Letter to the Editor: Blue Light Irradiation Induces Pollen Tube Rupture in Various Flowering Plants

In flowering plants, the pollen tube (PT) serves as a vehicle to transport two nonmotile sperm cells. PTs exhibit one of the fastest tip growth rates among cells (Williams 2012, Rabille et al. 2019). Various factors were identified to maintain proper balance between turgor pressure and cell wall (CW) synthesis at the PT tip, crucial for rapid growth, and any disruption can cause PT rupture (Ogawa and Kessler 2023). Herein, we reveal that blue light (BL) irradiation induces PT rupture in Arabidopsis, torenia and tobacco. Additionally, we observed Ca²⁺ influx after BL irradiation, accompanied by PT rupture or temporary elongation halt. These findings offer insights into the interplay between PT integrity and Ca²⁺ influx at the PT tip, presenting a novel method to control PT bursts.

Through hundreds of fluorescent observations of growing Arabidopsis PTs, we observed that PTs tend to rupture during excitation light irradiation. Therefore, we investigated the relationship between wavelength and duration of light irradiation and rupture using Arabidopsis PTs. When PTs of wild-type (WT) plants were continuously irradiated with excitation light of the CFP filter set, ∼60% of the PTs burst (Fig. 1A, Supplementary Movie S1). We then observed the accumulated frequency of PT ruptures over time. The earliest rupture was observed at 16 s after irradiation starts, with most ruptures occurring within 1 min (Supplementary Fig. S1). Frequent PT rupture could be induced even with a restricted duration of light irradiation to 15 s (Fig. 1B, Supplementary Movie S3), while PT rupture timing slightly delayed (Supplementary Table S1). Furthermore, PT rupture frequency increased with excitation light intensity in a dose-dependent manner (Fig. 1B, Supplementary Movie S2). To investigate wavelength dependence, we examined the response to excitation light irradiation with GFP and RFP filter sets for 15 s. The results showed that these conditions did not induce PT burst (Supplementary Movies S4, S5). In terms of light power, the photon flux density of RFP filter excitation light is comparable with CFP filter (Supplementary Table S2), indicating that these phenomena are not induced by every wavelength. We then restricted the BL irradiation field by narrowing the microscope’s aperture (Fig. 1C, Supplementary Fig. S2). We observed a 23% rupture in stimulated PT, including the tip region. However, no PTs ruptured when BL irradiated only the shank but not the tip region. These indicate a distinct sensitivity of the PT apical region to BL irradiation. To determine whether BL-induced rupture is common for growing PTs in different plants, we challenged PTs from Nicotiana benthamiana and Torenia fournieri with BL irradiation. Consequently, PT rupture was induced by continuous BL irradiation within 5 min (Fig. 1D, Supplementary Fig. S3, Table S1). Additionally, restricted duration of BL irradiation also induced PT rupture in Torenia (Supplementary Table S1). Thus, BL-induced PT rupture is not limited to Arabidopsis and it is probably a common phenomenon across plant species.

Fig. 1

BL irradiation induced PT burst and Ca²⁺ influx. (A) Montaged images of PTs before and after BL irradiation. Scale bars: 30 µm. Filled arrowheads indicate PT burst. (B) PT burst rate in different light intensities in 5 min. Box plots represent the means with 25th and 75th percentiles with minimum and maximum whiskers of PT burst rate in six–ten independent experiments (n = 53–136). (C) Limited irradiation of BL to the PTs. Dotted circle indicates irradiation area. Open arrowheads indicate PT burst. Filled arrowheads indicate PTs that did not rupture in the apical region-irradiated PTs. Arrows indicate PTs that did not rupture in shank region-irradiated PTs. Image was captured in 2 min after BL irradiation start. Scale bars: 30 µm. (D) BL irradiation in N. benthamiana and T.fournieri. Each image was captured 2 min after BL irradiation start. (E) Montaged images of PTs expressing GCaMP; steady state. Scale bars: 10 µm. (F) Montaged images of PTs expressing GCaMP; PT burst after BL irradiation. Scale bars: 10 µm.

In Arabidopsis, calcium dynamics are crucial in maintaining CW integrity during PT growth. The absence of Ca²⁺ influx through the calcium channels MILDEW RESISTANCE LOCUS O 1/5/9/15, triggered by RAPID ALKALINIZATION FACTOR 4/19 (RALF4/19) peptides binding to the ANXUR (ANX)/ BUDDHA PAPER SEAL (BUPS)/ LORELEI-LIKE GENE (LLG) complex, disrupts CW integrity, leading to PT burst (Gao et al. 2023). Earlier studies indicated that Ca²⁺ ionophore treatment increases PT CW thickness (Reiss and Herth 1979). Therefore, PT burst in the absence of Ca²⁺ influx in the mutant of the ANX/BUPS/LLG complex may result from decreased PT CW thickness, explaining the rupture as an imbalance between CW strength and turgor pressure. To monitor Ca²⁺ dynamics under BL irradiation, we observed calcium sensor protein GCaMP-expressed PTs using a microscope system that was constructed to irradiate the excitation light of the CFP filter while observing confocal microscopy with a 488 nm laser. Surprisingly, the high fluorescent intensity region extended to the basal side in ∼90% of the PTs (Fig. 1E, F, Supplementary Fig. S4, Movie S6). Similar Ca²⁺ influx was hardly observed when the GFP filter was used (Supplementary Movie S7). As the PTs ruptured during or after Ca²⁺ influx, BL-induced PT rupture might be caused by Ca²⁺ influx, contradicting our initial expectation of Ca²⁺ dynamics based on the mutant phenotype of the ANX/BUPS/LLG complex. The prerupture Ca²⁺ influx was also observed during PT reception and reactive oxygen species (ROS)-inducible burst in previous reports (Duan et al. 2014, Ngo et al. 2014). Most likely, excess Ca²⁺ influx could induce an imbalance between CW stiffness and turgor pressure, but the precise downstream effects remain debated. Among the PTs displaying Ca²⁺ influx, 66% did not rupture and showed significant inhibition of subsequent PT elongation (Supplementary Fig. S4F, G). Interestingly, the PT displayed temporal loss and partial reformation of the Ca²⁺ gradient during the unusual growth arrest. Weak BL irradiation induced growth arrest rather than rupture (Supplemental Movie S2). The apex of these quiescent PTs would halt CW synthesis and acquire sufficient stiffness against turgor pressure. Conversely, PTs accelerate tip growth before PT discharge in receptive synergid cells (Ngo et al. 2014). PTs can facilitate their burst by actively generating a nascent CW. To our knowledge, PT growth and burst were independently discussed in physiological and pharmacological studies. Our results elucidate the correlative aspect of PT growth and burst and suggest the importance of pleiotropic analyses.

The mechanism of Ca²⁺ influx induced by BL irradiation deserves more attention. In this study, we observed enhanced Ca²⁺ influx in most PTs after BL irradiation (Fig. 1E, F, Supplementary Fig. S4). Based on the fluorescent pattern of GCaMP and the photosensitive region (Fig. 1C, Supplementary Fig. S3), Ca²⁺ influx appears to be caused by calcium channels localized at the PT apex. Some of the tip-localized calcium channels are controlled by ligands, such as cAMP, cGMP and RALF peptides. BL irradiation might induce environmental changes that alter the associations of these channels and ligands. Alternatively, light irradiation, especially BL irradiation, might induce the generation of abiotic signals such as ROS considering the similar photon flux density of excitation light between the CFP and RFP filters (Supplementary Table S2). ROS can induce Ca²⁺ influx and it is indispensable for Ca²⁺ gradient formation at PT tips (Duan et al. 2014). Recently, Cho et al. proposed that light-induced partial membrane disruption and tiny pore formation increase membrane permeability to cations and a fluorescent dye in mammalian cells (Cho et al. 2023). Although direct evidence of transient pore formation is lacking, the model of light-induced membrane permeability changes offers a compelling explanation for the immediate enhancement of Ca²⁺ influx and suggest that BL irradiation can induce Ca²⁺ influx in other cell types in plants. Further analyses focusing on calcium channels, ROS, dynamic changes in the phospholipid bilayer at the PT tip and other cell types could help clarify the mechanism of Ca²⁺ influx in response to BL irradiation. BL-induced PT rupture is a potent tool for analyzing the rapid fertilization mechanism in flowering plants. Double fertilization in Arabidopsis is completed within 10 min after PT burst (Hamamura et al. 2011). Sperm cell-enclosing membrane removal and GENERATIVE CELL SPECIFIC 1 (GCS1)/HAPLESS 2 (HAP2) relocation must occur swiftly before gamete fusion (Sprunck et al. 2012, Sugi et al. 2023). To observe these events under a microscope, developing a new technique for controlling PT discharge timing and position was eagerly anticipated. Several methods exist for isolating sperm cells, such as physically cutting PTs and treatment with osmotic shock, ROS and RALF34 (Sprunck et al. 2012, Duan et al. 2014, Ge et al. 2017, Gilles et al. 2021). However, these often result in sample drifting, hindering continuous imaging. Conversely, our method allows the preparation of fresh sperm cells by changing the microscope channel without expensive equipment and sample drifting. Continuous imaging of identical sperm cells using our method elucidates the dynamics of sperm cell-enclosing membrane fragmentation and subtle quantitative changes in GCS1/HAP2 relocation upon PT discharge. These data will provide fundamental knowledge on sperm cell activation and contribute to understanding the double fertilization mechanism at the molecular level.

Supplementary Data

Supplementary Data are available at PCP online.

Data Availability

The data underlying this article are available in the article and in its online supplementary material.

Funding

The Japan Society for the Promotion of Science KAKENHI (Grant no. JP23K14214 awarded to N.S.; Grant nos JP20K21432, JP20H05778 and JP20H05781 to D.M.; Grant no. JP22K15145 and JP23H04749 to D.S.; Grant nos JP22H05172 and JP22H05175 to T.K.; and Grant nos JP18K14741 and JP20H05779 to Y.M.); Nagahisa Science Foundation (Grant no. G2023-01 to N.S.); Yokohama City University (Academic Research Grant to D.M.; Development Fund to D.M.; and Strategic Research Promotion Grant no. SK1903 to D.M.).

Acknowledgments

We thank H. Ikeda and H. Kakizaki for their technical support.

Author Contributions

N.S. and D.M. designed the study and wrote the manuscript. N.S. discovered the PT rupture induced by BL irradiation. Y.M. generated the pLAT52:GCaMP marker. D.S. contributed analysis in T. fournieri. N.S. collected other data used in this manuscript. T.K. provided critical advice and reviewed the manuscript. All authors have contributed to the manuscript and approved the manuscript submission.

Disclosures

The authors have no conflicts of interest to declare.