PUTATIVE INCOMPATIE3ILXTY PROTEINS IN DISTYLOUS TURNERA SPECIES: IMMUNOBLOT AND IMMUNOCYTOCHEMISTRY ANALYSES

DAVOOD KHOSRAVI

A thesis submitted to the Faculty of Graduate Studies in Partial fulfilment of the requirernents

For the degree of MASTER OF SCIENCE

Graduate Programme in Biology York University

North York, Ontario

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PUTATIVE INCOMPATLIBILITY PROTEINS IN DISTYLOUS T?2RNERA SPECIES: IMMUNOBLOT AND

IMMUNOCYTOCHEMISTRY ANALYSES

by Davood Khosravi

a thesis submitted to the Faculty of Graduate Studies of York University in partial fulfillment of the requirements for the degree of

Master of Science

2000 O

Permission has been granted to the LIBRARY OF YORK UNI- VERSITY to lend or seIl copies of this thesis, to the NATIONAL LIBRARY OF CANADA to microfilm this thesis and to lend or seIl copies of the film. and to UNIVERSITY MICROFILMS to publ ish an abstract of this thesis. The author reserves other publication rights. and neither the thesis nor extensive extracts from it may be printed or other- wise reproduced without the author's written permission.

Abstract

Self-incompatibility was investigated in distylous Tzrmercr species (Tumeraceae)

by using polyclonal antibodies raised against recombinant proteins specific to styles and

pollen of the short-styled morph. Style and polien immune serums were used in IEF-,

SDS-immunoblotting and immunocytochemisty experiments to study and localize

proteins specific to the short-styled norph. In KEF-immuncbotting experiments, style

immune serum reacted with proteins specific to styles, and pollen immune serum with

proteins specific to the pollen. Ln SDS-immunoblotting, style immune serum detected a

single 40 Kd band in styles of the short-styled morph. This 40 Kd band did not appear in

styles of the long-styled morph or any other floral tissues examined. Pollen immune

serum detected a single 55 Kd band in pollen of both morphs, but not in any other floral

tissues examined. In immunocytochemistry experiments, style immune serurn localized

the 40 Kd protein to the transmitting tissue (where poilen tube growth takes place). This

supports the hypothesis that the 40 Kd style protein is invohed in incompatibility.

Because the pollen protein appears in both morphs, it iikely plays no direct role in

incompatibility.

Using SDS-PAGE and comparing protein profiles of styles from short- and long-

styled morphs, a 68 Kd protein specific to the styles of the short-styled rnorph was

identified. Exploring 66 individuals gom different species of Tzrner-a showed the

consistent appearance of 568 in styles of the short morph but not in the long. Neither

style nor pollen immune semm reacted with 568 indicating that S68 is a novel protein

possibly involved in self-incompatibility .

ACKNOWLEDMENTS

1 would like to thank Dr- Joel Shore for giving me the opportunity to continue my

education and his guidance throughout my work. 1 wouid like to thank Dr. Andre

Bedard, Dr. Daphne Goring, Dr. Barry Loughton, Dr. Laurence Packer, and Dr-

Lawrence Licht for valuable advice and use of their equiprnent. 1 would like to thank

Andreas Athanasiou for his help in various areas, fiom technical to philosophical issues.

Without the help of Farshad Tamari, undoubtedly, 1 (we) would not be able to accompiish

as much as we did.

TABLE OF CONïXNTS

... ........................................................................................ List of Tables vu1

................................................................................... List of Lllustrations ix

................................................................................ List of Abbreviations. x

........................................................................................... Introduction - 1

............................................................................. Methods and Materiais -19

................................................................................................ Results 31

........................................................................................... Discussion -52

........................................................................................... References -68

vii

LIST OF TABLES

Table 1. Distylous species used in this study.. . . . . . . . . .. . . . .. . . . . . . . . .. . . . . . . . . . . . . . . . .... 20

Table 2. Survey of various population and species for the presence of S68.. . . . . . . - 3 O

Table 3 Presence and absence of S68 for the populatioas and species surveyed.--5 I

. . . Vlll

LIST OF ILLUSTRATIONS

Figure 1 .

Figure 2 .

Figure 3 .

Figure 4.

Figure 5 .

Figure 6 .

Figure 7 .

Figure 8 .

Figure 9 .

Figure 10 .

Figure 1 1 .

Figure 12 .

Figure 13 .

Figure 14.

Reciprocal arrangement of pistil and anthers in heteromorphic species . . - 3

Identification of the style and pollen specific proteins .... ... ................ 18

Identification of the style and &sion protein .................................. -32

Detection of the style specific protein .......................................... 35

The expression of S40 is specific to the sqles of short-styled morph ..... -36

... Detection of the style specific proteins using the style immune serum -38

......... Detection of the pollen specific proteins .... .......................... -40

Detection of the pollen specifrc proteins using SDS-immunobIotting ..... 41

Immunolocalizaition of S40 to tissue in the styles of BRY .................. 45

The Irnmunolocaiization of S40 to style tissue in 3 different species ...... 46

The imrnunolocalization of S40 to stigma tissue in 3 different species .. -47

......... The imrnunolocaiization of the pollen specific proteins to polien 48

....... Identification of a novel style protein ................................. ... 49

SDS-polyacrylarnide gel analysis of the S68 in MHOMO ................. -50

LIST OF ABBERVIATIONS

AEB SF, 4-(2-Aminoethy1)-bemenedonyl ff uoride HCL

ARC 1, A m repeat containing

BRY, T shrlata plant, from Arco Verde, Brazil

EDTq Ethylenediaminetetraacetic Acid

IEF, Isoelectric focusing

IPTG, isopropyl- l -trio-P-~galactopyranoside

Kd, Kilodaltons

MHOMO, Mutant homo style

NaDOC, Na Deoxycolate

NBP, Nitroblue tetrazoiium

NP, Nonidet P40

PBS, Phosphate buffered saline

PCP, Pollen coat protein

PI, Isoelectric point

PMSF, Phenylmethyl sulfonyl Auoride

PS55s, 55 Kd pollen specific proteins

PVDF, Polyvinylidene difluoride

S40, 40 Kd short-styled specific style protein

S68, 68 Kd short-styled specific style protein

SCR, S-locus cysteine-rich protein

SD S-P AGE, SD S-poly acrylamide gel electrophoresis

SL8, T.subzrlara individuais fiom the Sao Luis 8 population

SLG, S-locus glycoprotein

S-locus, Self-incompatibility locus

SRK, S-locus receptor kinase

TBA, Tertiary butyl alcohol

TBS, Tris buffered saline

1 Introduction

Anti-selfing mechanisms in the angiosperms range fiom simple physical baniers

between male and female reproductive organs (e-g. herkogamy) to cell-ceil interactions

between pollen and the pistil (Herrero and Honnaza 1996). This physiological interaction

that results in rejection of the self-pollen is termed self-incompatibility, in contrast to the

self-sterility where infertility is contributed fiom either pollen or pistil (Lundqvist 1964;

de Nettancourt 1977). A self-incompatibility system is an effective way of promoting

outbreeding, thus, maintainhg heterozygosity. It has been estimated that half of the

species of flowering plants possess self-incompatibility (Darlington and Msther 1 949;

Brewbaker 1959). In fact, the sudden nse and success of the angiosperms in the

Cretaceous penod, according to Whitehouse (1950), is because of the evolution of self-

incompatibility and the avoidance of the hannful effects of inbreeding depression (de

Nettancourt 1977; Barrett and Cruzan 1994).

Franklin et al. (1995) classify self-incompatibility systems on the basis of three

critena, narnely, the genetics of the pollen phenotype, floral morphology, and the nurnber

of genes involved. Based on the incompatibility phenotype of the pollen, d l of the

incompatible species are put into two groups: 1) in sporophytic incompatibility, the

incompatibility phenotype of the pollen is determined by the genotype of the plant

producing the pollen, 2) in gametophytic incompatibility, the genotype of the individual

pollen grain determines the incompatibility phenotype of the pollen (de Nettancourt

1977; Franklin et al., 1995; Dodd et al., 1996). With respect to the morphology of

flowers and the number of mating types in a population, self-incompatible plants can be

either homomorphic, having one type of flower, or heteromorphic having two (distyly) or

three (tristyly) types of flowers. In distylous species there are long-styled morphs, where

the style is long and anthers are positioned below the styles, and the short-styled morphs,

where the style is short and anthers are positioned above the style (Fig. 1). Compatible

mating involves crossing between the morphs. In tristyly, there are long-, rnid- and short-

styled morphs. Each morph has two whorls of stamens that are borne at the levels not

occupied by the sigma, e-g. in the mid-styled morph, anhers are positioned above and

below the stigma but not at the sarne level (Fig. 1). The arrangement of the styles and

anthers in distylous and tristylous species, is referred to as reciprocal herkogamy. A

compatible mating, in tristyly, takes place only when pollen f?om a given level in one

morph pollinates the stigmas at the same level in another (e-g. short level pollen fkom the

mid- and long-styled morphs pollinates stigmas of the short-styled morph; Barrett and

Cruan 1994; Franklin et al., 1995). In contrast to heterostylous species, homomorphic

species are cornposed of numerous morphologically identicai mating types (Barrett and

Cruzan 1994). Interestingly, the number of alleles involved is also different in

hornomorphic and heteromorphic systems. Homomorphic species with either

gametophytic or sporophytic self-incompatibility are controlled by a polyallelic series at

one, two, or, more rarely, several loci (de Nettancourt 1977; Franklin et al., 1995).

Heterostyly is controlled by two alleles per locus and one or two loci (Lewis and Jones

1992).

Heterostylous species comprise only a srna11 proportion of the self-incompatible

angiosperms (Barrett and Cruzan 1994). Despite their fascinating characteristics, little or

Fig. 1. Depicted here is the reciprocal arrangement of pistil and anthers in heteromorphic

plants. Distyly, with short- and long-styled morphs (S and L respectively), and Tristyly

with iong-, mid-, and short-styied morphs (L, M, and S, respectively). Compatible

pollinations are indicated by the arrows, al1 other combinations are incompatible and

usually result in reduced or no seed set (modified fiom Barrett 1992).

2. Tristyly

no molecular studies have been done on heterostylous plants until recently w o n g et al.,

1994 a; Athanasiou and Shore 1997). On the other hand, p a t e r attention has been given

to the molecular studies of homomorphic plants, which has resulted in identification of a

number of genes invoIved in the self-incompatibility system.

This study uses Turnera (Tumeraceae) as a mode1 group of species to study the

molecular biology of self-incompatibility and other characteristics that are common to

distylous plants. Extensive studies have been done on the population biology and genetics

of Turnera species by Barrett (1978), Shore and Barrett (1984, 1985% b 1986, 1990),

Shore (1 990, 199 1).

In the introduction to this thesis, 1 will bnefly review 1) the historical

observations and investigations made on self-incompatibility systems and heteromorphic

plants, 2) studies of the physiology and molecular biology of self-incompatibility in

hornornorphic sporophytic and gametophytic species, specifically, the best studied

families (E3 rassicaceae, Solanaceae, and Papaveraceae). Emp hasis will be given to studies

of the physiology and molecular biology of self-incompatibility in heteromorphic

systems, particularly in distylous T'era species.

1.1 History

Self-incompatibility was first discovered in Verbascum phoerticium in 1764 by

Kolreuter, accordhg to East and Part (1917, cited in Franklin et al. 1995). Heterostyly

was notea in Primula by Clusius (Van Dijk 1943) as early as the 1 6UL century, and

Hildebrand, working with Primula and 0SÉali.s rosea, was the first to establish an

association between heterostyly and self-incompatibility (l3arrett 1992). Hildebrand' s

data were not entirely convincing, since they merely suggested that intercrosses yielded

more seed than the self- and intra-morph crosses, however, his studies were precursors to

the fiiture work by Darwin. Studying different distylous species nom different genera

(eg. Fago-* PuZrnonma, Limrm, Hottunia, andPolygomrm)), Darwin (1877) showed

that the self-and intra-morph pollinations are incompatible and intercrosses are

compatible. Studies, such as these, by Darwùi and Hildebrand, established that the main

barrier to selfing results fiom the incapacity of self-pollen to promote seed-set and not

fkom the architecture of the flower. After the rediscovery of Mendelian genetics in the

fust decade of the 20* century, the study of the genetics of heterostyly began by initial

collaborative work of Bateson and Gregory (1905) and then by Althausen (1908),

Dahlgreen (1916, 1922), Gregory (1915), and Eghis (1925) (de Nettancourt 1977). These

workers showed that self-incompatibility and floral polymorphism are govemed by a

single gene complex which segregates as a simple Mendelian locus (de Nettancourt

1977).

1.2 Homomorphie Sporophytic Self-incompatibility Systems

In thts incompatibility system, BrasSica oleracea, B. campestris (also referred to

as rapa) and "their allotetrap!oid hybrid, B, nqs"(Brassicaceae) have been studied

extensively (Dodds et al. 1996). The pistils of BrasSica have dry stigmas covered with a

waxy cuticle and show the highest ievel of the incompatibility reaction with the opening

of the flower (de Nettancourt 1977; Dickinson et al. 1990, 1992; Hinata et al. 1993; Trick

and H e h a n n 1992; Nasrallah and Nasrallah 1993). During compatible pollinations, the

pollen tubes penetrate the stigmatic cuticle and grow through a specialized region of the

papillar cell wall. When pollinated with incompatible pollen, recognition and rejection

occurs rapidly on the surface of the stigrna where pollen is prevented fiom fully

hydrating.

1.2.1 Pollen phenotype determination in Brassica

Pollen phenotype determination and time of gene action in the sporophytic system

has been explained in the foilowing way. Heslop-Harrison et al. (1973) postdate that the

time of gene action occurs during the late phase of pollen maturation in the tapetuni,

where synthesized incompatibility constituents are transferred to the microspore. A

number of studies of the mo1ecuIar biology of the Brassicaceae strongly support this

postdate. The tapetum cells are sporophytic thus any self-incompatibility determinants

they produce would reflect the S-genotype of the diploid plant (Dodds 1996). Recently,

Schopfer et al., (1999) cloned a S locus-encoded cDNA fiom B. campesnis, which they

refemed to as the S locus cysteine-rich protein (SCR) gene, and provided strong evidence

that it is the pollen determinant of seif-incompatibility. The results of RNA gel blot

analysis showed that the SCR gene is active post-meiotically and gametophytically in

microspores (Schopfer et al., 1999). However, these data do not exclude the expression

of SCR in tapetum cells. Pnor to the identification of the SCR, it has been demonstrated

that application of pollen coat constituents fiom incompatible pollen on the stigma

surface prevents the compatible pollen f?om germinating (Dickinson and Elleman, 1994).

Studies such as these resulted in the identification of a 7 Kd pollen coat protein (PCP) by

Dickinson and colleagues (Franklin et al., 1995). It was demonstrated that the PCP

interacts specifically with S-locus glycoprotein (SLG, Doughty et al., 1993) making it a

candidate for the male S determinant (Stanchev et. al., 1996; Stephenson et al., 1997;

Doughty et al., 1998). However, as aforementioned, there is strong evidence that SCR is

the poilen (male) determinant of self-incompatibility in Brassica. Database searches did

not identfi any significant homologies between SCR and other genes (Schopfer et al.,

1999). Based on these results and the lack of both S-locus linkage and S haplotype-

associated polymorphism of PCPs analyzed to date, it is unlikely that PCPs fùnction in

the self-incompatibility reaction (Schopfer et al. 1999).

1.2.2 Cloning of the Pistil S Genes from Brassica

The first S-locus glycoprotein (SLG) was identified fiom B. oleracea (Nasrallah

and Wallace, 1967a,b; Nasrallah et al., 1970, 1972) and later the gene encoding this

protein was cloned (Nasraliah et al. 1985; 1995). Using the nucleotide sequence of the

two regions flanking SLG, Stein et al., (1991) cloned the S-receptor kinase (SRK) gene.

More recently, another gene was identified, ARCl, (Gu et al., 1998; Stone et al., 1999)

and evidence was provided that it plays a role in the female part of the self-

incompatibility reaction (Stone et al, 1999). The SLG is secreted in the stigmatic papilla

ce11 wall, the SRK is localized to the stigmatic papilla plasma membrane. The ARCl is

shown to interact specifically with SRK, in fact, it is proposed that ARCl is one of the

earliest components of the self-pollen rejection pathway (Gu et al., 1998; Stone et al.,

1999, Dickinson 1999).

1.3 Eomomorphic Gametophytie Self-incompatibility Systems

Self-incompatibility has been characterized in a number of different species. Here

I will concentrate on species Erom two families, the Solanaceae and Papaveraceae. In

these families, the physiology of pollen rejection diEers fkom the Brassicaceae in that

they have pollen tube growth inhibition. The Solanaceae has stylar inhibition, i-e., both

compatible and self pollen grains hydrate and germinate and as the pollen tubes reach the

style, incompatible pollen tube growth (Newbigin et. al., 1993; Frankin et. al., 1995)

slows down or stops. The Papaveraceae has a stigmat ic inhibition, incompatible pollen

tube grows and inhibition occurs on or just below the stigma (Frankin et. al., 1995).

Stylar inhibition is characteristic of the majority of self-incompatible plants with

binucleate pollen. Some of the exceptions with trinucleate pollen and stylar inhibition are

in the genera, Beta, Helimths, BDugaimiIIa and Fagopyrum. Stigrnatic inhibition is

mainly restricted to sporophytic incompatibility, while stylar inhibition is restricted, with

few exceptions, to garnetophytic incompatibility. The interesting outmme of stylar

inhibition is the growth of incompatible and compatible pollen tubes in the same style,

where compatible pollen tubes do not render incompatible pollen tubes compatible and

vice versa (de Nettancourt 1977). Based on the fact that incompatible and compatible

pollen tubes do not influence each other, Linskens (1965) proposed an interesting

hypothesis, placing the action of incompatibility substances on the surface of the pollen

tube,

1.3.1, Cloning of the Pistil S Gene from the Solanaceae

A number of genes encoding S allele glycoproteins have been cloned fiom

Solanaeous species, initially fiom Nicotimm alata (Anderson et al, 1986). McClure et al.

(1989) demonstrated that these glycoproteins have ribonuclease activity. Specific

antibodies raised against a synthetic peptide fiom the S-glycoprotein, labelled the

intercellular matrix in the stigma, transmitting tissue of the style, and the ce11 wall to the

epidermis of the placenta. Huang et al.. (1994) provided evidence, by transforming

Pefunia plants with a non-functional S3-RNase, that the ribonuclease activity of S-RNase

is essential for the expression of self-incompatibility. McClure et al., (1990)

demonstrated that in N. alaia the rRNA of incompatible, but not compatible, pollen tubes

is degraded in vivo. This provided insight into the nature of the substrate for the S-RNase

as well as the role of the ribonuclease in self-incompatibility. Reviews of this topic can be

found in the following papers: Anderson et aL, (1989); Ebert et al., (1989); Kao and

McCubbin (1996); Dodds et al,. (1996).

1.3 -2 Cloning of the Pistil S Gene from the Papaveraceae

In Pupaver rhoeas, it was shown that the addition of crude stigma extract to

pollen grown in vifro prevents pollen tube growth (Franklin-Tong et al., 1988), thus

enabling the detection of stigma S gene products. This method has been used to isolate

and characterize S gene products fiom several S genotypes of P. rhoeas. As expected,

recombinant S gene products also elicit genotype-specific inhibition of pollen tube

growth Franklin-Tong et al., 1988; Walker, 1994; Foote et al., 1994). The expression of

the S allele appears to be confined to the stigma tissue, and its highest level occurs 1 day

before anthesis. Protein and DNA data base searches, using the S allele sequences did not

detect any significant homology, indicating that the Papaver rhoeas S gene is different

fkom any of îhe other S genes cloned to date (Foote et al., 1994). This presents a new

class of S genes and it suggests that the self-incompatibility mechanisms in these species

is dBerent fium other species studied to date. Identification of the male d e t e e n a n t of

self-incompatibility in gametophytic systems has proven to be dificult, and it has yet to

be discovered.

1.4 Heteromorphic Self-incompatibility Systems

In addition to reciprocal herkogamy and self-incompatibility, heterornorphic

species possess several other floral traits that are polymorphic; these Uiclude pollen size,

pollen production, pollen exine sculpturing, and stigma morphology, collectivel y referred

to as ancillary polymorphisms (Shore and Barrett 1986; Dulberger 1992). With respect to

the role of reciprocal herkogamy and ancillary polymorphisms in promoting out-breeding

in heterostylous plants, two somewhat different hypotheses have been proposed. First,

Dulberger (1975a,b, 1992) proposed that the floral polymorphisms play a direct role in

the self-incompatibility mechanism, in other words, stnictural dserences somehow

provide the plant with tools necessary to recognize self and reject it. This mode1

fûnctionally integrates the morphological, developmental, and biochemical components

of the entire syndrome @arrett 1992; Duiberger 1992). Recentiy, identification of

putative incompatibility proteins (Athanasiou and Shore 1997) in pollen and styles of the

short-styled morph cast doubt on the direct involvement of structural components in the

self-incompatibility reaction. The second hypothesis, proposed by Lloyd and Webb

(1992), assigns different fùnctions to the morphological polymorphisms, each influencing

distinct aspects of the outbreeding mechanism. For example, according to this hypothesis,

reciprocal herkogamy is â functional adaptation to promote cross-pollination rather than a

self-rej ection mechanism (Lloyd and Webb 1992).

A number of self-incornpatibility reactions Ieading to self-pollen rejection have

been suggested in heteromorphic systerns, including lack of adhesion, hydration,

germination of pollen, inability of pollen tubes to penetrate the stigmatic zone, and arrest

of pollen tubes in style and ovary. For many species studied, a cumulative incompatibility

system has been suggested where a sequence of incompatibility barriers fùnctions in

concert (Dulberger 1992; Barrett and Cruzan 1994). The variation of pollen tube

inhibition sites in heteromorphic systems differs fiom the homomorphie systems where,

commonly, a specific rejection site is observed. In most of the species studied (e.g.

Pnniula spp, and Rubiaceae), variation of pollen tube inhibition sites not only occurs

among species but also differs between the rnorphs. The inhibition of pollen tubes

generally takes place in the style of the long-styled morph and in the stigmatic tissues of

the short-styled morph (Wedderbm and Richards 1990; Barrett and C m 1994). This

is also shown to be tme, in recent studies by Tamari, Athanasiou and Shore (unpublished

data), for most of the species of Tumera.

1.4.1 Genetics of Distyly

Heterostyly has been described in more than 100 species distributed in

approximately 24 families (Ganders 1979a; Lloyd and Webb 1992a) and the inheritance

of heterostyly has been determined for 23 species in 11 families (Barrett 1992). We know

now that a single gene cornplex, which segregates as a simple Mendelian factor governs

the floral polymorphism and incompatibility barriers to self- and intra-morph crossing, in

distyly. There is a diallelic locus S, s with one dominant allele S only to be found in the

short-styled morph and theoretically not obtainable in homozygous condition in natural

populations, and a recessive, s allele, present in the homozygous state in long-styled

plants. In fact, the unifonnity of the dialleiic locus and dominance of the short morph is

the most striking features of distyly although the genetics are reversed in H'jpericum

aegyficm and Armeriu maritirna @aker 1966; Lewis and Jones 1992). To explain the

genetic control of self-incompatibility and the other characters in the heterostyly system

via a single locus, the supergene model was proposed. Most of the evidence in support of

the supergene model cornes fiom the extensive work done by Ernst (1957) on the

Pnmulaceae and later, by Baker (1966) on the Plumbaginaceae. The supergene locus is

composed of at least three tightly linked Ioci that are rarely disturbed by crossing-over

(Ernst 1957; Dowrick 1956). In this respect, the supergene has been defined by

Darlington and Mather (1949) as "a group of genes mechanicdly held together on a

chromosome and usually inherited as a unit" (de Nettancourt et al., 1977).

1 A.2 Turnma Species as a Mode1 System

Tuntera (Tmeraceae) is a genus of tropical, hennaphroditic species that

commonly occur as weeds of roadsides and open waste ground (Barrett 1978).

Populations of Tuniera species exhibit the floral polymorphism cornmon to most

distylous species (e.g. reciprocal herkogarny, pollen size, and pollen production

dimorphisms (Barrett 1978; Shore and Barrett 1984). Stigmas of the floral morphs lack

papillae but their highly dissected nature provides a large surface for pollen capture. The

mature pollen is two-celled and three colporate (Mahalingappa 1975). Pollen of the long-

styled morph is smaller than that of the short-styled morph. The stigma surface is larger

for the long-styled than for the short-styled morph (Shore and Barrett 1985b). Pollination

is largely by insects (Barrett 1978). Flowers of Turneru, being bowl shaped, are atypical

for distyous species. Tumera species are either diploid ( 2 x = IO), tetrapIoid (4x = 20),

hexaploid ( 6 x = 30), or octaploid (8x = 40). A relationship exists between the ploidal

level of the plants and their self-incornpatibility phenotype and the occurrence of floral

polymorptiism (Shore and Barra, 1985a). Diploid (with genotype of short = Ss, and long

= ss) and tetraploid (with genotype of short = Ssss, and long = ssss) populations are

distylous with strong self-incompatibility whereas hexploid and octaploid species are

homostylous and self-compatible (Shore and Barrett 198Sb, 1987). Crosses made

between the distylous morphs and 12 populations comprising of three hexaploid varieties

of Tumeara ulmiflia provided evidence that the hexaploid varieties are "'long"

homostyles. Homostyle variants have long styles and long starnens. The styles have

incompatibility characteristics of the long-styled plants, but the pollen has incompatibility

characteristics of the short-styled plants (Barrett and Shore 1987).

Many features of Tumera make it a usefiil candidate for the study of distyly.

Some of these features are the ease of maintenance in the greenhouse, relatively small

size of plants, profbse flowering year round, large fiowers that are easily manipulated,

and there are some valuable self-compatible variants available (see below).

1.4.3 Self-compatible Variants of Turnera

A single short-styled individual, termed BRY, belonging to Tumera subulata

Smith was discovered on a roadside in Arco Verde, N.E. Brazil (Shore and Barrett

1985). BRY has a diploid chromosome number of 2n = 10. Crossing studies showed that

BRY is self-compatible, showing aberrant stylar behaviour. In contrast, pollen fkom BRY

exhibited the normal incompatibility phenotype of the short morph (Shore and Barrett

1986). Later studies using pollen competition experiments and examination of pollen tube

growth of short- and long-styled morph pollen on BRY styles, confirmed the existence of

a weakened incompatibility system in BRY, referred to as cryptic incompatibility

(Athanasiou and Shore 1997). BRY has been a valuable tool in previous studies (Shore

and Barrett 1986; Athanasiou and Shore 1997; Athanasiou and Shore unpublished data)

as well as in studies below. SL8 is another single short-styled plant of T. subulata fkom

Sao Luis B r e l , which is somewhat self-compatible. Investigation of crosses made

between SL8 and BRY has provided interesting results with respect to the control of gene

products of the distyly locus (Athanasiou and Shore 1997).

1.4.4 Molecular studies of heterostyly

Since Danirin drew attention to these groups of angiosperms, there have been

extensive studies of distylous species using ecological and genetic approaches. However,

there is very little knowledge of the rnolecular mechanisms responsible for morphological

differences and self-incompatibility. In this respect, de Nettancourt (1997) pointed out

that "the area of heterostyly self-incompatibility is wide open for an intensification of

research on the molecular biology of pollen-pistil interaction". Motecular studies c m

contribute to the current knowledge, and provide the necessary information to answer

questions ranging 6om molecular mechanisms to sequences of evolutionary events

Ieading to present day heterostyly. Molecular studies have been initiated to i den te

morph specific proteins, in an effort to identiEy the gene products involved in the

p hy siological or structural characteristics of het erosty lous systems (Stevens and Murray

1982; Wong et aL, 1994 a, b; Athanasiou and Shore 1997).

Comparing the protein profiles of pollen and styles of the short- and the long-

styled morphs in distylous Averrhûa carambola (ûxalidaceae), Wong et al., (1994a) have

shown a number of differences. For instance, they found that an 82 Kd protein is present

in the short-styled but not in die long-styled morph. In contrast, a 72 Kd protein is present

in the long- but not in the short-styled morph (Wong et al., 1994a). However, this shidy

only involved a single long-styled plant and two different clones of a short-styled plant;

thus a sound population study is required in order to assess the generality of these

results.

A parallel, but more complete work has been done by Athanasiou and Shore

(1997) on a number of individuals fiom distylous populations of T. scabra Millsp., T.

krqovickasïi Arbo, and T. subulutu Smith. By comparing the protein profiles [compared

using non-denaturing isoelectric focusing (IEF)] of polien and styles, obtained f?om open

flowers of the morphs, proteins specific to pollen and styles of the short-styled morph

were identified @ig. 2). These proteins were absent fkom long-styled plants. Styles of the

short-styled morph possess three protein bands with pIs of approximately 6.5, 6.3, and

6.1 and pollen of the short-styled plants possesses two protein bands with pIs of

approximately 6.7 and 6.8. None of the style specific proteins appear 24 h before flower

opening. However, a few hours before the opening of the flower, the expression of these

proteins begins, reaching to the maximum level when the flower opens. In pollen,

proteins with a pI of 6.7 are present 48 h pnor to fiowering, and proteins with a pI of 6.8

appear approxirnately 10- 18 h prior to flowering. These proteins were absent fiom al1 of

the other organs examined. Athanasiou and Shore (1997) showed that the morph specific

proteins are either linked tightly to or are the direct products of the distyly locus. They

also suggested possible regulatory effects of the distyly locus, which may control the

expression of the morph specific proteins that reside at loci elsewhere in the genome. In

summary, these results provided three main pieces of evidence suggesting a role for the

identified proteins in either physiological or structura1 features of distyly in Turneru

(Athanasiou and Shore 1997). The evidence is as folIows: 1) tissue specific expression. 2)

expression coincides with the opening of the flower. 3) tight association of the morph

specific proteins and one of the morphs.

Identification of morph specific proteins is an initial step toward the

understanding of these proteins and their function. Athanasiou and Shore (unpublished

data) adopted a reverse genetics approach, and by using the identified proteins, were able

to clone two genes, one fkom pollen and the other fiom styles of the short morph.

Database sequence searches of DNA and proteins of style and pollen showed homology

with the polygalacturonase genes. The pollen gene showed greater homology with pollen

polygalacturonase genes fiom the database than with the Tumera style polygalacturonase

gene (Athanasiou and Shore unpubfished data).

1-5 Objectives of the Thesis

This thesis extends the initial work of Athanasiou and Shore (1997) that resulted in

cloning of the putative pollen and style incompatibility genes. The objectives of the thesis

are as follows:

1. To show that the cloned genes do indeed encode the putative incompatibility proteins.

2. To raise polyclonal antibodies against the recombinant pollen and style proteins.

3. To use the polyclonal antibodies to detect the putative incompatibility proteins.

4. To characterize further the putative incompatibility proteins as well as to assess the

quality of the polyclonal antibody using SDS-immunoblotting.

5. To localize the putative incompatibility proteins to tissues.

Figure 2. Identification of the style and pollen specific proteins in Tzirnera using

isoelectrk focusing gel electrophoresis.

(A) Cmde extracts of styles and anthers fiom the short- and long-styled morphs (S, and

L respectively) were eIectrophoretically dzerentiated on an isoelectric focusing gel

and silver-stained. The proteins unique to the styles of short-styled morph are

indicated by three arrows.

@) Cnide extracts of poilen and anthers fiom the short- and long-styled morphs (S, and

L respectively) were electrophoretically differentiated on an isoelectric focusing gel

and silver stained. Proteins unique to the pollen of short-styled morph are indicated

by two arrows.

STYLES P'

B ANTHERS

Modified from Athanasiou and Shore (1 997)

2 Methods and Materials

2.1 Plant Material

Turnera species used in this study were T. scabra Millsp, T. krapovickasii Arbo,

and T, szibztlata Smith. These plants were obtained fkom seeds and stem cuttings collected

fÏom naturd populations (Table 1, Barrett 1978; Shore and Barrett 1983, l985a, 1986)

All plants were grown under glasshouse conditions as described in Shore and Barrett

(1985a). A mutant homostylous plant was identified (termed MHOMO, Tamari and

Shore unpublished data) as a single branch on an otherwise short-styled plant and it was

established by stem cuttings. In homostylous plants, styles have the phenotype of the long

morph and anthers are elevated to the same level as stigmas. MHOMO shares not only its

floral phenotype, but also its mating phenotype with the self-compatible homostylous

plants in Tzlrnera, SL8 and BRY are also somewhat self-compatible and are established

by stem cuttings (for details see table 1 and introduction).

2.2 Expression of style and p d e n genes in Escheric/tia coli

The protocol used here is fiom Invitrogen (refer to the Invitrogen manual: Xpress

Systern Protein Expression TrcHis). A clone of the style gene, termed TRCSTY18, was

digested with Kpni and Hind III to release 875 bp fiagment encoding 291 amino acids.

This fragment was subcloned into the Xpress vector TrcHis B that aIso had been cleaved

by Kpn 1 and Hind III. The plasmid with the insert in the correct reading fiame for

translation (experimental), and the plasrnid without the insert (negative control) were

used to transforrn E. coli BL2 1 pLys S (Novagen). CeIls harboring the füsion constmcts

Table 1. Distylous species of Tztrnera used in this shidy.

Code Population and Ploidal level Origin MIDC T, scabra 2x Margarita. Isle., Venezuela TAB DR7 MAN SL7 SLSa BRYa MHOMO~ Joelii

T, scabra 2x Costa Rica T. scabra 4x Dominican Republic T. scabra 2x Managua N i c a r a , ~ T. szrbulata 2x BraU1, Sao Luis T. sztbzrlara 2x Brazil, Sao Luis T. szrbztluta Smith 2x Brazil T. sztbzrlata x T. krapovickassi T, joelii 2 x Brazil

a A somewhat self-compatible short-styled plant (see introduction for detaiIs)

MHOMO refers to a homostylous plant resulted fkom crossing of T. subulatcz and 7'.

krapovickassi, which was initially a single branch bearing homostylous flowers on a

short-styled plant (see methods and materials).

were grown ovemight at 3 7 O C in 5 ml of 1xYT (8 g/L tryptone, 5 g/L yeast extract, 2.5

glL, NaCl) with ampiciflin (1 00 &ml). Then 50 ml of 1 xYT was hoculated with 100 pl

of the cells and cultured for 6.5 h; expression was induced by the addition of isopropyl-l-

trio-P-D-galactopyranoside (IE'TG) to 1 mM d e r 3.5 h of ceII growth. Afier induction, 1

ml of culture was removed hourly, for a time course investigation. The gene product

started to accumulate within the first hou after induction and reached its maximum Ievel

in 5-6 h.

A clone of the pollen gene, termed TRCPOLl8, was digested with EcoR I to

retease a 1 153 bp fragment encoding 384 arnino acids. This fiagment was cloned into the

Xpress vector TrcHis B that had been cleaved by EcoRI. The gene product was induced

and a tirne course investigation was done as above. The gene product started

accumulating 2-3 h after induction and reached its maximum level in 5-7 h.

2.3 Bacterial extract preparation

The trial sarnples of the bacterial culture (1 ml each) from both the negative

control and experimental trials were processed and two fractions were separated as

supernatant (soluble) and pellet (insoluble) according to the Xpress system manual. The

supernatant was rnixed with an equal volume of 2x Laemmli buffer (125 mM Tris-HC1,

pH 6.8, 20% glycerol, 5% SDS, 10% P-mercaptoethanol) and the pellet was resuspended

in 100 pl of Laemmli buffer by pipetting up and down. Note that pipetting was done in a

manner so as to avoid bubble formation, until the pellet was completely resuspended. The

suspension was heated at 100° C for 6 min, the insoluble material was removed by

centrifugation (14,000 xg, 10 min). The resulting supernatant was subjected to SDS-

polyacrylamide gel electrophoresis (SDS-PAGE, Mini-Protein II Cell fiom BioRad, 5%

stacking gel at 50V, 10% resolving gel at l4OV) foiiowing the procedure of Laemmli and

Favre (1973) and proteins were stained with Coomassie BriUiant Blue ovemight and

destained the next day. By comparing protein profiles fiom the experimental and negative

control samples, the fusion protein was identifïed. Most of the fusion protein, if not all,

was in the insoluble portion (inclusion bodies) of the bacterial extract. Fusion protein

concentrations for both pollen and style were deterrnined by comparing a known amount

of Bovine S e m Albumin with the fusion protein, subjected to SDS-PAGE.

2.4 Generation of antiserum against the style and pollen fusion proteins

The procedure described here was used for the style fusion protein and with some

modification, for the polIen fusion protein; the modifications have been pointed out

where required. Bacterial ceils harboring the fusion protein, either stored at -80° C or

freshly grown, were pelleted by centrifugation, at 3000 xg, and the pellet was

resuspended in lysis b a e r (NP-40, 135 rnM NaCl, 20 mM Tris pH 8.0, 1 rnM MgC12, 1

mM Ca&, 10% glycerol, and 1% NP-40). Protease inhibitor (PMSF 35 &ml, and

Aprotinin 5 pg/rnl or AEBSF 17.5 pg/xni, as an alternative for PMSF) was added prior to

sonication. The cells (5 ml) were lysed by sonication 12 x 15 s with 10 s of cooling afier

each sonication. Sonication was at the highest setting, using the smallest probe.

Temperature was kept below 8" C at all tunes. The lysate was cleared by centrifugation

(4" C, 12,000 xg, 10 min) and the pellet was resuspended in Herman's buffer (10 mM

Tris pH 8, 1% NP-40, 100 mM NaCl, 5% NaDOC, 1 mM EDTA) by pipetting. The

lysate was again cleared by centrifugation ( 4 O C , 12,000 xg, 10 min) and the pellet was

resuspended in 2 ml of l x Laemrnli b&er (without Bromphenol Blue) by pipetting. The

concentration of fusion proteins was estimated as described above. Aliquots of the

sample with 150-200 pg arnount of hsion protein were subject to SDS-PAGE to p-

the fiision protein. The Coomassie Brilliant Blue-stained fusion band was excised fiom

the gel and processed for injection or stored at -20" C for later use. Since the pollen

fusion protein concentration was low, a greater volume of the sarnple was loaded on the

gel to obtain the 150-200 pg protein. However, because of the very high concentration of

total proteins, the separation on SDS-PAGE was unsuccessfil. This problem was

circumvented by reducing the electrophoresis power by 50% for both stacking and

resolving gel (for large gel 60 V and 180 V constant respectively) and by diluting the

protein sarnple with 1 xLaemmli buffer.

Pnor to injection into the rabbits, protein bands were washed 4 x 1 h with l x PBS

(Phosphate buffered saline) , and crushed using a three way connector (Three-Way Large

Bore Stopcock with Male Luer Slip Adaptor, Boxter Healthcare Corporation) as follows:

the opening of the three way connector was reduced gradually as the sample was pumped

fiom one syringe to another, until opening was so small that sample could not be forced

fiom syringe to syringe. The resultant slurry, without adjuvant, was injected into the

rabbits (see below for details). Alternatively, protein samples were acetone precipitated

and the pellet was dried in speed-vac and stored at -20a C. For the first injection, the

pellet was resuspended in l x PBS and mked with an equal volume of Freund's complete

adjuvant (Sigma). Subsequent booster injections were made without adjuvant.

A total of 7 New Zealand White rabbits were used to generate antisenim; 4 rabbits

for the style fusion protein and the rest for the pollen fusion protein. Two of the rabbits

were injected with SDS-PAGE purified protein and the rest with acetone precipitated

total protein. The pre-immune serums f?om rabbits were examined for cross-reactivity

before injection and as a resuit, selected rabbits were used. Injections were made

subcutaneously at several sites with approximately 200 pg for the initial irnrnunization.

For the subsequent boosters the animals were injected with approximately 100 pg at

intervals of 2-weeks to 4 weeks. Bleeding was 5 days after each injection. Blood was

allowed to clot at room temperature (about 1 h), then it was lefi at + 4O C overnight- The

serum was collected and cleared by centrifugation for 15-30 min at 1500 g at 4 OC. A

bacterial inhibitor (0.1% sodium azide) was added and the senun was stored at 4 OC for a

short period or aliquoted and stored at - 80 OC for extended periods.

2.5 SDS-PAGE immunoblotting

Aliquots of protein sarnples (5-10 pg) were subjected to SDS-PAGE as indicated

above, and proteins were electrophoretically transferred to 0.2 pm Immuno-Blot PVDF

(polyvinylidene difluoride) membrane (BioRad) according to BioRad's manual

(ovemight, 30V, and the next day I h at lOOV in 20% methanol, 25 mM Tris-HCl, 0.192

M glycine, pH 8.3). The PVDF membrane was blocked with either 0.05% Tween-20 or

3% gelatin in Tris buffered saline (TBS; 20 mM Tris pH 7.5, 0.5 M NaCl) with slow

shaking at roorn temperature. Both blocking procedures provided simitar results. niree

prirnary antibodies were used in this study. The T7-Tag, a mouse monoclonal antibody

directed against the 11 amino acid gene (referred to as the 10 leader peptide) expressed

by the translation vector (Novagen). ï h k antibody was diluted 1/10,000 in blocking

solution and used to identie the fûsion protein. The prirnary immune semm directed

against pollen was diluted 1/2000 in blocking solution. Finally the primary immune

serum directed against the style fusion protein was diluted 113000 in blocking solution.

Pollen and style immune serums were used to screen pollen, style, and other floral tissue

extracts. Protein extracts were prepared separately in 90 p1 phosphate buffered saline

(PBS, 130 mM NaCI, 7 mM Na2HP04, 3 rnM NaH2P04, pH 7.4) using a cerarnic mortar

and a pestle on ice as follows: 21 short styles, 11 long styles, 10 anthers, 3 petals, 10

filaments. The protein extracts were mixed with 4x laemmli buffer with a 3:l ratio,

respectively. The insolub1e cellular material was pelleted by centrifugation (14,000 g)

and 20 pl of the supernatant was subjected to SDS-PAGE.

The immunoblotting procedure described below is identical for ail of the prïmary

antibodies (Ab). The prirnary Ab incubation was performed by slow shaking for 2 h, in at

least 25mI of solution for 5x5 cm of PVDF membrane. After 3x10 min washes with

blocking solution the membrane was incubated for 1 h with a secondary antibody (a

monoclonal goat anti-rabbit conjugated to akaline phosphatase, Sigma, diluted 1/10000,

or a monoclonal goat anti-mouse conjugated to alkaline phosphatase, Cedarlane). Finally,

the membrane was washed for 3x10 min with blocking solution and the chromogenic

substance, nitroblue tetrazoliurn (NBP, Sigma) and 5-bromo-4-chloro-3-indolyy

phosphate (BCIP, Sigma), were added in the buffer (100m.M Tris pH = 9.5, 100 mM

NaCl, 50 mM MgC12) The membrane was incubated, without shaking, at 37O C, until the

desired colour density was observed, The membrane was washed with distilled water and

air-dried.

2.6 IEF immunoblotting

Protein extracts of the tissues were prepared as mentioned above, without the

laemmli buffer, and subject to nondenaturing KEF gel electrophoresis (Ampholine

PAGplate pH 5.5-8.5, ampholine concentration of 2S%, Arnersham Pharrnacia Bio Tech

Ab) on a LKB Multiphore apparatus cooled to 10" C following the procedure of

Athanasiou and Shore (1997). Initially, the IEF gel was prefocused for 60 min at 6W. The

protein extracts were absorbed on micracloth (Calbiochem, Corp) wicks and were loaded

at the anodal end of the gel. Proteins were electrophoretically focused for 90 min at 6W.

Proteins were stained by either silver or Coomassie Brilliant Blue staining. For silver

staining, gels were fixed in 10% trichloroacetic acid, 3.5% sulphosalacylic acid, 30%

methanol, followed by 12% trichloroacetic acid, 30% methanol, for 1 h each. Then, gels

were rinsed for 2 h, silver stained in 0.15% AgN03, 0.056% formaldehycie for 30 min,

rinsed for 1 min and developed in 3% NaC03, 0.056% formaldehyde, and 0.4 mg/liter

sodium thiosulphate. The stain was made permanent by fixing in 10% acetic acid. For

immuoblotting, proteins were electrophoretically transferred to PVDF membrane

according to BioRad7s instruction manual. The rest of the immunoblotting procedure was

identical to the SDS-PAGE immunoblotting.

2.7 Tissue fixation and Immunocytochemistry

Styles were collected from open flowers and k e d for 4 h in Carnoy's solution

(75% ethanol, 25% glacial acetic acid), and vacuum-infiltrated for 45 min. Styles were

dehydrated through a praded series of ethano1:tertiary butyl alcohol (TBA), and were

equilibrated to 100Y0 TBA and infiltrated with Tissueprep wax (Fisher) according to the

foilowing schedule: 70%-2-12 h, 70%:10%-45 min, 70%:20%-45 min, 70%:30Y45

min, 60%:40%-45 rnin, 50%:50%-45 min, 40%:60%45 min, 30%:70%-45 min,

20%:80%-45 min, 100% TBA4S min, 100% TBA-45 min, 50% TBA:50% Wax-

overnight at 6 1 C, 100% Wax-overnight in 6 1" C, 100% Wax-overnight in 61" C, 100%

Wax-overnight in 61" C. The wax temperature did not exceed 63" C at any tirne. The

styles were embedded, trimmed, and sectioned (3 p); sections were stored at 4O C for

several weeks without any activity loss. Sections were expanded while floating on 37" C

water and placed on Biobond (British BioCell, CEDARLANE) coated glass slides. The

slides were then placed on a wanning tray at 35O C overniglit to adhere the sections to the

slides.

Anthers were collected fiom open flowers and fixed for 6 hr in Carnoy's solution for

6 hr without vacuum infiltration. The dehydration procedure was as described for styles;

in order to change the solutions during the dehydration procedure, pollen and anthers

were pelleted by centrifugation (1 000 g, 5 min) and new solution was added. During the

fixation and dehydration, pollen dissociates from the anthers. To separate the pollen fiom

the anthers, pollen was suspended in the solution by gentle shaking, and it was collected

by pipetting. The details of the embedding procedures were as described for styles.

During the wax infiltration, poilen sank to the bottom of the container, thus the wax was

changed by pouring off the old wax and adding fiesh wax. The embedded pollen was

sectioned (5 p) and place on BioBond coated slides as descnbed for styles.

In order to remove the wax fÎom sections and to reliydrate them, for both pollen

and styles, slides were passed through two changes of histoclear (Sigma), each for 15 min

and following the series: 1 : 1, histoclear: 100% EtOH-2min; 95% EtOH-2min; 80%

EtOH-2min; 70% EtOH-2min; 60% EtOH-2min; 50% EtOH-2min; 40% EtOH-2rnin;

30% EtOH-2min; distilled water-5 min; and washing buffer (Tris-HC1-NaCl, 100 mM

Tris, 120 mM NaCl) buffer-30 min. Non-specific binding sites were bIocked by

incubating the sections with 200 pl of blocking solution (normal goat senun was diIuted

1/30 in washing buffer) for 30 min. The blocking solution was shaken gently fiom the

slides and the slides were incubated with the primary antibody - the primary antibody

was diluted in washing buffer. Style immune and pre-immune serums were diluted to

1/100 and pollen immune and pre-immune serums were diluted to 1/200. Sections were

washed with washing buffer for 3x10 min and were incubated with secondary antibody

for immunodetection. The secondary antibody was CY3-conjugated aanipure goat anti-

rabbit IgG (H+L, Jackson LmmunoResearch). Sections were washed with washing buffer

for 3x10 min and aqueous mounting medium (Antifade Kit, ProLong), with fading-

preventive properties, was applied according to the company's instructions. For

population studies, styles fiom 4 populations of Turnera were collected and prepared for

immunocytochemisty as described above.

2-8 Identification of a novel short specific protein in styles

Extracts of styles fiom individuals of both Iongs and shorts fiom different

populations and species (Table 2) were prepared (see above), subjected to SDS-PAGE,

and stained with Coomassie Brillimt blue. The protein profiles were compared in order to

identie rnorph specific protein bands.

Table 2, Survey of different individuais in various populations and species of Tztrnera, for the presence of S68 (a 68 Kd protein specifïc to the short-styled morph).

Code Population No. No. No. short-styled long-styled homostyled

MIDC T. scabra 4 3 TAB T. scabra 8 5 DR7 T. scabra 3 7 MAN T. scabra 3 4 SL7 T. strbztlnra 1 1 SL8" T, s ub ulata 1 BRYa T. subzilata Smith 8 8 MHOMO~ T.subzdata xT. krapovickassi 1 1 Joelii T. joelii 4 4 Total nurnber of individuals 33 33 1

a A somewhat self-compatible short-styled plant (see introduction for details)

MHOMO refen to a homostylous plant resulted fiom crossing of T. sttbuZata and 7'.

krapovickassi, which was initially a single branch bearing hornostylous flowers on a short

styled plant (see methods and materials).

3. Results

3.1 Production of polyclonal antibodies directed against style and pollen specific

pro teins

A portion (875 bp of the style gene and 1153 bp of the pollen gene) of the cloned

genes were subcloned ïnto au expression vector, and transfonned into E- coli. Gene

product expression was then induced in vitro. The fùsion proteins were identified by two

methods. First, the total insoluble proteins tiom E. coli ceiis containing the expression

plasmid without an insert (negative control) and with the insert (the experimental) were

subjected to SDS-PAGE and the protein profiles were compared. Second, SDS-

immunoblotting experiments were carried out using monoclonal antibodies against the

leader region of the fùsion protein. The experimental with the style gene insert showed a

strong band, approximately 40 Kd (style fusion protein + 3 Kd leader region added by

expression vector), which was not observed in the negative control (Fig. 3A). By

comparing the experimental sample and negative control for pollen, a band was identified

in the experimental sample, approximateiy 48 Kd (pollen fusion protein + 3 Kd leader

region added by expression vector, Fig 3B). However, the pollen fùsion protein was in

close proximity and concentration to bactenal proteins. The identity of the style fision

protein was c o n h e d by using the immunoblotting tests against the leader region (Fig.

3C). The identity of pollen fiision protein was aiso confinned by immunoblotting tests

against the leader region (data not shown).

Polyclonal antibodies against the fusion proteins were raised using two different

methods. First, total proteins (fiision + bacterial) were injected en masse into rabbits.

Figure 3, Identification of the style ând poilen fùsion protein using SDS-PAGE and

irnmuzsb lotting.

(A) A Commassie Blue-stained 10% SDS-polyacrylamide gel of the inclusion body

proteins f?om E. coIi cells containiny the expression plasmid without an insert (No 1)

and with the style gene insert (I). The style fusion protein (SFP) is indicated by the

arro W.

(B) A Commassie Blue-stained 10% SDS-polyacrylamide gel of the inclusion body

protein fiom E. coli cells containing the expression plasmid without an insert (No I)

and with the pollen gene insert (1). The poilen fùsion protein (PFP) is indicated by

the arrow.

(C) An immunoblot of the inclusion body proteins fiom the E. coli cells containing the

expression plasrnid without a style gene insert (No 1) and with the insert (1). The

fusion protein is indicated by the arrow. The primary antibody was 1/10,000 T7-Tag,

a mouse monoclonal antibody directed against the 2 1 amino acid gene 10 leader

peptide expressed by the translation vector (Novagen). The secondary antibody was

1/1000 goat anti-mouse conjugated dkaline phosphatase (Cedarlane).

47 Kd-

SFP)

Second, the fusion proteins were purified using SDS-PAGE and the gel band containing

proteins was excised from the gel and injected into rabbits.

3.2 Assessing the quality of the immune serum

Lmmunoblotting experiments were carried out to assess the quality of the

polyclonal antibodies and the nature of their reactivity against fusion proteins and

proteins fiom extracts of style, poilen, and other floral tissues. Tissue extracts were

examined using both SDS-PAGE immunoblotting and IEF irnmunob lotting whereas

fusion proteins were only examined using SDS-PAGE immunoblotting. The style

immune semm, from al1 4 rabbits, showed strong reactivity to the style fusion protein and

weaker affinity to a number of bacterial proteins (Fig. 4A lane C). The style immune

serum also showed some reactivity to the pollen fùsion protein (Fig. 5 lane 5). The pre-

immune semm showed a weak activity to a number of bacterial proteins (data not shown)

- pre-immune serum was tested for any reactivity before injection. The pollen immune

serum reacted strongly to the poilen fusion protein and weakiy to a number of bacteriai

proteins (data not shown). The pre-immune serum showed a weak activity with a number

of bacterial proteins. These results indicate that the immune serum recognizes the &sion

protein; therefore, the experiment was extended to test the immune semrns against the

fioral tissue extracts (Fig 5).

3-3 Test of style immune serum against tissue extracts on SDS-PAGE

immuno blotting

In the SDS-PAGE immunoblotting experiments, extracts of styles fiom both

morphs were examined against the style inmune serurn raised by injection of the SDS-

PAGE-purified style fùsion protein. Two bands were stained in the styles of short-styled

morphs: a 120 Kd band common to both morphs, and a 35 Kd band specific to the styles

of short-styled morph, termed S35 (Fig. 4A). Immune serum fi-om the rabbit injected with

total protein (fusion protein + bacterial proteins) does not react with the 120 Kd protein

band, but it did recognize S35 (Fig. 4B). This indicates that the immune serum raised by

injecting total protein has no affmity to the 120 Kd protein or its affmity is so low that it

is not detectable by immunobloting tests (Fig 4B). Ln contrast, the immune serum raised

by injecting SDS-PAGE-purified protein shows a strong a m t y to this protein (Fig 4A).

Different concentrations of style extracts were used to estimate the detectable level of this

protein by using the immune serum; S35 was detected in extracts containing as iittle as

1-25 styles (Fig, 4A). This result verifies the relatively high concentration of S35 in the

style tissue andlor the high avidity of immune serum to the protein. Immune serum was

also tested against petals and filaments fiom both morphs and the results showed that

immune semm reacts with the 120 Kd protein aIthough S35 was missing from these

tissues (Fig. 5). This 120 Kd protein provides a convenient interna1 marker for loading

levels.

Figure 4. Detection of the style specific protein using SDS-immunoblotting.

(A)Three different concentrations, decreasing 5orn left to nght, of cmde extracts of

styles f?om the short- and long-styled (S and L respectively) progeny of BRY, as well

as the style fusion protein (C), were electrophoresed thorough 10% SDS-

polyacrylamide gel and electroblotted onto PVDF (Bio-Rad) membrane. The prima^

antibody was 1/2000 immune serum directed against the style fision protein. The

- secondary antibody was 1 /IO, 000 goat anti-rab bit conjugated to aikaline p hosphh-iase

(Sigma). The marker proteins CM) were peucil-marked on the membrane before

proceeding with immunoblotting procedures.

(B) Cnide extracts of styles from the short- and long-styled (S and L respectively)

progeny BRY and the style firsion protein (C), were electrophoresed thorough IO%

SDS-polyacrylamide ge1 and electroblotted onto PVDF membrane. The primary

antibody was 1/2000 immune senim directed against the style fusion protein but from

a rabbit that was injected with total protein (fusion protein + bacterial proteins). This

is different from other rabbits that were injected with SDS-purified fusion protein.

The secondary antibody was 1/10,000 goat anti-rabbit conjugated to alkaline

phosphatase. The marker protein is indicted by "My. Dashed ellipses indicate absence

of the 120 Kd band.

Figure 5. The expression of S35 is specific to the styles of short-styled morph.

The foilowing crude extracts were obtained from short- and long-styled progeny o f BRY:

styles (lanes I and 2), füaments (lanes 4 and S), perds (lanes 7 and 8). These extracts, as

well as the pollen fusion protein (PFP, lane 5) were electrophoresed thorough 10% SDS-

polyacrylamide gel and electroblotted onto a PVDF (Bio-Rad) membrane. The primary

antibody was 1/2000 immune serum directed against the style fusion protein (this

immune serum was raised by injecting the SDS-PAGE purified fusion protein). The

secondary antibody was 1/ 10,000 goat anti-rab bit conjugated to alkaline p hosphatase

(Sigma). S35 appeared only in styles of short-qled BRY, and the 120 kD band occurred

in ail of the tissues examined.

3.4 Test of the style immune serum against tissue extracts on IEF

immunoblotting

In the IEF immunoblotting experiments, the style antibody was tested against

extracts of styles and pollen from short- and long-styled selfed progeny of BRY, as weil

as the extracts of styles fiom short-styled selfed progeny of SL8. Two bands, in close

proximity, were detected in the styles of the short-styled morph of BRY and a single band

was detected in the styles of the short-styled rnorph of SL8 (Fig. 6A). The band in SL8 is

at a different position than the two bands in BRY. Immune serum did not react with any

proteins fi-om styles of long-styled morph of BRY. Pollen extracts also showed no

reactivity against the immune serurn. Ln another IEF gel, the sarne extracts and proteins

were electrophoretically separated and silver-stained. By comparing the profiles of the

immunostained and silver stained bands, the relative position of immunostained bands

was identified (Fig 6B). In fact, the Mmunostained bands correspond to the bauds that

were identified as proteins specific to the short-styled morph by Athanasiou and Shore

(1997). These proteins were originally used to clone the style gene.

3.5 Test of the pollen immune serum against tissue extracts on IEF

immuno bIotting

In the IEF immunoblotting experiments, pollen antibody was tested against the total

extracts of style, pollen, filament, and petals Eom both morphs of BRY. In the extracts of

pollen fiom both morphs, the immune serum reacted with a number of bands that are

Figure 6. Detection of the style specific proteins using the style immune serum.

(A) Au immunoblot of the crude extracts of pollen fiom short- and longstyled BRY

(lanes 1 and 2 respectively), styles fiom short- and long-styled BRY (lanes 3 and 5

respectively) and styles of short-styled SL8 (lane 4). The primary antibody was

1/2000 immune serum directed against the style fùsion protein. The secondary

antibody was 1110,000 goat anti-rabbit conjugated to alkaline phosphatase (Sigma).

The immunostained protein bands appear only in styles of short-styled BRY and

SL8. Protein bands are indicated by arrows-

(B) Cnide extracts of pollen from the short- and long-styled BRY (lanes 1 and 2

respectively), styles corn short- and long-styled BRY (lanes 3 and 5 respectively)

and styles of short-styled morphs of SL8 (Iane 4) were eIectrophoretically separated

on an isoelectric focusing gel and silver-stained. Short specific proteins are indicated

by the arrows.

specific to pollen of the short-styled morph, which are missing fiom long-styled morph

and a number of bands common to both morphs (Fig. 7A). Interestingly, immune serum

does not react with any protein fiom styles, filaments, and petals of either morph (Fig. 7A

and B), with exception of some background staining. Different trials showed that this

background staining can be eliminated by using different blocking reagents (see

methods). The iinmunostained, short polien specific proteins correspond to the pollen

specific proteins that were originally identified (Athanasiou and Shore 1997) and used to

clone the pollen gene; this was the gene used to raise the polyclonal antibodies.

3.6 Test of pollen immune serum against tissue extracts on SDS-PAGE

immunoblotting

In SDS-PAGE immunoblotting experiments, extracts of pollen and styles £?om

both morphs were tested against the pollen immune serurn. A single band or possibly two

bands, barely separated, appear at the same position in pollen of both morphs. Some

background staining was aiso observed, which was eliminated by optimizing the

immunoblotting procedures. Immune serum did not react with any proteins from styles of

either morph (Fig. 8). The polien immune serum did not detect any protein fiom other

floral tissues examined (data not shown). This indicates thzt the immune serum is specific

to the same protein or group of proteins in poilen of both short- and long-styled morphs

and not to proteins £tom the other tissues examined. In contrast to SDS-immunoblotting,

in IEF-imrnunoblotting, a number of bands were detected, only some of which were short

pollen specific.

Figure 7. Detection of the poilen specific proteins for BRY long- and short-styled

plants using the pollen immune serum on EF-immunoblot.

This panel shows an immunoblot of the crude extracts of styles fiom a long- (lane 1) and

two short-styled plants (lanes 2 and 9, anthers containhg pollen f7om short- (lanes 4 and

7), long-styled plants (lane 3) and Filaments (lane 6). The primary antibody was 1/2000

immune serum directed against the poilen hsion protein. The secondary antibody was

1/10,000 goat anti-rabbit conjugated to alkaline phosphatase (Sigma). The

immunostained protein bands appear in poilen of short- and long-styled BRY, however,

some of the bands are specific to the short-styled morph, these protein bands are enclosed

by braces. The styles and filaments do not show any reactivity.

Figure 8. Detection of the pollen specific proteins using SDS-immunoblotting.

Cnide extracts of pollen f?om the short- and long-styled BRY progeny (P-S and P-L

respectively) and £kom styles of short- and long-styled (S-S and S-L respectively) BRY

progeny were electrophoresed through 10% SDS-polyacrylamide gel and electroblotted

onto a PVDF (Bio-Rad) membrane. The primary antibody was 1/2000 immune semm

directed against the pollen fusion protein. The secondary antibody was 1/10,000 goat

anti-rabbit conjugated to alkaline phosphatase (Sigma). A single or perhaps doublet band,

approximately 55 Kd (referred to as PS55s) was immunostained in pollen of both

morphs. Styles do not show any reactivity.

P-S P-L S-S S-L

3 -7 Immunocytochemistry

To localize style specific proteins, immunocytochemistry experiments were

conducted on sectioned stigmas, and styles, using the style immune serum and pre-

immune serum. This investigation was extended to a number of populations and species

of Tumera to explore the generality of the results. In a cross section of a style fkom a

short-styled morph, epidennal tells@), cortical celis (C), transmitting tissue (TT), and

vascular bundles (V) have been labelled (Fig- 9A)-

Style pre-immune serum tested on sections of styles and stigmas fiom short- and

long-styled BRY progeny showed only weak background staining (Fig 9B). Style

immune serum tested against the sections of stigma and style of the long-styled morph

showed only background activity in every part of the sections except the vascular bundles

where staining was rnuch greater compared to the pre-immune serum staining (Fig 9D

and 9F). In contrast, style immune serum showed a distinct immunostaining of the

transmitting tissue of the stigmas and styles of short-styled BRY (Fig 9C and 9E).

Examination of different portions of style (lower, middle, and upper portions) showed

identical results (data not shown). Sections of styles and stigmas fiom other species of

firrnera; T. joelii, T scabra @R7), and T. srbzdafa (SL8) were tested against the style

immune serum. The results of immunostaining, for these species and populations (Fig

10A-F) were consistent with the results of BRY where vascular bundles were stained in

styles of both morphs and transmitting tissue was stained in only styles of the short-styled

morph. However, cornparhg the iwnunostaining intensity of the transmitting tissue

among the populations, DR7 (Fig 1 OB), SL8 (Fig I OD) and T. Jaelii (Fig 10F) examined

showed that DR7 has the greatest staining and BRY (Fig 9C) the least. The staining in

stigma transmitting tissue was simila. for the populations, DR7 (Fig 1 lB), SL8 (Fig 1 ID)

and T. h e f i (Fig 1 IF), except for BRY (Fig 9E) where staining was somewhat lesser.

The stigma fiom the long-styled did not show staining in any of the populations DR7 (Fig

1 lA), SL8 (Fig 1 lC), T. Joeiii (Fig 11E) and BRY (Fig 9F).

To localize the pollen specific proteins, imrnunocytochemistry experiments were

conducted on poilen sections fi-om both morphs using the pollen immune serum and

pre-immune serum. The pollen immune serum showed immunostaining in poilen of the

short-styled morph (Fig 12G) as weli as the long-styled morph (Fig 12F). The results

were as predicted since pollen immune serum detected proteins with identical MW in

pollen of both morphs using SDS-PAGE immunoblotting experiments. The pre-immune

serurn did not stain poUen from long-styled (Fig 12B and D) or short-styIed (Fig 12C and

El.

3 -8 Identification of a novel short specific protein in styles of Turnera species

Using SDS-PAGE, a conspicuous 68 Kd band (terrned S68) was identified from

the styles of short-sty1ed plants. ExpIoring styles of 33 short-styled and 33 long-styled

individuals fiom dEerent populations and species, (Table 3) showed the consistent

appearance of S68 in styles of short-styled plants but this band appears to be missing or

less abundant in the styles of long-styled plants (Fig. 13). In immunoblotting tests, neither

style nor pollen immune serum, raised against pollen and style fusion proteins (see

above), reacted with S68. Investigation of styles fiom short-styled and homostylous

flowers of MHOMO, using SDS-PAGE, showed that S68 is again missing or Less

abundant in styles of the homostylous flowers but it appears as a strong band in styles of

the short-styled flowers (Fig 14).

Figure 9. Tmmunolocaiizaition of S35 to tissues in the styles of BRY.

Stigmas and styles fiom short- and long-styled BRY were fixed, embedded in pa raEi

sliced into 7 pm thick sections and incubated with either the style pre-immune semm or

immune serum. Antigen and antibody complexes were detected by a CY3 conjugated

secondary antibody. Photographs were taken by bright-field and fluorescence

microscopy. (A) The photograph of a style section from a short-styled plant were taken

by bright-field microscopy; epidermal cells (E), cortical cells (C), tramsrnithg tissue

(TT), and vascular bundles (V). (B) A style sections hom a short-styled plant

immunostained with pre-immune serum. (C) A style section f?om a short-style plant

immunostained with immune serum. (D) A style section fiom a long-styled plant

immunostained with immune serum. (E) A stigma section hom a short-styled plant

immunostained with immune serum O A stigrna section fiom a long-styled plant

immunostained with immune serum. Bar = 0.1 mm; the scale is the same for al1 of the

photographs shown.

Figure 10. The Immunolocalization of S35 to style tissue in 3 dBerent Tzmera

species.

Styles fiom short- and long-styled DR7, T. helii , and SL8 were fixed, embedded in

paraffm, sliced into 7 pm sections and incubated with immune serum. Antigen and

antibody complexes were detected by a CY3 conjugated secondary antibody.

Photographs were taken by fluorescence microscopy. (A) Long-styled DR7 (B) Short-

styled DR7. (C) Long-s~led T. joelii. @) Short-styled T. joelii- (E) Long-styled SL8. (F)

Short-styled SL8. Bar = 0.1 mm; the scale for DR7 and SL8 is the same.

Figure 11. The imunolocalization of S35 to the stigmas tissue in 3 different

Tzrmera species-

Stigmas fiom short- and long-styled DR7, T, joelii, and SL8 were fixed, embedded in

para% sliced into 7 p sections and incubated with immune semm. Antigen and

antibody complexes were detected by a CY3 conjugated secondary antibody.

Photographs were taken by fluorescence microscopy. (A) Long-styled DR7. (B) Short-

styled DR7. (C) Long-styled Zjoelii. @) Short-styled T. joeiii- (E) Long-styled SL8. (F)

Short-styled SL8. Bar = 0.1 mm; the scale is the same for ail of the photographs shown.

Fiqure 12. hmunolocalizaition of the pollen specific proteins to poilen of BRY.

Pollen fiom short- and long-styled BRY was fixed, embedded in paraffin, sliced into 5

p m sections and incubated with either the pollen pre-immune serum or immune setum.

Antigen and antibody complexes were detected by a CY3 conjugated secondary antibody.

Photographs were taken by bright-field and fluorescence microscopy. The scale is the

same for the photographs A - C and D - G. (A) Photographs of poilen sections f?om a

short-styled plant were taken by bright-field microscopy. (B) Polien sections fiom a

long-styled plant immunostained with pre-immune semm; (C) Pollen sections fiom a

short-styled plant immunostained with pre-immune serum; @) same section as (B) under

higher magnification; (E) same section as in (C) under higher magnification. (F) Poilen

sections fkom a long-styled plant immunostained with immune serum. (G) Pollen sections

form a short-styled plant immunostained with immune serum. Bar = 0.1 mm; scale is the

same for A-C and D-G.

Figure 13. Identification of a novel style protein specific to the short-styled morph.

A Coomassie Blue-stained 10% SDS-polyacrylamide gel of crude extracts of styles from

8 different short- and long styled plant of T. joelii, short-styled SL8, and short-styled

SL7. Lanes 1-4 long-styled T joelii; lane 5 short-styled SL8; lane 6 short-styled SL7;

lanes 7-10 short-styled T. joelii;. Marker protein is identïfïed by "M". Styles of short-

styled plants show a 68 Kd protein band (referred to as S68).

Figure 14. SDS-polyacrylamide gel analysis of the S68 in MHOMO.

A Coomassie Blue-stained 10% SDS-polyacrylamide gel of cmde extracts of styles from

long- and short-styled BRY, and short-styled and homostyled mutant (MHOMO). Lane 1,

H O M O ; lane 2, long-styled BRY; lane 3 short-styled fiom original plant of MHOMO;

lane 4, MI-IOMO; Iane 5 short-styled BRY. Marker protein is identifïed by " M . S68 did

not appear in styles of the homostyle MHOMO. As expected, S68 appeared in styles of

the short-styled BRY and PVMOMO. S68 is identified by an arrow.

Table 3. Survey of different individuals in various populations and species of Trcmera, for the presence of S68 (a 68 Kd protein specific to the short-styled morph).

Plant Population S hort-styled long-sty led S68 S68 Present Absent Present. Ab sent

MlDC T. scabra 4 O O 4 TAB T. scabra 8 O O 5 DR7 T. scabra 3 O O 7 MAN T. scabra 3 O O 4 SL7 T. szrbztlata 1 O O 1 SL8" T. szrbzrlata 1 O - - BRYa T. strbzilata 8 O O 8 JO elii T. joelii 4 O O 4 Total number of individuds 33 O O O

" A somewhat self-compatible short-styled plant (see introduction for details)

b MHOMO refers to a short-styled plant resulted fkom crossing of T. szcbzrlaia and T.

krapovicknssi, wwhich was initially a single branch bearing homostylous flowers (see

methods and matenals).

4. Discussion

To investigate the mechanisms o f self-incompatibility, molecular studies have

been initiated to ident* gene products involved in this reaction. In gametophytic and

sporophytic homomorphic systems, a number of such proteins (SLG, SRK, SCR, and

ARCI) have been identified and analyzed (see introduction, Nasrallah et. al.,al. 1985,

1995; Anderson el al., 1986; Gu et aL, 1998; Stone et al., 1999; Schopfer et al., 1999).

Few molecular studies have been attempted in the heteromorphic systems, and these have

met with Lirnited success (Stevens and Murray 1982; Wong et. al., 1994a). The most

promising results came with the identification of the S associated short-styled specific

proteins in pollen and styles of Tzrmera species (Athanasiou and Shore 1997).

Subsequently, two genes were cloned, which showed homology to polygaiacturonase

genes; one is believed to encode the style specific proteins and the other pollen specific

proteins. A goal of this thesis has been to ver@ that the genes cioned do indeed encode

the proteins identified by Athanasiou and Shore (1 997).

4.1 Mechanism of self-incompatibility in homornorphic systems

Based on the available data, efforts have been made to elucidate the molecular

rnechanisms of self-incompatibility in the homomorphic systems, mainly in the families

Brassicaceae, Solanaceae, and Papaveraceae. Since the S gene products in these families

show no homology to one another, it is postuiated that the mechanisms of self-rejection

are also difierent. This suggests an independent evolution of self-rejection mechanisms a

number of times during the evolution of angiosperms. A thorough discussion of this issue

can be found in Newbigin (1996).

Here 1 present a bnef review of various rnechanisms through which

incompatibility reactions are postutulated to work. In the Brassicaceae. according to

Dickinson (1999), pollen grains land on the stigma, secrete S locus cysteine-rich protein

(SCR, the poilen determinant of self-incompatibility) and SCR passes through the stigma

ce11 wall - if SCR is corn self pollen - then it is able to bind to the S receptor complex

[S-locus glycoprotein (SLG) + S-receptor kinase, (SRK)] . This binding results in

autophosphorylation of SRK and phosphorylation of ARCI, the first component in the

self-pollen rejection pathway (Gu et al., 1998; Stone el al., 1999; Dickinson 1999). In the

Solanaceae, two different mechanisms have been proposed. The first mechanism

proposes that S-RNases are excluded fiom the pollen tube unless they are recognized and

allowed into the pollen tube by the S-locus product in pollen. The second mechanism

proposes that S-RNases are taken into the tube non-specifically and their activity is

controlled in the pollen tube, either by inactivating or preventing them fiom gaining

access to the substrate (Dodds et. al., 1996; Fr- et. al., 1995). In the Papaveraceae, it

is believed that the stigmatic S proteins act as a signal molecule, interacting with a

membrane-bound pollen receptor in an S allele-specific manner (Frankin et. al., 1995;

Franklin-Tong 1999). Although the nature of allelic specificity and interaction is as yet

unlinown, Franklin-Tong (1999) provides evidence that the interaction results in

triggering a ca2+-dependent signal transduction pathway that leads to the inhibition of

incompatible pollen.

No attempts have been made to explain in detaif how self-incompatibility may

operate in heteromorphic species because the molecular knowledge of the system is

lacking (de Nettancourt 1997). However, it has been suggested that the mechanism of

self-incompatibility in heteromorphic systems is not only different fkom other systems,

but also may differ between the morphs (Lloyd and Webb 1992) Based on new data

presented here, 1 attempt to outline what might occur in distylous Trmera species.

4.2 The style gene encodes the style specific proteins

To show that the style gene encodes the style specifk proteins identified by

Athanasiou and Shore (1997), polyclonal antibodies were raised against the style fusion

protein and used to screen various floral tissues. In IEF-immunobotting experîments, the

style immune semm reacted with only protein bands specific to the styles of the short-

styled morph. These bands correspond to the same protein bands identified and used for

cloning of the style gene (Athaoasiou and Shore unpubtished data). This result lends

evidence that the cloned gene indeed encodes the style specific proteins.

The style immune serum also reacted with a single protein band f?om styles of

short-styled SL8 (see introduction), although in a different position fiom the bands in

BRY. This result is significant since a population survey of the Tziniera species by

Athanasiou and Shore (1997), exploring the generality of the appearance of these

proteins, showed that SL8 and its 14 short-styled and 10 long-style progeny iacked the

style specific proteins. However, the appearance of the style protein band on IEF

immunoblotting indicates that SL8 also possesses the style specific protein but it has an

altered pI- This result indicates that the pI of the SL8 style specific protein is different

from the pI of the short specific proteins in other Tztrnera species examined. This

difference can perhaps be explained by change introduced via post-translational

modifications or mutation(s) in the codhg sequence. Since SL8 is sornewhat self-

compatible (see introduction), it is ternpting to suggest that any changes made to the style

specific protein in SL8 may have something to do with self-compatibility of SL8. For this

reason 1 believe that SL8 may provide some important information about the style

specific proteins as well as the mechanism of self-incompatibility reaction in distylous

Tztmern. As in previous studies, mutation(s) of the S-system has contributed to the

understanding of self-incompatibility systems (de Nettancourt 1997). For instance, a

study of a self-compatible Lycopersiczcm perzrvianzrm confirmed the role of ribonucleases

in self-incompatibility and more specificaiiy, the involvement of a histidine residue at the

catalytic site of this enzyme (de Nettancourt 1997; Royo el. al,. 1994). Also, in self-

compatibIe Brassica naplis, a 1-bp mutation in the SRK gene was identified which results

in a tnincated protein, demonstrating that plants defective in SRK protein expression are

self-compatible (Goring el. al., 1993).

The style immune serum did not react with any pollen proteins fi-om either morph

in IEF immunobloting (Fig GA). This result was somewhat surprising since the style

immune serum showed weak activity with the poilen fusion protein (Fig 5) . However, the

possibility that the concentration of fusion protein was greater, providing more antigenic

sites for antibody binding, cannot be disrnissed.

4.3 Characterisation of the styIe specific proteins

To characterize the style protein, the style immune serum was tested asainst styles

and other floral tissues using SDS-PAGE-immunoblotting. A 35 Kd band appeared only

in styles of the short-styled morph and a 220 Kd band occurred in styles as well as all of

the tissues examined. ~ u n o d e t e c t i o n of the 120 Kd band suggested that S35 and the

120 Kd protein might share a number of epitopes (possibly a consewed region like a

catalytic site)- However, the likelihood of this was questioned when the style immune

serum f?om a different rabbit imrnunostained S35 but not 120 Kd band. Fiom IEF-

immunoblotting results, it was concluded that the style immune serum is specific to the

identified style proteins and since this immune serum also reacted with S35 fi-om styles of

short-styled morph, 1 propose that S35 is the style specific protein. From the results of

IEF- and SDS-PAGE-iwnunoblotting, it can be concluded that the quality of style

immune serum is adequate for Mmunocytochemistry. Thus, style immune serum was

used to localize S35 to specific style tissues (see be1ow)-

4.4 Localization of S35 to the style tissues

In the homomorphie systems, the pistil S gene products are mainly located in the

tissue types that corne in close contact with polien andor pollen tubes. For systems with

stigmatic inhibition (e-g. Brassicaceae) the pistil S gene products are localized mainly to

the stigmatic papilla plasma membrane and ceil wail (Umbach et. al., 1990; Dodds et. al.,

1996). Ln the Solanaceae, with stylar inhibition, they are mainly expressed in the

transmitting tissue where pollen tubes grow (Newbigin et. al., 1993). Studies such as

these have provided valuable information on the tùnction, site of action, and to some

extent rnechanism of action of the S gene products.

In previous studies using IEF experiments (Athanasiou and Shore 1997), and in

this study, using KEF and SDS-PAGE immunoblotting, it was shown that the expression

of style specific proteins is restricted to the styles and the pollen specific proteins to the

pollen of Timera. Tmern possesses a stylar incompatibility where incompatibility

occurs after poilen germination and tube growth is inhibited in the stigma or in the upper

portion of the style (Tamari, Athanasiou and Shore, unpublished). If? the style protein is

involved in incompatibility 1 expect to see the expression of style specific proteins in the

transmitting tissues of the styles and stigmas of the short-styled morph. This would be

strong evidence that the style protein is indeed an incompatibility protein.

The style immune semm was used to localize S35 to the tissues in the pistil of 4

different species/populations of Ttirnera. In ail of the species examùied, the transmitting

tissue of the style and stigma of short-styled plants was stained (Fig. 9- 1 1). The

transmitting tissues of styles and stigmas of the long-styled plants did not show any

staining. Do style specific proteins play a role in the incompatibility response or not? The

characteristics of S35 suggest such a role because, 1) of transmitting tissue expression, 2)

expression coincides with the opening of the flower (Athanasiou and Shore 1997), 3)

tight association to the short-styled morph (Athanasiou and Shore 1997), and 4)

possessing a different pI in self-compatible SL8.

4.5 The polIen gene encodes the pollen specific proteins

To show that the pollen gene encodes the pollen specific proteins, polyclonal

antibodies were raised against the pollen firsion protein and used to screen various floral

tissues for the antigen. In IEF-immunoblotting experiments, the pollen immune serum

reacted with a number of protein bands fkom pollen of both short- and long-styled plants.

Some of these protein bands appeared in both short- and long-styled pollen (common

bands) and others appeared only in the pollen of the short-styled rnorph (poilen specific

proteins). The pollen specific protein bands correspond to the original bands identified

(Athanasiou and Shore 1997) and used for cloning the pollen gene (Athanasiou and Shore

unpublished data). This result provides evidence that the cIoned gene indeed encodes the

identified pollen proteins. Since the immune serum reacted with a number of protein

bands from pollen of both morphs, the specifrcity of immune serum to the antigen was

questioned. Since immune serum showed no activity with any of the proteins fiom styles

and other floral tissues, it can be assumed that the immune serum has a high specificity

and affrnity to the protein bands in polien of the morphs.

Polygalacturonase has been detected genetically and biochemicall y in the pollen

of maize and Oenothera organemis, as well as other plant species, and it has also been

shown that invasive plant pathogens secrete this enzyme to degrade the ceU wall of the

host ( M e n and Lonsdale 1993; Brown and Crouch 1990). Hence, it has been suggested

that polygalacturonase, in conjunction with other celi wall degrading enzymes (pectin

esterase and pectate lyase) may func50n in promoting anther dehiscence, penetration of

the stigma and growth of the pollen tube (Allen and Lonsdde 1993). The expression of

multiple polygalacturonase geries during later stages of pollen developmerit (afier

microspore mitosis), has been demonstrated in Oenothera ovganensis and in Zea mays

(maize, Allen and Lonsdale 2993; Hadfield and Bennett 1998). In Oenothera organensis,

cDNA clones were isolated, characterized and were shown to be expressed abundantly,

producing products of similar weight o d y in pollen, and thus represent a small gene

family (Brown and Crouch 1990). The nucleotide and inferred amino acid sequences of

the cDNA showed similarity to the published sequences of poly,oaiac~onases, therefore,

these authors suggested a possible role in development, germination, and tube growth of

poilen. Similarly, in maize, a total of seven different pollen polygalacturonase sequences,

highly conserved at the DNA level, appear to belong to a multigene family. Based on

these studies, the appearance of the protein bands in IEF immunostaining can perhaps be

explained in the foilowing manner. In Tzrrnera, a small gene family with sequence

similarities to polygalacturonase is expressed only in pollen (representing al1 of the

protein bands observed), and some members of this family have somewhat different

sequences (explaining the different pIs observed) and they are expressed only in the

pollen of short-styled morph. Alternatively, some of these genes products are modified

differently in short-styled morph (representing the short specific bands). M o ~ c a t i o n s

could be on either mEWA (alternative spiicins) or protein (post-translational, e.g.

addition ofglycoprotein). In summary these immunoblotting results support three points:

1. The cloned gene encodes the proteids that were originally extracted ti-om the IEF gel

and used for cloning the gene.

2. Proteins are expressed only in pollen, possibly representing a srnall gene family.

3. One or more of the pollen specific proteins are restricted to the short-styled morph.

4-5 Characterization of the polIen specific proteins

The pollen immune serurn was tested against pollen and other floral tissues using

SDS-PAGE-immunoblotting, in order to obtain more information about the pollen

specific proteins. A single -55 Kd band was immunostained in pollen of both short- and

long-styled morphs, in contrast to EF-immunoblotting where a number of protein bands

were immunostained. It is possible that the immune serum reacts with a number of

proteins with a similar MW but different PIS. Hence, these proteins can only be

differentiated by IEF gels. Again, these results are similar to the results obtained for

Oenothra orgcnlensis, where a pollen specific gene family, composed of approximately

six to eight members has gene products of almost the same size (Brown and Crouch

1990). This fùrther supports the existence of a gene family that is expressed in pollen

only, producing proteins of similar MW but different pIs in Tztmera species. Finally,

Shore and Athanasiou (unpublished data) have identified two clones, fiom Tzmera,

differing in their 3' untranslated sequences, possibly demonstrating the expression of

similar genes in this gene farnily.

4.6 Localization of P55s to Pollen

The pollen immune serum was used to localize the P55s to the pollen grains fiom

the short- and long-styled morphs of BRY. The pollen grains fiom both morphs were

stained and there was no obvious difference between them. These and immunoblotting

results clearly showed that the pollen antibody is specific to one or more proteins in the

pollen of both morphs, and concurs with IEF- and SDS-immunoblotting studies. This

coupled with unpublished results of Tamari and Shore, suggests that the pollen gene

might not be involved in self-incompatibility, but might be iinked and in disequilibrium

with the S-allele of distyiy.

4.7 The expression of S35 in self-compatible variants (BRY and SL8) of Turnera

A difference was observed in the intensity of staining between BRY and other

species; BRY showed a lower stainïng. Since all the conditions have been maintained

constant, this could indicate that the expression of S35 is lower in BRY compared with

other species. However, a sound quantitative study is necessary to provide fûrther support

for this claim. To justi@ the cryptic incompatibility in BRY (see introduction), it is

tempting to postulate that a low concentration of S35 in transmitting tissue may render

BRY sornewhat self-compatib le. This implies that the strength of self-rejection in the

pistil of fiinzercz depends on the concentration of S35. In other self-incompatibility

systems, it has been demonstrated that the concentration of S gene products in the pistil

have a direct relationship to the self-incompatibility reaction. Nasrallah et. al., (1992)

identified a mutation in B. campestris that drastically reduced the levels of stigma SLG

and led to the loss of the incompatibility response in the pistil but not in the pollen. In the

Solanaceae, self-incompatible and self-compatible species of Nicoiiutu were manipulated

and transformed with S-RNase genes or sense and anti-sense constructs producing plants

which expressed different concentration of stylar S-RNase (Murfett et. al,, 1996). In this

study, Mufiett et. a[., (1996) were able to show the involvement and the consequences of

the absence of S-RNases to the self-incompatibility reaction.

Immunostaining in the transmitting tract of SL8 is as strong as in the other species

of Tzirnera. Perhaps, the self-compatibility of SL8 is caused by the changes introduced to

the S35, rendering it somewhat less fùnctional, as aforementioned, the pI of S35 in SL8 is

dflerent fi-om other Tzimera populations. This is based on the assumption that SL8 has

aberrant style, however, no study has been done to determine the incompatibility

phenotype of pollen or style.

4.7 Pollen tube wall and growth

In cases of many gametophytic species, it has been shown that pollen tubes can

grow in an artificial medium. This is also true for species of Tumera that have been

tested to date (Shore, persona1 communication). Nonetheless, there is considerable

evidence for the interaction between the growing pollen tube and the transmitting tissue

of the style. The extracellular matrïx of transmitting tissue contains sugars,

polysaccharides, glycosylated proteins, and lipids (Lord and Sanders, 1992; Sanders and

Lord, 1992; Franklin-Tong 1999). With respect to pollen tube growth, several fùnctions

have been suggested for the stylar components, including, adhesion, nutrition, directional

guidance, and signalling (Franklin-Tong 1999). The poilen tube cell wall comprises

layers which correspond to the primary and secondary w d s of other plant cells. An outer

fibrillar layer present around the entire tube, that is mainly composed of pectin,

hemicellulose, and cellulose, and a second, inner layer of callose h g . The inner

cailosic layer is missing fiom the apical end of the polien tube. Depositions of visible

amount of callose start 10-30 jm back fkom the pollen tube tip in al1 of the species

studied (e-g. Brassica, Nicoiiana iabaaim, and Petunia hybrida; Heslop-Harrison 197 1 ;

Derksen 1995b; Geitmann 1997). Callose is synthesized by callose synthases located in

the membrane of pollen tube (Gibeaut and Carpita 1994). It is thought that pectins are

polymerized and esterified within the golgi compIex and then transported to the plasma

membrane, where secretory vesicles hse with the plasma membrane and release their

contents. Deposited esterified pectins are subjected to de-esterification by methyl-esterase

present in the transmittîng tissue or in the pollen tube wail, and cross-linked by the ca2+,

resulting in a ngid frarnework that provides the support for the growing pollen tube

(Geitmann and Cresti 1995; Geitmann 1997; Franklin-Tong 1999)- This is in contrast to

the esterified pectins, which are somewhat water soluble- Pectins have also been

postulated to play a role in cell wall hydration, filtration, adhesion between the ceils, and

wall plasticity during growth (Levy and Staeheiin 1992; Carpita and Gibeaut 1993;

Geitmann 1997). Pollen tube elongation is indeed confmed to the apical end of the cell

(the growth zone), and it depends on the constant supply with ce11 wall material and

membrane surface (Geitmann 1997). The force responsible for the elongation is believed

to be the hydrostatic pressure, equaiiy exerted at aU points of the ce11 surface. The celI

wall at the pollen tube tip is assumed to be weaker then at the flanks of the tube due to the

absence of callose and possibly its lower degree of cross-linking between polymers

(Derksen 1995b).

Polygalacturonases were discovered 37 years ago, and since then there have been

extensive studies of polygalacturonase-mediated pectin disassembly (Hadfield and

Bennett 1998). Polygalacturonases catalyze the random hydroIytic splitting of the interna1

glycosidic a-1,4 linkage in ~-gdactLLronm chahs of pectic substances (Tagawa and Kaji

1988). Studies showed that polygalacturonase participates in many plant developmental

processes (e.g. organ abscission, pod and anther dehiscence, and pollen grain maturation

and pollen tube growth, Hadfield and Bennett 1998). Two possible fùnctional roles have

been suggested for polygalacturonase in poll.en tubes: First, to degrade the wdls of the

stylar cells to allow penetration of the pollen tube or to provide wall precursors for tube

growth. Second, to act on the pollen tube wall to facilitate growth (Hadfield and Bennett

1998; Brown and Crouch 1990)

4-8 Self-incompatibility mechanism(s) in distylous Turnera

Zn Tzmera, poilen tubes of both morphs germinate and grow but incompatibIe

tubes stop - this is typical of many seIf-incompatibility systems with stylar inhibition (de

Nettancourt 1977). Considering the role polygalacturonases may play in pollen

germination and poilen tube growth (Brown and Crouch 1990; Allen and Lonsdale 1993),

and since both pollen and style genes show approximatery 77% similarity to

polygalacturonase genes, it is tempting to suggest that self-rejection mechanisms involve

polygalacturonase genes in Tztmera.

Here, 1 will attempt to discuss the various mechanisms through which

incompatibility reactions may function in Tumera. Two different types of hypotheses are

u s u d y considered to represent the events of seif-incompatibility system: absence of

stimulation by the stigma and style for poilen growth (complementary system) and

inhibition of growth of pollen tubes in the pistil (an active oppositional system, de

Nettancourt 1977). It has been suggested that heterornorphic seif-incompatibility results

fiom a complementary system (Barrett and Cruzan 1994) rather than an oppositional

system as in the case of homomorphic self-incompatibility (de Nettancourt 1997). There

is not enough evidence to reject either of these hypotheses- However, in Tzmzera, the

oppositional system seems more iikely for the folIowing reasons. First, Turnera pollen

fiom both morphs can grow in an artificial medium. Second, compatible and

incompatible pollen tubes can grow in the stigma and styles of both morphs untii the

incompatible pollen tubes corne to a halt (Tamari et al., unpublished). These data suggest

that poilen tubes of both morphs are able to grow without any specific requirement from

the pistil, hence, reinforcing the oppositional hypothesis; this has also been suggested for

other species (de Nettancourt 1977). Another way of testing the ability of poilen tubes

growth, without a specialized substance provided by style or stigma, would be to have

Tztmern pollen from both morphs germinate and pollen tubes grow in the foreign species.

The p o h a t i o n studies of self-compatible BRY demonstrated that this plant has a

normal polien incompatibility phenotype but the styles possess a cryptic incompatibility

(see introduction, Shore and Barrett 1986), This implies that the self-incompatibility

components of polien and pistil are distinct in fi~rnera and most likely there exists a

specific interaction and recognition mechanism between the two components. The

specific interaction may also explain the fact that incompatible and compatible pollen

tubes in the same style do not idluence one another. Since S35 is present in the

transmitting tissue (intracellular and extraceilular matrix) of the stigmas and styles of

short-styled morph, it is iïkely that it cornes in close contact with compatible (long-styled

morph) and incompatible tubes (short-styled morph). It is possibie that the interaction of

S3 5 with self-incompatibility component(s) of tubes from short-styled pollen initiate the

seIf-incompatibility response. S35 may act as a signal molecule, as in the Papaveraceae,

interacting with a membrarze-bound pollen receptor or it may be taken into the tube, as in

the Solanaceae and act on a specific substrate (e.g. pectin substances). The other

possibiiity is the digestion of short-styled pollen tube w d by S35, while long-styled

poilen tube is protected - alternatively, only the short-styled pollen tube wail may be

modified so that it is digested by S35. Neither of these scenarios explains the inhibition of

the long-styled pollen tubes growing in the styles of long-styled plants. Perhaps a

different, as yet undetected, molecule is in the styles of long-styled morph that interacts

with the pollen self-incompatibility components and initiates a self-rejection response.

This supports the hypothesis that different self-incompatibility mechanisms operate in the

two morphs (Lloyd and Webb 1992). In either case, my study provide strong support for

the occurrence of a novel iricompatibility protein, i-e. polygalacturonase.

4.9 Identification of a novel short specific sQle protein

Comparing the protein profdes of the styles of short- and long-styled morphs,

differentiated by SDS-PAGE, revealed a 68 Kd band in short-styled plants that was not

detected or weakiy detected in long-styled plants. In immunoblotting tests neither style

nor pollen immune serum reacted with S68, leading to the conclusion that S68 is different

Eom the style and poilen specific proteins identified by Athanasiou and Shore 1997. This

novel protein may play a role in physiological or morpholo~cal features of distylous

Trrmera species. In summary, there are two pieces of evidence that may support such

roles for S68. Its expression is restricted to the short-styled morph in all of the Tzrrnera

species examined to date. It is not expressed in MITOM0 (homostyled self-compatible

flower) but it is expressed in the styles of the short-styled flowers of the same plant.

Further studies will be required to understand its role in distyly.

4.10 Conclusions

Proteins specific to the pollen and styles of short-styled plants were discovered by

Athanasiou and Shore (1997) and later genes were cloned fiom BRY using these

proteins. Both poilen and style genes showed homology to polygalacturonase genes. 1

raised polyclonal antibodies against recombinant proteins from these genes. IEF-, SDS-

immunoblotting showed that these genes indeed encode the short specific proteins.

Immunocytochemistry provided strong evidence that at least the style protein rnay play a

role in incompatibility based on localization of this protein to the transmitting tissue of

style. In fact, this presents the first strong evidence in support of an incompatibility role

for a protein in any distylous species. If it is an incompatibility protein, it is novel in that

it is the first polygalacturonase irnplicated in self-incompatibility.

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