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Project Article | Ammonium Activates the bZIP20-APX2/CATA Transcriptional Module via ABA-Dependent Pathways to Respond to Cadmium Stress in Rice Seedlings
Release time:
2025-01-07
Cadmium (Cd²⁺) is a highly toxic heavy metal that enters the soil through human activities. Its long-term accumulation poses a threat to both plant and human health. Plants growing in Cd²⁺-contaminated environments suffer from oxidative stress, metabolic disorders, nutrient imbalances, and tissue necrosis. Moreover, Cd²⁺ can accumulate in humans through the food chain, leading to various diseases.
Rice, as an important global food crop, is also a major source of Cd²⁺ accumulation. The form of nitrogen fertilizer, especially ammonium nitrogen (NH₄⁺), has a significant impact on plant growth and Cd²⁺ uptake. Research has shown that abscisic acid (ABA) is involved in regulating plant responses to Cd²⁺ stress, and NH₄⁺ can promote ABA accumulation and activate related antioxidant pathways, thereby enhancing rice's tolerance to Cd²⁺ stress. However, the exact mechanism by which NH₄⁺ alleviates Cd²⁺ toxicity via the ABA pathway still requires further investigation.
To better understand how NH₄⁺ mitigates Cd²⁺ toxicity in rice through ABA-dependent pathways, the research aims to develop strategies for reducing Cd²⁺ accumulation in crops, protecting human health, and improving agricultural sustainability. Recently, a research paper titled "Ammonium mitigates cadmium toxicity by activating the bZIP20-APX2/CATA transcriptional module in rice seedlings in an ABA-dependent manner" was published in the Journal of Hazardous Materials (CAS Q1, IF=12.2). The study was led by Dr. Dongwei Di as the first author and Professor Weiming Shi as the corresponding author.
Research Content
The study found that, compared to nitrate (NO₃⁻) treatment, ammonium (NH₄⁺) significantly reduced Cd²⁺ uptake in rice roots, improved its antioxidant capacity, and enhanced resistance to Cd²⁺ stress. Exogenous ABA addition improved rice growth under Cd²⁺ stress, while the ABA biosynthesis inhibitor fluorinone (Flu) exacerbated the stress. NH₄⁺ alleviated Cd²⁺ toxicity by reducing the accumulation of Cd²⁺ and reactive oxygen species (H₂O₂) in an ABA-dependent manner. In short, NH₄⁺ enhances rice tolerance to Cd²⁺ stress by activating the ABA signaling pathway.
Figure 1: NH₄⁺ enhances plant tolerance to Cd²⁺ stress by influencing ABA signaling pathways.
The researchers analyzed the transcription levels of genes involved in ABA biosynthesis under different combinations of Cd²⁺ with NH₄⁺ and NO₃⁻. The results showed that under high Cd²⁺ stress, NH₄⁺ promoted the transcription of ABA biosynthesis genes more than NO₃⁻, increasing ABA accumulation in plants.
Figure 2: NH₄⁺ increases ABA accumulation by promoting ABA biosynthesis.
The study also analyzed the transcription levels of 89 bZIP transcription factors. The transcription level of bZIP factors under NO₃⁻ + Cd²⁺ conditions was set as "1," and the transcription factors induced or repressed by NH₄⁺ + Cd²⁺ and NO₃⁻ + Cd²⁺ + ABA were identified. A total of 12 upregulated and 12 downregulated genes were identified. Only the upregulation of OsbZIP20, 21, and 29 could be suppressed by exogenous Flu, while the downregulation of bZIP16 and 34 could be alleviated by Flu.
Figure 3: OsbZIP20 may be a key transcription factor for NH₄⁺-induced and ABA-dependent Cd²⁺ stress tolerance.
The root growth of Osbzip20 mutants and wild-type ZH11 was measured under NO₃⁻ and NH₄⁺ nitrogen sources with Cd²⁺ stress. Under NO₃⁻ + Cd²⁺ conditions, both Osbzip20 and ZH11 showed similar root growth inhibition. However, under NH₄⁺ + Cd²⁺ conditions, Osbzip20 showed more severe root growth inhibition than ZH11. Endogenous Cd²⁺ content in the roots under NH₄⁺ + Cd²⁺ conditions was similar between the two. H₂O₂ and malondialdehyde (MDA) levels were measured in their roots. Compared to ZH11, Osbzip20 roots accumulated higher levels of MDA and H₂O₂, suggesting that OsbZIP20 does not directly affect Cd²⁺ accumulation in rice roots. Further analysis of key antioxidant enzyme activity revealed that OsbZIP20 helps maintain the activity of antioxidant enzymes, particularly APX and CAT.
Figure 4: OsbZIP20 participates in Cd²⁺ stress tolerance by maintaining APX and CAT enzyme activities under Cd²⁺ stress.
qPCR analysis of the expression of antioxidant enzyme genes in rice Nip and OsbZIP20 overexpressors (OsbZIP20ox) revealed that OsbZIP20 significantly regulates OsAPX2 and OsCATA. Yeast one-hybrid (Y1H) and CUT&RUN-qPCR confirmed that OsbZIP20 interacts with the promoters of OsAPX2 and OsCATA. However, transient expression in tobacco showed that OsbZIP20 slightly upregulated the transcription of OsAPX2 and OsCATA in tobacco leaves, suggesting that OsbZIP20 regulates the transcription of downstream genes required for protein modification.
Figure 5: OsbZIP20 directly binds and activates the promoters of OsAPX2 and OsCATA.
Previous studies found that OsSAPK9 can phosphorylate and activate OsbZIP20 in rice. To determine whether phosphorylation of OsbZIP20 activates downstream gene transcription, potential phosphorylation sites on OsbZIP20 were predicted using Plant PTM Viewer 2.0, identifying Ser6 and Ser150. Phosphorylation simulations indicated that phosphorylation of OsbZIP20 is necessary for its transcriptional activity. Dual-Luciferase assays showed that phosphorylated OsbZIP20 significantly activated OsAPX2 and OsCATA transcription, and co-transfection with OsbZIP20 and OsSAPK9 also activated OsAPX2 and OsCATA transcription. When Ser6 and Ser150 were replaced with alanine, the activation of OsAPX2 and OsCATA was significantly reduced, indicating that these two sites are targets for OsSAPK9-mediated phosphorylation.
Figure 6: Phosphorylation of OsbZIP20 is necessary for its transactivation function.
To further explore the role of OsSAPK9 under high Cd²⁺ stress, Ossapk9 knockout mutants were used. The results showed that root growth inhibition under NH₄⁺ and NH₄⁺ + Cd²⁺ conditions was more severe in Ossapk9 than in Nip, suggesting that OsSAPK9 is involved in NH₄⁺-mediated alleviation of Cd²⁺ toxicity. Further analysis revealed that antioxidant gene expression of OsAPX2 and OsCATA and antioxidant enzyme activity (APX and CAT) were lower in Ossapk9, while H₂O₂ and MDA accumulation was higher. This indicates that OsSAPK9 regulates NH₄⁺-mediated alleviation of Cd²⁺ toxicity by upregulating antioxidant gene expression and maintaining antioxidant enzyme activity.
Figure 7: OsSAPK9 responds to Cd²⁺ stress by regulating APX and CAT enzyme activity.
This study reveals the molecular mechanism by which NH₄⁺ mitigates rice's response to Cd²⁺ stress through ABA-dependent signaling pathways. Specifically, NH₄⁺ significantly reduces Cd²⁺ accumulation in plants and alleviates Cd²⁺-induced oxidative damage by increasing antioxidant enzyme activity. The transcription factor OsbZIP20 plays a key role in this process by directly binding and activating the expression of antioxidant-related genes OsAPX2 and OsCATA, thereby enhancing the plant's antioxidant capacity. Moreover, OsSAPK9 kinase-mediated phosphorylation of OsbZIP20 is crucial for its transcriptional activation function, enabling OsbZIP20 to more effectively activate the expression of downstream antioxidant genes. These findings not only clarify the molecular basis of NH₄⁺ alleviating Cd²⁺ toxicity but also provide potential targets for genetic improvement to enhance crop tolerance to heavy metals.
Figure 8: Working model: NH₄⁺ alleviates Cd²⁺ toxicity by activating the OsSAPK9-OsbZIP20-OsAPX2/OsCATA pathway.
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