Flowering plants produce small particles known as pollen that – with the help of the wind, bees and other animals – carry male sex cells (sperm) to female sex cells (eggs) contained within flowers. When a grain of pollen lands on the female organ of a flower, called the pistil, it gives rise to a tube that grows through the pistil towards the egg cells at the base.

The surface of the pistil is covered in a layer of long cells named papillae. Like most plant cells, the papillae are surrounded by a rigid structure known as the cell wall, which is mainly composed of strands known as microfibrils. The pollen tube exerts pressure on a papilla to allow it to grow through the cell wall towards the base of the pistil. Previous studies have shown that the pistil produces signals that guide pollen tubes to the eggs. However, it remains unclear how pollen tubes orient themselves on the surface of papillae to grow in the right direction through the pistil.

Riglet et al. combined microscopy, genetic and chemical approaches to study how pollen tubes grow through the surface of the pistils of a small weed known as Arabidopsis thaliana. The experiments showed that an enzyme called KATANIN conferred mechanical properties to the cell walls of papillae that allowed pollen tubes to grow towards the egg cells, and also altered the orientation of the microfibrils in these cell walls. In A. thaliana plants that were genetically modified to lack KATANIN the pollen tubes coiled around the papillae and sometimes grew in the opposite direction to where the eggs were.

KATANIN is known to cut structural filaments inside the cells of plants, animals and most other living things. By revealing an additional role for KATANIN in regulating the mechanical properties of the papilla cell wall, these findings indicate this enzyme may also regulate the mechanical properties of cells involved in other biological processes.

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. How the pollen germinates a tube and how pollen tube growth is regulated have been the object of many investigations (Palanivelu and Tsukamoto, 2012; Mizuta and Higashiyama, 2018). 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 sac (Dresselhaus and Franklin-Tong, 2013; Higashiyama and Yang, 2017; Cameron and Geitmann, 2018). At the cellular level, cytoskeleton has been extensively studied during pollen tube elongation (Fu, 2015), 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 the pollen tube elongation 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 direction (Gossot and Geitmann, 2007).

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 ovary (Lennon and Lord, 2000). The transmitting tissue has an essential function in pollen tube guidance, providing chemical attractants and nutrients (Crawford and Yanofsky, 2008; Higashiyama and Hamamura, 2008). 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 migration (Discher et al., 2005; Fu et al., 2010; Ermis et al., 2018). 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) tracks (Paredez et al., 2006). While penetrating the cell wall, the pollen tube exerts a pressure onto the stigmatic cell (Sanati Nezhad and Geitmann, 2013). Physical forces are known to reorganise the cortical microtubules (CMTs), which by directing CSCs to the plasma membrane, reinforce wall stiffness by novel cellulose microfibril (CMF) synthesis (Paredez et al., 2006; Sampathkumar et al., 2014). Hence, there is an intricate interconnection between CMT organisation, CMF deposition and cell wall rigidity (Xiao and Anderson, 2016). A major regulatory element of CMT dynamics is the KATANIN (KTN1) microtubule-severing enzyme, which allows CMT reorientation following mechanical stimulation (Uyttewaal et al., 2012; Sampathkumar et al., 2014; Louveaux et al., 2016). 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 techniques, genetic tools, chemical analysis of the cell wall and atomic force microscopy. We show that isotropic reorientation of CMTs occurs in aged Col-0 and ktn1-5 papilla cells, which is accompanied by a change in the growth direction of pollen tubes that tend to make coils on papillae. We show that CMT reorganisation is associated with isotropic rearrangement of CMFs, high content of crystalline cellulose and particular cell wall mechanics of ktn1-5 stigmas. Altogether, our results indicate that cytoskeleton dynamics and mechanical properties of the cell wall, which both depend on KTN1 activity, have a major role in guiding early pollen tube growth in stigma papillae.

Previous work reported that rearrangements of actin microfilaments (Iwano et al., 2007; Rozier et al., 2020) and destabilization of CMTs (Samuel et al., 2011) occur in stigmas following compatible pollination in Brassicaceae species. However, CMT pattern and dynamics during papilla development have never been described. Our data show that during the course of stigma maturation, which is associated with papilla cell elongation, 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 differentiate (Baskin, 2005; Landrein and Hamant, 2013).

During invasion of plant cells by fungal or Oomycete pathogens or upon mechanical stimulation, the network of microtubules is rearranged, generally associated first with the depolymerization of CMTs at the contact site and later with the formation of an array of microtubules surrounding the invading organism or microneedle (Hardham, 2013). Interestingly, penetration of the pollen tube in the papilla cell wall does not lead to such microtubule reorganisation (Figure 3). This suggests that at the cellular level, the invading pollen tube does not elicit a defence-like response in the papilla, which accepts and supports pollen tube growth. In addition, we show that when the stigma is manually pressed between slide and coverslip, the mechanical stimulation applied perpendicularly to the wall leads to CMT fragmentation (Figure 3—figure supplement 1). This indicates that although the papilla microtubule network is sensitive to mechanical forces, the pressure exerted by the pollen tube is not strong enough to perturb CMT organisation. One possible explanation is that the penetration force developed by the growing pollen tube acts mainly in plane to the cell wall surface and hence would only have a little effect on the papilla cell, contrary to a pressure applied perpendicularly to the wall. Our data are in contradiction with the earlier observation that growing pollen tube provokes fragmentation of CMTs in the papilla (Samuel et al., 2011). This discrepancy is likely to relate to the microscopic approaches used between the two studies, live imaging and a fluorescent microtubule probe in our case, chemically fixed material and immunostaining of microtubules in Samuel et al., 2011.

The progressive randomisation of CMT orientation we observed in papilla cells during ageing 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 organisation and the trajectory that pollen tube takes while growing in the papilla cell wall, although the effects of oryzalin on cell wall properties remain unknown. 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 organisation 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 organisation, cell wall composition and CMF orientation (Burk et al., 2001; Burk and Ye, 2002; Ryden et al., 2003). 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 (Figure 6). This places mechanics of the cell wall as a likely component involved in the coiled phenotype. Surprisingly, of the six cell wall mutants we analysed, including xxt1 xxt2 and prc1 known to exhibit both abnormal CMT organisation and softer cell walls, none induced the coiled phenotype displayed by ktn1-5 papillae. More remarkably, despite the similar stiffness of xxt1 xxt2 and ktn1-5 papilla cell walls as measured by AFM, pollen tubes behaved differently on these two stigma types, pollen tubes not only coiling but also growing faster on ktn1-5 stigmas. This observation is in accordance with the recent findings that changes in indentation properties of plant cell walls do not correlate with changes in tensile stiffness as described in cell-free strips of onion epidermal walls (Zhang et al., 2019). In our experiments, indentations tell us about the elasticity of the wall to a pressure normal to the plane of the wall surface (i.e. out-of-plane mechanics), while pollen tube growth rates inform about the resistance of the wall to a force in-plane to the papilla wall (i.e. in-plane mechanics). Several lines of evidence lead us to suggest that mechanical properties of the cell wall are not identical in the two mutants. Previous work showed that cellulose crystallinity is one factor implicated in cell wall mechanics. Indeed, the epidermal cells of the inflorescence stem in the any1 mutant have the relative amount of crystalline cellulose significantly reduced, while the total content of cellulose in the cell wall is unaltered, and this is accompanied by reduced stiffness of the cell wall (Fujita et al., 2013; Altartouri et al., 2019). Our chemical analysis reveals that the cell wall composition is different between xxt1 xxt2 and ktn1-5 stigmas, with this latter having 25% more crystalline cellulose, and hence should exhibit stiffer cell walls (Figure 5—figure supplement 4). This data is at odds with AFM measurements but a possible explanation would be that the two layers detected by TEM in the papilla cell wall (Figure 6—figure supplement 1) display different mechanical properties, the inner layer being stiffer than the outer one in the ktn1-5 mutant. This may explain the protruding growth of pollen tubes observed in ktn1-5 papillae, the tube pushing away the softer outer layer but not the inner (Figure 6A–D). Interestingly, Zhang et al., 2019 reported that cellulose networks largely determine in-plane mechanics whereas out-of-plane mechanics depends on both homogalacturonan (pectin) and cellulose networks. Cell walls of the qua2-1 mutant are specifically affected in pectins, with a 50% reduction in homogalacturonan content compared with Col-0 without observable changes in other polysaccharides (Mouille et al., 2007). In light of these data, it is noteworthy that papilla cells of the qua2-1 mutant do not induce pollen tube turns (Figure 5—figure supplement 3), which suggests that pectins play no significant role in the pollen tube phenotype and that the main factor contributing to pollen tube guidance is in-plane mechanics, and hence relies on the cellulose network. The orientation of the rigid CMFs is another determinant of cell wall mechanics. The ktn1-5 mutant was described to have a severe reduction in cell length and an increase in cell width in all organs (Burk et al., 2001). 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 axis (Burk and Ye, 2002). Similarly, we found that orientation of CMFs is also altered in ktn1-5 papillae (Figure 7) and this was associated with larger papilla cells (Figure 5—figure supplement 5). Hence, we may assume that mechanical anisotropy, described as the cell wall anisotropy made by the orientation of the rigid CMFs (Sassi et al., 2014), is impaired in ktn1-5 papillae. Contrary to the ktn1-5 mutant, papillae of the xxt1 xxt2 double mutant display an anisotropic CMF pattern with parallel fibres approximatively oriented transversally to the papilla axis. These data are in agreement with those reported for etiolated hypocotyl cells, where CMFs are largely parallel to one another, straighter than in WT and oriented almost transversely to the long axis of the cell (Xiao et al., 2016). In addition, we found the papilla cell shape of xxt1 xxt2 stigmas to be closer to that of Col-0 than that of ktn1-5 papillae, which is consistent with an anisotropic growth of the papillae in the xxt1 xxt2 mutant compared with ktn1-5. Apart from the similar stiffness of papilla cell walls as deduced from AFM measurements, the main differences between ktn1-5 and xxt1 xxt2 cell walls are 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 7 (Takáč et al., 2017), whereas targeted quantitative RT-PCR showed that several Microtubule-associated protein (MAP) and wall signal receptor genes showed lower expression levels in xxt1 xxt2 (Xiao et al., 2016). 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. Remarkably, several lines of evidence reveal that papilla cells from stage 15 share common features with ktn1-5 papillae, such as CMT and CMF increased isotropy, decreased stiffness of the cell wall as deduced from AFM and less resistance of the wall to pollen tube growth. Isotropic orientation of the cytoskeleton at stage 15 is likely to relate to cell elongation, which is known to be accompanied by cytoskeleton reorganisation (Crowell et al., 2011; Zhang et al., 2014). At the organ level, it has been suggested that the mechanical anisotropy of the wall restrains organ emergence (Sassi et al., 2014). 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 orientation (Baskin, 2005). KATANIN, by acting on CMT dynamics and CMF organisation, would fine-tune the mechanical properties of the matrix through which the pollen tube grows. It is worth noting that growth rate of pollen tubes germinated in vitro is slowed down when pollen tubes pass through a microgap of a microfluidic device, the tubes adapting their invasive force to the mechanical constraints (Sanati Nezhad et al., 2013). Based on measurements of pollen tube growth rates, our data show that the 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.

Our study shows that mechanics plays a key role in early pollen tube guidance in the papilla cell. This role is mediated by a specific CMT/CMF organisation and mechanical anisotropy of the papilla cell, which both are dependent on KTN1. Importantly, these specific mechanical properties of the stigmatic cells prevent emerging pollen tubes to grow upward on papillae and straighten pollen tube direction, helping the tube to find its correct path to the stylar transmitting tract. Our findings also raise the question about the biological importance of the mechanical changes that occur during stigma development. Indeed, for aged stigmas, modifications of the mechanical properties of the cell wall, accompanied by an acceleration of pollen tube growth, could be seen as beneficial for the plant by favouring ultimate fertilization and seed set on old flowers, and hence supporting dissemination of the species. This assumption is particularly relevant given that the pistil length increases with ageing and similarly the journey the pollen tube has to travel to reach the ovules. A recent work showed that ageing is associated with decreased fertility due to stigma senescence, which is initiated at stage 16–17 (Gao et al., 2018). The faster the pollen tube will grow in old stigmas, the more chance it will have to circumvent papilla cell death and hence to fertilize the ovule. We may suggest that KTN1, by mediating mechanical anisotropy of stigmatic cells and promoting pollen tube growth in old stigmas, has played an evolutionary role in the success of fertilization.

To conclude, we uncovered a yet unexpected function for KTN1 in early pollen tube guidance on the stigma. In addition, our study also clearly unveils 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).

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 4, 5 and 6 to 5 and Figure 5—figure supplements.

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Author details

1. Lucie Riglet

   Laboratoire de Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, UCBL, INRAE, Allée d'Italie, France

   Present address

   Sainsbury Laboratory, University of Cambridge, Cambridge, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

2. Frédérique Rozier

   Laboratoire de Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, UCBL, INRAE, Allée d'Italie, France

   Contribution

   Resources, Investigation, Methodology

   Competing interests

   No competing interests declared

3. Chie Kodera

   Laboratoire de Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, UCBL, INRAE, Allée d'Italie, France

   Present address

   Institut Jean-Pierre Bourgin, INRAE, AgroParisTech, Université Paris-Saclay, Versailles, France

   Contribution

   Resources, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Simone Bovio

   Laboratoire de Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, UCBL, INRAE, Allée d'Italie, France

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

5. Julien Sechet

   Institut Jean-Pierre Bourgin, INRAE, AgroParisTech, Université Paris-Saclay, Versailles, France

   Contribution

   Formal analysis, Funding acquisition, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8398-4743

6. Isabelle Fobis-Loisy

   Laboratoire de Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, UCBL, INRAE, Allée d'Italie, France

   Contribution

   Conceptualization, Supervision, Visualization

   Competing interests

   No competing interests declared

7. Thierry Gaude

   Laboratoire de Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, UCBL, INRAE, Allée d'Italie, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   thierry.gaude@ens-lyon.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0804-9445

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