LIP1 and LIP2 are palmitoylation-anchored receptor-like cytoplasmic kinases at the tip of Arabidopsis pollen tubes whose simultaneous loss impairs micropylar guidance and reduces attraction to the ovule-secreted peptide AtLURE1. The original 2013 question asked which membrane receptor binds AtLURE1 and how LIP1/LIP2 participate in its perception; the receptor-identity part was resolved in 2016 by the discovery of PRK6 and the MDIS1-MIK1/2 heteromer as LURE1 receptors, with PRK6 shown to associate with LIP1/LIP2 and ROPGEFs and PRK1 with LIP1. The anchoring-enzymology part was largely resolved for LIP1 in 2024, when the redundant class A S-acyltransferases PAT1, PAT2, PAT3, PAT4 and PAT8 were shown to S-acylate LIP1 (and PRK1) and to be required for AtLURE1.2-directed guidance. What remains open is the transduction mechanism itself: how AtLURE1 binding regulates the assembly and kinase activities of the PRK6/PRK1-LIP1/LIP2-ROPGEF module, what LIP1/LIP2 phosphorylate, which S-acyltransferase anchors LIP2 and how the anchoring of LIP1/LIP2 is regulated during pollen-tube growth, which molecular components account for the attraction retained by lip1 lip2 pollen tubes, and what causes the in-vivo-specific branching phenotype of lip1 lip2 pollen tubes.

Background

Flowering plants deliver immotile sperm cells to the female gametophyte (embryo sac) through the polarized tip growth of the pollen tube. In the final, micropylar step of this journey (the micropyle is the narrow opening of the ovule through which the pollen tube enters), two synergid cells flanking the egg secrete small cysteine-rich defensin-like peptides that act as chemoattractants: LURE peptides in Torenia and the AtLURE1 family in Arabidopsis thaliana (Takeuchi and Higashiyama 2012, doi:10.1371/journal.pbio.1001449). A central question is how the pollen tube, the male partner, perceives these peptides at its tip and converts them into directional growth. Receptor-like kinases (RLKs) are single-pass membrane proteins with an extracellular ligand-binding domain and an intracellular kinase domain, whereas receptor-like cytoplasmic kinases (RLCKs) lack the extracellular and transmembrane parts and act inside the cell, often as signaling partners physically associated with RLK receptor complexes. Liu et al. (2013, doi:10.1016/j.cub.2013.04.043) identified two pollen-tube-expressed RLCKs of subfamily VII, LIP1 and LIP2 (Lost In Pollen tube guidance 1 and 2), that localize to the plasma membrane of the tube tip through S-palmitoylation (the enzyme-catalyzed attachment of the fatty acid palmitate to N-terminal cysteine residues; the responsible enzymes in plants are DHHC-motif protein S-acyltransferases). Because LIP1/LIP2 have no extracellular domains, they cannot bind the extracellular peptide themselves and were proposed to be intracellular components of an AtLURE1 receptor complex. Genetic evidence supports an essential role: lip1 lip2 double mutants show male-specific transmission defects (about 32-43% male transmission instead of the expected 50%), only about 44% of their pollen tubes enter the ovule micropyle (versus 94% for wild type), about 10% branch abnormally on the ovule surface in vivo, and in an in vitro bead assay only 70% respond to recombinant AtLURE1.2 (versus 95% of wild-type tubes). Five mechanistic threads were left open: the identity of the ligand-binding receptor(s), the interaction partners and assembly order of the receptor complex, the explanation of the residual attraction, the basis of the branching phenotype, and the enzymology of LIP1/LIP2 palmitoylation. Two of these have since been settled in part. The receptor-identity thread was resolved: the leucine-rich repeat RLK PRK6 is the essential tip-localized AtLURE1 receptor (Takeuchi and Higashiyama 2016, doi:10.1038/nature17413), the MDIS1-MIK1/MIK2 receptor heteromer (MALE DISCOVERER 1 with its interacting kinases MDIS1-INTERACTING KINASE 1 and 2) binds LURE1 as an additional perception module (Wang et al. 2016, doi:10.1038/nature16975), the AtLURE1.2-PRK6 extracellular complex has been crystallized (Zhang et al. 2017, doi:10.1038/s41467-017-01323-8), and PRK6 cooperates with the related PRK1, PRK3 and PRK8 kinases and associates with ROP guanine-nucleotide-exchange factors (ROPGEFs, activators of the ROP1 GTPase that drives tip growth). PRK6 has also been reported to associate with LIP1/LIP2 in planta, and PRK1 was shown to interact with LIP1 by split-luciferase and FRET assays, placing LIP1/LIP2 between receptor activation and the tip-growth machinery. On the anchoring side, Xiang et al. (2024, doi:10.1093/plcell/koae109) identified the redundant class A S-acyltransferases PAT1, PAT2, PAT3, PAT4 and PAT8 (collectively PENTAPAT) as enzymes whose loss reduces LIP1 S-acylation and plasma-membrane association and compromises AtLURE1.2-directed guidance. However, no study has examined how ligand binding changes the receptor-kinase associations or the kinases' activities, no phosphorylation substrate of LIP1/LIP2 has been identified, no S-acyltransferase has been assigned to LIP2, and the regulation of LIP1/LIP2 anchoring during pollen-tube growth remains unaddressed; functional work after 2013 has concentrated on the PRK6 and MDIS1-MIK branches and on the female side rather than on LIP1/LIP2 signaling.

Problem Statement

The goal is to determine the LIP1/LIP2-dependent signal-transduction mechanism by which the pollen tube converts AtLURE1 perception into guided tip growth in Arabidopsis thaliana. Its scientific objective is to complete the signaling branch from ligand perception to growth reorientation at the pollen-tube tip; the following open aspects (1)-(5) are facets of this mechanism question rather than a jointly required completion checklist: a solution must establish at least the ligand-regulated assembly and activation of the module (1) and the immediate downstream substrates of LIP1/LIP2 (2), whereas aspects (3)-(5) are related open problems whose treatment is not required to resolve the mechanism question. (1) Ligand-regulated assembly and activation: establish how AtLURE1 binding to PRK6 (and/or the MDIS1-MIK1/2 heteromer; PRK1, which also interacts with LIP1, may participate) changes the association, localization and kinase activities of the PRK6/PRK1-LIP1/LIP2-ROPGEF complex at the tube tip, including the assembly order of its components. (2) Downstream targets: identify the immediate protein substrates phosphorylated by LIP1 and LIP2 and how they connect to the ROP1-dependent tip-growth machinery. (3) Anchoring enzymology: identify the protein S-acyltransferase(s) responsible for palmitoylating the N-terminal cysteines of LIP2 (for LIP1 the class A enzymes PAT1/2/3/4/8 were assigned in 2024), verify the LIP2 modification biochemically, and establish how the anchoring of LIP1 and LIP2 is regulated during pollen-tube growth. (4) Residual attraction: determine which molecular components account for the LIP1/LIP2-independent attraction retained by lip1 lip2 pollen tubes. (5) Phenotype mechanism: explain the cellular and molecular basis of the branching that lip1 lip2 pollen tubes display on the ovule surface in vivo but not in vitro.

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