ABSTRACT
Successful fertilization in angiosperms depends on the proper trajectory of pollen tubes through the pistil tissues to reach the ovules. Pollen tubes start their path by progressing within the cell wall of the papilla cells, applying pressure to the wall. Mechanical forces are known to play a major role in plant cell shape by controlling the orientation of cortical microtubules (CMTs), and hence deposition of cellulose microfibrils (CMFs). Here, by combining cell imaging and genetic approaches, we show that isotropic orientation of CMTs in aged, katanin1-5 (ktn1-5) or oryzalin-treated papilla cells is accompanied by a tendency of pollen tubes to coil around the papillae. In addition, using atomic force microscopy, we uncover that aged and ktn1-5 papilla cells have a softer cell wall. Altogether, our results suggest that KATANIN-dependent control of microtubule dynamics, and associated mechanical anisotropy of stigmatic walls, mediate pollen tube growth directionality.
INTRODUCTION
Following deposition of dehydrated pollen grains on the receptive surface of the female organ, the stigma, pollen rehydrates, germinates and produces a pollen tube that carries the male gametes toward the ovules where the double fertilization takes place. This long itinerary through the different tissues of the pistil is finely controlled, avoiding misrouting of the pollen tube and hence assuring proper delivery of the sperm cells to the female gametes. In Arabidopsis thaliana, pollen tubes grow within the cell wall of papillae of the stigmatic epidermis, and then through the transmitting tissue of the style and ovary1. The transmitting tissue has an essential function in pollen tube guidance, providing chemical attractants and nutrients2,3. In contrast to these accumulating data showing the existence of factors mediating pollen tube growth in the pistil, whether guidance cues exist at the very early stage of pollen tube emergence and growth in the papilla cell wall remains largely unknown. The cell wall constitutes a stiff substrate and hence a mechanical barrier to pollen tube progression. There are numerous examples in animal cells demonstrating that mechanical properties of the cellular environment, and in particular rigidity, mediate cell signalling, proliferation, differentiation and migration4–6. In plant cells, cell wall rigidity depends mainly on its major component, cellulose, which is synthesized by plasma membrane-localized cellulose synthase complexes (CSCs) moving along cortical microtubule (CMT) tracks7. While penetrating the cell wall, the pollen tube exerts a pressure onto the stigmatic cell8. Such physical forces are known to reorganize the cortical microtubules (CMTs), which by directing CSCs to the plasma membrane, reinforce wall stiffness by novel cellulose microfibril (CMF) synthesis9,10. Hence, there is an intricate interconnection between CMT organization, CMF deposition and cell wall rigidity11,12. A major regulatory element of CMT dynamics is the KATANIN (KTN1) microtubule-severing enzyme, which allows CMT reorientation following10, 13, 14. mechanical stimulation10,13,14. Here we investigated whether the CMT network of papilla cells might contribute to pollen tube growth and guidance in stigmatic cells by combining cell imaging and genetic approaches. We found that CMT network of papilla cells is modified with ageing, CMT bundles being anisotropic at anthesis and becoming isotropic at later stages of stigma development. This change in CMT organization was accompanied by a modification of the direction of pollen tube growth, which passes from predominantly straight to coiled. In the ktn1-5 mutant, papilla cells exhibited a strong CMT isotropy associated with a marked tendency of wild-type (WT) pollen tubes to turn around the papilla. Coiled growth of pollen tubes was also observed on stigma papillae treated with the CMT-depolymerizing drug oryzalin, although at a lower extent. Faster growth of coiled pollen tubes was observed in aged WT as well as ktn1-5 papillae, which both were found to exhibit softer cell walls. Additionally, we tested a series of cell wall mutants, including mutants having CMT disorganization and decreased cell wall rigidity and, unexpectedly, none of them used as female induced coiled growth of WT pollen tubes. Altogether, our results strongly suggest that CMT organization and cell wall mechanical properties dependent on KTN1 have a major role in guiding early pollen tube growth in stigma papillae.
RESULTS
CMT dynamic pattern and pollen tube growth during stigma development
To assess the functional role of stigmatic CMTs in pollen – papilla cell interaction, we first analysed their organization in papillae at stages 12 to 15 of stigma development as described15 (Fig. 1a,b). We generated a transgenic line expressing the CMT marker MAP65.1-citrine under the control of the stigma specific promoter SLR116. Before (stage 12) and at anthesis (stage 13), the CMTs were aligned perpendicularly to the longitudinal axis of papilla cells and were highly anisotropic (median value of 0.40) (Fig. 1c,d). At stage 14, when anthers extend above the stigma, the CMT pattern became less organized, with a higher variability in anisotropy values. Finally, at stage 15 when the stigma extends above anthers, CMT anisotropy had a median value of 0.09 indicative of an isotropic orientation of CMTs (Fig. 1c,d). These findings reveal that the papilla CMT cytoskeleton is dynamic during development, with a change of CMT array orientation from anisotropy to isotropy. We then wondered whether this change in CMT organization could be correlated with pollen tube growth. To this end, we self-pollinated Col-0 papillae from stages 12 to 15 and examined pollen tube growth one hour after pollination by scanning electron microscopy (SEM) (Fig. 2a). At stage 12 and 13, we found most (~60%) pollen tubes to grow straight in the papillae, whereas about 30% and 10% of tubes made half-turn or one turn around stigmatic cells, respectively (Fig. 2b). At later stages of development, the tendency to coil around the papillae dramatically increased, with more than 35% of pollen tubes at stage 14 and more than 55% at stage 15 making one or more than one turn around papillae. These results suggest that CMT organization in the papilla impacts the direction of growth of pollen tubes and that loss of CMT anisotropy is associated with coiled growth.
Impaired CMT dynamics of papillae affects pollen tube growth direction
To confirm the relation between stigma CMTs and pollen behaviour, we examined pollen tube growth on stigmas of the katanin1-5 mutant, which is known to exhibit reduced CMT array anisotropy in root cells17–19. Because the CMT organization in ktn1-5 papillae is unknown, we crossed ktn1-5 with the MAP65-1-citrine marker line and found that CMT arrays were more isotropic in ktn1-5 papillae when compared with those of the WT (Fig. 3a,c). Using SEM, we then analysed Col-0 pollen behaviour on ktn1-5 stigmatic cells at stage 13 (Fig. 3b). We observed that Col-0 pollen tubes acquired a strong tendency to coil around ktn1-5 papillae, with above 60% of tubes making one or more than one turn around papillae, sometimes making up to 6 turns, before reaching the base of the cell (Fig. 3d and Supplementary Fig. 1a). In some rare cases, pollen tubes even grew upward in the ktn1-5 mutant and appeared blocked at the tip of the papilla (Supplementary Fig.1b). To test the direct impact of stigma CMTs on pollen tube growth direction, we examined whether the destabilization of CMTs in Col-0 papillae could affect pollen tube growth. To this end, we treated stigmas by local application of the depolymerizing microtubule drug oryzalin in lanolin pasted around the style. After 4 hours of drug treatment, no more CMT labelling was detected in papillae, while CMTs were clearly visible in mock-treated (DMSO) stigmas (Fig. 4a). Stigmas were then pollinated with Col-0 pollen and one hour later, pollen tubes were found turning on drug-treated but not on control papillae (Fig. 4b,c). However, the number of coils was significantly lower than on ktn1-5 papillae. Indeed, 25% of the pollen tubes made at least 2 coils in ktn1-5 papillae whereas this percentage represented only 4% on the oryzalin treated stigmas (Fig. 3d,4c). Altogether, these results confirm that stigmatic CMTs contribute to the directional growth of pollen tubes in papilla cells.
Mechanical properties of the cell wall are disturbed in ktn1-5 papilla cells
Because the main role of CMTs in plant cells is to guide the trajectory of CSCs, thereby impacting the mechanical anisotropy of the cell wall, we analysed the cell walls of Col-0 and ktn1-5 papillae following pollination. First, using Transmission Electron Microscopy (TEM), we found Col-0 pollen tubes to penetrate the cuticle and to grow between the two layers of the papilla cell wall, as previously described20, in both Col-0 and ktn1-5 papilla cells (Fig. 5a). We did not detect any significant difference in the ultrastructure of cell walls (Supplementary Fig. 2). Interestingly, as the pollen tube progresses through the papilla cell wall, it generates a bump (external deformation) and an invagination (internal deformation) in the cell wall. As such deformations could reflect differences in wall properties, we quantified the external (extD) and internal (intD) deformation following pollination of Col-0 and ktn1-5 stigmas. To visualize more clearly this deformation, we pollinated stigmas expressing the plasma membrane protein LTI6B fused to GFP (LTI6B-GFP) with pollen whose tube was labelled with the red fluorescent protein RFP driven by the ACT11 promoter21 (Fig. 5b). We found that Col-0 pollen tubes grew with almost equal extD and intD values in Col-0 cell wall. However, the ratio between extD and intD was about 3 when Col-0 pollen tubes grew in ktn1-5 papilla cells (Fig. 5b,c,d). These quantitative data are consistent with the observation of major protuberances caused by pollen tubes on ktn1-5 stigmatic cells using SEM (Fig. 3b and Supplementary Fig. 1). Assuming that stigmatic cells are pressurized by their turgor pressure, this may reflect the presence of softer walls in ktn1-5 papillae. To test this hypothesis, we assessed the stiffness of Col-0 and ktn1-5 papilla cell walls using Atomic Force Microscopy (AFM) with a 400 nm indentation. We found that cell wall stiffness in ktn1- 5 papilla cells was about 30% lower than that in stage 13 WT cells (Fig. 5e,f). To confirm this result, we investigated the stiffness of the papilla cell wall on WT stigmas at stage 15, where increased coiled pollen tubes were detected (Fig. 2 a,b). We found the cell wall to be softer than that of papillae at stage 13 but stiffer than that of the ktn1-5 (Fig. 5e,f). We then reasoned that the presence of softer walls should also affect the pollen tube growth rate, stiffer walls reducing growth. We thus monitored the growth rate of Col-0 pollen tubes in Col-0 and ktn1-5 papillae. We found that pollen tubes grew faster (~ x 1.8) within ktn1-5 papillae (Fig. 5g). Similarly, we found that pollen tube growth on stage 15 papillae was faster (~ × 1.6) than on stage 13 papillae (Fig. 5h). Altogether, these results suggest that KATANIN-dependent mechanical properties of papilla cell wall impact pollen tube growth.
Defects in papilla cell-wall composition are not sufficient to affect Col-0 pollen tube behaviour
The katanin1/fra2 mutant was initially described as a mutant impaired in cell wall biosynthesis and CMT array organization19. This prompted us to test whether other mutants affected in cell wall biogenesis might exhibit the coiled pollen tube phenotype. Indeed, in the simplest scenario, the presence of softer walls, whatever the cause, may be sufficient to induce extra coiling due to faster pollen tube growth. We selected mutants impaired in the cellulose synthase complex (kor1.1, prc1 and any1), hemicellulose biosynthesis (xxt1 xxt2, xyl1.4) and pectin content (qua2.1) (Supplementary Table 1). Strikingly, none of the 6 cell wall mutants displayed the coiled pollen tube phenotype (Supplementary Fig. 3). This suggests that the relation between CMT organisation, cell wall stiffness and pollen tube trajectory is stricter than anticipated. Because CMTs guide cellulose deposition, they also control the directional elongation of plant cells22. The contribution of stigmatic CMTs to pollen tube growth may thus be mediated by papilla cell shape only. For instance, wider cells in ktn1-5 would promote pollen tube coiling. To check that possibility, we measured the length and width of papilla cells in Col-0 (at stage 13 and 15), ktn1-5, xxt1 xxt2 and any1 (Supplementary Fig. 4). We did not find any correlation between papilla length and the coiled phenotype. Indeed, coiled phenotype was observed in stage-15 WT papillae that were longer than those at stage-13, as well as in ktn1-5 papillae that had a length similar to stage-13 WT papillae. The correlation between papilla width and coiled phenotype was also not clear-cut. As expected, ktn1-5 mutant exhibited wider papillae than Col-0. Stage-15 WT papillae were also significantly larger than stage-13 WT papillae, where coils were observed. However, xxt1 xxt2 and any1 papillae were also wider than the WT but did not display the coiled phenotype. Altogether, this suggests that papilla morphology is not sufficient to explain the coiled phenotype. Because CMT disorganization in papillae affects both wall stiffness and mechanical anisotropy, we next investigated the relative contribution of these two parameters in pollen tube growth. We focused our analysis on the xxt1 xxt2 double mutant. Indeed, this mutant was reported to display CMT orientation defects and a softer cell wall in hypocotyl cells, when compared with the WT12. As the pollen tube phenotype on xxt1 xxt2 stigmas was similar to the WT, we wondered whether the cell wall stiffness of stigmatic cells was actually affected in these mutant papillae. Using AFM, we found the papilla cell wall of the mutant to be about 30% softer than that of Col-0 papillae, i.e. very similar to that of ktn1-5 (Fig. 5f). However, despite this similarity, the pollen tube growth rate in xxt1 xxt2 stigmas was identical to Col-0 stigmas at stage 13 (Fig. 5h). In addition, contrary to ktn1-5, we did not observe external bumps on xxt1 xxt2 papillae when pollen tubes were growing (Supplementary Fig. 3g). These results indicate that alteration of the cell wall stiffness alone is not sufficient to induce the pollen tube coiled phenotype and promote tube growth speed, and suggest that, instead, the mechanical anisotropy of cell walls plays a key role in pollen tube trajectory.
DISCUSSION
How the pollen germinates a tube and how pollen tube growth is regulated have been the object of many investigations23,24. The use of in vitro pollen germination as well as semi-in vivo fertilization assays, together with the analysis of mutants defective in pollen or ovule functions, have rapidly expanded our knowledge of the mechanisms that sustain pollen tube growth, its guidance toward the ovule and the final delivery of male gametes within the embryo sac25–27. At the cellular level, cytoskeleton has been extensively studied during pollen tube elongation28, highlighting the critical role played by the actin microfilaments in pollen-tube tip growth through delivery of materials for the biosynthesis of the plasma membrane and cell wall. By contrast, CMTs seem to have a lower importance in this process, drugs affecting CMT polymerisation having no significant effects on pollen-tube growth rate, yet altering the capacity of pollen tubes to change their growth direction29. Rearrangements of actin microfilaments30 and destabilization of CMTs31 have been described in stigmas following compatible pollination in Brassica species. However, CMT pattern and dynamics during papilla development have never been reported. Our data show that during the course of stigma maturation, which is associated with papilla cell elongation (Supplementary Fig. 4a,b), CMT bundles progressively move from perpendicular (anisotropic) to the elongation axis at stage 12 to disorganised (isotropic) at stage 15. This correlates with the known CMT dynamics during elongation in plant cells, where CMT arrays are highly anisotropic in young cells and become more isotropic as cells differentiate22,32. The progressive randomisation of CMT orientation in papilla cells is accompanied by an increased coiled growth of pollen tubes in papillae. Similarly, when CMTs are destabilized by the microtubule depolymerizing drug oryzalin, coiled pollen tubes are more frequently observed compared with untreated control stigmas. These results reveal a link between the stigmatic CMT cytoskeleton organization and the trajectory that pollen tube takes while growing in the papilla cell wall. The fact that the most striking effect on pollen tube growth was found on ktn1-5 mutant suggests that the coiled phenotype depends not only on the CMT organization but implicates other factors. Among various phenotypic alterations described in loss-of-function mutants for KTN1, are the impaired cell mechanical properties and cell elongation, defects in CMT organization, cell18,19,33. wall composition and CMF orientation18, 19, 33. Interestingly, we found that the protuberance of pollen tubes at the surface of ktn1-5 papillae was associated with a faster growth rate and a lower rigidity of the cell wall compared with Col-0. This places mechanics of the cell wall as a likely component involved in the coiled phenotype. Surprisingly, cell wall mutants, such as xxt1 xxt2 and prc1, known to exhibit both abnormal CMT organization and softer cell walls, and somehow that may share mechanical properties with ktn1-5, did not induce the coiled phenotype displayed by ktn1-5 papillae. More remarkably, despite the similar stiffness of xxt1 xxt2 and ktn1-5 papilla cell walls measured in our AFM experiments, pollen tubes behaved differently on these two stigmas. This indicates that components other than cell wall stiffness are involved in the pollen tube coiled phenotype and that mechanical properties of the cell wall are likely not identical in the two mutants. Indeed, though sharing a similar alteration of cell wall stiffness, the global plant morphology of these mutants is clearly distinct. The ktn1-5 mutant was described to have a severe reduction in cell length and an increase in cell width in all organs19. This cellular phenotype was attributed to the distorted deposition of CMFs correlated with the isotropic orientation of CMTs, whereas in WT cells CMFs, like CMTs, are oriented perpendicularly to the elongation axis18. We also found larger papilla cells for ktn1-5 (Supplementary Fig. 4c,d), supporting the hypothesis that orientation of CMFs is also altered in ktn1-5 papillae. Hence, we may assume that mechanical anisotropy, described as the cell wall anisotropy made by the orientation of the rigid CMFs34, is impaired in ktn1-5 papillae. By contrast, the xxt1 xxt2 double mutant, although deprived of xyloglucans in its cell walls, shows a growth phenotype35,36 not as severe as ktn1-5. In etiolated hypocotyls, contrary to Col-0, CMFs are largely parallel to one another, straighter than in WT and oriented approximately transversely to the long axis of the cell12. Interestingly, we found the papilla cell shape of xxt1 xxt2 stigmas to be similar to Col-0, though with slightly wider papillae (Supplementary Fig. 3g,4e,f). However, xxt1 xxt2 papillae were less wide than ktn1-5 papillae, which is consistent with an anisotropic growth of the papillae in the xxt1 xxt2 mutant compared with ktn1-5. At the cell wall level, apart from a similar stiffness, the main differences between ktn1-5 and xxt1 xxt2 appear to be the cell wall composition, orientation of CMFs and possible changes in molecular connections between cell wall components and plasma membrane and/or cytoskeleton proteins. Indeed, a recent proteomic study revealed that loss of KATANIN function is associated with the decrease in abundance of several cytoskeleton proteins, such as profilin 1, actin-depolymerizing factor 3 and actin 737, whereas targeted quantitative RT-PCR unveiled that Microtubule-associated protein (MAP) and wall signal receptor genes are downregulated in xxt1 xxt212. In this latter study, KTN1 expression level was shown to be unchanged compared with Col-0. Altogether, our study suggests that KTN1, by maintaining the papilla mechanical anisotropy, has a key function in mediating early pollen tube guidance on stigma papillae.
The coiled phenotype was not only observed in ktn1-5 but also in the WT Col-0 papillae at stage 15. This is likely to be related to isotropic orientation of CMTs and CMFs known to occur during cell elongation38,39. At the organ level, it has been suggested that the mechanical anisotropy of the wall restrains organ emergence34. The authors propose that for the same wall stiffness, a cell wall with isotropic properties would lead to larger outgrowth than a wall with anisotropic properties. Our data are consistent with this hypothesis, albeit at the subcellular scale, since large protuberance of papilla wall following pollen tube growth is observed in ktn1-5 papilla cells, exhibiting walls with isotropic properties.
It remains unclear how mechanical anisotropy guides pollen tube growth. We can hypothesise that as pollen tube grows inside the wall, it encounters recently deposited CMFs on the inner side of the wall (facing the cytoplasm) and older CMFs on the outer side. It is likely that these layers have different mechanical properties related to CMF orientation22. Pollen tubes may grow helically by default, as is the case for climbing plants around a cylindrical substrate but the presence of a mechanically reinforced inner wall may slow down and bias the trajectory of the pollen tube. It is worth noting that growth rate is slowed down when pollen tubes pass through a microgap of a microfluiding device, the tubes adapting their invasive force to the mechanical constraints40. Our data show that the pollen tube tip, while progressing in the papilla wall, senses the mechanical features of its environment and reacts accordingly. Hence, it reveals some unanticipated internal and hidden properties of the cell wall. Interestingly, recent work showed that axons were capable of adapting their growth rate and direction according to mechanical constraints, growing straighter on rigid substrate underlining that mechanical signals are important regulators of pathfinding41.
In the last decades, many studies pointed out that chemical but also mechanical components must be considered to be implicated in pollen tube growth direction25. Implication of chemical cues for pollen tube guidance in the stigma remains largely unknown, although the blue copper protein plantacyanin, when overexpressed in Arabidopsis stigmas, was reported to be a possible guidance factor through an as yet undiscovered mechanism 42. However, no defect in pollen tube growth directionality was detected in a know-down plantacyanin mutant, questioning the actual role of this protein as a chemoattractant in Arabidopsis. In our study, we add mechanics as a key player in early pollen tube guidance in the papilla cell. Our results suggest that this role is mediated by a specific CMT organization and mechanical anisotropy of the papilla cell, which both are dependent on KTN1. Importantly, KTN1 prevents emerging pollen tubes to grow upward on papilla cells and straightens pollen tube direction, helping the tube to find its correct path to the stylar transmitting tract. This highlights an yet unexpected role for KTN1 in pollen tube guidance, which was until now mostly known to be involved in plant development and stress-response regulation. In addition, our study also clearly unveils the fact that the mechanical properties of one single cell (e.g., the stigmatic papilla) impact the behaviour of its neighbouring cell (e.g., the pollen tube).
MATERIALS AND METHODS
Plant Materials and Growth Conditions
Arabidopsis thaliana, ecotype Columbia (Col-0), Arabidopsis transgenic plants generated in this study and Arabidopsis mutants were grown in soil under long-day conditions (16 hours of light / 8 hours of dark, 21°C / 19°C) with a relative humidity around 60%. ktn1-5 (SAIL_343_D12), xxt1xxt2, prc1.1, qua2.1, xyl1.4, kor1.1 and any1 mutant lines were described previously35, 43–49. All mutants were in Col-0 background except kor1.1 which was in WS.
Plasmid construction
We used the Gateway® technology (Life Technologies, USA) and two sets of Gateway®-compatible binary T-DNA destination vectors50,51 for expression of transgenes in A. thaliana. The DNA fragment containing the Brassica oleracea SLR1 promoter was inserted into the pDONP4-P1R vector. The 165 bp-LTI6B fragment was introduced into the pDONR207 vector. MAP65 gene spanning the coding region from start to stop codons was introduced into the pDONR221 vector. CDS from citrine or GFP were cloned into the pDONP2R-P3 vector. Final constructs, pSLR1∷MAP65-citrine and pSLR1∷LTI6B-GFP were obtained by a three-fragment recombination system (Life Technologies) using the pK7m34GW and the pB7m34GW destination vectors, respectively. We generated a pAct11 ∷RFP construct by amplifying the promoter of the A. thaliana Actin 11 gene and cloning it into the pGreenII gateway vector in front of the RFP coding sequence.
Generation of transgenic lines and crossing
Transgenic lines were generated by Agrobacterium tumefaciens-mediated transformation of A. thaliana Col-0 as described52. Unique insertion lines, homozygous for the transgene were selected. We introduced the pSLR1∷LTI6B-GFP or pSLR1∷MAP65-citrine construct in ktn1-5 background by crossing and further selecting the progeny on antibiotic containing medium.
Microscopy
Confocal microscopy
Flowers at stages 12 to 1515 collected from fluorescent lines were emasculated and stigmas were observed under a Zeiss LSM800 microscope (AxioObserver Z1) using a 40x Plan-Apochromat objective (numerical aperture 1.3, oil immersion). Citrine was excited at 515 nm and fluorescence detected between 530 and 560nm. GFP was excited at 488 nm and fluorescent detected between 500 and 550 nm. RFP was excited at 561 nm and fluorescent detected between 600 and 650 nm.
Live imaging
Flowers from stages 12 to 15 were emasculated and pollinated on plants with mature pollen from the pACT11∷RFP line. Immediately after pollination, stigmas were mounted between two coverslips. To maintain a constant humidity without adding liquid directly on the stigma surface, we use a wet piece of tissue in contact with the base of the stigma. Pollinated stigmas were observed under a Zeiss microscope (AxioObserver Z1) equipped with a spinning disk module (CSU-W1-T3, Yokogawa) using a 40x Plan-Apochromat objective (numerical aperture 1.1, water immersion). Serial confocal images were acquired in the entire volume of the stigma every 1 μm and every minute. Images were processed with Image J software and pollen tube lengths were measured.
Atomic Force Microscopy
Pistils at stage 13 were placed straight in a 2% agar MS medium and 0.8% low-melting agarose was added up to the base of papilla cells. AFM indentation experiments were carried out with a Catalyst Bioscope (Bruker Nano Surface, Santa Barbara, CA, USA) that was mounted on an optical microscope (MacroFluo, Leica, Germany) equipped with a x10 objective. All quantitative measurements were performed using standard pyramidal tips (RFESP-190 (Bruker)). The tip radius given by the manufacturer was 8-12 nm. The spring constant of the cantilever was measured using the thermal tune method and was 35 N/m. The deflection sensitivity of the cantilever was calibrated against a sapphire wafer. All experiments were made in ambient air at room temperature. Matrix of 10×10 measurements (step 500 nm) was obtained for each papilla, with a 1μN force. The Young’s Modulus was estimated using the Nanoscope Analysis (Bruker) software, using the Sneddon model with a < 200nm indentation.
Environmental Scanning Electron Microscopy (SEM)
Flowers from stages 12 to 15 were emasculated and pollinated on plants with mature WT pollen. One hour after pollination, pistils were cut in the middle of the ovary, deposited on a SEM platform and observed under Hirox SEM SH-3000 at -20°C, with an accelerating voltage of 15kV. Images were processed with ImageJ software and pollen tube direction was quantified by counting the number of turns made by the tube, only on papillae that received one unique pollen grain.
Transmission Electron Microscopy
Stage 13 flowers were emasculated and pollinated on plants with mature WT pollen. One hour after pollination, pistils were immersed in fixative solution containing 2.5% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) and after 4 rounds of 30 min vacuum, they were incubated in fixative for 12 hours at room temperature. Pistils were then washed in phosphate buffer and further fixed in 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.2) for 1.5 hours at room temperature. After rinsing in phosphate buffer and distilled water, samples were dehydrated through an ethanol series, impregnated in increasing concentrations of SPURR resin over a period of 3 days before being polymerized at 70°C for 18 h, sectioned (65 nm sections) and imaged at 80 kV using an FEI TEM tecnaiSpirit with 4 k x 4 k eagle CCD.
Anisotropy estimation
Flowers from MAP65 lines were emasculated and stigmas were observed under confocal microscope. Images were processed with ImageJ software and quantitative analyses of the average orientation and anisotropy of CMTs were performed using FibrilTool, an ImageJ plug-in53. Anisotropy values range from 0 to 1; 0 indicates pure isotropy, and 1 pure anisotropy.
Membrane deformation estimation
Flowers from LTI6B lines were emasculated and pollinated with mature pollen from the PACT11∷RFP line. 20 minutes after pollination, stigmas were observed under confocal microscope. Serial confocal images every 1 μm encompassing the entire volume of the stigma were recorded and processed with ImageJ software. Plasma membrane deformation was estimated by choosing the slide from the stack that corresponded to the focus plan of the contact site with the RFP-labelled pollen tube. On the Bright field image corresponding to the selected slide, a line was drawn connecting the two ridges of the invagination of the papilla. Two perpendicular lines, one toward the exterior (ExtD) of the papilla to the maximum point of deformation, and the other toward the interior (IntD) on the GFP image were measured, respectively.
Statistical analysis
Graph and statistics were obtained with R software or Excel. Statistical tests performed are specified in figure legends.
Chemical treatment
To avoid contact of pollen grains with liquid, we performed local applications of oryzalin (Chemical service, Supelco) at 833 μg/mL (DMSO) in lanolin pasted around the style, just under the stigmatic cells, for 4 hours at 21°C. Oryzalin-treated pistils were pollinated with mature WT pollen and 1 hour after pollination observed under SEM.
Author contributions
L.R. performed all the experiments and the image analysis, except TEM and AFM, which were subcontracted. F.R. and C.K. contributed to cell imaging setup. L.R., T.G., and I.F.L., designed the study and analysed the data. L.R. and T.G. wrote the manuscript.
Competing interests
The authors declare no competing interests.
Acknowledgements
We thank O. Hamant for critical reading of the manuscript and fruitful discussion, A. Boudaoud, S. Bovio and V. Battu for advice on AFM experiments, and the Sice and MechanoDevo team members for discussion, P. Bolland, A. Lacroix and J. Berger for plant care and the PLATIM imaging facility of the SFR Biosciences Gerland-Lyon Sud. We thank the Bordeaux Imaging Centre especially L. Brocard and B. Batailler for TEM microscopy. We also thank the BioMeca® society and Pascale Milani for the AFM measurements. L.R. was funded by a fellowship from the French Ministry of Higher Education and Research. The work was supported by Grant ANR-14-CE11-0021.
Footnotes
↵* e-mail: thierry.gaude{at}ens-lyon.fr ; isabelle.fobis-loisy{at}ens-lyon.fr