Title: Biology of diffusible pollen wall compounds
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Title: Biology of diffusible pollen wall compounds
Physical Description: ix, 74 leaves : ill. ; 28 cm.
Language: English
Creator: Kirby, Edward George, 1947-
Publication Date: 1977
Copyright Date: 1977
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Subject: Pollen   ( lcsh )
Botany thesis Ph. D
Dissertations, Academic -- Botany -- UF
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non-fiction   ( marcgt )
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Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 68-73.
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Edward George Kirby, III.
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BIOLOGY OF DIFFUSIBLE POLLEN WALL COMPOUNDS


By

Edward George Kirby, III






















A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY














UNIVERSITY OF FLORIDA


1977





































To Mindy















ACKNOWLEDGEMENTS


I must express my sincere appreciation to Dr. Indra Vasil

(committee chairman) for his guidance and meaningful discussions during

the course of my studies. Also, I wish to express thanks to Dr. Willard

W. Payne, committee cochairman; Dr. Henry C. Aldrich; Dr. Ray E.

Goddard;and Dr. Rex L. Smith for their advisory help and for reviewing

the manuscript.

Sincere appreciation is extended to the group in Nijmegen including

Professor H.F. Linskens for support during my studies, stimulating

discussions,and for supplying samples of Petunia pollen. In addition,

thanks are given to Dr. M. Kroh for her help with initiating the study

of carbohydrates of Petunia pollen eluents and to Dr. M.M.A. Sassen

for support and for interesting discussions of pollen morphology and

physiology.

I would like to extend my most sincere thanks to Dr. C.A. Hollis

of the School of Forest Resources and Conservation for his many sug-

gestions and to Sarah G. Mesa and Joel E. Smith of the Forest Physiology

Laboratory for technical assistance. In addition, I gratefully

acknowledge Dr. John Gray and the School of Forest Resources and

Conservation and the Division of Sponsored Research for support during

the course of these studies. Thanks are also due to Dr. Dick Loeppert

of the Department of Soil Science for help with liquid chromatography

and to Margaret Costanten and Alma Lugo for preparing the figures.








Finally, 1 must express my humble gratitude to the late Professor

R.G. Stanley, under whose supervision these studies were initiated and

whose infectious enthusiasm for science must be perpetuated by us all.
















TABLE OF CONTENTS



Page

ACKNOWLEDGEMENTS. ......... . . . . . . .. iii

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

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

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

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

CHAPTER ONE ELUTABLE SUBSTANCES OF POLLEN GRAIN WALLS. . . 4

Introduction . ...... . . . . 4
Materials and Methods. . . . . . . 6
Results. ................. .. 11
Discussion. .... . . . . .... .13

CHAPTER TWO POLLEN WALL ELUENTS OF Petunia hybrida L.
THE CARBOHYDRATE COMPONENT . . . . . .. .15

Introduction.. ..... . . . . . . 15
Materials and Methods. .... . . ... 17
Results. ................. ... 21
Discussion .... . . . . . . . .31

CHAPTER THREE EFFECT OF POLLEN ELUENTS AND POLLEN ELUENT
FRACTIONS ON GERMINATION OF ELUTED POLLEN
SAMPLES OF Petunia hybrid L. IN VITRO . . .. .35

Introduction ....... . . . . .35
Materials and Methods. ... . . . . ... 38
Results. . . . . . . ... ..... .44
Discussion . . . . . . . . . 60

CONCLUSIONS . . . ..... ......... . .66

REFERENCES ...... ....... . . . . . . .68

BIOGRAPHICAL SKETCH .................. . .74














LIST OF TABLES


Table Page

1.1 Binucleate Pollen Species Used in Determinations of
Carbohydrates (a-Naphthol Procedure), Proteins (Lowry
Technique) and Total Elutable Compounds (Dry Weight
Basis) . . . . . . . . . . . . . 7

1.2 Trinucleate Pollen Species Used in Determinations of
Carbohydrates (a-Naphthol Procedure), Proteins (Lowry
Technique) and Total Elutable Compounds (Dry Weight
Basis). . . . . . . . . . . . . 9

1.3 Quantitative Differences Between Binucleate and
Trinucleate Pollen Conditions . . . . . . .. 12

2.1 Carbohydrate Contents of Eluents of Pollen of
Petunia hybrida . .... . . . . . . 22

2.2 Chromatographic Analyses of Carbohydrate Fractions of
Eluents of Petunia Pollen . .. .... . . ... 24

2.3 Pollen Germination Tests of Eluted Pollen Fractions . . 28

2.4 Pollen Tube Length in Compatible Pollinations with
Eluted Pollen Grains of Petunia . . . . . .. .29

3.1 The Effect of Pollen Eluents of Petunia hybrida on
Germination of Eluted Pollen Samples. . . . . ... 46

3.2 The Effect of Basic, Neutral, Acidic, and Non-Polar
Fractions on Eluents of Petunia hybrida Pollen, Pre-
pared by Ethyl Acetate Partitioning, on :termination
of Eluted Pollen Samples. .. . . . . . . . 47

3.3 The Effects of Amino Acid-Protein and Carbohydrate
Components of Dowex 50W-8X Effluents of Neutral and
Acidic Fractions of Pollen Eluents of Petunia hybrida
on Germination of Eluted Pollen Samples . . . ... .49

3.4 The Effect of Autoclaved Pollen Eluents of Petunia
hybrida on Germination of Eluted Pollen Samples . . . 50

3.5 Effect of Specific Molecular Weight Fractions of
Ammonium Sulfate Precipitated Proteins of Eluents of
Pollen of Petunia hybrida on the Germination of
Eluted Pollen . . . . . . . . . . . 54














LIST OF FIGURES


Figure Page

2.1 Effect of elution time on quantity of carbohydrate
eluted from pollen of Petunia hybrida. . . . . ... 23

2.2 Pollen grain of Petunia hybrida after elution with
10 ml 1% NaCI, pH 7.5. ...... . . . . . 26

2.3 Fresh, non-eluted pollen grain of Petunia hybrida ..... 27

3.1 Ultrafiltration procedure for the fractionation of
dialyzed ammonium sulfate precipitated proteins from
eluents of pollen of Petunia hybrida ........... 42

3.2 Effect of time of elution on quantity of protein eluted
from pollen of Petunia hybrida . . . . . . .. 45

3.3 Discontinuous polyacrylamide gel electrophoresis of
the acetone-precipitated protein fraction of 20 ml
(2 min) eluents of pollen of Petunia hybrida . ... .. . 51

3.4 Gel scan of discontinuous polyacrylamide gel electro-
phoresis of acetone precipitated protein fraction of
20 ml (2 min) eluents of Petunia hybrida . . . . 52

3.5 Discontinuous polyacrylamide gel electrophoresis of the
protein fraction with molecular weight between 50,000
and 100,000 daltons, as prepared by ultrafiltration. . 56

3.6 Gel scan of discontinuous polyacrylamide gel electro-
phoresis of the protein fraction with molecular weight
between 50,000 and 100,000 daltons, as prepared by
ultrafiltration. . .. . . . . . . . 57

3.7 Effect of varying quantities of acetone-precipitated
protein fraction of eluents of Petunia hybrida pollen
on germination capacity of eluted pollen . . . ... 58


vii















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


BIOLOGY OF DIFFUSIBLE POLLEN WALL COMPOUNDS


By

Edward George Kirby, III

June 1977

Chairman: Indra K. Vasil
Cochairman: Willard W. Payne
Major Department: Botany

Quantitative determinations of components eluted from pollen of

42 angiosperm species representing 25 families and 2 cytologically

distinct pollen types were performed. Analyses of the eluents for

proteins, carbohydrates,and total elutable substances indicated sig-

nificant differences between binucleate and trinucleate pollen con-

ditions regarding total elutable compounds as well as elutable

carbohydrates and proteins. In all cases binucleate pollen released

greater quantities of elutable components than trinucleate pollen.

Analyses of the carbohydrate fraction of pollen eluents of

Petunia hybrida L. demonstrated that eluted carbohydrates repre-

sent about 20% of the total pollen dry weight. Thin layer chromatography

of free sugars in Petunia eluents yielded mannose, glucose,and galactose.

In addition, rhamnose, fucose, xylose, arabinose, and glucuronic and

galacturonic acids were present in the oligosaccharide fraction.








Although eluted pollen of Petunia possesses poor germination

ability in vitro on a simple medium, it produces good pollen tubes on

compatible or incompatible styles. Germination of eluted pollen

in vitro is significantly restored by the addition of pollen wall

eluents to the germination medium. Neutral as well as acidic heat-

labile protein components of pollen eluents appear to restore the

germination capacity of eluted Petunia pollen. A linear relationship

exists between the quantity of eluted protein added and the germination

ability of eluted pollen samples.

Polyacrylamide gel electrophoresis and liquid chromatography

indicate that the protein component of Petunia pollen eluents contains

up to 19 protein components between the range of 5,000 to 61,000 daltons

molecular weight. Addition of protein fractions of pollen eluents

prepared by ultrafiltration to eluted pollen samples demonstrates that

proteins with molecular weights between 50,000 and 100,000 daltons are

likely responsible for restoring the germination capacity of eluted

pollen.














INTRODUCTION


The significance of mobile fractions of substances within pollen

walls has only recently come to the forefront of scientific research.

Although considerable knowledge has accumulated regarding allergenic

compounds of pollen contributing to hay fever (King and Norman, 1962;

Johnson and Marsh, 1966a, b; Underdown and Goodfriend, 1969), limited

reports are available concerning the biological and physiological nature

of substances that diffuse from pollen grain walls. Protein fractions

of pollen wall compounds may be involved as controlling factors in

intraspecific incompatibility in poplars (Knox et al., 1972) and inter-

specific incompatibility in Cosmos bipinnauts (Howlett et al., 1975).

Enzymes have been shown to diffuse from pollen grains of many species,

and cytochemical studies have demonstrated that these are associated

with pollen grain walls (Knox and Heslop-Harrison, 1969, 1970).

The studies presented in this dissertation take several approaches

to the study of pollen surface compounds. Angiosperm species have been

divided into two distinct groups based on when the division of the

generative cell of the pollen grain takes place. In species with

trinucleate pollen division of the generative cell occurs prior to

shedding; in binucleate pollen generative cell division takes place

after shedding. Trinucleate pollen is considered the derived condition

(Schnarf, 1939; Brewbaker, 1959; Rudenko, 1959). The cytological con-

dition of pollen at the time of shedding is well correlated with

physiological properties, such as germination ability in vitro,








viability during storage, the site of the self-incompatibility reaction

and gametophytic or sporophytic control of self-incompatibility

(Brewbaker, 1959). Based on studies of 42 angiosperm species repre-

senting 25 families, data in Chapter One indicate that the number of

pollen nuclei at the time of shedding is also correlated with the

quantity of elutable substances, including carbohydrates, proteins and

total elutable compounds; greater quantities of all three are present

in eluents of species with binucleate pollen.

Further qualitative analyses of the carbohydrate fraction of eluents

of Petunia pollen reported in Chapter Two reveal a wealth of mono-

saccharides and monosaccharide components of oligosaccharides. Elution

has very little effect on the germination of Petunia pollen grains on

compatible or incompatible styles, however the germination ability of

eluted pollen in vitro is significantly reduced. Experimental results

show that the germination ability of eluted pollen samples of Petunia

may be significantly restored if eluted compounds are added to the

germination medium (Chapter Three). Analyses of the eluent fractions

reveal that acidic and neutral heat-labile protein fractions of pollen

eluents are responsible for boosting the germination of eluted pollen

samples. Like the carbohydrate fraction of eluents of Petunia pollen,

the protein fraction is also quite diverse, consisting of 19 components

separable by electrophoresis and ranging in molecular weight from

approximately 5,000 to 61,000 daltons. The germination restoring

proteins present in pollen wall eluents seem to be limited to proteins

with a molecular weight range of 50,000 to 100,000 daltons.

Through the studies presented here the complex nature of pollen

surface compounds becomes apparent, specifically with regard to Petunia,




-3-



a species with binucleate pollen and gametophytic control of self-

incompatibility. Other available studies of pollen diffusates of Cosmos

(Howlett et al., 1975), a species with trinucleate pollen and sporophytic

control of self-incompatibility, indicate a less diverse nature of

pollen diffusates. The diverse nature of the readily mobile fraction

of Petunia pollen walls may be associated with phylogenetically primi-

tive binucleate pollen and gametophytic control of self-incompatibility.















CHAPTER ONE

ELUTABLE SUBSTANCES OF POLLEN GRAIN WALLS*



Introduction


The majority of angiosperm species shed their pollen in the bi-

nucleate condition. However, Brewbaker (1967) has shown that in 30%

of 2,000 species studied, division of the generative cell takes place

prior to shedding. Such pollen grains are shed in the trinucleate

condition. It was initially proposed by Schurhoff (1926) and supported

by subsequent studies (Schnarf, 1939; Brewbaker, 1959; Rudenko, 1959),

that trinucleate pollen is the derived condition and, thus, phylo-

genetically advanced.

In addition to the cytological difference between binucleate and

trinucleate pollen grains, Brewbaker and Hajumder (1961) list four dis-

tinct characteristics that are strongly correlated with the number of

pollen nuclei. Binucleate pollen is usually very easy to germinate

in vitro, whereas trinucleate pollen germinates in vitro with consider-

able difficulty. Binucleate pollen can normally be stored for con-

siderable periods of time under defined conditions, while trinucleate

pollen cannot be stored for any appreciable length of time. Intra-

specific incompatibility is generally observed in the style or ovary


*This work has appeared in an abbreviated form as: Kirby, E.G. and
Smith, J.E.: Elutahle substances of pollen grain walls. In "Fertiliza-
tion in Higher Plants" (ed. ll.F. Linskens). North-Holland, Amsterdam.
pp. 127-130 (1974).


-4-








for species having binucleate pollen, whereas species with trinucleate

pollen usually display incompatibility reactions in the stigma.

Binucleate species are usually characterized by gametophytic incompati-

bility systems and trinucleate species are characterized by sporophytic

incompatibility systems.

There are notable exceptions, however, to the listed physiological

differences. Some well known species have trinucleate pollen that

germinates readily in vitro (e.g. Brassica, Pennisetum; Vasil, 1960a,

1962) and in some species having trinucleate pollen the incompatibility

reaction takes place in the style (e.g. Beta). In addition, grasses,

which have trinucleate pollen, have a well defined gametophytic

incompatibility system.

Explanations of the above phenomena are generally based on meta-

bolic requirements for mitosis of the generative cell, which occurs

prior to shedding in trinucleate pollen. This mitotic activity appar-

ently deprives the pollen protoplast of substrates essential for

germination in vitro, extended storage, and growth of pollen tubes into

the style in incompatible matings. Required substrates may be made

available to pollen only be genetically compatible stigmas following

the action of enzymes or "recognition substances" released from the

pollen walls (Knox et al., 1972).

The present work was undertaken to clarify and establish the

chemical and physiological characteristics of binucleate and trinucleate

pollen in an attempt to further elucidate the biological function of

substances contained within the walls of pollen grains.










Materials and Methods


Pollen from 42 species representing 25 families of angiosperms was

collected during the 1973-1974 growing season or purchased from the

C.G. Blatt and Co., Independence, Missouri, U.S.A. (see Tables 1.1 and

1.2). Pollen was stored at -50C with silica gel as desiccant.

Samples of 50 mg were extracted for 1 hr at 40C on a shaker using

Coca's solution (Coca, 1922) minus phenol (5.0 g NaCI, 2.75 g NaHCO3 in

1000 ml distilled water).

Aliquots of the extracts were analyzed for proteins by the Lowry

procedure (Lowry et al., 1951) and for carbohydrates by a-naphthol-

sulfuric acid. In this procedure aliquots of eluents containing not

more than 100 og carbohydrate were added to 5.0 ml a-naphthol reagent

(2.0 g a-naphthol in 500 ml cone. H2SO ), mixed well and heated in

boiling water for 10 min. After cooling optical density readings were

taken at 555 nm on a Beckman DB spectrophotometer. The amount of total

elutable substances was determined on a dry weight basis. The number

of pollen grains in 10 mg samples of each of the 42 species under analy-

sis was determined by evenly suspending 10 mg pollen in 5.0 ml water

and counting pollen numbers using a hemacytometer. Calculations of the

number of pollen grains per milligram were made accordingly.









Table 1.1

Binucleate Pollen Species Used in Determinations of Carbohydrates (a-
Naphthol Procedure), Proteins (Lowry Technique) and Total Elutable Com-
pounds (Dry Weight Basis). All values are expressed as pg per one
million pollen grains.



Total
Binucleate Pollen Carbohydrates Proteins Elutable
Species Compounds


-------- g/106 pollen grains--------------

Betulaceae
Alnus sp. 10.38 5.20 360.04
Betula papyrifera 9.42 5.06 372.23

Euphorbiaceae
Ricinus communis 8.84 4.70 389.79

Fagaceae
Quercus virgiana 8.32 5.17 363.92
Fagus sp. 9.98 5.28 354.96

Juglandaceae
Juglans nigra 10.13 5.09 370.09
Carya glabra 8.46 4.44 384.91

Leguminosae
Acacia sp. 9.34 4.90 350.42

Magnoliaceae
Magnolia grandiflora 9.42 5.04 357.24

Moraceae
Cannabis sativa 9.43 4.76 378.84
Morus rubra 7.94 4.97 382.42

Myricaceae
Myrica sp. 9.55 5.37 376.96

Myrtaceae
Eucalyptus sp. 10.54 4.91 364.11

Oleaceae
Fraxinus sp. 9.12 4.73 374.44
Olea sp. 10.98 4.89 360.94

Palmae
Caryota sp. 9.15 5.50 391.84
















Binucleate Pollen
Species




Rosaceae
Rosa sp.
Pyrus communis

Salicaceae
Populus sp.

Simarubaceae
Ailanthus sp.

Urticaceae
Urtica sp.


Table 1.1
(Continued)




Carbohydrates


Total
Proteins Elutable
Compounds


------ -g/106 pollen grains---------------


4.90
4.81


4.42


5.13


10.05


366.92
364.78


376.92


369.41


5.31 369.41









Table 1.2

Trinucleate Pollen Species Used in Determinations of Carbohydrates (a-
Naphthol Procedure), Proteins (Lowry Technique) and Total Elutable Com-
pounds (Dry Weight Basis). All values are expressed as ug per one
million pollen grains.



Total
Trinucleate Pollen Carbohydrates Proteins Elutable
Species Compounds


--------g/106 pollen grains--------------

Amaranthaceae
Amaranthus sp. 6.18 3.38 343.21
Acnida tamariscina 3.98 3.41 319.82

Cactaceae
Opuntia sp. 6.24 4.29 328.31

Caprifoliaceae
Sambucus sp. 6.41 4.05 341.19

Chenopodiaceae
Kochia sp. 6.04 3.94 337.43
Beta vulgaris 5.16 3.67 320.04
Chenopodium album 4.68 3.81 331.17

Compositae
Chrysanthemum sp. 4.97 3.99 323.23
Artemesia dracunculus 6.58 3.49 334.42
Taraxicum sp. 5.24 3.27 315.01
Helianthus sp. 6.73 4.03 321.11
Baccharis sp. 6.74 4.38 322.81
Chrysothamnus sp. 5.86 3.86 324.56
Ambrosia elatior 5.40 4.20 331.19

Graminae
Festuca sp. 6.13 4.08 343.21
Zea mays 5.59 3.83 350.13

Plantaginaceae
Plantago lanceolata 6.62 3.95 317.21

Polygonaceae
Rumex sp. 5.78 3.64 330.07





-10-



Table 1.2
(Continued)


Trinucleate Pollen
Species




Typhaceae
Typha sp.

Ulmaceae
Ulmus americana
Celtis occidentalis


Total
Carbohydrates Proteins Elutable
Compounds


--------ig/106 pollen grains--------------


5.61


4.53
5.38


3.69


327.75


3.72 346.08
4.17 339.98





-11-





Results


All determinations of proteins, carbohydrates and total elutable

compounds have been calculated as micrograms per one million (10 )

pollen grains (Tables 1.1 and 1.2). This represents an effort to

minimize any differences related with pollen size. The results show

clearly significant differences between bjnucleate and trinucleate

conditions regarding total elutable compounds, as well as elutable

carbohydrates and proteins (Table 1.3). This is evident in the greater

quantities eluted from species with hinucleate pollen.











Table 1.3

Quantitative Differences Between Binucleate and
Trinucleate Pollen Conditions


Binucleate


Trinucleate


----------pg/106 pollen grains-----------


Carbohydrates
(a-naphthol)


Proteins
(Lowry)


Total elutable compounds
(dry weight basis)


9.40 + 1.83**


4.98 + 0.57**


370.97 20.48**


5.75 1.83


3.85 0.57


330.15 20.48


Significance:
** 0.01




-13-


Discussion


Tsinger and Petrovskaya-Baranova (1961) concluded from the presence

of proteins and other compounds that pollen grain walls are ". . living,

physiologically active structures playing a very responsible role in

the processes of interchange between pollen grain and its substrate" (p. 106).

Studies of Makinen and Brewbaker (1967) and Stanley and Search (1971)

showed that proteins and enzymes contained within pollen walls can be

eluted within 5 sec, indicating a rapid interchange between pollen grain

and substrate.

Cytochemical studies (Knox and Heslop-Harrison, 1969, 1970;

Vithanage and Knox, 1976) have shown that enzymes are associated with

the intine layer of the pollen wall. More recently, attention has

focused on gametophytic and sporophytic fractions of pollen wall com-

pounds. The difference between these two fractions is the site of their

synthesis. Heslop-Harrison et al. (1973), Howlett et al. (1975) and

Vithanage and Knox (1976) have reported that protein fractions as well

as allergenic and antigenic fractions may be associated with exine and

intine sites. Their observations indicate that intine-held compounds

are gametophytically synthesized, while compounds located in the exine

are sporophytically synthesized. Furthermore, it has been proposed

that control of gametophytic intraspecific incompatibility is mediated

through intine-held recognition substances, while exine-held compounds

control sporophtyic incompatibility systems (Heslop-Harrison et al.,

1973).

The'results of this preliminary study indicate that cytologically

distinct groups of angiosperm pollen (binucleate and trinucleate) that








have been reported to differ in several physiological aspects, including

type of incompatibility control, also display quantitative differences

in elutable components that have been both gametophytically and

sporophytically contributed. Values reported in Tables 1.1, 1.2 and

1.3 for total elutable substances, calculated on a dry weight basis,

are considerably higher than values reported for elutable carbohydrates

and proteins. Components, including flavinoids, organic acids, minerals

and others, are likely to form the bulk of the elutable substances. If,

indeed, control of incompatibility is regulated through "recognition

substances" carried within the walls of pollen grains, then qualitative

studies characterizing distinct fractions of these compounds could

elucidate possible control mechanisms involved in intraspecific in-

compatibility.















CHAPTER TWO

POLLEN WALL ELUENTS OF Petunia hybrida L. THE CARBOHYDRATE COMPONENT



Introduction


Recently, considerable attention has focused on wall components

of pollen grains, specifically with regard to possible functions of

readily mobile fractions in cell recognition and incompatibility on the

stigma, and pollinosis in man. Although the structure of pollen grain

walls has been studied in detail (for reviews see Heslop-Harrison,

1968a; Vasil, 1973; Stanley and Linskens, 1974), few recent studies have

focused on their chemical constituents, with the exception of work on

allergens (King and Norman, 1962; King et al., 1964; Johnson and Marsh,

1966a, b; King et al., 1967; Underdown and Goodfriend, 1969; Griffiths

and Brunet, 1971). The outer layer of the pollen wall, the exine, is

composed of oxidized carotenoid derivatives (Brooks and Shaw, 1968).

The wall layer clhI-.'t no the haploid pollen protoplast, the intine

layer, is generally thought to be cellulosic in nature (Sitte, 1953;

Heslop-Harrison, 1968b), but also contains non-cellulosir pect inseous

materials (Roland, 1971; Knight et al., 1972). The complex sporo-

pollenin component is derived from both microspore protoplast and from

polyploid or multinucleate tapetal cells of the anther sac (Heslop-

Harrison, 1971; Vasil, 1973).

In addition to structural components of pollen walls, a rapidly

diffusing fraction has been demonstrated in many species, as is shown





-16-


by the work of several groups. However, the major emphasis of the

majority of this work has been centered on protein components of pollen

diffusates. With the exception of the work of Howlett et al. (1975),

who found the major carbohydrate component of pollen diffusates of

Cosmos bipinnatus to be arabinose, little analytical work has focused

on the diffusible carbohydrates of pollen walls. Because of the

sizeable carbohydrate fraction found in eluents from pollen of 42

angiosperm species (Chapter One), the present study was undertaken to

further characterize the carbohydrate fraction of readily mobile pollen

wall compounds.




-17-


Materials and Methods


Pollen Collection. Pollen of Petunia hybrida, clone W166K with

self-incompatibility alleles SS2 was obtained from plants grown in the

green house. Anthers were removed prior to anthesis and allowed to

open in Petri plates. Pollen was then collected, separated from

extraneous material by the use of sieves and was used fresh or after

storage at -5C over silica gel.

Preparation of Eluents. To obtain eluents, 50 mg samples of pollen

were treated in the following four ways: (1) The pollen sample was

placed on a stainless steel sieve (5 jim mesh) and suspended in 10 ml

1% NaC1, pH 7.5 (Knox et al., 1972). Vacuum was applied from a water

aspirator and the extract filtered through the sieve and collected. No

pollen was present in the eluents. Total elution time was 1 min.

(2) The pollen sample was placed on a stainless steel sieve (5 pm mesh),

suspended in 20 ml 1% NaCl, pH 7.5,and the same procedure followed as

in (1) above. Elution time was 2 min. (3) The pollen sample was

placed in an Erlenmeyer flask with 10 ml 1% NaC1, pll 7.5, and shaken for

30 min at 40C. Pollen was separated from the eluent by centrifugation

at 500 x g for 10 min. (4) The pollen sample was suspended in 3.0 ml

1% NaCI, pH 7.5, and homogenized using a MSE 150 Watt ultrasonic

disintegrator for a total of 5 min in 30 sec intervals. Microscopic

examination revealed that nearly all pollen grains were broken after

this treatment. The homogenate was centrifuged at 10,000 x g for

30 min and the supernatant decanted. Pollen eluents and the super-

natant of (4) were evaporated to dryness on a flash evaporator and




-18-


taken up in 1 ml cold double distilled water. All above operations

were performed in the cold to minimize breakdown of eluted compounds

by enzymes present in pollen eluents.



Analytical Procedures


Separation of Neutral and Acidic Sugars. Immediately after pre-

paration eluents and the supernatant of (4) were passed through Dowex-50

(H+) short columns, evaporated to dryness and solubilized in 1 ml double

distilled water. Free neutral and acidic sugar fractions of Dowex-50

effluents were separated on a Dowex-l-formate column according to the

procedure of Kroh (1973) and aliquots were chromatographed directly.

Oligosaccharide and components of Dowex-50 effluents were hydrolyzed

with 2 N trifluoracetic acid (TFA) for 1 hr at 1210C, or with 80% TFA

(v/v) for 48 hrs at 1210C prior to separation of neutral and acidic

sugar fractions. TFA hydrolysates were evaporated and acid removed

by repeated addition of water and evaporation. Neutral and acidic

sugar components of hydrolysates were separated by Dowex-l-formate

columns and aliquots of Dowex-l effluents were chromatographed.

Chromatography. Thin layer chromatographs of neutral sugars were

developed in ethyl acetate-pyridine-water (80:20:10) (v/v). Chromato-

graphs of acidic sugars were run in acetone-n-butanol-0.1 M NaH2PO

(40:25:35) (v/v). Standards utilized were galactose, glucose, mannose,

arabinose, xylose, fucose, and rhamnose as neutral sugars and galac-

turonic and glucuronic acids as acid sugars.

Sugars were detected on chromatographs with methanolic AgNO3, KOH

and sodium thiosulfate reagents.








Total Carbohydrate Determination. Qunatitative determination of

total carbohydrates was performed according to the phenol-sulfuric acid

method of DuBois et al. (1956).

Electron Microscopy. Fresh and eluted pollen grains of Petunia

hybrida were fixed in 3.0% glutaraldohyde prepared in 0.1 N phosphate

buffer, pH 7.0, for 30 min to 1 hr, washed with two changes of buffer

and post fixed for 3 hrs in 3.0% Os4 or 3.0% KMnO4 prepared in the

same buffer. All fixations were at room temperature. Post-fixation

was followed by buffer and distilled water washes and agar embedding.

Dehydration was carried out in an ethanol series followed by propylene

oxide and pollen grains were embedded in Epon.

Thin sections of pollen grains were cut on a Porter-Blum MT-2

ultramicrotome using a diamond knife. Sections were post-stained with

saturated aqueous uranyl acetate followed by Reynolds' (1963) lead

citrate. Electron micrographs were made using a Philips EM 300 electron

microscope operating at 60 kV.



Pollen Germination Assays


In Vitro Assay. Fresh or eluted pollen grains of Petunia hybrida

were germinated in 10% sucrose, 100 ppm boron (supplied as H 80 ) for

3 hrs at 30C. The concentration of pollen was maintained at

5 mg/ml germination medium. Pollen grains were considered germinated

after 3 hrs if pollen tubes were extended to a length equal to or

greater than the diameter of the pollen grain.

In Vivo Assay. Fresh or eluted pollen was applied to compatible

styles of Petunia hybrid (clone TU). The pollinated styles plus





-20-


pedicels were first put in 2 ml water in small vials and placed in a

light box or growth chamber at 27C. Pollen tube length was measured

after 24 hrs by staining callose in pollen tubes in stylar squash mounts

with aniline blue (0.005% in 0,15 M KH2PO pH 8.2) (Jensen, 1962) and

observing under a Leitz ultraviolet microscope.











Results


Carbohydrate Content of Petunia Pollen Eluents. Total carbohydrate

determinations of pollen cluents and the supernatant of homogenized

pollen are shown in Table 2.1. Carbohydrates are very quickly eluted

from Petunia pollen in quantities that approach 20% of the total dry

weight of the pollen sample (Fig. 2.1).

Qualitative Analysis of Elutable Substances by Thin Layer Chroma-

tography. Separation by thin layer chromatography (TLC) of free sugars

as well as hydrolysis products of the pollen eluents and the supernatant

of homogenized polled are presented in Table 2.2. The composition of

free sugars of (1) and (2) is identical: both contain mannose, glucose

and galactose. These sugars are also present as free neutral sugars in

(3) and (4). Hydrolysis of the oligosaccharide components of (1) yields

rhamnose,fucose and xylose in addition to mannose, glucose and

galactose. In (2) arabinose was also found as an hydrolysis product.

Hydrolysis of oligosaccharide fractions yielded glucuronic and

galacturonic acids from both (1) and (2). In all chromatograms of

Dowex-l-formate column effluents developed in acetonc-n-butanol-0.1 M

NaH2PO (40:25:35) for the separation of acid sugars an additional spot,

which did not co-chromatograph with galacturonic or glucuronic acid

standards, was detected with alkaline silver nitrate. This spot may

represent an unknown uronic acid, sugar phosphate, aldonic acid or

other compound.

Aliquots from (3) and (4) contained identical free neutral and

acidic sugar fractions before and after hydrolysis (Table 2.2). Mannose,










Table 2.1

Carbohydrate Contents of Eluents of Pollen of Petunia hybrida


mg Carbohydrate
Per 50 mg Pollen


(1) (1 min elution)

(2) (2 min elution)

(3) (10 min elution)

(4) (disrupted pollen)


7.20

9.36

10.0


Per cent
of Total


60.0

78.0

83.3


Eluents from (1), (2), (3), and (4) refer to numbers used in the text
for 10 ml, 20 ml, shaken and eluents of disrupted pollen, respectively.
Carbohydrate determinations were performed according to the technique
of DuBois et al. (1956).


















mg CARBOHYDRATE
PER 50mq POLLEN


A
A


10 20 30 40

ELUTION TIME (min)


Figure 2.1. EFr[ unii ( lflY of vnrlwhycl cluiud
Froiii pollIn o i e hvl r i' Cii I- ho I v d l t ,hi del It, rallii -
nunol %,'n re lit, I o rmcd ic o rd i ii Lto L L, FLechn i (Iuo n fDk Ii n is
eL 1. (19)56).





-24-


U)



E1CO
U,



U)
4-L, ,-|


H C





4-
-.1l >^









h T]

mJ
-U)










ru
'-l




U)
U)

4-t
-U)
^l >
< U)

U)'-








(U

-U)
l-l

U)l


U)
4-


U)



+ + + 1 + + + + +
Ho








I I I I + + +
u-


U) I


0 4J


r ,ca
p U)




ru 0
o4-4



Lm *H 7m
-40 4-4




Q -. -H >,-
3 En u C
U) 4-4. (U .I




) -,1 w c)
r. Uc lI
E0 C + *




Id U) -w C-4

1u0 u0 -
C >4-U)

c- -- U U
4-f U)rd -U 4

-4. UO



10 t3 W 0

4 0 m

0 r (U ,
4- ,-r 03









drai o 0
* Ud U) U.Z
4-4U o r














U)U)UO p
(- 0 H U







E6o u)





0 ( i-id C
U) U 3 c l
4-4 1U 0 U |

















3 -a


U4.0 u











I 0 u
U) IU C

-HU)'U)U 4-44













r ) n I o
0 U)U)U)

ci t.O U



0 0 r
) U u) U)
4-1U o o00





" 0 U)
CO 00 O
- 0 I)







cn| ( 4c U) u

4- D oj L -












3 L (U C




-25-


glucose and galactose were present as free neutral monosaccharides, and

glucuronic and galacturonic acids were present as free uronic acids.

These acid sugars were not observed in the free sugar fractions of (1)

and (2). Hydrolysis of the oligosaccharide fraction of (3) and (4)

also yielded identical monosaccharide components, rhamnose, fucose,

xylose, arabinose, mannose, glucose, and galactose as well as glucuronic

and galacturonic acids (Table 2.2).

Since the solvent system employed for chromatography of neutral

sugars (ethyl acetate-pyridine-water) cannot unequivocally resolve

fucose and xylose, some question may be raised as to whether both

monosaccharides are present in hydrolysates of oligosaccharide

fractions of pollen eluents. However, in all chromatograms of hydroly-

sates, two spots were clearly distinguished which co-chromatographed

with xylose and fucose of standard sugar mixtures.

Electron Microscopy. Electron micrographs of eluted and non-

eluted pollen grains of Petunia hybrida display similar cytoplasmic

detail (Figures 2.2 and 2.3). Plasma membranes of eluted material

appear undisturbed by the elution procedure. No bursting was observed.

In addition no basic difference was observed in the ultrastructural

appearance of intine and exine layers of the pollen walls of eluted and

non-eluted samples. However, the arrows in Figure 2.2 point to empty

areas between the intine and the plasmalemma. Such areas were fre-

quently observed in sections from eluted pollen and occasionally seen

in sections from non-eluted pollen. These areas may represent sites

of eluted intine-held fractions, however, their occurrence being a

result of fixation procedures is not precluded.






-26-


Figure 2.2. Pollen grain of Petunia hybrida after elution with
10 ml 1% NaCI, pll 7.5. Cytoplasmic detail is similar
to that of non-eluted pollen; the plasmalemma is con-
tinuous. Spaces (arrows) within the intine layer are
frequently observed. (Magnification 19,200 x)

























p -


Figure 2.3. Fresh, non-cluted pollen grain of Petunia hybrida.
Exine (E) and intine (I) layers are clearly seen, as
well as typical cytoplasmic organelles. (Magnification
19,200 x)










Table 2.3

Pollen Germination Tests of Eluted Pollen
Fractions


Pollen Sample


Per cent of Control


(1) (1 min elution) 29.3%


(2) (2 min elution) 4.9%


(1) represents values for pollen eluted with 10 ml 1% saline, pH 7.5.

(2) represents values for pollen eluted with 20 ml 1% saline, pH 7.5.

Values are reported as percentages of control germinations of non-eluted
pollen samples. Values are means of 4 replications of two experiments.
For each replication 400 pollen grains were counted.











Table 2.4

Pollen Tube Length in Compatible Pollinations with
Eluted Pollen Grains of Petunia


Cross


Mean Tube Length (mm)


T2H x W166K

T H x W166K (1)

T H x W166K (2)
2


20.7 s = 1.89

20.7 s = 2.87

16.4 s = 7.07


Results are reported for female plants (T2H) crossed with pollen (W166K)
eluted with 10 ml saline, pH 7.5 (1); pollen eluted with 20 ml saline,
pH 7.5 (2); and non-eluted control pollen. Numbers (T2H and W166K)
refer to clones of Petunia hybrida. Values for standard deviations (s)
are also given. All pollinations were performed in quadruplicate.








Germination Tests. In vitro germination tests showed that eluted

pollen germinates poorly (Table 2.3). The values range from 4.9%

for pollen eluted with 20 ml saline solution to 29.3% for pollen

eluted with 10 ml saline. Values are expressed as per cents of control

germinations. The low values reported for germination of eluted pollen

may be caused by the effect of saline during the elution process, and/or

by the removal of wall-bound substances required for germination.

However, in vivo germination assays of eluted pollen samples performed

on compatible styles showed good pollen tube growth (Table 2.4). Pollen

tube length of samples eluted with 10 and 20 ml saline solution (20.7

and 16.14 mm respectively) compares favorably with values for non-eluted

controls (20.7 mm).











Discussion


From this study it is concluded that a wealth of carbohydrate

material is present in eluents of pollen grains of Petunia hybrida.

However, the origin of this diverse carbohydrate fraction remains a

mystery.

One may assume that the method and duration of elution determines

if substances present in eluents are derived from wall sites, or are

also contributed by haploid pollen protoplasts. It appears likely that

quantitatively less carbohydrate material is removed from the pollen

cytoplasm when pollen is eluted for 1 or 2 min, rather than shaken for

30 min. Both quantitative and qualitative sugar determinations support

this. Eluents prepared by washing pollen with 10 ml and 20 ml saline

solution contain monnose, glucose and galactose as free sugars. Eluents

prepared by shaking for 30 min and the supernatants of homogenized

pollen contain glucuronic and galacturonic acids in addition to the

above monosaccharides as free sugars. Since only supernatants of

homogenized pollen and 30 min eluents contain glucuronic and galacturonic

acids as free sugars, it is probable that these sugars and likely other

components found in 30 min eluents may be of cytoplasmic origin. Ten

and 20 ml eluents do not contain free glucuronic and galacturonic acids

which may indicate that cytoplasmic components are not present. In all

cases hydrolysates of oligosaccharide fractions contained rhamnose,

fucose, xylose, mannose, glucose, galactose, glucuronic acid, and

galacturonic acid. Arabinose was present in all hydrolysates, excepting

the 10 ml eluents. Electron micrographs of eluted pollen compared with




-32-


those of non-eluted pollen grains reveal no difference in morphological

appearance of the cytoplasm after elution. The plasma membranes of

eluted pollen grains appear undisturbed. This leads to further support

of the contention that cytoplasmic constituents are not present in

10 ml eluents.

The observation that eluted pollen is able to germinate in com-

patible pollinations also supports the contention that protoplasmic

integrity is not altered during elution. This has also been shown to

be the case with eluted pollen of Lilium longiflorum (Fett et al., 1976),

another example of gametophytic self-incompatibility. However, eluted

pollen grains of Petunia germinate poorly in vitro, while the removal

of loosely bound substances from lily pollen resulted in little decrease

in germination ability. In Petunia the decrease in pollen germination

ability may be due, in part, to effects of the eluting solution

(saline), or may be caused by the removal of specific factors from wall

sites (Chapter Three). Wall-held compounds may function as a source of

enzymes or nutrients required for pollen tube elongation. Such factors

could be supplied by female tissues to eluted pollen grains during

germination on compatible stigmas.

Monosaccharides present in eliients of Petunia pollen are commonly

found as components of higher plant cell walls (Lamport, 1970).

Hydrolysates of pollen wall diffusates from Cosmos bipinnatus (Howlett

et al., 1975) contained only arabinose and possibly glucose and galac-

tose. Glucuronic and galncturonic acids could not be detected.

However, procedures employed by Howlett's group eliminated compounds

with molecular weights less than 10,000 daltons. Eluents of Petunia

pollen contain diverse c;rhohledreir s, including free sugars and








oligosaccharides. It is quite possible that such diversity also is

present in the carbohydrate fraction of eluents of pollen of Cosmos

bipinnatus, but did not appear in analyses of wall diffusates.

Studies on homogenized rose pollen (Zolotovitch and Secenska,

1962 and Zolotovitch et al., 1964) have demonstrated that glucose,

galactose and rhamnose are present. Knight et al. (1972) have shown

that uronic acids, as determined by the microdecarboxylation method,

are major components of pollen of 53 angiosperm and 5 gymnosperm species.

This uronic acid fraction is likely the pectinaceous intine matrix

material reported by Roland (1971). Pectin fractions of intine mate-

rials probably function in the initial uptake of water by germinating

pollen grains, since pectins influence water distribution within plant

cell walls (Northcote, 1972). Galacturonorhamnans containing an a-D-

galacturonic acid-L-rhamnose backbone with side chains of L-fucose,

D-xylose and D-galactose are the major constituents of plant pectins

(Northcote, 1972). These monosaccharides are found as hydrolysis

products of wall eluents of Petunia pollen and it is conceivable that

elutable oligosaccharide fractions of wall eluents containing such

sugars are soluble precursors of pectinaceous material of the intine.

Intercellular material from stylar transmitting tissue of

Petunia hybrida contains a mixture of low molecular weight carbohydrates

(Kroh, 1973). This material provides nutrients for growing pollen

tubes. Analysis of the carbohydrate component of eluents of pollen

of Petunia hybrida revealed a mixture of acidic and natural mono-

saccharides as components of the oligosaccharide fraction. In addition

free neutral and acidic sugars are present. If the function of the





-34-



carbohydrate fraction of pollen eluents is similar to the function of

the carbohydrate fraction of intercellular stylar transmitting tissue,

we may expect some degree of homology of composition.














CHAPTER THREE

EFFECT OF POLLEN ELUENTS AND POLLEN ELUENT FRACTIONS ON GERMINATION
OF ELUTED POLLEN SAWMLES OF Petunia hybrida IN VITRO



Introduction


Much of the early work on protein components of pollen has come

from apiarists, since pollen functions as the major source of amino

acids for bees, and from allergists, since the causal factors in pollen

allergies are thought to be proteins. Pollen has been shown to contain

from 7.0% protein (lodgepole pine) to 35.1% protein (date palm) (Todd

and Bretherick, 1942). lydrolysates of pollen proteins contain all

amino acids commonly found in plant tissues (Auclair and Jamieson,

1948). Studies on pollen allergens have mainly focused on rye grass

and ragweed species. Allergenic proteins are generally low molecular

weight compounds (5,000-30,000 daltons) and contain carbohydrate

moieties (Abramson, 1947; Marsh et al., 1966).

Enzymes have been observed to diffuse from pollen, as determined

by starch liquification, and a function for diffusable enzymes in pollen

tube nutrition was postulated early (Green, 1894). More recently,

Haeckel (1951) studied amylasc, phosphatase and invertase activities

in pollen of 20 angiosperm species and found phosphatase activities to

be the highest prior to germination. Russian workers (Poddubnaya-

Arnoldi et al., 1959) described the release of many enzymes by angio-

sperm pollen and traced the localization of these enzymes to the pollen








grain wall. Tsinger and Petrovskaya-Baranova (1961) determined that

additional enzymes are present in pollen walls of peony and Amaryllis,

including dehydrogenases, cytochrome oxidase and acid phosphatase.

These workers concluded that, because of its wealth of typical cyto-

plasmic enzymes, the pollen wall must be termed a living physiological

structure.

Mfkinen and Lewis (1962) demonstrated that antigen-reacting enzymes

diffuse from the surfaces of pollen of Oenothera when placed on moist

agar. The pattern of diffusion of proteins from germinating Petunia

pollen has been described vby 'anlcy and Linskens (1964, 1965). These

workers also determined that sucrose metabolizing activity is localized

on the surface of Petunia pollen and diffuses rapidly into the growth

medium. Lewis et al. (1967) determined that washing pollen grains of

Oenothera with an aqueous, non-germination promoting medium releases

catalase, amylase, esterase, phosphatase and leucine aminopeptidase

activities. Furthermore, Stanley and Thomas (1967) reported the release

of high amounts of cellulase from pollen of cattail, pine and pear prior

to germination. It appears that pollen grains of many angiosperm

species are actively involved in releasing proteins, especially enzymes,

just prior to germination.

Recent cytochemical studies have revealed that protein components

are found in the walls of pollen grains, both in the pectocellulosic

intine, particularly in pore regions, and in sexine cavities of the

exine. These components include readily diffusible allergenic and

enzyme fractions (Knox and Heslop-Harrison, 1969, 1970, 1971a; Heslop-

Harrison et al., 1973; Vithanage and Knox, 1976). Furthermore, it has

been shown that antigenic proteins found in the exine layer of pollen





-37-


grains of Iberis (Cruciferae) are lost to the stigma within 5 to 10

minutes following compatible or incompatible pollinations (Heslop-

Harrison et al., 1974). It has also been suggested that mobile pollen

wall proteins are directly involved in incompatibility reactions (Knox

et al., 1970; Knox and Heslop-Harrison, 1971a, b; Knox, 1971; Knox

et al., 1972; Howlett et al., 1975). However, the work of Fett et al.

(1976) indicates that mobile proteins are not involved in the self-

incompatibility response in Lilium longiflorum.

The previous chapter described the complexity and possible func-

tions of the carbohydrate components of eluents of Petunia pollen walls.

The present chapter reports the stimulatory effect of proteinaceous

fractions of wall eluents of Petunia pollen on germination of eluted

pollen grains in vitro. In addition consideration is focused on

chemical properties of the stimulatory fraction and its mode of action.











Materials and Methods


Pollen Collection and Storage. Pollen of Petunia hybrida, clone

W166K, was collected and stored as described previously (Chapter Two).

Preparation of Eluents. Samples of pollen (25 mg) were placed on

a Millipore filter (0.4 pm pore diameter) mounted on a filter support

funnel. Pollen was suspended in 20 ml double distilled water, the water

drawn through the filter by vacuum and the resulting eluent collected

in a vacuum flask. The elution process was performed in an ice water

bath and using ice cold solutions. Eluted pollen grains remained on

the Millipore filter and were easily transferred to incubation flasks

for germination tests.

Water was removed from eluent samples using a flash evaporator.

Eluents prepared in this manner were used fresh in further analyses and

germination assays, or stored in the freezing compartment of a refriger-

ator at -50C.

Separation of Neutral, Basic, Acidic and Non-Polar Fractions of

Eluents. Eluents from 100 mg pollen (4 x 25 mg samples) were redissolved

in 20 ml double distilled water, pH adjusted to 3.0 with 0.5 N HC1, and

partitioned against re-distilled ethyl acetate (Hollis and Tepper, 1971)..

Under these conditions basic compounds are freely soluble in the aqueous,

pH 3.0 phase. The remaining ethyl acetate phase was partitioned

against water at pH 8.5 (adjusted with 0.4 N NH OH). The resulting

aqueous phase contained freely soluble acidic compounds. The remaining

ethyl acetate was lastly partitioned against double distilled water

(pH 6.0). The aqueous phase contained freely soluble neutral compounds,








while non-polar compounds were contained within the ethyl acetate phase.

The four fractions thus obtained (pH 3.0, 6.0, 8.5 and ethyl acetate)

were dried using a flash evaporator. Residues from the pH 3.0 and pH

8.5 phases were washed with double distilled water and repeatedly

evaporated to remove all traces of 1IC1 or NH3. All operations were

carried out in the cold. The four dry fractions were used directly to

test their effects on germination of eluted pollen, or used in further

preparative procedures.

Separation of Carbohydrates and Amino Acid-Protein Fractions.

Aliquots of pH 6.0 and pH 8.5 phases obtained by partitioning procedures

were placed on a short Dowex 50W-8X cation exchange column and eluted

with 0.4 N NH4OH, yielding the carbohydrate fraction, followed by

elution with 4.0 N NH OH, yielding the amino acid-protein fraction.

All Dowex effluents were dried in a flash evaporator, repeatedly washed

with double distilled water and evaporated to remove all traces of NH3.

The amino acid-protein and carbohydrate fractions were then redissolved

in pollen germination medium and their effects on the germination of

eluted pollen grains assayed.

Denaturation. Aliquots of eluents of Petunia pollen were sealed

in hydrolysis tubes and placed in an autoclave at 1210C for 90 min.

This procedure causes proteins to lose their tertiary structural

characteristics.

Preparation of Acetone Protein Precipitates. Proteins in eluents

of Petunia pollen were precipitated according to a modification of

Hare's (1970) procedure. Dry eluents were taken up in 70% acetone and

the creamy suspension centrifuged at 27,000 x g for 20 min. The

resulting pellet was washed two times with 70% acetone followed by two








washes with 100% acetone. All procedures were performed in the cold.

The final white precipitate was dried with an air stream. This material

is referred to as the acetone precipitated protein fraction.

The dried precipitate was dissolved in pollen germination medium

or in protein extracting solution modified from Hare (1970) as follows:


Urea 15 g

K2S205 0.5 g

Ascorbic Acid 1.0 g

Cleland's Reagent 0.1 g

Tween 20 (10% solution) 4.0 ml

Tris to pH 8.5

Double distilled water to 100 ml


Forty microliters of the extractive, which contained about 45 ug pro-

tein, were applied to precharged polyacrylamide gels and developed at

2.5 milliamperes per gel. Tris-glycine buffer (pH 8.5) containing

0.05% bromophenol blue was in the upper chamber and tris-HC1 buffer

(pH 8.1) was in the lower chamber. After 70 min gels were removed and

shaken in 10% aqueous trichloroacetic acid (TCA) for 30 min followed by

staining for 48 hrs with 0.25% Coomassie blue added to fresh TCA.

Molecular Weight Determination. The range of molecular weights

of proteins of eluents of Petunia pollen was determined using a Waters

AGL/GPC 502 liquid chromatograph equipped with a model 6000 solvent

delivery system and using a 1.5 m Porasil CX (Waters Associates)

molecular sieve column. Water was used as solvent. All operations

were performed at room temperature. Proteins in effluents were detected








with a UV flow detector and the flow rate was 0.2 ml per min. Column

operating efficiency was determined to be 1680 theoretical plates.

Molecular weight ranges were calculated relative to a mixture of

known molecular weight protein standards.

Total Protein Precipitation and Protein Fractionation as to

Molecular Weight. Lyophilized eluents from 250 mg pollen were taken up

in 20 ml ice cold water and placed in a 50 ml flask on ice. Ammonium

sulfate (7.2 g) was added and the mixture stirred on ice for 15 min.

The resulting suspension was centrifuged 20 min at 28,000 x g in a

refrigerated contrifuge. The supernatant was decanted and the protein

pellet taken up in 5 ml cold distilled water, transferred to a dialysis

bag and dialyzed against water overnight to remove all traces of

ammonium sulfate. The resulting dialysate was used directly for

further fractionation, or frozen at -200C for later use.

Total protein samples prepared in this manner were taken up in

100 ml cold distilled water and placed on an Amicon High Performance

Thin Channel Diafiltration System equipped with an LP-1 pump. This

system can be used to separate proteins as to approximate molecular

weight and the procedures for fractionating proteins of Petunia pollen

eluents is diagrammed in Figure 3.1.

Carbohydrate Assay. Total carbohydrates present in protein frac-

tions of pollen eluents were determined by the phenol-sulfuric acid

technique of DuBois et al. (1956).

Protein Assay. All protein determinations were performed according

to the procedure outlined by Lowry et al. (1951).

In Vitro Pollen Germination. Germination tests of eluted and non-

eluted pollen samples of Petunia hybrida were performed in 0.05 M






-42-













x ~
a -*4 .44)0 N
5. 0 H
0 0
a e o ZY "g ^ t;I



c 0~
2- 5 c 4 _
aIYLE >a-:
N S4 / CSC I 4 .5 A
4, ." 'd.C ..-4.-'.'a -. -' f i ^

a^ ~ ~ ~ r r./ u "
0 6
"S 2\ u oo c~ io C
C V NC- N C 00 ON -.0 Ca
2o'--' 3 S N a '1 0 N aO






Ai t
NC I 4 0*



-^ NC 44- 00* 44 4
U. B ~m So u ^ o
o~ ~ -^ vm c ^d \[3 c



















lg~~c a)^ \ 3
& 0 .4 0 .4440 -- \ n4 NC
44 .0 N a4 N \ < Ca'H
NC N.4. N 4.'> .. 4' -3






0.0 ^ 00 4) \4 '"-4 0.
w% 4 N lN .a


















3tZ
~~~~ an 0 N N
NO o 0 4'N 3 O Ca -







BO0^ \ ^K c *Ca
S0. ~ ~ a- -g! -* '
N" .S cS 3 NC" 44.4
"~ ~ U1
44-4 .0 .-- .4 Co aH









O in
O "" 0 0










So go s 3 ^S
0. 0 4 .0 C g0









Vlfj/. ') ^ 0 -' 0- .- "0 H-r'
.44 .4 ..OC4040CC









*....~ ^ ^ / uaa -i -
4)4' u N U N '- 'J -4001 0'-&-
-U .4--- 0 0. 04* 44 -' ''
'-' 0 s/ "N 4)00
4, C 0 -a NC ^ U











.34 'id) N .0 N SC
04 4,0i .004 NC c4. \ 1. Ca N -
m- ufx c:o u\ c -<,-U
NX 0o 4












Bl a a U" CI r( t. 0 ^ -^>
0 440 .-'.'.4N C 0










oI a ^ ^ ^ 3
NO NC0 NN




Ni Ca i -"(
04C 08 '.
,0C4C 40 ow~o-
ip :3 44-




.0 0
0NC wan
'-4t .4" N
4, 00 .0
an 4-. ONU




"-'"'. 4,-V 4 C C


444 "
0~4 "4442 t NC4-44

4)0 ] .44 044' NC H 4
,~ 2"o 22. ~ I '
4444 NC ,4 .4400 -4 I 444 44
.64) ~ 0 "'ICa '




-43-



calcium phosphate buffer (pH 6.0) containing 0.4 1M raffinose and 80 ppm

boron, supplied as H3 BO3 Eluents and protein fractions were dissolved

in germination medium prior to the addition of pollen. Germination

was at 270C for 3 hrs on a water bath shaker and pollen concentration

was maintained at 5 mg per ml (25 mg per 25 ml).




-44-


Results


The effect of duration of elution on the quantity of protein eluted,

as determined by the Lowry procedure, is shown in Figure 3.2. The

data show that 2 min elutions release 84.2% of the total protein eluted

in 10 min and are likely to contain almost all of the sporophytic

components of pollen wall eluents, but also much of the gametophytic

protein components of the pollen wall.

Table 3.1 presents the results of a germination test involving

non-eluted control pollen of Petunia hybrida and pollen after elution.

Such tests indicate that elution has greatly reduced the ability of

pollen to germinate. After three hours no noticible difference was

observed in pollen tube length between eluted and control samples.

Throughout all experiments pollen tube length averaged 97.6 after

3 hrs. The percentage of pollen grains that germinated varied somewhat

with the pollen sample used. If eluted compounds are added to eluted

pollen samples in amounts present in non-eluted controls, the germina-

tion capacity of eluted pollen is significantly restored (Table 3.1).

Non-eluted control pollen germinated at approximately 51-64%, the

variation being due to the pollen sample.

Results of adding acidic, basic, neutral and non-polar fractions

of pollen eluents to germinating eluted pollen are reported in Table 3.2.

The neutral fraction (phl 6.0) and the acidic fraction (pH 8.5) con-

siderably increase per cent germination of eluted pollen (83.4% and

101.4%, respectively). The acidic fraction boosts the percent germina-

tion of eluted pollen to the level of non-eluted controls. Addition of
























160




140

a
60
E 100


0

60




20



0 I 2 3 4 5 6 7 8 9 10

Time of Elution (min.)


Figure 3.2. Effect of time of elution on quantity of protein eluted
from pollen of Petunia hybrida. Proteins were determined
according to the Lowry procedure (Lowry et al., 1951).




-46-


Table 3.1

The Effect of Pollen Eluents of Petunia hybrida on
Germination of Eluted Pollen Samples



Treatment Germination (Per cent of Control)


Control 100

Eluted pollen 48.8
(20 ml water
for 2 min)

Eluted pollen 71.8
plus eluent



Values are reported as per cents of control germinations, which ranged
from approximately 51% to 64%, the variability being dependent on the
pollen sample. Total eluents were prepared as described in the text.
All germinations involved 25 mg pollen; in all cases eluent added is
that quantity derived from 25 mg pollen.











Table 3.2

The Effect of Basic, Neutral, Acidic, and Non-Polar Fractions of Eluents
of Petunia hybrida Pollen, Prepared by Ethyl Acetate Partitioning, on
Germination of Eluted Pollen Samples



Treatment Germination (Per cent of Control)


Control 100

Eluted Pollen 50.5

pH 3.0 phase 4.6
(basic fraction)

pH 6.0 phase 83.4
(neutral fraction)

pH 8.5 phase 101.4
(acidic fraction)

ethyl acetate phase 14.9
(non-polar fraction)



Values reported are per cents of control germinations. All germinations
involved 25 mg pollen; in all cases eluent added is that quantity
derived from 25 mg pollen. Values are means of 4 replications of three
experiments. For each replication 400 pollen grains were counted.








basic and non-polar fractions decreases the germination capacity of

eluted pollen.

Table 3.3 presents the effects of amino acid-protein and carbo-

hydrate components of neutral and acidic fractions of pollen wall

eluents on per cent germination of cluted pollen grains. Protein-amino

acid components eluted from Dowex 50W-8X columns with 4.0 N NH OH of

both acidic and neutral fractions are effective at boosting the per cent

germination of eluted pollen close to that of controls (97.4% and

80.9%, respectively). When carbohydrate components of acidic and

neutral fractions eluted from Dowex 50W-8X columns are added, no

stimulation of pollen germination was observed (Table 3.3).

Table 3.4 shows that after heat treatment, eluents cannot restore

the germination capacity of eluted pollen.

A diagram of the polyacrylamide gel electrophoretic pattern of

proteins present in acetone protein precipitates of pollen eluents of

Petunia hybrida redrawn from gel scans is presented in Figure 3.3. A

gel scan is presented in Figure 3.4. A total of 19 individual bands

was revealed by staining with Coomassie blue, indicating a strong

diversity of protein components with respect to both charge and molecular

weight. This conclusion is further supported by molecular weight range

determinations of eluted proteins. Analytical procedures using liquid

chromatography indicate a range in molecular weight between approximately

5,000 and 61,000 daltons. No sharp individual peaks were found, in-

dicating a continual diversity in molecular weight between the above

listed limits. No particular protein fraction constitutes a high

percentage of the total.




-49-


Table 3.3

The Effects of Amino Acid-Protein and Carbohydrate Components of Dowex
50W-8X Effluents of Neutral and Acidic Fractions of Pollen Eluents of
Petunia hybrida on Germination of Eluted Pollen Samples



Treatment Germination (Per cent of Control)


Control 100

Eluted pollen 49.0

Neutral eluent fraction, 51.2
0.4 N NH40 Dowex 50W-8X
effluent (carbohydrate
component)

Neutral eluent fraction, 80.9
4.0 N NH40H Dowex 50W-8X
effluent (amino acid-
protein component)

Acidic eluent fraction, 53.2
0.4 N NH40H Dowex 50W-8X
effluent (carbohydrate
component)

Acidic eluent fraction, 97.4
0.4 N NH40H Dowex 50W-8X
effluent (amino acid-
protein component)



Values reported are per cents of control germinations. All germinations
involved 25 mg pollen; in all cases eluent added is that quantity
derived from 25 mg pollen. Values reported are means of 4 replications
of three experiments. For each replication 400 pollen grains were
counted.











Table 3.4

The Effect of Antoclaved Pollen Eluents of Petunia hybrids
on Germination of Fluted Pollen Samples


Treatment


Germination (Per


cent of Control


Control


Eluted pollen


Untreated eluent

Autoclaved eluent


47.6

74.1

42.6


Values reported are per cents of control germinations. All germinations
involved 25 mg pollen; in all cases eluent added is that quantity
derived from 25 mg pollen. Values reported are means of 4 replications
of two experiments. For each replication 400 pollen grains were counted.

















ORIGIN






































Figure 3.3. 3. iscont inuouS poylv;eryliumido gel eloct roplhoresis of the
; c on -p rec i i Li t ed proLein frMct ion of 20 F il (2 niii)
e I Iunts t o[ p]o 1 1 ii ion f Ptn i y i i D'Ievel' I opCd l t 2.5
imiIii [iIImps per gI Upper buiFfur Lris-glyci lne (pH 8.5);
low l r Ill I ris-HCI (pli 8.1). Rudrriwni from trns 1 sclIn.































O



z

0












ORIGIN






Figure 3.4. Gel scan of discontinuous polyacrylamide gel electro-
phoresis of acetone precipitated protein fraction of
20 ml (2 min) eluents of Petunia hybrida. Developed
at 2.5 milliamps per gel and stained with Coomassie
blue. Upper buffer tris-glycine (pH 8.5); lower buffer
tris-HCI (pH 8.1).





-53-


Experiments 1, 2, and 3 testing the germination boosting capacities

of molecular weight fractions of ammonium sulfate precipitated proteins

greater than 10,000 daltons, greater than 50,000 daltons and greater

than 50,000 but less than 100,000 daltons, respectively, are reported

in Table 3.5. The results of these experiments indicate that proteins

between 50,000 and 100,000 daltons appear to be responsible for in-

creasing the per cent germination of eluted pollen.

When lyophilized 50,000 to 100,000 dalton fractions are taken up

in protein extracting solution and applied to pre-charged polyacrylamide

gels and subjected to electrophoresis under conditions identical to

those used for electrophoresis of the total protein fraction, staining

with Coomassie blue reveals only two bands (Figure 3.5). A gel scan is

presented in Figure 3.6. This indicates that the 50,000 to 100,000

dalton fraction responsible for restoring the germination capacity of

eluted pollen contains a maximum of two proteins. Since electrophoretic

separation is based on charge as well as molecular weight, it cannot be

concluded, however, that two molecular weight species are involved in

restoring germination capacity. In addition, cellulase assays per-

formed according to the procedure of Meyers et al. (1960), in which

cellulase activity is measured in terms of glucose release, revealed

no cellulolytic activity in the 50,000 to 100,000 dalton fraction.

Acetone precipitates of eluted pollen proteins contain approxi-

mately 16% carbohydrate, as determined by the phenol-sulfuric acid assay.

The effect of varying quantities of acetone precipitated protein from

total pollen eluents on the germination capacity of eluted pollen

samples is presented in Figure 3.7. This experiment was undertaken

to determine if germination restoring components are functioning in a










Table 3.5

Effect of Specific Molecular Weight Fractions of Ammonium Sulfate Pre-
cipitated Proteins of Eluents of Pollen of Petunia ihybrida on the
Germination of Fluted Pollen


EXPERIMENT 1: Effects of ammonium sulfate precipitated
than and less than 10,000 daltons on the germination of
Petunia hybrida


Treatment

Non-eluted pollen


Per cent
Germination

51.8


Eluted pollen

Eluted pollen plus proteins
greater than 10,000 daltons

Eluted pollen plus proteins
less than 10,000 daltons


EXPERIMENT 2: Effects of ammonium sulfate precipitated
than and less than 50,000 daltons on the germination of
Petunia hybrida


Treatment

Non-eluted pollen


Per cent
Germination

63.8


proteins greater
eluted pollen of


Per cent of
Control

100


64.7

101.3


74.4


proteins greater
eluted pollen of


Per cent of
Control

100


Eluted pollen

Eluted pollen plus proteins
greater than 50,000 daltons

Eluted pollen plus proteins
less than 50,000 daltons


60.1

96.9


63.7









Table 3.5
(Continued)



EXPERIMENT 3: Effects of ammonium sulfate precipitated proteins greater
than 100,000 daltons and proteins less than 100,000 daltons but greater
than 50,000 daltons on the germination of eluted pollen of Petunia
hybrida

Per cent Per cent of
Treatment Germination Control

Non-eluted pollen 64.8 100

Eluted pollen 40.6 62.7

Eluted pollen plus proteins 41.6 64.2
greater than 100,000 daltons

Eluted pollen plus proteins 54.8 84.6
between 50,000 and 100,000
daltons



Germinations performed for the above described experiments are reported
as means of 4 replications of 2 experiments. For each replication 400
pollen grains were counted. Variations in values for the germination
of eluted and control pollen is due to the diversity of pollen samples.

















ORIGIN








































Figure 3.5. Discontinuous polyacrylamide gel electrophoresis of the
protein fraction with molecular weight between 50,000 and
100,000 daltons, as prepared by ultrafiltration. Proteins
were prepared from 20 ml (2 min) eluents of pollen of
Petunia Ihybrida by precipitation with ammonium sulfate.
Gels were developed at 2.5 milliamps per gel. Upper buf-
fer tris-glycine (pH 8.5); lower buffer tris-HCI (ph 8.1).
Redrawn from gel scan.





























0



z
or
0









ORIGIN








Figure 3.6. Gel scan of discontinuous polyacrylamide gel electro-
phoresis of the protein fraction with molecular weight
between 50,000 and 100,000 daltons, as prepared by
ultrafiltration. Proteins were prepared from 20 ml
(2 min) eluents of pollen of Petunia hybrida by precipi-
tation with ammonium sulfate. Developed at 2.5 milliamps
per gel and stained with Coomassie blue. Upper buffer
tris-glycine (pIl 8.5); lower buffer tris-HCl (pH 8.1).





-58-


PERCENT INCREASE
IN GERMINATION
A
20





15





10





5
A





25 50 75 100

QUANTITY OF ELUENT ADDED (tl)





Figure 3.7. Effect of varying quantities of acetone-precipitated
protein fraction of eluents of Petunia hybrida pollen on
germination capacity of eluted pollen. Proetin pre-
cipitates from 125 mg pollen were redissolved in 500 pl
germination medium and added to 25 mg eluted pollen
samples prior to germination. 100 ml contains elutable
proteins from 25 mg pollen. Values are averages of 4
experiment s.








concentration independent manner, in which case their presence alone

may result in the restoration of germination, or in a concentration

dependent manner. One hundred twenty-five milligrams of pollen were

eluted and proteins precipitated as described previously. The protein

precipitate was redissolved in 500 ol pollen germination medium and

varying amounts were added to 5 ml germination medium containing 25 mg

eluted pollen. The resulting curve is a straight line. Linear regres-

sion analysis gives an equation of y = 9.21x 0.55, with an r value of

0.998, indicating a good linear relationship between the per cent

increase in germination of eluted pollen and the quantity of acetone

precipitated protein added, within the range zero to the amount present

in non-eluted pollen samples.





-60-


Discussion


Many factors have been implicated in controlling pollen germina-

tion, including calcium (Brewbaker and Kwack, 1963), boron (Schmucker,

1935), growth substances (Loo and Hwang, 1944; Bose, 1959; Raghavan and

Baruah, 1959; Ermsweller et al., 1960; Vasil, 1960b; Carmichael, 1971),

vitamins (Cooper, 1939; Vasil, 1960b), antibiotics (Vasil, 1960b),

colchicine (Sen and Verma, 1963), inorganic ions, such as manganese,

cobalt, sodium and potassium (Brink, 1924; Cooper, 1939; Yamada, 1958;

Vasil, 1960b; Zielinski and Olez, 1963), and plant tissue extracts (Sen

and Verma, 1963). The results presented here show that, in addition to

the above listed factors controlling pollen germination, protein com-

ponents contained within the pollen walls of Petunia hybrida are

critical in controlling pollen germination in vitro.

After elution with water for 2 min the germination capacity of

Petunia pollen is markedly decreased. However, the addition of eluted

substances back to eluted pollen samples significantly restores germina-

tion capacity. The germination level of control pollen samples can be

achieved by adding specific protein fractions back to eluted pollen

samples. Since addition of acidic and neutral fractions of pollen

eluents increases the germination acpacity, it is concluded that two

or more components of the pollen cluents are likely responsible for

restoration of germination capacity.

The addition of amino acid-protein and carbohydrate components

of neutral and acidic Fractions to eluted pollen shows that only

protein-amino acid components possess the ability to increase germina-

tion of eluted pollen. These experiments lend evidence that neutral








and acidic proteins are involved. This contention is further supported

by the fact that denatured eluent possesses no germination boosting

capacity. In addition, there is a linear relationship between the

added quantity of acetone precipitated protein prepared from pollen

eluent (between the range zero to the amount present in non-eluted

samples) and the capacity for germination of eluted pollen samples.

The addition of larger quantities of eluent protein results in higher

percentages of germination of eluted pollen. This indicates the

possible direct involvement of eluted proteins in the germination

process.

Gel electrophoresis of the protein fraction indicates a high

diversity with regard to both size and charge. Nineteen individual

bands were present (Figures 3.3 and 3.4). Electrophoresis of ammonium

sulfate precipitates of pollen wall proteins of Cosmos bipinnatus

(Howlett et al., 1975) revealed only six major fractions; preparative

procedures used in Howlett's study eliminated proteins with molecular

weights less than 10,000 daltons, while the present study included such

low molecular weight components.

Molecular weight determinations support the electrophoretic

observation of a diverse protein component of pollen walls of Petunia.

Liquid chromatography of acetone protein precipitates yields a range

of molecular weights. These ranges correspond to those obtained by

Howlett et al. (1975) using SDS polyacrylamide gel electrophoresis to

determine molecular weight. However, they found two major peaks at

11,500 and 30,000. Results with Petunia pollen proteins indicate a wide

range in molecular weight with no major peaks, i.e., without a large

percentage of the total protein compartmented within a narrow range of








molecular weight. This is clearly seen on polyacrylamide gels (Figures

3.3 and 3.4), where electrophoretic separation on the basis of both

charge and molecule size reveals many components.

Howlett et al. (1975) reported two faint PAS-staining bands after

electrophoretic separation of pollen wall proteins of Cosmos bipinnatus.

Techniques for localizing carbohydrate-containing proteins on poly-

acrylamide gels of Petunia pollen proteins have been unsuccessful, which

may indicate the absence of glycoproteins. However, analysis of the

total protein fraction of Petunia eluents indicated that it contains

approximately 16% carbohydrate. This value agrees favorably with the

value of 14.4% carbohydrate reported for short ragweed allergen

(Underdown and Goodfriend, L969). If glycosylation is a prerequisite

for secretion, as Eylar (1965) has suggested, it is logical that protein

components synthesized in the microsopre protoplast and deposited

extracellularly in wall sites are glycoproteins.

Preparation of 10,000, 50,000 and 100,000 dalton fractions by

ultrafiltration of ammonium sulfate precipitated proteins of Petunia

pollen eluents and addition of these fractions to samples of eluted

pollen germinating in vitro have shown that proteins between 50,000 and

100,000 daltons are likely responsible for resotring the germination of

eluted pollen to control levels. Electrophoresis of this fraction

yields only two bands indicating that the germination restoring capacity

is contained in one, or possibly two, molecular species. Table 3.5

reports that eluted protein fractions derived from 25 mg pollen and

within the molecular weight range of 50,000 to 100,000 daltons boost

the germination of 25 me samples of eluted pollen to 83.9% of the con-

trols. Separation techniques, especially a two-step procedure as was








used to prepare the 50,000-100,000 dalton fraction, result in some loss

of protein. Also, it should be kept in mind that the germination

restoration is dependent on the amount of the protein fraction added

(Figure 3.4). Protein loss during preparative procedures is likely to

contribute to the seemingly low value for per cent germination of eluted

pollen plus the 50,000 to 100,000 dalton fraction.

Since the addition of a fairly narrow range molecular weight

fraction can significantly restore the germination capacity, which was

decreased by elution, such factors may also be controlling germination

of Petunia pollen in vivo. Both acidic and neutral proteins appear

responsible. Since proteins active in germination restoration are heat-

labile, an enzymatic nature may be indicated. Possible candidates for

these proteins would include enzymatic proteins whose molecular weights

are within the range found to be active in boosting the germination of

eluted pollen. The possibility of the cellulase complex (molecular

weight range 52,000 to 76,000 daltons) being a key factor in the ger-

mination of Petunia pollen is attractive, since softening of the

pectocellulosic intine layer in pore regions of pollen grains is

required for pollen tubes to emerge. Stanley and Search (1971) found

considerable cellulose activity in pollen eluents of pine, cattail and

pear. However, assays for cellulase activity, measured as the release

of glucose from carboxymethyl cellulose, have shown no cellulase

activity in the 50,000 to 100,000 dalton fraction which promotes pollen

germination.

The work of Heslop-Iiarrison, Knox and co-workers has revealed a

possible function for diffusible proteins of pollen grains as "recogni-

tion substances" in both interspecific (Knox et al., 1972) and








intraspecific incompatibility (Howlett et al., 1975), although Fett

et al. (1976) have not been able to find such "recognition substances"

contributing to gametophytically controlled self-incompatibility in

Lilium longiflorum. The only available study somewhat comparable to

the work presented here focused on components of pollen eluents of

Cosmos bipinnatus (Howlett et al., 1975). Cosmos, a member of the

Compositae, has trinucleate pollen and a sporophytic incompatibility

system. The incompatibility reaction takes place on the surface of the

stigma within 30 min of pollination. On the other hand, Petunia, a

member of the Solonaceae, has binucleate pollen anda gametophytically

controlled incompatibility system. The incompatibility reaction takes

place after the pollen grain has germinated and the pollen tube has

entered the style. Pollen of Petunia hybrida germinates easily on

compatible or incompatible stigmas, regardless of the presence of

elutable components of the pollen wall (Chapter Two). These results

are similar to those obtained with Lilium (Fett et al., 1976). How-

ever, elutable components of Petunia pollen appear intimately involved

with germination in vitro.

Both carbohydrate and protein components of eluents of Petunia

pollen are diverse. It is interesting that carbohydrate and protein

components of pollen eluents of Cosmos are considerably less diverse.

Since in Cosmos the acceptance or rejection of a given pollen grain must

take place quickly and "recognition substances" are likely involved, it

is possible that a lack of diversity of pollen eluent components in

Cosmos indicates directed synthesis of specific molecules required for

the recognition reaction.





-65-


In Cosmos elutabLe proteins which are able to partially overcome

self-incompatibility are heat stable (Howlett et al., 1975). In pollen

of Petunia heat-labile proteins appear intimately involved in germina-

tion in vitro. Their absence results in poor germination ability,

which can be restored by the addition of eluted proteins. This adds

further support to the hypothesis that protein components of the walls

of pollen grains of Petunia and Cosmos are functionally distinct.















CONCLUSIONS


Analysis of the data concerning the quantities of carbohydrates,

proteins and total elutable components of pollen wall eluents of 42

angiosperm species forces the conclusion that greater amounts of all

of the above constituents are found in eluents of binucleate pollen

species. A closer look at pollen eluents of Petunia, a species with

binucleate pollen and whose physiology and genetics are fairly well

known, revealed a diverse carbohydrate component, including the presence

of several free monosaccharides and additional monosaccharide com-

ponents of oligosaccharides. It is likely that such carbohydrates

diffuse from "storage areas" of the pectocellulosic intine layer of the

pollen wall, or are products of the breakdown of tapetal cells and are

stored within the exine. Since some degree of similarity has been noted

between these carbohydrates and components of the transmitting tissue,

a possible nutritive function for these carbohydrates during early

pollen tube growth must be considered.

The observation that after elution pollen of Petunia germinates

poorly in vitro and that germination of eluted pollen can be restored

by the addition of eluted substances posed the question of which com-

ponents of Petunia pollen eluents were responsible for restoring

germination. Addition of the carbohydrate fraction of eluents to

eluted pollen germinating in vitro was not able to restore germination.

However, this does not indicate that carbohydrates of pollen eluents








are not serving roles as metabolic substrates during early pollen tube

growth on the stigma, but merely indicates the involvement of another

factors) in restoring pollen germination in vitro. This factor turned

out to be a heat-labile protein fraction, without detectable cellulase

activity, and with a molecular weight between 50,000 and 100,000

daltons. Since this fraction is a key in the control of germination

in vitro, it is also likely to be intimately involved in germination on

the stigma. Therefore, further work is suggested to identify the pre-

cise nature of this fraction in order to gain a more thorough knowledge

of the process of pollen germination and fertilization.

When comparing the analytical work described here, utilizing eluents

from a species having binucleate pollen, with a somewhat similar study

of pollen diffusates of a species having trinucleate pollen (Cosmos),

the most striking differences are seen in the diversity of eluent or

diffusate components. Petunia eluents contain higher diversity with

regard to both carbohydrate and protein components. Clearly, no

phylogenetic trends can be based on studies of only two species,

especially when analytical techniques used in determining character-

istics of eluents in the two studies differed somewhat. However, it

may be boldly suggested that elutable compounds from phylogenetically

primitive binucleate pollen species are not only greater in quantity

than those of species with trinucleate pollen, but may also contain more

diversity. Eluents of trinucleate pollen may have been derived by

reduction from the primitive binucleate condition and may reflect

synthesis of only specific molecules mediating sporophytically con-

trolled incompatibility responses.















REFERENCES


Abramson, H.A. Chemical, physical and immunological properties of
electrophoretically purified pollen extracts. Ann. Allergy 5,
19-26 (1947).

Auclair, J.L., Jamieson, C.A. Qualitative analysis of amino acids in
pollen collected by bees. Science 108, 357-358 (1948).

Bose, N. Effect of gibberellin on the growth of pollen tubes. Nature
(London) 184, 1577 (1959).

Brewbaker, J.L. Biology of the angiosperm pollen grain. Indian J.
Genet. Plant Breed. 19, 121-133 (1959).

Brewbaker, J.L. The distribution and phylogenetic significance of
binucleate and trinucleate pollen grains in the angiosperms.
Amer. J. Bot. 54, 1069-1083 (1967).

Brewbaker, J.L., Kwack, B.E. The essential role of the calcium ion
in pollen germination and pollen tube growth. Amer. J. Bot. 50,
859-865 (1963).

Brewbaker, J.L., Majumder, S.K. Incompatibility and the pollen grain.
Rec. Adv. Bot. 3, 1503-1508 (1961).

Brink, R.A. The influence of electrolytes on pollen tube development.
J. Gen. Physiol. 6, 677-682 (1924).

Brooks, J., Shaw, C. Chemical structure of the exine of pollen walls
and a new function for carotenoids in nature. Nature (London)
219, 523 (1968).

Carmicheal, J.W. The effect of gibberellic acid on in vitro pollen
germination in Digitaria pentzii Sent. Proc. Soil Crop Sci. Soc.
Fla. 30, 255-258 (1971).

Coca, A.F. Stulies in specific hypersensitiveness. V. Preparation of
fluid extracts and solutions for use in the diagnosis and treatment
of the allergies with notes on the collections of pollens. J.
immunol. 7, 163-178 (1922).

Cooper, W.C. Vitamins and the germination of pollen grains and fungus
spotes. Bot. Gaz. 100, 844-852 (1939).









DuBois, M., Giles, K.A., Hamilton, J.D., Rebers, P.A., Smith, F.
Colorimetric method for determination of sugars and related
substances. Anal. Chem. 28, 350-356 (1956).

Ermsweller, S.L., Uhring, J., Stuart, N.W. The role of naphthalene
acetamide and potassium gibberellate in overcoming self-incom-
patibility in Lilium longiflorum. Proc. Amer. Soc. Hort. Sci.
75, 720-725 (1960).

Eylar, E.11. On the biological role of glycoproteins. J. Theoret. Biol.
10, 89-113 (1965).

Fett, W.F., Paxton, J.D., Dickinson, D.B. Studies on the self-incom-
patibility response in Lilium longiflorum. Amer. J. Bet. 63,
1104-1108 (1976).

Green, J.R. On the germination of the pollen grain and the nutrition
of the pollen tube. Ann. Bet. 8, 225-228 (1894).

Griffiths, G.W., Brunet, R. Isolation of a basic protein antigen from
low ragweed pollen. Can. J. Biochem. 49, 396-400 (1971).

Haeckel, A. The enzymes of pollen. Planta 39, 431-459 (1951).

Hare, R.C. Physiology and biochemistry of pine resistance to the
fusiform rust fungus Cronartium fusiforme. Ph.D. Thesis,
University of Florida (1970).

Heslop-Harrison, .. Pollen wall development. Science 161, 230-237
(1968a).

Heslop-Harrison, J. Some fine structural features of intine growth
in young microspores of Lilium henryii. Port. Acta Biol. 10,
235 (1968b).

Heslop-Harrison J. The Pollen wall: Structure and development. In
"Pollen: Development and Physiology" (ed. J. Heslop-Harrison),
pp. 75-97, London, Butterworths. 1971.

Heslop-Harrison, J., Heslop-Harrison, Y., Knox, R.B., Howlett, B.
Pollen wall proteins: "Gametophytic" and "sporophytic" fractions
in pollen walls of the Malvaceae. Ann. Bot. 37, 403-412 (1973).

Heslop-Harrison, J., Knox, R.B., Heslop-Harrison, Y. Pollen wall
proteins: Exine held fractions associated with the incompatibility
response in the Cruciferae. Theoret. Appl. Genet. 44, 133-137
(1974).

Hollis, C.A., Tepper, II.B. Auxin transport within intact dormant and
active white ash shoots. Plant Physiol. 48, 146-149 (1971).









Howlett, B.J., Knox, R.B., Paxton, J.D., Heslop-Harrison, J. Pollen
wall proteins: Physiocochemical characterization and role in
self-incomatibility in Cosmos bipinnatus. Proc. R. Soc. Lond.
(B) 188, 167-182 (1975).

Jensen, W.A. Botanical Histochemistry, San Francisco, W.H. Freeman
and Co. 1962.

Johnson, P., Marsh, D.C. Allergens from common rye grass pollen
(Lolium perenne). I. Chemical composition and structure.
Immunochemistry 3, 91-100 (1966a).

Johnson, P., Marsh, D.C. Allergens from common rye grass pollen
(Lolium perenne). II. The allergenic determinants and carbo-
hydrate moiety. Immunochemistry 3, 101-110 (1966b).

King, T.P., Norman, P.S. Isolation studies of allergens from ragweed
pollen. Biochemistry 1, 709-720 (1962).

King, T.P., Norman, P.S., Connell, J.T. Isolation and characteriza-
tion of allergens from ragweed pollen. II. Biocehmistry 3,
458-468 (1964).

King, T.P., Norman, P.S., Lichtenstein, L.M. Studies on ragweed pollen
allergens. V. Ann. Allergy 25, 541-553 (1967).

Knight, A.H., Crooke, W.M., Shepherd, H. Chemical composition of
pollen with particular reference to cation exchange capacity and
uronic acid content. J. Sci. Fd. Agri. 23, 263-274 (1972).

Knox, R.B. Pollen wall proteins: Localization, enzymic and antigenic
activity during development in Gladiolus (Iridaceae). J. Cell
Sci. 9, 209-237 (1971).

Knox, R.B., Heslop-Harrison, J. Cytochemical localization of enzymes
in the wall of the pollen grain. Nature (London) 223, 92-94
(1969).

Knox, R.B., Heslop-Harrison, J. Pollen wall proteins: Localization
and enzymic activity. J. Cell Sci. 6, 1-27 (1970).

Knox, R.B., Heslop-Harrison, J. Pollen wall proteins: Localization
of antigenic and allergenic proteins in pollen grain walls of
Ambrosia spp. Cytobios 4, 49-54 (1971a).

Knox, R.B., Heslop-Harrison, J. Pollen wall proteins: The fate of
intine-held antigens on the stigma in compatible and incompatible
pollinations of Phalaris tuberosa L. J. Cell Sci. 9, 239-251
(1971b).

Knox, R.B., Heslop-Harrison, J., Reed, C. Localization of antigens
associated with the pollen grain wall by immunofluorescence.
Nature (London) 225, 1066-1068 (1970).








Knox, R.B., Willing, R.R., Ashford, A.E. Role of pollen wall proteins
as recognition substances in interspecific hybridization in
poplars. Nature (London) 237, 381-382 (1972).

Kroh, M. Nature of the intercellular substance of stylar transmitting
tissue. In Biogenesis of Cell Wall Polysaccharides (ed. F.
Loewus), pp. 195-206. New York, Academic Press. 1973.

Lamport, D.T.A. Cell wall metabolism. Ann. Rev. Plant Physiol. 21,
235-270 (1970).

Lewis, D., Burrage, S., Walls, D. Immunological reactions of single
pollen grains, electrophoresis and enzymology of pollen protein
exudates. J. Exp. Bot. 18, 371-378 (1967).

Loo, T., Hwang, T. Growth stimulation by manganese sulphate, IAA and
colchicine in pollen germination. Amer. J. Bot. 31, 356-367
(1944).

Lowry, O.H., Rosebraugh, N.J., Farr, A.L., Randall, R.J. Protein
measurement with the folin-phenol reagent. J. Biol. Chem. 193,
265-275 (1951).

Mlkinen, Y.L.A., Brewbaker, J.L. Isoenzyme polymorphism in flowering
plants. I. Diffusion of enzymes out of intact pollen grains.
Physiol. Plant. 20, 477-482 (1967).

Mikinen, Y.L.A., Lewis, D. Immunological analysis of incompatibility
(S) proteins and of cross reacting materials in a self-
incompatible mutant of Oenothera organensis. Genet. Res. 3,
352-363 (1962).

Marsh, D.G., Milner, F.H., Johnson, P. The allergenic activity and
stability of purified allergens from the pollen of common rye
grass. Int. Arch. Allergy 29, 521-526 (1966).

Meyers, S.P., Prindle, B., Reynolds, E.S. Cellulolytic activity of
marine fungi. Degradation of lignocellulose material. Tappi 43,
534-540 (1960).

Northcote, D.H. Chemistry of the plant cell wall. Ann. Rev. Plant
Physiol. 23, 113-132 (1972).

Poddubnaya-Arnoldi, V.A., Tsinger, N.V., Petrovskaya, T.P., Polunina,
N.N. Histochemical study of the pollen grains and pollen tubes
in the angiosperms. Rec. Adv. Bot. 1, 682-685 (1959).

Raghavan, V., Baruah, V. Effect of the time factor on the stimulation
of pollen germination and pollen tube growth by certain auxins,
vitamins and trace elements. Physiol. Plant. 12, 441-451 (1959).





-72-


Reynolds, E.S. The use of lead citrate at high pli as an electron
opaque stain in electron microscopy. J. Cell Biol. 17, 208-212
(1963).

Roland, F. Characterization and extraction of the polysaccharides of
the intine and the generative cell wall in pollen grains of some
Ranunculaceae. Grana 11, 101-106 (1971).

Rudenko, R.E. Significance of the male gametophyte for the taxonomy of
the Angiospermae. Biol. Zhurn. 44, 1467-1475 (1959).

Schmucker, T. Uber der Einfluss von Borsaure auf Pflanzen, insbesondere
keimende Pollenkdrner. Plant 23, 264-283 (1935).

Schnarf, K. Variation im Bau Des Pollenkbrnes der Angiospermen. Tabul.
Biol. 17, 72-89 (1939).

Schurhoff, P.N. Die Zytologie der Blutenpflanzen. Stuttgart, Enke
Pbl. Co. 1926.

Sen, B., Verma, G. In Plant Tissue and Organ Culture (eds. P.
Maheshwari and N.S. Ranga Swamy), p. 239, Delhi, University
of Delhi Press. 1963.

Sitte, P. Unterschungen zur Submikroskopischen Morphologie der Pollen
und Sporenmembranen. Mikroskopie 8, 290-299 (1953).

Stanley, R.G., Linskens, H.F. Enzyme activation in germinating Petunia
pollen. Nature (London) 203, 542-544 (1964).

Stanley, R.G., Linskens, H.F. Protein diffusing from germinating pollen.
Physiol. Plant. 18, 47-53 (1965).

Stanley, R.G., Linskens, H.P. Pollen: Biology, Biochemistry,
Management. New York, Springer-Verlag. 1974.

Stanley, R.G., Search, R.W. Pollen protein diffusates. In Pollen
development and physiology (ed. J. Heslop-Harrison), pp. 174-176,
London, Butterworths. 1971.

Stanley, R.G., Thomas, D. des S. Pollen enzymes and grwoth. Proc.
Assoc. So. Agric. Workers 64, 265-268 (1967).

Todd, F.E., Bretherick, 0. The composition of pollens. J. Econ.
Entomol. 35, 312-317 (1942).

Tsinger, N.V., Petrovskaya-Baranova, T.P. The pollen grain wall A
living physiologically active structure. Dokl. Akad. Nauk. SSSR
138, 466-469 (1961).

Underdown, B.J., Goodfriend, L. Isolation and characterization of a
protein allergen from ragweed pollen. Biochemistry 8, 980-989
(1969).





-73-


Vasil, I.K. Pollen germination in some Gramineae: Pennisetum
typhoideum. Nature (London) 187, 1134-1135 (1960a).

Vasil, I.K. Studies on pollen germination of some Cucurbitaceae.
Amer. J. Bot. 47, 239-247 (1960b).

Vasil, I.K. Studies on pollen germination of certain Leguminosae and
Cruciferae. Beit. Biol. Pfl. 38, 137-159 (1962).

Vasil, I.K. The new biology of pollen. Naturwissenschaften 60,
247-253 (1973).

Vithanage, H.T.M.V., Knox, R.B. Pollen wall proteins: Quantitative
cytochemistry of the origins of intine ane exine enzymes in
Brassica oleracea. J. Cell Sci. 21, 423-435 (1976).

Yamada, Y. Effect of cobalt on the growth of pollen. Kagaku (Science)
28, 257-258 (1958).

Zielinski, Q.B., Olez, H. Effects of the levels of manganese in the
culture medium on pollen germination and pollen growth of prune and
pear. Proc. Amer. Soc. liort. Sci. 83, 205-209 (1963).

Zolotovitch, G., Secenska, M. Biochemische Unterschungen des pollens
von Rosa damascena. C.R. Acad. Bulg. Sci. 15, 639-642 (1962).

Zolotovitch, G., Secenska, M., Deceva, R. Uber der VerAnderungen in
der Zusammensetzung der Saccharide and der Fermentaktivitat bei
der Lagerung von Rosenpollen. C.R. Acad. Bulg. Sci. 17,
295-298 (1964).















BIOGRAPHICAL SKETCH


Edward George Kirby, III was born 28 November, 1947, in Toledo,

Ohio. He attended elementary school in Birmingham, Michigan,and

graduated from Detroit Country Day School in 1965. He pursued a

Bachelor of Science degree in zoology at the University of Michigan

and was graduated in 1969. In addition, he received a secondary

teaching certificate in biology at that time.

He first attended the University of Florida in the fall of 1969

and was awarded the Master of Science degree in botany in 1971.

During the interim between his master's and doctoral studies he

spent two periods at the Botanisch Laboratorium, Universiteit Nijmegen

in Holland where part of his dissertation research was performed.

Currently he is a post-doctoral research associate at the Tree Genetics

Laboratory, Oregon Graduate Center, Beaverton, Oregon.

Edward G. Kirby, III is married to the former Melinda Scars Moody.


-74-











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.



\ \k- )

Indra K. Vasil, Chairman
Professor of 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.



/ '. /, ' I

Willard W. Payne, Co-chairman
Professor of 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.





Henry C.AAldrich
Professor of Microbiology












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.





Rayy. Coddard
Professor of Forest Resources and
Conservation







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.





Rex L. Smith
Associate Professor of Agronomy






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.

June 1977 \


Dean( college of Agriculre


Dean, Graduate School




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