The Crucial Role of Enzyme OsPLC4 in Mediating Ferroptosis in Rice

On November 10, 2025, a research team led by Professor Nam-Soo Jwa from Sejong University published a research paper titled "Rice phospholipase C4 enhances Ca²⁺ and ROS bursts during effector-triggered immunity leading to ferroptotic cell death" in Plant Communications. This study revealed the crucial role of rice phospholipase C4 (OsPLC4) in mediating ferroptosis in rice, providing a new perspective for understanding the molecular mechanisms of ferroptosis in plants.

Research Background

Ferroptosis is a type of programmed cell death regulated by iron ions and reactive oxygen species (ROS), playing a key role in plant immune responses. When rice is infected with avirulent rice blast strains, intracellular Ca²⁺ influx and ROS bursts synergistically trigger ferroptosis, thereby activating defense responses.

Phospholipase C (PLC) is an important enzyme connecting lipid signaling and Ca²⁺ regulation, but its mechanism of action in rice immunity is still unclear. This study found that rice phospholipase C4 (OsPLC4) promotes ferroptosis during effector-triggered immunity (ETI) by regulating Ca²⁺ and ROS levels, providing a new molecular mechanism for understanding the plant disease resistance signaling network.

Research Content

Avirulent M. oryzae infection triggers intracellular Ca²⁺ accumulation, OsPLC4 expression, and IP₃ generation in rice

Imaging of rice leaf sheath cells using Fluo-5F AM showed no significant difference in intracellular Ca²⁺ levels between PO6-6 (virulent) and INA168 (avirulent) treatments at 0–6 dpi; however, at 36–48 hpi, the green fluorescence in INA168-treated rice cells was significantly enhanced, accompanied by hypersensitive response (HR), while this phenomenon was not observed in PO6-6-treated rice cells, and extracellular (apoplast) Ca²⁺ significantly increased at 36 hpi. qRT-PCR results showed that OsPLC4 expression was significantly increased at 6–24 hpi after INA168 treatment, while it remained at a low level after PO6-6 treatment; simultaneously, the IP₃ content continuously increased at 6–48 hpi after INA168 treatment and was significantly higher than that after PO6-6 treatment, which is consistent with the OsPLC4-induced and continuously increasing Ca²⁺ content. In terms of disease resistance phenotype, ∆Osplc4 plants showed increased fungal hyphal growth in the leaf sheath, reduced cellular HR, increased leaf lesion area, and increased fungal biomass. At the molecular level, OsRbohB, OsMEK2, OsMPK1, and OsPAL1 were rapidly induced in the wild type (WT), while the expression of these genes was significantly suppressed in ∆Osplc4 (1H). These results indicate that OsPLC4 is a key member of the rice PLC family that mediates sustained Ca²⁺ influx and defense activation during ETI.

OsPLC4: A Key Gene in Ferroptotic Cell Death

Upon infection with a non-toxic strain of M. oryzae, wild-type rice exhibited significant intracellular Ca²⁺ influx, ROS and Fe³⁺ accumulation, lipid peroxidation, and HR cell death. These responses were significantly suppressed in the ΔOsplc4 mutant. The mutant showed decreased IP₃ levels, absence of ROS and Fe³⁺ signals, reduced MDA levels, and no HR, indicating that OsPLC4 mediates ferroptotic cell death by regulating the IP₃-dependent Ca²⁺ signaling pathway.

Different Chemical Substances Can Regulate Ca²⁺-Mediated Ferroptosis in Rice

Treatment with different chemical substances revealed that Ca²⁺ signaling is crucial in OsPLC4-mediated ferroptosis. The Ca²⁺ chelator EGTA significantly inhibited the accumulation of Ca²⁺, ROS, and Fe³⁺, as well as HR cell death; neomycin (a PLC inhibitor) and nifedipine (a Ca²⁺ channel blocker) partially reduced these responses. Conversely, TFP (a calmodulin binding agent) and ASM (a salicylic acid analog) significantly enhanced Ca²⁺ influx, ROS and Fe³⁺ accumulation, and HR cell death. These results indicate that Ca²⁺ influx inhibitors can suppress ferroptosis, while Ca²⁺ activators can promote ferroptosis.

Model of OsPLC4-Mediated Ferroptosis in Rice Plant Immunity

The study proposes a mechanistic model of OsPLC4-regulated Ca²⁺-dependent ferroptosis. In effector-triggered immunity (ETI), extracellular Ca²⁺ is released and enters the cytoplasm through resistance bodies and Ca²⁺ channels. OsPLC4, located on the plasma membrane, is activated and hydrolyzes PIP₂ to produce IP₃ and DAG; IP₃ promotes the release of Ca²⁺ from the endoplasmic reticulum, while the PA generated from DAG activates the MAPK and Rboh pathways, inducing ROS production. The interaction of Ca²⁺, ROS, and Fe²⁺ triggers lipid peroxidation and ferroptosis. EGTA, Neomycin, and NDP inhibit this pathway, while TFP and ASM enhance the response. This model reveals the crucial role of OsPLC4 in connecting Ca²⁺, ROS, and iron signaling and regulating plant immunity.

Figure 1. Proposed model of the phospholipase C4 pathway and its central role in the tight crosstalk between Ca²⁺ and ROS in Ca²⁺-mediated ferroptotic cell death downstream of resistosome activation. (Nguyen, et al. 2025)

Summary

This research represents the collective effort of Professor Nam-Soo Jwa's team at Sejong University. The team first discovered ferroptosis in rice during infection by the M. oryzae in 2019 and has since been dedicated to unraveling its molecular mechanisms, deeply exploring the synergistic mechanisms of calcium ions, reactive oxygen species, and iron signaling in regulating plant ferroptosis and immune responses.

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Reference

1. Nguyen, N. K., et al. (2025). Rice phospholipase C4 enhances Ca²⁺ and ROS bursts during effector-triggered immunity leading to ferroptotic cell death. Plant Communications. DOI: 1016/j.xplc.2025.101589.