Tip-localized receptors control pollen tube growth and lure sensing in arabidopsis

ABSTRACT Directional control of tip-growing cells is essential for proper tissue organization and cell-to-cell communication in animals and plants1,2. In the sexual reproduction of flowering

plants, the tip growth of the male gametophyte, the pollen tube, is precisely guided by female cues to achieve fertilization3. Several female-secreted peptides have recently been identified

as species-specific attractants that directly control the direction of pollen tube growth4,5,6. However, the method by which pollen tubes precisely and promptly respond to the guidance

signal from their own species is unknown. Here we show that tip-localized pollen-specific receptor-like kinase 6 (PRK6) with an extracellular leucine-rich repeat domain is an essential

receptor for sensing of the LURE1 attractant peptide in _Arabidopsis thaliana_ under semi-_in-vivo_ conditions, and is important for ovule targeting in the pistil. PRK6 interacted with

pollen-expressed ROPGEFs (Rho of plant guanine nucleotide-exchange factors), which are important for pollen tube growth through activation of the signalling switch Rho GTPase ROP1 (refs 7,

8). PRK6 conferred responsiveness to AtLURE1 in pollen tubes of the related species _Capsella rubella_. Furthermore, our genetic and physiological data suggest that PRK6 signalling through

ROPGEFs and sensing of AtLURE1 are achieved in cooperation with the other PRK family receptors, PRK1, PRK3 and PRK8. Notably, the tip-focused PRK6 accumulated asymmetrically towards an

external AtLURE1 source before reorientation of pollen tube tip growth. These results demonstrate that PRK6 acts as a key membrane receptor for external AtLURE1 attractants, and recruits the

core tip-growth machinery, including ROP signalling proteins. This work provides insights into the orchestration of efficient pollen tube growth and species-specific pollen tube attraction

by multiple receptors during male–female communication. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS

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institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS A RECEPTOR–CHANNEL TRIO CONDUCTS CA2+ SIGNALLING FOR POLLEN TUBE RECEPTION

Article 06 July 2022 PERSISTENT DIRECTIONAL GROWTH CAPABILITY IN _ARABIDOPSIS THALIANA_ POLLEN TUBES AFTER NUCLEAR ELIMINATION FROM THE APEX Article Open access 22 April 2021 PXL1 AND SERKS

ACT AS RECEPTOR–CORECEPTOR COMPLEXES FOR THE CLE19 PEPTIDE TO REGULATE POLLEN DEVELOPMENT Article Open access 07 June 2023 REFERENCES * Itofusa, R. & Kamiguchi, H. Polarizing membrane

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_Arabidopsis_. _EMBO J._ 34, 55–66 (2015) Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank M. Hasebe and S. Miyazaki for seeds of a part of mutant lines; Y.

Matsubayashi, H. Shinohara, D. Maruyama, M. Ohtsu and K. Motomura for valuable comments and discussions; D. Kurihara, Y. Hamamura and S. Nagahara for technical assistance with confocal

microscopy and physiological analyses; M. M. Kanaoka for agro-infiltration method using tobacco leaf; S. Oishi for reverse-phase high-pressure liquid chromatography (HPLC); and the Japan

Advanced Plant Science Network for use of some microscopes. This work was supported by grants from the Japan Science and Technology Agency (ERATO project to T.H.) and the Japan Society for

the Promotion of Science Fellowships (no. 5834 to H.T.). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Division of Biological Science, Graduate School of Science, Nagoya University,

Furo-cho, Chikusa-ku, Nagoya, 464-8602, Aichi, Japan Hidenori Takeuchi & Tetsuya Higashiyama * JST ERATO Higashiyama Live-Holonics Project, Nagoya University, Furo-cho, Chikusa-ku,

Nagoya, 464-8602, Aichi, Japan Hidenori Takeuchi & Tetsuya Higashiyama * Institute of Transformative Bio-Molecules (ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602,

Aichi, Japan Tetsuya Higashiyama Authors * Hidenori Takeuchi View author publications You can also search for this author inPubMed Google Scholar * Tetsuya Higashiyama View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS H.T. designed the study; H.T. performed experiments; H.T. and T.H. wrote the manuscript. CORRESPONDING

AUTHORS Correspondence to Hidenori Takeuchi or Tetsuya Higashiyama. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. EXTENDED DATA FIGURES AND

TABLES EXTENDED DATA FIGURE 1 PHYLOGENETIC RELATIONSHIP, EXPRESSION, GENE STRUCTURE AND FERTILITY OF _A. THALIANA_ PRK FAMILY PROTEIN MUTANTS. A, A neighbour-joining (NJ) tree constructed

using full-length amino-acid sequences of PRK1–PRK6 (ref. 14), PRK7 and PRK8 (assigned in this study). The bootstrap values as percentages and the scale for substitutions per site are shown.

B, _PRK_ expression during pollen germination and growth. Expression levels are shown using normalized values and standard deviation from microarray data (_n _= 4 for dry pollen, 30 min _in

vitro_ pollen tube (PT), and 4 h _in vitro_ PT; _n _= 3 for semi-_in-vivo_ PT)13. C, Structure and T-DNA insertion of _PRK_ genes. Grey boxes show exons, and black boxes show introns or

untranslated regions that are registered in The _Arabidopsis_ Information Resource (TAIR). The T-DNA insertion sites determined by genomic PCR and sequencing are drawn on the gene structure

and indicated in Extended Data Table 1. D, Reverse transcription PCR (RT–PCR) analysis of the _prk_ single mutants. Anther cDNA was used for the analysis. Positions of the primers are

indicated in the gene structure (C). _ACT2_ was used as the loading control. For gel source data, see Supplementary Fig. 1. E, F, The rate of developing seeds upon self-pollination of _prk_

single mutants (E) and upon reciprocal crosses with Col-0 and _prk_ multiple mutants (F). Asterisks in E indicate the mutants used for the _prk_ multiple mutants in this study. Note that, in

addition to multiple mutants of _PRK1_ and _PRK3_ subclass genes (shown in dark blue), multiple mutants of _PRK1_, _PRK4_ and _PRK6_, which are the top three most highly expressed in

semi-_in-vivo_ pollen tubes, and _PRK1_, _PRK2_, _PRK4_ and _PRK5_, which form another subclade, were analysed. The _prk1 prk2 prk4 prk5_ multiple mutant contains _prk1 prk2 prk5_ mutations

that cause reduced pollen tube growth _in vitro_14. Data are mean and s.d. of three (all samples in E; Col-0 pistil × _prk3-1 prk6-1 pr8-1_ and _prk3-1 prk6-1 pr8-2_ in F) or four (other

samples in F) pistils. G, Developing seeds in siliques 8 days after pollination with Col-0, _prk6_ and the _prk1 prk3 prk6_ triple mutant. The images are representative of four samples.

Scale bar, 1 mm. EXTENDED DATA FIGURE 2 EVALUATION OF ATLURE1-RESPONSIVE WAVY ASSAY. A–F, Semi-_in-vivo_ pollen tubes on medium containing the indicated concentrations of AtLURE1.2 peptide.

Entire (A–D) and magnified (E, F) images show wavy and swollen tip growth of wild-type, but not _prk6-1_ mutant, pollen tubes in a concentration-dependent manner. Scale bars, 200 μm (A–D)

and 20 μm (E, F). G, Growth of PRK6–mRuby2 pollen tubes that directly germinated on medium (that is, _in vitro_ pollen tubes) containing 1 μM AtLURE1.2 peptide. H, Growth of semi-_in-vivo_

pollen tubes from _chx21-s1/chx21-s1 chx23-4/CHX23_ plant18 on medium containing 1 μM AtLURE1.2 peptide. Roughly half the pollen tubes showed wavy growth as in the wild type (arrowheads),

but the rest did not (arrows). These results indicate that _in vitro_ pollen tubes and _chx21 chx23_ double-mutant pollen tubes have no or less ability to respond to the external AtLURE1

peptide. Scale bars, 200 μm. I, Semi-_in-vivo_ pollen tube growth and AtLURE1-responsive wavy assay for _prk_ mutants additional to those shown in Fig. 1f. Scale bars, 100 μm (top) and 10 μm

(bottom). J, Complementation of the growth defect in _prk3 prk6_ pollen tubes by expression of PRK3–mClover or PRK6–mClover. Note that PRK3–mClover expression restored the growth defect but

not the wavy response. The images of A–J are representative of at least three assays. K, Pollen tube tip localization of PRK3–mClover in a single-plane confocal image (top) and its

intensity image by pseudocolour (bottom). The data are representative of three samples. Scale bar, 10 μm. EXTENDED DATA FIGURE 3 POLLEN TUBE GROWTH OF _PRK_ MUTANTS IN THE PISTIL. A, Pollen

tubes of Col-0, _prk6_, _prk3 prk6_ and _prk1 prk3 prk6_ growing in the Col-0 pistils. Aniline blue staining was performed 12 or 24 HAP. White arrows indicate the tip of the longest pollen

tube in the transmitting tract. Data are representative of three samples for each genotype. Scale bar, 500 μm. B, Length from the top of the stigma to the tip of the longest pollen tube, 12

or 24 HAP with Col-0 and _prk_ mutants. About 2,700 μm is the maximum limit for the length in this measurement. The data are the mean and s.d. of three pistils. ND, no data. EXTENDED DATA

FIGURE 4 GROWTH AND OVULE-TARGETING OF _PRK_ MUTANT POLLEN TUBES ON THE SEPTUM SURFACE. A–G, Entire images of growth and ovule-targeting of wild-type (A), _prk6_ (B), _prk3 prk6_ (C, D),

_prk3 prk6 prk8-2_ (E, F), and _prk1 prk3 prk6_ (G) pollen tubes on the septum surface in wild-type pistils. Arrows indicate the tip of the longest pollen tube on the septum surface.

Asterisks mark ovules that did not attract near pollen tubes. The regions shown in A, B and D are shown in Fig. 2e, f and g, respectively, as higher magnification images. Data are

representative of 1–3 images for each genotype. Similar growth properties were observed in a total of 4 samples. Scale bar, 500 μm. Quantitative analysis is shown in Fig. 2d. No analysis was

performed for the _prk1 prk3 prk6_ mutant because almost no pollen tube reached the ovule. EXTENDED DATA FIGURE 5 INTERACTION OF PRK6 WITH POLLEN-EXPRESSED ROPGEFS, PRKS AND LIPS. A, Gene

expression of _ROPGEF_s during pollen germination and growth. The data are normalized expression values and standard deviation from microarray data (_n _= 4 for dry pollen, 30 min _in vitro_

PT, and 4 h _in vitro_ PT; _n _= 3 for semi-_in-vivo_ PT)13 as noted in Extended Data Fig. 1b. _ROPGEF8_, _ROPGEF9_, _ROPGEF11_, _ROPGEF12_ and _ROPGEF13_ are expressed specifically in the

dry pollen grain and pollen tube. B, BiFC assay showing the interaction between PRK6–cYFP and nYFP–GEF8, nYFP–GEF9, nYFP–GEF12, or nYFP–GEF13 (see Methods). C, A control experiment using

C-terminal-deleted ROPGEF12 (ROPGEF12ΔC). The C-terminal domain is suggested to mediate the interaction with PRK2 (ref. 8). D, BiFC assay showing interaction between PRK6–cYFP and PRK6–nYFP,

PRK3–nYFP, LIP1–nYFP or LIP2–nYFP. Scale bars, 50 μm. Images are representative of more than three experiments. E, Co-immunoprecipitation assay of PRK–mClover and ROPGEF12 proteins

expressed in _N. benthamiana_ leaf cells. ROPGEF12-3 × Flag protein was precipitated with full-length PRK3, PRK6 and kinase domain-deleted PRK6 (K-del), but not mClover control or cytosolic

domain-deleted PRK6 (cyto-del-1). Data are representative of three experiments. For gel source data, see Supplementary Fig. 2. EXTENDED DATA FIGURE 6 PRK6 PROTEIN STRUCTURE AND PRK PROTEINS

OF _A. THALIANA_, _A. LYRATA_ AND _C. RUBELLA_. A, Structures of the PRK6 protein and its deletion version used in this study. The PRK6 extracellular domain contains the N-terminal cap and

six LRRs. JM, juxtamembrane domain; N-cap, N-terminal cap; SP, signal peptide; TM, transmembrane domain. The numbers indicate the amino acid ranges of each domain. B, A 3D ribbon model of

the PRK6 extracellular domain, amino acid residues 28–231, was predicted using the homology modelling platform, SWISS-MODEL (http://swissmodel.expasy.org/), and the FLS2 crystal structure

(Protein Data Bank (PDB) accession 4MN8) as a template, and was drawn using Swiss-PdbViewer (http://spdbv.vital-it.ch/)38. The PRK6 extracellular domain contains the N-terminal cap and six

LRRs. C, A neighbour-joining tree constructed using PRK protein sequences from tomato (_Lycopersicon esculentum_, LePRK1-3), _A. thaliana_ (AtPRK1–AtPRK8), _A. lyrata_ (AlPRK1–AlPRK8), and

_C. rubella_ (CrPRK1–CrPRK8). The bootstrap values as percentages and the scale for substitutions per site are shown. Accession numbers for _A. lyrata_ and _C. rubella_ PRKs: AlPRK1

(XP_002868416), AlPRK2 (XP_002883746), AlPRK3 (XP_002877261), AlPRK4 (XP_002883234), AlPRK5 (XP_002891583), AlPRK6 (XP_002871954), AlPRK7 (XP_002867307, modified according to the genome

sequence), AlPRK8 (XP_002887434), CrPRK1 (EOA19015), CrPRK2 (EOA32286, partial sequence), CrPRK3 (EOA25493), CrPRK4 (EOA31871), CrPRK5 (EOA37472), CrPRK6 (EOA23063), CrPRK7 (EOA18255), and

CrPRK8 (EOA34527). D, A sequence alignment of AtPRK3, AtPRK6 and CrPRK6. Signal peptide, N-terminal cap, LRR1–LRR6, transmembrane domain and kinase domain are indicated beneath the

alignment. EXTENDED DATA FIGURE 7 SEMI-_IN-VIVO_ POLLEN TUBE GROWTH AND RESPONSE TO THE ATLURE1 PEPTIDE OF PRK6 VARIANT MUTANTS. Pollen tubes of _prk6_, _prk3 prk6_, _prk3 prk6 prk8-2_ and

_prk1 prk3 prk6_ mutants were assessed in this assay. Full-length PRK6, the PRK6 orthologue of _C. rubella_ (CrPRK6), kinase-domain-deleted PRK6 (K-del), and cytosolic-domain-deleted PRK6

(cyto-del-2) were expressed as mRuby2 fusion proteins under the control of their own promoters. Upper differential interference contrast images show semi-_in-vivo_ pollen tube growth in the

medium containing the AtLURE1 peptide at 6 HAP. Yellow arrowheads mark some of the pollen tubes showing apparent wavy phenotype. The bottom two images are a blight field image and a confocal

image for mRuby2 of a representative pollen tube in the wavy assay. The data are representative images of at least three assays for one or two lines of each genotype. Scale bars, 200 μm

(top) and 20 μm (bottom). EXTENDED DATA FIGURE 8 A CONSERVED BASIC AMINO ACID PATCH OF LURE IS ESSENTIAL FOR ATTRACTION. A, The sequence of full-length AtLURE1.2 accompanied by

lysine/arginine residues (yellow highlight) mutated to glycines for AtLURE1.2(GGGG). Cysteine residues in the mature peptide are shown in red. B, C, Semi-_in-vivo_ attraction assay using

gelatine beads containing 5 μM His–AtLURE1.2(GGGG) (B) and wavy assay using 10 μM His–AtLURE1.2(GGGG) in the medium (C). The AtLURE1.2(GGGG) peptide showed no activity in these assays. The

data are representative of 14 or 3 samples for B or C, respectively. Scale bars, 20 μm (B) and 200 μm (C). SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION This file contains a

Supplementary Discussion and Supplementary Figures 1-2 which show uncropped scans with size marker indications. (PDF 289 kb) SUPPLEMENTARY TABLE 1 This table contains a list of primers used

in the study. (XLS 34 kb) TIME LAPSE IMAGING OF PRK6 MRUBY2 DURING POLLEN TUBE TIP GROWTH PRK6 mRuby2 was observed predominantly at the plasma membrane of the tip and detected in cytoplasmic

granules with cytoplasmic streaming. Scale bar, 10 μm. (MOV 1215 kb) TIME LAPSE IMAGING OF SEMI-_IN VIVO_ POLLEN TUBE GROWTH AND RESPONSE TO THE ATLURE1 PEPTIDE Pollen tubes of wild-type

(Col-0), _prk6_, and _prk6_ expressing PRK6-mRuby2 (PRK6-mRuby2) grew straight in the absence of AtLURE1 peptides in the medium (-). In this observation, the pollen tube growth rate at 3-4 h

after pollination (180-240 min) was 200 ± 25 µm/h in the wild type and 151 ± 18 µm/h in _prk6_. Col-0 and PRK6-mRuby2, but not _prk6_, showed the wavy growth phenotype in the presence of 1

µM AtLURE1.2 in the medium (+). (MOV 2319 kb) TIME LAPSE IMAGING OF PRK6 MRUBY2 AND ALEXA488-LABELED ATLURE1.2 DURING POLLEN TUBE REORIENTATION Time-lapse images captured every 5 s shown in

Fig. 4a-f. Overlaid images of PRK6-mRuby2 and Alexa488-labeled AtLURE1.2 (left) and intensity images for PRK6-mRuby2 (right) are shown. A gelatine bead containing 5 µM Alexa488-labeled

AtLURE1.2 was used in this assay. Scale bar, 10 μm. (MOV 3680 kb) AN ADDITIONAL EXAMPLE OF TIME LAPSE IMAGING OF PRK6 MRUBY2 AND ALEXA488-LABELED ATLURE1.2 DURING POLLEN TUBE REORIENTATION

An additional example of the assay shown in Supplementary Video 3. Scale bar, 10 μm. (MOV 5087 kb) POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE

FOR FIG. 3 POWERPOINT SLIDE FOR FIG. 4 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Takeuchi, H., Higashiyama, T. Tip-localized receptors control

pollen tube growth and LURE sensing in _Arabidopsis_. _Nature_ 531, 245–248 (2016). https://doi.org/10.1038/nature17413 Download citation * Received: 09 June 2015 * Accepted: 15 February

2016 * Published: 09 March 2016 * Issue Date: 10 March 2016 * DOI: https://doi.org/10.1038/nature17413 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this

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