
==== Front
Plant Commun
Plant Commun
Plant Communications
2590-3462
Elsevier

S2590-3462(24)00204-9
10.1016/j.xplc.2024.100934
100934
Research Article
Pollen-expressed RLCKs control pollen tube burst
Xu Yin-Jiao 14
Luo Ting 14
Zhou Peng-Min 14
Wang Wei-Qi 1
Yang Wei-Cai 1
Li Hong-Ju hjli@genetics.ac.cn
23∗
1 Center for Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
2 Center for Molecular Agrobiology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
3 Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
∗ Corresponding author hjli@genetics.ac.cn
4 These authors contributed equally to this article.

30 4 2024
12 8 2024
30 4 2024
5 8 10093425 9 2023
13 2 2024
26 4 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
In angiosperms, the pollen tube enters the receptive synergid cell, where it ruptures to release its cytoplasm along with two sperm cells. This interaction is complex, and the exact signal transducers that trigger the bursting of pollen tubes are not well understood. In this study, we identify three homologous receptor-like cytoplasmic kinases (RLCKs) expressed in pollen tubes of Arabidopsis, Delayed Burst 1/2/3 (DEB1/2/3), which play a crucial role in this process. These genes produce proteins localized on the plasma membrane, and their knockout causes delayed pollen tube burst and entrance of additional pollen tubes into the embryo sac due to fertilization recovery. We show that DEBs interact with the Ca2+ pump ACA9, influencing the dynamics of cytoplasmic Ca2+ in pollen tubes through phosphorylation. These results highlight the importance of DEBs as key signal transducers and the critical function of the DEB–ACA9 axis in timely pollen tube burst in synergids.

This study reports the identification of three pollen tube–expressed RLCKs, DEB1/2/3, that control pollen tube burst through interacting with the Ca2+ pump ACA9 on the plasma membrane to influence the dynamics of cytoplasmic Ca2+ in pollen tubes.

Key words

Pollen tube burst
RLCKs
Calcium signaling / transport
Double fertilization
Published: April 30, 2024
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pmcIntroduction

Flowering plants employ sophisticated fertilization strategies, allowing sperm cells to encounter female gametes. The journey begins with the germination of pollen on the stigma, leading to the growth of a pollen tube that navigates through the extracellular matrix of the pistil (Cheng and Li 2023). Guided by attractants secreted by the synergid cells, the pollen tube aims for the receptive synergid within the embryo sac (Okuda et al., 2009; Meng et al. 2019, 2023), setting the stage for intricate cell–cell communication and signaling events upon contact. These events culminate in pollen tube burst and degeneration of the synergid cell, facilitating fusion of sperm cells with the female gametes, thus initiating development of the zygote and endosperm (Li et al., 2018b; Hafidh and Honys 2021).

Several synergid cell surface proteins pivotal for pollen tube reception have been characterized. These include receptor kinases such as FERONIA (FER), HERCULES RECEPTOR KINASE 1 (HERK1), and ANJEA (ANJ), as well as MILDEW RESISTANCE LOCUS O 7 (MLO7)/NORTIA (NTA) and GPI-anchored proteins like LORELEI (LRE) and early nodulin-like proteins (ENDOLs), among others (Escobar-Restrepo et al., 2007; Capron et al., 2008; Amien et al., 2010; Kessler et al., 2010; Hou et al., 2016; Galindo-Trigo et al., 2020). These proteins play essential roles in regulating pollen tube burst in the synergid cell, with NTA being recruited by the FER/LRE receptor complex to the synergid plasma membrane, facilitating the Ca2+ influx necessary for pollen tube burst and synergid cell degeneration (Jones et al., 2017; Ju et al., 2021). Notably, Ca2+ within the synergid serves as a secondary messenger for pollen tube burst (Denninger et al., 2014; Hamamura et al., 2014; Ngo et al., 2014). In addition, pollen-derived RALFs, including RALF6, 7, 16, 36, and 37, interact with the extracellular domains of FER, ANJ, and HERK1, triggering pollen tube rupture within the receptive synergid cell (Zhong et al., 2022). Deficiencies in these components result in pollen tube overgrowth within the receptive synergid, failure of pollen tube growth arrest and burst, and entry of multiple pollen tubes, termed polytubey, due to persistent pollen tube attraction by the intact, persistent synergid cell, a phenomenon known as fertilization recovery (Kasahara et al., 2012; Meng et al., 2023).

Contrastingly, few components have been identified within the pollen tube that contribute to its burst within the synergid. In maize, the Zea mays Embryo Sac 4 (ZmES4) peptide, produced by synergids, activates a plasma membrane–localized potassium channel, KZM1, leading to pollen tube burst (Amien et al., 2010). In Arabidopsis, the transcription factors MYB97, MYB101, and MYB120, expressed in the pollen tube, are essential for this process upon synergid contact (Leydon et al., 2013; Liang et al., 2013). The receptor kinases ANXUR1 (ANX1) and Buddha’s Paper Seal 1 (BUPS1), present on the pollen tube plasma membrane, have been proposed to mediate pollen tube burst by detecting RALF34 from the inner integument (Miyazaki et al., 2009; Ge et al., 2017; Li and Yang 2018). The Ca2+ pump ACA9, localized to the pollen tube plasma membrane, is also crucial for pollen tube growth and subsequent burst within the synergid (Schiøtt et al., 2004). ACA9 is autoinhibited and belongs to the P2B-ATPase family, whose activity is often regulated by phosphorylation (Hwang et al., 2000; Sze et al., 2000; Frei dit Frey et al., 2012; Giacometti et al., 2012).

Although significant progress has been made, the precise mechanism by which the pollen tube detects synergid-derived signals to cease its elongation and initiate bursting remains unclear. Our research contributes to unraveling this enigma by highlighting the crucial roles of three receptor-like cytoplasmic kinases (RLCKs) expressed in the pollen tube—DEB1, DEB2, and DEB3—in timely burst of the pollen tube within the synergid. We found that DEB1 directly interacts with and phosphorylates ACA9. Importantly, introduction of a phosphomimic version of ACA9 counteracts the delayed pollen tube burst and abnormal growth pattern in the deb1 deb2 deb3 triple mutant, but a kinase-dead form of DEB1 is unable to rescue the mutant phenotype. Moreover, Ca2+ dynamics are altered in pollen tubes of the mutant, underscoring the intricate interplay between RLCKs and calcium signaling in the orchestration of pollen tube burst. This study provides insights into the complex molecular interactions that enable fertilization in flowering plants, enhancing our understanding of plant reproductive biology.

Results

deb1/2/3 mutants show defective pollen tube reception

Our study focused on the roles of RLCKs, which are crucial in signal transduction processes including plant development, immunity, and reproduction (Liang and Zhou 2018). To identify RLCKs required for pollen tube burst in the synergid, we investigated the transcriptome of pollen and pollen tubes available at the Genevestigator database (https://genevestigator.com). A comprehensive analysis revealed 51 RLCKs expressed in pollen and pollen tubes (Supplemental Figure S1A and S1B). Among them, three homologous genes, AT3G07070/PBL26, AT3G24790/PBL25, and AT4G13190/PBL24, were chosen for further analysis and renamed Delayed Burst (DEB) in reference to the phenotype of their mutants. The functions of these genes were assessed by evaluating the T-DNA insertion mutants SALK_023374 (deb1), SALK_072589 (deb2), and SALK_152499 (deb3), which produce no full-length transcripts, as shown by RT–PCR (Supplemental Figure 1C and 1D). Investigation of their male function via reciprocal crosses revealed a significant decrease in male transmission efficiency for single deb1 (75%), deb2 (76%), and deb3 (80%) mutants, as well as the double mutants deb1+/−deb2−/− (61%), deb1−/−deb2+/− (54%), and deb2−/−deb3+/− (16%), but their female transmission efficiency remained unaffected (Supplemental Table 1). These findings suggest compromised fertilization efficacy of deb mutant pollen tubes.

To examine the cause of the reduced male transmission efficiency, we first observed pollen tube behavior in the pistil. Wild-type pistils pollinated with pollen of the single and double mutants, as well as a triple deb1 deb2 deb3-2 mutant generated by gene-editing technology in the deb1 deb2 background (Rao et al., 2018), were stained at 24 h after pollination (HAP). Polytubey and pollen tube overgrowth in the synergid were observed for the deb mutants but not for wild-type pollen tubes (Figure 1A–1F). The percentage of defective pollen tube reception (polytubey and overgrowth) in the deb1, deb2, and deb3-1 single, deb1 deb2 double, and deb1 deb2 deb3-2 triple mutant pollen tubes increased with increasing time after pollination (5, 8, 12, 20, and 24 HAP) (Figure 1G). During the early phase (5 and 8 HAP), ovules pollinated with deb or wild-type pollen exhibited a comparable percentage of defective pollen tubes, but this percentage was significantly higher upon pollination by deb rather than wild-type pollen during the later phase (20 and 24 HAP) (Figure 1G and 1H), suggesting the effect of fertilization recovery after the defect in timely pollen tube reception. Compared with the single mutants, the double and triple mutants had a higher percentage of defective pollen tubes at 24 HAP (Figure 1G and 1H). There were no differences in seed setting rate, anther and pollen development, pollen tube growth rate, or ovule-targeting ratio between the deb mutants and the wild type (Supplemental Figures 2 and 3). Together, these results suggest that DEB1, 2, and 3 may play redundant roles in timely pollen tube burst in the synergid.Figure 1 deb pollen tubes show a polytubey and overgrowth phenotype.

(A–F) Pollen tubes of different genotypes in wild-type (WT) ovules. Arrows, more than one pollen tube entering the micropyle. Asterisk, pollen tube overgrowth. Bars, 20 μm.(G) Statistics of ovules with defective pollen tubes (combined count of pollen tube overgrowth and polytubey) upon pollination by deb single, double, and triple mutants. Each dot shown is mean ± SEM. ANOVA, ∗p < 0.1, ∗∗∗p < 0.001. n > 150 ovules per time point (hours after pollination, HAP). ns, no statistical significance.(H) Statistics of three categories of phenotype (polytubey, overgrowth, overgrowth and polytubey). n = 718, 1005, and 1023 ovules for WT, deb1 deb2, and deb1 deb2 deb3-2.

To confirm the defective pollen tube burst of the deb mutants, pollen tubes of the wild type and the deb mutants were labeled with green fluorescent protein (GFP) driven by the pollen-tube-specific promoter LAT52 and pollinated on wild-type pistils (Twell et al., 1989). At 8 HAP, the wild-type pollen tubes released the GFP-labeled pollen tube content in most ovules (<5% failed burst) (Figure 2A and 2E), whereas a large proportion of deb1 deb2 mutant pollen tubes failed to burst (Figure 2B–2E). The failed bursting phenotype included two types: failed burst of the sole inserted pollen tube and two inserted pollen tubes with at least one burst (Figure 2B–2D). Statistical analysis showed that the deb1 deb2 pollen tubes failed to burst in approximately 36% of ovules at 8 HAP, and this percentage dropped to 20% at 20 HAP (Figure 2E). By contrast, almost all the wild-type pollen tubes burst in the synergid at different time points (Figure 2E). These results suggest a delayed burst for the deb mutant pollen tubes. Given the increased proportion of polytubey in deb-pollen-tube-inserted ovules with time, the potential defect in fertilization of deb mutants was likely compensated by the delayed burst of the early-arriving pollen tube and the entry of additional pollen tubes into the embryo sac. Moreover, the expression of GFP-fused DEB1, DEB2, and DEB3 genomic sequences under their native promoters in deb single mutants rescued the delayed pollen tube burst (Figure 2F). Collectively, these findings demonstrate that DEB1, 2, and 3 function redundantly in regulating timely pollen tube burst.Figure 2 Pollen tube burst in the synergid cell is delayed in the deb1 deb2 mutant.

(A) A WT pollen tube expressing LAT52:GFP burst in the WT synergid cell at 8 HAP.(B) A deb1 deb2 pollen tube expressing LAT52:GFP did not burst in the WT synergid cell at 8 HAP.(C) One deb1 deb2 pollen tube burst in the synergid cell, and another deb1 deb2 pollen tube next to the embryo sac is still intact.(D) Two deb1 deb2 pollen tubes penetrated the synergid cell. Arrows, pollen tubes; asterisks, released pollen tube content labeled by GFP in the synergid cells. Bar, 20 μm.(E) Statistics of delayed pollen burst in the deb1 deb2 mutant at different time points (HAP). Data shown are mean ± SEM. ANOVA, ∗∗p < 0.001. n > 100 ovules.(F) Genomic sequences fused with GFP completely rescued the pollen tube defect. Pollen tubes were examined at 24 HAP. Student’s t-test, p > 0.05. ns, no significant difference. Data shown are mean ± SEM. n > 300 pollen tubes; more than 15 pistils were examined for each genotype.

Lipid modification determines the plasma membrane localization of DEB1/2/3 in pollen tubes

To explore the role of DEBs in pollen tube burst, we assessed the subcellular distribution of DEBs tagged with GFP in pollen tubes. Confocal microscopy showed that DEB1 was primarily found on the plasma membrane, particularly at the apical and subapical zones (Figure 3A). DEB2 and DEB3 were localized both on the plasma membrane and in the cytosol of the pollen tube tips (Figure 3B and 3C). Comparison of DEB1, 2, and 3 sequences retrieved from the TAIR database revealed that the N terminus of DEB3 was truncated compared with those of DEB1 and DEB2. A closer examination of the DEB3 sequence revealed a start codon located 54 bp before the annotated start codon, which was significant because of the presence of two conserved lipid modification sites in the newly identified 18-amino-acid sequence (Figure 3D and 3E). The importance of this sequence was confirmed by experiments in which a truncated version of DEB3 (DEB3ΔN) fused to GFP and expressed under the LAT52 promoter was localized exclusively in the cytosol (Figure 3F). These findings indicate that the N-terminal 18 amino acids of DEB3 are critical for its association with the plasma membrane.Figure 3 DEBs are localized on the plasma membrane of pollen tubes.

(A–C) Confocal images of DEB1-GFP (A), DEB2-GFP (B), and DEB3-GFP (C) in pollen tubes stained with FM4-64. The profiles in the right panels are the fluorescence intensity along the yellow lines, showing the enrichment of the fusion protein on the plasma membrane. Bars, 10 μm.(D) The gene structure of DEB3 and DEB3ΔN; line, intron; darker gray box, exon; lighter gray box, UTR.(E) DEBs contain putative palmitoylation and myristoylation sites at the N-terminal 3 and 6 sites.(F) Confocal imaging showing that DEB3ΔN-GFP shows no plasma membrane localization.(G–I) Site mutation of the two putative modification sites disrupts the plasma membrane localization of the corresponding DEB proteins in pollen tubes. Bars, 3 μm.(J) Site-mutated DEB1, DEB2, and DEB3 fused to GFP cannot rescue the defective pollen tube phenotype of the corresponding mutants at different time points after fertilization (HAP). Each dot shown is mean ± SEM. n > 300 pollen tubes for each sample at each time point. ANOVA, ∗∗∗p < 0.001.

The third cysteine in all three DEB proteins and the specific sixth cysteine in DEB1/2 and a glycine in DEB3 were identified as potential sites for palmitoylation and myristoylation, respectively, via GPS-Palm prediction (http://gpspalm.biocuckoo.cn). To determine the significance of these sites, we engineered mutations at these positions, replacing the third and sixth cysteines with serine and the sixth glycine with alanine, resulting in DEB1C3S, C6S, DEB2C3S, C6A, and DEB3C3S, G6S mutants. These mutated proteins, tagged with GFP and driven by their respective native promoters, failed to localize to the plasma membrane in pollen tubes (Figure 3G–3I). Moreover, they did not ameliorate the impaired pollen tube reception phenotype (Figure 3J), underscoring the necessity of lipid modifications for plasma membrane localization of the DEBs and their role in facilitating pollen tube reception.

DEB1 specifically interacts with and phosphorylates ACA9 to modulate pollen tube reception

Given the known function of RLCKs in membrane signaling through phosphorylation of downstream targets (Liang and Zhou 2018), we focused on ACA9, a calcium pump at the plasma membrane that is known to be involved in pollen tube burst (Schiøtt et al., 2004). ACA9 is characterized by two cytosolic regions: the N-terminal domain (1–197 aa) and a larger cytoplasmic loop (466–859 aa) (Figure 4A). To assess the interaction between DEB1 and ACA9, we performed co-immunoprecipitation (Co-IP) experiments with DEB1 and both the full-length ACA9 and its individual cytosolic domains, ACA91–197 and ACA9466−859. DEB1 showed a much stronger affinity for ACA9466−859 and a weaker association with ACA91−197 and full-length ACA9 (Figure 4B). The mild interaction between DEB1 and full-length ACA9 was likely due to the low amount of ACA9 protein (Figure 4B). Yeast two-hybrid assay confirmed the interactions between DEB1 and the two domains of ACA9, but DEB2 and DEB3 were excluded from this analysis because of their self-activation in the assay (Figure 4C). Co-IP results showed no interaction between DEB1 and the intracellular domains (ICDs) of ANX1 and BUPS1 (Supplemental Figure 4). Collectively, these results suggest a direct regulatory relationship between the DEBs and ACA9 in pollen tube reception.Figure 4 DEB1 interacts with ACA9.

(A) A diagram of the topology of ACA9 on the plasma membrane and N-terminal ACA91−197 and cytosolic loop ACA9466−859. Arrow, direction of the Ca2+ flux.(B) ACA9466−859 interacts with DEB1 in Arabidopsis protoplasts as shown by anti-FLAG Co-IP.(C) Yeast two hybrid assay showing the interaction between DEB1 and the two ACA9 fragments.(D) Genetic rescue of the overgrowth and polytube phenotypes of the deb1/2/3 mutant by phosphomimics of ACA9, wild-type ACA9, and kinase-dead DEB1. n > 1200 ovules for each genotype. Significant differences were determined by one-way ANOVA. Multiple comparisons were performed using Tukey’s honestly significant difference (HSD) (p < 0.05). Data are mean ± SD. Different and shared letters indicate statistically significant (p < 0.05; Tukey HSD) and non-significant (p > 0.05; Tukey HSD) differences, respectively.(E) Imaging of G-CaMP5 showing the cytosolic Ca2+ oscillation in WT pollen tubes within 15 min. Upper panel, kymograph images. Lower panel, profiling of Ca2+.(F) Imaging of G-CaMP5 showing the cytosolic Ca2+ oscillation in deb1 deb2 deb3-2 pollen tubes within 15 min. The profiling and kymographs are representative of 10 pollen tubes of the corresponding genotype.

Next, purified DEB1 protein was incubated with ACA91−197 and ACA9466−859, and a Phos-tag assay showed that DEB1 was able to phosphorylate both ACA91−197 and ACA9466−859 (Supplemental Figure S5). Subsequent mass spectrometry analysis of the co-incubated proteins identified nine phosphorylated threonine/serine (T/S) residues in ACA9 (Supplemental Figure 6). To investigate the functions of these residues, we simultaneously substituted these nine T/S sites with aspartic acid (D) to create the phosphomimic ACA9 (ACA99S>D) and expressed it under the control of the LAT52 promoter in the deb1/2/3 triple mutant. Phenotypic analysis showed that ACA9S9>D could partially restore the defective pollen tube reception phenotype in the deb1 deb2 deb3-2 mutant in different transgenic lines (Figure 4D). However, overexpression of ACA9 driven by the LAT52 promoter did not alleviate the pollen tube defect of the deb1 deb2 deb3-2 triple mutant (Figure 4D). This result confirmed the functional relevance of ACA9 phosphorylation by DEBs in pollen tube burst. Conversely, a kinase-dead variant of DEB1 (DEB1K108E) failed to complement the deb1 deb2 deb3-2 mutant phenotype (Figure 4D), underscoring the necessity of DEB kinase activity in this signaling pathway.

Given that ACA9 regulates Ca2+ dynamics in the pollen tube, we examined the Ca2+ dynamics in the deb1 deb2 deb3-2 mutant. Live imaging of the Ca2+ sensor G-CaMP5 (Li et al., 2018a; Meng et al., 2020) revealed that Ca2+ oscillations were altered in deb triple mutants compared with the wild type (Figure 4E and 4F, Supplemental Figure 7, and Supplemental Videos 1, 2, 3, and 4), consistent with the hypothesis that DEBs, through ACA9, modulate the Ca2+ dynamics required for pollen tube burst. Although deb mutants and aca9 both display a defect in pollen tube burst in the synergid, pollen tube overgrowth and polytubey were not reported in aca9 in a prior study (Schiøtt et al., 2004). To resolve this discrepancy, we examined the pollen tube phenotypes of aca9-1 and aca9-4 mutants at a longer time after artificial pollination, as their pollen tube growth rate is much slower than that of the wild type (Schiøtt et al., 2004). Pollen tube staining of wild-type pistils pollinated with aca9-1 and aca9-4 pollen for 30 HAP showed a higher ratio of overgrowth and polytubey compared with those pollinated by wild-type pollen (Figure 5A–5I). Although this conditional polytubey-and-overgrowth phenotype is not apparent under natural conditions and within a shorter time window after pollination (Schiott et al., 2004; Zhong et al., 2022), it supports the interplay between DEBs and ACA9.Figure 5 The aca9 mutant shows a polytubey and overgrowth phenotype

(A) Diagram of the T-DNA insertion sites in ACA9.(B–D) Pollen tube growth of WT (B) and aca9-1(C) and aca9-4(D) pollen tubes displaying polytubey in WT ovules. Bars, 0.5 mm.(E–G) Enlarged view of pollen tubes of WT (E) and aca9-1(F and G) in WT ovules. White arrow, polytubey. Yellow, overgrown pollen tube. Bars, 20 μm.(H) Ratio of polytubey in aca9-1 and aca9-4. Data shown are mean ± SEM. n ≥ 20 pistils, Student’s t-test, ∗∗∗p < 0.001.(I) Statistics of overgrown pollen tubes in aca9-1 and WT at 24 HAP. n ≥ 8 pistils, Student’s t-test, ∗∗∗p < 0.001.(J and K) A working model of the role of DEB in pollen tube reception. In the WT pollen tube, DEBs on the plasma membrane transduce signals from the synergid, which are perceived by unknown receptors, and modulate Ca2+ oscillations through interaction with ACA9. In the deb mutant pollen tubes, the extracellular signal cannot be transduced to the ACA9–Ca2+ signaling pathway, causing impaired pollen tube burst.

Supplemental Video 1. WT1+2

Supplemental Video 2. WT3

Supplemental Video 3. dtr123-4-1-crop

Supplemental Video 4. dtr123-4-2-crop

Collectively, these results shed light on the molecular mechanism by which DEB1, DEB2, and DEB3 regulate timely pollen tube burst in the synergid, emphasizing their redundant and critical roles. This study delineates a signaling pathway involving lipid modification–dependent localization of DEBs, their interaction with and phosphorylation of ACA9, and the consequent modulation of Ca2+ dynamics that orchestrates the intricate process of pollen tube reception in plants (Figure 5J and 5K).

Discussion

Our research has clarified the role of DEB1/2/3 in pollen tube reception. Notably, the absence of these proteins results in compromised pollen tube burst, leading to overgrowth and polytubey phenotypes. Typically, synergid reception of a wild-type pollen tube is characterized by a timely burst, which releases two sperm cells. However, deb mutant pollen tubes exhibit a markedly delayed burst and continued elongation, diverging significantly from the expected behavior. This delay aligns with the polytubey phenotype, which stems from prolonged synergid attraction due to fertilization recovery (Beale et al., 2012; Kasahara et al., 2012; Meng et al., 2023), suggesting a disruption in mutual communication and pointing toward a complex network of factors, possibly beyond the DEBs. Intriguingly, seed setting rates in deb mutants are close to those of wild-type plants, indicating an underlying robust mechanism that compensates for the delayed pollen tube burst and underscoring a sophisticated, resilient system that ensures reproductive success despite genetic alterations that affect pollen tube dynamics.

Another key finding of our study is the identification of ACA9 as the target of DEB in modulation of synergid–pollen tube interactions, particularly through its effect on Ca2+ dynamics. The direct interaction between DEB1 and ACA9, followed by phosphorylation of ACA9 by DEB1, reveals a regulatory mechanism that shapes Ca2+ signatures vital for pollen tube reception. The precise ACA9 phosphorylation sites in the pollen tube, among the nine identified sites, remain to be verified owing to the limitations of in vitro assays. This interaction suggests that additional components may act downstream of DEBs, as evidenced by the partial phenotypic rescue via ACA9 phosphorylation mimicry. Moreover, although overall Ca2+ levels in deb mutants did not differ from those of the wild type, the altered Ca2+ spikes suggest a role for the DEBs in modulating Ca2+ oscillations, which are required for synergid–pollen tube communication. Despite the technical challenges in capturing high-resolution spatiotemporal images of these Ca2+ dynamics at pollen tube–synergid contact (Iwano et al., 2012; Ngo et al., 2014), the observed changes in the deb mutants provide insight into the mechanisms behind delayed pollen tube burst.

This work supports the notion that RLCKs in pollen tubes have specific roles in different aspects of pollen tube function. This is backed by evidence showing that LIP1 and LIP2, close paralogs of the DEBs, are crucial for guiding the pollen tube into the embryo sac (Liu et al., 2013), whereas MARIS plays a role in maintaining pollen tube integrity during the early stages (Boisson-Dernier et al., 2015; Liao et al., 2016). One unresolved aspect is the identity of the upstream receptor kinases that interact with DEB1, 2, and 3. RLCKs lack an extracellular domain for signal perception outside the cell (Liang and Zhou 2018), and our examination of potential interactions with known receptor kinases like ANX1 and BUPS1 did not establish direct links, hinting at a novel signaling pathway critical for pollen tube reception. In conclusion, our study highlights the roles of DEB1, 2, and 3 in pollen tube reception and identifies ACA9 as a key downstream effector in this signaling cascade.

Methods

Plant materials and growth conditions

Arabidopsis thaliana wild type (Col-0 ecotype) and the mutants deb1 (SALK_023374), deb2 (SALK_ SALK_072589), and deb3-1 (SALK_152499) were obtained from the ABRC. The double mutants were obtained by crossing these mutants. The deb1 deb2 deb3-2 triple mutant was reported previously (Rao et al., 2018), as was the aca9-1 mutant (Schiøtt et al., 2004), and the aca9-4 mutant (GK_782B07) was obtained from the ABRC. Seeds were sterilized and germinated on MS medium. Seven-day-old seedlings were transferred to soil and grown at 22°C under a 16-h light/8-h dark cycle.

Constructs

The 1541-, 1557-, and 1400-bp promoter sequences upstream of the start codons of DEB1, DEB2, and DEB3, respectively, were cloned as the native promoters for genomic GFP fusion constructs. For subcellular localization, genomic sequences—including promoters, coding sequences, and native 3′ terminators—were inserted into the pCAMBIA1300-GFP backbone. For the LAT52-promoter-driven constructs, the coding sequences were inserted into the pCAMBIA1300-LAT52:GFP-OCST1 terminator backbone. For protoplast transformation, the coding sequences were inserted into the pBSK-35S:HA/FLAG-OCST1 backbone. For yeast two-hybrid assays, the coding sequences were inserted into the pBT3N and pPR3N plasmids (dual membrane system). For protein expression and purification, the coding sequences were cloned into pGEx4T-2 or pET28a.

Protein purification and in vitro phosphorylation assay

His- or glutathione S-transferase (GST)-tagged proteins were expressed in Trans BL21(DE3) pLysS strain and were induced to express the fusion proteins by incubation with 1 mM IPTG at 22°C. The cells were lysed with PBS buffer (135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM K2HPO4, 1 mM phenylmethyl sulfonyl fluoride, 1 mM dithiothreitol [DTT], and 1% Triton X-100, pH 7.5). The supernatants of the two proteins were mixed with glutathione agarose beads (GE Healthcare) and rotated at 4°C for 3 h. The beads were washed with PBS buffer five times, boiled in SDS loading buffer in a 100°C water bath for 10 min, and then subjected to SDS–PAGE and immunoblotting with anti-GST (CWbiotech) and anti-His (CWbiotech) antibodies. The split-ubiquitin-based membrane yeast two-hybrid assay (Dualsystems Biotech) was performed for interactor screening and pairwise protein–protein interaction analysis according to the manufacturer’s protocol.

For the phosphorylation assay, 2 μL 10× kinase buffer (10 mM DTT, 100 mM MgCl2, 20 mM CaCl2, 5 mM ATP, 500 mM HEPES, pH 7.5), 0.5 μg kinase, and 5 μg substrate were mixed and adjusted to a final volume of 20 μL. The mixture was incubated at 30°C for 1 h, and 6.672 μL 4× SDS–PAGE loading buffer was added to stop the reaction. The sample was boiled at 100°C for 5 min and centrifuged at 14 000 g for 10 min.

Protoplast transformation and Co-IP

The full-length coding sequences of genes were cloned for fusion with C-terminal FLAG or HA in the pBSK backbone under the control of the 35S promoter and terminated by the poly(A) terminator to generate the fusion constructs. Leaves from plants grown at 22°C under long-day conditions (16-h light/8-h dark) for 4 weeks were cut into fine strips, placed into cell-wall digestion buffer, and shaken for more than 4 h to prepare Arabidopsis protoplasts. The transient expression constructs were co-transformed into Arabidopsis protoplasts using the polyethylene glycol method (Iwata et al., 2011). The protoplasts were harvested 12 h after transformation and lysed in lysis buffer (0.05 M HEPES–KOH [pH 7.5], 150 mM KCl, 1 mM EDTA, 0.1% Triton X-100, and 1 mM DTT with freshly added proteinase inhibitor cocktail). The lysate was centrifuged at 10 000 g at 4°C for 10 min, and the supernatant was subjected to Co-IP with anti-FLAG magnetic agarose beads (Thermo Fisher Scientific) for 4 h with 360° shaking at 4°C. The beads were washed with lysis buffer six times, diluted in 10× loading buffer, and boiled for 5 min before SDS–PAGE. The immunoblotting reaction was performed according to standard procedures with anti-FLAG (CWBIO) and anti-HA (CWBIO) antibodies.

In-gel digestion and LC–MS/MS analysis

Proteins in the gel were reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide in the dark. After alkylation, the proteins were digested with trypsin in 25 mM ammonium bicarbonate at 37°C overnight. Peptides were extracted from the gel with buffers containing 5% trifluoroacetic acid and 50% acetonitrile by ultrasonication twice, freeze dried with a SpeedVac, and desalted by StageTip.

For MS analyses, the resuspended peptides were analyzed with an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) coupled online to an Easy-nLC 1000 chromatograph (Thermo Fisher Scientific) in data-dependent mode. The peptides were separated by reverse phase LC with a 150 μm (ID) × 250 mm (length) analytical column packed with C18 particles of 1.9 μm diameter. Precursor ions were measured in the Orbitrap analyzer at 240 000 resolution (at 400 m/z) and a target value of 106 ions. The twenty most intense ions from each MS scan were isolated, fragmented, and measured in the linear ion trap with MSA neutral losses of m/z 98, 49, and 32.6. The CID normalized collision energy was 35. The data were analyzed using Thermo Scientific Proteome Discoverer software version 1.4. The proteome sequences of Arabidopsis thaliana from TAIR were used for database searching. The mass tolerance for precursor ions was set to 20 PPM, and the mass tolerance for fragment ions was set to 0.6 Da. Serine, threonine, and tyrosine phosphorylation and methionine oxidation were included in the search as variable modifications. Cysteine carbamidomethylation was set as a static modification. The false discovery rates for both peptide and protein identification were set to 0.01.

Aniline blue staining

Flowers at the 12c stage were emasculated and left to grow for 12–24 h to achieve pistil maturation. Pollen grains from wild-type or mutant plants were dispersed on the stigma. After 24 h, the pistils were excised and fixed in Carnoy’s fixative (75% ethanol and 25% acetic acid) for 2–4 h, washed three times in 50 mM PBS buffer (NaHPO4/NaH2PO4, pH 7.0), and immersed in 2 M NaOH overnight for softening. After three washes with PBS, the pistil was stained with 0.1% aniline blue (0.1% in KPO4, pH 8.0) for 6 h. The stained pistils were observed under an Axioskop 2 microscope (Zeiss) equipped with an ultraviolet filter.

Alexander’s staining

Flower buds were fixed in Carnoy’s fixative (75% ethanol and 25% acetic acid) with vacuum infiltration for 2 h. After fixation, anthers were dissected from the bud on a microscope slide and placed into Alexander’s staining solution (10 mL 95% ethanol, 1 mL of 1% malachite green in 95% ethanol, 25 mL glycerol, 5 mL of 1% acid fuchsin in water, 0.5 mL of 1% Orange G in water, 4 mL glacial acetic acid, and distilled water to a total of 100 mL) overnight at 55°C.

DAPI staining

The pollen grains were placed onto a slide and incubated in DAPI (5 mg/mL, 1:1000 or 1:100 dilution; Fisher) for 5 min, then washed with PBS three times. The stained pistils were observed under an Axioskop 2 microscope (Zeiss) equipped with an ultraviolet filter set.

Confocal microscopy

Images were acquired using a Zeiss 980 laser scanning confocal microscope (CLSM; Zeiss). The excitation/emission wavelengths for GFP and FM4-64 staining were 488 nm/505–530 nm and 543 nm/560–600 nm, respectively. For FM4-64 staining, pollen tubes were stained in 5 μM of FM4-64 for 5–10 min and washed twice with liquid pollen germination medium before imaging. Ca2+ imaging was performed as described previously (Meng et al., 2020). Kymograph analysis was performed using ImageJ.

Bioinformatics

The transcriptome data were downloaded from the Genevestigator database (http://genevestigator.com). RLCK sequences were aligned using Muscle, subjected to phylogenetic analysis with the maximum likelihood method in MEGA X software, and processed with iTol (http://itol.embl.de). Lipid modification sites were predicted using GPS-Palm (https://gpspalm.biocuckoo.cn).

Funding

This work was supported by 10.13039/501100001809 NSFC grants (31991203 , 32130032 ), the 10.13039/501100012166 National Key Research and Development Program of China (2022YFF1003500 ), the CAS Project for Young Scientists in Basic Research (no. YSBR-078 ), and the Strategic Priority Research Program of the Chinese Academy of Science (XDA24020306 ).

Supplemental information

Document S1. Figures S1–S7 and Table S1

Document S2. Article plus supplemental information

Acknowledgments

We thank Prof. Jianmin Zhou (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for kindly providing the deb1 deb2 deb3-2 triple mutant and for critical comments, Alice Cheung (University of Massachusetts) for critical comments, Shanjin Huang (Tsinghua University) for the G-CaMP5 plasmid, and the staff of the microscopy and proteomics platform (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for assisting in imaging and mass spectrometry. The authors declare that they have no competing interests.

Author contributions

Y.-J.X., T.L., P.-M.Z., and W.-Q.W. designed and performed the experiments and analyzed the results. H.-J.L. and W.-C.Y. conceived and supervised the projects. H.-J.L. wrote the manuscript.

Published by the Plant Communications Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.

Supplemental information is available at Plant Communications Online.
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