setting 5, rather than with a glass-Teflon homogenizer. Hybridization probes were labeled by random priming to a specific activity of 1 to 5 x 10’ cpm/pg DNA. The genomic DNA blot was probed with a 190- bp Styl/Hindlll fragment from a highly conserved part of the Arabidopsis PALl second exon. For RNA blot hybridization of PAL transcripts, a 1.2-kb Bglll fragment containing part of the PALl coding region was used as probe, and for GUS transcripts, a 2.2-kb Smal/EcoRI fragment from pBll O1 was used as a probe.

Nucleotide Sequence Analysis

A 3.1-kb EcoRI/Hindlll fragment of the genomic PALl clone was subcloned blunt in both orientations into the EcoRV site of plB125 (International Biotechnologies, New Haven, CT). A series of nested deletions was generated from these clones by unidirectional ex- onuclease III/Sl nuclease digestion. Single-stranded DNA was sequenced by the dideoxy chain-termination method (Sanger et al., 1977).

METHODS

S1 Nuclease Protection

Plant Material

Arabidopsis thaliana ecotype Columbia was used in all gene transfer experiments because Columbia regenerates more effi- ciently than Landsberg during the transformation procedure. Seeds were surface sterilized by soaking for 1 min in 70% ethanol, then for 10 min in a 1:3:0.01 solution of bleach:H,O:Tween 20 followed by five or six rinses in sterile water. Sterilized seeds were grown on MS medium (Murashige and Skoog, 1962) supplemented with 1% sucrose. To select for transformed plants, kanamycin (50 Ig/mL) was added to the medium. Sterile, grown plants were maintained in a 16-hr light/8-hr dark cycle at 22°C. To harvest seeds, plants were transferred to soil and grown in a greenhouse.

Unless otherwise stated, all experiments were performed with 3-week-old to 5-week-old sterile, grown plants. Plant material was wounded by cutting whole seedlings with a razor blade into pieces (about 3 mm) and kept in a Petri dish at high humidity until harvest. For HgCI, treatment, whole plants were submerged in a 1 O0 fiM HgCI, solution until harvest. For light induction, plants were dark- adapted for 4 days and then transferred to constant white light until harvest.

Screening of Genomic Library

We used a previously characterized PAL cDNA clone from bean (Edwards et al., 1985) to screen 4 x 104 pfu (equivalent to four haploid genomes) from a genomic Arabidopsis library (Chang and Meyerowitz, 1986). Positive clones were isolated by two additional rounds of plaque purification.

The S1 nuclease protection assay was performed as described by Ryder et al. (1987).

PAL1-GUS Gene Fusions

The EcoRI/Hindlll fragment of the genomic PAL clone was subcloned into the polylinker of pUCl9 to yield pAP-4. Recombinant plasmids containing the chimeric PALl promoter-GUS fusions were generated by ligating a 181 6-bp EcoRI/Bglll fragment (pS0- l ) , an 832-bp SnaBI/Bglll fragment (pS0-2), a 540-bp EcoRV/ Bglll fragment (pS0-3), and a 290-bp Hincll/Bglll fragment (pS0- 4) of pAP-4 between the Sal1 and the BamHl site of pBll 01.1 (Jefferson, 1987). The EcoRl site of pAP-4 and the Sal1 site of pBllOl were polished with the Klenow fragment of DNA polym- erase I before ligation. The binary vectors were transferred from Escherichia coli strain DH5a into Agrobacterium tumefaciens strain LBA4404 by direct DNA transfer (An, 1987).

Transformation

Arabidopsis was transformed with A. tumefaciens strain LBA4404 using the root transformation method (Valvekens et al., 1988) with one modification. Root explants were taken from 1 O-day-old to 15-day-old seedlings, which were grown in liquid MS medium supplemented with sucrose on a shaker (1 1 O min-’).

Histochemical Staining

Whole plants or detached organs were fixed in 0.3% formalde- hyde, 10 mM [2-morpholino]ethanesulfonic acid, pH 5.6, 0.3 M


mannitol for 45 min at room temperature with a brief initial vacuum infiltration, washed severa1 times in 50 mM Na phosphate buffer, pH 7.0, and incubated in phosphate buffer containing 0.2 mg to 1 .O mg of X-Gluc/mL at 37OC for 3 hr to 12 hr (Jefferson, 1987).

Tissue Sectioning

Plant tissue stained for GUS activity was dehydrated in an ethanol series (10%, 30%, 50%, 70%, 90%, 100%) within 24 hr. Ethanol was then replaced by incubation in 25%, 50%, 75%, and 100% xylene in ethanol each for 2 hr. Within the next 2 days to 3 days, xylene was gradually replaced by Paraplast tissue embedding medium (Monoject Scientific, St. Louis, MO). Fifty percent to 100% (v/v) Paraplast pellets were added to the xylene submerged tissue and incubated overnight. The tissue was then heated to 45OC and Paraplast was added until no more dissolved. The tissue was subsequently brought to 57OC and the Para- p1ast:xylene mixture was replaced by 100% molten Paraplast. Paraplast was changed five to six times until the xylene smell was gone. The tissue Paraplast mixture was cast in molds and allowed to solidify at 4OC. Sections were cut on a microtome (Minotome Cryostat, lnternational Equipment Co., Needham Heights, MA) and the Paraplast was removed by incubating the mounted sections in ethanol. The sections were photographed on a microscope (Diaphot-TMD, Nikon, Tokyo, Japan) using phase contrast.

Fluorimetric GUS Assay

GUS enzyme activity was determined according to Jefferson (1 987) by measuring the fluorescence of methylumbelliferone produced by GUS cleavage of methylumbelliferyl-0-D-glucuronide. GUS activity is expressed as nanomoles of methylumbelliferone per minute per milligram of protein. Protein was determined by the method of Bradford (1976).

ACKNOWLEDGMENTS

S.O. was supported by a fellowship from the Deutscher Akadem- ischer Austauschdienst. This work was funded by a grant from the Samuel Roberts Noble Foundation to C.J.L. We gratefully acknowledge the contributions of Michel Dron and Xiaowu Liang in initiating this research.

Received May 23, 1990; revised June 29, 1990.

REFERENCES

An, G. (1987). Binary Ti vectors for plant transformation and promoter analysis. Methods Enzymol. 153, 292-305.

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1987). Current Protocols in

Molecular Biology (New York: Green Publishing Assoc./Wiley Interscience).

Benfey, P.N., and Chua, N.-H. (1989). Regulated genes in transgenic plants. Science 244, 174-1 81.

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

Bleecker, A.B., Estelle, M.A., Somerville, C., and Kende, H. (1 988). lnsensitivity to ethylene conferred by a dominant muta- tion in Arabidopsis thaliana. Science 241, 1086-1089.

Bolwell, G.P., Bell, J.N., Cramer, C.L., Schuch, W., Lamb, C.J., and Dixon, R.A. (1 985). L-Phenylalanine ammonia-lyase from Phaseolus vulgaris: Characterization and differential induction of multiple forms from elicitor-treated cell suspension cultures. Eur. J. Biochem. 149, 411-419.

Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

Breathnach, U., and Chambon, P. (1981). Organization and expression of eukaryotic split genes coding for proteins. Annu. Rev. Biochem. 50,349-384.

Castresana, C., Garcia-Luque, I., Alonso, E., Malik, V.S., and Cashmore, A.R. (1 988). Both positive and negative regulatory elements mediate expression of a photoregulated CAB gene from Nicotiana plumbaginifolia. EMBO J. 7, 1929-1 936.

Chang, C., and Meyerowitz, E.M. (1986). Molecular cloning and DNA sequence of the Arabidopsis thaliana alcohol dehydroge- nase gene. Proc. Natl. Acad. Sci. USA 83, 1408-1412.

Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156-1 59.

Chory, J., Peto, C., Feinbaum, R., Pratt, L., and Ausubel, F. (1 989). Arabidopsis thaliana mutant that develops as a light- grown plant in the absence of light. Cell 58, 991-999.

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.

Davis, K.R., and Ausubel, F.M. (1 989). Characterization of elicitor-induced defense responses in suspension-cultured cells of Arabidopsis. MOI. Plant-Microbe Interact. 2, 363-368.

de Vlaming, P., and Kho, K.F.F. (1976). 4,2',4',6'-Tetrahydrox- ychalcone in pollen of Petunia hybrida. Phytochemistry 15,

Dixon, R.A., Dey, P.M., and Lamb, C.J. (1983). Phytoalexins: Enzymology and molecular biology. Adv. Enzymol. Relat. Areas MOI. Biol. 55, 1-135.

Downie, J.A., and Johnston, A.W.B. (1986). Nodulation of leg- umes by Rhizobium: The recognized root? Cell 47, 153-1 54.

Dron, M., Clouse, S.D., Dixon, R.A., Lawton, M.A., and Lamb, C.J. (1988). Glutathione and funga1 elicitor regulation of a plant defense gene promoter in electroporated protoplasts. Proc. Natl. Acad. Sci. USA 85, 6738-6742.

Edwards, K., Cramer, C.L., Bolwell, G.P., Dixon, R.A., Schuch, W., and Lamb, C.J. (1985). Rapid transient induction of phenylalanine ammonia-lyase mRNA in elicitor-treated bean cells. Proc. Natl. Acad. Sci. USA 82, 6731-6735.

J. 8, 1899-1906.

348-349.

848 The Plant Cell

Estelle, M.A., and Somerville, C. (1 987). Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology. MOI. Gen. Genet. 206,200-206.

Jefferson, R.A. (1987). Assaying chimeric genes in plants: The GUS gene fusion system. Plant MOI. Biol. Rep. 5, 387-405.

Jefferson, R.A., Kavanagh, T.A., 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

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 funga1 elicitor, wounding, and infec- tion. MOI. Cell. Biol. 7, 335-341.

Lawton, M.A., Clouse, S.D., and Lamb, C.J. (1990). Glutathione- elicited changes in chromatin structure within the promoter of the defense gene chalcone synthase. Plant Cell Rep. 8,

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). Developmental and environmental regulation of a phenylalanine ammonia-lyase-P-glucuronidase gene fusion in transgenic tobacco. Proc. Natl. Acad. Sci. USA 86, 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 cis- acting elements. EMBO J. 8, 1641 -1 648.

23,1349-1 359.

56 1 -564.

Meyerowitz, E.M. (1 987). Arabidopsis thaliana. Annu. Rev. Genet.

Meyerowitz, E.M. (1989). Arabidopsis: A useful weed. Cell 56,

Minami, E.I., Ozeki, Y., Matsuoka, M., Koizuka, N., and Tanaka, Y. (1989). Structure and some characterization of the gene for phenylalanine ammonia-lyase from rice plants. Eur. J. Biochem.

Murashige, T., and Skoog, F. (1 962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473-497.

Ryder, T.B., Hedrick, S.A., Bell, J.N., Liang, X., Clouse, S.D., and Lamb, C.J. (1 987). Organization and differential activation of a gene family encoding the plant defense enzyme chalcone synthase in Phaseolus vulgaris. MOI. Gen. Genet. 210,

Sachs, T. (1986). Cellular interactions in tissue and organ development. Symp. SOC. Exp. Biol. 40, 181-210.

Sanger, F., Nicklen, S., and Coulson, A.R. (1 977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA

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.

Stachel, S.E., and Zambryski, P.C. (1 986). Agrobacterium tumefaciens and the susceptible plant cell: A nove1 adaptation of extracellular recognition and DNA conjugation. Cell 47,

Valvekens, D., van Montagu, M., and van Lijsebettens, M. (1 988). Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selec- tion. Proc. Natl. Acad. Sci. USA 85, 5536-5540.

21, 93-1 1 1 .

263-269.

185,19-25.

219-233.

74,5463-5467.

155-1 57.

DOI 10.1105/tpc.2.9.837 1990;2;837-848Plant Cell

S Ohl, S A Hedrick, J Chory and C J LambFunctional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis.

This information is current as of February 26, 2021

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

http://www.plantcell.org/cgi/alerts/ctmain

http://www.plantcell.org/cgi/alerts/ctmain

http://www.aspb.org/publications/subscriptions.cfm

functional properties of a phenylalanine ammonia-lyase ... · and meyerowitz, 1986) with bean pal...

Documents