Plant Reprod (2014) 27:121–127 DOI 10.1007/s00497-014-0245-z
ORIGINAL ARTICLE
High humidity partially rescues the Arabidopsis thaliana exo70A1 stigmatic defect for accepting compatible pollen Darya Safavian • Muhammad Jamshed • Subramanian Sankaranarayanan • Emily Indriolo Marcus A. Samuel • Daphne R. Goring
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Received: 21 March 2014 / Accepted: 22 June 2014 / Published online: 29 June 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract We have previously proposed that Exo70A1 is required in the Brassicaceae stigma to control the early stages of pollen hydration and pollen tube penetration through the stigmatic surface, following compatible pollination. However, recent work has raised questions regarding Arabidopsis thaliana Exo70A1’s expression in the stigma and its role in stigma receptivity to compatible pollen. Here, we verified the expression of Exo70A1 in stigmas from three Brassicaceae species and carefully re-examined Exo70A1’s function in the stigmatic papillae. With previous studies showing that high relative humidity can rescue some pollination defects, essentially bypassing the control of pollen hydration by the Brassicaceae dry stigma, the effect of high humidity was investigated on pollinations with the Arabidopsis exo70A1-1 mutant. Pollinations under low relative humidity resulted in a complete failure of wild-type compatible pollen acceptance by the exo70A1-1 mutant stigma as we had previously seen. However, high relative humidity resulted in a partial rescue
Communicated by Zhenbiao Yang.
Electronic supplementary material The online version of this article (doi:10.1007/s00497-014-0245-z) contains supplementary material, which is available to authorized users. D. Safavian E. Indriolo D. R. Goring (&) Department of Cell and Systems Biology, University of Toronto, Toronto M5S 3B2, Canada e-mail: [email protected] M. Jamshed S. Sankaranarayanan M. A. Samuel (&) Department of Biological Sciences, University of Calgary, Calgary T2N 1N4, Canada e-mail: [email protected] D. R. Goring Centre for the Analysis of Genome Evolution and Function, University of Toronto, Toronto M5S 3B2, Canada
of the exo70A1-1 stigmatic papillar defect resulting is some wild-type compatible pollen acceptance and seed set. Thus, these results reaffirmed Exo70A1’s proposed role in the stigma regulating compatible pollen hydration and pollen tube entry and demonstrate that high relative humidity can partially bypass these functions. Keywords Exo70A1 Compatible pollen acceptance Pollen hydration
Introduction In angiosperms, successful pollinations are dependent on the particular properties of stigmatic surfaces at the time of pollen receptivity. Stigmas have been divided into two broad categories termed wet and dry stigmas (HeslopHarrison and Shivanna 1977), though an intermediate category of semidry has been used for Senecio squalidus (Allen et al. 2011). There are differences in the surface properties of wet and dry stigmas which result in different outcomes during the early stages of pollen–pistil interactions (Sanchez et al. 2004; Swanson et al. 2004; Hiscock and Allen 2008; Firon et al. 2012). Following pollination, desiccated pollen grains must hydrate to become metabolically active, then germinate and grow a pollen tube through the pistil towards an ovule for fertilization. Wet stigmas have free-flowing surface secretions that are aqueous in nature or composed of lipids, both of which can allow for indiscriminate capture, hydration, and germination of pollen grains. Thus, wet stigmas have little control over these initial stages of pollen–stigma interactions (Heslop-Harrison and Shivanna 1977; Sanchez et al. 2004; Swanson et al. 2004; Hiscock and Allen 2008).
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Dry stigmas are defined by the absence of these surface fluid secretions, and as a result, exert a tighter regulation over the early stages of pollen capture, hydration, and germination. For example, compatible interactions lead to the formation of an interface at the contact site derived from pollen coat and stigma surface components, followed by the movement of water from the dry stigma to the pollen grain for hydration (Heslop-Harrison and Shivanna 1977; Dickinson 1995; Swanson et al. 2004; Chapman and Goring 2010; Firon et al. 2012). In the Brassicaceae, a highhumidity environment can allow for some pollen hydration and germination to occur without compatible pollen– stigma interactions. High relative humidity has been found to rescue pollen hydration and germination (and fertility) of Arabidopsis thaliana pollen coat mutants that are unable to form a proper compatible pollen–stigma interface (Preuss et al. 1993; Hulskamp et al. 1995; Fiebig et al. 2000) or cause precocious germination of A. thaliana pollen developmental mutants in the absence of stigma contact (Johnson and McCormick 2001; Wang et al. 2012). We have previously proposed a role for Exo70A1 in the stigmatic papillae where it is required for the basal compatible pollen response, controlling the early stages of pollen hydration and germination in two Brassicaceae species, Brassica napus and A. thaliana (Samuel et al. 2009; Safavian and Goring 2013). The loss of Exo70A1 function in the stigma caused an absence of pollen hydration, pollen tube growth, and seed set following compatible pollinations (Samuel et al. 2009). However, Li et al. (2010, 2013) raised questions about A. thaliana Exo70A1’s expression in the stigma and inability of the exo70A1-1 mutant stigma to promote pollen hydration and germination. Li et al. (2010, 2013) proposed that A. thaliana Exo70A1 has a specific expression pattern and function in developing tracheary elements, and presented data showing pollen tube growth and seed set for exo70A1-1 mutant pistils following hand-pollinations. Here, we carefully reexamined Exo70A1’s function in accepting compatible pollen, using the well-characterized exo70A1-1 mutant, and investigated if increased levels of humidity could account for discrepancies between the two studies (Samuel et al. 2009; Li et al. 2013).
Materials and methods Plant growth conditions Wild-type A. thaliana Col-0 plants and exo70A1-1 mutant plants (Salk_014826, T-DNA insertion mutant in the Col-0 background; Synek et al. 2006; Samuel et al. 2009; Li et al. 2013) were grown in growth chambers under a long-day cycle of 16-h light/8-h dark at 22 °C with a relative
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humidity roughly around 40 or 80 % (monitored with a digital hygrometer). The exo70A1-1 mutant has been previously shown to be a T-DNA knockout mutant, lacking full length Exo70A1 mRNA (Synek et al. 2006; Samuel et al. 2009). Images of whole plants were captured using a Canon digital camera, and images of whole flowers were taken with a Nikon sMz800 microscope and NIS-elements imaging software at magnifications of 2.09, 3.09 and 6.09. PCR and microarray analyses RT-PCR and genomic PCR were conducted as described by Samuel et al. (2009). The exo70A1-1 mutant genotype was confirmed by genomic DNA PCR using Exo70A1 genespecific primers with the LBA1 T-DNA-specific primer (see Supplemental Table 1 online). For the RT-PCR analyses, RNA was extracted from stigmas harvested from stage 12 flowers for A. thaliana (Smyth et al. 1990), and freshly opened unpollinated flowers for Arabidopsis lyrata and Brassica rapa R-o-18. All plants were grown under low humidity conditions (B40 %), unless otherwise indicated. RNA samples were treated with DNase I to remove any contaminating genomic DNA prior to cDNA synthesis, and primers were selected to span introns for the RT-PCR analyses. Primer sequences and predicted cDNA versus genomic DNA band sizes are listed in Supplemental Table 1 online. Accession numbers are A. thaliana Exo70A1 At5g03540, Exo70A2 At5g52340, Exo70A3 At5g52350; A. lyrata Exo70A1 939738; and B. rapa Exo70A1 Bra009523. For the microarray expression analyses of the exocyst complex subunit genes, A. thaliana stigmas were harvested from stage 10 and stage 12 flowers for RNA extraction. The Whole Transcriptome Amplification kit was used to amplify the transcriptome prior to microarray analysis. Pollinations under low and high humidity To test the stigma receptivity of flowers from exo70A1-1 plants grown under 80 % humidity and wild-type Col-0, the largest flower buds (stage 12; Smyth et al. 1990) were emasculated and left overnight covered in plastic wrap to prevent dehydration. On the following day (when stage 12 flower buds would normally open), all emasculated pistils were hand-pollinated with Col-0 pollen by brushing 1–4 anthers across the stigmatic surface. For 40 % humidity exo70A1-1 mutant plants, variability in stigmatic papillar elongation was observed, and therefore, freshly opened or 1-day old flowers with elongated stigmatic papillae were emasculated, covered in plastic wrap, and pollinated the next day with Col-0 pollen. Following all pollinations, the plants were placed in either the 40 or 80 % relative humidity chambers as indicated in Fig. 3. For viewing
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Results and discussion
Fig. 1 Exo70A1 is expressed in the stigmas of three Brassicaceae species. a–e RT-PCR analysis for the Exo70A1-3 subclade genes in A. thaliana (a, b), and Exo70A1 in A. lyrata (c) and B. rapa (d). Actin was included as a positive control. Stigmas were collected from plants grown under low humidity conditions (B 40 %; a, c, d) or at 80 % relative humidity (b), and were from stage 12 flowers for A. thaliana, and freshly open unpollinated flowers for A. lyrata and B. rapa. DNA marker sizes are shown on the left of each panel, and the PCR products correspond to the predicted cDNA sizes (see Supplemental Table 1 online). d Microarray expression analysis of the exocyst complex subunit genes in A. thaliana stigmas. The exocyst complex consists of 8 subunits encoded by the predicted Exo70, Sec3, Sec5, Sec6, Sec8, Sec10, Sec15 and Exo84 genes (Synek et al. 2006; Chong et al. 2010). The samples, 1 and 2, represent replicates for each flower stage. The values are raw intensities after subtracting the background
pollen tubes, the pollinated pistils were left overnight, collected the next day, aniline blue stained, and viewed on an epifluorescence microscope as previously described (Samuel et al. 2009). For seed set, emasculated pistils from Col-0 and exo70A1-1 plants grown under 40 and 80 %, humidity were hand-pollinated with Col-0 pollen and left for 10 days for the siliques to mature. The pre-dehiscent siliques were dissected using fine-tipped forceps, and the seeds per silique were counted (n = 10 for each sample).
Previously, we demonstrated the expression of Exo70A1 in mature stigmas with A. thaliana Exo70A1 promoter:GUS transgenic plants and a B. napus RNA blot analysis (Samuel et al. 2009). To re-confirm the stigma expression of Exo70A1, RNA was extracted from unpollinated mature stigmas for three Brassicaceae species that we use to study pollen–stigma interactions: A. thaliana, A. lyrata and B. rapa (Samuel et al. 2009; Indriolo et al. 2012; Safavian and Goring 2013). RT-PCR analyses were conducted, and Exo70A1 was clearly detected in the stigma RNA samples from all three species (Fig. 1a, c, d). All the PCR band sizes corresponded to the predicted sizes for cDNA and were not due to any putative genomic DNA contamination (See Supplemental Table 1 online for predicted sizes). Exo70A1 belongs to a subclade with two other members, Exo70A2 and Exo70A3 (Synek et al. 2006; Chong et al. 2010), and only a specific RT-PCR product for Exo70A1 was detected in the A. thaliana stigma RNA (Fig. 1a). The dominant expression of A. thaliana Exo70A1, out of the three Exo70A subclade genes, remained consistent in stigmas exposed to 80 % relative humidity (Fig. 1b). Microarray expression analyses with A. thaliana stigma RNA confirmed these RT-PCR results with high expression levels detected for only Exo70A1 of the Exo70A subclade genes (Fig. 1e). The microarray datasets also showed that the genes encoding the remaining seven exocyst subunits were expressed in the stigma as well (Fig. 1e). To study the effects of relative humidity on pollinations with the A. thaliana dry stigmas, wild-type A. thaliana Col0 and exo70A1-1 plants were grown in chambers with either 40 or 80 % relative humidity. As previously published (Synek et al. 2006; Samuel et al. 2009; Li et al. 2013), the exo70A1-1 plants displayed a dwarf phenotype, and this was observed at both 40 and 80 % humidity (see Supplemental Fig. 1 online). The exo70A1-1 mutant was previously described to have defects in stigmatic papillar elongation (Synek et al. 2006; Li et al. 2013), and this defect was reported to be rescued in grafting experiments (Li et al. 2013). At 40 % humidity, the stigmatic papillar elongation was found to be variable as we had previously described with plants grown under these low humidity conditions (Samuel et al. 2009), but flowers with elongated papillae were clearly visible (Fig. 2b, c, e, f). Interestingly, at 80 % humidity, the exo70A1-1 flowers were wild-type in appearance with elongated stigmatic papillae in freshly opened flowers (Fig. 2h, i, k, l). Thus, in the exo70A1-1 mutant, the stigmatic papillar elongation trait is influenced by relative humidity in the growth conditions. To investigate whether Exo70A1 is required for accepting compatible pollen, freshly opened exo70A1-1 flowers with elongated stigmatic papillae were hand-
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Fig. 2 Elongated stigmatic papillae are present on both wild-type A. thaliana Col-0 and exo70A1-1 mutant pistils from freshly open flowers. a–f Stigmas from plants grown under 40 % relative humidity. d–f are enlarged images of the stigmas shown in a–c, respectively. d– e Stigmas from plants grown under 80 % relative humidity. j–l are enlarged images of the stigmas shown in g–i, respectively. Scale bar = 400 lm for a–c, g–i; 1 nm d–f, j–l
pollinated with wild-type Col-0 pollen and observed for pollen grain attachment and pollen tube growth by aniline blue staining (Fig. 3). Both 40 and 80 % relative humidity conditions were tested, and plants from one condition were pollinated under the reciprocal condition as well. Under both humidity conditions, wild-type Col-0 pistils showed abundant pollen tubes growth in the aniline blue-stained pistils (Fig. 3a, d, g, j) and seed set (Fig. 3n). In contrast, there was a complete absence of Col-0 pollen adhesion or pollen tube growth on exo70A1-1 pistils when pollinations were conducted at 40 % relative humidity using plants grown under either condition (Fig. 3b, e, h, k, m). These results are consistent with our previous study where these plants grown in low humidity conditions (Samuel et al. 2009). Interestingly, some Col-0 pollen adhesion and pollen tube growth was observed when pollinations were
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conducted at 80 % relative humidity using plants grown under either condition (Fig. 3c, f, i, l, m). Thus, the high environmental humidity was able to partially restore compatible Col-0 pollen acceptance on the exo70A1-1 stigma. In our previous study, this pollination defect was rescued under low humidity growth conditions through the stigma-specific expression of an RFP:Exo70A1 construct, resulting in good seed set (*32 seeds/silique; Samuel et al. 2009). Here, we found that when the exo70A1-1 plants were grown at 80 % humidity, some siliques with seeds did develop (see Supplemental Fig. 1 online). For the exo70A1-1 pistils manually pollinated with Col-0, the seed set data were consistent with the aniline blue staining results where no seeds were produced at 40 % humidity, but partial seed set occurred at 80 % humidity (6.6 seeds/ silique; Fig. 3n). Thus, the partial rescue of compatible pollen acceptance at 80 % humidity resulted in fertilization and seed production. In conclusion, the results presented here confirm that Exo70A1 is expressed in the stigma and is required in the stigmatic papillae for the acceptance of compatible pollen. Further evidence supporting this conclusion is seen in our previous study where the stigma-specific expression of RFP:Exo70A1 in A. thaliana exo70A1-1 plants restores fertility, and the expression of a stigma-specific Exo70A1 RNAi knock-down construct in B. napus resulted in reduced acceptance of compatible pollen while the stigmatic papillae were wild-type in appearance (Samuel et al. 2009). These constructs were expressed by the stigmaspecific SLR1 promoter which is expressed as the flower reaches maturity (Franklin et al. 1996; Foster et al. 2005; Fobis-Loisy et al. 2007), and all plants were tested under low humidity growth conditions (Samuel et al. 2009). In addition, as predicted for a dry stigma species, high humidity can partially overcome this response and rescue this defect in the exo70A1-1 mutant. These results may help to explain some of the discrepancy between our results and those published by Li et al. (2013). In Li et al. (2010, 2013), the authors proposed that Exo70A1 has a tissue-specific expression pattern and function in developing tracheary elements. Exo70A1 promoter:GUS expression was shown to be localized to developing tracheary elements, and once fully formed, mature tracheary elements no longer displayed GUS expression (Li et al. 2013). However, in the data presented in the Li et al. (2010) study, weaker GUS staining is visible outside of the developing tracheary elements in the pistil samples. For our RT-PCR analysis (Fig. 1), the stigmas were harvested from mature pistils where the tracheary elements would be fully formed and, therefore, would not be a potential source of tissue contamination. Li et al. (2013) also reported pollen tube growth and seed set for exo70A1-1 mutant pistils following hand-pollinations. The
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Fig. 3 High humidity partially rescues the acceptance of compatible pollen by exo70A11 mutant stigmas. a–l Pollen grain attachment and pollen tube growth in wild-type A. thaliana Col-0 and exo70A1-1 mutant pistils following manual pollinations with wild-type Col0 pollen. DIC and aniline bluestained images are shown for each pollinated pistil. The humidity levels for growth and pollination conditions are indicated above the images. Scale bar = 100 lm. m Mean adhered pollen grains/stigma and n mean seeds/silique for wild-type A. thaliana Col-0 and exo70A1-1 mutant plants following manual pollinations with wild-type Col-0 pollen at either 40 % humidity or 80 % humidity. The different letters represent means that are significantly different at P \ 0.001 (one-way ANOVA with Tukey-HSD post hoc tests). Error bars indicate SE. n = 10 stigmas or siliques, respectively
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relative humidity levels for these experiments were not reported, and it was difficult to evaluate stigmatic papillar elongation due to the absence of DIC images for the pollinated pistils (Li et al. 2013). Although our evidence does not preclude the possibility that hydraulic transport through tracheary elements can influence stigmatic papillar elongation (depending on the relative humidity levels in the growth environment), it reinforces the necessity for Exo70A1 expression in A. thaliana stigmas to regulate compatible pollen acceptance. In addition to our proposed function for Exo70A1 in the stigma for the basal compatible pollen response (Samuel et al. 2009; Safavian and Goring 2013), and its proposed role in tracheary element development (Li et al. 2013), Exo70A1 has been implicated in seed coat mucilage formation (Kulich et al. 2010), cell plate formation during cytokinesis (Fendrych et al. 2010), and recycling of the PIN auxin efflux carriers (Drdova et al. 2013). With the range of functions that have been uncovered for Exo70A1 in A. thaliana, Za´rsky´ et al. (2013) concluded that Exo70A1 likely has broader roles beyond tracheary element development. A survey of the public microarray expression data suggests that Exo70A1 is expressed across a large range of tissues, including tissues lacking developing tracheary elements (Chong et al. 2010). Thus, an alternative model for Exo70A1 is that it represents the canonical Exo70 function in plants, as part of the exocyst complex to tether secretory vesicles to target membranes for fusion by SNARE proteins (reviewed in Heider and Munson, 2012; Za´rsky´ et al. 2013). Consistent with this model, disruptions in vesicle trafficking have been observed (Fendrych et al. 2010, 2013; Li et al. 2013; Safavian and Goring 2013; Indriolo et al. 2014). Other members of the large plant Exo70 gene family may have subfunctionalized to have specific roles such as Exo70B2 and Exo70H1’s proposed roles in plant responses to pathogens (Pecenkova et al. 2011; Stegmann et al. 2012) or neofunctionalized such as the association of Exo70E2 with the novel EXPO organelles (Wang et al. 2010). Acknowledgments DS was supported by an Ontario Graduate Scholarship (OGS). This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada to MAS and DRG, University start-up grants to MAS, and a Canada Research Chair to DRG.
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ORIGINAL ARTICLE
High humidity partially rescues the Arabidopsis thaliana exo70A1 stigmatic defect for accepting compatible pollen Darya Safavian • Muhammad Jamshed • Subramanian Sankaranarayanan • Emily Indriolo Marcus A. Samuel • Daphne R. Goring
•
Received: 21 March 2014 / Accepted: 22 June 2014 / Published online: 29 June 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract We have previously proposed that Exo70A1 is required in the Brassicaceae stigma to control the early stages of pollen hydration and pollen tube penetration through the stigmatic surface, following compatible pollination. However, recent work has raised questions regarding Arabidopsis thaliana Exo70A1’s expression in the stigma and its role in stigma receptivity to compatible pollen. Here, we verified the expression of Exo70A1 in stigmas from three Brassicaceae species and carefully re-examined Exo70A1’s function in the stigmatic papillae. With previous studies showing that high relative humidity can rescue some pollination defects, essentially bypassing the control of pollen hydration by the Brassicaceae dry stigma, the effect of high humidity was investigated on pollinations with the Arabidopsis exo70A1-1 mutant. Pollinations under low relative humidity resulted in a complete failure of wild-type compatible pollen acceptance by the exo70A1-1 mutant stigma as we had previously seen. However, high relative humidity resulted in a partial rescue
Communicated by Zhenbiao Yang.
Electronic supplementary material The online version of this article (doi:10.1007/s00497-014-0245-z) contains supplementary material, which is available to authorized users. D. Safavian E. Indriolo D. R. Goring (&) Department of Cell and Systems Biology, University of Toronto, Toronto M5S 3B2, Canada e-mail: [email protected] M. Jamshed S. Sankaranarayanan M. A. Samuel (&) Department of Biological Sciences, University of Calgary, Calgary T2N 1N4, Canada e-mail: [email protected] D. R. Goring Centre for the Analysis of Genome Evolution and Function, University of Toronto, Toronto M5S 3B2, Canada
of the exo70A1-1 stigmatic papillar defect resulting is some wild-type compatible pollen acceptance and seed set. Thus, these results reaffirmed Exo70A1’s proposed role in the stigma regulating compatible pollen hydration and pollen tube entry and demonstrate that high relative humidity can partially bypass these functions. Keywords Exo70A1 Compatible pollen acceptance Pollen hydration
Introduction In angiosperms, successful pollinations are dependent on the particular properties of stigmatic surfaces at the time of pollen receptivity. Stigmas have been divided into two broad categories termed wet and dry stigmas (HeslopHarrison and Shivanna 1977), though an intermediate category of semidry has been used for Senecio squalidus (Allen et al. 2011). There are differences in the surface properties of wet and dry stigmas which result in different outcomes during the early stages of pollen–pistil interactions (Sanchez et al. 2004; Swanson et al. 2004; Hiscock and Allen 2008; Firon et al. 2012). Following pollination, desiccated pollen grains must hydrate to become metabolically active, then germinate and grow a pollen tube through the pistil towards an ovule for fertilization. Wet stigmas have free-flowing surface secretions that are aqueous in nature or composed of lipids, both of which can allow for indiscriminate capture, hydration, and germination of pollen grains. Thus, wet stigmas have little control over these initial stages of pollen–stigma interactions (Heslop-Harrison and Shivanna 1977; Sanchez et al. 2004; Swanson et al. 2004; Hiscock and Allen 2008).
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Dry stigmas are defined by the absence of these surface fluid secretions, and as a result, exert a tighter regulation over the early stages of pollen capture, hydration, and germination. For example, compatible interactions lead to the formation of an interface at the contact site derived from pollen coat and stigma surface components, followed by the movement of water from the dry stigma to the pollen grain for hydration (Heslop-Harrison and Shivanna 1977; Dickinson 1995; Swanson et al. 2004; Chapman and Goring 2010; Firon et al. 2012). In the Brassicaceae, a highhumidity environment can allow for some pollen hydration and germination to occur without compatible pollen– stigma interactions. High relative humidity has been found to rescue pollen hydration and germination (and fertility) of Arabidopsis thaliana pollen coat mutants that are unable to form a proper compatible pollen–stigma interface (Preuss et al. 1993; Hulskamp et al. 1995; Fiebig et al. 2000) or cause precocious germination of A. thaliana pollen developmental mutants in the absence of stigma contact (Johnson and McCormick 2001; Wang et al. 2012). We have previously proposed a role for Exo70A1 in the stigmatic papillae where it is required for the basal compatible pollen response, controlling the early stages of pollen hydration and germination in two Brassicaceae species, Brassica napus and A. thaliana (Samuel et al. 2009; Safavian and Goring 2013). The loss of Exo70A1 function in the stigma caused an absence of pollen hydration, pollen tube growth, and seed set following compatible pollinations (Samuel et al. 2009). However, Li et al. (2010, 2013) raised questions about A. thaliana Exo70A1’s expression in the stigma and inability of the exo70A1-1 mutant stigma to promote pollen hydration and germination. Li et al. (2010, 2013) proposed that A. thaliana Exo70A1 has a specific expression pattern and function in developing tracheary elements, and presented data showing pollen tube growth and seed set for exo70A1-1 mutant pistils following hand-pollinations. Here, we carefully reexamined Exo70A1’s function in accepting compatible pollen, using the well-characterized exo70A1-1 mutant, and investigated if increased levels of humidity could account for discrepancies between the two studies (Samuel et al. 2009; Li et al. 2013).
Materials and methods Plant growth conditions Wild-type A. thaliana Col-0 plants and exo70A1-1 mutant plants (Salk_014826, T-DNA insertion mutant in the Col-0 background; Synek et al. 2006; Samuel et al. 2009; Li et al. 2013) were grown in growth chambers under a long-day cycle of 16-h light/8-h dark at 22 °C with a relative
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humidity roughly around 40 or 80 % (monitored with a digital hygrometer). The exo70A1-1 mutant has been previously shown to be a T-DNA knockout mutant, lacking full length Exo70A1 mRNA (Synek et al. 2006; Samuel et al. 2009). Images of whole plants were captured using a Canon digital camera, and images of whole flowers were taken with a Nikon sMz800 microscope and NIS-elements imaging software at magnifications of 2.09, 3.09 and 6.09. PCR and microarray analyses RT-PCR and genomic PCR were conducted as described by Samuel et al. (2009). The exo70A1-1 mutant genotype was confirmed by genomic DNA PCR using Exo70A1 genespecific primers with the LBA1 T-DNA-specific primer (see Supplemental Table 1 online). For the RT-PCR analyses, RNA was extracted from stigmas harvested from stage 12 flowers for A. thaliana (Smyth et al. 1990), and freshly opened unpollinated flowers for Arabidopsis lyrata and Brassica rapa R-o-18. All plants were grown under low humidity conditions (B40 %), unless otherwise indicated. RNA samples were treated with DNase I to remove any contaminating genomic DNA prior to cDNA synthesis, and primers were selected to span introns for the RT-PCR analyses. Primer sequences and predicted cDNA versus genomic DNA band sizes are listed in Supplemental Table 1 online. Accession numbers are A. thaliana Exo70A1 At5g03540, Exo70A2 At5g52340, Exo70A3 At5g52350; A. lyrata Exo70A1 939738; and B. rapa Exo70A1 Bra009523. For the microarray expression analyses of the exocyst complex subunit genes, A. thaliana stigmas were harvested from stage 10 and stage 12 flowers for RNA extraction. The Whole Transcriptome Amplification kit was used to amplify the transcriptome prior to microarray analysis. Pollinations under low and high humidity To test the stigma receptivity of flowers from exo70A1-1 plants grown under 80 % humidity and wild-type Col-0, the largest flower buds (stage 12; Smyth et al. 1990) were emasculated and left overnight covered in plastic wrap to prevent dehydration. On the following day (when stage 12 flower buds would normally open), all emasculated pistils were hand-pollinated with Col-0 pollen by brushing 1–4 anthers across the stigmatic surface. For 40 % humidity exo70A1-1 mutant plants, variability in stigmatic papillar elongation was observed, and therefore, freshly opened or 1-day old flowers with elongated stigmatic papillae were emasculated, covered in plastic wrap, and pollinated the next day with Col-0 pollen. Following all pollinations, the plants were placed in either the 40 or 80 % relative humidity chambers as indicated in Fig. 3. For viewing
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Results and discussion
Fig. 1 Exo70A1 is expressed in the stigmas of three Brassicaceae species. a–e RT-PCR analysis for the Exo70A1-3 subclade genes in A. thaliana (a, b), and Exo70A1 in A. lyrata (c) and B. rapa (d). Actin was included as a positive control. Stigmas were collected from plants grown under low humidity conditions (B 40 %; a, c, d) or at 80 % relative humidity (b), and were from stage 12 flowers for A. thaliana, and freshly open unpollinated flowers for A. lyrata and B. rapa. DNA marker sizes are shown on the left of each panel, and the PCR products correspond to the predicted cDNA sizes (see Supplemental Table 1 online). d Microarray expression analysis of the exocyst complex subunit genes in A. thaliana stigmas. The exocyst complex consists of 8 subunits encoded by the predicted Exo70, Sec3, Sec5, Sec6, Sec8, Sec10, Sec15 and Exo84 genes (Synek et al. 2006; Chong et al. 2010). The samples, 1 and 2, represent replicates for each flower stage. The values are raw intensities after subtracting the background
pollen tubes, the pollinated pistils were left overnight, collected the next day, aniline blue stained, and viewed on an epifluorescence microscope as previously described (Samuel et al. 2009). For seed set, emasculated pistils from Col-0 and exo70A1-1 plants grown under 40 and 80 %, humidity were hand-pollinated with Col-0 pollen and left for 10 days for the siliques to mature. The pre-dehiscent siliques were dissected using fine-tipped forceps, and the seeds per silique were counted (n = 10 for each sample).
Previously, we demonstrated the expression of Exo70A1 in mature stigmas with A. thaliana Exo70A1 promoter:GUS transgenic plants and a B. napus RNA blot analysis (Samuel et al. 2009). To re-confirm the stigma expression of Exo70A1, RNA was extracted from unpollinated mature stigmas for three Brassicaceae species that we use to study pollen–stigma interactions: A. thaliana, A. lyrata and B. rapa (Samuel et al. 2009; Indriolo et al. 2012; Safavian and Goring 2013). RT-PCR analyses were conducted, and Exo70A1 was clearly detected in the stigma RNA samples from all three species (Fig. 1a, c, d). All the PCR band sizes corresponded to the predicted sizes for cDNA and were not due to any putative genomic DNA contamination (See Supplemental Table 1 online for predicted sizes). Exo70A1 belongs to a subclade with two other members, Exo70A2 and Exo70A3 (Synek et al. 2006; Chong et al. 2010), and only a specific RT-PCR product for Exo70A1 was detected in the A. thaliana stigma RNA (Fig. 1a). The dominant expression of A. thaliana Exo70A1, out of the three Exo70A subclade genes, remained consistent in stigmas exposed to 80 % relative humidity (Fig. 1b). Microarray expression analyses with A. thaliana stigma RNA confirmed these RT-PCR results with high expression levels detected for only Exo70A1 of the Exo70A subclade genes (Fig. 1e). The microarray datasets also showed that the genes encoding the remaining seven exocyst subunits were expressed in the stigma as well (Fig. 1e). To study the effects of relative humidity on pollinations with the A. thaliana dry stigmas, wild-type A. thaliana Col0 and exo70A1-1 plants were grown in chambers with either 40 or 80 % relative humidity. As previously published (Synek et al. 2006; Samuel et al. 2009; Li et al. 2013), the exo70A1-1 plants displayed a dwarf phenotype, and this was observed at both 40 and 80 % humidity (see Supplemental Fig. 1 online). The exo70A1-1 mutant was previously described to have defects in stigmatic papillar elongation (Synek et al. 2006; Li et al. 2013), and this defect was reported to be rescued in grafting experiments (Li et al. 2013). At 40 % humidity, the stigmatic papillar elongation was found to be variable as we had previously described with plants grown under these low humidity conditions (Samuel et al. 2009), but flowers with elongated papillae were clearly visible (Fig. 2b, c, e, f). Interestingly, at 80 % humidity, the exo70A1-1 flowers were wild-type in appearance with elongated stigmatic papillae in freshly opened flowers (Fig. 2h, i, k, l). Thus, in the exo70A1-1 mutant, the stigmatic papillar elongation trait is influenced by relative humidity in the growth conditions. To investigate whether Exo70A1 is required for accepting compatible pollen, freshly opened exo70A1-1 flowers with elongated stigmatic papillae were hand-
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Fig. 2 Elongated stigmatic papillae are present on both wild-type A. thaliana Col-0 and exo70A1-1 mutant pistils from freshly open flowers. a–f Stigmas from plants grown under 40 % relative humidity. d–f are enlarged images of the stigmas shown in a–c, respectively. d– e Stigmas from plants grown under 80 % relative humidity. j–l are enlarged images of the stigmas shown in g–i, respectively. Scale bar = 400 lm for a–c, g–i; 1 nm d–f, j–l
pollinated with wild-type Col-0 pollen and observed for pollen grain attachment and pollen tube growth by aniline blue staining (Fig. 3). Both 40 and 80 % relative humidity conditions were tested, and plants from one condition were pollinated under the reciprocal condition as well. Under both humidity conditions, wild-type Col-0 pistils showed abundant pollen tubes growth in the aniline blue-stained pistils (Fig. 3a, d, g, j) and seed set (Fig. 3n). In contrast, there was a complete absence of Col-0 pollen adhesion or pollen tube growth on exo70A1-1 pistils when pollinations were conducted at 40 % relative humidity using plants grown under either condition (Fig. 3b, e, h, k, m). These results are consistent with our previous study where these plants grown in low humidity conditions (Samuel et al. 2009). Interestingly, some Col-0 pollen adhesion and pollen tube growth was observed when pollinations were
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conducted at 80 % relative humidity using plants grown under either condition (Fig. 3c, f, i, l, m). Thus, the high environmental humidity was able to partially restore compatible Col-0 pollen acceptance on the exo70A1-1 stigma. In our previous study, this pollination defect was rescued under low humidity growth conditions through the stigma-specific expression of an RFP:Exo70A1 construct, resulting in good seed set (*32 seeds/silique; Samuel et al. 2009). Here, we found that when the exo70A1-1 plants were grown at 80 % humidity, some siliques with seeds did develop (see Supplemental Fig. 1 online). For the exo70A1-1 pistils manually pollinated with Col-0, the seed set data were consistent with the aniline blue staining results where no seeds were produced at 40 % humidity, but partial seed set occurred at 80 % humidity (6.6 seeds/ silique; Fig. 3n). Thus, the partial rescue of compatible pollen acceptance at 80 % humidity resulted in fertilization and seed production. In conclusion, the results presented here confirm that Exo70A1 is expressed in the stigma and is required in the stigmatic papillae for the acceptance of compatible pollen. Further evidence supporting this conclusion is seen in our previous study where the stigma-specific expression of RFP:Exo70A1 in A. thaliana exo70A1-1 plants restores fertility, and the expression of a stigma-specific Exo70A1 RNAi knock-down construct in B. napus resulted in reduced acceptance of compatible pollen while the stigmatic papillae were wild-type in appearance (Samuel et al. 2009). These constructs were expressed by the stigmaspecific SLR1 promoter which is expressed as the flower reaches maturity (Franklin et al. 1996; Foster et al. 2005; Fobis-Loisy et al. 2007), and all plants were tested under low humidity growth conditions (Samuel et al. 2009). In addition, as predicted for a dry stigma species, high humidity can partially overcome this response and rescue this defect in the exo70A1-1 mutant. These results may help to explain some of the discrepancy between our results and those published by Li et al. (2013). In Li et al. (2010, 2013), the authors proposed that Exo70A1 has a tissue-specific expression pattern and function in developing tracheary elements. Exo70A1 promoter:GUS expression was shown to be localized to developing tracheary elements, and once fully formed, mature tracheary elements no longer displayed GUS expression (Li et al. 2013). However, in the data presented in the Li et al. (2010) study, weaker GUS staining is visible outside of the developing tracheary elements in the pistil samples. For our RT-PCR analysis (Fig. 1), the stigmas were harvested from mature pistils where the tracheary elements would be fully formed and, therefore, would not be a potential source of tissue contamination. Li et al. (2013) also reported pollen tube growth and seed set for exo70A1-1 mutant pistils following hand-pollinations. The
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Fig. 3 High humidity partially rescues the acceptance of compatible pollen by exo70A11 mutant stigmas. a–l Pollen grain attachment and pollen tube growth in wild-type A. thaliana Col-0 and exo70A1-1 mutant pistils following manual pollinations with wild-type Col0 pollen. DIC and aniline bluestained images are shown for each pollinated pistil. The humidity levels for growth and pollination conditions are indicated above the images. Scale bar = 100 lm. m Mean adhered pollen grains/stigma and n mean seeds/silique for wild-type A. thaliana Col-0 and exo70A1-1 mutant plants following manual pollinations with wild-type Col-0 pollen at either 40 % humidity or 80 % humidity. The different letters represent means that are significantly different at P \ 0.001 (one-way ANOVA with Tukey-HSD post hoc tests). Error bars indicate SE. n = 10 stigmas or siliques, respectively
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relative humidity levels for these experiments were not reported, and it was difficult to evaluate stigmatic papillar elongation due to the absence of DIC images for the pollinated pistils (Li et al. 2013). Although our evidence does not preclude the possibility that hydraulic transport through tracheary elements can influence stigmatic papillar elongation (depending on the relative humidity levels in the growth environment), it reinforces the necessity for Exo70A1 expression in A. thaliana stigmas to regulate compatible pollen acceptance. In addition to our proposed function for Exo70A1 in the stigma for the basal compatible pollen response (Samuel et al. 2009; Safavian and Goring 2013), and its proposed role in tracheary element development (Li et al. 2013), Exo70A1 has been implicated in seed coat mucilage formation (Kulich et al. 2010), cell plate formation during cytokinesis (Fendrych et al. 2010), and recycling of the PIN auxin efflux carriers (Drdova et al. 2013). With the range of functions that have been uncovered for Exo70A1 in A. thaliana, Za´rsky´ et al. (2013) concluded that Exo70A1 likely has broader roles beyond tracheary element development. A survey of the public microarray expression data suggests that Exo70A1 is expressed across a large range of tissues, including tissues lacking developing tracheary elements (Chong et al. 2010). Thus, an alternative model for Exo70A1 is that it represents the canonical Exo70 function in plants, as part of the exocyst complex to tether secretory vesicles to target membranes for fusion by SNARE proteins (reviewed in Heider and Munson, 2012; Za´rsky´ et al. 2013). Consistent with this model, disruptions in vesicle trafficking have been observed (Fendrych et al. 2010, 2013; Li et al. 2013; Safavian and Goring 2013; Indriolo et al. 2014). Other members of the large plant Exo70 gene family may have subfunctionalized to have specific roles such as Exo70B2 and Exo70H1’s proposed roles in plant responses to pathogens (Pecenkova et al. 2011; Stegmann et al. 2012) or neofunctionalized such as the association of Exo70E2 with the novel EXPO organelles (Wang et al. 2010). Acknowledgments DS was supported by an Ontario Graduate Scholarship (OGS). This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada to MAS and DRG, University start-up grants to MAS, and a Canada Research Chair to DRG.
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