miR396b/GRF6 module contributes to salt tolerance in rice

Summary

Salinity, as one of the most challenging environmental factors restraining crop growth and yield, poses a severe threat to global food security. To address the rising food demand, it is urgent to develop crop varieties with enhanced yield and greater salt tolerance by delving into genes associated with salt tolerance and high‐yield traits. MiR396b/GRF6 module has previously been demonstrated to increase rice yield by shaping the inflorescence architecture. In this study, we revealed that miR396b/GRF6 module can significantly improve salt tolerance of rice. In comparison with the wild type, the survival rate of MIM396 and OE‐GRF6 transgenic lines increased by 48.0% and 74.4%, respectively. Concurrent with the increased salt tolerance, the transgenic plants exhibited reduced H₂O₂ accumulation and elevated activities of ROS‐scavenging enzymes (CAT, SOD and POD). Furthermore, we identified ZNF9, a negative regulator of rice salt tolerance, as directly binding to the promoter of miR396b to modulate the expression of miR396b/GRF6. Combined transcriptome and ChIP‐seq analysis showed that MYB3R serves as the downstream target of miR396b/GRF6 in response to salt tolerance, and overexpression of MYB3R significantly enhanced salt tolerance. In conclusion, this study elucidated the potential mechanism underlying the response of the miR396b/GRF6 network to salt stress in rice. These findings offer a valuable genetic resource for the molecular breeding of high‐yield rice varieties endowed with stronger salt tolerance.

Introduction

Soil salinity has become an increasingly severe global problem, exacerbated by natural environment deterioration, poor irrigation practices and climate changes (Park et al., 2016; Zhao et al., 2021). It is estimated that roughly 20% of the world's irrigated agricultural lands are currently affected by salinity, and this is possibly increasing to 50% by 2050 (Ahmad et al., 2022; Kumar et al., 2020; Zhang et al., 2023). Generally, high‐salinity will greatly inhibit plant growth and development, including the seed germination, root length, plant height, and fructification of plant (Liang et al., 2014, 2018). Rice stands out as one of the most vital cereal crops worldwide, with a salt stress threshold of 3 dS/m, beyond which its yield decreases by 12% per unit (dS/m) (Deng et al., 2022; Negrão et al., 2011). Therefore, it holds significant importance to cultivate salt‐tolerant, high‐yield rice varieties to combat soil salinization and ensure food security.

Due to internal trade‐off mechanisms, high yields and strong biotic/abiotic resistance are usually hard to be compatible in plants (Deng et al., 2017). BIG RICE GRAIN1 (BIRG1), encoding a detoxification efflux carrier/multidrug and toxic compound extrusion (DTX/MATE) family transporters, mediating grain size and salt tolerance by controlling Cl⁻ homeostasis in rice cells (Ren et al., 2021). Loss‐of‐function of BRIG1 resulted in increased grain size but decreased salt tolerance (Ren et al., 2021). Conversely, overexpressing quiescent‐centre‐specific homeobox (OsQHB) led to an increase in plant height, tiller number, panicle length, grain length and grain width, but at the cost of decreased salt tolerance (Zhou et al., 2022). Interestingly, successful breeding practice have produced elite rice varieties with both high‐yield and strong disease resistance, indicating that a balance between growth and immunity can be achieved through genetic recombination (Li et al., 2021; Wang et al., 2018). For example, ARGONAUTE2 (AGO2) has been found to not only increase grain length by modifying the histone methylation level of BIG GRAIN3 (BG3) but also enhance salt tolerance by elevating cytokinin levels in roots (Yin et al., 2020). Additionally, ectopic overexpression of AtHDG11 has been shown to improve both drought and salt tolerance as well as yield in cotton (Cao et al., 2009; Yu et al., 2015). This highlights the pivotal role of certain genes in enhancing both plant yield and resilience to adverse environmental conditions. Despite significant progress in understanding the genetic mechanisms of salt stress response in rice, there is still much to learn about the molecular mechanisms governing rice's response to salt tolerance, especially the positive regulatory factors that could potentially be utilized for producing high‐yield, salt‐tolerant rice.

Plant microRNAs (miRNAs) are a class of conserved endogenous small non‐coding RNAs, play pivotal roles in various developmental processes, signal transduction, plant hormone responses and stress resistance (Begum, 2022; Liu et al., 2021; Ma and Hu, 2023; Meyers and Axtell, 2019; Sun et al., 2023). MiR156, the first miRNA identified in plants (Duan et al., 2022; Jerome Jeyakumar et al., 2020; Zheng et al., 2019), has been found to regulate both grain yield and salt tolerance in rice by targeting the key gene Ideal Plant Structure 1 (IPA1) (Jia et al., 2022; Jiao et al., 2010; Wang and Wang, 2017). Additionally, the miR172‐IDS1 regulatory module coordinates salt tolerance and grain development in rice, overexpression of miR172 significantly enhancing rice grain weight and salt tolerance (Cheng et al., 2021).

MiR396 is a well‐studied miRNA family known for its extensive targeting of growth‐regulating factor (GRF) genes (Gao et al., 2022; Liebsch and Palatnik, 2020; Wang et al., 2023). In rice, there are eight pri‐miR396s and twelve GRF family members (Gao et al., 2016; Liebsch and Palatnik, 2020). Increasing references have well documented that miR396‐GRFs act as a molecular module to regulate rice grain yield (Liu et al., 2023; Omidbakhshfard et al., 2015). Both miR396e and miR396f target OsGRF8, knockout of miR396e and miR396f leads to enhanced OsGRF8 expression, resulting in higher grain yield due to larger grain size and more panicle branching (Zhang et al., 2020a). Interestingly, miR396 members have also been implicated in regulating salt tolerance in various plant species, including tobacco, cotton, rice, and creeping bentgrass (Chen et al., 2015; Gao et al., 2010; Wang et al., 2013; Yuan et al., 2019). It is noteworthy that overexpression of miR396c in rice has been reported to reduce salt tolerance, suggesting that the miR396/GRF module may also play a role in modulating salt tolerance in rice (Gao et al., 2010).

Previously, we discovered that miR396b/GRF6 module enhances rice grain yield by influencing inflorescence architecture and the number of grains per panicle (Gao et al., 2016). Nevertheless, it remains unclear whether this miR396b/GRF6 module is also involved in salt tolerance. In the current research, we observed a significant change in the expression of the miR396b/GRF6 module in response to salt stress. Compared to the wild type, seedlings from MIM396 transgenic lines exhibited a 48.0% increase in survival rate under salt stress, while the GRF6 overexpression line showed a remarkable 74.4% increase. Moreover, we identified that the upstream regulator ZNF9 and downstream factor MYB3R are involved in transmitting salt stress signals. This discovery highlights the critical role of the miR396b/GRF6 module in coordinating the response to salt stress and grain yield in rice. These findings pave the way for the molecular design of new super rice varieties with high yield and robust salt tolerance.

Materials and methods

Plant materials and salt treatment

The rice cultivar Yuetai B (YB, Oryza sativa L. ssp. indica) and Zhong‐Hua‐11 (ZH11, Oryza sativa L. ssp. japonica) seeds were initially submerged in distilled water at 37 °C for approximately 36 h to facilitate germination. Following germination, the seedlings were transplanted into a bottomless 96‐well plate filled with Yoshida's culture solution and then grown at a temperature of 28 °C, with 14‐h light/10‐h dark, maintaining a relative humidity of 60%. To evaluate rice salt tolerance, 14‐day rice plants were treated with 200 mM NaCl for 4 days and then followed by a 5‐day recovery with NaCl‐free Yoshida's culture solution. The survival rate was then calculated by comparing the treated plants to control plants.

For the analysis of fresh and dry weight, the 14‐day seedlings from the treatments and controls were gently dried with absorbent paper after 4 days of salt treatment. Subsequently, root and shoot tissues were separated, and the fresh weight of the shoot was measured. Each group had three replicates, with eight plants randomly collected from each group. All materials were then dried at 70 °C for 3 days, and the dry weight of the materials was determined.

Generation of transgenic plants

The transgenic plants in this article were in the YB and ZH11 background. MIM396‐1, MIM396‐3, OE‐GRF6‐1 and OE‐GRF6‐3 transgenic lines were obtained from a previous study (Gao et al., 2016). GRF6‐knockout (KO‐GRF6‐1 and KO‐GRF6‐2) and ZNF9‐KO mutants (KO‐ZNF9‐1 and KO‐ZNF9‐2) were generated by CRISPR/Cas9‐based gene editing strategy (Ma et al., 2015). Specifically, two specific protospacer adjacent motif (PAM) sequences were designed in the exons of GRF6 and ZNF9. Subsequently, the corresponding two single guide RNAs (sgRNAs) were introduced into the pYLCRISPR/Cas9Pubi‐H vector. This recombinant plasmid was then introduced into the Agrobacterium strain (EHA105), which was subsequently used to transform the YB cultivar. The mutations at the targeted sites were confirmed through PCR amplification and sequencing.

For OE‐ZNF9 and OE‐MYB3R lines, the full‐length coding sequence (CDS) of ZNF9 and MYB3R was driven by ubiquitin (UBI) promoter within the pCAMBIA1301‐UBI vector. Then, the same transformation method was used to transfer into wild type, and the positive OE transgenic lines were obtained and identified by hygromycin screening and qRT‐PCR. As for the KO‐MYB3R mutants, they were obtained from the mutant library within the ZH11 background at Biogle Company (Biogle, Hangzhou, China).

Quantitative real‐time PCR (qRT–PCR) analysis

Total RNAs were extracted from various rice tissues using TRIzol Reagent following the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). Subsequently, cDNA was synthesized using HiScript III First Strand cDNA Synthesis Kit (+gDNA Wiper) (Vazyme, Nanjing, China). Ubiquitin was used as the internal control. The qRT‐PCR analysis was performed on a Roche LightCyclerr 480II instrument using the HieffqPCR SYBR Green Master Mix (No Rox) (Yeasen, Shanghai, China). The relative expression levels were calculated using the 2^−ΔΔCT method.

RNA‐seq analysis

For the RNA‐seq, total RNA was extracted from the shoots of YB and OE‐GRF6 transgenic plants with or without 200 mM NaCl for 9 h, and 3 μg of total RNA was used for construction of the RNA libraries. The library construction and RNA‐sequencing were performed by igenecode company (Beijing, China).

Following sequencing, the raw data obtained from sequencing underwent a filtration process, and the filtered clean reads were then aligned to the Nipponbare genome sequence (http://rice.uga.edu/index.shtml). Following this, differentially expressed genes (DEGs) were identified (|log2(FoldChange)| > 1 and q‐value <0.001) using the PossionDis method. To further analyse the data, Gene Ontology (GO) Biological Process term enrichment analysis was carried out using OmicShare tools (https://www.omicshare.com/tools) with default parameters. Each analysis was based on three independent biological replicates.

ROS staining

The 4‐day seedlings were treated with or without 100 mM NaCl for 12 h, and then, roots were collected for ROS staining. Roots were incubated to H2DCFDA staining buffer (10 μM H2DCFDA, sodium phosphate buffer, pH 7.4) for 10 min in the dark, followed by washing PBS buffer for three times. The samples fluorescence observed with a Leica DM7i8 confocal microscope, and the fluorescence intensity was quantified using Image J.

Endogenous substance measurement

For the analysis of endogenous substances, 14‐day rice seedlings were treated with or without NaCl (200 mM) for 24 h. The upper part of roots that incubated with or without NaCl were collected and subjected to proline content (Pro), H₂O₂ content, and malondialdehyde (MDA) content and ROS‐scavenging enzyme activities (SOD, POD and CAT) measurement. The levels of these endogenous substances were determined using spectrophotometry with the corresponding assay kits (Beijing Boxbio Science Technology, Beijing, China).

Transcriptional activation analysis

To assess the transcriptional activation of ZNF9 and MYB3R in yeast, the CDS region was cloned into the pGBKT7 and pGADT7 vectors. The recombinant plasmids were separately introduced into yeast strain AH109 chemically competent cells (Weidi Biotechnology, Shanghai, China). Transformants were cultured on SD/−Trp‐Leu or SD/−Trp‐Leu‐His‐Ade medium plate and incubated for 3–5 days at 30 °C for observation. For transcriptional activity assays in rice protoplasts, the CDS region of ZNF9 and MYB3R was fused with both the GAL4 DNA binding domain (GAL4BD‐ZNF9, GAL4BD‐MYB3R) and the GAL4BD‐VP16 domain (GAL4BD‐VP16‐ZNF9, GAL4BD‐VP16‐MYB3R). These recombinant plasmids were transiently expressed in rice protoplasts, and the LUC/REN activity was quantified using the dual‐luciferase reporter assay system (Promega, Madison, WI, USA) (Cheng et al., 2023; Yuan et al., 2022).

Subcellular localization

The CDS region of ZNF9 and MYB3R, both without a stop codon, was cloned into HBT‐sGFP vector to generate 35S::ZNF9‐sGFP and 35S::MYB3R‐sGFP fusion vector. These fusion vectors, along with the empty 35S::sGFP vector, were subsequently transfected into rice protoplasts and cultured at 28 °C overnight. Then, the FV1000 confocal system was used for observing GFP fluorescence.

Yeast one‐hybrid assay

To establish the yeast one‐hybrid assay, the yeast one‐hybrid library was constructed using Matchmaker® Gold Yeast One‐Hybrid Library Screening System User Manual (Clontech). Total RNA was extracted from YB and subjected to purification using a Poly A+ column to obtain mRNA. Reverse transcription was conducted to generate dsDNA, followed by purification of dsDNA molecules exceeding 400–600 bp using a CHROMA SPIN+TE‐400 column. The miR396b promoter was then inserted into pAbAi vector, resulting in pAbAi‐bait. Co‐transformation of the dsDNA and linearized pGADT7‐Rec vector into pAbAi‐bait Y1HGold yeast competent cells was performed, and 150 μL per plate was plated onto SD/−Leu plates supplemented with Aureobasidin A (AbA; Coolaber, Beijing, China). Clones were counted after 3 days of incubation at 30 °C.

For detect protein–DNA interactions, the CDS of ZNF9 and GRF6 was ligated into the pGADT7 vector to generate pGADT7‐ZNF9 and pGADT7‐GRF6 constructs, respectively. The promoter sequences of miR396b and MYB3R were amplified from YB and subsequently cloned them into the pAbAi vector. These constructs were co‐transformed into the yeast strain Y1HGold and cultured on a plate containing SD/−Leu/‐Ura medium for 3 days at 30 °C. Then, the yeast cells, in two dilutions, were transferred and grown plates containing SD/‐Ura/−Leu added with or without AbA at 30 °C for 3 days.

Electrophoretic mobility shift assay (EMSA)

The CDS region of ZNF9 and GRF6 was cloned into the pCold‐TF and pMAL‐c2X vector, respectively. These recombinant constructs, His‐ZNF9 and MBP‐GRF6, were expressed in E. coli BL21 (DE3) cells and subsequently purified following the manufacturer's instructions. FAM‐labelled and unlabelled probes or primers were synthesized with both forward and reverse strands by GenScript (Nanjing, China). Non‐labelled probe, as the competitive, was added to the reactions. For the DNA EMSA, the purified protein was incubated with FAM‐labelled probes in a binding buffer at 25 °C for 25 min. The resulting complexes were then separated by 5% Native PAGE electrophoresis in 0.5 × TBE buffer for 1 h. After electrophoresis, visualization was carried out using a Typhoon Trio Imager (GE, Amersham Typhoon).

ChIP–qPCR analysis

ChIP–qPCR experiments using the OE‐ZNF9‐Flag and OE‐GRF6‐Flag transgenic plants were performed as described with minor modifications (Wang et al., 2019). Anti‐Flag antibodies (MBL, PM020) were used for detection. Approximately 2 g leaves of 14‐day seedlings of transgenic rice were cross‐linked for 20 min by immersing in 1% (v/v) formaldehyde (Sigma, 47608) under vacuum. The genomic DNA was then randomly sheared into small fragments ranging from 100 to 400 bp through sonication. The protein/chromatin complexes were then incubated with anti‐Flag antibodies (MBL, PM020) overnight at 4 °C. The precipitated DNA was used for qRT‐PCR analysis.

Dual‐luciferase transcriptional activity assay

To analyse the transcriptional activity of ZNF9 and MYB3R in rice protoplasts, the OE‐ZNF9 and OE‐GRF6 vectors were constructed to serve as effectors. Reporter constructs were generated by cloning the promoter region of miR396b and MYB3R into pGreenII0800‐LUC vector. The recombinant plasmids were co‐transfected into rice protoplasts, which were cultured at 28 °C overnight. The LUC and REN activities were measured using the dual‐luciferase reporter assay system (Promega, Madison, WI, USA). For transcriptional activity assays in tobacco (Nicotiana benthamiana), the recombinant plasmids transformed with the GV3101 (pSoup‐p19) chemically competent cells. Then, the tobacco leaves were transfected with both the effector and the reporter, as previously described (Yuan et al., 2022), and images were captured using a Tanon 5200 Multi imaging system.

Haplotype and evolutionary analysis

Single nucleotide polymorphisms (SNPs) within the ZNF9 gene were extracted from a 2 kb promoter region. Haplotypes with a frequency greater than 5% and represented by more than 10 varieties were selected for association analysis. Phylogenetic trees of ZNF9 were constructed from 2767 rice accessions using the maximum likelihood method with MEGA 7.0 software. The construction was based on a distance matrix with 1000 bootstrap replicates, and the resulting phylogenetic tree was visualized using iTOL. For haplotype network analysis, the DnaSP6 and PopART version 1.7 were employed to align and organize haplotypes based on variations within ZNF9 across the 2767 rice accessions.

A total of 3056 rice accessions were used for evolutionary analysis from the Rice SNP‐Seek Database (https://snpseek.irri.org/_snp.zul) and OryzaGenome (http://viewer.shigen.info/oryzagenome/). This dataset included 3024 rice varieties and 32 wild rice genome sequences. The genetic differentiation parameters between indica and japonica varieties of ZNF9 and its flanking regions were analysed using the PopGenome R software package. These parameters included haplotype and nucleotide F _ST , Nei's G _ST , and Hudson's G _ST and H _ST .

Statistical analysis

A Student's t‐test was performed using GraphPad Prism 9 software to determine significance, which was defined: *P < 0.05; **P < 0.01 and ***P < 0.001, respectively. All the assays were carried out three biological replicates. The primers used for vector construction, yeast transformation, transcriptional activity and qRT‐PCR analysis were listed in Table S1.

Results

miR396b/ GRF6 module enhances rice salt tolerance

To investigate the response of the pre‐miR396a–h to salt tolerance, we firstly carried out qRT–PCR analysis of the expression of pre‐miR396 members under salt treatment. Except for pre‐miR396c, both pre‐miR396a and pre‐miR396b were induced by salt treatment as early as 1 h (Figure 1a). Compared with pre‐miR396a, pre‐miR396b showed continuous induction up to 6 h (Figure 1a). Hence, our attention is primarily directed towards the function of miR396b. To confirm whether miR396b contributes to salt tolerance, after NaCl treatment, the target mimicry of miR396b (MIM396) lines showed increased salt tolerance and a higher survival rate compared with the wild type (Figure 1b,c), suggesting miR396b is an important regulator of salt tolerance in rice. Previously, we demonstrated that miR396b targets GRF6 to modulate inflorescence development (Gao et al., 2016). To explore the significance of GRF6 in salt stress, we examined the expression of GRF6 and found that it was markedly induced by salt stress (Figure 1d). Additionally, the expression of GRF6 was significantly elevated in MIM396 transgenic lines (Figure 1e). Further examination revealed that the survival rate of GRF6 overexpression lines was notably higher than the wild‐type plants under salt stress, accompanied by a significant up‐regulation of GRF6 expression in overexpression lines (Figure 1f–h). On the contrary, when GRF6 was knocked out using CRISPR/Cas9 genome editing (Figure S1), the KO‐GRF6 lines exhibited greatly reduced survival rate compared to the wild type (Figure 1g,h), suggesting that GRF6 is responsible for the salt tolerance. Taken together, these results underscore the critical role of the miR396b/GRF6 module in enhancing salt tolerance in rice.

Figure 1.

miR396b/GRF6 module regulates salt tolerance. (a) qRT–PCR analysis of the pre‐miR396 transcripts after NaCl treatment. Data are mean ± SD (n = 3). (b, c) Salt tolerance phenotypes and statistical quantification of survival rates of the wild‐type (YB) and MIM396 plants. The salt stress treatment involved at least 24 plants, and individual experiment was repeated five times. Data are mean ± SD (n = 5). (d) Transcript levels of GRF6 were calculated at 1,3, 6, 9, 12 and 24 h under 200 mM NaCl treatment compared with the expression value at 0 h. Data are mean ± SD (n = 3). (e) Transcript levels of GRF6 were analysed in wild‐type (YB) and MIM396 transgenic lines. Data are mean ± SD (n = 3). (f) The expression levels of GRF6 in wild‐type (YB), OE‐GRF6 and KO‐GRF6 lines. 14‐day plants were with or without 200 mM NaCl to salt stress and collected for qRT‐PCR analysis. Data are mean ± SD (n = 3). (g, h) Phenotype and the survival rates of wild‐type (YB), overexpression lines (OE‐GRF6) and knockout mutants (KO‐GRF6) after 4‐day of salt stress and then 5‐day of recovery. Data are mean ± SD (n = 5). Scale bars, 5 cm. Different asterisks in a, c, d, e, f and h indicate significant differences in wild‐type (YB), MIM396 and GRF6 transgenic lines determined by Student's t‐test (*P < 0.05; **P < 0.01; ***P < 0.001).

MDA, Pro and ROS are usually suggested as critical indicators of plant response to environmental stress (Copenhaver et al., 2021; Jia et al., 2022; Wang et al., 2021a; Zhao et al., 2022). 2′,7′‐dichlorodihydrofluorescein diacetate (H2DCFDA) staining of miR396b and GRF6 transgenic seedling root tips revealed that the fluorescence signal in MIM396 and OE‐GRF6 lines decreased under salinity stress (Figure 2a,b), consistent with the changing trend of H₂O₂ accumulation (Figure 2c). Meanwhile, the ROS‐scavenging enzyme activities of catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) were drastically enhanced in MIM396 and OE‐GRF6 lines under salt stress compared to wild type (Figure 2d,e; Figure S2a). In accordance with the enhanced salt tolerance, Pro was significantly elevated but MDA levels were markedly decreased in MIM396 and OE‐GRF6 lines than that in wild type (Figure 2f,g). Moreover, relative to the wild type, damage of the salt treatment on fresh and dry weight of the MIM396 and OE‐GRF6 lines were significantly alleviated (Figure S2b,c). In summary, the dataset strongly suggests that the miR396b/GRF6 module serves as a vital regulator of salt tolerance, augmenting a plant's ability to adapt to osmotic stress.

Figure 2.

ROS scavenging in root tips of MIM396 and OE‐GRF6 transgenic lines under salt stress. (a) H2DCFDA staining analyses of wild‐type (YB), MIM396, OE‐GRF6 and KO‐GRF6 root tips under salt treatment. Scale bars, 500 μm. (b) Fluorescence intensity analyses of wild‐type (YB), MIM396, OE‐GRF6 and KO‐GRF6 transgenic lines under salt treatment. (c–g) Measurements of H₂O₂ content (H₂O₂, c), catalase activity (CAT, d), superoxide activity (SOD, e), malondialdehyde content (MDA, f) and proline content (Pro, g). Different asterisks in b‐g indicate significant differences in MIM396, GRF6 transgenic lines and the wild type (YB) determined by Student's t‐test (*P < 0.05; **P < 0.01; ***P < 0.001). Data are mean ± SD (n = 5).

ZNF9 regulates miR396b/ GRF6 expression in response to salt stress

In order to identify the key factors regulating the expression of miR396b/GRF6 in response to salt stress, we amplified the promoter fragments of miR396b using YB genomic DNA as a template, designating them as miR396b_Pro521, miR396b_Pro981, miR396b_Pro1410 and miR396b_Pro1918, respectively (Figure S3a). Dual‐luciferase reporter assay indicated the miR396b promoter activity peaking at 981 bp and decreasing thereafter, meaning this fragment plays a crucial role in regulating miR396b expression (Figure S3b). This is consistent with bioinformatic annotation that it contains multiple cis‐acting elements associated with plant growth and developmental responses (Table S2). Then, we performed a yeast one‐hybrid assay using the miR396b_Pro981 fragment as bait (Figure S4), and 27 candidate regulating genes were obtained (Table S3). Among which, one gene encoding C3HC4 type RING zinc finger (Table S3), known to play an essential in the regulation of abiotic stress including cold, salt and dehydration in Brassica rapa (Jung et al., 2013), may serve as regulators of miR396b. Further analysis revealed a cis‐acting element, AtMYB84 (CACCtACC), was predicted to interact with ZNF9 using the Plant Cis‐acting Regulatory DNA Elements (PlantCARE) database (Figure 3a). To validate the direct regulation of ZNF9 to miR396b promoter, yeast one‐hybrid assay was carried out. Results showed that ZNF9 could bind the ‘CACCAACC’ motif (Figure 3b). When the ‘CACCAACC’ was mutated, the binding ability of ZNF9 disappeared (Figure 3b), implying that ZNF9 specifically bind to ‘CACCAACC’ in the miR396b promoter. This is consistent with the electrophoretic mobility shift assay (EMSA) assay that ZNF9 markedly reduced the migration of the ‘CACCAACC’ motif (Figure 3c). Correspondingly, chromatin immunoprecipitation (ChIP)‐qPCR analysis showed that ZNF9 was significantly enriched in the promoter of miR396b (Figure 3d). Furthermore, a dual‐luciferase reporter assay showed that the luciferase activity of miR396b::LUC was significantly elevated when ZNF9 was co‐expressed in rice protoplasts and tobacco (Figure 3e–g). Collectively, these results convincingly demonstrate that the ZNF9 activated the expression of miR396b by directly binding to its promoter.

Figure 3.

ZNF9 directly activates the expression of miR396b. (a) Localization of putative ZNF9 binding sites S and mS (mutated S site) in the miR396b promoter. (b) Yeast one‐hybrid analysis of ZNF9 binding the promoter of miR396b. Growth of yeast cells transformed with prey (pGADT7‐ZNF9) and bait (S or mS), along with negative control (pGADT7 + pAbAi) and positive control (pGADT7‐Rec‐p53 + p53‐Abai), on selective medium without or with AbA (600 ng/mL). (c) EMSA analysis of the interaction between ZNF9 and the CACCAACC motif. The mutant probe of mS (CACCGACC) eliminated binding. (d) ChIP‐qPCR validation of the ZNF9 binding on CACCAACC sites in (a). P denotes the loci of the DNA fragments subjected to ChIP‐PCR analysis. The red triangle represents the binding motif ‘CACCAACC’. (e) Schematic diagrams of the effector and reporter used for dual‐luciferase transcriptional activity assay in rice protoplasts and tobacco. (f) The bioluminescence image of LUC signal from tobacco leaf section infiltrated by designated different effectors and reporters. (g) Promoter activity analysis, based on the LUC/REN ratio, different effector and report on their combination in rice protoplasts. Different asterisks in d and g indicate significant differences determined by Student's t‐test (**P < 0.01; ***P < 0.001). Data are mean ± SD (n = 3).

ZNF9 is a salt‐induced transcriptional activator

Bioinformatics analysis showed that ZNF9 encodes a zinc finger protein with a conserved C3HC4‐type RING finger domain (Figure 4a; Figure S5). Spatiotemporal expression analysis indicated that ZNF9 was expressed ubiquitously in all tissues, including the roots, shoot, young leaves and panicles, with the highest expression levels observed in young leaves (Figure 4b). C3HC4‐type RING finger subfamily genes, which have been proposed to be significantly induced by abiotic stress in plants such as Arabidopsis thaliana, Brassica rapa and Artemisia desertorum (Baxter et al., 2007; Jung et al., 2013; Yang et al., 2008). Consistent with this, our results indicated that ZNF9 mRNA abundance progressively increased upon exposure to salt treatment, peaking at 3 h (Figure 4c), confirming that ZNF9 is induced by salt treatment.

Figure 4.

ZNF9 functions as a salt stress induced transcriptional activator. (a) Analysis of the C3HC4‐type RING finger domain in ZNF9 protein sequence. The black box indicates the C3HC4‐type RING finger domain. (b) qRT–PCR analysis of the ZNF9 expression in various organs. R, roots; C, culms; L, leaves; YP0, YP1, YP2, YP3, YP4 and YP5, represent young panicles about 0–0.5 cm, 0.5–1 cm, 1–2 cm, 2–3 cm, 3–4 cm, and 4–5 cm, respectively. (c) ZNF9 expression in 14‐day seedlings treated with 200 mM NaCl. (d) Subcellular localization of ZNF9‐sGFP fusion protein in rice protoplasts. D53–mCherry was used as a nuclear marker. Scale bars, 5 μm. (e) Transcriptional activation analysis of ZNF9 in yeast. pGADT7‐T + pGBKT7‐53 and pGADT7‐T + pGBKT7‐Lam were used as positive and negative control, respectively. (f) Schematic diagrams of the effector and reporter used for transcriptional activation analysis in rice protoplasts. (g) Quantitative analysis of ZNF9 transcriptional activation in rice protoplasts. Different asterisks in g indicate significant differences determined by Student's t‐test (***P < 0.001). Data are mean ± SD (n = 3).

Transient expression assay in rice protoplasts demonstrated that ZNF9 is a nuclear protein (Figure 4d). To assess whether ZNF9 possesses transcriptional activation capability, the transcriptional activity assays in yeast cells and rice protoplasts were conducted. Results indicated that ZNF9 displayed no self‐activation activity in yeast (Figure 4e). However, when compared to the control constructs BD and BD‐VP16, BD‐ZNF9 and BD‐VP16‐ZNF9 induced significantly high luciferase activity in rice protoplasts (Figure 4f,g). These observations suggest ZNF9 likely acts as a salt stress induced transcriptional activator.

ZNF9 negatively regulates rice salt tolerance by activating miR396b expression

ZNF9 functions as a salt‐induced transcription factor, activating the expression of miR396b, which suggests its potential role as a negative regulator of salt tolerance. To characterize the role of ZNF9 in salt tolerance, ZNF9 overexpression and knockout mutants were generated (Figure 5a). qRT‐PCR results revealed that endogenous ZNF9 mRNA levels were dramatically elevated in OE‐ZNF9 plants relative to wild type, especially under salt stress (Figure 5b). When subjected to salt stress conditions, the survival rates of OE‐ZNF9 transgenic seedlings showed significantly lower than the wild type, while those of KO‐ZNF9 seedlings were significantly increased (Figure 5c,d). In accordance with the phenotype of seedlings, MDA contents were drastically higher, while Pro was significantly lower in the overexpression lines compared to the wild type (Figure 5e,f). The fluorescence intensity of KO‐ZNF9 root tips was weaker than that of the wild type under salt stress (Figure 5g,h). Expectedly, the changing trend of ROS‐scavenging enzyme activities (CAT, SOD and POD) and H₂O₂ accumulation in KO‐ZNF9 lines was opposite to that of the ZNF9 overexpression lines (Figure S6a–d). Consistent with the salt tolerance phenotype, the fresh and dry weight of KO‐ZNF9 were significantly higher than those of the wild type when treated with salt (Figure S6e,f). These results indicate that ZNF9 functions as a negative regulator of salt stress.

Figure 5.

ZNF9 negatively regulates salt tolerance in rice. (a) Mutation in KO‐ZNF9‐1 and KO‐ZNF9‐2 mutants. (b) ZNF9 expression level in the wild‐type (YB) and ZNF9 transgenic lines under normal condition and 200 mM NaCl treatment. Data are mean ± SD (n = 3). (c, d) The phenotypes and survival rate of the ZNF9‐koncout (KO) and ZNF9‐overexpression (OE) lines under salt stress. Data are mean ± SD (n = 5). Scale bars, 5 cm. (e, f) Measurement of MDA (e) and Pro (f) contents in ZNF9 transgenic plants with or without 200 mM NaCl treatment. Data are mean ± SD (n = 5). (g, h) H2DCFDA staining and fluorescence intensity analyses of wild‐type (YB), OE‐ZNF9 and KO‐ZNF9 root tips of 4‐day seedlings under normal condition and 100 mM NaCl treatment. Data are mean ± SD (n = 5). Scale bars, 500 μm. (i, j) The phenotypes and survival rate of the wild type (YB), OE‐ZNF9‐4, MIM396‐3 and OE‐ZNF9‐4/MIM396‐3 under salt stress. Data are mean ± SD (n = 5). Scale bars, 5 cm. Different asterisks in b, d, e, f, h and j indicate significant differences between wild‐type (YB) and ZNF9 transgenic lines determined by Student's t‐test (*P < 0.05; **P < 0.01; ***P < 0.001).

In order to explore the genetic relationship of ZNF9 and miR396b, the expression of miR396b and GRF6 was examined in YB, OE‐ZNF9 and KO‐ZNF9 lines. The expression of miR396b was up‐regulated in the OE‐ZNF9 plants but down‐regulated in the KO‐ZNF9 plants under salt treatment (Figure S7a), which was consistent with the opposite trend of GRF6 expression level (Figure S7b). This result suggested that the function of ZNF9 might be partially dependent on miR396b. Furthermore, the OE‐ZNF9‐4 line was crossed with MIM396‐3 line to get double transgenic plants (OE‐ZNF9‐4/MIM396‐3). After NaCl treatment, the survival rates of OE‐ZNF9‐4/MIM396‐3 line were increased compared with OE‐ZNF9‐4 or YB lines (Figure 5i,j). Simultaneously, in comparison with OE‐ZNF9‐4 line, in the OE‐ZNF9‐4/MIM396‐3 line, the expression of miR396 decreased, while GRF6 expression increased (Figure S8a‐c). Moreover, the damage of fresh and dry weight was alleviated in OE‐ZNF9‐4/MIM396‐3 line (Figure S8d,e). These biochemical and genetic evidences indicate that miR396b was one direct target gene of ZNF9. These results collectively demonstrated that ZNF9, as a key regulatory factor of rice salt tolerance, modulating the expression of miR396b/GRF6 module.

MYB3R is a downstream target of miR396b/ GRF6 module in response to salt stress

To dissect the downstream signalling pathways regulated by the miR396b/GRF6 module in response to salt stress, transcriptome analysis between the OE‐GRF6 and wild‐type plants was carried out by using 14‐day seedling leaves treated with 200 mM NaCl. A total of 5347 differentially expressed genes (DEGs) were identified, with 2502 genes were up‐regulated and 2845 genes were down‐regulated in the OE‐GRF6 plants during salt stress (Figure S9). GO analysis revealed that the DEGs were primarily enriched in biological processes related to ‘cellular process’, ‘metabolic process’, ‘regulation of biological process’ and ‘response to stimulus’. These processes are all crucial for plant cell survival and in vivo enzyme activities (Figure S10). More interestingly, GO terms related to abiotic stress processes, including ‘antioxidant activity’, ‘electron carrier activity’, ‘transporter activity’ and ‘signal transducer activity’ were specifically found in molecular function term (Figure S10), which was consistent with the positive role of GRF6 in salinity treatment.

To identify the specific target of the miR396b/GRF6 module in regulating salt stress, a combined analysis revealed that 31 genes were overlapped between the up‐regulated genes of RNA‐seq and our previous ChIP‐seq data of OE‐GRF6 line (Gao et al., 2016) (Figure 6a). Of which, one gene was annotated to be involved in ‘positive regulation of response to salt stress’ in GO analysis (Figure 6b). Further analysis revealed that the gene encoded MYB3R responsible for tolerance to freezing, drought, and salt stress in transgenic Arabidopsis (Dai et al., 2007). qRT‐PCR analysis demonstrated that MYB3R was up‐regulated in the GRF6 overexpression lines but down‐regulated in the KO‐GRF6 plants, especially under salt treatment (Figure 6c), indicating that MYB3R is the potential target of miR396b/GRF6 module.

Figure 6.

miR396b/GRF6 target MYB3R to enhance salt tolerance in rice. (a) Overlap analysis of the GRF6 target genes with RNA‐seq and ChIP‐seq. (b) Gene ontology (GO) enrichment analyses of 31 overlapping genes. The red box indicates signalling pathways directly responding to salt stress. (c) Expression level of MYB3R in the wild‐type (YB) and GRF6 transgenic lines with and without 200 mM NaCl. (d) Yeast one‐hybrid assay for GRF6 binding to the regulatory regions of MYB3R promoter. (e) ChIP‐qPCR analysis of enrichment of GRF6 in the MYB3R promoter. P1 and P2 represent the loci of the DNA fragments subjected to ChIP‐PCR analysis. (f) EMSA assay for the interaction between GRF6 and MYB3R promoter. (g) Genome browser traces of MYB3R ChIP‐seq from GRF6. Black lines indicate binding site. (h–j) Schematic diagram of the effector and reporter constructs used for dual‐luciferase transcriptional activity assay (i) and transcriptional activity of MYB3R activated by GRF6 was analysed in tobacco (h) and rice protoplasts (j). Different asterisks in c, e and j indicate significant differences determined by Student's t‐test (*P < 0.05; **P < 0.01). Data are mean ± SD (n = 3).

Furthermore, we confirmed the DNA‐binding specificity of GRF6 to the MYB3R promoter through a yeast one‐hybrid assay (Figure 6d). EMSA combined with ChIP‐qPCR analysis revealed that GRF6 could directly bind to the ‘CGSMR’ cis‐element of the MYB3R promoter (Figure 6e,f), consistent with the ChIP‐seq results (Figure 6g). These were further confirmed by the luciferase reporter assay in rice protoplasts and tobacco (Figure 6h–j), demonstrating that GRF6 enhances the expression of MYB3R (Figure 6j), is consistent with the observation of a stronger fluorescence signal in tobacco leaves (Figure 6h). Collectively, these findings illustrate that the miR396b/GRF6 module directly targets MYB3R to enhance its expression, thereby regulating salt tolerance in rice.

MYB3R positively regulates salt tolerance in rice

MYB3R encodes a protein that belongs to the R1R2R3 type MYB family of transcription factors in rice. Transcriptional activity assays, along with subcellular localization studies, confirmed that MYB3R acts as a transcriptional activator in response to salt stress (Figure 7a,b; Figure S11). Notably, MYB3R showed a similar expression pattern as that of GRF6 under salt treatment (Figure 7c). Spatiotemporal expression analysis revealed that MYB3R was ubiquitously expressed in all organs, especially in the leaves and roots (Figure S12). These results demonstrated that MYB3R could be associated with regulation processes governing the salinity stress.

Figure 7.

MYB3R positively regulates salt tolerance in rice. (a) Subcellular localization of MYB3R in rice protoplasts. D53–mCherry was used as a nuclear marker. Scale bar, 5 μm. (b) Transcriptional activity assay of MYB3R with dual‐luciferase reporter system in rice protoplast. Data are mean ± SD (n = 3). (c) qRT‐PCR analysis of MYB3R expression under 200 mM NaCl treatment at different time point. Data are mean ± SD (n = 3). (d) The phenotype of MYB3R overexpression (OE‐MYB3R‐1 and OE‐MYB3R‐2), mutant (KO‐MYB3R) and their corresponding wild‐type (YB and ZH11, respectively) rice seedlings treated with 200 mM NaCl for 4 days. Data are mean ± SD (n = 5). (e) ROS signals in MYB3R transgenic lines and their corresponding wild‐type (YB and ZH11, respectively) rice seedling roots detected with H2DCFDA staining. Scale bars, 500 μm. (f, g) Measurement of MDA (f) and Pro (g) in the shoots of MYB3R transgenic lines and their corresponding wild type (YB and ZH11, respectively) under normal condition or 200 mM NaCl treatment. Data are mean ± SD (n = 5). (h, i) The phenotypes and survival rate of the wild type (YB), KO‐GRF6‐2, OE‐MYB3R‐2 and KO‐GRF6‐2/OE‐MYB3R‐2 under 200 mM NaCl treatment. Data are mean ± SD (n = 5). Scale bars, 5 cm. Different asterisks in b, c, f, g i indicate significant differences between YB and MYB3R transgenic lines determined by Student's t‐test (*P < 0.05; **P < 0.01; ***P < 0.001). (j) A proposed model of the miR396b/GRF6 module for the regulation of rice salt tolerance. ZNF9 directly binds to and activated the expression of miR396b in OE‐ZNF9 plants, the increased expression of miR396b cleaved the GRF6 transcripts and repressed the downstream regulator for salt tolerance. On the other hand, in KO‐ZNF9 plants, the transcription factor GRF6 was up‐regulated, and promote the downstream MYB3R expression and the activity of reactive oxygen scavenging enzymes which eliminate the reactive oxygen species, and ultimately enhance salt tolerance of rice.

To verify the biological function of MYB3R to salt response in rice, MYB3R transgenic plants were treated with salt, and the overexpression lines exhibited enhanced salt tolerance, resulting in a higher survival rate among the OE‐MYB3R lines when subjected to salt treatment (Figure 7d; Figure S13a). Concurrently, endogenous MYB3R mRNA levels in OE‐MYB3R plants increased in response to salt stress (Figure S14). Conversely, the CRISPR/Cas9‐engineered mutants exhibited a salt‐sensitive phenotype, along with significantly decreased survival rate under salt stress (Figure 7d; Figure S13a). Correspondingly, OE‐MYB3R exhibited weaker fluorescence intensity and H₂O₂ accumulation, accompanied by higher ROS‐scavenging enzyme activities (CAT, SOD and POD) and increased accumulation of fresh and dry weight in seedlings (Figure 7e; Figures S13b and S15). In contrast, the MYB3R knockout seedlings showed an opposite trend to the MYB3R overexpression lines on the antioxidant biochemical indexes and the fresh and dry weight (Figure 7f,g; Figures S13b and S15). Collectively, these results indicate that MYB3R, functions as a positive regulator of salt stress, positively regulates rice salt tolerance.

In order to understand the genetic interaction between GRF6 and MYB3R, we crossed the KO‐GRF6‐2 and OE‐MYB3R‐2 lines, and the KO‐GRF6‐2/OE‐MYB3R‐2 hybrid line showed a similar survival rate as the OE‐MYB3R‐2 lines (Figure 7h,i), but significantly higher than the wild‐type and the KO‐GRF6‐2 lines, corresponding to an up‐regulation in the expression of MYB3R and an increase in the accumulation of fresh and dry weight in seedlings (Figure S16), confirming that MYB3R could rescue the salt tolerance character of GRF6.This suggests that MYB3R is a direct target of GRF6, actively participating in the regulation of salt tolerance in rice.

Discussion

As sessile organisms, plants must cope with various stresses in changing environments such as soil salinity, drought and extreme temperatures (Yang et al., 2022; Zhu, 2016). The increasing salinity land seriously endangers human food security and sustainable development, making it urgent to create high‐yield, salt‐tolerant crop varieties (Ahmad et al., 2022; Zhao et al., 2021). A comprehensive understanding of the intricate mechanisms governing salt stress responses is crucial for the development of salt‐tolerant crops, which in turn promotes agricultural sustainability and food security, especially given the constraints of diminishing agricultural land and a burgeoning global population (Mickelbart et al., 2015; Yang et al., 2022; Zhu, 2016). In this article, we elucidate the pivotal role of the miR396b/GRF6 module in the rice plant's response to salt stress (Figure 7j). More importantly, ZNF9, identified as a salt‐induced regulator negatively influencing rice salt tolerance (Figures 4c and 5b‐d), was found to directly bind to the ‘CACCAACC’ element in the miR396b promoter (Figure 3). This interaction modulates the expression of miR396b/GRF6 in response to salt stress (Figures 3 and 7j). In rice, there are a total of 56 homologous genes of ZNF9, with most of which have unknown functions (Figure S17). The functional analysis of ZNF9 establishes a robust foundation for the study of this gene family. Furthermore, the high degree of conservation of ZNF9 across different species implies its significance in mediating salt stress responses in various organisms (Figure S18). While current studies have highlighted the significant role of miRNAs in salt stress responses, the upstream regulatory factors of miRNAs in salt stress have remained relatively unexplored. In this study, we identified a new regulator of salt tolerance response, ZNF9, which directly modulates the expression of miR396b, consequently regulating the salt tolerance response (Figures 3 and 5b–d) and grain yield in rice (Figure S19). Haplotypic analysis based on the promoter sequence combined with evolutionary analysis revealed that ZNF9 was mainly comprised by the ZNF9 ^XI and ZNF9 ^GJ subtypes, showing apparent genotypic differentiation between indica and japonica subspecies (Figure S20; Table S4 and S5). Correspondingly, the ZNF9 ^XI and ZNF9 ^GJ haplotypes showed distinct regional distribution differentiation, highlighting its significant breeding potential (Figures S21 and S22). In the future, directed editing of ZNF9 may hold the promise of enhancing rice yields while improving salt tolerance within the miR396b/GRF6 module (Zhang et al., 2021).

MYB transcription factors play a pivotal role in plant response to stress resistance (Li et al., 2019b; Wang et al., 2021b). Overexpression of MYB49 enhances tolerance to salt stress in both Arabidopsis and tomato (Cui et al., 2018; Zhang et al., 2020b). Loss of the AtMYB73 results in the hyper‐induction of SOS1 and SOS3 genes, leading to improved survival rates under high salt conditions (Kim et al., 2013). In the present study, we identified MYB3R, a member of the R1R2R3‐type MYB gene family, as a target of the miR396b/GRF6 module (Figure 6). MYB3R is known to be involved in responding to cold, drought, and salt stress in Arabidopsis (Dai et al., 2007). Moreover, overexpression of OsMYB3R‐2 enhanced tolerance to chilling stress in rice by modulating the cell cycle and a putative DREB/CBF pathway (Ma et al., 2009). In our research, we found that MYB3R serves as a downstream target of GRF6 and plays a critical role in salt tolerance in rice (Figures 6 and 7d). These results further broaden our understanding of the pivotal roles of the MYB transcription factors involving in resilient adaption of rice to adverse environments.

miR396/GRF is an important module affecting plant growth including root, leaf and floral organ development, heading date and grain size and number (Kim and Tsukaya, 2015; Liebsch and Palatnik, 2020). In Pitaya, the expression of miR396b/GRF6 was significantly induced by drought, low temperature, high temperature and ABA treatment (Li et al., 2019a). Meanwhile, GRF6 interacts with DELLAs to respond to cold stress in plants (Kim and Kende, 2004; Lantzouni et al., 2020). In the current investigation, we established that the miR396b/GRF6 module significantly enhances salt tolerance (Figure 1). It is worth noting that the control of salt tolerance by miR396/GRFs has also been reported in other crop species, including tobacco, cotton and creeping bentgrass (Chen et al., 2015; Wang et al., 2013; Yuan et al., 2019). However, previous studies have only identified the significant role of miR396 and the potentially involved GRFs under salt stress. Here, we systematically elaborated the molecular mechanism of miR396/GRF6 module in rice for salt tolerance, revealing the upstream ZNF9 and downstream MYB3R are key regulatory factors of miR396/GRF6 module responding for salt stress (Figures 3 and 6). Additionally, we found that the miR396b/GRF6 module maintains ROS homeostasis through MYB3R to confer salt tolerance in rice (Figure 7e; Figures S12b and S14a–d). Previously, we found that the miR396b/GRF6 module regulates auxin synthesis and activates expression of the auxin response related genes including OsYUCCA and OsARFs, thereby promoting secondary branching to improve rice yield (Gao et al., 2016). MYB3R, as a target of the GRF6‐regulated salt tolerance pathway, exhibited no significant change in yield (Figure S23), indicating that the pathways regulating grain yield and salt tolerance through GRF6 are independent (Figures S23 and S24). These findings greatly broaden our understanding on the signalling transduction of miR396/GRF6 module of rice involving in environmental stress.

MiR396/GRF has been known a key hub to modulate grain yield in rice, and it is also highly conserved in different crops (Axtell et al., 2012; Gao et al., 2016; Liu et al., 2014; Zhang et al., 2018). Interestingly, the fresh and dry weights of MIM396 and OE‐GRF6 were significantly higher than those of the wild type under salt stress (Figure S2b,c). This implies that miR396b/GRF6 has a greater growth potential under salt stress, and the miR396b/GRF6 module may possess a conserved function related to both high yield and salt tolerance. This discovery holds promise for the development of molecular breeding strategies aimed at creating new crop varieties with elevated grain yields and enhanced salt tolerance at the miRNA level. Thus, miR396b/GRF6 module may represent a valuable target for manipulating rice salt tolerance to improve regional adaptability and grain yield.

In summary, our results have revealed a mechanistic framework that elucidates the regulation of salt response in rice by the miR396b/GRF6 module. We have also demonstrated that ZNF9 plays a direct role in regulating miR396b/GRF6 expression, and MYB3R serves as a direct downstream target of the miR396b/GRF6 module, influencing salt tolerance in rice. Collectively, this study reveals a novel mechanism by which high‐yield miR396b/GRF6 modules regulate salt tolerance in rice. The findings advance our understanding of the molecular mechanisms of high‐yield salt tolerance genes and may provide a new idea to engineer high‐yield and salt‐tolerant rice.

Accession numbers

Sequence from this article can be found in the GeneBank/EMBL databases under the following accession numbers: ZNF9, Os09g0504700; GRF6, Os03g0729500; MYB3R, Os01g0841500; UBI, Os03g0234200.

Author contributions

S.L. and H.Y. designed the research project. H.Y., M.C., R.W., Z.W. and F.F. performed the experiments and analysed the data. H.Y., M.C., W.W., F.S. and F.G. work on transgenic lines and developed materials. M.C. performed yeast one‐hybrid assay analysis. H.Y. and S.L. wrote the paper. All authors have read and agreed to the published version of the manuscript.

Conflict of interests

The authors declare no competing interests.

Supporting information

Figure S1 CRISPR/Cas9‐mediated targeted mutagenesis of GRF6 in YB.

Figure S2 Peroxidase (POD) activity, fresh and dry weight in shoots of MIM396，OE‐GRF6, KO‐GRF6 and wild type (YB).

Figure S3 Activity analysis of the different miR396b promoter fragment.

Figure S4 Construction of yeast one‐hybrid library using YB cDNA.

Figure S5 The gene structure of ZNF9.

Figure S6 ROS‐scavenging enzyme activity, fresh and dry weight in the ZNF9 transgenic lines.

Figure S7 miR396b and GRF6 expression levels in wild‐type (YB) and ZNF9 transgenic lines.

Figure S8 Analysis of expression level, fresh and dry weight in OE‐ZNF9‐4, MIM396‐3 and OE‐ZNF9‐4/MIM396‐3 lines under NaCl treatment.

Figure S9 Venn diagrams of the DEGs up‐regulated and down‐regulated in salt‐treated wild‐type (YB) and OE‐GRF6 rice seedlings.

Figure S10 GO term analysis of GRF6‐regulated genes.

Figure S11 Analysis of MYB3R transcriptional activation in yeast.

Figure S12 MYB3R expression profile detected by qRT‐PCR.

Figure S13 Survival rate and ROS fluorescence intensity in MYB3R transgenic lines under salt stress.

Figure S14 Expression levels of MYB3R in the wild‐type (YB) and MYB3R overexpression lines under 200 mM NaCl treatment.

Figure S15 Activity of the ROS‐scavenging enzymes, fresh and dry weight of the MYB3R transgenic lines treated with salt.

Figure S16 Analyses of expression level, fresh and dry weight in KO‐GRF6‐2, OE‐MYB3R‐2 and KO‐GRF6‐2/OE‐MYB3R‐2 lines under NaCl treatment.

Figure S17 Phylogenetic analysis of the ZNF9 homologous protein in rice.

Figure S18 Alignment of the C3HC4‐type RING finger domain of ZNF9 protein sequence in different plant species.

Figure S19 Analysis of yield‐related traits in the ZNF9 transgenic lines.

Figure S20 Genetic variation analysis of ZNF9.

Figure S21 Haplotype analysis of ZNF9.

Figure S22 Geographical distribution of ZNF9 ^XI and ZNF9 ^GJ haplotypes.

Figure S23 Yield‐related traits of the MYB3R transgenic lines.

Figure S24 Yield‐related traits of the MIM396, OE‐GRF6 and KO‐GRF6 lines.

Table S1 Primers and probes used in this study.

Table S2 Identification of cis‐acting elements in miR396b promoter sequence.

Table S3 The screening results of the yeast one‐hybrid assay.

Table S4 The Pi value of ZNF9 and its flanking region.

Table S5 The Fst value of ZNF9 and its flanking genomic regions.

Data availability

The RNA‐seq data that the findings of this study have been deposited in the National Genomics Data Center (NGDC) with the accession code PRJCA020403. All other relevant data are available from the corresponding author upon request.