The Plant Cell, Vol. 5, 263-275, March 1993 O 1993 American Society of Plant Physiologists

Ablation of Papillar Cell Function in Brassica Flowers Results in the Loss of Stigma Receptivity to Pollination

Muthugapatti K. Kandasamy, Mary K. Thorsness,’ Sabine J. Rundle, Michael L. Goldberg, June B. Nasrallah, and Mikhail E. Nasrallah2

Section of Plant Biology, Division of Biological Sciences, Cornell University, Ithaca, New York 14853

Plant reproduction in crucifers is dependent on interactions that occur at the stigma surface between the male gameto- phyte (pollen and pollen tube) and papillar cells. To dissect these complex interactions, papillar cells were genetically ablated by targeting the expression of a toxin to appropriate cells of the flower with a flower-specific and developmentally regulated promoter. In transgenic Brassica plants that expressed the toxic gene fusion, flower morphology was normal except for aberrant papillar cell development and partia1 pollen sterility. Microscopic, biochemical, and functional analy- ses, mainly focused on papillar cell responses, revealed that papillar cells lost their ability to elongate, to synthesize cell-specific proteins, and to support pollen germination after self- or cross-pollination. This loss of stigma receptivity to pollination was mimicked by treating pistils with protein phosphatase inhibitors. Differences in the effects of genetic and chemical ablation on the pollination responses of Brassica and Arabidopsis flowers are discussed and are ascribed in part to a requirement for phosphorylationldephosphorylation events in Brassica but not in Arabidopsis.

INTRODUCTION

Specific cell-cell interactive events are important components of pollination and fertilization processes. In a successful polli- nation, shortly after pollen capture, the interaction between a pollen grain and the stigma surface leads to hydration and germination of the pollen grain (Heslop-Harrison, 1975). In crucifers such as Brassica and Arabidopsis, the emerging pol- len tube invades the wall of one of the numerous papillar cells at the stigma surface and grows within the papillar cell wall before reaching the extracellular matrix of the transmitting tis- sues of the pistil.

To probe the function of papillar cells in the pollination re- sponses of crucifers, our approach has been to ablate these cells genetically by driving the expression of the diphtheria toxin A chain (DT-A), a potent inhibitor of translation (reviewed in Colher, 1977), under the control of the promoter of the Bras- sica SLocus Glycoprotein (SLG) gene. The SLG gene is a tightly regulated gene that is expressed exclusively in pistils and an- thers and is highly active in the papillar cells of Brassica and Arabidopsis (Nasrallah et al., 1988; Sato et al., 1991; Toriyama et al., 1991). We initiated these genetic ablation experiments simultaneously in the two distantly related crucifer genera Arabidopsis (Thorsness et al., 1993) and Brassica (this paper) to decipher any common features of stigma responses in crucifers. We were also interested in the fact that Brassica,

Current address: Department of Molecular Biology, University of

To whom correspondence should be addressed. Wyoming, Laramie, W 8207l.

unlike Arabidopsis, has a genetic self-incompatibility system- an intraspecific pollen recognition mechanism that acts as a barrier to self-pollination. Therefore, although not predicted a priori, it was conceivable that ablation studies could shed light on differences in pollen-stigma interactions in the two genera.

In this paper, we describe the results of SLG::DT-A expres- sion in Westar, a self-fertile cultivar of 6. napus that can be transformed by Agrobacterium (Fry et al., 1987). B. napus is an amphidiploid species derived from B. campestris and B. oleracea, two species that are by and large self-incompatible. We show that, as was the case with Arabidopsis SLG::DT-A transformants, toxic gene expression in transgenic Brassica affected the development and viability of pollen and resulted in the ablation of papillar cells. Most significantly, however, and in contrast to Arabidopsis, the ablated papillar cells lost their capacity to sustain pollen tube development following self- or cross-pollination, and the transgenic stigmas were effectively sterile at anthesis. This finding together with recent data im- plicating a transmembrane protein kinase in self-incompatible pollination responses in Brassica (Stein et al., 1991; Goring and Rothstein, 1992; Stein and Nasrallah, 1993) prompted us to attempt the chemical ablation of stigmas through the use of protein phosphatase inhibitors. We show that treatment of pistils with these inhibitors mimicked the phenotype of genet- ically ablated stigmas and substantiated the differences in pollination responses between Brassica and Arabidopsis. We propose that compatible pollinations in Brassica are based on the operation in flowers of a specific pollen-papillar cell

264 The Plant Cell

signaling system that requires biochemically active papillarcells.

RESULTS

Plants Transformed with the SLGi3::DT-A Fusion ExhibitAberrant Pistil and Anther Development

A chimeric gene consisting of a 3.65-kb SLG13 promoter frag-ment and the DT-A gene (Thorsness et al., 1991) was introducedinto cultivar Westar plants by Agrobacterium-mediated trans-formation of floral stems. A total of 25 kanamycin-resistant plantswere obtained. Of these, 15 independent transformants werefound to carry the toxic gene fusion by DNA gel blot analysis.Figure 1 shows that the transgenic plants (numbered lanes)carried varying numbers of integrations of the transgene. Anegative control of DNA isolated from an untransformed Westarplant did not hybridize to the probe (Figure 1, lane W).

Earlier work (Sato et al., 1991) has shown that in transgenicB. napus cv Westar plants, the SLG13 promoter was active ex-clusively in pistils and anthers. In pistils, promoter activity wasdetected at high levels in the stigmatic papillar cells and atlow levels in the transmitting tissue of the style and ovary. Inthe anthers, low levels of promoter activity were detected inthe haploid microspores and in the tapetum, a cell layer of theanther that plays a nutritive role during pollen development.As a consequence, SLGjs.'.DT-A transformants exhibited aber-rant pistil and pollen development. Figure 2 shows that, whilethe flowers of SLGi3'.:DT-A transformants resembled theflowers of untransformed Westar plants in gross morphology

1 23 4 5 6 8 9 10 11 12 14 15 16 W

(Figure 2A), they exhibited specific aberrations in pistils andanthers. In eight transformants analyzed, the pistil terminatedin an underdeveloped flattened surface instead of a roundedbilobed dome (Figure 2B). In addition, pollen sterility was ob-served in most of the transformants expressing an ablatedstigma phenotype. In plants with severe pollen sterility, the ma-ture anthers were smaller than normal and failed to shed pollen(Figures 2A and 2C). The effects of SLG^r.DT-A expressionwere analyzed in detail in 11 transgenic plants. The resultsof this analysis are summarized in Table 1 and described inthe following sections.

Toxic Gene Expression Affects the Development ofPapillar Cells

The pistils in flowers of SLG13::DT-A transformants were ex-amined by light and electron microscopy to identify the celltypes affected by toxic gene expression. Figure 3 shows thatthe expression of the transgene primarily affected the struc-ture and development of the papillar cells of the stigma surface.When compared to wild-type pistils (Figure 3A), the ablatedpistils (Figure 3B) exhibited a normal arrangement of internalcell layers. At the light microscope level, the transmitting tis-sue, which forms the path for the growth of pollen tubes throughthe pistil, appeared to be unaffected. In contrast, the papillarcells, which in the pistils of wild-type flowers average 100 urnin length (Figure 3C), appeared stunted in ablated pistils (Fig-ure 3D). Table 1 shows that papillar cell length was affectedto varying extents in the flowers of different transgenic plants,and papillar cells were only half as long as untransformed con-trol cells in some cases. At the ultrastructural level, thecytoplasm of ablated stigmatic papillae was found to containabnormal clusters of organelles like mitochondria and plastids(Figures 3E to 3G). This clustering of organelles was cell au-tonomous and did not extend to the adjacent subepidermalcells of the stigma (Figure 3G).

im21.7-

9.4-

6.7-

3.4-

1.7-

Figure 1. DNA Gel Blot Analysis of Primary SLG,3::DT-ATransformants.

Genomic DNA from SLG13::DT-A transformants (numbered lanes) andthe untransformed Westar (W) was digested with BamHI and hybrid-ized with a 32P-labeled DT-A fragment. Molecular length markers inkilobases are indicated at left.

Toxic Gene Expression Affects Pollen Development

The effect of SLG13::DT-A expression on pollen fertility variedconsiderably between transformants. Of 11 plants analyzedby pollination, the percentage of sterile pollen grains producedby transgenic anthers was variable, with three transformantsshowing ~50°/o pollen sterility (Table 1). In some but not alltransgenic plants, the severity of pollen phenotype correlatedwith the severity of stigma phenotype. For example, plant 14exhibited severe ablation of stigmatic papillae and 65% pol-len sterility. Further, pollen sterility was inherited in correlationwith the transgene. In the T2 progenies of plant 14, pollensterility varied from 12 to 96% with seven plants showing morethan 45% sterility (data not shown).

The effect of toxic gene expression on anther and pollendevelopment was analyzed in detail in several transgenicplants. Figure 4 shows cross-sections through anthers at thebinucleate microspore stage from an untransformed control

Ablation of Papillar Cell Function in Brassica 265

Figure 2. Comparison of Wild-Type Westar and Ablated Flowers.(A) Photograph of wild-type (W) and ablated (DTA) flowers showing morphological abnormalities in stigma (St) and anther (An) of SLG,3::D7-Xtransformants. Flower development in transformants was normal except for the reduced, flattened stigmas and the reduced amounts of pollerproduced. Bar = 3 mm.(B) Close-up view of an ablated and a wild-type stigma. Bar = 0.4 mm.(C) Close-up view of a severely ablated anther and a wild-type anther. Bar = 0.4 mm.

plant (Figure 4A) and from an SLG^r.DT^A transformant (Fig-ure 4B). Examination of the sections indicated that, althoughtransgenic anthers contained arrested microspores, the orga-nization of anther tissues in general, and of the tapetum inparticular, was visibly unaffected by DT-A expression. Figure5 shows that subtle differences in the tapetum between wild-type and transgenic anthers were nevertheless observed dur-ing the course of anther development. Wild-type and transgenicanthers were very similar at the late uninucleate microsporestage (Figure 5A, panels 1 and 4). In untransformed controlanthers (Figure 5A, panels 1 to 3), tapetal cell disintegrationbecame visible at the binucleate microspore stage (Figure 5A,panel 2) and was clearly evident at the early trinucleate micro-spore stage (Figure 5A, panel 3). In anthers of transgenic plantswith high levels of pollen sterility (Figure 5A, panels 4 to 6),however, an intact tapetum was still visible in anthers at thebinucleate microspore (Figure 5A, panel 5) and the early

trinucleate microspore stages (Figure 5A, panel 6). Tapetal celldisintegration was only delayed in transgenic anthers, howeverbecause the tapetum eventually degenerated and was com-pletely absent in pre-dehiscence anthers.

More severe effects of the SLG13::DT-A transgene were evi-dent in microspores within the anther locules starting at thelate uninucleate microspore stage (Figure 5A, panel 4). Adetailed view of microspore development was obtained by ex-amining suspensions of microspores (Figure 5B) isolated fromanthers of an untransformed plant (Figure 5B, panels 1 to 3)and a transgenic plant exhibiting pollen sterility (Figure 5B,panels 4 to 6). Early microspore development apparentlyproceeded normally: microspores at the early uninucleate stagefrom untransformed anthers (Figure 5B, panel 1) and from trans-genic anthers (Figure 5B, panel 4) were morphologically similar.The first signs of abnormal development were evident at thebinucleate microspore stage, at which time a fraction of

266 The Plant Cell

Table 1. Summary of the Effects of SLG13::DT-A Expression on Stigmatic Papillae, Pollen Sterility, and Pollination Response

Pollination Responsea

Transgenic Stigmas Untransformed Stigmas Papillar Cells

Length Percent Reduction Pollen Plant (km f SD) in Lengthb (O/o sterility) Self-Pollinated Cross-Pollinatedc Self-Pollinated Cross-Pollinatedd

we 101 f 10 - 2 >300 3 95 f 8 6 1 1 >300 >300 N D' 5 67 f 6 34 28 7 2 5 6 f 5 >300 6 65 f 6 36 50 4 f 1 8 f 2 >300 7 79 f 9 22 3 80 f 40 50 f 30 ND 8 95 f 8 6 15 >300 >300 ND 9 61 f 5 40 12 12 f 7 70 f 37 ND 10 96 f 9 5 20 140 f 86 >300 ND 1 1 66 f 6 35 47 13 f 9 10 f 2 >300 12 69 f 8 32 3 2 f 1 1 1 f 9 ND 14 50 f 8 50 65 13 f 8 42 f 29 >200 15 54 f 6 47 6 3 2 2 39 f 30 ND

aNumber of pollen tubes that invaded the papillae; the data are an average of pollen tube counts on six stigmas. Percent reduction in papillar cell length relative to untransformed Westar. Cross-pollinated with pollen from untransformed Westar. Cross-pollinated with pollen from the SLG13::DT-A transformant indicated.

ND, not determined. eW, Westar.

microspores in transgenic anthers (Figure 56, panel 5) ap- peared smaller than those in untransformed anthers (Figure 56, panel 2). Later in development, these affected microspores remained small and became the collapsed sterile grains evi- dent at anthesis (Figure 58, panel 6). In contrast, the unaffected microspores within transgenic anthers, like the microspores within untransformed anthers (Figure 56, panel 3), completed their development into oval-shaped trinucleate pollen grains.

Papillar-Specific Proteins Are Not Synthesized in Ablated Cells

To determine whether the ablated papillar cells of the SLGl3::DT-A transformants were biosynthetically active, we as- sayed transgenic stigmas for the presence and synthesis of the S Locus-Related (SLR7) gene product, the SLRl glyco- protein. The SLRl gene (Lalonde et al., 1989; Trick and Flavell, 1989) is highly conserved in Brassica species. Like SLG, the ubiquitous SLRl glycoprotein is synthesized specifically in stig- matic papillae and is developmentally regulated during stigma maturation. Anti-SLR1 antibodies have been used to detect SLRl protein in a wide variety of self-incompatible and self- compatible Brassicas including Westar (Umbach et al., 1990). These studies have shown that SLRl protein migrates on SDS-polyacrylamide gels as a cluster of immunoreactive bands corresponding to different SLRl glycoforms. Figure 6 shows the immunoblot analysis of stigma proteins from un- transformed Westar (lane W) and from severa1 transgenic plants

(numbered lanes) with anti-SLR1 antibodies. Transformants exhibiting reductions in papillar cell length of more than 30% (plants 5, 6, 9, 11, 12, 14, and 15; Table 1) produced very low or no detectable levels of SLRl protein, whereas transformants with negligible or no reduction in papillar cell length (plants 3, 8, and 10; Table 1) produced levels of SLRl protein com- parable to the untransformed control. Plant 7, in which papillar cell length was reduced by .u20%, produced significant lev- els of SLRl protein. The stunted papillae in this plant did not appear to be associated with extensive papillar cell dysfunc- tion, and may be due in part to factors other than toxic gene expression.

The conclusion that severely ablated papillar cells were bio- synthetically deficient was further confirmed by in vivo incorporation experiments in which 3%-methionine was ap- plied to the surface of intact stigmas as described in Methods. This method of application allows the direct uptake of label by the stigma surface and was found to be effective in the meta- bolic labeling of proteins expressed in papillar cells (Nasrallah et al., 1985). Figure 7shows the results of this analysis as per- formed on buds and flowers from untransformed Westar (W), from an SLG13::DT-A transformant with no papillar cell abla- tion (plant 3), and from another transformant with severe papillar cell ablation (plant 15). The labeled proteins were sepa- rated by isoelectric focusing and visualized by autoradiography, and the position of the SLRl protein band was identified by immunoblot analysis of a duplicate gel (data not shown). La- beled SLRl protein (Figure 7, arrowhead) was detected in untransformed Westar stigmas from flower buds and flowers.

Ablation of Papillar Cell Function in Brassica 267

Figure 3. Microscopic Analysis of Wild-Type and Ablated Pistils.

(A) and (B) Light micrographs of longitudinal sections through the stigma and upper portion of the style. Pistils were fixed, embedded, and cutinto 1-nm sections. The sections reveal differences in the length of the papillar cells (P) between wild-type (A) and ablated (B) stigmas. Thecortex (C), epidermis (E), and transmitting tissue (TT) appear unaffected. Bars = 200 urn.(C) and (D) Light micrographs of stigma squashes from wild-type (C) and ablated (D) flowers showing the effects of ablation on papillar cellelongation. Bars = 100 urn.(E) to (G) Transmission electron micrographs of ablated papillar cells and adjacent subepidermal cells. Mitochondria are distributed uniformlyin the subepidermal cells but are clustered in the ablated papillar cells. (F) is an enlarged portion of the mitochondrial cluster shown in (E).MIT, mitochondria; P, papillar cells; SE, subepidermal cells. Bars = 2 urn.

268 The Plant Cell

w

Figure 4. Light Micrographs of Transverse Sections of Anthers fromWild-Type and SLG,3::DT-A Transformants.

(A) Wild-type anther.(B) Ablated anther.Anthers at the binucleate microspore stage were fixed, embedded,and cut into 1-nm sections. Ablated anthers show normal organiza-tion of sporophytic tissues but partial microspore sterility. Two of thefour anther lobes are shown in each case. C, connective; E, epidermis;En, endothecium; M, microspore; S, stromium; Sp, sterile microspore;T, tapetum; V, vascular tissue. Bar = 100 urn.

In transformant 3, stigmas from flower buds at 2 days beforeanthesis (-2 buds), stigmas from buds at 1 day before anthe-sis (-1 buds), and stigmas from flowers (F) produced labeledSLR1 protein at levels comparable to the untransformed con-trol. In transformant 15, the label was incorporated largely intoproteins that focused in the acidic region of the gel. We be-lieve that these proteins are synthesized, not in papillar cells,but in the underlying cells of the stigma and style. In supportof this notion, no incorporation into SLR1 protein was detectedin the extracts of the ablated pistils from transformant 15. The^S-methionine incorporation into additional proteins (Figure7, arrows) was also drastically reduced in the severely ablatedstigmas relative to untransformed Westar and transformant 3.The nature of these proteins is not known. However, their ab-sence from ablated stigma extracts and their increasedsynthesis at later stages of flower development in wild-typestigmas suggest that they are papillar cell-specific proteinspresumably involved in the function of mature papillae.

SLG13::DT-A Expression Affects Stigma Receptivityin a Developmentally Regulated Manner

We performed a series of reciprocal pollinations with the openflowers of transgenic and untransformed plants. In one set ofpollinations, the behavior of the transgenic pollen of plants 5,6, 11, and 14 on untransformed Westar stigmas was tested.As shown in Table 1, these pollinations produced high pollentube counts even when the pollen was derived from transgenicplants exhibiting 65% pollen sterility. In wide crosses, untrans-

formed Westar stigmas supported the development of pollerfrom 6. oleracea and B. campestris, but not Arabidopsis. Ab-lated stigmas did not support any pollen tube developmentin these wide pollinations (data not shown). Moreover, thereceptivity of transgenic stigmas to intraspecific pollen tubegrowth was dramatically impaired by expression of the toxicgene. The pollen tube counts shown in Table 1 demonstratethat the impairment of stigma receptivity was correlated withthe severity of ablation phenotype. Thus, transgenic flowerswith marginally ablated stigmas (plants 3, 8, and 10) retainedthe capacity to support the growth of self-pollen and pollenfrom untransformed Westar; in general, more than 300 pollentubes were routinely observed on these stigmas, and pollina-tion resulted in normal seed set. Transformant 7, in whichpapillae were stunted but biosynthetically active, supportea somewhat lower number of pollen tubes, indicating that papilar cell function was only partially impaired in this plant. Verlow pollen tube counts were noted when transgenic flowerwith severely ablated stigmas were self- or cross-pollinatedOf the transformants listed in Table 1, four transformants (56,11, and 12) allowed on average fewer than 10 pollen tubes/stigma. In addition, pollination of flower stigmas in these plantsresulted in very poor or no seed set in severe cases. Interest-ingly, however, self- or cross-pollination of stigmas fromimmature flower buds at 3 to 4 days before anthesis resultedin high pollen tube counts and seed set, thus providing a meansto maintain these severely ablated plants. Figure 8 illustrates,for transformant 14, the absence of pollen tubes upon pollina-tion of the mature stigmas of open flowers (Figure 8A) and thedevelopment of pollen tubes on the immature stigmas of youngflower buds (Figure 8B).

To identify the developmental stage at which the effects oftoxic gene expression on stigma receptivity became detect-able, we analyzed the pollination response of stigmas at variousstages of bud and flower development in several transgenicplants. Figure 9 shows the pollen tube counts obtained afterself-pollination in two transgenic plants and in untransformedWestar. In 2- to 3-mm buds (uninucleate microspore stages),large numbers (100 to 200) of pollen tubes were observed ontransgenic and control stigmas alike. Starting at the 5-mm budstage, however, the receptivity of transgenic stigmas to pollentube growth declined progressively and was eventually lostat 1 day before anthesis and at the flower stage. A differentdevelopmental progression was observed in untransformedWestar. Untransformed stigmas derived from buds at thebinucleate and early trinucleate microspore stages maintainedtheir ability to support high levels of pollen tube growth. In-terestingly, however, untransformed stigmas supported veryfew if any pollen tubes at 1 day before anthesis, prior to regain-ing their receptivity in flowers.

The decline in stigma receptivity observed during the de-velopment of transgenic stigmas can be correlated with theeffects of toxic gene expression on papillar cell elongation.The growth patterns of developing papillae in two severely ab-lated transformants and an untransformed control are shownin Figure 10. Early in development, in 2-mm buds at the

Ablation of Papillar Cell Function in Brassica 269

PO

Figure 5. Developmental Analysis of Anther and Pollen Maturation in Wild-Type and SLG,3::DT-A Transformants.

(A) Light micrographs of developing wild-type (W, panels 1 to 3) and ablated anthers (DTA, panels 4 to 6) at the uninucleate microspore stage(UNI), the binucleate microspore stage (Bl), and the trinucleate pollen stage (TRI). One anther lobe is shown from each stage. Sterile microspores(arrowheads) become evident in ablated anthers at the late uninucleate microspore stage. The tapetum in ablated anthers is intact at all develop-mental stages shown, while dissolution of the tapetum commences at the binucleate microspore stage in wild-type anthers. M, microspores;Po, trinucleate pollen grains; T, tapetum. Bars = 50 urn.(B) Light micrographs of developing microspores. Microspores were isolated from wild-type (W, panels 1 to 3) and ablated anthers (DTA, panels4 to 6) at the early uninucleate microspore stage (E-UNI), the binucleate microspore stage (Bl), and the trinucleate pollen stage (TRI). Differencesin the size of normal and sterile microspores within ablated anthers become evident at the binucleate stage. Large arrowheads point to micro-spores and pollen grains of normal size, and small arrowheads indicate the ablated microspores. M, microspores; Po, trinucleate pollen grains.Bars = 50 urn.

270 The Plant Cell

W 3 56 7 8 9 10 11 12 14 15

t

97-

66-I

43-

31-

Figure 6. Immunoblot Analysis of SLR1 Proteins in Stigma Extracts.Dramatic differences in the levels of SLR1 protein in ablated and nonab-lated plants are shown. Stigma extracts of untransformed Westar (W)and SLG13::DT-A transformants (numbered lanes) were separated bySDS-PAGE, transferred to nitrocellulose membrane, and reacted witha rabbit SLR1-specific polyclonal antibody. Molecular mass markersin kilodaltons are shown at left.

uninucleate microspore stage, the length of papillar cells intransformants with ablated stigmas was equivalent to that inuntransformed controls. Discrepancies in papillar cell lengthbecame visible in ~3-mm buds at the early binucleate micro-spore stage, a stage at which wild-type papillae had attainedonly ~50% of their full length. In both transformants, the elon-gation of papillar cells ceased during later stages of flowerdevelopment, whereas the papillae of untransformed controlplants continued to elongate until anthesis. As a result, thediscrepancies in papillar cell length between ablated and con-trol stigmas increased progressively during development andwere most pronounced in flowers.

Treatment of Flowers with Phosphatase InhibitorsBlocks Pollen Tube Development in Brassica butNot in Arabidopsis

The failure of wild-type pollen tubes to develop on ablated stig-matic papillae cannot be ascribed to the toxic effects ofstigmatic DT-A molecules on pollen grains for two reasons.First, DT-A molecules are not secreted and are not internal-ized by eukaryotic cells in the absence of the diphtheria toxinB subunit (Pappenheimer and Gill, 1972; Collier, 1977). Sec-ond, in Arabidopsis SLG13::DT-A transformants, ablatedpapillar cells retained the capacity to support pollen tubegrowth (Thorsness et al., 1993). Thus, successful pollen tubedevelopment in Brassica requires biochemically active papil-lar cells. In view of recent data implicating the operation of

phosphorylation cascades in the recognition of "self pollenin self-incompatible strains of Brassica (Stein et al., 1991;Goring and Rothstein, 1992; Stein and Nasrallah, 1993), wereasoned that changes in the phosphorylation state of papil-lar cell proteins may also be important for compatiblepollinations in Brassica. To investigate the relationship betweenprotein phosphorylation and pollen-stigma interactions, wetreated pistils with inhibitors of protein serine/threonine phos-phatases and observed the effects of inhibitor treatment onpollination response. At low concentrations, the inhibitorsokadaic acid or microcystin-LR inhibit type 1 and type 2A pro-tein phosphatases specifically (Bialojan and Takai, 1988;MacKintosh et al., 1990) and do not affect other protein phos-phatases or protein kinases (reviewed in Cohen et al., 1990).To gain some insight into the discrepancy between the geneticablation results obtained in Brassica and Arabidopsis, we ex-tended the phosphatase inhibitor studies to Arabidopsis. Theresults of this analysis are presented in Table 2. The effects

3.5)

15-1 -2 F -1 -2

9.5Figure 7. ^S-Methionine Incorporation in Stigmas of Wild-Type andSLG,3::DT-A Transformants.

Stigmas from untransformed Westar (W), a transformant that did notexpress the transgene (plant 3), and a transformant with ablated papillarcells (plant 15) were labeled, and the labeled extracts were separatedby isoelectric focusing. F, stigmas from flowers; -1, stigmas from budsat 1 day prior to anthesis; -2, stigmas from buds at 2 days prior toanthesis. The arrowhead marks the position of the SL.R1 protein asdetermined by immunoblotting of parallel lanes. SLR1 was synthesizedat all developmental stages in Westar and plant 3, but not in plant 15.Other developmental^ regulated proteins that were also reduced inablated stigmas are indicated by arrows. The origin of electrophoresisand the pH gradient are shown at left.

Ablation of Papillar Cell Function in Brassica 271

Figure 8. Fluorescent Micrographs Depicting Pollen Tube Growth onMature and Immature Stigmas of an SLG13::DT-A Transformant.(A) A self-pollinated mature stigma from a flower of transformant 14.A uniform layer of pollen was applied to the stigmas and resulted incallose deposition and intense fluorescence of papillar cells. P, papil-lae. Bar = 100 urn.(B) Self-pollination of an immature stigma at 3 days prior to anthesisfrom transformant 14 showing pollen tube development into the papil-lar cells. Po, pollen; Pt, pollen tube.

of inhibitor treatment on pollen tube development were dra-matically different in Brassica and Arabidopsis. In B. napus,the germination and growth of Westar pollen tubes were in-hibited on Westar pistils treated with either okadaic acid ormicrocystin-LR. In contrast, treatment of Arabidopsis flowerswith the phosphatase inhibitors had no effect on the growthof Arabidopsis pollen tubes in our assay system. The success-ful pollinations observed on inhibitor-treated Arabidopsis pistils

argue that the inhibitors were not imported into the pollengrains.

DISCUSSION

The observed effects of SLG13::DT-A expression on the devel-opment of anthers and stigmas are consistent with the patternof SLG13 promoter activity previously described in Brassicaplants transformed with an SLGj3::p-glucuronidase (SLG13::GUS) gene fusion (Sato et al., 1991). In anthers, SLG13::GUSexpression was detected in cells of the tapetum and in the de-veloping microspores, but not in other cells of the anther.Consequently, the expression of the SLG,3::DT-A fusion in an-thers affected specifically the development and viability ofpollen. The first detection of aberrant microspore developmentfollowed closely the onset of promoter activity in anthers atthe late uninucleate microspore stage. The arrest in subse-quent microspore development resulted, at anther maturity,in abnormal grains with the collapsed appearance typical ofsterile pollen. In all cases analyzed, however, a variable pro-portion of pollen grains escaped the effects of toxic geneexpression, and as a result, the transgenic plants exhibitedpartial pollen sterility. Although the specific contributions oftoxic gene expression in the tapetum and in microspores aredifficult to unravel, pollen sterility may be attributed to thecombined sporophytic and gametophytic expression of thetransgene. In our transgenic plants, the cells of the tapetumwere not visibly killed. Nevertheless, tapetal expression of theSLG13::DT-A gene was evident in the delay of tapetal cell dis-integration relative to wild type. Presumably, the accumulationof tapetum-encoded degrading enzymes responsible for thebreak-down of cellular components was affected.

unlransformcd Westar

transgenic plant #5

transgenic plant #14

1_§ 200.

t"oJj 100-

2mm 3mm 4mm 5mm 6mm 7mm 1 ;

Flower Development

Figure 9. Pollen Tube Counts after Self-Pollination of DevelopingStigmas in Untransformed Westar and Two Ablated SLG,3::DT-ATransformants.Pollinations were performed on inflorescences bearing 2- to 7-mm budsand mature flowers (F).

272 The Plant Cell

120

110

100

90

f - 80 o - - .E m n 5 80

f

Lo

O 5 0

9) -I 40

30

7.0

3 4 S 8 F

Bud size (mm)

'"L, Figure 10. Papillar Cell Length at Different Stages of Stigma Devel- opment in Untransformed Westar and Transformants 5 and 14.

Stigmas from different developmental stages were fixed, softened, and examined by light microscopy. While the papillar cells from untrans- formed stigmas continued to elongate up to anthesis, papillar cell elongation was arrested at the 3- to 4-mm bud stage in ablated plants. (O), untransformed Westar; (O), transformant 5; (M), transformant 14; F, mature flowers.

We concluded that, in SLG13::DT-A Brassica transformants, as was the case in SLG13::DT-A Arabidopsis transformants (Thorsness et al., 1993), toxic gene expression produced only a partia1 impairment of tapetal function. This result may be ascribed to the fact that the SLG13 promoter becomes active relatively late during tapetal cell development and maintains avery low level of activity in the anthers of both Brassica and Arabidopsis. Although it has been suggested that a single mol- ecule of DT-A can cause cell death (Yamaizumi et al., 1978), it is doubtful that such is the case in plant cells. In any event, our results can be contrasted with the complete disabling of tapetal function and abortion of all pollen development obtained when expression of DT-A (Koltunow et al., 1990) or RNase (Mariani et al., 1990) was driven by a highly active tapetal- specific promoter.

We had previously shown that the major site of SLG13 pro- moter activity was in the papillar cells of the stigma (Sato et al., 1991), where promoter activity was first detected after the onset of papillar cell differentiation in buds at the late uninucle- ate microspore stage. The expression of the SLG13::DT-A gene predictably affected the development of papillar cells. In con- trast, the much lower level of promoter activity in the transmitting tissues of the style and ovary did not result in any

visible aberrations in these tissues. In ablated stigmas, papil- lar cell differentiation was initiated normally and the number of papillar cells was equivalent to that in untransformed stigmas. However, the elongation and maturation of papillar cells failed to proceed in buds after the uninucleate microspore stage. These latter processes apparently required continued protein synthesis, a process inhibited by DT-A. As a result of toxic gene expression, the stigmatic papillae of ablated flowers appeared stunted and exhibited marked cytoplasmic abnormalities. In addition, the ablated papillar cells were biosynthetically defi- cient as judged by their failure to synthesize and accumulate papillar cell-specific proteins. Significantly, the biochemical dysfunction of the ablated papillar cells was correlated with the loss by these cells of the capacity to support pollen tube growth. Whereas the stigmas of flowers in untransformed Westar plants are self- and cross-fertile, the flower stigmas of ablated SLG13::DT-A transformants were self- and cross-sterile. We found that the stigmas of ablated plants gradually lost their receptivity in correlation with SLG13 promoter activity and SLG13::DT-A expression. In contrast, the developing stigmas of untransformed Westar were receptive to pollen tube growth throughout their development except for a narrow window of -24 hr, during which very few pollen tubes developed. This transient pollen incompatibility, which was evident in buds at 1 day before anthesis and in buds with expanded petals whose anthers were not yet dehisced, appears to be common in Bras- sicas and has been observed in some B. oleracea and B. campestris self-incompatible and self-compatible strains (M. E. Nasrallah, unpublished data).

The phenotype brought about by ablating stigmas leads to the conclusion that metabolically active papillar cells are re- quired for successful in plant pollen tube growth in Brassica. Although the inability of ablated papillar cells to carry out any one of a number of biochemical reactions may account for their loss of receptivity, one possible explanation is suggested by the results of experiments in which papillar cell function was ablated by treatment of flower pistils with protein phosphatase inhibitors. We observed an inhibition of pollen tube develop- ment on inhibitor-treated Brassica stigmas, suggesting that a protein dephosphorylation event occurs during pollen-stigma interaction for successful pollen tube development to proceed.

Table 2. Effect of Protein Phosphatase lnhibitors on the Receptivity of Flower Stigmas to Pollen Tube Growth

Number of Pollen Tubes/Stigmaa

Okadaic Acid Microcystin-LR

Treated Control Treated Control

B. napus O >300 5 >300 Arabidoosis >100 >I O0 >I O0 >I O0

BNumbers shown are based on five independent pollination experiments.

Ablation of Papillar Cell Function in Brassica 273

Ablated papillar cells would fail to carry out such a reaction and would therefore be unable to support pollen tube devel- opment. We propose that the phosphorylated state of one or more papillar cell proteins is crucial to the fate of pollen after it contacts the stigma surface. The developmentally regulated changes in stigma receptivity observed in untransformed stigmas would possibly reflect the balance of the activities of specific protein kinases and protein phosphatases on these proteins during the normal course of stigma development.

components are related to those involved in the specific sig- naling between papillar cells and pollen in self-incompatible pollinations remains to be seen.

METHODS

‘Iant lansformation

The stigma sterility induced in Brassica by expression of the SLGI3::DT-A gene and protein phosphatase inhibitor treat- ments is in sharp contrast with the consequences of genetic and biochemical ablation of papillar cells in Arabidopsis. In Arabidopsis as in Brassica, expression of the SLGI3::DT-A gene produced ablated papillar cells that were biochemically impaired (Thorsness et al., 1993). Nevertheless, the ablated Arabidopsis stigmas were still receptive and allowed pollen tube development in appropriate crosses, indicating that the impairment of biosynthetic activity in papillar cells is not of crit- ical importance to pollen tube growth in this genus. In addition, unlike Brassica stigmas, Arabidopsis stigmas treated with pro- tein phosphatase inhibitors remained receptive to pollen tube growth. The different results obtained in Brassica and Arabidopsis were unexpected because the processes of pol- len hydration, pollen germination, and invasion of the papillar cell wall by the developing pollen tube, as well as the path followed by pollen tubes through the tissues of the pistil, ap- pear similar in these two distantly related genera (Ellernan et al., 1992). Our data nevertheless suggest that different mech- anisms of pollen perception operate in Brassica and Arabidopsis. As noted earlier, a well-docurnented difference in the reproductive biology of these two distantly related genera is the operation in Brassica of a self-incompatibility system un- der the control of the S locus. Arabidopsis lacks such a pollen recognition system, and we have failed to demonstrate that the equivalent of an S locus exists in this genus (K. G. Dwyer and M. E. Nasrallah, unpublished data). Recent work has indicated that the inhibition of pollen development in self- incompatible pollinations in Brassica is based on the activity of an S locus-encoded transmembrane receptor protein ki- nase (Stein et al., 1991; Goring and Rothstein, 1992; Stein and Nasrallah, 1993). We have proposed that this kinase is acti- vated in response to self-pollination and would couple the initial recognition event at the pollen-papillar cell interface to a phos- phorylation cascade, thereby leading to pollen rejection (Stein et al., 1991). By demonstrating the importance of the phos- phorylation state of stigmatic proteins to the success of compatible pollinations in Brassica, our results indicate that pollen acceptance in self-fertile strains is dependent on the

Plasmid pMKT17, a Ti plasmid that carried an SLGf3::DT-A toxic gene fusion and a kanamycin resistance gene for selection of transformants (Thorsness et al., 1991), was introduced into Bressica napus cv Westar (a commercial oilseed rape cultivar) by Agrobacterium tumefaciens- mediated transformation of floral stems (Fry et al., 1987). Flowering- stem discs were infected with Agrobacterium strain pCIB542/A136 harboring pMKT17, and selection for transformants was on medium containing 10 mglL kanamycin. Green kanamycin-resistant shods were rooted, transferred to soil, and maintained in the greenhouse.

DNA Gel Blot Analysis

Genomic DNA was prepared from leaves of transgenic plants by a minipreparation procedure (Mettler, 1987). The DNA was digested with BamHI, fractionated on 0.9% (whr) agarose gels, denatured, and trans- ferred to GeneScreen Plus membranes (Du Pont-New England Nuclear). Membranes were hybridized to a fragment containing the DT-A coding region and labeled with 32P-dATP using a random primer labeling kit (Boehringer Mannheim). Hybridization was performed at 65OC in 10% (wlv) dextran sulfate, 0.3 M sodium phosphate, pH 7.0, 5% (whr) SDS, 10 mM EDTA, and 0.14 mg/mL denatured salmon sperm DNA.

Light Microscopy and Transmlsslon Electron Microscopy

Stigmas and anthers at various stages of development were fixed in a mixture of 4% (w/v) paraformaldehyde and 2.5% (vhr) glutaraldehyde in 0.05 M phosphate buffer, pH 7.2, at 2OoC for 3 hr. The samples were washed in buffer and postfixed in buffered 1% (whr) OsO, at 4 O C over- night. After washing in buffer, the samples were dehydrated in an ethanol series and embedded in Spurr‘s epoxy resin. For light micros- copy, 0.5- to l y m sections were cut with glass knives, stained with toluidine blue, and observed and photographed with an optical mi- croscope (Zeiss, Oberkochen, Germany). For ultrastructural analysis, thin sections (70 nm) were cut with a diamond knife, stained with ura- nyl acetate and lead citrate, and examined in an electron microscope (model EM 300; Philips Netherland, Eindhoven, The Netherlands). To measure the length of the papillar cells, stigmas were fixed briefly in a3:l mixtureof ethanol/acetic acid, softened in 1 N NaOH, and exam- ined by light microscopy.

operation of yet another signal transduction system. However, such a signaling system does not apparently operate in lmmunoblot Analysis of Stigma Extracts

- - - . . . .

Arabidopsis. The molecular components Of this proposed “Stigm$samples consisted of the stigma proper and a portion ofthe signaling system remain unknown, but appear to be develop- upper style. Proteins were extracted from stigmas in 10 mM Tris-HCI mentallY regulated as judged bY the changes in stigma receptivity observed during the COUrSe of normal flOwer de- velopment in untransformed plants. Whether any of these

buffer, pH 7.2. The concentration of the protein in the extracts was es- timated by using the Coomassie protein assay reagent (Bio-Rad). Samples containing 5 vg of total protein were subjected to SDS-PAGE

274 The Plant Cell

on 10% (wh) resolving gels according to the method of Dreyfuss et al. (1984). Molecular weight markers were a mixture of six reference proteins from Bio-Rad. The fractionated proteins were electrophoreti- cally transferred to nitrocellulose membranes according to the method of Towbin et al. (1979). The nitrocellulose membranes were subjected to immunoblot analysis with rabbit polyclonal antibodies raised against the SLRl protein as described previously (Umbach et al., 1990).

Metabolic Labellng of Stlgmatlc Paplllae with 35S-Methlonine

For metabolic labeling of stigmas, we used three developmental stages: flowers, buds at 1 day before anthesis, and buds at 2 days before anthe- sis. Groups of 20 flowers or buds from each stage were labeled. The flowers or buds were mounted upright on solidified agar medium after removal of sepals, petals, and anthers. One microliter of a =S-methi- onine solution (prepared by mixing 30 pL 35S-methionine [New England Nuclear], 70 pL distilled H20, and 1 pL Tween 20) was ap- plied to the surface of each stigma under a stereoscope. The flowers or buds were incubated overnight at 25OC, and the stigmas, with a portion of the style attached, were excised, washed three times in 10 mM Tris-HCI buffer, pH 7.2, frozen in liquid NP, and homogenized in 40 pL of distilled water. After centrifugation at 14,0009 for 10 min, the total protein content of the supernatant was determined by using a Coomassie protein assay reagent.

Stigma homogenates containing 10 pg of total protein were subjected to isoelectric focusing for 2 hr on a polyacrylamide gel slab with a pH gradient of 3.5 to 95 (Nasrallah et al., 1985). In any one experiment, samples were electrophoresed in duplicate sets. Following electropho- resis, a portion of the polyacrylamide gel containing one set of samples was stained with Coomassie Brilliant Blue R 250 and photographed. The stained gel was then impregnated with ENHANCE (Du Pont-New England Nuclear), dried, and exposed to x-ray film (Eastman Kodak) at -7OOC for 1 to 2 days to visualize the newly synthesized proteins. The portion of the gel containing the second set of samples was used to identify the position of the SLRl protein band. After electrophore- sis, the gel was blotted by capillary action to nitrocellulose membrane prewetted in Tris-buffered saline (10 mM Tris-HCI, pH 8.0, and 150 mM NaCI); immunoblot analysis of the transferred proteins was then per- formed, as described above for proteins resolved by SDS-PAGE.

Analysis of Polllnation Response and Pollen Viability

To examine the pollination response of transgenic plants, stigmas were either self-pollinated or reciprocally crossed to untransformed control plants. Four hours after pollination, the stigmas were fixed in etha- nol/acetic acid (3:1), softened in 1 N NaOH, stained with decolorized aniline blue, and pollen tube growth was monitored by UVfluorescence microscopy (Kho and Baer, 1968).

An initial assessment of pollen viability was obtained by examining the morphology of pollen grains with the light microscope. Pollen from five just-opened flowers of each transformant was observed. The per- centages of normal oval-shaped and abnormal (sterile) collapsed grains were calculated from pollen grain counts in a total of 20 microscopic fields. In vivo assays to test if pollen from transgenic plants was func- tional involved pollinating untransformed control stigmas with pollen from transformed plants and monitoring pollen tube growth by the above procedure. Self- and cross-pollinations were also monitored by seed set and the production of progeny plants.

Treatment of Flowers with Protein Phosphatase lnhibltors

Flowers were removed from plants either on the day of anthesis for B. napus cv Westar or on the morning of the day before anthesis for Arabidopsis thaliana. The flowers were emasculated to prevent self- pollination, and the flower pedicel was cut under water with a razor blade. Brassica flowers were treated directly with phosphatase inhibi- tors. In the case of Arabidopsis, the inhibitor treatment was preceded by an incubation period in which the flower was allowed to mature by planting it via its pedicel in solid Murashige and Skoog medium (Murashige and Skoog, 1962) for 12 hr. Treatment with phosphatase inhibitors consisted of placing the flowers in microtiter wells with their pedicels submerged in 200 pL of water containing either okadaic acid or microcystin-LR. Care was taken to ensure that the stigma was never in contact with the inhibitor or control solutions. In initial experiments, we assayed severa1 concentrations of inhibitors. The lowest effective concentrations were 2 pM for okadaic acid and 4 pM for microcystin- LR and were used in subsequent experiments. These inhibitor con- centrations were achieved by diluting stock solutions prepared by solubilizing okadaic acid in dimethyl sulfoxide (DMSO) and microcystin- LR in 10% (vh) methanol. Controls therefore involved parallel treat- ments of flowers with either DMSO at a final concentration of 0.065% (wh) or methanol ata final concentration of 0.15% (vh). Inhibitor-treated and control flowers were incubated in a humid chamber for 16 to 24 hr. The flowers were then pollinated with pollen from untreated flowers and incubated for an additional 4 hr in the presence of inhibitors for inhibitor-treated flowers and of DMSO or methanol for control flowers. The pistils were then excised from the flowers and processed as de- scribed above for microscopic analysis of pollen tube growth.

ACKNOWLEDGMENTS

This work was supported by grants from CIBA-GEIGY Corporation (Re- search Triangle Park, NC) and from the U.S. Department of Agriculture. M.K.T. and S.J.R. were supported by National Science Foundation post- doctoral fellowships awarded in 1988 and 1992, respectively. We acknowledge Kent Loeffler for the cover photograph.

Received January 15, 1993; accepted January 25, 1993.

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Receptivity to Pollination.Ablation of Papillar Cell Function in Brassica Flowers Results in the Loss of Stigma

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