The endoplasmic reticulum-localized Ca2+-ATPase OsACA5 regulates immunity and the seed setting rate in rice

Calcium signaling plays a central role in plant immunity and development, and its homeostasis relies on the precise regulation of calcium transporters such as Ca²⁺-ATPases. However, the mechanisms by which Ca²⁺-ATPases coordinate disease resistance and reproductive development in rice remain largely unclear. In this study, we investigated the function of the endoplasmic reticulum (ER)-localized Ca²⁺-ATPase gene Oryza sativa autoinhibited Ca²⁺-ATPase 5 (OsACA5). The expression of OsACA5 was induced by infection with the rice blast fungus Magnaporthe oryzae and by the pathogen-associated molecular pattern (PAMP) flg22. In contrast, loss-of-function osaca5 mutants exhibited significantly enhanced resistance to rice blast, as evidenced by reduced lesion areas, increased reactive oxygen species (ROS) production, and elevated expression of defense-related genes, indicating that OsACA5 acts as a negative regulator of plant immunity. Further analyses revealed that OsACA5 negatively regulates early PAMP-triggered immunity (PTI)-associated Ca²⁺ influx induced by flg22 and chitin, thereby suppressing immune activation. In addition to enhanced disease resistance, osaca5 mutants displayed notable agronomic changes, including reduced seed setting rate and plant height, as well as increased thousand-grain weight and grain length. Together, these findings demonstrate that OsACA5 plays a critical role in balancing disease resistance and reproductive development in rice by modulating PTI-associated calcium signaling, providing new insights into the regulatory function of ER-localized Ca²⁺-ATPases and offering a potential strategy for breeding rice varieties with stable disease resistance and optimized yield-related traits.

1 Introduction

Calcium signaling is involved in physiological processes during plant growth, and affects cell growth, development, and stress responses and triggers systemic defense reactions (Tian et al., 2020). Ca²⁺ influx is among the earliest signaling events in response to stimulation by pathogen/microbe-associated molecular patterns (Grant et al., 2000). Ca²⁺ is a key second messenger in plant immunity, and increases in the cytosolic Ca²⁺ concentration occur in response to both PTI and effector-triggered immunity (ETI). These Ca²⁺ signals are subsequently decoded by Ca²⁺-binding sensor proteins, regulating multiple cellular processes involved in plant immunity (Koster et al., 2022). Ca²⁺-dependent phosphorylation of respiratory burst oxidase homologs (RBOHs) leads to ROS bursts, increasing plant resistance to biotic stress (Gilroy et al., 2016; Wang et al., 2019). ROD1(resistance of rice to diseases 1), a Ca²⁺ sensor, suppresses rice immunity by activating catalase to scavenge ROS; disruption of ROD1 results in broad-spectrum disease resistance in rice Gao et al., 2021). Cytosolic Ca²⁺ bursts not only activate ROS but also regulate the expression of related disease resistance genes and MAPK kinase activity (Kim et al., 2009; Ranf et al., 2011). Under specific conditions, leaf infiltration with Ca²⁺ ionophores can induce high levels of PR gene expression in cpn1-1(COPINE1) plants (Lee and McNellis, 2009). Recent transcriptome-based analyses of rice responses to Magnaporthe oryzae infection further highlight Ca²⁺-related signaling genes as central immune hubs, emphasizing the importance of Ca²⁺ dynamics in rice blast interactions (Salem et al., 2025a).

In plants, calcium ions are transported primarily by three types of proteins: channels, pumps, and exchangers. In addition, several nonspecific calcium transport proteins, including cyclic nucleotide-gated channels (CNGCs), glutamate receptor-like proteins (GLRs), and annexins (ANNs), have been identified (Goel et al., 2011). Ca²⁺-ATPases are divided into two subfamilies: P-type IIA and P-type IIB. Oryza sativa ER-type Ca²⁺-ATPases (OsECAs), which belong to the P-type IIA subfamily, are primarily localized to the plasma membrane. They transport ions such as calcium, manganese, and cadmium and are associated with stress responses (Li et al., 2008). The OsACA family includes type IIB P-type autoinhibited Ca²⁺-ATPases, which have been identified in multiple species Axelsen and Palmgren, 2001; Taneja and Upadhyay, 2018; Jiang et al., 2023). Previous studies have identified twelve AtACA genes Chandan et al., 2024). AtACA family genes have diverse functions. The first gene identified, AtACA1 was confirmed to be localized to the chloroplast inner envelope membrane and plays a role in controlling the stomatal aperture and signal transduction between chloroplasts and the cytoplasm (Harper et al., 1998; Rahmati Ishka et al., 2021). AtACA7, AtACA9, and AtACA13 regulate fertility in Arabidopsis by participating in pollen and pollen tube development (Schiott et al., 2004; Yu et al., 2018; Rahmati Ishka et al., 2021). AtACAs can also regulate stress resistance in Arabidopsis; furthermore, AtACA12 expression is significantly increased after flg22 treatment (Garcia Bossi et al., 2020). The expression levels of AtACA4, AtACA8, and AtACA10 change significantly under abiotic stresses such as salt and cold (Yang et al., 2017; Yu et al., 2018). The simultaneous loss of Arabidopsis AtACA4 and AtACA11 leads to decreased disease resistance Boursiac et al., 2010), suggesting that OsACAs might regulate rice immunity through a similar mechanism; however, this hypothesis awaits experimental verification.

Research on the OsACA family in rice has focused primarily on resistance to abiotic stress. OsACA1 and OsACA8 increase drought resistance by regulating lignin synthesis and stomatal closure in rice (Kan et al., 2017; Bang et al., 2019). OsACA6 is regulated by miR1432 to modulate resistance to cold, salt, and drought stress in rice Dai et al., 2024). Moreover, the expression of OsACA6 and OsACA7 increases under acid rain stress (Liang and Ma, 2025). While genes in the OsACA family play regulatory roles in various aspects of abiotic stress, reports on their role in biotic stress are limited, as only OsACA9 has been reported to regulate resistance to bacterial blight through the accumulation of ROS and to positively regulate leaf senescence in rice (Wang et al., 2024). OsACA family proteins primarily regulate plant growth by modulating cytosolic and extracellular Ca²⁺ concentrations, but their functions in disease resistance remain to be elucidated.

Although the role of calcium signaling in plant immunity has been extensively studied, the specific function of ER-localized calcium ATPases, such as OsACA5, in immune responses remains unclear. Integrative multitranscriptomic analyses of rice–M. oryzae interactions have also revealed endoplasmic reticulum (ER)-associated processes as important components of defense responses, suggesting that a closer examination of ER-localized Ca²⁺ transport machinery during infection is warranted (Salem et al., 2025b). Previous research has focused primarily on the role of calcium ATPases in stress responses, whereas the dual function of OsACA5 in both immunity and reproductive development has not been thoroughly explored.

This study addresses this gap by revealing the negative regulatory role of OsACA5 in immune responses, in addition to its positive regulation of reproductive development. We found that OsACA5 is localized to the ER and that osaca5 mutants exhibit enhanced resistance to blast disease, which is accompanied by increases in calcium influx, ROS accumulation, and the expression of defense-related genes. These mutants presented significantly reductions in seed setting rate and plant height, among other changes in agronomic traits. By elucidating how OsACA5 influences both immunity and reproduction through the regulation of calcium homeostasis, this research provides new insights for crop breeding aimed at increasing disease resistance and improving seed setting rates.

2 Results

2.1 OsACA5 expression is induced by M. oryzae and flg22 treatment

The presence of calcium transport elements in the rice genome has been reported previously (Singh et al., 2014). To identify calcium signaling components involved in the rice immune response, we analyzed the expression patterns of different calcium transporter gene families, including cation/H⁺ exchangers (CAXs), autoinhibited Ca²⁺-ATPases (ACAs), annexins (ANNs), glutamate receptor-like channels (GLRs), and cyclic nucleotide-gated channels, in rice following M. oryzae infection and flg22 treatment. Upon infection with M. oryzae, the expression of genes belonging to the GLR and CNGC families was not induced, whereas several genes in the other three families were upregulated to varying degrees. Among them, the expression of OsCCX2, OsACA5, and OsANN3 was most strongly induced. Under flg22 treatment, several members of all five gene families were upregulated to varying extents, with OsCNGC16, OsCAX4, OsACA5, OsGLR1.1, and OsANN3 exhibiting the greatest degree of induction (Figures 1A, B). Taken together, these results indicate that several calcium-related genes respond to both M. oryzae infection and flg22 treatment. Although OsANN3 (LOC_Os05g31750) was also induced under both conditions, this gene has been previously characterized and shown to regulate ROS production and Ca²⁺ influx dynamics, and was therefore not further analyzed in this study (Zhang et al., 2021). Within the OsACA family, the expression of OsACA3, OsACA5, and OsACA7 was induced by both treatments. Among these genes, OsACA5 exhibited the highest expression level, whereas OsACA3 showed the second-highest expression. On the basis of their strong and consistent transcriptional responses, OsACA5 and OsACA3 were selected for further functional analysis.

Figure 1

Figure 1. The expression of OsACA5 is induced by Magnaporthe oryzae and the pathogen-associated molecular pattern flg22. (A) Heatmap and corresponding bar plots showing the relative expression levels of selected calcium transporter genes in NIP plants at 24 h postinoculation with M. oryzae strain 70–15 compared with NIP plants at 0h (B) Heatmap and corresponding bar plots showing the relative expression levels of the same genes in NIP plants at 1 h after treatment with 1 μM flg22 compared with those in the water control. Gene expression levels were determined by RT–qPCR and are presented as relative expression values. Bar plots show the mean ± SD of three independent biological replicates (n = 3). The heatmaps and adjacent bar plots represent the same datasets. Red and blue colors indicate upregulation and downregulation, respectively, as shown in the color scale.

2.2 OsACA5 is localized to the ER

To investigate the potential functions of OsACA3 and OsACA5, we performed a comprehensive analysis of the rice Ca²⁺-ATPase gene family, including an analysis of phylogenetic relationships and domain architecture, cis-acting element prediction, and spatiotemporal expression profiling (Figures 2A; Supplementary Figures S2, 3). While promoter cis-acting element analysis, spatiotemporal expression profiling, and conserved motif distribution analysis revealed considerable diversity among family members, domain architecture analysis revealed that OsACA3, OsACA5, and OsACA10 share an identical domain structure. This specific domain organization differs notably from that of OsACA6 and OsACA7; previous studies reported that OsACA6 is localized to the plasma membrane, whereas OsACA7 is localized to the Golgi apparatus (Huda et al., 2013; Singh et al., 2014). To further validate the subcellular localization of OsACA6, we co-expressed OsACA6–GFP with the ER marker mCherry–HDEL in rice protoplasts and performed a co-localization analysis (Supplementary Figure S5). OsACA6–GFP fluorescence was mainly detected at the cell periphery, whereas mCherry–HDEL showed a typical reticulate ER network pattern. Consistent with these distinct distribution patterns, only limited overlap between OsACA6–GFP and mCherry–HDEL signals was observed, supporting that OsACA6 is unlikely to be ER-localized. To verify the subcellular localization of the candidate genes, we constructed OsACA3–GFP and OsACA5–GFP fusion protein and transiently expressed them in rice protoplasts. The dataconfirmed that OsACA3 and OsACA5 are localized to the ER, which is consistent with the localization of the ER marker protein HDEL (Napier et al., 1992). Additional experiments revealed that the structurally similar gene OsACA10 is also localized to the ER (Figure 2B). Moreover, fluorescence intensity line-scan analysis along the indicated regions showed highly overlapping profiles between the GFP signal and the HDEL marker signal, with coincident peaks across the scanned distances (Figure 2C), further supporting ER localization of OsACA3, OsACA5, and OsACA10. Together, these results support the ER localization of OsACA3, OsACA5, and OsACA10. Given that OsACA10 shares an identical domain architecture with OsACA3 and OsACA5 (Figure 2A), we propose that this specific domain organization featuring the CaATP_NAI and ATPase_IIB_Ca domains may be associated with ER targeting in the OsACA family. Future studies are needed to test whether other members possessing this architecture (OsACA1, OsACA8, and OsACA9) are also ER-localized.

Figure 2

Figure 2. Subcellular localization of rice OsACA family members in protoplasts. (A) Phylogenetic relationship and domain architecture of the rice OsACA family. The phylogenetic tree is shown on the left, and the corresponding domain organization of each protein is shown on the right. Colored boxes indicate conserved domains. (B) Confocal images of OsACA3–GFP, OsACA5–GFP and OsACA10–GFP (green) co-expressed with the endoplasmic reticulum marker mCherry–HDEL (red) in rice protoplasts. The GFP channel, mCherry–HDEL channel, merged images (Merged), and bright-field images (BF) are shown. Scale bar = 10 μm. (C) Fluorescence intensity profiles of GFP (green) and mCherry–HDEL (red) were extracted using the entire image area as the region of interest (ROI). Gray values are plotted against distance (μm) to assess the degree of spatial overlap between the two signals.

2.3 OsACA5 negatively regulates rice blast resistance

To explore the potential roles of OsACA3 and OsACA5 in rice immunity, we generated mutants on the NIP background, obtaining two independent mutant lines for each gene, namely, osaca5-1, osaca5-2, osaca3–1 and osaca3-2 (Supplementary Figure S1). Lesion areas were investigated seven days after wound inoculation with M. oryzae strain 70-15. The lesion area in NIP plants was significantly larger (by 40%) than that in osaca5 plants indicating that OsACA5 deficiency increases resistance to blast. Supsequent spray inoculation confirmed these results, with the diseased area in osaca5 plants reaching only nearly 15% of that in NIP plants (Figures 3A–D). Both the wound and spray inoculation experimental results revealed that osaca5 increases rice blast resistance.

Figure 3

Figure 3. The osaca5 mutant exhibits increased resistance to M. oryzae and potentiated immune responses. (A) Phenotypes of NIP and osaca5 plants at 7 days after inoculation (by wounding) with the compatible M. oryzae isolate 70-15 (n = 15 lesions). (B) Quantitative analysis of lesion areas from the wound inoculation experiment shown in (A). (C) Phenotypes of NIP and osaca5 plants at 7 days after spray inoculation with the compatible M. oryzae isolate 70-15 (n = 15 lesions). (D) Quantitative analysis of diseased leaf area from the spray inoculation experiment shown in (C). (E) Detection of hydrogen peroxide (H₂O₂) accumulation by 3,3’-diaminobenzidine (DAB) staining in the leaves of NIP and osaca5 plants at 48 hours post-inoculation (hpi) with M. oryzae; reddish-brown staining indicates H₂O₂. (F) Quantification of the DAB-stained area in (E). (G) Detection of superoxide anion accumulation by nitroblue tetrazolium (NBT) staining in the leaves of NIP and osaca5 plants at 48 hpi with M. oryzae; blue staining indicates superoxide. (H) Quantification of the NBT-stained area in (G). (I) Kinetics of reactive oxygen species (ROS) bursts, determined by measuring luminescence, in the leaf discs of NIP and osaca5 plants after treatment with 1 μM flg22. (J–N) Relative expression levels of the defense-related genes OsNAC4 (J), OsWRKY45 (K), OsPR3 (L), OsPR1a (M), and OsPAL1 (N) in NIP and osaca5 plants at 0 and 24 hours post-inoculation with M. oryzae, as determined by qRT–PCR. The data in (B, D, F, H, I) are presented as the mean ± SD (n ≥ 3). Asterisks above the bars (or at the peak time points in I) indicate statistically significant differences according to one-way ANOVA followed by Dunnett’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ns>0.05). Scale bar in (E) = 200 µm.

In contrast, wound-inoculation assays of two independent osaca3 mutant lines (osaca3–1 and osaca3-2) revealed lesion areas that were not significantly different from those of NIP plants (Supplementary Figures S4A, B), indicating that OsACA3 deficiency does not alter rice blast resistance under these conditions. After establishing the resistant phenotype of osaca5, we assessed the transcript levels of other OsACA family members in osaca5.Compared with that of NIP, although the expression of most OsACA genes did not significantly change, and only minor decreases were observed for a few members, the expression of OsACA5 markedly decreased (Supplementary Figure S4C–K).

To clarify the role of OsACA5 in rice blast resistance, immune physiological indicators were detected in NIP and osaca5 plants after inoculation with 70-15. DAB and NBT staining at 48 hours post-inoculation revealed significantly larger areas stained dark brown and blue–purple in osaca5 plants than in NIP plants (Figures 3E–H). We also used chemiluminescence to assess the ROS burst in osaca5 plants after stimulation. Upon treatment with flg22, compared with NIP plants, osaca5–1 and osaca5–2 plants presented stronger ROS bursts (Figure 3I). We subsequently examined the expression levels of defense-related genes to further investigate the immune response and found that a series of defense-related genes were activated; the expression levels of Oryza sativa NAC domain-containing transcription factor 4 (OsNAC4), Oryza sativa WRKY transcription factor 45 (OsWRKY45), Oryza sativa pathogenesis-related protein 3 (OsPR3), Oryza sativa pathogenesis-related protein 1a (OsPR1a), and Oryza sativa phenylalanine ammonia-lyase 1 (OsPAL1) were significantly greater in osaca5 plants than in NIP plants at both 0 hours and 24 hours post-inoculation (Figures 3J–N). The increase in ROS activity, oxidative bursts, and defense-related genes expression in osaca5 plants upon pathogen inoculation indicate that OsACA5 negatively regulates the immune response in rice.

2.4 OsACA5 negatively regulates PAMP-triggered calcium influx and affects the expression of calcium signaling-related genes

To investigate whether OsACA5 mediates Ca²⁺ influx under PAMP stimulation, noninvasive microtest technology (NMT) was used to detect dynamic changes in Ca²⁺ in mesophyll cells after treatment with PAMPs (flg22 and chitin), which have been shown to trigger PTI signaling in plants Chinchilla et al., 2007). Under untreated conditions, both NIP and osaca5 plants showed small Ca²⁺ fluxes, maintaining a stable state of Ca²⁺ influx/efflux. After the addition of 1 µM flg22, Ca²⁺ influx increased sharply. Compared with that in NIP plants, the Ca²⁺ flux in osaca5 plants was significantly greater, peaking rapidly within a short time and slowly returning to the initial state after 8 minutes (Figures 4A, B). After the addition of 1 µM chitin, Ca²⁺ influx increased sharply in both NIP and osaca5 plants, with the Ca²⁺ influx in osaca5 plants being significantly greater than that in NIP plants, although the intensity of Ca²⁺ influx was less than that after flg22 treatment (Figures 4C, D). Both flg22 and chitin caused Ca²⁺ influx, and the degree of influx was significantly greater in osaca5 plants than in NIP plants. Calcium influx is an early signal stimulated by PAMPs (Upadhyay, 2022). The stronger and more sustained Ca²⁺ influx observed in osaca5 plants may contribute to the heightened activation of immune responses and ultimately to the enhanced resistance of osaca5 plants to M. oryzae.

Figure 4

Figure 4. OsACA5 negatively regulates calcium signaling. (A) Net Ca²⁺ flux in rice mesophyll cells measured by noninvasive microtest technology (NMT) after treatment with 1 μM flg22. The arrow indicates the time of flg22 application. (B) Statistical analysis of the Ca²⁺ flux rates at the initial, peak (magnitude), and final stages from the experiment shown in (A). (C) Net Ca²⁺ fluxes in rice mesophyll cells after treatment with 1 μM chitin. The arrow indicates the time of chitin application. (D) Statistical analysis of the Ca²⁺ flux rates at the initial, peak (magnitude), and final stages from the experiment shown in (C). (E) Relative expression levels of calcium signaling-related genes (OsCPK12, OsCML16, OsCIPK31, OsCBL2, OsPLC1, OsRLCK185, OsCNGC9, and OsCPK20) in NIP and osaca5 plants, as determined by qRT–PCR. Asterisks indicate statistically significant differences between NIP and osaca5 plants at the indicated time points (Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001). The data in (E) are presented as the mean ± SD (n = 3). Different lowercase letters above the bars indicate statistically significant differences according to one-way ANOVA followed by Dunnett’s multiple comparisons test (p < 0.05).

To validate the above results, we selected several calcium transporter genes for qRT–PCR analysis. We compared the expression levels of genes encoding calcium-dependent protein kinases (Oryza sativa calcium-dependent protein kinase 12 (OsCPK12) (Tavu and Redillas, 2025) and OsCPK20 Fu et al., 2013)), calcium signaling sensors (Oryza sativa calmodulin-like protein 16 (OsCML16) (Liu et al., 2025) and Oryza sativa calcineurin B-like protein 2 (OsCBL2) (Qin et al., 2024)), and calcium channel proteins (OsCNGC9) (Wang et al., 2021), and calcium signaling-related genes (Oryza sativa phosphoinositide-specific phospholipase C1 (OsPLC1) (Li et al., 2017), Oryza sativa CBL-interacting protein kinase 31 (OsCIPK31) (Piao et al., 2010), and Oryza sativa receptor-like cytoplasmic kinase 185 (OsRLCK185) (Yamaguchi et al., 2013)) between NIP and osaca5 plants. Compared with those in NIP plants, the expression levels of OsCPK12, OsCML16, OsCIPK31, OsCBL2, and OsPLC1 significantly decreased in osaca5 plants, whereas the expression levels of OsRLCK185 and OsCNGC9 significantly increased. OsCPK20 expression did not significantly differ (Figure 4E), indicating that OsACA5 may indirectly influence the activity of other calcium channels.

2.5 OsACA5 regulates agronomic traits in rice

An increase in disease resistance in plants is often accompanied by a reduction in yield (Kim et al., 2024b). Therefore, we investigated the agronomic traits of the osaca5 mutants. The height of the NIP plants was 84 cm, whereas those of the osaca5–1 and osaca5–2 plants were 76 cm and 78 cm, respectively. The height of the NIP plants was significantly greater than that of the osaca5 plants (Figures 5A, B). The seed setting rate of the osaca5 plants, below 60%, was significantly lower than that of the NIP plants (Figure 5C). The effective tiller number did not significantly differ between NIP and osaca5 plants (Supplementary Figure S6A). With respect to grain length, compared with NIP plants, osaca5 plants had significantly longer grains (Figures 5E, G). However, no significant difference in grain width was detected between the osaca5 and NIP plants (Figured 5F, S6B). Additionally, the thousand-grain weight of the osaca5 plants was significantly greater than that of the NIP plants (Figure 5D). A comparison of the total number of grains per panicle revealed that NIP plants produced far more grains than did osaca5 plants, with osaca5 plants did producing a greater proportion of empty grains (Figure 5H). These findings suggest that osaca5 mutation severely affects the seed setting rate in rice. However, no significant differences in pollen viability or other traits were detected (Supplementary Figures S6C, D).

Figure 5

Figure 5. OsACA5 is required for maintaining normal agronomic traits in rice. (A) Architecture of NIP and two independent osaca5 mutant lines (scale bar = 10 cm). (B) Plant height of NIP and osaca5 plants. (C) Seed setting rate of NIP and osaca5 plants. (D) Thousand-grain weight of NIP and osaca5 plants. (E) Grain length of NIP and osaca5 plants. (F) Grain width phenotype of NIP and osaca5 seeds (scale bar = 1 cm). (G) Grain length phenotype of NIP and osaca5 seeds (scale bar = 1 cm). (H) Single panicle Grains arranged in circular patterns showing the numbers of filled and empty grains (scale bar = 1 cm). (I) Relative expression levels of Ca²⁺ signaling- and fertility-related genes in NIP and osaca5 plants determined by qRT–PCR.Data in (B–E, I) are presented as mean ± SD (n ≥ 15 plants for B-E; n = 3 biological replicates for I). Statistical significance was determined as described in the Methods. *P < 0.05, **P < 0.01, and ***P < 0.001.

To determine whether disrupted Ca²⁺ signaling contributes to the reduced seed setting rate of osaca5 plants, we examined the expression of several Ca²⁺ signaling- and fertility-related genes by qRT–PCR. These genes included the cyclic nucleotide-gated channel family members OsCNGC4 and OsCNGC13, as well as the Ca²⁺-dependent protein kinase family members OsCPK9, OsCPK21, and OsCDPK23. Previous studies have shown that OsCNGC4 and OsCNGC13 influence panicle fertility and the seed setting rate by regulating pollen germination and pollen tube growth within the pistil (Xu et al., 2017; Kim et al., 2024a), whereas OsCPK9, OsCPK21, and OsCDPK23 act as typical Ca²⁺ signal “decoders” that finely modulate reproductive processes such as pollen development, panicle development, and grain formation Asano et al., 2002; Wei et al., 2014; Wen et al., 2019). Compared with those in NIP plants, the transcript levels of OsCNGC4, OsCNGC13, OsCPK9, OsCPK21, and OsCDPK23 were significantly lower in both osaca5–1 and osaca5-2, with the two allelic mutants displaying a highly consistent downward trend (Figure 5I). These results indicate that Ca²⁺ signaling pathways that are closely associated with reproductive development are globally suppressed in the absence of OsACA5, which may be an important cause of the markedly reduced seed setting rate observed in the osaca5 mutants.

3 Discussion

Calcium signaling plays a crucial role in plant immune responses and is tightly regulated by calcium transporters, including autoinhibited Ca²⁺-ATPases (Tian et al., 2020). In this study, OsACA5 expression was strongly induced by M. oryzae infection and flg22 treatment (Figure 1), suggesting that OsACA5 may function in the early phase of PTI activation. Domain analysis of OsACA family members revealed that OsACA5 shares conserved CaATP_NAI and ATPase-IIB_Ca superfamily domains with OsACA1, OsACA3, OsACA8, OsACA9, and OsACA10. In contrast, OsACA6 lacks the CaATP_NAI domain but contains a CaATP_NAI superfamily domain, whereas OsACA7 has two distinct domains, the HAD_like superfamily and Cation_ATPase_C domains (Figure 2A). To assess whether domain composition is correlated with subcellular localization, we examined the expression of both OsACA3 and OsACA10,which have the same domains as OsACA5 does. Our analysis revealed that both OsACA3 and OsACA10 localized to the ER (Figure 2B). In contrast, OsACA6 and OsACA7, which have distinct domain structures, were localized to the plasma membrane (Supplementary Figure S5) and Golgi apparatus, respectively. On the basis of these results, we hypothesize that other members of the OsACA family, such as OsACA1, OsACA8, and OsACA9, may also localize to the ER and that the conserved CaATP_NAI domain could play a role in determining ER localization. However, further studies are needed to verify this hypothesis. The ER is a crucial site for protein synthesis, folding, and calcium storage. OsACA5 plays a critical role in maintaining ER calcium homeostasis, which is essential for both immune signaling and reproductive development. osaca5 mutation may disrupt ER calcium homeostasis, potentially triggering the unfolded protein response (UPR) and thereby amplifying plant immune signaling (Senft and Ronai, 2015). Although osaca5 mutation altered calcium flux (Figures 4A–D), the precise mechanism through which ER-localized calcium ATPases modulate calcium signaling through membrane-associated effector components remains unclear.

In osaca5 plants, the expression of OsCPK12, OsCML16, OsCIPK31, OsCBL2, and OsPLC1 was downregulated, whereas that of OsRLCK185 and OsCNGC9 was upregulated, suggesting that the Ca²⁺ signaling network was disrupted (Figure 4E). The decreased expression of Ca²⁺ sensors and kinases may weaken calcium decoding, whereas the induction of OsRLCK185 and OsCNGC9 may represent a compensatory response to sustain Ca²⁺ influx and defense activation. Together, these findings imply that OsACA5 acts as an ER-localized Ca²⁺ pump that connects ER calcium homeostasis with cytosolic Ca²⁺ signaling and immune regulation in rice.

To investigate the function of OsACA5, the lesion area of the osaca5 plants inoculated with M. oryzae was investigated and found to be significantly smaller than that of the NIP plants (Figures 3A–D). osaca5 mutation enhanced disease resistance. osaca5 plants exhibited greater H₂O₂ accumulation after M. oryzae infection (Figures 3E–H) and exhibited stronger ROS bursts under flg22 treatment (Figure 3I), and the expression of a series of core defense-related genes was significantly greater in osaca5 plants than in NIP plants (Figures 3J–N). Analysis of the phenotypes and physiological behaviors of both types of plants confirmed that OsACA5 is a negative immune regulator. OsACA5 likely negatively regulates rice blast resistance by modulating calcium signaling. An increase in the cytosolic Ca²⁺ concentration is closely related to ROS burst (Tan et al., 1998). Using NMT, we found that Ca²⁺ influx in osaca5 plants was significantly greater than that in wild-type plants after flg22 and chitin treatment (Figures 4A–D). osaca5 mutation may impair the active transport of Ca²⁺ into the ER lumen. Upon PAMP stimulation, massive Ca²⁺ influx leads to a substantial increase in the cytosolic Ca²⁺ concentration, subsequently triggering a downstream ROS burst and sustained elevation of the expression of defense genes.

However, the enhanced immunity in osaca5 was accompanied by a decreased seed setting rate, reduced plant height, and fewer grains per panicle (Figure 5). osaca5 mutation might directly cause these defects by disrupting specific growth and development processes, particularly reproductive development. Previous studies have shown that inhibiting the ER calcium pump in Arabidopsis leads to depletion of ER calcium stores and disruption of cytosolic calcium homeostasis and consequently blocks pollen tube growth (Iwano et al., 2009). Similarly, research on Oryza sativa has shown that RALF signaling establishes a calcium gradient by activating MLO calcium channels to maintain pollen tube integrity and directional growth (Gao et al., 2023). Through pollen iodine staining assays, we found that pollen viability was not impaired in osaca5 plants (Supplementary Figures S6C, D). We therefore speculate that the reduced seed setting rate in the mutants may have been caused by altering processes such as pollen tube guidance or early postfertilization development rather than directly regulating pollen viability.

In this study, we found that compared with wild-type plants, OsACA5 mutants exhibit enhanced immune responses, likely due to increased calcium influx and ROS bursts, but their seed setting rate is lower (Figure 5). This suggests that enhanced immunity may come at the cost of reproduction, reflecting a physiological trade-off where resources are allocated to immune responses during pathogen infection, which may suppress reproductive processes. We speculate that OsACA5 deficiency disrupts ER calcium homeostasis, affecting pollen tube calcium balance and impairing pollen tube growth or embryo development, ultimately leading to reduced seed setting. These findings indicate that while OsACA5 enhances immunity, its normal function is essential for reproductive development, and plants may need to balance immunity and reproduction under stress conditions. Future studies will further explore how OsACA5 coordinates these processes and regulates calcium signaling.

Taken together, the results of this study reveal the significant role of the ER-localized Ca²⁺-ATPase OsACA5 in regulating immunity and development in rice. OsACA5 prevents the activation of immune responses by negatively regulating pathogen signal-triggered calcium influx and ROS bursts. Moreover, its normal function is vital for maintaining calcium homeostasis during reproductive processes, thereby ensuring yield formation. osaca5 mutation results in increased disease resistance but a reduced seed setting rate. This study demonstrates that the ER-localized Ca²⁺-ATPase OsACA5 modulates the regulation of disease resistance and the seed setting rate in rice. The osaca5 mutants exhibited increases in calcium influx, and disease resistance, and, consequently, a reduced seed setting rate.

4 Materials and methods

4.1 Plant materials and growth conditions

Transgenic lines were developed on the Nipponbare (NIP) background and maintained in our laboratory. Two independent osaca5 knockout mutants (osaca5–1 and osaca5-2) were generated using CRISPR/Cas9. Guide RNAs targeting OsACA5 were designed using the E-CRISP web tool and cloned and inserted into a Cas9 expression vector. Two sgRNAs were used to target OsACA5, and the resulting mutants contained indels at least two target sites, as shown in Supplementary Figure S1. The constructs were introduced into NIP calli via Agrobacterium tumefaciens EHA105-mediated transformation, and T₀ plants were screened by PCR and sequencing of the OsACA5 target site. T₁ and T₂ plants of the two knockout mutants and wild-type NIP were grown under field conditions at the Yazhou Bay Certification and Breeding Base in Sanya, Hainan Province. At maturity, the agronomic traits of ten randomly selected plants from each genotype were assessed. After harvest, the grains were dried at 37 °C for four days, after which the seed setting rate and thousand-grain weight were measured.

4.2 Phylogenetic tree construction, domain analysis, cis-acting element prediction, and spatiotemporal expression profiling

To investigate the evolutionary relationships and functional characteristics of the rice Ca²⁺-ATPase gene families OsACA and OsECA, we performed gene family identification and phylogenetic analysis, domain analysis, cis-acting element prediction, and spatiotemporal expression profiling for both families. First, a phylogenetic tree was constructed using the full-length amino acid sequences of members from the OsACA and OsECA families. The tree was generated in MEGA using the Neighbor-Joining (NJ) method, and its stability was evaluated by 1000 bootstrap resamplings. The resulting phylogeny reveals the evolutionary relationships among members of the two families and provides a basis for further functional inference. For domain analysis, the amino acid sequences of OsACA and OsECA proteins were analyzed using InterPro, SMART, and NCBI CD-Search (Conserved Domain Database, CDD) to identify conserved domains, such as the P-type ATPase domain and the CaATP_NAI domain. The domain information was integrated with the phylogenetic tree to further characterize the potential functional features of different gene members. In addition, cis-acting element prediction was conducted on the 2.0 kb promoter regions upstream of each OsACA and OsECA gene using the PlantCARE database. The identified cis-acting elements were categorized and summarized, and the results were visualized using bar graphs. Finally, to investigate the spatiotemporal expression patterns of the OsACA and OsECA family members, we retrieved expression data from the RiceXPro database. A heatmap was generated based on transcript abundance (TPM) across different tissues and developmental stages, revealing the dynamic expression profiles of these Ca²⁺-ATPase genes throughout rice development.

4.3 Subcellular localization

The coding sequence of OsACA5 lacking its stop codon was amplified and fused in frame to the N-terminus of GFP under the control of the CaMV 35S promoter in the pCAMBIA1390 vector. Recombinant and empty constructs were delivered into rice mesophyll protoplasts isolated from 10-day-old etiolated seedlings using a Plant Protoplast Preparation and Transformation Kit (Coolaber, Beijing, China; Cat. PPT111-5T) and transfected via a PEG-CaCl₂ solution at room temperature. After overnight incubation at 28°C in the dark, GFP fluorescence was observed in live protoplasts on a Zeiss LSM880 confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany).

4.4 Pathogenicity assays

Seven- to ten-day-old cultures of M. oryzae strain 70–15 were washed from agar plates with 0.5% (v/v) Tween-20, and the conidial suspension was adjusted to 2.0 × 10⁵ spores/mL. For the wound inoculation assays, 15 leaves per genotype from three-week-old rice seedlings were gently pricked with a sterile needle to generate three wound sites per leaf, and the wound sites were inoculated with 10 µL drops of the conidial suspension, after which the inoculation sites were covered with moist cotton soaked in a preservative solution. For the spray inoculation assays, two-week-old seedlings were uniformly misted with a conidial suspension (2.0 × 10⁵ spores/mL) prepared by eluting spores from culture plates with 5 g/L gelatin. In both assays, the plants were incubated in a moist chamber at 28°C in the dark for 24 h and then subjected to a 12 h light/12 h dark cycle at the same temperature and humidity for six days.

4.5 RNA extraction and qRT–PCR

Total RNA was extracted from two-week-old Nipponbare seedlings spray-inoculated with M. oryzae strain 70-15 (treatment) or 5 g/L gelatin (mock control) after 24 h of incubation at 28°C in the dark (≥ 90% humidity) by grinding liquid nitrogen-frozen leaf tissue in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and following the manufacturer’s protocol; RNA integrity was confirmed by 1% agarose gel electrophoresis, and the purity (A₂₆₀/A₂₈₀) was determined on a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). For resistance gene expression, osaca5 seedlings were processed similarly after 2 h of inoculation, and 1 µg of total RNA was reverse transcribed and preamplified in a single tube using HiScript IV All-in-One Ultra RT SuperMix (Vazyme, Nanjing, China). qRT–PCR was performed on a LightCycler 480 II (Roche, Basel, Switzerland) with ChamQ Universal SYBR qPCR Master Mix (Vazyme) in 20 µL reactions (1 µL of cDNA, 200 nM primers) using the following conditions: 95°C for 30 s and 40 cycles of 95°C for 10 s/60°C for 30 s, followed by melt-curve analysis; each of three biological replicates was assayed in technical triplicates.

4.6 Measurements of net Ca²⁺ flux

Ca²⁺ movement in and out of rice mesophyll cells, known as Ca²⁺ flux, was measured in real time using noninvasive microtest technology equipment (NMT Physiolyzer, YoungerUSA LLC; Xuyue (Beijing) Sci. & Tech. Co., Ltd.). The leaf samples were torn, and the resulting pieces were fixed to the bottom of a culture dish with double-sided tape. Measuring solution was added to submerge the samples, and after a 30-minute incubation, the solution was discarded and replaced with 5 ml of fresh measuring solution for analysis. A mesophyll cell was identified under a microscope, and the Ca²⁺ flux microsensor was positioned approximately 10 μm from the cell surface before detection. Data were collected on each mesophyll cell for 5 minutes, and a treatment solution was added to the culture dish to reach a final concentration of 1 μM. Ca²⁺ flux was continuously monitored until the signal stabilized and did not significantly increase or decrease. Each test group consisted of 6 replicates. Ca²⁺ flux data were recorded using imFluxes V2.0 software (YoungerUSA LLC, Amherst, MA 01002, USA), and the expressed as picomoles • cm⁻² • s⁻¹; a positive value indicates efflux, and a negative value indicates influx.

4.7 DAB and NBT staining

To detect the accumulation of reactive oxygen species in rice following inoculation with M. oryzae, histochemical staining using DAB and NBT was performed to assess the localization of hydrogen peroxide (H₂O₂) and superoxide anion (O₂⁻), respectively, and quantify their concentrations. Rice leaves from two-week-old seedlings were harvested at 24 hours (for NBT) or 48 hours (for DAB) postinoculation with M. oryzae strain 70-15 (at a spore concentration of 1.5 × 10⁵/mL) and immersed in the respective staining solutions (DAB: 1 mg/mL, pH 3.5; NBT: 0.1%, pH 7.8). The samples were vacuum-infiltrated at –0.08 MPa to –0.1 MPa for 30 minutes and subsequently incubated in the dark at 25 °C for 8 hours (DAB) or 12 hours (NBT), after which the DAB-treated samples were gently agitated at 75 rpm. Chlorophyll was removed by boiling the leaves in 95% ethanol for 20 minutes. DAB-stained samples were imaged using a Smartzoom 5 stereomicroscope (Carl Zeiss AG, Oberkochen, Germany), where reddish-brown precipitates indicated sites of H₂O₂ accumulation, and the pixel intensity of the stained areas was quantified using ImageJ. NBT-stained samples were photographed with a smartphone, with blue deposits revealing O₂⁻ production, and the percentage of stained area per leaf was calculated in ImageJ using the formula: stained area (%) = (blue pixels area/total leaf pixels) × 100%.

4.8 Detection of ROS levels

ROS were detected using the luminol method. Rice plants grown on 1/2 MS medium for 10 days were selected, and sheaths approximately 3 mm in length were excised and equilibrated in ddH₂O for 12 hours. The sheaths were then treated with 1 µM flg22 in a solution containing 20 µM luminol (Sigma) and 10 µg/ml horseradish peroxidase (Sigma). The reaction was continuously monitored at 5-second intervals for 15 minutes using a microplate reader (Tecan, Switzerland).

4.9 Statistical analysis

Lesion area percentages and lesion lengths were measured on digital images using ImageJ software. Agronomic traits such as plant height and tiller number were recorded and compiled for statistical evaluation. Sample normality for agronomic trait measurements and lesion data was assessed by the Shapiro–Wilk test in GraphPad Prism 8.0. Quantitative datasets were subjected to one-way analysis of variance (ANOVA) to detect overall differences among treatment groups, followed by Dunnett’s multiple comparisons test to compare each treatment with the control and report multiplicity-adjusted p values. All the statistical analyses were performed using GraphPad Prism. Differences at p < 0.05 and p < 0.01 were considered significant and highly significant, respectively.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Author contributions

MZ: Visualization, Project administration, Resources, Validation, Formal Analysis, Data curation, Investigation, Writing – review & editing, Writing – original draft, Conceptualization. YZ: Writing – review & editing, Formal Analysis, Writing – original draft, Data curation, Conceptualization. ZP: Funding acquisition, Conceptualization, Data curation, Supervision, Writing – original draft. ST: Conceptualization, Investigation, Writing – original draft. ZZ: Investigation, Writing – original draft, Formal Analysis, Data curation. CL: Conceptualization, Writing – original draft, Investigation. XL: Writing – review & editing, Project administration, Supervision, Data curation, Writing – original draft. JX: Supervision, Writing – review & editing, Funding acquisition, Writing – original draft, Project administration.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This study was supported by the Agricultural Science and Technology Innovation Fund project of Hunan Province (2025CX94) and the Open Project Program of the State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China (SKL-KF202413).

Acknowledgments

We sincerely thank Dr. Jinglei Li (Hunan University), Dr. Xinpeng Li (Hunan Agricultural University), Dr. Pengpeng Fang (Hunan University), and Dr. Weijun Chen (Hunan Hybrid Rice Research Center) for their valuable assistance and helpful suggestions during the experiments.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2026.1758629/full#supplementary-material

References

Asano, T., Kunieda, N., Omura, Y., Ibe, H., Kawasaki, T., Takano, M., et al. (2002). Rice SPK, a calmodulin-like domain protein kinase, is required for storage product accumulation during seed development: phosphorylation of sucrose synthase is a possible factor. Plant Cell. 14, 619–628. doi: 10.1105/tpc.010454

PubMed Abstract | Crossref Full Text | Google Scholar

Axelsen, K. B. and Palmgren, M. G. (2001). Inventory of the superfamily of P-type ion pumps in Arabidopsis. Plant Physiol. 126, 696–706. doi: 10.1104/pp.126.2.696

PubMed Abstract | Crossref Full Text | Google Scholar

Bang, S. W., Lee, D. K., Jung, H., Chung, P. J., Kim, Y. S., Choi, Y. D., et al. (2019). Overexpression of OsTF1L, a rice HD-Zip transcription factor, promotes lignin biosynthesis and stomatal closure that improves drought tolerance. Plant Biotechnol. J. 17, 118–131. doi: 10.1111/pbi.12951

PubMed Abstract | Crossref Full Text | Google Scholar

Boursiac, Y., Lee, S. M., Romanowsky, S., Blank, R., Sladek, C., Chung, W. S., et al. (2010). Disruption of the vacuolar calcium-ATPases in Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway. Plant Physiol. 154, 1158–1171. doi: 10.1104/pp.110.159038

PubMed Abstract | Crossref Full Text | Google Scholar

Chandan, K., Gupta, M., Ahmad, A., and Sarwat, M. (2024). P-type calcium ATPases play important roles in biotic and abiotic stress signaling. Planta 260, 37. doi: 10.1007/s00425-024-04462-7

PubMed Abstract | Crossref Full Text | Google Scholar

Chinchilla, D., Zipfel, C., Robatzek, S., Kemmerling, B., Nurnberger, T., Jones, J. D., et al. (2007). A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448, 497–500. doi: 10.1038/nature05999

PubMed Abstract | Crossref Full Text | Google Scholar

Dai, Y., Feng, X., Liu, Z., Wang, M., Zhou, Y., Cui, L., et al. (2024). miR1432 negatively regulates cold tolerance by targeting OsACAs. Plant Cell Environ. 47, 5443–5456. doi: 10.1111/pce.15109

PubMed Abstract | Crossref Full Text | Google Scholar

Fu, L., Yu, X., and An, C. (2013). Overexpression of constitutively active OsCPK10 increases Arabidopsis resistance against Pseudomonas syringae pv. tomato and rice resistance against Magnaporthe grisea. Plant Physiol. Biochem. 73, 202–210. doi: 10.1016/j.plaphy.2013.10.004

PubMed Abstract | Crossref Full Text | Google Scholar

Gao, M., He, Y., Yin, X., Zhong, X., Yan, B., Wu, Y., et al. (2021). Ca(2+) sensor-mediated ROS scavenging suppresses rice immunity and is exploited by a fungal effector. Cell 184, 5391–404 e17. doi: 10.1016/j.cell.2021.09.009

PubMed Abstract | Crossref Full Text | Google Scholar

Gao, Q., Wang, C., Xi, Y., Shao, Q., Hou, C., Li, L., et al. (2023). RALF signaling pathway activates MLO calcium channels to maintain pollen tube integrity. Cell Res. 33, 71–79. doi: 10.1038/s41422-022-00754-3

PubMed Abstract | Crossref Full Text | Google Scholar

Garcia Bossi, J., Kumar, K., Barberini, M. L., Dominguez, G. D., Rondon Guerrero, Y. D. C., Marino-Buslje, C., et al. (2020). The role of P-type IIA and P-type IIB Ca2+-ATPases in plant development and growth. J. Exp. Bot. 71, 1239–1248. doi: 10.1093/jxb/erz521

PubMed Abstract | Crossref Full Text | Google Scholar

Gilroy, S., Bialasek, M., Suzuki, N., Gorecka, M., Devireddy, A. R., Karpinski, S., et al. (2016). ROS, Calcium, and Electric Signals: Key Mediators of Rapid Systemic Signaling in Plants. Plant Physiol. 171, 1606–1615. doi: 10.1104/pp.16.00434

PubMed Abstract | Crossref Full Text | Google Scholar

Goel, A., Taj, G., Pandey, D., Gupta, S., and Kumar, A. (2011). Genome-wide comparative in silico analysis of calcium transporters of rice and sorghum. Genom Proteomics Bioinf. 9, 138–150. doi: 10.1016/S1672-0229(11)60017-X

PubMed Abstract | Crossref Full Text | Google Scholar

Grant, M., Brown, I., Adams, S., Knight, M., Ainslie, A., and Mansfield, J. (2000). The RPM1 plant disease resistance gene facilitates a rapid and sustained increase in cytosolic calcium that is necessary for the oxidative burst and hypersensitive cell death. Plant J. 23, 441–450. doi: 10.1046/j.1365-313x.2000.00804.x

PubMed Abstract | Crossref Full Text | Google Scholar

Harper, J. F., Hong, B., Hwang, I., Guo, H. Q., Stoddard, R., Huang, J. F., et al. (1998). A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain. J. Biol. Chem. 273, 1099–1106. doi: 10.1074/jbc.273.2.1099

PubMed Abstract | Crossref Full Text | Google Scholar

Huda, K. M., Banu, M. S., Garg, B., Tula, S., Tuteja, R., and Tuteja, N. (2013). OsACA6, a P-type IIB Ca(2)(+) ATPase promotes salinity and drought stress tolerance in tobacco by ROS scavenging and enhancing the expression of stress-responsive genes. Plant J. 76, 997–1015. doi: 10.1111/tpj.12352

PubMed Abstract | Crossref Full Text | Google Scholar

Iwano, M., Entani, T., Shiba, H., Kakita, M., Nagai, T., Mizuno, H., et al. (2009). Fine-tuning of the cytoplasmic Ca2+ concentration is essential for pollen tube growth. Plant Physiol. 150, 1322–1334. doi: 10.1104/pp.109.139329

PubMed Abstract | Crossref Full Text | Google Scholar

Jiang, Q., Wu, X., Zhang, X., Ji, Z., Cao, Y., Duan, Q., et al. (2023). Genome-Wide Identification and Expression Analysis of AS2 Genes in Brassica rapa Reveal Their Potential Roles in Abiotic Stress. Int. J. Mol. Sci. 24. doi: 10.3390/ijms241310534

PubMed Abstract | Crossref Full Text | Google Scholar

Kan, C. C., Chung, T. Y., Wu, H. Y., Juo, Y. A., and Hsieh, M. H. (2017). Exogenous glutamate rapidly induces the expression of genes involved in metabolism and defense responses in rice roots. BMC Genomics 18, 186. doi: 10.1186/s12864-017-3588-7

PubMed Abstract | Crossref Full Text | Google Scholar

Kim, E. Y., Kim, M. H., Yun, S. D., Lee, S. K., Kim, E. J., Kim, J. H., et al. (2024a). Redundant role of OsCNGC4 and OsCNGC5 encoding cyclic nucleotide-gated channels in rice pollen germination and tube growth. Plant Physiol. Biochem. 208, 108522. doi: 10.1016/j.plaphy.2024.108522

PubMed Abstract | Crossref Full Text | Google Scholar

Kim, J. S., Kidokoro, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (2024b). Regulatory networks in plant responses to drought and cold stress. Plant Physiol. 195, 170–189. doi: 10.1093/plphys/kiae105

PubMed Abstract | Crossref Full Text | Google Scholar

Kim, M. C., Chung, W. S., Yun, D. J., and Cho, M. J. (2009). Calcium and calmodulin-mediated regulation of gene expression in plants. Mol. Plant 2, 13–21. doi: 10.1093/mp/ssn091

PubMed Abstract | Crossref Full Text | Google Scholar

Koster, P., DeFalco, T. A., and Zipfel, C. (2022). Ca(2+) signals in plant immunity. EMBO J. 41, e110741. doi: 10.15252/embj.2022110741

PubMed Abstract | Crossref Full Text | Google Scholar

Lee, T. F. and McNellis, T. W. (2009). Evidence that the BONZAI1/COPINE1 protein is a calcium- and pathogen-responsive defense suppressor. Plant Mol. Biol. 69, 155–166. doi: 10.1007/s11103-008-9413-6

PubMed Abstract | Crossref Full Text | Google Scholar

Li, X., Chanroj, S., Wu, Z., Romanowsky, S. M., Harper, J. F., and Sze, H. (2008). A distinct endosomal Ca2+/Mn2+ pump affects root growth through the secretory process. Plant Physiol. 147, 1675–1689. doi: 10.1104/pp.108.119909

PubMed Abstract | Crossref Full Text | Google Scholar

Li, L., Wang, F., Yan, P., Jing, W., Zhang, C., Kudla, J., et al. (2017). A phosphoinositide-specific phospholipase C pathway elicits stress-induced Ca(2+) signals and confers salt tolerance to rice. New Phytol. 214, 1172–1187. doi: 10.1111/nph.14426

PubMed Abstract | Crossref Full Text | Google Scholar

Liang, C. and Ma, Y. (2025). Impact of Exogenous Ca2+ on Maintaining Intracellular Ca2+ Homeostasis in Rice Roots Under Acid Rain Stress. J. Plant Growth Regul., 1–19. doi: 10.1007/s00344-025-11832-2

Crossref Full Text | Google Scholar

Liu, S., Wang, S., Zheng, Y., Zhao, P., Jin, H., and Xie, G. (2025). The OsCaM1-OsCAMTA1 module confers salinity tolerance by enhancing transcripts of the targets OsDREB1B and OsCML16 in rice. Plant J. 123, e70420. doi: 10.1111/tpj.70420

PubMed Abstract | Crossref Full Text | Google Scholar

Napier, R. M., Fowke, L. C., Hawes, C., Lewis, M., and Pelham, H. R. (1992). Immunological evidence that plants use both HDEL and KDEL for targeting proteins to the endoplasmic reticulum. J. Cell Sci 102, 261–271. doi: 10.1242/jcs.102.2.261

PubMed Abstract | Crossref Full Text | Google Scholar

Piao, H. L., Xuan, Y. H., Park, S. H., Je, B. I., Park, S. J., Park, S. H., et al. (2010). OsCIPK31, a CBL-interacting protein kinase is involved in germination and seedling growth under abiotic stress conditions in rice plants. Mol. Cells 30, 19–27. doi: 10.1007/s10059-010-0084-1

PubMed Abstract | Crossref Full Text | Google Scholar

Qin, X., Zhang, X., Ma, C., Yang, X., Hu, Y., Liu, Y., et al. (2024). Rice OsCIPK17-OsCBL2/3 module enhances shoot Na(+) exclusion and plant salt tolerance in transgenic Arabidopsis. Plant Physiol. Biochem. 215, 109034. doi: 10.1016/j.plaphy.2024.109034

PubMed Abstract | Crossref Full Text | Google Scholar

Rahmati Ishka, M., Brown, E., Rosenberg, A., Romanowsky, S., Davis, J. A., Choi, W. G., et al. (2021). Arabidopsis Ca2+-ATPases 1, 2, and 7 in the endoplasmic reticulum contribute to growth and pollen fitness. Plant Physiol. 185, 1966–1985. doi: 10.1093/plphys/kiab021

PubMed Abstract | Crossref Full Text | Google Scholar

Ranf, S., Eschen-Lippold, L., Pecher, P., Lee, J., and Scheel, D. (2011). Interplay between calcium signalling and early signalling elements during defence responses to microbe- or damage-associated molecular patterns. Plant J. 68, 100–113. doi: 10.1111/j.1365-313X.2011.04671.x

PubMed Abstract | Crossref Full Text | Google Scholar

Salem, F., ElGamal, A., Tang, X., Yang, J., and Kong, W. (2025a). Transcriptional Dynamics of Receptor-Based Genes Reveal Immunity Hubs in Rice Response to Magnaporthe oryzae Infection. Int. J. Mol. Sci. 26. doi: 10.3390/ijms26104618

PubMed Abstract | Crossref Full Text | Google Scholar

Salem, F., ElGamal, A., Zhang, Z., and Kong, W. (2025b). Integrative multi-transcriptomic analysis uncovers core genes and potential defense mechanisms in rice-Magnoporthe oryzae interaction. Plant Cell Rep. 44, 114. doi: 10.1007/s00299-025-03490-1

PubMed Abstract | Crossref Full Text | Google Scholar

Schiott, M., Romanowsky, S. M., Baekgaard, L., Jakobsen, M. K., Palmgren, M. G., and Harper, J. F. (2004). A plant plasma membrane Ca2+ pump is required for normal pollen tube growth and fertilization. Proc. Natl. Acad. Sci. U. S. A. 101, 9502–9507. doi: 10.1073/pnas.0401542101

PubMed Abstract | Crossref Full Text | Google Scholar

Senft, D. and Ronai, Z. A. (2015). UPR, autophagy, and mitochondria crosstalk underlies the ER stress response. Trends Biochem. Sci. 40, 141–148. doi: 10.1016/j.tibs.2015.01.002

PubMed Abstract | Crossref Full Text | Google Scholar

Singh, A., Kanwar, P., Yadav, A. K., Mishra, M., Jha, S. K., Baranwal, V., et al. (2014). Genome-wide expressional and functional analysis of calcium transport elements during abiotic stress and development in rice. FEBS J. 281, 894–915. doi: 10.1111/febs.12656

PubMed Abstract | Crossref Full Text | Google Scholar

Tan, S., Sagara, Y., Liu, Y., Maher, P., and Schubert, D. (1998). The regulation of reactive oxygen species production during programmed cell death. J. Cell Biol. 141, 1423–1432. doi: 10.1083/jcb.141.6.1423

PubMed Abstract | Crossref Full Text | Google Scholar

Taneja, M. and Upadhyay, S. K. (2018). Molecular characterization and differential expression suggested diverse functions of P-type II Ca(2+)ATPases in Triticum aestivum L. BMC Genomics 19, 389. doi: 10.1186/s12864-018-4792-9

PubMed Abstract | Crossref Full Text | Google Scholar

Tavu, L. E. J. and Redillas, M. (2025). Oxidative Stress in Rice (Oryza sativa): Mechanisms, Impact, and Adaptive Strategies. Plants (Basel) 14. doi: 10.3390/plants14101463

PubMed Abstract | Crossref Full Text | Google Scholar

Tian, W., Wang, C., Gao, Q., Li, L., and Luan, S. (2020). Calcium spikes, waves and oscillations in plant development and biotic interactions. Nat. Plants 6, 750–759. doi: 10.1038/s41477-020-0667-6

PubMed Abstract | Crossref Full Text | Google Scholar

Upadhyay, S. K. (2022). Calcium Channels, OST1 and Stomatal Defence: Current Status and Beyond. Cells 12. doi: 10.3390/cells12010127

PubMed Abstract | Crossref Full Text | Google Scholar

Wang, J., Liu, X., Zhang, A., Ren, Y., Wu, F., Wang, G., et al. (2019). A cyclic nucleotide-gated channel mediates cytoplasmic calcium elevation and disease resistance in rice. Cell Res. 29, 820–831. doi: 10.1038/s41422-019-0219-7

PubMed Abstract | Crossref Full Text | Google Scholar

Wang, J., Ren, Y., Liu, X., Luo, S., Zhang, X., Liu, X., et al. (2021). Transcriptional activation and phosphorylation of OsCNGC9 confer enhanced chilling tolerance in rice. Mol. Plant 14, 315–329. doi: 10.1016/j.molp.2020.11.022

PubMed Abstract | Crossref Full Text | Google Scholar

Wang, X., Wang, Z., Lu, Y., Huang, J., Hu, Z., Lou, J., et al. (2024). OsACA9, an Autoinhibited Ca(2+)-ATPase, Synergically Regulates Disease Resistance and Leaf Senescence in Rice. Int. J. Mol. Sci. 25. doi: 10.3390/ijms25031874

PubMed Abstract | Crossref Full Text | Google Scholar

Wei, S., Hu, W., Deng, X., Zhang, Y., Liu, X., Zhao, X., et al. (2014). A rice calcium-dependent protein kinase OsCPK9 positively regulates drought stress tolerance and spikelet fertility. BMC Plant Biol. 14, 133. doi: 10.1186/1471-2229-14-133

PubMed Abstract | Crossref Full Text | Google Scholar

Wen, K., Chen, Y., Zhou, X., Chang, S., Feng, H., Zhang, J., et al. (2019). OsCPK21 is required for pollen late-stage development in rice. J. Plant Physiol. 240, 153000. doi: 10.1016/j.jplph.2019.153000

PubMed Abstract | Crossref Full Text | Google Scholar

Xu, Y., Yang, J., Wang, Y., Wang, J., Yu, Y., Long, Y., et al. (2017). OsCNGC13 promotes seed-setting rate by facilitating pollen tube growth in stylar tissues. PLoS Genet. 13, e1006906. doi: 10.1371/journal.pgen.1006906

PubMed Abstract | Crossref Full Text | Google Scholar

Yamaguchi, K., Yamada, K., Ishikawa, K., Yoshimura, S., Hayashi, N., Uchihashi, K., et al. (2013). A receptor-like cytoplasmic kinase targeted by a plant pathogen effector is directly phosphorylated by the chitin receptor and mediates rice immunity. Cell Host Microbe 13, 347–357. doi: 10.1016/j.chom.2013.02.007

PubMed Abstract | Crossref Full Text | Google Scholar

Yang, D. L., Shi, Z., Bao, Y., Yan, J., Yang, Z., Yu, H., et al. (2017). Calcium Pumps and Interacting BON1 Protein Modulate Calcium Signature, Stomatal Closure, and Plant Immunity. Plant Physiol. 175, 424–437. doi: 10.1104/pp.17.00495

PubMed Abstract | Crossref Full Text | Google Scholar

Yu, H., Yan, J., Du, X., and Hua, J. (2018). Overlapping and differential roles of plasma membrane calcium ATPases in Arabidopsis growth and environmental responses. J. Exp. Bot. 69, 2693–2703. doi: 10.1093/jxb/ery073

PubMed Abstract | Crossref Full Text | Google Scholar

Zhang, Q., Song, T., Guan, C., Gao, Y., Ma, J., Gu, X., et al. (2021). OsANN4 modulates ROS production and mediates Ca(2+) influx in response to ABA. BMC Plant Biol. 21, 474. doi: 10.1186/s12870-021-03248-3

PubMed Abstract | Crossref Full Text | Google Scholar