Plant Physiol. (1980) 66, 171-174
0032-0889/80/66/017 1/04/$00.00/0
Comparison between Aging of Slices and Ethylene Treatment of
Whole White Potato Tubers1
Received for publication July 20, 1979 and in revised form March 24, 1980
HARRY W. JANES AND STEVEN C. WIEST
Department of Horticulture and Forestry, Cook College, New Jersey Agricultural Experiment Station, Rutgers
University, New Brunswick, New Jersey 08903
ABSTRACT
Cyanide-resistant 02 consumption can be stimulated by either treating
whole white potato tubers (Norchip) with ethylene, in the presence of 100%
02, or aging slices obtained from untreated potato tubers. A comparison of
alternative pathway activity elicited by either treatment was undertaken.
The proportion of electrons flowing through the alternative path in the
presence of intermediate concentrations of KCN and at various concentrations of salicylhydroxamic acid was identical in both cases. However, the
respiration of slices from ethylene-treated tubers was in every case stimulated by KCN, whereas the aged slices never exhibited this phenomenon.
Furthermore, the metabolism of D-IU-'4Clglucose was several hundred
times greater in aged slices than in fresh slices from C2H4-treated tubers.
These results, along with the respiratory kinetics of aged slices from
ethylene-treated tubers, suggest that aged slices and fresh slices from
ethylene-treated tubers are biochemically dissimilar.
Slices obtained from white potato tubers exhibit a respiratory
rise above that of the whole tuber (11). However, this type of
respiration is not cyanide-resistant (9). Upon aging, these slices
develop an additional respiratory increment and also show the
was monitored using the Warburg manometric technique (8).
The oxidation of exogenously applied glucose was measured as
described elsewhere (7). Two g of slices were placed in 4 ml 100
mm K-phosphate, pH 5.5, with 2 ACi (0.75 x 10-'0 mol) D-[U'4CJglucose. 14CO2 was collected by suspending plastic cups containing 10% NaOH in the flasks. These cups were removed after
15, 30, 60, 90, and 120 min and placed into Hydromix scintillation
cocktail (Yorktown Research, South Hackensack, N.J.), and new
cups containing NaOH were placed in the sample chambers. Rates
of "CO2 evolution were determined by regression analysis of the
resulting time course.
Total glucose was measured as described by Bergmeyer et al.
(2) based on the reduction of NADP by glucose-6-P dehydrogenase. Five g of potato slices were homogenized in 25 ml hot 80%
ethanol. The homogenate was centrifuged at 20,000g for 30 min
and the supernatant was reduced to dryness. The residue was
redissolved in H20 and passed through Dowex H+ and Cl- to
remove ionic materials. The pH of the solution of unbound
material was adjusted to 7. An aliquot of this neutral fraction was
used for the analysis of glucose (2). Experiments were repeated at
least two times.
RESULTS AND DISCUSSION
Figure I demonstrates that exposure of fresh slices to concentrations
of KCN between 0.01 and 0.1 mm resulted in varying
development of an alternative, cyanide-resistant pathway (5).
Recently (3, 8), it was demonstrated that fresh slices obtained amounts of inhibition, with intermediate inhibition shown at 0.03
from potato tubers previously treated with C2H4 in air or 100%0 02 mM. Slices which exhibited cyanide-resistant respiration were not
would possess the alternate CN--insensitive respiration prior to inhibited by cyanide alone, as was shown previously (5, 8). However, in the presence of SHAM2, the inhibitory effect of various
any aging.
To gain a more complete understanding of the development of cyanide concentrations was similar to that of fresh slices in the
CN- resistance under these two conditions (i.e. C2H4 treatment in presence or absence of SHAM (data not shown). Therefore, 0.03
100%0 02 or aging), this study was undertaken. It is apparent that, mM KCN was used as an "intermediate" concentration and 0.1
although alternative pathway activity is the result of both treat- mm was assumed to be maximally inhibitory (10) for the purposes
ments (3, 5, 8), the metabolic processes stimulated by them are of this study. Finally, Figure I demonstrates that I mm SHAM in
the absence of KCN has no inhibitory effect on slices.
different.
Figure 2, A to D, presents the inhibition of 02 uptake by various
concentrations of SHAM in the presence of 0.03 or 0.1 mm KCN.
MATERIALS AND METHODS
Several marked differences in the response of the slices to SHAM
+
KCN can be noted. Slices freshly obtained from untreated
Potatoes (Solanum tuberosum cv. Norchip) were stored at 7 C
for about 4 months prior to use. The tubers were preconditioned tubers show no sensitivity to SHAM even in the presence of 0.1
at room temperature for 10 days before treatment. Whole tubers mM KCN (Fig. 2A). However, SHAM sensitivity appears to
were stored in 4-liter jars continuously flushed with 10 ,ul/l C2H4 develop upon aging of slices from these tubers for 24 h (Fig. 2B).
in 100% 02 or 100%0 02 alone for 24 h prior to slicing and Aged slices also appear to be somewhat sensitive to KCN even in
respiratory determinations (8). Respiration of fresh slices was the absence of SHAM, although a much lower percentage of the
always determined within 3 h of slicing. Alternatively, slices were total 02 uptake is inhibited solely by KCN in aged slices. The
cut from whole tubers and aged in 0.1 mM CaSO4 for 24 h prior
2
to making respiratory measurements. Respiration of potato slices
Abbreviations: SHAM: salicylhydroxamic acid; V,v,: velocity of 02
uptake attributable to cytochrome oxidase; V.,,: velocity of 02 uptake
' Paper of the Journal Series, New Jersey Agricultural Experiment
attributable to the alternate oxidase; V,..: velocity of 02 uptake not
Station, Cook College, Rutgers University, New Brunswick, New Jersey.
attributable to cytochrome oxidase or the alternate oxidase.
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Plant Physiol. Vol. 66, 1980
JANES AND WIEST
172
2B) in that these fresh C2H4-treated slices are sensitive to SHAM
in the presence of KCN and both the basal respiratory rate and
residual respiration (about 20 ,ld 02/g. h) are quantitatively similar
to that observed in aged slices from untreated tubers. However,
unlike the response of aged slices, KCN alone appeared to enhance
pC'
the uptake of 02 in slices prepared from C2H4-treated potatoes.
cew
Aged slices from tubers which had been treated with C2H4 + 02
(aged C2H4-treated; Fig. 2D) possess properties dissimilar to both
aged and fresh C2H4-treated. The basal respiratory rate of about
78 ,u 02/g. h was significantly higher than the values obtained for
both aged and fresh C2H4-treated slices. Likewise, the residual
0
.01
.1
respiration of aged C2H4-treated slices (about 34 t1l 02/g. h) was
.0001
.001
substantially
higher than that observed in any other treatment.
CN CONCENTRATION
Addition of KCN in the absence of SHAM appeared to inhibit
(MM)
slightly the 02 uptake rate in aged C2H4-treated slices, as was
FIG. 1. The effect of various CN- concentrations in the presence (0) observed in aged slices. The stimulation of 02 consumption by
or absence (0) of I mM SHAM on the respiration of fresh slices from KCN observed in fresh C2H4-treated slices was not apparent in
aged C2H4-treated slices.
untreated potatoes.
It was reported earlier (6) that aged slices do at times show a
basal respiratory rate (in the absence of inhibitors) of aged slices stimulation due to CN- treatment. However, we have never noted
was about twice that of fresh slices, which confirms previous this phenomenon in aged slices of this cultivar. It has been
reports (10). In addition, the residual respiration (02 consumption speculated (3) that the increase in respiration due to CN- could
not inhibited by 0.1 mm KCN + I mm SHAM) was 19,u1 02/g occur by the Pasteur effect if the energy charge is altered. It may
tissue h in aged tissue, nearly twice the value of 10 Al 02/g h also be possible that CN- is acting to stimulate an oxidative
observed in untreated slices. The response of slices prepared from enzyme system. It has been reported that 02 uptake in human
tubers treated with C2H4 + 02 for 24 h prior to slicing (fresh C2H4- polymorphonuclear leukocytes is stimulated by CN- by way of
treated; Fig. 2C) superficially resembles that of aged slices (Fig. stimulating a peroxidase-like enzyme system which oxidizes
40
~~~~~[KC
~
_
40~0
N]
=,\
401
~~~~~~~~
-020
0
*
*
.03
*
._
W
*
*
*
*
A .1 OmM
CL °
00)
80
C
DAGED ETHYLENE
FRESH ETHY LENE
40
.2
.4
.6
.8
1.0
0
SHA M
(mM)
.2
.4
.6
.8
.o
FIG. 2. Effect of various SHAM concentrations in the presence of 0 (Ol), 0.03 mM (), or 0.1 mM (*) KCN on the respiration of potato slices. Each
graph presents the respiration of the following types of slices: A, fresh slices; B, aged slices; C, fresh slices from C2H4-treated tubers; and D, aged slices
from C2H4-treated tubers.
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AGING OF POTATO SLICES AND ETHYLENE
Plant Physiol. Vol. 66, 1980
NADPH (4). The data presented here indicate the stimulation in
the respiration of slices by KCN from C2H4-treated potatoes is
mainly due to increased Vast, whereas the V,yt increased only
slightly and V, remained similar to that in aged slices.
Bahr and Bonner (1) devised a component analysis for distinguishing between CN-sensitive and CN-insensitive respiratory
pathways in isolated mitochondria. Their analysis consisted of
fitting data to the model represented by the equation:
Vt = p g(i) + Vcyt
g(i) + V,yt
[2]
where Vres represents this third category of 02 consumption. This
model, which contains several assumptions that have yet to be
experimentally confirmed, is nevertheless a useful analytical tool
by which the various treatments can be compared. This analysis
provides a method for determining the potential of the slices to
respire via a CN-resistant pathway, but it is unable to separate the
contribution of the two pathways when respiration is not inhibited.
Figure 3 (A-C) shows such an analysis based on titration with
SHAM of slice respiration in the presence of partially or totally
inhibitory KCN concentrations.
Figure 3, A to C, represents our data fitted to the model given
by equation 2. The linearity of the data (correlation coefficients
for aged and fresh C2H4-treated slices are significant at P = 0.01
and that for aged C2H4-treated slices is significant at P = 0.05)
suggest that all 02 uptake can be accounted for by the three
categories of 02 utilization inherent in equation 2. Table I demonstrates that the values of p and V,. are essentially identical for
aged and fresh C2H4-treated slices. However, Vcy, was elevated
above that for fresh slices. The value of V,y, in aged C2H4-treated
slices was even slightly greater than that in the other treatments.
If an increased value for Vcyt indicated solely a greater flux of
electrons through the respiratory chain prior to the branch point,
then p should increase as well (assuming that, once the Cyt
pathway was saturated, excess electrons would be shunted to the
alternative pathway). This was observed in the cases of aged and
fresh C2H4-treated slices. However, aged C2H4-treated slices elicited a larger V,y, and a smaller p than the corresponding values
for aged and fresh C2H4-treated slices. Several possible explanations exist to account for this observation. First, a third type of
respiratory pathway (not inhibited by KCN or SHAM) may
develop more fully when slices from C2H4-treated tubers are aged.
This would account for the elevated value of Vres. However, this
alone would not explain the altered values of p and V,y, An
alternative explanation would be that the capacity of the cytochrome pathway is altered so that it possesses the capacity to
handle an increased flux of electrons, and the ratio of the current
in the alternative pathway to that in the Cyt pathway is reduced.
This would account for the altered values of V,,, and p but not for
the increased V,,. The data can be explained only by invoking a
more complex scheme, such as a combination of the above two
possibilities. Nevertheless, the data do indicate that the kinetics of
02 uptake in aged C2H4-treated slices is unlike that of aged slices
=
0.615g(i)+
13.3
r= 0 923
20-
0
60-
F
B
re
sh C2
H4
40
Vt' =0.654g(Si)+
20
19.6
o.919
~~~~~~~~~~r=
0
= p
fresh C2H4-treated slices.
In a further effort to compare 02 uptake in aged and fresh
C2H4-treated slices, the metabolism of ['4C]glucose was investi-
or
Vt'
40
p
tration; g(i) is the rate of 02 uptake when the Cyt pathway is
completely inhibited as a function of i, the concentration of
alternate oxidase inhibitor; and V,,, represents the rate of 02
utilization by Cyt oxidase at a given KCN concentration. Theologis and Laties (10) recognized a third category of 02 consumption observable in tissue slices which was insensitive to classical
inhibitors of either Cyt oxidase or the alternate oxidase and
appropriately modified equation 1 to the following form:
Vt = Vt- Vres
A ged
A
Ill]
represents the capacity
Of 02 utilization via the alternate oxidase at a given KCN concen-
where: V, is the total rate of 02 uptake;
173
0
C2H4
Aged
C
40
Vt
20-
' =
0.316
r=
0
I10
30
20
40
g(i)+
23.5
o. 760
50
60
9(i)
FIG. 3. V,' as a function of g(i) in slices from the sources noted in the
figure. g(i) was derived from Figure 2. V' and g(i) are in,l 02/fresh weight.
h.
Table I. Kinetic Parameters of Respiration as Calculatedfrom Figure 3
V,
Treatments
p
V,Yt
ILI 02/g. h
0 aa
9.7
Fresh
7.9 a
18.6
0.61 c
13.3 ab
Aged
0.66 c
18.7 bc
19.6
C2H4
0.32 b
23.5 c
35.0
Aged, C2H4
aMeans within a column followed by the same letter are not significantly
different according to Duncan's New Multiple Range Test at P = 0.05.
gated (Table II). It is immediately obvious that glucose is utilized
at least 2 orders of magnitude more rapidly in aged and aged
C2H4-treated slices than in fresh or fresh C42H-treated slices.
Concomitant with increased utilization was an increased uptake
of labeled glucose by aged and aged C2H4-treated slices. This may
have been due to a differential permeability of the membranes to
glucose or due to the increased utilization causing a greater intraextracellular concentration gradient. In either case, the conclusion
that aged slices are different than C2H4-treated slices remains
valid. The interpretation of this data also requires several additional assumptions, such as (a) all glucose taken up entered a
utilizable pool, (b) all endogenous glucose was utilizable, and (c)5
the formation of glucose during the course of the experiment was
negligible. The data of Table II represent minimal values for the
release of C02/unit 02 uptake. Nevertheless, aged slices oxidize
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174
JANES AND WIEST
Table II. Metabolism of D-[U-'4CCGlucose by Potato Slices Treated as
Indicated
Glucose Oxidation
CO2
Treatments
Released
% Up-
take
Endogenous
Glucose
C02 Releaseda
CO2 Released'/
02 Taken
Up
cpm/g h
Fresh
96
19.5
Aged
93
31,503
C2H4
162
21
Aged, C2H4
95
36,630
a C02 derived from glucose.
umol/g
4.8
9.0
0.9
7.4
pmol/g- h ,umol/ymol
0.51
0.40
65.52
25.1
0.15
0.54
61.38
18.0
Plant Physiol. Vol. 66, 1980
activity in response to C2H4 may lead to the development of
enhanced respiration (9). Aging does, in fact, result in the increased ability to metabolize glucose regardless of previous treatment of the tubers. However, although C2H4 treatment results in
an increase in respiration, fresh C2H4-treated slices behave similarly to fresh slices from the standpoint of glucose uptake and
utilization.
The development of cyanide-resistant respiration is an interesting, albeit confusing, phenomenon. The recent demonstration that
C2H4 treatment of tubers induces its development, as does the
aging of slices, does not mean that the two processes are synonymous. Rather, our data suggest that the substrate for CN-resistant
respiration in slices from C2H4-treated tubers may be different
than that for CN-resistant respiration in aged slices.
LITERATURE CITED
glucose at a much greater rate than fresh slices. The purpose of
this study was to compare fresh slices from C2H4-treated tubers
with aged slices from untreated tubers. For this purpose, confirmation of the above assumptions is not necessary. Rather, any
differences in the amount of 14CO2 released will demonstrate a
difference between the tissues. Such differences between fresh and
aged slices were observed (Table II). Whether the observed differences were actually due to differences in glucose metabolism or
due to the above assumptions being incorrect will not be addressed
in this paper. However, it is interesting to speculate on the cause
of the observed differences if the above assumptions are valid.
Table II shows, as well as previous studies (7), that aging of slices
results in the increased ability to metabolize glucose. C2H4 treatment does not induce an increased glucose metabolism even
though O2 uptake is dramatically increased. Thus, it appears that
the aging process is quite distinct from the response of tubers to
C2H4.
C2H4 treatment of whole potato tubers is not synonymous with
aging of slices (Table II). Each process leads to CN-resistance, but
the metabolic source of this resistance may differ. Substrate mobilization via increased glycolytic and tricarboxylic acid cycle
1. BAHR JT, WD BONNER JR 1973 Cyanide-insensitive respiration. 1. The steady
states of skunk cabbage spadix and bean hypocotyl mitochondria. J Biol Chem
248: 3441-3445
2. BERGMEYER HV, K BRENDT, F SCHMIDT, H STORK 1974 Determination of
glucose. In HU Bergmeyer, ed. Methods of Enzymatic Analysis, Vol 3.
Academic Press, New York, pp 1196-1201
3. DAY DA, GP ARRON, RE CHRISTOFFERSEN, GG LATIES 1978 Effect of ethylene
and carbon dioxide on potato metabolism. Plant Physiol 62: 820-825
4. DECHATELET LR, LC MCPHAIL, PS SHIRLEY 1977 Effect of cyanide on NADPH
oxidation by granules from human polymorphonuclear leukocytes. Blood 49:
445-454
5. HACKETT DP, DW HAAS, SK GRIFFITHS, DJ NIEDERPRUEM 1960 Studies on the
development of cyanide-resistant respiration in potato tuber slices. Plant Physiol 35: 8-19
6. LANGE H 1970 Respiratory pathways in subein-synthesizing and proliferating
potato tuber tissue after derepression. Planta 90: 119-132
7. LATIES GG, C HOELLE 1965 Malonate and cyanide insensitivity in relation 1o
respiratory compensation in potato slices. Plant Physiol 37: 757-764
8. RYCHTER A, HW JANES, C FRENKEL 1978 Cyanide-resistant respiration in freshly
cut potato slices. Plant Physiol 61: 667-668
9. SOLOMos T 1977 Cyanide-resistant respiration in higher plants. Annu Rev Plant
Physiol 28: 279-297
10. THEOLOGIs A. GG LATIES 1978 Relative contribution of cytochrome-mediated
and cyanide-resistant electron transport in fresh and aged potato slices. Plant
Physiol 62: 232-237
11. VAN STEVENICK RFM 1975 The "washing" or "aging" phenomenon in plant
tissues. Annu Rev Plant Physiol 26: 237-258
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