Sterols in germinating seeds and developing seedlings of longleaf pine, Pinus palustris Mill.

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Title:
Sterols in germinating seeds and developing seedlings of longleaf pine, Pinus palustris Mill.
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Vu, Cu Van, 1942-
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Longleaf pine   ( lcsh )
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Thesis--University of Florida.
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Includes bibliographical references (leaves 66-82).
Statement of Responsibility:
by Cu Van Vu.
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Typescript.
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Vita.

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University of Florida
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STEROLS IN GERMINATING SEEDS AND DEVEL.C-PINC,
SEEDLINGS OF LGNGLEAF PITE, Pinus palustris Mill.







By

CU VAN V0


A DISSERTATION "DSE7.TF.D TO TE GiADUATE COUNCIL OF
THE UNIVERSITY CF FL.'FilEA
IN PARTIAL FULFILL' 1iT OF THE Rf,'U T LE EJTS FOR THE
DEGREE OF' DOCTOR OF PHILOSOPHY









UNIVERSITY OF FLORIDA
1976












ACKNOWLEDGMENTS

I wish to express my sincere appreciation and gratitude to Dr. R.

E. Goddard, Chairman of the Committee, for his advice and assistance

throughout the graduate program, and his valuable help in preparation

of this manuscript My gratitude, in particular, is expressed to

Dr. R. H. Biggs, who served as Co-Chairman of the Committee, for his

moral support, his foresight in initiating the experiments, his ex-

cellent guidance and help throughout the course of the research, and his

patient assistance in preparation of the whole manuscript. My special

thanks also is given to Dr. L. A. Garrard, Dr. T. E. Humphreys, Dr. C.

M. Kaufman, and Dr. R. M. Roberts, members of the Dissertation Committee,

for their valuable criticism and aid in correction and improvement of

the manuscript.

I am deeply indebted to Dr. J. L. Gray, Director of the School of

Forest Resources and Conservation, for his recommendation for my

admission to the Graduate School, and for his support for completion of

my graduate program.

My deep acknowledgment is made to the late Professor R. G. Stanley

for his excellent supervisorship in guiding my professional development,

his valuable suggestions and stimulations of my interest in this prob-

lem, and his patient guidance during my early stages of research.

Numerous others extended invaluable assistance at crucial periods

of this study, in ways large and small. Dr. R. W. King, Dr. M. H.

Gaskins, Dr. C. A. Hollis IIT, Mr. J. E, Smith, Mrs. S. G. Mesa,






hMr E. G. Kirby III, Mr. J. K. Peter, to mention but a few, will always

be remembered for their meaningful contributions. The financial supports

provided by the U.S. AID and the School of Forest Resources and Con-

servation are gratefully acknowledged. Also the assistance of Mrs.

Ann Barry in typing the manuscript is truly appreciated.

My deep gratitude should be given to my parents and sisters, whose

hardwork, sacrifice, and encouragement made my college education

possible.

Finally, I gratefully acknowledge the needed long-suffering

patience and encouragement provided by my wife, Tieng Tran, and the

love and enjoyment given me by my son, Thien, which made all my disser-

tation possible.











TABLE OF CONTENTS

Page

AC KOi-.LE DGEN S .......................... ........................ ii

LIST OF TABLES .. ............... .............................. vi

LIST OF FIGURES ............................................. vii

ABSTRACT ............................ ....... ........ ...... ...... viii

INTRODUCTION ...................................................... 1

LITERATURE REVIEW ........................... ............. 4

Biochemical Aspects ....................................... 7
Physiological Aspects .................................. .... 12
Sterols as Membrane Components ....................... 13
Sterols as Plant Hormones ............................. 15
Sterols in Developing and Germinating Seeds ............. 17
Sterols as Protective Agents .............. ....... 18

MATERIALS AND MIETHODS .................... .................. 20

Chemicals .................. ,.........o......................... 20
Sources of Seeds ........... ................ ................ 20
Preparation and Germination of Seeds ........... ............. 21
Sterol Extraction and Fractionation ........................ 21
Identification of Sterols ................................... 24
Effects of Inhibitors on the Biosynthesis of
Sterols, Reducing Sugars, and Chlorophyll ................ 26
Sterol Extraction and Identification .................... 27
Determination of Reducing Sugars ...... .............. 27
Chlorophyll Determination ............................... 27
Isocitrate Lyase Assay ................... ................ 28
Germination of Seeds ........................o.......... 28
Extraction and Assay of the Enzyme .................... 29
Steroid Inhibitor Treatment .......................... 29
Sterol Treatment .............. ............. ........ 29

RESULTS ............ ................. ............ ....... ........ 31

Identification of Sterols Isolated from Germinating
Pine Embryos .............................................. 31
Changes in Sterol Content upon Germination ............... 37
Effects of SKF 7997-A3, ABA, and Cyclohexiinide on
Total Sterol Biosynthesis oo ............................... 42







Page


Effects of the three Inhibitors on the Level of
Reducing Sugars ............................................ 46
Effects of the three Inhibitors on Total Chlorophyll
Biosynthesis ................................................. 48
Effects of SKF 7997-A3 on the Activity of
Isocitrate Lyase ................... o ........... 48

DISCUSSION ............ ......................................... 52

SUMMARY .......................................................... 64

REFERENCES .............................................................. 66

BIOGRAPHICAL SKETCH ....................................... ...... 83











LIST OF TABLES


Table Page

1 Relative retention, with respect to 5 a-cholestane,
of known sterols and those from 11-day-old longleaf pine
seedlings ............................ .................... 33

2 Mass spectrum data of campesterol of germinating
longleaf pine embryos ................................. 34

3 Mass spectrum data of stigmasterol of germinating
longleaf pine embryos .................................... 35

4 Mass spectrum data of B-sitosterol of germinating
longleaf pine embryos ...................................... 36












LIST OF FIGURES


Figure Page

1 A possible biosynthetic pathway of plant sterols .......... 11

2 Flow chart for procedures used in the extraction and
fractionation of longleaf pine sterols .................. 25

3 Gas-liquid chromatogram of sterols of 11-day-old
longleaf pine seedlings ................................... 32

4 Changes in total sterol content and composition
during germination of longleaf pine seeds ................. 38

5 Changes in steryl ester content and composition
during germination of longleaf pine seeds ................. 39

6 Changes in steryl glycosides during germination
of longleaf pine seeds .................................... 40

7 Changes in free sterols during germination of
longleaf pine seeds ..................... ............... 41

8 Effects of ABA, SKF 7997-A3 and cycloheximide on total
sterol content during germination of longleaf pine
seeds ..................................................... 43

9 Effects of ABA, SKF 7997-A3 and cycloheximide on the
campesterol level during germination of longleaf
pine seeds ......................... ..................... 44

10 Effects of ABA, SKF 7997-A3 and cycloheximide on the
A-sitosterol level during germination of longleaf
pine seeds .......................................... 45

11 Effects of ABA, SKF 7997-A3 and cycloheximide on the
content of reducing sugars during germination of
longleaf pine seeds ...................... .......... ...... 47

12 Effects of ABA, SKF 7997-A3 and cycloheximide on the
chlorophyll content during germination of longleaf
pine seeds .................... ..... ............... 49

13 Changes of specific activity of isocitrate lyase in
megagametophytes of germinating longleaf pine seeds ....... 50











Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy



STEROLS IN GERMINATING SEEDS AND DEVELOPING
SEEDLINGS OF LONGLEAF PINE,Pinus palustris Mill.

By

Cu Van Vu
March 1976
Chairman: Ray E. Goddard
Co-Chairman: Robert H. Biggs
Major Department: Agronomy

The presence and possible functions of sterols in germinating

embryos and young seedlings of longleaf pine (Pinus palustris Mill.)

were studied at different periods after germination. Sterol analyses

were performed by gas-liquid chromatography (GLC) and verified by

combination of GLC-mass spectrometry. Campesterol and 8-sitosterol

were two major sterols which accounted for most of the sterol composi-

tion while stigmsterol was present only in very small amounts. No

cholesterol was revealed by GLC-mass spectrometry although there was a

minor peak appearing on the sterol gas-liquid chromatograms with a re-

tention time close to that of authentic cholesterol.

By fractionation, three different forms of sterols were obtained:

steryl esters, steryl glycosides, and free sterols. The sterols were

mainly found in the esterified fraction, while steryl glycosides and

free sterols only made up a small portion of the total sterol value.

The total sterol content in general increased during seedling

development, and this increase reflected mainly a change in steryl

viii






esters. The low levels of both free and glycosidic sterols remained

nearly unchanged throughout the experimental germination period.

These results were in agreement with the suggestion that sterols

increase during germination as a consequence of increased membrane and

organelle production.

Cycloheximide, abscisic acid (ABA), and the animal steroid inhi-

bitor SKF 7997-A3 had significant effects on the biosynthesis of sterols,

the content of chlorophyll, and the concentration of reducing sugars.

The activity of isocitrate lyase, a key enzyme necessary for convert-

ing fatty acids to carbohydrates in germinating fatty seeds, was

sharply reduced when decoated pine seeds were treated with SKF 7997-A3

prior to germination. This depression in enzymatic activity was not

repaired by administration of sterols to the steroid inhibitor-

treated seeds.

Results indicate the involvement of sterols, either directly or

indirectly, with the metabolism of germinating seeds and the growth

and development of young seedlings of longleaf pine.










INTRODUCTION

Although steroids are known to play very important roles in animal

life, their significance in higher plants is still obscure [111, 118].

Considerable information has been gathered on the biochemistry and

physiology of steroids in animals, but investigations of the function

and metabolism of plant steroids have only recently begun [113, 117,

120, 154].

In recent years, modern analytical methods have been used to iden-

tify and quantify steroids from plants [153]. These analyses have led

to the discovery that most steroids found in animals are also present

in plants [114, 154]. The major sterols found in germinating seeds of

different species have been reported as B-sitosterol, stigmasterol,

campesterol, and cholesterol [111]. The latter was once thought to

occur only in higher animals.

Very little is known about the exact role of steroids in plants

[111]. Some plant steroids have a very profound effect on animals, and

it is unlikely that they have no effect at all on the plants in which

they occur [111, 118].

It has been suggested that steroids may function similarly in

plants and animals [117, 120]. Various exogenously applied steroids

have been reported to affect growth and development of shoot and root

[14, 16, 123], influence sex expression in dioecious plants [172],

accelerate water absorption and germination [15, 19, 119, 120, 185],

and influence chlorophyll synthesis [171o Inhibitors of animal steroid

biosynthesis, when applied, also inhibit flowering in certain plants

1







[41, 89, 206]. An inhibition of sterol biosynthesis and a retardation

of stem growth were observed in tobacco seedlings when they were treated

with three familiar plant-growth retardants: 2'-isopropyl-4'-

(trimethylammonium chloride)-5'-methylphenylpiperidine carboxylate

(Amo 1618), B-chloroethyltrimethylammonium chloride (CCC), and tributyl-

2,4-dichlorobenzyl-phosphonium chloride (Phosfon D) [67]. These obser-

vations indicate the possible involvement of sterols, either directly

or indirectly, in plant growth and development. These metabolites are

widely distributed in the plant kingdom and should no longer be con-

sidered "waste products" [210].

One of the best systems in plants for steroid studies is that of

developing and germinating seeds [111]. However, studies of sterols,

the major group of plant steroids, have usually dealt with seeds of

species that are important in medicine [59, 118, 135, 136] and agri-

culture [11, 12, 13, 44, 47, 69, 96, 107]. Few such studies have been

done with seeds of forest trees, particularly the coniferous seeds

which have been used as the main seed sources for forest regeneration

throughout the world. Reports on sterols in these coniferous species

are very rare, possibly limited to less than a dozen [22, 111]. Thus,

the study of sterols in coniferous seeds could contribute to an under-

standing of tree physiology.

Longleaf pine, Pinus palustris, was selected for this study

because of the wide distribution of this commercially important species

in the southeastern portion of the United States. Also the seeds of

this species of known origin can be obtained in sufficient quantities,

permitting the design of properly controlled experiments.

The objectives of the present investigation were to study the pre-

sence and variation in amounts of sterols in longleaf pine seeds during




3


germination, the effects of inhibitors on sterol biosynthesis during

germination, and to investigate the possible functions) of sterols on

growth and development of germinating seeds.












LITERATURE REVIEW

The study of sterols began with investigations on the dissolution

of gallstones [38]. Isolation of the main constituent of gallstones

from alcoholic solution was first carried out by Poulletier de la

Salle, circa 1769 [195]. Similar experiments were repeated by

Dietrich in 1788 [66] and DeFourcroy in 1789 [65]. In 1816, Chevreul

gave the name "cholesterine" (chole, bile; stereos, solid) to the waxy,

scaly substance obtained by digesting gallstones with alcohol [52].

With the discovery of "cholesterine", its occurrence in man and other

higher animals was investigated extensively. Berthelot in 1859 [36]

prepared esters of "cholesterine", establishing its alcoholic nature,

and in usage today this is indicated by the termination, -ol, thus,

cholesterol [38].

In parallel to numerous studies of cholesterol in animals, research

on plant sterols probably began with Braconnot (1811) [42] who iso-

lated from mushrooms an "adipocire" that Vauquelin (1813) [230] and

Gobley (1856) [102] termed "agaricine" and Tanret (1889) [218] named

"ergosterine" ergosteroll). In 1878, Hesse [126] prepared from

Calabar beans and peas a sterol indistinguishable from the "cholesterol"

that Beneke (1862) [26] had previously obtained from peas. Hesse [126]

observed that while it seemed to be isomeric with animal cholesterol,

it was clearly not identical with it. Because cholesterol had been

associated generally with animal sources, Hesse named the new vegetable

substance phytosterol. Later, Windaus and Hauth (1906) [234] proved

that the Calabar bean phytosterol prepared by Hesse was actually a mix-

ture of sitosterol and stigmasterol.

4







Studies on sterol synthesis during germination of seeds started

with the work of Schulze and Barbieri in 1882 [211]. They found that

the sterol content of whole seedlings of lupine after germination in

the dark was greater than that of the ungerminated seeds. In 1918,

Ellis [86] reported that the main phytosterol present in whole wheat and

in the embryo was sitosterol. Also the percentage of phytosterol pre-

sent in the embryo was much higher than in the plant, suggesting an

essential function of sterol in germination and growth. Since its

discovery by Burian in wheat and rye embryo in 1897 [43], sitosterol

had usually been regarded as a single entity. In 1926, Anderson et al

[2, 3] demonstrated that sitosterol exists in at least three isomeric

forms, a, B, and Y, which are so intimately associated and so dif-

ficult to separate it is not surprising that they were considered as a

single substance in the initial analysis.

In 1927, Terroine et al. [220] demonstrated that the sterol con-

tent of seeds usually increased during germination, even in the dark.

They assumed that the sterols originated at the expense of fats stored

in the seeds. Their assumption was verified in 1936 by MacLachlan

[174] who observed that the germination of soybeans was accompanied by

a marked reduction in total fat, followed by an increase in sterols.

Also during this rapid mobilization and utilization of fat, esterifi-

cation of the sterols showed a marked increase.

Up to the 1940's, no function had been ascribed to the universally

distributed phytosterols in plants. In a series of papers published

between 1943 and 1946, Balansard et al. [14, 15, 16, 17, 18, 19] showed

that saponins, a group of steroids of wide occurrence in the plant

kingdom, were able to elicit plant growth responses when added to







plants in certain stages of growth. The growth rate of isolated wheat

embryos was approximately doubled by optimal concentrations of saponin,

although higher concentrations were toxic [16]. Saponin applications

also increased the rate of development of shoots and roots in begonia

[14]. Treatment of seeds of cereals with dilute solutions of saponins

accelerated germination and increased the subsequent rate of seedling

growth [18]. Seeds of peas and corn also had increased water uptake in

the presence of saponins, with a subsequent increase in the rate of

germination [15, 19].

In the early 1950's, the important discovery that the steroid,

cortisone, was of significant value in the treatment of arthritis and

other diseases encouraged many chemists to undertake the arduous task

of increasing this commercial product through partial synthesis from

plant steroids such as ergosterol, stigmasterol, and diosgenin [35, 212].

This search for suitable steroids caused an increase in interest in the

occurrence and distribution of steroids in plants. In 1953, Bergmann

[35] compiled much of the information known on plant sterols. He

listed the different sterols occurring in fungi, lichens, algae, and

higher plants; details on their molecular formula, melting point, and

specific rotation were also clearly described.

Although isotopic tracers were used by Arigoni in 1958 to follow

the biosynthesis of sterols in germinating soybeans [4], his work was

hampered by the lack of techniques to separate plant sterol mixtures.

The sterols of such mixtures differ only in their small degree of

unsaturation and in the shape of their side chain at C-24, showing such

similarities in their solubilities as to make their separation difficult

by conventional chromatographic techniques [35, 90, 112]. Since the

report in 1960 by Beerthuis and Recourt [23] that gas-liquid chromato-







graphy (GLC) could be used for separation and partial identification of

sterols, many studies using GLC techniques have been conducted on

sterols in germinating seeds of different species [145, 200].

The application of GLC to isolation and identification of sterols

has revealedthe heterogeneous nature of many plant sterol fractions,

some of which are too complex to resolve even by GLC [58, 171, 222,

227, 228]. Difficulties and problems are still evident when a positive

identification of a particular plant sterol is desired [58, 204].

Some attempts have been made to use other techniques, such as mass

spectrometry, to identity phytosterols, but the results obtained were

not always satisfactory due to the difficulty in obtaining pure samples

in sufficient quantities for an analysis [153, 167]. Knights in 1967

[153] used a combination of GLC and mass spectrometry for identification

of fifteen plant sterols and closely related compounds. Since then,

more advances and improvements have been made in the extraction and

identification of sterols in different plant species [44, 47, 69],

facilitating the studies of their possible functions in the plants in

which they occur.

Biochemical Aspects

It has now become clear that the de novo biosynthesis of sterols

occurs essentially by the same pathways regardless of the organism in

which it takes place [183]. It is generally accepted that mevalonic

acid (MVA) serves as the first intermediate of isoprenoid biosynthesis

in mammals [219], higher plants [13, 184, 226], algae [100, 101], and

fungi [216]. Langdon and Bloch in 1953 [164, 165] demonstrated that

squalene was a sterol precursor in animals. Later, the conversion of

MVA-2-14C to squalene was demonstrated in germinating seeds of Pisum
sativum [48, 49], and that squalene is the possible sterol precursor in

higher plants [27].







In theory, all trans-squalene may be the starting material from

which cyclic triterpenes will be formed [115]. Both plants and animals

convert squalene to squalene 2,3-oxide [31, 199, 233] from which there

are two major routes by which steroids are biosynthesized, through

lanosterol and cycloartenol [115, 118]. The former appears to operate

in animals [40] and fungi [231], while in plants the first cyclic

product is often cycloartenol [34, 87, 180, 197]. The conclusion that

sterols are synthesized via lanosterol in animals and via cycloartenol

in plants comes from the fact that lanosterol has been isolated from

plants less often than has cycloartenol [114]. The incorporation of

labeled acetate and MVA into cycloartenol rather than lanosterol, both

in vivo and in vitro, has been demonstrated in many plant species using

both photosynthetic and nonphotosynthetic tissues [5, 150, 151, 175,

196, 202]. Cycloartenol also appears to be the key triterpene in algae

since they incorporate MVA7 and squalene 2,3-oxide into this triterpene

rather than into lanosterol [95, 100, 190, 198, 201].

At one time, lanosterol was thought to be a precursor in plant

sterol biosynthesis; this came from the reports of isolation of lano-

sterol from tobacco [32] and Euphorbia latex [193]. Recent studies

have shown that the component that was thought to be lanosterol in

tobacco is 24-methylene cycloartenol [33]. The Euphorbia latex con-

tains both cycloartenol and lanosterol, but lanosterol is not actually

found in the plant tissue and is considered an end-product in latex

[194]. Labeled cycloartenol is converted to lanosterol, but not vice

versa, in Euphorbia latex, and the biosynthesis of triterpenes in the

latex is independent of that in plant tissue [194].

The last phases in sterol biosynthesis are the conversion of

cycloartenol to different sterols. Although cholesterol has been found






to be distributed in small amounts in higher plants [104], the major

plant sterols that contain additional alkyl groups at C-24 have been

identified as sitosterol, campesterol, and stigmasterol [104, 111].

There are many reports of incorporation of labeled acetate or MVA into

different sterols in different plant species [4, 13, 29, 30, 135, 138,

139]. When seeds of Pinus pinea were germinated in presence of MVA-

2-14C, a high yield of labeling in campesterol, isofucosterol, and

sitosterol was obtained [226].

The conversion of cycloartenol to the major sterols requires

introduction of an alkyl group at C-24, demethylations at the C-4 and

C-14 positions, opening of the 96-19-cyclopropane ring, and introduction

of the A5 bond [111]. The introduction of an alkyl group at C-24 does

not occur for cholesterol.

In the alkylation process, the C-28 and C-29 of the major plant

sterols are derived from S-adenosyl methionine, either by transmethyla-

tion for C-28 (campesterol) or double transmethylation for both C-28 and

C-29 (sitosterol and stigmasterol) [10, 51, 167, 236]. Although the

exact stage and mechanism of alkylation is still not completely under-

stood [114], the intermediate which is alkylated must have a double

bond at C-24 position [205]. This is substantiated by the presence of

24-methylene cycloartanol [1, 34,.122, 181, 192], 24-methylene lophenol,

and 24-ethyldiene lophenol in numerous plants (99). The 24-methylene

cholesterol, differing from 24-methylene lophenol by a double bond at

C-7 and an extra methyl group at C-4, has also been known as a key

intermediate in the biosynthesis of C-28 and C-29 sterols [20, 118, 225].

Reduction of the 24(28) double bond produces either 24 a-methylcholes-

terol (campesterol) or 246-methylcholesterol (22-dihydrobrassicasterol).

When a second methyl group is introduced into methylene cholesterol,







fucosterol or isofucosterol is formed. Reduction of the 24(28) bond of

this fucosterol will produce 24a-ethylcholesterol (sitosterol) [118].

These processes have been shown to occur in pine tissues. When the

germinating seeds of Pinus pinea were incubated with 28-1C-24-methylene

cholesterol, there was labeling in campesterol, isofucosterol, and

sitosterol [226].

The incorporation of MVA-2-14C into triterpenes and sterols of

various plants indicates that, as in lanosterol demethylation in animals,

the loss of the 4a-methyl group is followed by epimerization of the

4B-methyl to the 4a position before removal [97, 150, 151]. The

isolation of many plant triterpenes lacking a methyl group at C-4

indicates that the overall demethylation sequence in plants may be

C-4a-- C-14a---C-4a (after epimerization), rather than that visualized

as operative in rat liver homogenates, C-14a --C-4a-> C-4B (after

epimerization) [98, 180].

In the final stages of sterol biosynthesis, a rearrangement of the

double bonds must occur. Although the sequence of these arrangements

has not been well established, the possible pathway would be A 7_

A 5,7 A5, and these steps seem to be irreversible [99, 111].

The possible general pathway for biosynthesis of free plant sterols

is shown in Fig. 1.

In higher plants, sterols have been found to occur in three dif-

ferent forms: free sterols, steryl esters of fatty acids, and steryl

glycosides which are acylated and non-acylated [111, 118]. The esters

of major plant sterols have been isolated from different tissues of

many plant species [44, 143, 146, 149, 235], as well as from

cellular organelles [144]. The most important acid components of the

steryl esters have been shown to be palmitic, oleic, linoleic, and











MEVALONIC ACID


d:1

SQUALENE



0NE -OXIDE
SQUALENE 2,3-OXIDE


C---- Ee-L ----H

HO CHOLESTEROL


CAMPESTEROL


STIGMASTA-7, 22-DIEN-30-OL
I (SPINASTEROL)


S' OBTUSIFOLIOL




LDENE
PHENOL



,24(28)-
N-38 -OL

STIGMASTA-
HO
5,7,24(28)-TRIEN-30 -OL


^;-


tj ,- STIGMASTA-5,7-DIEN- 3 -OL H ST
\ HO STIGMAST
STIGMASTA- 5,7,22-TRIEN- 1-DIEN-30-
30-OL (28'ISOFUC(






STIGMASTEROL ( SITOSTEROL
HO O HO

.A possible biosynthetic pathway of plant sterols.
Adapted from [101, 111, 175, 181].


A-5,24(28)
*OL
OSTEROL)


Figure 1







linolenic acid [44, 186]. Although steryl esters have been isolated

from plants, their biosynthetic pathway is still in question, and no

localization for esterification has been established [111]. When

4C-labeled MVA was used as a substrate, the radioactivity recovered in

the steryl esters was much higher than when six-day-old tobacco seedlings

were incubated with 14C-labeled cholesterol or sitosterol [45, 46].

The isolation of steryl glycosides in both forms has also been

reported from a variety of plants [44, 46, 88, 169]. Any 4-demethyl

sterol such as sitosterol, campesterol, stigmasterol, and cholesterol

can be the moiety of both steryl glycosides [46, 169]. The sugar com-

ponents have also been shown to be commonly glucose and in some cases

mannose and galactose [81, 82, 147, 169, 221, 237], and the main acyl

moieties in the acylated forms are palmitic, stearic, oleic, linoleic,

and linolenic acid [147, 169, 221]. The biosynthesis of steryl glyco-

sides has been obtained by particulate enzyme preparations from seeds of

different species [46, 131, 132, 148, 191]. The most active glycosyl

donor is UDP-Glucose, and the reaction is stimulated by ATP [78, 111,

132]. The pH of the incubation medium varies, depending on tissues

[79, 80], but optimal conditions are near pH 7.0 [46, 187].

Two different pathways have been proposed for the formation of

acylated steryl glycosides: through steryl glycoside or acyl glycoside

[111]. Experiments with a cell-free system support the former [9, 132]

while in vivo experiments favor the latter pathway [46, 82].


Physiological Aspects

The almost universal occurrence of sterols in plants would suggest

that these compounds have some definite role in plant metabolism. As

yet the nature of this function is quite obscure, and little physio-







logical significance has been attached to the universally distributed

phytosterols [44, 118].

In animal systems, sterols serve at least three functions: as

membrane components, as hormones, and as precursors of other steroids

[111, 117, 120]. It has been suggested that sterols may act in a

similar manner in both plants and in animals [117, 120].


Sterols as Membrane Components

The role of sterols as components of plant cell membranes was

suggested in 1918 by Ellis [86]. Recently this role of sterols was

re-emphasized by many authors [44, 105, 107, 109, 130, 144, 145]. In

general, the level of sterols was reported to increase with time of

germination in many plant species, leading to the suggestion that

actively growing tissues, such as germinating seeds and developing seed-

lings, accumulate sterols as a result of increased membrane synthesis

[44, 111, 145].

In a study of the distribution of sterols in organelles, it was

found that all membranes contain fractions of free sterols, steryl

glycosides, and steryl esters [107, 144]. Furthermore, when cholesterol -
14C was added to the isolation medium, it became distributed intracellu-

larly, leading to the conclusion that significant amounts of sterols in

plants are associated with membranes, including those of organelles,

and that sterols play an important role as structural and functional

components [107, 111].

The forms and categories of sterols that may constitute an inte-

gral part of the lipid layer of the cellular membranes are topics of

many discussions [111, 130, 145]. Grunwald [105] noticed that choles-

terol was more effective than CaC12 in preventing the leakage of B-







cyanin from red beet root cells treated with methanol. Other sterols,

such as g-sitosterol and stigmasterol, were less effective. Choles-

terol was also found more effective than the other sterols in restor-

ing the K+ and NO- uptake capability in etiolated Pisum sativum stem

sections treated with filipin [124]. For membrane stabilizing effec-

tiveness, cholesterol palmitate and cholesterol glycoside, both lacking

a free hydroxyl group at the C-3 position of the cholestene nucleus,

were found ineffective in preventing electrolyte leakage from barley

root cells, whereas free cholesterol was effective in this regard [109].

Also, when plant sterols were permitted to incorporate in vitro into

erythrocyte membranes, it was found that cholesterol entered more

readily than sitosterol, while campesterol was intermediate. Campe-

sterol and sitosterol contain an extra methyl and ethyl group in the

chain, respectively, which could reduce the ability of a sterol to

enter the phospholipid membrane. Also, the presence of the double bond

in the side chain of ergosterol and stigmasterol apparently reduces

their flexibility, resulting in a reduction of their capacity to

dissolve into the erythrocyte phospholipid complex [22, 77].

All these facts have led to the suggestion that only sterols having

a free hydroxyl group at the C-3 position and a flat molecular con-

figuration similar to cholesterol could be absorbed into the membrane

phospholipids and be active [110, 111]. The level of free sterols

which has been reported to increase with time of germination in seed of

a number of species may partially support this suggestion and may ex-

plain the possible function of free sterols in higher plants [44, 69,

145].

Information on the physiological role of steryl esters and steryl

glycosides is very limited. By analogy with cholesterol esters in








animal tissues, sterols were postulated to be transported intracellu-

larly as esters from their site of synthesis to the various organelles

[1451. The steryl glycosides probably represent either storage or

transport forms of sterols in plants [8, 132, 224]. The addition of a

sugar moiety to the nonpolar free sterol facilitates its solubility in

the cytoplasm for possible tr'iP.o -t to different parts of the develop-

ing seedlings [1321.


Sterols as Plant Hormones

Sterols may have hormonal activity in plants as well as in animals

[117]. Through oxidation and aromatization, plant sterols were reported

to be transformed to other steroids which have been identified as

hormones in animals [22, 28, 111, 116, 120, 207].

Studies on steroid hormones in higher plants so far have been

largely concerned with their function in the insect molting process

[22, 116]. Recent isolations of sterols with insect molting hormonal

activity contributed to the proof of the capacity of steroid biosynthesis

in higher plants which in many ways resembles that in animals [121,

127].

The status of our -kowledge of the influence of animal steroid hor-

mones on growth and reproduction of plants began in 1945 with Love and

Love [172]. They found that they could produce male or female flowers

on Melandrium dioecum at will by applying either androgens or estrogens

to the stems before flowering.

In 1971, Gawienowski et al. [94] reported that treating monoecious

cucumber plants with either 178-estradiol or testosterone caused the

induction of pistillate flowers. An increase in estrogen biosynthesis

during flowering in Phaseolus vulgaris was also reported in the same

year by Kopcewicz [160]. Quantitative investigations of the content of







the estrogen substance in the developing bean plants showed that this

compound appears as flower buds emerge, reaching a maximum at the

period of flower bud development and pod formation. Estrogens in-

creased the number of flowers when applied to Ecballium elaterium [161]

and Cichorium intybus [1581, These sex hormones also influenced the

female to male sex ratio in favor of female flowers, while androgens

changed the sex ratio in favor of maleness [161]. In addition to the

effect of animal steroids on flowering in plants, the major plant

sterol, sitosterol, was also active in the initiation of flower buds in

Chrysanthemum [39].

The suppression of floral induction by steroid biosynthesis

inhibitors was reported in Pharbitis nil [41, 206], Xanthium

pensylvanicum [41], and Lolium temulentum [89]. The animal steroid

inhibitor SKF 7997-A3 inhibits effectively the flower-inducing pro-

cesses only if applied to the leaves and shortly before the beginning

of the dark period in two short-day plants Xanthium and Pharbitis.

Application to buds or to leaves after a long night is without effect.

Since the substance is a steroid biogenesis inhibitor in animal tissues

and since it also blocks sitosterol and stigmasterol biosynthesis in

Xanthium leaves, there may be participation of steroidal substances in

floral induction and sex expression in plants. However, their mechanism

of action has not been determined. It has been suggested that steroids

may constitute one of the components of the hypothetical flowering hor-

mone [41, 160].

Besides the effect of steroids on flowering and sex expression,

there are also additional responses on plant growth [123, 178].

Sitosterol, estrone, and 17B-estradiol were reported to stimulate growth

in six-day-old dwarf seedlings of Pisum sativum [155]. Also sitosterol,





17

stigmasterol, and cholesterol, like gibberellic acid, could overcome

completely the effect of the retardant Amo 1618 on stem growth of

tobacco seedlings [67]. It had been shown that estrone increased the

endogeneous level of gibberellins in dwarf Pisum sativum [156]. Further-

more, auxin content of the seedlings of Pinus silvestris and Pisum

sativum also showed a sharp increase over the control when both were

treated with estrone and 178-estradiol [157, 159]. This indicates the

possibility that steroids by themselves do not act as hormones, but may

have an effect on the biosynthesis of gibberellins which, in turn,

influences flowering and plant growth [158, 159].


Sterols in Developing and Germinating Seeds

The presence and accumulation of sterols during seed development

were reported in such important agronomic crops such as soybean [141],

pea [12], and corn [63, 64]. In general, the biosynthesis of sterols

was highest in the least mature seeds and decreased with seed maturity,

with formation of 8-amyrin as a final regulation of sterol biosynthesis

in mature seeds [12].

During corn seed development, free sterols and steryl esters were

the major sterol fractions in both low and high oil content varieties.

Steryl glycosides accounted for only a small portion of the total

sterol content [63, 64]. Also in corn, while the free sterol level

rapidly decreased between 15 and 26 days after pollination, the steryl

esters continued to increase, reached a maximum level at the late

stages of linear kernel growth, and then decreased during the final

part of the growth period [63].

In soybean, during seed maturation the free form of sterols was

the most abundant and amounted to 70% of the total sterols 1141].







Steryl esters and steryl glycosides also accumulated during maturation,

but free sterols accumulated for a period longer than that for steryl

glycosides and steryl esters.

The presence of sterols in mature dry seeds was also reported

[12, 73, 133]. In the case of peas, B-sitosterol was present at a

concentration as high as 0.67 mg/g of dried seed [12]. It is possible

that large amounts of sterols are synthesized during seed development

and accumulate in dry mature seeds to satisfy the initial demands

made by growth during germination, probably either by production of

new membranes or transformation of the existing membranes to be more

permeable to gases and water [12, 111].

Sterol synthesis was not found to occur to any significant extent

during the first 2 to 4 days following germination [12, 44, 69, 145].

Perhaps sterol reserves were sufficient in early stages of germina-

tion and biosynthesis was required only after reserves were depleted

[12, 111].

As previously discussed, an increase in total sterol content was

observed in different species during germination [44, 69, 70, 71, 72,

135]. This increase in sterols was probably a result of increased

cellular organelle production and new membrane formation [44, 111,

145]. Sterol biosynthesis was also proportional to an increase in dry

matter and protein, and was higher in seedlings grown in nutrient

solution than those grown in distilled water [68, 69].


Sterols as Protective Agents

It has been suggested that sterols could be used to control fungi

and insects [22]. When the cotton leafworm insect was grown on a

medium containing 8-sitosterol, a high level of insect sterility was







observed, suggesting that the use of naturally occurring plant sterols

may be valuable as a means of biological control [208]. Reports also

showed that sterols may cause an inhibition in growth and reproduction

of some fungi [84]. Thus, the presence and level of sterols in plant

tissues could play a role in disease resistance and may be involved in

affording plant protection against fungi and insects [6, 117, 125, 137].

By manipulation of the sterol content of the host through breeding,

more resistant crops may be obtained [85]. The application of

methodology for altering the sterol level in plants could lead to more

efficient crop production and could assist in the response to the

demands of the world population for increased food productivity [22].













MATERIALS AND METHODS

Longleaf pine seeds were germinated under controlled conditions

in a growth chamber, and the germinating embryos and young seedlings

were used for extraction of sterols, reducing sugars, and chlorophyll.

The effects of inhibitors on the metabolism of err~inatingpine seeds

were also studied. All chemical determinations were made in duplicate,

and the results reported were means having reproducibility within + 5%.


Chemicals

Cholesterol, stigmasterol, 5 a-cholestane, digitonin, trisodium

DL-isocitrate, sodium glyoxylate, Tris-HCI, Tris-Base, and cyclo-

heximide were purchased from Sigma Chemical Company. Campesterol,

6-sitosterol, the U-tube glass column, and the packing material OV-

101 5% coated on Gas-Chrom Q 80-100 mesh were purchased from Applied

Science Lab., Inc. Solvents were reagent grade and were obtained from

Fisher Scientific Co.

Tris-(2-diethylaminoethyl)-phosphate trihydrochloride (SKF 7997-A3)

was obtained from Smith, Kline, and French Laboratories. Abscisic acid

was obtained from the Department of Fruit Crops, University of Florida.

Mercuric acetate was purchased from Fisher Scientific Co. Silica gel

for column chromatography was from Baker Chemical Co. Radioactive

14C-cholesterol, 14C-cholesteryl palmitate, and 3H-cholesterol were

purchased from New England Nuclear Company.


Sources of Seeds

Longleaf pine seeds (Pinus palustris Mill.) were purchased from

20







Resource Operations, Inc., Birmingham, Alabama. The seeds were

collected in 1973 in the region of Escambia County, Alabama. The

percentage of germination was approximately 70%. Seeds were stored in

a cold room at 50C until used.


Preparation and Germination oF Seeds

Seeds were soaked in double distilled water under aerated condi-

tions in a cold room (50C) for 24 hours. They were planted 1.5 cm deep

in moist vermiculite in glass trays and then placed in a growth chamber

at 25 1C and a 12-hour day (fluorescent light 400 ft-c). The seeds

were checked daily and double distilled water added, if necessary, to

ensure adequate moisture. Many seeds started to germinate radiclee

protrusion) on the 5th day of incubation.

Seeds showing radicle protrusion were selected and used for

experiments. These seeds were considered to be in the 1st day of

germination and were placed in moist vermiculite and returned to the

growth chamber. At the end of each predetermined period of growth and

development, the seeds or seedlings were removed from vermiculite,

washed with double distilled water, and only those having approximately

the same length of radicle were used. The germinating embryos were

removed from the megagametophytes, washed with double distilled water,

and used for sterol extraction.


Sterol Extraction and Fractionation

An extraction technique based on the procedures of Stedman and

Rusaniswkyi [215] and Keller, Bush and Grunwald [44, 107, 142] was

used. The germinating pine embryos were homogenized in 5 ml acetone

with an Omni-mixer for 5 minutes at full speed. A small amount of

fine glass beads was added to facilitate homogenization. The result-







ing homogenate was extracted with 200 ml acetone in a Soxhlet apparatus

for 24 hours. The acetone extract was cooled to room temperature and

divided into two equal aliquots. The first aliquot was used for

extraction of total sterols, and the second aliquot was used for frac-

tionation of three different forms of sterols.

For total sterols, the acetone extract was taken to dryness under

vacuum, and 50 ml of 95% ethanol containing 0.15 ml concentrated H2SO4

were added and refluxed for 15 hours to cleave the steryl glycosides.

Fifteen ml of 10% KOH in 95% ethanol (w/v) were then added and the

solution was refluxed for 30 minutes to hydrolyze the steryl esters.

The resulting mixture was cooled to room temperature and was

neutralized to pH 7.0 with H2SO4 ethanol solution. The sterols were

then extracted three times with 30 ml of n-hexane. A small amount of

double distilled water was added in the first extraction to give two

separate layers in the separatory funnel. The n-hexane fractions were

combined and extracted three times with 50 ml of 90% methanol. The

methanol fraction was then back extracted twice with 30 ml of n-

hexane. All the n-hexane fractions were combined and taken to dryness

under vacuum. The resultant residue was dissolved in 20 ml boiling

absolute ethanol and transferred to a centrifuge tube in a 1000C water

bath. Ten ml of hot 2% digitonin in 80% ethanol (w/v) were added to

each tube, and the bath was boiled continuously for a few minutes,

after which 5 ml of hot double distilled water were added, and the

mixture allowed to cool and remain overnight at room temperature.

The tubes were centrifuged at 15,000 g for 30 minutes and the pre-

cipitates were washed three times with 30 ml of 80% ethanol and three

times with 30 ml of diethyl ether. The white digitonide precipitates

were dried overnight at room temperature, and then were hydrolyzed at







700C for 2 hours with 2 ml of pyridine, containing a known amount of

5 a-cholestane as an internal standard. The pyridine mixture was left

at room temperature for 12 hours, and the digitonin was removed by

precipitation with 30 ml of diethyl ether followed by centrifugation

at 10,000 g for 30 minutes. The ether layer was recovered, taken to

dryness under an air stream, and the residue dissolved in ethyl acetate

for injection into a gas-liquid chromatograph.

The second acetone aliquot was also taken to dryness under vacuum;

50 ml of 95% ethanol were added and partitioned three times against

an equal volume of n-hexane. The ethanol fraction contained the steryl

glycosides, and the free sterols and the steryl esters were in the n-

hexane fractions. The method of Goodman [103] was used for separation

of free sterols and steryl esters. A 2 x 10 cm glass column was

packed with 5 g of silica gel (70-325 mesh) and the n-hexane fraction

was applied as a slurry to the column. A serial elution was carried

out as follows: 50 mlof n-hexane followed by 50 ml of 10% benzene in

n-hexane (discarded); 100 ml of 40% benzene in n-hexane (steryl esters);

50 ml of benzene (discarded); and 100 ml of chloroform (free sterols).

The glycosidic, esterified, and free sterol fractions were dried

under vacuum. The glycosidic fraction was refluxed for 15 hours in 50

ml of 95% ethanol containing 0.15 ml of concentrated H2S04, neutralized

with KOH-ethanol, and partitioned three times against n-hexane. The

esterified fraction was hydrolyzed for 30 minutes in 15 ml of 10% KOH

in 95% ethanol (w/v), neutralized with H2SO4-ethanol, and partitioned

three times against n-hexane. The hexane fractions were dried under

vacuum. These fractions were transferred to plastic centrifuge tubes

with boiling absolute ethanol and precipitated with digitonin as

described above to obtain free sterols.







Radioactive 14C-cholesterol was used for determination of the

recover) of the total sterol extraction. H-cholesterol and 1C-

cholesteryl palmitate were added to the silica gel column for correc-

tion of any losses through fractionation. Radioactivity was measured

on a Packard Tri-Carb liquid scintillation spectrometer.

The overall flow chart of procedures used for extraction and

fractionation of longleaf pine sterols was summarized in Fig. 2.


Identification of Sterols

The sterol analysis was performed with a Packard Gas Chromatograph

Series 7300. The GLC system consisted of a dual flow controller,

Model 824, equipped with a flame ionization detector; a '"" column air

oven console, Model 805; a deviation temperature controller, Model 873;

a dual electrometer, Model 843; a dual bipolar high voltage supply,

Model 834; and a recorder, Model 562. The column used was a 6-foot

glass U-tube with a 3.5 mm i.d. packed with Gas-Chrom Q, 80-100 mesh,

coated with 5% dimethyl silicone liquid OV-101 [106, 108]. Helium was

the carrier gas at a flow rate of 100 ml/minute at 30 psi of column

inlet pressure. The flow rates of hydrogen and air were 100 ml/minute

and 350-400 ml/minute, respectively.

The column was operated isothermally at 2500C and the temperatures

of the injector and detector were 2750C. The internal standard 5 a-

cholestane was chromatographed with each sample and the retention times

of sterols were determined relative to cholestane. For quantitative

analysis of individual sterols, peak areas were measured. Total sterol

values were summations of the individual sterol values.

The GLC-mass spectrometry of the different sterol components was

performed with a combination of a double-beem, double-focusing AEI








GFRMINATING PINE. T.RYOS

HOCOGE-E1'TE IN ACETONE WITH CMNI-MIXER
(5 MINUTES)
HOCl iI.NLATE

EXTRACT IN SOXHLET APP. WITH ACETONE
(24 HOURS)

ALIQUOT 1 ALIQUOT 2

DRY UNlV REDII FPSSURE DRY UIJTELR FJ:iLUCI-) PRESSURE
ADD 50 ML 95% ETOH + 0.15 ML H2SO4 ADD 50 ML 95% ETOH
REFLUX FOR 15 HOURS PA',TI1 1Cr AGAINST n-HEXANE


EXTRACT SOLUTION


ADD 15 ML 10% KOH IN 95% ETOH
REFLUX FOR 30 MINUTES
NEUTRALIZE WITH H2SO4-ETOH

TOTAL FREE STEROL HYDROLYSATE

PARTITION AGAINST n-HEXANE

HEXANE FRACTION

DRY UNDER REDUCED PRESSURE
PRECIPITATE WITH DIGITONIN
COOL OVERNIGHT AT ROOM TEMPERATURE

STEROL DIGITONIDE

WASH WITH 80% ETOH AND ETHER
DRY OVERNIGHT AT ROOM TEMPERATURE

DRY STEROL DIGITONIDE

ADD 2 ML PYRIDINE, HEAT AT
700C FOR 2 HOURS
LEAVE OVERNIGHT AT ROOM TE PER4TURE
REMOVE DIGITONIN WITH ETHER

STEROLS IN ETHER

DRY UNDER AIR STREAM
ADD ETHYL ACETATE

GAS LIQUID CHROMATOGRAPHY


ETOH FRA
(STERYL GL


ACTION
YCOSIDES)


HEXANE FRACTION

COLUMN CHROMATTOG]

(5g SILICA

FREE STEROLS STERYL ESTERS


RAPH

GEL)


Flow chart for procedures used in the extraction and fractionation
of longleaf pine sterols.


Figure 2.





26

MS-30 Mass Spectrometer coupled by a silicone membrane separator to a

Pye Gas Chromatograph. The GC was fitted with a 5-foot glass column

with 6.4 mm o.d. packed with Supelcoport 100-120 mesh coated with 3%

methyl silicone SP 2100. The column was operated isothermally at 2700C,

and helium was used as carrier gas at 30 ml/minute. The ion source was

operated at 2200C, and the electron beam was at a potential of 70 eV.

An AEI DS-30 digital computer which was hooked up to the combined GLC-

MS system was employed to acquire and process the data.


Effects of Inhibitors on the Biosynthesis of Sterols,
Reducing Sugars, and Chlorophyll

The following inhibitors were tested on germinating pine seeds for

their effects on biosynthesis of sterols, reducing sugars, and chlo-

rophyll: tris-(2-diethylaminoethyl)-phosphate trihydrochloride (SKF

7997-A3), abscisic acid (ABA), and cycloheximide. The inhibitors

were dissolved in double distilled water. The pH's of the solutions

were immediately adjusted to 7.2-7.4 either with 0.1 N HClor0.1 N

NH40H [41, 178]. The final concentrations of the inhibitors to be

tested were SKF 7997-A3 5 mg/ml; ABA 0.25 mg/ml; cycloheximide 0.25

mg/ml.

The seeds were prepared and germinated as mentioned above. Only

the seeds showing radicle protrusion were selected. The seeds were

immersed in the solutions (200 seeds/100 ml) under aerated conditions

for 30 minutes. The seeds were then removed, blotted, planted in

moist vermiculite and placed in growth chamber under the same condi-

tions as stated above. At the end of 3, 7, and 12 days, the germinat-

ing seeds and seedlings were removed and washed with double distilled

water. The embryos were separated from the megagametophytes, washed

with double distilled water, and used for extraction and determination
of total sterols, chlorophyll content, and reducing sugars.







Sterol Extraction and Identification

The total sterols were extracted and identified by GLC as described

above. The different sterol components were also verified by GLC-MS

to ensure that there was no change in the identity of the different

sterols.


Determination of Reducing Sugars

The samples were dried in an oven at 700C for 48 hours, then ground

to pass a 60-80 mesh screen and stored at 700C in open glass vials

until the plant material came to a constant weight. The vials were

transferred to a desiccator to cool. Approximately 2-g samples were

extracted overnight in a Soxhlet apparatus with 80% ethanol. The

ethanol extract was taken to a syrupy stage under vacuum, and approxi-

mately 50 ml of double distilled water were added. The water solution

was filtered under vacuum in a Buchner funnel using Ii-atrman No. 1

filter paper and a double layer of activated charcoal and cellulose

powder. The clear aqueous solution was brought to 10"' ml with double

distilled water, and the reducing sugars were determined according to

a modified method of Nelson [57, 129, 182]. The transmissions were

read at 500 nm [129] in a Beckman DB spectrophotometer, and the

amounts of reducing sugars in the samples were calculated on a dry

weight basis.


Chlorophyll Determination

Total chlorophyll was extracted and content calculated by a modi-

fied method of Starnes and Hadley [214]. Approximately 5 g of ger-

minating embryos were macerated for 10 minutes at full speed in an Omni-

mixer in 20 ml of 80% aqueous acetone and vacuum filtered through a

Buchner funnel using Whatman No. 1 filter paper. The residue was homo-







genized a second time for 5 minutes and refiltered to ensure that all

chlorophyll had been extracted. The filtrate was brought to 100 ml

and allowed to incubate for 1 hour. A 10-ml aliquot was taken and

centrifuged at 2,500 g for 5 minutes. The absorbance of the superna-

tant was measured at 663 nm and 645 nm with a Beckman DB spectrophoto-

meter. The concentrations of chlorophyll a (Ca) and b (Cb) respec-

tively, were calculated by the formulas

Ca = 12.717 x A2 2.584 x A1 = mgs chl. a/liter

Cb = 22,869 x A1 4.670 x A2 = mgs chl. b/liter
where A1 and A2 were the absorbances at wavelengths 645 nm and 663 nm,

respectively.

These values were then used to determine the total chlorophyll

content on a dry weight basis.


Isocitrate Lyase Assay

Germination of Seeds

Seeds were soaked in a cold room (50C) in double distilled water

under aerated conditions for 36 hours. The seed coats, inner membranes,

and nucellar caps were removed, and the decoated seeds were planted

in moist vermiculite and transferred to a growth chamber for germina-

tion. The seeds were maintained at 25 1C under a 12-hour day (white,

fluorescent tubes, 400 ft-c). Most of the seeds started to germinate

radiclee protrusion) on the second day. The germinating seeds were

removed from the vermiculite and washed with double distilled water.

The germinating embryos were removed and the megagametophytes were

used for enzyme studies.

For certain experiments, the megagametophytes were split length-

wise, and the embrycs were removed and discarded at 0 day and at 2 days.






Extraction and Assay of the FEn:_yv

Isocitrate lyase was extracted according to the method of Ching

[53]. A sample of 10 gametophytes was ground in 10 ml of 0.05 M

Tris-HCl buffer, pH 7.5, containing 10 mM mercaptoethanol and 1 mM

disodium EDTA. The extract was centrifuged at 30,000 g for 10 minutes

and the supernatant treated with charcoal.

The enzyme was assayed in a total volume of 1 ml (0.1 ml of the

extract and 0.9 ml of a mixture of 0.10 M Tris-HC1 buffer pH 7.4,

10 mM cysteine hydrochloride, 10 mM MgCl2, and 20 nm trisodium DL-

isocitrate) at 300C for 5 minutes. Glyoxylate was determined by the

mercuric acetate method of Jacks and Alldridge [134] with a comparable

reaction mixture without substrate for the blank. Protein was ex-

tracted by a modified method of West [232] and was estimated by the

method of Lowry et al. [173].


Steroid Inhibitor Treatment

After removing the seed coats and nucellar caps, the seeds were

dipped in the solution of SKF 7997-A3 (5 mg/ml, pH 7.2-- 7.4) for 30

minutes under aeration. The seeds were then blotted and planted in

moist vermiculite under the germination conditions described above

until harvested for enzyme extraction.


Sterol Treatment

A mixture of sterols (65% B-sitosterol and 35% campesterol) at a

concentration of 5 mg/ml was used. The sterols were dissolved in 0.1%

Tween 80 before adding distilled water [159]. The decoated seeds,

after treatment for 30 minutes with the steroid inhibitor SKF 7997-A3,

were blotted and submersed in the sterol solution under aeration for

another 30 minutes. The seeds were then removed, blotted, placed in




30

moist vermiculite, and transferred to a growth chamber for germination.

The isocitrate lyase was extracted and assayed at the end of each de-

sired period.













RESULTS

Identification of Sterols Isolated from Germinating
Pine Embryos

A typical GLC retention pattern and'characterization of sterols

isolated from 11-day-old longleaf pine seedlings are shown in Fig. 3

and Table 1. The relative retention of the two major peaks present

in the total sterol fraction corresponded to the standards campesterol

(peak 4) and -sitosterol (peak 6), and the two minor peaks to the

standards cholesterol (peak 3) and stigmasterol (peak 5). Peak 2 did

not correspond to any available authentic sterol.

The mass spectrum data are presented in Tables 2, 3, and 4. The

GLC peaks 4, 5, and 6 which corresponded respectively to campesterol,

stigmasterol, and B-sitosterol had the m/e values specific for these

sterols. These m/e values also corresponded to the ions obtained with

the authentic standard sterols (Tables 2, 3, and 4) and with those

published by Knights in 1967 [153].

The GLC peak 2 did not have any significantly high m/e values from

227 to 428. Most of the m/e ions in this region came from background

values having intensities smaller than 1%. The most intense peaks were

at m/e 225, 120, 106, 91, 77, and 57, having relative intensities 26%,

100%, 21%, 13%, 22%, and 13%, respectively. Based on these observa-

tions, it was concluded that the GLC peak 2 was not a sterol but prob-

ably was an unknown long chain hydrocarbon.

The GLC peak 3, which corresponded to the relative retention of

the standard cholesterol, also did not show any high relative intensity

31












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z 0














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Table 1. Relative retention, with respect to 5a-cholestane,
of known sterols and those from 11-day-old long-
leaf pine seedlings,


Component Relative retention (r)1

Longleaf pine peak 2 1.44

Cholesterol 1.88
Longleaf pine peak 3 1.87

Campesterol 2.46
Longleaf pine peak 4 2.44

Stigmasterol 2.71
Longleaf pine peak 5 2.71

B-sitosterol 3.16
Longleaf pine peak 6 3.15

Column characteristics: 5% OV-101 on Gas-Chrom Q (80-100
mesh), column temperature 2500C, carrier gas helium at a
flow rate of 100 ml/minute; average retention time of 5a-
cholestane was 13.6 minutes.














Table 2. Mass spectrum data of campesterol of germinating long-
leaf pine embryos.


CAMPESTEROL
Expected Relative Intensity
Fragmentation m/e Standard Pine Sterol

Molecular ion [M+] 400 95 93
M 15 385 57 54
M 18 (HOH) 382 95 96
M [15 + 18] 367 64 65
M [18 + 67(C5H7)] 315 58 58
M [18 + 93(C7H9)] 289 93 98
M [18 + 121(C9H13)] 261 45 41
M [18 + 108(C8H12)] 274 31 18
M side chain 273 42 42
M [side chain + 18] 255 83 70
M [side chain + 42] 231 52 51
M [side chain + 18 + 42] 213 100 100
M [side chain + 27] 246 18 13
M [side chain + 27 + 17] 229 51 44















Table 3. Mass spectrum data of stigmasterol of germinating long-
leaf pine embryos.


STIGMASTEROL
Expected Relative Intensity
Fragmentation m/e Standard Pine Sterol

Molecular ion [M+] 412 53 48
M- 15 397 11 6
M 18 394 26 29
M [15 + 18] 379 12 10
M [18 + 67] 327 6 12
M [18 + 93] 301 18 15
M [18 + 121] 273 23 25
M [18 + 108] 286 5 12
M side chain 273 23 25
M [side chain + 18] 255 100 100
M [side chain + 42] 231 18 27
M [side chain + 42 + 18] 213 49 55
M [side chain + 27] 246 5 8
M [side chain + 27 + 17] 229 24 28














Table 4. Mass spectrum data of 6-sitosterol of germinating long-
leaf pine embryos.


8-Sitosterol
Expected Relative Intensity
Fragmentation m/e Standard Pine Sterol

Molecular ion [M+] 414 97 97
M 15 399 55 54
M 18 396 93 90
M [15 + 18] 381 59 62
M [18 + 67] 329 54 52
M [18 + 93] 303 93 92
M [18 + 121] 275 39 36
M [18 + 108] 288 15 13
M side chain 273 42 42
M [side chain + 42] 231 54 53
M [side chain + 18] 255 79 76
M [side chain + 42 + 18] 213 100 100
M [side chain + 27] 246 21 16
M [side chain + 27 + 171 229 47 50







above the 299 m/e value. All the values from 299 to 420 were smaller

than 1% of relative intensity and were suspected to arise from the

background. The most intense peaks were at m/e 297 (8% of relative

intensity), 157 (21%), 111 (19%), 97 (55-), 85 (26%), 83 (89%), 71 (49%),

69 (64%), 57 (100%), and 55 (63%). Because the mass spectrum data did

not show any m/e value specific for cholesterol [153], it was concluded

that the GLC peak 3 was not cholesterol but was probably an unknown

long chain hydrocarbon.

From the results obtained by GLC and verified by combination of

GLC-MS, it was therefore concluded that germinating longleaf pine

embryos contained two major sterols, campesterol and 6-sitosterol, and

one minor sterol, stigmasterol. The esterified, glycosidic, and free

sterol fractions contained the three above mentioned sterols. Some

total and esterified sterol fractions also contained one additional

minor peak having a relative retention value of 4.03.


Changes in Sterol Content upon Germination

All the sterol values reported here were the corrected values

based on the recovery of radioactive materials through the extraction

and fractionation procedures. The average recovery of total sterols

was 82%. The recoveries of steryl esters and free sterols through

silica gel were 91% and 90%, respectively.

On a dry weight basis, the total sterol content increased during

seedling development (Fig. 4). By fractionation, three different

forms of sterols were found in longleaf pine seedlings: steryl esters,

steryl glycosides, and free sterols (Figs. 5, 6, and 7). The total

sterol content was always higher than the summation of free, esteri-

fied, and glycosidic sterols. These total sterol values not only



















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9 c o
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0 w I ;--
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4(M Aip b,'bw S\ O vwS














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included the three forms mentioned above but also other sterol con-

jugates, such as themono- and disulfates [47]. Fractionation also

revealed that the sterols were found mainly in the esterified fraction

which increased in amount during germination in nuch the same pattern

as the total sterol (Figs. 4 and 5). Steryl glycosides and free sterols

only made up a small portion of the total sterol content throughout

the germination period (Figs. 6 and 7).

In general, in the total and esterified sterol fraction, the

campesterol content increased with seedling development while the f-

sitosterol level remained approximately the same. The stigmasterol

content was very low and remained fairly constant throughout the whole

period of germination (Figs. 4 and 5). The data in Figs. 6 and 7

represented, respectively, the total glycosidic and total free sterol

content which occurred in very small amounts.


Effects of SKF 7997-A3, ABA, and Cycloheximide on
Total Sterol Biosynthesis

The germinating pine seeds were treated with SKF 7997-A3, ABA,

and cycloheximide. In general, all the three inhibitors depressed

the biosynthesis of total sterol (Fig. 8). At 3 days, ABA slightly

increased the total sterol content over the control. At 7 and 12 days,

ABA depressed total sterol biosynthesis to 77% and 79% of the control,

respectively.

SKF 7997-A3 had an effect on total sterol content during the whole

testing period. The sterol level was reduced to 50% of the control

at 3 days, 30% at 7 days, and 65% at 12 days.

Among the three inhibitors, cycloheximide had the greatest effect

on sterol biosynthesis. After cycloheximide treatment, the total




















[ CONTROL
E ABA (0.25mg/ml)
E3 SKF 7997-A3(5mg/ml)
H CYCLOHEXIMIDE (0.25mg/ml)


3 7 12


TIME (days)


Effects of ABA, SKF 7997-A3 and cycloheximide on
total sterol content during germination of long-
leaf pine seeds.


3.00



2.50



2.00



1.50



1.00



.50


Figure 8.





















CONTROL
ABA (0.25mg/ml)

SKF 7997-A3(5mg/ml)
CYCLOHEXIMIDE (0.25 mg/ml )


3 7 12


TIME (days)


Effects of ABA, SKF 7997-A3 and cycloheximide on
the campesterol level during germination of long-
leaf pine seeds.


3.00



2.50



2.00



1.50



1.00



.50


Figure 9.





















CONTROL
ABA (0.25mg/ml)
SKF 7997-A3(5mg/ml)
CYCLOHEXIMIDE (0.25 mg/ml)


3 7 12


TIME (days)


Figure 10.


Effects of ABA, SKF 7997-A3 and cycloheximide on
the B-sitosterol level during germination of
longleaf pine seeds.


3.00



2.50



2.00



1.50



1.00



.50





46

sterol level was only 14% of the control after 3 days and 21% after 7

days. The germinating seeds treated with cycloheximide started to

degenerate and rot after 9 to 10 days and were discarded.

ABA treatment increased the level of campesterol after 3 days but

later inhibited and after 12 days, the campesterol level was only 60%

of the control. ABA treatment, on the other hand, depressed B-

sitosterol synthesis during the first 3 days, but increased it over the

control at 7 days and 12 days (Figs. 9 and 10).

SKF 7997-A3 caused a reduction of campesterol and B-sitosterol

levels during the whole testing period. The greatest effect of SKF on

campesterol was found at the 7-day stage when the campesterol level

was only 24% of the control. After SKF treatment, the S-sitosterol

level was only 38% of the control after 3 days and 58% of the control

after 12 days.

Cycloheximide reduced the levels of both campesterol and 8-

sitosterol. The most pronounced effect was on B-sitosterol at the 3-

day stage, with only 5% of the control, and on campesterol at the 7-

day stage, with only 6% of the control.


Effects of the three Inhibitors on the Level of
Reducing Sugars

All three inhibitors sharply reduced the level of reducing sugars

throughout the test period (Fig. 11). The degree of inhibition at

the 3-day stage was 24% (ABA), 36% (SKF 7997-A3), and 47% (cyclohexi-

mide). At the 7-day stage, the inhibitions were 54% (ABA), 69% (SKF),

and 87% (cycloheximide). After 12 days, the inhibitions were 80% (ABA)

and 83% (SKF).























e-- CONTROL
o----o ABA (0.25mg/ml )
S---A SKF 7997-A3(5mg/ml)

-- CYCLOHEXIMIDE
(0.25mg/ml)


N5,

"NC


0 3 7 12
0 3 7 Il


TIME (days)


Figure 11.


Effects of ABA, SKF 7997-A3 and cycloheximide
on the content of reducing sugars during
germination of longleaf pine seeds.








Effects of the three Inhibitors on Total Chlorophyll
Biosynthesis

The effect of ABA on chlorophyll biosynthesis was less than that

of SKF 7997-A3 and cycloheximide (Fig. 12). Both ABA and SKF depressed

the total chlorophyll content to the same level of 94% of the control

on the 3rd day, but at day 12, the SKF-treated seedlings contained

only 76% as much chlorophyll as the control, whereas ABA-treated

seedlings contained 3~9 as much as the control.

Cycloheximide-treated seedlings contained approximately 48% as

much chlorophyll as the control both at 3-day and 7-day stage.


Effects of SKF 7997-A3 on the Activity of Isocitrate
Lyase

The participation of one of the key enzymes of the glyoxylate

cycle, isocitrate lyase, and its relation to germination of longleaf

pine seeds is presented in Fig. 13. When imbibed seeds were initially

removed from the seed coats, no activity of the enzyme was detected

in megagametophytes. The specific activity, however, increased

greatly by day 2 when protrusion of the radicle occurred, reached the

maximal level at day 8, remained unchanged for two additional days,

and then greatly declined by day 12. When the germinating embryos

were removed from the gametophytes on the 2nd day, the specific acti-

vity of the enzyme increased to the same level as the control at the

4th day, then the activity declined. But when the embryos were

removed from the gametophytic tissues immediately after imbibition at

0 day, isocitrate lyase was almost nil at the 2-day period, and then

increased and reached a maximal level at day 4 which was only 34% of

the control.

When the decoated seeds were treated at 0 day with the steroid































0-@-


0 3 7 I1


TIME (days)


Figure 12.


Effects of ABA, SKF 7997-A3 and cycloheximide
on the chlorophyll content during germination
of longleaf pine seeds.


4.0 r


3.0 1


2.0







1.0


CONTROL
ABA (0.25 mg /ml)
SKF 7997-A3(5mg/ml)


o---- CYCLOHEXIMIDE
(0.25mg /mi )


p--













e--- CONTROL
o---o SKF 7997-A3(5mg/ml)
a---a EMBRYOS REMOVED
AT 2-DAY
o--- EMBRYOS REMOVED
AT 0 -DAY
x----x SKF 7997-A3(5mg/ml)
---STEROLS (5mg/ml)


2 4 6 8


10 12


TIME (days)


Figure 13.


Changes of specific activity of isocitrate lyase
in megagametophytes of germinating longleaf pine
seeds.


120


100





80





60





40





20





51

inhibitor SKF 7997-A3, the activity of the enzyme at day 2 was only

about 50% of the control, but at day 4 was nearly the same as the con-

trol; the activity then declined slowly thereafter. After treatment

of the SKF-treated seeds with a suspension of campesterol and B-

sitosterol, the pattern of development of the enzymatic activity was

approximately the same as for the SKF-treated seeds. No recovery of

the activity of the enzyme was observed.













DISCUSSION

With the use of gas chromatography (GLC) and a combination of gas

chromatography-mass spectrometry (GLC-MS), three sterols were isolated

and identified in germinating longleaf pine embryos. Campesterol and

B-sitosterol were the two major sterols, accounting for most of the

sterol fraction, while stigmasterol only made up a very small portion

of the total sterols. No trace of cholesterol was detected by GLC-MS,

although there was one peak corresponding fairly closely to the reten-

tion time of authentic standard cholesterol.

By fractionation, three different forms of sterols, namely,

steryl esters, steryl glycosides, and free sterols, were demonstrated

to make up the total sterol content in germinating longleaf pine

embryos. Steryl esters were the main sterol form, while steryl glyco-

sides and free sterols were only minor fractions.

The total sterol content increased with time of germination, from

1.0 mg/g dry weight at 3 days to 3.1 mg/g dry weight at 20 days.

The pattern of change in steryl esters was parallel to that in total

sterols and accounted for most of the increase in total sterol content,

while free sterols and steryl glycosides were low and remained fairly

unchanged throughout the experimental period.

Changes in sterol content with time of germination have been

studied in many different species of higher plants, and plant sterols

were reported to occur mainly as free, esterified, and glycosidic

sterols [44, 69, 111, 130, 145]o Sterol analyses during germination

and seedling development in most cases showed that the total sterol

52








content increased [44, 68, 70, 71, 72, 136]. These increases in

sterol content suggest that sterols by themselves could play some role

during growth and development of longleaf pine seedlings and in higher

plants, in general. The definite roles of sterols and of different

sterol forms in plants have not been elucidated, and many suggestions

of function have been made in relation to the presence of different

sterol forms in plants [44, 111, 130, 145]. Correlation analyses of

different sterol forms by fractionation have been made as related to

development and differentiation processes in order to find some clues

of their physiological significance in plants. Experimental data

obtained by different workers for different species are somewhat con-

flicting and make it difficult to answer some fundamental questions

such as intracellular distribution of sterol forms, their function and

their evolution in developing tissues [130, 145, 223].

In many reported cases, free sterols accounted for a large por-

tion of the increase in total sterol content during germination

[44, 73, 145], leading to the suggestion that free sterols are inte-

gral parts of plant cell membranes and that an increase in free sterol

content during seedling development is a consequence of increased orga-

nelle genesis and new membrane synthesis [44, 111]. This hypothesis

was mainly based on the observation by Grunwald that cholesterol in

its free form was most effective in influencing the permeability of

beet root and barley root cells as measured by the rate of leakage of

a-cyanin and electrolytes, respectively [105, 109]. Stigmasterol,

B-sitosterol, cholesterol esters and cholesterol glycosides were virtu-

ally ineffective in modifying leakages. Also, cholesterol was much

more readily incorporated into erythrocyte membranes than the other

plant sterols, due to its flat molecular configuration and to its side

chain flexibility [77].







Cholesterol is a minor sterol in many plant species and is some-

times found only in traces [47, 62, 63, 69, 154, 181]. The most pro-

minent higher plant sterols are 3-sitosterol, stigmasterol, and camp-

esterol [44, 47, 62, 63, 133]. The presence of cholesterol in higher

plants was first reported in 1963 by Johnson et al. [1381 who used

thin-layer and gas-liquid chromatography. Since then, cholesterol was

shown to occur in a number of plant species [44, 47, 62, 63, 69, 181].

The gas-liquid chromatograms of sterol fractions, in most cases, showed

a minor peak appearing before that of campesterol and having a reten-

tion time corresponding closely to authentic standard cholesterol.

This minor component, supposed to be cholesterol, accounted for

less than 1%, or a trace, of the total sterol composition [47, 62,

63, 69]. Attempts to trap this minor GLC fraction for further analysis

with mass spectrometry were not successful because the fraction was

either absent or below the level of detection by mass analysis [47].

Thus, the reports on identity of cholesterol in tissues of Hordeum vul-

gare, Triticum aestivum, and Zea mays were tentative, based on the

retention time of a component on the gas-liquid chromatograms [47, 62,

631. Investigations on cholesterol in germinating seeds of Arachis

hypogeaPhaseolus vulgaris, Pisum sativum, and Secale cereale also

failed to demonstrate the presence of cholesterol [69].

Longleaf pine seedlings contain very little, if any, cholesterol,

with the major sterols being 3-sitosterol and campesterol, in addi-

tion to a small portion of stigmasterol. In conifers, B-sitosterol

was identified from extracts of pollen from Pinus sylvestris and Pinus

mugo [21], and both B-sitosterol and campesterol are present in the

seeds of P. pinea [226] and P. elliottii [166] and in the barks of P.

banksiana, P. contorta, P. lanbertiana and P. taeda [203]. The major








sterol in pine stems was B-sitosterol, which accounted for 60-70% of

the sterol composition and occurred as the free form, as esters of

various fatty and aromatic acids, and as glycosides. No trace of

stigmasterol was detected, and the gas chromatograms of samples from

P. taeda and P. banksiana showed the presence of a very minor peak

with the retention time of cholesterol [203]. A minor component was

also identified in seeds and seedlings of P. elliottii [166].

The experiment on incorporation of cholesterol was not conducted

using plant cell membranes, but using erythrocyte membrane in which

cholesterol alone comprises approximately 25% of the total lipid mem-

brane composition [1621. It is therefore not hard to understand why

cholesterol is readily absorbed into erythrocyte membrane and not

sitosterol, campesterol or stigmasterol which are completely absent in

this animal membrane.

Young seedlings contain not only free sterols but also steryl

esters and steryl glycosides [111]. Esterified sterols accounted for

83% of the total sterol content in tobacco leaves [107]. Cellular

fractionation studies using these tobacco leaves also demonstrated

that the steryl ester content was highest in the 20,000-46,000 g

pellet, suggesting that most of the sterols in the tobacco leaves were

associated with membrane-containing organelles and occurred in the

esterified forms. In scutellum of maize, a rapid increase in steryl

esters during germination was observed [145]. Also in Phaseolus

vulgaris and Raphanus sativus, steryl ester content on a dry weight

basis increased, although to a lesser extent than free sterols [69].

Bush [44], working on sterol changes during germination of tobacco

seeds, found that the total sterols increased. This increase parallel-

ed the increase in free sterols. The steryl esters also increased,







but to a lesser extent, while steryl glycoside content was low and

decreased with time of germination. During gemination of barley

seeds, a marked increase in free sterols, esterified sterols, and

esterified steryl glycosides was observed [130]. It was proposed that

esterified steryl glycosides, with both polar and nonpolar regions of

the molecule, were ideally suited as membrane constituents [130]. In

the present study, the longleaf pine sterols were found mainly in the

esterified forms which increased during germination in a way similar

to the total sterol content. Similar patterns of increase in sterol

content in different plant species were also reported [69, 70, 71, 72,

136]. Based on these observations, it could be that sterols may have

a structural role, possibly as an integral part of the lipid layer of

plant cell membranes [107, 111].

The evolution and localization of sterols in the needles of Pinus

maritima was studied by David et al. in 1962 [61]. A gradual accumu-

lation of sterols and fatty acids as lipid droplets during development

of pine needles was registered during their first two years. There

was a great increase in total sterol content from 0.3 mg/g fresh

weight at the beginning of needle formation in the first year to 2.16

mg/g fresh weight at the end of the second year. This increase in

sterol content paralleled an increase in fatty acids, showing the

possibility that sterols may accumulate in droplets either in free or

esterified forms [61]. In animal systems, cholesterol esters were

reported to constitute a large portion of the intracellular lipid

droplets and serve as intermediates in the biosynthesis of steroids

in the adrenal cortex of rats [24, 25, 60, 170, 179]. By analogy, it

was also proposed in tobacco seedlings that stigmasterol and choles-

terol were synthesized as, or rapidly converted to, esterified sterols








and then became associated with the lipid droplets of the cell where

they may serve as a reserve pool for further steroidogenesis [45]. In

germinating barley embryos, the free sterol content was rather high;

however, the most striking observation was the increase in steryl

esters within 5 days of germination [130]. The function of steryl

esters in barley embryos was suggested to be involved in the transport

of sterols and fatty acids released from triglycerides to the growing

areas of the seedling for further synthesis and for membrane formation

[130]. In plants, no evidence is available on the possible transport

of sterols and fatty acids either in separate or combined forms from

one site to another (e.g. from the reserve tissues to the germinating

embryos). There is a report on intracellular sterol transport prob-

ably from their site of synthesis to other organelles [145]. In long-

leaf pine seeds, there are some interesting relationshipsbetween the

high level of fats in megagametophytic tissues and large amounts of

steryl esters present during the germination period. All of these

facts, including the observation of lipid droplets in Pinus maritima

needles, may provide a partial answer as to the possible function of

steryl esters in growing pine tissues, in particular, and in higher

plants, in general.

In addition to the possible structural role of sterols in plant

cell membranes, the almost universal occurrence of these components

in pines and other plants suggests that they might have some other

definite functions. As yet, the nature of these possible functions

remains quite obscure. Sterols are present in mitochondria of higher

plants [74, 152], but no information is available on their possible

function, other than structural, in these organelles. However, in







yeast, sterols are required for mitochondrial function [117]. Inhibi-
tion of sterol synthesis in yeast reduces respiratory competency which

can be restored by addition of ergosterol [189].

Other effects of sterols on the metabolism of germinating seeds

have been suggested: Sterols may operate at the level of transport of

essential materials, such as a possible association of sterols with

the transport of saccharides across the membrane [154]. Sterols may

have an effect on the development of chloroplasts and photosynthesis

[154]. Sterols may interact with proteins, such as enzymes, to con-

trol their functions [154].

Attempts to find further evidence of such effects in germinating

longleaf pine seeds were made by using the animal steroid inhibitor

SKF 7997-A3, abscisic acid (ABA) and cycloheximide to determine their

effects on the synthesis of sterols, chlorophyll content, and tissue

concentrations of reducing sugars.

The average total chlorophyll content in green leaves of Pinus

spp. is approximately 2.6 mg/g dry weight [163]. During germination

of Pinus banksiana seeds, an increase in total chlorophyll content

with time of germination was reported; at 9 days, the chlorophyll con-

tent was 0.9 vg/embryo [76].

Changes in reducing sugars in longleaf pine embryos occurred in a

manner similar to that reported in Pinus luchuensis, Pinus taiwanensis

and Pinus morrisonicola [217] and in the hardwood Acacia confusa [140]

seeds during germination. In these three pine species, the level of

reducing sugars increased, reached the maximum at 8-10 days, and then

decreased thereafter [217].

SKF 7997-A3 was reported to inhibit growth in peas [178] and to

suppress floral induction in two short-day plants Xanthium pensylvanicum







and Pharbitis nil [41], and in a long-day plant, Lolium temulentum

[89]. In vitro, this animal steroid inhibitor inhibits the conversion

of lanosterol to cholesterol, but in Xanthium leaves, although two

principal compounds whose production was inhibited by SKF 7997-A3 were

identified as g-sitosterol and stigmasterol, the block is probably at

more than one site and remains to be elucidated [41]. As has been

shown experimentally, the biosynthesis of B-sitosterol and campesterol

was depressed significantly in SKF 7997-treated pine seedlings. A

change in sterol content in the membrane could strongly affect the

transport of metabolites across the membrane. The results obtained

showed that SKF 7997-A3 sharply depressed the level of reducing sugars

and significantly reduced the total chlorophyll content.

Abscisic acid CABA) also inhibited the sterol content in long-

leaf pine, but to a lesser degree than SKF 7997-A3 and cycloheximide.

Campesterol was decreased with a slight increase in 8-sitosterol. The

ability of ABA to induce dormancy in seedlings of a number of species

has been reported [93, 111]. In longleaf pine seedlings, decreased

levels of campesterol and increased levels of B-sitosterol with ABA

treatment had similar patterns at the 3-day and 5-day germination

period. No information is available on the possible relationship of

the individual sterols such as B-sitosterol to seed dormancy; however,

-sitosterol in dry mature Pisum sativum seeds was reported to be the

predominant sterol in all fractions [177] and was present to the extent

of 0.67 mg/g of dried seed [12, 13]. In potato tubers (Solanum

tuberosum) during storage, B-sitosterol was the predominant sterol

[75]. The relatively high level of 8-sitosterol in these situations

may indicate the initiation of dormancy by ABA.







Abscisic acid (ABA) also depressed the synthesis of reducing

sugars and chlorophyll in longleaf pine seedlings. Its effect was

comparatively less than that caused by SKF 7997-A3. The effects of

ABA on the synthesis of sterols and chlorophyll were reported in corn

shoots [176]. By measuring the incorporation of labeled mevalonic acid

into the sterol fraction of ABA-treated and control corn shoots over a

finite experimental period, it was shown that ABA had no inhibitory

effect on sterol biosynthesis in corn. After incubating etiolated corn

shoots in the light with and without ABA and subsequently comparing

the levels of chlorophyll in the tissues, it was found that ABA de-

pressed the synthesis of chlorophyll by 42% [176]. Also ABA greatly

decreased the chlorophyll level of excised embryos of Fraxinus ornus

[213]. In isolated leaf discs, ABA accelerated the loss of chlorophyll

in all species examined [7, 83, 209].

ABA reduced the level of reducing sugars nearly as much as SKF

7997-A3 did. The inhibition of ABA on a-amylase and ribonuclease was

reported in barley aleurone layers [54, 56]. In the case of longleaf

pine, ABA may inhibit the synthesis of certain specific en:)-mes

required for formation of reducing sugars. ABA may exert its action

in a manner similar to that in barley grains by inhibiting the syn-

thesis of the enzyme-specific RNA molecules that are required for the

expression of the gibberellic acid effect, or by preventing the in-

corporation of these RNA molecules into an active enzyme-synthesizing

unit [56, 93].

The antibiotic cycloheximide is of considerable interest since it

is a potent inhibitor of protein synthesis at the translational level

[168]. In cycloheximide-treated longleaf pine seedlings, a strong

suppression of sterols, reducing sugars and chlorophyll was observed.








Cycloheximide was reported in isolated barley aleurone layers to inhi-

bit the production of a-amylase and ribonuclease to a greater extent

than did ABA [55, 56]. ABA inhibited the synthesis of a-amylase after

a lag of 2 to 3 hours while the addition of cycloheximide (10 pg/ml)

to barley aleurone layers resulted in an immediate cessation of a-

amylase synthesis and also the incorporation of 1C-leucine into the

cellular proteins of aleurone layers [56].

The suppression of the level of reducing sugars by SKF 7997-A3

suggests that sterols may have an influence on some particular enzyme

in steps transforming fatty acids to sugars. Pine seeds contain an

embryo deeply embedded in gametophytic tissues which serve as a lipid

reservoir supporting the early growth of the young seedling [53].

However, lipids are not transported directly from megagametophyte to

the embryo and must be transformed into sugars via the glyoxylate

cycle [53]. Isocitrate lyase, a key enzyme of the glyoxylate cycle,

which is almost nil in ungerminated seeds, increases in activity many-

fold during germination, accompanied by a decrease in lipid content of

the gametophyte. Once lipids are exhausted, the enzyme activity is

markedly reduced [37, 53, 91, 92].

The participation of isocitrate lyase in converting lipolytic

products of acetyl-CoA to carbohydrate in germinating megagametophytes

of longleaf pine seeds was assayed. No isocitrate lyase activity was

found in the unge-rTdnted seeds. During the first few days of germi-

nation, the activity of the enzyme increased dramatically with a peak

at 8 days, approximately the time lipid breakdown and carbohydrate

synthesis was most rapid. Later on, as the lipid content declined in

the later stages of germination, so did the activity of the enzyme.

Similar patterns of enzymatic activity development was observed in







the germinating seeds of Pinus ponderosa [37, 53], Pinus pinea [92],

and in other fatty germinating seeds such as Citrullus vulgaris [128]

and Cucurbita pepo [50].

When longleaf pine seeds were treated with SKF 7997-A3, there

was a sharp suppression of enzyme activity after the fourth day of

germination. It is suggested therefore that sterols may have an

effect on the regulation and developmental activity of this enzyme.

No information is available on the possible interaction of sterols and

enzymes occurring in plant systems. It is reported that there is the

formation of a complex between the enzyme trypsin and ergosterol,

yielding a more stable and more reactive enzyme towards c.: albumin

than the uncorplexed enzyme [188]. The lack of recovery of enzyme

activity after treatment with sterols may be due to the insolubility

of sterols, the probability of SKF 7997 to be a noncompetitive inhi-

bitor, or to produce irreversible inhibition of the enzyme through

chemical modification of its structure, as the action of diisopropyl-

phosphofluoridate (DFP) on acetylcholineesterase and other enzymes

possessing an essential reactive serine residue at their active sites

[168], or the possibility of SKF 7997-A3 changing the expression of

genetic information stored in the chromosomes that cannot be restored

by addition of sterols.

Further attempts were made by removal of the embryos from the

megagametophytes at 0-day and at 2-day and following the change in

isocitrate lyase activity as related to the embryonic induction fac-

tors. This experiment was based fundamentally on the observations by

Bilderback in Pnus ponderosa [37] and by Young and Varner in Pisum

sativum [229, 238]. In ponderosa pine, the isocitrate activity con-

tinued to develop normally when the embryo was removed two days, but








not immediately, after stratification [37]. The same result was obtain-

ed with phosphatase in peas [229,238]. It has been suggested that

events occurring in the reserve tissues are under embryonic control,

and that the embryo could play an active regulatory role by producing

same substance resulting in enhanced enzyme production in pine megagame-

tophytes [37]. In ponderosa pine seeds,, this unknown embryonic factor

cannot be replaced by gibberellic acid (GA3), indoleacetic acid (IAA),

and benzyladenine (BA); however, when isolated gametophytes were

incubated with the embryo diffusate, a substantial increase in activity

of isocitrate lyase nearly to the control was observed [37]. Observa-

tions of limited increase of enzyme activity in longleaf pine gameto-

phytic tissues when embryos were removed 2 days after germination did

not permit further work on the theoretical embryo substance, supposed

to be a sterol. Also the insolubility of sterols in aqueous solution

did not allow testing of the possible interaction of sterols with the

enzyme isocitrate lyase.














SUMMARY

Very little work has been done on sterols in germinating coniferous

seeds. The object of this study was to follow the occurrence and

variations of different forms of sterols and to investigate their

possible physiological functions during germination of longleaf pine

seeds (Pinus palustris Mill.).

Pine seeds were germinated in a growth chamber under controlled

conditions, and the germinating embryos or young seedlings were used for

extraction and fractionation of sterols and their derivatives. By

using gas-liquid chromatography (GLC) and combination of GLC-mass

spectrometry (GLC-MS) for sterol identification and analysis, three

different components, campesterol, stigmasterol and B-sitosterol, were

detected in the pine sterol composition. Campesterol and B-sitosterol

were two major sterols while stigmasterol was only present in small

amounts throughout the whole experimental period. Fractionation

revealed three different forms of sterols in pine tissues: steryl esters,

steryl glycosides, and free sterols. Steryl esters were the main sterol

fraction, while free sterols and steryl glycosides were only minor ones.

All the three sterol components were present in these fractions.

The total sterol content increased with time of seedling develop-

ment, from 1.0 mg/g dry weight at 3 days to 3.1 mg/g dry weight at 20

days. The changes in steryl esters paralleled total sterols, while

steryl glycosides and free sterols were low and remained fairly constant

throughout the germination period. The campesterol content increased
with seedling development in both the total sterol content and the

64








steryl ester fraction, while the levels of B-sitosterol and stigmasterol

were nearly the same.

Cycloheximide, abscisic acid (ABA) and SKF 7997-A3 significantly

inhibited the biosynthesis of sterols, reducing sugars, and total

chlorophyll in longleaf germinating pine seeds. SKF 7997 and cyclo-

heximide reduced the levels of both B-sitosterol and campesterol, while

the application of ABA caused a decrease in campesterol and a slight

increase in B-sitosterol. A depression of reducing sugars and chloro-

phyll by SKF 7997 could indicate that there is a possible operation of

sterols at the transport level of essential materials, such as

saccharides across membranes, and that the formation and development of

chloroplasts may be under the partial control of sterols.

SKF 7997 also sharply reduced the activity of the enzyme iso-

citrate lyase. The application of campesterol and B-sitosterol to the

SKF 7997-treated seeds did not reverse the inhibition caused by the

animal steroid inhibitor. The depression of isocitrate lyase activity

by SKF 7997 indicates that this steroid inhibitor has an influence

on a catalytic protein that may influence germination, but it will

require further experimentation to determine if sterols have a direct

regulatory role in pine seed germination.













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BIOCIRAPHICAL SKETCH

Cu Van Vu was born June 29, 1942 in Lam Dong, South Vietnam. In

1961 he graduated from Dalat High School and registered as a pre-med

student at the University of Saigon. He earned the certificate of

Physics, Chemistry and Biology in 1962 and was admitted to the School

of Medicine, University of Saigon. Because of health conditions, he

dropped out of the Medical School a short period after and was admitted

to the College of Agriculture in Saigon in November 1962. He continued

his studies at the same time at the College of Basic Sciences, University

of Saigon, and earned the certificate of Physics, Chemistry and Natural

Sciences in 1963. In November 1965, he received the degree of Engineer

in Forest Sciences from the College of Agriculture, Saigon and joined

the staff of this College as an instructor assistant from 1966 to 1970.

In August 1970 he was awarded a fellowship for going to the U.S. for

higher education. From September 1970 to present, doctoral work has

been pursued at the University of Florida in forest tree physiology.

Cu Van Vu is a member of XI STICl PI and of the Society of

American Foresters. He is married to the former Tieng Tran and has a

two-ycar-old son, Thien.







I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



ay Goddard, Chairman
Ass ciate Professor, Forestry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy,

/0 i ..,
WMM-r l-r^^-- t
Professor, Fruit C ops
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

+- (? .. -

Yfeon A. Garrard
Associate in Plant Physiology

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.


"l + i'+ c ++ f-, -/7 ^"-
Thomas E. Humphreys
Professor, Botany

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.


Professor, Forestry






I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




KRoert M. Roberts
Associate Professor, Biochemistry

This dissertation was submitted to the Graduate Faculty of the College of
Agriculture and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy,

March, 1976


Dea', /College of Agr&tIlture


Dean, Graduate School

































UNIVERSITY OF FLORIDA
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3 1262 08554 1117