Nature 407 , 649 - 651 (2000) © Macmillan Publishers Ltd.
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05 October 2000
DOAN-TRUNG LUU, XIKE QIN, DAVID MORSE & MARIO CAPPADOCIA
Biology Department, University of Montreal, Montreal , Quebec H1X 2B2, Canada
Correspondence and requests for materials should be addressed to M.C. (e-mail: mario.cappadocia@umontreal.ca).
Many flowering plants avoid inbreeding through a genetic mechanism termed selfincompatibility. An extremely polymorphic S-locus 1 controls the gametophytic selfincompatibility system that causes pollen rejection (that is, active arrest of pollen tube growth inside the style) when an S-allele carried by haploid pollen matches one of the
S-alleles present in the diploid style. The only known product of the S-locus is an S-
RNase expressed in the mature style 2 . The pollen component to this cell–cell recognition system is unknown and current models 3 , 4 propose that it either acts as a gatekeeper allowing only its cognate S-RNase to enter the pollen tube, or as an inhibitor of non-cognate S-RNases. In the latter case, all S-RNases are presumed to enter pollen tubes; thus, the two models make diametrically opposed predictions concerning the entry of S-RNases into compatible pollen. Here we use immunocytochemical labelling of pollen tubes growing in styles to show accumulation of an S-RNase in the cytoplasm of all pollen-tube haplotypes, thus providing experimental support for the inhibitor model.
We have used a monospecific polyclonal antibody directed against a specific S-RNase to label pollen tubes growing in incompatible styles. S-RNases contain an RNase activity domain required for pollen rejection allele-specific recognition 7 , 8
5 , 6 and a hypervariable region involved in
. The 15-amino-acid peptide used to prepare the antibody corresponded to the hypervariable region of the S
11
-RNase, and the antibody recognizes the S
11
-RNase but does not recognize the S
13
-RNase, which differs from the former by only 3 amino acids in this region 8 . In self-pollinated styles of plant V22 (genotype
S
11
S
13
; expressing both S
11
and S
13
RNases), the arrest of pollen-tube growth occurs between the upper third and upper half of the style and is complete by 48 hours postpollination. Styles collected 18 hours post-pollination ( Fig. 1a ), which contain stillgrowing pollen tubes, were thus selected for the immunolabelling. Light microscopy was used to identify the stylar regions containing the apical tips of pollen tubes, where elongation occurs, and transverse sections from these regions were then stained for transmission electron microscopy (TEM) with the anti-S
11
and gold-labelled secondary antibodies. Pollen tubes appear as small, irregularly shaped cells amid the larger, rounder cells of the transmitting tissue, and their identity was determined by comparing the morphology of unpollinated and pollinated styles 9 , 10 and by in situ hybridization with a pollen-specific gene probe (data not shown). In these samples (not stained with osmium, in order to preserve their antigenicity), apical regions of pollen tubes contain

darker-staining cytoplasm at their peripheral regions surrounding either a paler electronlucent vacuole or numerous electron-lucent secretory vesicles, depending on the position of the section relative to the pollen tip 11 .
Figure 1 S
11
-RNase entry into incompatible pollen tubes.
Full legend
High resolution image and legend (47k)
Pollen-tube labelling ( Fig. 1b and c ) occurs principally in the cytoplasm. More distal regions of pollen tubes (not shown) appear collapsed, devoid of cytoplasm, and are not labelled. In contrast to the pollen tubes, cells of the transmitting tissue generally show labelling over the lighter-staining regions. The presence of S-RNases inside membranebounded compartments is expected for a secreted protein and is consistent with the label found in the extracellular matrix ( Fig. 1b and c ). For the experiments shown in Fig. 1 , label density in the extracellular matrix (6 2 gold particles per µm substantially less than that inside the pollen tube cytoplasm (32 10 gold particles per µm 2
2 ) is twice that found inside the transmitting tissue cells (3 0.6 gold particles per µm 2 ) and
). All label densities are greater than the background (0.5 0.3 gold particles per µm 2 ), measured either in the absence of the primary antibody (not shown) or in stylar epidermal tissues stained with the anti-S
11
antibody ( Fig. 1d ). Epidermal tissues are known not to express S-RNases 12 , 13 , and are not expected to cross-react with the antibody.
To confirm genotype-independent S-RNase uptake by pollen tubes, we labelled sections from V22 styles following a fully compatible cross. Pollen was obtained from the Shomozygous plant VF60, so that all have an identical S
12
-allele constitution. All pollen tubes are stained with the anti-S
11
antibody, and the pattern of label accumulation inside the cytoplasm ( Fig. 2a ) is indistinguishable from that found in fully incompatible crosses ( Fig. 1 ). The label density inside S
12
-pollen tubes (31 15 gold particles per µm per µm 2
2 ) is higher than that found in the extracellular matrix (3.8 0.8 gold particles
) or inside the transmitting tissue cells (2.6 0.8 gold particles per µm 2 ). Thus, the S
11
-RNase enters S
12
pollen tubes. In another experiment, styles of V22 were pollinated with S
13
and S
14
pollen (a semi-compatible cross). Once again, the S
11
-RNase was found in the cytoplasm of all pollen tubes ( Fig. 2b ) even though none of the pollen was S
11
. Despite a lower overall intensity of label, the label density in pollen cytoplasm
(12 4 gold particles per µm 2 ) was again fivefold higher than that found in the extracellular matrix (2.2 0.9 gold particles per µm 2 ) and tenfold higher than the transmitting tissue cells (1.4 0.7 gold particles per µm 2 ). As a last control for the specificity of the reaction, sections containing compatible S
11
and S
13
pollen tubes growing in S
12
S
14
styles were stained with the anti-S
11 antibody ( Fig. 2c ). As expected, given the absence of an S
11
-RNase, only background label density was measured (0.6
0.3 gold particles per µm 2 ). We note that the lack of label in the pollen tube cytoplasm indicates that the antibody does not cross-react with a pollen-specific antigen. Taken

together, these results show an active uptake or accumulation of the S
11
-RNase in pollen tube cytoplasm. We also note that, as all pollen tubes are similarly labelled, S-RNase accumulation in pollen tubes is genotype-independent. All these observations are inconsistent with a gatekeeper model requiring specific entry of an S-RNase only into pollen tubes carrying the corresponding S-allele.
Figure 2 S
11
-RNase entry into compatible pollen tubes.
Full legend
High resolution image and legend (15k)
To place in perspective our findings showing S-RNase uptake in both compatible and incompatible pollen tubes growing in vivo , we note that genotype-independent accumulation of S-RNases in pollen tubes germinated in vitro in solutions of purified S-
RNases has been previously reported
RNA degradation 14 and growth arrest
14
15
. However, in these studies, both pollen-tube
were genotype-independent, and thus the genotype-independent S-RNase uptake was attributed to an artefact of in vitro germinated pollen 16 .
Our studies support a model for self-incompatibility in which an RNase inhibitor must be present inside pollen tubes. The current inhibitor model of gametophytic selfincompatibility 3 identifies pollen-S with this RNase inhibitor. Pollen-S, like stylar S-
RNase, would contain two domains, one binding in an allele-specific way to the recognition domain of its corresponding S-RNase, the other binding to (and inhibiting) the activity domain of any other S-RNase. For the self-incompatibility system to work, one must assume that binding to the two domains is mutually exclusive, and that binding to the recognition domain is thermodynamically favoured over binding to the
RNase activity domain. Thus, both binding of pollen-S to the recognition domain (for incompatible crosses) or to the RNase activity domain (for compatible crosses) would result in S-RNase accumulation in pollen tubes, as we show here.
How does the inhibitor model deal with the breakdown of self-incompatibility in pollenpart mutations? In most cases 1 , 17 the breakdown has been associated with the presence of two different S-alleles in the same pollen grain, a situation similar to the compatibility of tetraploids derived from self-incompatible diploids (also termed competitive interaction). To explain the competitive interaction, the inhibitor model requires a mechanism enabling pollen-S to bind to the RNase activity domain even in the presence of its cognate S-RNase recognition domain. In other cases, where competitive interaction had been ruled out 18 , 19 , pollen compatibility was presumed to result from deletion or inactivation of the pollen S-allele. However, the inhibitor model for pollen-S predicts that deletions of pollen-S should be lethal 17 . To reconcile the inhibitor model with the compatibility of pollen-S deletions, we propose that the two functions of RNase inhibition and allele-specific recognition are carried by separate proteins, RNase inhibitor and pollen-S, respectively. In our view, all pollen contain a general RNase inhibitor that can potentially bind and inactivate any S-RNase. This binding can only be prevented when pollen-S binds to the recognition domain of its cognate S-RNase, which would lead to the self-incompatibility reaction by activation of the RNase activity. Our version of the inhibitor model predicts that deletion of pollen-S would produce fully compatible pollen, and could best be confirmed by recovery of

self-compatible pollen-part mutants generated by mutagenesis of S-homozygous plants.
The compatibility of S-heteroallelic pollen could now be due either to a mechanism impeding pollen-S binding to the recognition domain of its cognate S-RNase or to a simple lack of pollen-S expression.
Methods
Plant material Two fully self-incompatible but cross-compatible diploid Solanum chacoense Bitt. lines were obtained from the Potato Introduction Station (Sturgeon Bay,
Wisconsin). The self-incompatibility alleles in the parental line PI458314 were identified by genetic crosses and gene sequence as S
11
and S
12
(ref. 20 ) and the alleles in the parental line PI230582 identified as S
13
and S
14
(ref. 21 ). The F
1
hybrids used in the present study were obtained by genetic crosses of the parental lines, and include G4
(S
12
S
14
) (ref. 22 ) and V22 (S
11
S
13
) (ref. 23 ), whereas VF60 (S
12
S
12
) was a selfed line from the parental line PI458314 (ref. 24 ).
Microscopy and immunolabelling Styles from pollinated flowers were harvested
18 hours post-pollination and either fixed immediately in 0.5% glutaraldehyde in 0.1 M
PBS (pH 7.4) as preparatory for immunogold labelling 25 , or squashed and stained with a
0.2% solution of aniline blue to estimate the location of the pollen tube tips by ultraviolet microscopy 26 . Samples fixed with glutaraldehyde for 1.5 hours at room temperature were dehydrated and embedded in LR White (Electron Microscopy
Services). Regions of the blocks containing the tips of growing pollen tubes were selected by observation of sections stained with toluidine blue. Regions behind the growing pollen tips do not contain cytoplasm and are not stained with the antibody. The selected regions were then thin-sectioned (150–170 nm 'purple' sections) and placed on
Formvar-coated grids. Sections were incubated with a 1/50 dilution of a rabbit anti-S
11 antibody for 1 hour at room temperature, washed with PBST (PBS containing 0.05% w/v Tween 20) then incubated with a secondary goat anti-rabbit conjugated to 20-nm gold particles (Ted Pella Inc.). To confirm identification, some samples were hybridized to an antisense pollen-specific Solanum gene probe homologous to the Petunia hybrida
PGP35 (Accession number AF161330). The single-strand-RNA probe was prepared using T7 RNA polymerase and Digoxigenin (DIG)-labelled UTP (Boehringer
Mannheim). The probe was hybridized to the sections for 2 hours at 42 °C in a solution containing 40% formamide as described 27 . The DIG label was detected using a sheep anti-DIG conjugated to 10-nm gold particles (Ted Pella Inc). The sequence of antibodies for double labelling subsequent to the in situ hybridization was either (1) 10nm anti-DIG, anti-S
11
, 20-nm anti-rabbit or (2) anti-S
11
followed by a mixture of 10-nm anti-DIG and 20-nm anti-rabbit. Results were identical with both protocols. Sections were observed using a JEOL JEM 100S operating at 80 kV. The rabbit anti-S11 antibody was prepared by Cocalico Biological Inc. against the peptide
KPKLTYNYFSDKMLN synthesized as a branched multiple-antigen peptide (Research
Genetics). On standard western blots, the antibody reacts only with the S
11
-RNase 8 .
Received 5 June 2000; accepted 19 July 2000
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Acknowledgements.
We thank L. Pelletier and N. Nassoury for technical assistance, G.
Teodorescu for plant care, and S. McCormick, A. Cheung, T.-H. Kao and V. de Luca for helpful discussions. The work was supported by a fellowship from Programme
Québecois de Bourses d'Excellence, Québec (D.-T.L.) and by grants from NSERC
(M.C.) and Fonds pour la Formation de Chercheurs et l'Aide à la Recherche (D.M.,
M.C.).
Figure 1 S
11
-RNase entry into incompatible pollen tubes. a , Self-pollinated styles of an S
11
S
13
plant were fixed 18 hours post-pollination, sectioned in region II, and stained sequentially with the anti-
S
11
antibody and a 20-nm gold-labelled secondary antibody. b , c , The apical tips of all growing pollen tubes (P) are labelled to a greater extent than either the transmitting tissue cells (TT) or the extracellular matrix (ECM) separating the cells. Label inside pollen tubes appears associated with the cytoplasm (Pc) rather than the vacuolar spaces (Pv), unlike the labelling found in transmitting tissue cells. d , Only background labelling is present in the cytoplasm, the surrounding cell wall or the cuticle (Cu) of epidermal cells (EP), which do not express S-RNases. All scale bars are 1 µm.

Figure 2 S
11
-RNase entry into compatible pollen tubes. Pollinated styles were treated as described in Fig. 1. a , A fully compatible cross of S
11
S
13
styles pollinated with S
12
pollen. b , A semicompatible cross of S
11
S
13
plants pollinated with S
13
and S
14
pollen. The region observed is close to the middle of the style and is deduced to contain only fast-growing compatible S
14
pollen tubes.
The several electron-lucent regions correspond to secretory vesicles (Psv). c , A fully compatible cross of S
12
S
14
styles pollinated with S
11
and S
13
pollen, showing the background labelling of the pollen tubes in the absence of an S
11
-RNase. Cells are labelled as in Fig. 1 and all scale bars are
1 µm