ARRHENIUS PLOTS OF MITOCHONDRIALRESPIRATION IN PIMA COTTON VARIETIES

OF DIFFERING TEMPERATURE TOLERANCE.

Item Type text; Dissertation-Reproduction (electronic)

Authors CENTNER, MICHAEL STEPHEN.

Publisher The University of Arizona.

Rights Copyright © is held by the author. Digital access to this materialis made possible by the University Libraries, University of Arizona.Further transmission, reproduction or presentation (such aspublic display or performance) of protected items is prohibitedexcept with permission of the author.

Download date 26/05/2021 02:04:28

Link to Item http://hdl.handle.net/10150/184792

INFORMATION TO USERS

This reproduction was made from a copy of a document sent to us for microfilming. While the most advanced technology has been used to photograph and reproduce this document, the quality of the reproduction is heavily dependent upon the quality of the material submitted.

The following explanation of techniques is provided to help clarify markings or notations which may appear on this reproduction.

1. The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting through an image and duplicating adjacent pages to assure complete continuity.

2. When an image on the film is obliterated with a round black mark, it is an indication of either blurred copy because of movement during exposure, duplicate ·copy, or copyrighted materials that should not have been filmed. For blurred pages, ·a good image of the page can be found in the adjacent frame. If copyrighted materials were deleted, a target note will appear listing the pages in the adjacent frame.

3. When a map, drawing or chart, etc., is part of the material being photographed, a definite method of "sectioning" the material has been followed. It is customary to begin filming at the upper left hand corner of a large sheet and to continue from left to right in equal sections with small overlaps. If necessary, sectioning is continued again-beginning below the first row and continuing on until complete.

4. For illustrations that cannot be satisfactorily reproduced by xerographic means, photographic prints can be purchased at additional cost and inserted into your xerographic copy. These prints are available upon request from the Dissertations Customer Services Department.

5. Some pages in any document may have indistinct print. In all cases the best available copy has been filmed.

University MicrOfilms

International 300 N. Zeeb Road Ann Arbor, MI4B106

8303382

Centner, Michael Stephen

ARRHENIUS PLOTS OF MITOCHONDRIAL RESPIRATION IN PIMA COTTON VARIETIES OF DIFFERING TEMPERATURE TOLERANCE

The University of Arizona PH.D. 1982

University Microfilms

Intern ati 0 n al 300 N. Zeeb Road, Ann Arbor, MI 48106

PLEASE NOTE:

In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark_,f_.

1. Glossy photographs or pages V

2. Colored illustrations, paper or print ~

3. Photographs with dark background V

4. Illustrations are poor copy __

5. Pages with black marks, not original copy __

6. Print shows through as there is text on both sides of page __

7. Indistinct, broken or small print on several pages __

B. Print exceeds margin requirements __

9. Tightly bound copy with print lost in spine __

10. Computer printout pages with indistinct print __

11. Page(s) lacking when material received, and not available from school or author.

12. Page(s) seem to be missing in numbering only as text follows.

13. Two pages numbered . Text follows.

14. Curling and wrinkled pages __ _

15. Other ______________ . ______________________________________ _

University Microfilms

International

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

contained herein.

iv

TABLE OF CONTENTS

LIST OF ILLUSTRATIONS

LIST OF TABLES

LIST OF PHOTOGRAPHIC PLATES . . . . . ABSTRACT

CHAPTER

1. INTRODUCTION . . . . . . . . 2. LITERATURE REVIEW

On Membranes

On Cotton Sensitivity to Chilling Injury and the Prevention of such Injury • •• • • • • • • •

Phase Changes and Chilling Injury •

3." MATERIALS AND METHODS •••

4.

Cold Germination Experiment •

Electrophoretic Protein Characterization • • • •

Mitochondrial Extraction

Arrhenius Plots • • • • •

RESULTS AND DISCUSSION

. . . . . . . .

Cool Temperature Germination and Growth

Electrophoresis • • • • •

v

. .

Page

vii

ix

x

xi

1

3

3

S

9

13

19'

16

18

21

2S

27

28

TABLE OF CONTENTS--Continued

Respiration Parameters • .. . . . . State 3 Respiration . . . . . . . . . . State 4 Respiration . . . . . . . . . . . . . .

Respiration Efficiency Index •

ADP:O Ratios • • • • . . . . . Respiratory Control Ratios • . . .

Arrhenius Plots

5. Sp-MMARY • • • • • • • • • • • • • • • •

APPENDIX 1:

APPENDIX 2:

REFERENCES •

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 • • • • • •

Urea soluble proteins stained for esterase activity • ". • • • . . . . . . . . . . . . . .

x

Page

29

29

ABSTRACT

Mitochondria were extracted from seedling radicles of

Pima S-5 and Pima E-14 cottons and the state 3 respiration,

state 4 respiration, ADP:O ratio and respiratory control (RC)

ratio were measured in vitro over a range of temperatures

from 6 to l8C.

Mitochondria from E-14 seedlings exhibited a mean

state 3 respiration rate of 13.42 ~Mo2/min/gm tissue while

mitochondria from S-5 seedlings showed a mean state 3 rate

of 17.94 ~Mo2/min/gm tissue. Mean state 4 respiration ex­

hibited a similar trend with measurements of 73.4 ~M02/min/gm

tissue and 11.73 ~Mo2/min/gm tissue for E-14 and S-5.

Mitochondria from E-14 seedlings exhibited a mean

ADP:O ratio of 3.73 compared to an ADP:O of 3.28 for S-5,

across all assay temperatures. Mean respiratory control

ratio was 1.79 for: E-14 and 1.53 'for S-5. These lower

respiration rates of E-14 coupled with higher ADP:O ratios

and RC ratios: support a greater respiratory efficiency at

low temperatures of this variety compared to S-5. Addi­

tionally, the E-14 mitochondrial membranes exhibited an

ability to remain in a fluid state to a lower temperature

than Pima S-5 mitochondrial membranes as judged by Arrhenius

plots of respiration. Since mitochondrial respiration

xi

xii

is considered to be regulated by membrane-bound enzymes, any

change in membrane fluidity would conceivably affect mito­

chondrial enzyme activity and thus alter respiration rates.

Changes in respiration rates will be reflected as a break in

an Arrhenius plot. The mean break point temperature of state

3 respiration was IO.7C for E-14 and 13.4C for S-5. The mean

break point temperature for state 4 respiration was IO.9C for

E-14 and 13.6C for S-5. The ability of E-14 to withstanQ a

greater degree of chilling under field conditions ca~ be

attributed, in part, to the greater fluidity of s~edling

mitochondrial membranes at low temperatures and a concomitant

conservation of respiratory energy through a lower rate of

respiration. Assays of mitochondrial respiration and

Arrhenius plots of mitochondrial respiration versus temper­

atures could be used to select cotton lines more tolerant to

chilling temperatures.

CHAPTER 1

INTRODUCTION

A major limitation in the production of food and

fiber in many areas of the world is cool temperatures early

in the growing season. Many important crop plants are of

tropical origin but are grown in regions where they may be

subject to temperature cooler than those to which they are

adapted. Cotton (Gossypium sp.) is a tropical perennial

that is grown as an annual crop in many regions' of the world.

Cotton, as with other tropical plants, can be adversely af­

fected by above freezing low temperatures, termed chilling

temperatures. If soil temperature falls below about 15C

cotton growth is severely slowed; and physical damage to the

plant will result if soil temperatures remain low for an ex­

tended period [19]. Recent research has shown that the cell

membrane is a primary site of chilling injury in plants

[48]. In chilling sensitive plants, the membrane undergoes

a phase shift from a more fluid to a less fluid state with

decreasing temperature [50]. This phase change resu1ts'in

the disruption of the membrane function as a selectively

permeable barrier to solute flow and as a matrix for enzyme

attachment and activation. A major factor in determining a

1

membrane propensity to go t~rough a phase change at some

temperature is in the degree of unsaturation in the fatty

acids making up the lipids in the membrane [78]. Membranes

containing a higher proportion "of unsaturated fatty acids

remain in a more fluid state at lower temperatures than do

membranes with only saturated, or a lesser proportion of

unsaturated fatty acids [78].

A useful tool in the study of membrane phase change

is the Arrhenius plot which graphs the log of a reaction

rate against the inverse of the temperature. The slope of

an Arrhenius plot of a membrane dependent function can be

related to the activation energy of the associated enzymes

[14]. In chilling sensitive species an Arrhenius plot will

have a line with a break with one or more changes in slope

at those temperatures where the phase change ~ccurs. The

present study was designed to show if one significant factor

in determining"differences in chilling sensitivity in cotton

is the mitochondrial membranes resistance to phase change

at chilling temperatures.

2

CHAPTER 2

LITERATURE REVIEW

On Membranes

The modern concept of biological membranes is based

on the fluid mosaic model of Singer and Nicholson [74]. This

model visualizes a membrane composed of a lipid bilayer with

hydrophyllic polar head groups oriented toward the exterior

surface of the membrane and hydrophobic fatty acid tail

groups of the interior of the bilayer. Associated with the

lipid bilayer are proteins, some of which are associated

with the polar face of the bilayer while others are confined

to the hydrophobic core or extended through the bilayer [44,

78]. See Figure 1. The lipids in the membrane are normally

in a fluid state and exhibit vibrational and rotational move­

ments within an individual lipid molecule and vibrational,

rotational and translational movements of entire lipid mole­

cules, both literally and. from one side of the bilayer to the

other [78]. Membrane fludity also allows for the movement of

the membrane associated proteins. Glycerophospholipids,

which make up the mitochondrial and plasma membranes [71],

undergo a phase transition from a liquid crystalline to a

solid gel and back, characterized by a marked increase.

3

X I o I o=p-o· I o' I

CH-CH-CH I 2 I 2

o 0 I I C=O C-O I I

(C H2)n (CH2)n r I

CH3 CH3

POLAR HEAD

NON-POLAR TAIL

a)

H'H«R~ ««ftf;1«ft~~ mftH~ ~ "He uu Q(!J"a OU~ ~~ gy~ ~g ~~« H Y

b)

Figure 1. The fluid-mosaic model of membrane structure. Based on Singer and Nicolson.

a) structural formula of phosphoglyceride(X = alcohol) •

b) Schematic diagram of membrane (p = proteins).

4

5.

in viscosity [78] due to the loss of the liquid nature of the

tail groups in the core of the bilayer. The tendency of a

membrane to go through a phase change at some temperature is

dependent on several factors. The temperature of phase

change is decreased; with shorter fatty acid chain lengths

[44]; with unsaturation of the fatty acid chains [44, 78],

and with membrane hydration [78]. As the membranes go

through a phase change the associated proteins and enzymes

are also affected. The membrane proteins themselves are not

involved in the phase change; the temperature at which a

membrane will gel is the same if the proteins are denatured

or in their native state [48]. Raison [64] has stated that

temperature-induced changes in the activity of respiratory

enzymes are "not a property of the enzyme itself but a re­

flection of the changes in the molecular ordering of the

surrounding lipids. These temperature induced changes in

membrane fluidity and the activity of associated enzymes are

a basic cause of chilling injury in plants [48].

On Cotton Sensitivity to Chilling Injury and the Prevention of such Injury

Lehman [45] in 1925 reported 15C as the temperature

which limited cottonseed germination. Ludwig [47] in 1932

reported l2C as the limiting temperature while Arndt [4] in

1935 stated that temperatures below 18C were adverse to

germination and emergence and that temperatures of 30-32C

were optimal.

6

Christiansen [20] noticed that there are two periods

of chilling susceptibility in upland cotton (Gossypium

hirsutum L.). The first occurs at the initiation of im­

bibition and the second occurs 18-30 hours after germination.

Buston, Sprenger and Pegelow [12] reported a similar timing

of chilling sensitivity for Pima cotton (Gossypium barbadense

L.). There is evidence to explain these two periods of

sensitivity.

One of the characteristics of the initial imbibition

of many seeds is the leakage of solutes [10, 71, 73]. In

cotton, these solutes include sugar, amino acids, and nucleo­

tides [23, 36, 76, 77]. Christiansen, Carns and Slyter [23]

have stated that much of the solute loss is from the area of

the root tip. Simon [72] reported that in dry seeds the

membrane phospholipids are a hexagonal arrangement and that

as moisture content increases the membrane reorients to a

lamellar (bilayer) arrangement. Reorientation normally

causes a rapid decrease in the rate of solute leakage [10,

73]. If seeds of the lima beans (Phaseolus lunatus L.)

[63], cotton [23] or soybean (Glycine'~ L.) [10] are

allowed to imbibe at chilling temperatures, solute leakage

does not decrease with hydration but increases with the

duration of chilling. This indicates that reorientation at

chilling temperatures is incomplete. It may be that at

temperatures below that of phase change the membrane re­

orients into the solid gel phase. Thus, initial inbibition

at chilling temperatures appears to allow solute leakage to

proceed to an injurious degree.

7

In a study of nucleic acid metabolism during germi­

nation of Pima cotton, Clay, Katterman and Hammet [25] re­

lated a second period of chilling sensitivity (28 to 32 hours)

to the periods of DNA and RNA synthesis. Ribosomal RNA

synthesis peaks at 18 hours, messenger RNA synthesis peaks

at 36 hours and DNA synthesis peaks at both of these times.

Guinn [36] has noted that RNA and proteins decrease in

chilled cotton plants. DeVay and Paldi [27], studying wheat

(Triticum aestivum L.) found that one difference between

cold hardy winter wheat and non-hardy spring wheat is the

relative ability to synthesize ribosomal RNA under cold

conditions. Periods of growth, (i.e., the elongation of the

embryonic axis, etc.) are characterized by cell division and

the necessary replication of DNA and RNA, and any inter­

ference of this replica~ion could be a factor in chilling

injury. Christiansen [19] noted two symptoms of imbibitional

chilling injury which he later [21] associated with these

two periods of sensitivity. Radical tip abortion was damage

attributed to chilling during initial imbibition, and root

cortex disintegration was caused by chilling during the

elongation of the embryonic axis. . If chill damaged cotton

plants are placed in optimal conditions, radical tip abor­

tion causes a short lag in growth followed by normal growth

rates while root cortex disintegration causes a severe re­

duction of growth. Radical tip abortion appears to be re­

lated to a chilling induced stelar lesion reported in corn

by Cohn and Obendorf [26].

8

prehydration of seeds at optimal temperatures has

been shown to improve the cold temperature germination of

lima beans [63], cotton [22], and soybean [10]. As hydration

increases seed moisture content to 17-45% in peas [73], 35-

50% in soybean [10], and 13% in cotton [22], imbibitional

solute loss is greatly decreased or halted. Optimal tem­

perature hydration may permit the membrane to reorient while

at a temperature where the membrane is in its fluid liquid­

crystalline conformation.

In addition to hydration, chemical compounds, espe­

cially adenine nucleotides, have been shown to improve cot­

tonseed germination under chilling conditions [15]. Since a

constituent of solute loss in cotton is nucleotides [77] this

treatment may serve to reduce the loss of, or replace these

compounds. Another possibility is that treatment with

adenine nucleotides may increase the poll of ATP in seed

tissues. ATP content has been related to seed vigor [17],

and soybean axes incubated in AMP have been shown to accmulate

9

ATP [3]. Calcium and magnesium [23], as well as talc (native

hydrous magnesium silicate) [2] have been shown to reduce

chilling injury in cotton. Overlath and Thilo [60] state

that divalent cations such as Ca++ and Mg++ form stochio­

metric complexes with acidic phospholipids and may play a

role in membrane stabilization. Calcium is also known as an

activator for membrane associated ATPases [24].

Chemical modification of membranes has been shown to

reduce chilling susceptibility in tomato (Solanum escultatum

L.). Phospholipid composition and saturation of the fatty

acids were modified with ethanolamine. Both the head and

the tail groups were modified [80]. In a later paper [39]

it was reported that treated tomato seedling cotyledons

showed less cold damage than untreated seedling cotyledons.

Light and electron microscopy revealed that membrane modi­

fication was ·most pronounced for the cytoplasm, the mito­

chondria and cell walls.

Phase Changes and Chilling Injury

The specific site of chilling injury appears to be

the various cell membranes including those of organelles.

Lyons, Wheaton and Pratt [50] found that chilling sensitive

plants had less flexible mitochondrial membranes, as judged

by their ability to swell in hypotonic solutions, than did

chilling resistant plants. The more flexible mitochondria

10

had a higher double bond index for their fatty acids than did

the less flexible mitochondria.· Nobel [57] reported similar

osmotic responses in isolated cholorplasts.

Raison et ale [65] detected changes in membrane

properties using spin labeling techniques. The authors re­

ported that·the mitochondrial membranes in chilling sensitive

S''leet pot·atos (Ipomea batatos L.) and rat liver (Ratus ~)

underwent a phase change from a liquid crystalline to a solid

gel at the temperature at which chilling injury took place.

Chilling insensitive potato (Solarum tuberosum L.) and trout

(Ictalurus punctaius) mitochondria showed no such alternation.

Duke, Schrader and Miller [28] studied chilling ef­

fects on mitochondrial respiration and associated enzymes and

found that the activity of NADP isocitrate dehydrogenase,

(a Krebs cycle enzyme), showed activity changes at the

same temperature as respiration. Raison et ale [66] found

that succinate oxidase, succinate dehydrogenase, and cyto­

chrome c oxidase associated with chilling sensitive mito­

chondria showed activity changes at about the same

temperature at which oxidative activity was affected. When

the membrane lipids were. solubilized with detergent these

enzymes ceased to show changes in activity, indicating that

the alteration of membrane structure caused these changes in

activity. Similar alterations have been shown in the mem­

branes of glyoxysomes, mitochondria and proplastids of castor

11

bean endosperm [79], and the mitochondria of soybean [28].

Raison [64] emphasized the relation between the activation

energy of enzymes and manbrane phase change. He stated that

membrane properties and not'enzyme properties are the cause

of an increase activation energy at temperatures below phase

change. 'Data showing that cytoplasmic enzymes for glycolysis

do not show these increases in activation energy suppor't this

concept. Metabolic imbalances occur when respiratory activity

can not compensate for the increases in activation energy at

low temperatures.

Stewart and Guinn [76], noted that cotton plants

show decreases in ATP content with chilling and that they

can recover from one day of chilling but not two, suggesting

that the decrease in ATP supply is caused by a relatively

greater use of ATP in metabolic functions than production

by mitochondria. After two days ATP pools in the cell would

probably be too low to maintain ATP-dependent cellular in­

tegrity. This fits well with the idea that non-membrane de­

pendent functions proceed at a relatively constant rate

(determined by the QIO of the enzymes) while membrane de­

pendent functions, such as ATP production, are decreased

disproportionally by membrane phase change.

Wilson [81], divides chilling sensitive plants into

two categories. Category one plants are those that can not

be hardened against chilling injury and category two plants

12

are those that can be hardened. Hardening apparently in­

volves, in part, a modification of the membrane lipids.

Harris and James [37] found that the biosynthesis of un­

saturated fatty acids increased with decreasing temperature

in castor bean, sunflower, and flax. Peoples et a1. [62]

correlated the ability of alfalfa chloroplasts to.resist a

change in photosynthetic activity at lOC with the double

bond index of the fatty acids of the chloroplast membrane.

St. John and Christiansen [68] found that linolenic acid, an

unsaturated fatty acid, increased as cottonseed were germi­

nated at lower temperatures and that using an inhibitor of

linolenic acid synthesis reduced the plants ability to

withstand chilling temperatures. Bartkowski et a1. [8]

showed tHat Pima cotton emergence at low soil temperature

was correlated with the unsaturated/saturated fatty acid

ratios. Higher unsaturation was correlated with higher

emergence.

This study focuses on the behavior of Pima cotton

mitochondria over a range of temperatures to determine if

the resistance of mitochondria to phase change as shown by

Arrhenius plots of respiration rates is a factor in a cu1ti··

var's response to chilling temperature. I\1itochondria \<Jere

chosen as the organelle of interest as they are membrane

bound and are the main producer of ATP, a major determiner

of seedling vigor [17].

CHAPTER 3

MATERIALS AND METHODS

The two cotton germp1asms (Gossypium barbadense L.)

used in this study, Pima E-14 and Pima S-5, were chosen as

examples of two cottons with nearly inverse lint-yield

versus altitude relationships (Figure 2). Pima E-14 was

derived from Hybrid B, one of the parents of Pima S-3 cotton

lines and developed for lint production at high elevation

(above 750 m) [82]. Pima S-5 was a F4 selection from a

cross between an experimental strain and Pima S-4, and is

adapted to elevations below 450 m [31]. The major factor in

determining adaptation to elevation is temperature tolerance.

Three different types of experiments were utilized

in this project to characterize the two cotton varieties

and the relative tolerance of these varieties to-chilling

temperatures. The first experiment was a comparison of the

germination at ~hi1ling temperatures of these two varieties

of cotton. The second experiment was a comparison of the

mitochondrial respiration of these varieties over a range

of temperatures, as evaluated b~ an Arrhenius plot. The

third experiment was an electrophoretic comparison of the

urea soluble seed proteins of the two cotton varieties.

13

"'0 -Q> .->c ;.I fJ) Q> ~ 110 -Cb

E.~Z ~ ------~~ 100~ -~~

uS 90 ~

\;:9 ...---"'0 '-" _ 80

Q) .-)4 70

c: .-ctS 1-1 60

Ci) "'til' ,

1000 2000 3000 4000

Elevation (feet)

Figure 2. Yield vs altitude relationships of Pima S-5 and Pima E-2.

Pima E-2 was a direct forerunner of Pima E-14. Adapted from data. supplied by C. A. Feaster.

I-' ~

15 .

Cold Germination Experiment

In order to characterize the differing temperature

tolerance of Pima S-5 and Pima E-14, a cool temperature

germination test was run. An additional lot of Pima S-5 was

included in this experiment to compare to the Pima S-5 used

for mitochondrial extraction and temperature assay. Four

replications of 100 seeds each were surface sterilized by

immersing them in a 0.25% (v/v) solution of NaHC10 5H20. for

five minutes. Each 100 seed replication was placed between

folds of unbleached Kim towe1s™ (Kimberly-Clark Corp.)

moistened with 70 m1 of distilled, deionized H20, and placed

between facing clear plastic meat trays (A and E Plastics,

Industry, CA). The trays were partially sealed with masking

tape and randomly placed on a Fiberglass CamtrayTM (Cambro

Manufacturing Company, Huntington Beach, CA). The entire

fiberglass tray was enclosed in a clear plastic bag and

placed in a Burrows Model 1831 (Burrows Equipment Corp.,

Evanston, Illinois) seed germinator set to maintain a tem­

perature of 18 + 1C. Six days later the trays were removed

from the plastic enclosure, opened, and a count was made to

determine perc;ent germination. Twenty radicles were randomly

selected from each replication and weighed to determine fresh

weight, dried for 72 hours at 60C and weighed again to

determine dry weight.

Electrophoretic Protein Characterization

16

E1ectorphoresis of urea soluble proteins was used to

detect a~y subtle genetic differences between seeds of the

two varieties of cotton used in this study. Electrophoresis

has been used by numerous laboratories to characterize dif-

ferent varieties within a species and to indicate contamina-

tion of one variety by another [1,16,53].

Six cottonseed of each variety were decorticated and

soaked in Urea buffer (2.0M (NH2)2CO, 0.005M EDTA, 0.005M

deoxycholic acid, 0.02M tris base, Ph 8.9) overnight. Each

group of six seeds were then homogenized in 2 m1 of Urea

buffer by a Brinkmann Po1ytron at speed setting five for

five .seconds. An additional 3 m1 of Urea buffer was added to

each tube and the tubes centrifuged at 1000 x ~ for 10

minutes. The resulting supernatant was a clear yellow liquid

with a floating layer of lipids. The lipids were aspirated

from the supernatant and 1 m1 of the supernatant removed for

further treatment. To this 1 m1 of supernatant 0.03 g of

ascorbic acid was added (a modification of the methods of

Mohamed [56]). A precipitate formed which was removed by

centrifugation at 1000 x g for 10 minutes. The resulting

supernatant was a clear, colorless liquid. One hundred

microliters of this supernatant per gel was used as a

sample.

17

Disc gels of 7% acry1amide buffered at pH 8,.9 with

a 3% acry1amide stacking gel buffered at pH 6.7 were prepared

from stock solutions by a procedure modified from Ornstein

and Davis [59]. The gels were cast in 100 rom x 7 mm

(OD) x 5 mm (ID) glass tubing. The 7% gel was poured to a

depth of 70 mm and allowed to polymerize before the 3%

stacking gel was poured to a depth of 10 rom.

A tris-g1ycine solution at pH 8.91 was used as the

anode electrolyte and a tris solution buffered at pH 8.07 was

used as the cathode electrolyte. Precise pH was attained

using a Bec~an Model 1019 research pH meter accurate to

+ 0.002 pH units. The 100 ~1 sample for each gel was loaded

directly on top of the stacking gel through the upper buffer.

Bromophenol blue (0.001%) (v/v) was added to the upper buffer

as a marker dye. An Instrumentation Specialities (Lincoln,

Nebraska) Co. Model 493 electrophoresis power supply was con­

nected to the unit and the electrophoresis run at a constant

power of 0.4 watts per disc gels. The electrophoresis was

stopped when the marker dye band had traveled 55 to 60 mm

into the running gel. The gels were removed from the glass

tubes by rimming with water injected via ,a syringe., The

proteins were fixed and stained at room temperature by im­

mersion for 12 hours in 12% trichloroacetic acid to which

0.25% (w/v) coomassie blue G-250 has been added in the ratio

of 1 m1 coomassie blue: 5 m1 TCA. Esterase activity was

18

detected as per the method of Brewbaker [11]. Following

staining, the gels were transferred for storage into sealed

15 ml test tubes containing 7% acetic acid. Following

staining, the protein gels were scanned at 590 nm using a

Beckmann Model 3600 sp:ectrophotometer. The esterase gels

were scanned at 400 nm.

Mitochondrial Extraction

Three hundred to five hundred grams of acid delinted

cottonseed were first surface sterilized by soaking in a

0.25% (v/v) NaHclO.SH20 solution for five minutes. The seed

were then spread between moistened Kim towels™ (Kimberly~

Clark Mfg., Neenah, WI) and placed between two facing fiber-

glass trays. The trays were sealed with masking tape and in­

cubated for 48 hours in a Labline Instruments Inc. Co.

(Melrose Park, IL) incubator set at 3lC. At the end of this

germination period the radicle of the cottonseed had grown

to a length of 3 to 6 centimeters, depending on the variety,

and only the nonglanded portion of the radicle below the

hypocotyl was taken as the tissue to be extracted. One gram

of radicle tissue was used for each mitochondrial extraction.

All steps of the mitochondrial extraction were carried out

over ice or at 4C at all steps.

One gram of radicle tissue was weighed out into 20 ml

glass beakers containing a volume of grinding buffer (0.07-sM

K2HP04

, 0.018M KH2P04 , O.OO~M EDTA, O.sM sucrose, pH 7.2)

such that the weight of the beaker plus buffer was 20 gr.

The grinding buffer was then decanted and the radicles

19

chopped into approximately 1 mrn pieces with a new steel razor

blade. These pieces were placed in a 15 ml plastic centri-

fuge tube in 5 ml of grinding buffer with 205 mg BSA (Frac­

tion V, Sigma) and ground at speed setting 5 for five seconds

with a Brinkman™ Polytron. Five additional ml of grinding

buffer were added to the tissue slurry and the centrifuge

tube placed in a Sorvall RC-5 centrifuge fitted with a SM-24

rotor and ceintrifuged at 1000 x g for 5 minutes. The super­

natant was then filtered through a single layer of 50~ nylon

mesh (Tetko Inc.) and an additional 100 mg of BSA added. The

filtrate was then centrifuged at 40,000 x ~ for five minutes.

The result of this centrifugation was a dense pellet of

starch surrounded by a less dense mitochondrial pellet. The

supernatant was poured off and 0.5 ml of wash buffer (0.0014M

K2HP04' 0.5M sucrose, pH 7.2) was added to the centrifuge

tube and the mitochondrial pellet carefully dislodged with a

glass stirring rod. The mitochondrial pellet resuspended

in 0.5 ml of wash buffer was used as the sample.

Respiration studies of the extracted mitochondria

were conducted using a Clark type oxygen electrode (Yellow

Springs Instrument Co.). The 0.5 ml wash buffer plus pellet

was decanted into a round glass cuvette containing 1.0 ml of

reaction buffer (0.3M mannitol, 0.015M TRIS, 0.015M KH 2P04 ,

20

0.0002M thiamine pyrophosphate, 0.0163M MgC1 2 ) and a round

magnetic stir bar fitted into the bottom of the cuvette'. The

cuvette was immersed in a water bath connected to a Lauda

K-2/R circulating water refridgerator (Brinkman Inst.) to

maintain the desired temperature ± O.lC. At the start of

the respiration run 5.0 ~1 of aketog1utarate (akg) was in­

jected via a Hamilton #702 microliter syringe through a

groove in the oxygen electrode directly into the sample con­

taining cuvette. The akg was formulated to give a 12 ~M

solution in 1.5 m1. After 30-180 seconds 1.0 ~1 of ADP

formulated to give a 150 ~M solution in 1.5 m1 was injected.

Upon the addition of ADP, the mitochondria showed active

state 3 respiration, an increase due to the oxidative

phosphorylation of ADP to ATP. When the added ADP had been

phosphorylated to ATP the respiration rate dropped to a

resting, or state 4, respiration rate. The ratio between

the rate of state 3 (acceptor present) and state 4 (acceptor

absent) respiration is known as the respiratory control ratio

and can be used as one criterion of the relative intactness

of functional integrity of mitochondria [46]. If a known

concentration of ADP is injected and the amount of O2 con­

sumed in state 3 respiration is carefully measured then an

ADP:O ratio'can be calculated and used as a measure 0::15 'the

intactness of the phosphorylating steps of the respiratory

electron transport chain. Respiratory control ratios and

ADP:O ratios were calculated according to Estabrook [30].

21

Mitochondrial respiration was determined for seven

different temperatures from 6 to l8C in two degree increments.

Due to a decrease of 4% per degree in the sensitivity of the

oxygen probe and the increase in oxygen solubility at low

temperature~ a correction factor was calculated for each

temperature. Oxygen solubility was calculated from the table

"Solubility of Gases in water" on page 1707 of the 44th edi­

tion of ·the Handbook of Chemistry and Physics (Chemical

Rubber Company). The correction factor for each temperature

can be seen in Table 1.

Arrhenius Plots

About the year 1880, [14], Svante Arrhenius observed

through experimental evidence that the rate of a chemical re­

action increased exponentially with increases in temperature.

Because of this relationship, a rate constan~ k, can be cal­

culated for a reaction at a given temperature by the following

equation:

k = Ae-E/ RT (1) .

where A is a constant in moles per liter per second, e is

2.71828, (the natural log base), E is the activation energy,

22

Table 1. Correction factors used to account for increased O2

solubility and decreased oxygen probe sensitivity.

Solubility of O2

Temp. in H2O Probe Sensitivity Correction (C) ].10 2/1 % full scale factor

6 387.6 61.2 2.53

8 368.7 66.4 2.22

10 351.5 72.1 1.94

12 335.6 78.3 1.72

14 321.1 84.9 1.51

16 307.5 92.2 1.33

18 295.2 100.0 1.18

R is the gas constant (1.987 calories per degree per mo~e)

and T is the temperature in degrees Kelvin. If logarithms

are taken of both sides of the equation, the equation becomes

ink - in A - E/Rt. (2)

This transformed to base ten becomes

log k = log A - EV2.303 RT. (3)

23

Thus, a graph of the rate of a reaction at different tempera­

. tures plotted as the log of the reaction rate vs ~ will be a

straight line with a slope of 2~3~~ R. Since R is known,

the Arrhenius activation energy, E t can be calculated from

the slope. Conversely, if a biological system exists whose

reaction rate is enzyme dependent, each enzyme with its own

E (or E), and a temperature 'dependent change occurs a·

such that the Ea of the enzymes are affected, then the rate

of that reaction will be affected and this change will be

manifested as a change in the slope of the Arrhenius plot.

In the case of this study the temperature dependent change

is membrane fluidity of the mitochondria and the measured

rate change is that of respiration.

The Arrhenius plots of the log of the state 3 and

state 4 respiration rates versus the inverse of the 'tempera-

ture generated in this study were derived according to the

following procedure:

1. Regression equations were calculated for the log of the

state 3 and state 4 respiration rates versus the inverse

of temperature from 6 to l8C and 18 to 6C. Each re-

gression series in either direction consisted of five

regression equations containing at least three, to all

seven, consecutive temperature points. A total of ten

regression equations,five in increasing temperature and

five in decreasing temperature were calculated in this

mannel;.

24

2~ The line of best fit from among the ten regression equa­

tions, as judged by the highest regression coefficient,

was taken as one linear segment of the Arrhenius plot.

3. The line of next best fit was chosen from the remaining

five regression equations as second linear segment of

the Arrhenius plot with the ~ priori conditions that no

more than one point could be shared between the ·two

linear segments, and that all seven temperature points

must lie on one or the other segment.

4. In this manner, an Arrhenius plot was generated that

consisted of two linear segments; one segment the best

fit from high to low temperatures, and one segment the

best fit from l.ow to high temperatures.

CHAPTER 4

RESULTS AND DISCUSSION

This study was an attempt to determine if a signifi­

cant factor in the adaptation of cotton to low temperatures

is the behavior of the plant's mitochondria in chilling con­

ditions. Mitochondrial respiration was measured using a

polarographic method which monitors oxygen over time. An

oxygen sen'sitive electrode is connected to a recorder which

shows oxygen consumption as a pen trace across a roll of

ruled paper that is unwinding at a set rate. The rate of

oxygen consumption is measured by the slope of the line

traced by the pen. If the rate of oxygen consumption in­

creases or decreases, the slope of the pen trace will in­

crease or decrease. If, as in the case of mitochondrial

respiration, the respiration alternates from a resting state

of low oxygen consumption, to an active state of higher

respiration, depending on the presence or absence of a suit­

able substrate (ADP plus cofactors), the resulting trace

will be a line with greater and lesser slopes corresponding

to greater and lesser oxygen consumption respectively

(Figure 3). Four parameters of mitochondrial respiration

can be measured from a trace of oxygen consumption in the

presence and absence of ADP plus cofactors. The first is

25

100

z o ~

O'§ ~ UJ

~

O(KG

l-t 1 MINUTE

~~----------------------------------------------TIME

Figure 3. Typical oxygraph trace of mitochondrial respiration.

~ m

the rate of oxygen consumption in the presence of ADP plus

cofactors, or state 3 respiration. The second is the rate

of oxygen consumption after the ADP added previously is ex­

hausted, or state 4 respiration. The third parameter that

can be measured is the ratio of state 3 to state 4 respira-

tion, the respiratory control ratio. The fourth parameter

is a measure of the amount of oxygen consumption per fixed

amount of exogenous ADP added, called the ADP:O ratio.

In addition to simple value comparisons of these

parameters between Pima 5-5 and Pima E-l4, the state 3 and

state 4 respiration parameters can be graphically analysed

through the use of an Arrhenius plot to yield information

concerning the temperature at which the mitochondrial mem­

branes are thought to undergo a phase change from a liquid-

crystalline structure to a solid gel.

These four' parameters of mitochondrial respiration

will be compared and contrasted between'the Pima 5-5 and

Pima E-l4 to suggest a physiological basis for the greater

27

performance of Pima E-l4 over Pima 5-5 at high altitudes/low

temperatures.

Cool Temperature Germination and Growth

Following incubation at laC for six days, the Pima

E-l4 cottonseeds exhibited greater germination and growth

compared to the two Pima 5-5 seed lots. Pima E-l4 had a

mean germination percentage of 94.75 compared to 60.25 for

the Pima S-5 used for mitochondrial extraction. The bulk

Pima S-5 seed lot had a germination percentage of 81.5

28

(Table 2). The Pima E-14 cottonseed also had greater radicle

fresh weight and dry weight than either Pima S-5 seed lot al­

though these differences were significant only for the

radicle fresh weights of the Pima E-14 seed lot compared to

the bulk Pima S-5 seed lot. These results are a demonstra­

tion of the Pima E-14 cottonseeds' superior tolerance to

chilling temperature compared to Pima S-S cottonseed.

Electrophoresis

Photographs of the pH 8.9 acry1amide gels of Pima

S-5 and Pima E-14 cottonseed proteins can be seen in Plates

1 and 2. The gels in Plate 1 have been stained for total

proteins with .025% (w/v) coomassie blue. The gels in

Plate 2 have been stained to reveal esterase activity. Gels

stained with coomassie blue show only slight differences in

banding pattern such as presence or absence of bands, that

is their "protein fingerprints" are nearly identical. The

gels stained for esterase do show some discrete differences.

Respiration Parameters

State 3 Respiration

The state 3 respiration rates increase significantly

with temperature at each tr:::mperature level for both varieties

plate 1. Urea soluble proteins stained with coomassie blue G-250.

Plate 2. Urea soluble proteins stained for es:terase activity.

29

Table 2. Germination and growth of Pima S-5 and Pima E-14 following incubation at 18C for six days.

Fresh wt. Dry wt.

30

Germination 20 radic1es 20 radic1es - -% + sx g + sx g + sx

Pima S-5 bulk sample 81. 5 + 5.5 1.0 + .09 .09 + .01

Pima E-14 94.75 + 3.5 1.7 + .49 .13 + .05

Pima S-5 60.25 + 9.9 1.2 + .17 .09 + .02

(Table 3). At all temperature levels the Pima S-5 exhibits

significantly greater respiration than the Pima E-14. The

mean state 3 respiration rate across all temperatures is

17.9~M 02/min/g tissue for Pima S-5 and 13.4~M 02/min/g

tissue for the Pima E-14.

State 4 Respiration

Like the state 3 respiration rates, the state 4

respiration rates· increase with increasing temperature, but

the increases are not significant with each temperature in­

crease (Table 4). At each temperature, the Pima S-5 exhibits

significantly greater state 4 respiration than Pima E-14

with mean respiration rate of 11.7~M 02/mir,Vg tissue for

the Pima S-5 and 7.3~M 02/minfg tissue for ·the Pima E-14.

31

Table 3. The increase of Pima S-5 and Pima E-14 mitochondrial State 3 respiration with increasing temperature.

Assay Temp. Pimaa S-5 Pima E-14 (e) uMo2/min/g tissue

6 8.04 a 6.37 a

8 10.46 b 8.20 b

10 13.73 c 9.94 c·

12 16.90 d 12.12 d

14 19.59 e 14.93 e

16 22.67 f 19~62 f

18 30.18 g 21.01 g

Means in columns followed by the same letter are not sig­nificantly different at the .05 level according to a Duncans Multiple Range Test.

Respiration ,Efficiency Index

As mitochondria are the primary site of cell respi-

ration and ~he function of mitochondrial respiration is ATP

formation [46], one could assume that a greater rate of

mitochondrial respiration would result in greater ATP form-

ation and higher germination and growth rates. The data of

this present study ,do not support this concept. A possible

reason for this lies in the partitioning of mitochondrial

respiration. Mitochondrial respiration involves both cata-

bolic and 'anabolic processes; catabolic processes including

32

Table 4. The increase of Pima S-5 and Pima E-14 mitochondrial State 4 respiration with increasing temperature.

Assay Temp. Pima S-5 Pima E-14 (C) llM02!min!g tissue

6 6.65 a 4.06 a

8 7.52 ab 5.08 ab

10 9.55 bc 5.98 bc

12 10.92 cd 6.85 cd

14 12.00 d 7.80 d

16 13.94 e 9.21 e

18 18.79 f 11.10 f

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

obtain measurable respiratio~restricting respiration measure­

ments to only one temperature per gradient centrifigation.

However, there is some evidence to suggest that mitochondrial

respiration is severely limited at temperatures below that at

which membrane phase change occurs (Appendix 1). The nearly

63

level slopes shown by Pima E-l4 state 4 mitochondria respira­

tion (Figures 7 and 9) lend some support to this conjecture

although the same results were not obtained with Pima E-l4

state 3 mitochondrial respiration or with Pima S-5 state 3

or state 4 mitochondrial respiration.

If mitochondrial respiration is severely limited at

temperatures below that required for mitochondrial membrane

phase change and the respiration at these temperatures is

found to be due to non-mitochondrial enzymatic activity such

as lipoxygenase, this is indirect evidence of ATP utilization

in the absence of ATP production. Stewart and Guinn [77]

observed that cotton seedlings would recover from one day of

chilling but not two. Thus, at chilling temperature the

cotton seedlings would take up oxygen in nonmitochondrial

respiration, a utilization of ATP without production. After

one day the ATP pool would be sufficient to maintain meta­

bolic processes. However, after two days of chilling the

ATP pool would be depleted and metabolic imbalances occur.

Raison [64] stated that an aspect of chilling injury is the

metabolic imbalance that occurs when respiratory activity

(i.e., ATP production) cannot compensate for the increase in

enzyme activation energy caused by membrane phase change at

low temperature.

The temperature at which membranes undergo a phase

change is determined to a large extent by the ratio of

64

unsaturated fatty acids to saturated fatty acids making up

the lipids of the membranes °[78]. Bartkowski et ale [8]

found that the field emergence of Pima cotton varieties under

low soil temperatures was correlated to the unsaturated/satu­

rated fatty acid ratios of the total seed. Pima E-2, a fore­

runner of Pima E-14, had the highest ratio, 2.81, and had the

highest field emergence. Pima S-4, used as one parent in

the development of Pima S-5, pad theOlowest."ratio of 2.43 and

also showed the lowest field emergence. Bartkowski [9] re­

pcYr.ted a fatty acid ratio of 2.46 for Pima S-5,; very close

to that of Pima S-4. It is this difference in unsaturated

to saturated fatty acid ratios that is responsible for the

difference in the temperature of membrane phase change

demonstrated by the Arrhenius plots of mitochondrial respira­

tion as affected by temperature. Pima E-14, the variety with

the higher unsaturated/saturated fatty acid ratio has a lower

break point temperature for its mitochondrial membranes than

Pima S-5, the variety with the lower unsaturated/saturated

fatty acid ratio.

The unsaturated fatty acid considered important to the

maintenance of membrane fluidity is linolenic acid, although

loinleic acid has been implicated in alfalfa (Medicago

sativa L.) [33] and corn (Zea mays L.) [35]. Low temperature

apparently induces the synthesis of unsaturated fatty acids

in accumulating seed. Harris and James [37] demonstrated

65

that unsaturated fatty acids increase in castor bean, sun­

flower, and flax at reduced temperatures and stated that the

increased solubility of oxygen at low temperatures caused

this increased synthesis. However Marten et ale [51] stated

that the fatty acid desatruase activity was regulated by

membrane fluidity and not temperature per~. In either case

cold acclimation has been shown to increase the unsaturated/

saturated fatty acid ratios in membrane fluidity -to a lower

temperature.

The literature also suggests that there can be dif­

ferences in the enzymes associated with the cellular mem­

branes between varieties as well as differences in the fatty

acid composition of cellular membranes. Grimwood and

McDaniel [34] reported variant malate dehydrogenase isoen­

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.

REFERENCES

1. A1exandrescu, Victoria, 1974. Water soluble proteins from maize in the first growing stages: electrophoretic and immonoe1ectric study. Rev. Roum. Bichim, 11, 2, 77-85.

2. Amin, J. V., 1969. Some aspects of respiration and respiration inhibitors in low temperature effects of the cotton plant. Physio1. Plant. 22: 1184-1191.'

3. Anderson, J. D., 1977. Responses of adenine nuc1eotides in germinating soybean axes to exogenously applied adenine and adenosine. Plant Physio1. 60: 689-692.

4. Arndt, C. H., 1935. A study of some of the factors that may influence cottonseed germination and seed­ling growth. South Carolina Agr. EXp. Sta. Report 45: 46-49.

5. Axelrod, B., 1974. Lipoxygenase~ in Food Related Enzymes. Am. Chem. Soc., Wash., D-.-C., 324-348.

6. Bagnall, D. J. and J. Wolfe, 1982. Arrhenius plots: information or noise? 'Cryo-1etters l, 7-16.

7. Baldwin, J. and P. W. Hochachka, 1970. Functional significance of isoenzymes in thermal acclimation. Acty1cho1inesterase from trout brain •. Biochem~ J. 116: 883-887.

8. Bartkowski, E. J., D. R. Buxton, F. R. H. Katterman and H. W. Kircher, 1977. Dry seed fatty acid compo­sition and seedling emergence of Pima cotton at low soil temperatures. Agron. J. 69: 37-40.

9. Bartkowski, E. J., 1978. Mechanisms of chilling injury in cotton. Ph.D. Dissertation, University of Arizona.

10. Brqm1age, W. J., A. C. Leopold and D. J. Parrish, 1978. Chilling stress to soybeans during inhibition. Plant Physio1. 61: 525-529.

11. Brewbaker, J. L., M. D. Upadhya, Y. Makunen and T. MacDonald, 1968. Isoenzyme polymorphism in flowering

77

plants III. Gel electrophoretic methods and applica­tions. Physiol. Plant., 21: 930-940.

12. Buxton, D. R., P. J. Sprenger and E. J. Pege1ow, 1976. Periods of chilling sensitivity in germinating Pima cottonseed. Crop. Sci. 16: 471-474.

13. Buxton, D. R. and P. J. Sprenger, 1976. Genetic variability for cottonseed germination at favorable and low temperatures. Crop Sci. 16: 243-246.

78

14. Casey., E. J., 1962. Biophysics-Concepts and Mechanisms. Van Nostrand-Reinho1d~ 195-199.

15. Centner, M. S., 1977. The effect of pregermination seed treatments on the germination and growth of Aca1a 1517-70 cottonseed at sub-optimal temepratures. M. S. Thesis, New Mexico State University.

16. Cherry, oJ. P., F. R. H. Katterman and J. E. Endrizzi, 1970. Comparative studies of seed proteins of species of Gossypium by gel electrophoresis. Evolution 24: 431-447.

17. Ching, T. M., 1973. Adenosine triphosphate content and seed vigor. Plant Physiol. 51: 400-402.

18. Ching, T. M. and K. K. Ching, 1972. Content of adeno­sine phosphates and adeny1ate energy charge in germinating ponderosa pine seedlings. Plant Physio1. 50: ·536-540.

19. Christiansen, M. N., 1963. Influence of chilling upon seedling development of cotton. Plant Physio1. 38: 520-522.

20. Christiansen, M. N., 1963. Periods of sensitivity to chilling in germinating cotton. Plant Physio1. 43: 431-433.

21. Christiansen, M. N., 1968. Induction and prevention of chilling injury to radicle tips of imbibing cotton-seed. Plant Physio1. 43: 743-746. .

22. Christiansen, M. N., 1969. Seed moisture content and chilling injury to imbibing cottonseed. Proc. 1969 Be1twide Cotton Prod. Res. Conf., 50-51. Pub1. Na tiona1 Cotton Council, r.1emphis, Tenn.

79

23 •. Christiansen, M. N., H. R. Carns and D. J. Slyter, 1970. Stimulation of solute loss from radic1es of Gossypium hirsutum L. by chilling, anaerobiosis, and low pH. Plant Physio1. 46: :53-56.

24. Christiansen, M. N. and C. D. Foy, 1979. Fate and function of calcium in tissue. Comm. in Soil Science and Plant Analysis 10, (1 & 2): 427-442.

25. Clay, W. F., F. R. Katterman and J. R. Hammett, 1975. Nucleic acid metabolism during germination of Pima cotton (Gossypium barbadense L.). Plant Physio1. 5.5: 231-236.

26. Cohn, M. A. and R. L. Obendorf, 1978. Occurance of a ste1ar 1eison during imbibitiona1 chilling of Zea mays L. Am. J. Botany 65: 50-56. ---

27. Devay, M. and E. Pa1di, 1977. Cold induced rRNA synthesis in wheat cu1tivars during the hardening period. Plant Science Letters 8: 191~195

28. Duke, S. H., L. Schrader and M. G. l-li11er, 1970. Low temperature effects on Soybean (Glycine max [L.] Merr. cv. Wells) mitochondrial respiration and several de­hydrogenases during inhibibition and germination. Plant Physio1. 60: 716-722.

29. Ernster, L. and R. Luft, 1964. Mitochondrial respira­tory control: biochemical, physiological, and patho­logical aspects. in Advan. Metab. Disord. 1: 95-123.

30. Estabrook, R. W. and M. E. Pullman, 1967. Oxidation and Pho~phory1ation. in r1ethods in EnZj~ology X: 41-47: S. P. C010\,lick and N •. 0. Kaplan eds. ~cademic Press, New York.

31. Feaster, C. u., E. L. Turcotte and E. F. Young, Jr., 1976. Registration of Pima S-5 cotton. (Reg. No. 60). Crop Sci. 16:604.

32. Goldstein, A. H., J. o. Anderson and R. G. McDaniel, 1980. Cyanie-insensitive and cyanid-sensitive 02 . update in wheat. I. Gradient-purified mitochondria. Plant Physio1. 66: 488-493.

33. Grenier, G. and C. Willemot, 1974. Lipid changes in roots of frost hardy and less hardy alfalfa under hardening conditions. Cryobiology 11: 324-331.

34. Grimwood, B. G. and R. G. McDaniel, 1970. Variant malate dehydrogenase iso,zymes in mitochondrial populations. Biochimica et Biophysica Acta. 220: 410-415.

35. Gubbles, G. H., 1974. Growth of corn seedlings under low temperature as affected by genotype, seed size, total oil and fatty acid content of the seed. Can. J. Plant Sci. 54: 425-426.

36. Guinn, G., 1979. Changes in sugars, starch, RNA, protein, and lipid soluble phosphate in leaves of cotton plants at low temperature. Crop Sci. 11: 262-264.

37. Harris, P. and A. T. James, 1969. Effect of low tem­perature on fatty acid biosynthesis in seeds. Bio­chimica et Biophysica Acta. 187: 13-18.

38. Hobbs, P. R. and R. L. Obendorf, 1972. Interaction of initial seed moisture and imbibitional temperature on germination and productivity of soybean. Crop Sci. 12: 664-667.

80

39. Ilker, R., A. J. Waring, J. M. Lyons and R. W. Briedenbach, 1976. The ~hysiological responses of tomato-seedling colyledons to chilling and the in­fluence of membrane modifications upon these responses. Protoplasma 90: 229-252.

40. Keiner, C. M. and W. J. Bramlage, 1981. Temperature effects on the activity of the alternative respiratory pathway in chill-sensitive Cucumis sativus Physiol. 68: 1474-1478.

41. Kidd, F. and C. West, 1927. Physiological pre­determination: the influence of the physiological condition of the seed upon the course of subsequent growth and upon the yield. IV. Review of Literature, Chapter III. Ann. Appl. BioI.

42. Killion, D. D., 1969. Response of cotton and soybean mitochondria to inhibitors and phytotoxicants. Ph. D. Dissertation, University of Arkansas.

43. Kuiper, P. J. C., 1972. Temperature responses of ATPase of bean roots as related to growth temperature and to lipid requirement of ATPase. Physiol. Plant. 26: 200-205.

44. Lee, A. G., 1975. Interactions within biological membranes. Endeavour, May, Vol. XXXIV.

45. Lehman, S. G., 1925. Studies on the treatment of cottonseed. North Carolina Agr. Exp. Sta. Tech. Bull. 26.

46. Lehinger, A. L., 1975. Biochemistry, 2nd ed. Worth Publishers Inc., New York.

47. Ludwig, C. A., 1932. The germination of cottonseed at low temperature. Journal of Agricultural Research 44,4: 367-380.

48. Lyons, J. M., D. Graham and J. K. Raison, 1979. Low temperature stress in crop plants: the role of the membrane. Academic Press, New York.

49. Lyons, J. M., J. K. Raison and J. Kumamoto, 1981. Polarographic determination of phase changes in mito­chondrial membranes in response to temperature. in Methods in Enzymology XXXII: 258-262. S. Fleischer and L. Packer, eds. Academic Press, New York.

50. Lyons, J. M., T. A. Wheaton and H. K. Pratt, 1964. Relationship between the physical nature of mito­chondrial membranes and chilling sensitivity in plants. Plant Physiol. 39: 262-268.

81

51. Martin, C. E., K. Hiramitsu, Y. Kitajima, V. Nozawa, L. Skriver and G. A. Thompson, Jr., 1976. Molecular control of membrane properties during temperature acclimation. Fatty acid desaturase regulation of membrane fluidity in acclimating Tetrahymena. cells. Biochemistry 15,24: 5218-5227.

52. McCland, R. W. and H. M. Kolenbrander, 1974. Resolu­tion of temperature dependent conformers of histidine ammonia-lyase on disc gel electrophoresis: correlation with Arrhenius discontinuities. Experimentia 30/7. 15.7. 1974. 730-731.

53. McDaniel, R. G., 1970. of proteins in Hordeum.

Electrophoretic characterization J. of Heredity 61: -243-247.

54. McDaniel, R. G., 1978. Annual research report to the American Seed Research Foundation. Search, pp. 1-7

55. Myers, B. D. and G. O. Throneberry, 1966. Effect of gossypol on some oxidative respiratory enzymes. Plant Physio1. 41: 787-791.

56. Mohamed-Osman, A. M., 1973. Comparative studies of physiological and mitochondrial photoresponses in soybean (Glycine max L. Merr.) cu1tivars. Ph. D. Dissertation, University of Arizona.

57. Nobel, P •. S., 1974 •. Temperature dependence of the" permeability of chloroplasts from yhi11ing-sensitive and chilling-resistant plants. P1anta (Ber1.) 115: 369-372.

58. Holan, W. G., 1980. Effect of temperature on electron transport activities of isolated chloroplasts. Plant Physio1. 66: 234-237.

82

59. Ornstein, L. and B. J. Davis, 1962. Disc e1ectro­phor.esis. Distillation Products Industries, Rochester, N. Y.

60. Over1ath, P. and L. Thi10, 1978. structural and func­tional aspects of biological membranes revealed by lipid phase transitions. in International Review of Biochemistry. BiochemistrY-of Cell Walls and Membranes, II. Vol. 19. J. C. Metcalf, ed. University Park Press, Baltimore.

61. Panday, K. K., 1972. Isozyme specific~ty to tempera­ture. Nature New Biology 239: 27-28.

62. Peoples, T. R., D. W. Koch and S. C. Smith, 1978. Re­lationship between chloroplast membrane f~tty acid composition and photosynthetic response to a chilling temperature in four alfalfa cu1tivars. Plant Physio1. 61: 472-473.

63. Pollack, B. M. and V. K. To~le, 1966. Inbibition period as the critical temperature sensitive stage in germination of lima bean seed. Plant Physio1. 41: 221-229.

64. Raison, J. K., 1980. Effect of low temperature on respiration. Chapter 15 in The Biochemistry of Plants, Vol. 2. Academic Press.

65. Raison, J. K., J. M. Lyons, R. J. Mehlhorn and A. D. Keith, 1971. Temperature-induced phase change in mitochondrial membranes detected by spin-labeling. J. of Biologi-cal Chemistry 246, 12: 4036-4040.

66. Raison, J. K., J. M. Lyons and W. W. Thomson, 1971'.

83

The influence of membranes of the temperature-induced changes in the kinetics of some respiratory enzymes of mitochondria. Archives of Biochemistry and Biophysics, 142: 83-90.

67. Rosinger, C. H., J. M. Wilson and M. W. Kerr, 1982. Changes in the temperature response of Hill reaction activity of chilling sensitive and chilling resistant plants after hardening. J. Exper. Bot. 33: 321-331.

68. st. John, J. B. and M. N. Christansen, 1976. Inhibition of linolenic acid synthesis and modification of chilling resistance in cotton seedlings. Plant Physiol. 57: 257-259.

69. Schmidt, K. R., 1973. Characterization of mitochondrial populations and mitochondrial DNA in cotton (Gossypium barbadense L.). M. S. Thesis, University of Arizona.

70. Silvius, J. R., B. D. Read and R. N. McElhaney, 1978. Membrane enzymes: Artifacts in Arrhenius plots due to temperature dependence of substrate-binding affinity. Science 199: 902-904.

71. Simon, E. W., 1974. Phospholipids and plant membrane permiabil~ty. New Phytol. 73: 377-420.

72. Simon, E. W., 1978. Plant membranes under dry condi­tions. Pestic. Sci. 9: 169-172.

73. Simon, E. ~v. and H. H. Wiebe, 1975. Leakage during imbibition, resistance to damage at low temperature and water content of peas. New Phytol. 74: 407-411.

74. Singer, S. J. and G. L. Nicolson, 1972. The fluid mosaic model of the structure of cell membranes. Science 175: 720-731.

75. Somers, G.-N., 1975. in Isoenzymes. II Pysiological function. C. L. Marke~ Academic Press, N. Y.

76.

77.

78.

stewart, J. McD. and G. Guinn, 1969. Chilling injury and changes in adenosine triphosphate of cotton seedlings. Plant Physio1. 44: 605-608.

Stewart, J. McD. and G. Guinn, 1971. Chilling injury and nucleotide changes in young cotton plants. Plant Physio1. 48: 166-170. '

84

Thompson, T. E. and C. Huang, 1978. Dynamics of lipids in biomembranes. Chapter 2 in Physiology of Membrane Disorders. T. E. Andreoli M-.-D., J. F. Hoffman, Ph. D. and D. D. Fanesti11 M.D., eds. Plenum Publishing Corporation.

79. Wade, N. L., R. W. Breidenbach, J. M. Lyons, A. D. Keith, 1974. Temperature induced phase changes in ' the membranes of glyoxysomes, mitochondria, and prop1astids from germinating castor bean endosperm. Plant Physio1. 54: 320-323.

80. Waring, A. J., R. W. Breidenbach and J. M. Lyons, 1976. In vivo modification of plant membrane phospholipid composition. Biochimica et Biophysica Acta. 443: 157-168.

81. Wilson, J. M., 1978. Leaf respiration and ATP levels at chilling temperatures. New Phytol. 80:325-334.

82. Young, E. F., Jr., C. V. Feaster and E. L. Turcotte, 1976. Registration of Pima S-5 cotton. (Reg. No. 58.) Crop Sci. 16: 604.