Increased template activity in chromatin from cadmium chloride treated pea tissues

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol.50,No.4,1973

INCREASED TEMPLATE ACTIVITY IN CHROMATIN FROM CADMIUM CHLORIDE TREATED PEA TISSUES Lee A. Hadwiger,

Sharon von Broembsen,

and Robert

Eddy, Jr.

Department of Plant Pathology, Washington State University, Pullman, Washington 99163

Received

January

3,

1973 SUMMARY

Increases in the synthesis of the isoflavonoid, pisatin, and the activity of phenylalanine arnnonia lyase (PAL) are induced in excised pea pods by low synthesis and concentrations (5X10'4M) of CdC12. The induction of pisatin PAL are suppressed if RNA-synthesis-inhibiting concentrations of 6-methyl purine, actinomycin D or =-amanitin are applied within 1 h of inducer application. Cycloheximide (0.1 mg/ml) blocks the induction of these responses if applied to tiss es within 6 h after inducer application. Within 1 h after CdCl (5X10' 1 M) is applied to pods there is an increase in the rate of synthesis 8f all sizes of RNA as well as an increase in the template activity and dye binding capacity of pea chromatin. The results support the hypothesis that conformational changes in DNA are associated with the induction process. Precise

biological

of the multiplicity

effects

of sites

Low concentrations

of other erties

plant

regarded

as inhibitors

as massive (PAL),

controlled (7),

plants

In general,

plants

DNA-intercalating

(366 nm) in combination

0 19 73 by Academic Press, of reproduction iu an-v form

Inc. reserved.

with

, actually

(5,6).

by certain

psoralen

certain

production compounds,

RNA synthesis

of pisatin

(9).

as well ammonia

These gene-

DNA-alkylating

UV light

prop-

legumes from

of phenylalanine

compounds (lo),

1120

as the production

of phytoalexin

stimulate

pathway

of high

The antifungal

in that

and the activity

plant

heavy metals.

the production

the induction

in peas can also be induced (7),

with

in protecting

(8) has been a paradox

of RNA synthesis

react

because

such as CdC12, CuC12, HgC12, and

to be essential

in both pisatin

responses

which

in peas as well

in various

an enzyme in the biosynthetic

base analogs

UV light

Copyright All rights

increases

(7).

to decipher

in inducing

pisatin,

are believed

pathogens

(2-4)

organisms

phytoalexins

of phytoalexins

the cell

salts

of the "phytoalexin",

in peas (7) and other

lyase

pathogenic

isoflavonoid

certain

(1) within

of heavy metal

CrC13 can mimic plant concentrations

of cadmium are difficult

agents

(260 nm) (ll),

compounds (12),

fungal

BIOCHEMICAL

Vol. 50, No. 4, 1973

mycelial

extracts

lysine

(13),

numerous

(2,4)

effecters

and DNA-complexing

spermine

cytotoxic

by compounds

(10)

(16-18).

whose

This

template

provides

increase

activity

pisatin

Actinomycin

treatments

2 cm long)

were

The pods

covered

sterile

petri

Treatments

(1.0

of 1 g pod.

hour

incubation

respectively, aseptically

from

enclosed were

ml of inducer

solution)

then

placed

as described

previously.

pea seedlings .--The

were

in the dark (13)

Heavy metal

to induce

relative

rates

pisatin

pods

split

(less

to minimize

and placed

in

erotic

acid

respectively.

Orotic

applied

to the endocarp

surface

CdC12

of 3 g of split,

(6X10-4M).

(30 UC) and subsequently were

combined

and fractionated

at 22°C.

of RNA synthesis

and

-5-3H

Another treated

to the

1121

After

endo-

18 and 24

was quantitated

in

were

(7)

sprayed

acid

imnature 3 g sample

(15 m later)

(5-40%

induced

pea tissue

on

and non-

with

6-14C

(3.0

erotic

acid

PC) was

pea pods 15 m prior was labelled with

and the RNA was extracted by linear

exposed

production.

-6-14C

(19)

L.)

blossom

solutions

the

method

sativum

and pisatin

by pulse-labeling

phenol

and the

N.Y.

applied

determined

30 m the two samples

RNA synthesis

irmnediately

were

-5-3H

low concentrations

in the

tissue

acid

documented

dishes.

PAL was assayed

with

the effects

are well

Orangeburg,

induced

to treatment

of DNA.

Max-Plank-Institute.

pea (Pisum --

still

periods

-RNA synthesis

exclusively

of chromatin.

by TH. Wieland,

(1 g/treatment)

Pods were

grown

to test

PAL activity,

Alaska

while

almost

in pea tissues

sites

are not

the conformation

system

Schwarz-Mann,

.--Immature

harvested

contamination.

carps

ways,

hormones,

AND METHODS

was donated

D-3H was obtained

Induction than

amanitin

that

D binding

MATERIALS

induced

properties

synthesis,

and actinomycin

Materials.--=

is

induction

evidence

steriod

such as wounding

in various

well-defined

such as poly-L-

hormones,

DNA conformation-changing

report

of heavy metals

(Plant

production

to change,

this

molecules

stimuli

Pisatin

(14-16)

We have employed of heavy metals,

and simple

responses).

known

basic

and RNase (13).

compounds

of these

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

sucrose)

water.

with

erotic

After

at 60°C by the density

gradient

Vol.50,No.4,

1973

centrifugation.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Centrifugation

32,000 rpm at 0 to 4 C. and relative

counts

were obtained

Bonner (20) except and CdC12-treated water

extracted

administered

Assay:

these assays

that

MgC12 was deleted

tissues

were thoroughly

which does not contain

UTP-3H into

ratios

to Huang &

medium".

The H20

washed 7 times in 10X volumes of to chromatin

extraction.

gradient

ug DNA.

The

purifications

The E. e

and was

polymerase

peak" described

used in

by Burgess

(22)

sigma factor.

of RNA synthesis a trichloroacetic

DNA and 0.1 unit

the addition

was measured by determining acid-insoluble

fraction.

the incorporation Incubation

volume of 0.2 ml containing

was filtered

washed 3 times with was dried

of E. coli

of 5% trichloroacetic

The mixture

Dye-bindinq chromatin

was carried

equivalent -treated and after centrifuged

measured

Chicago liquid

an additional for

with

tissues.

according

D binding

based scintillation

capacity

of pea

to the methods of Beato

preparations

Each combination

chromatin

15 h at 39,000 r-pm in a SW 40 Spinco rotor.

(the

from CdC12 (5X1De4M)

was stirred

on a 5-20% sucrose

1122

The filter

counter.

pH 7.5)

15 m was layered

at 0 C.

15 ml chloroform.

CL3H (1 uc/O.63 mg) was mixed with

of 1 mg DNA in 2 ml Tris,

was stopped by

(PHWP 02500) and the filter

The actfnomycfn

as above was determined

and water-treated

1.0 Pg DNA or 1.0 pg of

in 10 ml of Triton-X

scintillation

0.04

0.1 mgfml pyrophosphate) filter

15 ml of 5% TCA and twice

Actinomycin

(23).

a milipore

of pea chromatin.--

isolated

(with

of

10 vmoles Tris-HCl

Incubation

RNA polymerase.

acid

through

and the radioactivity

in a Nuclear

al.

Control

1.0 umole MgC12, 0.25 umole MnC12, 3 umoles B-mercaptoethanol,

chromatin

et.

efficiencies

according

pmoles of ATP, GTP and CTP, and 0.04 umoles UTP-3H (5pc),

fluid

18 h at

pods.

from the "grinding

phosphocellulose

10 m at 37°C in a final

(pH 7.5),

(7).

was extracted

two density

of actual

for

counting

previously

CdC12 prior

was run through

for

of water-treated

Pea chromatin

was the "pooled

The rate

were corrected

two sets

(21) on the basis

in a SW-50-L rotor

as described

to remove external

chromatin

quantitated

out for

The ratios

by dual labeling

RNA Polymerase

sterile

was performed

15 m at 4°C

gradient

and

The respective

was

Vol.50,No.4,1973

BIOCHEMICAL

Table

1.

Effect

(PAL)

and pisatin

of metals

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on the

levels

of phenylalanine

PAL-nmoles Cinnamic Acid

Cont.

Pisatin uglg

CdC12

1,157

f

46

107

CdC12

1,242

t

28

156

HgCl 2

1,036

t 101

123

ZnC12

345 2

50

155

NaC12

102t

2

0

MnC12

892

4

0

MN1 2

912

15

0

CoC12

146 +

14

11

6

0

972 + 114

365

902

H20 Actinomycin

10 Wml

D

One ml aqueous

solution

of each chemical

was applied

the pods

to being

incubated

dark

prior

or 24 h (pisatin

assay).

at 37°C.

of metal

from optimal

reSUltS

pellets

were

plate

and for

in the

PAL activity

PAL and pisatin

Concentrations

layered

lyase

in pea pod cells.

Chemical Applied

pod/h

ammonia

employed

were

salts

to establish

that

most

tions

pure

DNA and only

the protein

fractions

from

Also,

of the

included

in the

aliquots

part

of the

rpm for

Actinomycin

a minimal upper

in this

cinnamic

to lX10'5M.

(7,13). only

the

table. bound

resuspended

72 h in a Spinco

of radioactivity

to the

tem-

pellets

Dm3H was associated

portion

acid/g

previously

of radioactivity

38,000

bound

1X10-2

of

18 h (PAL assay)

as nmoles

as described

ranged are

on 6M CsC12 and centrifuged

of the

expressed

the determination

UV measurements.

at 22°C for

assayed

tested

concentrations for

is

to the endocarp

were

Ti 50 rotor with

frac-

occurs

with

of the gradient.

RESULTS AND DISCUSSION Increased

amounts

of pisatin

can be induced

1123

when low molar

concentrations

Vol.50,No.4,

1973

BIOCHEMICAL

of the heavy metal immature

tissue

level

tissue

of pea tissue

in 24 h.

(1X10-l

are

were

solutions

from

Under

to lX10'4M)

activity

either

could

in the

reaction

ammonia

lyase

obtained

within

inherent

mixture.

level

increases

of this

only

in the

pod tissue

enzyme is

already

maximal

are approached

induction

responses

are

detectable

by high

such as 6 methylpurine

(1 mg/ml),

a-amanitin

(1.0

within

mg/ml)

The protein

metal. these

responses

if

synthesis

if

applied

Cadmium chloride tissue

as indicated

to result species

from

within

a differential

rate

in phenylalanine apparently

the

In general,

at 3 and 8 h, respectively,

and peak

after

inducer

D (0.6

(0.1

application

shown

rate

of synthesis

or

of the

heavy

mg/ml)

of the

inhibits

inducer.

of RNA synthesis

in Fig.

These

mg/ml)

application

cycloheximide

the

application.

of RNA-synthesis

actinomycin

1 h of the

increases

salts

tissues.

6 h after

(6X10-4iil)

metal

1.

in pea

The stimulation

throughout

appears

the various

sized

of RNA.

accessibility

chloride

treatment

of the chromatin

We compared

the

template

and non-induced

The data

chromatin

extracted

from

that

of chromatin

than

to affect

2 indicate

CdC12 (6X10-4M)-treated extracted

the

from

1124

RNA synthesis

the template

(1 h) tissue H20-treated

D.

from CdC12 (6X10'4M)-

relative that

the

and actinomycin

chromatin

to evaluate

in Table

significantly

RNA polymerase

of purified

pea tissue

--in vitro.

was found

DNA to both

activity

potential

higher

inhibitor

by the experiments

The cadmium

induced

applied

to 1X10e3M non-induced

of peas since

concentrations

inhibitors

in PAL

of these

in seedling

18 and 30 h, respectively, were suppressed

MnC12 and CoC12

from

increases

pis-

100 pg per g

of 6X10e4

concentrations 10 fold

the

increases

of PAL extracted

than

increase

NaCl,

10 fold

the application

by comparable

pea seedlings.

than

salts,

than

of excised,

on intact (sx~o-~M)

the metal

The activity

obtained

surface

(~3 ug) to more

18 h after

in PAL and pisatin

levels

or Z&12

Greater

The greater

endocarp

1) or sprayed

(lX10m3M)

ineffective.

be increased

were

(Table

the same conditions

were

not

to the

non-detectable

of CdC12 or HgC12.

tissues

applied

of oea pod halves -4 (5X10 M), HgCl2

Cadmium chloride atin

salt

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tissues.

is

activity consistently When the

of

Vol.50,No.4,

BIOCHEMICAL

1973

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1

AA

I

5

9

13

17

21

25

FRACTIONS Fig.

1.

various

Relative

incorporation

fractions

of RNA extracted

pea pod tissue.

Orotic

of 3 g of split,

imnature

Another

3 g sample

treated

(15 m later)

with

two sets

with

water.

erotic After

previously circles

acid

versus

(7)

acid

water-treated

with

two samples gradient

CdC12 (6XlCm4M).

were

combined

The solid

5s RNA; II = light

line ribosomal

and

centrifugation

(see

and relative

dpm l4 C to dpm 3H ('4C/3H) ratios

surface

(30 IJC) and subsequently

efficiencies

indicate

into

to the endocarp

-5-3H

30 m the

counting

erotic

to treatment

by density

for

controls.

I = 4s and/or

at 254 nm.

CdC12-treated

pea pods 15 m prior

The open

of water-treated

labeled

6- 14C (3 UC) was applied

(corrected

as described

by the triangles.

from

and fractionated

The ratios

administered

acid

was labeled

the RNA was extracted

Methods).

of radioactively

obtained

by dual

indicates RNA; III

counts are

indicated

labeling

the optical

density

= heavy ribosomal

RNA.

reaction with

mixture

CdC12 (6X10-4

tracted D binding

from

containing

chromatin

to 6X10e5M),

CdC12 and H20-treated

capacity.

The chromatin

from

polymerase tissues

H20-treated

tissue

was supplemented

activity

decreased.

was also

shown to differ

containing

1125

pellet

from

Chromatin

ex-

in actinomycin

the CdC12-treated

Vol.50,No.4,

Table 2. induced

1973

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Template activity pea pod tissue

of freshly

assayed

with

prepared I.

coli

Labelled UTP-3H incorporated (uumol/lO min/mg DNA) Prep I Prep II

-Ent

H20-chranatin

from H20 or CdC12

RNA polymerase.

Deletions from Reaction Mixture

Template

chromatin

1,059

1,052

H20-chromatin

3,866

2,505

DdCl2-chromatin

6,255

5,162

111,975

27,045

DNA H20-chromatin

-Mg-Mn

451

em

Cd612-chromatin

-Mg-Mn

393

--

-Mg-Mn

364

DNA Split

I imnature

pea pods (609) were treated

1 h and the chromatin

was extracted

that MgC12 was deleted

as described

designated

"Prep I" and "Prep II"

extraction

and assay

procedure

procedures

was not interrupted

saturation

of the enzyme.

tissue

complexed with

H20-treated

The chromatin

were from different except

assaying. level

22% more actinomycin

effects

of RNA synthesis,

of pea chromatin tion

Template activity

harvests that

of chromatin

preparations of pea pods.

the Prep II extraction

Also Prep I was assayed

whereas

The

with

Prep II was assayed with

D-3H than did the chromatin

a 50%

from

tissue.

The remarkable rate

to Huang & Banner (20) except

in Methods.

before

of the saturation

H20 or CdC12 (6X10m4M) for

medium.

were similar

enzyme in excess level

according

from the grinding

and DNA were determined

with

0

suggest

of gene-controlled

of cadmium in peas in increasing

and the template that

this

responses.

activity

heavy metal

and actinomycin is directly

Since increases

1126

PAL activity, D binding

the sites

involved

in the activa-

in RNA synthesis

occur follow-

Vol. 50,No.4,

ing

the

application

"inhibitors"

of low

induction

have a specific other

of pea tissue.

stimulatory

function

effects

low concentrations viewed

both

as well

as their

It

is

and the

polymerase

Previously

denaturation

stabilizing

one explanation in the

"melting-in

in certain

(1,16,17,18)

effects

which

induce

regions

of DNA occurs. do not

induce,

the double-helical for

the action process" of the

(15)

appear

that

cellular

the

at

D should

D binding

sites

tissue

to the

are

known to bind

the

hydrogen

Tm, the mean temperature

of polymerase

be

on DNA in vitro.

of DNA, disrupting

other

ions,

bond-

at which

including

Nat,

to the sugar-phosphate and raising

in this

of

of DNA.

production

conformation

exists

on the DNA of chromatin

of heavy metals

to bind

25)

heavy metals

action

from CdC12-treated

In contrast,

of cadmium (14)

the

in actinomycin

and lowering

(24,

at low concentrations

phytoalexin bases

no 'paradox" suggest

transcription

increase

or RNA synthesis

such as actinomycin

of chromatin

DNA conformation

Mn2+ and Co2+ which bond,

the

results

effects

on the

to the heterocyclic

changing

thermal

to relate

and Hg2+ ions

preferentially

physical

effects

that

Similarly,

compounds

availability

reported

Cadmium2+

ing,

blocking

tempting

These

are minimal.

of their

heavy metals

we propose

in the cell

of DNA-specific

in terms

of either

D (7),

system

toxic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

concentrations

such as actinomycin

in this

which

BIOCHEMICAL

1973

report

initiation

back-

the Tm.

is that

it

by lowering

Thus,

could

assist

the Tm with-

DNA. ACKNOWLEDGEMENTS

We appreciate the technical help of Diane Payne, Myra Smart, - - Joe Kraft, Jaci Ralston and Diane Law and thank C. A. Ryan, C. Sander and H. Krakauer for help preparing the manuscript. Research was supported in part by NIH grant GM18483. REFERENCES :: i: 5. 6. i: 1::

Vallee, B. L. and Ulmer, D. D., Ann. Rev. Biochem. 41, 91(1972). Cruickshank, I. A. M. and Perrin, Dawn R., Aust. J.-Biol. Sci. 16, lll(1963) Perrin, Dawn R. and Cruickshank, I. A. M., Aust. J. Biol. Sci. E, 803(1965). Schwochau, M. E. and Hadwiger, L. A., Arch. Biochem. Biophys. 126, 731(1968). Cruickshank, I. A. M. and Perrin, Dawn R., Phytopath. 2, 60, 335(1967) Klarman, W. L. and Sanford, J. B., Life Sci. 7, 1095(1968r Schwochau, M. E. and Hadwiger, L. A., Arch. Biochem. Biophys. 134, 34(1969). Hess, S. L. and Hadwiger, L. A., Plant Physiol. 4& 197(1971). Hadwiger, L. A., Phytochem, 5, 523(1966). Hadwiger, L. A. and Schwochau, M. E., Plant Physiol. 47-, 346(1971).

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