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 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
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
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
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
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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