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Literature Sharing | OsbZIP27 coordinates with OsHUB1 and OsHUB2 to modulate drought tolerance in rice
Release time:
2025-02-21
Histone H2B ubiquitination (H2Bub) is positively linked to transcriptional activation, but the genetic programs affected by H2Bub to enhance drought tolerance remain largely unknown. Here, we show that OsbZIP27 interacts directly with OsHUB1/2 to regulate drought tolerance in rice by binding to the promoters of OsHAK1 and OsGLN1 to achieve H2Bub and transcriptional activation. Consistently, mutations in OsbZIP27 reduce transcription of OsHAK1 and OsGLN1, resulting in increased sensitivity to drought stress. Moreover, loss of OsHUB1 and OsHUB2 function causes hypersensitivity to drought stress, whereas OsHUB2 overexpression enhances drought tolerance. Together, our results indicate that OsbZIP27 coordinates with OsHUB1/2 to enhance rice drought tolerance by increasing H2Bub and expression of OsHAK1 and OsGLN1.
In our previous studies, oshub1-3 oshub2-2 and oshub1-4oshub2-3 mutants displayed early flowering phenotypes. To investigate whether OsHUB1 and OsHUB2 contribute to drought tolerance, we treated the mutants and wild type plants with PEG4000. Prior to treatment, no visible differences were noted between wild type and mutant plants. After PEG4000 treatment, all plants exhibited wilting, and during recovery, leaves of approximately half of the wild type plants regained green coloration, whereas oshub1-3 oshub2-2 and oshub1-4 oshub2-3 mutants demonstrated.
Fig. 1. OsHUB1 and OsHUB2 contribute to drought tolerance. A, C, E, and G: Phenotypes of WT and oshub1-3 oshub2-2
To identify proteins in the drought response pathway that interact with OsHUB1/2, we conducted a yeast two-hybrid screening using OsHUB1 as a bait. Sequencing of positive colonies revealed OsbZIP27, a bZIP transcription factor, as an OsHUB1 interactor (Table S1), and a follow-up yeast two-hybrid assays confirmed that OsbZIP27 interacts with both OsHUB1 and OsHUB2 (Fig. 2A). Pull down assays validated these results, as beads linked to MBP-His OsHUB1/2 successfully bound to GST-OsbZIP27 (Fig. 2B). Likewise, beads linked to GST-OsbZIP27 bound to MBP-His-OsHUB1/2 (Fig. 2C). Additionally, luciferase complementation assays in Nicotiana benthamiana leaves showed strong signals when OsbZIP27 was fused to the LUC C-terminus (OsbZIP27-cLUC) and OsHUB1/OsHUB2 to the LUC N-terminus (OsHUB1/OsHUB2-nLUC) (Fig. 2D and 2E). Finally, co-immunoprecipitation (Co-IP) assays indicated that HA-OsbZIP27 co-precipitated with FLAG-OsHUB1/2 in N. benthamiana leaves (Fig. 2F and 2G). Together, these findings establish that OsbZIP27 directly interacts with OsHUB1/2.
Fig. 2. OsbZIP27 directly interacts with OsHUB1/2.
To understand the functions of OsbZIP27 in the stress response, we examined its expression across tissues subjected to various abiotic stresses. OsbZIP27 was highly expressed in root tissues of both seedlings and mature plants (Fig. S4), and OsbZIP27 expression further increased in response to PEG4000 treatment but were not in response to ABA, NaCl, H2O2, or low-temperature treatment (Fig. S4). Subcellular localization assays revealed that OsbZIP27- GFP localized to the nucleus, whereas GFP alone localized to both the nucleus and cytosol (Fig. S3). To assess the role of OsbZIP27 in drought tolerance, we generated OsbZIP27 mutants using CRISPR/Cas9, which yielded one mutant with a cytosine insertion (osbzip27-1) and another with a seven-nucleotide deletion (osbzip27-2) (Fig. S5). osbzip27-1 caused a frameshift, producing a stop codon in the 30 UTR region, whereas osbzip27-2 produced a premature stop codon (Fig. S5). While pretreatment phenotypes were similar to wild type for both knock-out lines after PEG4000 treatment, osbzip27-1 and osbzip27-2 mutants exhibited more severe wilting and chlorosis as well as a lower survival rate (Fig. S4). Soil drought testing also demonstrated that osbzip27-1 and osbzip27-2 were more sensitive to water withdrawal, showing severe chlorosis, compared to green leaves in wild type plants upon rewatering (Fig. 3A and 3C) and had reduced survival rates (Fig. 3B and 3D). We then tested the effect of ABA treatment, finding osbzip27 mutants had similar sensitivity to ABA as wild type plants, with both displaying reduced shoot length (Fig. S6). These results collectively suggest that OsbZIP27 primarily mediates drought tolerance through an ABA-independent pathway.
Fig. 3. osbzip27 mutants exhibit sensitivity to drought stress
To determine whether OsbZIP27 directly binds the promoters of OsHAK1 and OsGLN1, an electrophoretic mobility shift assay (EMSA) was conducted. As bZIP factors primarily bind G-box motifs, we scanned the promoter regions of OsHAK1 and OsGLN1 using PlantCARE and identified one G-box motif in the S1 region of the OsHAK1 promoter and two in the S2 region of the OsGLN1 promoter (Figs. 5A, 6C, 6D). Diminishing bands observed upon incubation of OsbZIP27 with increasing concentration of competitive DNA probes indicated direct binding of OsbZIP27 to the OsHAK1 and OsGLN1 promoters in vitro (Fig. 5B and 5C). Next, we performed a luciferase assay to confirm transcriptional activation. Constructs with luciferase driven by either the OsHAK1 or OsGLN1 promoter (ProOsHAK1::LUC or ProOsGLN1::LUC) (Fig. 5D) were cotransformed with 35S::OsbZIP27-GFP or 35S::GFP in tobacco leaves. Co-expression with 35S::OsbZIP27-GFP yielded stronger bioluminescence signals (Fig. 5E and 5F), suggesting OsbZIP27 activates OsHAK1 and OsGLN1 transcription by directly binding to their promoters.
Fig. 5. OsbZIP27 binds to OsHAK1 and OsGLN1 promoters to activate gene expression.
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