Regulation of the Cinnamate 4-Hydroxylase (CYP73A1) in

Plant Physiol. (1997)

113:

951-959

Regulation

the Cinnamate 4-Hydroxylase (CYP73Al) in

Jerusalem Artichoke Tubers in Response to Wounding and

Chemical Treatments

Yannick Batard, Michel Schalk, Marie-Agnès Pierrel, Alfred Zimmerlin, Francis Durst,

and Danièle Werck-Reichhart*

Département d’Enzymologie Cellulaire et Moléculaire, lnstitut de Biologie Moléculaire des Plantes, Centre

National de Recherche Scientifique,

frans-Cinnamate 4-hydroxylase (C4H)

a plant-specific cyto-

chrome (P450) that

encoded by the gene

CYP73A

and catalyzes

the second step of the multibranched phenylpropanoid pathway.

lncreases in C4H activity in response to physical and chemical

stresses have been well documented, but the mechanism of these

increases has never been studied in detail. This paper reports on the

regulatory mechanism controlling C4H activity in Jerusalem arti-

choke (Helianfhus tuberosus) tubers

response to wounding and

chemical treatments. We compared induction of C4H and other

P450-catalyzed activities. C4H was moderately induced by chemi-

cals relative to other P450s. lncreases in enzyme activity, C4H

protein, and transcripts were quantified and compared in tuber

tissue

h after wounding and chemical treatments. Our data

suggest that induction of the enzyme activity results primarily from

gene activation. lime-course experiments were performed after

wounding and aminopyrine treatment. Compared with wounded

tissues, aminopyrine triggered an additional and delayed peak of

transcript accumulation. lhe timing of

the

induced changes in

activity, protein, and transcripts confirms that C4H induction re-

sults primarily from an increase

CYP73Al

mRNA, in both

wounded and aminopyrine-treated tissues. However, posttranscrip-

tional mechanisms might also contribute to the regulation of C4H

activity.

P450s

are ubiquitous enzymes that catalyze the activa-

tion of molecular oxygen and the insertion of one of its

atoms into physiological and artificial substrates (Porter

and Coon, 1991).

plants P450s perform many oxygen-

ation reactions in secondary metabolism, in sterols and

fatty acid derivative synthesis, and in the detoxification of

xenobiotics (Bolwell et al., 1994; Durst and OKeefe, 1995).

More than 16 P450-catalyzed reactions have been reported

in the pathway leading to the biosynthesis of phenylpro-

panoids (Werck-Reichhart, 1995). The influx of metabolites

into the pathway

controlled by a sequence of three

catalytic steps leading from Phe to activated 4-coumaroyl

COA. The second reaction of the sequence is catalyzed by a

P450 called C4H. Complete cDNA sequences were recently

reported for the enzymes from Jerusalem artichoke

(Heli-

anthus

tuberosus;

Teutsch et al., 1993), mung bean

(Vigna

radiata;

Mizutani et al., 1993), and alfalfa

(Medicago sativa;

Corresponding author; e-mail

[email protected]

strabg.fr; fax 33-

-35- 84

84.

Rue Goethe,

67000

Strasbourg, France

Fahrendorf and Dixon, 1993). In accordance with the pro-

posed nomenclature for P450s (Nebert et al., 1993), the

tuberosus

enzyme was termed CYP73A1. Enzymes of the

general phenylpropanoid pathway are usually expressed

in a coordinate manner, but the regulatory mechanisms

underlying coordinated expression are unclear. Both the

substrate (cinnamic acid) and the product (p-coumaric

acid) of C4H have been implicated in the regulation of

upstream and downstream enzymes of the phenylpro-

panoid pathway (Dixon and Paiva, 1995; Werck-Reichhart,

1995). Whether C4H activity plays a role in the process,

however, remains to be determined.

A characteristic common to many P450s from a11 organ-

isms is their inducibility by exogenous chemicals. The in-

duction mechanisms of bacterial and animal P450s by xe-

nobiotics have been extensively studied. In some cases (i.e.

induction by polyaromatic hydrocarbons or hypolipidemic

drugs) the induction was shown to involve gene activation,

and the signal transduction pathways involved have been

elucidated (Denison and Whitlock, 1995). It is believed that

the transduction pathways activated by these molecules

are normally triggered by endogenous signaling molecules

the nature and function of which are not yet understood

(Nebert and Feyereisen, 1994; Okey et al., 1994). Aside from

activated transcription, the regulation of animal P450s has

also been shown to involve regulation at several posttran-

scriptional levels, depending

the effector and particular

P450. These include stabilization

mRNA and protein,

modification of intranuclear transcript processing, and en-

hanced translation (Porter and Coon, 1991; Roberts et al.,

1994). Little information is available concerning the mech-

anism of the regulation of plant P450s. C4H, which

easily

detected in many plant tissues and catalyzes a reaction

important for defense and lignification, has been one of the

most extensively studied P450s. C4H activity is induced by

a number of stresses, including wounding and chemical

effectors (Werck-Reichhart, 1995).

addition, several re-

ports suggested that the enzyme might be glycosylated

(Kochs et al., 1992; Fahrendorf and Dixon, 1993; Smith et

Abbreviations: C4H, trans-cinnamate 4-hydroxylase (NADPH:

oxygen oxidoreductase [4-hydroxylating], EC 1.14.13.11); DEHP,

bis(2-ethylhexy1)-phthalate;

ECOD, 7-ethoxycoumarin O-deethyl-

ase; EROD, 7-ethoxyresorufin

(or

7-ethoxyphenoxazone) O-deeth-

ylase; NA, naphthalic anhydride; P450, Cyt P450.

951

952 Batard et al. Plant

Physiol.

Vol.

1997

al., 1993; Werck-Reichhart et al., 1993), thus suggesting a

possible regulatory effect of this glycosylation. In previ-

ous studies, increases in protein or steady-state mRNA

concomitant with enzymatic activity have been reported

(Fahrendorf and Dixon, 1993; Teutsch et al., 1993; Werck-

Reichhart et al., 1993; Hotze et al., 1995; Logemann et al.,

1995; Frank et al., 1996); however, induction of C4H ac-

tivity has never been correlated with C4H protein and

mRNA levels in an attempt to determine if this enzyme is

subject to posttranscriptional regulation.

This paper describes the effects of wounding and of

different chemical effectors, including those triggering

gene activation and important signal transduction path-

ways in animals or bacteria (e.g. phenobarbital, benzo-

[alpyrene, and clofibrate), on C4H activity and accumula-

tion of CYP73A1 protein and transcripts. The impact of

these effectors on C4H is compared with their effect on

other P450 (ECOD and ER0D)-catalyzed reactions in the

same plant. The effect of wounding and of aminopyrine,

the most potent P450 inducer in

tuberosus,

was studied

in more detail and in a time-dependent manner. Our re-

sults suggest that induction of C4H in response to all

effectors tested results primarily from transcriptional

regulation.

MATERIALS AND METHODS

Chemicals

tran~-[3-~~C]Cinnamate was from Isotopchim (Ganobie,

France). NA and isosafrole were purchased from Aldrich,

phenobarbital

(5-ethyl-5-phenyl-barbituric

acid, sodium

salt) from Fluka, 1,8-ethoxyresorufin from Pierce, and

DEHP from EGA-Chemie (Steinheim, Germany). All other

chemicals were obtained from Sigma.

Plant Material

Jerusalem artichoke

(Heliantkus tuberosus

var Blanc

commun) tubers were grown in an open field, harvested in

November, and stored in plastic bags at 4°C in the dark.

For aging experiments tubers were sliced (1.5 mm thick),

washed, and incubated for 48 h in aerated (4 dm3 min-l),

distilled water containing various chemicals, as described

previously (Reichhart et al., 1980). Chemicals tested as C4H

effectors were prototype P450 inducers known to trigger

different signal transduction pathways in animals and

bacteria (phenobarbital, aminopyrine, P-naphthoflavone,

isosafrole, 3-methylcholanthrene, benzo[a]pyrene, clofi-

brate, and DEHP) (Bresnick, 1993), plant metabolites (fla-

vone and 8-methoxypsoralen), metals (MnCl,, HgCl,, and

CdCl,), and agrochemicals (biphenyl, lindane, and NA),

a11 known to induce animal or plant P450s (Reichhart et

al., 1979; Fujita, 1985; Letteron et al., 1986; Fonne-Pfister et

al., 1988; Borlakoglu and John, 1989; Siess et al., 1989;

McFadden et al., 1990; Batard et al., 1995). The MnCl,

solution was adjusted to pH 7. Water-insoluble com-

pounds (P-naphthoflavone, flavone, biphenyl, NA, 8-me-

thoxypsoralen, and benzo[a]pyrene) were first dissolved

in 4 mL of DMSO and then added to the aging medium

(1.5

L).

Preparation

Microsomal Fractions

described by Werck-Reichhart et al. (1990).

Plant tissues were extracted and microsomes prepared as

Enzyme Assays

C4H was assayed, as described previously (Reichhart et

al., 1980). ECOD and EROD activities were determined

fluorimetrically (Werck-Reichhart et al., 1990). The results

are means

of duplicates or triplicates.

Analytical Methods

P450 and microsomal protein contents were determined

as described by Gabriac et al. (1991). CYP73A1 content was

quantified by differential spectrophotometry (presence

versus absence of saturating concentration of substrate)

using an absorption coefficient

(E)

for the

A,,,

difference of 128 mM-' (Urban et al., 1994).

SDS-PAGE (10% total monomer, 0.3% cross-linker), elec-

troblotting onto nitrocellulose membranes (Hybond C,

Amersham), and immunoblot staining using the rabbit

polyclonal antibody C4Hpa2

were performed as de-

scribed by Werck-Reichhart et al. (1993). The immunoreac-

tive protein was quantified using a densitometer (model

SC930, Shimadzu, Columbia, MD) and comparison with

known amounts of purified protein run on the same gel.

Total RNA was isolated according to the method of Lesot

et al. (1990). Denatured RNA was separated in the presence

of formaldehyde through a 1.2% agarose gel and trans-

ferred to a nylon membrane (Hybond N+, Amersham)

(Sambrook et al., 1989). RNA blot prehybridization and

hybridization with a full-length

CYP73A1

nucleotidic

probe radiolabeled with

[

a-32P]dCTP by random priming

was carried out at 65°C according to established proce-

dures. Membranes were washed twice for 10 min with 2~

SSC, 0.1% SDS, once for 10 min with

0.2X

SSC,

0.1%

SDS at

room temperature, and then twice for 30 min with 0.2~

SSC, 0.1 SDS at 55°C. Hybridization signals were quanti-

fied using a phosphor imager (model BAS1000, Fuji, Tokyo,

Japan). RNA amounts were standardized by hybridization

at 55°C to a 300-bp

Capsicum annuum

25s rRNA probe.

RESULTS

Modulation

C4H by Chemical Treatments

A preliminary experiment was performed to determine

the constitutive levels of C4H activity and P450 content and

the effect of DMSO on these levels in tuber tissues (Table I).

C4H activity, just detectable in dormant tissues, was in-

creased 14-fold after 48 h of aging in aerated water. A

slightly greater induction of activity was observed in the

presence of 0.2570 DMSO (approximately 18-fold). In com-

parison, P450 content was induced only approximately

4-fold in response to DMSO. Increases in C4H activity were

more than twice the corresponding increases in total P450,

which is indicative of a selective induction of CYP73A1

relative to other P450 forms.

Regulation

Cinnamate 4-Hydroxylase 953

Table

C4H

activity

microsomes from dormant and control

tuberosus

tuber

tissues

Microsomes were prepared from dormant tubers

from

tissues

sliced

and

aged for

aerated,

distilled

water or

water

plus

Microsome C4H Activity P450 Content

0.25%

(v/v)

DMSO.

pkat

mg-'

protein

pmol

mg-

protein

Dormant

tuber

0.8

0.07

922

Water

DMSO

14.1

Wounding (water)

10.9

0.3

The modulation of C4H activity in tubers treated with

compounds previously documented to induce P450 activi-

ties was examined (Table

11;

Figs.

and

2).

Addition of some

chemicals to the incubation medium resulted in a decrease

in C4H activity. Clofibrate and DEHP, inducers of fatty acid

o-hydroxylase in animals and plants (Salaün et al., 1986),

did not significantly alter the total P450 content (Table

II),

but at 5 mM, reduced C4H activity by 80 and 50%, respec-

tively (Fig.

1).

8-Methoxypsoralen (or xanthotoxin), a cou-

marin accumulated by Apiaceae that was previously shown

to induce P450s in animals (Letteron et al., 1986), increased

the total P450 content but decreased the C4H activity.

Iso-

safrole, benzo(a)pyrene, 3-methylcholanthrene, and

naphthoflavone, prototype inducers of animal P450s

(Bresnick, 1993), did not alter significantly the activity at the

concentrations assayed, even though an increase in the total

P450 content of the tissues was observed (Table

11).

HgCI,

and CdCI,, which were previously reported to induce P450

and the accumulation of terpenes in sweet potato discs

(Fujita, 1985), were apparently toxic at the concentrations

used in our experiments (Fig.

2).

All other chemicals, includ-

ing Mn2+, phenobarbital, aminopyrine, lindane, biphenyl,

NA, and flavone, increased both C4H activity and P450

content 1.6- to 3.3-fold. The highest induction was obtained

with chemicals (MnCI,, aminopyrine, and phenobarbital)

that had been previously tested for dose response (Reichhart

et al., 1979; Fonne-Pfister et al., 1988), which were therefore

used at optimal concentrations, but severa1 aromatic planar

molecules

such

NA,

biphenyl, and flavone also appeared

good inducers of C4H.

We previously reported the induction of alkoxycouma-

rin and alkoxyphenoxazone O-dealkylating activities in

tubevosus

tuber tissues by the same xenobiotics (Batard

et al., 1995). Figure

shows that the induction patterns of

ECOD and

EROD

activities measured in the same plant

microsomes are clearly different from that of C4H. On the

whole, O-dealkylating activities are more susceptible to

chemical stress than is C4H. They are, in particular,

strongly induced by aminopyrine, phenobarbital, benzo-

(a)pyrene, or 8-methoxypsoralen. In contrast, C4H activity

is inhibited by 8-methoxypsoralen treatment. However,

some planar aromatic molecules (biphenyl, NA, and fla-

vone) seem to induce C4H equally or better than dealky-

lase activities.

Expressed in yeast microsomes, C4H has been shown to

be fdly low-spin, i.e. with maximum

A,,,

resulting from

the presence

one molecule of water as the sixth ligand

the heme iron (Urban et al., 1994). Binding of the substrate

releases the water molecule and shifts the maximum

389 nm. A differential coefficient of absorption for the

A,,,

difference measured in the presence versus the

absence of saturating substrate was determined. Assuming

that the enzyme is also low-spin in plant microsomes, we

made a rough estimation of the contribution of C4H to the

total P450 in wounded and xenobiotic-treated tissues. Ta-

ble

I11

shows that in wounded tuber tissues, C4H is by far

the major P450. Its relative abundance, however, is less in

tissues treated with phenobarbital, MnCI,, and, in particu-

lar, aminopyrine. This is in good agreement with the data

shown in Figure

and Table

11,

which indicate that other

P450s are more strongly induced by these treatments than

is C4H.

CYP73A1 Protein and Transcript Levels in Treated Tissues

Figure

shows the leve1 of C4H protein immunodetected

in the control and treated microsomes, and the correlation

between the amount of C4H protein present in the tissues

and the C4H activity. The correlation

imperfect, probably

resulting from imprecise immunoblot quantification of the

protein, or it may reflect some sort of posttranslational

regulation. In a few cases, discrepancies between activity

and protein content can be explained. Clofibrate and DEHP

are peroxisome proliferators and induce the production

activated oxygen species and lipid peroxidation in both

animal and plant tissues (Palma et al., 1991). These mole-

cules were described as inducers

fatty acid hydroxy-

lases, and sometimes

C4H,

plants (Salaiin et al.,

1986).

Table

II.

lncreases

P450

content

microsomes from

tu-

berosus

tuber

tissues after

chemical

treatments

Dormant tissues were

sliced

and

aged

for

aerated, distilled

water

containing

the

indicated concentrations of chemicals.

P450

induction

given

ratio to the activity

P450

content

measured

tissues

aged

distilled

water

plus

DMSO

(Table

I).

P450

Treatment Abbreviated Concentration Relative

lnduction

Name

MnCI,

CdCI,

Aminopyrine

Phenobarbital

P-Naphthoflavoneb

Clofibrate

Diethyl hexyphthalate

Benzo(a)pyreneb

3-Methyl~holanthrene~

Lindaneb

Biphenylb

Naphthalic

anhydrideb

Flavoneb

8-Metho~ypsoralen~

HgCI2

lsosafrole

CIO

DEHP

B(a)P

3-MC

Lin

Bip

Flav

8-MP

Iso

2.5

1.7

0.26

0.24

6.5

0.1

0.2

1.5

0.05

0.1

2.5

0.2

n.d.a

n.d.

3.1

rf-

0.3

2.6

0.2

2.0

0.1

0.8

rf-

0.3

0.9

0.09

1.5

0.3

1.4

0.2

1.8

0.2

2.3

0.3

2.6

rf-

0.1

1.9

0.4

2.3

0.3

1.4

0.2

0.8

0.1

0.9

0.3

n.d.,

Not detectable.

Chemicals

solubilized

DMSO

(0.25%

of the total

volume

the

aging

medium).

954

Batard et al.

Plant

Physiol.

Vol.

1997

mo7

effector

Figure

Comparison

of the relative increases

C4H,

ECOD, and

EROD

activities

microsomes

from

tuberosus

tuber

tissues

after

different

chemical

treatments. Tuber tissues

were

sliced

and aged

for

aerated, distilled water

containing

the

different chemicals

before extraction

and

preparation

the

microsomes.

Abbreviated

names

and

concentrations

the

chemical effectors are defined

Table

II.

C4H,

ECOD,

and

EROD

were measured

using

radiochemical

fluorimetric

assays, as

described

"Materials

and

Methods." Relative activities

are

given

ratios

the activities

measured

tissues

aged

distilled water

water

plus

DMSO.

C4H

activities

are

given

Table

ECOD

and

EROD activities measured

microsomes

from

tissues

aged

water

were

0.01

0.002

and

0.028

0.004

pkat

mg-'

protein,

respectively,

and

water

plus

DMSO were

0.061

0.005

and

0.035

0.005

pkat

mg-'

protein, respectively.

In our experiments, we used concentrations more than

twice those previously reported to induce P450. Such con-

centrations still promote an increase in immunodetectable

C4H protein, but the oxidative stress seems to result in

protein inactivation. If the data concerning clofibrate and

DEHP are omitted from Figure 2B, the coefficient of corre-

lation between C4H activity and protein is shifted from

0.58 to 0.81.

CYP73A1

mRNA abundance in some of the same treated

tuber tissues is shown in Figure 3. Both wounding and

chemical treatments clearly resulted in an accumulation of

CYP73A1

transcripts (Fig. 3A). A good correlation between

the amounts of immunodetected C4H protein and the

abundance of the corresponding mRNA was also observed

(Fig. 3B). C4H induction by wounding and chemical treat-

ments thus seems to result primarily from increases in the

steady-state levels of

CYP73A1

mRNA.

Time-Course lnduction of CYP73A1 after Wounding and

Aminopyrine Treatment

To further test the hypothesis of transcriptional control

CYP73A1

gene expression, two separate experiments

were performed to monitor C4H enzyme activity, the leve1

of the C4H protein, and steady-state levels of C4H mRNA

after wounding and aminopyrine treatment.

After wounding, two successive waves of induction

were observed (Fig. 4). Transcript accumulation pro-

ceeded without a detectable lag phase. A first peak of C4H

transcript accumulation was observed at approximately

10 h following wounding. A second maximum occurred

after another 10 h. The steady-state content of C4H mes-

senger then decreased until 50 h after wounding, and

stayed relatively constant (transcripts still being detect-

able) for the next 2 d. C4H protein and enzyme activity

grossly followed the same pattern of induction, but the

maxima were shifted 10 to 20 h later than that observed

for the mRNA. Activity appeared slightly delayed com-

pared with protein accumulation.

In a second experiment, tuber slices were aged in water

or in water containing 20

aminopyrine (Fig.

5).

The two

waves

induction elicited by wounding were less appar-

ent in this experiment since

(a)

the tubers were not used at

exactly the same stage of dormancy release and dormancy

stage influences induction (amplitude and time course),

and (b)

smaller set of samples was analyzed. In

aminopyrine-treated tissues, the initial induction consis-

tent with the wounding response was slightly delayed and

reduced, but still obvious. It was followed by another

increase in transcripts and protein. C4H mRNA peaked

after 45 h in the presence of aminopyrine, then decreased

steadily. C4H protein increased until60 h and remained

until84 h. Transcripts and protein levels during this second

phase of induction never exceeded 140% of the maxima

observed after wounding.

DISCUSSION

P450s in animals and microorganisms are highly induc-

ible by exogenous molecules. This inducibility is usually

related to the detoxifying function of these enzymes, and

P450 forms very active in the metabolism of xenobiotics are

usually more susceptible to exogenous inducers than are

isozymes involved in essential physiological pathways

(Porter and Coon, 1991). Our findings suggest that this may

also

be the case in plants. We have shown that C4H, a

central enzyme in the phenylpropanoid pathway, is in-

duced by

wide range of chemicals, but

induced less

Regulation

Cinnamate

4-Hydroxylase

955

Mrxur

58-

48.5-

48.S-

200

100

2.2x

17.9

0.58

C4H

activity (pkat.mg-1 protein)

Figure

Immunoquantification

of the

CYP73A1

protein

micro-

somes

from tuber

tissues

treated

with

different chemicals.

A, Ten

micrograms

microsomal

protein

from

dormant

tuberosus

tuber

tissue

tissue that

had

been aged

for 48 h in

water,

solutions

chemicals (full names

and

concentrations given

Table

II),

or 100

immunopurified

C4H

were analyzed

SDS-PAGE,

transferred

onto nitrocellulose membrane,

and

immunostained

for

C4H. Staining

was

performed using rabbit polyclonal anti-C4Hpa2/2 serum

diluted

1:10,000 (Werck-Reichhart

al.,

1993). Standard indicates

prestained

molecular

mass

markers.

Immunoreactive

C4H was

then

quantified

densitometry

with

reference

to the

immunopuri-

fied enzyme. Data were

plotted

against

C4H

activity measured

in the

same

microsomes.

than

other P450s

in the

same plant. Several

of the

potent

inducers

animal P450s (phenobarbital, aminopyrine, fla-

vone, biphenyl,

and

DMSO) also trigger

increase

in C4H

activity

plants. Other chemical agents,

particular those

capable

inducing

CYP1A1 (i.e.

benzo[«]pyrene,

3-methylcholanthrene,

and

/3-naphthoflavone),

do not af-

Table

III.

C4H to

P450 ratio

in H.

tuberosus tuber

tissues

after

wounding

and

chemical

treatments

Microsomes were prepared from tuber

tissues

aged

for 24 h in

water,

for 48 h in 8 mM

phenobarbital,

or 20 mM

aminopyrine,

or for

h in 25 mM

MnCI

(aging times previously

reported

to be

optimal

for

induction

each

case).

P450

and C4H

contents were determined

using

spectrophotometrical

methods,

described

in the

text.

Microsome

Water

MnCI

P450 Content

pmol

nr,

± 2

240

± 16

347

± 43

427

± 49

C4H

Content

ig~

protein

± 2

102

± 2

106

± 5

125

± 5

C4H/P450

feet

C4H,

but do

increase P450 content

and

P450-dependent

dealkylase

activities. Common induction mechanisms,

re-

ceptors,

signal transduction pathways

are

possibly con-

served

animals

and

plants.

was one of the

best inducers

of C4H in H.

tuberosus.

This

was

unexpected, since this compound, which

was the

first

commercial herbicide

safener,

usually

thought

exert

selective

action

monocots

(Hatzios, 1991).

mung

bean,

NA has no

effect

on C4H

activity

and

increases

lauric

acid

herbicide metabolism only when applied

combination

with other inducers (Moreland

al., 1995).

grass crops

induces

the

metabolism

various

herbi-

cides

but

represses

C4H

activity

(Zimmerlin

al., 1992;

Persan

and

Schuler, 1995).

The

absence

of a

response

some

of the

chemicals

tested

our

experiments

does

not

exclude

the

possibility

of a

response

other

concentrations,

since

many

concentra-

tions

used

this work were chosen

from

studies

ani-

mals

other plant materials

and

were

not

optimized

for H.

tuberosus.

Clofibrate,

for

example,

may

induce

C4H at

lower

concentrations (Salaiin

al., 1986).

CYP73A1

25S

200

100

1.3x

22.9

0.91

40 60 80

CYP73A1

transcripts

(relative

amounts)

100

Figure

Accumulation

CYP73A1 transcripts

tuber

tissues

treated

with

different chemicals.

Twenty micrograms

total

RNA

extracted

from dormant

tuberosus tuber tissue

tissue that

had

been aged

for 48 h in

water

or in

solutions

chemicals (full names

and

concentrations given

Table

II) was

electrophoresed through

denaturing

formaldehyde

gels

and

transferred

capillarity

onto

nylon membrane.

The RNA

blot

was

successively hybridized

with

full-length CYP73A1

DNA

probe

high stringency

and

with

300-bp pepper probe coding

for a 25S

rRNA

at low

stringency,

described

"Materials

and

Methods."

CYP73A1

transcripts

were

quantified using

phosphor imager. Transcript abundance

was

cal-

culated

comparison

with

the

MnCI

-treated sample,

which

was

arbitrarily

assigned

value

100.

956

Batard

et al.

Plant

Physiol.

Vol. 113, 1997

A. 100

B._ 200

hours

after

wounding

0 1 2 4 8 12 16 20 24 30 36 50 60 72

CYP73A1

25S

20 40 60

Hours

Houra

atler

wounding

0 2 4 6 8

121(202430364248

Figure

Time

courses

induction

of C4H

activity,

CYP73A1

protein,

and

CYP73A1 transcripts

wounded

tubemsus

tuber

tissues.

Tuber

tissues

were

peeled,

sliced, washed,

and

aged

aerated,

distilled

water.

Accumulation

CYP73A1

transcripts.

Twenty micrograms

total

RNA

extracted

from

the

tissues

was

fractionated, transferred

membrane,

and

hybridized successively

with

CYP73A1

and 25S

rRNA nucleotidic probes,

described above. CYP73A1

transcripts

were

quantified using

phosphor imager. Transcript abundance

was

calculated

comparison

with

the

sample prepared after

8 h

aging,

which

was

arbitrarily assigned

value

100.

Immunoquantification

of the

CYP73A1

protein.

Ten

micrograms

microsomal protein

immunopurified

C4H was

submitted

immunoblot

analysis,

described above.

Standard

indicates

prestained

molecular

mass

markers.

Immunoreactive

C4H was

then

quantified

densitometry

with

reference

the

immunopurified

enzyme.

C, C4H

activity detected

microsomal preparations.

animals changes

P450 levels resulting

from

expo-

sure

xenobiotics

are

primarily

the

result

gene acti-

vation.

Depending

on the

P450

form

and on the

effector,

however,

all

types

posttranscriptional regulation have

been observed (Porter

and

Coon, 1991; Denison

and

Whit-

lock, 1995).

plants,

the

only data available

are

indica-

tive

transcriptional activation. Light-, wounding-,

infection-promoted

increases

in C4H

activity occur

after

lag

period

of 1 to 3 h and are

prevented

treatment with

cycloheximide,

puromycin,

cordycepin,

actinomycin

which indicates

novo synthesis

of the

enzyme resulting

from

gene activation (Hyodo

and

Yang, 1971; Tanaka

al.,

1974; Durst, 1976; Rhodes

al., 1976; Benveniste

al.,

1977; Lamb, 1977;

Oba and

Conn, 1988). Results recently

obtained

by RNA

blot hybridization

confirmed

increases

steady-state levels

of C4H

mRNA

after

wounding,

treatment

with xenobiotics,

fungal

elicitation

cell

suspension cultures

(Fahrendorf

and

Dixon, 1993; Teutsch

al., 1993; Hotze

al., 1995; Logemann

al., 1995).

Our

results

corroborate these data

and are

consistent with

the

suggestion that enhanced transcription

primarily

re-

sponsible

for the

induction

of C4H in

response

wound-

ing

and

chemical inducers. Wounding

followed

by two

peaks

transient

mRNA

accumulation. These

two

peaks

are

also observed

at the

level

of the C4H

protein

and

enzyme

activity

in the

microsomes. Such

biphasic

re-

sponse

wounding

has

already been observed (Morelli

al., 1994; Logemann

al., 1995)

and

seems

to be

cor-

with

two

distinct phases

stimulation

trans-

lational

activity synchronized with

the

expression

of two

classes

wound-induced genes involved

wound heal-

ing

and

prevention

pathogen invasion.

tubers,

important

function

these genes

is the

synthesis

suberin

for the

wound periderm.

It has

been

proposed

Regulation

Cinnamate 4-Hydroxylase 957

Hours

200-

aminopyrine

40 60

Hours

Figure

Time course of induction of

C4H

activity,

CYP73A1

pro-

tein,

and

CYP73A1

transcripts

tuber

tissues

aged

water or

aminopyrine. Tubers were peeled, sliced, washed,

and

aged

aerated, distilled

water

alone or containing

aminopyrine.

Accumulation of

CYP73A7

transcripts.

Twenty micrograms of

total

RNA

extracted from the tissues

was fractionated,

transferred to mem-

brane,

and

hybridized

with

CYP73A1

and

25s

rRNA

nucleotidic

probes,

described

above.

CYP73A

transcripts

were

quantified

using

phosphor

imager.

Transcript

abundance

was

calculated

comparison

with

the

sample prepared after 48

aging

the

presence

aminopyrine,

which

was

arbitrarily

assigned

value of

100.

lmmunoquantification

the

CYP73A1

protein.

Ten

micro-

grams

microsomal

protein

immunopurified

C4H

was

submitted

to immunoblot

analysis,

described above. lmmunoreactive

C4H

was

quantified

densitometric

scanning

comparison

with

the

reference immunopurified enzyme.

C4H

activity

detected

mi-

crosomal preparations.

that these two gene classes are triggered by either imme-

diate injury and cell division (Morelli et al., 1994). Amin-

opyrine treatment triggers an additional phase of tran-

script accumulation, delayed by about 38 h compared

with the wound response. This response to chemical treat-

ment is apparently stronger than, and independent from,

the wound-induced gene activation. It occurs a long time

after the beginning of the treatment and follows a slight

inhibition of wound induction. Aminopyrine induction

does not seem to result from gene activation directly

triggered by the exogenous molecule, but rather from the

onset of a cascade of events and signals that apparently

promote CYP73A1 gene transcription 30 h after the begin-

ning

the treatment.

Considering the successive waves of increases in steady-

state levels

CYP73A1 mRNA elicited by wounding and

aminopyrine treatment, it is tempting to speculate that

there is an activation of two or three independent CYP73A1

genes in

tuberosus.

The presence of at least two variant

cDNAs has been detected (Teutsch et al., 1993). These two

variants, however, showed 98.25 and 100% identity in nu-

cleic acids and amino acids, respectively. Noncoding se-

quences were not available for comparison.

A delay of about 15 to

observed between maximal

increases in CYP73A1 mRNA and in the accumulation of

the C4H protein or enzyme activity. This

a surprisingly

long delay compared with the lag observed in cell cultures

(Hotze et al., 1995; Logemann et al., 1995) and

difficult to

understand at this time. It is unlikely to reflect the time

necessary for protein maturation and correct membrane

targeting of the C4H inserted into the ER. Transcript pro-

cessing or availability of the components of the transla-

tional apparatus in wounded tuber tissues might slow the

expression of the enzyme (Morelli et al., 1994).

Although some correlation between accumulation of

CYP73A1 transcripts and increases in C4H protein and

activity was observed in our experiments, detailed analysis

of our results reveals severa1 discrepancies. For example,

Mn2+-, clofibrate-, or DEHP-treated tissues show a high

C4H protein content relative to enzymic activity. Such data

could be indicative of a posttranslational regulation. It may

be pointed out, however, that all three chemicals are likely

to favor an oxidative degradation of the protein. The time-

course experiments also indicate that the regulation of C4H

activity in tuber tissues brings into play mechanisms more

complex than simple activation

the CYP73AZ gene(s)

expression. For example, the elevated levels of C4H protein

recorded after long aging on aminopyrine (Fig. 5) and the

progressive increase in the delay between message and

protein accumulation observed in all experiments suggest

that transcript maturation and translation, but also protein

stabilization or inactivation, possibly have an impact on

C4H regulation.

Direct measurements of the rates

CYP73A1 transcrip-

tion, determination of the number of CYP73A1 copies in

the

tuberosus

genome, and evaluation of C4H stability

after different tuber treatments will be needed to obtain a

more accurate picture of the mechanisms regulating C4H

expression.

ACKNOWLEDGMENTS

thank

Dr.

Michael

Barrett

for

his

critica1

reading

the

manuscript. We

are

grateful

Monique

Ret for the preparation

immunopurified

C4H,

and to Marie Luce Schantz for the

gift

pepper 25s

rRNA

probe. The technical help of Marie-France

Castaldi is also gratefully acknowledged.

958 Batard et al. Plant Physiol.

Vol.

1997

Received July 15, 1996; accepted December 3, 1996.

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