On February 9, 2026, the research team led by Professor Jianfu Zhang from the Rice Research Institute of Fujian Academy of Agricultural Sciences published a research article entitled “The IPA1–NRT1.1B regulatory module regulates ROS homeostasis to confer cadmium tolerance in rice” in Plant Stress.
During the study, rice seedlings were cultivated in a JIUPO plant growth chamber under controlled environmental conditions, with a 16 h light/8 h dark photoperiod cycle. The temperature was maintained at 28°C during the light period and 26°C during the dark period. This study revealed a previously unknown regulatory layer involved in rice responses to cadmium stress, providing potential molecular targets for developing cadmium-tolerant rice varieties through molecular breeding strategies.
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Cadmium (Cd) is a highly toxic, non-essential heavy metal that poses severe threats to plants, animals, and human health. It can accumulate within plant tissues and subsequently enter edible grains, causing risks to human health through the food chain.
As one of the world’s major staple crops, rice serves as a primary dietary source of cadmium exposure. Long-term intake of cadmium-contaminated rice can cause kidney damage, neurotoxicity, and even cancer. Furthermore, cadmium has a biological half-life of up to 30 years in the human body. Therefore, developing low-cadmium rice varieties is of great significance for ensuring food safety and promoting sustainable agricultural development.
This study systematically elucidated the molecular mechanisms by which IPA1 and NRT1.1B respond to cadmium stress. Based on previous studies, site-directed mutations were introduced at the S201 and S213 phosphorylation sites of IPA1. The results demonstrated that phosphorylation at these sites weakened the binding ability of IPA1 to the NRT1.1B promoter, thereby regulating its transcriptional activation activity.
This mechanism establishes a connection between stress kinase signaling and reactive oxygen species (ROS)-mediated transcriptional regulation of cadmium tolerance, providing new insights into the molecular regulatory network underlying cadmium stress responses in rice.
The research team conducted hydroponic experiments using 100 μM Cd²⁺ treatment to simulate cadmium stress. The results showed that cadmium stress inhibited shoot and root growth of FH7185 (wild type), while the growth inhibition was more severe in the ipa1 mutant.
Further analysis revealed that under control conditions, no significant differences in cadmium content were observed among different genotypes. However, after treatment with 100 μM Cd²⁺, cadmium accumulation in the roots and shoots of the ipa1-1 mutant was significantly higher than that in the wild type, with particularly pronounced accumulation in roots.
In addition, ROS levels showed no significant differences between the two genotypes under control conditions. Following cadmium exposure, ROS accumulation increased in all tested lines. The ipa1 mutant exhibited the strongest DAB and NBT staining signals, accompanied by reduced CAT and POD activities and increased MDA content, indicating enhanced oxidative stress.
Figure 1: Temporal expression analysis of IPA1, morphological differences between ipa1 mutant and wild-type rice, and analysis of related physiological indicators
Furthermore, the research team performed a time-course expression analysis of antioxidant defense-related genes and metal transport-related genes in FH7185 and ipa1 mutant plants under 200 μM CdCl₂ treatment. The results indicated that IPA1 may regulate antioxidant defense mechanisms and cadmium homeostasis under stress conditions.
Taken together, these findings demonstrate that IPA1 enhances cadmium tolerance in rice by maintaining ROS balance and regulating the expression of cadmium transport-related genes.
Figure 2: Expression analysis of antioxidant genes and cadmium transporter genes in FH7185 and ipa1 mutant plants
To identify downstream targets of IPA1 under cadmium stress, the research team performed RNA sequencing (RNA-seq) analysis on rice seedlings of the ipa1 mutant treated with 200 μM CdCl₂ for 3 hours and untreated control seedlings.
The results revealed that loss of IPA1 function caused extensive transcriptional reprogramming in rice under cadmium stress. Gene Ontology (GO) enrichment analysis indicated that IPA1 may participate in the early activation of NRT1.1B and maintain its transcriptional response under cadmium stress.
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further showed that differentially expressed genes were significantly enriched in defense-related pathways, including phenylpropanoid biosynthesis and plant hormone signal transduction pathways, which are closely associated with ROS homeostasis and detoxification mechanisms.
Figure 3: Analysis of IPA1 binding to the NRT1.1B promoter and transcriptional regulation process
To verify the transcriptional regulatory role of IPA1 on NRT1.1B, the research team employed multiple molecular approaches, including yeast one-hybrid assays, electrophoretic mobility shift assays (EMSA), dual-luciferase (LUC) reporter assays, and quantitative reverse transcription PCR (qRT-PCR).
The combined molecular, genetic, and biochemical evidence demonstrated that IPA1 directly activates NRT1.1B expression by binding to its promoter, thereby potentially regulating oxidative stress responses under cadmium exposure.
Figure 4. Interaction analysis between IPA1 and NRT1.1B and relative expression analysis of NRT1.1B
The researchers further analyzed the expression pattern of NRT1.1B, as well as the physiological characteristics of nrt1.1b mutant rice, under cadmium stress.
The results showed that disruption of NRT1.1B significantly affected rice responses to cadmium exposure. Compared with wild-type plants, nrt1.1b mutants exhibited enhanced sensitivity to cadmium stress, accompanied by increased cadmium accumulation and elevated oxidative damage.
Analysis of antioxidant enzyme activities and ROS-related indicators further demonstrated that NRT1.1B plays an important role in maintaining ROS homeostasis and improving cadmium tolerance in rice.
Figure 5. Temporal expression analysis of NRT1.1B, morphological differences between nrt1.1b mutant and wild-type rice, and analysis of related physiological indicators
OsSAPK6 phosphorylates IPA1 at two key sites, Ser201 and Ser213, which is essential for IPA1-mediated regulation of stress responses in rice.
In this study, the two phosphorylation sites were substituted with aspartic acid residues to mimic phosphorylation. EMSA and dual-luciferase assays showed that the mutant IPA1 proteins exhibited significantly reduced binding affinity toward the NRT1.1B promoter and decreased transcriptional activation ability compared with the native IPA1 protein.
These results indicate that phosphorylation at these sites is required for maintaining the normal DNA-binding activity and transcriptional activation function of IPA1.
Collectively, these findings demonstrate that OsSAPK6 regulates IPA1 transcriptional activity through phosphorylation of the Ser201 and Ser213 residues, thereby modulating downstream cadmium stress responses.
Figure 6. Expression analysis of antioxidant genes and cadmium transporter genes in ZH11 and nrt1.1b mutant plants
The study revealed that OsSAPK6 expression is induced by cadmium treatment.
Phenotypic analysis showed that compared with wild-type ZH11, the Ossapk6 mutant displayed enhanced sensitivity to 100 μM CdCl₂ treatment, characterized by more severe leaf chlorosis and stronger growth inhibition.
Cadmium content measurements demonstrated that under control conditions, no significant differences in cadmium accumulation were observed between the mutant and wild-type plants in either roots or shoots. However, following cadmium treatment, the Ossapk6-1 mutant exhibited significantly increased cadmium accumulation, particularly in roots, indicating that loss of OsSAPK6 promotes cadmium uptake and accumulation in rice.
DAB and NBT staining analyses, together with physiological measurements, showed that ROS levels were comparable between mutant and wild-type plants under non-stress conditions. However, under cadmium stress, the mutant accumulated higher levels of ROS, exhibited increased MDA content, and showed reduced POD and CAT activities.
These results indicate that OsSAPK6 enhances cadmium tolerance in rice by reducing ROS accumulation and maintaining oxidative homeostasis.
Figure 7. Binding affinity of different IPA1 phosphorylation variants to the NRT1.1B promoter and expression pattern of OsSAPK6
Cadmium contamination poses a serious threat to food security and human health. Due to its high toxicity and tendency to accumulate in major food crops such as rice, understanding the molecular mechanisms underlying cadmium uptake, detoxification, and tolerance in rice is essential for developing low-cadmium rice varieties.
This study identified that the SAPK6–IPA1–NRT1.1B regulatory module enhances cadmium tolerance in rice by maintaining reactive oxygen species (ROS) homeostasis.
The findings reveal a novel mechanism of cadmium stress response involving the coordinated interaction of transcriptional regulation, protein phosphorylation, and ROS scavenging systems, providing valuable molecular targets for breeding cadmium-resistant rice varieties through molecular breeding approaches.
Author Information
Doctoral student Feihe Chen, master’s student Zhijia Wu, and doctoral student Haomin Zhang from the College of Plant Protection, Fujian Agriculture and Forestry University, are co-first authors of this research article.
Master’s students Yifei Zhang and Yingchun Zhang from the same institution, together with researchers from the Rice Research Institute of Fujian Academy of Agricultural Sciences, including Dr. Yongsheng Zhu, Dr. Yanjia Xiao, Senior Agronomist Hongguang Xie, Associate Researcher Qiuhua Cai, Dr. Liping Chen, and Academician Hua’an Xie, participated in this study.
Professor Jianfu Zhang from the Rice Research Institute of Fujian Academy of Agricultural Sciences served as the corresponding author.