Characterization of calcium-dependent protein kinases involved in ROS signaling during fertilization in the red alga Bostrychia moritziana (Ceramiales, Rhodomelaceae)
Article information
Abstract
The interplay between calcium signaling and reactive oxygen species (ROS) production is important in many cellular activities, yet its involvement in biological processes in red algae is mostly not studied. Fertilization in red algae is unique, and while it is known that ROS is a necessary signal, its producing and interplay with calcium and calcium-dependent protein kinases (CDPKs) is unknown. Through experimental assays, we visualized calcium influx and ROS accumulation during fertilization in the red alga Bostrychia moritziana. Through transcriptomics, five CDPK family genes were identified in B. moritziana, with structural analysis revealing variation in these homologs. Protein structure prediction shows similarities and differences in CDPK structures, likely reflecting their different roles in cellular processes. The regulatory role of CDPKs in ROS production was confirmed by the increased expression of CDPKs genes during fertilization. The inhibition experiments showed that reciprocal positive feedback between CDPK expression and ROS production, emphasizing the intricate regulatory mechanisms underlying calcium-dependent signaling pathways. The fertilization process of B. moritziana could also be an excellent model to study the role of CDPKs in the downstream signaling pathways of the interaction of calcium and ROS signaling.
INTRODUCTION
The interplay between calcium signaling and reactive oxygen species (ROS) production is crucial for many aspects of plant development and their responses to environmental stresses (Görlach et al. 2015). Calcium ions and ROS are intimately linked in plant cells, with calcium acting as a secondary messenger in ROS signaling pathways (Demidchik et al. 2018). ROS can regulate cellular calcium signaling by activating calcium channels, and cytosolic Ca2+ regulates ROS production by activating plasma membrane NADPH oxidase or by activating the mitochondrial electron transport chain and peroxidases (Gordeeva et al. 2003, Heyno et al. 2008, Gilroy et al. 2014, Görlach et al. 2015, De Nicolo et al. 2023). This leads to a positive feedback loop that amplifies the ROS signal, activating many downstream processes (Moon et al. 2022).
Calcium signaling involves dynamic changes in cytosolic Ca2+ concentration in response to diverse stimuli (Aldon et al. 2018, Marce et al. 2019), detected by specific calcium sensors, including calcium-dependent protein kinases (CDPKs), calmodulin, and calmodulin-like proteins (CMLs) (Reddy et al. 2011). CDPKs have emerged as crucial mediators of calcium-dependent regulation of ROS signaling (Li et al. 2008), phosphorylating target proteins involved in ROS metabolism and signaling (Boudsocq and Sheen 2013), and regulating ROS-responsive transcription factors, influencing stress response, growth, and development (DeFalco et al. 2010, Javed et al. 2020). Interactions between CDPKs and calcium sensors such as CMLs and calcineurin B-like proteins (CBLs) allow for precise spatiotemporal control of CDPK activity in response to specific stimuli (Rudd and Franklin-Tong 2001, Batistič and Kudla 2012). CDPKs regulate ROS production in cells through phosphorylation of NADPH oxidases, leading to their activation or inhibition, depending on the specific context (Lecourieux et al. 2006, Kobayashi et al. 2007, Dubiella et al. 2013, Mittler et al. 2022).
While extensively studied in higher plants, research on CDPK-mediated calcium signaling and its interaction with ROS in red algae remains limited (Myers et al. 2009, Matschi et al. 2013, Brawley et al. 2017). Red algae, possessing unique characteristics, show varied responses to ROS-mediated signaling, including regulation of lipid accumulation, wound repair, fertilization, and spore release (Seo et al. 2012, Moon et al. 2022, Teng et al. 2023). NADPH oxidases have been identified in several red algal species, although their functional properties are still being characterized (Hervé et al. 2006, Luo et al. 2015, Shim et al. 2022, Hong et al. 2024). Genomic studies reveal differences in red algal NADPH oxidases compared to plants, with the absence of the calcium-binding EF-hand domain (Brawley et al. 2017), suggesting the involvement of CDPKs in the calcium transmission to the enzyme.
The unique and complex patterns of red algae fertilization provide a model for studying ROS-mediated signaling (Shim et al. 2021, Kim et al. 2022). Previous research has shown that hydrogen peroxide is a primary signaling molecule in Bostrychia moritziana during fertilization, that blocking H2O2 leads to fertilization failure, and that Ca2+ is also critical for ROS generation and fertilization completion (Shim et al. 2022). How Ca2+ can affect NAPDH oxidases when they lack a Ca2+-binding domain, EF-hand, raises the possibility of a more complex interplay with other Ca2+-dependent protein kinases. In this study, we identified and investigated the role of CDPKs in B. moritziana fertilization and the interplay between ROS and calcium signaling.
MATERIALS AND METHODS
Algal cultures
Bostrychia moritziana gametophytes of both sexes were acquired from culture #2746, maintained in the John A. West collection. Thalli were maintained in unialgal cultures in IMR medium (Klochkova et al. 2005) at 20°C on a 16 h light / 8 h dark cycle with illumination of >20 μmol photons m−2 s−1 provided by cool white fluorescent lighting. Plants were transferred to fresh IMR medium every 10–14 d. One week before experimental use, fresh medium was prepared in 100 mL containers, into which 5–10 apical segments (1 cm) were transferred. The cultures were monitored for growth and reproductive development. Male and female gametophytes with actively developing spermatangia or carpogonial branches with visible trichogynes were used.
Preparations for fertilization and inhibition experiment
The fertilization process followed procedures as described in Shim et al. (2020). To induce sperm release, male gametophytes were exposed to a distilled water for 30 seconds. Male gametophytes were removed from the sperm suspension, and female gametophytes with visible trichogyne were placed in a petri dish and gently stirred to promote mixing and contact between sperm and the trichogyne (egg receptive area). NADPH-oxidase activity was inhibited using diphenylene iodonium (DPI). The inhibitory mechanism of DPI specifically targets activated NADPH-oxidases by forming stable complexes with the ROS-generating heme groups located on the membrane’s exterior surface (Reis et al. 2020). Caffeine was used to block calcium influx through caffeine sensitive Ca2+ channels (Taylor et al. 1996). Staurosporine was used to inhibit CDPKs during fertilization. As for the concentration of the inhibitors, the minimum dose required for inhibition obtained from preliminary experiments was used; 1 mM caffeine, 10 μM DPI 1 μM Staurosporine. Male or female plants were incubated with inhibitors for 30 min prior to gamete mixing.
Histochemical visualization of ROS
ROS detection was performed using 3,3′-diaminobenzidine (DAB) staining. When exposed to H2O2 and peroxidases, DAB undergoes oxidation, generating a visible reddish-brown precipitate (Daudi and O’Brien 2012, Kumar et al. 2014). The staining solutions were prepared by dissolving 1 mg DAB in 1 mL autoclaved sea water in microfuge tubes. Tips of female plants developing visible trichogynes were mixed with the spermatial solutions and immersed in DAB staining solutions. The tube was wrapped with aluminum foil to block light and kept at 10°C for 12 h until the reaction finished. Therefore, DAB staining enabled the detection of ROS that had accumulated within cells throughout the incubation process. To remove autofluorescence and enhance the staining, the blades were washed twice with ethanol and transferred to microfuge tubes containing 100% methanol, and incubated at 50°C for 3–5 h, then observed with under a light microscope.
Actin, calcium, and nuclei staining
To visualize the actin cytoskeleton, fertilized female gametophytes were fixed for 30 min in 3.7% (w/v) formaldehyde diluted in microfilament-stabilizing buffer (MFSB) consisting of 10 mM EGTA, 5 mM MgSO4, and 100 mM PIPES-KOH (pH 6.9) (Hong et al. 2023). After fixation, plants were rinsed three times with phosphate-buffered saline (PBS; 8 mM Na2HPO4, 2 mM NaH2PO4, 140 mM NaCl; pH 7.4), and then placed in 0.5% (v/v) Triton X-100 diluted in MFSB. The cells were then washed again three times with PBS before incubation in a staining solution of FITC-phalloidin (Sigma-Aldrich, St. Louis, MO, USA) for 0.5–1 h at 20°C in the dark. FITC-phalloidin was prepared as a stock solution of 0.05 mg mL−1 in methanol and was stored at −20°C in the dark. The stock was diluted with MFSB to a final concentration of 2.5 μg mL−1. Then the filaments were placed in Hoechst 33342 (Sigma-Aldrich) for 30 min to stain nuclei. A stock solution was prepared by dissolving Hoechst 33342 in sterile distilled water at 1 mg mL−1 concentration and diluted 1,000 times for working solution. The cells were washed three times using seawater before observation using a blue filter for actin and UV filter for nuclei. For calcium staining, female plants incubated with spermatia were immediately transferred to a solution containing 10 μM Fluo-8 AM (Abcam, Cambridge, UK) and 1 μg mL−1 Hoechst 33342 for 1 h and observed under a fluorescence microscope.
RNA extraction and cDNA synthesis
Total RNA was extracted from female gametophytes before and after exposure to spermatial solution. After gamete binding, total RNA was extracted from female plants at critical stages of the fertilization process, such as spermatial nuclear division (10 min post-attachment), formation of the fertilization channel (30 min post-attachment), spermatial nuclear migration into the carpogonium after cytoplasmic fusion (60 min post-attachment), and early post-fertilization development (180–360 min) (cite your paper here for ‘critical stages’). As a control, total RNA was extracted from female plants that were not exposed to the spermatial solution. Total RNA extraction was conducted with TRIzol reagent (iNtRON Biotechnology Inc., Seongnam, Korea), and subsequently, poly-A RNA was isolated using the Oligotex mRNA purification kit (QIAGEN, Hilden, Germany). RNA concentration was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The quality of RNA was evaluated using both the RNA 6000 Nano assay kit and Bioanalyser2100 system (Agilent Technologies, Palo Alto, CA, USA). Libraries were generated from one microgram of total RNA using TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. In brief, poly-A RNA was purified using poly-T oligo attached magnetic beads. After purification, the total poly-A-RNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved mRNA fragments were reverse transcribed into first strand cDNA using random primers. Short fragments were purified with a QiaQuick PCR extraction kit and eluted with buffer for the end preparation and addition of poly(A). Subsequently, the short fragments were connected with sequencing adapters. Each library was separated by distinct multiplexing indexing tag. The final cDNA preparations were diluted to 10 μg μL−1 using autoclaved milli-Q water and maintained at −20°C.
Identification and protein structure prediction of CDPK genes
To identify the CDPK family genes in B. moritziana, we used the conserved domains of plant CDPK family genes. Local BLAST searches were performed by comparing the transcriptome data of B. moritziana (GEO series accession No. 155429) with the genomes of previously reported plant genes in NCBI GenBank. Information on five CDPK genes was obtained. Each CDPK gene identified in B. moritziana was again local BLASTed against the genome information of red algae registered in NCBI GenBank and transcriptome datasets of red algae established in our laboratory to identify homologues.
Protein structure prediction was performed using the AlphaFold program (Varadi et al. 2022). Sequences for full-length CDPK sequences were submitted to one of the Google Collaboratory notebook implementations of AlphaFold 2.1.0 available at (https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb). The structures in PDB format were arranged according to their average pLDDT values. The protein structures analyzed by AlphaFold were visualized using the Mol* 3D viewer (https://www.rcsb.org/3d-view).
Quantitative polymerase chain reaction
To observe whether the CDPKs are differentially regulated during the fertilization process, real-time quantitative polymerase chain reaction (qPCR) was performed using iQ SYBR Green supermix (Bio-Rad Laboratories, Hercules, CA, USA) in a real-time qPCR detection system (Bio-Rad CFX96; Bio-Rad Laboratories). The amplification of cDNA was performed in triplicate, using three independent RNA preparations. Female gametophytes were harvested at 10, 30, 60, 180, and 360 min after incubation with a spermatial solution, and were frozen in liquid nitrogen. RNA was extracted and reverse transcribed to cDNA using the aforementioned methods. The final qPCR reaction volume was 20 μL and included 5 μL of diluted cDNA, 10 μL of iQ SYBR Green supermix and 200 nM of each primer (Supplementary Table S1). The reaction protocol was an initial 95°C for 3 min and then 40 cycles of 95°C for 15 s and 60°C for 20 s. The delta-delta Ct method was employed to determine relative gene expression levels, with glyceraldehyde 3-phosphate dehydrogenase serving as the housekeeping reference gene (Shim et al. 2016).
RESULTS
The fertilization process in B. moritziana begins with the attachment of a non-flagellated spermatium to a long trichogyne projecting from a carpogonium embedded in the female gametophyte (Fig. 1). DAB staining showed that large amounts of H2O2 accumulated in the attached spermatia, the trichogyne and the carpogonial cells during fertilization (Fig. 1A). Treatment with caffeine, a calcium channel blocker, prevented the accumulation of H2O2 at these sites and the fertilization did not occur (Fig. 1B). Caffeine treatment also inhibited the formation of actin filaments, which are produced in attached spermatia and trichogyne during fertilization (Fig. 1C & D).

Effects of caffeine on reactive oxygen species (ROS) production and microfilaments during fertilization of Bostrychia moritziana. (A) Apical part of female plant stained with 3,3′-diaminobenzidine (DAB). H2O2 accumulation was observed at the spermatium (s) attached to trichogyne (t) as well as at the carpogonial cells (c). (B) Caffeine-treated female plant was not stained with DAB. (C) Microfilaments develop within the attached spermatium and trichogyne. (D) Caffeine treatment inhibited microfilament formation inside the reproductive cells. Scale bars represent: A–D, 10 μm.
No fluo-8 staining was observed when spermatium was first attached to trichogyne (Fig. 2A), but calcium accumulation was observed in spermatium starting 5 min after attachment (Fig. 2B), inside trichogyne after 30 min (Fig. 2D), and in both spermatia and trichogyne after 45 min, when a fertilization channel was formed between spermatium and trichogyne (Fig. 2E).

Fluo-8 staining during fertilization of Bostrychia moritziana. Calcium signals were first observed in the cytoplasm of the spermatium and later in the trichogyne. N, spermatial nucleus; N’, first nucleus to move inside the trichogyne. Scale bars represent: A–E, 5 μm.
Five different CDPK family genes were identified in B. moritziana, of which only calcium-dependent tyrosine kinase (CDTK) and calcium-dependent serine/threonine kinase (CDSTK) genes had EF-hand domains (Fig. 3, Supplementary Table S2). The CDTK gene exhibited the most complex regulatory domain structure, containing two EF-hand domains coupled with a tyrosine kinase domain. The CDSTK gene had a single EF-hand domain followed by a serine/threonine kinase domain, indicating a simplified calcium-sensing mechanism compared to CDTK (Fig. 3).

Two-dimensional molecular structures and locations of active domains of Bostrychia moritziana calcium-dependent protein kinases (CDPKs). CDTK, calcium-dependent tyrosine kinase; CDSTK, calcium-dependent serine/threonine kinase; CAMK, calcium-calmodulin-dependent kinase; CIPK, CBL-interacting protein kinase; CDPKL, calcium-dependent protein kinase-like gene.
The remaining three CDPK family genes such as calcium-calmodulin-dependent kinase (CAMK), CBL-interacting protein kinase (CIPK), and calcium-dependent protein kinase-like gene (CDPKL) lacked EF-hand domains (Fig. 3). CAMK showed a distinctive architecture with an N-terminal pleckstrin-homology domain, suggesting that CAMK might respond to phospholipid signaling in addition to calcium-mediated regulation. CIPK contained unique regulatory domains, including the NAF domain and KA1 domain, suggesting its interaction with CBL proteins and potential involvement in stress response pathways. CDPKL showed the most basic domain structure with only a serine/threonine kinase domain, and the absence of additional regulatory domains might indicate its involvement in constitutive cellular processes rather than in complex calcium-dependent signaling cascades. Staurosporine, an inhibitor of CDPKs, blocked ROS production as well as nuclear movement during the fertilization of B. moritziana (Supplementary Fig. S1 & Video S1).
The 3D molecular structure of EF-hand containing CDTK and CDSTK genes in B. moritziana was analyzed (Fig. 4). Analysis of the protein structure prediction revealed a highly conserved calcium binding sites within the EF-hand domains of CDTK and CDSTK (Fig. 4). The B. moritziana CDTK with two EF-hand domains, both of which contained D-x-D motifs known to play a critical role in calcium binding (Fig. 4A & B). Another motif involved in calcium binding, E-xx-E, was also observed in the EF-hand domain of CDTK (Fig. 4C). The B. moritziana CDSTK had an EF-hand, where E-xx-E (Fig. 4D) and E-x-E (Fig. 4E) were identified as calcium-binding motifs.

Structure of the calcium-binding motifs of EF-hand in calcium-dependent protein kinase (CDTK) and calcium-dependent serine/threonine kinase (CDSTK) of Bostrychia moritziana analyzed by the AlphaFold program.
The expression of CDPK gene homologues was examined using qPCR to determine how they were regulated at each critical stage of the fertilization process (Fig. 5). CDTK and CDSTK had the most pronounced changes in expression during the fertilization process (Fig. 5A). CDTK showed a steady increase in expression after spermatia attachment until the time of spermatial nucleus migration into the carpogonium (60 min post-attachment), after which expression decreased rapidly (Fig. 5A), whereas CDSTK expression increased until 30 minutes after the formation of the fertilization channel, after which expression decreased gradually (Fig. 5A). No significant changes in expression were observed for CAMK before and after fertilization (Fig. 5A). CIPK expression increased significantly at the time of spermatial nucleus entry into the carpogonium, with high expression for several hours during the post-fertilization development, although there was little change in expression during the early stages of fertilization (Fig. 5A). CDPKL expression increased approximately 2-fold after spermatia attachment and remained steady thereafter (Fig. 5A).

Relative expression of Bostrychia moritziana calcium-dependent protein kinases (CDPKs) during fertilization and early post-fertilization development. Quantitative PCR was performed using mRNAs collected over time after gamete binding. Typically, spermatial nuclei migrate into the female carpogonium within 60 min after gamete binding. CDTK, calcium-dependent tyrosine kinase; CDSTK, calcium-dependent serine/threonine kinase; CAMK, calcium-calmodulin-dependent kinase; CIPK, CBL-interacting protein kinase; CDPKL, calcium-dependent protein kinase-like gene; DPI, diphenylene iodonium.
The expression of CDPKs during fertilization was significantly affected by ROS and calcium inhibitors (Fig. 5B & C). When ROS production by NADPH oxidase was blocked, by pretreating female plants with DPI, no CDPK gene was upregulated during fertilization except CIPK which was still upregulated, and the time point of upregulation was accelerated by 30 min compared to the control (Fig. 5B). When the influx of calcium ions was blocked by treatment with caffeine, the expression of all CDPKs was significantly downregulated during the fertilization (Fig. 5C). However, the timing of downregulation of these genes was slightly different at each stage of the fertilization process. For example, CDTK and CDSTK were sharply downregulated 30 min after gamete binding (Fig. 5C), CAMK was downregulated 180 min after gamete binding (Fig. 5C), and CIPK was downregulated for the first 10 min and then gradually recovered over time (Fig. 5C). CDPKL expression was largely unaffected by caffeine treatment (Fig. 5C).
DISCUSSION
In this study, we identified and investigated the expression of CDPKs in ROS signaling during fertilization in the red alga B. moritziana. Our results suggest a complex molecular mechanism underlying the interplay between calcium and ROS signaling pathways in this important biological process, where disruption of either ROS production, or calcium influx prevented fertilization from proceeding. Red algae offer a unique platform to study ROS-mediated signaling pathways. While previous studies suggested the involvement of ROS in processes such as lipid accumulation (Seo et al. 2012), wound repair (Moon et al. 2022, Hong et al. 2024), and fertilization (Shim et al. 2022), the specific mechanisms, particularly the role of CDPKs, remained unclear. Our Fluo-8 staining results showed that calcium influx in the spermatia occurs 5 min after gamete binding in B. moritziana. Calcium influx into the trichogyne occurs at the point where the fertilization channel is formed and the spermatial nuclei move into it.
We identified five genes of CDPK family in B. moritziana, of which only two (CDTK and CDSTK) possess a calcium-binding site (EF-hand). The inhibition of NADPH oxidase activity significantly altered the expression patterns of CDTK and CDSTK, highlighting the regulatory role of ROS in modulating calcium signaling pathways. Conversely, blocking calcium influx led to downregulation of most CDPK genes, indicating that CDPK gene expression is dependent on intracellular calcium levels during early stages of fertilization, and this enhances ROS (H2O2) production, by membrane-bound NAPDH-oxidase, which in turn increases Ca influx. These results support the existence of a positive feedback loop between Ca2+, CDPK and ROS production as summarized in Fig. 6.

The role of calcium-dependent protein kinases (CDPKs) in reactive oxygen species (ROS) production and calcium signaling during fertilization of Bostrychia moritziana. CDTK, calcium-dependent tyrosine kinase; CDSTK, calcium-dependent serine/threonine kinase; CAMK, calcium-calmodulin-dependent kinase; CIPK, CBL-interacting protein kinase.
CDPKs play diverse roles in mediating ROS signaling cascades in plants (Boudsocq and Sheen 2013). These kinases are activated in response to changes in cytosolic calcium levels, enabling them to modulate the activity of downstream targets involved in ROS metabolism and signaling pathways (Li et al. 2008). By phosphorylating NADPH oxidases, CDPKs regulate the production of ROS, particularly superoxide anion (O2-), in plant cells (Lecourieux et al. 2006). NADPH oxidases are integral membrane proteins responsible for the regulated production of ROS in plants (Kärkönen and Kuchitsu 2015). In plants, NADPH oxidases are mostly localized to the plasma membrane and the apoplast, where they contribute to ROS-mediated signaling (Jiménez-Quesada et al. 2016). In red algae, it has also been reported that fertilization and wound healing processes are mediated by ROS signals generated by NADPH oxidase (Moon et al. 2022, Shim et al. 2022, Hong et al. 2024). In these studies, inhibition experiments with caffeine showed distinct effects on ROS production, but did not investigate factors that directly connect calcium signaling and NADPH oxidase activity.
The activity of NADPH oxidases is tightly controlled by CDPKs to prevent excessive ROS production, which can lead to oxidative damage and cell death (Mhamdi and Von Breusegem 2018). This dynamic regulation allows plants to finely tune ROS levels in response to diverse environmental stimuli, ensuring appropriate cellular responses to stress (Kobayashi et al. 2007). When calcium influx into cells was blocked by caffeine, the expression of the five B. moritziana CDPKs mostly changes. Of these, CDTK and CDSTK, which have EF-hand domains, showed the most pronounced decrease in expression. When NADPH oxidase activity was inhibited by DPI treatment, no upregulation of CDTK and CDSTK was observed in response to fertilization. These results demonstrate that the expression of these CDPKs is regulated by intracellular calcium, which in turn activate NADPH-oxidase produced ROS levels. Interestingly, in the case of CIPK, there was a distinct increase in expression when ROS generation by NADPH oxidase was inhibited, suggesting that different CDPKs may have different sensitivities to ROS levels or different regulatory mechanisms. Considering that the phosphorylation of NADPH oxidase by CDPKs can either activate or inhibit its activity depending on the specific context and cellular conditions (Mittler et al. 2022), it is not surprising that the expression of each CDPK gene increases or decreases in response to different ROS levels.
Plant calcium signals are decoded by a vast array of Ca2+ sensors and Ca2+ responder proteins (Harmon et al. 2001). Ca2+ sensor proteins undergo conformational changes upon binding Ca2+, activating their respective Ca2+ responders such as CaM-dependent protein kinases and CIPKs which phosphorylate specific downstream proteins (Harmon et al. 2001). CDPKs are unique because they function as Ca2+ sensors as well as responders (DeFalco et al. 2010). CDTK and CDSTK showed the strongest and earliest response to ROS production during fertilization. Their expression increased between 30 and 60 min after gamete binding, when the female gamete cells produce the most ROS, and decreased by the end of fertilization, when ROS production decreased (Shim et al. 2022). In this study, a homolog of CIPK, a Ca2+ responder was identified. CIPK showed a distinct increase in expression between 180 and 360 min after gamete binding, when fertilization ends and post-fertilization development begin (Shim et al. 2021, 2022). The presence of EF-hand domains exclusively in CDTK and CDSTK suggests their Ca2+ sensors for activation, while the other three genes likely respond to calcium through alternative mechanisms. The expression pattern of CIPK is interesting because most Ca2+ responder proteins drive changes downstream of calcium signaling. The domain organisation suggests that CIPKs may function in complex signalling networks involving multiple protein-protein interactions. While some red algal CDPKs possess EF-hand domains, similar to those found in land plant CDPKs, CIPK lack this characteristic feature. This structural diversity among red algal CDPKs underscores the evolutionary divergence of calcium signaling mechanisms in these organisms.
CDPK-mediated phosphorylation of NADPH oxidases contributes to the spatial and temporal control of ROS production in plants (Miller and Mittler 2023). By targeting specific residues on NADPH oxidase proteins, CDPKs can modulate enzyme activity in a precise manner, enabling localized ROS production at distinct cellular locations (Lecourieux et al. 2006). This spatial regulation is essential for coordinating ROS signaling pathways involved in various physiological processes, including growth, development, and defense responses (Boudsocq and Sheen 2013). To understand the role of each of the CDPK homologs, it is necessary to know where each of these proteins are transported to in the cell after synthesis. Further immunocytochemical studies of the subcellular localization of each of the CDPK homologs will provide a more detailed understanding of their roles in the ROS signaling pathway.
CDPKs ensure the proper abundance and activity of NADPH oxidases in response to changing environmental conditions through transcriptional and post-translational mechanisms (Li et al. 2008). Given the unique characteristics of red algae, such as their complex life cycles and fertilization process, understanding the molecular mechanisms underlying ROS signaling mediated by CDPKs could provide valuable insights into their adaptation strategies and stress responses. While our study provides some insights into the role of CDPKs in ROS signaling during fertilization in B. moritziana, several limitations warrant further investigation. Treatment with staurosporine blocked ROS production after gamete binding and resulted in spermatial nuclear division and gamete fusion, but more direct evidence using specific inhibitors for each CDPK-like gene is needed to confirm this, as staurosporine is a broad-spectrum inhibitor of kinase activity (Lecourieux et al. 2006). Future studies should focus on elucidating the downstream targets and cytoskeletal effector proteins regulated by CDPKs to decipher the molecular mechanisms underlying their function in red algal fertilization. Additionally, comparative analyses across diverse red algal species and other algal lineages will deepen our understanding of the evolutionary dynamics of calcium-dependent signaling pathways.
ACKNOWLEDGEMENTS
We sincerely thank to Prof. John A. West providing Bostrychia strains. This work was supported by the research grant of Kongju National University in 2022 and Marine Fishery Bio-resources Center (2024) funded by the National Marine Biodiversity Institute of Korea (MABIK) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2022R1A2C1091633).
Notes
The authors declare that they have no potential conflicts of interest.
SUPPLEMENTARY MATERIALS
Primers for qPCR of Bostrychia moritziana (https://www.e-algae.org)
Five different calcium-dependent protein kinase family genes have been identified in Bostrychia moritziana (https://www.e-algae.org)
Inhibition of reactive oxygen species production during the fertilization of Bostrychia moritziana after treatment with 1 μM staurosporine (https://www.e-algae.org)
Inhibition of the fertilization of Bostrychia moritziana after treatment with 1 μM staurosporine (https://www.e-algae.org)