Histone Acetyltransferase GCN5 Regulates Rice Growth and Development and Enhances Salt Tolerance

doi:10.1016/j.rsci.2024.06.002

Abstract

Abstract:

Histone acetylation is indispensable in the process of crops resisting abiotic stress, which is jointly catalyzed by histone acetyltransferases and deacetylases. However, the mechanism of regulating salt tolerance through histone acetyltransferase GCN5 is still unclear. We revealed that GCN5 can catalyze the acetylation of canonical H3 and H4 lysine residues both in vivo and in vitro in rice. The knockout mutants and RNA interference lines of OsGCN5 exhibited severe growth inhibition and defects in salt tolerance, while the over-expression of OsGCN5 enhanced the salt tolerance of rice seedlings, indicating that OsGCN5 positively regulated the response of rice to salt stress. RNA-seq analysis suggested OsGCN5 may positively regulate the salt tolerance of rice by inhibiting the expression of OsHKT2;1 or other salt-responsive genes. Taken together, our study indicated that GCN5 plays a key role in enhancing salt tolerance in rice.

Key words: GCN5, histone acetyltransferase, salt tolerance, Oryza sativa

Xue Chao, Zhao Xinru, Chen Xu, Cai Xingjing, Hu Yingying, Li Xiya, Zhou Yong, Gong Zhiyun. Histone Acetyltransferase GCN5 Regulates Rice Growth and Development and Enhances Salt Tolerance[J]. Rice Science, 2024, 31(6): 688-699.

Figures/Tables 5

Fig. 1. Phylogenetic tree and cis-acting element of OsGCN5. A, Phylogenetic tree analysis of histone acetyltransferase (HAT) genes in Arabidopsis thaliana (At), Oryza sativa (Os), Triticum aestivum (Traes), and Zea mays (GRMZM). Plant HATs are classified into four families: TATA-binding protein associated factor (TAFII250) family, general control non-repressible 5-related N acetyltransferase (GNAT) family, MOZ, Ybf2/Sas3, Sas2, and Tip60 (MYST) family, and p300/cAMP responsive element-binding protein (p300/CBP) family.B, Alignment of amino acids of GCN5 from Oryza sativa and its homologs from other species, highlighting conserved domains and regions of variability. C, cis-Acting element analysis of OsGCN5 promoter sequence (2 000 bp upstream of the transcription start site, TSS). The analysis reveals the presence of various regulatory elements, including abscisic acid responsive elements (ABRE), transcriptional activator MYB recognition elements (MYB), light response module elements (AE-box, CAG-motif, and G-box), low temperature response element (LTR), cis-regulatory elements of anaerobic induction (ARE), elements involved in drought induction by binding MYB (MBS), and cis-regulatory elements involved in circadian rhythm control (circadian).

Fig. 2. Expression pattern of OsGCN5 in rice and subcellular localization and histone acetyltransferase activity of OsGCN5. A, Relative expression levels of OsGCN5, quantified by qRT-PCR. OsUBQ was used as an internal reference gene. Data are Mean ± SD (n = 3). B, Subcellular localization of OsGCN5-GFP (Green fluorescent protein) fusion protein in rice protoplasts. DIC, Differential interference contrast. Scale bars, 10 μm. C, SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. D and E, Western blotting results of histone acetylation assay performed in vitro. GST protein was used as a negative control. H3 antibody was used to detect histone H3 purified with a GST tag. Acetyl coenzyme A (Ac-CoA) was added to the reaction system in vitro as a donor of acetyl groups. Antibodies specific to histone acetylation at H3K18, H3K27, and H3K36 were used to assess the catalytic activity in vitro.

Fig. 3. Characterization of OsGCN5 genetic transformed plants. A, Sequencing results of OsGCN5 knockout lines (KO-1 and KO-2). PAM, Protospacer adjacent motif; WT, Wild type, Nipponbare. B, Expression levels of OsGCN5 in 14-day-old seedling leaves of wild type (WT, Nipponbare), OsGCN5 RNA interference lines (RNAi-1 and RNAi-2), and over-expression lines (OE-1 and OE-2). Data are Mean ± SD (n = 3). RNAi-1 and RNAi-2 lines were generated by interference of sequences on Exon 1 and Exon 5, respectively. UBQ gene was selected as an internal reference. C, Plant phenotypes of WT, OsGCN5 RNAi lines (RNAi-1 and RNAi-2), over-expression lines (OE-1 and OE-2), and knockout lines (KO-1 and KO-2). Scale bar, 10 cm. D and E, Plant height (D) and panicle length (E) of WT, OsGCN5 over-expression lines (OE-1 and OE-2), RNAi lines (RNAi-1 and RNAi-2), and knockout lines (KO-1 and KO-2). Data are Mean ± SD (n = 3). Different lowercase letters above the bars indicate significant differences at P < 0.05 (one-way analysis of variance followed by Tukey’s multiple-comparison test).F and G, Detection of histone acetylation levels in WT, OsGCN5 knockout lines (KO-1 and KO-2), over-expression line (OE-1), and RNAi line (RNAi-1) by Western blot (F), and analysis of band density by grayscale value (G). H3 was used as a loading control. Data are Mean ± SD (n = 3). * and **, P < 0.05 and P < 0.01 (t-test), respectively.

Fig. 4. Phenotypes and physiological changes of OsGCN5 transgenic lines under salt stress. A, Relative expression levels of OsGCN5 and some salt-responsive genes from rice seedlings (grown for 2 weeks) under normal conditions (0 mmol/L) and different NaCl concentrations (50, 100, and 150 mmol/L) for 24 h, quantified by qRT-PCR. UBQ gene was selected as an internal reference. B, Seedling (grown for 2 weeks) phenotypes of over-expression lines (OE-1 and OE-2), RNA interference lines (RNAi-1 and RNAi-2), and wild type (WT) under salt untreated (0 d) and 150 mmol/L NaCl treatments for 3 d. C and D, Seedling (grown for 2 weeks) (C) and survival rate (D) of over-expression line (OE-1), knockout line (KO-1), and wild type (WT) under salt untreated (0 d) and 150 mmol/L NaCl treatments for 5 d and recovery for 5 d. Data are Mean ± SD (n = 3). **, P < 0.01 (t-test). E, Reactive oxygen species accumulation in OsGCN5 over-expression line (OE-1), RNAi line (RNAi-1), knockout line (KO-1), and WT after 150 mmol/L NaCl stress detected by NBT (nitrotetrazolium blue chloride) staining. The color depth represents the amount of reactive oxygen species accumulation. F, Determination of malondialdehyde (MDA) content and peroxidase (POD) activity in WT and OsGCN5 transgenic lines before (CK) and after 150 mmol/L NaCl stress. Data are Mean ± SD (n = 3). Different lowercase letters above the bars indicate significant differences at P < 0.05 (One-way analysis of variance followed by Tukey’s multiple-comparison test).

Fig. 5. Transcriptome sequencing analysis and qRT-PCR verification. A, Venn diagram of the number of up-regulated genes. B, Gene Ontology (GO) analysis of the 2 651 co-upregulated genes between over-expressed lines and WT under 150 mmol/L NaCl treatment for 24 h. C, GO enrichment analysis of 662 unique up-regulated genes in OE lines under salt stress (150 mmol/L NaCl treatment for 24 h). D, qRT-PCR verification of some differentially expressed genes in WT and OE-1 rice seedlings (grown for 2 weeks) under normal conditions (0 mmol/L) and different NaCl concentrations (50, 100, and 150 mmol/L) for 24 h. The rice ubiquitin gene (UBQ) was selected as an internal reference gene. Data are Mean ± SD (n = 3). E, Heat maps of differentially expressed genes associated with salt stress response. F, Immunoblot analysis of the histone acetylation levels under salt stress. H3 was used as a loading control. CK, Control check; Salt, 150 mmol/L NaCl treatment for 24 h; WT, Wild type; OE, Over-expressed lines; FDR, False discovery rate.

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