3 - repositories

COOL TEMPERATURE-INDUCED CHANGES IN METABOLISM

RELATED TO CELLULOSE SYNTHESIS IN COTTON FIBERS

by

LAUAL KIRT MARTIN, B.S., M.S.

A DISSERTATION

IN

BIOLOGY

Submitted to the Graduate Faculty of Texas Tech University in

Partial Fulfillment of the Requirements for

the Degree of

DOCTOR OF PHILOSOPHY

Approved

Accepted

May, 1999

ACKNOWLEDGEMENTS

I would like to thank Dr. Candace H. Haigler for her never-ending guidance,

encouragement, patience, and instmction in the work and preparation of this study and

this paper. I want to thank her for her example of perfection, excellence, and integrity

that she strives for and has well demonstrated to me and other students at Texas Tech

University. I would also like to thank Drs. A. Scott Holaday and Larry Blanton for their

guidance and assistance in laboratory procedures. I also appreciate the guidance and

encouragement of Drs. A. Scott Holaday, Daniel R. Krieg, Michael San Francisco, and

James R. Mahan as members of my graduate committee.

I also appreciate the guidance and assistance of my co-workers Dr. Jim Taylor, Dr.

Linda Koonce, Dr. Wuzi Xie, and Nirmali Perrera. They have given many hours in

helping me with laboratory procedures to which I am tmly grateful.

Most of all, I am grateful to my wife Nancy and my daughters, Lucy and Livia, for

their love, patience, support, and understanding throughout all of the many years I have

been working on this degree. They have made numerous sacrifices and have given much

to the accomplishment of this work that can never be completely realized nor repaid.

11

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

ABSTRACT vii

LISTOFTABLES viii

LIST OF HGURES ix

LIST OF ABBREVIATIONS xi

CHAPTERS

I. INTRODUCTION AND LITERATURE REVIEW 1

1.1 The Cotton Fiber 1

1.1.1 An Overview of Fiber Development 1

1.1.2 Primary Cell Wall Synthesis 1

1.1.3 Secondary Cell Wall Synthesis 1

1.2 Cool Temperature and Fiber Development 2

1.2.1 Field Suidies 2

1.2.2 In Vitro Studies 3

1.2.3 Implications of Cool Temperature In Vitro Studies 5

1.3 Cellulose Biosynthetic Pathways in the Cotton Fiber 7

1.3.1 Previously Proposed Pathway of Cellulose Synthesis 7

1.3.2 Membrane-Associated Sucrose Synthase 7

1.3.3 Sucrose Movement to the Fiber Cell 8

1.3.4 Hexose Sugar Movement to the Fiber Cell 10

1.3.5 Additional Considerations of Carbon Movement 10

1.4 Metabolite Pools in the Cotton Fiber Related to Sue and Glc Metabolism and Cellulose Synthesis 12

1.5 Enzyme Activities in Fiber Cells Related to Sucrose and

Glucose Metabolism and Cellulose Synthesis 15

1.5.1 Invertase Activity 15

1.5.2 Glucokinase Activity 18

1.5.3 Sucrose Synthase Activity 18

iii

1.5.4 UDP-Glucose Pyrophosphorylase 19

1.5.5 Sucrose Phosphate Synthase Activity 20

1.5.6 Glucose 6-Phosphate Dehydrogenase Activity 20

1.5.7 Phosphoglucoisomerase 21

1.5.8 Phosphofmctokinase 21

1.5.9 Malate Dehydrogenase 21

1.5.10 Peroxidase 23

1.5.11 Overview of Enzyme Activity in the Cotton Fiber 23

1.6 Cool Temperatures and Plant Metabolism 24

1.6.1 Reduced Growth and Photosynthesis 24

1.6.2 Hexose Phosphate Increases and Enzyme Activities 24

1.6.3 Reduced Respiration at Cool Temperatures 27

1.7 Cool Temperatures and Fiber Metabolism 28

1.8 Research Objectives 28

n. EFFECTS OF COOL TEMPERATURE ON METABOLISM POOLS

IN HBERS OF CULTURED OVULES 29

2.1 Introduction 29

2.2 Materials and Methods 30

2.2.1 Plant Growth 30

2.2.2 Ovule Culture 31

2.2.3 Fiber Removal and Extraction 32

2.2.4 Enzymic Analysis 33

2.2.5 High Pressure Liquid Chromatography (HPLC) 33

2.2.6 Radiolabeling of Ovule Cultures 33

2.3 Results 34

2.3.1 Hexose Phosphates and UDP-Glc at 1, 4, and 7 h 34

2.3.2 Glc 6-P and Temperature 42

2.3.3 Fm 6-P and Temperature 44 2.3.4 UDP-Glc and Temperature 44

2.3.5 Glc 1-P and Temperature 47

IV

2.3.6 Cellulose Synthesis and Respiration Rates In

Acala SJ-1 and Paymaster HS200 48

2.3.7 HPLC Separation of Fiber Sue, Glc, and Fm 53

2.3.8 Sue Pool Sizes At 15°C Compared to 34°C 56

2.3.9 Glc and Fm Pool Sizes At 15°C Compared to 34°C 56

2.3.10 Sugar Pool Sizes From 18 to 24 DPA 56

2.3.11 Fiber Dry Weight Per Ovule 63

2.3.12 Comparisons Among Cultivars 63

2.3.12.1 Acala SJ-1 63

2.3.12.2 Coker 312 66

2.3.12.3 TM-1 66

2.3.12.4 JDX 69

2.3.12.5 Paymaster HS200 69

2.3.12.6 YG 72

2.3.12.7 Paymaster HS26 72

2.4 Discussion 75

2.4.1 Hexose Phosphates and Cool Temperature 75

2.4.2 UDP-Glc and Cool Temperature 76

2.4.3 Sugar Pool Sizes and Secondary Cell Wall Synthesis 77

2.4.4 Secondary Cell Wall Synthesis in Cultured Ovules 78

m. SPECIFIC ACTIVITY OF SUGAR POOLS DURESIG

SECONDARY CELL WALL SYNTHESIS 80

3.1 Introduction 80

3.2 Materials and Methods 81

3.2.1 Cotton Plant Growth 81

3.2.2 Cotton Ovule Culture 82

3.2.3 Radiolabeling of Ovules 82

3.2.4 Sugar Extraction From Top Fibers 83

3.3 Results 84

3.3.1 Carbon Entry into Top Fibers 84

3.3.2 Pulse Labeling of Acala SJ-1 84 V

3.3.3 Pulse Labeling of Paymaster HS200 86

3.3.4 Pulse/Chase Labeling of Acala SJ-1 86

3.3.5 Pulse/Chase Labeling of Paymaster HS200 89

3.4 Discussion 89

3.4.1 Sucrose Synthesis Within the Fiber 89

3.4.2 Paymaster HS200 Synthesizes Sue More EfficienUy 91

3.4.3 Possible Enzymic Restriction at Cool Temperature 92

3.4.4 Importance of the Pathways Involving Sue Synthesis 92

rv. CONCLUSIONS 94

REFERENCES 96

VI

ABSTRACT

Fiber secondary wall thickening associated with cellulose synthesis in cotton

(Gossypium hirsutum L.) is greatly hindered under the cool night temperatures that occur

during the growing season on the Southem High Plains of West Texas. Fibers on

cultured cotton ovules exposed to a 34/15°C temperature cycle during secondary wall

synthesis were used as an experimental system to investigate the biochemical causes of

this hindrance. Pool sizes of the metabolites related to cellulose synthesis were measured

at cool temperature (15°C) and at the optimum temperature for fiber cellulose synthesis

(34°C) in the fibers of cultured ovules from seven cultivars.

Glucose 6-phosphate levels increased during the 15°C part of the temperature cycle,

with 40- 70% increases observed in all cultivars compared to 34°C. Partially cool-

tolerant cultivars (such as Paymaster HS2(X)) demonstrated the ability to increase glucose

6-phosphate levels greatly at 15°C on the first day of exposure to cycling temperature;

however, more cool-sensitive cultivars (such as Acala SJ-1) did not demonstrate this

ability until 6 days after the cycling temperature commenced. Fmctose 6-phosphate

levels at 15°C within the fiber gradually increased above that at 34°C during the 6 days

of cycling.

UDP-Glucose levels decreased or only slightly increased at 15°C during the 6

days of cycling, although glucose 6-phosphate levels increased dramatically. Therefore,

a possible enzymic restriction at cool temperatures may occur subsequent to glucose 6-

phosphate in the cellulose synthesis pathway. Pulse/chase studies supported the

hypothesis of an enzymic restriction subsequent to glucose 6-phosphate, with partially

cool-tolerant Paymaster HS200 showing less restriction than more cool-sensitive Acala

SJ-1.

Vll

LIST OF TABLES

1.1 Cellulose Synthesis and Respiration at 15°C and 22°C 6

1.2 Summary of Reported Quantitative Changes in Hexose Sugar Pools of Cotton Fibers 16

2.1 Amount of Glc 6-P at 15°C and 34°C at 4-7 Hours Into Cycling and 15°C Percent Increase Over 34°C 43

2.2 Amount of Fm 6-P at 15°C and 34°C at 4-7 Hours Into Cycling and 15°C Percent Increase Over 34°C 45

2.3 Amount of UDP-Glc at 15°C and 34°C at 4-7 Hours Into Cycling and 15°C Percent Increase Over 34°C 46

2.4 Amount of Sucrose at 15°C and 34°C at 4-7 Hours Into Cycling and 15°C Percent Increase Over 34°C 57

2.5 Amount of Glucose at 15°C and 34°C at 4-7 Hours Into Cycling and 15°C Percent Increase Over 34°C 58

2.6 Amount of Fmctose at 15°C and 34°C at 4-7 Hours Into Cycling and 15°C Percent Increase Over 34°C 59

vm

LIST OF HGURES

1.1 Diagram of Carbon Movement Within the Fiber Cell If Carbon Enters the Cell by Sucrose Only 9

1.2 Diagram of Carbon Movement Within the Fiber Cell If Carbon Enters the Cell by Glucose Only 11

1.3 Diagram of Carbon Movement Within the Fiber Cell Considering

Both Glucose and Sucrose Import 13

1.4 Changes in Enzyme Activities During Cotton Fiber Cell Development 17

2.1 Metabolite Levels from Two Replications of Acala SJ-1 35

2.2 Metabolite Levels from Two Replications of Coker 312 36

2.3 Metabolite Levels from Two Replications of TM-1 37

2.4 Metabolite Levels from Two Replications of JDX 38

2.5 Metabolite Levels from Two Replications of Paymaster HS200 39

2.6 Metabolite Levels from Two Replications of YG 40

2.7 Metabolite Levels from Two Replications of Paymaster HS26 41

2.8 Cellulose Synthesis Rates in the Fibers of Acala SJ-1 and Paymaster HS200 . . . 49

2.9 Respiration Rates in the Fibers of Acala SJ-1 and HS200 at 34°C 50

2.10 Cellulose Synthesis Rates in the Fibers of Coker 312 and YG Compared to Paymaster HS200 and Acala SJ-1 51

2.11 Respiration Rates in the Fibers of Coker 312 and YG Compared to Paymaster HS200 and Acala SJ-1 52

2.12 Cellulose Synthesis and Respiration Rates Compared to Metabolite Levels of Acala SJ-1 at 34°C 54

2.13 Cellulose Synthesis and Respiration Rates Compared to Metabolite Levels of Paymaster HS200 at 34°C 55

2.14 Changes in Sugar Levels at 15°C and 34°C From 16 to 24 DPA In Acala SJ-1, Paymaster HS200, Coker 312, and YG 60

2.15 Comparison of Glucose Levels at 15°C and 34°C for Acala SJ-1,

Paymaster HS200, and Coker 312 62

2.16 Fiber Weight Per Ovule 64

2.17 Metabolites and Sugars in Fibers of Acala SJ-1 65

2.18 Metabolites and Sugars in Fibers of Coker 312 67

ix

2.19 MetaboUtes in Fibers of TM-1 68

2.20 Metabolites in Fibers of JDX 70

2.21 Metabolites and Sugars in Fibers of Paymaster HS200 71

2.22 Metabolites and Sugars in Fibers of YG 73

2.23 Metabolites in Fibers of Paymaster HS26 74

3.1 Changes in the Specific Activity of Sugar Pools of Acala SJ-1 During Pulse Labeling wiUi [ C]Glc 85

3.2 Changes in the Specific Activity of Sugar Pools of Paymaster HS200 During Pulse Labeling with ['' CJGlc 87

3.3 Changes in the Specific Activity of Sugar Pools of Acala SJ-1 During Pulse/Chase Labeling with [ ' CJGlc 88

3.4 Changes in the Specific Activity of Sugar Pools of Paymaster HS200 During Pulse/Chase Labeling with [ ' CJGlc 90

LIST OF ABBREVIATIONS

ATP-PFK

DPA

Fm

Fm6-P

Fm 1,6-BP

GK

Glc

Glc 1-P

Glc 6-P

Glc 6-PDH

iP

MDH

NADPH

PGI

POD

PPi

PPi-PFK

RuBisCO

SJ-1

SPP

SPS

Sue

SuSy

TM-1

Triose P

UDP-Glc

ATP-dependent phosphofmctokinase

Days Post-Anthesis

Fmctose

Fmctose 6-Phosphate

Fmctose 1,6-BisPhosphate

Glucose kinase

Glucose

Glucose 1-Phosphate

Glucose 6-Phosphate

Glucose 6-Phosphate dehydrogenase

inorganic phosphate

Malate dehydrogenase

Reduced nicotinamide adenine dinucleotide phosphate

Phosphoglucoisomerase

Peroxidase

Inorganic pyrophosphorylase

Pyrophosphate: fmctose 6-Phosphate 1-phosphotransferase

Ribulose 1,5-bisphosphate carboxylase oxygenase

Acala SJ-1

Sucrose phosphate phosphatase

Sucrose phosphate synthase

Sucrose

Sucrose Synthase

Texas Marker-1

Triose Phosphate

Uridine diphosphoglucose

UDPG-PP UDP-Glc pyrophosphorylase

XI

CHAPTER I

INTRODUCTION AND LITERATURE REVIEW

1.1 The Cotton Fiber

1.1.1 An Overview of Fiber Development

Cotton (Gossypium hirsutum L.) is the most important crop plant on the Southem

High Plains of West Texas. More acres are planted in cotton than any other crop, yielding

approximately 2 million bales of cotton lint to the area each year. The cotton fiber is an

elongated epidermal cell of the cotton ovule with a thickened secondary cell wall containing

almost pure cellulose (Haigler et al., 1991). The development of the fiber is completed in

42-58 days post anthesis (dpa) (Haigler, 1985) and is characterized by 2 partly overlapping

phases, elongation accompanied by primary wall synthesis and fiber thickening due to

secondary cell wall synthesis (Ryser, 1985). Secondary wall synthesis begins at 14-20

dpa and is characterized by the deposition inside the primary cell wall of cellulose

microfibrils oriented helically at a 20-30° angle to the longitudinal cell axis (Haigler, 1985;

Seagull, 1992). Wall thickening persists for 24-30 days (Haigler, 1985).

1 • 1.2 Primary Cell Wall Synthesis

Primary cell wall synthesis begins at anthesis and continues until 15-21 dpa in

Gossypium hirsutum L. as the fiber elongates and deposits a thin primary wall. Cellulose

microfibrils are deposited in a random orientation as the fiber cell begins to bulge and,

parallel to microtubules, reorient into flat helices as elongation continues (Seagull, 1992).

The 6-1,4 glucan chains within the cellulose microfibrils exhibit a lower average degree of

polymerization (or molecular weight) during primary wall synthesis as compared to

secondary cell wall synthesis (Timpa and Triplett, 1993). The primary cell wall contains

only 20-30% cellulose (Meinert and Delmer, 1977) with large amounts of glucose,

arabinose and uronic acid residues in the pectic and hemicellulose fractions and large

amounts of protein (Huwyler, Franz, and Meier, 1979).

1.1.3 Secondary Cell Wall Synthesis

Secondary cell wall synthesis is characterized by high rates (100-fold greater than

primary cell wall synthesis) of cellulose deposition inside the primary cell wall (Meinart and

Delmer, 1977). Microtubule orientation switches to a steeply pitched helical pattem

accompanied by a four fold increase in microtubule number (Seagull, 1992). The

secondary cell wall consists of 95% cellulose (Haigler, 1985) with a high degree of

1

polymerization (Timpa and Triplett, 1993). The amount of noncellulosic 6-1,3-glucan,

commonly referred to as callose, increases rapidly in the fiber at the onset of secondary cell

wall synthesis, then decreases as secondary cell wall deposition continues (Maltby,

Carpita, Montezinos, Kulow, and Delmer, 1979). Callose has both wound-defense and

developmental roles in other plant cells, but its exact function in the cotton fiber is

unknown, and reports differ on 6-1,3-glucan tumover in the cotton fiber as a possible

precursor to cellulose (Francey, Jaquet, Cairoli, Buchala, and Meier, 1989; Maltby et al.,

1979).

1.2 Cool Temperatures and Fiber Development

1.2.1 Field Studies

The effects of cool temperatures on cotton fiber development have been studied for

decades. Fibers exposed to cool temperatures (below about 25°C, which occurs generally

at night during the growing season on the Texas High Plains) have reduced overall rates of ^

secondary cell wall thickening (Gipson and Joham, 1968; Thaker, Saroop, Vaishnav, and

Singh, 1989). Diurnal "growth rings" are evident in the secondary walls corresponding to^

the reduced fiber thickening during the cool temperature periods (Grant, Orr, and Powell,

1966). Gipson and Joham (1968) reported the effects of low temperature on several

properties of the cotton fiber when exposed to night temperatures varying from 25°C to 5°C

during development Micronaire values of fibers harvested from open bolls were affected

the greatest, decreasing from 4.2 to 2.6 for both Acala and Paymaster cultivars. Micronaire

is a value referring to the flow rate of air passing through 3.24 grams of cotton compressed

in a cylinder of constant volume at 2.5 PSI (Kerby, 1991). It is an indication of both the

fmeness (diameter) of the fiber and the maturity (amount of secondary cell wall thickening

inside the primary cell wall) of the fiber, and optimum values are 3.7 to 4.2. Smaller

diameter and lower maturity both promote packing of fiber with less air space and

therefore, decreases air flow and the assigned micronaire value. Since the fiber weight gain

per boll per day also decreased 55% in the study of Gipson and Joham (1968), from 35 to

15 mg boir^ day' averaged over both cultivars, there is indication of a less mature fiber

resulting from insufficient cellulose deposition. Thaker and coworkers (Thaker et al.,

1989) reported that decreasing the minimum growth temperature in a cycling temperature

regime decreased the fmal fiber dry weight per seed and the rate of dry weight

accumulation, although final fiber length was not affected. Coimer and coworkers

(Conner, Krieg, and Gipson, 1972) report low concentrations of soluble sugars in the

developing cotton boll with a 10°C night temperature regime, whereas increasing amounts

2

were found in the bolls as the night temperature increased to 15°C, 20°, and 25°C during the

first 15 days of fiber development After the initiation of secondary wall formation at 15

dpa, the concentrations of soluble sugars in the bolls at all temf)eratures began to decrease,

however, lower amounts were found in the bolls grown at the higher temperatures. The

principle sugars found in the developing boll were glucose (Glc), fmctose (Fm), and

sucrose (Sue), with Glc being the principle sugar in the boll until 30 dpa (Conner et al.,

1972).

1.2.2 72 V/fr^Smdies

Haigler et al. (1991) reported delayed onset and prolonged periods of elongation and

secondary wall thickening in fibers of Gossypium hirsutum L. cv Acala SJ-1 grown in

vitro at cycling temperattires of 34°C/22°C and 34°C/15°C, but not at 34°C constant or a

cycling temperature of 34°C/28°C. These pattems of fiber elongation and fiber weight

accumulation are consistent with field observations of fiber development under fluctuating

temperatures. Fibers developing in vitro under cycling temperatures also responded

similarly to those of field-grown plants under diurnal temperature fluctuations in forming a

"growth ring" each time the temperature cycled to 22°C or 15°C. It is noteworthy that field

and in vitro fibers behaved similarly even though the substrate Glc was never limiting in

vitro. (Glc is used as the carbon source in in vitro cultures because ovules grown on Sue

form extensive callus and few fibers [Beasley, 1992].) This imphes that neither slowed

translocation of Sue nor slowed activity of a possible ovule/fiber invertase or soluble

sucrose synthase is sufficient explanation for the field effect The similarity of in vitro

results (Glc as the substrate) with field results (Sue as the transport sugar) also suggests

that the major cool temperature hindrances are not related to this difference.

In a similar study involving cycling temperatures, Roberts and coworkers (Roberts,

Rao, Huang, Trolinder, and Haigler, 1992) investigated rates of cellulose synthesis,

respiration, and long term Glc uptake in fibers of cultured cotton ovules (cv Acala SJ-1).

Respiration increased between 18°C and 34°C, then remained constant as temperature

increased to 40°C. The rate of cellulose synthesis also increased above 18°C, however, a

plateau was reached at 28°C above which synthesis was constant to 37°C before decreasing

at 40°C. These data indicate a difference in the responses of cellulose synthesis and

respiration to temperature. A comparison of the linear slopes between the two processes

from 18°C to 28°C shows that cellulose synthesis is more temperature responsive than

respiration; QJQ values equal 3.13 and 4.28 for respiration and cellulose synthesis,

respectively.

3

They (Roberts et al., 1992) also reported an increasing rate of Glc uptake from the

medium after day 15 of culture due to the onset of secondary wall deposition. The rate of

uptake after day 15 was much greater at 34°C constant and 34°C/28°C cycling than for

34°a22°C or 34°ai5°C cycling. When ovules were culttired at 34°C for 18 days and then

shifted to cycling temperamies, there were no changes in the rate of glucose uptake for

34°C/28°C cycling compared to 34°C constant. However, uptake almost ceased for the

34°C/15°C cycling condition between day 18 and day 21 before resuming at a reduced rate

by day 24. Therefore, there seems to be some sort of adjustment period for the fibers

when switched from 34°C to a 34°C/15°C cycle before cellulose deposition resumes, albeit

at a reduced rate. Also, Glc uptake for the 34°C/22°C cycling condition was reduced to a

lesser extent between day 18 and day 21.

Previous work with cultured cotton ovules in the Haigler lab (Haigler, Taylor, and

Martin, 1994) suggested that some cultivars may be more cool sensitive in cellulose

synthesis, including Acala SJ-1 and Texas Marker-1 (TM-1), than other more tolerant

cultivars, which included a Yugoslavian cultivar, YG. This cultivar was developed for

growth in Yugoslavia, an extreme northerly latitude for growing cotton, and it

demonstrated better metabolic activity at 15°C than other cultivars in germination studies

(Dr Brad Waddle, 1990, personal communication with Dr. Candace H. Haigler). Glc

uptake pattems were similar for SJ-1 and TM-1, showing reduced uptake at 34°C/22°C

cycling compared to the 34°C constant and 34°C/28°C cycling. For YG, however, glucose

uptake at the cooler 34°C/22°C cycling was no different from the 34°C constant and

34°C/28°C cycling. Also, neither the onset of secondary wall deposition nor the rate of

accumulation of fiber weight were delayed in YG under 34°C/28°C or 34°C/22°C cycling

compared to 34°C constant as they were for SJ-1 and TM-1. These results indicate that YG

is more cool-tolerant in several ways. It must be noted, however, that the rate of weight

gain and final fiber weight per ovule for YG was much lower than that of SJ-1, indicating

that factors other than temperature limit the rate of cellulose deposition for YG.

Haigler and coworkers (Haigler et al., 1994) investigated nineteen cultivars of cotton

for temperature dependence of cellulose synthesis and respiration. These cultivars included

past and current commercial cultivars, cultivars from breeding programs, YG, and JDX.

JDX is a cross between, G. hirsutum L. cv YG (cool-tolerant) and G. hirsutum L cv

[(1517-75 X Stahman 05-2B)-2-7621] (cool-sensitive) taken through 4 cycles of field

selection for cool tolerance (a gift to the Haigler lab from Dr. Jane Dever, Texas A&M

Agricultural Experiment Station, Lubbock, TX). Ovules were removed at 1 dpa and

cultured in vitro for 18 days before being ti-ansferred to cycling temperatures (34°C/15°C,

34°a22°C, or 34°a28°C) or left at 34°C constant. After 3 days, they were placed on

labeled [1^]-Glc medium for 4 hours prior to harvesting and measurement of '*C02 and

14C-crystalline cellulose. Table 1.1 is a partial summary of the results from their

experiment Three of the cultivars, Acala SJ-1, Texas Marker -1 (TM-1), and Coker 312

were shown to be cool-sensitive cultivars, with the rate of cellulose synthesis at 15°C

ranging from 11 to 15% Uiat at 34°C. Four cultivars. Paymaster HS26, YG, JDX, and

Paymaster HS200, were considered partially cool-tolerant based on the higher rates of

cellulose synthesis at 15°C ranging from 21 to 24% that at 34°C. Differences between the

cool-sensitive and partially cool-tolerant cultivars were also evident in the rates of

respiration at 15°C, when expressed as a percentage of the rate at 34°C. At 15°C,

respiration rates for the cool-sensitive cultivars were higher (17-20%) than most of the

partially cool-tolerant cultivars (14-19%). One exception was Paymaster HS26, which

behaved similar to the cool-sensitive cultivars at 15°C (19% of 34°C respiration rate), but

then behaved similarly to the partially cool-tolerant cultivars at 22°C. Therefore, it was

considered a partially cool-tolerant cultivar. Also, cellulose synthesis/respiration ratios on

21 dpa for the partially cool-tolerant cultivars at 15°C were 3.1 to 8.4 compared to 2.2 to

2.8 for the cool-sensitive cultivars, indicating that the partially cool-tolerant cultivars may

regulate carbon flow more towards cellulose synthesis than to respiration. The differences

occurring at 15°C were basically maintained at 22°C. The only exception was YG, which

had a lower percentage of 34°C cellulose synthesis at 22°C than the other partially cool-

tolerant cultivars. The YG percentage at 22°C was equal to the percentages of the cool-

sensitive cultivars. This indicates that existing cultivars can have individualistic responses

to the series of cool temperatures of this experiment.

1.2.3 Imphcations of Cool Temperature In Vitro Studies

The differential response of respiration and cellulose synthesis to cool temperatures

(Roberts et al., 1992) and the differential response among existing cotton cultivars to cool

temperatures (Gibson and Joham, 1968; Thaker, 1989; Haigler et aL, 1994) suggests that a

particular enzymatic step in the cellulose biosynthetic pathway may exist that is most

severely affected by cool temperatures (Haigler et al., 1991; Roberts et al., 1992).

Investigations of the effects of temperature on the metabolite pools of the pathway and flux

through them may be useful in determining an enzymatic step in the cellulose synthetic

pathway that is limited by cool temperature.

Table 1.1. Cellulose Synthesis and Respiration at 15°C and 22°C. Percent values are given as a percentage of that at 34°C. The cultivars for each temperature designation are listed in order of increasing cellulose synthesis/respiration ratios.

Cultivar %34°C Cellulose Synthesis %34°C Resou-ation C/R* at 22°C at 15°C 15°C

Cool-Sensitive: Acala SJ-1 TM-1 Coker312

Partially Cool-Tolerant: Yugoslavian (YG) JDX Paymaster HS26 Paymaster HS200

at 22°C

37% 38% 37%

37% 39% 44% 42%

at 15°C

11% 12% 15%

24% 21% 23% 24%

42% 38% 32%

33% 34% 31% 26%

19% 20% 17%

15% 14% 19% 14%

2.2 2.2 2.8

3.1 6.2 6.4 8.4

* cpm cellulose/cpm respiration

1.3 Cellulose Biosynthetic Pathways in the Cotton Fiber

1.3.1 Previously Propo.sed Pathway of Cellulose Synthesis

Carpita and Delmer (1981) characterized a possible metabolic pathway for cellulose

synthesis as

Glc - > Glc-6-Phosphate - > Glc-1-Phosphate - > UDP-Glucose - > Cellulose

showing UDP-Glucose (UDP-Glc) as the most likely substrate for the cellulose synthase

enzyme complex. This pathway was based on studies of plant grown fibers and on

[I'HDJGIC pulse-chase experiments with fibers from in vitro ovule culture using Glc as the

exogenous carbon source. In the plant grown fibers, UDP-Glc was the most abundant

nucleotide in the fiber, and it increased dramatically from 0.2 |imol/boIl at 13 dpa to 1.2

fimol/boll at 17 dpa, along with the onset of secondary cell wall formation. The

concentration increased again to 2.1 p,mol/boll at 24 dpa just prior to the maximum rate of

secondary cell wall synthesis. Fibers from cultured ovules exhibited a similar response in

metabolite concentration. Using exogenous [**C]Glc, it was determined that the rate of

synthesis and tumover of UDP-Glc was sufficient to account for the synthesis of cell wall

glucans, indicating that UDP-Glc was the probable precursor to secondary cell wall

cellulose in the cotton fiber. The pulse-chase experiments were used to characterize the

pathway from Glc through the sugar-phosphates, glucose 6-phosphate (Glc 6-P) and

glucose 1-phosphate (Glc 1-P), to UDP-Glc. A similar pathway through hexose

phosphates toward cell wall glucan synthesis was described by Kanabus and coworkers

(Kanabus, Bresson, and Carpita, 1986) for cultured carrot cells using radiolabeled Glc or

Sue as the carbon source. The Sue was hydrolyzed extracellularly, and Glc was

preferentially taken up by the cells.

1.3.2 Membrane Associated Sucrose Synthase

Recently, it has been proposed that a membrane-associated sucrose synthase (SuSy)

plays a major role in secondary cell wall synthesis by hydrolyzing Sue at the cell membrane

and supplying UDP-Glc directiy to the cellulose synthase complex (Amor, Haigler,

Johnson, Wainscott and Delmer, 1995). This is supported by the presence of large

amounts of SuSy in cotton fiber cells (Nolte, Hendrix, Radin, and Koch, 1995; Ruan,

Chourey, Delmer, and Perez-Grau, 1997) and the increased synthesis of 6-1,4-glucan in

detached, permeabilized cotton fibers when Sue rather than UDP-Glc is the supplied

substrate in the medium (Amor et al., 1995). The pathway of carbon flow described

earlier, however, was characterized from in vitro ovule culture using Glc as the carbon

source, which moves readily into the fiber cell, and did not include the synthesis of Sue for

use by SuSy. The direct entry of Sue into the fiber cell and its subsequent hydrolysis by

SuSy would appear to be more energy efficient than Glc entry into the fiber cell following

Sue hydrolysis in the seed coat and the resynthesis of Sue from metabolites within the cell

(Wagner and Backer, 1992). However, other factors, such as the fate of the Fm released

by the direct use of Sue by SuSy, need to be considered in determining energy efficiency

within the cell. Nevertheless, carbon movement from the region of phloem unloading in

the seed coat of the ovule to the fiber cells and carbon flow within the fiber cell have not

been fully determined. A careful consideration of evidence related to carbon movement is

necessary to determine the possible pathway for cellulose biosynthesis in the cotton fiber

cell.

1.3.3 Sucrose Movement to the Fiber Cell

One possibility of carbon movement to the fiber cell involves the direct symplastic

movement of Sue from the phloem to the fiber cells, entering the fiber cells through

plasmadesmata located in the foot of the fiber cell (Ruan et al., 1997). Evidence for this

pathway is demonstrated first by the presence of large amounts of SuSy in the fiber cells,

including the fact that much of it is soluble supplying UDP-Glc and Fm for possible use in

glycolysis and other metabolic processes in the fiber cell (Amor et al., 1995). Second,

there is rapid movement of the membrane impermeant dye, 5 (6)-carboxyfluorescein (CF)

from the transfer cells of the inner seed coat to the epidermal fiber cells (Ruan et al., 1997).

The movement of CF has been shown to be similar to that of the soluble sugars Glc and

Fm (Ruan and Patrick, 1995), which could be analogous to Sue. Third, there is also a

high frequency of plasmadesmata at the base of the fiber cell to account for the symplastic

movement of Sue which cannot move apoplastically through the heavily suberized cell wall

at the base of the fiber cell (Ryser, 1992). Finally, only small amounts of both soluble and

insoluble invertase are reported in the developing ovule to hydrolyze Sue (Hendrix, 1990),

and SuSy activity is undetectable in the seed coat (Ruan et al., 1997; Nolte et al., 1995).

Figure 1.1 diagrams the possible pathways of carbon flow within the fiber cell from

only Sue transported into the fiber cell (Delmer, in press). Invertase activity was added to

the diagram because of the high invertase activity reported to be present in the cotton fiber

cell (Basra and Malik, 1990; Waffler and Meier, 1994).

8

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1.3.4 Hexo.se Su^ar Movement to the Fiber Cell

Another possibility of carbon movement to the fiber cell involves the hydrolysis of

Sue after phloem unloading in the seed coat, the symplastic movement of the resulting

hexose sugars into the fiber cell, and the resynthesis of Sue in the fiber cell prior to use by

SuSy at the membrane surface. Evidence for this pathway is demonstrated by the presence

of large pools of Glc and Fm not only in the seed coat, but also in Uie fiber cells themselves

(Hendrix, 1990; Jaquet, Buchala, and Meier, 1982; Ruan et al., 1997). Also, starch is

present in the seed coat (Hendrix, 1990), implying Sue hydrolysis via invertase if the

observations showing lack of seed coat SuSy activity are correct. This pathway, however,

is not likely to be the dominant pathway for carbon movement in cotton ovules in vivo ,

rather the flux of large amounts of Sue through the seed coat to the epidermal cells likely

predominates (Ruan et al., 1997). The small amount of invertase activity reported in the

developing ovule by Hendrix (1990) may account for the levels of starch and hexose

sugars reported in the seed coat, but would not likely be adequate to supply the carbon

necessary for the high cellulose synthesis rates during secondary cell wall formation

(Meinert and Delmer, 1977).

Figure 1.2 diagrams the possible pathways of carbon flow from Glc transported into

the fiber cell, the most likely pathway for fibers of ovules cultured on exogenous Glc

(Haigler et al., 1994). It should be noted in this pathway that the large amounts of Sue

necessary for secondary cell wall synthesis must be synthesized only from metabolites in

the cell by the activity of sucrose phosphate synthetase (SPS) and sucrose phosphate

phosphatase (SPP). Therefore, the activities of these enzymes in vitro may possibly be

greater, and the flux of carbon through the metabolites in the cytoplasm may be altered to

some degree.

1.3.5 Additional Considerations of Carbon Movement

In addition to the two possibilities described above, it should be noted that the

metaboUtes involved in the pathways from Glc import are almost identical to those of the

diagram for Sue import into the cell. It should also be noted that cell-surface located acid

invertase activity was reported in the cotton fiber itself with maximum activity of the

invertase corresponding to the time of maximum Glc and Fm levels in the fiber and to the

onset of secondary cell wall synthesis (Buchala, 1987). They also reported rapid

movement of sugars into the fiber cell and incorporation of labeled carbon into 6-glucans

when isolated seed clusters were fed apoplastically witii [I'HllJGlc or [14C]Suc. The Sue

10

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11

was readily hydrolyzed by an invertase at the cell membrane. Experiments with a Sue

analogue, however, did show that Sue can enter the cell directiy without being hydrolyzed.

This study does show the possible movement of sugars, whether Glc or Sue, into the fiber

cell directiy from the extracellular fluid surrounding the fibers in the locule.

There is no reason to ignore the high possibility that all of the above pathways are

functioning in carbon movement into the fiber cell of the developing ovule. Figure 1.3

diagrams the possible pathways of carbon flow when both Glc and Sue are imported into

the fiber cell. Darkened arrows indicate paths that are common to both Suc-import-only

pathways (Figure 1.1) and Glc-import-only pathways (Figure 1.2).

Another interesting possibility to consider is that the different pathways described

may be used altemately under differing environmental conditions, such as warm and cool

temperatures, or at different stages of development of the fiber.

It must be noted that although Sue may the transported sugar into the cell, high levels

of invertase activity are reported in the fiber cell peaking at 15 to 16 dpa (Basra et al., 1990;

Waffler and Meier, 1994) with its activity making it the most active enzyme in the fiber

extracts (Waffler and Meier, 1994). The hydrolysis of much of the transported Sue by the

invertase would then account for the large amounts of Glc and Fm reported in the fiber cell

at the onset of secondary cell wall deposition (Basra et al., 1990; Jaquet et al., 1982; Ruan

et al., 1997). The extent to which Sue is hydrolyzed has not been elucidated, yet the high

activities of invertase throughout secondary cell wall deposition imply that a greater portion

is hydrolyzed than is used directiy by SuSy. Indeed, the relative amounts of Glc, Fm, and

Sue in fibers from cultured ovules (as reported in this study) are extremely similar to those

reported in fibers grown in vivo (Basra et al., 1990; Jaquet et al., 1982; Ruan et al., 1997),

indicating that cellular metabolism is very similar for both plant grown and cultured ovules.

1.4 Metabolite Pools in the Cotton Fiber Related to Sue and Glc Metabolism and Cellulose Synthesis

Carpita and Delmer (1981) reported the levels of metabolites in fibers of cultured

cotton (Gossypium hirsutum L.) ovules to be 4.1,0.8, and 7.1 nmol per ovule for Glc 6-

P, Glc 1-P, and UDP-Glc, respectively. The fibers used in the determination were at the

early stages of secondary cell wall synthesis at 15 or 16 dpa. In labeling studies, the sugar-

phosphates and UDP-Glc pools rapidly approached a near steady state level of radioactivity

within one hour of labeling, whereas Glc and Sue pools showed much lower specific

activities, which continually increased during the labeling period. Also, only 11% of the

12

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13

total reducing sugars were released by treatment of the fibers with 1.5% dimethylsulfoxide,

a treatment that alters the permeability of the plasma membrane but not the vacuolar

membrane. These results indicate the possibility of large storage pools of Glc and Sue

within the vacuole of the fiber cell. Computer simulation estimated the size of the Glc and

Sue storage pools in the fibers to be 1880 and 110 nmol per ovule, respectively.

The sizes of the metabolic pools reported by Carpita and Delmer were determined

from cultured ovules and, therefore, are smaller than those from field- or greenhouse-

grown ovules. Fibers from culmred ovules tend to be half the length that of plant-grown

fibers and the dry weight per boll equivalent of cultured fibers is considerably less than half

that of plant-grown fibers (Meinert and Delmer, 1977), indicating that fiber number or

secondary wall thickening per fiber may be reduced in cultured ovules. The development

of the fibers from cultured ovules, however, is similar to that of field-grown fibers (Haigler

et al., 1991), and the compositional analyses of cell walls derived from culture-grown

fibers indicate they are very similar to those derived from plant-grown fibers (Meinert and

Delmer, 1977). For comparison purposes, values for the metabolic pool sizes in the next

two reviews will be given not only in the value reported by the authors, but also in nmol

per fibers of one ovule indicated in parentheses.

Jaquet, Buchula, and Meier (1982) reported increasing levels of 6-1,3-glucan in

greenhouse-grown (26°C day/ 20°C night) cotton fibers (Gossypium hirsutum L.)

beginning at 9 dpa, peaking around 24 to 27 dpa at the time of increased rates of cell wall

dry weight accumulation, and dropping dramatically after 27 dpa. Cell wall dry weight

accumulation (due to cellulose synthesis) increased steadily after 17 dpa. Glc and Fm were

the most abundant hexoses during the transition from primary to secondary wall formation

and during early secondary wall deposition, but levels began to drop after 17 dpa as the

level of Sue increased to be the most abundant sugar by 37 dpa. At 17 dpa, Glc, Fm, and

Sue levels in the fibers of one ovule were reported to be approximately 6 mg (33,300

nmol), 4.2 mg (23,300 nmol), and less than 1 mg (2920 nmol), respectively. By 27 dpa,

the levels of Glc and Fm had dropped to approximately 1.9 mg (10,540 nmol) and 2.5 mg

(13,870 nmol), respectively, while Sue levels increased to 1.3 mg (3800 nmol). Sue levels

continued to increase while the levels of Glc and Fm continued to decrease until 37 dpa.

Glc 6-P and Fm 6-P, which were present in much lower concentrations than the hexose

sugars, also increased dramatically from 9 to 17 dpa, and continued to increase until 32

dpa. Glc 6-P was the sugar phosphate with the greatest concentration of 20 ug (71 nmol)

at 17 dpa and 26 ug (92 nmol) at 32 dpa. Frc 6-P concentrations were 4 ug (11.9 nmol)

and 10 ug (29.8 nmol) at 17 and 32 dpa, respectively.

14

Basra and coworkers (Basra, Sariach, Nayyar and Malik, 1990), working witii

field-grown Gossypium hirsutum L., also report the Sue content to be considerably less

than that of Glc or Fm at all stages of cotton fiber development. In contrast to that reported

by Jaquet et al. (1982), they report increasing Glc and Fm pools from 10 to 25 dpa, which

stayed relatively high until 35 dpa. Glc pool sizes were reported to increase from 2.2 to

5.1 mg g' dry wt (36.6 to 1300 nmol ovule" ) and Fm pool sizes increased from 1.4 to 5.2

mg g' dry wt (23.3 to 1327 nmol ovule'*). Sue concentrations decreased from 0.85 to

0.68 mg g'* dry wt during the same time period, however, concentrations actually increased

on a per ovule basis (7.5 to 92.7 nmol ovule*'). Fiber dry wt per ovule increased from 3

mg at 10 dpa to 46 mg at 35 dpa.

For comparison purposes, the hexose sugar levels from the above three studies are

listed in Table 1.2 in nmol per fibers of one ovule. The Sue content was considerably less

than that of Glc or Fm and increased during secondary cell wall development in each of the

studies. The studies differed, however, in the amount of hexose sugars and sucrose in the

cotton fibers and in the direction of change in pool sizes of the hexose sugars during

secondary cell wall development.

From these studies, the major carbohydrates in the cotton fiber appear to be Sue and

the hexose sugars, with Glc and Frc concentrations being considerably higher than that of

Sue. The concentrations of the sugar-phosphates Glc 6-P, Fm 6-P, and Glc 1-P, and the

sugar-nucleotide UDP-Glc are hundreds of times lower than the hexose sugars.

1.5 Enzyme Activities in Fiber Cells Related to Sucrose and Glucose Metabohsm and Cellulose Synthesis

1.5.1 Invertase Activity

Invertase (EC 3.2.1.26) activity was reported to be the most active enzyme in the

cotton fiber cell (Gossypium hirsutum L.), with activities increasing up to 25 }ikat/mg

protein at 16 dpa, the onset of secondary cell wall formation, and then decreasing thereafter

(Waffler and Meier, 1994). Figure 1.4 provides an overview of the changes in the

activities of invertase and the other enzymes that will be discussed in this section. (It must

be noted that the enzyme activities reported in Figure 1.4 were extracted from ovules of

either plant-grown, greenhouse-grown, or growth chamber cotton plants.) Basra and

coworkers (Basra et al., 1990) also reports high activities of three isozymes of invertase in

developing fibers of field-grown cotton. Soluble acid invertase activity, presumed to be

compartmentalized in the vacuole, increased rapidly to 54 |ig Glc released min'* g'* fresh wt

15

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16

Invertase (Waffler)

Invertase, soluble acid (Basra)

Invertase, alkaline (Basra)

POD (Waffler)

SuSy (Basra)

UDPG-PP (Waffler)

SPS (Tummala)

Glc 6-PDH (Waffler)

PPi-PFK (Waffler)

MDH (Waffler)

30 p.kat/mg protein

Fiber Elongation Trans. I Secondary Cell Wall Syn.

80 ig Glc/min/g fibeij

80 ig Glc/min/g fibeii

5 |i.kat/mg protein „ _ |

1500 nmol/min/g fiber

50 nkat/mg protein

3 |imol/g fiber

50 nkat/mg protein

20 nkat/mg protein

" ' 600 nkat/mg protein :

'.".•.'.•.•.•.•.•.

^irfMkdhri -f .%.•.•.•.•••.•.•.•.•

r T 0 10 15 20 25 30

Days Post-Anthesis (dpa)

35 40

Figure 1.4. Changes In Enzyme Activities During Cotton Fiber Cell Development. The maximum activity for each graph frame is given in the top left hand comer for comparison. The reference for each activity is given in parentheses.

17

at 15 dpa, decreased to 10 |ig at 25 dpa, then rose again to approximately 30 |ig at 30 dpa

(see Figure 1.4). Wall bound acid invertase activity, possibly indicative of apoplastic

hydrolysis of Sue, had lower activities than soluble acid invertase (10 p.g at 10 dpa

increasing to 20 .g at 20 dpa before decreasing to 7 |ig at 25 dpa), yet it demonstrated

similar pattems and constituted a significant proportion of the total activity in the fiber

(graph not shown in Figure 1.4). The acid invertase was thought to play a significant role

in cell elongation by increasing hexose vacuolar storage and, hence, the maintenance of

turgor for expansion of the cell (King, Lunn, and Embank, 1997). Basra and coworkers

(Basra et al., 1990) also reported activities of alkaline invertase (cytoplasmic) increasing to

45 |Xg at 15 dpa, then decreasing to 12 p.g by 30 dpa before increasing again to 30 |ig in a

pattem similar to acid invertase (see Figure 1.4).

From both of these studies, it is clear that much of the Sue entering the fiber cell is

rapidly hydrolyzed to Glc and Fm for use in the metabolic activities of the cell or for

storage, especially at the onset of secondary cell wall deposition. Waffler and Meier (1994)

reported the activity of invertase in |ikat/mg protein, as was the activity of peroxidase,

while the activities of the other enzymes examined were reported in nkat or pkat mg"

protein. This high invertase activity correlates with the relative sizes of the Sue, Glc, and

Fm pools in the fiber cell.

1.5.2 Glucokinase Activity

Although large pools of Glc are reported in the fiber cell (Basra et al., 1990; Jaquet et

al., 1982), suprisingly small levels of glucokinase (EC 2.7.1.2) activity were reported by

Waffler and Meier (1994). Glucokinase phosphorylates Glc to form Glc-6-P (see Figure

1.3). However, it was possible to obtain a stimulation of the activity by adding Fmctose-

1-P to the reaction mixture, indicating activity may not have been fully detectable with the

assay method used. Basra and Malik (1984) reported increasing activity of hexose kinase

during fiber elongation, however, the level of activity and the substrate involved were not

stated. The possible low activity of glucokinase indicates that much of the Glc suppUed by

invertase activity may be directiy routed to the large vacuole present in the developing fiber

(Ryser, 1992) for storage (Carpita and Dehner, 1981).

1.5.3 Sucro.se Synthase Activity

Another important enzyme in the cotton fiber cell is sucrose synthase (SuSy, EC

2.4.1.13). SuSy functions mainly in non-photosynthetic tissues to hydrolyze sucrose into

UDP-Glc and Fm (Heldt, 1997). A substantial amount of fiber SuSy is membrane-

18

http://Sucro.se

associated in Uie fiber cell (Nolte et al., 1995; Amor et al., 1995), and this SuSy has been

proposed to hydrolyze sucrose at the plasma membrane to supply UDP-Glc directiy to

cellulose syntiiase (Amor et al., 1995). UDP-Glc has been shown to be tiie most probable

substrate for cellulose synthase in the cotton fiber (Carpita and Delmer, 1981), which is

consistent with the proven substrate role for UDP-Glc in bacterial cellulose synthesis

(Kawague and Delmer, 1997; Ross, Mayer, and Benzima, 1991). The activity of soluble

SuSy increases rapidly from 20 nmol min"' g' fresh wt at 10 dpa to 770 nmol at 15 dpa,

plateaus until 20 dpa, then increases again to 1300 nmol at 25 dpa, corresponding to the

time of increasing rates of cellulose synthesis (Basra et al., 1990) (see Figure 1.4).

Activities then decreased to 800 nmol at 35 dpa. Interestingly, Waffler and Meier (1994)

reported no convincing SuSy activity, but they did indicate a possible detection problem

due to use of an alkaline assay buffer. Ruan and coworkers (Ruan et al., 1997) reported

activities of soluble SuSy in cotton fibers to be 147 nmol reducing sugar mg" protein min"

at 16 to 18 dpa. The role of soluble SuSy in fiber development is unknown. The

association of soluble SuSy with secondary cell wall development gives indication that the

soluble SuSy and the membrane-associated SuSy may be coupled to generate sufficient

UDP-Glc for cellulose synthesis (Ruan et al., 1997). Also, there could be rapid,

regulatory transitions between soluble and membrane-bound SuSy; phosphorylation has

been proposed as one such regulatory mechanism (Reimholz, Geigenberger, and Stitt,

1994).

1.5.4 UDP-Glucose Pvrophosphorylase

In contrast to invertase activity, which peaks at the onset of secondary wall

development and then decreases, the activity of UDP-Glucose pyrophosphorylase

(UDPG-PP, EC 2.7.7.9) increases throughout fiber development (Waffler and Meier,

1994). UDPG-PP catalyzes the synthesis of UDP-Glc from Glc 1-P and UTP (see Figure

1.3). Activities increased from 25 nkat mg" protein at 12 dpa to approximately 45 nkat at

26 to 36 dpa (see Figure 1.4). It was suggested that this enzyme may provide the UDP-

Glc for the synthesis of cellulose since the activity of UDPG-PP was at a maximum at the

same time as the maximum rate of cellulose deposition and because of the low activity

detected for SuSy (Waffler and Meier, 1994). However, the increasing activity of SuSy

reported by Basra et al. (1990) at the time of increasing rates of cellulose deposition may

also provide the UDP-glc necessary for the synthesis of cellulose.

It must also be noted that UDPG-PP is a major enzyme in the pathway to syntiiesize

Sue in the cytoplasm of the cell, which may then by hydrolyzed by SuSy at the plasma

19

membrane. It has been suggested that perhaps half of the Sue suppHed to the membrane-

associated SuSy results from the synthesis of Sue within the cell through the pathway

involving UDPG-PP, SPS, and SPP (Delmer, in press) (see Figure 1.3). This patiiway is

part of a larger pathway that uses the Fm released from the membrane-associated SuSy to

provide intermediates for other metabolic processes in the cell and to recycle as much

carbon as possible into cellulose synthesis (Delmer, in press).

1.5.5. Sucrose Phosphate Synthase Activity

Sucrose Phosphate Synthase (SPS, E.C. 2.4.1.14) activity catalyzes the reaction

combining UDP-Glc and Fm 6-P to form Sue-phosphate, which is then dephosphorylated

by sucrose phosphate phosphatase to form sucrose (see Figure 1.3). SPS activities in the

fibers of cultured ovules were reported to increase from less than 1 [imol g" fresh wt hr' at

8 dpa to 1.4, 1.7, and 2.25 jimol at 24 dpa for Acala SJ-1, Coker 312, and Paymaster

HS200, respectively (Tummala, 1996) (see Figure 1.4). Higher activities of up to 17 jimol

g' fresh wt hr' have been reported in the fibers of cultured ovules in the cultivar Coker 312

during secondary cell wall synthesis (Jaradat and Haigler, unpublished). In conjunction

with UDPG-PP, SPS functions as a major enzyme in the synthesis of Sue within the

cytoplasm of the fiber cell.

1.5.6. Glucose 6-Phosphate Dehydrogenase Activity

Glucose 6-phosphate dehydrogenase (Glc 6-PDH, EC 1.1.1.49) is a key enzyme in

the cotton fiber (Waffler and Meier, 1994), catalyzing the oxidation of Glc 6-P into 6-

phosphogluconopyranose-l,5-lactone (Rawn, 1989). This is the first step of the pentose

phosphate pathway (see Figure 1.3), which generates a source of reducing power in the

form of reduced nicotinamide adenine dinucleotide phosphate, NADPH (Hvazdina and

Jensen, 1992). NADPH is used in many biosynthetic reactions in nonphotosynthetic tissue

(Basra and Malik, 1984), one possible reaction being the synthesis of glutathione by

glutathione reductase, which is present in the developing fiber (Waffler and Meier, 1994).

Glutathione may then act as a hydrogen donor in peroxidase reactions (Waffler and Meier,

1994). Activities of 30-40 nkat mg"l protein were reported for Glc 6-PDH with two peaks

in activity at 12 dpa and 24 dpa (Waffler and Meier, 1994) (see Figure 1.4). Activities

decreased after 30 dpa below 30 nkat. Basra and Malik (1984) report increasing activity

during elongation and decreased activity when the rate of growth slows in the fiber. It

must be noted that although glucokinase levels are low in the cotton fiber, the substrate for

20

Glc 6-PDH, Glc 6-P, can be generated directiy from Fm 6-P by the action of

phosphoglucoisomerase.

1.5.7. Phosphoglucoisomerase

Phosphoglucoisomerase (PGI) catalyzes the reversible reaction converting Fm 6-P to

Glc 6-P. To my knowledge, the activity of PGI has not been measured in the cotton fiber

cell. This enzyme is important in the fiber cell linking two key metaboUtes that are

components of several important metabolic pathways (Figure 1.3). Glc 6-P is the

beginning metabolite for the pentose phosphate pathway, an early intermediate in the

glycolytic pathway, and can be converted to UDP-Glc, an intermediate in the pathway for

Sue synthesis. Fm 6-P is converted to fmctose-1,6-bisphosphate in the glycolytic

pathway, to Glc 6-P in the pathway for UDP-Glc synthesis and to Sue-phosphate in the

reaction catalyzed by SPS.

1.5.8. Phosphofmctokinase

Both ATP-dependent phosphofmctokinase (ATP-PFK, EC 2.7.1.11) and

pyrophosphate:fmctose 6-P 1-phosphotransferase (PPi-PFK) show maximum activity at

18 dpa, then activities decrease as fiber development continues (Basra and Malik, 1982;

Waffler and Meier, 1994). Both ATP-PFK and PPi-PFK catalyze the phosphorylation of

Fm 6-P to fmctose-1,6-bisphosphate in the glycolytic pathway of cellular respiration (see

Figure 1.3). The activity of PPi-PFK fluctuated between 8 and 9 nkat mg" protein from 10

to 30 dpa, with a peak at 18 dpa, before decreasing to 6 nkat (see Figure 1.4). The activity

of PPi-PFK was shown to be 4 to 5 times greater than that of ATP-PFK.

1.5.9 Malate Dehydrogenase

Two enzymes with activities that surpass those of the other enzymes in the fiber cell

except invertase and possibly SuSy are malate dehydrogenase (MDH, EC 1.1.1.37) and

peroxidase (POD, EC 1.11.1.7) (Waffler and Meier, 1994). MDH catalyzes the synthesis

of malate from oxalacetic acid (OAA), which is the product of the reaction catalyzed by

phosphoenolpymvate carboxylase (PEPC) combining PEP and COj. The COj is presumed

to be a by-product of the oxidation reactions of the Kreb's cycle of respiration (Basra and

Malik, 1983) and is "re-fixed" into malate in a series of reactions referred to as the dark

metabohsm ofCOj (Basra and Malik, 1983). Malate thus formed is either stored in the

vacuole (Geza and Jensen, 1992), enters the Kreb's cycle, or is converted to pymvate via

21

malic enzyme (ME)(Basra and Malik, 1984). These reactions are diagrammed as:

C 0 2 ^ NADP NAJ^PH-HH* CO2 PEP ^ ^ •OAA • Malate ^ = ^ - ^ ^^-•Pymvate

(PEPC) (MDH) V ^ ^ (ME)

Vacuole Kreb's cycle.

MDH activity is high (250 nkat mg' protein) during fiber cell elongation and increases

dramatically at the onset of secondary cell wall synthesis to 375 nkat mg" protein at 16 dpa

(Waffler and Meier, 1994) (see Figure 1.4). A gradual increase in activity continues until

32 dpa where the activity is approximately 480 nkat mg" protein. Basra and Malik (1983),

however, report elevated activities of MDH during the period of rapid fiber elongation and

lowered activity by 20 dpa during secondary cell wall synthesis. The malate content of the

fiber cell, however, rises with increasing age and shows a continuous tumover, thereby

favoring a rise in MDH activity with age (referenced in Waffler and Meier, 1994).

The storage of malate and potassium ions is thought to be largely responsible for the

maintenance of turgor in the fiber (Dindsa, Beasley, and Ting, 1975), and turgor is one

possible control point for fiber elongation. Kloth (1992), however, reported varying

specific activities of MDH among the cotton fibers of 24 cotton cultivars, ranging from

13.8 to 18.8 p.mol NADH min' mg' protein, with no correlation of MDH activity to fiber

length. However, this is not an indication that malate is not involved with turgor and fiber

extension because perhaps it is at or above an optimum level in many modem cultivars.

Interestingly, significant negative correlation's were found between MDH activity and

maturity, micronaire value, and wall thickness, but cultivar x environment interactions were

also significant for these three fiber qualities.

Elevated activities of ME were reported after 15 dpa in the cotton fiber cell (Basra

and Malik, 1983). The conversion of malate to pymvate would feed respiration while

generating NADPH + H* and releasing COj (Basra and MaUk, 1984). Increasing MDH

activity in later stages of secondary wall deposition may indicate that malate becomes a

source of carbon for respiration during cotton fiber maturation (Basra and Malik, 1984). It

should also be noted the PEP, OAA, and pymvate are important substrates for the

formation of various amino acids (Heldt, 1997).

22

1.5.10 Peroxida.se

The activity of cytoplasmic POD was also high, increasing gradually from 1 |ikat

mg' protein at 10 dpa to 2.25 |ikat mg"* protein at 32 dpa, with several peaks of activity

(Waffler and Meier, 1994). NaiUiani, Ramo Rao, and Singh (1982) reported a similar

response. The cytoplasmic POD may be responsible for the detoxification of hydrogen

peroxide in the fiber cell, using glutathione as the hydrogen donor (Waffler and Meier,

1994). The cell wall-bound POD activity increased dramatically during secondary cell wall

synthesis and remained high throughout fiber development (Naithani et al., 1982; Waffler

and Meier, 1994). This may be an indication of hydrogen peroxide production related to

the formation of dityrosine bridges in the cell wall and reduced fiber extension (Waffler and

Meier, 1994). POD activity has been shown to be associated with reduced hypocotyl

growth (Zheng and van Huystee, 1992) and crosslinking between macromolecules such as

lignin, protein, hemicellulose, and femhc acid (Bowler, Montague, and Inze, 1992; Cassab

and Vamer, 1988).

1.5.11 Overview of Enzyme Activity in the Cotton Fiber

In the cotton fiber, it is likely that two enzyme systems exist for newly imported Sue

metabolism, as evidenced by the activities of the Sue hydrolyzing enzymes present in the

cell (Basra and Malik, 1990). One system involves high activities of invertase, both

alkaline and acid, resulting in high concentrations of both Glc and Fm in the fiber cell as

substrates for metabolic activities and possibly for the maintenance of mrgor by vacuolar

storage of these sugars (Carpita and Delmer, 1981). The others system involves both

membrane associated SuSy, supplying UDP-Glc directiy to the cellulose synthase

complex, and soluble SuSy, which supplies UDP-Glc for Sue synthesis within the

cytoplasm of the cell. The product Fm from the membrane associated SuSy may then be

phosphorylated by fmctokinase to Fm 6-P, which may then be converted to Fm 1,6-

bisphosphate (Fm 1,6-BP) in the pathways of respiration or to Glc 6-P, the beginning

substrate for both the pentose phosphate pathway and the pathway for UDP-Glc and

subsequentiy sucrose synthesis. GK activity may also play a role in the synthesis of Glc 6-

P directiy from Glc.

The other enzymes with high activities are G6-PDH and MDH, which are involved in

the energy-producing pathways and dark respiration of C02, and POD, a very active

enzyme with possible involvement in reduced fiber elongation or crossUnking in the cell

wall (Waffler and Meier, 1994). It should be noted that changes in adenylate [Adenosine

triphosphate (ATP), Adenosine diphosphate (ADP), and Adenosine monophosphate

23

http://Peroxida.se

(AMP)] concenttation and energy charge occur as the fiber begins secondary cell wall

synthesis. Low energy charges associated with dephosphorylation of ATP during

secondary wall synthesis is attributed to higher energy requirements for cellulose synthesis

and increased use of ATP (Basra and Malik, 1982).

It should also be noted that the fiber cell metabolites discussed earlier-Sue, Glc, Frc,

Glc 6-P, Frc 6-P, Glc 1-P, and UDP-Glc~are the substrates and products for the above

mentioned enzymes involved in Glc and Sue metabohsm and cellulose synthesis. Cool

temperatures may affect an enzyme related to the use of these pools for energy production

or for the synthesis of cellulose in the secondary cell wall.

1.6 Cool Temperatures and Plant Metabolism

1.6.1 Reduced Growth and Photosynthesis

Gipson and Joham (1968) reported that root and shoot growth in the cotton plant

were reduced when temperatures fell below 20°C. They also reported delayed flowering

when the night temperatures were below 20°C. Burke, Mahan, and Hatfield (1988) also

reported reduced biomass production when temperatures fell below the crop-specific

thermal kinetic window of 23.5°C and 32°C determined for glutathione reductase.

Perrera, Hartmann, and Holaday (1995) showed that leaf net photosynthetic rates dechned

40% in 2 days after cotton was transferred from 30°C to 15°C temperamres and continued

to decline another 20% from 2 to 8 days after transfer. Warner and Burke (1993) reported

that low night temperatures of 20°C elevated the predawn starch levels by two- to four-fold

depending on the cultivar, and the elevated predawn starch levels were correlated with

reduced photosynthetic activity the following day. A Unear relationship existed for

predawn starch levels and photosynthetic activity in cotton. However, a subsequent study

revealed that photosynthetic rates in warm-night (28°C day/28°C night) plants were greater

(by 30%) than those of cool-night (28°C day/20°C night) plants with similar starch levels

(Warner, Holaday, and Burke, 1995), indicating that cool temperatures per se decrease

photosynthesis. These authors also suggested reduced Sue export from the leaves during

the 20°C night, and proposed that carbohydrate supply (photosynthesis) may be balanced

with sink demand at the low temperatures in cotton.

1.6.2 Hexose Phosphate Increases and Enzvme Activities

In addition to reduced starch accumulation during the day and reduced starch

degradation at night for the cool-night plants, Warner et al.(1995) also reported reduced

24

triose phosphate (TP) and Fm-1,6-BP levels in cotton leaves at 20°C compared to those at

28°C, but higher levels of Glc 6-P Uiat could support Sue synthesis from UDPG-PP. The

pathway for Sue synthesis from triose phosphates exported into the cytosol from the

chloroplast is diagrammed as

TP—>-Fm 1,6BP—• Fm 6-P -^Suc-P—•Sue.

I PPi / Glc 6-P — ^ Glc 1-P T^^UDP-Glc

UTP

UDP-Glc levels remained low during the 20°C night and did not increase as did the levels

during the 28°C night They concluded that some restriction of Sue synthesis may occur

subsequent to Fm 1,6-BP and suggested a possible restriction in the removal of

pyrophosphate (PPi) resulting from UDPG-PP activity. PPi accumulation could result in

product-inhibition of UDPG-PP. The subsequent restriction in UDP-Glc synthesis may

have resulted in the increase in Glc 6-P levels at the lower temperature and a restriction in

the availabiUty of UDP-Glc for SPS. Labate and Leegood (1989) also reported increasing

levels of hexose phosphates at cool temperatures in darkened barley leaves that had been

previously illuminated. They also showed that, contradictory to the 'energy-overflow'

hypothesis, the increases in Glc 6-P and Fm 6-P did not stimulate increases in respiration.

They (Labate and Leegood, 1989) proposed that increases in metabolic pool size may

compensate for temperature-dependent changes in enzyme velocities. Indeed, low

temperatures decrease the affinity of cytosolic Fm 1,6-BP for its substrate (Stitt and

Grosse, 1988).

Hurry, Keerberg, Pamik, Gardestrom, and Oquist (1995) used cold hardened and

non-hardened rye plants at similar physiological stages of development to investigate

enzyme activities and metabolic pool sizes related to photosynthetic acclimation. It was

clear that the immediate response to cold temperatures (5°C) by the non-hardened plants

was a reduction in photosynthetic activity (60%) and Sue synthesis in correlation with

inhibited ribulose bisphosphate carboxylase/oxygenase (RuBisCO) and SPS activity. The

reduction in Sue synthesis may have reduced the levels of inorganic phosphate for transport

back into the chloroplast and, therefore, may have caused the reduction in photosynthesis.

In response to the reduction in SPS activity, hexose phosphate levels increased 30%. The

Glc 6-P/Fm 6-P ratio decreased indicating more accumulation of carbon in the stroma of

the chloroplast.

25

However, after cold-hardening for ten weeks, the transfer of the rye plants to the

lower temperature of 5°C resulted in not only an increase in activation state of the enzymes

involved in carbon fixation [RuBisCO and stromal Fmctose 1,6-bisphosphatase (Fm 1,6-

BPase)], but also an increase in their total activity by 50 to 100% above that of the non-

hardened plants. Photosynthetic rates increased to equal that at 20°C. The enzymes of Sue

syntiiesis (SPS and cytosolic Fm 1,6-BPase) also increased in total activity (100 to 240%

over the non-hardened) and in the degree of activation. Hexose phosphate pools were 25%

greater than those in the non-hardened plants and 120% greater than those at 20°C to

compensate for the Hmitations imposed by product inhibition from the accumulation of Sue,

plus there was no limitation in inorganic phosphate supply for triose-phosphate transport

into the cytoplasm. Glc 6-P/Fm 6-P ratios were back up to 3 or 4, which indicated a

higher rate of Sue synthesis in the cytoplasm. The increased enzymatic activity for both

photosynthesis and Sue synthesis accompanied by increases in substrate levels to overcome

higher threshold concentrations necessary for enzyme activation at low temperatures allows

for continued photosynthesis and for the accumulation of sugars. In addition, the sugars

provide important cryoprotective functions as well as provide for the maintenance of basal

metabolism within the cell (Guy, Huber, and Huber, 1992).

A similar increase in hexose phosphates with decreasing temperature was found in

cold-hardened wheat plants along with an increase in RuBisCO activity (Kobza and

Edwards, 1987). Guy et al. (1992) also reports significant increases in sugar levels,

particularly Sue, accompanied by increases in SPS activity in spinach after 14 days of

exposure to cool temperature. Parallel to the increase in SPS activity was an increase (four-

to fivefold) in the synthesis of the SPS subunit, indicating not only an increase in activation

state of the enzyme, but also an increase in the amount of enzyme. They concluded that

Sue accumulated at low temperature due to a decrease in utilization of photosynthate and

served as an adjustment to overcome the slower enzyme kinetics associated with low

temperatures and as a cryoprotectant for increasing tolerance to freezing stress. Holaday,

Martindale, Aired, Brooks, and Leegood (1992) investigated photosynthesis and enzyme

activity in spinach and bean leaves for 10 days after transfer from 27°C to 10°C. They

reported increased hexose phosphate levels in spinach at 10°C over that of control plants at

24°C along with increases in the total activity of RuBisCO and Fm 1,6-BPase (stromal) by

20% and 67%, respectively. The enzymes were also fully activated, compared to only 65

to 83% activated in leaves at 24°C. Activities of three enzymes supporting Sue synthesis-

SPS, cytosolic Fm 1,6-BPase, and PGI~aIso increased between 58 and 87% in the

spinach leaves. The glycolytic enzymes, pymvate kinase and hexokinase, also

26

demonstt^ted increased activities after exposure to cool temperamre. In bean leaves,

however, photosynthetic rates decreased considerably after exposure to cool temperatures.

The total activities of RuBisCO and stromal Fm 1,6-BPase increased, although not to die

same extent as those in spinach, and the percentages of enzyme activation were much

lower. Cytosolic Fm 1,6-BPase activity increased 64%, but SPS activity decreased

substantially. The percentage of SPS enzyme activated also decreased with time. This

study clearly demonstrates that enzyme activities are altered with exposure to cool

temperatures and that species vary considerably in their abihty to increase enzyme activation

at cool temperatures.

Very little is known of the activities of the enzymes in cotton at chiUing

temperatures below 25°C. Perrera et al. (1995) reported a 40% decUne in photosyntiiesis

when cotton plants were transferred to 15°C, however the capacities of RuBisCO and

stromal Fm 1,6-BPase activity remained high. They atttibuted the 40% decUne in

photosynthetic activity to an increasing phosphate limitation due possibly to a slowing

down of Sue and starch synthesis because the total pool of hexose phosphates increased

only 11% and SPS activity had decreased by 40%.

1.6.3 Reduced Respiration at Cool Temperatures

Viola and Davies (1994) also reported increases in hexose phosphate levels at cold

temperatures in potato tubers along with a reduced glycolytic flux. Dixon and ap Rees

(1980) indicated that lowering temperatures from 25°C to 2°C in potato tubers restricts the

partitioning of carbon to glycolysis and increased the partitioning of carbon to the hexose

phosphates and Sue. Although glycolysis and respiration are reduced at low temperatures,

Labate and Leegood (1989) reported a high degree of energization (provision of ATP and

NADPH) in the darkened barley leaves at the low temperatures, indicating that the high

degree of energization may be responsible for the restriction in respiration at low

temperatures.

A similar response was found in immature cotton leaves (Cogbum, 1998) that were

exposed to a cycUng temperature of 30°C day/15°C night. Respiration rates were reduced

and did not recover with time at 15°C, however, respiratory ATP production was greater

than ATP utilization, resulting in higher ATP concentrations than those at 28°C. Low

temperatures may result in lower respiration rates, however, use of the respiratory products

seem to be lowered even more. Interestingly, when the cotton was transferred to a cycUng

temperature of 30°C day/19°C night, respiration rates decreased the first night, but with

time recovered to the rates at 28°C. Rates of leaf expansion and the dark respiration of

27

immature leaves of plants at 19°C also were similar to those at 28°C, whereas those at 15°C

were considerably reduced. There was an indication that enzyme activity and/or substrate

use are substantially altered as the temperature drops from 19°C to 15°C in cotton leaves.

1.7 Cool Temperatures and Fiber Metabolism

Very Uttie information is available on fiber enzyme activity at low temperatures.

Clearly, metabolism in the cotton fiber involves pathways of cellulose synthesis,

respiration, the dark metabolism of COj, and the pentose phosphate pathway. There is no

indication that cool temperatures affect any of the latter three energy pathways in such a

manner to reduce the synthesis of cellulose. In photosynthetic tissue of cotton seedhngs

there is indication of lowered SPS activity and the inability to supply inorganic phosphate

to the chloroplast, resulting in reduced photosynthetic activity (Perrara et al., 1995). Other

restrictions may also occur at any of the enzymes involved with cellulose synthesis.

1.8 Research Objectives

The research presented in this report investigates and compares the levels of the

metabolites involved in cellulose synthesis that were extracted from the fibers of cultured

cotton ovules grown at 15°C or at 34°C. The metabolites investigated were Glc, Sue, Fm,

Glc 6-P, Glc 1-P, Fm 6-P, and UDP-Glc. The goal was to determine whether differences

in the levels of each metabolite at the two temperatures could indicate a particular cool

temperature-sensitive block in the pathway. Glc 6-P, Glc 1-P, Fm 6-P, and UDP-Glc

levels were measured enzymatically for four cool-sensitive and three partially cool-tolerant

cultivars that were described earUer (see Table 1.1). For two cool-sensitive and two

partially cool-tolerant cultivars, these results were extended to measure Glu, Fm, and Sue

by High Performance Liquid Chromotography (HPLC). Using [ ' C] glucose in the

medium, pulse and pulse-chase experiments were also conducted on the cool-sensitive

cultivar Acala SJ-1 and the partially cool-tolerant cultivar Paymaster HS200 to determine

change in flux through metabolite pools related to cellulose synthesis and to identify

possible acclimation mechanisms to cool temperatures.

28

CHAPTER n

EFFECTS OF COOL TEMPERATURE ON METABOLITE

POOLS IN FIBERS OF CULTURED OVULES

2.1 Introduction

Fiber development in cotton (Gossypium hirsutum L.) is greatiy reduced during the

cool night temperatures that occur during the latter part of the growing season on the

Southem High Plains of West Texas. Fiber weight gain per day and micronaire are

significantiy reduced at night temperatures below 25°C (Gipson and Joham, 1968), and

final dry matter accumulation decreased with decreasing minimum temperature for growth

of tiie fiber (Thaker et al., 1989).

Studies of fiber development in vitro have been useful in showing that physiological

responses to cool temperatures occur in the fiber itself, separate from the effects on whole

plant physiology (Haigler et al., 1991; Roberts et al., 1992), and that responses to cool

temperatures in fibers of cultured ovules mimic those of field-grown fibers with reduced

rates of cellulose deposition manifested in the formation of "growth rings" (Haigler, 1991).

The rates of both respiration and cellulose synthesis during secondary cell wall deposition

in vitro decreased as the temperature decreased, however, the QJQ values between 18°C and

28°C of 3.13 for respiration and 4.28 for cellulose synthesis show that the latter is more

temperature sensitive (Roberts et al., 1992). Also, the responses of cellusose synthesis to

temperatures above 28°C differed from respiration in that cellulose synthesis plateaued at

28-37°C and respiration continued to increase as temperatures increased to 40°C. In

addition, upon rewarming to 34°C from cool temperatures, respiration recovered to control

rates within the first hour, whereas cellulose synthesis was depressed for several hours,

indicating that the two processes are uncoupled within the cotton fiber. It was suggested

that a rate-limiting enzymatic step in the pathway of cellulose synthesis may exist in the

fiber during periods of cool temperature below 25 °C.

The pathway leading to cellulose synthesis in the fiber cell was first described by

Carpita and Delmer (1981) as:

Glc •Glc 6-P • Glc 1-P •UDP-Glc • Cellulose

Although UDP-Glc has been shown to be the immediate substrate for cellulose synthesis

(Ross et al., 1991), it has been recentiy proposed that membrane-associated SuSy

hydrolyzes sucrose at the plasma membrane to directiy supply UDP-Glc to the cellulose

29

syntiiase complex (Amor et al., 1995), releasing Fm back into the cytoplasm. High SuSy

activity has been reported in the cotton fiber cell (Basra et al., 1990; Nolte et al., 1995;

Ruan et al., 1997) and evidence has been presented for the movement of Sue from the

ovule seed coat into die fiber cell (Ruan et al., 1997).

The Sue suppHed to the proposed SuSy and cellulose synthase complex may result

not only from direct import into the fiber cell, but also from Sue synthesis within the

cytoplasm of the cell. Large Pools of Glc and Fm exist in the fiber cell (Basra et al., 1990;

Jaquet et al., 1982; Ruan et al., 1997) possibly as a result of the Fm released from

membrane-associated SuSy activity and the substantial invertase activity present within the

cell (Basra et al., 1990; Waffler and Meier, 1994). The synthesis of Sue from these hexose

pools would channel more of the carbon present within the cell towards cellulose synthesis.

The pathways involved in sucrose synthesis within the cytoplasm of the fiber cell are

outiined in Figure 1.4 and include the phosphorylation of the hexose sugars, the synthesis

of UDP-Glc from the hexose phosphates, and the synthesis of Sue from Fm 6-P and UDP-

Glc. Indeed, high levels of UDPG-PP and SPS are reported in the cotton fiber cell

(Tummala, 1996; Waffler and Meier, 1994).

The purpose of the research presented in this chapter is to investigate the changes in

pool sizes of the metaboUtes involved in carbon metabolism in the cotton fiber upon

exposure to cool temperatures during secondary ceU waU synthesis. The changes in pool

size will be used to determine which step or steps in the metabolic pathways related to

cellulose synthesis may be affected most by the cool temperatures. In addition, metaboUte

pool sizes wiU be compared among several cool-sensitive and partially cool-tolerant

cultivars to denote differences among cultivars to cool temperature stress during secondary

cell waU synthesis.

2.2 Materials and Methods

2.2.1 Plant Growth

Seven cultivars of cotton (Gossypium hirsutum L.) with differing responses to cool

temperatures (see Figure 1.1) were grown in a greenhouse at a cycling temperature of 32-

37°C day/22-25°C night, with variation related to winter and summer conditions. Plants

were watered three times a day with an automatic drip system and fertiUzed one time each

week with a complete fertilizer (Rapid Gro 23-19-17, plus micronutrients).

30

2.2.2. Ovule Culture

Ovules were removed from flowers of the greenhouse-grown plants of cotton at 1

dpa and culttired by floating on top of Beasley medium (Beasley and Ting, 1973), except

120 mM Glc was included in the medium as the sole carbon source instead of Glc plus Fm

(Haigler et al., 1991). Gibberellic acid (0.5 |iM) and a-naphthaleneacetic acid (0.5 |iM)

were included to promote more uniform fiber initiation and elongation under cycling

temperature regimes (Haigler et al., 1991). Altiiough tiie concentt-ation of the hormone

a-naphthaleneacetic acid in the culture medium (0.5 |iM) was much lower tiian that used by

Haigler and coworkers (5.0 .M) (Haigler et al., 1991), no differences were found in fiber

weights, ovule weights, or glucose uptake from the culture medium. Ovules from different

flowers and locules were randomized in the culture vessels, 150-mL Erlenmeyer flasks

with 50 mL medium. Twenty-four to twenty-eight ovules were placed in each flask.

Ovules were cultured in a dark incubator at 34°C constant until 18 dpa when fibers

were actively engaged in secondary cell waU synthesis (Haigler et al., 1991) and then

transferred to dark incubators set for temperature regimes of 34°C/15°C on a 12 h/12 h

cycle. (Incubating aU flasks in the 34°C constant incubator until 18 dpa aUows

comparisons of fibers from ovules close to the same physiological age, since cycling delays

progression through aU developmental stages.) Half of the flasks were transferred on 18

dpa to an incubator beginning the high temperature of the cycle and half were transferred to

an incubator beginning the low temperature of the cycle for the purpose of extraction and

analysis at the same time and under the same conditions. Ovules were then harvested at

three time points-1, 4, and 7 h-on three separate days-18, 21, and 24 dpa-from both the

high and low temperatures of the cycle. These days were chosen to span 15°C shock and

an apparent 15°C accUmation observed in Acala SJ-1 (Haigler et al., 1991); the hours refer

to time after the temperature shift

In one experiment to compare cellulose synthesis and respiration rates to hexose

phosphate and UDP-Glc pool sizes at optimal growth temperature, ovules of Acala SJ-1

and Paymaster HS200 were cultured at constant 34°C and harvested on 12, 14, 16, 18, 21,

24, and 27 dpa. On each day, ovules from one flask were used for radiolabeUng

experiments to determine ceUulose synthesis and respiration rates and ovules from another

flask cultured at the same time were used for metaboUte extraction. In addition, ceUulose

synthesis and respiration rates were determined for the two cultivars at the 15°C side of

cycling temperatures on 18, 21, and 24 dpa for ovules switched to cycling temperatures on

18 dpa.

31

2.2.3. Fiber Removal and Extraction

At each of the three time points on either 18, 21, or 24 dpa, fibers were removed and

extracted from 20 ovules from both high and low temperatures. The ovules were removed

from the flasks with forceps and quickly placed for washing in a small perforated transfer

basket in a Petri dish filled with a temperature-adjusted medium lacking Glc, but

osmotically adjusted with 120 mM pentaerythritol. After three consecutive 10 sec washes,

the ovules were gentiy and quickly blotted three times on a paper towel (total time of 15

seconds) and placed in a mortar containing liquid nitrogen. While frozen, the fibers were

scraped from ovules using forceps and a flat-ended scalpel, then the ovules were discarded.

In the experiments involving enzymic analysis of the hexose phosphates and UDP-Glc on a

fresh weight basis, any fibers associated with frozen medium were also removed

(approximately 30% of die total) before weight determination. All of the fibers from die 20

ovules were used in experiments involving sugar analysis by high pressure liquid

chromatography (HPLC). The frozen fibers were quickly transferred to a cold mortar

containing 5 mL ice-cold 10% (w/v) trichloroacetic acid (TCA) for extraction according to

the method of Kanabus et al. (1986). Following 15 minutes of intermittent grinding while

on ice, the homogenate was centrifuged in four 1.7 mL microcentrifuge tubes in an

Eppendorf Microcentrifuge 5414 at 14,000 rpm for 4 minutes at 4°C. The four

supematants were coUectively combined and those fibers extracted for sugar analysis by

HPLC were washed and freeze-dried to determine fiber dry wt per ovule. A 600 |iL

portion of the supernatant was placed in a microcentrifuge tube with 800 |iL of a 3:1 (v/v)

mixture of 1,1,2-trichlorotriflouroethane and n-trioctylamine and shaken for 1 minute to

raise the pH of the supernatant to approximately 5.0. Suspensions were then centrifuged

as before and the upper aqueous phase (pH=5.0) was collected and kept on ice for

quantification of either the sugar-phosphates and UDP-Glc by enzymic analysis or the

neutral sugars by HPLC. In some experiments, known amounts of Glc 6-P and UDP-Glc

were added to the TCA solutions used for fiber extraction to assess recovery of sugars or

the extent of nucleotide decomposition. Upon enzymic analysis, 85 to 90% of the Glc 6-P

and 83 to 87% of the UDP-Glc were recovered, verifying that no major losses were

occurring from the extraction procedures.

In addition experiments, cotton fibers were also extracted with 80% (v/v) ethanol at

80°C, according to the method of Carpita and Delmer (1981), and with formic acid

saturated with 1-butanol, according to the method of Olempska-Beer and Freese (1984) to

compare extraction and recovery with TCA. Recovery of Glc 6-P and UDP-Glc after

extraction with 80% ethanol was only 78 and 76%, respectively, that of TCA extraction.

32

After extraction with formic acid saturated with 1-butanol, recovery of Glc 6-P and UDP-

Glc was only 79-85% and 75-81% tiiat of TCA, respectively.

2.2.4 Enzymic Analv.si.s

All enzymic analyses were conducted on a Uvikon 930 spectrophotometer (Research

Instmments International, San Diego) at 30°C. Hexose phosphates were measured as

described by Lowry and Passonneau (1972) in a reaction mixture containing 50 mM Tris

(pH=8.0) and 0.5 mM NADP, plus the enzymes added in sequence, 1.5 units of Glc-

6PDH for the determination of Glc 6-P, 1.5 units of phosphoglucomutase for the

determination of Glc 1-P, and 1.5 units of PGI for the determination of Fm 6-P.

UDP-Glc was also determined spectrophotometricaUy in an assay solution containing

50 mM Tris (pH=8.7), 2 mM MgCl2, 1 mM NAD\ and .03 units of UDP-Glc

dehydrogenase.

2.2.5 High Presure Liquid Chromatography (HPLC)

Glc, Sue, and Fm extracted from the fibers in the aqueous phase were separated by

High Pressure Liquid Chromatography (HPLC) using a cation exchange 300 x 6.5 mm

Waters Sugar-Pak 1 column , according to the method of Tobias, Boyer, and Shannon

(1992). The Sugar-Pak I column was maintained at 85-90°C, and the sugars were eluted

with a 50 mg L* Na2CaEDTA solution flowing at the rate of 0.6 mL min" and monitored

by a Waters model R401 differential refractometer (Altex Model 156). The hexose sugars

and sucrose pool sizes were determined only for two of the cool-sensitive cultivars, Acala

SJ-1 and Coker 312, and for two of the partially cool-tolerant cultivars. Paymaster HS200

and YG. Determinations were made at 1,4, and 7 h on 18, 21, and 24 dpa.

2.2.6 RadiolabeUng of Ovule Cultures

Cellulose synthesis and respiration rates from 12 dpa to 27 dpa at 34°C were

determined in one experiment for cultured ovules of Acala SJ-1 and Paymaster HS200.

Hexose phosphate and UDP-Glc pool sizes were also determined for the fibers of ovules

cultured at the same time. In addition, ceUulose synthesis and respiration rates were

determined at 15°C on 18, 21, and 24 dpa for ovules transferred to cycUng temperatures on

18 dpa. Ovules were labeled by adding [U- '*C]Glc to the culture medium (1.324 |iCi mL' ;

final specific activity 2.45 x 10' Bq fxmol"*) according to the method of Roberts et al.

(1992). Ovules were removed from the culture flasks, washed three times as described

previously in pentaerythritol medium minus Glc, and briefly blotted on paper towels before

33

being placed in 60 x 15 mm plastic Petri dishes containing 4 mL of labeled medium. The

Petri dishes had been equiUbrated to the incubation temperamre in the bottom of 300 mL

sealed plastic beakers. A filter paper (2.5 cm diameter) was suspended by a plastic paper

cUp from the inside of the plastic Ud and saturated with 1 N KOH before sealing for

trapping ^ COj to determine respiration rate.

Following labeUng for 4 h in a 34°C incubator, ovules were rinsed in temperamre-

equilibrated water three times, blotted on paper towels two times, and quickly frozen in

liquid nitrogen. All of the fibers were removed from the ovules while frozen as described

previously, freeze-dried, weighed, and then frozen at -20°C until extraction according to the

method of Roberts et al. (1992). Briefly, fiber samples were extracted in 5 mL of acetic-

nitric reagent for 2 h at 100°C to isolate radiolabeled crystalline ceUulose, collected onto

Whatman GF/C glass fiber filters, and rinsed five times with distiUed water. After drying,

the filters were added to 2.75 mL Scintiverse BOA scintillation cocktail (Fisher Scientific)

and counted in a Beckman LS 7500 scintiUation counter. CeUulose synthesis was

expressed as counts per minute incorporated per mUUgram dry weight of fiber. The filter

papers suspended to the inside of the plastic lids were aUowed to air dry foUowing the 4 h

incubation, mixed with 2.75 mL scintiUation cocktaU as described above, and analyzed by

scintiUation counting after 18 h (to exhaust chemiluminescence) in scintUlation cocktaU.

Respiration was expressed as counts per minute incorporated into COj per mUUgram dry

weight of fiber.

2.3 Results

2.3.1 Hexose Phosphates and UDP-Glc at 1. 4. and 7 h

The amounts of hexose phosphates and UDP-Glc present in the cotton fiber at 18,

21, and 24 dpa for each cultivar are presented in Figures 2.1 through 2.7. The top portion

of each figure graphs the changes in pool size (nmol g' fresh wt) for Glc 6-P and Fm 6-P

for each of two sets of experiments, whUe the lower portion graphs pool size changes for

UDP-Glc and Glc 1-P. To allow comparisons, Y-axis values are the same for all of the

graphs. The shaded portions of the graphs represent the 15°C side of the cycle and data

points represent 1,4, and 7 h after entry into that particular side of the temperature cycle.

Pool sizes are given for 18, 21, and 24 dpa for all seven cultivars with the addition of 22

and 23 dpa given for Acala SJ-1.

The maximum change in pool size of a metaboUte as a result of a change in

temperature generaUy occurred by 4 h into the cycle. There were no significant differences

in pool size between that at 4 h and that at 7 h for a metaboUte on any day in 94 percent of

34

J5

B

C/}

e c

,18 dpa. .....•....^l flnq ?4 dpa

Glc 6-P Glc 6-P Fm6-P Fm6-P

•'.•••.'

*. ••.-. •'

•." • .* I. ••.•. •' •.' . .*

12 0 —r^"*^1—

36 48 60 72 84 120 132 144 156 450

-12 0

^ = 1 5 ° C

120 132 144 156

Hours After Cycling Begins

Figure 2.1. Metabolite Levels from Two Replications of Acala SJ-1. Shaded areas indicate the 15°C side of the cycle. Each point is the average of two measurements for one replication.

35

450

400

350

4—•

t 300 D

r^ 2 5 0 ^ 200 e ^ 150

100

50

0

•21 dpa.

Glc 6-P

Glc 6-P

Fm6-P

Fm6-P

24 dpa

.••••.•*

I..

Iv/.;^:^

r^^oV^ '-.• •r-'-T

^ • . • • •

7

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0 12 24 36 48 60 72 84 96 108 120 132 144 156 J

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4

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•

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A

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-12 0 12 24 36 48 60 72 84 96 108 120 132 144 156

=15°C Hours After Cycling Begins

Figure 2.2. Metabolite Levels from Two Replications of Coker 312. Shaded areas indicate the 15°C side of the cycle. Each point is the average of two measurements for one replication.

36

J3

J3 c/3

'o

18 dp.a. ....,21 dpa,

Glc 6-P

Glc 6-P

Fm6-P

Fm6-P

24 dpa.

'.*• *'. I.--'.

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r .*.• • . ' • ' . ' ' •'.* • .'.'.' r •',• *.•'. '.• •'. «••.'• ' . • • • . 1- •.•.

if:': t • •. • * • " . • . •

»• •. • !• • 1 • , * . .

• . * l ^ fc"l] • > wH ^ M'*

/ r • .• ^ < • - . ' • 1 '.••'.

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V j ^ n

'.'.''.' • •'.'.. *'.•.' • '.*.'

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• f - ^ ^ - ^ - i — r ^ * - ^ r -n -12 0 12 24 36 48 60 72 84 96 108 120 132 144 156

UDP-Glc r^^ U * • - • • . ' . • • . ' . • *

UDP-Glc

Glc 1-P

Glc 1-P

^>:':':>-i

I . . •

. 1 .1

.1

V .;.• •:::-1 • : • : >

• \ >

i:-:'. r ..'.• f . . •. ' • . '•• •

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. .*! , .*• •.0

••• • . • •

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r,. f..' r..

r^-^ r 12 0 12 24 36 48 60 72 84 96 108 120 132 144 156


Figure 2.3. Metabolite Levels from Two Replications of TM-1. Shaded areas indicate the 15°C side of the cycle. Each point is the average of two measurements for one replication.

37

650

600

550

500

% 450

-S 400

^ 350

;^ 300 o I ^

200 150

100

50

0

450

400

350

t 300

^ 250

^ 200 S

^ 150

100

50

0

18 dpa. ...21 dpa, Glc 6-P

Glc 6-P

Fm6-P

Fm6-P

•.'..

24 dpa.

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' . * . l •.• • ' i ( .". V •'.' • . ' • '^^ i *'**^% J ."• 'ix 1 ' *'* m

- ^ A

r . . • . 1 . • • . .

1. •.•. • m 1 • , ' .

*'.' •', • '.'• ' .

'V'-v

*•*•/•" (A-v 1 • •. •

^ ' • ' • . " l

# 1 • ' ^ • * ^ fc',.

B • fc*.*..'

# • • ' .

# '."' »# r .'.• P * •.'" • 4 r •*.• • 1 \ 1. •

V - ' A . "

; / . - . j

• ' . • • *

•••% • ' : • *

• • • . »

. ^ . • . • 1

. ' . ' • • • • . • »

• • ; • • *

•!•'*

^ . . • j > . 1

^:* ."••.» .\M •. • •»

k' '•* W' 1 • .• • ! • . • • 1

• .* .| •.•.•« k . ' . ' l ^ • . ' l * . . . ' . 1

. ". • *.• •'* '. . • ". •.•".• ". • • '•':'* • . . 1 ".•.. A . • • • . » .•.•4 •. • . »

12 0 12 24 36 48 60 72 84 96 108 120 132 144 156

I • 11' 11' I =15°C Hours After Cycling Begins

Figure 2.4. Metabolite Levels from Two Replications of JDX. Shaded areas indicate the 15°C side of the cycle. Each point is the average of two measurements for one replication.

38

o B

650

600

550

500

450

400

350

300

250

200

150

100

50

0

18 dp.a. 21 dpa.

• 'A 1

".* t J

, 1 4

. 1 4

4

4

<• 4

>• 1

•• ^ H

r,-• ; •

•., K'\ • •. k • I • . K \ 1 • . fc •,

</• r.--. *'M r.l I.J r'i • • 1 *I

• • • . • • • • . • < . . . . . , i - • • . • • ' • . <

•. .•. .• • ' . ' • • • * '

• . . . . , 1 • " • . ' • ' • •»

'.• •'.• .• • • . • • • . " »

"• • •.".• •.*.

v^Vvt

i^ •• •'.•' v". •.•> •\ •'.• * 1^.'* •'•.'' ^ .'* • • . • • * . • «

Glc 6-P

Glc 6-P

Fm6-P

Fm6-P

r-. I . .

<• . •

v..

. . .» •.I

. • • •>

' . I .••4

*.l .•* . ' . I . • 4 ^y;^^y:v^

-r-'-t-

r..

•. • ' ,

' • . • • •

P:

• f - ^ - ^

24 36 48 60 72 84 96 108 120 132 144 156

UDP-Glc

UDP-Glc

Glc 1-P

r. •

mf:\

Glcl

p . - : r.'. P.-:

\y: f..-,

ryi

.••1

3"trj

I ' . t . •

• . 'J - . 1

1—-1 1—T r - 12 0 12 24 36 48 60 72 84 96 108 120 132 144 156


Figure 2.5. Metabolite Levels from Two Replications of Paymaster HS200. Shaded areas indicate the 15°C side of the cycle. Each point is the average of two measurements for one replication.

39

X3 (D

B

650

600

550

500

450

400

350

300

250

200

150

100

50

0

450

-

• •

18 dpa. •• p • . • • • . • • • .

;p i . " . • . • . • ; .

•J r-CJ.-V' i— •* t-ti-''.'-' i iiv"- — i r > L So- ^ uT"^"'-! iWl^i^ •I h :•'.•'.'•'•'>!i

iWiPI i ^ Z '.'T.'.V-v.j < o r.-'.•'.•"••".•'• I ' t®&3

. • ^ • • . • • • • . • • • • . • *

L 1 •••••• 1

»' •,'.' •, f ' . / ' . • ' ' . • ' \

i.\V.\'

p. . • • .

< • • . • • • • . • • • . ' . .

i- • : • •

' • ' ' . ' • • ' . ' •

'•''•.'•'': p". . *.

• ' . .•'. •''. •'". p.'. . • . ' . • * . " ' . P." • • . •

\y.'y f-.VV rV'VV-

—r-

** *.*• • ' . p

•.'•.4 • . • •4

.•••••1 . - . • I

•'.••..•I

' . " ' • ' l

." . .1

• •. I .*.•• J . ". •

•'.•* .-.•^i ••"••"•»

.• '•.••*

21 dpa Glc 6-P Glc 6-P

Fm6-P Fm6-P

* :'•' •.'•• •.'•

^''.-'y.-'.y.-^ \

i-y-':''n t-:-::':-si

' ' • • ' ' • • . ' • . ' ' • • . ' • • '

' ' • . ' • • ' • . ' • • * • . ' »

« • ; • • . • • • . • •

r,- .;• .;• t • • . ' . • , • . ' . 'p p * . ^ * ' ' . * «

P ' . ^ ^ H ' « *.*.f.4v***

*•'•%•'•'y-' «. •i\*.'*.'-'p

i T T - * . .'P p.fce."-. 4

tpi * f 1 • . ' . * . " *

^•1 ^lir**.*,*p H Tm*.'.''

MJM ft V * J . * * * . * *

^J0 *.1.''.V'.'•'•

V » • ' • * " ' • . ' • '

• • • ' • iV^ W- ' J ^ * P p • .• • • . .«

i:/:/:.pj^W^:A p. . ; . •.•.•.« • • • . • • . . I X-...-...\.i

t-v'-.'.-'-.v; 1 — 1 -

1 • . * . • ; . ..'p

• • . • • ' . • • ' . • * » • . ' • • . ' • • . " • 1 " . . . . . " . J

• * ' . ' • ' * . ' • '* . 'p

1' ' '1

r-.-V.

p . . \

< • • . • • - •

Ivv

* ' • • ; • • .

* ' . • * • • ' .

» . • • • . . ** *. k .• • • .

r.-v-.

^ .• '• . 1 • •«

k ,• '• ,

••. '* *. ^•fy. r.-.-.

f . . . .

•".•• ' f

• ^

.'•"•' ^ . ' • / I

/ •, . ' I

.* • ••

'• / '

i •'.•• 4. •.:

I'-::' ' .* • r,.

• *.' .* r. •.•. • ' • ' . * • I , ' . ' •.•

• • *. • • . ' • ' . • •

t.' •• •." • .' • .* 1 • • . " .

' • * . •

• .' .**_ .*

r *. •.

. •'. « • • • . " r. •.•. 1 ' . . . '

p*.*'. '

• *.*.'

"•'••."••" < . ' . . ' •• • ' .•• p . ' . .• t , . . • .

p. ' .* • ' < • • . • •

*'•'•!'•

24 ". •. •'. t

.'••*.*•' ' . ' • ' J

•' •"'

• • • • ' > _

M •••••vr ••••• \ j

V J V

IT •'.'"•7

.. P j

w

i 1 ,. 7

/ i

A / - V m /f^wi 'rv-vvj

•.-••.••.-.••!

1 • ",'• '.'• '.'• '.'• ".'• ".'• • ' . * • " . ' • ' . * ^

r ' ' i ' 12 0 12 24 36 48 60 72 84 96 108 120 132 144 156

r • p\ '

t;'

UDP-Glc

UDP-Glc

Glc 1-P

Glc 1-P

r,. ».* r. •' t.'

p*. •

••.1 •A

.-'••A

i:'y>;>A

I'.. p..,

+ 12 0 12 24 36 48 60

•:/i

J^^^vl f • 1

96 108 120 132 144 156


Figure 2.6. MetaboUte Levels from Two Replications of YG. Shaded areas indicate the 15°C side of the cycle. Each point is the average of two measurements for one replication.

40

B

650

600

550

500

450

400

350

300

250

200

150

100

50

0

450

400

350

300

250

200

150

100

50

0

18 dp.a.. ,21 dpa.

Glc 6-P

Glc 6-P

Fm6-P

Fm6-P

«• • . • ' • • • . • ' • • ' . • ' '

r-.

r.-

1-:

..1

.1

X-.

l ^oy J

^yi'

T

r.. *•., r.."

• • , •

t •' «*. ' r • •

X-. p.-X-. p. • X-.

•r-^n-

• • >

• .1

'.•I

. .1

If-r-:'. » . . '

• 1 ' . ' I

• .p ' . •J . . p

• f - ^ * ^

-12 0 12 24 36 48 60 72 84 96 108 120 132 144 156

-

^

J

;• 4 . 1 4

4 . 1 4

> l 4 • •

• •

•• ,'%

• p ' j

*4

« .P

« *' 1 ' o ' .p ^ ^

, ^ ^ ^ k

"P •p

•,p , 1

> p

> > p

X T V

1—

!•.• * • . ' •

p.. . '

r; -p . . .

i . • . •• .*• •• p - •.

* . • • '

*'.'• p • . *

^ • *. p . . '

p . . . •

1 .•'.•'

i.-'-' t • • ' . • p •• •.

^ '•'.•'

^^^v .* V p - ^ ' . 4 • •.

»'.••' "•'••V p.*."*. 1 .

K';'.

'•' •.' « . • ' • •

—r^

.• . • • . p

•.'• '.^

^ . . . • ; . •

• .• . p

. • • ' . ' p

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• ' • . • ' • • ' • '

. ' . * " . ' . p

.... • • . • . • ' . p

• • . • . • ' . »

y-'-) • • : • • • >

^ ' ' • f

f j . ' ' V ^ . P ' • • • . • ' i' ' • p

' . • • • . • *

•.'.• • j^ . 'J ".* • .'" '.'* . ' . . ' . " . p

.'..•,'«

.''.'.*'• • ' . ' ' * . p

•.'• * *'•'.*'•• ._• '. \

U - ^ 1 —

p . p .

A

j.-.-.v *•'.••'•'.••

*•''.'•'•'.'•

'•''•.'•'•. P ' • . " ' • C . . . ' . .

• ' . ." ' .

*'.'• •".*• • ' ' . . ' ' . P.'. .•.". • ' . . " .

'.••'.• '.'. *.'• <.••'.• f •'•.'•'• I.- • .• p'-V'"-' }•.••".••.;• p . . * • . *

* : • • ' : '

p • . • •.

*•'.'•'.'• p ' . • •.

p . •

.•;> • • / . " '

• • • • . • » . ' . .P . ' . ,P

".'•' .*•"

M •."•J • . • . 1

'.••* .••"•1

'.•i .'..'•I • • ' • . " • ^

. . •':•* ::'i •".••"'

UDP-Glc

UDP-Glc

Glc 1-P

Glc 1-P

t:'::':>i »•.'•• •.'•• •."' rv".V.\-1 n '•f^-fy-^ r '•• .• •• .• •• . " i - X '•.•':• •.rO ••. .• .• •. .KJ^ ' . " • • ' . • • • ' . * • *

J'•.•-•'•.•••'•.•*

0 ••.•• ••.•• ••» A

' • • • • • • • • • • • • 4

•••.•••.••W A • • • • • • . • • • . ' \ /

*•.•••.•••.•• M '••.•••.•••.• 9 ' • . - . • . • . " . • p

* • . . • • . • • • • . • • '

^ • • • • • . ' • • • • . ' • . »

* .' • .•' .*• » • . • . • • . • • ' • . • . >

l •^^^/^^ i^ ' • ; • • • . • • • • . ' * * ^

p • ' . • • ' . • • ' . •

».*. ".*...'. • » . * . • . " . • . • . p

• •'.' •'.' • ' . ' » . ' • • . ' • • . ' • p

p * . . ' • . • • • . " * < : .• •. .• •. .• p

' • . • • • . • • • . • »

*-.-y:.y-t

1* • . • • . • ' t

* • . • • • . • • • . * » p ' • . • . • . • . • *

p ' . * • ' . ' • ' . " '

''•':'•',•'•'* p' . * ' . ' . " ' . . ' . • ' *

J-riVv* t ^A^^ V ^ / K . . ' \ r . f - .SJ. ' \ l • •• •• •• •' rk.- •:• -l P-V-.V-'

. F^^--^ . r - ^ A - i SAJ^T- . 'V •^•;'\'« ^ . • • . * . *

I • ' . • • . • • !

•*,' •'/ •*.' ' 4 " , ' . • , ' . ' . ' •

fc'*,".''.".'','l

'*•'.•'•>'•'.* •.'.TjtA-.» ^W':^ Sr % v:

—r ' P L •!

< . • ' , . • . •

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r. •*/• •.' •. t . ' . • .

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*V '•*. ••. •* *. *'.* '•*. r. .*

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7^vl— ••V *

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A'."* •**.•'•* '•/•*i .* • * .•' •*'*.'•*' .' •'.'•

•. ' . • 1 .* • . ' 1

••V ^

*. • \ •V/{ •'/.'/ . • *.*• •» .* * .' ;.*• '* • ' . * • '

.* •'*.'< •'.' •* •'.'•'t

•''. •'•

• ' . • • * /

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1 / ' . / '

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r . • / . •

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i' •.'•'. ••'.* •*. t ' . ' •

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. •/ • . • . • 1 ^ ^

• ' .* 1 V H

'•' ' * J

- . ' . ' 1

• • - ' < ^

'-'-' w •• •'• ^ :*J . - . A

. -.W . • • . • »

•.-.» •'•.! A

•'.- A • • • 4 / ; .••••• A ' . / 1

• ^

r..". p . . . " . •

r...'.. p . • . • . •

1 • •. •

1 • ' . •

fc'.* • ' .

' *. '• '.

r*. *. •'.

Mil' -/ r • •

/ * •

^ 1 ' .

<J ' • '• ( ' • ' • . ' • •

^ ' • • i

\::\ Jd mr ^ W fc-.-.'.

* '.*• ' fc','

• *.'• * • ' . '

i-A • A L . ' / • »

V ' f ' i ^^^

*r • .•

*Cp

.'.•' '.•.• • • • . »

•••X . • ' • \

". .* 1 . ' • • • .•»

-• . * . ' P

•.;.•! • ' . ' 1

.':'*

ii' .'•' • • • ' !

'.' •'. 1

• . ' • ' . •«

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•. - . "P

'.**•*.' / . ' i

. ' ; A

'Jy* V-.' H * . . J

. 1

-12 0 12 24 36 48 60 72 84 96 108 120 132 144 156


Figure 2.7. Metabolite Levels from Two Replications of Paymaster HS26. Shaded areas indicate the 15°C side of the cycle. Each point is the

average of two measurements for one replication.

41

the determinations. Consequentiy, 4 h and 7 h measurements were considered together as

repUcations in analyses to determine differences between pool sizes at 15°C and 34°C. The

few significant differences that did occur between 4 h and 7 h had no affect on mean

differences between temperatures.

Pool sizes at 1 h into the 15°C side of the cycle differed significantiy from 4 h and/or

7 h pool sizes many times, particularly with Glc 6-P in which pool size variation was the

greatest between temperattires. At 1 h into the 15°C side of die cycle, the levels of

metaboUtes generaUy had not changed significantiy from levels on die 34°C side of die

cycle. Indeed, the temperature of the medium of die cultured ovules did not lower

completely to 15°C until after one and one-half hours into the cycle (Haigler et al., 1991).

In contrast, metaboUte levels at 1 h into the 34°C side of die cycle only differed from Uiose

at 4 h and 7 h 16 percent of the time, with most of those occurring on 18 dpa before cycling

had begun. There appeared to be a quick recovery to 34°C pool sizes within 1 h upon

rewarming to 34°C. In fact, the pool sizes at 21 and 24 dpa were lower at 1 h than those at

4 h and 7 h on the 34° side of the cycle. One possible explanation for these observations is

that the critical temperature affecting pool size may be close to 15°C and a change in pool

size would occur rapidly as the temperature began to increase. The data are also consistent

with a temperature-related restriction in metabohsm foUowing a metaboUte that elevates at

15°C and/or an elevation in a metaboUte to compensate for exposure to 15°C.

2.3.2 Glc 6-P and Temperature

The amount of Glc 6-P at 15°C and at 34°C at 4-7 h after cycling for all cultivars at

18, 21, and 24 dpa and the percent increase in pool size at 15° over that at 34°C for each

day are given in Table 2.1. The amount of Glc 6-P (nmol g'' fresh wt) in fibers of ovules

after 4 h at 15°C was significantly greater than that at 34°C for aU days in aU of the

cultivars. The percent increase in Glc 6-P at 15°C over that at 34°C was greater for the

partiaUy cool-tolerant cultivars on 21 dpa (57-71%) than for the cool-sensitive cultivars

(26-57%). By 24 dpa, Glc 6-P levels were at least 55% greater at 15°C for aU of tiie

cultivars, except for TM-1 in which levels at 34°C were also increasing from 18 to 24 dpa.

The partially cool-tolerant cultivars Paymaster HS200, JDX, and YG exhibited

greater absolute amounts of Glc 6-P at 15°C and 34°C at the shock treatment (18 dpa) and at

21 dpa than did the cool-sensitive cultivars, but the amounts decreased at 24 dpa to levels

close to those of the cool-sensitive cultivars. These partially cool-tolerant cultivars

appeared to have a greater abUity to increase Glc 6-P levels immediately in response to cool

temperatures. The other partiaUy cool-tolerant Paymaster HS26 did not exhibit greater

42

Table 2.1. Amount of Glc 6-P at 15°C and 34°C at 4-7 Hours Into Cycling and 15°C Percent Increase Over 34°C. The amount represents the mean ± SE (nmol g" fresh wt) from 4 replications with 20 ovules in each replication and two measurements of each replication. Significant differences between the means of each pair are indicated by different letters foUowing the mean.

Cultivar Temp.

Cool-Sensitive:

SJ-1

C-312

TM-1

Ave

15°C 34°C

15°C 34°C

15°C 34°C

. 15°C Ave. 34°C

18 dpa

Amount %

253.8 ± 7.3a 41 179.9 ±10.8b

232.0 ± 9.4a 34 173.6 ± 6.0b

276.0 ± 7.8a 28 215.0 ± 9.6b

253.9 34 189.5

Partially Cool-Tolerant:

HS200

YG

HS26

JDX

Ave

15°C 34°C

15°C 34°C

15°C 34°C

15°C 34°C

. 15°C Ave. 34°C

366.4 ±19.9a 27 287.5 ±20.0b

429.5 ±21.3a 59 270.2 ±19.1b

261.6 ±18.8a 33 196.5 ± 4.8b

342.0 ± 7.6a 54 222.0 ±10.2b

349.9 43 244.1

Days Post-Anthesis

21 dpa

Amount %

(nmol g" fresh wt)

263.6 ±11.la 48 177.6 ±7.3b

303.8 ±11.2a 57 193.2 ± 4.2b

298.0 ±10.8a 26 237.2 ± 7.3b

288.5 42 202.7

373.9 ±13.2a 61 232.8 ±12.1b

481.4 ±29.3a 71 282.0 ±19.7b

328.0 ±21.4a 57 208.8 ±17.0b

342.0 ±21.5a 61 211.6 ±18.9b

381.3 63 233.8

24 dpa

Amount %

305.2 ±18.7a 60 190.3 ± 8.3b

307.6 ±17.0a 60 192.4 ± 6.3b

357.6 ± 9.6a 37 261.8 ±15.3b

323.5 52 214.8

320.8 ±20.0a 60 201.0 ±18.6b

408.9 ±24.5a 64 249.1 ±10.5b

367.5 tlO.la 100 184.2 ± 4.7b

327.5 ±21.3a 55 211.1 ±18.1b

356.2 69 211.4

43

absolute values of Glc 6-P at 18 dpa, however, by 21 dpa, levels at 15°C were similar to

tiie odier partially cool-tolerant cultivars and were 57% greater dian diat at 34°C. Levels of

Glc 6-P were 100% greater by 24 dpa.

2.3.3 Fm 6-P and Temperamre

The amount of Fm 6-P at 15°C and at 34°C at 4-7 h after cycling for aU cultivars at

18, 21, and 24 dpa and the percent increase in pool size at 15°C over that at 34°C for each

day are given in Table 2.2. In all cultivars, the amount of Fm 6-P at 15°C was higher than

that at 34°C on every day. Significant differences (p<0.05), however, between the levels at

15°C and 34°C became more apparent as the days into cycUng increased. Fm 6-P levels

were relatively high at 15°C and 34°C on all days for the cool-sensitive TM-1 and the

partiaUy cool-tolerant YG; and therefore, no significant differences were found between

levels at 15°C and 34°C in these two cultivars on any day. The data from the other

cultivars, however, indicate that if the levels of Fm 6-P at 34°C were fairly low

(approximately 120 nmol g ' fresh weight or less), then significant differences were found

between the two temperatures. This data, therefore, indicates that a possible threshold level

of Fm 6-P must be attained that may be important to partiaUy compensate for reduced SPS

activity that occurs at low temperatures (Stitt and Gross, 1988). SPS has been shown to

have a relatively high Qio of 3.7 in darkened barley leaves (Stitt and Gross, 1988), and

SPS activity in cotton leaves decreased 40% when temperatures were decreased from 28°C

to 15°C (Perrera et al., 1995). Also, SPS extracts from spinach leaves were controUed to

remain inactive untU a threshold concentration of hexose phosphates was reached (Stitt,

WiUce, Feil, and Heldt, 1988).

Although no differences in Fm 6-P levels were found between the cool-sensitive and

partiaUy cool-tolerant cultivars, it was apparent that levels at 15°C became increasingly

greater than that at 34°C from 18 to 24 dpa. The increase in Fm 6-P may compensate for

the lower SPS activity at the lower temperature and/or may be the result of a reduced

glycolytic flux diat may occur at 15°C (Cogbum, 1996; Viola and Davies, 1994).

2.3.4 UDP-Glc and Temperature

The amount of UDP-Glc at 15°C and at 34°C at 4-7 h after cycling for all of die

cultivars at 18, 21, and 24 dpa and the percent increase in pool size at 15°C over that at

34°C for each day are given in Table 2.3. There were no significant differences between

UDP-Glc pool sizes at 15°C and 34°C for any cultivar on any day. At 18 dpa (shock

treatment), UDP-Glc levels for Coker 312 and Paymaster HS26 were 8-9% lower than

44

Table 2.2. Amount of Fm 6-P at 15°C and 34°C after 4 Hours Into Cycling and 15°C Percent Increase Over 34°C. The amount represents the mean ± SE (nmol g' fresh wt) from 4 replications with 20 ovules in each replication and two measurements of each replication. Significant differences between the means of each pair are indicated by a different letter foUowing the mean.

Cultivar Temp.

Cool-Sensitive:

SJ-1

C-312

TM-1

Ave

15°C 34°C

15°C 34°C

15°C 34°C

. 15°C Ave. 34°C

18 dpa

Amount

77.9 ± 3.7 66.2 ± 4.9

103.7 ± 4.5a 88.7 ± 2.8b

153.3 ± 8.0 141.5 ± 5.6

111.6 98.8

Partiallv Cool-Tolerant:

HS200

YG

HS26

JDX

Ave

15°C 34°C

15°C 34°C

15°C 34°C

15°C 34°C

. 15°C Ave. 34°C

149.0 ±18.0 146.2 ±21.0

188.1 ±25.8 167.2 ±30.4

97.6 ± 9.6 90.7 ± 6.6

132.9 ± 5.5 120.0 ± 9.9

141.9 131.0

%

18

17

9

13

2

13

8

11

8

Days Post-Anthesis

21 dpa

Amount %

(nmol g' fresh wt)

90.4 ± 5.1a 42 63.8 ±3.3b

128.3 ± 6.4a 33 96.4 ± 1.9b

151.9 ± 7.2 13 134.3 ± 8.1

123.5 26 98.2

126.3 ±22.6 13 111.9 ± 7.7

215.8 ±40.2 7 202.1 ±30.6

119.5 ± 4.3a 27 93.9 ± 6.0b

160.6 ± 6.7 4 155.5 ±11.8

155.5 10 140.8

24 dpa

Amount %

98.0 ± 8.7a 33 73.8 ±6.9b

120.9 ±7.0a 25 96.5 ±3.3b

164.6 ±10.1 12 145.6 ±12.9

127.8 21 105.3

130.9 ± 2.5a 28 102.1 ± 8.3b

201.0 ±20.8 23 163.4 ±15.5

135.9 ± 5.4a 57 86.6 ± 4.0b

181.6 ±21.3a 55 117.0 ±14.0b

162.4 38 117.3

45

Table 2.3. Amount of UDP-Glc at 15°C and 34°C after 4 Hours Into Cycling and 15°C Percent Increase Over 34°C. The amount represents the mean ± SE (nmol g" fresh wt) from 4 replications with 20 ovules in each replication and two measurements of each replication. There were no significant differences between means at 15°C and 34°C for any of the pairs on any day.

Days Post-Anthesis

18 dpa 21 dpa 24 dpa

Cultivar Temp. Amount % Amount % Amount %

Cool-Sensitive: (nmol g' fresh wt)

SJ-1 15°C 158.3 ±11.0 12 170.4 ±18.0 8 173.4 ±17.3 8 34°C 141.7 ±10.0 158.6 ±14.2 161.4 ±15.8

C-312

TM-1

Ave Ave

15°C 34°C

15°C 34°C

. 15°C

.34°C

122.3 ± 3.0 133.6 ± 4.2

166.7 ± 7.9 161.8 ± 3.5

149.1 145.7


HS200

YG

HS26

JDX

Ave Ave

15°C 34°C

15°C 34°C

15°C 34°C

15°C 34°C

. 15°C

.34°C

217.3 ±10.0 216.1 ± 7.0

322.9 ±25.9 320.6 ±18.1

144.2 ± 8.6 158.0 ± 8.1

224.2 ±17.4 186.3 ±15.9

227.2 220.3

-8

3

2

0

1

-9

20

3

148.9 ± 4.7 144.9 ± 2.9

223.7 ±11.3 211.5 ± 8.9

181.0 171.7

255.4 ± 8.2 226.7 ±14.1

370.7 ±16.5 347.5 ±30.5

180.5 ±15.6 170.6 ±16.6

208.8 ±10.9 202.7 ± 8.1

253.9 236.9

3

6

3

13

7

6

3

7

160.1 ± 7.8 144.2 ± 4.9

245.4 ±19.7 200.0 ±10.6

193.0 168.5

223.0 ±13.4 202.0 ±18.4

325.4 ±11.6 328.2 ±21.1

201.3 ±14.4 177.5 ±12.7

200.5 ±14.2 184.7 ±16.7

237.6 223.1

11

23

15

10

-1

13

9

6

46

tiiose at 34°C, and levels of Paymaster HS200, YG, and TM-1 were only sUghtiy higher

(1-3%), indicating that a possible enzmic restriction may occur in the padiway prior to

UDP-Glc syntiiesis. Levels at 15°C for the cool-sensitive Acala SJ-1 and die partiaUy cool-

tolerant JDX at 18 dpa, however, were 12 and 20% greater, respectively, than those at

34°C. This, however, may indicate that the use of UDP-Glc in metabolic pathways was

restricted in these cultivars.

At 21 and 24 dpa, UDP-Glc levels at 15°C were never over 13% greater than those at

34°C for any cultivar, except TM-1 at 24 dpa in which levels were 23% greater at 15°C.

Since the levels of Glc 6-P at 15°C at 21 and 24 dpa were at least 48% greater dian those at

34°C for almost aU of the cultivars, there is the implication that either the synthesis of UDP-

Glc may be somewhat restricted at cool temperatures or the use of UDP-Glc may be almost

equal to its synthesis. Certainly, Glc 6-P is an aUosteric activator of SPS (HUl and ap

Rees, 1995; Huber and Huber, 1992; Reinholz, Geigenberger, and Stitt, 1994), and tiiis

would increase the use of its substrate UDP-Glc in the synthesis of Sue within the fiber.

It must be noted that UDP-Glc levels at both 15°C and 34°C increased an average of

15% from 18 to 21 dpa in aU of the cultivars, with the exception of JDX at 15°C, and

smaUer increases or decreases were observed from 21 to 24 dpa. This does imply that

UDP-Glc levels may have been increasing to partially compensate for a restriction in SPS

activity at the lower temperatures.

Although botii Fm 6-P and UDP-Glc are substrates for SPS, Fm 6-P levels at 15°C

graduaUy increased over those at 34°C from 18 dpa to 24 dpa, whereas UDP-Glc levels at

15°C only increased slightly. However, UDP-Glc/Fm 6-P ratios at 15°C ranged from 1.1

to 2.0 for the cool-sensitive cultivars and 1.5 to 1.7 for the partiaUy cool-tolerant cultivars.

Higher concentrations of UDP-Glc compared to Fm 6-P may be another explanation for the

lack of significant increases at 15°C over those at 34°C, and may indicate that they may not

be as restrictive to SPS activity as those of Fm 6-P.

2.3.5 Glc 1-P and Temperature

The levels of Glc 1-P were very low compared to the other hexose phosphates, and

the curves for the 2 temperature conditions were generaUy overlapping. The amount of Glc

1-P detected in the fibers varied greatly between experiments for each cultivar and among

days within a cultivar. This suggests that flux through this metabolite is rapid and that the

activity of the reversible phosphoglucomutase favors the synthesis of Glc 6-P over Glc 1-

P. Therefore, Glc 1-P levels were omitted from this report.

47

2.3.6 Cellulose Synthesis and Respiration Rates in Acala SJ-1 and Paymaster H.9900

The average absolute amounts of Glc 6-P and Fm 6-P in the fibers of die cool-

sensitive cultivars were similar to or approaching those of the partially cool-tolerant

cultivars on 24 dpa. This suggested the possibUity that cultivars may differ in metaboUc

pool size because of differences in the rates of secondary cell wall synthesis. This

experiment was conducted to compare the rates of cellulose synthesis and respiration

between two cool-sensitive cultivars, Acala SJ-1 and Coker 312, and two partially cool-

tolerant cultivars. Paymaster HS200 and YG, and at the same time, measure the amounts of

hexose phosphates and UDP-Glc in the fibers of Paymaster HS200 and Acala SJ-1.

The rates of cellulose syntiiesis for Acala SJ-1 and Paymaster HS200 are given in

Figure 2.8. Both cultivars exhibit simUar pattems of ceUulose synthesis at 34°C-a

moderately increasing rate followed by a rapid rate of incorporation of [ '*C]Glucose into

crystaUine cellulose. The rate of incorporation began to increase dramatically after 16 dpa

for Paymaster HS2(X) and after 18 dpa for Acala SJ-1. There is a 2-3 day delay in the

onset of rapid ceUulose synthesis for Acala SJ-1 compared to Paymaster HS200. At 15°C

on 18 dpa, cellulose synthesis rates were the same for both cultivars. However, after a 3

day period of accUmation to 15°C, Paymaster HS200 synthesized ceUulose about two times

more efficientiy than Acala SJ-1 and continued the higher rate of synthesis through 24 dpa.

The amount of ' COj released at 34°C and at 15°C and the dry weight of die fibers per

ovule are given in Figure 2.9. Acala SJ-1 and Paymaster HS200 demonstrated simUar

patterns of respiration, with a decrease in respiration at 16 and 18 dpa corresponding to the

onset of rapid secondary ceU wall synthesis. It must be noted that although ceUulose

synthesis rates increased at 15°C on 21 dpa in the fibers of Paymaster HS200, there was

not the same increase in respiration rates. Paymaster HS200 appears to have the abUity to

increase flux of carbon preferentially towards cellulose synthesis after 3 days into cycUng.

The rates of ceUulose synthesis for Coker 312 and YG at 34°C are given along with

the rates for Paymaster HS200 and Acala SJ-1 in Figure 2.10. Coker 312 and YG began

rapid secondary ceU wall synthesis at 18 dpa, similar to Acala SJ-I, however, the rate for

Coker 312 at 18 dpa is 33% higher than that for Acala SJ-1. Respiration rates for Coker

312 and YG are also given along with the rates for Paymaster HS200 and Acala SJ-1 in

Table 2.11. The same decrease in respiration observed in Paymaster HS2(X) and Acala SJ-

1 was found in Coker 312 at 18 dpa and in YG from 14 to 18 dpa. Paymaster HS200

began to increase respiration rates after 16 dpa, and the other cultivars began to increase

rates after 18 dpa.

48

D

OH O

O

U <L> C

C3 Vi

u c a> Vi O o :3

5 u

33000

30000-

27000-

24000-

21000-

18000

15000-

12000-

9000-

6000-

3000-

0

. - -O -

SJ-1,34°C

HS200-34°C

SJ-1, 15° side of cycle

HS200, 15° side of cycle

12 "T" 15 18

—r 21 24 27 30

Days Post-Anthesis

Figure 2.8. Cellulose Synthesis Rates in the Fibers of Acala SJ-1 and Paymaster HS200. Values are presented as means (± standard deviations) of 3 replications with 6 ovules in each replication.

49

' ^

? o CU o

O u 1

r 1 u Tt 1—H

'""'

33000

30000

27000

24000

21000

18000

15000

12000

9000

6 0 0 0 -

3 0 0 0 -

0

•ffl— S J-1, fiber weight

- • — HS200, fiber weight

SJ-1, 34°C

— - O — HS200, 34°C

SJ-1, 15° side of cycle

HS200, 15° side of cycle

24

-22

-20

"T" 12

'TIS

1-18 21

- I r-24 27

18

16

14

12

10

8

6

4

ex) B

3 >

O iM (L>

cu •t-J

-C t>0

• 1 - <

>> VH

Q t-i

o x> UH

- 2

30 0

Days Post-Anthesis

Figure 2.9. Respiration Rates in the Fibers of Acala SJ-1 and HS200 at 34°C. Values are presented as means (± standard deviations) of 3 replications with 6 ovules in each repUcation.

50

>

ex

3

U

o o

5 r—1 u

39000

36000-

33000-

30000

27000-

24000-

21000-

18000-

15000-

12000-

9000-

6000-

3000-

0-

-0--

SJ-1, 34°C

HS200, 34°C

Coker 312, 34°C

YG, 34°C

1 ^

12 1 ^

15 18 21 - r -24 27 30

Days Post-Anthesis

Figure 2.10. Cellulose Synthesis Rates in The Fibers of Coker 312 and YG Compared toPaymaster HS200 and Acala SJ-1. Values are presented as means (± standard deviations) of 3 replications with 6 ovules in each replication.

51

(D

I OH

CN o u I

u

33000

30000

27000

24000-1

21000

18000

15000

12000-

9000-

6000-

3000-

0 12

-0--

SJ-1, 34°C

HS200, 34°C

Coker 312, 34°C

YG, 34°C

15 'TIS 21

1 ^

24 -r-27 30

Days Post-Anthesis

Figure 2.11. Respiration Rates in the Fibers of Coker 312 and YG Compared to Acala SJ-1 and Paymaster HS200. Values are presented as means (± standard deviations) of 3 replications with 6 ovules in each replication.

52

Figures 2.12 and 2.13 show tiiat tiie pools of Glc 6-P, Fm 6-P, and UDP-Glc began

to increase on 16 dpa for both cultivars at 34°C to support higher rates of ceUulose

syntiiesis and respiration. Pool sizes (nmol g" fresh weight) for botii Acala SJ-1 and

Paymaster HS200 in this one experiment were approximately the same and demonstt-ated

simUar pattems of change from 12 to 27 dpa. The relative proportions of tiie metaboUtes

were also similar for botii cultivars. It must also be noted tiiat die highest level of Glc 6-P

and UDP-Glc was observed 3 days earlier for Paymaster HS200, corresponding to die

earlier rapid rates of cellulose synthesis.

2.3.7 HPLC Separation of Fiber Sue. Glc. and Fm

The separation of the sugars extracted from the fibers was accompUshed by HPLC.

The amount of Sue, Glc, and Fm in the fibers of the cultured ovules was much greater than

that of the hexose phosphates and UDP-Glc. Sucrose levels varied from 140 to 400 nmol

oyule'\ and the Glc and Fm levels varied from 300 to 2720 nmol ovule'\ It must be noted

here that the cultivar YG is a poor producer of fibers, and the fiber weight per ovule of YG

was only 62, 47, and 49% that of the other 3 cultivars at 18, 21, and 24 dpa, respectively.

Therefore, the amount of sugars reported in the fibers of YG wiU be much less because

they are reported on a per ovule basis. (The sugar levels are presented on a per ovule basis

for better comparisons with the hexose phosphates and UDP-Glc, which were presented on

a per gram fresh weight basis. The fresh weight of 20 ovules was approximately 1 gram

for all of the cultivars at 18 to 24 dpa, with the exception of YG. All of the means

presented for the hexose phosphates and UDP-Glc can, therefore, be divided by 20 for an

approximate per ovule determination.) Glc 6-P and UDP-Glc levels were approximately 7

to 24 nmol ovule' and Fm 6-P levels were approximately 3 to 10 nmol ovule"\

For aU three sugars investigated, no significant differences were found between the

amounts at 1 h, 4 h, and 7 h on either side of the temperature cycle for any of the 3 sugars

in any of the cultivars on any of the 3 days, with 1 exception at 21 dpa for Acala SJ-1 at

34°C. The levels of Glc and Fm at 34°C in die fibers of Acala SJ-1 at 4 h on 21 dpa were

simUar to the levels at Ih and had not yet increased to the level observed at 7 h at 34°C.

There was no significant difference, however, between levels at 15°C and 34°C for Glc and

Fm at 21 dpa for Acala SJ-1 if either the 7 h level, the 4 h level, or if both 4 h and 7 h was

used in the statistical analysis. Therefore, simUar to the statistical analyses with the hexose

phosphates and UDP-glc, 4 h and 7 h wiU be considered together as separate replications to

determine differences between amounts at 15°C and 34°C for Sue, Glc, and Fm.

53

30000

<u

I ex

o 3

U

C<5

<N o u .s o o 3 3 u

25000-

^ 20000-

15000-

10000-

5000-

0

CeUulose

Respiration

Glc-6-P

UDP-Glc

Fm-6-P/

*

250

-200

-150 ^ 3 c

"T" 12

'TIS

-T" 18 21

—I— 24 27

-100

- 50

0 30

Days Post-Anthesis

c/3

3 o OH

B ;ra o

X) i3 (U

Figure 2.12. Cellulose Synthesis and Respiration Rates Compared To Metabolite Levels of Acala SJ-1 at 34°C. CeUulose synthesis and respiration rates are means (± standard deviations) of 3 replications with 6 ovules in each replication. Metabolite levels are the means of 2 measurements of 1 replication.

54

1 I ex

C<3

O

30000

25000-

3 20000^ I—H

3 U

^ 15000-u o <N

o u a

• »H

(U CO

O o

5 cJ 5000-]

10000-

0

Cellulose

Respiration

" D — Glc-6-P

. . . ^ _ . . . uDP-(5rcS •-A— Frrf-6-P

\ /

-200

-150 ^

T-12 15

- r -18 21

- I — 24 27

250

-100

- 50

0 30

CO

1 o

X)

B

Days Post-Anthesis

Figure 2.13. Cellulose Synthesis and Respiration Rates Compared To Metabolite Levels of Paymaster HS200 at 34°C. CeUulose synthesis and respiration rates are means (±standard deviations) of 3 replications with 6 ovules in each replication. Metabolite levels are the means of 2 measurements of 1 replication.

55

2.3.8 Sue Pool Sizes At 1 S°r Comp;^rpri To 34°r

The amount of Sue at 15°C and 34°C at 4 and 7 h after cycUng began and die percent

difference at 15°C from 34°C are given in Table 2.4. Sue levels were slightiy greater (up to

20% greater) at 15°C over 34°C for aU of die cultivars except Coker 312. Sue levels at

15°C for Coker 312 at 21 and 24 dpa were 17-18% less dian diat at 34°C, indicating diat

eitiier Sue is not syntiiesized as efficientiy at 15°C or that the use of Sue is much greater

than its synthesis at the cooler temperature.

2.3.9 Glc And Fm Pool Sizes At 15°C Compared To 34°C

The amounts of Glc and Fm at 15°C and 34°C at 4 and 7 h after cycling began and the

percent difference at 15°C from 34°C are given in Tables 2.5 and 2.6. Generally, Glc and

Fm pool sizes at 15°C were lower than those at 34°C for aU of the cultivars, but they were

significantiy lower (p <.05) only in Coker 312 at 21 dpa. Glc and Fm pool sizes in

Paymaster HS200 were greater at 15°C at 18 dpa, however, by 24 dpa, the pool sizes at

15°C were lower than those at 34°C. The same general trend of greater decreases in Glc

and Fm levels at 15°C compared to 34°C from 18 dpa to 21 or 24 dpa was evident among

aU of the cultivars and may indicate a possible adaptation to the cycling temperatures by

directing more carbon towards sucrose synthesis.

2.3.10 Sugar Pool Sizes From 18 To 24 DPA

A comparison of the amounts of Sue, Glc, and Fm within the fibers of all four

cultivars at 15°C and 34°C is given in Figure 2.14. Sue pool sizes at 18 dpa were

significantiy greater (p <.05) in the cool-sensitive cultivars than those in the partiaUy cool-

tolerant cultivars at both 15°C and 34°C, but there were no significant differences in Sue

levels at 24 dpa, except for that of YG. Sue levels at both I5°C and 34°C in the partiaUy

cool-tolerant HS200 graduaUy rose from 18 dpa to 24 dpa, but decreased in the cool-

sensitive Acala SJ-1 and Coker 312. Also, a dramatic change in the pool sizes of Glc and

Fm from 18 dpa to 24 dpa was evident in the fibers of all four cultivars. Glc and Fm

levels decreased 40 to 70% in the fibers of Coker 312 from 18 dpa to 21 dpa and 35 to

45% in the fibers of Acala SJ-1 from 21 to 24 dpa. A smaUer decrease in Glc and Fm

levels (7 to 26%) was found in the fibers of Paymaster HS200. Since the onset of rapid

secondary ceU wall synthesis began earUer for Paymaster HS200, sugar pool sizes were

also determined for Paymaster HS200 at 16 dpa and are included in Figure 2.14. Sue,

Glc, and Fm levels at 34°C at 16 dpa were 302, 2279, and 2292 nmol ovule\ simUar to

tiiose of Coker 312 at 18 dpa (see Tables 2.4, 2.5, and 2.6).

56

Table 2.4. Amount of Sucrose at 15°C and 34°C at 4-7 Hours Into Cycling and 15°C Percent Increase Over 34°C. The amount represents the mean ± SE (nmol ovule" ) from 4 repUcations with 20 ovules in each repUcation. Significant differences between the means of each pair are indicated be a different letter following the mean.

Cultivar Temp.

Cool-Sensitive:

SJ-1 15°C 34°C

C-312 15°C 34°C

Ave. 15°C Ave. 34°C

18 dpa

Amount

379 ± 14 342 ± 13

343 ±21 327 ±20

361 335


HS200 15°C 34°C

YG 15°C 34°C


224 ± 13 219 ± 15

158 ± 17 157 ± 9

191 188

%

11

5

8

2

0

2

Days Post-Anthesis

21 dpa

Amount

(nmol ovule')

372 ± 6a 311 ±19b

203 ± 10a 248 ± 15b

288 280

288 ±31 240 ±23

153 ± 5 145 ± 9

221 192

%

20

-18

1

20

6

15

24 dpa

Amount

301 ±25 273 ±27

218 ±26 262 ± 9

260 268

308 ±51 257 ±49

167 ± 5 157 ± 5

238 207

%

10

-17

-3

20

6

15

57

Table 2.5. Amount of Glucose at 15°C and 34°C at 4-7 Hours Into Cycling and 15°C Percent Increase Over 34°C. The amount represents the mean ± SE (nmol ovule" ) from 4 replications with 20 ovules in each replication. Significant differences between the means of each pair are indicated be a different letter foUowing the mean.

Cultivar Temp.

Cool-Sensitive:

SJ-1 15°C 34°C

C-312 15°C 34°C


18 dpa

Amount

2705± 93 2719 ±166

2198± 93 2132 ±200

2452 2426


HS200 15°C 34°C

YG 15°C 34°C


1348 ± 169 1192± 70

617 ± 65 666 ± 1

983 929

%

-1

3

1

13

-7

6

Days Post-Anthesis

21 dpa

Amount

(nmol ovule'*)

2205 ± 73 2276 ±122

643± 41a 1057 ±137b

1424 1666

1004 ±132 976 ±123

300± 26 397 ± 48

652 687

%

-3

-40

-15

3

-25

-5

24 dpa

Amount

1222 ± 100 1377 ±157

713 ±109 872± 73

968 1125

1162 ±302 1254 ±352

300± 42 369± 44

731 812

%

-11

-18

-14

-7

-19

-10

58

Table 2.6. Amount of Fmctose at 15°C and 34°C at 4-7 Hours Into Cycling and 15°C Percent Increase Over 34°C. The amount represents the mean ± SE (nmol ovule'*) from 4 repUcations with 20 ovules in each replication. Significant differences between the means of each pair are indicated be a different letter following the mean.

Days Post-Anthesis

18 dpa 21 dpa 24 dpa

Cultivar Temp. Amount % Amount 7o Amount %

Cool-Sensitive: (nmol ovule'*)

SJ-1 15°C 2690±152 -4 2279± 52 -10 1455±150 -11 34°C 2810 ±159 2529 ±107 1644 ±162

C-312 15°C 34°C


2219 ±126 2311 ±173

2455 2560


HS200 15°C 34°C

YG 15°C 34°C


1533 ±147 1400± 70

688± 75 794± 18

1110 1097

-4

-4

10

-13

1

825 ± 30a 1379 ±119b

1552 1954

1201±127 1306 ±154

433± 34 523± 36

817 914

-18

-21

-8

-17

-11

815 ±120 1070± 96

1135 1357

1232 ±297 1441 ±421

356± 25 395± 36

794 918

-17

-16

-15

-10

-14

59

3 > o

3 > o 3 e

1000

9 0 0 -

8 0 0 -

7 0 0 -

6 0 0 -

5 0 0 -

4 0 0 -

3 0 0 -

2 0 0 -

100-

ioo8

9 0 0 -

8 0 0 -

7 0 0 -

6 0 0 -

5 0 0 -

4 0 0 -

3 0 0 -

2 0 0 -

100-

0

Sue, 15°C

Sue, 34°C

- I 1 1 r-16 18 21 24

dpa

3000

2700-

2400-

2100

1800-

1500-

1200-

9 0 0 -

6 0 0 -

300-

0

3000-

2700-

2400-

2100-

1800

1500-

1200-

9 0 0 -

600-

300-

0

Glc, 15°C

i ' I

Fru,15°C

Glc, 34°C T

I I I

TFru,34^

- i 1 1 1 1 1 1 r-16 18 21 24 16 18 21 24

dpa dpa

Figure 2.14. Changes in Sugar Levels at 15°C and 34°C from 16 to 24 DPA in Acala SJ-1, Paymaster HS200, Coker 312, and YG. Values are presented as means (± SE) of 4 replications, with the exception of HS2(X) at 16 dpa with only one replication.

60

The levels of Glc and Fm in Paymaster HS200 at 34°C, dierefore, decreased 40 to

48% from 16 to 18 dpa, and Sue levels also decreased 28% during this same time period.

This corresponds to the onset of rapid secondary cell wall syntiiesis tiiat begins at 16 dpa in

Paymaster HS200, but is delayed untU 18 dpa for Coker 312, Acala SJ-1, and YG (see

Figure 2.10). Sue, Glc, and Fm pool sizes at 15°C in Paymaster HS200 at 16 dpa (246,

1669, and 1624 nmol ovule"*, respectively) were also greater than those at 18 dpa. It must

be noted tiiat the pool sizes of Paymaster HS200 at 16 dpa are die results of one experiment

only, and although the same trend occurs tiiat is evident in die other cultivars, caution must

stiU be appUed in the interpretation of this data.

From these data, it appears that the shift to rapid secondary cell waU synthesis that is

delayed in Coker 312 and Acala SJ-1 compared to Paymaster HS200 is likely responsible

for the decreasing sugar levels that are observed within the fibers of all of the cultivars.

Although both Coker 312 and Acala SJ-1 begin rapid ceU wall syntiiesis at 18 dpa, die

levels of sugars in the fibers of Acala SJ-1 do not really decrease to levels simUar to those

of Coker 312 until 24 dpa. The hexose sugar levels, however, were much higher (21 to

28%) for Acala SJ-1 at 18 dpa, and they were decreasing rapidly from 18 to 21 dpa;

therefore, the higher sugar levels in the fibers of Acala SJ-1 at the onset of rapid cell waU

synthesis may account for the difference observed. It must also be noted that the rate of

ceUulose synthesis in the fibers of Coker 312 were 33% greater than Acala SJ-1 at 18 dpa

and sugar levels may have started to decrease earlier than what was experienced in Acala

SJ-1.

Figure 2.15 compares pool sizes of Glc at both 15°C and 34°C in the fibers of Acala

SJ-1, Paymaster HS200, and Coker 312 from 16 to 24 dpa. This graph shows that the

dramatic decrease in sugar levels occurred at both 15°C and 34°C for aU 3 cultivars. Again,

the amounts at 16 dpa for Paymaster HS200 were the results of only one experiment. In

addition, the amount of Glc in the fibers of Acala SJ-1 and Paymaster HS200 at 15°C were

similar to those at 34°C for each day. There was a significant decrease (p <.05), however,

at 21 dpa in the amount at 15°C compared to tiiat at 34°C in Coker 312 and a similar 15°C

decrease at 24 dpa. The smaUer Glc and Fm pool sizes at 15°C for Coker 312 also

correspond to smaller Sue pool sizes at 15°C at 21 and 24 dpa that were not observed in the

other cultivars. The lower sugar levels at 15°C in Coker 312 may result from a more

efficient use of the sugars at 15°C and/or it may result from a possible reduction in tiie

amount of carbon entering the fiber ceU from the culture medium or from die surrounding

epidermal cells during this period of increasing carbon use for secondary ceU wall

synthesis.

61

3300-

3000-

2700-

2400-

2100-

3 1800-> o § 1500-1

1200'

900-

600-

300-

0

Glucose SJ-1, 15°C HS200, 15°C C-312, 15°C

-ffl--- SJ-1,34°C

- • - " HS200,34°C

-©--- C-312, 34°C

16 1 ^

18 1^

21 1 — 24

Days Post-Anthesis (dpa)

Figure 2.15. Comparison of Glucose Levels at 15°C and 34°C for Acala SJ-1, Paymaster HS200, and Coker 312. Values are presented as means (±SE) of 4 replications, witii the exception of Paymaster HS200 on 16 dpa with only one replication.

62

2.3.11 Fiber Drv Weight Per Ovule

The weights of the fibers (mg dry weight ovule"*) of the four cultivars obtained from

HPLC analysis are diagramed in Figure 2.16. There is a substantiaUy greater increase in

fiber weight for Paymaster HS200 from 18 to 21 dpa compared to the other cultivars. This

would be expected because Paymaster HS200 began rapid secondary ceU wall syntiiesis at

16 dpa, earUer than the other cultivars and experienced greater levels of Glc 6-P at 15°C

during the cycling temperatures. It is interesting to note that there were no significant

differences in fiber weight among Acala SJ-1, Paymaster HS200, and Coker 312 by 24

dpa. This indicates that there may have been a delay in fiber development at 15°C in the

cool-sensitive cultivars Acala SJ-1 and Coker 312 at the onset of cycUng temperatures, but

after a short period of acclimation (3 days), fiber development at 15°C was resumed to be

similar to that of Paymaster HS200. This figure also demonstrates the low productivity of

the cultivar YG.

The same differences in fiber weight between Paymaster HS200 and Acala SJ-1 in

this figure do not correspond to the lack of difference in fiber weight noted earUer in Figure

2.9. The ovules in the earUer experiment were kept at 34°C constant temperature. At 34°C,

the rates of cellulose synthesis were comparable for the two cultivars. However, when the

ovules were switched to cycUng temperatures. Paymaster HS200 had a greater abiUty to

increase Glc 6-P levels immediately on the day cycUng began and likely had a greater abUity

to synthesize ceUulose at 15°C at the onset of cycling.

2.3.12 Comparisons Among Cultivars

The material presented in this section diagrams all of the metaboUc pools at 15°C and

34°C for each cultivar and aUows comparisons among the cultivars in this study.

2.3.12.1 Acala SJ-1. The changes in pool size of the sugar-phosphates, UDP-Glc,

and sugars in the fibers of Acala SJ-1 from 18 to 24 dpa are diagrammed in Figure 2.17.

Hexose phosphate levels at 15°C increased significantiy above those at 34°C in the fibers of

Acala SJ-1, but the levels of the hexose phosphates were generaUy lower than those in the

other cultivars, which may indicate Sue synthesis was delayed because of lower substrate

levels and less activation of SPS. UDP-Glc levels increased only slightiy from 18 dpa to

24 dpa at bodi 15°C and 34°C, and die amount of UDP-Glc at 15°C was 12% higher tiian

that at 34°C at 18 dpa. Both of these factors indicate that Acala SJ-1 may not be very

efficient in synthesizing Sue from UDP-Glc. Sue levels were greater at 15°C than at 34°C

at 18 dpa and 21 dpa before the levels of sugars started to rapidly decline in the fibers of

Acala SJ-1 between 21 and 24 dpa. Sue levels did not decline as rapidly as the levels of

63

3 > o

4—>

iH

e

4-

3-

SJ-l

• HS200

• C-312

• YG

T T

T 1 .*.'.'

•:•:•: .'•'•' yy,'

!*!"!'

.*.'.*

•:•:•: *.*•*• yy,' ''yy.

yy,'

^ T

T • X

' '

T

• . , \ •

vV \ ••

\ \ \ \

•^K; -A

T *

- i ^H

'\''H '-.^^^H

'- ^ 1 ' ^ H

.\.x^H ''-^^1 \ \ H

—

1 '^.

:•:•:•: • ! ' ! " ! *

; • : • : • :

'.'.'.'

!'!'!*! •yy. .'.*.*

, X

• ^ ^

1 "

: -:>; • -.••; f s ;

•

•

•

'. : • " - • ' •

• - „ ;

;

: ",:;

' ,

"

\ • • •

•

] • '

T :^-

\ ' • • .

\ \

• • , . \

•••N ••. \

, \ ' - T

'N' H xX^^H-

' ^^^H \ "^^H

^ ^ H ^ ^^H

^ ' ^ H sV^H \N^H x\^l \ ^^^H

'• ^ ^ ^ H '^^^^^H ' B^^l

18 21 24 Days Post-Anthesis (DPA)

Figure 2.16. Fiber Weight Per Ovule. Fiber weights are given at 18, 21, and 24 dpa for Acala SJ-1, Paymaster HS200, Coker 312, and YG. The values are means (±SE) of 10-12 replications with fibers from 20 ovules in each replication.

64

'ao ^

3-== 6 2

3 > o

3 > o 3 e

> O

3

Acala SJ-1

500

4 0 0 -

300-

200

100 H

0 2 0 0 -

150-

100-

5 0 -

0 4 0 0 -

3 0 0 -

2 0 0 -

100-

5o8: 400-1

300

200

100-

3000-

2000-

1000-

3000-

2000-

1000-

0

Glc 6-P

40 rrr^-rrr^

Fm6-P

18

UDP-Glc

12

11

1 1 1 1 1 1 1 1

18

48

H 1 5 ° C n 34°C 60

42

20

- 3 •

•10

^^^^^^9^^^

33 ^^•rrwvr

12

Sucrose 10

Glucose

-11

Fmctose

21 Days Post-Anthesis

Figure 2.17. Metabolites and Sugars in Fibers of Acala SJ-1. Pool sizes are presented as the means of 3-8 replications ± SE. Numbers above the 15°C bars represent the % change in pool size of the mean at 15°C from that at 34°C.

65

Glc and Fm at 24 dpa, possibly indicating a less efficient use of the Sue in secondary ceU wall synthesis.

2.3.12.2 Coker 312. The changes in pool size of the sugar-phosphates, UDP-Glc,

and sugars in die fibers of Coker 312 from 18 to 24 dpa are diagrammed in Figure 2.18.

Similar to Acala SJ-1, levels of the hexose phosphates were generaUy lower than the other

cultivars and levels at 15°C were significantiy higher than tiiose at 34°C. Fm 6-P levels,

however, were about 20 nmol g* fresh weight higher than Acala SJ-1 at 34°C, and

increases at 15°C elevated the levels to over the possible 120 nmol threshold by 21 dpa,

possibly increasing the efficiency of Coker 312 in synthesizing Sue. Also similar to Acala

SJ-1, UDP-Glc levels rose only slightly from 18 to 24 dpa for botii 15°C and 34°C,

however, levels at 15°C at 18 dpa were 8% lower than those at 34°C. This may indicate

that Coker 312 either uses UDP-Glc more efficientiy in synthesizing Sue or is less efficient

in the synthesis of UDP-Glc from the hexose phosphates. The faster reduction in Glc and

Fm levels by 21 dpa in Coker 312 suggests that the former is more likely to be occurring.

There is a sharp decline in both Glc and Fm levels from 18 dpa to 21 dpa corresponding to

the onset of rapid secondary cell waU synthesis. This decline was not observed in Acala

SJ-1 until 21 dpa although both cultivars began rapid secondary cell wall synthesis at 18

dpa. Also, the levels of Glc and Fm in Acala SJ-1 did not decrease as much as the levels in

Coker 312. These two factors suggest the possibility that synthesis of Sue within the

cytoplasm and synthesis of cellulose may be occurring at a slightly faster rate in Coker 312.

Another interesting difference between Coker 312 and Acala SJ-1 is the lower Glc and Fm

levels at 15°C at 21 and 24 dpa tiian those at 34°C in Coker 312. Levels at 15°C in Acala

SJ-1 at 21 and 24 dpa were relatively the same as those at 34°C. Again, this may relate to

the more efficient use of UDP-Glc in synthesizing sucrose at 15°C. Indeed, the fiber

weights of Coker 312 were greater tiian Acala SJ-1 at 18 and 21 dpa.

2.3.12.3 TM-1. The changes in pool size of the sugar-phosphates and UDP-Glc in

the fibers of TM-1 from 18 to 24 dpa are diagrammed in Figure 2.19. SimUar to the other

cool-sensitive cultivars mentioned earUer, the levels of Glc 6-P graduaUy increased at 15°C

from 18 to 24 dpa and were significantly higher than those at 34°C. However, Fm 6-P

levels at both 15°C and 34°C were significantly higher (p <.05) tiian those of Acala SJ-1 on

aU three days and significantiy higher tiian those of Coker 312 on 18 and 24 dpa. Amounts

of UDP-Glc at 15°C and 34°C were also significantiy higher (p <.05) than tiiose of Acala

SJ-1 and Coker 312 at 21 and 24 dpa. This, along witii die fact tiiat Glc 6-P levels at 34°C

were significantiy higher (p <.05) than Acala SJ-1 and Coker 312 on aU three days

possibly indicates that TM-1 may have a greater capacity to synthesize Sue within the fiber

66

3*^ E P

00 ^ 3' 2 t's

3-

3 > o

> O

> O

500'

400'

300

200

100

0

200

150

100

50 0

400

- Fm 6-P

'ao ^ 300 -

200

100

5o8: 400

300

200

100

3000'

2000

1000

3C

2000

1000

0

Glc 6-P

34

UDP-Glc

-8

WW^ WW w w w

18

Coker 312

57 ^ ^ W ^ F W ^ * «

33

-18

-40

-40 ^ ^ ^ ^ • ^ ^ ^ • ^

M 15°Cn 34°C 60

11

Sucrose

•17

Glucose

-18 1 1 1 1 1 1 1

Fmctose

-24


24

Figure 2.18. Metabolites and Sugars in Fibers of Coker 312. Pool sizes are presented as the means of 3-8 replications ± SE. Numbers above the 15°C bars represent the % change in pool size of the mean at 15°C from that at 34°C.

67

I 4-^

00 ^

• ^

00 >

00 >

2 " e ^

3 > o

3 > o

3 > o

500

400

300

200

100

0

200- Fm6-P 150

100

50

0 400

300

200

100

5o8: 400

300

200'

100

3008'

2000-

1000-

30o8'

2000

1000

0

Glc 6-P 28

^ ^ ^ ^ ^ W « l ^

't - r m T T T

UDP-Glc

2 1 1 1 1 1 1 1

18

TM-1

26

13 ^ ^ ^ ^ ^ ^ ^ ^ v

15°Cn 34°C

37 ^••^^^^^^^^

13 I I I I I I T T

23 ^ ^ ^ ^ ^ • ^ ^ ^

Sucrose

Glucose

Fmctose

1 21

Days Post-Anthesis 24

Figure 2.19. Metabolites in Fibers of TM-1. Pool sizes are presented as the means of 3-8 replications ± SE. Numbers above the 15°C bars represent the % change in pool size of the mean at 15°C from that at 34°C.

68

ceUs, however, die higher Fm 6-P and UDP-Glc levels may also indicate tiiat TM-1 is not

as efficient in synthesizing Sue. Additional information on tiie rates of ceUulose syntiiesis

and sugar pool size changes would make it less difficult to speculate on die efficiency of

TM-1.

2.3.12.4 JDX, JDX is the first partiaUy cool-tolerant cultivar in which hexose

phosphates and UDP-Glc were measured. The changes in pool size of the sugar-

phosphates and UDP-Glc in die fibers of JDX from 18 to 24 dpa are diagrammed in Figure

2.20. SimUar to the other cultivars, Glc 6-P levels at 15°C were significantiy greater (p

<.05) than those at 34°C on all three days. Glc 6-P levels at 15°C at 18 dpa were

significantiy greater (p <.05) than those of the cool-sensitive cultivars, and the levels of Glc

6-P at 15°C stayed relatively the same from 18 to 24 dpa. SimUar to the cool-sensitive TM-

1, Fm 6-P levels and UDP-Glc levels were greater than those of Acala SJ-1 and Coker

312. Again, it is difficult to speculate on the abUity of JDX to synthesize Sue within the

fiber ceU because greater Glc 6-P, Fm 6-P, and UDP-Glc levels indicate tiiat JDX may

have a greater capacity to synthesize Sue, but may also indicate it is less efficient since pool

sizes are greater.

2.3.12.5 Pavmaster HS200. The changes in pool size of the sugar-phosphates,

UDP-Glc, and sugars in the fibers of the partiaUy cool-tolerant Paymaster HS200 from 18

to 24 dpa are diagrammed in Figure 2.21. The Glc 6-P levels at 15°C are significantly

higher than those of the cool-sensitive cultivars, with the exception of JDX, at 18 and 21

dpa. Whereas Glc 6-P levels are increasing from 18 to 24 dpa in the cool-sensitive

cultivars, Glc 6-P levels are decreasing in Paymaster HS200. Fm 6-P levels are also

higher at 18 dpa than the cool-sensitive cultivars, with the exception of TM-1, and are also

decreasing from 18 to 24 dpa. UDP-Glc levels at 15°C and 34°C also are higher at 18 dpa

and remain at the same high level through 24 dpa. The abUity to synthesize ceUulose at

15°C at a higher percentage of the 34°C rate than the cool-sensitive cultivars may be the

result of the abUity to increase hexose phosphate and UDP-Glc substrate levels high

enough to activate SPS and partiaUy compensate for temperature-induced enzyme

limitations at the cooler temperatures. Although Sue, Glc, and Fm concentrations were

much lower at 18 dpa than the cool-sensitive Acala SJ-1 and Coker 312, they were simUar

to Coker 312 concentrations at 21 dpa and Acala SJ-1 at 24 dpa. Therefore, the decreased

sugar levels seen at 18 dpa in Paymaster HS200 may simply be the result of rapid

secondary cell wall synthesis beginning earlier at 16 dpa rather than 18 dpa, which was

observed for the cool- sensitive cultivars. At 16 dpa, the Sue, Glc, and Fm concentrations

at 15°C were much lower than tiiose at 34°C, indicating tiiat Paymaster HS200 may be

69

00 >

1/3

00 >

1 -

00 >

o e

1

3 > o

.1-1

fre

3 > o

3 > o

JDX 500

400

300-

200-

100-

Ea i5°cn 34°c

0 200

150

100

50

- Fm 6-P - 11

0 400-

300-

2 0 0 -

100-

5' 400 300 200 100

30o8:

2000

1000

3oo8:

2000'

1000'

0-

Glc 6-P 54 62

* r f l * * d i * *

UDP-Glc

20 1 1 1 1 1 1 1 1

18

55

55-^ ^ ^

Sucrose

Glucose

Fmctose


24

Figure 2.20. Metabolites in Fibers of JDX. Pool sizes presented as the means of 3-8 replications ± SE. Numbers above the 15°C bars represent tiie % change in pool size of the mean at 15°C from that at 34°C.

70

'oo ^ 3-^ 2 ^

00 >

2 <«

3 - ^ 2 "

3 > o

o

3 > o

Paymaster HS2(X)

500

400

300

200

100

0 200

150

100

50

0 400

- Fm 6-P

'oo ^ 300 -

200

100

5o8: 400-300-200-100-

3000-

2000"

1000-

3000-

2000-

1000-

0

Glc -P

UDP-Glc

0

13

10

61 I T T T T T T "

13

20

1 1 1 1 1 1 1

-8 1 1 1 1 1 1 1 1

15°Cn 34°C 60

28

10 ^ T ^ - ^ F ^ ^ ^ ^

2j) Sucrose

^^^^^^^^m

! I I I I I I •

Glucose

^mk^- ±

Fmctose

-15

•>*.:•: : : : :


24

Figure 2.21. Metabolites and Sugars in Fibers of Paymaster HS200. Pool sizes are presented as the means of 3-8 replications ± SE. Numbers above the 15°C bars represent the % change in pool size of the mean at 15°C from that at 34°C.

71

more efficient in synthesizing ceUulose than tiie other cultivars at die lower temperature.

Coker 312 also had lower sugar levels at 15°C at 21 dpa than those at 34°C and may also be

efficient in synthesizing ceUulose at cooler temperatures. Therefore, the partial cool-

tolerance observed in HS2(X) may, again, just be die abUity to increase hexose phosphate

and UDP-Glc concentrations more efficientiy at the onset of cool temperature cycling.

2.3.12.6 YG- The changes in pool size of the sugar-phosphates, UDP-Glc, and

sugars in the fibers of YG from 18 to 24 dpa are diagrammed in Figure 2.22. Similar to

Paymaster HS200, Glc 6-P and Fm 6-P levels were higher tiian those of tiie cool-sensitive

cultivars, and they were even higher than Paymaster HS200. The amounts were the

highest at 21 dpa, possibly corresponding to the delay in the onset of rapid secondary ceU

wall synthesis which occurred at 18 dpa, similar to Acala SJ-1 and Coker 312. Sue, Glc,

and Fm levels were much lower than the other cultivars because of the low amount of

fibers produced by YG. However, there was a large decrease in Glc and Fm levels from

18 to 21 dpa, similar to that found in Coker 312. Again, the partial cool-tolerance may be

the result of increased hexose phosphate and UDP-Glc levels observed in YG.

2.3.12.7 Paymaster HS26. The changes in pool size of the sugar-phosphates and

UDP-Glc in the fibers of Paymaster HS26 from 18 to 24 dpa are diagrammed in Figure

2.23. SimUar to the cool-sensitive cultivars, Glc 6-P, Fm 6-P, and UDP-Glc levels at

15°C were low and increased from 18 to 24 dpa. By 21 dpa, however, the levels of Glc

6-P at 15°C were 57% greater tiian those at 34°C. UnlUce Paymaster HS200 and YG, die

partial cool-tolerance observed in Paymaster HS26 does not correlate to higher hexose

phosphate levels at 18 dpa, however, levels did greatly increase by 21 dpa, which is the

time that the partial cool-tolerance was observed. Unfortunately, the time that rapid

secondary cell wall synthesis begins and the amounts of Sue, Glc, and Fm were not

determined for Paymaster HS26, and both of these factors would make it easier to

speculate on the possible factors related to partial cool-tolerance.

72

'oo 3 * ^ 2 "*

00 > 3 JS 2 t'i

00 ^

3 -C3 2 «« 6 2

3 > o

3 > o

>

o

500"

400-

300-

200-

100-

0-200

150

100

50

0 400

300

200

100

508 400

300

200

100

3000'

2000'

1000'

3(

2000'

1000

0

59

X>.

0

^ ^ ^ ^ T ^ ^ -

•13

18

YG ES 15°Cn 34°C

:7i: - Glc 6-P 64

Fm6-P

Sucrose

6 6 • 1 1 1 1 1 1 1 1

Glucose

-25 -f - - - - - - - f

-. -19 -1

Fmctose

-17

i i ' i ' i ' i ' i ' i ' i ' i " 1 r -10

21 24 Days Post-Anthesis

Figure 2.22. Metabolites and Sugars in Fibers of YG. Pool sizes are presented as the means of 3-8 repUcations ± SE. Numbers above or below the 15°C bars represent the % change in pool size of the mean at 15°C from that at 34°C.

73

oo > W5

00 > 3 -fi 2 t's

00 > 3" 2 ^

3 > o

o 3 > o

> o

Paymaster HS26

500

400

300

200

100

0 200

150

100

50

0 400

300

200

100

5o8: 400'

300-

200-

100-

30o8"

2000-

1000-

- Fm6-P

1

30o8'

2000

1000

0

x) 15°CD 34°C Glc 6-P

33 57

100

27 57

w^^^^^rw

UDP-Glc

13 T T T T T T T

Sucrose

Glucose

Fmctose

-r-18

1 21

Days Post-Anthesis

1 ^

24

Figure 2.23. MetaboUtes in Fibers of Paymaster HS26. Pool sizes are presented as the means of 3-8 repUcations ±SE. Numbers above the 15°C bars represent the % change in pool size of the mean at 15°C from that at 34°C.

74

2.4 Discussion

2.4.1 Hexose Phosphates and Cool Temperamre

The increase of hexose phosphates, particularly Glc 6-P, as a response to cool

temperatures is seen in photosyntiietic tissues (Guy et al., 1992; Kobza and Edwards,

1987; Holaday et al., 1992; Hurry et al., 1995; Warner et al., 1995) and in non-

photosyntiietic tissues, such as potato (Dbcon and ap Rees, 1980; Viola and Davies, 1994).

The increases of hexose phosphates in most photosynthetic tissues result from the changes

in enzyme kinetics and activation at low temperatures in pathways related to photosynthesis

or sugar syntiiesis (Guy et al., 1992; Holaday et al., 1992; Hurry et al., 1995). In potato

tubers, the increase in hexose phosphate levels at cold temperatures correlated to a

partitioning of less carbon to glycolysis and more to sugar or starch synthesis (Dixon and

ap Rees, 1980; Viola and Davies, 1994). Less carbon was directed towards glycolysis

because of the cold-labiUty of the glycolytic enzymes, therefore, more carbon was available

for carbohydrate synthesis. In this study, the large increase in Glc 6-P at 15°C may be

associated with increasing Sue synthesis since Glc 6-P is a known aUosteric activator of

SPS (HiU and ap Rees, 1995; Huber and Huber, 1992; Reinholz et al., 1994) and die

substrate levels for the synthesis of UDP-Glc were increased. The large increase in Glc 6-

P levels at 18 dpa in the fibers of the partiaUy cool-tolerant cultivars was not observed in

those of the cool-sensitive cultivars untU 24 dpa and likely is the major contribution to the

partial cool-tolerance observed in these cultivars (Haigler et al., 1994). This may also

explain the apparent acclimation period of 3 days reported in the cool-sensitive Acala SJ-1

in earUer studies (Haigler, 1991). After a period of at least 3 days, the hexose phosphate

levels in the cool-sensitive cultivars at 15°C may have increased enough to increase

cellulose synthesis similar to that of the partiaUy cool-tolerant cultivars. Certainly, fiber

weight in the cool-sensitive cultivars by 24 dpa was similar to that of the partially cool-

tolerant Paymaster HS200. Further investigations on the activity of SPS in the fibers of

these cultivars at 15°C are necessary to speculate on the influence of hexose phosphates on

Sue synthesis within the fibers of cotton.

Although Glc 6-P levels at 15°C increased 55 to 60% by 24 dpa, Fm 6-P levels at

15°C only increased significantiy over that at 34°C in cultivars in which the absolute

amounts of Fm 6-P in the fiber were low, indicating a possible threshold level must be

attained to aid in the reduction in SPS activity at lower temperatures (Neuhaus, Quick,

Siegl, and Stitt, 1990). SPS is relatively temperature sensitive with a high Q^Q of 3.7 in

darkened leaves, and higher levels of hexose phosphate are needed to attain a given flux

through SPS (Stitt and Gross, 1988). Indeed, SPS activity has been controlled to remain

75

inactive in extracts of spinach leaves until a direshold concentration of hexose phosphates

has been reached, and tiien activity is stimulated by further increases of hexose phosphates

(Stitt et al., 1988). In tiiis study, the increased activity of SPS for tiie syntiiesis of Sue in

the fibers of some cultivars at 15°C may not be reaUzed until die levels of the substrate Fm

6-P have been raised above this threshold level.

It is interesting to note tiiat the Qio of 3.7 for SPS in darkened barley leaves (Stitt and

Gross, 1988) is very simUar to that observed for ceUulose synthesis in cotton fibers (QK, of

4.28) from 18°C to 28°C (Roberts et al., 1992). The possibility exists diat die reduction in

cellulose synthesis at lower temperatures in the cotton fiber is directiy related to the

reduction in SPS activity that occurs at lower temperatures.

2.4.2 UDP-Glc and Cool Temperature

The decrease in UDP-Glc or the lack of increasing amounts of UDP-Glc at 15°C over

that at 34°C in the fibers of most of the cultivars at 18 dpa (shock treatment) indicates that a

possible enzymic restriction may exist in the pathway immediately before the synthesis of

UDP-Glc as a result of cool temperatures (see Figure 1.4). The enzyme UDPG-PP may

be affected the most by cooler temperatures in the pathway towards cellulose synthesis.

Although the absolute amounts of UDP-Glc increased at 21 dpa, there were stUl no

significant differences between those at 15°C and those at 34°C. This indicates that levels

may be sUghtly increasing simply in response to the onset of secondary cell waU synthesis

and the increasing need of Sue for ceUulose synthesis. Certainly, the elevated Glc 6-P

levels observed at 21 dpa at 15°C would likely result in an increase in substrate levels for

UDP-Glc synthesis. However, the elevated Glc 6-P pools would also stimulate the activity

of SPS, thus driving reactions towards Sue synthesis. A greater flux towards Sue

synthesis would therefore result in very Uttie increases in UDP-Glc pool sizes.

It must also be noted that a considerable amount of soluble SuSy has been reported to

be present in the fibers of cotton (Amor et al., 1995; Basra et al., 1990; Ruan et al., 1997).

Although the exact function of the soluble SuSy within the fiber is unknown (Amor et al.,

1995; Ruan et al., 1997), it has been suggested tiiat it may play a role in supplying UDP-

Glc and Fm for metaboUc functions witiiin the ceU (Delmer, unpublished). Soluble SuSy

activity would thus affect the levels of UDP-Glc within the cell. There is indication,

however, that tiie relative amounts of soluble SuSy in the fiber cell may have been

overstated due to extraction procedures (Delmer, unpubUshed), and it seems unlikely that

there would be such a need for soluble SuSy within the cell because of the high levels of

invertase activity that would supply the necessary sugars for metabolism. Further studies

76

of SuSy activity within the fiber wUl aid in understanding die exact functions of this

enzyme in the cytoplasm of the ceU.

2.4.3 Sugar Pool Sizes and Secondarv Cell Wall Synthesis

HPLC analyses revealed appreciable amounts of Sue, Glc, and Fm in the fibers of

the cultured cotton ovules compared to the hexose phosphates and UDP-Glc, and the

amounts were in proportions simUar to those of greenhouse-grown and field-grown fibers

(Basra et al., 1990; Jaquet et al., 1982) (see Table 1.2). In tiiis study, Glu and Fm levels

were 6 to 7 times greater than that of Sue. The absolute amounts of Glu and Sue were very

simUar to those reported by Carpita and Delmer (1981) in the fibers of cultured ovules at 16

to 18 dpa.

In this study. Sue levels decreased 30% and Glc and Fm levels decreased 40 to 48%

at 34°C from 16 to 18 dpa in the fibers of Paymaster HS200, which began rapid secondary

cell waU synthesis at 16 dpa. For the fibers of Coker 312, which began rapid secondary

ceU wall synthesis at 18 dpa. Sue levels also decreased (25%) and Glu and Fm levels

decreased simUarly (40 to 70%) from 18 dpa to 21 dpa (see Figure 2.12). SimUar

substantial decreases (40 to 50% for Glc and Fm) occurred in the fibers of Acala SJ-1,

which also began rapid ceUulose synthesis at 18 dpa, but the decreases were not fully

reaUzed untU 24 dpa, possibly due to greater concentrations of the hexose sugars in Acala

SJ-1. In ovules of greenhouse grown cotton (Gossypium hirsutum L.), fiber Glu and Fm

levels also decreased dramaticaUy (60 to 68%) from 17 to 27 dpa (Jaquet et al., 1982) (see

Table 1.2), indicating that the growth and development of fibers in vitro are simUar to those

of greenhouse-grown or field-grown plants (Carpita and Delmer, 1981; Haigler, 1991).

The decrease in hexose sugars experienced at the onset of secondary ceU wall

synthesis may play a role in regulation of SPS and SuSy activity within the fiber. In

cotyledon development of Viciafaba L., high invertase activity at the phloem unloading

region of the seed coat elevated the hexose sugars/sucrose ratio, which activated SPS

activity and the subsequent synthesis of Sue during the prestorage stage of cotyledon

development(Weber, Buchner, Borisjurk, and Wobus, 1996). The free hexoses inhibited

the SuSy activity. However, during the storage phase, invertase activity decreased with a

resulting lower hexose/sucrose ratio, which stimulated SuSy activity and deactivated SPS.

In a similar manner, the decrease in invertase activity reported in the cotton fiber at die

onset of secondary cell waU synthesis (Basra et al., 1990; Waffler and Meier, 1994)

corresponds to the decrease in hexose sugars observed in this study. The greater hexose

sugar levels at the onset of secondary ceU wall sythesis may stimulate the activity of SPS

77

and die reduced sugar levels may tiien stimulate SuSy activity to provide UDP-Glc for

ceUulose synthesis at the membrane. Further studies are necessary to evaluate the

importance of sugars in die regulation of enzyme activity witiiin the fiber ceU.

Interestingly, in this study, tiiere was no significant difference (p <.05) in the levels of die

hexose sugars and Sue at 24 dpa among die tiiree cultivars Paymaster HS2(X), Acala SJ-1,

and Coker 312. It is possible that a certain sugar concentration must be maintained within

the fiber ceU that is necessary for the activation of the enzymes involved in both Sue

synthesis and cellulose synthesis.

In developing canola sUiques, carbon is stored as Sue rather than as starch and much

of the Sue is hydrolyzed by acid invertase to hexose sugars for vacuolar storage before

rapid seed growth (King, Lung, and Furbank, 1997). Later, during seed growth and

increasing siUque wall cellulose synthesis. King and coworkers proposed that the carbon

was supplied from the pool of Sue resulting from photosynthesis, sucrose import, and the

hexose vacuolar reserves. Similarly, both the presumed vacuolar hexose storage in the

cotton fiber (Carpita and Delmer, 1981) and imported Sue into the fiber may be used for the

synthesis of Sue, and hence, cellulose, during rapid secondary cell wall synthesis. The

rapid depletion of hexose sugars from the vacuole would likely decrease mrgor in the fiber

cell, however, the extremely large increase in MDH activity seen at the onset of secondary

ceU waU synthesis may play a role in supplying malate for vacuolar storage and hence,

maintaining turgor (Waffler and Meier, 1994) (see Figure 1.4).

2.4.4 Secondarv Cell Wall Svnthesis in Cultured Ovules

Although the Sue necessary for cellulose synthesis in the cotton fiber in vivo is likely

derived from both Sue import and from Sue synthesis within the ceU itself (Delmer,

unpublished; Ruan et al., 1997), there is likely little Sue import into the fiber ceU of

cultured ovules. Therefore, the fuU extent of fiber development is not Ukely realized for

ovules grown in vitro. This is demonstrated by the reduced fiber length and weight

observed in vitro (Meinert and Dehner, 1977). However, in vitro culture does provide an

excellent opportunity to observe the changes in die metabolic padiways related to Sue

synthesis within the ceU separate from direct Sue import into the cell.

From this study, the changes in development of the cotton fiber are shown to differ

among cultivars, i.e., the onset of secondary cell wall synthesis began earlier after anthesis

for Paymaster HS200 and the reduction in hexose pool sizes occurred at different times

during secondary cell waU development. Also, the rate of secondary ceU wall synthesis at

cooler temperatures may also differ among cultivars, which may be affected by differences

78

in Sue syntiiesis witiiin tiie ceU. The rates observed in Acala SJ-1 as measured by

decreases in Glc and Fm pool sizes appeared to be slower dian tiiose of Coker 312, botii of

which began rapid secondary ceU wall syntiiesis at 18 dpa. Furtiier investigations are

necessary, however, to determine differences in carbon flow through die metaboUtes

related to Sue synthesis.

79

CHAPTER m

SPECmC ACnVITY OF SUGAR POOLS DURING

SECONDARY CELL WALL SYNTHESIS

3.1 Introduction

The development of die cotton fiber is adversely affected during die cool night

temperatures experienced during the growing season of die Texas High Plains. Fibers

exposed to cool temperatures (below 25°C) have reduced overaU rates of secondary ceU

wall thickening (Gipson and Johan, 1968; Haigler et al., 1991), lower micronaire values

(Gipson and Johan, 1968), and reduced fiber dry weight per seed (Thaker et al., 1989).

In a similar manner, fibers of ovules cultured in vitro also show delayed onset of

secondary ceU waU thickening and the formation of "growth rings" corresponding to times

when temperatures cycled below 22°C (Haigler et al., 1991). Also, the rates of ceUulose

synthesis in fibers of cultured cotton ovules are reduced considerably as temperamres are

reduced below 25°C (PiUonel and Meier, 1985; Roberts et al., 1992). CeUulose syntiiesis

in the fibers of cultured ovules has a higher Qj than respiration, suggesting that a p)ossible

block may occur specificaUy in the pathways related to ceUulose synthesis within the fiber

(Roberts et al., 1992).

Recently, it has been hypothesized that a membrane-bound SuSy plays an essential

role in hydrolyzing Sue and supplying UDP-Glc directiy to ceUulose synthase at the ceU

membrane for the synthesis of cellulose (Amor et al., 1995; Ruan et al., 1997). While

imported Sue is likely a source of Sue for SuSy activity (Ruan et al., 1997), the synthesis

of Sue from metaboUtes within the fiber is also a likely source considering the high

invertase activity reported in the fiber at aU developmental stages (Basra et al., 1990;

Waffler and Meier, 1994) and the abundant Glc and Fm pools that exist in the fiber

(Carpita and Delmer, 1981; Jaquet et al., 1982). The synthesis of Sue witiiin the fiber

would not only use the expected hexose sugar pools but also recycle into ceUulose

synthesis the large amount of Fm that is presumably released from the activity of the

membrane-bound SuSy. Therefore, we propose that the pathways that are related to

ceUulose syntiiesis in the fiber in plants include the phosphorylation of the hexose sugars,

the synthesis of UDP-Glc from the hexose phosphate pool, and the syntiiesis of Sue from

UDP-Glc and Fm 6-P, as outiined in Figure 1.3.

An investigation of the metaboUtes related to these padiways in fibers of ovules

cultured in vitro from seven different cultivars showed a significant increase in the levels of

Glc 6-P upon transfer from 34°C to 15°C (see Table 2.1) and slight increases in die levels

80

of Fm 6-P (see Table 2.2). PartiaUy cool-tolerant cultivars, as indicated by a greater

abiUty to retain tiieir 34°C rate of ceUulose syntiiesis at 15°C (Haigler et al., 1994), had

elevated Glc 6-P levels at 15°C on the very day tiiat die cultured ovules were transferred to

cycling temperatures (shock treatment) diat were not attained by the cool-sensitive cultivars

untU after 3 days into cycUng temperatures. Thus, a response that may confer partial cool-

tolerance in cultivars is a rapid increase in the levels of Glc 6-P witiiin the fiber.

Despite the elevation in Glc 6-P at die onset of cycling to cooler temperamres (shock

treatment), UDP-Glc levels were reduced, or changed only slightiy, within the fiber (see

Table 2.3). Therefore, reduced activity of PGM or UDPG-PP may hinder the synthesis of

UDP-Glc from the Glc 6-P pool. (A reduction in UDP-Glc pool size may also be an

indication of increased enzyme activity in the pathway after UDP-Glc synthesis, but the

opposite occurs for cellulose synthesis and it would be unexpected for any enzymatic

reaction.) Further investigations of carbon flow through the pathway may give indications

of how cool temperamres affect flux through the pathways related to Sue and cellulose

synthesis.

Also, it was shown that the onset of secondary ceU waU synthesis was accompanied

by a 40 to 70% reduction in the hexose sugar pools (see Figures 2.12 and 2.13) in the

fibers of the cultured ovules at both 34°C and 15°C. This indicates a possible

developmental shift within the fibers likely related to the increased synthesis of ceUulose at

the onset of secondary cell wall development In addition, the hexose sugars have been

hypothesized to exist in two separate pools within the fiber cell, a smaller metaboUc pool

and a larger storage pool, presumably the vacuole since the cotton fiber is highly vacuolated

(Carpita and Delmer, 1981). Similar metaboUc and storage pools also are beUeved to exist

for Sue. Therefore, the reduction in the overaU pool size of a particular sugar at the onset

of rapid secondary cell waU synthesis likely involves dramatic changes in one or both of

these pools.

The purpose of this research is to investigate the effects of cool temperatures (15°C)

on metabolite flux through the sugars during secondary cell wall formation using pulse-

labeUing and pulse-chase analyses at botii warm (34°C) and cool (15°) temperatures.

3.2 Materials and Methods

3.2.1 Cotton Plant Growth

Two cultivars of cotton (Gossypium hirsutum L.) with different abUity to retain their

34°C rate of ceUulose syntiiesis at 15°C (Haigler et al., 1994) were chosen for tiiis study.

Paymaster HS200 synthesized ceUulose at 15°C at a rate of 24-26% that at 34°C, whereas

81

Acala SJ-1 syntiiesized cellulose at a rate of only 12% tiiat at 34°C (Haigler et al., 1994).

Botii cultivars were grown in tiie greenhouse at cycling temperatures of 32 to 37°C during

the day and 22 to 25°C during die night, watered three times a day, and fertiUzed widi a

complete fertiUzer once a week.

3.2.2 Cotton Ovule C^hure.

Ovules from each cultivar were removed from flowers at 1 dpa, randomly divided

among culture flasks containing cotton medium described previously (Haigler et al., 1991;

Roberts et al., 1992), and culttired in 34°C dark incubators. On 18 dpa, ovules were

transferred to dark incubators set for temperature regimes of 34°C/15°C on a 12 h/12 h

cycle. At the same time on 18 dpa, half of the flasks for each cultivar were transferred to

an incubator beginning the high temperature of the cycle and half were transferred to an

incubator beginning the low temperature of the cycle. This facUitated simultaneous

comparisons at 15°C and 34°C on subsequent days.

3.2.3 Radiolabeling of Ovules

At 21 dpa (3 days after cycling temperatures began), ovules were allowed to adjust to

the cycled temperature for 4 hours before being placed in a culmre medium containing

[U-*'*C]Glc (1.324 ^iCi/mL; final specific activity 9.8 x 10 Bq/^imol) witii a final Glc

concentration of 10 mM. For each cultivar at each temperamre, ovules were removed from

the culture flasks, placed in smaU plastic transfer baskets, and washed three times in

temperature-adjusted medium without Glc in which 0.12 M pentaerythritol was added as an

osmotic substitute. The plastic transfer basket was blotted briefly on paper towels between

each wash. Ovules were then removed one at a time with forceps, blotted briefly on paper

towels, and randomly placed into four 60 x 15 mm plastic Petri dishes containing 5 mL of

labeled medium, with a total of 10 ovules in each Petri dish. The Petri dishes were placed

in the bottom of 300 mL sealed plastic beakers and placed in the incubator for the

appropriate time period.

In pulse-labeling studies, the Petri dishes were removed from the incubator at 30 min,

1 h, 1.5 h, and 2 h after labeling began, and ovules were killed by freezing in Uquid

nitrogen. In pidse-chase studies, the Petri dishes were removed after 45 minutes of

labeUng, and the ovules in three of the Petri dishes were quickly tt-ansferred using plastic

transfer baskets into four consecutive washes of incubation medium containing 10 mM

unlabeled glucose as the carbon source. Again, the transfer baskets were briefly blotted on

paper towels after each wash. Ovules were then placed into plastic sealed containers

82

containing only unlabeled medium and incubated as a chase for 1, 2, or 4 additional hours

after tiie 45 minute pulse. The ovules in tiie fourth Petri dish were removed after die 45

min pulse and frozen in liquid nitrogen.

3.2.4 Sugar Extraction From Top Fibers

WhUe frozen, only the top aerial fibers were removed from die ovules and extracted

with TCA as described earUer (Kanabus et al., 1986). Analysis of only aerial non-

submerged fibers impUed that labeled glucose had entered the fiber cell only from the ceUs

of the seed coat ratiier than from tiie external medium (PUlonel et al., 1980; Roberts et al.,

1992). After extraction and retention of the aqueous phase, the fibers were washed 3 times

with distUled water, freeze-dried, and weighed to the nearest tenth of a mUUgram.

The sugars extracted from the fibers in the aqueous phase were separated by HPLC

using a Waters Sugar-Pak I cation exchange column (300 x 6.5 mm) according to the

method of Tobias et al. (1992). The Sugar-Pak I column was maintained at 85 to 90°C,

and the sugars were eluted widi a 50 mg L* Na2CaEDTA solution flowing at the rate of 0.6

mL min* and monitored by a differential refractometer (Altex Model 156). From the

refractometer, the effluent flowed through a radioactive flow detector (Packard Radiomatic

A-100 FLO-ONE, Meriden, CT), which mixed the effluent with scintillation cocktaU

(Packard Ultima-Flo M, Meriden, CT) at a ratio of 1:2, respectively. Counts per minute

were recorded and graphed over time. Peaks of radioactivity were matched to the sugar

concentration peaks from HPLC and the specific activity of each sugar was calculated as

counts per minute (cpm) nmol* of sugar.

In addition to the Sue, Glc, and Fm peaks present on the sugar profile during HPLC

separation, an additional large peak was present at the beginning of die profUe containing

material that did not bind to the column. This peak did contain radioactivity, and it was

determined that aU of the hexose phosphates and UDP-Glc eluted at diis point The peak

was as large as that of Glc and Fm and, therefore, contained many other compounds

besides the hexose phosphates and UDP-Glc, including possibly malate, which is known

to be in the fiber cell in concentrations similar to the hexose sugars (Basra and Malik, 1984;

Dhindsa et al., 1975). Therefore, because of die uncertainty of die labeling and because

exact pool sizes of the hexose phosphates and UDP-Glc were not exu-acted in these

experiments, tiiese data were omitted from the specific activity graphs.

83

3.3 Results 3.3.1 Carbon Entry in(^ Top Aerial Fibff

Sugars were only extracted from die aerial fibers in order to investigate carbon flow

into the fiber from seed coat ceUs radier dian from die medium. Glc is die carbon source

that is die most effective m cotton culmre systems (Beasley, 1992), and after 20 minutes of

labeling, Glc was the only sugar tiiat was labeled in the top aerial fibers (data not shown).

From this fmding, it was concluded that Glc moves readUy and rapidly from the medium

through the ovule and into the aerial fibers. Thus, carbon enters the fiber cells mainly as

Glc in cultured ovules and not as Sue, which is presumed to be the major source of carbon

entering die fiber ceU in vivo (Ruan et al., 1997). Therefore, Sue diat can be detected later

within the fibers of cultured ovules must be subsequently synthesized within them

following the proposed pathways described in Figure 1.2.

3.3.2 Pulse Labeling of Acala SJ-1

The changes in the specific activity of Glc, Sue, and Fm pools in the aerial fibers of

Acala SJ-1 at both 15°C and 34°C at 21 dpa during pulse labeling are diagrammed in Figure

3.1. Glc and Fm attained the same specific activity at 15°C and at 34°C within 1 h of

labeUing and maintained what appeared to be a steady state content of radioactivity.

Certainly, the cotton fiber ceU contains both a metaboUc and a storage pool of Glc and Fm

(Carpita and Delmer, 1981) and this would suggest that some radioactivity may have

initially entered the non-metaboUc storage pool. Attaining a steady-state level of activity

indicates an active flux into other pools.

The specific activity in Sue, however, was greatly reduced at 15°C compared to 34°C

at aU time points during pulse labeling, indicating that some restriction of carbon movement

must occur in Acala SJ-1 subsequent to Glc entering the fiber cell, but before the synthesis

of Sue. Since the Glc 6-P pool dramaticaUy increased in size at 15°C (see Table 2.1), the

restriction must occur after the synthesis of Glc 6-P. The specific activity in Sue at 15°C

did increase during the pulse, indicating that carbon movement was flowing from Glc to

Sue. At 34°C, there was a rapid increase in radioactive carbon in the Sue pool from 30

mmutes to 1 h. However, the Sue pool reached steady state content of radioactivity by 1.5

h, supportmg die idea that carbon is flowing through the Sue pool, potentiaUy to the

synthesis of ceUulose.

The specific activity of Fm is smaU compared to Glc or Sue. Perhaps die labeled Glc

tiiat enters tiie fiber ceU is mainly used to synthesize UDP-Glc rather tiian Fm 6-P (see

Figure 1.2). Since Fm is released from the SuSy activity, it seems lUcely that it wUl be

84

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85

constantiy recycled to Fm 6-P for die syntiiesis of Sue and, dierefore, tiiere would be less

need to syntiiesize Fm 6-P from Glc 6-P. Therefore, only a small portion of die

radioactivity entering tiie ceU wUl be located in die Fm 6-P pool. The Uttie radioactivity tiiat

was measured in die Fm pool was Ukely tiie result of some labeled Glc 6-P isomerized to

Fm 6-P, which was used for the synthesis of Sue.

3.3.3 Pulse Labeling of Pavmaster HS200


Paymaster HS200 at botii 15°C and 34°C at 21 dpa are diagrammed in Figure 3.2. SimUar

to Acala SJ-1, the specific activity in the Glc and Fm pools at 15°C was the same as that at

34°C. Also simUar to Acala SJ-1, die specific activity in Sue at 34°C was much greater than

that at 15°C, also indicating that a cool temperature restriction may occur in the pathway of

Sue syntiiesis after Glc 6-P. The specific activity in Sue of Paymaster HS200 at 34°C

continued to increase over the labeling period and was greater than that of Acala SJ-1 at 1.5

and 2 h. This indicates that Paymaster HS200 may be more efficient in synthesizing Sue

than is Acala SJ-1.

The specific activity of Glc graduaUy increased after 1 h of labeUng in Paymaster

HS200, possibly indicating that more label was entering the non-metaboUc storage pool

than was observed in Acala SJ-1. At this developmental stage, the pools of Glc and Fm in

Acala SJ-1 are rapidly decreasing (Figure 2.14), presumably to provide carbon for the

synthesis of Sue in addition to the carbon entering the cell, but they have already decreased

for Paymaster HS200. Therefore, more label may be directed into rather than out of the

storage pool of Paymaster HS200. Also, the specific activity of Glc in Paymaster HS200

was slightly higher than Acala SJ-1 at all 4 time points in pulse labeling. This may indicate

that carbon flow into the fibers of Paymaster HS200 may be greater than that into the fibers

of Acala SJ-1. However, the pool sizes of Sue and Glc in the fibers of Paymaster HS200

were lower than in Acala SJ-1 at 21 dpa, and part of the difference in specific activity can

simply be attributed to concentration differences.

3.3.4 Pulse/Chase Labeling of Acala SJ-1

The changes in die specific activity of Glc, Sue, and Fm pools in the aerial fibers of

Acala SJ-1 at botii 15°C and 34°C at 21 dpa during pulse/chase labelmg are diagrammed in

Figure 3.3. During the chase, the specific activity of Glc began to decrease as the specific

activity of Sue began to increase, clearly indicating that Sue is synthesized at the expense of

86

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88

Glc in tiie cotton fibers. Also, die specific activities of Glc and Fm at 15°C were similar to

tiiose at 34°C, but die specific activity of Sue at 34°C was much higher tiian tiiat at 15°C.

This indicates again diat cool temperamres (15°C) restrict die syntiiesis of Sue in tiie fibers

of Acala SJ-1.

3.3.5 Pulse/Cha.se Laheling of Pavmaster HS200


Paymaster HS200 at botii 15°C and 34°C at 21 dpa during pulse/chase labeling are

diagrammed in Figure 3.4. SimUar to Acala SJ-1, the specific activities of Glc and Fm at

15°C were simUar to 34°C during die chase, whereas die specific activity of Sue at 15°C

was much lower than at 34°C. Again, this indicates a possible cool temperature restriction

in the synthesis of Sue within the cotton fiber.

The specific activity of Sue at 34°C in the fibers of Paymaster at tiie end of the pulse

was much greater than that in the fibers of Acala SJ-1. Also, the specific activities

decreased during the chase in the fibers of Paymaster HS200 whereas they increased for 2

h of the chase in the fibers of Acala SJ-1 before decreasing at 4 h after the pulse. These

factors suggest that Paymaster HS200 may be more efficient than Acala SJ-1 in

synthesizing Sue.

Specific activities of Glc decreased rapidly after 1 h into the chase to an apparent

steady state level, presumably what is in the storage pool. It must be noted that the specific

activity of Fm at 34°C increased during the chase in the fibers of both Paymaster HS200

and Acala SJ-1. Again, this indicates that some of the Glc 6-P is isomerized to Fm 6-P and

used to synthesize Sue phosphate by the action of SPS in the pathway towards Sue

synthesis (see Figure 1.2). The Fm is then released back into the cell by the activity of the

membrane-bound SuSy. The constant recycling of the Fm released from the activity of

SuSy within the fiber ceU would likely explain the lower specific activity found in the large

Fm pool compared to that of Sue or Glc. Movement of label into the storage pool, again,

is likely not rapid.

3.4 Discussion

3.4.1 Sucrose Svnthesis Within the Fiber

The results of the radioactive labeUng studies at 21 dpa clearly indicated tiiat much of

the carbon entering the cotton fiber ceU as Glc is used to syntiiesize Sue. This supports the

proposal that Sue is the substrate needed in the fiber cell for the activity of SuSy to directiy

provide UDP-Glc to the ceUulose synthease complex at the membrane (Amor et al., 1995;

89

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90

Ruan et al., 1997). In this study. Sue was synthesized at die expense of Glc within the

cotton fibers of ovules cultured in vitro during secondary cell wall syntiiesis. It has been

shown previously that approximately 80-90% of the carbon entering tiie ovules in vitro was

directed towards ceUulose synthesis and less dian 20% was directed towards respiration

(Roberts et al., 1992). This study therefore shows that much of the carbon entering die

fiber cell is directed towards the syntiiesis of Sue, which wiU be hydrolyzed by SuSy at die

membrane.

In the pulse labeling studies, it appeared that Glc and Fm approached a near steady-

state content of radioactivity witiiin 1 h of labeUng. This would seem to indicate that most

of the label was found in the active metabolic pool rather than the vacuolar storage pool

within the fiber, and this was an indication of flux to other pools. However, in the

pulse/chase studies, specific activity of Glc during the chase decreased to what appeared to

be a steady state level, especiaUy in Paymaster HS200, which would indicate that some

label was directed towards the storage pool. In previous studies, the Glc pool never

reached a steady state content of radioactivity, but increased for 4 hours of pulse labeUng

(Carpita and Delmer, 1981). They were labeling cultured ovules of Acala SJ-1, however,

at 15 to 16 dpa in which most of the Glc entering the fiber was probably being stored in the

vacuole and rapid secondary cell waU synthesis had not yet been initiated. In our study, the

ovules were at 21 dpa, after the onset of rapid cell wall development, in which the

incoming Glc was presumably used primarily for the synthesis of ceUulose (Roberts et al.,

1992). Some label, however, did appear to be entering the storage pool of Glc, indicating

that active exchange between the two pools of each carbohydrate is occurring within the

fiber cell.

3.4.2 Pavmaster HS200 Svnthesizes Sue More Efficientiv

In this study. Paymaster HS200 was more efficient in synthesizing Sue than Acala

SJ-1. The specific activity of Sue was higher earUer in Paymaster HS200 than in Acala SJ-

1 at both 15°C and at 34°C. This may explain, in part, the differences in the ceUulose

synthesis/respiration ratios previously reported for these two cultivars (Haigler et al.,

1994), assuming Sue is synthesized and then directiy hydrolyzed by the membrane-

associated SuSy. Paymaster HS200 had a cellulose synthesis/respiration ratio of 8.4 at

15°C, and the ratio for Acala SJ-1 was only 2.2. These ratios were determined at 21 dpa

after 3 days of cycling temperatures.

The differences in specific activities of Sue observed in this study indicate that many

of the differences in fiber growth and development observed among cultivars may be

91

related to die abiUty to syntiiesize Sue widiin die fiber. Also, tiiis implies tiiat genetic

manipulations to upregulate the activities of enzymes involved in Sue synthesis, such as

UDPG-PP or SPS, may prove to be effective in improving fiber quality and/or fiber yields

in cotton.

It should be noted diat the Glc 6-P pool sizes in Acala SJ-1 were probably stiU lower

than tiiat of Paymaster HS200 (see Table 2.1), which had a greater capacity for elevating

Glc 6-P levels in the fiber as soon as the ovules were placed in cool temperatures. Higher

levels of Glc 6-P in Paymaster HS200 would not only provide more substrate to increase

enzyme activity, but it is also an aUosteric activator of SPS, thereby increasing tiie

synthesis of Sue. It is possible that the large increase in the pool size of Glc 6-P at cool

temperatures is an adaptive measure to maintain tiie synthesis of Sue within tiie fiber. It

can not be determined from this study, however, whether the increase in Glc 6-P pool size

would be enough to account for the increase in specific activity of Sue demonstrated by

Paymaster HS200.

3.4.3 Possible Enzvmic Restrictions At Cool Temperatures

This study provides more evidence for a possible cool-sensitive enzymic restriction at

the synthesis of Sue within the fiber. In both cultivars, the specific activity of Sue was

much lower at 15°C than at 34°C in the pulse labeling studies and in the pulse/chase studies,

whereas the specific activity of Glc at 15°C was simUar to that at 34°C. A block or

restriction before the synthesis of Sue would reduce the amount of substrate available for

SuSy activity and would, therefore, potentiaUy reduce the synthesis of cellulose at cool

temperatures.

3.4.4 Importance of the Pathways Involving Sue Synthesis

FinaUy, the pathways related to Sue synthesis within the fiber cell may be more

important in ceUulose synthesis than is generaUy realized and may play a partial role in

establishing cool tolerance within the fiber. This study indicates that the movement of

carbon into the fiber ceU is fairly rapid and is not hindered by the cool temperatures. The

similar rapid entry of Sue into the fiber cell in vivo (Ruan et al., 1997) would lUcely not

hinder its use by the membrane SuSy at cooler temperatures. It therefore seems lUcely that

differences among cultivars in the synthesis of ceUulose at cooler temperatures can likely be

attributed to differences tiiat may occur in the pathways related to Sue synthesis within the

fiber. It must be noted that the composition of the ceU wall of cultured fibers was

remarkably simUar to that of plant-grown fibers (Meinert and Delmer, 1977) and the

92

response of die fibers of cultured ovules to cool temperature was very simUar to tiiat of

field-grown fibers (Haigler et al., 1991). In vitro culture of cotton ovules, dierefore, plays

an important role in studying the metaboUc reactions that occur within the ceU by

eUminating direct entry of Sue into the ceU. This phenomenon, however, may also have

Umitations as the ovule itself is removed from die enclosure of the carpel, and this may

effect other processes widiin die cell, such as die dark metabohsm of the COj released from

respiration (Basra and Malik, 1983; Dhindsa, 1980).

93

CHAPTER rv

CONCLUSIONS

The synthesis of ceUulose in die fiber cells of cotton (Gossypium hirsutum L.) likely

involves tiie hydrolysis of Sue by membrane-bound SuSy to provide UDP-Glc directiy to

the cellulose synthase presumably localized in the membrane. The source of Sue for

hydrolysis is lUcely Sue import directiy into tiie fiber cell from tiie surrounding ceUs in the

ovule seed coat and Sue synthesis from metabolites within the fiber. Therefore, die

pathways involved in cellulose synthesis within the ceU are those pathways involved with

the synthesis of Sue. These pathways include the phosphorylation of the hexose sugars,

which are in great abundance widiin the fiber at die onset of secondary cell waU synthesis,

the synthesis of UDP-Glc, and the synthesis of Sue from UDP-Glc and Fm 6-P.

Analysis of the changes in metabolite concentrations that occur when the ovules were

transferred at 18 dpa to cycUng temperattires (34°C/15°C) show that Glc 6-P levels

increased significantiy (40 to 70%) within die fiber when cycled to 15°C in all cultivars.

The partially cool-tolerant cultivars. Paymaster HS200, YG, and Paymaster HS26, as

defined by their ability to retain higher percentages of 34°C ceUulose synthesis at 15°C,

were shown to have the abUity to greatly increase levels of Glc 6-P within the ceU

immediately upon exposure to cool temperatures and thereby increase the substrate levels

for the synthesis of UDP-Glc and Sue. In addition, Glc 6-P is an activator of SPS. The

cool-sensitive cultivars, however, did not show the same elevated Glc 6-P levels until 24

dpa (six days after cycling temperatures began).

Also, Fm 6-P levels at 15°C increased in all of the cultivars (12-55%) above that at

34°C, however, there was only a significant difference between levels at 15°C and 34°C in

cultivars where the levels at 34°C were lower than approximately 120 nmol g'' fresh

weight. This indicated that there may be a possible threshold level of Fm 6-P that is

particularly critical at lower temperatures, possibly to help maintain activities of enzymes

such as SPS.

UDP-Glc levels decreased, or only slightiy increased, upon transfer to cooler

temperatures, indicating that a possible temperature sensitive restriction may occur in the

pathway of UDP-Glc syntiiesis at PGM or UDPG-PP. This was supported by pulse-

labeUng and pulse/chase studies in which die specific activity of Sue, which requires UDP-

Glc for synthesis, at 15°C was much lower than that at 34°C in botii the partiaUy cool-

tolerant Paymaster HS200 and in the cool-sensitive Acala SJ-1. Paymaster HS200,

however, seemed to be more efficient in die synthesis of Sue than Acala SJ-1. This may be

94

a result, in part, of tiie abiUty of Paymaster HS200 to greatiy increase Glc 6-P levels

immediately upon exposure to cool temperatures that may activate SPS witiiin tiie fibers.

Also from this study, the levels of hexose sugars decreased dramatically (40 to 70%)

at die onset of secondary ceU wall synthesis, which occurred at 16 dpa in Paymaster

HS200 and at 18 dpa in Coker 312, YG, and Acala SJ-1. This dramatic decrease may be a

response to the accelerated synthesis of ceUulose widiin the fiber and die high demand for

substrate and/or it may play a developmental function in stimulating tiie activity of SuSy

within the fiber ceU (Weber et al., 1996). Clearly, die use of hexose sugars in tiie fiber will

be greatiy enhanced with no Sue import mto the fiber ceU of cultured ovules, and die

dramatic decreases may be accelerated in vitro.

The use of cotton ovule culture in these studies has been effective in allowing the

study of pathways within the cotton fiber aside from the import of Sue into the fiber ceU.

This provides opportunity for measurements of metabolites and enzyme activity within the

cell in controlled situations in efforts to improve the quaUty and/or yield of the fiber in

cotton. The response of cultured ovules in this study seemed to mimic that of greenhouse-

grown ovules (Jaquet et al., 1982) and, therefore, have proven useful in understanding

metabohsm within the fiber cell.

95

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