Background Introduction
Rice, as a globally important food crop, is significantly affected by salt stress, which impacts its growth and yield. Salt stress can have multiple effects on rice at morphological, physiological, biochemical, and molecular levels, making it crucial to analyze the molecular mechanisms of salt tolerance in rice for agricultural production. Histone deacetylases (HDACs) play a key role in plant responses to abiotic stress. For instance, OsHDA706 regulates histone acetylation of the OsPP2C49 promoter to influence salt stress response, and the OsHDA710 knockout mutant enhances salt tolerance by regulating stress-related genes. However, the specific role and mechanism of OsHDAC1 in salt stress remain unclear. Aldehyde dehydrogenases (ALDHs) are involved in aldehyde metabolism and reactive oxygen species (ROS) scavenging. In rice, OsALDH7 and OsALDH22 are induced by salt stress, while OsALDH2B1 has been found to participate in seed size and brassinosteroid signaling regulation, but its function as a transcription factor in abiotic stress is still not well understood. Glutathione reductases (GRs) maintain redox balance by catalyzing the conversion of GSSG to GSH. The expression of OsGR3 in rice increases under salt stress and affects GSH/GSSG levels, but its regulatory mechanism and association with stress response require further investigation. Previous studies have revealed some salt stress signaling pathways (e.g., IDS1-TPR1-HDA1 module), but the intersection of epigenetic and metabolic regulation mechanisms still needs to be supplemented. Based on this, this article focuses on the interaction between OsHDAC1, ALDH2B1, and GR3, exploring their regulatory network in rice salt stress response.
Article Information
On April 11, 2025, Professor Li Lijia’s team from the School of Life Sciences at Wuhan University published a research paper titled “OsHDAC1 deacetylates the aldehyde dehydrogenase OsALDH2B1, repressing OsGR3 and decreasing salt tolerance in rice” in the journal Plant Physiology. This study reveals the cascade regulatory mechanism of the OsHDAC1-OsALDH2B1-OsGR3 module in rice salt stress response.
Original link: https://doi.org/10.1093/plphys/kiaf14
Research Content
Histone deacetylases (HDACs) play a role in plant responses to various environmental stresses. To elucidate the involvement mechanism of OsHDAC1 in stress response, researchers analyzed its 2000 bp promoter region using the PLANTCARE database. This analysis revealed multiple cis-acting elements related to abiotic stress and plant hormone responses. Notably, these elements include abscisic acid response elements, anaerobic induction response elements, ethylene response elements, MYB recognition elements, MYC binding elements, stress response elements, salicylic acid response elements, and wound response elements, indicating that OsHDAC1 may play a role in abiotic stress and hormone responses. To verify this hypothesis derived from computational analysis, various abiotic stress and hormone treatments were applied to 14-day-old Nipponbare (NIP) seedlings, followed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis. The results showed that the expression of OsHDAC1 was dynamically regulated under these treatments (Figure 1).
Figure 1: Expression analysis of the OsHDAC1 gene in rice under abiotic stress and hormone stress.
After treating OsHDAC1 RNA interference (Ri), overexpression (OE), and NIP plants with 150 mM NaCl, the seedlings were allowed to recover under normal conditions, with survival rate as the final criterion for salt tolerance. Under non-stress conditions, no significant differences were observed among the lines. However, after 10 days of NaCl treatment, OsHDAC1-Ri plants exhibited better growth status, while OsHDAC1-OE plants showed more severe wilting compared to NIP. The recovery survival rate indicated that OsHDAC1-Ri plants reached 58%, significantly higher than NIP (20%) and OsHDAC1-OE (10%). Additionally, abiotic stress conditions typically induce the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), leading to changes in cellular physiological pathways and membrane properties. After salt treatment, the intensity of DAB staining (a marker for H2O2 accumulation) and nitro blue tetrazolium (NBT) staining (an indicator of ROS content) in OsHDAC1-OE lines was higher than that in NIP seedlings, while OsHDAC1-Ri lines were lower than NIP. Under control conditions, the MDA levels of transgenic plants were comparable to those of NIP plants. However, salt stress significantly increased the MDA levels in NIP and OsHDAC1-OE plants, while no such phenomenon was observed in OsHDAC1-Ri plants. After salt stress, the H2O2 content increased in both transgenic and NIP plants. The H2O2 content in OsHDAC1-OE plants significantly increased compared to pre-treatment, while OsHDAC1-Ri plants maintained relatively low levels of H2O2. In summary, these results suggest that OsHDAC1 may reduce salt tolerance in rice by affecting the accumulation of ROS and MDA (Figure 2).
Figure 2: OsHDAC1 reduces salt tolerance in rice plants.
To elucidate the molecular mechanism of OsHDAC1’s response to salt stress, yeast two-hybrid screening experiments were conducted to identify its interacting proteins. The results showed that OsHDAC1 strongly interacts with the aldehyde dehydrogenase family member OsALDH2B1. Luciferase (LUC) complementary imaging (LCI) experiments indicated that signals could only be detected when OsHDAC1-nLuc was co-transformed with OsALDH2B1-cLuc in tobacco leaves. Bimolecular fluorescence complementation (BiFC) experiments showed that the OsHDAC1-nYFP/OsALDH2B1-cYFP complex specifically formed and localized in the nucleus. The MBP-OsALDH2B1-His protein purified from Escherichia coli BL21(DE3) was co-incubated with OsHDAC1-GST protein, using MBP-OsALDH2B1-His and GST as negative controls. The results indicated that OsHDAC1-GST successfully pulled down MBP-OsALDH2B1-His, while the standalone GST could not. Finally, the interaction was verified in the rice protoplast system using co-immunoprecipitation (Co-IP) experiments. When OsHDAC1-GFP was co-transformed with OsALDH2B1-Myc in rice protoplasts, OsALDH2B1-Myc was detected to co-precipitate with OsHDAC1-GFP. In conclusion, both in vitro and in vivo evidence strongly suggests that OsALDH2B1 physically interacts with OsHDAC1, and this interaction specifically occurs in the nucleus (Figure 3).
Figure 3: OsALDH2B1 interacts with OsHDAC1 in vitro and in vivo.
Based on the observed interaction between OsHDAC1 and OsALDH2B1, further investigation was conducted to determine whether OsHDAC1 could deacetylate OsALDH2B1. To ascertain the enzymatic specificity of OsHDAC1 towards OsALDH2B1, in vitro deacetylation experiments were performed using recombinant OsALDH2B1-His and OsHDAC1-GST proteins. Compared to the GST control group, co-incubation with OsHDAC1-GST significantly reduced the acetylation level of OsALDH2B1-His. This result was validated in vivo: in rice protoplasts, co-expressing OsALDH2B1-Myc and OsHDAC1-GFP significantly reduced the acetylation level of OsALDH2B1-Myc compared to the control expressing GFP. These experiments demonstrate that OsHDAC1 can deacetylate OsALDH2B1 both in vitro and in vivo. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of deacetylated OsALDH2B1-His identified two specific sites (K311 and K531) as targets of OsHDAC1. Evolutionary conservation analysis showed that K311 is highly conserved among rice and Arabidopsis ALDH family members, while K531 is a modification site unique to OsALDH2B1. These data confirm that OsALDH2B1 is a substrate of OsHDAC1, with modification sites that include both conserved and lineage-specific regulatory sites (Figure 4).
Figure 4: OsHDAC1 deacetylates OsALDH2B1.
To explore the impact of OsHDAC1-mediated deacetylation on the stability of OsALDH2B1, the mRNA and protein levels of OsALDH2B1 in OsHDAC1-OE and Ri plants were first assessed. Immunoblot analysis showed that compared to NIP, the protein abundance of OsALDH2B1 was reduced in OsHDAC1-OE plants, while it was increased in OsHDAC1-Ri plants, although mRNA levels did not change. To verify whether OsHDAC1 participates in the degradation of endogenous OsALDH2B1, the protein synthesis inhibitor cycloheximide (CHX) was applied to the plants. Consistent with in vitro results, CHX treatment accelerated the degradation of OsALDH2B1 in OsHDAC1-OE lines, while degradation was slowed in OsHDAC1-Ri lines. Subsequently, the effects of deacetylation at K311 and K531 on the stability of OsALDH2B1 were investigated. Two mutant proteins were constructed: OsALDH2B1-KQ (K311 and K531 mutated to glutamine Q to simulate acetylation state) and OsALDH2B1-KR (mutated to arginine R to simulate deacetylation state). Cell-free degradation experiments showed that OsALDH2B1-KQ-His was more stable than OsALDH2B1-KR-His. These results suggest that OsHDAC1 may influence the stability of OsALDH2B1 by regulating the deacetylation state of specific lysine residues (K311 and K531) (Figure 5).
Figure 5: OsHDAC1 reduces the stability of OsALDH2B1 protein.
To investigate the role of OsALDH2B1 in salt tolerance, overexpression lines (OsALDH2B1-OE) and RNAi lines (OsALDH2B1-Ri) were constructed, and salt tolerance experiments were conducted on these lines and the osaldh2b1 mutant. The results showed that the osaldh2b1 mutant was highly sensitive to salt stress, with survival rates rapidly declining, while treatment with the HDAC inhibitor TSA alleviated this sensitivity. The survival rate of OsALDH2B1-OE lines was significantly higher than that of the wild-type NIP, while OsALDH2B1-Ri8 and Ri11 lines exhibited salt sensitivity similar to that of the osaldh2b1 mutant. Under 150 mM NaCl conditions, after 6 days of germination growth, the staining intensity of DAB and NBT in OsALDH2B1-Ri lines was higher than that in NIP, while the staining intensity of OsALDH2B1-OE lines was lower. MDA content increased in NIP and OsALDH2B1-Ri lines, indicating higher levels of cellular stress. Under untreated conditions, there were no significant differences in H₂O₂ content among the lines, but all increased after salt stress. The increase in H₂O₂ content in OsALDH2B1-OE lines was smaller, while the H₂O₂ content in OsALDH2B1-Ri lines significantly increased (Figure 6).
Figure 6: OsALDH2B1 enhances salt tolerance in rice.
To elucidate the genetic interaction relationship between OsHDAC1 and OsALDH2B1 in rice salt tolerance, OsALDH2B1/OsHDAC1 double interference lines (OsALDH2B1/OsHDAC1-Ri7 and Ri9) were constructed under the OsHDAC1-Ri3 background. Assessment of salt stress treatment revealed that OsALDH2B1-Ri lines exhibited extreme sensitivity, with a recovery survival rate of only about 15%; while OsHDAC1-Ri lines had the highest survival rate among all tested lines. Notably, in the OsALDH2B1/OsHDAC1 double interference lines, the suppression of OsALDH2B1 expression completely eliminated the salt tolerance phenotype originally present in OsHDAC1-Ri lines. These results indicate that the function of OsHDAC1 in regulating rice salt tolerance is genetically dependent on the presence and function of OsALDH2B1, and vice versa (Figure 7).
Figure 7: The function of OsHDAC1 in rice salt tolerance depends on OsALDH2B1.
Previous studies have confirmed that the core binding sequence of OsALDH2B1 is GCCGCCGCC/G and TTTTTTTTT. Analysis of the 2000 bp upstream region of OsGR3 using the PLANTCARE website showed that there are three potential OsALDH2B1 binding motifs in this promoter region, suggesting that OsALDH2B1 may regulate the expression of OsGR3 by binding to its promoter. The researchers used three corresponding probes labeled with 5-carboxyfluorescein (FAM) for electrophoretic mobility shift assay (EMSA), and the results showed that recombinant MBP-OsALDH2B1-His could specifically bind to the OsGR3 probe but not its mutant form. OsALDH2B1 selectively bound to the two distal motifs upstream of the OsGR3 promoter, with no binding activity to the motif near the start codon, and as the concentration of unlabeled competitor probes increased, the signal intensity of the MBP-OsALDH2B1-His-probe complex gradually weakened. Yeast one-hybrid experiments further confirmed that OsALDH2B1 specifically interacts with the ProA/B region. These results indicate that OsALDH2B1 can directly bind to the OsGR3 promoter through the two distal motifs. To further elucidate the mechanism by which OsALDH2B1 regulates the transcription of OsGR3, the pGreenII-0800-luc system was used for luciferase reporter gene detection. After cloning the 2000 bp OsGR3 promoter fragment into the reporter vector, dual luciferase experiments in rice protoplasts showed that OsALDH2B1-Myc could activate the LUC activity driven by the OsGR3 promoter, while co-expression of OsHDAC1-HA inhibited this activation effect (Figure 8).
Figure 8: OsALDH2B1 binds to the OsGR3 promoter and activates the transcription of OsGR3.
To assess the changes in the lysine deacetylation state of OsALDH2B1 in plants under salt stress conditions, the dynamic abundance of OsHDAC1 and OsALDH2B1 proteins during stress was first examined. Immunoblot analysis using OsHDAC1 and OsALDH2B1 antibodies showed that as the duration of salt stress increased, the content of OsHDAC1 gradually decreased, while the protein level of OsALDH2B1 continuously increased, with its acetylated form also increasing synchronously. These results indicate that the deacetylation/acetyation modification of OsALDH2B1 is involved in the salt stress response process, and the salt stress-induced suppression of OsHDAC1 expression promotes the transition of OsALDH2B1 to an acetylated state. To further explore the regulatory role of OsHDAC1 on the in vivo deacetylation of OsALDH2B1 under salt stress, the levels of acetylated OsALDH2B1 were compared between NIP and OsHDAC1-Ri lines through immunoblotting. The results showed that at the initial stage of salt stress (especially after 12 hours of treatment), the relative intensity of the acetylated OsALDH2B1 band in OsHDAC1-Ri lines was significantly higher than that in NIP plants (Figure 9).
Figure 9: Salt-induced accumulation of acetylated OsALDH2B1 is mediated by OsHDAC1.
Research Conclusion
This study reveals the mechanism by which the OsHDAC1/OsALDH2B1/OsGR3 module regulates salt tolerance in rice: under salt stress, the expression of OsHDAC1 is downregulated, reducing the deacetylation of OsALDH2B1 at K311/K531 sites, preventing its ubiquitin-proteasome degradation. The stable OsALDH2B1 binds to the OsGR3 promoter, activating its transcription to enhance ROS scavenging ability. OsHDAC1-Ri and OsALDH2B1-OE plants exhibit significantly improved salt tolerance, while the inhibition of OsALDH2B1 eliminates the salt tolerance effect of OsHDAC1 knockdown. This module provides new targets for improving salt tolerance in rice through epigenetic and redox metabolic cooperation.
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Current Contributor
Wu Hejing
Shanxi Agricultural University, College of Agriculture
Master’s student, Class of 2024