Eur. J. Biochem. 204,621 -629 (1992) Q FEBS 1992

Phenylalanine ammonia-lyase in potato (Solanum tuberosum L.) Genomic complexity, structural comparison of two selected genes and modes of expression IIans-Jiirgen JOOS and Klaus HAHLBROCK Max-Planck-Institut fur Ziichtungsforschung, Abteilung Biochemie, Koln, Federal Republic of Germany

(Received October 21, 1991) - EJB 91 1415

Potato (Sofanum tuberosum L. cv. Datum) contains approximately 40 - 50 phenylalanine ammonia-lyase (PAL) genes/haploid genome. Considerable cDNA heterogeneity indicates that at least about 10, and probably more, of these genes are potentially active. One subfamily, represented by one selected member (PAL-I), was analyzed with respect to genomic complexity, nucleotide and deduced amino acid sequence, and mode of constitutive or induced expression. For comparison, a second gene (PAL-2), representing several subfamilies that are easily distinguished from PAL-I, was included in these studies. Extensive structural similarities were observed both between the TATAproximal portions of the PAL-I and PAL-2 promoters, particularly in the areas containing putative cis-acting elements, and among all presently known PAL proteins from various higher and lower plants. The relative abundance of PAL mRNA varied greatly in several major potato organs. However, the patterns obtained with probes detecting either total PAL mRNA or more specifically, PAL-Irelated or PAL-2-related mRNA species, were the same within experimental error. Mature leaves contained particularly low levels of PAL mRNA. Infection of these leaves with the pathogenic fungus, Phytophthora infestans, resulted in a large, transient induction of PAL mRNA. The relative timing of PAL-I and PAL-2 mRNA expression, however, differed in compatible (fungus virulent, plant susceptible) but not in incompatible interactions (fungus avirulent, plant resistant). Wounding of leaves caused an extremely rapid and transient induction of both PAL mRNA species.

Phenylalanine ammonia-lyase (PAL) is one of the most extensively studied enzymes in higher plants. It converts Lphenylalanine to trans-cinnamicacid, a universal intermedate in the formation of a large variety of plant-specific phenylpropanoid derivatives,including lignin, flavonoids, stilbenes,coumarins and numerous soluble as well as wall-bound esters and amides. Since the first discovery of PAL 30 years ago by Koukol and Conn (1961), the presence of this enzyme has been demonstrated in all higher plants tested, as well as in some yeast species (Hanson and Havir, 1981; Camm and Towers, 1973; Hahlbrock and Scheel, 1989). In higher plants, PAL activity varies greatly with the stage of development, with cell and tissue differentiation and upon exposure to various kinds of stress (Hahlbrock and Scheel, 1989). In all cases so far investigated, changes in PAL activity are regulated at the transcriptional level. Particularly wellstudied systems, in which individual PAL genes and their Correspondence to K. Hahlbrock, Max-Planck-Institut fur Zuchtungsforschung, Abteilung Biochemie, W-5000 Koln 30, Federal Republic of Germany Abbreviation. PAL, phenylalanine ammonia-lyase. Enzymes. Phenylalanine ammonia-lyase (EC 4.3.1.5);4-coumarate:CoA ligase (EC 6.2.1.12).

responses to endogenous or exogenous signals have been analyzed, are parsley (Chappell and Hahlbrock, 1984; Lois et al., 1989; Schulz et al., 1989; Lois and Hahlbrock, 1991), bean (Edwards et al., 1985; Cramer et al., 1989; Liang et al., 1989) and Arabidopsis thaliana (Oh1 et al., 1989). In all three plants, PAL is encoded by small families of about 3 -4 genes (Cramer et al., 1989; Lois et al., 1989; Oh1 et al., 1989). Potato is a fourth plant in which PAL has been studied extensively, not only with regard to tissue differentiation and various kinds of stress (Hahlbrock and Scheel, 1989), but also as a molecular-genetic marker in restriction-fragment-length polymorphism analyses (Gebhardt et al., 1989). Rapid and local PAL gene activation has been demonstrated in fungusinfected potato leaves (Fritzemeier et al., 1987; Cuypers et al., 1988). However, the genetic background of PAL has not been elucidated in this plant and seems to be unusually complex (Hahlbrock et al., 1989), in contrast to that of a closely related enzyme, 4-coumarate: CoA ligase, which in potato is encoded by two structurally similar genes (Becker-Andri?et al., 1991). Here, we present data on the approximate number of PAL genes in potato, and on some structural features and modes of expression of two selected members of subfamilies.

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Fig. 1. Partial restriction maps of 12 selected genomic PAL clones from potato cv. Datura. Genes were numbered arbitrarily beginning with the two investigated most extensively. Identification, orientation and alignment of the genes was based on hybridization analyses with the two EcoR1 fragments of the PAL cDNA from potato cv. Isola (Fritzemeier et al., 1987) and a genomic fragment referred to in the text as PAL-2 5' probe, as indicated on the bottom line. Note that an internal EcoRI site of the cDNA is present in all PAL genes, except P A L - ] . The following restriction sites are shown: B = BurnHI; E = EcoRT; EV = EcoRV; H = HindIII; K = KpnI; P = PsiI; S = Srnul: ss = SstI.

EXPERIMENTAL PROCEDURES General This work was carried out in parallel to similar investigations on the closely related 4-coumarate: CoA ligase gene family in the potato cultivar Datura. Most of the materials and methods were therefore the same as those reported recently by Becker-Andrk et al. (1991), including the various RNA samples from plant tissues. Exceptions were as follows.

PAL cDNA A new cDNA library was constructed according to the method of Gubler and Hoffmann (1983) using poly(A)-rich RNA obtained (Maniatis et al., 1982) from suspensioncultured potato cv. Datura cells treated for 2.5 h with fungal-culture-filtrate elicitor (Rohwer et al., 1987). The two EcoRI fragments of a previously isolated potato cv. Isola PAL cDNA (Fritzemeier et al., 1987) and a genomic PAL-2

fragment described below in Results served as hybridization probes.

RESULTS Genomic complexity A genomic DNA library was established from leaves of the potato cv. Datura and screened with a previously identified PAL cDNA from another cultivar, potato cv. Isola (Fritzemeier et al., 1987). From a total of 595 PAL clones, 34 were further analyzed by restriction digestion with EcoRI. On this basis, the 34 clones fell into 12 different classes. One representative from each class is shown in Fig. 1. The inserts of all 12 genomic PAL clones, with the exception of clone 1, could be aligned with respect to an internal EcoRI site which is also present in the potato cv. Isola cDNA (Fig. 1). The combined criteria for inclusion of a clone in this list were efficient hybridization with three different probes: a 1-kb EcoRV - EcoRI fragment from the putative promoter and 5' coding region of clone no. 2 (see below), and the

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Fig. 3. Genomic DNA blot hybridized with a 79-bp fragment from the 3’ end of the PAL-1 cDNA. DNA from the PAL-1 clone (Fig. 1) or from potato cv. Datura leaves was digested as indicated. Arrows indicate sizes deduced from the restriction map of PAL-I. Fig.2. Genomic PAL DNA blot. DNA from potato cv. Datura leavcs was digcsted with EcoRT, separated on agarose gels, blotted and hybridized either with the PAL-1 cDNA comprising 11 77 bp from the 3’ portion of the gene (A), or a 1330-bp XbuI-EcoRV fragment containing the 5’ half of the PAL- l gene (B). The precise position of this fragment is indicatcd in Fig. 8. Positions of DNA size markers (left) and of fragmcnts predicted from the restriction map in Fig. 1 (right) are indicated.

two EmR1 fragments comprising the potato cv. Isola cDNA (0.6 kb and 0.9 kb), the relative positions of which are indicated in Fig. 1. Since clone 1 lacked the central EcoRI site, this clone, as well as the corresponding cDNA, was expected to be easily distinguished from all other genomic and cDNA clones. It was therefore chosen for further study and designated PAL-1. For comparison, clone 2 (PAL-2) was selected from the other P A L genes. As a first step in the analysis of PAL-1, the corresponding cDNA was isolated by screening a cDNA library prepared from KNA from elicitor-stimulated potato cv. Datura cells. RNA-blot hybridization demonstrated that 2.5 h after the addition of elicitor, these cells contained about 30 times morc PAL mRNA than untreated control cells. Using the potato cv. Isola cDNA as hybridization probe and the absence of the internal EcoRI site as a further selective criterion, 11 putative PAL-] cDNA clones with inserts of about 1200 bp were obtained which were all identical by partial sequence analysis. One of them was fully sequenced and found to be identical to the 3’ portion of the PAL-1 gene (see below). This insert will be referred to in the following as P A L - ] cDNA. Apart from this selected class, approximately 10 distinct types of P A L cDNA seemed to occur in a total of 64 clones. The high degree of restriction-fragment-length polymorphism that became apparent from analyses of both the genomic DNA (Fig. 1 ; Gebhardt et al., 3 989) and the various PAL cDNA clones (data not shown) indicated the presence of a large number of P A L genes in potato. Judging from the relative frequency of occurrence of individual types of P A L cDNA, as defined by partial sequencing as well as restriction fragment patterns, P A L - ] mRNA was most abundant (z30%) among all types present in elicitor-stimulated potato cells.

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Fig. 4. Copy number estimation of P A L 4 genes in potato cv. Datura. Leaf DNA was digested with restriction enzymes as indicated and hybridized with the 1177-bp PAL-I cDNA. A titration series of the cloned EcoRI - Xbal fragment containing 1666 bp from the 3’ end of the PAL-I gene (see below in Fig. 8) was based on a recent determination of thc haploid potato genome size (Becker-Andre et al., 1991). The expected positions of PAL-l fragments from different restriction digests are indicated by arrows.

We used different portions of the cloned PAL-1 gene as hybridization probes to obtain a rough estimate of the actual copy number of genes within the entire P A L family, as well as the PAL-I subfamily. Fig. 2 shows the hybridization patterns of genomic potato cv. Datura DNA with the PAL-I cDNA representing the 3‘ portion, and with a 1330-bp Xhal EcoRV fragment comprising the proximal part of the promoter as well as the first exon of the PAL-I gene. As expected, different and very complex banding patterns were obtained. Although the complexity was greatly reduced when a small, 79-bp fragment from the extreme 3’ end of the PAL-I cDNA

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Fig. 6. Putative cis-acting elements in the TATA-proximalportions of the potato PAL-I and P A L 2 promoters. Regions with similarity to elements previously identified in PAL genes from parsley (P, A, L and C; Lois et al., 1989) and bean (B1 and B2; Cramer et al., 1989) are shown. Not included is an AT-rich region of the PAL-I promoter with extensive similarity in length, nucleotide sequence and position (-370 to -409) to a putative cis-acting element of a bean PAL promoter (Cramer et al., 1989). The nucleotide sequence of the PAL-I gene up to position - 1156 is available on request; the PAL-2 promoter was not analyzed upstream from position -278.

625 was used for hybridization, more than the single band predicted from the PAL-1 gene structure (see Fig. 8) was observed (Fig. 3 ) , suggesting that PAL-1 belongs to a sizable subfamily of P A L genes in potato. For more precise determinations, reveral different hybridization probes were used in copy number reconstructions either of the entire P A L gene family or of various subfamilies. As an example, Fig. 4 shows the results obtained with the PAL-I cDNA, which by definition detects the 3’ portions of all PAL genes. On the basis of this experimental approach, a large number of genomic DNA bands contained between one and at least 20, or even more, P A L gene copies, and the sum of all fragments appeared to be about 40 - 50 copies/haploid genome. Somewhat lower numbers were obtained with more selective 3’ or 5’ fragments of the PAL-1 gene, but even the 79-bp probe which had been used above (Fig. 3) to detect fragments with similarity only to the 3‘ untranslated region of PAL-1 revealed the presence of a minimum of about 30 copies of this relatively large subfamily (data not shown). In view of such a high degree of genomic complexity, we decided to confine all further studies to the two selected PAL-1 and PAL-2 genes.

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I111111111111l111111111l11l11111111111l1llllll1lll Nucleotide sequences were determined for the entire YYNNGLPSNLTAGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSA 490 PAL-1 gene, the 1200-bp PAL-I cDNA, as well as most of EQHNQDVNSLGLISARKTAEAVDILKLMSSTYLVALCQAIDLRHLEENLK 537 the PAL-2 gene, and are presented in part in Figs 5 and 6. lll11ll1111ll1l11111111llIlIlIIIlIIIll11lIIIIIIIII EQHNQDVNSLGLISARKTAEAVDILKLMSSTYLVALCQAIDLRHLEENLK 540 The numbering of nucleotides refers to the putative transcription start site of the PAL-I gene, which was determined by SVVKNTVSQVAKRTLTIGAIGELHPARFCEKELLRVVDREYLFTYADDPC 587 primer-extension analysis. These results were only partially III1II11IIII1IIIII.:1I1I1111IIIIII1IIIIIIII.IIIIII SVVKNTVSQVAKRTLTIGVLGELHPARFCEKELLRVVDREYLFAYADDPC 590 conclusive, as indications for two major (positions + 1 and + 9) and nine minor sites (positions - 1, + 2 to + 8, and SSTYPLMQKLRQVLVDHAMKNGESEKNINSSIFQKIGAFEDELNAVLPKE 637 + 10) were obtained (arrowheads in Fig. 5). The first ................................................ of the two major sites was arbitrarily defined as position VESARALLESGNPSIPNRITECRSYPLYRLVRQELGTELLTGEKVRSPGE 687 +I. .................................................. Fig. 5 shows the entire nucleotide sequence of the structural portion of the PAL-I gene. This sequence contains a short EIEKVFTAMCNGQINDPLLECLKSWNGAPLPIC*720 5’ untranslated region of 42 bp, two exons of 413 bp and ................................. 1747 bp, an intron of 114 bp and a 3’ untranslated region of Fig. 7. Comparison of amino acid sequencesof PAL4 and PAL-2 from 138 bp, including a putative poly(A) addition signal. The exon/intron borders were determined by nuclease S1 mapping. potato, as deduced from the respectivenucleotide sequences. The arrow They coincide with breaks in an otherwise continuous open head indicates interruptions of the nucleotide sequences by introns. reading frame. The border sequences conform to the consen- The C-terminus of PAL-2 was not determined. sus postulated by Breathnach and Chambon (1981) and are located at positions identical to those of two other well-characterized P A L genes from bean and parsley (see below). The PAL-I cDNA comprises 1177 bp, which are completely identi- transcriptional regulation of PAL. genes from parsley (Lois et cal to the corresponding portion of the PAL-I gene, and a al., 1989; da Costa e Silva, unpublished results) and bean (Liang et al., 1989). poly(A) tail of approximately 50 bp. The deduced amino acid sequences of PAL-1 and PAL-2 Also available, but not shown, is the nucleotide sequence from potato are shown in Fig. 7. The PAL-I protein consists of most of the corresponding parts of the PAL-2 gene, excludof 720 amino acids with a relative molecular mass of 78617. ing the 3’ end which was not analyzed. Sequence similarity between the two genes is 75% for the 5’ untranslated regions, Of the 35 amino acid differences occurring within the 82% of 92% for the first exons, 85% for the last 43 bp of the introns the sequence that have been established for both genes, 22 are (which are otherwise very dissimilar) and 95% for the 1349 bp localized in the first quarter (24%) which is encoded by the first exon, in good agreement with a relatively high degree of determined for both second exons. The proximal portions of the PAL-I and PAL-2 promoters sequence conservation in the large, second exons of all known (264 bp each, starting at the putative transcription start site PAL proteins from higher plants (E. Logemann and K. of PAL-1) are compared in Fig. 6. Three major regions of Hahlbrock, unpublished results). A schematic diagram of some of the major structural extended similarity are apparent, two from positions - 191 to - 136 (region 1) and - 122 to -40 (region 2), and a third features established for the PAL-I and PAL-2 genes is from the putative TATA box to the start site of transcription depicted in Fig. 8. The scheme also indicates the fragments and beyond. Regions 1 and 2 contain several partially overlap- used above for DNA-blot and below for RNA-blot hyping sequence motifs (Fig. 6) that have been associated with bridizations.

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Time after onset of t r e a t m e n t ( h ) Fig. 9. Timing of P A L mRNA accumulation in elicitor-treated potato cv. Datura cell cultures. Three different PAL mRNA populations were measured using the indicated hybridization probes (see text for further explanation). Total extractable RNA (1 5-40 pg, depending on signal strcngth within each set of experiments) from elicitor-treated (0)or water-treated control cells ( 0 )was separated by gel electrophoresis, blottcd, and hybridized with the respective 32P-labeled (random-primed) probe. Signals obtained after autoradiography were quantified by laser scanning densitometry. Note that each set of data was plotted on a separate 100% scale. max., maximum.

Modes of expression We then addressed the question of whether the selected PAL-I subfamily and the group of subfamilies measured with the PAL-2 probe were differentially expressed in individual plant organs or in response to stress. Pathogen and wound stress have been investigated with particular emphasis on P A L in parsley (Schmelzer et al., 1989; Lois and Hahlbrock, 1991) and bean (Liang et al., 1989), and with emphasis on the coordination of P A L and 4-coumarate - CoA ligase genes in potato (Fritzemeier et al., 1987; Cuypers et al., 1989; Becker-Andrk et al., 1991). Both types of stress were therefore included in our present investigations. The three probes used for these studies (Fig. 8) were the 1177-bp P A L - ] cDNA (by definition detecting all P A L mRNA species under the stringency conditions used), the 79-bp PAL-I 3' probe (operationally defined as specific for the PAL-I subfamily), and the 247-bp PAL-2 5' probe (detecting a major portion, but not all, of the various types of PAL mRNA). Since signal intensities obtained with

the three probes were not directly comparable, all individual sets of data were plotted on separate 100% scales. We also analyzed the behavior of PAL in cell culture, because the PAL-I cDNA was generated from a mRNA preparation from elicitor-stimulated cells. As shown in Fig. 9, the P A L mRNA species detected by all three probes were induced in cultured potato cells very rapidly and transiently by this treatment, with sharp peaks occurring approximately 0.5 h after addition of elicitor. Preliminary results indicated that a second wave of transient induction commenced at 2 h, but details were not investigated further. The relative P A L mRNA levels in various potato organs, as measured with the three different probes, are shown in Fig. 10A-C. Depending on whether equal amounts of total extractable RNA or RNA content of the respective cells, which varied greatly (Fig. lOD), were used as a basis for calculating relative concentrations, P A L mRNA was most abundant either in stems and roots or in corolla, stems and

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tubers. Particularly low levels were observed in mature leaves, where fungal infections (compatible as well as incompatible interactions) caused an accumulation of considerable amounts of PAL mRNA, in agreement with earlier results reported by Fritzemeier et al. (1987). In all cases tested, the hybridization patterns obtained with the three PAL probes were identical within experimental error, except that little, if any signal was detectable with the PAL-I 3' probe in infected leaves (Fig. 10A). The possibility that the

Fig. 11. Timing of PAL mRNA accumulation during compatible (0) or incompatible (A) interactions of potato cv. Datura leaves with P. infestans (Pi1 and Pi4, respectively), compared with mock-inoculated water controls ( 0 ) .See legends of Fig. 9 and Fig. 10 for further details. max., maximum.

latter result was due to differential expression of the PAL genes monitored with the PAL-1 3' and the PAL-2 5' probes was further tested in a more detailed time-course experiment (Fig. 11). Most or all P A L mRNA species were found to accumulate more in compatible than in incompatible interactions, but only the subpopulation detected by the PAL-I 3' probe clearly accumulated more rapidly in the incompatible interaction. The accumulation of P A L mRNA was even more rapid and transient upon wounding of potato leaves (Fig. 12).Sharp peaks were detected with all three probes 2 h after wounding, returning to the original levels about 6 h later.

DISCUSSION The existence of 40 or more P A L genes in the haploid potato genome is surprising for two reasons. The number of P A L genes present in several other plant species is smaller by approximately one order of magnitude (Lois et al., 1989;

628

PAL- t 0 PAL-2

I 80 E

al

cc

Time a f t e r wounding(h)

Fig. 12. Timing of PAL mRNA accumulation upon wounding of potato cv. Datura leaves. See legend of Fig. 9 for additional details. Data were obtained in parallel on the same filters and therefore plotted on the same scales as used in Fig. 11. Rel., relative; max., maximum.

Cramer et al., 1989; Oh1 et al., 1990). Secondly, in the same potato cv. Datura, only two genes were found to encode 4-coumarate: CoA ligase, the closely related, last enzyme of general phenylpropanoid metabolism (Becker-Andre et al., 1991). The occurrence of several different types of PAL cDNA, as distinguished by restriction profiles and partial sequencing, indicates that at least about one fourth of the P A L genes, and probably more, are potentially active in elicitor-stimulated potato cv. Datura cells. It remains open as to whether one or more of the various genomic or cDNA clones represent allelic forms, as might be suggested by the tetraploid nature of potato. However, using similar methods, only three out of eight possible alleles were detected for the two 4-coumarate:CoA ligase genes from the same potato cv. Datura (Becker-Andri: et al., 1991). By analogy, we consider it very likely that most of the different P A L clones shown here are derived from distinct genes. The unequivocal demonstration that the two genes analyzed encode PAL is based on structural comparison of the deduced polypeptides with several PAL proteins from other species. The enzymatic activity of two of these, PAL-1 and PAL-4 from parsley, was measured in extracts of Escherichia coli cells transformed with the respective cDNA species (Schulz et al., 1989). Moreover, the promoters of both the PAL-I and P A L 2 genes from potato contain characteristic cis-acting elements, whose involvement in P A L gene regulation has been demonstrated by several independent methods (Liang et al., 1989; Lois et al., 1989; da Costa e Silva, unpublished results). Thus, we are confident that the two genes from potato encode different isoforms of PAL. Given the large number of P A L genes, we addressed the general question of whether differential expression of subsets of these genes occurs at all in potato, rather than the more specific, but very difficult, question of which gene is expressed under what conditions. A clearly positive answer was obtained with fungus-infected leaves (Fig. 11B and C). This result was surprising, because no difference was observed between the behavior of the two 4-ccumarate: CoA ligase genes when their expression was examined with the same RNA preparations as used in this study (Becker-Andrt et al., 1991). However, although individual gene family members may be expressed

differentially, the overall patterns of constitutive or induced expression of P A L (Figs 10 and 11A) and 4-coumarate: CoA ligase (Becker-Andre et al., 1991) mRNA appear to be the same under all conditions tested in potato, as they are in parsley (Lois and Hahlbrock, 1991). The large differences observed here in the timing and magnitude of P A L mRNA induction by the three types of stress, compatible and incompatible interactions with a pathogenic fungus and wounding, have also been reported for 4-coumarate: CoA ligase mRNA. In that connection, possible reasons for the different extent to whch phenylpropanoid pathways are activated in compatible and incompatible interactions with appropriate fungal races have been discussed (Fritzemeier et al., 1987; Becker-Andrk et al., 1991).The slow and asynchronous spread of the fungus from small infection sites in both types of interaction, in contrast to the narrow window of time during which the healing of relatively large wound sites commences, is an obvious explanation for the differential timing of P A L mRNA induction, as well as 4-coumarate: CoA ligase mRNA induction (Becker-AndrC et al., 1991), upon infection and wounding of potato leaves. Taken together, our results add another striking example of the previously observed, highly coordinated regulation of P A L and 4-coumarate: CoA ligase genes and enzyme activities (Hahlbrock and Scheel, 1989). With regard to potential differences in the regulation of individual genes, the high degree of similarity of the proximal portions of the potato PAL-I and PAL-2 promoters may indicate additional requirements for differential gene activity. For example, it is possible that as yet unidentified cis-acting elements in the TATA-distal regions of the promoters or downstream from the transcription start site (Douglas et al., 1991) have decisive functions in differential gene regulation. Alternately, distinct or specifically modified trans-acting factors, provided by the respective cellular environment, could act differentially on similar or identical promoter structures. A third possibility would be a combination of these two alternatives. The recent identification of a pathogen-inducible protein that binds specifically to one of the putative cis-acting elements present in various PAL promoters (Pin Fig. 6 ) opens up new experimental approaches to these questions (da Costa e Silva, unpublished results). We thank Drs K. H. Fritzemeier and Isolde Hauser for initial work on the total genomic and PAL-2 clones from potato, Elke Logemann for technical assistance and Drs W. Sacks, D. Scheel and G. Strittmatter for valuable comments on the manuscript. This work was supported by grant no. 1.02 from the Bundesminister fur Forschung und Technologie.

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