Plant, Cell and Environment (1985) 8, 117-127

The dual role of oxygen in avocado fruit respiration:

Kinetic analysis and computer modelling of
diffusion-affected respiratory oxygen isotherms
M. L. TUCKER* & G. G. LATIES Department of Biology, and Molecular Biology Institute, University of
California, Los Angeles, CA 90024, U.S.A.

Received 23 July 1984; accepted for publication 10 October 1984

Abstract. Oxygen plays a dual role in affecting the

rate of respiration of avocado fruit (Persea
americana Mill. cv. Hass). The respiration rate
v. oxygen concentration curve for steady state
avocado fruit respiration is biphasic. The curve
becomes monophasic. however. when measured
under conditions of rapidly changing oxygen concentration in a closed circulating system. The results
are interpreted as indicating that oxygen at relatively
high concentrations modulates respiration independent of its interaction with the terminal oxidasepresumably cytochrome oxidase.
A computer model is presented which takes into
account the effect of diffusion barriers on the kinetics
of oxygen utilization as a function of concentration
in avocado fruit. The model is used to make
predictions concerning the apparent Km of the
terminal oxidase or oxidases in avocado fruit. It is
concluded that the apparent Km of the terminal
oxidase of uninhibited avocado fruit is that of
cytochrome oxidase, and that the alternative,
cvanide-resistant oxidase of avocado fruit does not
contribute appreciably to the uninhibited respiration
of preclimacteric or climacteric avocado fruit.
Key-words: Persea americana Mill. cv. Hass; avocado; respiration;
diffusion; oxygen; kinetics; regulation; feedback; alternative
oxidase: cytochrome oxidase; energy charge.

Introduction

An inspection of the literature indicates that a drop

in oxygen concentration from that in air (i.e.
21 KPa) to levels that fully saturate cytochrome
oxidase (e.g. 15 KPa) causes a decrease in the
respiration rate of most plant storage organs and
fruits (Biale, 1946; James, 1953; Blackman, 1954;
Ben-Yehoshua, Robertson & Biale, 1963; Forward,
1965; Burton, 1974). Furthermore, the respiration
rate of several plant storage organs is stimulated by
oxygen concentrations greater than in air (Mapson &
*Present address: Department of Molecular Plant Biology,
University of California, Berkely, CA 94720, U.S.A.
Correspondence: G. G. Laties, Department of Biology,
University of California, Los Angeles, CA 90024, U.S.A.

Burton, 1962; Theologis & Laties, 1982). Three

basicallv different historical rationalizations of the
foregoing observations are discussed below as an
introduction to the explanation proposed herein for
the effect of relatively high oxygen concentrations on
the respiration of avocado fruit.
Blackman (1954), more than three decade.^ ago,
suggested that oxygen plays a dual role in affecting
the respiration rate of apples. He proposed that the
terminal oxidase directly involved in respiration in
apples has a high affinity for oxygen, and that it is
not inhibited until the external oxygen concentration
drops below approximately 5 KPa, at which point
fermentation begins, and carbon dioxide production
increases relative to oxygen consumption. The
gradual increase in respiration with oxygen concentrations greater than 5 KPa was postulated to be
due to what he termed 'carbon activation'. i.e.
substrate mobilization. He envisioned an early step
in respiratory metabolism regulated by oxygen at
relatively high concentrations, independent of the
involvement of the terminal oxidase.
Mapson & Burton (1962) analysed the kinetics of
the change in respiration rate with oxygen concentration in potato tubers and concluded that two or
more terminal oxidases contribute to the overall
respiration. They proposed that cytochrome oxidase,
with a calculated apparent Km for oxygen of
0.4 mmol m 3 , mediates 70% and 50% of oxygen
use by potato tubers in air and pure oxygen,
respectively. The residual 30% and 50% oxygen use
was attributed to the operation of an undefined
oxidase having a low affinity for oxygen. Using a
double reciprocal plot of respiration rate versus
intercellular oxygen concentration, they calculated
that the putative second oxidase had an apparent K-.,
for oxygen of approximately 150 mmol m 3 ,
considerably greater than that for cytochrome
oxidase.
However, Chevillotte (1973) disputed Mapson
and Burton's conclusion and developed a mathematical model in which diffusion influences the
apparent oxygen isotherm for cytochrome oxidase in
a way that gives the appearance of two oxidases with
different K,s'
operating concomitantly. Chevillotte

118

M. L. TUCKER & G. G. LATIES

concluded that Mapson and Burton's data fit a

model in which cytochrome oxidase, with a high
affinity for oxygen, is the terminal oxidase, but where
the creation of an anaerobic centre in individual
potato cells leads to an apparent biphasic isotherm
that seemingly represents two or more different
oxidases with very different Km's for oxygen.
A fruit or plant storage organ poses several
barriers to the passage of oxygen into the cell (e.g.
skin, flesh, cell wall, membranes, and cytoplasm),
each barrier exerting its own effect on the respiration
rate as a function of oxygen concentration. The most
obvious effect is an increase in the apparent Km for
oxygen of any given oxidase (James, 1953;
Chevillotte, 1973). The presence of diffusion barriers
also causes a more subtle change in the oxygen
concentration v . respiration rate relationship
(isotherm) such that it no longer conforms to typical
Michaelis-Menten
kinetics (Chevillotte, 1973).
Chevillotte's work emphasizes the importance of
diffusion barriers in the analysis of oxygen kinetics in
respiration.
In what follows careful consideration is given to
the possible effect of diffusion on the respiration rate
as a function of oxygen concentration. To this end
oxygen isotherms are compared from fruit maintained at atmospheric pressure (101 KPa), as well as
25 KPa to reduce the effects of gas diffusion barriers
(e.g. skin and flesh). In addition, a computer
program is used which models the effect of diffusion
barriers on the oxygen isotherm for avocado fruit
respiration at both 101 and 25 KPa. The construction of respiration rate v. oxygen concentration
curves at reduced pressure has permitted a meaningful comparison of the apparent respiratory oxygen
Km's in the presence and absence of cyanide, and has
established-contrary
to an early suggestion by
Solomos & Laties (1974)-that the cyanide-resistant,
alternative path does not contribute significantly to
the respiration of either preclimacteric or climacteric
fruit.
Evidence is presented that the respiration of
preclimacteric avocado fruit is affected by oxygen
tension at relatively high oxygen concentrations
( > 5 KPa OJ, much as Blackman (1954) demonstrated more than three decades ago for apples.
Blackman's theory that oxygen at relatively high
concentrations regulates some event (e.g. substrate
mobilization) other than the participation of the
terminal oxidase is reaffirmed. In particular, we have
established that negative feedback due to reduced
oxygen concentrations well above those saturating
cytochrome oxidase takes place in times commonly
associated with measurements of respiratory activity
and not solely in prolonged periods such as the 50 h
or more used by Blackman in his studies of
respiratory behaviour of apples during storage. We
have further extended Blackman's concepts, and
provided a framework for understanding respiration
behaviour of bulky plant storage organs that takes

into account external oxygen tension, internal

oxygen tension, intrinsic respiration rates, diffusion
resistance, and the time-constants and oxygen
sensitivity of the proposed feedback loop. Our
modelling and hypobaric experiments have allowed
us to deduce the operational terminal oxidase, and to
provide a reasonable explanation, based on the
operation of but one terminal oxidase, of the often
noted biphasic respiratory isotherm in fruits and
storage organs.
Materials and methods

Mature fruit (Persea americana Mill. cv. Hass) were

obtained from a local tree. Figure 1 shows the
apparatus for respiration measurements. Experiments were conducted at 25 OC. Fruit were placed in
specified containers (see below), and a flow of watersaturated air passed through at 0.83 cm3 s 1 at
atmospheric pressure. When required, a circulating
closed system maintained at atmospheric pressure
(101 KPa) was created as illustrated in Fig. 1 by
diverting the gas flow through a flow regulated gas
pump (Mace model 921-0001). Hypobaric conditions
of 25 KPa were achieved in both the closed and flowthrough svstem with a Cartesian diver vacuum
control ( ~ i l m o n t )in line with a hermetically sealed
metal bellows vacuum pump (Cole-Parmer model K7065-20). Flow rates in the closed svstem were
increased to assure rapid mixing of gases around the
fruit (Table 1). Steady state carbon dioxide production in the open flow-through system was monitored
automatically every hour by passing the effluent gas
through an infra-red gas analyser (Anarad, model
AR500). Oxygen concentration in the circulating
closed system was monitored continuously by
passing the gas through a paramagnetic oxygen
analyser (Beckman model 755). Instruments measuring oxygen, carbon dioxide or gas flow were placed
on the atmospheric pressure side of the vacuum
pump so as to reduce possible gas leaks and errors in
measurements due to reduced pressure.
Fruit containers
All containers were adequate for the open flowthrough system as described. In the circulating closed
system in which a large gas volume was desired (slow
depletion of oxygen), a single fruit was placed inside
a 1 m3 glass jar fitted with a two-hole rubber
stopper. In the circulating closed system in which a
small gas volume was desired (quick depletion of
oxygen), two different types of containers were used.
At normal ambient pressure when the smallest
possible gas volume was desired, a fruit was sewn
into a rubber mesh mat, and the encased fruit placed
into a balloon as follows. A large round rubber
balloon was prepared with an airtight outlet (a
fixed into
modified disposable 1 cm3 plastic syringe)
place opposite the normalballoon opening with a
- ,

DUAL ROLE O F OXYGEN

119

Flow-through

r----------l

flow
gas
mixture

--i^
'flow
req'~lator

Closed System

L----

analyser

gas
circulating
pump

r-l

recorder

Figure 1. Diagram of respiration measurement system. The vacuum control (Cartesian diver), stainless steel bellows vacuum
pump and flow regulator (enclosed in dashed lines) were required only when less than atmospheric pressure was desired. The
gas circulating pump was not required in the closed system when the vacuum pump was being used.

rubber patch kit. The modified end of the balloon

was closed and the balloon turned inside out. The
balloon was then blown up and the fruit pushed into
the modified end of the balloon while slowly
releasing the trapped gas, thus causing the balloon to
encase the fruit and re-exposing the outside of the
balloon. Once the fruit was inside the balloon the
normal opening was plugged with a one-hole rubber
stopper, and the surface of the balloon covered with
a layer of petroleum jelly and wrapped in plastic to
reduce oxygen diffusion from the outside. Air was
passed in through a tube in the rubber stopper and
out through the plastic syringe. The rubber mesh
between the balloon and fruit permitted air
circulation around the fruit. If hypobaric conditions
were to be used, three fruits were placed inside a
glass tube open at both ends, and the gas space
inside the tube not taken up by the fruit was filled
with 3-mm glass beads. The tube was then closed
with rubber stoppers pierced by glass tubing to
permit gas flow.
Cyanide and ethylene treatment
Fruit were treated with ethylene or cyanide in an
open flow-through system. Ethylene at 10 cm3 m - 3
in air was administered using a mixture of
compressed gases. Cyanide was added to the gas
stream by bubbling air through 500 cm3 of buffered
solution (Tes 20 mol m 3 , pH 7.0) containing 5 mol
Table

1.

Flow

rates

Experimental conditions

and pressures
measurements

during

respiratory

Flow rate
(cm3 s"')
--

Flow-through
101 KPa
25 KPa
Closed system
101 KPa
25 KPa

0.83
0.83
3.3
1.7

m 3 KCN, which is in equilibrium with 500 cm3

m P 3 HCN in air at 25OC (Robbie, 1946).
Computer model
The program is written in APL and is available upon
request. The conceptual design of the model is
described in the Results section.
Results and discussion
Biphasic oxygen isotherm
Two basically different methods were used to study
the effect of oxygen concentration on the respiration
of avocado fruit. One approach was to pass oxygen
of known concentration in nitrogen over avocado
fruit at a regulated rate, and measure the rate of CO,
evolved. The rates reported herein for this flowthrough method were measured 12 h after a change
in oxygen concentration (Fig. 2). A time interval of
12 h was required for re-establishment of a constant
respiration rate after a change in oxygen concentration. Thus, the flow-through method yields steady
state respiration values. In the second method an
avocado fruit was held at atmospheric pressure in a
closed system wherein the gas mixture was continuously circulated past the fruit and through an
oxygen analyser at a rate of 3.3 cm3 s l . Oxygen
used in respiration in the closed system was replaced
stoichiometrically by respiratory carbon dioxide,
thus continuously depleting the oxygen in the gas
mixture while maintaining a relatively constant
ambient pressure. The effect of the differences in the
solubilities of 0, and CO-, are considered in the
following discussion.
The steady state flow-through system gave a
biphasic oxygen isotherm, whereas in the closed
system the oxygen isotherm depended on the gas
volume. More precisely, if the oxygen concentration
in the closed system changed relatively slowly, from
21 to 5 KPa oxygen in more than 5 h (large gas
volume), the isotherm appeared to be biphasic much

120

M. L. TUCKER & G. G. LATIES

F-5'-

slow
slow

A,,, = 13 rnrnol m-3

Oxygen ( k P a )

Figure 2. Respiration rate of preclimacteric avocado fruit

v. oxygen concentration with an open and closed system,
respectively. The solid symbols refer to the rate of oxygen use in a
closed system in which the concentration of oxygen drops
continuously. Oxygen concentration in the closed system dropped
quickly (circles) or slowly (triangles), depending on the gas
volume. The open triangles and squares refer to the rate of steady
state CO., evolution in an open flow-through system maintained at
101 and 25 KPa, respectively. Respiration rates were normalized
to that of the given fruit in air at atmospheric pressure. Actual
respiration rates varied for individual fruit in air between 8 x 1 0 3
cm3 kg"' s"'.
and 15 x

as that for the steady state flow-through system (Fig.

2). However, if the oxygen concentration changed
relatively quickly, from 21 to 5 KPa oxygen in less
than 1.5 h (small gas volume), the isotherm
approached that expected of a single oxidase having
a high affinity for oxygen (e.g. cytochrome oxidase;
Figs 2 & 3). Climacteric fruit under similar
circumstances (data not shown) demonstrated a shift
from biphasic to monophasic oxygen isotherms,
similar to that observed with preclimacteric fruit
(Figs 2 & 3).
A double reciprocal plot of the biphasic oxygen
isotherm for preclimacteric fruit slowly depleted of
oxygen yields an apparent Km of 74 mmol m 3 for
the low affinity oxygen response, and an apparent K,,,
of 9 mmol m 3 for the high affinity response (Fig.
3). The reciprocal plot of the monophasic respiratory
isotherm for preclimacteric fruit rapidly depleted of
oxygen yields an apparent Km for oxygen of 13 mmol
m-3.
Basic principles of diffusion
Whereas the effect of diffusion barriers
kinetics of respiration as a function of
concentration in an intact fruit is complex,
purpose a basic, simple concept is all
necessary.

on the
oxygen
for our
that is

Figure 3. Double reciprocal plots of respiratory oxygen isotherms

shown in Fig. 2 where oxygen was depleted quickly (circles) or
slowly (triangles) in a closed system. K,,s' are expressed in mmol
m 3 and are calculated from the negative reciprocal of the
abscissa intercept and converted from partial pressure oxygen in
the gas phase to the equilibrium concentration in water at 25 'C.

Fick's first law of diffusion of a gas through a

plane sheet (Equation 1; Crank, 1956) indicates that
the flux (J,pmol s 1 c m 2 ) depends on the diffusion
coefficient of the gas in the barrier (D, cm2 s l ) , the
thickness of the barrier (X, cm), and the difference in
gas concentration across the barrier (Cl -C?, pmol
c m 3 ) . Fick's first law is rearranged in Equation 2 to
accentuate the direct relation of flux to concentration
difference and the inverse relation of concentration
difference to the diffusion coefficient. This simple
concept of proportionalities is important to the
following discussion, and remains basically true
independent of the diffusion model being used:
J = (D/Y)(Cl-

(CI- C2)

CZ)

(planar sheet) (1)

= [J/D{X)}

(Eqn 1 rearranged) (2)

Diffusion of oxygen across the skin and flesh of a

fruit is better represented by diffusion equations for a
hollow sphere and solid sphere, respectively.
(Ci - C2) = (Q/47iD)[(R - r)/(Rr)] (hollow sphere) (3)
(Cl- C2) = (a/6D)(R2)
(solid sphere) (4)
Equation 3 describes the diffusion of oxygen across
the thickness of a non-respiring hollow sphere (e.g.
the skin, which is taken to be non-respiring as an
approximation) in which oxygen is being used for
respiration inside the hollow sphere (viz. by the flesh;
Crank, 1956). The difference in oxygen concentration
across the thickness of the hollow sphere is related to
the total flux of oxygen across its surface (Q, pmol
s l ) (calculated by multiplying the specific respiration rate by the volume of tissue), the diffusion

DUAL ROLE O F OXYGEN

coefficient for oxygen in the thickness of the hollow
sphere (D, cm2 s l ) , and the outside (R, cm) and
inside (r, cm) radii. Equation 4 describes diffusion of
oxygen through a solid sphere having a uniform rate
of respiration (a, pmol 0, c m 3 s l ) throughout
(Crank, 1956). The rate of respiration in the solid
sphere (Eqn 4) is related to the flux (J)in Pick's first
law (Eqn l), and the total flux (Q) in Eqn 3. In each
case there is direct proportionality of concentration
gradient to flux (a direct function of respiration rate)
and inverse proportionality to the diffusion
coefficient.
Interpreting the biphasic response
Two interpretations of the biphasic response may
be considered. The first is that the biphasic isotherm
for the slow depletion of oxygen (Fig. 2) is due to a
diffusion effect on the oxygen isotherm similar to
that proposed by Chevillotte (1973) for potato tuber
oxygen
isotherms.
In
this
view,
the
Michaelis-Menten type isotherm under conditions of
rapid depletion (Fig. 2) is due to the fact that oxygen
moves into the fruit more rapidly on rapid than on
slow depletion, the presumed reason being that the
higher solubility of C 0 2 compared with 0, creates a
negative pressure across the skin and flesh that
facilitates oxygen uptake. If true, the oxygen gradient
across the fruit radius would be much diminished.
The second interpretation is that oxygen at
relatively high concentrations has an intrinsic effect
on the rate of respiratory metabolism through some
means other than the terminal oxidase, which
requires more than 1.5 h to be effective. To be
convinced of the second interpretation one must
eliminate the first. To this end the rate of oxygen
diffusion at a fixed partial pressure of oxygen was
increased by lowering the total ambient pressure
since diffusion depends on mean free path, which in
turn is inversely dependent on pressure. Accordingly
at 25 KPa (0.25 atm) pure oxygen, the rate of
diffusion of oxygen through the skin and intercellular
gas space is increased four-fold over that in air at
normal pressure, at the same time that the oxygen
concentration gradient is diminished approximately
four-fold at any given respiration rate (Eqn 2-4).
The respiration rate of an unripe avocado at a
given oxygen tension was first determined at
101 KPa, following which the pressure was reduced
to 25 KPa for 12 h and the respiration rate
determined once again. The process was repeated
using a series of concentrations of oxygen in
nitrogen. In this way oxygen isotherms were
determined at 101 and 25 KPa. Within experimental
error, the isotherms are similar (Fig. 2, open
triangles and squares, respectively) and both reflect
the biphasic oxygen isotherm obtained for the slow
depletion of oxygen in a closed system (Fig. 2). This
indicates that the oxygen concentration gradient
across the skin and flesh of preclimacteric avocado
fruit is not large, and cannot account for the

121

difference between the isotherms for the quick and

slow depletion of oxygen in the closed system
(Fig. 2). Oxygen at relatively high concentrations
must therefore have an intrinsic effect on the rate of
respiratory metabolism through some means, other
than the terminal oxidase, that requires more than
1.5 h to be effective.
Description of computer model
A computer program was written that describes
avocado fruit respiration as a function of oxygen
concentration and oxygen permeation. The model
avocado fruit is assumed to be spherical in shape
weighing approximately 0.2 kg. The seed of an
avocado fruit has a much lower respiration rate than
the flesh (mesocarp) and the respiration rate of the
seed does not change appreciably upon ripening,
whereas the flesh respiration rate changes markedly
(Ben-Yehoshua, 1964). The model seed has a radius
of 2.1 cm and a maximum respiration rate of
6 x l o 3 cm3 O2 k g 1 s l . The flesh has an inside
radius of 2.1 cm and an outside radius of 3.6 cm.
The maximum respiration rate of the flesh is set at a
value which gives the desired maximum respiration
rate for the entire fruit including the seed (e.g.
maximum respiration: seed = 6 x l o 3 cm3 0, k g 1
s-l., flesh = 6 x lo-'
cm3 0, kg-'
scl;
fruit = 5 x l o p 2 cm3 O2 kg-' s-l). The skin is
treated as a non-respiring tissue.
The goal of the diffusion model, which begins with
the assumption of a single terminal oxidase having
typical Michaelis-Menten kinetics, is to simulate the
effects of diffusion barriers on the apparent oxygen
isotherm. The model begins at the centre of the fruit
(centre of seed) with a given oxygen concentration
and repeatedly calculates the rate of respiration and
oxygen concentration at 36 equi-distant points
(0.1 cm apart) along the radius of the fruit. The
necessity of beginning at the centre and moving
outward will become apparent as the model is
further described.
The oxygen concentration at the periphery of a
solid sphere, of radius 0.1 cm in the centre of the
fruit (centre of the seed) is calculated using the
diffusion equation for a solid sphere (Eqn 4). The
rate of respiration of this 0.1-cm radius sphere at the
centre of the fruit is calculated using an initial given
oxygen concentration of, for example, l o 5 pmol
cm3
in
the
Michaelis-Menten
equation
[V = Vm[OJ(Km + [O,])], where the maximum rate of
respiration, (Im), is 6 x l o 3 cm3 0, k g 1 s 1 (seed
respiration). The selection of the diffusion coefficient
and Km for 0, in Eqn 4 is discussed below.
The oxygen concentration 0.1 cm from the centre
of the fruit, calculated as just described, is now used
to calculate the approximate respiration rate of a
hollow sphere (shell) of tissue having an inside radius
(4 of 0.1 cm and an outside radius (R) of 0.2 cm.
The respiration rate is calculated using the
Michaelis-Menten equation. The concentration of

122

M. L. TUCKER & G. G. LATIES

oxygen at the outside surface of this hollow sphere,

or shell, is now calculated using the diffusion
equation for a hollow sphere (Eqn 3). The total flux
of oxygen across this hollow sphere (Q) is calculated
by combining the oxygen used (pmol s l ) in the
centre 0.1 cm radius sphere with the oxygen used in
the surrounding hollow sphere. This hollow sphere
calculation is repeated for 34 successive concentric
hollow spheres in which the oxygen concentration at
the surface of the preceding hollow sphere is used to
calculate the rate of respiration of the hollow sphere
under consideration, and the oxygen concentration
at the surface of the latter is calculated using a flux
for oxygen determined by summing the rate of
oxygen use by all the preceding hollow spheres and
the centre sphere. The only exceptions to this series
of calculations are when the radius of the concentric
hollow spheres attains a value of 2.1 cm-the radius
of the seed-at which point the maximum rate of
respiration used in the Michaelis-Menten equation is
changed to that required of the flesh (e.g. 6 x
cm3 0, k g 1 s l ) ; and again, when the radius
attains a value of 3.6 cm, where the skin begins, the
diffusion equation for a hollow sphere (Eqn 3) is
slightly modified (Eqn 5) to use a permeability
coefficient (P, cm s l ) instead of a diffusion
coefficent (D, cm2 s l ) for a skin of an undetermined
thickness. However, although undetermined, the
thickness of the hollow sphere modelling the skin
(Eqn 5) is much less than its radius so that the square
of the inside radius (r2) closely approximates the
product of outside and inside radii (i.e. Rr) used in
Equation 3.
(CI - C2) = [(Q/4nP)(l/r2)l

(skin) (5)

The respiration rate of the hollow sphere which

models the skin is given a value of zero. This
computer model is not an exact solution, but an
attempt to approximate the actual values. It is
assumed that a calculation of the oxygen concentration and respective respiration rates at 36 equidistant steps along the radius of the fruit will closely
approximate the actual continuous change in oxygen
concentration and respiration rate along the radius.
The need to begin at the centre and move outward
is that at each step the rate of respiration is
calculated with the Michaelis-Menten equation,
which requires that one knows the oxygen concentration. The oxygen concentration at any given shell can
be calculated only if the flux across its outer
boundary is known and the latter is equivalent to the
summed rate of oxygen consumption of all tissue
centripetal to that boundary.
The series of calculations starting from the centre
of the fruit and proceeding along the radius toward
the surface is repeated in the program for different
oxygen concentrations at the center of the fruit (e.g.
KT5,
. . ., 200 mmol m-3). This concept is
illustrated in Fig. 4. in which the series of
calculations has been carried out in turn with four
u

10
Rate max = 0-05 cm3 O n k g ' s '

Respiration
rate
Flesh

Skin

(x103) -

Radius of fruit (crn)

Figure 4. Computer-generated oxygen profiles along the radius of

a model avocado fruit respiring at the indicated overall rate
expressed as cm3 0, k g 1 s 1 (see text). The model avocado fruit
has an approximate weight of 0.2 kg. The maximum respiration
rate in saturating oxygen concentrations (e.g. pure oxygen) of the
seed and the flesh are 6 x 1 0 3 and 6 x 1 0 ' cm3 O., kg*' s - '
respectively, which gives rise to a maximum whole fruit rate of
5 x 1 0 ' cm3 0, k g 1 s ' . The model assumes a single oxidase
having an apparent K,,, for oxygen of 3.0 mmol m 3 , and a
diffusion coefficent of 1 . 0 l~ o 3 cm2 s ' for the seed and flesh
and a permeability coefficient of 3.Ox l o 3 cm s 1 for a 0.1-cm
thick skin. The dashed line at 0.25 KPa marks the concentration
of oxygen in the gas phase which is in equilibrium with 3 mmol
m 3 oxygen in water at 25'C.

different concentrations at the center of the fruit. The

result is four distinct oxygen profiles along the fruit
radius, linked to four different oxygen concentrations
at the fruit surface. The respiration rate depicted at
the surface of the fruit is the specific respiration rate
for the entire fruit having the oxygen profile. The K,,,
for O,, the diffusion and permeability coefficients,
and Vm for the seed and flesh are the same for each
profile in Fig. 4. The skin thickness is set at an
approximate value of 0.1 cm in Fig. 4 simply for the
purpose of plotting the data. If these calculations are
repeated for a range of initial oxygen concentrations
at the centre of the fruit, and the surface oxygen
concentration versus whole fruit respiration plotted
for each series of calculations, a respiratory oxygen
isotherm is obtained as shown by any one of the
dashed lines in Fig. 5.
Diffusion and permeability coefficients, and a Km
for oxygen assuming a single respiratory oxidase,

DUAL ROLE O F OXYGEN

0 - 3 rnmol K 3

I
5
Oxygen ( k Pa)

10

15

20

Oxygen ( k P a )

Figure 5. Actual. and computer generated respiratory oxygen

isotherms for avocado fruits of different respiration rates. Elevated
respiratory rates were achieved by ethylene treatment in a period
wherein respiration rate was proportional to time of ethylene
treatment. Measured oxygen isotherms are from a single fruit in a
small-volume closed system maintained at approximately
101 KPa, in which oxygen concentration decreased quickly (solid
lines). Computer generated oxygen isotherms (dashed lines) were
calculated assuming a single oxidase having an apparent Km of
3.0 mmol m 3 . and diffusion coefficient of 1.0 x l o 3 cm2 s 1 for
the seed and flesh and a permeability coefficient of 3.0 x L O 3 cm
s for a 0.1-cm thick skin. The maximum respiration of the seed
remained constant at 5.5 x l o 3 cm3 Oi k g ' s ' in all computer
generated isotherms while the maximum respiration rate of the
flesh varied for each isotherm.

were derived to give the best fit to actual data (Figs 5

& 6). In order to obtain fruit of different restoration
rates in air, fruit were treated with ethylene for
varying intervals. Within a period of approximately
16 h the ethylene induced respiratory climacteric
is roughly proportional to the time of ethylene
exposure (Tucker & Laties, 1984). The diffusion
characteristics of avocado do not change until the
fruit have begun to soften at or near the peak of the
respiratory climacteric (Ben-Yehoshua et al., 1963).
Accordingly, respiratory oxygen isotherms constructed best to fit the computer model were
obtained with firm fruit prior to the respiratory peak.
Once the best fits were found, subsequent calculations simulating hypobaric conditions or treatment
with cyanide were performed using the derived
diffusion and permeability coefficients, these
coefficients being multiplied by 4 at a simulated
u

Figure 6. Comparison of an actual respiratory oxygen isotherm

(solid line) measured in a climacteric avocado fruit with computer
generated oxygen isotherms (dashed lines) assuming a single
oxidase with an oxygen Km of either 0.3, 3.0, or 10.0 mmol m 3 .
Other parameters for the computer model are the same as in
Fig. 4.

ambient pressure of 25 KPa. The Km for oxygen

which gives the best fit to several avocado respiratory
isotherms with different maximum respiration rates
is 3 mmol m 3 . Similarly, the values that give the
best fit to respiratory oxygen isotherms at
atmospheric pressure are a diffusion coefficient of
. O x l o 3 cm2 s 1 for the seed and flesh, and a
permeability coefficient of 3.0 x l o 3 cm s 1 for the
skin. The accuracy of these coefficients and the Km
for the terminal oxidase is further substantiated by
the relatively good fit to actual isotherms obtained at
normal and reduced pressure (Fig. 8).
The apparent K,,, of 3 mmol m 3 derived from the
computer model is that for the surface of an avocado
fruit cell. The diffusion modeling program does not
take into account the effect of diffusion barriers
within the cell (e.g. membranes, cell wall, etc.). The
true Km of the terminal oxidase would be well below
3 mmol m 3 ( < 0.01 mmol m 3 ; see Lloyd &
Edwards, 1977).
Oxygen isotherm predictions using the computer model
Figures 6 and 7 show model predictions for
respiratory oxygen isotherms when diffusion

124

M. L. TUCKER & G. G. LATIES

[oxygen, k Pa]

-'

Figure 7. Double reciprocal plot of respiratory oxygen isotherms

shown in Fig. 6.

coefficients remain constant and the Km of the model

oxidase is varied (i.e. 0.3, 3.0 and 10.0 mmol m 3 ) . It
is obvious that when the K,,, for oxygen of the model
oxidase increases 10-fold from 0.3 to 3.0 mmol m 3
the difference between the two isotherms (Fig. 6) is
not large. This is because in constructing a
Michaelis-Menten curve the true oxygen affinity of
the terminal oxidase is distorted by the large oxygen
concentration gradient across skin and flesh due to
diffusion resistance and oxygen utilization with the
result that the apparent Kmfor 0, is erroneously high
and the shape of the isotherm is ultimately
significantly modified by diffusion kinetics. For
example, assuming a reasonable diffusion resistance,
a 10-fold difference in apparent Km (viz. 0.3
compared with 3 mmol m 3 ) at the cell surface,
would lead to but a two-fold difference in the
external oxygen concentration sustaining a respiration rate of 90% V,,,.,,.

Circulating closed system at normal and reduced

pressure
Figure 8 (solid lines) shows the respiratory oxygen
isotherm for the quick depletion of oxygen by
ethylene-treated fruits measured at an ambient
pressure of 101 and 25 KPa, respectively. As
expected, there is a shift towards lower oxygen
concentrations in the isotherm at 25 KPa compared

Oxygen ( k Pa 1

Figure 8. Measured (solid line) and computer generated (dashed

lines) respiratory oxygen isotherms for climacteric fruit at 101 and
25 KPa ambient pressure. Oxygen isotherms measured at 101 and
25 KPa are from two separate fruits. The apparent K,,, used in the
computer model for both isotherms was 3 mmol m 3 . The
diffusion and permeability coefficients used for the model isotherm
at 101 KPa are those used in Fig. 5. Diffusion coefficients used in
calculation of the 25 KPa isotherms are the foregoing multiplied
by four.

with that at 101 KPa. The model isotherms in Fig. 8

were calculated using the same Km3s.However, a
maximum model respiration rate was chosen for the
flesh in each case to best match the maximum
measured rates, and the diffusion and permeability
coefficients at 25 KPa were taken to be four times
those at 101 KPa. The fit of the computer model
isotherms to the actual data is relatively good,
confirming that the postulated apparent K,,, and
diffusion and permeability coefficients selected for
the computer model constitute reasonable values.

Cyanide resistant respiration

Figure 9 shows the respiratory oxygen isotherms at
25 KPa for two fruit treated for 24 h with 10 cm3
m 3 ethylene (control fruit) and 500 cm3 m p 3
cyanide, respectively. Cyanide at 500 cm3 m 3 in the
gas stream fully inhibits cytochrome oxidase in
whole fruit after a 4-h exposure (Tucker & Laties,
1984). In addition to inhibiting cytochrome oxidase,
cyanide evokes the respiratory climacteric (Tucker &
Laties, 1984). The oxygen isotherms shown in Fig. 9

DUAL ROLE OF OXYGEN

125

cyanide

0 - 3 mmol m-'

0-5

[Oxygen, k Pa]

Oxygen ( k Pa)

Figure 9. Respiratory oxygen isotherms of climacteric avocado

fruit kept at 25 KPa, and exposed to 10 cm3 m 3 ethylene
(control) or 500 cm3 m 3 cyanide. Computer generated oxygen
isotherms (dashed lines) assume a single oxidase having the
specified K,,, for oxygen 0.3, 3.0, 10.0 mmol m 3 . Diffusion and
permeability coefficents used are those in Fig. 5 multiplied by four
to correct for the reduced pressure (i.e. 25 KPa).

were obtained prior to the peak of the respiratory

climacteric and onset of fruit softening. It was
necessary to measure oxygen isotherms at reduced
nressure because as noted above. diffusion barriers
otherwise make it difficult to distinguish between two
oxidases with a relatively high affinity for oxygen
e.g. KLs of 3.8 and 11.5 mmol m 3 for whole yeast
cytochrome oxidase and alternative oxidase, respectively (Henry, de Troostembergh & Nyns, 1977)l.
At reduced pressure the effect of diffusion barriers is
minimized, and it becomes possible to distinguish
between the two oxidases. At a total pressure of
25 KPa treatment of fruit with cvanide. which
selectively engages the cyanide-resistant alternative
oxidase, causes a significant shift of the oxygen
isotherm to high oxygen concentrations.
Figure 10 shows the double reciprocal plot of the
data of Fig. 9. The oxygen isotherm shifts upon
addition of cyanide from one suggestive of a single
oxidase with an apparent Km of 3 mmol m 3 to one
with an apparent K,,, of > 10 mmol m 3 , as judged
by comparison with the double reciprocal plots of
oxygen isotherms generated by the computer model.
Although the double reciprocal plots are not ideally

1.0

1.5

2-0

'

Figure 10. Double reciprocal plot of respiratory oxygen isotherms

shown in Fig. 9.

linear (see also Fig. 7), they indicate that the

cyanide-resistant oxidase does not contribute
appreciably to the uninhibited respiration of
climacteric fruit since the measured oxygen isotherm
in the absence of cyanide conforms to the computer
generated isotherm describing a single oxidase with
an apparent Km for oxygen of 3 mmol m'3.

Concluding remarks
Basis of the biphasic isotherm
The biphasic nature of the respiratory oxygen
isotherm under steady-state conditions, or conditions of slow depletion of oxygen, is neither
diffusion related nor due to the operation of two
terminal oxidases, but rather is ascribable to
cytochrome oxidase mediated respiration in conjunction with regulation of an early stage in respiratory
metabolism by oxygen at relatively high concentrations (apparent Km of
74 mmol m 3 0J.
Apparent Kmlsof 9 and 13 mmol m 3 for slow and
quick oxygen depletion experiments, respectively,
were calculated for cytochrome oxidase from double
reciprocal plots of actual oxygen isotherms at
atmospheric pressure for preclimacteric avocado
fruit. These apparent Km's are diffusion modified
Km's, greater in value than the true Km's. Diffusion
barriers cause a shift in the apparent Km to higher

126

M. L. TUCKER & G. G. LATIES

values, the apparent K,,, being indirectly influenced

by the respiration rate as well. Computer simulation
of the effect of diffusion barriers on the respiratory
oxygen isotherm in avocado fruit predicts that the
apparent Km measured at the surface of a freely
accessible cell for the high affinity oxygen response
would be approximately 3.0 mmol m 3 . This value is
to be compared with an apparent K,,, at the cell
surface of 3.8 mmol m 3 reported for cytochrome
oxidase in yeast (Henry et al., 1977). Thus, it is
concluded that the apparent Km for the high affinity
oxygen response in avocado reflects the affinity for
oxygen of cytochrome oxidase.
Kinetic analysis of respiratory isotherms of
preclimacteric and climacteric avocado fruit both at
25 and 101 KPa in the presence and absence of
cyanide indicate that the cyanide-resistant alternative
path, which has a lower affinity for oxygen than does
cytochrome oxidase (Solomos, 1977; Henry et al.,
1977), does not contribute appreciably to fruit
respiration in the absence of the inhibitor.
Mechanism of
concentrations

the

oxygen

response

at

high

That the effect on respiration of oxygen at high

concentrations requires more than 1.5 h may be
important in understanding the mechanism by which
oxygen regulates respiration other than by way of
the terminal oxidase. In this connection, Blackman
(1954) observed that when apples were removed
from air to 5 KPa oxygen in nitrogen, it took up to
24 h to establish a lower, constant rate of respiration. On the presumption that oxygen inside the fruit
comes to equilibrium with external oxygen quite
rapidly, he proposed that the slowness to respond to
a drop in 0, concentration was due to a slow change
in the level of one or more endogenous substrates or
regulators.
Purpose of the feedback response at high oxygen
concentrations
If the respiration rate of a fruit or large storage
organ is allowed to increase inordinately, or if the
organ grows to an excessive volume, the oxygen
gradient across the skin, flesh, etc., will increase until
the centre of the organ becomes anaerobic, with
attendant fermentation (James, 1953). With an
oxygen responsive negative feedback system such as
pictured in Fig. 2, however, a modest drop in
internal oxygen levels lowers the respiration rate, and
thereby prevents 0, depletion below the level that
sustains cytochromg oxidase activity. This concept is
illustrated for preclimacteric avocado fruit in Fig. 11
in which a hypothetical respiratory CO, isotherm
overlays the measured oxygen isotherm. Respiratory
CO, isotherms reported for apple (James, 1953;
Blackman, 1954) appear very similar to the hypothetical CO, isotherm shown in Fig. 11 for the slow
depletion of oxygen in the environment of an

10

15

20

Oxygen ( k P a )

Figure 11. Overlay of hypothetical respiratory C 0 2 isotherms with

measured respiratory oxygen isotherms shown in Fig. 2 for
preclimacteric fruit. Arrows indicate postulated critical oxygen
concentration at which fermentation would begin at the centre of
the fruit.

avocado fruit. The critical external concentration of

oxygen at which fermentation and an increase in
CO, evolution begin is displaced to a lower value
when respiration is decreased by feedback regulation
at high oxygen concentrations (slow depletion,
Fig. 11).
Another consequence of feedback repression may
be to limit the extent of the ethvlene induced
respiration climax in air in a variety of fruits and
storage organs due to a drop in tissue oxygen
concentration as the respiration rises. When ethylene
is given together with pure oxygen, the normal
constraint on respiration is over-ridden, and the rate
rises to two- and three-fold that in ethylene in air
(Theologis & Laties, 1982). In this case, however, the
high oxygen tension that overrides the constraint
serves in turn to sustain the elevated rate and thus
prevent an anaerobic centre.
Further, feedback repression by diminishing
oxygen concentration may serve to lower the
respiration rate in enlarging fruits or storage organs,
thus avoiding anaerobiosis with increasing size. It is
possible, moreover, that growth is itself suppressed
in consequence of the lowered respiration rate,
thereby doubly insuring against disaster. Avocado
fruit are of particular interest in this regard since cell
division, which may be expected to be accompanied
by a high rate of respiration, occurs throughout
volume growth (Biale & Young, 1971).
Implications of
concentrations

the

oxygen

response

at

high

The theory that oxygen regulates respiration at an

early stage in the respiratory path has important

DUAL ROLE O F OXYGEN

consequences on our understanding of how biosynthesis and respiratory metabolism interact to maintain homeostatic balance. It is the generally accepted
view that biosynthetic requirements dictate the
respiratory rate. This view is exemplified by
Atkinson's energy charge model for regulation of
metabolic processes (Atkinson, 1977). For energy
charge to be maintained respiration (more broadly,
metabolism) must be regulated by the relative
concentration of ATP and one or both of the other
adenylate nucleotides, so that when energy charge
drops below a given high value, respiratory
metabolism is stimulated, whereas when energy
charge increases above the same high value,
respiratory metabolism is inhibited. Nevertheless,
however stringent the control of relative rates of
utilization (biosynthesis) and regeneration (ATP
formation)-U
and R in Atkinson's parlance-by
energy charge, it must be recognized that absolute
rates may vary markedly in a given narrow range of
energy charge as a function of substrate level, for
example (Atkinson, 1977). Thus, an oxygen responsive negative feedback loop may determine respiration rate without changing or abrogating control by
energy charge. Alternatively, if biosynthesis is less
affected than ATP formation by negative feedback,
the cross-over point of U and R curves will be
displaced to a lower energy charge value while
control by energy charge continues.
In this connection it was reported that when
oxygen was diminished in the environment of
germinating lettuce seeds (Raymond & Pradet, 1980)
energy charge dropped concomitantly with a drop in
oxygen use. In excised maize root tips (Saglio,
Raymond & Pradet, 1983) as well, reduced oxygen
concentration was accompanied by a simultaneous
lowering of respiration and the ATPIADP ratio.
These results suggest that under certain conditions in
plant tissues energy charge is not maintained at a
constant value, but rather is either directly regulated
by oxygen concentration, or changes as a consequence of oxygen regulation of respiration rate.
Acknowledgments

The authors wish to thank June Baumer for her

expert assistance with the computer programming.
This work was supported by Research Grant
GM 19807 from the United States Public Health
Service.

127

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