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ARRHENIUS PLOTS OF MITOCHONDRIALRESPIRATION IN PIMA COTTON VARIETIES
OF DIFFERING TEMPERATURE TOLERANCE.
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Centner, Michael Stephen
ARRHENIUS PLOTS OF MITOCHONDRIAL RESPIRATION IN PIMA COTTON VARIETIES OF DIFFERING TEMPERATURE TOLERANCE
The University of Arizona PH.D. 1982
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ARRHENIUS PLOTS OF MITOCHONDRIAL
RESPIRATION IN PIMA COTTON
VARIETIES OF DIFFERING
TEMPERATURE TOLERANCE
by
Michael Stephen Centner
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF PLANT SCIENCES
In Partial Fulfillment of the Requirements For the Degree of
DOCTOR OF PHILOSOPHY WITH A MAJOR IN AGRONOMY AND PLANT GENETICS
In the Graduate College
THE UNIVERSITY OF ARIZONA
1 9 8 2
THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE
As members of the Final Examination Committee, we certify that we have read
the dissertation prepared by Michael Stephen Centner
entitled Arrhenius Plots of Mitochondrial Respiration in
Pima Cotton Varieties of Differing Temperature
Tolera'nce ,
and recommend that it be accepted as fulfilling the dissertation requirement
for the Degree of Doctor of Philosophy
D~ /]
I
Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.
D1ssertat10n D1rector
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements fo~ an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission of extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
This dissertation is dedicated to my parents
Richard Leo Centner and Margaret Josephine
Hanlon Centner, whose love, patience and
support are seemingly unlimited.
iii
ACKNOWLEDGMENTS
First and foremost, the author wishes to thank God,
the Almighty, the Most Merciful, for His grace and blessing
throughout the course of this study.
The author wishes to thank the State of Arizona, and
The University of Arizona for the financial support allowing
graduate study at a. superior research institution.
The author wishes to thank his advisor and mentor,
Dr. Robert G. McDaniel for his guidance and direction
throughout the course of this study.
The author wishes to thank his dissertation committee
for their. input into this study and the suggestions leading
to this manuscript's final form.
Special acknowledgment goes to Sarah Oordt who typed
this dissertation and Sedley ~. Josserand who drew the figures
PERCOLL GRADIENT PURIFICATION OF MITOCHONDRIA ••••
ARRHENIUS PLOTS OF ACALA 1517-D MITOCHONDRI~ ••••
. . . . . . . . . . . . .
vi
Page
28
28
30
31
35
37
41
67
69
72
77
Figure
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
LIST OF ILLUSTRATIONS
The fluid-mosaic model of membrane structure. Based on Singer and Nicolson.. . . . . . . . . . . . .
Pima E-14 and Pima S-5 lint yield vs. a1tit;ude. Adapted from data supplied by c. A. Feaster · · · · Typical oxygraph trace of mito-chondrial respiration · · · · · · Arrhenius plot of Pima E-14 state 3 mitochondrial respiration. First experiment · · · · · · · · · Arrhenius plot of Pima E-14 state 4 mitochondrial respiration. First experiment · · · · · · · · · Arrhenius plot of Pima E-14 state 3 mitochondrial respiration. Second experiment · · · · · · · · Arrhenius plot of Pima E-l4 state 4 mitochondrial respiration. Second experiment · · · · · · · · Arrhenius plot of Pima E-14 state 3 mitochondrial respiration. Third experiment · · · · · · · · · Arrhenius plot of Pima E-14 state 4 mitochondrial respiration. Third experimen:t · · · • · · · · · Arrhenius plot of Pima E-l4 state 3 mitochondrial respiration. Fourth experiment · · · · · · Arrhenius plot of Pima E-14 state 4 mitochondrial respiration. Fourth experiment · · · ·
vii
Page
· . . . . . . 4
· · · · · · · 14
· · · · • · · 26
· · · · · · · 42
· · · · · · · 43
· · · · · · · 44
· · · · · 45
· · · · · · · 46
· · · · · · · 47
· · · · · · · 48
· · · · · · · 49
LIST OF ILLUSTRATIONS--Continued
Figure
12. Arrhenius plot of Pima S-5 state 3 mitochondrial respiration. First experiment • • • • • • • • . . . . . . . .
13.
14.
15.
16.
17.
B1.
B2.
B3.
Arrhenius plot of Pima S-5 state 4 mitochondrial respiration. First experiment • '. • .' • • • •
Arrhenius plot of Pima 8-5 state 3 mitochondrial respiration. Second experiment • • •
Arrhenius plot of Pima S-5 state 4 mitochondrial respiration. Second experiment • • •
Arrhenius plot of Pima S-5 state 3 mitochondrial respiration. Third experiment • • • • • • • •
Arrhenius plot of Pima S-5 state 4 mitochondrial respiration. Third experiment • • • • • • • • •
Arrhenius plots of the respiration of isolate mitochondria from seea1ings of Aca1a cotton (Grosspium hirsudum L.)
Densitometer tracing o,f representative stained electrophoresis gels of the soluble seed proteins extracted from Pima S-5 and E-14 •
Densitometer tracings of representative 'electrophoresis gels of soluble seed proteins of Plrna S-5 and E-14 incubated with esterase specific enzyme stain .' •••••••••••
. . . . . . . .
viii
Page
50
51
52
53
54
55
74
75
76
Table
1.
2.
3.
4.
5.
6.
7.
8.
AI.
LIST OF TABLES
Correction factors used to account for increased O2 solubility and decreased oxygen probe sensitivity • . . . . . . . . . . . Germination and growth of Pima S-5 and Pima E-14 following incubation at l8C for six days • • • • • • • • . . . . . . . . . . The increase of Pima S-5 and Pima E-14 mitochondrial State 3 respiration with increasing temperature • • • • • • • • •
The increase of Pima S-5 and Pima E-14 mitochondrial State 4 respiration with increasing temperature • • • • • • • • •
A comparison of the difference between mean State 3 and State
. . . . . . . . .
4 mitochondrial respiration rates of Pima S-5 and Pima E-14 at each assay temperature • • • • • • •
A comparison of the respiratory efficiency index of Pima S-5 and Pima E-14 at each assay temperature
The increase of Pima S-5 and Pima E-14 mitochondrial ADP:O ratio with increasing temperature • • • •
The increase of Pima S-5 and Pima E-14 mitochondrial respiratory control with increasing temperature
Mean respiratqry control ratios of : gradient purified Pima S-5 mitochondria • • • • •
ix
Page
22
30
31
32
33
34
36
38
71
Plate
1.
2.
LIST OF PHOTOGRAPHIC PLATES
Urea soluble proteins stained with coomassie blue G-2S0 • • • • • •
Means followed by the same letter are not significantly different at the .05 level according to a Duncans Multiple Range Test.
the oxidation of the products of glycolysis, as well as fatty
acids and amino acids [46], and the anabolic processes of ATP
and the reduction of oxygen to water. If one assumes that
the increase in respiration from state 4 to state 3 is respi-
ration required to phosphonylate ADP to ATP then the mito-
chondrial respiration can be partitioned by subtracting the
state 4 respiration rate from the state 3 respiration rate.
If this is done using the data of mean E-14 and S-5 respira-
tion rates,it is found that the differences are approximately
equal (Table 5). However, if these differences are expressed
33
Table 5. A comparison of the difference between mean State 3 and State 4 mitochondrial respiration rates of Pima S-5 and Pima E-14 at each assay temperature.
Assay Temp. Pima.E-14 Pima S-5 (e) .. llMo2/min/g
6 2.3 1.4
8 3.1 2.9
10 4.0 4.2
12 5.3 6.0
14 7.1 7.6
16 10.4 8.7
18 9.9 11.4
as a percentage of state 3 respiration according to the
equation:
state 3 - state 4 x 100 state 3
then a respiration efficiency index in percent can be gen-
era ted. Table 6 shows both Pima E-14 and Pima S-5 respira-
tion efficiency indices for each assay temperature. The
higher the index, the greater proportion of oxidative
metabolism available for ATP synthesis. At each assay temp
erature the Pima E-14 is more efficient than the Pima S-5.
34
Table 6. A comparison of the respiratory efficiency index of Pima S-5 and Pima E-14 at each assay temperature.
Assay Temp. Pima S-5 (%) Pima E-14 (%) C
6 17.3 36.3
8 28.2 38.0
10 30.4 39.8
12 35.4 43.5
14 38.7 47.8
16 38.5 53.0
18 37.7 47.2
The Pima E~14 has an efficiency of 36.3% at 6C while at the
same temperature Pima S-5 showed an efficiency of 17.3%. At
the highest assay temperature, l8C, the Pima E-14 respiration
efficiency was 47.2% while the Pima S-5 respiration effi
ciency 37.7%. It should be noted that the respiration ef- o
ficiency of Pima S-5 mitochondria increases more" rapidly than
the respiration efficiency of the Pima E-14 mitochondria
This trend might continue so that at the high temperatures
at which the Pima S-5 is adapted its mitochondrial respira
tion efficiency could be expected to equal or exceed that of
the Pima E-14.
35
ADP:O Ratios
The ADP:O ratios db not increase significantly with
each increase in temperature but rather increase in steps,
remaining stable over a range of temperatures and then in
creasing significantly with the next increase in temperature.
These steps are evident in both germplasm although statis
tically more distinct in the Pima S-5 (Table 7).
At each assay temperature the Pima E-14 mitochondrial
ADP:O ratio is numerically greater than the Pima S-5 mito
chondrial ADP:O ratio, and significantly greater at all
temperatures except 6, 14, and l6C. The Pima E-14 exhibit
lower respiration at an assay temperature than does Pima S-5,
and it synthesises more ATP per unit oxygen, as indicated by
the ADP:O ratios. This 'reflects a greater immediate meta
bolic efficiency for the Pima E-14 compared to the Pima S-5.
It is commonly accepted that ADP:O ratios have a
maximum value of 4 [46]. However the Pima E-14 exhibits an
ADP:O ratios greater than 4 at l4C and values approaching 4
at 16 and laC. A possible source of these values is in the
correction factors ~sed in accounting for the increased solu
bility of oxygen wi~h decreasing temperatures. These were
calculated using the solubility of 02 in H20 and not in the
Reaction buffer, which contains solutes that would act to de
crease this solubility. Since the same correction factor was
used for both cotton varieties, deviations from absolute
36
Table 7. The increase of Pima S-5 and Pima E-14 mitochondrial ADP:O ratio with increasing temperature.
Assay Temp. (e)
6
8
10
12
14
16
18
Pima S-5
2.49 a
3.01 ab
3.12 b
3.05 b
3.62 c
3.58 c
3.60 c
Pima E-14
2.93 a
3.60 bc
3.65 bc
3.59 b
4.08 c
3.98 bc
3.93 bc
Means followed by the same letter are not significantly different at the .05 level according to a Duncans Multiple Range Test.
values, which may have resulted from the correction factors,
would have had no effect on the comparison of parameters
between the two varieties.
The ADP:O ratios obtained in this study are consider
ably better than those obtained by Killion [42], who reported
an ADP:O ratio of 1.03 for mitochondria extracted from Rex
glandless cotton using aketoglutarate as a substrate.
Lee [44] has stated that both lipids and proteins
show movement in membranes and that the nature of the lipids
surrounding an enzymatic protein is important in determining
37
its activity. He suggests that this relationship might ex
plain the selective loss of enzymatic activity with decreasing
temperature. Lipid is required for ATPase activity [43] and
if these lipids are not homogeneous as to degree of unsatura
tion or c~in length, the stepwise increase in ADP:O ratios
demonstrated in this study (Table 7) could be explained by
slight differences in the temperature of membrane phase change
of the ATPase associated lipids.
Respiratory Control Ratios
As with ADP:O ratios, the increases are not signifi
cant with each temperature increase but occur in statistically
distinct groups (Table 8). With both varieties the respira
tory control ratios increase with temperature to l4C. At
this temperature and above the respiratory control ratios
remain constant.
As with ADP:O ratios, the Pima E-14 respiratory con
trol ratios are greater than those for Pima S-5, averaging
1.79 for Pima E-14 and 1.53 for Pima S-5. At each assay
temperature the Pima E-14 respiratory control ratio is sig
nificantly greater than the Pima S-5 respiratory control
ratio •.
The respiratory control ratios for both varieties of
cotton were relatively low, ranging from 1.19 to 1.65 for
Pima S-5 and from 1.58 to 1.96 for Pima E-·14. Schmidt [69]
38
Table 8 •. The increase of Pima S-5 and Pima E-14 mitochondrial respiratory control with increasing temperature.
Assay Temp. (C)
6
8
10
12
14
16
18
Pima S-5
1.2 a
1.4 b
1.5 bc
1.6 cd
1.6 d
1.6 d
1.6 d
Pima E-14
1.6 a
1.6 a
1.7 ab
1.8 b
1.9 c
2.0 c
1.9 c
Means followed by the same letter are not significantly different at the .05 level according to a Duncans Multiple Range test.
reported a respiratory control ratio of 2.55 for cotton mito-
chondria isolated from cotyledons. One cause of this dif-
ference may be the difference in assay temperatures.
Schmidt assayed mitochondria at 27C rather that at the 6 to
18C range in this-study. Another factor is that the Schmidt
study used glandless cottonseed (R. G. McDaniel, personal
communication, 1982). Glandless cottonseed was utilized to
avoid the high gossypol normally present in cotton cotyle
dons. Gossypol is the po1ypheno1ic known to uncouple mito
chondria when present during extraction [55]. This project
39
used portions of the radicle tissue of glanded seed selected
to be relatively free from gossypol but may have contained
cells with enough to cause some loss of respiratory control.
The respiratory control ratios from the Pima E-14 are
in general significantly higher at all assay temperatures
than those of Pima S-5. This is a reflection of the lower
state 3 and state 4 respiration rates of Pima E-14 compared
to Pima 5-5. McDaniel [54] in a study of wheat (Triticum
aestivum L.) noted the same relationship of respiration rates
to respiratory control. One variety, "Cajame" exhibited .
greater respiratory control ratios and ADP:O ratios than the
"Jori", (Triticum durum L.).
This increase in respiratory control ratio with a~.
decrease in respiration rate may be a result of a simple
mathematical relationship, namely that the ratio between
the numbers with a given difference is inversely related
to the size of the numbers (the law of relative change).
Thus, as the difference between state 3 and state 4 respira-
tion rates for Pima E-14 and Pima S-5 is ?lPproxima;te1y the
same the E-14 variety, which exhibits lower respiration
rates will have a greater respiratory control ratio. This
raises the possib1ity that the-Pima S-5 respiration rates
are greater because of uncoupling, a phenomenon that is
characterized by a stim~lation of oxygen uptake by in
tact mitochondria in the absence of ADP [46], with a
40
lowering or loss of respiratory control and ADP:O ratios [29].
If this were true, then the differences in respiration param
eters would be artifactual and not a true reflection of the
in vivo respiratory efficiency of the two cotton varieties.
However, given the fact that the extraction procedures were
identical for both varieties, and the germination perform
ance of the Pima S-5 was poor compared to Pima E-14, it seems
reasonable to consider the respiration data represent a valid
estimation of the respiratory efficiency of the two varieties.
In a recent paper, Kiener and Bramlage [40], at
tempting to explain the post-chilling respiratory burst
exhibited by many chilling sensitive plant species, describe
an alternate, non cytochrome mediated respiratory pathway in
Cucumis sativ.a. When ~hey investigated the direct effect of
temperature on the respiration of hypocotyl sections they re
ported a slight change in the QlO of uninhibited oxygen up
take between 15 and 20C. Within this same temperature range
they noted a distinct change in the response of respiration
to two respiration inhibitors, SHAM (Salicyl-hydroxamic acid)
and KCN. At temperatures below the change in uninhibited
oxygen uptake Ql the sensitivity of the respiration to KCN
decreased. Kiener and Bramlage propose that membrane phase
change is responsible for a severe restriction of membrane de
pendent cytochome mediated respiration and that the alterna
tive pathway is compensatory for this restriction. Goldstein
et a1. [32] has demonstrated with wheat that gradient puri
fied mitochondria do not exhibit cyanide insenstive respri
ation and that SHAM inhibited 1ipoxygenase activity.
Lipoxygenase is responsible for the peroxidation of polyun
saturated fatty acids [5], and Simon [71] has stated that
41
.. high oxygen tensions increase membrane permeability due to
increased preoxidation of fatty acids by 1ipoxygenase. This
evidence taken together suggests that mitochondrial respira
~ion is severely restricted at temperatures below that at
which membrane phase change takes place and oxygen uptake
below that temperature is in part due to l;:poxygenase ac
tivity. The greater efficiency of Pima E-14 mitochondrial
respiration compared to Pima S-5 mitochondrial respiration at
low temper.atures might then be due to a l:esser 1ipoxygenase
activity in the Pima E-14 compared to Pima S-5.
Arrhenius Plots
The Arrhenius plots shown in Figures 4 through 17
represent the results of individual experiments run on
separate days and analyzed according to the procedure given
in the "Materials and Methods" section.
Experiment 4 of the Pima S-5 mitochondrial respira
tion versus- temperature was run at only six temperatures and
would arbitrarily have been broken into two three point seg
ments by the procedure outlined in the IIMateria1s and Methods".
1.8
1.5
.Q)
~ a.. , 1.2
C o . ..,. "S a.. .9 . ..,. ~ CI) Q) a..
Ol o ,.....
.a
.3 Break Point = 9.9°C
o Y - -a.98x + 1.71
6 y - -3.79x + 1.39
42
o .03 .oa .09 .12 .18
~ T
Figure 4. Arrhenius plot of Pima E-l4 state 3 mitochondrial respiration. First experiment.
43
1.8
Q.) 1.5
..... «S 1-4
C 1.2
0 ..... ..... «S 1-4 .9 ..... Q.. CIJ Q.) 1-4
" .6 Ol 0 ~
.3
o . 03 .06 . .12 .15
Figure 5. Arrhenius plot of Pima E-l4 state 4 mitochondrial respiration. First experiment. No break point calculated.
Q)
"S 1-04
C 0 .~
-S 1-04 .~
~ ~ t:n 0 ......
1.8
1.5
1.2
.9
.6
.3
o
Break Point - 10.86°C
o Y - -7.5)( + 1.7
6 y = -2.14x "" 1.225
.03 .06 .09
~
44
~12
Figure 6. Arrhenius plot of Pima E-l4 state 3 mitochondrial respiration. Second experiment.
Q) ..... «S a.. C 0 ..... is a.. ..... Q.. CIJ Q) a.. Ol 0 ......
1.8
1.5
1.2
.9
.6
.3
o
Break Point = 11.9°C
o Y = -8.52)( + 1.51
8 Y = -0.75x + 0.86
.03 .06
45
:12 .15 .18
Figure 7. Arrhenius plot of Pima E-l4 state 4 mitochondrial respiration. Second experiment.
Q)
~ $.t
C 0 ..... ... ctI $.t ..... c.. CIJ Q) $.t
t:n 0 ......
1.5
1.2
.9
.s
.3 Break Point = 11.23°C
o y,. e12.2x .... 1.98
6 y. "1.4Sx... 1.03
46
o .03 .OS .12 35 :18
Figure 8 •. Arrhenius plot of Pima E-14 state 3 mitochondrial respiration. Third experiment.
c o ..... e ..... Q.. (IJ a> .,.. t:n o ......
1.8
1.5
oS
.3
o
47
Bre2k Point = 10.00C
0 y- -7.2x ... 1.36
6 y= -.38x ... .68
.03 .06 .15 .. 18
Figure 9. Arrhenius plot of Pima E-l4 state 4 mitochondrial respiration. Third experiment.
Q) ..... ItS $001
C 0 .-S .9 .-0. (J) Q) $001 .6
Ol 0 -
t:J y - -5.27)( -t 1.62
o .03 .06
Figure 10. Arrhenius plot of Pima E-14 state 3 mitochondrial respiration. Fourth experiment.
48
o y = -4.4x + 1.27
o .03 .06 .12 ;15 ;18
Figure 11. Arrhenius plot of Pima E-14 state 4 mitochondrial respiration. Fourth experiment.
49
50
1·8
1.5
a> "S :..
1.2
C 0 ...... .... ~ .9 ~ .... Q.. en a> ~ .6
Ol 0 ... Break Point·c 13.79° C
.3 0 Y= -11.5x + 2.0179
6 Y= -3.2)( + 1.41
0 .03 .06 D9 ~12 .15 ·.:18
l/T
Figure 12. Arrhenius plot of Pima 8-5 state 3 mitochondrial respiration. First experiment.
Q) ... «' ,. C 0 ..... ~ ,. ..... ~ f/j
.a> ,.. t:J 0 ......
1.8
1.5
1.2
.9
.S
.3
o
Break Point = 14.28°C
o Y = -14.45)( ... 2.00S
6 y III -2.0Sx + 1.14
.03 .OS .12 .15
Figure 13. Arrhenius plot of Pima 8-5 state 4 mitochondrial respiration. First experiment.
51
1.8
1.5
1.2
.S
.6
.3 Break Point = 13.33°(
o Y = -S.583x'" 2.03
6 y~. -4.24x + 1.63
52
o .03 .06 .12 l5 .18
Figure 14. Arrhenius plot of Pima S-5 state 3 mitochondrial respiration. Second experiment.
53
1.8
1.5
Q.) ...... ~ ,...
1.2
C 0 ..... ...... ~ .9 ,... ..... Q. en Q.)
.6 ,... Ol 0 Break Point = 13.98°C ,.....
.3 0 y= -11.5x + 1.95
6 y= -3.18x + 1.37
o .03 .06 .09 .15 .18
o} ~ T
Figure 15. Arrhenius plot of Pima S-5 state 4 mitochondrial respiration. Second experiment.
1.8
1.5
1.2
.9
.6
.3
o
Break Point = 12.98°C
o Y = -11.21)( + 2.13
6 y - -4.55.)( + 1.63
.03 .06 .09
~
54
.12 ·.15 :18
Figure 16. Arrhenius plot of Pima 8-5 state 3 mitochondrial respiration. Third experiment.
1.8
a> 1.5 ....
. (tI ,.. C 1.2
0 •• .... «' ,..
.9 •• ~ en a> ,..
.6
Ol 0 ~
.3
o
Break Point = 12.7° C
0 Y = -10.78)( + 1.94
l:l Y = -3.52x ... 1.38
.03 .06 ~09
~ T
.12
55
;15 ;18
Figure 17. Arrhenius plot of Pima S-5 state 4 mitochondrial respiration. Third experiment.
56
An Arrhenius plot was not generated for this experiment' al
though the data were included in the mean respiration values
for Pima S-5 state 3 and state 4 respiration rates as af
fected by temperature. The plot of state 4 respiration
versus temperature for experiment 1 of the Pima E-14 (Figure
5) had two three point segments with a standard error of
0.0000, however the use of these two lines would have ex
cluded one data point. The best fit for the state 3 and
state 4 respiration of experiment 4, Pima E-14, (Figures 10
and 11) was a single straight line.
The Pima E-14 Arrhenius plots show a mean state 3
respiration rate discontinuity of 10.7C with a standard de
viation of 0.7C and mean state 4 respiration rate discon
tinuity at 10.9C with a standard deviation of 1.3C while the
Pima S-5 shows its mean state 3 and state 4 respiration rate
discontinuities at l3.4C w:Lth a standard deviation of O'.4C
and l3.6C with a standard deviation of 0.8C res~ective+y. The
qualitative aspects of these differences in Arrhenius break
points are as expected, as more chil+ing resistant varieties
of plants a~d species of animals characteristically exhibit
lower break points than chilling sensitive varieties of
species [28, 65, 66].
The use of an Arrhenius plot to detect differences in
chilling sensitivity between varieties was suggested by the
work of Lyons et al. [49] who used polarographic techniques
57
to determine phase changes in the membranes of mitochondria.
Several researchers have studied the effect of temperatures
on the enzymes associated with mitochondrial membranes and
found that the activation energies (Ea) of these enzymes
changed over temperature. Duke et al. [28] found that the
Arrhenius slopes (Ea) for NADP-isocitrate dehydrogenase
closely paralleled the slopes for respiration and had the
same break point in soybeans (Glycine~. L.). Kuiper [43]
noted that the enzyme ATP synthetase had a higher Ea at
lower temperatures and a lower Ea at high temperatures in
bean roots (Phaseolus ~.). He also noticed that the ATP
synthetase required the presence of lipid activity. Temper
ature adapted plants did not show as great a difference in
ATP synthetase activity as a function of temperature.
Other researchers have done similar studies with
chloroplasts. Peoples et al. [62] found that the membrane
unsaturation was correlated with a reduction of photosyn
thetic response at lOC in four alfalfa cultivars and Nobel
[57] found that chloroplast membranes underwent a phase
change in chilling sensitive Phaseolus vulgarus L. and
Lycopersicon esculenturn Mill., as indicated by permeability
changes, while chilling resistant ~isurn sativ~ L., and
Spinacia oleracea L. did not show these changes. Other re
searchers [58, 67] have been unable to show that these
changes in chloroplast membranes, and thus their photo
synthetic.activity, are related to chilling injury, indi
cating that the chloroplast membrane changes are not a
primary cause of chilling injury~
58
An objection to the use of Arrhenius plots in evalu
ating enzyme activity over a range of temperatures has been
raised by Silvius et ale [70], namely that a temperature de
pendent Km of enzymes can give Arrhenius plots with breaks
if assayed at fixed substrate levels. This argument appears
to be invalidated by the work of Raison et ale [66] who found
that the temperature dependent activity of succinate oxidase,
succinate dehydrogenase and cytochrome £ oxidase showed
breaks (discontinuities) in Arrhenius plots at about the
same temperature as did the oxidative activfty, but when the
membrane lipids were solubilized with detergent, the enzymes
ceased to exhibit these breaks in plotted activity. Raison
[64] has been definitive on this point s.tating that "The
change in the thermodynamic and kinetic properties of
the respiratory enzymes of these (tropical) plants is
not an intrinsic property of the enzyme protein but is
induced by changes in the molecular ordering of the mito
chondrial membranes with which the enzymes are associated".
He further supports this contention by pointing out that the
change in Ea for cytoplasmic enzymes is constant while the
change in Ea for membrane dependent mitochondrial enzymes
is not.
In a recent paper, Bagnall and Wolfe [6] also
criticize the use of Arrhenius plots in the study of plant
chilling sensitivity. The authors base their criticism on
.severa1 arguments:
1. That Arrhenius plot "break points" of whole growth
versus temperature are not correlated with chilling
sensitivity;
59
2. that the statistical argument for fitting two straight
intersecting segments to data, producing a "break point",
rather than fitting a smooth curve to the same data,
producing no "break point", is weak; and
3. that the slopes of nonlinear Arrhenius plots do not
reflect the true enthalpy' of activation of the enzyme
substrate complex.
This last argument is supported by the authors by showing
that an Arrhenius plot of plant growth versus temperature
will have a break where plant growth ceases and the slope
goes to minus infinity. They state that a slope of minus
infinity is clearly not proportional to the activation
energy of a reaction with a finite rate, and thus the slope
of an Arrhenius plot of growth versus temperature would not
accurately represent a mathematical relation to growth.
60
Bagnall and Wolfe's objections to the use of
Arrhenius plots to characterize a plant's suseptibility to
chilling stress are primarily directed to the use of whole
growth data. This study did not use whole growth data but
considered mitochondrial respiration rates and oxidative
phosphorylation ~fficiency data in relation tq known 'growt'h
responses of cotton cultivars to ,temperature stress. Mito
chondria are membrane bound organelles with the function of
ATP production. The membranes of mitochondria have been.shown
to undergo phase change [65, 79] and some membrane associated
enzymes show a change in activity at the temperature of phase
change [66]. Although respiration decreases with decreasing
temperature, as growth does, respiration does not cease at a
definite temperature, as growth does. Plant tissues remain
viable at the temperature of liquid nitrogen vapor (-196C),
this viability implies that life processes continue at this
temperature; far below any imaginable temperatue of membrane
phase change.
A temperature induced membrane phase change in mitro
chondria, followed by the resulting change in the respiration
and ATP productio~would have a direct effect on seed vigor.
ATP content of seeds 'and the adenylate energy change of the
cell have both been related to seed vigor [17, 18]. For
these reasons, an Arrhenius plot of mitochondrial respiration
versus temperature would be expected to provide useful
information on the chilling sensitivity and response to
chilling temperatures.
61
Bagnall and Wolfe's criticism of the use of two
straight lines to form a "break point" rather than a smooth
curve through all points may be entirely valid. However,
Bagnall and Wolfe themselves point out that other researchers
attach importance to the area of a "sharp change in curva
ture". It seems reasonable that this point of curvature
change can be approximated by the intersection of two staight
lines.
The inverse relationship between the rate of change
in enzyme activation energy and the rate of change in an
enzyme dependent function, such as respiration, is apparent
upon examination of the slopes of the Arrhenius plots in
Figures 4 to 17. The slope of 'an Arrhenius plot of enzyme
activity is thought to be equal to the activation energy of
the-assayed enzymes. Kuiper's [43] work with- bean ATP
synthetase showed a high Ea at low temperatures and a low
Ea at high temperatures. This observation agrees with those
of Duke et al. [28], Raison et al. [66] and Raison [64] whose
Arrhenius plots or enzyme activity show a steeper slope (i.e.,
a greater Ea> at temperatures below the break point compared
to the slope at temperatures above the break point. Since
mitochondrial respiration is dependent upon the activity of
membrane bound enzymes [46], an increase in the activation
energy of a respiration related enzyme would be ~~pected to
cause a decrease in the rate of respiration change. There~
fore, at temperatures below the point of membrane phase
change, (with the resulting increase in the Ea for the
associated enzymes), the slope of respiration rate increase
will be less than for temperatures above the break point
where the enzymatic Ea is less and the respiration rate
increases more rapidly.
62
The mitochondrial fractions used in these experiments
were isolated by differential centrifugation and consisted of
a crude pellet. This fraction primarily consists of mito
chondria with a lesser amount of mitochondrial membrane
fragments and extramitochondrial membrane fragments [32].
Extramitochondrial fractions have been reported to exhibit
respiratory control [46], and may have contributed to the
oxygen uptake by the crude pellet. An attempt was made to
reduce this contamination by gradient purification of the
crude pellet. The results obtained were inconclusive due to
many technical difficulties including inconsistant gradient
formation and the necessity of pooling purified samples to
zymes in the sucrose gradient purified mitochondria of barley
(Hordeum vulgare L.). Other researchers have reported the
presence of isoenzymes in a wide variety of organisms in
cluding fish [7], bacteria [52], and plants [61]. These
isoenzymes are thought to aid in the enzymatic adaptation of
metabolic system to a wide range of environmental conditions.
Preliminary results indicate a difference in the electro
phoretic banding pattern of esterase isoenzymes between
pima E-14 and Pima 8-5 (see Plate 2). Other enzymes may
differ as well including those responsible for the steps in
the Krebs cycle. Enzyme differences could be such that the
activity at a set temperature of two isoenzymes differ·s
significantly and suggests another possible mechanism for
respiratory adaptation or compensation at low temepratures.
66
Due to the variation in both lipid composition and
membrane associated enzymes, a potential for the genetic
improvement of chilling tolerance exists in cotton. Buxton
and Sprenger [13] evaluated a broad base of genetic lines of
both upland and long staple cotton and found significant
interactions between genotype and environment. The authors
concluded that a genetic potential for development of cotton
·lines resistant to chilling temperatures exists. Bartkowski
et ale [8], studying membrane composition came to the same
conclusion, stating that one basis for selection could be a
relatively high unsaturated/saturated fatty acid ratio.
CHAPTER 5
SUMMARY
It is becoming increasingly clear that the nature of
plant cell membranes and their associated enzymes is of prime
importance in determining plants' adaptability to, and toler-
ance of, low temperatures. As plant cells can be defined as
a dynamic interaction of membrane bound and enzyme regulated
metabolic systems of differing composition, the exact temper-
ature at which a cell's activity is limited is poorly defined.
In this study of differing temperature tolerances of two .
Pima cottons, mitochondria were chosen as the organelle of
interest as they are membrane bound and are the primary site
of energy production of the cell. This study has shown that
one aspect determining the differing temperature tolerances
between these two cottons is a greater respiratory efficiency
at low temperatures and a greater resistance to mitochondrial
membrane phase change as shown by Arr~enius plots of state 3 )
and state 4 mitochondrial respiration.
Although these results correlate well with the known
temperature tolerance of these two cottons, mitochondrial
membrane phase change per ~ cannot be considered to be the
. whole story of low temperature limitation of plant growth.
67
68
Preliminary evidence indicates that mitochondrial respiration
is severely restricted at temperatures below that at which
membrane phase change occurs and that respiration below this
point is in part due to oxygen uptake by lipoxygenase
activity. Lipoxygenase will oxidize polyunsaturated fatty
acids and this might lead to a change in the ratio of un
saturated to saturated fatty acids causing an unfavorable
upward shift in the temperature of membrane phase change.
Further research into the relationship of mitochondrial
respiration and chilling injury should include the study of
purified mitochondria, free from lypoxygenase activity and
the role of lipoxygenase in low temperature respiration.
APPENDIX 1
PERCOLL GRADIENT PURIFICATION
OF MITOCHONDRIA
In order to further define the effect of temperature
on mitochondrial respiration, attempts were made to purify
mitochondria by Percoll gradient centrifugation. This tech
nique has been shown to allow the isolation of intact, puri
fied mitochondria [32]. The materials and methods used were
identical to those for crude pellet mitochondrial isolation
with the additional steps of placing the crude pellet on top
of a 2% to 60% Percoll gradient contained in a 5 ml cellulose
nitrate centrifuge tube~ The gradient was obtained by di
luting an isotonic 100% Percoll solution with wash buffer:-.to
make two solutions of 2% and 60% Percoll respectively. These
solutions were placed in equal weights into a plastic grad
ient former and mixed with a vibrating stirrer to form the
gradient in the cellulose nitrate tubes. The gradients with
the crude pellet floated on top were carefully placed into
SW-50l rotor and centrifuge buckets and centrifuged at 37,000
x gofor 25 minutes in a sorvall OTD-2 oil turbine drive
ultracentrifuge. At the conclusion of the centrifuge run
the tubes were removed and the separated mitochondrial bands
69
70
removed and placed into 15 m1 plastic centrifuge tubes,
brought up to 10 mL:tota1 volume with wash buffer and repe1-
1eted at 40,000 x g-for 10 minutes in a Sorva11 RC-S- SUDer - ~
speed refrigerated centrifuge. The resulting pellets were
removed and run on the oxygraph-the same as a crude pellet.
This procedure has been shown to yield two populations of
mitochondria; an upper band of mitochondria and other mem-
brane fragments and a lower band of purified intact
mitochondria [32}.
Due to difficulty in controlling the total yield of
mitochondria and the total volume of reaction mixture, ADP:O
ratios were not calculated. Respiratory control ratios were
calculated as this ratio is independent of mitochondrial
population denisty past some threshold density. In general
the results indicate that mitochondrial respiration is
sever·e1y restricted at low temperature (Table A1). These
results cannot be considered to be definitive due to a lack
of experimental continuity. The lack of respiration.at low
temperature was interpreted to mean that the mitochondria
were inviable and the experiment was terminated. Later ex-
periments run at higher temperatures yielded oxygraph traces
indicating that intact, viable mitochondria were extracted
by this procedure. These results are now tentatively in-
terpreted to indicate that respiration of purified
Table AI. Mean respiratory control ratios of gradient purified Pima S-5 mitochondria.
Assay Temp. (e)
9.0
9.5
24.0
24.0
Mitochondrial population assayed
N/A
N/A
Upper
Lower
Upper
Lower
N/A data not available.
Mean respiratory control ratio
0
0
2.0
3.7
2.0
2.6
mitochondria is severely restricted at temperatures below
phase change.
71
APPENDIX 2
ARRHENIUS PLOTS OF ACALA 1517-0 MITOCHONDRIA
When this study of correlating mitochondrial respira
tion parameters with low temperature tolerance in cotton was
concieved, attempts were made to procure those cottons which
demonstrated the greatest difference in low temperature toler
ance. Dr. M. N. Christiansen, the director of the Plant
Stress Lab at Beltsville, Md., personally communicated that
his information indicated that two G. hirsutum L. varieties,
Acala 1517-0 and Deltapine l4M-8, demonstrated the greatest
difference to low temperature. small lots of these cotton
seeds were obtained but were not of great enough quantity to
allow the replication of definitive experiments. For this
reason two Pima cotton types were eventually chosen for study
as they were readily available in relatively large quantities.
In the course of developing a suitable protocol for
measuring mitochondrial respiration over a range of tempera
tures, a preliminary set of experiments was run with Acala
1517-0 cottonseed. The materials and methods used were
identical to those in the'main study except for the type of
germplasm extracted and that no correction for temperature
was used. These experiments indicated that it is possible to
72
73
attain Arrhenius plots by the procedures used. Figure Bl is
an Arrhenius plot of three replications of Acala l5l7-D
mitochondrial state 3 respiration.
Proteins were extracted from ungerminated seeds of
Pima S-5and E-14 and were separated by polyacrylamide
gel electrophoresis. Densit~meter tracings of representative
protein gels are illustrated in Figure Bl, where a general
protein stain was used: and in Figure B3, where an esterase
enzyme stain was utilized. Slight, but noticeable differ
ences were evident in the tracings of the two Pima varieties.
100
,.... o 10
r:E =.
5
Break Point ::: 12"C
1~ __ ~~ __ ~~ __ ~~ __ ~ ____ ~ ____ ~ 12
Figure BI. Arrhenius plots of the respiration of isolated mitochondria from seedlings of Acala cotton (Grosspium hirsudurn L.).
74
5-5
E-14
Figure B2. Densitometer tracing of representative stained electrophoresis gels of ·the soluble seed proteins extracted. from Piam 8-5 and E-l4.
75
Figure 3B.
76
rn I =
Densitometer tracings of representative electrophoresis gels of .soluble seed proteins of Pima S-5 and E-l4 incubated with ·esterase specific enzyme stain.
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