The Plant Cell, Vol. 3, 829-840, August 1991 O 1991 American Society of Plant Physiologists

Phenylpropanoid Pathway lntermediates Regulate Transient Expression of a Chalcone Synthase Gene Promoter

Gary J. Loake,' Arvind D. Choudhary,",' Maria J. Harrison," Mehrdad Mavandad,'92 Christopher J. Lamb,b and Richard A. Dixonas3

a Plant Biology Division, The Samuel Roberts Noble Foundation, P.O. Box 21 80, Ardmore, Oklahoma 73402

La Jolla, California 92037 Plant Biology Laboratory, The Salk lnstitute for Biological Studies, 10010 North Torrey Pines Road,

A chimeric gene construct containing a bean chalcone synthase (CHS) promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene was strongly expressed when electroporated into alfalfa protoplasts that were then exposed to a fungal elicitor. Low concentrations (5 x 10-6 to l O V 4 M) of exogenously applied trans- cinnamic acid (CA), the first intermediate of the phenylpropanoid pathway, slightly stimulated elicitor-induced CAT expression, whereas high concentrations ( P ~ O - ~ M) severely reduced expression to below the levels observed in the absence of elicitor. In contrast, frans-p-coumaric acid (4-CA, the second intermediate in the pathway) stimulated expression from the CHS promoter up to 4.5-fold at 5 x 10-4 M. Expression of CAT driven by the promoters of other elicitor-inducible defense response genes was not rnarkedly affected by CA or 4-CA. Stimulation of CHS promoter expression by low concentrations of CA and 4-CA was completely abolished by 5' deletion to position -130, but not -174. When the -180 to -130 region of the CHS15 promoter was coelectroporated into elicited protoplasts on a separate plasmid along with the intact -326 CHS-CAT construct, the decreased CAT expression as a function of CA or 4-CA concentration was consistent with the coelectroporated sequence cornpeting in frans with the intact promoter for the binding of a factor(s) involved in the up regulation of CHS transcription by 4-CA and low concentrations of CA. Our data support the hypothesis that phenylpropanoid compounds may act as natural and specific regulators of plant gene expression and define the location of a cis-acting element in the CHS15 promoter involved in the induction by phenylpropanoid pathway intermediates.

INTRODUCTION

A major defense response of leguminous plants to fungal infection is the rapid accumulation of antimicrobial isofla- vonoid phytoalexins in the area of attempted penetration (Dixon, 1986). This has been shown in a number of sys- tems to involve rapid transcriptional activation of genes encoding phytoalexin biosynthetic enzymes (reviewed by Dixon and Harrison, 1990). Chalcone synthase (CHS; EC 2.3.1.74) catalyzes the first reaction in the biosynthesis of flavonoid/isoflavonoid compounds from the phenylpro- panoid precursor 4-coumaroyl coenzyme A. CHS is en- coded by a family of seven genes in bean (Ryder et al., 1987) and at least this number in alfalfa (K. Dalkin and R.A. Dixon, unpublished results). Functional analysis of the promoter of a bean CHS gene (CHS15), which is

' Current address: Department of Botany, Nagpur University Campus, Nagpur 440010, India. * Current address: Department of Vegetable Crops, University of California-Davis, Davis, CA 95616. To whom correspondence should be addressed.

inducible by fungal elicitor or reduced glutathione in elec- troporated soybean and alfalfa protoplasts, has defined the location of both positive and negative cis-acting ele- ments, which affect quantitatively the expression of the gene, and sequences essential for elicitor inducibility within -326 bp of the transcription start site (Dron et al., 1988; Harrison et ai., 1991a; Lawton et ai., 1991). Gel-shift and in vitro footprinting techniques have demonstrated the presence of binding sites for bean and alfalfa nuclear proteins within these regulatory elements (Harrison et ai., 1991a, 1991b; Lawton et ai., 1991). Sequences homolo- gous to the CHS cis-acting elements are also found in the promoter of a bean L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) gene, which is coordinately induced with CHS at the onset of phytoalexin synthesis (Cramer et al., 1985, 1989).

The appearance of increased activities of phenylpro- panoid biosynthetic enzymes and levels of their transcripts in plant cell suspension cultures is both rapid and transient, implying tight control of both initiation and cessation of

830 The Plant Cell

transcription (Dixon and Harrison, 1990). Transcription of PAL and CHS genes in bean cell suspension cultures is prevented by simultaneous application of relatively high concentrations (10-4 to 10-3 M) of trans-cinnamic acid (CA), the immediate product of the PAL reaction (Bolwell et al., 1988; Mavandad et al., 1990). Furthermore, low concentrations of CA stimulate elicitor-induced PAL en- zyme activity in bean cell cultures (Dixon et al., 1980) and excision-induced PAL enzyme activity in pea epicotyls (Shields et al., 1982). An increasing body of evidence suggests that CA may act in vivo to regulate expression of the phenylpropanoid pathway, both at the level of gene transcription and by way of posttranslational effects (Bolwell et al., 1986, 1988; Mavandad et al., 1990). In particular, inhibition of PAL enzyme activity in elicitor- treated cells in vivo by the specific inhibitor L-a-aminooxy- P-phenylpropionic acid (Amrhein and Gerhardt, 1979) re- sults in a delayed superinduction of transcripts encoding PAL and CHS (Bolwell et al., 1988), suggesting that CA pools may exert both feedback and feed-forward effects on transcript levels. However, the exact molecular mech- anisms underlying these effects are not understood, and transcriptional effects of other phenylpropanoid com- pounds have not been studied.

tion of phenylpro-

thetic derivatives on the exDreSsion of eleCtrODOrated

with relief from cinnamic acid down regulation (Mavandad,

Figure 1 shows the CHS15 promoter constructs used in our study. We have previously described the charac- teristics of an alfalfa protoplast system with respect to expression of the bean CHSl5 promoter-chloramphenicol acetyltransferase (CAT) construct pCHCl (Choudhary et al., 1990a, 1990b). These protoplasts are derived from suspension-cultured cells that are responsive to elicitors, and in which elicitor-induced PAL and CHS expression is affected by CA in a manner identical to that reported for bean cells (Mavandad et al., 1990). Alfalfa protoplasts retain viability after electroporation, in contrast to the very labile protoplasts obtained from bean cell suspensions. The act of digesting away the cell walls may be the reason for the apparent elicitation of the protoplasts (as measured by expression of electroporated construct pCHC1 [Figure 1 A]), and this response is increased 1.5-fold (statistically significant) on exposure to elicitor from the cell walls of Colletotrichum lindemuthianum (Choudhary et al., 1990a, 1990b). The relatively high level of basal expression of electroporated CHS promoter constructs facilitates ex- amination of both down regulation and stimulation of expression by phenylpropanoid pathway intermediates in identically prepared batches of protoplasts.

1990).

H3 E R E R R K R 1 yf ITATAJ X J i CAT .i N O S 3 1 1 1 chimeric genes in elicitor-responsive alfalfa proioplasts

(Choudhary et al., 1990a). We describe the selectivity of A pCHC1 1 1

phenylpropanoid pathway intermediates in regulating +120

data define the location of a cis-acting element involved in B PCHCP -173 puc19

-326 -29 +1

expression from different promoters in this system. Our

the quantitative regulation of CHS promoter expression by CA and trans-p-coumaric acid (4-CA) and suggest a role for these compounds as natural regulators of plant gene PCHC3

expression.

RESULTS E pRChlOGl

, ,-

puc19

-1512 Transient Promoter Analysis in Elicited Alfalfa Protoplasts

The bean CHS15 promoter is used as a model in our laboratories for studies on cis elements and trans-acting factors mediating developmental and environmental the NOS 3' region. expression of a plant defense response gene (Dron et ai,, 1988; Harrison et al., 1991 a, 1991 b; Lawton et al., 1991). Previous studies have shown that exogenously applied CA inhibits the appearance of CHS15 transcripts in elicitor- treated bean cells, and that inhibition of flow into the phenylpropanoid pathway by application of the SPeCifiC PAL inhibitor L-a-aminooxy-P-phenylpropionic acid causes a delayed superinduction of CHS15 transcripts consistent

Figure 1. Promoter-Reporter Gene Constructs.

(A) pCHCl , which contains a transcriptional fusion of the CHSl5 Prc"ter (from -326) to the bacterial CAT gene, terminated by

(B) pCHC2, in which the CHS15 PromOtet' is 5'-deleted to Position -173. (C) pCHC3, in which the CHS15 promoter is 5'-deleted to -130. (D) pHRGPGl , which contains a translational fusion of the bean HRGP 4,1 prometer to the CAT gene, (E) pRChloG1, contains a transcriptional fusion of a rice chitinase gene promoter to CAT. AI, Accl; B, BamHI; Hf, Hinfll; H2, Hincll; H3, Hindlll; K, Kpnl; R, EcoRI; SI, Sphl; X, Xbal.

Regulation of CHS by Phenylpropanoids 831

Table 1. Effects of CA Derivatives on CHS-CAT-NOS (pCHC1) Expression in Electroporated Alfalfa Protoplasts

Relative CAT Activity (%p at Concentrations (M) below

Compound o 5~ 10-6 10-4 5 x 1 0 - ~

trans-Cinnamic acid 100 119 f 16 71 -t 27 13 f 1.4 Hydrocinnamic acid 1 O0 105 f 4 92 * 20 50 20 trans-p-Coumaric acid 1 O0 126 k 2 21 5 5 15 456 f 41 trans-Caffeic acid 100 1 0 5 f 2 97,O.l 9 6 k 1 . 5 trans-Sinapinic acid 1 O0 104 f 1 11 7 * 3 11 3 f 7

a Measured in protoplasts harvested 1 O hr after application of elicitor plus CA derivative. Results ( ~ s D ) are the average of two to four replicate determinations using separate protoplast batches.

The variation of CAT expression between replicate elec- troporations of pCHCl into aliquots of the same batch of protoplasts is & ~ O / O (n = 7). However, variation of absolute values of expression of the same construct electroporated into different batches of protoplasts is much greater (often as much as 1 O-fold). For this reason, the following experi- mental design was used. For each experiment, protoplasts from the same batch were divided into a number of equal aliquots sufficient for examining the full range of CA or 4-CA concentrations used. Whatever the construct being examined, pCHCl was always electroporated into one of the aliquots as interna1 control. Levels of expression for each treatment within an experiment were then normalized relative to the leve1 of pCHCl expression in the same batch of elicited protoplasts in the absence of phenylpro- panoid compounds. Each experiment was then repeated (from two to four times) using a new batch of protoplasts each time, and, unless stated otherwise, the data plotted show the average values plus standard deviation for rep- licated, normalized results. We chose this strategy of replication and normalization rather than coelectroporation of a control promoter linked to a different reporter gene into each individual aliquot of protoplasts (with expression of results as the ratio of test:control) because of (1) the low variability of expression of the same construct within a single batch of protoplasts and (2) the possibility of titration of transcription factors involved in expression of the test promoter by a control promoter in trans.

Relative Effects of Phenylpropanoid Compounds on Expression of the CHS15 Promoter

Table 1 shows the effects of a range of CA derivatives on CAT expression from pCHC1 in elicited alfalfa protoplasts. The structures of these compounds are shown in Figure 2. The concentrations used (5 x 1 O-6, 1 O-4, and 5 x 1 O-4 M) were those that gave stimulation, partia1 inhibition, and

near maximum inhibition, respectively, of CHS promoter expression by CA. In the experiments reported below, low concentrations of CA resulted in a stronger stimulation of CAT expression than in the experiments in Table 1. Re- duction of the side chain of CA (to yield hydrocinnamic acid) significantly reduced its inhibitory activity. Of the three biosynthetic products of CA tested, caffeic and sinapinic acids did not inhibit CHS promoter expression, even at 5 x 10-4 M. In contrast, 4-CA, the direct product of the microsomal CA 4-hydroxylase and thus the second inter- mediate in the phenylpropanoid pathway, stimulated expression of the chimeric CHS-CAT-nopaline synthase (NOS) gene construct, up to 4.5-fold at 5 x lOW4 M. These data indicate that CA derivatives have a selective, struc- ture-dependent biological activity unrelated to their general properties as hydrophobic weak acids. They also suggest a potential regulatory role for 4-CA.

Promoter Specificity of the Effects of CA and 4-CA

To investigate the selectivity of the effects of CA on promoter expression in the alfalfa protoplast system, we compared in Figure 3A the effects of increasing CA con- centrations on expression from three elicitor-inducible pro- moters, the bean CHSI 5 and hydroxyproline-rich glyco- protein (HRGP) 4.1 promoters and the rice chitinase RChlO promoter as CAT-NOS fusions in pUC19 (Figure 1). Expression of the CHS promoter was stimulated at low CA concentrations but was strongly down regulated at CA concentrations of 5 x 1 O-5 to 1 O-4 M and higher, whereas

R1 = R2 = R3 = H; frans - Cinnamic acid

R1 = R3 = H , R2 = OH; trans - p - Coumaric acid

R1 = H, R2 = R3 = OH; trans - Caffeic acid

-

R2= OH, R1 = R3 = OCH3; trans - Sinapinic acid

0 CH2 - CH2 - COOH

Hydrocinnamic acid

Figure 2. Structures of Phenylpropanoid Compounds Used in This Study.

832 The Plant Cell

log,, [clnnamlc acld] (1M)

1

.-

- c 8 % e o.

1 .o 2.0 3.0 log,, [cinnamic acid] (pM)

Figure 3. Dose-Response Curves for Effects of CA on Expres- sion of Promoter-CAT Constructs in Alfalfa Protoplasts and on Protoplast Viability.

(A) Alfalfa protoplasts were electroporated in the presence of 40 pg of pCHCl (bean CHS15 promoter) (W), pHRGPG1 (HRGP 4.1 promoter) (O), or pRChl OG1 (rice chitinase promoter) (O), treated with elicitor from C. lindemuthianum (final concentration 50 pg of glucose equivalents per milliliter) plus a range of concentrations of CA, and cultured for an additional 1 O hr before extraction and assay of CAT activity. All values are normalized to the CAT activity determined in protoplasts from the same batch electroporated with pCHCl in the absence of CA (100%, internal control, dashed line). The error bars represent the spread of values in four (pCHC1) or two (pHRGPG1, pRChlOG1) replicate experiments; in each replicate, data are normalized to the pCHC1 internal control. The average CAT activity (in the standard assay) from pCHCl in protoplasts exposed to elicitor but not CA was 3662 dpm in acetylated chloramphenicol. The expression levels of pHRGPGl and pRChlOGl in the absence of CA were approximately 20% that of pCHCl. (B) Viability, determined by Evans blue staining, of the protoplasts analyzed in (A) at the time of harvest (1 O hr after the addition of elicitor plus CA).

expFession from the chitinase promoter remained only slightly depressed at all CA concentrations. In contrast, expression of the HRGP 4.1 promoter was stimulated at all CA concentrations, to a maximum of about 1.6-fold at 5 x 1 O-4 M. High concentrations of CA did not appear to affect the viability of the protoplasts (Figure 3B). The effects of CA are, therefore, promoter specific rather than reflecting a blanket effect such as would occur if CA were simply toxic to the cells.

In Figure 4, the effects of 4-CA were examined on the above set of promoter-CAT constructs. Expression from the CHS15 promoter was strongly stimulated by 4-CA (4.5-fold at 5 x 10-4 M), whereas the HRGP 4.1 promoter was only weakly stimulated at 5 x 1 O-4 M (1.25-fold), and at 10-3 M 4-CA was expressed at levels below that observed with the minus 4-CA water control. Expression of the chitinase promoter was stimulated by 4-CA (maxi- mum of 1.75-fold) but to a considerably lesser extent than was observed for the CHSl5 promoter. These data imply selective action of phenylpropanoid compounds on stress- inducible promoters.

In initial experiments, we investigated the cauliflower mosaic virus (CaMV) 35s promoter (linked to the CAT reporter gene) as a possible control promoter for the effects of CA and 4-CA. However, this promoter is stimu- lated by 4-CA in a manner similar to that of the CHS15

J0 t oo 1 .o 2.0 3.0

log,, [clnnamlc acld] (VM)

Figure 4. Dose-Response Curves for Effects of 4-CA on Expres- sion of Promoter-CAT Constructs in Alfalfa Protoplasts. Experimental design was as given for Figure 3. Promoters were bean CHS15 (pCHC1) (W), bean HRGP 4.1 (pHRGPG1) (O), and rice chitinase (pRChl OG1) (O). Error bars represent standard deviation from four independent experiments.

Regulation of CHS by Phenylpropanoids 833

O ’ I

1 .o 2.0 3.0 log,, [clnnemic scld] (pM)

o - f 6 0 -

o LI: 4 0 -

- m -

2 0 I

O I

1 .o 2.0 3.0 log, o [cinnamic acldl (PM)

400 . 3 350 . C - 5 3 0 0 . z U

+ 250 . Ll a

c

50 I

0 - I I I

1 .o 2.0 3.0 log,, [pcoumaric acid] (p M)

Figure 5. cis Elements Required for Stimulation of CHSl5 Pro- moter Expression by CA or 4-CA.

promoter and is also down regulated by CA, although the dose-response curve for inhibition indicates that the CaMV 35s promoter is less sensitive to CA than is the CHS15 promoter. Thus, in an experiment where equal absolute levels of expression from each promoter were measured in the absence of CA, 10-4 M CA inhibited CHS15 expres- sion by 70%, whereas at this concentration the CaMV 35s promoter was expressed at 125% of its level in the ab- sence of CA. lnhibition of CaMV 35s promoter expression paralleled that of the CHS15 promoter at CA concentra- tions of 7.5 x 1 O-4 M and higher (data not shown).

Because the strongest stimulation by 4-CA and down regulation by CA was observed with the CHSl5 promoter, we performed further experiments aimed at identifying putative cis-acting elements for phenylpropanoid regula- tion in this promoter.

Functional Analysis of cis-Acting Elements for Regulation of the CHSlS Promoter by Phenylpropanoid Compounds

The level of expression of the bean CHS15 promoter in alfalfa protoplasts is in part regulated by cis-acting ele- ments located between positions -326 and -1 30 (Harri- son et al., 1991a). Figure 5 shows the effects of deletions

(A) Alfalfa protoplasts were electroporated in the presence of 40 pg of pCHC1 (M) or pCHC2 (O), treated with elicitor from C. lindemuthianum (final concentration 50 pg of glucose equiva- lents per milliliter) and a range of concentrations of CA, and cultured for an additional 10 hr before extraction and assay of CAT activity. The points represent average values from four independent experiments using different batches of protoplasts, with data normalized to the CAT activities observed in elicited protoplasts in the absence of phenylpropanoid compounds. The absolute values for expression of pCHC2 in the absence of CA were from 50.0% to 57.0% of the values for pCHCl . The insets (a and b, axes same as in [A]) show the two extremes of variation in the dose-response curves included in the averaged data in (A). Note that the variation in behavior of the different protoplast batches used in the replications of this particular experiment was characterized by lateral displacement of the CA dose-response CuNes. (B) Protoplasts were electroporated in the presence of 40 pg of pCHCl (M) or pCHC3 (O), treated with elicitor from C. lindemu- thianum and a range of concentrations of CA, and cultured for an additional 1 O hr before extraction and assay of CAT activity. The points are average values from two independent experiments using different batches of protoplasts, with data normalized to the CAT activities observed in elicited protoplasts from the internal control pCHCl in the absence of CA. The absolute values for expression of pCHC3 in the absence of CA in the two experiments were 26 and 32% of the values for pCHC1. (C) Effects of 4-CA on expression of pCHCl (m) and pCHC3 (O). Experimental design was as given in (A). Error bars are standard deviations for triplicate independent experiments, with data from each independent experiment normalized to the internal control value for pCHCl in the absence of 4-CA.

834 The Plant Cell

in the -326 to -1 30 region of the promoter on the expres- sion of CAT in electroporated protoplasts in the presence of a range of concentrations of CA or 4-CA. The data in Figure 5A and insets a and b show the behavior of a -173 deletion (pCHC2) compared with the -326 promoter. Dele- tion to -1 73 had little effect on the dose-response curve for CA. However, as shown in Figure 58, further deletion of the CHS15 promoter to -1 30 (construct pCHC3) led to a complete loss of responsiveness to the stimulatory ef- fects of low CA concentrations. However, the -130 con- struct exhibited down regulation by high CA concentra- tions similar to that seen with the -326 promoter.

Because the region of the CHS15 promoter between -130 and -173 appears to be absolutely required for stimulation by low concentrations of CA, we investigated whether it might also be involved in the stimulation of expression by 4-CA. Figure 5C shows that the -130 deletion pCHC3 exhibited a significant reduction in respon- siveness to 4-CA. At levels higher than 5 x 10-5 M, the stimulation by 4-CA was considerably lower than that observed for the intact promoter, and the dose-response curve for the -130 deletion was very similar to those observed for the HRGP and -chitinase promoters (Figure 4).

To obtain further evidence to support the hypothesis that the decreased responsiveness to CA and 4-CA of the -130 deletion resulted from the removal of a functional cis-acting element(s) that can interact with a frans-acting factor, we first examined the effects of coelectroporating in t m s with pCHCl the plasmid pMael-I, which contains the CHS promoter sequence from position -326 to the Mael site at position -141, as shown in Figure 6. As seen in Figure 7A, this had little or no effect on the responsive- ness of the promoter in pCHC1, resulting in a CA dose- response curve similar to that observed in Figure 5A with the pCHC2 deletion. The -326 to -141 fragment of the CHSl5 promoter contains severa1 binding sites for alfalfa nuclear proteins, including a region between positions -250 and -213 (Figure 6D, box 111) which contains both silencer and activator elements (Harrison et al., 1991a). Coelectroporation of pCHP1 (Figure 6B), containing a te- tramer of the box 111 element alone, in trans with pCHC1 again gave a small, and perhaps not significant, decrease in stimulation in response to CA similar to that seen on coelectroporation of pMael-1 or on comparing expression of pCHC2 with pCHCl (data not shown). Coelectropora- tion of pCHPl had no effect on the down regulation of pCHC1 expression by CA. These data suggest that the box 111 element, while affecting expression quantitatively, is not involved in specific regulation of the CHS15 promoter by phenylpropanoids.

The involvement of a putative frans-acting factor(s) es- sentia1 for stimulation of CHS15 expression by CA or 4-CA and specific to a region of the promoter between -130 and -183 was tested by coelectroporating in t m S with pCHC1 the plasmid pCHP2 (Figure 6C), which con-

* *

r D. i i I , i1

~ ~ o c n u ~ c i d ~ ' c - a n ~ Y X*CI C A T A ~ O T A O A O A T O ~ A T M A

. '1..

* C C M A ~ T C ~ T A C C ~ C ~ C O M C ~ ~ O O ~ ~ C A O A C ~ ~ O O T A O O C ~ O T ~ ~ T A ~ ~ ~ ~ = C ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ IXI

ATITC l O U T O U l O U T ~ O ~ A T A T O T O ~ l ~ A O A ~ ~ l C ~ C ~ l ~ l ~ C

2" y ."- t In * Deu

0 i ."F

7- .vil R O C ~ C O T O * T * C 1 ~ ~ C C T * C C C T ~ C R C C T * T C t * C C C ~ O C ~ ~ A C ~ ~ O C ~ M C l ~ ~ C C M C ~ l C ~ C ~ C ~ C ~ C P.

7 1 9u-r Y n . . . M C ~ C A O C M C C M A C C M C M C C C T C T C T * C C A C l C M O T O C l O l O M C ~ O O l C A C ~ C ~ C A ~ C ~ M C C M A ~ C O C A

7,- W" . 7. .b OTOCROK c a o i a n c i ~ c c * i c a i c ~ ~ c ~ c c ~ c n c ~ n a a n c ~ ~ n c ~ a ~ c i ~ a ~ a ~ ~ a ~ a ~ ~ ~ c c a I ._

r..-Mny* 1D.C*(I.CIT M I

Figure 6. Bean CHS15 Promoter and Constructs Used in the Coelectroporation Experiments Shown in Figure 7.

(A) pMael-1, which contains a fragment of the CHSl5 promoter from -326 to -140 in plasmid plB1.24. (6) pCHP1, which contains a tetramer of a double-stranded synthetic oligonucleotide homologous to box III of the CHS pro- moter (see (D), below), in the plasmid pSP65. (C) pCHP2, which contains the region of the CHS promoter from -183 to -130 in plasmid pGEM3. Note that the BamHl (B) sites are lost during the ligation, and that the inserts in pCHPl and pCHP2 can only be excised by cutting in the respective polylink- ers. The designation of the BamHl site refers to the ligation- cloning strategy only. (D) The nucleotide sequence of the CHS15 promoter and the transcriptional fusion to CAT. The 5' end points for pCHC1, pCHC2, and pCHC3 are marked by small arrowheads. The TATA box is boxed (around -29). The two underlined regions (-74 to -52 and -242 to -194) share extensive homology to regions within the promoter of an elicitor-inducible bean PAL gene (Cramer et al., 1989). The other underlined regions (orientation marked with arrows) are the three CCTACC(N),CT motifs. Boxes I , II, and 111, which bind proteins from bean nuclear extracts (see text) are boxed (boldface). The overlined regions (boldface) are binding sites for alfalfa nuclear proteins.

Regulation of CHS by Phenylpropanoids 835

tains three copies of the -130 to -183 sequence of the CHS15 promoter. The sequence was extended to -183 because recent in vitro DNase I footprinting experiments have defined a protein binding site extending 5’ of the -1 73 deletion end point (Harrison et al., 1991a). Figure 7B shows that coelectroporation of this region completely eliminated stimulation of pCHC1 expression by low con-

3 U C

centrations of CA but did not prevent down regulation of pCHC1 by high concentrations of CA. Coelectroporation

tion was greater than that observed in Figure 5C on

; a o . 4

$ 6 0 .

U 4 0 .

2 0 .

of the -1 83 to -1 30 sequence also inhibited the stimula- tion of pCHCl expression by 4-CA (Figure 7C); this inhibi-

deletion of the CHS15 promoter to -130. Within the variability inherent in this experimental approach, coelec- troporation of the -1 83 to -1 30 region or deletion to -1 30 has similar effects on responsiveness to CA or 4-CA.

quences did not decrease overall expression or affect the dose response to phenylpropanoid compounds (data not shown), whereas, although pMael-1, pCHPl, and pCHP2 each decreased overall expression, only pCHP2 abolished CA/4-CA stimulation. Our data, therefore, strongly suggest

u - m Q

e -

O I I I , Coelectroporation of control plasmids lacking CHS se- 1 0 2.0 3.0

log, o [cinnamic acid] (pM)

1 4 0

1 -? e 1 2 0

2 100

.- c ao a o

6 0

- - m a 4 0

C o .- - U

the presence of a cis element for CA/4-CA-mediated stim- ulation of the CHS15 promoter between positions -130 and -1 83. Because of the lack of effect of coelectropora- tion of pMael-1 (which extends to -141), it is most prob- able that the element is either situated between positions -130 and -140 or at the very 3’ end of the Mael-1

3’ nucleotide (see below).

.- fragment, where a putative cis element is cut at its most

o

2 0

O 1 .o 2.0 3.0 log,p [cinnamic acid) (pM)

Coelectroporation of CHS Promoter Sequences in trans with pCHC1.

(A) Alfalfa protoplasts were electroporated in the presence of 40 bg of pCHC1 (m) or 40 qg of pCHC1 plus 50 bg of pMael-1 (O) and treated with elicitor and CA as above. The points represent average values from four independent experiments using diff erent

observed in elicited protoplasts in the absence of CA. This exper- iment was performed with the same batches of protoplasts as used in Figure 5A. Values for pCHC1 plus pMael-1 were 20% and 50% of the corresponding values for pCHCl alone. (6) Protoplasts were electroporated in the presence of 40 kg of pCHC1 (B) or 40 bg of pCHCl plus 40 qg of pCHP2 (O) and treated as in (A). The points are average values from two inde- pendent experiments. The absolute values for expression of pCHCl coelectroporated with pCHP2 in the absence of CA in the two experiments were 31% and 39% of the values for pCHC1

(C) Protoplasts were electroporated as given in (B), but showing the effects of coelectroporation of pCHP2 on the responsiveness of the CHSl5 promoter to 4-CA. ., pCHCl ; O, pCHCl plus pCHP2.

400 - A I 6 300 - - c

3 O batches of protoplasts, with data normalized to the CAT activities

I

I 10 2 0 3 0 alone. O

lopo [pcoumarlc acid] (JLM)

Figure 7. lnvolvement of trans-Acting Factors Required for Stim- ulation of the CHS15 Promoter by CA or 4-CA, Suggested by

836 The Plant Cell

DlSCUSSlON

Phenylpropanoid Compounds Can Regulate Expression of the Bean CHSl5 Promoter

The role of plant phenolic secondary metabolites as regu- lators of microbial gene expression is now firmly estab- lished with respect to the effects of flavonoids/isoflavo- noids on the nod genes of Rhizobium (Firmin et al., 1986) and wound-induced phenolics such as acetosyringone on the vir genes of Agrobacterium (Stachel et al., 1986). These conclusions were reached by testing the effects of phenolic compounds on nod or vir promoter-lacZ fusions, a strategy similar to that used in the present work to show the effects of CA derivatives on plant gene promoters.

Variability in the dose-response curves for effects of phenylpropanoids on expression of the CHS promoter- CAT constructs in replicate experiments using different protoplast batches was most noticeable at the lower con- centrations, at least in the cases where the constructs were responsive to these concentrations. This could be explained by different endogenous levels of phenylpro- panoids in different protoplast preparations because this variation was typified by lateral displacement of the curves (e.g., Figure 5, insets a and b).

Because the same reporter gene (CAT) was used with all the constructs tested, it is unlikely that the effects of phenylpropanoid compounds occur at the leve1 of mRNA stability in the experiments reported in this paper. Close structural derivatives of CA (caffeic and sinapinic acids) did not modulate expression from the CHS promoter, and hydrocinnamic acid was a significantly weaker inhibitor than was CA. There is, thus, a specific structural require- ment, in both the aromatic ring and side chain, for modu- lation of CHS promoter activity by phenylpropanoid com- pounds. The addition of a single phenolic hydroxyl group para to the side chain results in stimulation of the CHS15 promoter, even at high 4-CA concentrations. Further struc- ture-function experiments are in progress.

The stimulation of CAT expression by low concentra- tions of CA in the electroporated protoplasts resembles the effects of CA on elicitor-mediated induction of PAL activity (Dixon et al., 1980) and PAL transcript levels (Mavandad et al., 1990) in cultured bean cells. Deletion and coelectroporation experiments suggest that the same cis element might be involved in stimulation by 4-CA or low concentrations of CA. Thus, the stimulatory effects of low CA concentrations could result from metabolism to 4-CA, or CA and 4-CA could act independently through the same element(s). Higher concentrations of exoge- nously applied CA could saturate the substrate turnover capacity of the hydroxylase, thereby leaving inhibitory concentrations of CA in the cells.

Of the three elicitor-inducible promoters tested in this study (CHS15, HRGP 4.1, and chitinase), only the CHS15

promoter was strongly up regulated by 4-CA. The other two promoters, in common with the -130 deletion of CHSl5, exhibited similar 4-CA dose responses, charac- terized by a weak (1.25-fold to 1.8-fold) stimulation at 5 x lW4 M. This may, therefore, reflect a nonspecific effect of 4-CA. Unlike the CHSl5 promoter, the HRGP 4.1 and chitinase promoters were not down regulated by high CA concentrations. This further indicates that CA is not a blanket inhibitor of transcription in alfalfa protoplasts. It is, however, interesting that CA has opposite effects on the CHS and HRGP promoters; although both drive expres- sion of defense response proteins, the latter is not involved in phenylpropanoid metabolism. The CaMV 35s promoter behaves in a manner similar to that of the CHSl5 promoter in response to CA and 4-CA, although it is not as sensitive to CA-mediated down regulation as the CHS15 promoter (G.J. Loake, A.D. Choudhary, and R.A. Dixon, manuscript in preparation). It has been shown recently that the CaMV 355 promoter contains distinct domains conditioning tis- sue specificity, one of which appears to control preferential expression in vascular tissue (Benfey et al., 1989). It would, therefore, be reasonable to propose that if a specific phenylpropanoid regulatory element were to be found in other promoters, the CaMV 35s promoter might be a good candidate by virtue of its expression in cells where the phenylpropanoid pathway is active for the supply of lignin precursors during xylem differentiation. Because the mini- mal (5' deletion to -46) CaMV 35s promoter is not up regulated by 4-CA (G.J. Loake, unpublished results), it is likely that specific 4-CA responsive cis element(s) do in- deed exist in the CaMV 35s promoter.

cis-Acting Elements for Regulation of the CHS15 Promoter by Phenylpropanoid Compounds

The stimulation of expression of the CHS15 promoter by low concentrations of CA and 4-CA appears to occur through specific cis-acting sequences because this stim- ulation is totally abolished by deletion of the promoter to position -130. The effects of this deletion appear to be because of the removal of a binding site for a trans-acting factor because coelectroporation of the sequence from -1 83 to -1 30 in trans with pCHGl also results in inhibition of stimulation by CA and 4-CA, presumably by competition for this factor.

DNase I footprinting has revealed a binding site for alfalfa nuclear proteins between positions -177 and -164 of the CHSl5 promoter and binding sites further upstream with functionally defined activator and silencer properties (Harri- son et al., 1991a) (Figure 6D). In addition, the region from -1 73 to -130 contains the motif CCACCAAACTCCTAC, close variants of which are found in a number of light- induced and elicitor-induced genes (Lois et al., 1989) and one full copy and a half copy (in the opposite orientation) of the sequence CCTACC(N),CT (the full copy of which

Regulation of CHS by Phenylpropanoids 837

overlaps the motif of Lois et al., 1989), which occurs again in the CHS promoter (centered around -52) in a region with close homology to sequences within the coordinately regulated bean PAL2 promoter (Figure 6D). The down- stream CCTACC(N),CT motif may be an important element in determining the elicitor inducibility of the CHS promoter (L. Yu, R.A. Dixon, and C.J. Lamb, unpublished results), but deletion of the upstream copies does not affect relative elicitor inducibility in alfalfa protoplasts (Harrison et al., 1991a). It should be noted that pMael-1 (-326 to -141) contains the most 5’ of the CCTACC(N),CT elements intact (although cut at the final T residue), but that coelec- troporation of pMael-1 does not abolish stimulation by CA, in contrast to the effects of coelectroporation of the -183 to -1 30 sequence. Finer deletion mapping has now shown total loss of 4-CA stimulation on deletion to -143, and approximately 50% loss on deletion to -155 (G.J. Loake, O. Faktor, C.J. Lamb, and R.A. Dixon, unpublished re- sults). These data, therefore, strongly implicate the CCTACC(N),CT motif between positions -154 to -140 and its immediate flanking sequences in 4-CA-mediated stimulation of CHS promoter expression. It should be noted that this element is the 5‘ half of an inverted repeat, the 3‘ half being located between positions -135 and -1 21 (Figure 6D). The coelectroporation data are consist- ent with the involvement of this 5‘ CCTACC(N)7CT element because only a small portion of the 3’ half of the repeat is present in the -183 to -130 fragment. It would appear, from the lack of effect of coelectroporation of pMael-1, that some of the sequence between -140 and -130 is necessary for binding of factor(s) to the -154 to -140 element .

Because deletion of the CHS15 promoter to -1 30 does not abolish down regulation by CA, any putative cis ele- ments responsible for this phenomenon must lie down- stream of -1 30. Evidence that down regulation at high CA concentrations is a specific transcriptional effect rather than a result of toxicity or inhibition of elicitor-mediated signal transduction is provided by the observations that expression from the HRGP 4.1 and chitinase promoters is not blocked by CA (Figure 3). Moreover, 10-3 M CA does not inhibit appearance of transcripts encoding the elicitor- induced p-1,3-glucanase or the constitutively expressed transcript H1 in bean cells (Mavandad et al., 1990).

Phenylpropanoids as Regulators of Plant Gene Expression

Plant phenolic compounds recently have been implicated in the control of plant hormone transport and cell division (Binns et al., 1987; Jacobs and Rubery, 1988). We now conclude that CA and 4-CA regulate the transcription of a gene encoding a key enzyme in plant phenylpropanoid biosynthesis, and that this phenomenon requires specific

cis-acting elements in the CHS promoter and cognate trans-acting factors.

The simplest model for phenylpropanoid-mediated plant gene regulation would invoke a flux-sensory system, pos- sibly responsive to the relative levels of CA and 4-CA (as sensors of the rates of CA production and removal, re- spectively). Such a mechanism appears necessary be- cause flux into this pathway is regulated mainly by in- creases in enzymatic capacity resulting from de novo gene expression. PAL and CHS gene activation is characteris- tically rapid but transient. Stimulation by low CA and 4-CA may facilitate rapid acceleration of pathway activation, with the down regulation by high CA being a key element of the deinduction phase. This model would predict the ex- istence of a regulatory protein(s) that binds CA/4-CA and that then interacts with the transcriptional apparatus, whether it be directly, indirectly by protein-protein interac- tions, or indirectly by modifying the activity of a transcrip- tion factor(s). Stimulatory effects of 4-CA and low CA concentrations and down regulatory effects of high CA concentrations would appear to require separate cis ele- ments. Work is now in progress to characterize the cis elements for down regulation and to identify factors that may mediate the responses to phenylpropanoids.

Transcriptional stimulation by 4-CA is not large, in con- trast to the effects of flavonoids as nod gene inducers, supporting a “fine-tuning” rather than a major “induction” role. In previous studies, we have determined the levels of free CA in bean cell cultures to be in the range of 4 x 1 O-6 to 2.5 x 10-5 M, within the concentration range for the stimulatory effects reported here. Concentrations of 4-CA were approximately 1 O-fold higher, again within the stim- ulatory range. The addition of 10-3 M CA to bean cells is followed by rapid uptake to give a maximum intracellular concentration of about 5 x 10-4 M, followed by metabolism to 4-CA, caffeic acid, and glucose conjugates (Edwards et al., 1990). It will now be critical to study carefully the kinetics of changes in interna1 CA and 4-CA pools during induction of phenylpropanoid biosynthesis in the absence of exogenously added CA to determine whether they change in a manner consistent with a potential regulatory role.

METHODS

Chemicals

CA, 4-CA, and caffeic acid were purchased from Sigma. Hydrocin- namic and sinapinic acids were purchased from Aldrich.

DNA Constructs

pCHCl contains a transcriptional fusion of the 5’ upstream region to position -326 of the bean (Phaseolus vulgaris) CHS15 pro-

838 The Plant Cell

moter (Ryder et al., 1987) to the bacterial CAT gene and NOS 3' terminator in pUC19 (Figure 1A). pCHC2 and pCHC3 are similar constructs in which the CHS promoter is deleted to -173 and -1 30, respectively.

pMael-1 (Figure 6A) contains the region from -326 to -1 41 of the bean CHS15 promoter in plasmid plB1.24. A 2.4-kb Hindlll- EcoRl fragment of the CHSl5 gene was digested with Mael; the Hindlll-Mael fragment was blunt ended with deoxynucleotide tri- phosphates using the Klenow fragment of DNA polymerase and cloned into the Smal site of plB1.24. pCHP1 (Figure 6B) contains four copies of the box 111 sequence of the CHS15 promoter in plasmid pSP65. The two complementary oligonucleotides:

5 ' - S A T C A C C A A T T R T T S S T T A C T A A A T T T A A C A S T T 6 6 T T A A T A A C C A A T 6 A T T T A A A T T S T C A C T A 6 ~ 5 '

were synthesized using a Du Pont Generator DNA synthesizer and ligated, and the tetramer, after separation by gel electropho- resis, was cloned into the BamHl site of plasmid pGEM7zf+. The fragment was then excised with Sacl/Hindlll and religated into pSP65 cut with Sacl/Hindlll. Sequencing confirmed the presence of four tandem head-to-tail copies of the box 111 sequence.

pCHP2 (Figure 6C) contains three copies of the -183 to -130 region of the CHS15 promoter in plasmid pGEM3zf+. The two complementary oligonucleotides:

5 ' . S A T C A T 6 6 T C T T C A A A A A T A T 6 C C A C C . T A C C A 6 A A G T T T T T A T A C 6 6 T 6 6 -

A A A C T C C T A C T C A C 6 A A C T A 6 6 6 A A 6 C A 6 T T T 6 A 6 6 A T 6 A 6 T 6 C T T 6 A T C C C T T C 6 T C T A S . 5 '

were synthesized, ligated, and cloned into the BamHl site of pGEM3 as described above. Sequencing confirmed the presence of three tandem head-to-tail copies of the above sequence.

pHRGPGl contains a translational fusion of the bean HRGP promoter from -927 to +37 to the bacterial CAT gene and NOS 3' terminator in pUC19. A 964-bp Hindlll-BarnHI fragment of the HRGP promoter from plasmid p3.13 (P. Powell, K. Wycoff, R.A. Dixon, and C.J. Lamb, unpublished results) was ligated to a 4597- bp fragment resulting from a complete Hindlll and a partial BamHl digestion of pCHC1.

pRChlOG1 contains a transcriptional fusion of the rice (Oryza sativa) chitinase promoter from -1512 to +15 to the bacterial CAT gene and NOS 3' terminator in pUCl9. A 1527-bp chitinase promoter fsagrnent from plasmid pRCH10 (Zhu and Lamb, 1991), resulting from a complete Hindlll and partial Accl digest, was ligated into Hindlll-digested and Accl-digested pUCl9. A 1542-bp Hindlll-BamHI fragment from the resulting plasmid was then li- gated into pCHC1 digested as above.

Growth and Elicitation of Bean Cell Cultures

Cell suspension cultures of bean cv lmuna were grown in a modified Schenk and Hildebrandt medium as described previously (Dixon et al., 1981). They were exposed to elicitor from the cell walls of Colletotrichum lindemutbianum (20 pg of glucose equiv- alents per milliliter of culture) (Dixon and Lamb, 1979), harvested by filtration, frozen with liquid NP, and stored at -70°C.

lsolation and Electroporation of Protoplasts

Rapidly growing cell suspension cultures of alfalfa (Medicago sativa) cv Calwest 475 were maintained as described previously

(Choudhary et al., 1990a). Suspensions that had been through at least 12 seria1 subcultures since initiation from callus were har- vested on the fifth day after subculture for preparation of proto- plasts. Protoplasts were released from 1-g batches of cells by incubation in 100 x 15 mm Petri dishes with 10 mL of driselase (1'10, w/v, Sigma), Onozuka cellulase RS (l0/o, w/v, Karlan, Torrance, CA), Onozuka macerozyme R1 O (0.5%, w/v, Karlan), hemicellulase (0.5%, w/v, Sigma) and 0.4 M mannitol in suspen- sion cell culture medium, pH 5.8, for 9 to 10 hr in the dark at 25°C with gentle shaking (40 strokes per minute). Protoplasts were passed through 70-, 40-, and 30-pm nylon mesh filters, collected by centrifugation at 1 OOg for 1 O min, and washed three times with W5 medium, pH 5.8 (Menczel et al., 1981).

Protoplasts were resuspended at a density of 2.0 x 1 07/mL in MS salts (Murashige and Skoog, 1962), 0.4 M mannitol, 30 mM MgCI2, 0.1 YO (w/v) 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.8, heat shocked for 5 min at 45OC, and put on ice for 2 min. Protoplast samples (500 pL) were transferred to 1.5-mL plastic cuvettes containing up to 90 pg of plasmid DNA plus 50 pg of carrier calf thymus DNA, and incubated at room temperature for 1 O min. The cuvettes were then placed on ice for 10 min after the addition of 200 pL of 40% PEG (40% w/v PEG, 0.1% w/v MES, 30 mM MgC12, 0.4 M mannitol, pH 7.0), and the samples were then electroporated on ice using a BTX Transfector 300 unit (50 pF, 150 V, BTX; San Diego, CA). After 10 min more on ice and 1 O min at room temperature, the suspensions were slowly diluted with 5 volumes of 0.2 M CaCI2, 0.4 M mannitol, centrifuged at 509 for 5 min, and washed, and the protoplasts were then cultured in 2 mL of protoplast medium (Kao and Michayluk, 1980) contain- ing 0.4 M mannitol at 25OC in the dark.

Elicitation and Analysis of Protoplasts

C. lindemutbianum cell wall elicitor (50 pg of glucose equivalents per milliliter final concentration) (Dixon and Lamb, 1979) and phenylpropanoid compounds (final concentration from 1 O-6 to 10-3 M, solutions adjusted to pH 5.8 before addition) were added to protoplasts 2 hr after electroporation. Protoplasts were har- vested by centrifugation 1 O hr postelicitation, frozen in liquid NP, and stored at -7OOC.

Viability of protoplasts after electroporation, elicitation, and addition of CA was determined by the Evans blue stain exclusion procedure (Graff and Okong O-Ogola, 1971).

Assay of CAT activity was performed as described by Fromm et al. (1 985). After identifying reaction products by autoradiogra- phy, spots corresponding to 1-, 3-, and 1,3-acetyIated chloramph- enicols were scraped off, and radioactivity was determined by liquid scintillation counting.

ACKNOWLEDGMENTS

We thank Dr. Michel Dron for providing pCHC constructs, Dr. Qun Zhu for the rice chitinase promoter, Dr. Keith Wycoff for the bean HRGP 4.1 promoter, and Scotty McGill and Allyson Wilkins for preparation of the manuscript. G.J. Loake and A.D. Choudhary contributed equally to this work. A.D. Choudhary was the recipient of a Government of lndia Scholarship for study abroad. G.J. Loake is a Noble Foundation Plant Biology Postdoctoral Fellow.

Regulation of CHS by Phenylpropanoids 839

This paper is No. Xlll in the series “Stress Responses in Alfalfa (Medicago sativa L.).

Received April 16, 1991 ; accepted June 26, 1991.

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DOI 10.1105/tpc.3.8.829 1991;3;829-840Plant Cell

G J Loake, A D Choudhary, M J Harrison, M Mavandad, C J Lamb and R A Dixongene promoter.

Phenylpropanoid pathway intermediates regulate transient expression of a chalcone synthase

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