Stigma plasma membrane H + -ATPase is involved in pollen hydration and pollen tube penetration in Brassicaceae self-incompatibility

Stigma plasma membrane H + -ATPase is involved in pollen hydration and pollen tube penetration in Brassicaceae self-incompatibility | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Stigma plasma membrane H + -ATPase is involved in pollen hydration and pollen tube penetration in Brassicaceae self-incompatibility Maki Hayashi, Kazuki Fukushima, Hiromi Masuko-Suzuki, Shin-ichiro Inoue, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6981590/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract During pollination in self-incompatible Brassicaceae plants, the female papilla cells selectively transport water to non-self pollen after self or non-self discrimination through pollen-pistil interaction, and this process is crucial for fertilization. However, the mechanism of the selective water transport has not been elucidated. In this study, we determined that the stigma autoinhibited plasma membrane H + -ATPase (AHA) is involved in the selective water transport from papilla cells to pollen. We characterized AHA isoforms that are expressed in the stigma of self-incompatible plant, Brassica rapa by expression analysis. A chemical activator of AHA applied to stigmas suppressed pollen hydration and pollen tube penetration into stigma in cross-pollination of B. rapa . In contrast, an AHA inhibitor allowed pollen hydration and pollen tube penetration in self-pollination. Consistent with these pharmacological effects, stigma AHA was activated through the phosphorylation of penultimate Thr in response to self-pollination compared with cross-pollination. Furthermore, speed of pollen hydration and elongation of pollen tubes were delayed on the stigmas of ost2-2D , a constitutive active mutant of AtAHA1 in the self-compatible Arabidopsis thaliana . These results indicate that regulation of AHA activity in the stigma is involved in selective water transport for pollen hydration, possibly by regulating osmotic pressure in Brassicaceae self-incompatibility. Biological sciences/Plant sciences/Plant physiology Biological sciences/Plant sciences/Plant reproduction Plasma membrane H+-ATPase Brassica rapa Arabidopsis thaliana Pollen hydration Pollen tube penetration Self-incompatibility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Most angiosperms have hermaphrodite flowers, with a female pistil and plural male stamens in the same flower 1 . The mature pollen from the anthers attaches to the stigmatic papilla cells of the pistil, where it absorbs water and hydrates 2 – 4 . The hydrated pollen germinates a pollen tube on the papilla cells and the pollen tubes elongate and penetrate into the pistil to carry the sperm cells to the ovules, achieving fertilization 5 , 6 . The transport of water from the papilla cells to the pollen is a primary step important for the post-pollination processes including pollen hydration, pollen tube germination, pollen tube penetration into the stigma, and pollen tube elongation in the pistil. However, the molecular mechanisms underlying water transport from female to male cells have not been fully elucidated. Approximately half of angiosperms exhibit self-incompatibility, rejecting fertilization by self-pollen and thus avoiding inbreeding depression within a species 7 . In Brassicaceae plants showing self-incompatibility, papilla cells block water transport to the self-pollen, suggesting that this water transport is not passive but is selectively controlled by the papilla cells 8 , 9 . Self-incompatibility in Brassicaceae species is controlled by the multi-allelic genes of the S locus, including S locus protein 11 (SP11)/ S locus cysteine-rich protein (SCR) and S receptor kinase (SRK), the male and female determinate factors, respectively 10 – 16 . The female factor SRK is expressed and localized in the plasma membrane of papilla cells, and the male factor SP11 is expressed in the anther tapetum and secreted onto the pollen surface 14 , 17 , 18 . When SP11 and SRK have the same S haplotype, these proteins bind 19 – 22 . Using this receptor-ligand binding system, papilla cells recognize and reject self-pollen through suppression of post-pollination processes. Thus, signaling from SRK inhibits selective water transport from the papilla cells to the pollen 5 . Some intracellular processes have been reported regarding the water transport from papilla cells to pollen in Brassicaceae plants. Vacuole-derived vesicles/ multivesicular bodies (MVBs) appear in the cytosol of papilla cells near the pollen attachment site only in cases of compatible pollination 9 , 23 – 26 . In the self-incompatible Arabidopsis lyrata and Brassica napus , self-pollination induces autophagic degradation of the vesicles/MVBs in papilla cells, and this process is required for the rejection of self-pollen on the stigma 25 , 27 , 28 . These results suggest that vesicles/MVBs contribute to pollen hydration, probably by secreting water-laden vesicles from papilla cells to pollen. On the other hand, it was shown in the pistil of A. thaliana that plasma membrane aquaporin PIP1;2 and ER-bound aquaporin SIP1;1 coordinately contribute to pollen hydration by mediating the direct water efflux from the papilla cells 29 . Thus, some independent phenomena in the papilla cells have been shown to play important roles in the compatible pollen hydration; however, the relationship between these phenomena remains unclear. Plant cells often control the direction of water transport across the plasma membrane through regulation of intracellular osmotic pressure by ion transports, which is utilized for some physiological responses. For example, in hypocotyl cells during elongation growth and in guard cells during stomatal opening, the phytohormone auxin and light increase the osmotic pressure of the cells, promoting water uptake and cell expansion, respectively 30 – 33 . Conversely, in guard cells during stomatal closure and in pulvinar motor cells of legumes during dia-heliotropic leaf movement, the phytohormone abscisic acid (ABA) and light decrease the osmotic pressure of the cells, promoting water excretion and cell shrinkage, respectively 34 – 37 . It is well established that an autoinhibited plasma membrane H + -ATPase (AHA) plays a key role in these responses through osmoregulation in the cells 38 , 39 . AHA is the primary transporter in plant cells and uses the ATP hydrolysis energy to pump H + out of the cell, thereby generating the driving force for various secondary transports on the plasma membrane 38 , 40 , 41 . For example, AHA in guard cells is activated by light and increase the osmotic pressure of guard cells to open stomata. Conversely, during stomatal closure, ABA signaling inactivates AHA, decreasing the osmotic pressure in guard cells 42 – 44 . Thus, cellular osmoregulation controlled by AHA activity is crucial for the direction of water transport across plasma membrane in plant cells. From these observations, we hypothesized that osmoregulation by AHA is also involved in the regulation of water transport from the papilla cells to the pollen in Brassicaceae species. In this study, to identify novel regulatory intracellular events in post-pollination processes, we examined the contribution of AHA in the stigma to pollen hydration, pollen tube penetration into the stigma, and pollen tube elongation in the pistil in Brassicaceae plants. Pharmacological and physiological experiments using self-incompatible B. rapa and self-compatible A. thaliana provided new insights into post-pollination processes in Brassicaceae plants, in which AHA regulates osmotic pressure in stigma cells and is involved in the selective water transport from papilla cells to pollen. Results Autoinhibited plasma membrane H + -ATPases expression in Brassica rapa stigmas To verify the function of plasma membrane H + -ATPase in papilla cells, we first annotated genes of the autoinhibited plasma membrane H + -ATPases (AHAs) in self-incompatible Brassica rapa genome, and termed these BrAHAs. Based on a blast search using the full-length amino acid sequences of Arabidopsis thaliana AHAs (AtAHAs) as the query, we found that the B. rapa genome contains 18 homologous BrAHA genes (Fig. 1 a). To examine the expression of BrAHAs in the stigma, RNA was extracted from the stigma of the S 9 strain of self-incompatible B. rapa , and RT-qPCR was performed. Among the 18 BrAHA genes, two BrAHAs , BrAHA1a ( Bra038835 ) and BrAHA2c ( Bra011172 ), were strongly expressed in the stigma (Fig. 1 b). A similar expression profile was obtained using stigma of another strain of self-incompatible B. rapa , that is S 8 (Fig. S1 ). The BrAHA proteins (BrAHA1a and BrAHA2c) are encoded by two genes belonged to the same clade as AtAHA1 and AtAHA2, which are known to be the main isoforms in A. thaliana (Fig. 1 a) 45 . Next, we cloned the coding sequences of these genes, and compared the deduced amino acid sequences with AtAHA1 and AtAHA2. AHA proteins in seed plants possess a penultimate Thr (pen-Thr) residue, region I, and region II in the C-terminal region that are important for the autoinhibitory effects of H + -ATPase 46 , and all of these are highly conserved in BrAHA1a and BrAHA2c (Fig. S2). We then investigated the intracellular localization of the two BrAHAs by transient expression assay of GFP-fused proteins with a plasma membrane marker (PIP2A-mCherry) in Nicotiana benthamiana leaves. The result showed that the fluorescence signals from GFP-BrAHA1a and GFP-BrAHA2c were well merged with the fluorescent signal from PIP2A-mCherry in the plasma membrane (Fig. 1 c). These results indicate that BrAHA1a and BrAHA2c are plasma membrane H + -ATPases highly expressed in the stigma. Involvement of stigma AHA in pollen tube penetration into the stigma of B. rapa To investigate whether AHA activity in the stigma is important for post-pollination processes in B. rapa , we observed pollen tube penetration into stigma treated with the AHA activator fusicoccin (FC) or the AHA inhibitor vanadate. AHA is activated by phosphorylation of the pen-Thr residue in the C-terminal YTV motif and subsequent binding of 14-3-3 proteins to the phosphorylated YTV motif (Fig. S2). FC activates AHAs by stabilizing the physical interaction between the C-terminal phosphorylated motif and 14-3-3 proteins 47 – 51 . The stigmas of B. rapa were treated with FC for 3 h, and activation of stigma AHAs was tested by immunoblotting using a specific antibody recognizing phosphorylated pen-Thr. We confirmed that the phosphorylation level of pen-Thr in AHA of stigmas was strongly increased by FC compared with the solvent control (Mock) (Fig. 2 a). We further confirmed the effect of FC treatment by detecting the change in apoplast pH in the stigma using the pH indicator bromocresol purple (BCP). FC-treated stigmas extruded more H + , and changed the color of the medium containing BCP, indicating that it was more acidic compared to mock treatment (Fig. 2 b). On the other hand, vanadate binds to the E2 form of AHA, and inhibits its activity independently of the pen-Thr phosphorylation 52 . Thus, the effect of vanadate treatment was confirmed only by the pH change in the stigma apoplast. As expected, vanadate-treated stigmas showed less color change in the BCP medium resulting from apoplast acidification compared to mock treatment (Fig. 2 c). From these results, we confirmed that treatment with FC or vanadate had the expected effect on AHA activity in the stigmas of B. rapa . To dissect the relationship between AHAs and post-pollination processes, we next investigated the effects of FC and vanadate on self-incompatibility in B. rapa . Stigmas of the self-incompatible S 9 strain were treated with FC or vanadate for 3 h before pollination, and then pollinated with pollen from the S 9 strain (self-pollination) or S 8 strain (cross-pollination). In the S 9 stigmas treated with the solvent only (mock), self-pollen and its pollen tubes showed the typical responses of self-incompatibility, including inhibition of pollen adhesion, pollen hydration, pollen tube germination, pollen tube penetration into stigma, and pollen tube elongation (Fig. 3 a). In contrast, when the S 9 stigmas were cross-pollinated with S 8 pollen, the pollen attached to the stigma germinated pollen tubes, and the pollen tubes penetrated into the stigma and elongated to the style. When S 9 stigmas were treated with FC, pollen tube penetration into the stigma was strongly inhibited not only in self-pollination but also in cross-pollination (Fig. 3 a). Furthermore, when S 9 stigmas were treated with vanadate, pollen tube penetration was observed in self-pollination as well as in cross-pollination. To quantify these results, we counted the number of pistils that were penetrated and not penetrated by the pollen tube, based on whether the pollen tube had passed through the stigma-style boundary and entered the style (Fig. 3 b: left diagram). FC treatment reduced the ratio of “Penetrated/Not penetrate” pistils in cross-pollination, and vanadate increased the ratio of “Penetrated/Not penetrate” pistils in self-pollination (Fig. 3 b). Thus, our results indicate that FC inhibited pollen tube penetration into the style in compatible pollination, whereas vanadate induced pollen tube penetration in incompatible pollination. The effects of FC and vanadate were similarly observed in stigmas of another self-incompatible strain, S 8 in B. rapa (Fig. S3). We next tested the effects of FC and vanadate on the pollen tube penetration using a self-compatible B. rapa yellow sarson C634 strain. In this strain, FC inhibited the penetration of self-pollen tubes into the stigma, whereas vanadate did not (Fig. 3 c). The self-compatibility in yellow sarson results from abnormal transcription of SRK and SP11 genes and disruption of the membrane-anchored protein kinase, M locus protein kinase (MLPK), which interacts with SRK 53 – 57 . These results indicate that AHA activity in the stigma is involved in post-pollination processes in B. rapa and suggest that its involvement is independent, or downstream, of self/non-self-discrimination by the self-incompatibility system in B. rapa . Involvement of stigma AHA in pollen hydration in self-incompatible B. rapa The results of self/non-self-discrimination in Brassica species self-incompatibility is manifested as failure or success of pollen hydration on the papilla cells 9 . We next tested by pharmacological experiments whether AHA activity in papilla cells is important for water supply to pollen during pollen hydration in B. rapa with observations using a time-lapse system. In the solvent control (mock), pollen from the S 9 strain (self-pollination) showed little size change within 60 min after pollination (MAP) on the S 9 papilla cells (Fig. 4 a), whereas pollen from the S 8 strain (cross-pollination) rapidly expanded within 20 MAP onto the papilla cells of the S 9 strain, and pollen tubes germinated at 30 to 60 MAP (Fig. 4 b). In contrast, vanadate treatment of papilla cells induced pollen expansion within 20 MAP and pollen tube germination at 60 MAP in the self-pollination (Fig. 4 a: vanadate), and FC treatment of papilla cells showed a delay in pollen expansion during cross-pollination (Fig. 4 b: FC). Although pollen on the FC-treated papilla cells swelled to the similar extent as those on mock-treated cells at 60 MAP, little pollen tube germination was observed at that time (Fig. 4 b). These results indicate that regulation of AHA activity is involved in pollen hydration via efficient water transport from papilla cells to pollen during the post-pollination processes. This suggests that AHA-mediated active osmoregulation in papilla cells may control the water transport to pollen after self/non-self-discrimination. Changes in the phosphorylation status of stigma AHA in response to self- and cross-pollination in self-incompatible B. rapa Next, to examine the activity of AHA in the stigmas of self-incompatible B. rapa , the pen-Thr levels of AHA were compared after pollination with self- or non-self-pollen. Pollinated stigmas (and unpollinated control stigmas) were washed to remove pollen, and proteins extracted from the stigmas were subjected to SDS-PAGE. The phosphorylated pen-Thr levels of AHA were determined by immunoblot analysis using a specific antibody against phosphorylated pen-Thr (Fig. 5 ). Signal of pen-Thr of AHA was observed to some extent in the unpollinated stigma. In contrast, self-pollination induced phosphorylation of pen-Thr of AHA in the stigmas, increasing its phosphorylation level approximately two-fold compared to the unpollinated stigmas. The levels of phosphorylated pen-Thr did not change in cross-pollinated stigmas compared to unpollinated stigmas. These results indicate that AHA activity in the stigma is regulated via the phosphorylation level of pen-Thr in response to self- and non-self-pollen in B. rapa . In addition to pen-Thr, the C-terminal region of AHA contains multiple phosphorylation sites that affect AHA activity. In A. thaliana , phosphorylation of pen-Thr and Thr-881 was characterized as AHA activation sites, while phosphorylation of Ser-899 and Ser-931 was characterized as inhibition sites (Fig. S2) 33 , 58 – 61 . In the major AHA isoforms in B. rapa stigmas, BrAHA1a and BrAHA2c, the amino acid corresponding to Ser-899 was not conserved, but the other phosphorylation sites were conserved (Fig. S2). To identify phosphorylation sites important for regulating stigma AHA activity in the inhibition of post-pollination processes, we performed quantitative phosphoproteomic analysis using self- and cross-pollinated B. rapa stigmas (Table S1 ). Protein samples were prepared in a similar manner as for immunoblot analysis (Fig. 5 ). Only two phosphorylation sites, Thr-881 and Thr-924 were detected at the C-terminus of BrAHAs in our phosphoproteomic analysis. Although amino acid substitutions at both sites are known to affect AHA activity 58 , 62 , 63 , the phosphorylation levels of these sites did not change in response to self- and non-self-pollination (Table S1 ). The peptide including pen-Thr was not detected in this analysis. Thus, we concluded from these results that phosphorylation of the pen-Thr of AHA may be important for regulation of stigma AHA activity in self-incompatibility of B. rapa . Physiological and genetic verification of the involvement of stigma AHA in post-pollination processes in A. thaliana In both self-incompatible and self-compatible B. rapa , AHA was found to be involved in the post-pollination processes in the pistils (Fig. 2 – 5 , S3). To verify the involvement of AHA in post-pollination processes in other Brassicaceae species, A. thaliana was used in a pharmacological experiment. Because A. thaliana shows self-compatibility, like the yellow sarson strain of B. rapa , we only examined the effect of FC and not vanadate. Pistils of A. thaliana were treated with FC in a similar manner to those of B. rapa , and then the pistils were pollinated to observe the effect of FC on pollen tube penetration into the stigma. In stigmas treated with the solvent only control (mock), pollen tubes penetrated into the stigma, whereas in stigmas treated with FC, pollen tubes stopped elongating on the papilla cells and did not penetrate (Fig. S4). These results suggest that AHA activity in the stigma is involved in post-pollination processes in A. thaliana as in B. rapa . To identify AHA genes expressed in the stigma of A. thaliana , we next searched the Arabidopsis eFP Browser database ( https://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi ) and found that AHA1 , AHA2 , and AHA11 are predominantly expressed in the stigma (Fig. S5a, b). In particular, AHA1 and AHA2 comprise around 47% and 27% of the total AHA transcripts, respectively, and their expression accounts for approximately 74% of the expression of all AHA genes in the stigma (Fig. S5c). To study the expression and subcellular localization of AHA1 and AHA2 proteins in papilla cells, we generated stable transgenic A. thaliana plants expressing GFP-fused AHA1 or AHA2 proteins under the control of their respective native promoters. Fluorescence signals from GFP-AHA1 and GFP-AHA2 were clearly observed in the peripheral region of papilla cells (Fig. S5d). From these results, we concluded that AHA1 and AHA2 are the major isoforms of AHA proteins in A. thaliana papilla cells, as reported in other tissues 45 . To genetically verify the involvement of AHA in post-pollination processes, we used a dominant-positive mutant of ost2-2D that expresses constitutively active AHA1 with a G867S mutation 64 . Pollen from the WT was pollinated onto WT or ost2-2D papilla cells of pistils, and pollen tube penetration into the pistils was observed 2 and 4 h after pollination (HAP). At the time of observations, all pollen tubes penetrated the stigmas of the pistils, and there was no difference in the pollen tube penetration between the two plant lines (Fig. 6 a). However, at both 2 HAP and 4 HAP, the length of pollen tubes penetrating the pistils was clearly different between WT and ost2-2D pistils, that is the pollen tubes in the ost2-2D pistils elongated less than those in the WT pistils (Fig. 6 a, S6). Because pollen hydration affects pollen tube germination and elongation 29 , 65 , we speculated that the difference in pollen tube length might result from differences in the speed of pollen hydration in the WT and ost2-2D papilla cells. We therefore compared the speed of pollen hydration after the pollination of WT pollen on WT or ost2-2D stigmas. WT pollen on the WT papilla cells gradually hydrated within 15 MAP, whereas the hydration of WT pollen on the ost2-2D papilla cells was slower compared to that on the WT papilla cells (Fig. 6 b). These results indicate that the constitutively active AHA1 in the stigma partially inhibits pollen hydration on ost2-2D papilla cells. The delay in pollen hydration on ost2-2D stigmas is likely responsible for the shorter pollen tube elongation compared to WT. These results provide genetic support for the effects FC. In contrast to the phenotype of ost2-2D , pollen tube penetration and elongation in the pistils of aha1-9 and aha2-5 knockout mutants were similar to those in the WT, and no phenotypic differences were observed between the WT and these mutants (Fig. S7a, b). In addition, we generated aha11 mutants by genome editing and examined its phenotype (Fig. S8). The aha11 mutants, similar to the aha1-9 and aha2-5 mutants, showed no phenotypic differences in pollen tube penetration and elongation compared to the WT (Fig. S8b, c). These results are consistent with the results that vanadate treatment of B. rapa pistils did not suppress pollen tube penetration into the stigmas in compatible pollination, again providing genetic support for the results of the pharmacological experiments (Fig. 3 ). Discussion In self-incompatible Brassicaceae plants, selective water transport from female papilla cells to male pollen is clearly observed only after non-self-pollination 9 , 66 . Based on these findings, we hypothesized that osmoregulation in the papilla cells regulates water transport from papilla cells to pollen during the post-pollination process, and we focused on AHA, a primary transporter that plays a major role in osmoregulation of plant cells. In pharmacological experiments in B. rapa , the application of FC, which promotes AHA activity, to stigmas stopped post-pollination processes in cross-pollination (Fig. 3 , 4 , S3), whereas the application of vanadate, which inhibits AHA activity, to the stigmas allowed post-pollination processes in self-pollination to proceed. Furthermore, FC had a similar effect on A. thaliana stigmas, suppressing post-pollination processes (Fig. S4). Although AHA possesses multiple phosphorylation sites in its C-terminal region that affect activity, phosphorylation of the pen-Thr residue of AHA is commonly observed in many stimuli and serves as the primary activation switch 39 , 58 , 59 , 63 . The level of this pen-Thr phosphorylation in the stigmas of B. rapa increased in response to self-pollination compared with non-pollination and cross-pollination (Fig. 5 ). Furthermore, a constitutively active mutant of AHA1, ost2-2D , in A. thaliana showed delayed pollen hydration and suppressed pollen tube elongation (Fig. 6 , S6). These results suggest that activation of AHA in the stigma by self-pollination may promote water retention through an increase in osmotic pressure in the papilla cells, which inhibit the post-pollination processes (Fig. 7 ). Activation of AHA is most likely regulated by SP11-SRK signaling because stigma AHA was activated in response to self-pollination (Fig. 5 ). The effects of FC and of an AHA1 constitutively active mutation were also observed in the self-compatible plant A. thaliana (Fig. 6 , S4, S6). Therefore, the status of AHA activity in the stigma may generally affect post-pollination processes through the control of osmotic pressure in papilla cells in Brassicaceae plants, regardless of whether the plant is self-compatible or incompatible. A. thaliana exhibits interspecies incompatibility when crossed with some other Brassicaceae species 67 , and the AHA-mediated regulatory system in A. thaliana might be utilized during interspecific incompatibility. As shown in Fig. 5 , levels of pen-Thr phosphorylation in AHA were at a steady state before pollination and the level did not change after pollination with compatible pollen. This result suggests that papilla cells may be ready to provide water to pollen before compatible pollination occurs. In this case, the osmotic pressure in papilla cells is kept relatively low, and the water-retaining capacity of the papilla cells may not be strong. In guard cells, ABA inhibits light-induced stomatal opening by suppressing phosphorylation of the pen-Thr of AHA 42 , 43 . The suppressed phosphorylation of AHA reduces osmotic pressure of guard cells, leading to water efflux. Similar to the guard cells, this low osmotic pressure in the papilla cells may provide an efficient force for water transport by widening the difference in osmotic pressure between the papilla cells and the pollen when compatible pollen is attached to the stigma. Since AHA is inactivated by dephosphorylation of pen-Thr, in unpollinated or cross-pollinated papilla cells, members of the type 2C protein phosphatase clade D family that remove the phosphate group, or unknown protein kinases that phosphorylate sites leading to AHA inactivation, may function to maintain some level of pen-Thr phosphorylation low 39 , 68 . It has also been reported that transport of vesicles/MVBs to the plasma membrane promotes the water supply to pollen 9 , 23 , 24 , 26 , but it is currently unclear what is carried in the transport vesicles/MVBs during this process. The transport vesicles/MVBs may carry transporters, including aquaporins localized to the plasma membrane, which may support water transport from papilla cells to pollen. Generally, aquaporins transport water according to the difference in osmotic pressure between inside and outside the cell 69 , so it makes sense that papilla cells would keep AHA activity low and maintain a low osmotic pressure to supply water to pollen. Taken together, our results suggest that osmoregulation via AHA, as well as vesicle transport in the papilla cells, play a crucial role in efficient water supply during pollination with compatible pollen. Pollen tube penetration was not inhibited in ost2-2D mutant pistils, unlike in FC-treated WT pistils (Fig. 6 a, S4). The difference in the effects of the ost2-2D mutation and FC treatment could be explained by differences in the activated AHA isoforms in the stigma by the genetic mutation and chemical treatment. The major isoforms, AHA1 and AHA2 , are expressed throughout the plant and have redundant functions 45 . In the stigmas of A. thaliana , AHA1 , AHA2 , and AHA11 are predominantly expressed (Fig. S5). FC treatment promotes the activity of all the AHA isoforms. Therefore, all AHAs expressed in the stigma may induce a strong increase in osmotic pressure in papilla cells, and completely inhibit the penetration of pollen tubes into the stigma (Fig. S4). In contrast, only AHA1 is constitutively active in the ost2-2D mutant. Thus, in the ost2-2D mutant, the total amount of activated AHA in the stigma is less than that observed with FC treatment, which may explain why inhibition of pollen tube penetration was not observed in the mutant stigmas. Similarly, no differences were observed between aha single mutants and the WT in pollen tube penetration or elongation (Fig. S7, S8), which may also be due to the functional redundancy of AHAs. We were unable to test this possibility in this study because the aha1 aha2 double mutant is embryonic lethal 45 . Vanadate treatment of B. rapa stigmas did not affect pollen tube penetration into the stigma in pollinations with compatible pollen (Fig. 3 ). Therefore, another possibility to explain these results, besides functional redundancy, is that AHA activity in papilla cells was already low enough that the effects of aha1, aha2 , and aha11 mutations in A. thaliana and vanadate treatment in B. rapa stigma were not clearly observed (Fig. 3 , S3, S7, S8). Restriction of AHA activity in the stigma to a relatively low level may be necessary for post-pollination processes to proceed. To date, several signaling components have been identified that induce post-pollination processes and control self-compatibility/-incompatibility 6 , 70 . RAPID ALKALINIZATION FACTOR (RALF) peptide and its receptor-like protein kinase FERONIA (FER) have been reported to play important roles in water transport from papilla cell to pollen during post-pollination processes in Brassicaceae 65 , 71 . In A. thaliana roots, RALF-FER signaling suppresses AHA activity probably via Ser-899 phosphorylation (Fig. S2) and induces alkalinization of the apoplast 72 , 73 . Therefore, it was expected that there are some relationships between RALF-FER signal and AHA activity during post-pollination processes in self-incompatibility. Some A. thaliana AHA members, including AHA1, 2, 5, and 10, retained a Ser residue at position 899, whereas AHA3 retained a Gly residue 39 . Similar to the AtAHA3, two AHAs predominantly expressed in the stigma of B. rapa lack the conserved Ser-899 and is instead Gly (Fig. S2). Among BrAHAs, only BrAHA2d and BrAHA2e possess a Ser residue corresponding to Ser-899, but these genes were not as strongly expressed in papilla cells (Fig. 1 b, S1). In addition, RALF peptides are perceived not only by FER but also by other receptor-kinases of the CrRLK family. One member of the CrRLK family, ERULUS, has been shown to be involved in phosphorylation of Ser-904 in AtAHA1 and AtAHA2 74 , and phosphomimic of Ser-904 enhances AHA activity in yeast cells 58 . Therefore, FER signaling may not suppress AHA activity in B. rapa papilla cells, and the regulation of AHA activity by RALF peptides may be diverse and differ between cells and tissues. Unlike its action in Arabidopsis roots, RALF peptides may even promote AHA activity in B. rapa stigmas by binding with an unknown member of the CrRLK family. In summary, regulation of AHA activity in papilla cells is involved in water transport to pollen for pollen hydration in Brassicaceae plants. In self-incompatible plants of Brassicaceae, pollination with self-pollen increased AHA activity in the stigma. The AHA activity enhances the osmotic pressure and water retention in the papilla cells and inhibits water release to pollen. Our work provides new insights into the molecular mechanisms underlying post-pollination processes. Materials and methods Plant material and growth conditions In this study, S homozygotes ( S 8 , S 9 , and S 12 ) and S heterozygote (cv. Gokurakuten; unknown S haplotypes) were used as self-incompatible strains of Brassica rapa 11 and the cv. yellow sarson C634 was used as a self-compatible strain 53 . Seeds were germinated on water-wetted filter paper, and seedlings were transplanted to soil in pots. All B. rapa plants were grown in a greenhouse at Tohoku University. Flowers that opened on the day of the experiment were used and emasculated just before the experiments. Self- and cross-pollination were performed by hand. Arabidopsis thaliana ecotype Col-0 was used as a wild type (WT). T-DNA insertion mutant lines, aha1-9 (SAIL_1285_D12) and aha2-5 (SALK_022010), were obtained from the Arabidopsis Biological Resource Centre. Homozygotes of both mutants had been isolated and used in a previous study 43 , 75 . We used ost2-2D as a constitutive active mutant of AtAHA1 64 . Arabidopsis plants were grown in soil in a growth room, under white light emitting photodiode (200 µmol m − 2 s − 1 ) with a 16-h/8-h light/dark cycle. The growth temperature was approximately 22°C, and the relative humidity was 40–60%. Flowers from Arabidopsis plants grown for 4–5 weeks were emasculated and hand-pollinated to observe plasma membrane H + -ATPase phosphorylation and activity, pollen hydration, and pollen tube elongation (Fig. 6 , S4, S6 to S8). Nicotiana benthamiana plants were grown in soil in a growth chamber, under a white light fluorescent lamp (90 µmol m − 2 s − 1 ) with a 16-h/8-h light/dark cycle. The growth temperature was approximately 28°C, and the relative humidity was 40–60%. For the GFP-fused proteins transient expression assay, 3-week-old plants were used 76 . Construction of the phylogenetic tree The amino acid sequences of AtAHA and BrAHA homologs were obtained from TAIR10 and Brapa_1.0. Full-length amino acid sequences were aligned using MAFFT software ( https://mafft.cbrc.jp/alignment/software/ ). The phylogenetic tree was constructed via the Neighbor-Joining method 77 using full-length amino acid sequences of AHA homolog proteins. Gene expression analysis of BrAHAs in B. rapa stigmas by reverse transcription-quantitative PCR (RT-qPCR) Total RNAs were extracted from five stigmas of S 9 or S 8 haplotype using the RNeasy RNA plant Mini kit (Qiagen). First-strand cDNAs were synthesized from the total RNAs using PrimeScript IV 1st strand cDNA Synthesis Kit (TaKaRa). RT-qPCR was performed by Bio-Rad CFX Connect using SsoFast EvaGreen Supermix (Bio-Rad). Melting curves were generated using specific primers (Table S2). Expression of each gene was quantified with Bio-Rad CFX Maestro 2.3 and normalized to ubiquitin-conjugating enzyme 21 78 . Subcellular localization of BrAHA proteins in Nicotiana benthamiana leaves GFP-BrAHA1a, GFP-BrAHA2c, and AtPIP2A-mCherry under control of the 35S promoter were transiently expressed in leaves of Nicotiana benthamiana . PIP2A-mCherry was used as a localization marker for the plasma membrane. The full-length B. rapa and A. thaliana cDNA of each gene was amplified by PCR using the specific primers (Table S1 ). The amplified fragments were inserted downstream of the 35S promoter in pRI101-AN DNA vector (TaKaRa) for GFP - BrAHA1a and GFP - BrAHA2c and of pCAMBIA1302 (CAMBIA) for AtPIP2A - mCherry using the In-Fusion cloning system (Clontech). The resulting construct was introduced into N. benthamiana leaves using an Agrobacterium -mediated transformation method 76 . Agrobacterium GV3101 strain was transformed with the vectors and cultured at 28°C for about 20 h. The agrobacteria were collected and resuspended in infection buffer including 10 mM Mes-KOH (pH 5.6) and 10 mM MgCl 2 . GFP and mCherry fluorescent signals from epidermal cells were observed 4 days after infiltration using a confocal laser microscope LSM 710 (Zeiss). GFP fluorescence was detected at 488 nm excitation and 493–550 nm emission. mCherry fluorescence was detected at 561 nm excitation and 593–650 nm emission. Chemical treatment of pistils Pistils were excised at a length of 4 mm from the stigma apex of opened flowers of B. rapa , and the pistils were placed on 1% agar medium containing fusicoccin (FC) or sodium orthovanadate (vanadate) with the cut-end buried in the medium and left at room temperature for 3 h. After chemical treatment, the stigmas were self- or cross-pollinated and left for 1 h on the chemical-containing medium. The stigmas were then transferred to plant agar medium without chemicals and incubated overnight at room temperature. The treatments of A. thaliana pistils were performed similarly to those for B. rapa with minor modifications. The opened flowers were cut at the inflorescence stems and emasculated. The tips of the inflorescence stem containing pistils were treated with chemicals as above. Six hours after the start of treatment, pollen was placed on the stigma. After 2 days, the pistil samples were transferred to 1% agar medium without chemicals and incubated overnight for elongation of the pollen tube. Apoplast pH measurements in B. rapa stigmas Acidification of media around stigmas was monitored using a pH indicator dye, bromocresol purple. Four pistils of B. rapa cv. Gokurakuten were excised from the pedicel and placed with the stigma in contact with the solid medium (0.008% bromocresol purple, 1% low-melting-point agarose adjusted to pH 7.0). Samples were incubated at room temperature for 30 min and then images were taken. Observation of pollen and pollen tube behavior on the stigma Pistils of B. rapa and A. thaliana were placed on 1% agar medium and pollinated. In the case of Figs. 3 and 4 , in self-pollination, pollen from the S 9 strain was pollinated onto the stigma of the S 9 strain (Self-pollination), and in cross-pollination, pollen from the S 8 strain was placed on the stigma of the S 9 strain (Cross-pollination). After the pollination, at each time point pistils were fixed in 3:1 ethanol: acetic acid for at least 30 min. The pistils were washed 3 times with distilled water and hydrolyzed in 1N NaOH for 1 h at 60°C. To visualize pollen tubes, samples were stained with aniline blue solution (0.1% aniline blue, 0.1 M K 3 PO 4 ) overnight in the dark 79 , and observed by fluorescence microscopy. Pollen tube penetration was determined by whether the pollen tube had passed through the stigma-style boundary and entered the style (Fig. 3 b: left diagram). Observation of pollen hydration Pistils and stamens were dissected from freshly opened flowers, placed on a glass cover slip, and held in place with tape. Under an inverted microscope (Axio Observer, Zeiss), pollen from the stamen was placed on the papilla cells using manipulators (NM-4 and MMO-4, NARISHIGE) equipped with a glass rod. Images of pollen hydration were taken at each time point after the start of pollination. The short axis of the pollen was measured in each image by the “ellipse” mode of Fiji ImageJ software ( https://imagej.net/software/fiji/downloads ). The degree of pollen hydration was calculated as the percentage change in the short axis length from the point of pollination (0 MAP). Detection of phosphorylation and amount of AHA protein in B. rapa stigmas The amount of AHA and its phosphorylation were measured by immunoblot analysis, using previously described methods 43 , 76 with slight modifications. Pistils of B. rapa cv. Gokurakuten were excised and placed on 1% agar medium. The pistils were pollinated with self (Gokurakuten) or non-self ( S 8 strain) pollen for 30 min. The pollinated and unpollinated pistils were washed with 0.1% sodium dodecyl sulfate (SDS) in MilliQ water for 10 sec to remove pollen from the stigmas. The water was wiped off the pistils, and stigmas were excised with a razor. The stigmas were immediately frozen in liquid nitrogen and disrupted using zirconia beads. Proteins of 6 stigmas in each treatment were solubilized by adding the SDS buffer (10 mM Tris-HCl [pH 6.8], 2% SDS, 1 mM EDTA, 20% glycerol, 80 mM DTT, 0.02% bromophenol blue, 200 µM sodium fluoride, 5 µM phenylmethylsulfonyl fluoride, 20 µM leupeptin). The solubilized samples were centrifuged at 13,000 rpm for 5 min at 4 o C. The supernatant was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and then immunoblotting. Specific antibodies against the catalytic domain of Arabidopsis plasma membrane H + -ATPase (AtAHA) and the phosphorylated pen-Thr residue of AtAHA were used as first antibodies, as previously described 80 . Anti-actin antibody (Sigma) was also used as a first antibody. Goat anti-rabbit or mouse IgG-horseradish peroxidase conjugate (Bio-Rad) was used as a secondary antibody. The chemiluminescence signal was detected using an imaging system (ChemiDoc Touch, Bio-Rad). Signal intensity was analyzed using Fiji ImageJ software. Quantitative phosphoproteomic analysis S 12 stigmas after self-pollination or cross-pollination (pollinated with S 8 pollen) were prepared as described above for immunoblot analysis. The stigma samples (n = 70/treatment) were lysed in 150 µL of 6 M guanidine-HCl, 100 mM HEPES-NaOH, pH 7.5, 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 × g for 15 min at 4 ºC. The supernatants were recovered, and proteins (400 µg each) were purified by methanol–chloroform precipitation and solubilized in 150 µL of 0.1% RapiGest (Waters) in 50 mM triethylammonium bicarbonate. After sonication, the protein solutions were digested with 8 µg trypsin/Lys-C mix (Promega) at 37 ºC overnight. The resulting peptide solutions were acidified with TFA, centrifuged, and subjected to the High-Select Fe-NTA phosphopeptide enrichment kit (Thermo Fisher Scientific). The eluates were acidified, desalted using GL-Tip SDB (GL Sciences), evaporated in a SpeedVac concentrator, and re-dissolved in 0.1% TFA and 3% acetonitrile. LC-MS/MS analysis of the resultant peptides was performed on a nanoElute 2 coupled with a timsTOF HT mass spectrometer (Bruker). The peptides were separated on a 75-µm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Peptides were loaded onto the column at a flow rate of 0.2 µL/min starting at 3% B, which was linearly ramped to 32% B over 90 min, then raised to 95% B at 91 min, and held at that level until 101 min. The mass spectrometer was operated in parallel accumulation–serial fragmentation (PASEF) mode. The m/z range for both MS1 and MS2 spectra was 100–1700, and the ion mobility range was 0.6–1.6 V·s/cm 3 . The ramp time was 100 ms, with a duty cycle of 100%. Each acquisition cycle consisted of 10 PASEF MS2 scans. A polygon filter was applied in the m/z and ion mobility space to exclude low m/z , singly charged ions from precursor selection. The raw data were processed using the FragPipe (v22.0). Database searches were performed with the MSFragger (v4.1), employing the default parameters of the LFQ-phospho workflow against the Brassica rapa peptide database (Brapa15 pep; 41,020 entries). Carbamidomethylation of cysteine (+ 57.0215 Da) was set as a fixed modification. The following variable modifications were included: acetylation of protein N-terminus (+ 42.0106 Da); oxidation of methionine (+ 15.9949 Da); phosphorylation (+ 79.9663 Da) of serine, threonine, or tyrosine. The resulting identifications were filtered using Philosopher with default parameters (MS Booster was disabled), and IonQuant (v1.10.27) was used for quantification with default software settings. Expression and subcellular localization of AtAHAs in stigma Genomic A. thaliana AHA1 and AHA2 genes including each 5′ and 3′ noncoding sequence were amplified by PCR from genomic DNA of the wild type (Col-0) using their respective specific primers (Table S1 ) according to previous methods 45 , 75 . These amplified DNA fragments were cloned into the gene transfer vector pCAMBIA1300 (CAMBIA) by the In-Fusion cloning system at the Hin dIII/ Bam HI sites (for AHA1 gene) and Xba I site (for AHA2 gene). The cDNA of GFP was amplified and inserted into the site between the end of the 5′ noncoding sequence and the start codon of each AHA gene using In-Fusion cloning system (Table S1 ). The constructed vectors were verified by DNA sequencing. The resulting vectors were used for transformation of Agrobacterium tumefaciens GV3101 strain and introduced into A. thaliana WT (Col-0) using the floral dip method 81 . Transgenic plants were selected by resistance to hygromycin and used for analysis. The pistils were emasculated in the evening of the day before observation. Emasculated pistils were placed on glass-bottom dishes, and a 1% plant agar medium block was placed on top of the pistil to bring the papilla cells in contact with the surface of the glass-bottom dish. GFP fluorescence (excitation 488 nm/emission 483–530 nm) signal derived from the papilla cells was observed with a confocal microscope LSM 710 (Zeiss). Generation of aha11 mutants by genome editing To generate the aha11 mutants by genome editing, we constructed the pKAMA-ITACHI(pKI)1.1R vector bearing the CRISPR/Cas9 82 . The single guide RNA (sgRNA) for AHA11 was designed by CRISPR-P v2.0 ( http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR ) (Table S1 ). The hybridized sgRNA oligo DNA was inserted into the Aar I site of the pKI1.1R vector, and the constructed plasmid was verified by DNA sequencing. The resulting vector was introduced into A. tumefaciens GV3101 strain and the bacteria was used to transform A. thaliana WT (Col-0) using the floral dip method. T 1 generation of the transgenic plants were selected based on hygromycin resistance and aha11 mutation. T 2 seeds that showed no RFP fluorescence were selected to remove the CRISPR/Cas9 cassette, and homozygous aha11 mutation was confirmed by DNA sequencing. The resulting mutant lines were used for the phenotypic analysis. Accession numbers AHA1 (At2g18960), AHA2 (At4g30190), AHA3 (At5g57350), AHA4 (At3g47950), AHA5 (At2g24520), AHA6 (At2g07560), AHA7 (At3g60330), AHA8 (At3g24640), AHA9 (At1g80660), AHA10 (At1g17260), AHA11 (At5g62670), UBC21 (At3g25760), PIP2A (At3g53420) Declarations CONFLICT OF INTEREST The authors declare no conflict of interest. Funding This work was supported, in part, by JSPS KAKENHI (22K14867 and 24K17874 to M. H., 23K18058 to M. W., 22K05581 to Y. T.) and by MEXT KAKENHI (22H05172 and 22H05179 to M.W.). Author Contribution M.H. and M.W. conceived and designed the experiments. M.H., K.F., H.-M.S., S.I. and H.K. performed the experiments. M.H., S.I., T.K., Y.T., H.K., and M.W. analyzed the data. M.H. and M.W. drafted the manuscript. All authors edited the manuscript. Acknowledgement We thank Dr. Nathalie Leonhardt (University of Aix Marseille, CEA, CNRS, BIAM, UMR7265, SAVE, Saint Paul-Lez-Durance) for kindly providing ost2-2D seeds, Dr. Hiroki Tsutsui and Dr. Tetsuya Higashiyama (University of Tokyo) for providing pKAMA-ITACHI vector, and Dr. Kohei Nishino (Tokushima University) for his technical assistance in phosphoproteomic analysis. We also thank Dr. Seiji Takayama and Dr. Sota Fujii (The University of Tokyo) for helpful comments, and Kana Ito, Sadayoshi Ogata, Tai Takemoto, Toko Kanomata, Yuta Takahashi, and Temari Endo (Tohoku University) for technical assistance. 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The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4 , 406–425 (1987). Osaka, M. et al. Cell type-specific transcriptome of Brassicaceae stigmatic papilla cells from a combination of laser microdissection and RNA sequencing. Plant Cell Physiol. 54 , 1894–1906 (2013). Wong, K. C., Watanabe, M. & Hinata, K. Fluorescence and scanning electron microscopic study on self-incompatibility in distylous Averrhoa carambola L. Sex. Plant Reprod. 7 , (1994). Hayashi, Y. et al. Biochemical characterization of in vitro phosphorylation and dephosphorylation of the plasma membrane H+-ATPase. Plant Cell Physiol. 51 , 1186–1196 (2010). Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium ‐mediated transformation of Arabidopsis thaliana : Floral dip transformation of Arabidopsis. Plant J. 16 , 735–743 (1998). Tsutsui, H. & Higashiyama, T. PKAMA-ITACHI vectors for highly efficient CRISPR/Cas9-mediated gene knockout in Arabidopsis thaliana. Plant Cell Physiol. 58 , 46–56 (2017). Okuda, S. et al. jPOST environment accelerates the reuse and reanalysis of public proteome mass spectrometry data. Nucleic Acids Res. 53 , D462–D467 (2025). Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6981590","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":484180464,"identity":"67679990-8358-4e06-b621-20d1965d0204","order_by":0,"name":"Maki Hayashi","email":"data:image/png;base64,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","orcid":"","institution":"Tohoku University","correspondingAuthor":true,"prefix":"","firstName":"Maki","middleName":"","lastName":"Hayashi","suffix":""},{"id":484180469,"identity":"f62b0ca0-6677-4bdf-8f3f-06fc2fddaf7d","order_by":1,"name":"Kazuki Fukushima","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Kazuki","middleName":"","lastName":"Fukushima","suffix":""},{"id":484180471,"identity":"1e7042ce-5eb0-407c-bf0f-7e1be4962b15","order_by":2,"name":"Hiromi Masuko-Suzuki","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Hiromi","middleName":"","lastName":"Masuko-Suzuki","suffix":""},{"id":484180472,"identity":"e9138e0e-0e83-482b-a5ff-414dcc22313c","order_by":3,"name":"Shin-ichiro Inoue","email":"","orcid":"","institution":"Saitama University","correspondingAuthor":false,"prefix":"","firstName":"Shin-ichiro","middleName":"","lastName":"Inoue","suffix":""},{"id":484180473,"identity":"05046abc-cafa-4d26-942c-f13bf40d192f","order_by":4,"name":"Toshinori Kinoshita","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Toshinori","middleName":"","lastName":"Kinoshita","suffix":""},{"id":484180474,"identity":"7a90a1ec-b9c0-4f58-a0fb-195707a00650","order_by":5,"name":"Yoshinobu Takada","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Yoshinobu","middleName":"","lastName":"Takada","suffix":""},{"id":484180477,"identity":"470657b6-dc8c-4d62-8bc4-7c82d559331b","order_by":6,"name":"Hidetaka Kosako","email":"","orcid":"","institution":"Tokushima University","correspondingAuthor":false,"prefix":"","firstName":"Hidetaka","middleName":"","lastName":"Kosako","suffix":""},{"id":484180483,"identity":"b4457ea5-a44d-43a2-84b0-1d9fe4223921","order_by":7,"name":"Masao Watanabe","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Masao","middleName":"","lastName":"Watanabe","suffix":""}],"badges":[],"createdAt":"2025-06-26 09:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6981590/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6981590/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86704224,"identity":"90bfa8ec-c50c-47d6-9262-4c88d31d89c9","added_by":"auto","created_at":"2025-07-14 16:57:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":878781,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBrAHAs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expressed in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBrassica rapa \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003estigmas.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Phylogenetic relationships of AHA proteins from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eBrassica rapa.\u003c/em\u003e Full-length amino acid sequences of AHA proteins were used to construct the tree. The alignment and tree were constructed using MAFFT software with the neighbor-joining method. \u003cem\u003eB. rapa\u003c/em\u003e AHAs are indicated in blue and shown as geneID names in Brara Chiifu V1.5. Scale bar = 0.1 substitutions per site.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Expression of \u003cem\u003eBrAHA\u003c/em\u003e genes in \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e9\u003c/em\u003e\u003c/sup\u003e stigmas by reverse transcription-quantitative PCR (RT-qPCR). Expression of each\u003cem\u003e AHA \u003c/em\u003egene relative to that of \u003cem\u003eubiquitin-conjugating enzyme 21 \u003c/em\u003e(\u003cem\u003eUBC21\u003c/em\u003e) is shown. Data are mean ± SD (n = 3).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Intracellular localization of GFP-BrAHA proteins. GFP-BrAHA1a and GFP-BrAHA2c were co-expressed with PIP2A-mCherry in leaf epidermal cells of\u003cem\u003e Nicotiana benthamiana\u003c/em\u003e. Fluorescence images were obtained using a confocal laser microscope. PIP2A-mCherry was used as a localization marker for the plasma membrane. Scale bars indicate 20 μm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6981590/v1/f50b5618943d4210a69c073a.png"},{"id":86705890,"identity":"2cb221c0-36bb-4e16-b88a-79efff8fe5d3","added_by":"auto","created_at":"2025-07-14 17:13:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":410440,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of the chemicals on plasma membrane (PM) H\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-ATPase phosphorylation and activity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. rapa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e stigmas.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Phosphorylation of the penultimate Thr (pen-Thr) residue of the stigmatic PM H\u003csup\u003e+\u003c/sup\u003e-ATPase in response to FC. Pistils were treated with the PM H\u003csup\u003e+\u003c/sup\u003e-ATPase activator FC (10 µM) for 4 h, and proteins were extracted from the stigmas and used for SDS-polyacrylamide gel electrophoresis (PAGE). Immunoblotting was then performed using antibodies against the phosphorylated pen-Thr residue of PM H\u003csup\u003e+\u003c/sup\u003e-ATPase (phosphorylated pen-Thr), total AHA proteins (AHA), and actin (Actin).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003ec\u003c/strong\u003e) Effects of FC (\u003cstrong\u003eb\u003c/strong\u003e) and vanadate (\u003cstrong\u003ec\u003c/strong\u003e) on \u003cem\u003eB. rapa\u003c/em\u003e stigma AHA activity. Stigmas were cut from the pistils treated with FC or vanadate (1 mM) as described in (\u003cstrong\u003ea\u003c/strong\u003e). Media acidification around the stigmatic apoplast was monitored using a pH indicator dye, bromocresol purple. Experiments repeated on three occasions gave similar results. The color change reflects a change in media pH from 7 to 5. “Mock” indicates the corresponding solvent control.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6981590/v1/12ac90309b8e4c0ae2680265.png"},{"id":86705011,"identity":"567b1751-94bb-4672-a74a-d52dbdda37a4","added_by":"auto","created_at":"2025-07-14 17:05:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1586540,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of stigma PM H\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-ATPase activity on pollen tube penetration into stigmas of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. rapa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Pollen tube penetration into the pistil of a self-incompatible \u003cem\u003eB. rapa S\u003c/em\u003e\u003csup\u003e\u003cem\u003e9\u003c/em\u003e\u003c/sup\u003e strain. Stigmas were treated with FC (10 µM) or vanadate (1 mM), as described in Fig. 2. “Mock” indicates the corresponding solvent control. The strains used in the crossing were indicated to the left of the panels. White arrowheads indicate the positions of the tip of the pollen tubes that penetrated into the pistil. Scale bars indicate 200 µm.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Number of pistils penetrated/not penetrated by the pollen tube.\u003c/p\u003e\n\u003cp\u003e“Penetrated” or “Not penetrated” pistil was determined based on whether the pollen tube had passed through the stigma-style boundary and entered the style (left diagram). The dashed line in the diagram indicates the position of stigma-style boundary. Stacked bar graphs show the number of pistils penetrated/not penetrated by the pollen tube; n=18. (\u003cstrong\u003ec\u003c/strong\u003e) Pollen tube penetration into the pistil of a self-compatible \u003cem\u003eB. rapa \u003c/em\u003estrain, \u003cem\u003eyellow sarson\u003c/em\u003e. The stigmas were treated with chemicals as in Fig. 2 and then pollinated with self-pollen. Stacked bar graphs show the number of pistils penetrated/not penetrated by the pollen tube; n=12. White arrowheads and graph were similarly shown as in (\u003cstrong\u003ea\u003c/strong\u003e) and (\u003cstrong\u003eb\u003c/strong\u003e). Scale bars indicate 200 µm.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6981590/v1/5753ccf8146778a22fb72551.png"},{"id":86704227,"identity":"76fca0c3-36b5-4a38-be3e-675fb9f9339f","added_by":"auto","created_at":"2025-07-14 16:57:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":496121,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of stigma PM H\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-ATPase activity on pollen hydration of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. rapa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003eb\u003c/strong\u003e) Pollen hydration on vanadate-treated stigmas in self-pollination (\u003cstrong\u003ea\u003c/strong\u003e) and FC-treated stigmas in cross-pollination (\u003cstrong\u003eb\u003c/strong\u003e). Chemical treatments were performed as in Fig. 2. MAP indicates minutes after pollination. Scale bars indicate 20 µm. The graphs show the time course of pollen hydration for 1 h after pollination. Pollen hydration was measured as the rate of expansion of pollen every 10 min, and shown as percentage values relative to that at 0 min. Data are mean ± SE (Mock, FC; n = 5, Vanadate; n =7). Asterisks show statistically significant differences between each chemical treatment and the corresponding mock treatment using Student’s \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003e*p\u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**p\u003c/em\u003e\u0026lt; 0.01, \u003cem\u003e***p\u003c/em\u003e\u0026lt; 0.001; ns, not significance).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6981590/v1/9e40aded269225210639bcbb.png"},{"id":86703532,"identity":"273d398a-9e67-4b25-8a14-3d43b815af0e","added_by":"auto","created_at":"2025-07-14 16:49:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":166242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhosphorylation status of the stigmatic BrAHAs in response to self- and cross-pollination.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePistils of a self-incompatible\u003cem\u003e B. rapa\u003c/em\u003e were unpollinated (UP), crossed with the same strain pollen (Self), or crossed with the non-self-strain pollen (Cross). After the pollination, proteins were extracted from the stigmas, and subjected to SDS-PAGE. Immunoblottings of phosphorylated penultimate Thr (pen-Thr) of PM H\u003csup\u003e+\u003c/sup\u003e-ATPase (phosphorylated pen-Thr) and total AHA proteins (AHA) were performed. The intensities of BrAHA phosphorylation (pen-Thr/AHA) are shown as relative values to that of UP stigmas. Data were averaged from five independent experiments. Error bars indicate SD. Asterisks indicate statistically significant differences using Tukey’s multiple comparisons test (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; ns, not significance).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6981590/v1/f54139e2fe62b33c87906b9b.png"},{"id":86703534,"identity":"3c8d110d-9313-44bb-a897-47b704ef27d8","added_by":"auto","created_at":"2025-07-14 16:49:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":426275,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePollen tube penetration and pollen hydration on the stigma of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eost2-2D\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant with a constitutively active PM H\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-ATPase isoform.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) WT pollen tube penetration into WT and \u003cem\u003eost2-2D\u003c/em\u003e pistils. White arrowheads and graph are similarly indicated as in Fig. 3. Scale bars indicate 200 µm. Stacked bar graphs show the number of pistils penetrated/not penetrated by the pollen tube (n=11).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Time course of WT pollen hydration on WT and \u003cem\u003eost2-2D\u003c/em\u003e stigmas. Stigmas of WT and \u003cem\u003eost2-2D\u003c/em\u003e were pollinated with pollen from the WT. Pollen hydration was measured every 1 min for 15 min after pollination (MAP) and shown as percentage pollen hydration, as in Fig. 3. Data are mean ± SE (n = 19). Asterisk shows statistically significant differences between WT and\u003cem\u003e ost2-2D\u003c/em\u003e pistils using Student’s \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003e*p\u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**p\u003c/em\u003e\u0026lt; 0.01). The photographs show the hydration of pollen at 0 and 10 MAP.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6981590/v1/7ce8fef4ba15a65dbaff9d49.png"},{"id":86703535,"identity":"72a2a6dc-0e1a-4d79-80f4-8e946008772c","added_by":"auto","created_at":"2025-07-14 16:49:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":380548,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic presentation of a suggested model of AHA-mediated osmoregulation in papilla cells for self-incompatibility in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. rapa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(left) When self-pollen attaches to the papilla cells, papilla cell AHA is activated through pen-Thr phosphorylation after recognition of self-pollen. Activated AHA promotes various secondary transports and accumulates osmolytes, thereby increasing the osmotic pressure in the papilla cells. These papilla cells have high water retention capacity, so that water is not transferred to the pollen. (right) When non-self-pollen attaches to the papilla cells, the AHA activation in the papilla cells does not occur. In this case, secondary transports and accumulation of osmolytes are not promoted, and the osmotic pressure of the papilla cells remains at the level before pollination. As a result, water is transferred to the pollen.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6981590/v1/7b6196f2f6ff95509db0c0de.png"},{"id":90510804,"identity":"ec568537-1af4-49a8-8b8a-995b610e9fd4","added_by":"auto","created_at":"2025-09-03 13:32:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6161456,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6981590/v1/f1ffd3e6-2285-48a6-be5e-edf2e3834552.pdf"},{"id":86703527,"identity":"1ce7fceb-9859-4559-9a66-ed0c7a8e7d2d","added_by":"auto","created_at":"2025-07-14 16:49:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1718436,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6981590/v1/dfd3d270e11981953d34e397.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Stigma plasma membrane H + -ATPase is involved in pollen hydration and pollen tube penetration in Brassicaceae self-incompatibility","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMost angiosperms have hermaphrodite flowers, with a female pistil and plural male stamens in the same flower \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The mature pollen from the anthers attaches to the stigmatic papilla cells of the pistil, where it absorbs water and hydrates \u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The hydrated pollen germinates a pollen tube on the papilla cells and the pollen tubes elongate and penetrate into the pistil to carry the sperm cells to the ovules, achieving fertilization \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The transport of water from the papilla cells to the pollen is a primary step important for the post-pollination processes including pollen hydration, pollen tube germination, pollen tube penetration into the stigma, and pollen tube elongation in the pistil. However, the molecular mechanisms underlying water transport from female to male cells have not been fully elucidated.\u003c/p\u003e\u003cp\u003eApproximately half of angiosperms exhibit self-incompatibility, rejecting fertilization by self-pollen and thus avoiding inbreeding depression within a species \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In Brassicaceae plants showing self-incompatibility, papilla cells block water transport to the self-pollen, suggesting that this water transport is not passive but is selectively controlled by the papilla cells \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Self-incompatibility in Brassicaceae species is controlled by the multi-allelic genes of the \u003cem\u003eS\u003c/em\u003e locus, including \u003cem\u003eS\u003c/em\u003e locus protein 11 (SP11)/\u003cem\u003eS\u003c/em\u003e locus cysteine-rich protein (SCR) and \u003cem\u003eS\u003c/em\u003e receptor kinase (SRK), the male and female determinate factors, respectively \u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The female factor SRK is expressed and localized in the plasma membrane of papilla cells, and the male factor SP11 is expressed in the anther tapetum and secreted onto the pollen surface \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. When SP11 and SRK have the same \u003cem\u003eS\u003c/em\u003e haplotype, these proteins bind \u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Using this receptor-ligand binding system, papilla cells recognize and reject self-pollen through suppression of post-pollination processes. Thus, signaling from SRK inhibits selective water transport from the papilla cells to the pollen \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSome intracellular processes have been reported regarding the water transport from papilla cells to pollen in Brassicaceae plants. Vacuole-derived vesicles/ multivesicular bodies (MVBs) appear in the cytosol of papilla cells near the pollen attachment site only in cases of compatible pollination \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In the self-incompatible \u003cem\u003eArabidopsis lyrata\u003c/em\u003e and \u003cem\u003eBrassica napus\u003c/em\u003e, self-pollination induces autophagic degradation of the vesicles/MVBs in papilla cells, and this process is required for the rejection of self-pollen on the stigma \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. These results suggest that vesicles/MVBs contribute to pollen hydration, probably by secreting water-laden vesicles from papilla cells to pollen. On the other hand, it was shown in the pistil of \u003cem\u003eA. thaliana\u003c/em\u003e that plasma membrane aquaporin PIP1;2 and ER-bound aquaporin SIP1;1 coordinately contribute to pollen hydration by mediating the direct water efflux from the papilla cells \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Thus, some independent phenomena in the papilla cells have been shown to play important roles in the compatible pollen hydration; however, the relationship between these phenomena remains unclear.\u003c/p\u003e\u003cp\u003ePlant cells often control the direction of water transport across the plasma membrane through regulation of intracellular osmotic pressure by ion transports, which is utilized for some physiological responses. For example, in hypocotyl cells during elongation growth and in guard cells during stomatal opening, the phytohormone auxin and light increase the osmotic pressure of the cells, promoting water uptake and cell expansion, respectively \u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Conversely, in guard cells during stomatal closure and in pulvinar motor cells of legumes during dia-heliotropic leaf movement, the phytohormone abscisic acid (ABA) and light decrease the osmotic pressure of the cells, promoting water excretion and cell shrinkage, respectively \u003csup\u003e\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. It is well established that an autoinhibited plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPase (AHA) plays a key role in these responses through osmoregulation in the cells \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. AHA is the primary transporter in plant cells and uses the ATP hydrolysis energy to pump H\u003csup\u003e+\u003c/sup\u003e out of the cell, thereby generating the driving force for various secondary transports on the plasma membrane \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. For example, AHA in guard cells is activated by light and increase the osmotic pressure of guard cells to open stomata. Conversely, during stomatal closure, ABA signaling inactivates AHA, decreasing the osmotic pressure in guard cells \u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Thus, cellular osmoregulation controlled by AHA activity is crucial for the direction of water transport across plasma membrane in plant cells. From these observations, we hypothesized that osmoregulation by AHA is also involved in the regulation of water transport from the papilla cells to the pollen in Brassicaceae species.\u003c/p\u003e\u003cp\u003eIn this study, to identify novel regulatory intracellular events in post-pollination processes, we examined the contribution of AHA in the stigma to pollen hydration, pollen tube penetration into the stigma, and pollen tube elongation in the pistil in Brassicaceae plants. Pharmacological and physiological experiments using self-incompatible \u003cem\u003eB. rapa\u003c/em\u003e and self-compatible \u003cem\u003eA. thaliana\u003c/em\u003e provided new insights into post-pollination processes in Brassicaceae plants, in which AHA regulates osmotic pressure in stigma cells and is involved in the selective water transport from papilla cells to pollen.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eAutoinhibited plasma membrane H\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e-ATPases expression in\u003c/b\u003e \u003cb\u003eBrassica rapa\u003c/b\u003e \u003cb\u003estigmas\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo verify the function of plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPase in papilla cells, we first annotated genes of the autoinhibited plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPases (AHAs) in self-incompatible \u003cem\u003eBrassica rapa\u003c/em\u003e genome, and termed these BrAHAs. Based on a blast search using the full-length amino acid sequences of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e AHAs (AtAHAs) as the query, we found that the \u003cem\u003eB. rapa\u003c/em\u003e genome contains 18 homologous \u003cem\u003eBrAHA\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). To examine the expression of \u003cem\u003eBrAHAs\u003c/em\u003e in the stigma, RNA was extracted from the stigma of the \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e strain of self-incompatible \u003cem\u003eB. rapa\u003c/em\u003e, and RT-qPCR was performed. Among the 18 \u003cem\u003eBrAHA\u003c/em\u003e genes, two \u003cem\u003eBrAHAs\u003c/em\u003e, \u003cem\u003eBrAHA1a\u003c/em\u003e (\u003cem\u003eBra038835\u003c/em\u003e) and \u003cem\u003eBrAHA2c\u003c/em\u003e (\u003cem\u003eBra011172\u003c/em\u003e), were strongly expressed in the stigma (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). A similar expression profile was obtained using stigma of another strain of self-incompatible \u003cem\u003eB. rapa\u003c/em\u003e, that is \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The BrAHA proteins (BrAHA1a and BrAHA2c) are encoded by two genes belonged to the same clade as AtAHA1 and AtAHA2, which are known to be the main isoforms in \u003cem\u003eA. thaliana\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we cloned the coding sequences of these genes, and compared the deduced amino acid sequences with AtAHA1 and AtAHA2. AHA proteins in seed plants possess a penultimate Thr (pen-Thr) residue, region I, and region II in the C-terminal region that are important for the autoinhibitory effects of H\u003csup\u003e+\u003c/sup\u003e-ATPase \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, and all of these are highly conserved in BrAHA1a and BrAHA2c (Fig. S2). We then investigated the intracellular localization of the two BrAHAs by transient expression assay of GFP-fused proteins with a plasma membrane marker (PIP2A-mCherry) in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. The result showed that the fluorescence signals from GFP-BrAHA1a and GFP-BrAHA2c were well merged with the fluorescent signal from PIP2A-mCherry in the plasma membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). These results indicate that BrAHA1a and BrAHA2c are plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPases highly expressed in the stigma.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInvolvement of stigma AHA in pollen tube penetration into the stigma of\u003c/b\u003e \u003cb\u003eB. rapa\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate whether AHA activity in the stigma is important for post-pollination processes in \u003cem\u003eB. rapa\u003c/em\u003e, we observed pollen tube penetration into stigma treated with the AHA activator fusicoccin (FC) or the AHA inhibitor vanadate. AHA is activated by phosphorylation of the pen-Thr residue in the C-terminal YTV motif and subsequent binding of 14-3-3 proteins to the phosphorylated YTV motif (Fig. S2). FC activates AHAs by stabilizing the physical interaction between the C-terminal phosphorylated motif and 14-3-3 proteins \u003csup\u003e\u003cspan additionalcitationids=\"CR48 CR49 CR50\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. The stigmas of \u003cem\u003eB. rapa\u003c/em\u003e were treated with FC for 3 h, and activation of stigma AHAs was tested by immunoblotting using a specific antibody recognizing phosphorylated pen-Thr. We confirmed that the phosphorylation level of pen-Thr in AHA of stigmas was strongly increased by FC compared with the solvent control (Mock) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). We further confirmed the effect of FC treatment by detecting the change in apoplast pH in the stigma using the pH indicator bromocresol purple (BCP). FC-treated stigmas extruded more H\u003csup\u003e+\u003c/sup\u003e, and changed the color of the medium containing BCP, indicating that it was more acidic compared to mock treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). On the other hand, vanadate binds to the E2 form of AHA, and inhibits its activity independently of the pen-Thr phosphorylation \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Thus, the effect of vanadate treatment was confirmed only by the pH change in the stigma apoplast. As expected, vanadate-treated stigmas showed less color change in the BCP medium resulting from apoplast acidification compared to mock treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). From these results, we confirmed that treatment with FC or vanadate had the expected effect on AHA activity in the stigmas of \u003cem\u003eB. rapa\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo dissect the relationship between AHAs and post-pollination processes, we next investigated the effects of FC and vanadate on self-incompatibility in \u003cem\u003eB. rapa\u003c/em\u003e. Stigmas of the self-incompatible \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e strain were treated with FC or vanadate for 3 h before pollination, and then pollinated with pollen from the \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e strain (self-pollination) or \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e strain (cross-pollination). In the \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e stigmas treated with the solvent only (mock), self-pollen and its pollen tubes showed the typical responses of self-incompatibility, including inhibition of pollen adhesion, pollen hydration, pollen tube germination, pollen tube penetration into stigma, and pollen tube elongation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In contrast, when the \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e stigmas were cross-pollinated with \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e pollen, the pollen attached to the stigma germinated pollen tubes, and the pollen tubes penetrated into the stigma and elongated to the style. When \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e stigmas were treated with FC, pollen tube penetration into the stigma was strongly inhibited not only in self-pollination but also in cross-pollination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Furthermore, when \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e stigmas were treated with vanadate, pollen tube penetration was observed in self-pollination as well as in cross-pollination. To quantify these results, we counted the number of pistils that were penetrated and not penetrated by the pollen tube, based on whether the pollen tube had passed through the stigma-style boundary and entered the style (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb: left diagram). FC treatment reduced the ratio of \u0026ldquo;Penetrated/Not penetrate\u0026rdquo; pistils in cross-pollination, and vanadate increased the ratio of \u0026ldquo;Penetrated/Not penetrate\u0026rdquo; pistils in self-pollination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Thus, our results indicate that FC inhibited pollen tube penetration into the style in compatible pollination, whereas vanadate induced pollen tube penetration in incompatible pollination. The effects of FC and vanadate were similarly observed in stigmas of another self-incompatible strain, \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e in \u003cem\u003eB. rapa\u003c/em\u003e (Fig. S3).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next tested the effects of FC and vanadate on the pollen tube penetration using a self-compatible \u003cem\u003eB. rapa yellow sarson\u003c/em\u003e C634 strain. In this strain, FC inhibited the penetration of self-pollen tubes into the stigma, whereas vanadate did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The self-compatibility in \u003cem\u003eyellow sarson\u003c/em\u003e results from abnormal transcription of \u003cem\u003eSRK\u003c/em\u003e and \u003cem\u003eSP11\u003c/em\u003e genes and disruption of the membrane-anchored protein kinase, \u003cem\u003eM\u003c/em\u003e locus protein kinase (MLPK), which interacts with SRK \u003csup\u003e\u003cspan additionalcitationids=\"CR54 CR55 CR56\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. These results indicate that AHA activity in the stigma is involved in post-pollination processes in \u003cem\u003eB. rapa\u003c/em\u003e and suggest that its involvement is independent, or downstream, of self/non-self-discrimination by the self-incompatibility system in \u003cem\u003eB. rapa\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInvolvement of stigma AHA in pollen hydration in self-incompatible\u003c/b\u003e \u003cb\u003eB. rapa\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe results of self/non-self-discrimination in \u003cem\u003eBrassica\u003c/em\u003e species self-incompatibility is manifested as failure or success of pollen hydration on the papilla cells \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. We next tested by pharmacological experiments whether AHA activity in papilla cells is important for water supply to pollen during pollen hydration in \u003cem\u003eB. rapa\u003c/em\u003e with observations using a time-lapse system. In the solvent control (mock), pollen from the \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e strain (self-pollination) showed little size change within 60 min after pollination (MAP) on the \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e papilla cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), whereas pollen from the \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e strain (cross-pollination) rapidly expanded within 20 MAP onto the papilla cells of the \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e strain, and pollen tubes germinated at 30 to 60 MAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In contrast, vanadate treatment of papilla cells induced pollen expansion within 20 MAP and pollen tube germination at 60 MAP in the self-pollination (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea: vanadate), and FC treatment of papilla cells showed a delay in pollen expansion during cross-pollination (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb: FC). Although pollen on the FC-treated papilla cells swelled to the similar extent as those on mock-treated cells at 60 MAP, little pollen tube germination was observed at that time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). These results indicate that regulation of AHA activity is involved in pollen hydration via efficient water transport from papilla cells to pollen during the post-pollination processes. This suggests that AHA-mediated active osmoregulation in papilla cells may control the water transport to pollen after self/non-self-discrimination.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eChanges in the phosphorylation status of stigma AHA in response to self- and cross-pollination in self-incompatible\u003c/b\u003e \u003cb\u003eB. rapa\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNext, to examine the activity of AHA in the stigmas of self-incompatible \u003cem\u003eB. rapa\u003c/em\u003e, the pen-Thr levels of AHA were compared after pollination with self- or non-self-pollen. Pollinated stigmas (and unpollinated control stigmas) were washed to remove pollen, and proteins extracted from the stigmas were subjected to SDS-PAGE. The phosphorylated pen-Thr levels of AHA were determined by immunoblot analysis using a specific antibody against phosphorylated pen-Thr (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Signal of pen-Thr of AHA was observed to some extent in the unpollinated stigma. In contrast, self-pollination induced phosphorylation of pen-Thr of AHA in the stigmas, increasing its phosphorylation level approximately two-fold compared to the unpollinated stigmas. The levels of phosphorylated pen-Thr did not change in cross-pollinated stigmas compared to unpollinated stigmas. These results indicate that AHA activity in the stigma is regulated via the phosphorylation level of pen-Thr in response to self- and non-self-pollen in \u003cem\u003eB. rapa\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn addition to pen-Thr, the C-terminal region of AHA contains multiple phosphorylation sites that affect AHA activity. In \u003cem\u003eA. thaliana\u003c/em\u003e, phosphorylation of pen-Thr and Thr-881 was characterized as AHA activation sites, while phosphorylation of Ser-899 and Ser-931 was characterized as inhibition sites (Fig. S2) \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan additionalcitationids=\"CR59 CR60\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. In the major AHA isoforms in \u003cem\u003eB. rapa\u003c/em\u003e stigmas, BrAHA1a and BrAHA2c, the amino acid corresponding to Ser-899 was not conserved, but the other phosphorylation sites were conserved (Fig. S2). To identify phosphorylation sites important for regulating stigma AHA activity in the inhibition of post-pollination processes, we performed quantitative phosphoproteomic analysis using self- and cross-pollinated \u003cem\u003eB. rapa\u003c/em\u003e stigmas (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Protein samples were prepared in a similar manner as for immunoblot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Only two phosphorylation sites, Thr-881 and Thr-924 were detected at the C-terminus of BrAHAs in our phosphoproteomic analysis. Although amino acid substitutions at both sites are known to affect AHA activity \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, the phosphorylation levels of these sites did not change in response to self- and non-self-pollination (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The peptide including pen-Thr was not detected in this analysis. Thus, we concluded from these results that phosphorylation of the pen-Thr of AHA may be important for regulation of stigma AHA activity in self-incompatibility of \u003cem\u003eB. rapa\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhysiological and genetic verification of the involvement of stigma AHA in post-pollination processes in\u003c/b\u003e \u003cb\u003eA. thaliana\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn both self-incompatible and self-compatible \u003cem\u003eB. rapa\u003c/em\u003e, AHA was found to be involved in the post-pollination processes in the pistils (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, S3). To verify the involvement of AHA in post-pollination processes in other Brassicaceae species, \u003cem\u003eA. thaliana\u003c/em\u003e was used in a pharmacological experiment. Because \u003cem\u003eA. thaliana\u003c/em\u003e shows self-compatibility, like the \u003cem\u003eyellow sarson\u003c/em\u003e strain of \u003cem\u003eB. rapa\u003c/em\u003e, we only examined the effect of FC and not vanadate. Pistils of \u003cem\u003eA. thaliana\u003c/em\u003e were treated with FC in a similar manner to those of \u003cem\u003eB. rapa\u003c/em\u003e, and then the pistils were pollinated to observe the effect of FC on pollen tube penetration into the stigma. In stigmas treated with the solvent only control (mock), pollen tubes penetrated into the stigma, whereas in stigmas treated with FC, pollen tubes stopped elongating on the papilla cells and did not penetrate (Fig. S4). These results suggest that AHA activity in the stigma is involved in post-pollination processes in \u003cem\u003eA. thaliana\u003c/em\u003e as in \u003cem\u003eB. rapa\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eTo identify \u003cem\u003eAHA\u003c/em\u003e genes expressed in the stigma of \u003cem\u003eA. thaliana\u003c/em\u003e, we next searched the Arabidopsis eFP Browser database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi\u003c/span\u003e\u003cspan address=\"https://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and found that \u003cem\u003eAHA1\u003c/em\u003e, \u003cem\u003eAHA2\u003c/em\u003e, and \u003cem\u003eAHA11\u003c/em\u003e are predominantly expressed in the stigma (Fig. S5a, b). In particular, \u003cem\u003eAHA1\u003c/em\u003e and \u003cem\u003eAHA2\u003c/em\u003e comprise around 47% and 27% of the total \u003cem\u003eAHA\u003c/em\u003e transcripts, respectively, and their expression accounts for approximately 74% of the expression of all \u003cem\u003eAHA\u003c/em\u003e genes in the stigma (Fig. S5c). To study the expression and subcellular localization of AHA1 and AHA2 proteins in papilla cells, we generated stable transgenic \u003cem\u003eA. thaliana\u003c/em\u003e plants expressing GFP-fused AHA1 or AHA2 proteins under the control of their respective native promoters. Fluorescence signals from GFP-AHA1 and GFP-AHA2 were clearly observed in the peripheral region of papilla cells (Fig. S5d). From these results, we concluded that AHA1 and AHA2 are the major isoforms of AHA proteins in \u003cem\u003eA. thaliana\u003c/em\u003e papilla cells, as reported in other tissues \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTo genetically verify the involvement of AHA in post-pollination processes, we used a dominant-positive mutant of \u003cem\u003eost2-2D\u003c/em\u003e that expresses constitutively active AHA1 with a G867S mutation \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Pollen from the WT was pollinated onto WT or \u003cem\u003eost2-2D\u003c/em\u003e papilla cells of pistils, and pollen tube penetration into the pistils was observed 2 and 4 h after pollination (HAP). At the time of observations, all pollen tubes penetrated the stigmas of the pistils, and there was no difference in the pollen tube penetration between the two plant lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). However, at both 2 HAP and 4 HAP, the length of pollen tubes penetrating the pistils was clearly different between WT and \u003cem\u003eost2-2D\u003c/em\u003e pistils, that is the pollen tubes in the \u003cem\u003eost2-2D\u003c/em\u003e pistils elongated less than those in the WT pistils (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, S6).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBecause pollen hydration affects pollen tube germination and elongation \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, we speculated that the difference in pollen tube length might result from differences in the speed of pollen hydration in the WT and \u003cem\u003eost2-2D\u003c/em\u003e papilla cells. We therefore compared the speed of pollen hydration after the pollination of WT pollen on WT or \u003cem\u003eost2-2D\u003c/em\u003e stigmas. WT pollen on the WT papilla cells gradually hydrated within 15 MAP, whereas the hydration of WT pollen on the \u003cem\u003eost2-2D\u003c/em\u003e papilla cells was slower compared to that on the WT papilla cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). These results indicate that the constitutively active AHA1 in the stigma partially inhibits pollen hydration on \u003cem\u003eost2-2D\u003c/em\u003e papilla cells. The delay in pollen hydration on \u003cem\u003eost2-2D\u003c/em\u003e stigmas is likely responsible for the shorter pollen tube elongation compared to WT. These results provide genetic support for the effects FC.\u003c/p\u003e\u003cp\u003eIn contrast to the phenotype of \u003cem\u003eost2-2D\u003c/em\u003e, pollen tube penetration and elongation in the pistils of \u003cem\u003eaha1-9\u003c/em\u003e and \u003cem\u003eaha2-5\u003c/em\u003e knockout mutants were similar to those in the WT, and no phenotypic differences were observed between the WT and these mutants (Fig. S7a, b). In addition, we generated \u003cem\u003eaha11\u003c/em\u003e mutants by genome editing and examined its phenotype (Fig. S8). The \u003cem\u003eaha11\u003c/em\u003e mutants, similar to the \u003cem\u003eaha1-9\u003c/em\u003e and \u003cem\u003eaha2-5\u003c/em\u003e mutants, showed no phenotypic differences in pollen tube penetration and elongation compared to the WT (Fig. S8b, c). These results are consistent with the results that vanadate treatment of \u003cem\u003eB. rapa\u003c/em\u003e pistils did not suppress pollen tube penetration into the stigmas in compatible pollination, again providing genetic support for the results of the pharmacological experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn self-incompatible Brassicaceae plants, selective water transport from female papilla cells to male pollen is clearly observed only after non-self-pollination \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Based on these findings, we hypothesized that osmoregulation in the papilla cells regulates water transport from papilla cells to pollen during the post-pollination process, and we focused on AHA, a primary transporter that plays a major role in osmoregulation of plant cells. In pharmacological experiments in \u003cem\u003eB. rapa\u003c/em\u003e, the application of FC, which promotes AHA activity, to stigmas stopped post-pollination processes in cross-pollination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, S3), whereas the application of vanadate, which inhibits AHA activity, to the stigmas allowed post-pollination processes in self-pollination to proceed. Furthermore, FC had a similar effect on \u003cem\u003eA. thaliana\u003c/em\u003e stigmas, suppressing post-pollination processes (Fig. S4). Although AHA possesses multiple phosphorylation sites in its C-terminal region that affect activity, phosphorylation of the pen-Thr residue of AHA is commonly observed in many stimuli and serves as the primary activation switch \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. The level of this pen-Thr phosphorylation in the stigmas of \u003cem\u003eB. rapa\u003c/em\u003e increased in response to self-pollination compared with non-pollination and cross-pollination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Furthermore, a constitutively active mutant of AHA1, \u003cem\u003eost2-2D\u003c/em\u003e, in \u003cem\u003eA. thaliana\u003c/em\u003e showed delayed pollen hydration and suppressed pollen tube elongation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, S6). These results suggest that activation of AHA in the stigma by self-pollination may promote water retention through an increase in osmotic pressure in the papilla cells, which inhibit the post-pollination processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eActivation of AHA is most likely regulated by SP11-SRK signaling because stigma AHA was activated in response to self-pollination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The effects of FC and of an AHA1 constitutively active mutation were also observed in the self-compatible plant \u003cem\u003eA. thaliana\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, S4, S6). Therefore, the status of AHA activity in the stigma may generally affect post-pollination processes through the control of osmotic pressure in papilla cells in Brassicaceae plants, regardless of whether the plant is self-compatible or incompatible. \u003cem\u003eA. thaliana\u003c/em\u003e exhibits interspecies incompatibility when crossed with some other Brassicaceae species \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, and the AHA-mediated regulatory system in \u003cem\u003eA. thaliana\u003c/em\u003e might be utilized during interspecific incompatibility.\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, levels of pen-Thr phosphorylation in AHA were at a steady state before pollination and the level did not change after pollination with compatible pollen. This result suggests that papilla cells may be ready to provide water to pollen before compatible pollination occurs. In this case, the osmotic pressure in papilla cells is kept relatively low, and the water-retaining capacity of the papilla cells may not be strong. In guard cells, ABA inhibits light-induced stomatal opening by suppressing phosphorylation of the pen-Thr of AHA \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The suppressed phosphorylation of AHA reduces osmotic pressure of guard cells, leading to water efflux. Similar to the guard cells, this low osmotic pressure in the papilla cells may provide an efficient force for water transport by widening the difference in osmotic pressure between the papilla cells and the pollen when compatible pollen is attached to the stigma. Since AHA is inactivated by dephosphorylation of pen-Thr, in unpollinated or cross-pollinated papilla cells, members of the type 2C protein phosphatase clade D family that remove the phosphate group, or unknown protein kinases that phosphorylate sites leading to AHA inactivation, may function to maintain some level of pen-Thr phosphorylation low \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIt has also been reported that transport of vesicles/MVBs to the plasma membrane promotes the water supply to pollen \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, but it is currently unclear what is carried in the transport vesicles/MVBs during this process. The transport vesicles/MVBs may carry transporters, including aquaporins localized to the plasma membrane, which may support water transport from papilla cells to pollen. Generally, aquaporins transport water according to the difference in osmotic pressure between inside and outside the cell \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e, so it makes sense that papilla cells would keep AHA activity low and maintain a low osmotic pressure to supply water to pollen. Taken together, our results suggest that osmoregulation via AHA, as well as vesicle transport in the papilla cells, play a crucial role in efficient water supply during pollination with compatible pollen.\u003c/p\u003e\u003cp\u003ePollen tube penetration was not inhibited in \u003cem\u003eost2-2D\u003c/em\u003e mutant pistils, unlike in FC-treated WT pistils (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, S4). The difference in the effects of the \u003cem\u003eost2-2D\u003c/em\u003e mutation and FC treatment could be explained by differences in the activated AHA isoforms in the stigma by the genetic mutation and chemical treatment. The major isoforms, \u003cem\u003eAHA1\u003c/em\u003e and \u003cem\u003eAHA2\u003c/em\u003e, are expressed throughout the plant and have redundant functions \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In the stigmas of \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eAHA1\u003c/em\u003e, \u003cem\u003eAHA2\u003c/em\u003e, and \u003cem\u003eAHA11\u003c/em\u003e are predominantly expressed (Fig. S5). FC treatment promotes the activity of all the AHA isoforms. Therefore, all AHAs expressed in the stigma may induce a strong increase in osmotic pressure in papilla cells, and completely inhibit the penetration of pollen tubes into the stigma (Fig. S4). In contrast, only AHA1 is constitutively active in the \u003cem\u003eost2-2D\u003c/em\u003e mutant. Thus, in the \u003cem\u003eost2-2D\u003c/em\u003e mutant, the total amount of activated AHA in the stigma is less than that observed with FC treatment, which may explain why inhibition of pollen tube penetration was not observed in the mutant stigmas. Similarly, no differences were observed between \u003cem\u003eaha\u003c/em\u003e single mutants and the WT in pollen tube penetration or elongation (Fig. S7, S8), which may also be due to the functional redundancy of AHAs. We were unable to test this possibility in this study because the \u003cem\u003eaha1 aha2\u003c/em\u003e double mutant is embryonic lethal \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Vanadate treatment of \u003cem\u003eB. rapa\u003c/em\u003e stigmas did not affect pollen tube penetration into the stigma in pollinations with compatible pollen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Therefore, another possibility to explain these results, besides functional redundancy, is that AHA activity in papilla cells was already low enough that the effects of \u003cem\u003eaha1, aha2\u003c/em\u003e, and \u003cem\u003eaha11\u003c/em\u003e mutations in \u003cem\u003eA. thaliana\u003c/em\u003e and vanadate treatment in \u003cem\u003eB. rapa\u003c/em\u003e stigma were not clearly observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, S3, S7, S8). Restriction of AHA activity in the stigma to a relatively low level may be necessary for post-pollination processes to proceed.\u003c/p\u003e\u003cp\u003eTo date, several signaling components have been identified that induce post-pollination processes and control self-compatibility/-incompatibility \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. RAPID ALKALINIZATION FACTOR (RALF) peptide and its receptor-like protein kinase FERONIA (FER) have been reported to play important roles in water transport from papilla cell to pollen during post-pollination processes in Brassicaceae \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eA. thaliana\u003c/em\u003e roots, RALF-FER signaling suppresses AHA activity probably via Ser-899 phosphorylation (Fig. S2) and induces alkalinization of the apoplast \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. Therefore, it was expected that there are some relationships between RALF-FER signal and AHA activity during post-pollination processes in self-incompatibility. Some \u003cem\u003eA. thaliana\u003c/em\u003e AHA members, including AHA1, 2, 5, and 10, retained a Ser residue at position 899, whereas AHA3 retained a Gly residue\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Similar to the AtAHA3, two AHAs predominantly expressed in the stigma of \u003cem\u003eB. rapa\u003c/em\u003e lack the conserved Ser-899 and is instead Gly (Fig. S2). Among BrAHAs, only BrAHA2d and BrAHA2e possess a Ser residue corresponding to Ser-899, but these genes were not as strongly expressed in papilla cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, S1). In addition, RALF peptides are perceived not only by FER but also by other receptor-kinases of the CrRLK family. One member of the CrRLK family, ERULUS, has been shown to be involved in phosphorylation of Ser-904 in AtAHA1 and AtAHA2\u003csup\u003e74\u003c/sup\u003e, and phosphomimic of Ser-904 enhances AHA activity in yeast cells \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Therefore, FER signaling may not suppress AHA activity in \u003cem\u003eB. rapa\u003c/em\u003e papilla cells, and the regulation of AHA activity by RALF peptides may be diverse and differ between cells and tissues. Unlike its action in \u003cem\u003eArabidopsis\u003c/em\u003e roots, RALF peptides may even promote AHA activity in \u003cem\u003eB. rapa\u003c/em\u003e stigmas by binding with an unknown member of the CrRLK family.\u003c/p\u003e\u003cp\u003eIn summary, regulation of AHA activity in papilla cells is involved in water transport to pollen for pollen hydration in Brassicaceae plants. In self-incompatible plants of Brassicaceae, pollination with self-pollen increased AHA activity in the stigma. The AHA activity enhances the osmotic pressure and water retention in the papilla cells and inhibits water release to pollen. Our work provides new insights into the molecular mechanisms underlying post-pollination processes.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003ePlant material and growth conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn this study, \u003cem\u003eS\u003c/em\u003e homozygotes (\u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e) and \u003cem\u003eS\u003c/em\u003e heterozygote (cv. Gokurakuten; unknown \u003cem\u003eS\u003c/em\u003e haplotypes) were used as self-incompatible strains of \u003cem\u003eBrassica rapa\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and the cv. \u003cem\u003eyellow sarson\u003c/em\u003e C634 was used as a self-compatible strain \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Seeds were germinated on water-wetted filter paper, and seedlings were transplanted to soil in pots. All \u003cem\u003eB. rapa\u003c/em\u003e plants were grown in a greenhouse at Tohoku University. Flowers that opened on the day of the experiment were used and emasculated just before the experiments. Self- and cross-pollination were performed by hand.\u003c/p\u003e\u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e ecotype Col-0 was used as a wild type (WT). T-DNA insertion mutant lines, \u003cem\u003eaha1-9\u003c/em\u003e (SAIL_1285_D12) and \u003cem\u003eaha2-5\u003c/em\u003e (SALK_022010), were obtained from the Arabidopsis Biological Resource Centre. Homozygotes of both mutants had been isolated and used in a previous study \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. We used \u003cem\u003eost2-2D\u003c/em\u003e as a constitutive active mutant of AtAHA1 \u003csup\u003e64\u003c/sup\u003e. Arabidopsis plants were grown in soil in a growth room, under white light emitting photodiode (200 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with a 16-h/8-h light/dark cycle. The growth temperature was approximately 22\u0026deg;C, and the relative humidity was 40\u0026ndash;60%. Flowers from Arabidopsis plants grown for 4\u0026ndash;5 weeks were emasculated and hand-pollinated to observe plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPase phosphorylation and activity, pollen hydration, and pollen tube elongation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, S4, S6 to S8).\u003c/p\u003e\u003cp\u003e\u003cem\u003eNicotiana benthamiana\u003c/em\u003e plants were grown in soil in a growth chamber, under a white light fluorescent lamp (90 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with a 16-h/8-h light/dark cycle. The growth temperature was approximately 28\u0026deg;C, and the relative humidity was 40\u0026ndash;60%. For the GFP-fused proteins transient expression assay, 3-week-old plants were used \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConstruction of the phylogenetic tree\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe amino acid sequences of AtAHA and BrAHA homologs were obtained from TAIR10 and Brapa_1.0. Full-length amino acid sequences were aligned using MAFFT software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mafft.cbrc.jp/alignment/software/\u003c/span\u003e\u003cspan address=\"https://mafft.cbrc.jp/alignment/software/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The phylogenetic tree was constructed via the Neighbor-Joining method \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e using full-length amino acid sequences of AHA homolog proteins.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene expression analysis of\u003c/b\u003e \u003cb\u003eBrAHAs\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eB. rapa\u003c/b\u003e \u003cb\u003estigmas by reverse transcription-quantitative PCR (RT-qPCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNAs were extracted from five stigmas of \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e haplotype using the RNeasy RNA plant Mini kit (Qiagen). First-strand cDNAs were synthesized from the total RNAs using PrimeScript IV 1st strand cDNA Synthesis Kit (TaKaRa). RT-qPCR was performed by Bio-Rad CFX Connect using SsoFast EvaGreen Supermix (Bio-Rad). Melting curves were generated using specific primers (Table S2). Expression of each gene was quantified with Bio-Rad CFX Maestro 2.3 and normalized to \u003cem\u003eubiquitin-conjugating enzyme 21\u003c/em\u003e \u003csup\u003e78\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSubcellular localization of BrAHA proteins in\u003c/b\u003e \u003cb\u003eNicotiana benthamiana\u003c/b\u003e \u003cb\u003eleaves\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGFP-BrAHA1a, GFP-BrAHA2c, and AtPIP2A-mCherry under control of the \u003cem\u003e35S\u003c/em\u003e promoter were transiently expressed in leaves of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e. PIP2A-mCherry was used as a localization marker for the plasma membrane. The full-length \u003cem\u003eB. rapa\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e cDNA of each gene was amplified by PCR using the specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The amplified fragments were inserted downstream of the 35S promoter in pRI101-AN DNA vector (TaKaRa) for \u003cem\u003eGFP\u003c/em\u003e-\u003cem\u003eBrAHA1a\u003c/em\u003e and \u003cem\u003eGFP\u003c/em\u003e-\u003cem\u003eBrAHA2c\u003c/em\u003e and of pCAMBIA1302 (CAMBIA) for \u003cem\u003eAtPIP2A\u003c/em\u003e-\u003cem\u003emCherry\u003c/em\u003e using the In-Fusion cloning system (Clontech).\u003c/p\u003e\u003cp\u003eThe resulting construct was introduced into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves using an \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation method \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eAgrobacterium\u003c/em\u003e GV3101 strain was transformed with the vectors and cultured at 28\u0026deg;C for about 20 h. The agrobacteria were collected and resuspended in infection buffer including 10 mM Mes-KOH (pH 5.6) and 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e. GFP and mCherry fluorescent signals from epidermal cells were observed 4 days after infiltration using a confocal laser microscope LSM 710 (Zeiss). GFP fluorescence was detected at 488 nm excitation and 493\u0026ndash;550 nm emission. mCherry fluorescence was detected at 561 nm excitation and 593\u0026ndash;650 nm emission.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChemical treatment of pistils\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePistils were excised at a length of 4 mm from the stigma apex of opened flowers of \u003cem\u003eB. rapa\u003c/em\u003e, and the pistils were placed on 1% agar medium containing fusicoccin (FC) or sodium orthovanadate (vanadate) with the cut-end buried in the medium and left at room temperature for 3 h. After chemical treatment, the stigmas were self- or cross-pollinated and left for 1 h on the chemical-containing medium. The stigmas were then transferred to plant agar medium without chemicals and incubated overnight at room temperature.\u003c/p\u003e\u003cp\u003eThe treatments of \u003cem\u003eA. thaliana\u003c/em\u003e pistils were performed similarly to those for \u003cem\u003eB. rapa\u003c/em\u003e with minor modifications. The opened flowers were cut at the inflorescence stems and emasculated. The tips of the inflorescence stem containing pistils were treated with chemicals as above. Six hours after the start of treatment, pollen was placed on the stigma. After 2 days, the pistil samples were transferred to 1% agar medium without chemicals and incubated overnight for elongation of the pollen tube.\u003c/p\u003e\u003cp\u003e\u003cb\u003eApoplast pH measurements in\u003c/b\u003e \u003cb\u003eB. rapa\u003c/b\u003e \u003cb\u003estigmas\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAcidification of media around stigmas was monitored using a pH indicator dye, bromocresol purple. Four pistils of \u003cem\u003eB. rapa\u003c/em\u003e cv. Gokurakuten were excised from the pedicel and placed with the stigma in contact with the solid medium (0.008% bromocresol purple, 1% low-melting-point agarose adjusted to pH 7.0). Samples were incubated at room temperature for 30 min and then images were taken.\u003c/p\u003e\u003cp\u003e\u003cb\u003eObservation of pollen and pollen tube behavior on the stigma\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePistils of \u003cem\u003eB. rapa\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e were placed on 1% agar medium and pollinated. In the case of Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, in self-pollination, pollen from the \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e strain was pollinated onto the stigma of the \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e strain (Self-pollination), and in cross-pollination, pollen from the \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e strain was placed on the stigma of the \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e strain (Cross-pollination).\u003c/p\u003e\u003cp\u003eAfter the pollination, at each time point pistils were fixed in 3:1 ethanol: acetic acid for at least 30 min. The pistils were washed 3 times with distilled water and hydrolyzed in 1N NaOH for 1 h at 60\u0026deg;C. To visualize pollen tubes, samples were stained with aniline blue solution (0.1% aniline blue, 0.1 M K\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e) overnight in the dark \u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e, and observed by fluorescence microscopy. Pollen tube penetration was determined by whether the pollen tube had passed through the stigma-style boundary and entered the style (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb: left diagram).\u003c/p\u003e\u003cp\u003e\u003cb\u003eObservation of pollen hydration\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePistils and stamens were dissected from freshly opened flowers, placed on a glass cover slip, and held in place with tape. Under an inverted microscope (Axio Observer, Zeiss), pollen from the stamen was placed on the papilla cells using manipulators (NM-4 and MMO-4, NARISHIGE) equipped with a glass rod. Images of pollen hydration were taken at each time point after the start of pollination. The short axis of the pollen was measured in each image by the \u0026ldquo;ellipse\u0026rdquo; mode of Fiji ImageJ software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/software/fiji/downloads\u003c/span\u003e\u003cspan address=\"https://imagej.net/software/fiji/downloads\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The degree of pollen hydration was calculated as the percentage change in the short axis length from the point of pollination (0 MAP).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetection of phosphorylation and amount of AHA protein in\u003c/b\u003e \u003cb\u003eB. rapa\u003c/b\u003e \u003cb\u003estigmas\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe amount of AHA and its phosphorylation were measured by immunoblot analysis, using previously described methods \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e with slight modifications. Pistils of \u003cem\u003eB. rapa\u003c/em\u003e cv. Gokurakuten were excised and placed on 1% agar medium. The pistils were pollinated with self (Gokurakuten) or non-self (\u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e strain) pollen for 30 min. The pollinated and unpollinated pistils were washed with 0.1% sodium dodecyl sulfate (SDS) in MilliQ water for 10 sec to remove pollen from the stigmas. The water was wiped off the pistils, and stigmas were excised with a razor. The stigmas were immediately frozen in liquid nitrogen and disrupted using zirconia beads. Proteins of 6 stigmas in each treatment were solubilized by adding the SDS buffer (10 mM Tris-HCl [pH 6.8], 2% SDS, 1 mM EDTA, 20% glycerol, 80 mM DTT, 0.02% bromophenol blue, 200 \u0026micro;M sodium fluoride, 5 \u0026micro;M phenylmethylsulfonyl fluoride, 20 \u0026micro;M leupeptin). The solubilized samples were centrifuged at 13,000 rpm for 5 min at 4\u003csup\u003eo\u003c/sup\u003eC. The supernatant was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and then immunoblotting.\u003c/p\u003e\u003cp\u003eSpecific antibodies against the catalytic domain of \u003cem\u003eArabidopsis\u003c/em\u003e plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPase (AtAHA) and the phosphorylated pen-Thr residue of AtAHA were used as first antibodies, as previously described \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. Anti-actin antibody (Sigma) was also used as a first antibody. Goat anti-rabbit or mouse IgG-horseradish peroxidase conjugate (Bio-Rad) was used as a secondary antibody. The chemiluminescence signal was detected using an imaging system (ChemiDoc Touch, Bio-Rad). Signal intensity was analyzed using Fiji ImageJ software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantitative phosphoproteomic analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e stigmas after self-pollination or cross-pollination (pollinated with \u003cem\u003eS\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e pollen) were prepared as described above for immunoblot analysis. The stigma samples (n\u0026thinsp;=\u0026thinsp;70/treatment) were lysed in 150 \u0026micro;L of 6 M guanidine-HCl, 100 mM HEPES-NaOH, pH 7.5, 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 min at 4 \u0026ordm;C. The supernatants were recovered, and proteins (400 \u0026micro;g each) were purified by methanol\u0026ndash;chloroform precipitation and solubilized in 150 \u0026micro;L of 0.1% RapiGest (Waters) in 50 mM triethylammonium bicarbonate. After sonication, the protein solutions were digested with 8 \u0026micro;g trypsin/Lys-C mix (Promega) at 37 \u0026ordm;C overnight. The resulting peptide solutions were acidified with TFA, centrifuged, and subjected to the High-Select Fe-NTA phosphopeptide enrichment kit (Thermo Fisher Scientific). The eluates were acidified, desalted using GL-Tip SDB (GL Sciences), evaporated in a SpeedVac concentrator, and re-dissolved in 0.1% TFA and 3% acetonitrile. LC-MS/MS analysis of the resultant peptides was performed on a nanoElute 2 coupled with a timsTOF HT mass spectrometer (Bruker). The peptides were separated on a 75-\u0026micro;m inner diameter \u0026times; 150 mm C18 reversed-phase column (Nikkyo Technos). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Peptides were loaded onto the column at a flow rate of 0.2 \u0026micro;L/min starting at 3% B, which was linearly ramped to 32% B over 90 min, then raised to 95% B at 91 min, and held at that level until 101 min. The mass spectrometer was operated in parallel accumulation\u0026ndash;serial fragmentation (PASEF) mode. The \u003cem\u003em/z\u003c/em\u003e range for both MS1 and MS2 spectra was 100\u0026ndash;1700, and the ion mobility range was 0.6\u0026ndash;1.6 V\u0026middot;s/cm\u003csup\u003e3\u003c/sup\u003e. The ramp time was 100 ms, with a duty cycle of 100%. Each acquisition cycle consisted of 10 PASEF MS2 scans. A polygon filter was applied in the \u003cem\u003em/z\u003c/em\u003e and ion mobility space to exclude low \u003cem\u003em/z\u003c/em\u003e, singly charged ions from precursor selection. The raw data were processed using the FragPipe (v22.0). Database searches were performed with the MSFragger (v4.1), employing the default parameters of the LFQ-phospho workflow against the \u003cem\u003eBrassica rapa\u003c/em\u003e peptide database (Brapa15 pep; 41,020 entries). Carbamidomethylation of cysteine (+\u0026thinsp;57.0215 Da) was set as a fixed modification. The following variable modifications were included: acetylation of protein N-terminus (+\u0026thinsp;42.0106 Da); oxidation of methionine (+\u0026thinsp;15.9949 Da); phosphorylation (+\u0026thinsp;79.9663 Da) of serine, threonine, or tyrosine. The resulting identifications were filtered using Philosopher with default parameters (MS Booster was disabled), and IonQuant (v1.10.27) was used for quantification with default software settings.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression and subcellular localization of AtAHAs in stigma\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGenomic \u003cem\u003eA. thaliana AHA1\u003c/em\u003e and \u003cem\u003eAHA2\u003c/em\u003e genes including each 5\u0026prime; and 3\u0026prime; noncoding sequence were amplified by PCR from genomic DNA of the wild type (Col-0) using their respective specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) according to previous methods \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. These amplified DNA fragments were cloned into the gene transfer vector pCAMBIA1300 (CAMBIA) by the In-Fusion cloning system at the \u003cem\u003eHin\u003c/em\u003edIII/ \u003cem\u003eBam\u003c/em\u003eHI sites (for \u003cem\u003eAHA1\u003c/em\u003e gene) and \u003cem\u003eXba\u003c/em\u003eI site (for \u003cem\u003eAHA2\u003c/em\u003e gene). The cDNA of GFP was amplified and inserted into the site between the end of the 5\u0026prime; noncoding sequence and the start codon of each \u003cem\u003eAHA\u003c/em\u003e gene using In-Fusion cloning system (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The constructed vectors were verified by DNA sequencing. The resulting vectors were used for transformation of \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101 strain and introduced into \u003cem\u003eA. thaliana\u003c/em\u003e WT (Col-0) using the floral dip method \u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. Transgenic plants were selected by resistance to hygromycin and used for analysis.\u003c/p\u003e\u003cp\u003eThe pistils were emasculated in the evening of the day before observation. Emasculated pistils were placed on glass-bottom dishes, and a 1% plant agar medium block was placed on top of the pistil to bring the papilla cells in contact with the surface of the glass-bottom dish. GFP fluorescence (excitation 488 nm/emission 483\u0026ndash;530 nm) signal derived from the papilla cells was observed with a confocal microscope LSM 710 (Zeiss).\u003c/p\u003e\u003cp\u003e\u003cb\u003eGeneration of\u003c/b\u003e \u003cb\u003eaha11\u003c/b\u003e \u003cb\u003emutants by genome editing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo generate the \u003cem\u003eaha11\u003c/em\u003e mutants by genome editing, we constructed the pKAMA-ITACHI(pKI)1.1R vector bearing the CRISPR/Cas9 \u003csup\u003e82\u003c/sup\u003e. The single guide RNA (sgRNA) for \u003cem\u003eAHA11\u003c/em\u003e was designed by CRISPR-P v2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR\u003c/span\u003e\u003cspan address=\"http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The hybridized sgRNA oligo DNA was inserted into the \u003cem\u003eAar\u003c/em\u003eI site of the pKI1.1R vector, and the constructed plasmid was verified by DNA sequencing. The resulting vector was introduced into \u003cem\u003eA. tumefaciens\u003c/em\u003e GV3101 strain and the bacteria was used to transform \u003cem\u003eA. thaliana\u003c/em\u003e WT (Col-0) using the floral dip method. T\u003csub\u003e1\u003c/sub\u003e generation of the transgenic plants were selected based on hygromycin resistance and \u003cem\u003eaha11\u003c/em\u003e mutation. T\u003csub\u003e2\u003c/sub\u003e seeds that showed no RFP fluorescence were selected to remove the CRISPR/Cas9 cassette, and homozygous \u003cem\u003eaha11\u003c/em\u003e mutation was confirmed by DNA sequencing. The resulting mutant lines were used for the phenotypic analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAccession numbers\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eAHA1\u003c/em\u003e (At2g18960), \u003cem\u003eAHA2\u003c/em\u003e (At4g30190), \u003cem\u003eAHA3\u003c/em\u003e (At5g57350), \u003cem\u003eAHA4\u003c/em\u003e (At3g47950), \u003cem\u003eAHA5\u003c/em\u003e (At2g24520), \u003cem\u003eAHA6\u003c/em\u003e (At2g07560), \u003cem\u003eAHA7\u003c/em\u003e (At3g60330), \u003cem\u003eAHA8\u003c/em\u003e (At3g24640), \u003cem\u003eAHA9\u003c/em\u003e (At1g80660), \u003cem\u003eAHA10\u003c/em\u003e (At1g17260), \u003cem\u003eAHA11\u003c/em\u003e (At5g62670), \u003cem\u003eUBC21\u003c/em\u003e (At3g25760), \u003cem\u003ePIP2A\u003c/em\u003e (At3g53420)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported, in part, by JSPS KAKENHI (22K14867 and 24K17874 to M. H., 23K18058 to M. W., 22K05581 to Y. T.) and by MEXT KAKENHI (22H05172 and 22H05179 to M.W.).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.H. and M.W. conceived and designed the experiments. M.H., K.F., H.-M.S., S.I. and H.K. performed the experiments. M.H., S.I., T.K., Y.T., H.K., and M.W. analyzed the data. M.H. and M.W. drafted the manuscript. All authors edited the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Dr. Nathalie Leonhardt (University of Aix Marseille, CEA, CNRS, BIAM, UMR7265, SAVE, Saint Paul-Lez-Durance) for kindly providing ost2-2D seeds, Dr. Hiroki Tsutsui and Dr. Tetsuya Higashiyama (University of Tokyo) for providing pKAMA-ITACHI vector, and Dr. Kohei Nishino (Tokushima University) for his technical assistance in phosphoproteomic analysis. We also thank Dr. Seiji Takayama and Dr. Sota Fujii (The University of Tokyo) for helpful comments, and Kana Ito, Sadayoshi Ogata, Tai Takemoto, Toko Kanomata, Yuta Takahashi, and Temari Endo (Tohoku University) for technical assistance. This work was supported, in part, by JSPS KAKENHI (22K14867 and 24K17874 to M. H., 23K18058 to M. W., 22K05581 to Y. T.) and by MEXT KAKENHI (22H05172 and 22H05179 to M.W.). A part of this study was supported by the Medical Research Center Initiative for High Depth Omics and Research Equipment Sharing System, Tohoku University (090, Carl-Zeiss LSM 710). This work used research equipment shared in the MEXT Project for promoting public utilization of advanced research infrastructure (Program for Supporting Construction of Core Facilities) grant numbers JPMXS0440600022, JPMXS0440600023, and JPMXS0440600024.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe MS proteomics data have been deposited to the ProteomeXchange Consortium via the jPOST partner repository83 with the dataset identifier PXD065620.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBarrett, S. C. H. \u0026amp; Hough, J. Sexual dimorphism in flowering plants. \u003cem\u003eJ. Exp. Bot.\u003c/em\u003e \u003cstrong\u003e64\u003c/strong\u003e, 67\u0026ndash;82 (2013).\u003c/li\u003e\n\u003cli\u003eTaylor, L. P. \u0026amp; Hepler, P. K. Pollen germination and tube growth. \u003cem\u003eAnnu. Rev. Plant Physiol. Plant Mol. 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However, the mechanism of the selective water transport has not been elucidated. In this study, we determined that the stigma autoinhibited plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPase (AHA) is involved in the selective water transport from papilla cells to pollen. We characterized AHA isoforms that are expressed in the stigma of self-incompatible plant, \u003cem\u003eBrassica rapa\u003c/em\u003e by expression analysis. A chemical activator of AHA applied to stigmas suppressed pollen hydration and pollen tube penetration into stigma in cross-pollination of \u003cem\u003eB. rapa\u003c/em\u003e. In contrast, an AHA inhibitor allowed pollen hydration and pollen tube penetration in self-pollination. Consistent with these pharmacological effects, stigma AHA was activated through the phosphorylation of penultimate Thr in response to self-pollination compared with cross-pollination. Furthermore, speed of pollen hydration and elongation of pollen tubes were delayed on the stigmas of \u003cem\u003eost2-2D\u003c/em\u003e, a constitutive active mutant of AtAHA1 in the self-compatible \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. These results indicate that regulation of AHA activity in the stigma is involved in selective water transport for pollen hydration, possibly by regulating osmotic pressure in Brassicaceae self-incompatibility.\u003c/p\u003e","manuscriptTitle":"Stigma plasma membrane H + -ATPase is involved in pollen hydration and pollen tube penetration in Brassicaceae self-incompatibility","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 16:49:53","doi":"10.21203/rs.3.rs-6981590/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d2468829-521c-4bf5-b5bb-f809c5f8c2b1","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51412497,"name":"Biological sciences/Plant sciences/Plant physiology"},{"id":51412498,"name":"Biological sciences/Plant sciences/Plant reproduction"}],"tags":[],"updatedAt":"2025-09-03T13:24:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-14 16:49:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6981590","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6981590","identity":"rs-6981590","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}