Plant Reproduction

The male germ unit (MGU) in Arabidopsis pollen is comprised of one vegetative nucleus (VN) and two sperm nuclei (SN). It is evolutionarily specialized to deliver immotile sperm nuclei to an ovule for fertilization. Despite some progress in research on MGU, its organization and transport remain only partially understood. Here, we identified Nucleoporin1/136 as a new player in the structural organization and positioning of MGU in pollen tubes. We and others have previously reported the reduced fertility of nup1-1 plants; however, the mechanism remains unknown. In this work, we further examined the role of NUP1 in fertility using two mutant alleles, nup1-1 and nup1-2-/+. The reciprocal crosses between the nup1 mutants and the Col-0 wild type indicate that the nup1 mutant pollen is defective. To study the effect of a complete NUP1 knockout on pollen, we generated a transgenic line that produces pollen with and without NUP1 expression. This work led to the surprising discovery that the NUP1 protein is inherited from the pollen mother cell to the daughter cell during microgametophyte development. Subsequent in vitro experiments showed that NUP1 is required for pollen germination and pollen tube elongation. Further microscopic studies demonstrated that NUP1 is highly expressed in VN and essential for maintaining nuclear shape and size. We also demonstrated that NUP1 is required for proper MGU organization and transport, likely through maintaining VN morphology. Notably, our finding of nuclear morphology-mediated regulation of MGU may also explain the mechanistic details underlying the defective MGU movement in previously reported mutants such as kaku4, wit, and wip, which have abnormal nuclear morphology.

Following pollination, Arabidopsis pollen grains rapidly hydrate through the transfer of water from the stigma to the pollen. Several stigma regulators of pollen hydration have been identified, and the corresponding mutants generally support milder defects in wildtype Col-0 pollen hydration, signifying the involvement of other unidentified factors in this process. Here, we uncovered a role for the stigma-specific mechanosensitive channel gene, MscS-Like 7 (MSL7), in supporting pollen hydration. While the msl7 mutant stigmas were found to support reduced hydration of wildtype Col-0 pollen, the phenotype was quite mild and very similar to that observed for other published pollen hydration mutants. Thus, we conducted a detailed comparison of different pollen hydration mutants on the stigma side (receptor kinases, PIP aquaporins) and pollen (PCP-Bs, MLS8) to compare pollen hydration mutant phenotypes and look for any additive effects of combining different mutants. Overall, all combinations resulted in the same mild hydration defect with no additional reductions in pollen hydration and no impact on pollen germination. This is in contrast to that observed for self-incompatible (SI) pollen from a transgenic Arabidopsis SI Col-0 line which shows very little pollen hydration and no pollen germination as part of the SI pollen rejection response. Together, these findings suggest that the regulation of compatible pollen hydration is quite complex and that there are likely other unknown mechanisms involved. Key MessageThe same mild pollen hydration defect is observed across very different Arabidopsis stigma mutants and it does not prevent pollen germination and pollen tube growth.

In the Brassicaceae, the process of accepting compatible pollen is a key step in successful reproduction and highly regulated following interactions between the pollen and the stigma. Central to this, is the initiation of secretion in the stigma, which is proposed to provide resources to the pollen for hydration and germination and pollen tube growth. Previously, the eight exocyst subunit genes were shown to be required in the Arabidopsis stigma to support these pollen responses. One of the roles of the exocyst is to tether secretory vesicles at the plasma membrane for membrane fusion by the SNARE complex to enable vesicle cargo release. Here, we investigate the role of Arabidopsis SNARE genes in the stigma for pollen responses. Using a combination of different knockout and knockdown SNARE mutant lines, we show that VAMP721, VAMP722, SYP121, SYP122 and SNAP33 are involved in this process. Significant disruptions in pollen hydration were observed following pollination of wildtype pollen on the mutant SNARE stigmas. Overall, these results place the Arabidopsis SNARE complex as a contributor in the stigma for pollen responses and reaffirm the significance of secretion in the stigma to support the pollen-stigma interactions. Key MessageThe VAMP721, VAMP722, SYP121, SYP122 and SNAP33 SNAREs are required in the Arabidopsis stigma for pollen hydration, further supporting a role for vesicle trafficking in the stigmas pollen responses.

In Brassicaceae self-incompatibility (SI), self-pollen rejection is initiated by the S-haplotype specific interactions between the pollen SCR/SP11 ligand and the stigma S Receptor kinase (SRK). In Brassica SI, a member of the Plant U-Box (PUB) E3 ubiquitin ligases, ARC1, is then activated by SRK in this stigma and cellular events downstream of this cause SI pollen rejection by inhibiting pollen hydration and pollen tube growth. During the transition to selfing, Arabidopsis thaliana lost the SI components, SCR, SRK, and ARC1. However, this trait can be reintroduced into A. thaliana by adding back functional copies of these genes from closely related SI species. Both SCR and SRK are required for this, though the degree of SI pollen rejection varies between accessions, and ARC1 is not always needed to produce a strong SI response. For A. thaliana C24, only transforming with A. lyrata SCR and SRK confers a strong SI trait, and so here we investigated if ARC1-related PUBs were involved in the SI pathway. Two close ARC1 paralogs, PUB17 and PUB16, were selected, and CRISPR/Cas9 technology was used to generate pub17 and pub16 mutations in the C24 accession. These mutants were then crossed into a transgenic A. thaliana SI-C24 line and their potential impact on SI pollen rejection was investigated. Overall, we did not observe any significant differences to implicate PUB17 and PUB16 functioning in the transgenic A. thaliana SI-C24 stigma to reject SI pollen.

Pollen tubes are dynamic tip-growing cells that deliver sperm nuclei to female gametes in flowering plants, allowing for sexual reproduction and seed formation. Actin and microtubule cytoskeletons both play important roles in directional pollen tube growth and guidance. While actin dynamics are well-studied in pollen tubes, the role of microtubules and the interactions between these two cytoskeletal filaments are less well understood. To address this knowledge gap, we imaged growing Arabidopsis thaliana pollen tubes co-expressing fluorescently-labeled tubulin and actin markers and observed partial co-localization of actin and microtubule filaments. We found that treatment with microtubule disrupting drugs did not affect the actin cytoskeleton. In contrast, when actin filaments were depolymerized, microtubules in the medial region of pollen tubes were disrupted, while microtubules at the cell cortex remained intact. Thus, the microtubule cytoskeleton in A. thaliana pollen tubes relies on the actin cytoskeleton in a spatially dependent manner. Furthermore, we utilized native expression of the microtubule plus-end binding protein EB1b to track microtubule orientation in growing pollen tubes. We found the microtubule array to be largely parallel, with plus ends growing away from the tube apex. Together, these findings offer new insights into the dynamics and organization of microtubules in growing pollen tubes and the interactions between actin filaments and microtubules.

Previous studies have alternately supported and discounted the hypothesis that the maternal genome plays a predominant role in early embryogenesis in plants. We used 24 embryo defective (emb) mutants of Arabidopsis thaliana to test for maternal and paternal effects in early embryogenesis. 5 emb mutants had equal maternal and paternal effects, 5 showed maternal effects and weak paternal effects, and the remaining 14 emb mutants conditioned only maternal effects, demonstrating a more important role for the maternal allele for most of these EMB genes. To assess genome-wide maternal and paternal contributions to early embryos, we produced allele-specific transcriptomes from zygote to mature stage embryos derived from reciprocal crosses of Columbia-0 and Tsu-1, a hybrid combination we show to be a faithful proxy for isogenic Columbia-0. Parent-of-origin analysis of these transcriptomes revealed a reciprocal maternal bias in thousands of genes from the zygote to octant stage. This bias greatly diminished by the globular stage, and was absent at later stages. Comparison with egg cell transcriptomes revealed no correlation between transcript levels in the egg and maternal bias in pre-globular embryos, suggesting that the maternal bias observed in early embryos is due to preferential zygotic transcription of maternal alleles. Taken together, the functional and transcriptome data presented here support a predominant role for the maternal genome in early Arabidopsis embryogenesis. SignificanceIn both animals and plants, the zygote is produced by the union of the egg and sperm cells. In animals, it is well accepted that mRNAs and proteins from the egg direct the first steps of embryogenesis. Here we present genetic and genomic experiments that support a predominant role for the maternal genome in early embryogenesis of plants, as well. In contrast to animals, our data suggest that this maternal influence is primarily derived not from inheritance of egg transcripts, but from preferential transcription of maternal alleles in the zygote and early embryo. This transient maternal zygotic bias may reflect an ancestral condition to diminish paternal influence on early embryogenesis in outcrossing plants.

Arabidopsis lines with loss-of-function mutations in the gene encoding Embryo sac Pectin MethylEsterase Inhibitor (Atepmei) were found to have short silique and high seed sterility. Examination of tissue-cleared mature ovules (FG7-stage) revealed irregularly positioned nuclei within the embryo sacs. Instead of horse-shoe-shaped ovules, defective globular ovules without proper micropylar and chalazal ends were found. Embryo sac cell-type-specific GFP marker expression studies confirmed gamete and accessory cell identity alterations. Egg cell-specific marker (DD45) expression analysis confirmed the presence of multiple egg cells in the mutant embryo sacs, possibly due to defect in embryo sac cellularization. These supernumerary egg cells were functional as evident from the production of twin embryos when supernumerary sperm cells were provided. The results of Ruthenium red and tannic acid-ferric chloride staining of Atepmei mutant developing ovules, conferred its interaction with the specific PME in proper cell wall formation and maintenance around embryo sac nuclei which also coincide with its fate as a specific gamete. This is the first report implicating role of cell wall in gamete cell fate determination by altering cell-cell communication. Our analysis of the twin-embryo phenotype of epmei mutants also sheds light on the boundary conditions for double fertilization in plant reproduction.

Hybrid crops are agriculturally desirable due to heterosis but are costly or difficult to produce. Synthetic apomixis, clonal reproduction through seed, offers the ability to fix hybrid vigor. Two components are needed to achieve this goal: the formation of diploid gametes identical to the maternal parent through apomeiosis, and the induction of embryogenesis in the egg cell without fertilization, known as parthenogenesis. In maize, parthenogenesis was achieved by egg cell expression of the transcription factor ZmBABY BOOM 1 but a viable apomeiosis strategy has not been reported. In the MiMe (Mitosis instead of Meiosis) system, in addition to mutants that skip recombination and sister chromatid adhesion, mutation of genes involved in cell cycle control during meiosis is needed to skip the second division and ensure diploid gametes. In this report we describe the effect of mutation of maize A-type cyclin genes with similarity to Arabidopsis TARDY ASYNCHRONOUS MEIOSIS (TAM). In double mutant plants, we find that diploid gametes are formed with high efficiency and that the progeny are tetraploid. These genes provide a viable route towards creating synthetic apomixis in maize.

In flowering plants, pollen tubes communicate with ovular cells to achieve precise one-to-one pollen tube reception. The final step of this communication between the pollen tube and synergid cells has been extensively investigated and visualized by calcium imaging. Synergid cells exhibit characteristic cytoplasmic calcium concentration oscillations, which are thought to play a critical role in pollen tube reception. However, their significance and relationship with calcium dynamics in the entire ovule remain unclear. Here, we show, using the calcium sensor GCaMP6s, that proteins involved in asparagine-linked glycosylation (N-linked glycosylation) are required for normal calcium oscillations in synergid cells but are not essential for pollen tube reception. Using a semi-in vivo assay in Arabidopsis thaliana, we found that the amplitude of these oscillations prior to rapid pollen tube growth across the filiform apparatus was reduced in mutants lacking the oligosaccharyltransferase (OST) 3/6 subunit or alpha1,2-glucosyltransferase (ALG) 10, both of which are involved in N-linked glycosylation. Notably, these mutants did not exhibit reduced fertility attributable to defects in the female gametophyte but instead showed a polytubey phenotype due to a sporophytic defect. These findings suggest that N-linked glycans mediate communication between synergid cells and the pollen tube and indicate that the typical pattern of calcium oscillations in synergid cells is not essential for triggering pollen tube rupture. Furthermore, we show that sporophytic tissues of the ovule exhibit calcium waves that propagate toward the funiculus in correlation with pollen tube contact and rupture, implying that ovular tissues can potentially transmit these signals distantly beyond the ovule. Together, these findings reveal previously unrecognized intercellular calcium signaling and its significance in pollen tube reception by the ovule.

Brassinosteroids (BRs) are steroid hormones identified in plants. Besides promoting cell elongation and division, BRs facilitate the development of both male and female reproductive tissues. In animals, reproductive steroid hormones play an essential role in reproductive tissue development by regulating gene expression. Here, we focused on the function of BRs during fertilization. We measured the content of biologically active BRs, brassinolide (BL) and castasterone (CS), in the reproductive tissues of Arabidopsis thaliana. Both BL and CS accumulated abundantly in pollen grains and in larger amounts in pistils than in leaves. To evaluate BL function during fertilization, we used an in vitro guidance assay with exogenously applied BL. Although pollen tubes need to be elongated through the pistils for efficient capacitation, BL treatment promoted pollen tube capacitation and improved attraction to ovules in vitro. Transcriptome analysis demonstrated that BL treatment induced the expression of half of the genes expressed in pollen tubes that elongated through the pistils. These results indicated that BL supplied from pistils is a key factor for pollen tube capacitation. However, using the bri1 mutant for the guidance assay resulted in reduced pollen tube capacitation, suggesting that BRI1-signaling in pistils is also important. Furthermore, BRs act on ovules. Exogenous BL application to ovules maintained guidance capacity by promoting the expression of small secreted proteins involved in pollen tube attraction and gamete fusion. Overall, BRs play a significant role as male and female reproductive hormones throughout the plant fertilization process.

The angiosperm ovule is characterized by the close association of the two generations, with the haploid female gametophyte or embryo sac being encapsulated by the diploid sporophyte, which usually forms two integuments. How the gametophyte and sporophyte coordinate their development has long been of interest. However, the function of the inner integument in embryo sac development has remained elusive. Here, we addressed this question. We applied a genetic ablation strategy to achieve an early block in inner integument outgrowth. We generated plants expressing BARNASE under the control of an early acting endothelium-specific promoter. Corresponding lines carried ovules lacking most of the inner integument. The genetic and cell biological data revealed that in the near absence of an inner integument embryo sac development is blocked at the mono-nuclear embryo sac stage in most pre-fertilization ovules. Approximately 10 percent of the ovules developed a functional embryo sac and underwent fertilization. Subsequent embryo and endosperm development appeared unperturbed and viable seeds were produced albeit of altered shape. Our results show that the inner integument plays an important role in early embryo sac development as well as ovule and seed shape, but is dispensable for embryo and endosperm development. Key words: embryo sac, embryo, endothelium, female gametophyte, integument, seed development HighlightGenetic ablation of the inner integument demonstrates its role in embryo sac development and its irrelevance for embryogenesis.

The plant male reproductive system is very sensitive to high temperature stress leading to a reduction in fertility. Damage caused by heat stress is restored by the activation of transcription and the synthesis of chaperones that regulate the heat stress response. Here we report that AtRRB1 is a homolog of the yeast ribosome chaperone protein Rrb1p. AtRRB1 is an essential gene and a T-DNA insertion in the coding region impairs male and female gametogenesis. The heterozygous rrb1-1 mutant shows decreased expression of AtRRB1 and increased transcription of the 60S ribosome proteins RPL3B and RPL4, in line with a chaperone role of AtRRB1 in ribosome biogenesis. Embryo sac development across ovules of a single pistil occurs uncoordinated and about half of the ovules abort. Half of rrb1-1 pollen is substantially smaller and produce shorter pollen tubes than WT pollen. In contrast to the Col-0 pollen, smaller pollen is overly sensitivity to high temperature (24h at 32{degrees}C) treatment, specifically during the early bicellular microspore development stage. Heat stressed rrb1-1 bicellular microspores accumulated excessively rough endoplasmic reticulum stacks, suggesting that loss of AtRRB1 activity causes an arrest in ER associated protein biosynthesis. These findings support a critical requirement for ribosome biogenesis and protein synthesis in bicellular microspores to recover from high temperature stress.

Sexual reproduction in flowering plants relies on double fertilization, a process marked by two fusion events between the male and female gametes that lead to seed formation. Because this process unfolds within the embryo sac embedded deep inside the ovule, direct observation remains technically demanding, especially in maize, where the large size of female reproductive organs presents additional obstacles. The described method enables high-resolution visualization of cellular events unfolding during maize double fertilization. The approach integrates optimized fixation, clearing and confocal imaging of embryo sacs from ears pollinated with fluorescent pollen marker lines. Precise timing of embryo sac fixation is critical, allowing capture of key events such as pollen peri-germ cell membrane break-down or gamete karyogamy. The protocol provides detailed guidance for ovule dissection, fixation, preparation and renewal of the clearing solution and confocal imaging of embryo sacs. This method offers unprecedented access to the cellular events of double fertilization in maize, establishing a robust framework for studying reproductive processes and supporting future discoveries in plant reproduction.

The pollen tube plays a pivotal role in double fertilization by delivering sperm cells (SCs) to the ovule. In Arabidopsis thaliana, a pair of SCs tightly connects with the vegetative nucleus (VN) to form the male germ unit (MGU), which is located in the apical region during pollen-tube growth, keeping the VN ahead of the SCs. MGU transport relies on independent motility of VN and SC pairs. However, the complexity of this dual motive force has hindered our understanding of MGU behavior, including its positioning and nuclear order. We used Arabidopsis mutants or transgenic plants that produced semi-motile MGUs with aberrant VNs or SCs to analyze the positioning of VN or SCs after stochastically disconnecting the MGU. In pollen tubes with an immotile SC pair, the VN was [~]70 m away from the tip, whereas in pollen tubes with an immotile VN, the SC pair was [~]100 m away from the tip, implying that the VN and SCs have independent home positions. The position of MGU moved forward owing to the loss of the microtubule-destabilizing kinesin KINESIN-13A. Conversely, microtubule depolymerization by oryzalin treatment or introducing mutations in TUBULIN BETA 4 (TUB4) deregulated the position of the MGU and shifted its position backward. In addition, tub4 plants exhibited reduced fertility. These data indicate a significant role of microtubules in stable MGU positioning to ensure reproductive success.

Hydroxyproline O-arabinosylation is a highly-conserved and plant-specific post-translational modification found on extensins and other structural proteins in the cell wall, and is catalyzed by Hydroxyproline O-arabinosyltransferases (HPATs). In Arabidopsis, loss of HPAT1 and HPAT3 (hpat1/3) causes reorganization of components in the pollen tube (PT) cell wall, which compromises cell wall structural integrity and decreases PT growth and fertility. We have previously shown that reduced secretion (caused by loss-of-function mutations in secretory genes EXO70A2, SEC15A, and SEC1A) suppressed cell wall defects and strongly rescued poor growth and fertility in hpat1/3 PTs. Here, we show that a missense mutation in PHOSPHOLIPASE C6 (PLC6) also rescues hpat1/3 PT growth and fertility. Transgenic insertion mutations that disrupt PLC6 expression did not improve hpat1/3 pollen fertility, and did not affect PT growth or fertility in the wild type background. This data suggests that our missense allele (plc6-4) does not function like a true loss-of-function allele, and that PLC6 is not required for wild type PT growth. However, in the absence of hpat1/3, plc6-4 PTs have defects in transmission and polarized growth, as indicated by meandering growth paths and a resulting crooked appearance. plc6-4 PT elongation and straightness are more sensitive to elevated levels of calcium than wild type. This may be due the nature of the plc6-4 mutation, which causes an E569K amino acid substitution in the lipid-binding C2 domain. The 569 position is located among conserved residues that bind calcium. The resulting charge inversion caused by the E569K substitution may disrupt PLC6s lipid binding and phospholipase activities. Here, we show that PLC6 influences polarized PT growth and HPAT-mediated PT growth and fertility, and future studies are necessary to better understand the relationship between calcium and PLC6 in PT growth.

In flowering plants, continuous cell-cell communication between the compatible male pollen grain/growing pollen tube and the female pistil is required for successful sexual reproduction. In Arabidopsis thaliana, the later stages of this dialogue are mediated by several peptide ligands and receptor kinases that guide pollen tubes to the ovules for the release of sperm cells. Despite a detailed understanding of these processes, a key gap remains on the nature of the regulators that function at the earlier stages. Here, we report on two groups of A. thaliana receptor kinases, the LRR-VIII-2 RK subclass and the SERKs, that function in the female reproductive tract to regulate the compatible pollen grains and early pollen tube growth, both essential steps for the downstream processes leading to fertilization. Multiple A. thaliana LRR-VIII-2 RK and SERK knockout mutant combinations were created, and several phenotypes were observed such as reduced wild-type pollen hydration and reduced pollen tube travel distances. As these mutant pistils displayed a wild-type morphology, the observed altered responses of the wild-type pollen are proposed to result from the loss of these receptor kinases leading to an impaired pollen-pistil dialogue at these early stages. Furthermore, using pollen from related Brassicaceae species, we also discovered that these receptor kinases are required in the female reproductive tract to establish a reproductive barrier to interspecies pollen. Thus, we propose that the LRR-VIII-2 RKs and the SERKs play a dual role in the preferential selection and promotion of intraspecies pollen over interspecies pollen.

Evidence from the model bryophytes Physcomitrium and Marchantia suggests that a BELL-KNOX genetic module acts as a master regulator controlling sporophyte identity. Investigating any conservation of this system in flowering plants has proved challenging, but studies of the Arabidopsis eostre mutant and naturally apomictic angiosperms point to ectopic activation of KNOX and BELL transcription factors mediating the switch from sexual to apomictic development. We show here that in Arabidopsis, ectopic expression, under a germline-specific promoter, of KNOX and BELL genes not normally expressed in the gametophytes both disrupts germ cell specification and causes defects in cell identity throughout gametophyte development - some mirroring events seen in naturally apomictic plants. A better understanding of this TALE-HD genetic module in flowering plants may thus help to unravel the molecular control of higher plant life cycles, while providing a route to engineering synthetic apomixis in crops. This study also highlights the utility of applying data from bryophytes, where the ontogeny transitions are spatio-temporally distinct, to apomixis research in angiosperms.

In angiosperms, pollen tubes deliver sperm cells to the ovule and communicate with the external environment as they elongate through the pistils. Although pollination alters Ca2+ conditions within the pistil, the effects of extracellular Ca2+ fluctuations on pollen tube growth and guidance remain largely unknown. In this study, we visualized intracellular Ca2+ dynamics using a semi-in vivo assay with the Ca2+-sensitive fluorescent protein GCaMP6s to investigate how pollen tubes respond to changes in extracellular Ca2+ levels. We found that the Ca2+ levels in the apical region of the pollen tubes reflected the extracellular Ca2+ concentrations. The pollen tube growth rate increased depending on the Ca2+ concentration in the growth medium. However, excessive Ca2+ affected the polar growth of pollen tubes. At elevated Ca2+ concentrations of 10 mM, the pollen tube exhibited coiling behavior and failed to maintain directional growth toward the ovule. Moreover, we provided the first evidence that Ca2+ oscillations are not restricted to the apical region but propagate as a wave, reaching 30-50 m from the apex toward the basal regions. As the pollen tube approached the ovule, it coincided with a substantial elevation in Ca2+ levels, which appeared to drive the accelerated nuclear migration toward the tube apex. Our findings demonstrate that the extracellular Ca2+ environment directly regulates intracellular Ca2+ levels in pollen tubes, thereby influencing their growth and guidance.

Quantitative pollen viability analysis is a critical but labor-intensive step in plant reproductive biology. Existing deep-learning Segment Anything Models (SAM) fail to reliably segment viable pollen in Alexander-stained anthers. To address this, we fine-tuned an existing Cellpose-SAM model for pollen segmentation. We integrated it into PAT (Pollen Analysis Tool), a cross-platform desktop application. PAT features instance segmentation with interactive quality control, an in-app model retraining module, and publication-ready statistical outputs. We deployed PAT in an EMS suppressor screen of semi-sterile Arabidopsis smg7-6 mutants, enabling efficient candidate prioritization for whole genome sequencing and mapping candidate mutation. This screen led to the identification of a point mutation in CAP-D2 (capd2-2), a Condensin I subunit, that rescues the smg7-6 meiotic phenotype. Notably, mutation in a Condensin II subunits (CAP-D3 and CAP-H2) does not confer rescue. Further characterization suggests the capd2-2 allele is hypomorphic, showing no defects in vegetative growth, chromocenter compaction, or transposable element silencing. Collectively, we demonstrate that accessible AI tools have the potential to bridge gaps in plant phenotyping and accelerate the pace of biological discovery. HighlightWe combined AI-powered image analysis with an easy-to-use desktop app to automate plant pollen counting, then used it to identify a new genetic suppressor of meiotic defects.

Flowering plants contain tightly controlled pollen-pistil interactions required for promoting intraspecies fertilization and preventing interspecies hybridizations. In Arabidopsis, several receptor kinases (RKs) are known to regulate the later stages of intraspecies pollen tube growth and ovular reception in the pistil, but less is known about RK regulation of the earlier stages. The Arabidopsis RKF1 cluster of Leucine-Rich Repeat Malectin (LRR-MAL) RKs was previously found to function in the stigma to promote intraspecies pollen hydration. Here, we tested additional combinations of up to seven Arabidopsis LRR-MAL RK knockout mutants for the RKF1 cluster, LIK1, RIR1 and NILR2. These LRR-MAL RKs were discovered to function in the female stigma to support intraspecies Arabidopsis pollen tube growth and to establish a pre-zygotic interspecies barrier against Capsella rubella pollen. Thus this study uncovered new biological functions for these poorly understood group of RKs in regulating the early stages of Arabidopsis sexual reproduction.