Induction by ethylene of cyanide-resistant respiration

BIOCHEMICAL

Vol. 70, No. 2, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

INTXJCJ?ION BY ETAYIENEOF CYANIDE-RESISTAtXC RESPIRATION T. Solomosl and G. G. Laties Departmsnt of Biology and Molecular Biology Institute, University of California, Los Angeles, California 90024 Received

March

25,1976

Sumnary: Ethylene and cyanide induce an increase in respiration in a v.sxiety of plant tissues, whereas ethylene has no effect on tissues whose respiration is strongly inhibited by cyanide. It is suggested that the existence of a cyanide-insensitive electron transport path is a prerequisite for stimulation of respiration by ethylene. Introduction:

Throughthe

years two appazentlyunrelatedbasic

problems in

plant physiology

have been the focus of unabated investigation.

intense

has centered

ation

interest

(1, 2). transport increasing

Quite separately, in certain

on the cause and nature of the burst

- which attends

- the climacteric

ripening

and plant

Our studies

(3).

point

of respir-

in a great variety

the phenomenon of cyanide-insensitive

plant tissues

attention

On one hand

mitochondria to an intimate

of fruits

electron

has received

ever-

relationship

between the two phenOmeaS. Material University

and Methods: omharrl.

Citrus

am3 avocado fruits

Other fruits,

roots,

were obtained

tubers and seeds were purchased.

Ethylene and cyanide were applied

as previously

were isolated,

assayed, according

and their

Sweet potato

(5).

distilled

described

prepared with a hand slicer

water arki suspended in air-saturated

measurements. electrode.

slices

activity

framthe

(4).

Mitochondria

to published methogs were rinsed with

O.lmM CaSO4 for respiratary

Oxygen uptake by slices was measured with a Clark oxygen Mitochondrial

respiration

was measured with a Rank (Bottisham,

Cambs. U.K.) oxygen electrcde. Results and Discussion: ethylene

We have described

and cyanide in initiating

the su

effectiveness

of

seemingly identicalphysiologicaland

Gr esent address: College of Agriculture, Department of Horticulture, University of Maryland, College Park, MaryLand 20742. Copyright AN rights

0 I976 by Academic Press, Inc. of reproduction in any form reserved.

663

Vol. 70, No. 2, 1976

BIOCHEMICAL

AND BIOPHYSICAL

biochemical

changes in avocado fruits

enhance=&

of respiration

of pre-existing respiratory

capacity

enzynrzs (4, 6), it

anaerobiosis

oxidase,

and in fact

in such tissues

rather

of cyf~~hranre oxidase,

in tissues

where c@chr~

(7).

The existence however, permits

(4, 6, 7, 8).

the aerobic

Thus, it

acts sGnil~~ly,

it

is generally

affected

by cyanide,

inhibited

by cyanide should be unaffected respiration

tissues

observations

(9).

Fig.

Table 1 compares the effects tissues.

bothbyethylene

The respiration andby

cyanide.

cherimoyas and apples as readily rutabagas,

beet roots,

carruts,

should be is strongly

That is,

for ethylene

and ethylene

have been reported

IB showsthatbothethylene of intact

and

red sweet potatoes.

of cyanide and ethylene of lemons and grapefruits

on a variety is stimul&ed

Cyanide induces the climacteric as does ethylene, sweet potatoes

in

and the respiration

andpotatoesis

with the evolution Furthermare, 664

of ethylene,

with rutabagas,

of

stimulated

cyanide is camnonly associated lags behind.

IA

has no effect.

Whereas the enhancement of respiration

usually

Fig.

green peas,

bybothcyanideandethylene.

evolution

If

respiration.

whose respiration

to aside and ethylene

cyanide enhance the rate of respiration

Of plant

path (3).

path must be present.

oxygen uptake,

with respect

in resistant

by ethylene

by ethylene.

the alternate

inhibits

for segments of pea internodes

Finally

or alternate

that

of cyanide on the oxygen uptake of fresh

where cyanide strongly Similar

while

path

of respiratory

recognized

is stinndated

slmiLar4

the effects

electron

should induce cyanide-resistant

Acccnxling4, tissues whose respiration

depicts

oxidation

Pasteur effect

Cyanide may even stimulate

cyanide evokes the cyanide-resistant,

plant

oxidase is the main terminal

of a cyazdde-insensitive

in the presence of cyanide (3).

to stimulate

release

may be expected to

cyanide is known to imroke a conventional

substrates

ethylene

of

is assumed that both agents similar4

plants,

tissue

Since the

than to -de nova synthesis

in certain

respiration

(4, 6).

on respiration.

Cyanide, as an inhibitor simulate

tubers

in each case is seemingly due to the realization

enzymatic

one or more restraints

and potato

RESEARCH COMMUNICATIONS

by

ethylene and in one

Vol. 70, No. 2, 1976

BIOCHEMICAL

0” 0

-

1.

The effect rate

of

A.

RESEARCH COMMUNICATIONS

CzH4

4-

;...

0

Fig.

AND BIOPHYSICAL

20’

.x44--.. 40’

of ethylene

. .. . . . . . . . . . .._._.. !y. 60’

80’

loo’

and cyanide

fresh peas and intact &) &/L

Fresh peas:

I ’ 120

’ 140

reswxtively

on the rOOtS.

red sweet potato ethylene

~lr 100 fi/t

respiration

~~

pr0videdatsrrOw.

Sweet potato

B.

root:

17 pt./L ethylene

or 400 u.444 HCN

proviaedatarr0w.

experimnt

with lemons, no detectable

cyanide-induced

respiration

rise

respiration

may be independent

production

is a secondary effect

that

where cyanide partially

peas, ethylene

ethylene

- suggesting of ethylene

evolution that

has but a mrginal

production,

respiration

stirmiLstory

and in high RQ values

1.6-2).

operative.

enhances respiration, In consequence it

the cyanide-resistant decontrols

path.

both aerobic

crease in the levels

the cyanide-resistant is proposed that

respiration

case, as with

of glycolytic

end-products

the presumption electron

that

where

path must be

evokes or imple8.k?nts

of the alternate despite

with the climacteric

665

shows

germinating

In this

ethylene

and glycolysis

of ATP associated

ethylene

Table 1 further

The data imrite

The evocation

and that

effect.

in a large accumulation

ethylene

of cyanide on

in six-day-old

green peas, cyanide results (e.g.

the effect

of cyanide action.

inhibits

accom@nied the

path apparently the frequent (lo),

in-

an& with

Vol. 70, No. 2, 1976

Table 1.

BIOCHEMICAL

The effectsof

of a variety

AND BIOPHYSICAL

cyanide and ethylene

of plant

RESEARCH COMMUNICATIONS

on the respiration

rate

tissues. Respiration Control

Tissue

02

Rate HCN

W& co2

02

co,

02

co,

IN&3fr WMw 6

6

1.6

16

18

23

Avocados

35

35

150

140

150

IA0

Cherimoyas

35

35

I.60

150

152

145

7

7

I.6

15

21

20

Apples

Lemons Grapefruit

ll

ll

30

29

40

40

Beetroots

ILL

IJ.

22

22

24

28

Carrots

12

lo

20

19

30

41

14

ll

14

Il.

2.5

Potatoes

2.5

18

17

22

22

24

28

Rutabagas

9

9

18

15

23

20

Green Peas

8

a

2

2

60

85

Sweet Potatoes

w

&day-old

pea seedlings

Ethylene

and HCN provided

XL0

at levels

7.5 150

7.5 150

evoking optimsl

response in each

case.

the rise studies

in respiration

tubers

treated

with ethylene

with cherimoyas we have foundthatthe

appreciably ethylene

in potato

with the rise

treatments

have suggested that and the alternate the restriction

In our

of ATP increase

in response to both cyanide and

(ll).

Cur data suggest that is the determination

in respiration

levels

(6).

a primary

of the electron the apportioning path is regulated

of electron

effect transport

of ethylene path.

of electrons

666

respiration

Bahr and Bonner (12)

between the cytochrarre

by an equilibrium

flow through

on plant

nmxhanism.

In this

cytochrome cxidase evokes the

path view

Vol. 70, No. 2, 1976

alternate

BIOCHEMICAL

path.

However, ethylene

whereas diversion

of electrons

can explain

resistance

of ethylene

or the stimulation

sweet potatoes of fresh

that

("yams")

slices,

cytochrou~

it

of the intact

tion

of electron

paths are in limited

of the alternate

organ (Table 2, experiments cytochrcme

path --in vivo.

slice,

respiration

whereas antimycin,

an equally

the cytochro~

4 and 5).

by cyanide and ethylene

substrate

stimulates

potato

CO enhances fruit

respiration

forxr&ion

The similarity slice

sweet potato.

(13).

as well

to an additional In this

connection

cytochrolne

(9).

Furthermore,

(14) and in Chlorella

effect

cells

of ant-in

667

as the main role

of cyanide

we have found

axidase (viz.

400

unpublished).

azide stimulates (13) - thus ruling

of cyanide and m-CLAM on

(Table 2, experiments

either

out

for cyanide stimul.ation.

as the locus of the alternate

To show inhibitionby

transport

The failure

(Solomos and Isties,

as an explanation

the mitochondrion

protuthecoides

where cyanide does, rules

and points

respiration

respiration

b)

of cyanide

in response to a Pasteur effect

of the synergistic

and mitochondrial

establishes

tuber

of the role

of electron

far too low to inhibit

in avocado slices

out cyanohydrin

inhibitor

of cy-tochron~ oxidase.

CO at concentrations

of root,

(Table 2, Fig.

of Chlorella

in respiration

of cyanide stimulation,

beyond the inhibition

respiration

effective

mobilization

fully

resptiation.

the respiration

increase

Thus, the restric-

oxidase does not in itself

system, has no effect

to produce a sustained

&l)

rate

is almost 10 times

In consequence the stimulation

in cyanide-insensitive

Cyanide markedly increases

that

use in intact

the respiration

path (X2),

must be taken into account in any fuJA understanding

explanation

the effect

by cyanide.

- as evidenced by the fact that

the alternate

glycolytAc

and

oxidase inhibition

cannot account for either

of respiration

flow through

and occasionally

through

oxidase,

whether in the presence of cyanide or m-chlorobenzhydroyamic

that

(and ethylene)

RESEARCH COMMUNICATIONS

in response to cytochroms

both electron

acid (m-CLAM), an inhibitor

activate

does not inhibit

to cyanide,

It is nateworthy

AND BIOPHYSICAL

1, 2, 3, 4) path in red

cyanide or m-CLAM singly,

both

The effects

81.0

Control

Intact

5

roots

Slices (1 mu thick)

4

15

0.2mMKCN+3mMm-CLAM

10

98

3mMm-CLAM

Control

98 99

0.2 mM KCN

llOlX

$ Od/g F.W./h

150

0.5 mM rn-l.lXdiM+ 0.2 KCH

340

0.2 mM KCN M

730

0.5 l&l m-CLAM

(17 RM succinate)

90

0.5 m-CLAM+ 0.2 mMKCN

3

450

0.5 m-CLAM

2

520 475

nitrogen/mln Control (17 mM malate) 0.2 n&l KCN

Mitochondda

rate

natoms axygen/mg mltochodrial

Respiration

of mitochondrla,

1

Experinmxhl. material Addit ions

of cyanide and m-CIAU on the respiration

of red sweet potato.

Experiumt number

roots

Table 2.

85

0

0

58 81

1.2

1.1

0

1.56

l 53

1.50

A.DP/O

end Intact

lo

80.

ion

slices

percent

Inhibit

tissue

Vol. 70, No. 2, 1976

pathsmstbe

BIOCHEMICAL

operating

considerable

disparity

of the two paths, other.

must be less than the potential must enhance or sctlvste

is activated tional (4, 6).

ethylene tissues

tion

of glycolytic

and lactate,

intermsdiates

an3 ATP (ll).

is stWd.ated

by ethylene

The la&

the "activation"

Glycolysle

(6, 11) seemingly

increase

decontrol

by ethylene

involves

electron

Pasteur effect

transport

phosphorylative

efficiency

path

chain continues

kinase.

capscity

leads

educed by cyanide and ethylene

to operate

whose respiration

in phosphorylative Huwever, while the site

I of the

in the presence of

Is enhanced by cyanide,

may drop sharply while the absolute

669

of two

by cyanide.

the expected reduction

Thus in tissues

of the alternate

and pyruvate

path per se Is not coupled to phosphorylation,

(15).

of glycolytic

the activation

alternate

cyantie

the accumula-

suggest that where respiration

due to the by-pass of the cytochrame path.

respiratory

Inthese

respiration.

efficiency

conventional

Table 1)

as it evokes an extensive

phosphofructokinsse

in ATP levels

contradicts

pea seedlings,

(e.g.

- hence enhancement - of both aerobic respira-

of a conventional

The frequent

is strongly

nor changes in the levels

of adequate cyanide-insensitive

to the evocation

or elevated

other hand, causes neither

These observations

enzymes - viz.

unaltered

affects

insofsr

glycolysis

decreases ATP and doubles the rate of

onthe

is the basis far the decontrol

rate-controlling

(e.g.

or marginally

end-products

tion and glpolysis.

and cyanide

in terms of a conven-

where respiration

anaerobiosis

Ethylene,

are either

inhibited

to affect,

of ethanol

glucose breakdown.

cyanide

path,

which canuot be construed

lA) ar psrtlally

cyanide stinulates

acc~tion

cyanide to take

without

of the alternate

since the ATP levels

fails

transport

frcm one

and cyanide enhance respiration,

On the other hand in tissues

either

diverted

the latter.

under ccmditlons

green peas, Fig.

electrons

For stlmulationby

capacity

where both ethylene

Pasteur effect,

ortheremustbe

so that

on the other hand, the rate of electron

In tissues

RESEARCH COMMUNICATIONS

at ~capa~tiysimultsneously

path cannot be accamnodatedbythe place,

AND BIOPHYSICAL

levels

of ATP

Vol. 70, No. 2, 1976

may not only fail electron

flow through

branch-point

proposition

ati by the persisting

is reaffirmed

fresh slices respiration

is often x

e

tissues

strongly

inhibited

respiration

Thus, slicing

cyanide sensitive

(16, 18).

in the latter,

isolated

(16, 17).

alluded

to above precludes

in intact

in mitochondria

in several plant

stkulated

respiration

tissues

an operative

tissues,

as

is associated

(Theologis slices

and

restores

degree in mitochondria p&h

of cyanide resistance the fact that resistance

tissue

suggesl~thatcyanide

slices

and mito-

(and ethylene)-

in origin.

show that ethylene

path is a prerequisite Purthermore,

is raised

of the alternate

tissues,

stimulates

in which cyanide acts simzilarly.

alternate

plant respiration.

in intact

is mitochondrial

In sunmmry our results in plant

breakdown

demonstration intact

tuber

and beet root

and to a considerable

tissues,

tubers or

cyanide-resistant

The loss of CN resistance

from cyanide-resistant

the ADP:O

is mitochondrial.

that mitochor&ial

Whereas the lability

to cyanide can be demonstrated chondria

tubers

alone makes potato

the regular

attending

In the latter

from potato

On the other hand, aging of potato

cyanide resistance therefrom

1).

by cyanide (16, 17) while

the evidence indicates

unpublished).

instance

by cyanide (6, 8) the question

is labile.

of the

3).

isolated

with Wised ~mbrane phospho- and galactolipid Laties,

The location

(Table 2, experiment

is stlnarlated

connection

respiration

14).

of ATP in tissues

In the first

of mitochodria

to whether cyanide-stimulated In this

levels

drops to zero (Table 2, experiment

Since the respiration

level

of cyanide on the ADP:O ratio

respectively.

is decreased by two-thirds

in m&rate

axxi before the second, phosphory-

by the effect

oxidation

because of the increased

is supported by measurements

solpewhere beyond the first,

case the ratio

RESEARCH COMMUNICATIONS

with an increase

is enhanced by cyanide (ll,

malate and succinate ratio

I, together

incOrpOratiOn

whose respiration

site

site

The foregoing

of both [%P]

AND BIOPHYSICAL

to drop, but may in fact increase,

phosphorylation.

lative

BIOCHEMICAL

the ability

670

respiration

only

That is, the presence of

for ethylene of ethylene

enhancement of to enhence plant

Vol. 70, No. 2, 1976

respiration ethylene Neither

is widespread "activates"

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. ll. 12. 13. 14. 15. 16. 17. 18.

AND BIOPHYSICAL

and not confined

the alternate

feedback modulator

This work was supported 61 to G.G.L.

RESEARCH COMMUNICATIONS

to climacteric

path directly

is the mechanism by which the evocation

as a positive

Project

BIOCHEMICAL

of key glycolytic

fruits.

or indirectly of the alternate

Whether is not clear. path acts

enzymes.

by an Atomic Energy Commission Contract

AT(O4-3)-31

Biale, J.B. (1960) Handb. Pflanzenphysiol, I2 536-592. Sacher, J. A. (1973) Ann. Rev. Plant Physiol, 21, 197-224. Bendall, D. S. and Bonner, W. D. Jr. (197'1) Plant Physiol, 47, 236-245. Solomos, T. and Laties, G. G. (1974) Plant Physiol, 54, 506-51.1.. Hobson, G. E., Lance, C. E., Young, R. E., and Biale, J. B. (1966) Nature, 209, 1242-1243. S~lomos, T. and Iaties, G. G. (1975) Plant Physiol, 55, 73-78. James, W. 0. (1953) Plant Respiration, Oxford University Press, Oxford. Hams, C. S. and Bar-J. (1931) Proc. Roy. Sot, B108, 95-118. Burg, S. P. and Burg, E. A. (1967) Plant Physiol, 42, 144-152. Young, R. E. and Biale, J. B. (1967 Plant Physiol, 42, 1357-1362. Solomos, T. and Laties, G. G. (1976 Plant Physiol, In press. Bahr, J. T. and Bonner, W. D. Jr. (1973) J. Biol. Chem, 248, 3441-3445. Grant, N. G. and Hommersand, M. H. (1974) Plant physiol, 54, 50-59. Lips, Ii. S. and Biale, J. B. (1966) Plant Physiol, 41, 797-802. Tomlinson, P. F. Jr. and Moreland, D. E. (1975) Plant Pbysiol, 55, 365-369. S. K. and Niederpruem, D. J. (1960) Hackett, D. P., Haas, D. W., Griffiths, Plant Physiol, 35, 8-19. Banner, W. D., Christensen, E. L. and Bahr, J. T. (1972) In Biochemistry and Biophysics of Mitochondrial Membranes, G. F. Azzone, ed. PP. JJ-3119, Academic Press. Robertson, R. N., Turner, J. S., and Wilkins, M. T. (194‘7) Aust. J. Exptl. viol. &a. sci, 25, 1-8.

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