Zinc Finger Homeobox Transcription Factors OsMIF1 and OsMIF2 Regulate Grain Size and Panicle Development in Rice

Zinc Finger Homeobox Transcription Factors OsMIF1 and OsMIF2 Regulate Grain Size and Panicle Development in Rice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Zinc Finger Homeobox Transcription Factors OsMIF1 and OsMIF2 Regulate Grain Size and Panicle Development in Rice Jinpyo So, Kyoungwon Cho, Jong-Yeol Lee, Don-Kyu Kim, Oksoo Han This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8286035/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Mar, 2026 Read the published version in Rice → Version 1 posted 9 You are reading this latest preprint version Abstract Mini zinc finger (MIF) proteins are plant-specific zinc finger-homeodomain (ZF-HD) transcription factors lacking a homeodomain, whose biological functions are critical for normal plant development and the response to environmental stress. Here, CRISPR-Cas9 was used to engineer null alleles of rice OsMIF1 and OsMIF2, and the resulting OsMIF1- and OsMIF2-deficient knockout lines were used to identify the biological roles of OsMIF1 and OsMIF2. The results suggest that OsMIF proteins transcriptionally regulate grain size by controlling the size of epidermal cells and the length and branching of rice panicles. RNA-seq analysis of OsMIF-knockout cells revealed altered expression of genes involved in development, the response to environmental stress and grain size. In addition, 10 protein-interacting partners of OsMIF1 were identified using a yeast two-hybrid screen: these proteins play roles in diverse developmental, hormonal, stress response, and metabolic processes, suggesting that OsMIF1 is effectively a regulatory hub, whose role is to integrate signals as they propagate through rice development- and stress response pathways. The results presented here support the conclusion that OsMIF1 and OsMIF2 are master transcription factors that regulate development throughout the adult plant life cycle and contribute significantly to plant resilience in the presence of environmental stressors. Rice Zinc finger-homeobox Mini zinc finger OsMIF1 OsMIF2 Grain size Panicle development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Rice is a critically important agricultural crop that is the primary dietary staple and source of calories for nearly half of the human population (Fukagawa & Ziska, 2019 ; Mohidem et al., 2022 ). Therefore, the quality and quantity of every rice harvest is critical and ways to increase rice yield and quality are a major focus of ongoing agricultural research (Alam et al., 2024 ; Hori & Sun, 2022 ). Panicle and seed number, architecture/structure, size and composition (Gengmi Li et al., 2021 ; Gangling Li et al., 2021 ), which are regulated by a complex network of genetic, hormonal, and environmental factors, are key determinants of rice yield and quality. Moreover, the regulation of these critical rice traits is intricately and coordinately influenced by resource allocation (Gengmi Li et al., 2021 ; Li et al., 2022 ; Ren et al., 2023 ) as well as phytohormones ( i.e. , abscisic acid (ABA), auxin, cytokinin, and ethylene) and MAPK and G-protein signaling pathways and relevant transcription factors (TFs) (Gengmi Li et al., 2021 ; Zhao et al., 2022 ). TFs play pivotal roles in integrating and coordinating hormonal and other signaling pathways that regulate rice grain and panicle quality and quantity (Gengmi Li et al., 2021 ; Zhao et al., 2022 ). For instance, OsSPL family TFs regulate rice panicle architecture and grain traits (Dai et al., 2018 ; Hu et al., 2021 ; Lian et al., 2020 ; Segami et al., 2017 ; S. Wang et al., 2015 ), while WRKY TFs orchestrate and integrate both developmental processes and stress response-related pathways and their impact on rice grain and panicle traits (Li et al., 2025 ; Yang et al., 2025 ). The zinc finger-homeodomain (ZF-HD) protein family is a group of plant-specific TFs that regulate plant growth, development, flowering and the response to environmental stressors (Bollier et al., 2022 ; Jang et al., 2023 ; Shalmani et al., 2019 ). ZF-HD proteins harbor both zinc finger (ZF) and homeodomain (HD) motifs, while mini zinc finger (MIF) proteins (a subfamily of ZF-HD proteins) lack the HD motif (Bollier et al., 2022 ; Islam et al., 2022 ; Niu et al., 2021 ). In general, ZF-HD TFs bind to specific cis-acting regulatory motifs in gene promoter regions as monomeric, homo-/hetero- dimeric or higher order protein complexes. These TFs ultimately regulate expression of genes that in turn regulate various biological processes including organogenesis, hormone signaling, and the response to abiotic and biotic stressors (Bollier et al., 2022 ; Shen et al., 2025 ). Arabidopsis ZF-HD TFs play diverse development-related roles in hormone signaling and light-mediated morphogenesis (Bueso et al., 2014 ; Kim et al., 2019 ; Perrella et al., 2018 ); for example, ZHD5 promotes shoot regeneration and cytokinin-associated phenotypes, ATHB25 (also known as ZFHD2/ZHD1) regulates gibberellin biosynthesis and seed longevity (Bueso et al., 2014 ; Kim et al., 2019 ), and ZFHD10 coordinates and integrates light signaling to promote elongation of hypocotyls (Perrella et al., 2018 ). Rice ZF-HD genes play significant roles in reproductive development including floral initiation and seed formation (Jain et al., 2008 ; Shalmani et al., 2019 ; Shen et al., 2025 ); for example, OsZHD1 and OsZHD2 redundantly regulate grain size, modulate cell proliferation in spikelets and glumes, OsZHD2 also regulates biosynthesis of ethylene which in turn regulates root development (Guo et al., 2025 ; Yoon et al., 2020 ), while OsZHD1, OsZHD2, OsZHD4, and OsZHD8 form homo- and heterodimers that directly repress transcription of target genes in plants/cells in the presence of abiotic stressors (Figueiredo et al., 2012 ). MIF proteins, which possess only a single zinc finger domain (Hu & Ma, 2006 ; Thiaw & Gantet, 2024 ), play roles in cell division, meristem state transitions, and reproduction-related processes ( i.e. , development of vegetative and floral organs) (Thiaw & Gantet, 2024 ). In Arabidopsis , MIF1 integrates multiple phytohormone signals, and its overexpression results in developmental defects such as dwarfism, loss of apical dominance, and dark-green spoon-shaped cotyledons (Hu & Ma, 2006 ). Furthermore, overexpression of MIF1 or MIF3 induces ectopic shoot meristems along leaf margins, disrupts leaf growth, and modulates auxin and gibberellin-regulated processes (Hu et al., 2011 ). The tomato MIF homolog SlIMA regulates floral meristem termination, carpel number and fruit development (Bollier et al., 2018 ), while the Gerbera hybrida MIF protein GhMIF directly activates expression of the GEG gene, a member of the GASA family, to suppress ray petal elongation (Han et al., 2017 ). A recent study demonstrated that PASPRO1 (OsMIF3) and PASPRO2 (OsMIF4) interact with and inhibit the transcriptional function of rice ZHD TFs, thereby regulating the surface material patterns of the production of anther and pollen. OsMIF3- and OsMIF4 knockout lines exhibit abnormal cuticle formation, defective pollen surface structures and reduced fertility (Jang et al., 2023 ). Furthermore, overexpression of OsMIF1 enhances drought tolerance by regulating developmental processes and interacting with ZHD TFs and OsDIP1, thereby improving resilience of plants exposed to stress (Thiaw, 2024 ). Many studies support a role for MIFs in reproductive development. Despite their potential importance, the biological roles of rice MIFs remain poorly understood and warrant further study. Here, CRISPR-Cas9 gene editing was used to generate and then characterize OsMIF1 and OsMIF2 knockout (KO) plants and to elucidate the biological roles of these rice TFs. These studies revealed that OsMIF1 and OsMIF2 KO plants produce enlarged seeds and aberrant panicles. Furthermore, RNA-seq data showed that the phenotypic changes in the engineered KO plants correlate with altered patterns of gene and gene pathway expression. Interestingly, library-scale yeast two-hybrid (Y2H) screening identified 10 candidate protein-interacting partners of OsMIF1; we postulate that these proteins could potentially mediate some of the downstream effects of OsMIF1. Methods Sequence Alignment and Prediction of Protein Structure Fifteen Zinc Finger-Homeodomain (ZF-HD) family proteins were selected as described previously (Hu et al., 2008 ). The corresponding DNA and protein sequences were obtained from the MSU Rice Genome Annotation Project (MSU RGAP; https://rice.uga.edu/ ). Protein sequences were aligned using Clustal Omega ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ), and visualized with ESPript ( https://espript.ibcp.fr/ESPript/ESPript/ ). A phylogenetic tree was constructed from the aligned sequences using the Maximum Likelihood method with 1,000 bootstrap replications in MEGA12. Motif analysis was performed using InterPro ( https://www.ebi.ac.uk/interpro/ ) to predict conserved domains and functional motifs. In addition, the MEME suite ( https://meme-suite.org/meme/tools/meme ) was used to identify conserved motifs among the ZF-HD proteins. Sequence logos generated by MEME were used to illustrate the positional conservation and amino acid composition of the predicted motifs. To further investigate structural features, the three-dimensional structures of ZF-HD proteins were predicted using AlphaFold ( https://alphafold.ebi.ac.uk/ ). The predicted protein models were subsequently visualized and analyzed using PyMOL to examine structural features such as conserved domains and secondary structures. Plant Materials and Growth Conditions Seeds of Oryza sativa L. ssp. japonica cv. Ilmi were obtained from the Korea Seed & Variety Service. The rice plants used in this study were cultivated in paddy fields under standard agronomic conditions. RNA Isolation and Quantitative Real-Time PCR (qRT-PCR) Total RNA was extracted from each rice tissue using a previously published method (Li & Trick, 2005 ). cDNA was synthesized from 1 µg total RNA using the QuantiTect Reverse Transcription Kit (Cat. #205311, Qiagen, Hilden, Germany), following the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was performed using the QuantiTect SYBR Green PCR Kit (Cat. #204343, Qiagen, Hilden, Germany) on a Qiagen Rotor-Gene Q real-time PCR cycler. The thermal cycling conditions were as follows: 95°C for 15 min; 40 cycles of 94°C for 15 s, 60°C for 30 s, and 72°C for 30 s; followed by a melting curve analysis from 72 to 95°C. OsUBI5 was used as an internal control gene to normalize gene expression levels (Jain et al., 2006 ). Relative expression levels were calculated using the 2 –ΔΔCt method (Livak & Schmittgen, 2001 ). All primers used for qRT-PCR are listed in Supplementary Table 1. GUS Staining Assay Genomic DNA was extracted from 21-day-old seedlings as described by (Dellaporta et al., 1983 ). A 1.5 kb upstream promoter region of the OsMIF1 gene was amplified from 5 ng of genomic DNA and cloned into the pCAMBIA1201 vector. The construct was introduced into the Agrobacterium tumefaciens strain EHA101 and used to transform embryogenic rice calli derived from mature seeds (Kim et al., 2012 ). Transcription from the cloned OsMIF1 promoter was then quantified in various tissues harvested from transgenic lines using a previously described GUS reporter staining technique (Dedow et al., 2022 ). More specifically, tissue samples were pretreated in ice-cold 90% acetone for 5–15 min, incubated in staining buffer [50 mM sodium phosphate buffer (pH 7.2), 2 mM each of K 4 [Fe(CN) 6 ] and K 3 [Fe(CN) 6 ], 2% Triton X-100, 2 mM X-Gluc] under vacuum for 5 min, and then incubated overnight at 37°C in the dark. Samples were rinsed in 70% ethanol before storage at 4 ℃ or immediate quantification of GUS activity. Hormone Treatment To investigate hormone responsiveness, leaf discs were prepared from leaves of 28-day-old rice plants. The leaves were excised and cut into uniform leaf discs, which were then floated on a hormone-containing solution in 6-well plates. The leaf discs were immersed in the treatment solution and incubated at room temperature. Samples were immediately frozen in liquid nitrogen and stored at -70°C until further analysis. Prediction of Cis-acting Regulatory Elements The 1.5 kb upstream promoter regions of OsMIF1 and OsMIF2 were retrieved from the Rice Genome Annotation Project ( http://rice.plantbiology.msu.edu/ ). Cis-regulatory elements within these promoter sequences were predicted using the PlantCARE database ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) (Lescot et al., 2002 ). For visualization, the PlantCARE results were processed and visualized in R (version 4.5.1) using the tidyverse and ggplot2 packages. Generation of OsMIF1 and OsMIF2 Knockout Rice Knockout (KO) lines of OsMIF1 and OsMIF2 were generated in Oryza sativa ssp. japonica cv. Ilmi through the CRISPR-Cas9 system. To target both OsMIF1 and OsMIF2 , sgRNA (5’-GCGGCCAAGCCGTACGCGAACGG-3’) was designed using CRISPR-P 2.0, an online tool ( http://crispr.hzau.edu.cn/CRISPR2/ ). The sgRNA was first cloned into the pRGE31 entry vector and subsequently transferred into the pCAMBIA-Cas9 binary vector containing the Cas9 expression cassette and the rice U3 promoter for Agrobacterium-mediated transformation (Chandra et al., 2023 ; Pham et al., 2024 ). The pCAMBIA-Cas9-sgRNA construct was introduced into rice via Agrobacterium tumefaciens strain EHA101. Agrobacterium-mediated transformation was performed according to a previously described method (Kim et al., 2012 ). Screening and Characterization of KO Mutant Rice Plants To verify mutations at the target site in T 0 and T 1 generations, genomic DNA was extracted from 30-day-old rice plants following a previously described protocol (Dellaporta et al., 1983 ). The target region was amplified by PCR using specific primers (Supplementary Table 1). The resulting amplicons were subjected to deep sequencing using the MiniSeq platform (KAIST, Daejeon, Republic of Korea). The sequencing data were analyzed using RGEN Tools ( http://www.rgenome.net ). For the T 2 generation, PCR was performed using specific primers (Supplementary Table 1), and the amplified products were sent to Cosmogenetech (Seoul, Republic of Korea) for sequencing. Scanning Electron Microscopy (SEM) To examine the cell size of mature rice spikelets, scanning electron microscopy (SEM) was performed using a JSM-IT300 (JEOL Ltd., Tokyo, Japan). Samples were mounted on aluminum stubs, sputter-coated with a thin layer of platinum, and observed under SEM. The length and width of epidermal cells on the outer surface of spikelet hulls were measured using ImageJ software ( https://imagej.net/ ). Transcriptome Analysis Total RNA extracted from immature seeds (14 days after flowering) and developing panicles (10 cm) of non-transgenic (NT) and OsMIF1 and OsMIF2 KO mutant lines was sent to DNALINK Biotechnology Company (Seoul, Republic of Korea) for RNA sequencing. Libraries were prepared using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina Inc., San Diego, CA, USA) and sequenced on the Illumina NovaSeq 6000 platform with paired-end reads (2×101 bp). Each sample was assigned a unique barcode index, and sequencing was performed according to the manufacturer’s instructions. Transcript quantification was performed using Kallisto, which performs pseudo-alignment of reads to the reference transcriptome and estimates transcript abundance (Bray et al., 2016 ). The resulting expression matrix was normalized using TMM (trimmed mean of M-values) normalization. Differentially expressed genes (DEGs) between KO and control samples were identified using both the edgeR and DESeq2 packages in R. Statistical significance was determined by setting the threshold at p < 0.05 (Love et al., 2014 ; McCarthy et al., 2012 ). Venn diagrams for DEG comparisons among the mutants were constructed using the online tool Venny 2.1.0 ( https://bioinfogp.cnb.csic.es/tools/venny/ ). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the functional annotation tool DAVID (Database for Annotation, Visualization, and Integrated Discovery, https://davidbioinformatics.nih.gov ). GO and KEGG terms with p-value ≤ 0.1 were considered significant. For visualization, the data were imported into R (v4.5.1), and bar plots were generated using the ggplot2, dplyr, and forcats packages. Heatmaps for visualization of gene expression patterns were generated using Heatmapper ( http://www.heatmapper.ca/expression/ ), an online tool for expression data clustering and heatmap construction. Protein–protein interaction networks were predicted using the STRING database ( https://string-db.org/ ). The corresponding protein identifiers were retrieved and queried against the Oryza sativa Japonica Group database in STRING. Interactions were filtered using a minimum required interaction score of 0.4, and the resulting network was visualized in Cytoscape for clustering and pathway annotation. KEGG pathway enrichment of the mapped proteins was assessed using STRING’s built-in functional enrichment tool. Y2H Library-scale Screening Assay Yeast Two-Hybrid screening of OsMIF1 was conducted using the Saccharomyces cerevisiae strain AH109, which contains two reporter genes, HIS3 and ADE2, under the control of distinct GAL promoters. The full-length OsMIF1 was amplified using gene-specific primers containing Eco RI and Bam HI sites (Supplementary Table 1), and cloned into the corresponding sites of the pGBKT7 vector for expression as a myc-tagged GAL4 DNA-binding domain fusion. The construct was confirmed by DNA sequencing and subsequently co-transformed into the yeast strain with a rice cDNA activation domain (AD) library. Transformants were selected on synthetic dropout (SD) medium lacking leucine, tryptophan, histidine, and adenine (SD-LWHA), which permits the growth of yeast cells expressing interacting bait and prey proteins. To confirm protein–protein interactions, DNA fragments encoding the prey proteins from 60 initial candidate clones were isolated via PCR or by transformation into E. coli . These prey clones were then reintroduced into yeast AH109 along with either the OsMIF1 bait plasmid or an empty bait vector as a negative control. Interactions were assessed based on yeast growth on SD-LWHA selection medium. Candidates were further validated through DNA sequencing and restriction enzyme digestion. Results Structural and Evolutionary Features of rice ZF-HD proteins OsMIF1 and OsMIF2 To elucidate the structural characteristics of OsMIF1 and OsMIF2 and determine the percent similarity/divergence among rice ZF-HD proteins, the amino acid sequences of 11 rice ZF-HD and 4 rice MIF proteins were aligned (Supplementary Table 2) (Hu et al., 2008 ) and subjected to phylogenetic tree, domain prediction, and three-dimensional structure prediction analyses (Fig. 1 ). In the phylogenetic tree for this subset of rice ZF-HDs, OsMIF1 and OsMIF2 clustered together with a bootstrap value of 100%, indicating a highly reliable evolutionary relationship (Fig. 1 A). The aligned full-length amino acid sequences of OsMIF1 and OsMIF2 demonstrate 98.1% identity and only two amino acid differences (Fig. 1 B). OsMIF3 and OsMIF4 cluster with OsMIF1 and OsMIF2 (bootstrap value 71%) to form a distinct MIF subfamily within the larger rice ZF-HD protein family. OsZHD1–4 are more closely related to OsMIF proteins than to other rice ZF-HD proteins (Fig. 1 A). Similar results were obtained when the alignment was limited to the ZF domain in this subset of rice ZF-HD proteins (Supplementary Fig. 1). The domain structure of rice ZF-HDs was predicted using AlphaFold ( https://alphafold.ebi.ac.uk/ ). The results reveal that rice ZF-HD proteins (ZHDs) possess both a zinc finger domain and a homeobox domain, whereas rice MIFs, including OsMIF1 and OsMIF2, possess a zinc finger domain but do not possess a homeobox domain (Fig. 1 B, Supplementary Table 3). Zinc finger domains contain a conserved C 5 H 3 motif and homeobox domains are comprised of a characteristic three-helix fold (Fig. 1 B). AlphaFold-based 3D structural prediction confirmed that both of these features are present in the rice ZF-HDs (Fig. 1 C, Supplementary Fig. 2), and that OsMIF1, belonging to the MIF subclass of ZF-HDs, is predicted to lack a homeodomain but to contain a zinc finger domain with the characteristic conserved cysteine (C38, C54, C71, C74, and C76) and histidine (H42, H77 and H81) residues configured for zinc coordination. In contrast, OsZHD1 is predicted to harbor a characteristic zinc finger domain (with conserved cysteines C60, C76, C93, C96, and C98 and histidine residues H64, H99 and H103) and a homeobox with a typical three α-helix domain (Fig. 1 C). These results indicate that OsMIF1 and OsMIF2 represent a distinct conserved MIF subgroup in the rice ZF-HD family. The bioinformatic tool OrthoDB ( https://www.orthodb.org/ ) identified 65 plant MIF proteins orthologous to OsMIF1 and OsMIF2. Among them, three proteins from wheat ( Triticum aestivum L.) showed the highest similarity to and formed a distinct subgroup from OsMIF1 and OsMIF2. In addition, eight proteins from maize ( Zea mays L.) exhibited considerable similarity to OsMIF1 and OsMIF2 (Supplementary Fig. 3). Tissue- and Stage-specific Expression of OsMIF1 and OsMIF2 During Rice Developmen To investigate the tissue- and stage specificity of OsMIF1 and OsMIF2 expression, qRT-PCR and GUS staining assays were performed in diverse tissues of plants at different stages of development (Fig. 2 ). qRT-PCR data indicate that OsMIF1 and OsMIF2 are expressed at a low level in 14 DAG seedlings but are expressed at a high level in developing panicles before flowering and at an early developmental stage (1–5 cm); their expression then decreased gradually as panicle development progressed. At 0 DAF, their expression was high in stems and roots but low in flag leaves, while OsMIF1 peaked at 3 DAF and OsMIF2 peaked at 1 DAF during seed development and both declined from their peak expression level until 7 DAF. Expression of both genes increased from 7 DAF to 14 DAF and then reached a plateau and remained high until 21 DAF (Fig. 2 A). A 1,500 bp fragment of the upstream promoter region of OsMIF1 was cloned into a β-glucuronidase (GUS) reporter gene vector, and GUS expression from the resulting plasmid was quantified in various tissues and at different stages of rice plant development. These data were used to confirm the tissue- and developmental stage-specificity of OsMIF1 expression (Fig. 2 B–I, Supplementary Fig. 4–5). The results suggest that OsMIF1 is not expressed in flag leaves at 0 DAF (Fig. 2 B), but is expressed in the inner vascular tissues of the stem and root at 0 DAF (Fig. 2 C–D). Furthermore, the pattern of GUS reporter gene expression suggests that OsMIF1 is expressed in panicles during early (0–1 cm) and intermediate stages (5–10 cm), gradually diminishing during the late stages of panicle development (Fig. 2 E–G), while OsMIF1 expression in spikelets is weak early in development, localized to the distal end during intermediate development, broadly distributed during late development and gradually decreases towards the onset of maturity (Fig. 2 H). During seed development, reporter gene expression is concentrated initially at the distal end, throughout the seed during the middle stages of development, peaking at 14 DAF and remaining strong until 21 DAF (Supplementary Fig. 5). While the results suggest that OsMIF1 is not expressed in cells on the surface of mature seeds, the data demonstrate that OsMIF1 is strongly expressed in endospermal cells of mature rice seeds (Fig. 2 I). Transcripts of OsMIF1 and OsMIF2 were also quantified in hormone-treated leaf discs using qRT-PCR (Supplementary Fig. 6). The results showed that OsMIF1 is induced by ABA and NAA but suppressed by MeJA and GA 3 , while OsMIF2 is slightly induced by ABA and NAA and repressed by MeJA and ACC (Supplementary Fig. 6). Analysis of the 1.5 kb promoters of OsMIF1 and OsMIF2 using PlantCARE revealed the presence of multiple cis-acting regulatory motifs ( i.e. , hormone-regulating motifs ABA, MeJA and GA; motifs that mobilize responses to stressors including drought, hypoxia, wounding) (Supplementary Fig. 7). Overall, these results indicate that OsMIF1 and OsMIF2 are expressed across diverse tissues during reproductive stages, implying their putative roles in panicle development and seed maturation. The fact that transcription of OsMIF1 and OsMIF2 is induced/repressed in response to plant hormones suggests that these MIFs mediate hormone signaling during reproductive development or other reproductive processes as well as the response to exogenous stress. Phenotypic Characterization of OsMIF1, OsMIF2 and OsMIF1/OsMIF2 Mutant Lines CRISPR-Cas9 technology was used to engineer OsMIF1 and OsMIF2 knockout (KO) plant lines, which were then used to elucidate the functional roles of these rice MIFs (Fig. 3 ). Three mutant lines were generated and selected for further study: osmif1 harbors a 1 bp insertion mutation, osmif2 also harbors a 1 bp insertion mutation, while the third line, osmif1/osmif2 , is a double mutant harboring a 1 bp insertion in each CRISPR-Cas9- edited MIF gene (Fig. 3 B, Supplementary Fig. 8, Supplementary Table 4–6). qRT-PCR data confirmed efficient knockout of OsMIF1 and/or OsMIF2 in a pattern consistent with the genotype of all three mutant lines (Fig. 3 C). The morphological traits of the panicles were compared in osmif1 , osmif2, osmif1/osmif2 and wild-type control (NT) plants; the goal was to test the prediction that the mutant plants would exhibit defects during reproductive stages (Fig. 4 A–E, Supplementary Fig. 9). Plant height increased in all mutant lines relative to NT, with osmif1 , osmif2 , and osmif1/osmif2 plants being on average 6.7, 5.1, and 3.9% taller than NT, respectively (Fig. 4 A). The number of panicles per plant decreased in osmif1 relative to NT by 17.3%, while the number of panicles per plant was similar in NT, osmif2 and osmif1/osmif2 plants (Fig. 4 B). Panicles were 5.9% and 6.5% shorter in osmif1 and osmif1/osmif2 plants but only slightly shorter in osmif2 than in NT plants (Fig. 4 C). In addition, there were 6.1% fewer primary branches in osmif1/osmif2 plants than in NT plants (Fig. 4 D), and the number of secondary branches was 19.5 and 24.1% lower in osmif1 and osmif1/osmif2 plants than in NT plants, respectively. In contrast, primary branching was comparable in osmif1 , osmif2 and NT plants, and secondary branching was similar in osmif2 and NT controls (Fig. 4 D and E). These results reveal defects in panicle development, including reduced panicle elongation and branching, in osmif1 mutant plants. Our results also provide evidence that OsMIF1/2 influence seed development and morphology (Fig. 4 F–J). For example, 100-grain weight of osmif1 and osmif1/osmif2 seeds was 10.3% and 17.9% higher than NT seeds, respectively, while 100-grain weight of osmif2 seeds was 0.6% lower than NT seeds. Consistent with these data, osmif1 seeds were 3.2% longer, 4.5% wider, and 5.3% thicker than NT seeds, while osmif2 seeds showed relatively minor (0.8 to 1.6) differences from NT and osmif1/osmif2 double mutant seeds diverged even more strongly from the control seed, being 5.9% longer, 5.7% wider, and 7.2% thicker than NT seed (Fig. 4 F–H). When the outer surface of the spikelets of NT and osmif1/osmif2 seeds were examined by scanning electron microscopy (SEM), the results show that the epidermal cells of osmif1/osmif2 double mutant seeds were 12.4% wider and 11.0% longer than NT epidermal cells (Fig. 4 I–J). Therefore, osmif1 , osmif2 and especially osmif1/osmif2 plant lines exhibit increased seed size and weight, which is consistent with the observed increased size of plant epidermal cells. Furthermore, the above results implicate OsMIF1 and OsMIF2 as critical regulators of reproductive development, structures and processes, for example, by modulating panicle architecture and seed morphology. Knockout of OsMIF1 alone caused phenotypic changes, whereas knockout of OsMIF2 alone does not, while double knockout plants exhibit more pronounced effects than osmif1 mutant plants. The results suggest that OsMIF1 plays a primary role while OsMIF2 plays secondary supportive roles in reproductive development in rice. Global Gene Expression Profiling of osmif1 and osmif2 Panicles and Immature Seeds Global transcriptomic analyses were performed to correlate OsMIF1/OsMIF2 genotype with phenotype and associated changes in expression of relevant biological pathways. To this end, RNA-seq data were collected using samples from 10 cm developing panicles and 14 DAF immature seeds from NT, osmif1 , osmif2 , and osmif1/osmif2 plant lines (Fig. 5 , Supplementary Fig. 10). In 10 cm developing panicles, RNA-seq yielded 39–51 million reads per sample with Q30 values above 91%. In 14 DAF seeds, 25–34 million reads per sample were obtained, and all libraries showed Q30 values above 87% (Supplementary Table 7). Correlation coefficients varied from 0.975 to 0.988, indicating that the RNA-seq data were derived from the same developmental stage and tissue (Fig. 5 A, Supplementary Fig. 10A). The RNA-seq data were subjected to differential expression analysis using edgeR and DESeq2 software packages (significance threshold was p < 0.05). In samples from 10 cm developing panicles, 1,124 genes were upregulated, including 55 genes expressed in all mutant lines, while 1,403 genes were downregulated, of which 325 transcripts were consistently detected independent of the genotype of the sample (Fig. 5 B). GO analysis was performed using the DAVID tool ( https://davidbioinformatics.nih.gov ), which links each up- or down-regulated gene with annotations that assign an established or putative biological function, e.g. Biological Process (BP), Cellular Component (CC), Molecular Function (MF), KEGG pathway (Fig. 5 C). GO enrichment analysis of the 325 commonly downregulated genes revealed significant enrichment in a subset of BP terms including post-embryonic plant morphogenesis, mRNA transcription, response to light stimulus, hydrogen peroxide catabolic process, and response to oxidative stress. Enriched CC terms included nucleus, extracellular region, plant-type cell wall, and lipid storage body, while enriched MF terms included DNA-binding transcription factor activity, oxidoreductase activity, and peroxidase activity; enzyme inhibitors were also significantly overrepresented. KEGG analysis revealed enrichment of pathways involved in biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, (general) metabolism, starch and sucrose metabolism, and cyanoamino acid metabolism (Fig. 5 C). To validate the RNA-seq data, qRT-PCR was performed for a subset of downregulated DEGs, including those associated with carotenoid biosynthesis, cyanoamino acid metabolism, MAPK signaling, and phenylpropanoid biosynthesis (Fig. 5 D–E, Supplementary Fig. 11–12, Supplementary Table 8–9). Expression values for DEGs were normalized to an appropriate control using the 2 −ΔΔCT method and results are presented as log₂ values. qRT-PCR data confirmed that downregulated DEGs outnumber upregulated DEGs in this dataset (Fig. 5 E). The results demonstrate that the phenotypes associated with knockout mutations in OsMIF1 and OsMIF2 are characterized by suppression of genes involved in morphogenesis, stress response, transcriptional and enzymatic activities, and hormone and metabolic pathways; this supports the conclusion that OsMIF genes play essential roles in rice development and environmental adaptation. GO enrichment analysis was also performed on RNA-seq data from immature rice seeds (14 DAF). These data revealed that downregulated DEGs are enriched in the following GO terms: photosynthesis, carbon fixation, cell wall modification, photosystem components, thylakoid membrane proteins, and chloroplast-associated factors (Supplementary Fig. 10C). qRT-PCR analysis of photosynthesis-related genes confirmed the prediction that they would be down-regulated in osmif1 (KO) rice relative to NT controls, thus validating the RNA-seq findings described above (Supplementary Fig. 10D–E, Supplementary Table 10). Functional Roles of OsMIF1 and OsMIF2 during Seed Development When the seeds from osmif1 and osmif2 KO mutant rice plants were compared to NT control seeds, we observed that the mutant seeds are larger than control seeds, and similarly, SEM data showed that KO epidermal cells on the surface of spikelets are enlarged relative to control cells (Fig. 4 F–J). These observations were confirmed and validated using qRT-PCR to quantify expression of genes that modulate the size of mature seeds and the size of cells in developing panicles of KO mutant plants (Supplementary Table 11) (Lee & Kende, 2002 ; Li et al., 2020 ; Shim et al., 2022 ; Shin et al., 2005 ; Si et al., 2016 ; S. Wang et al., 2015 ; Yu et al., 2018 ; Zhan et al., 2022 ; Zhou et al., 2015 ; Zuo et al., 2021 ). Our RNA-seq data had revealed higher expression of these genes/pathways in the KO mutants than in NT control plants (Fig. 6 A); furthermore, qRT-PCR data revealed that all of the corresponding genes are upregulated in the KO mutant lines (Fig. 6 B). These findings implicate OsMIF1 and OsMIF2, and the level at which these genes are expressed, as determinants of seed and spikelet cell size. Previous studies reported that OsMIF1 and OsMIF2 are co-expressed with major seed storage proteins (SSPs) (So et al., 2025 ). Furthermore, data presented above (Fig. 2 ) demonstrate that both genes are highly expressed in immature seeds with peak expression at 14 DAF. To further explore potential crosstalk between SSPs (and/or co-expression of SSPs) and OsMIF1/OsMIF2, expression of these genes was quantified and compared in MIF1/2 KO and NT seeds (Supplementary Fig. 13). For this analysis, SSP genes were grouped according to their previously reported classification (Supplementary Table 12) (Chandra et al., 2023 ; Pham et al., 2024 ). First, expression of SSPs was quantified using RNA-seq data from 14-DAF immature seeds. Although no consistent trend was observed in these data, transcripts from the Pro13b-II subgroup of prolamin genes were generally downregulated (Supplementary Fig. 13A). This result was confirmed by performing qRT-PCR on Pro13b-II genes using cDNA and primers reported in previous studies (Supplementary Fig. 13B). Identification of Putative OsMIF1 Protein-Interacting Partners by Yeast Two-Hybrid Screening In the initial discovery of the ZF-HD protein family, Y2H assays revealed that ZF-HD proteins form both homo- and heterodimers (Windhövel et al., 2001 ). Based on these findings, we conducted a Y2H screen for protein-interacting partners of OsMIF1, in which OsMIF1 was the “bait” and the “prey” were protein products expressed from a whole rice genome cDNA library (Supplementary Fig. 14). The screen identified 18 candidate protein-interacting partners of OsMIF1, of which 10 were in-frame (Supplementary Table 13–14). The 10 in-frame proteins included OsSIZ1 (NM_001420071), OsCIPK14 (NM_001404631), akin-beta (XM_015783664), OsMIF1 (NM_001423141), OsBIG (NM_001422705), Snf7 family protein (XM_015782163), OsMORF8b (XM_015756999), OsDjA6 (NM_001402407), OsNBR1 (NM_001416698), and OsELF3.2 (XM_015795402). These putative OsMIF1-interacting proteins are involved in diverse processes, including growth and development, hormone signaling, stress responses, and RNA metabolism; this result suggests that OsMIF1 acts as a regulatory hub to integrate signals from developmental and stress-related pathways (Table 1 , Supplementary Table 15). One of the interacting partner proteins was OsMIF1 itself, which is consistent with the fact that OsMIF1 forms homodimers; this result also predicts the potential for interactions between OsMIF1 and other ZF-HD proteins (Table 1 ). GO enrichment analysis revealed that three candidate protein-interacting partners of OsMIF1, i.e. , OsSIZ1, OsBIG, and OsDjA6, are predicted to contain a zinc finger motif. Thus, OsMIF1 may interact not only with ZF-HD proteins but also with other ZF proteins (Supplementary Fig. 15). Table 1 Candidate OsMIF1-interacting proteins identified by Y2H library screening No Gene symbol NCBI accession number Function Reference 1 OsSIZ1 NM_001420071 Regulates growth and development, and mediates responses to phosphate/nitrogen status and environmental stresses. (Mishra et al., 2018 ; Mishra et al., 2017 ; Park et al., 2010 ; Thangasamy et al., 2011 ; Wang et al., 2011 ; H. Wang et al., 2015 ) 2 OsCIPK14 NM_001404631 Mediates Ca²⁺-dependent MAMP-induced defense signaling. (Kurusu et al., 2010 ) 3 akin-beta XM_015783664 Is suppressed by OsTZF1 and is induced under cold stress in tolerant rice. (Ding et al., 2025 ; Jan et al., 2013 ) 4 OsMIF1 NM_001423141 Enhances drought tolerance by regulating rice growth and development. (Thiaw, 2024 ) 5 OsBIG NM_001422705 Is essential for rice growth and development; loss of function causes seedling lethality. (Cheng et al., 2019 ) 6 Snf7 family protein XM_015782163 Is upregulated in leaf and root under direct-sown drought stress. (Kumar et al., 2022 ) 7 OsMORF8b XM_015756999 Acts as a multiple organellar RNA-editing factor interacting with other OsMORFs, and is downregulated by cold and salt stress. (Zhang et al., 2019 ) 8 OsDjA6 NM_001402407 Negatively regulates rice innate immunity, likely via the ubiquitin–proteasome degradation pathway. (Sarkar et al., 2013 ; Zhong et al., 2018 ) 9 OsNBR1 NM_001416698 Mediates pest resistance and enhances cold tolerance via autophagy and reduced ubiquitination. (Guo et al., 2023 ; Zhang & Chen, 2020 ) 10 OsELF3.2 XM_015795402 Controls heading date, circadian rhythm, and stress tolerance in rice. (Fu et al., 2009 ; Wang et al., 2021 ; Zhao et al., 2012 ) Defects in Root Development and the Response to Salt Stress in OsMIF1- and OsMIF2-KO Mutants The ten-candidate protein-interacting partners of OsMIF1 include proteins associated with the response to drought, salinity and other environmental/water stressors (Table 1 ). In addition, a previous study reported that overexpression of OsMIF1 affected root growth (Thiaw, 2024 ). These observations suggest that OsMIF1 could influence plant stage-specific changes in root morphology after germination (Fig. 7 ). Consistent with this hypothesis, phenotypic analysis revealed enhanced root elongation during the early vegetative stage in osmif1 (KO) plants. Relative to NT controls, osmif1 plant roots were 14–16% longer at 7 DAG, 14–20% longer at 14 DAG, and 21–44% longer at 21 DAG. Furthermore, this phenotypic trait was exacerbated in roots of osmif1/osmif2 plants (Fig. 7 D). In contrast, root number was slightly (6–11%) higher at 7 DAG, but 16–46% and 18–38% lower than NT controls in 14 and 21 DAG plants. This phenotypic trait was also exacerbated in osmif1/osmif2 double mutant plants (Fig. 7 E). The effects of salt stress on germination rate were also compared in osmif1 , osmif2 , osmif1/osmif2 and NT control plants. The results showed that NT seeds consistently germinate more rapidly than OsMIF mutant seeds. In the absence of salt stress, NT seeds reached 93.3% germination by 96 h, whereas osmif1 , osmif2 , and the osmif1/osmif2 double mutants achieved 70.0%, 55.2%, and 82.1% germination, respectively. In the presence of 75 mM NaCl, ( e.g. , conditions of salt stress), germination was 55.2% in NT at 96 h, but only 22.2–26.1% for osmif1 mutant seeds. Higher salt stress ( i.e. , 150 mM NaCl) or the osmif1/osmif2 double mutant genotype exacerbated this phenotype. thus, germination rate was 25.0% for NT seeds at 96 h but 0–5.6% in osmif 1/2 mutants (Supplementary Fig. 16), with the lowest germination rate in osmif1/osmif2 double mutant seeds under high salt stress. In summary, these results suggest that OsMIF1 and OsMIF2 knockout plants exhibit increased sensitivity to water/salt stress; this trait manifests as root elongation, likely reflecting a compensatory response to the adverse environmental conditions. Discussion osmif1/2 KO Genotype Associated with Defects in Reproductive Development This study exploits transcription profiling and CRISPR-Cas9 gene editing technology to elucidate the biological roles of mini zinc finger TFs OsMIF1 and OsMIF2. Previous studies showed that plant-specific MIFs play critical roles regulating plant growth and development, hormone signaling, and stress response pathways (Hu et al., 2008 ; Shen et al., 2025 ; Tran et al., 2007 ). More specifically, MIF proteins are critical during reproductive development, regulating floral meristem termination, carpel number, petal elongation, and anther and pollen development (Bollier et al., 2018 ; Han et al., 2017 ; Thiaw & Gantet, 2024 ); the mechanisms by which OsMIF1/2 regulate these processes are diverse. OsMIF3 (PASPRO1) and OsMIF4 (PASPRO2) are specifically expressed in pollen and the anther wall, where they play important roles during the reproductive stage by regulating the surface morphology of anthers and pollen to ensure normal reproductive development (Jang et al., 2023 ). OsMIF3 and OsMIF4 are not expressed in panicles and seeds, whereas OsMIF1 and OsMIF2 are expressed in panicles, seeds, stems and roots (Fig. 2 ) (Jang et al., 2023 ). Loss-of-function mutants of OsMIF1 and OsMIF2 generated by CRISPR-Cas9 showed reduced panicle branching and increased grain size due to increased cell size, and higher abundance of SSPs reflecting increased transcription of SSP genes (Fig. 3 – 4 , Supplementary Fig. 13). While OsMIF1/2 and OsMIF3/4 play distinct functional roles, each of the two paired MIFs is redundant to each other. This is consistent with the fact that the transcriptional profiles of OsMIF1/2 and OsMIF3/4 differ. Functional Connections between MIF and Zinc Finger Proteins Rice ZHD proteins OsZHD1 and OsZHD2, which belong to another branch of the ZF-HD family, also share roles in root development and seed size (Guo et al., 2025 ; Yoon et al., 2020 ). In addition, OsZHD1, OsZHD2, OsZHD4, and OsZHD8 bind to the promoter region of OsDREB1B (Figueiredo et al., 2012 ). The functional redundancy of ZF-HD proteins likely reflects their origin from gene duplication and the evolutionary maintenance of paralogs through active compensation mechanisms (Iohannes & Jackson, 2023 ; Jang et al., 2023 ). While MIF and ZHD proteins exhibit functional redundancy within their respective subgroups, MIF TFs appear to work in opposition to and effectively suppress ZHD protein activity. Results presented here demonstrate that the phenotype of osmif1/2 KO mutants includes larger seeds and longer roots; in contrast, KO mutants of OsZHD1 and OsZHD2 have the opposite effects i.e. , smaller seeds and shorter roots (Guo et al., 2025 ; Yoon et al., 2020 ). KO mutants of OsMIF3 and OsMIF4 also exhibit reduced expression of OsDREB1A and OsDREB1B , previously reported to be transcriptionally repressed by ZHD TFs. Furthermore, OsMIF3 and OsMIF4 interfere with binding of OsZHD1 and OsZHD9 to the promoters of OsDREB1A and OsDREB1B (Jang et al., 2023 ). In general, the zinc finger motif confers the ability to bind directly to DNA and to interact with other zinc finger proteins, thereby regulating transcription of target genes (Mackay & Crossley, 1998 ). In addition, ZF-HD proteins tend to homodimerize or form heterodimers (Windhövel et al., 2001 ). Consistent with this, OsMIF1 self-associates to form homodimers (Table 1 , Supplementary Table 13–14). Arabidopsis MIFs interact with ZHDs ( e.g. , ZHD5), inhibiting their nuclear localization and DNA-binding activity (Hong et al., 2011 ), and four rice ZF-HD TFs form homo- or heterodimers (Figueiredo et al., 2012 ). We also note that a previously reported Y2H study documents potential interaction between OsMIF1 and OsZHD proteins (Thiaw, 2024 ). AtMIF2 and tomato SlIMA directly interact with the C 2 H 2 zinc-finger protein KNU to form a transcriptional repressor complex with corepressors TOPLESS and HDA19. These protein–protein interactions repress expression of WUS/SlWUS, thereby regulating floral meristem termination and carpel number (Bollier et al., 2018 ). In the present study, the Y2H screen for OsMIF1 identified three ZF proteins as putative protein-interacting partners (Table 1 , Supplementary Table 15). Collectively, these findings suggest that MIF proteins are members of large networks of ZF proteins ( e.g. these networks include ZFs that lack the HD domain). Additional studies on the mechanisms and interactions between MIF genes and ZF/ZHD proteins are warranted; these studies are expected to discover novel protein-protein interactions, elucidate their downstream effects, and provide insight into the structure and activities of plant regulatory networks. ZF-HD Proteins as Mediators of Hormonal Cross-Talk and Stress Adaptation in Rice ZF-HD TFs integrate environmental signals, such that ZFHD1 enhances expression of drought-responsive genes under water deficit and in Arabidopsis ZFHD10 promotes blue light–induced hypocotyl elongation by recruiting TZP (Barth et al., 2009 ; Perrella et al., 2018 ). Overexpression of MIF1 causes reduced root growth and abnormal root hair formation. Furthermore, it leads to altered responses to phytohormones such as gibberellin (GA), abscisic acid (ABA), and auxin (Hu & Ma, 2006 ). In amaranth roots, MIF1 is transcriptionally up-regulated by drought stress, leading to reduced root growth and altered expression of growth-related genes (Huerta-Ocampo et al., 2011 ). Four rice ZF-HD TFs bind to the OsDREB1B promoter and repress its expression under multiple stress conditions (Figueiredo et al., 2012 ). OsZHD2 enhances root meristem activity through ethylene-induced auxin signaling, thereby improving nutrient uptake and stress resilience, linking ZF-HD activity to auxin- and ethylene-mediated regulation of plant architecture (Yoon et al., 2020 ). Overexpression of OsMIF1 also alters root growth, and OsMIF1 overexpressing transgenic plants display dark-green, curled leaves and exhibit enhanced tolerance to water deficit (Thiaw, 2024 ). Here, we show that KO mutants of OsMIF1 and OsMIF2 exhibit increased root length but reduced root number and are more sensitive to salt stress during germination (Fig. 7 , Supplementary Fig. 16); moreover, the expression of both genes was induced by ABA and NAA (Supplementary Fig. 6). RNA-seq analysis of 10-cm developing panicles in osmif1 and osmif2 KO lines revealed downregulation of ethylene signaling and light, oxidative, and abiotic stress response pathways (Fig. 5 ). Taken together, these observations indicate that ZF-HD family proteins play important roles in stress response pathways and root development, suggesting a strong association with hormonal pathways involving auxin, ethylene, and ABA. Therefore, we postulate that ZF-HD proteins modulate and/or mediate hormonal cross-talk and hormone-regulated developmental processes. Water stress inhibits photosynthesis by stomatal closure, metabolic suppression, and increased oxidative stress ( i.e. , accumulation of reactive oxygen species). However, these effects can be mitigated in plants through adaptive mechanisms such as enhanced photosynthetic efficiency and induction of drought tolerance pathways (Feng et al., 2023 ; Iqbal et al., 2022 ; Lv et al., 2024 ; Martínez-Goñi et al., 2024 ; Qiao et al., 2024 ; Zhang et al., 2022 ). Thus, the increased sensitivity of OsMIF1 and OsMIF2 KO lines to salt stress and their enhanced root elongation suggests a compensatory drought-avoidance strategy in which reduced photosynthetic capacity and stress tolerance are offset by deep water foraging (Fig. 7 , Supplementary Fig. 16). Importantly, RNA-seq analysis revealed that photosynthesis-related and carotenoid biosynthesis genes are downregulated in osmif1 and osmif2 KO plants (Supplementary Fig. 10). These results highlight the need for further investigation into the mechanistic link between photosynthesis and root development in rice, particularly to elucidate how OsMIF1 and OsMIF2 may coordinate these processes. Moreover, the hypothesis that OsMIF1-interacting proteins play roles in light, heat, and drought signaling warrants further study, as does OsMIF protein roles in stress adaptation and developmental regulation (Table 1 ). Conclusions In summary, the results presented here support the proposed hub protein model in which OsMIF1 and OsMIF2 collaborate with and/or regulate ZF-HD proteins to fine-tune rice reproductive development. Moreover, we propose that OsMIF1 and OsMIF2 also play critical roles in the responses to drought and salt stress as well as the regulation of photosynthesis. In particular, the increasing frequency of droughts and floods caused by ongoing climate change poses a serious threat to global rice production and food security. Therefore, understanding the multifaceted roles of OsMIF1 and OsMIF2 will yield valuable insight into the molecular mechanisms underlying climate adaptability in rice. Such insight is critical as a foundation for engineering climate-resilient rice cultivars and to develop strategies for stable food production in the face of environmental fluctuations. Abbreviations ABA Abscisic acid ACC 1-Aminocyclopropane-1-carboxylic acid CRISPR-Cas9 Clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 DAF Days after flowering DAG Days after germination DEGs Differentially expressed genes GA Gibberellic acid GO Gene Ontology GUS β-Glucuronidase HD Homeodomain KEGG Kyoto Encyclopedia of Genes and Genomes KO Knockout MAPK Mitogen-activated protein kinase MeJA Methyl jasmonate MIF Mini zinc finger NAA 1-Naphthaleneacetic acid NT Non-transgenic SEM Scanning electron microscopy SSP Seed storage protein TF Transcription factor Y2H Yeast two-hybrid ZF Zinc finger ZF-HD Zinc finger–homeodomain Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials All data generated and analyzed during this study are included in this published article and its Additional files. Competing interests The authors declare that they have no competing interests. Funding This research was supported by a grant from the National Institute of Agricultural Science, Rural Development Administration, Republic of Korea (PJ013149), and the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (RS-2023-00248217 to K Cho, 2023R1A2C1002936, 2024H1A7A2A02000017, and BK21 FOUR (Fostering Outstanding Universities for Research, No. 4120240915070) to O Han). Authors' contributions JP So, KW Cho and OS Han conceived and designed the research; JP So performed the experiments; JP So, JY Lee and KW Cho analyzed the data; JP So wrote the manuscript; KW Cho, DK Kim, OS Han reviewed and revised the manuscript. All authors have read and agreed to the published version of the manuscript. Acknowledgements This research was supported by the NRF and RDA, Republic of Korea. 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Int J Mol Sci 23(1). https://doi.org/10.3390/ijms23010125 Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.zip Cite Share Download PDF Status: Published Journal Publication published 10 Mar, 2026 Read the published version in Rice → Version 1 posted Editorial decision: Revision requested 15 Jan, 2026 Reviews received at journal 15 Jan, 2026 Reviewers agreed at journal 06 Jan, 2026 Reviews received at journal 31 Dec, 2025 Reviewers agreed at journal 15 Dec, 2025 Reviewers invited by journal 08 Dec, 2025 Editor assigned by journal 08 Dec, 2025 Submission checks completed at journal 05 Dec, 2025 First submitted to journal 05 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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16:36:33","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":813997,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-8286035/v1/185356313f46d6ac43c212e9.png"},{"id":98010058,"identity":"73ea7f04-77e5-4b0b-b3bd-b92974b13cf1","added_by":"auto","created_at":"2025-12-11 18:15:56","extension":"xml","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":271421,"visible":true,"origin":"","legend":"","description":"","filename":"28db24cf27ea46d8b2007579c1e8ad7e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8286035/v1/f724143c9e57e8c1baa50b62.xml"},{"id":98426270,"identity":"b41a77de-f33b-48d8-8700-25da94ac210c","added_by":"auto","created_at":"2025-12-17 16:35:58","extension":"html","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":284879,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8286035/v1/8575378473fcef6be145a5ce.html"},{"id":98010027,"identity":"134c00e6-8d9f-42fa-8a12-d628f54b1dff","added_by":"auto","created_at":"2025-12-11 18:15:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8756120,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic and Structural Features of Rice ZF-HD Transcription Factors. \u003cstrong\u003e(A)\u003c/strong\u003e Phylogenetic tree and domain organization of the ZF-HD family proteins. \u003cstrong\u003e(B)\u003c/strong\u003e Multiple sequence alignment showing conserved motifs within the zinc finger and homeobox domains. \u003cstrong\u003e(C)\u003c/strong\u003e Predicted 3D structures of OsMIF1 and OsZHD1 proteins.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8286035/v1/dcb356b909f5fe473073552f.png"},{"id":98424695,"identity":"e987474e-9e0f-4a4c-a768-74c8c85ef448","added_by":"auto","created_at":"2025-12-17 16:33:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9895357,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiling of OsMIF1 and OsMIF2 in various rice tissues. \u003cstrong\u003e(A)\u003c/strong\u003e qRT-PCR analysis of \u003cem\u003eOsMIF1\u003c/em\u003eand \u003cem\u003eOsMIF2\u003c/em\u003e across different developmental stages and tissues. \u003cem\u003eOsUBI5\u003c/em\u003ewas used as the internal control. Values are mean ± SD (n = 3). \u003cstrong\u003e(B-I) \u003c/strong\u003eHistochemical staining in different tissues of \u003cem\u003epOsMIF1::GUS\u003c/em\u003e transgenic rice plant. \u003cstrong\u003e(B)\u003c/strong\u003eFlag leaf. \u003cstrong\u003e(C)\u003c/strong\u003e Stem. \u003cstrong\u003e(D)\u003c/strong\u003e Root. \u003cstrong\u003e(E-G)\u003c/strong\u003e Developing panicles. \u003cstrong\u003e(H)\u003c/strong\u003eDeveloping spikelets. \u003cstrong\u003e(I)\u003c/strong\u003e Mature seeds.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8286035/v1/c083c731a59d6da7eb11ff36.png"},{"id":98010033,"identity":"977085d5-206e-4bf9-8688-38a0f5639a6f","added_by":"auto","created_at":"2025-12-11 18:15:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11688159,"visible":true,"origin":"","legend":"\u003cp\u003eGeneration of OsMIF1 and OsMIF2 knockout (KO) lines using CRISPR-Cas9 system. \u003cstrong\u003e(A) \u003c/strong\u003eSchematic representation of the CRISPR-Cas9 binary vector used for genome editing.\u003cstrong\u003e (B)\u003c/strong\u003e Identification of mutations in T\u003csub\u003e2\u003c/sub\u003e OsMIF1 and OsMIF2 KO lines. \u003cstrong\u003e(C)\u003c/strong\u003e qRT-PCR analysis of \u003cem\u003eOsMIF1\u003c/em\u003e and \u003cem\u003eOsMIF2\u003c/em\u003e expression levels in the KO lines compared to the non-transgenic (NT) control. \u003cem\u003eOsUBI5\u003c/em\u003e was used as the internal control. Error bars represent SD (n = 3).\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8286035/v1/6839fc609671873810b5ae49.png"},{"id":98010061,"identity":"ed8685f2-fffe-4f46-aca2-e7b11e751636","added_by":"auto","created_at":"2025-12-11 18:15:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":48887999,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic characterization of panicle and grain morphology in \u003cem\u003eosmif1\u003c/em\u003e, \u003cem\u003eosmif2\u003c/em\u003e, and \u003cem\u003eosmif1/osmif2\u003c/em\u003eKO lines. \u003cstrong\u003e(A–E)\u003c/strong\u003e Morphological traits of panicles in the NT and KO mutants. \u003cstrong\u003e(A)\u003c/strong\u003e Plant height, \u003cstrong\u003e(B) \u003c/strong\u003eNumber of panicles per plant, \u003cstrong\u003e(C) \u003c/strong\u003ePanicle length,\u003cstrong\u003e (D) \u003c/strong\u003eNumber of primary branches, and \u003cstrong\u003e(E) \u003c/strong\u003eNumber of secondary branches. Values are mean ± SD (3 ≤ n ≤ 6). \u003cstrong\u003e(F–H)\u003c/strong\u003e Seed morphology analysis in NT and KO mutants. \u003cstrong\u003e(F)\u003c/strong\u003e Grains with husk (top) and de-husked grains (bottom), \u003cstrong\u003e(G)\u003c/strong\u003e weight of 100 grains, and \u003cstrong\u003e(H)\u003c/strong\u003ecomparison of grain length, width, thickness. Values are mean ± SD (n = 30). \u003cstrong\u003e(I-J) \u003c/strong\u003eScanning electron microscopy (SEM) analysis of the outer surface of NT and \u003cem\u003eosmif1/osmif2\u003c/em\u003e KO mutant line spikelet hulls. \u003cstrong\u003e(I)\u003c/strong\u003eRepresentative SEM images of the outer surface of spikelet hulls from the NT and the \u003cem\u003eosmif1/osmif2\u003c/em\u003e double mutant. \u003cstrong\u003e(J) \u003c/strong\u003eAverage cell length and width of outer epidermal cells in spikelet hulls (n = 10). \u003cem\u003eP\u003c/em\u003e-values were calculated using Student’s t-test (*p\u0026lt; 0.1, **p\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8286035/v1/6ae5234ce1f330d229821862.png"},{"id":98010052,"identity":"811265d7-8834-4aab-8548-b0114a8fbb76","added_by":"auto","created_at":"2025-12-11 18:15:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":33984954,"visible":true,"origin":"","legend":"\u003cp\u003eRNA-seq-based transcriptome analysis of 10-cm developing panicles from \u003cem\u003eosmif1\u003c/em\u003e, \u003cem\u003eosmif2\u003c/em\u003e, and \u003cem\u003eosmif1/osmif2\u003c/em\u003e KO plants. \u003cstrong\u003e(A) \u003c/strong\u003ePearson correlation matrix of transcriptomic profiles of NT, \u003cem\u003eosmif1\u003c/em\u003e, \u003cem\u003eosmif2\u003c/em\u003e, and \u003cem\u003eosmif1/osmif2\u003c/em\u003e lines. \u003cstrong\u003e(B)\u003c/strong\u003e Venn diagrams of differentially expressed genes (DEGs) that are up-regulated (left) or down-regulated (right) in each KO line (the non-transgenic (NT) line was used as the control/reference line). \u003cstrong\u003e(C)\u003c/strong\u003e Gene ontology (GO) classification of down-regulated DEGs. \u003cstrong\u003e(D-E)\u003c/strong\u003e Expression profiling of DEGs associated with KEGG pathways significantly enriched according to STRING database. \u003cstrong\u003e(D) \u003c/strong\u003eHeatmap based on RNA-seq data. Relative expression is represented using normalized Z-score values. \u003cstrong\u003e(E) \u003c/strong\u003eqRT-PCR analysis of selected DEGs. Expression of target genes was normalized using the 2\u003csup\u003e−ΔΔCT\u003c/sup\u003e method; relative expression in each \u003cem\u003eosmif1\u003c/em\u003e and \u003cem\u003eosmif2\u003c/em\u003e KO lines using NT as control is shown as log\u003csub\u003e2\u003c/sub\u003e of the average fold-change using \u003cem\u003eOsUBI5\u003c/em\u003e as an internal control. Error bars represent SD (n = 3). \u003cem\u003eP\u003c/em\u003e-values were calculated using Student’s t-test (*p\u0026lt; 0.1, **p\u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8286035/v1/31a6cd65d66c7e14ab243a32.png"},{"id":98425368,"identity":"3e136760-c6ab-4fcb-8296-def385a6655d","added_by":"auto","created_at":"2025-12-17 16:34:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3173404,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiling of genes that regulate grain size in 10-cm developing panicles. \u003cstrong\u003e(A) \u003c/strong\u003eHeatmap based on RNA-seq data. Expression levels were calculated using TMM-normalized TPM values and are shown using normalized Z-scores. \u003cstrong\u003e(B)\u003c/strong\u003e qRT-PCR of genes that regulate cell and grain size. Expression of target genes was normalized using the 2\u003csup\u003e−ΔΔCT\u003c/sup\u003e method; normalized expression values are represented as log\u003csub\u003e2\u003c/sub\u003e of the average fold-change using \u003cem\u003eOsUBI5\u003c/em\u003e as the internal control. Error bars represent SD (n = 3). \u003cem\u003eP\u003c/em\u003e-values were calculated using Student’s\u0026nbsp;t-test (*p\u0026lt; 0.1, **p\u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-8286035/v1/84aa7b490c0abbac581366a2.png"},{"id":98010035,"identity":"5ba0851d-a8f0-424f-9d73-e5f2b86319f8","added_by":"auto","created_at":"2025-12-11 18:15:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4833381,"visible":true,"origin":"","legend":"\u003cp\u003eDefects in root morphology and development in young \u003cem\u003eosmif1\u003c/em\u003e and \u003cem\u003eosmif2 \u003c/em\u003eKO seedlings. \u003cstrong\u003e(A–C)\u003c/strong\u003eRepresentative roots in NT, \u003cem\u003eosmif1\u003c/em\u003e, \u003cem\u003eosmif2\u003c/em\u003e, and \u003cem\u003eosmif1/osmif2\u003c/em\u003eseedlings at 7 days after germination (DAG) \u003cstrong\u003e(A)\u003c/strong\u003e, 14 DAG \u003cstrong\u003e(B)\u003c/strong\u003e, and 21 DAG \u003cstrong\u003e(C)\u003c/strong\u003e. \u003cstrong\u003e(D) \u003c/strong\u003eRoot length and \u003cstrong\u003e(E)\u003c/strong\u003e root number in NT and mutant seedlings at 7, 14, and 21 DAG. Error bars represent SD (n = 20).\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-8286035/v1/a8809f7f2565a5e82c203539.png"},{"id":98010063,"identity":"4a91e060-7196-4ed7-aa28-5bc8cea8e2f9","added_by":"auto","created_at":"2025-12-11 18:15:58","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":80375166,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.zip","url":"https://assets-eu.researchsquare.com/files/rs-8286035/v1/e4ca9da2936b5af9c7ab8726.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Zinc Finger Homeobox Transcription Factors OsMIF1 and OsMIF2 Regulate Grain Size and Panicle Development in Rice","fulltext":[{"header":"Background","content":"\u003cp\u003eRice is a critically important agricultural crop that is the primary dietary staple and source of calories for nearly half of the human population (Fukagawa \u0026amp; Ziska, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mohidem et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, the quality and quantity of every rice harvest is critical and ways to increase rice yield and quality are a major focus of ongoing agricultural research (Alam et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Hori \u0026amp; Sun, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Panicle and seed number, architecture/structure, size and composition (Gengmi Li et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gangling Li et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which are regulated by a complex network of genetic, hormonal, and environmental factors, are key determinants of rice yield and quality. Moreover, the regulation of these critical rice traits is intricately and coordinately influenced by resource allocation (Gengmi Li et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ren et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) as well as phytohormones (\u003cem\u003ei.e.\u003c/em\u003e, abscisic acid (ABA), auxin, cytokinin, and ethylene) and MAPK and G-protein signaling pathways and relevant transcription factors (TFs) (Gengmi Li et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTFs play pivotal roles in integrating and coordinating hormonal and other signaling pathways that regulate rice grain and panicle quality and quantity (Gengmi Li et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For instance, OsSPL family TFs regulate rice panicle architecture and grain traits (Dai et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lian et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Segami et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; S. Wang et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), while WRKY TFs orchestrate and integrate both developmental processes and stress response-related pathways and their impact on rice grain and panicle traits (Li et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe zinc finger-homeodomain (ZF-HD) protein family is a group of plant-specific TFs that regulate plant growth, development, flowering and the response to environmental stressors (Bollier et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Jang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shalmani et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). ZF-HD proteins harbor both zinc finger (ZF) and homeodomain (HD) motifs, while mini zinc finger (MIF) proteins (a subfamily of ZF-HD proteins) lack the HD motif (Bollier et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Islam et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Niu et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In general, ZF-HD TFs bind to specific cis-acting regulatory motifs in gene promoter regions as monomeric, homo-/hetero- dimeric or higher order protein complexes. These TFs ultimately regulate expression of genes that in turn regulate various biological processes including organogenesis, hormone signaling, and the response to abiotic and biotic stressors (Bollier et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shen et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eArabidopsis\u003c/em\u003e ZF-HD TFs play diverse development-related roles in hormone signaling and light-mediated morphogenesis (Bueso et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Perrella et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e); for example, ZHD5 promotes shoot regeneration and cytokinin-associated phenotypes, ATHB25 (also known as ZFHD2/ZHD1) regulates gibberellin biosynthesis and seed longevity (Bueso et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and ZFHD10 coordinates and integrates light signaling to promote elongation of hypocotyls (Perrella et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Rice ZF-HD genes play significant roles in reproductive development including floral initiation and seed formation (Jain et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Shalmani et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shen et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2025\u003c/span\u003e); for example, OsZHD1 and OsZHD2 redundantly regulate grain size, modulate cell proliferation in spikelets and glumes, OsZHD2 also regulates biosynthesis of ethylene which in turn regulates root development (Guo et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yoon et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), while OsZHD1, OsZHD2, OsZHD4, and OsZHD8 form homo- and heterodimers that directly repress transcription of target genes in plants/cells in the presence of abiotic stressors (Figueiredo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMIF proteins, which possess only a single zinc finger domain (Hu \u0026amp; Ma, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Thiaw \u0026amp; Gantet, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), play roles in cell division, meristem state transitions, and reproduction-related processes (\u003cem\u003ei.e.\u003c/em\u003e, development of vegetative and floral organs) (Thiaw \u0026amp; Gantet, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In \u003cem\u003eArabidopsis\u003c/em\u003e, MIF1 integrates multiple phytohormone signals, and its overexpression results in developmental defects such as dwarfism, loss of apical dominance, and dark-green spoon-shaped cotyledons (Hu \u0026amp; Ma, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Furthermore, overexpression of MIF1 or MIF3 induces ectopic shoot meristems along leaf margins, disrupts leaf growth, and modulates auxin and gibberellin-regulated processes (Hu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The tomato MIF homolog SlIMA regulates floral meristem termination, carpel number and fruit development (Bollier et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), while the \u003cem\u003eGerbera hybrida\u003c/em\u003e MIF protein GhMIF directly activates expression of the GEG gene, a member of the GASA family, to suppress ray petal elongation (Han et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA recent study demonstrated that PASPRO1 (OsMIF3) and PASPRO2 (OsMIF4) interact with and inhibit the transcriptional function of rice ZHD TFs, thereby regulating the surface material patterns of the production of anther and pollen. OsMIF3- and OsMIF4 knockout lines exhibit abnormal cuticle formation, defective pollen surface structures and reduced fertility (Jang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, overexpression of OsMIF1 enhances drought tolerance by regulating developmental processes and interacting with ZHD TFs and OsDIP1, thereby improving resilience of plants exposed to stress (Thiaw, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Many studies support a role for MIFs in reproductive development. Despite their potential importance, the biological roles of rice MIFs remain poorly understood and warrant further study.\u003c/p\u003e\u003cp\u003eHere, CRISPR-Cas9 gene editing was used to generate and then characterize OsMIF1 and OsMIF2 knockout (KO) plants and to elucidate the biological roles of these rice TFs. These studies revealed that OsMIF1 and OsMIF2 KO plants produce enlarged seeds and aberrant panicles. Furthermore, RNA-seq data showed that the phenotypic changes in the engineered KO plants correlate with altered patterns of gene and gene pathway expression. Interestingly, library-scale yeast two-hybrid (Y2H) screening identified 10 candidate protein-interacting partners of OsMIF1; we postulate that these proteins could potentially mediate some of the downstream effects of OsMIF1.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSequence Alignment and Prediction of Protein Structure\u003c/h2\u003e\u003cp\u003eFifteen Zinc Finger-Homeodomain (ZF-HD) family proteins were selected as described previously (Hu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The corresponding DNA and protein sequences were obtained from the MSU Rice Genome Annotation Project (MSU RGAP; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rice.uga.edu/\u003c/span\u003e\u003cspan address=\"https://rice.uga.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Protein sequences were aligned using Clustal Omega (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/Tools/msa/clustalo/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/Tools/msa/clustalo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and visualized with ESPript (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://espript.ibcp.fr/ESPript/ESPript/\u003c/span\u003e\u003cspan address=\"https://espript.ibcp.fr/ESPript/ESPript/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A phylogenetic tree was constructed from the aligned sequences using the Maximum Likelihood method with 1,000 bootstrap replications in MEGA12.\u003c/p\u003e\u003cp\u003eMotif analysis was performed using InterPro (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/interpro/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to predict conserved domains and functional motifs. In addition, the MEME suite (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to identify conserved motifs among the ZF-HD proteins. Sequence logos generated by MEME were used to illustrate the positional conservation and amino acid composition of the predicted motifs.\u003c/p\u003e\u003cp\u003eTo further investigate structural features, the three-dimensional structures of ZF-HD proteins were predicted using AlphaFold (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.ebi.ac.uk/\u003c/span\u003e\u003cspan address=\"https://alphafold.ebi.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The predicted protein models were subsequently visualized and analyzed using PyMOL to examine structural features such as conserved domains and secondary structures.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePlant Materials and Growth Conditions\u003c/h3\u003e\n\u003cp\u003eSeeds of \u003cem\u003eOryza sativa\u003c/em\u003e L. ssp. japonica cv. Ilmi were obtained from the Korea Seed \u0026amp; Variety Service. The rice plants used in this study were cultivated in paddy fields under standard agronomic conditions.\u003c/p\u003e\n\u003ch3\u003eRNA Isolation and Quantitative Real-Time PCR (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from each rice tissue using a previously published method (Li \u0026amp; Trick, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). cDNA was synthesized from 1 \u0026micro;g total RNA using the QuantiTect Reverse Transcription Kit (Cat. #205311, Qiagen, Hilden, Germany), following the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was performed using the QuantiTect SYBR Green PCR Kit (Cat. #204343, Qiagen, Hilden, Germany) on a Qiagen Rotor-Gene Q real-time PCR cycler. The thermal cycling conditions were as follows: 95\u0026deg;C for 15 min; 40 cycles of 94\u0026deg;C for 15 s, 60\u0026deg;C for 30 s, and 72\u0026deg;C for 30 s; followed by a melting curve analysis from 72 to 95\u0026deg;C. \u003cem\u003eOsUBI5\u003c/em\u003e was used as an internal control gene to normalize gene expression levels (Jain et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Relative expression levels were calculated using the 2\u003csup\u003e\u0026ndash;ΔΔCt\u003c/sup\u003e method (Livak \u0026amp; Schmittgen, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). All primers used for qRT-PCR are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\n\u003ch3\u003eGUS Staining Assay\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from 21-day-old seedlings as described by (Dellaporta et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). A 1.5 kb upstream promoter region of the \u003cem\u003eOsMIF1\u003c/em\u003e gene was amplified from 5 ng of genomic DNA and cloned into the pCAMBIA1201 vector. The construct was introduced into the \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA101 and used to transform embryogenic rice calli derived from mature seeds (Kim et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Transcription from the cloned \u003cem\u003eOsMIF1\u003c/em\u003e promoter was then quantified in various tissues harvested from transgenic lines using a previously described GUS reporter staining technique (Dedow et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). More specifically, tissue samples were pretreated in ice-cold 90% acetone for 5\u0026ndash;15 min, incubated in staining buffer [50 mM sodium phosphate buffer (pH 7.2), 2 mM each of K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e] and K\u003csub\u003e3\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e], 2% Triton X-100, 2 mM X-Gluc] under vacuum for 5 min, and then incubated overnight at 37\u0026deg;C in the dark. Samples were rinsed in 70% ethanol before storage at 4 ℃ or immediate quantification of GUS activity.\u003c/p\u003e\n\u003ch3\u003eHormone Treatment\u003c/h3\u003e\n\u003cp\u003eTo investigate hormone responsiveness, leaf discs were prepared from leaves of 28-day-old rice plants. The leaves were excised and cut into uniform leaf discs, which were then floated on a hormone-containing solution in 6-well plates. The leaf discs were immersed in the treatment solution and incubated at room temperature. Samples were immediately frozen in liquid nitrogen and stored at -70\u0026deg;C until further analysis.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePrediction of Cis-acting Regulatory Elements\u003c/h2\u003e\u003cp\u003eThe 1.5 kb upstream promoter regions of \u003cem\u003eOsMIF1\u003c/em\u003e and \u003cem\u003eOsMIF2\u003c/em\u003e were retrieved from the Rice Genome Annotation Project (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rice.plantbiology.msu.edu/\u003c/span\u003e\u003cspan address=\"http://rice.plantbiology.msu.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Cis-regulatory elements within these promoter sequences were predicted using the PlantCARE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Lescot et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). For visualization, the PlantCARE results were processed and visualized in R (version 4.5.1) using the tidyverse and ggplot2 packages.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGeneration of OsMIF1 and OsMIF2 Knockout Rice\u003c/h3\u003e\n\u003cp\u003eKnockout (KO) lines of OsMIF1 and OsMIF2 were generated in \u003cem\u003eOryza sativa\u003c/em\u003e ssp. \u003cem\u003ejaponica\u003c/em\u003e cv. Ilmi through the CRISPR-Cas9 system. To target both \u003cem\u003eOsMIF1\u003c/em\u003e and \u003cem\u003eOsMIF2\u003c/em\u003e, sgRNA (5\u0026rsquo;-GCGGCCAAGCCGTACGCGAACGG-3\u0026rsquo;) was designed using CRISPR-P 2.0, an online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispr.hzau.edu.cn/CRISPR2/\u003c/span\u003e\u003cspan address=\"http://crispr.hzau.edu.cn/CRISPR2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The sgRNA was first cloned into the pRGE31 entry vector and subsequently transferred into the pCAMBIA-Cas9 binary vector containing the Cas9 expression cassette and the rice U3 promoter for Agrobacterium-mediated transformation (Chandra et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Pham et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The pCAMBIA-Cas9-sgRNA construct was introduced into rice via \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA101. Agrobacterium-mediated transformation was performed according to a previously described method (Kim et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eScreening and Characterization of KO Mutant Rice Plants\u003c/h3\u003e\n\u003cp\u003eTo verify mutations at the target site in T\u003csub\u003e0\u003c/sub\u003e and T\u003csub\u003e1\u003c/sub\u003e generations, genomic DNA was extracted from 30-day-old rice plants following a previously described protocol (Dellaporta et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). The target region was amplified by PCR using specific primers (Supplementary Table\u0026nbsp;1). The resulting amplicons were subjected to deep sequencing using the MiniSeq platform (KAIST, Daejeon, Republic of Korea). The sequencing data were analyzed using RGEN Tools (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rgenome.net\u003c/span\u003e\u003cspan address=\"http://www.rgenome.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). For the T\u003csub\u003e2\u003c/sub\u003e generation, PCR was performed using specific primers (Supplementary Table\u0026nbsp;1), and the amplified products were sent to Cosmogenetech (Seoul, Republic of Korea) for sequencing.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eScanning Electron Microscopy (SEM)\u003c/h2\u003e\u003cp\u003eTo examine the cell size of mature rice spikelets, scanning electron microscopy (SEM) was performed using a JSM-IT300 (JEOL Ltd., Tokyo, Japan). Samples were mounted on aluminum stubs, sputter-coated with a thin layer of platinum, and observed under SEM. The length and width of epidermal cells on the outer surface of spikelet hulls were measured using ImageJ software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/\u003c/span\u003e\u003cspan address=\"https://imagej.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eTranscriptome Analysis\u003c/h2\u003e\u003cp\u003eTotal RNA extracted from immature seeds (14 days after flowering) and developing panicles (10 cm) of non-transgenic (NT) and OsMIF1 and OsMIF2 KO mutant lines was sent to DNALINK Biotechnology Company (Seoul, Republic of Korea) for RNA sequencing. Libraries were prepared using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina Inc., San Diego, CA, USA) and sequenced on the Illumina NovaSeq 6000 platform with paired-end reads (2\u0026times;101 bp). Each sample was assigned a unique barcode index, and sequencing was performed according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003eTranscript quantification was performed using Kallisto, which performs pseudo-alignment of reads to the reference transcriptome and estimates transcript abundance (Bray et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The resulting expression matrix was normalized using TMM (trimmed mean of M-values) normalization. Differentially expressed genes (DEGs) between KO and control samples were identified using both the edgeR and DESeq2 packages in R. Statistical significance was determined by setting the threshold at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Love et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; McCarthy et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eVenn diagrams for DEG comparisons among the mutants were constructed using the online tool Venny 2.1.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinfogp.cnb.csic.es/tools/venny/\u003c/span\u003e\u003cspan address=\"https://bioinfogp.cnb.csic.es/tools/venny/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the functional annotation tool DAVID (Database for Annotation, Visualization, and Integrated Discovery, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://davidbioinformatics.nih.gov\u003c/span\u003e\u003cspan address=\"https://davidbioinformatics.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). GO and KEGG terms with p-value\u0026thinsp;\u0026le;\u0026thinsp;0.1 were considered significant. For visualization, the data were imported into R (v4.5.1), and bar plots were generated using the ggplot2, dplyr, and forcats packages. Heatmaps for visualization of gene expression patterns were generated using Heatmapper (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.heatmapper.ca/expression/\u003c/span\u003e\u003cspan address=\"http://www.heatmapper.ca/expression/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), an online tool for expression data clustering and heatmap construction.\u003c/p\u003e\u003cp\u003eProtein\u0026ndash;protein interaction networks were predicted using the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The corresponding protein identifiers were retrieved and queried against the \u003cem\u003eOryza sativa\u003c/em\u003e Japonica Group database in STRING. Interactions were filtered using a minimum required interaction score of 0.4, and the resulting network was visualized in Cytoscape for clustering and pathway annotation. KEGG pathway enrichment of the mapped proteins was assessed using STRING\u0026rsquo;s built-in functional enrichment tool.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eY2H Library-scale Screening Assay\u003c/h2\u003e\u003cp\u003eYeast Two-Hybrid screening of OsMIF1 was conducted using the \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e strain AH109, which contains two reporter genes, HIS3 and ADE2, under the control of distinct GAL promoters. The full-length \u003cem\u003eOsMIF1\u003c/em\u003e was amplified using gene-specific primers containing \u003cem\u003eEco\u003c/em\u003eRI and \u003cem\u003eBam\u003c/em\u003eHI sites (Supplementary Table\u0026nbsp;1), and cloned into the corresponding sites of the pGBKT7 vector for expression as a myc-tagged GAL4 DNA-binding domain fusion. The construct was confirmed by DNA sequencing and subsequently co-transformed into the yeast strain with a rice cDNA activation domain (AD) library. Transformants were selected on synthetic dropout (SD) medium lacking leucine, tryptophan, histidine, and adenine (SD-LWHA), which permits the growth of yeast cells expressing interacting bait and prey proteins.\u003c/p\u003e\u003cp\u003eTo confirm protein\u0026ndash;protein interactions, DNA fragments encoding the prey proteins from 60 initial candidate clones were isolated via PCR or by transformation into \u003cem\u003eE. coli\u003c/em\u003e. These prey clones were then reintroduced into yeast AH109 along with either the OsMIF1 bait plasmid or an empty bait vector as a negative control. Interactions were assessed based on yeast growth on SD-LWHA selection medium. Candidates were further validated through DNA sequencing and restriction enzyme digestion.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eStructural and Evolutionary Features of rice ZF-HD proteins OsMIF1 and OsMIF2\u003c/h2\u003e\u003cp\u003eTo elucidate the structural characteristics of OsMIF1 and OsMIF2 and determine the percent similarity/divergence among rice ZF-HD proteins, the amino acid sequences of 11 rice ZF-HD and 4 rice MIF proteins were aligned (Supplementary Table\u0026nbsp;2) (Hu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and subjected to phylogenetic tree, domain prediction, and three-dimensional structure prediction analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the phylogenetic tree for this subset of rice ZF-HDs, OsMIF1 and OsMIF2 clustered together with a bootstrap value of 100%, indicating a highly reliable evolutionary relationship (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The aligned full-length amino acid sequences of OsMIF1 and OsMIF2 demonstrate 98.1% identity and only two amino acid differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). OsMIF3 and OsMIF4 cluster with OsMIF1 and OsMIF2 (bootstrap value 71%) to form a distinct MIF subfamily within the larger rice ZF-HD protein family. OsZHD1\u0026ndash;4 are more closely related to OsMIF proteins than to other rice ZF-HD proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Similar results were obtained when the alignment was limited to the ZF domain in this subset of rice ZF-HD proteins (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe domain structure of rice ZF-HDs was predicted using AlphaFold (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.ebi.ac.uk/\u003c/span\u003e\u003cspan address=\"https://alphafold.ebi.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The results reveal that rice ZF-HD proteins (ZHDs) possess both a zinc finger domain and a homeobox domain, whereas rice MIFs, including OsMIF1 and OsMIF2, possess a zinc finger domain but do not possess a homeobox domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Supplementary Table\u0026nbsp;3). Zinc finger domains contain a conserved C\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e3\u003c/sub\u003e motif and homeobox domains are comprised of a characteristic three-helix fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). AlphaFold-based 3D structural prediction confirmed that both of these features are present in the rice ZF-HDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Supplementary Fig.\u0026nbsp;2), and that OsMIF1, belonging to the MIF subclass of ZF-HDs, is predicted to lack a homeodomain but to contain a zinc finger domain with the characteristic conserved cysteine (C38, C54, C71, C74, and C76) and histidine (H42, H77 and H81) residues configured for zinc coordination. In contrast, OsZHD1 is predicted to harbor a characteristic zinc finger domain (with conserved cysteines C60, C76, C93, C96, and C98 and histidine residues H64, H99 and H103) and a homeobox with a typical three α-helix domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These results indicate that OsMIF1 and OsMIF2 represent a distinct conserved MIF subgroup in the rice ZF-HD family.\u003c/p\u003e\u003cp\u003eThe bioinformatic tool OrthoDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.orthodb.org/\u003c/span\u003e\u003cspan address=\"https://www.orthodb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) identified 65 plant MIF proteins orthologous to OsMIF1 and OsMIF2. Among them, three proteins from wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) showed the highest similarity to and formed a distinct subgroup from OsMIF1 and OsMIF2. In addition, eight proteins from maize (\u003cem\u003eZea mays\u003c/em\u003e L.) exhibited considerable similarity to OsMIF1 and OsMIF2 (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eTissue- and Stage-specific Expression of OsMIF1 and OsMIF2 During Rice Developmen\u003c/h2\u003e\u003cp\u003eTo investigate the tissue- and stage specificity of OsMIF1 and OsMIF2 expression, qRT-PCR and GUS staining assays were performed in diverse tissues of plants at different stages of development (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). qRT-PCR data indicate that OsMIF1 and OsMIF2 are expressed at a low level in 14 DAG seedlings but are expressed at a high level in developing panicles before flowering and at an early developmental stage (1\u0026ndash;5 cm); their expression then decreased gradually as panicle development progressed. At 0 DAF, their expression was high in stems and roots but low in flag leaves, while \u003cem\u003eOsMIF1\u003c/em\u003e peaked at 3 DAF and \u003cem\u003eOsMIF2\u003c/em\u003e peaked at 1 DAF during seed development and both declined from their peak expression level until 7 DAF. Expression of both genes increased from 7 DAF to 14 DAF and then reached a plateau and remained high until 21 DAF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA 1,500 bp fragment of the upstream promoter region of \u003cem\u003eOsMIF1\u003c/em\u003e was cloned into a β-glucuronidase (GUS) reporter gene vector, and GUS expression from the resulting plasmid was quantified in various tissues and at different stages of rice plant development. These data were used to confirm the tissue- and developmental stage-specificity of OsMIF1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;I, Supplementary Fig.\u0026nbsp;4\u0026ndash;5). The results suggest that OsMIF1 is not expressed in flag leaves at 0 DAF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), but is expressed in the inner vascular tissues of the stem and root at 0 DAF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u0026ndash;D). Furthermore, the pattern of GUS reporter gene expression suggests that OsMIF1 is expressed in panicles during early (0\u0026ndash;1 cm) and intermediate stages (5\u0026ndash;10 cm), gradually diminishing during the late stages of panicle development (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u0026ndash;G), while OsMIF1 expression in spikelets is weak early in development, localized to the distal end during intermediate development, broadly distributed during late development and gradually decreases towards the onset of maturity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). During seed development, reporter gene expression is concentrated initially at the distal end, throughout the seed during the middle stages of development, peaking at 14 DAF and remaining strong until 21 DAF (Supplementary Fig.\u0026nbsp;5). While the results suggest that OsMIF1 is not expressed in cells on the surface of mature seeds, the data demonstrate that OsMIF1 is strongly expressed in endospermal cells of mature rice seeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI).\u003c/p\u003e\u003cp\u003eTranscripts of \u003cem\u003eOsMIF1\u003c/em\u003e and \u003cem\u003eOsMIF2\u003c/em\u003e were also quantified in hormone-treated leaf discs using qRT-PCR (Supplementary Fig.\u0026nbsp;6). The results showed that \u003cem\u003eOsMIF1\u003c/em\u003e is induced by ABA and NAA but suppressed by MeJA and GA\u003csub\u003e3\u003c/sub\u003e, while \u003cem\u003eOsMIF2\u003c/em\u003e is slightly induced by ABA and NAA and repressed by MeJA and ACC (Supplementary Fig.\u0026nbsp;6). Analysis of the 1.5 kb promoters of OsMIF1 and OsMIF2 using PlantCARE revealed the presence of multiple cis-acting regulatory motifs (\u003cem\u003ei.e.\u003c/em\u003e, hormone-regulating motifs ABA, MeJA and GA; motifs that mobilize responses to stressors including drought, hypoxia, wounding) (Supplementary Fig.\u0026nbsp;7).\u003c/p\u003e\u003cp\u003eOverall, these results indicate that OsMIF1 and OsMIF2 are expressed across diverse tissues during reproductive stages, implying their putative roles in panicle development and seed maturation. The fact that transcription of \u003cem\u003eOsMIF1\u003c/em\u003e and \u003cem\u003eOsMIF2\u003c/em\u003e is induced/repressed in response to plant hormones suggests that these MIFs mediate hormone signaling during reproductive development or other reproductive processes as well as the response to exogenous stress.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003ePhenotypic Characterization of OsMIF1, OsMIF2 and OsMIF1/OsMIF2 Mutant Lines\u003c/h2\u003e\u003cp\u003eCRISPR-Cas9 technology was used to engineer OsMIF1 and OsMIF2 knockout (KO) plant lines, which were then used to elucidate the functional roles of these rice MIFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Three mutant lines were generated and selected for further study: \u003cem\u003eosmif1\u003c/em\u003e harbors a 1 bp insertion mutation, \u003cem\u003eosmif2\u003c/em\u003e also harbors a 1 bp insertion mutation, while the third line, \u003cem\u003eosmif1/osmif2\u003c/em\u003e, is a double mutant harboring a 1 bp insertion in each \u003cem\u003eCRISPR-Cas9-\u003c/em\u003eedited MIF gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Supplementary Fig.\u0026nbsp;8, Supplementary Table\u0026nbsp;4\u0026ndash;6). qRT-PCR data confirmed efficient knockout of \u003cem\u003eOsMIF1\u003c/em\u003e and/or \u003cem\u003eOsMIF2\u003c/em\u003e in a pattern consistent with the genotype of all three mutant lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe morphological traits of the panicles were compared in \u003cem\u003eosmif1\u003c/em\u003e, \u003cem\u003eosmif2, osmif1/osmif2\u003c/em\u003e and wild-type control (NT) plants; the goal was to test the prediction that the mutant plants would exhibit defects during reproductive stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;E, Supplementary Fig.\u0026nbsp;9). Plant height increased in all mutant lines relative to NT, with \u003cem\u003eosmif1\u003c/em\u003e, \u003cem\u003eosmif2\u003c/em\u003e, and \u003cem\u003eosmif1/osmif2\u003c/em\u003e plants being on average 6.7, 5.1, and 3.9% taller than NT, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The number of panicles per plant decreased in \u003cem\u003eosmif1\u003c/em\u003e relative to NT by 17.3%, while the number of panicles per plant was similar in NT, \u003cem\u003eosmif2\u003c/em\u003e and \u003cem\u003eosmif1/osmif2\u003c/em\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Panicles were 5.9% and 6.5% shorter in \u003cem\u003eosmif1\u003c/em\u003e and \u003cem\u003eosmif1/osmif2\u003c/em\u003e plants but only slightly shorter in \u003cem\u003eosmif2\u003c/em\u003e than in NT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In addition, there were 6.1% fewer primary branches in \u003cem\u003eosmif1/osmif2\u003c/em\u003e plants than in NT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), and the number of secondary branches was 19.5 and 24.1% lower in \u003cem\u003eosmif1\u003c/em\u003e and \u003cem\u003eosmif1/osmif2\u003c/em\u003e plants than in NT plants, respectively. In contrast, primary branching was comparable in \u003cem\u003eosmif1\u003c/em\u003e, \u003cem\u003eosmif2\u003c/em\u003e and NT plants, and secondary branching was similar in \u003cem\u003eosmif2\u003c/em\u003e and NT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and E). These results reveal defects in panicle development, including reduced panicle elongation and branching, in \u003cem\u003eosmif1\u003c/em\u003e mutant plants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOur results also provide evidence that OsMIF1/2 influence seed development and morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u0026ndash;J). For example, 100-grain weight of \u003cem\u003eosmif1\u003c/em\u003e and \u003cem\u003eosmif1/osmif2\u003c/em\u003e seeds was 10.3% and 17.9% higher than NT seeds, respectively, while 100-grain weight of \u003cem\u003eosmif2\u003c/em\u003e seeds was 0.6% lower than NT seeds. Consistent with these data, \u003cem\u003eosmif1\u003c/em\u003e seeds were 3.2% longer, 4.5% wider, and 5.3% thicker than NT seeds, while \u003cem\u003eosmif2\u003c/em\u003e seeds showed relatively minor (0.8 to 1.6) differences from NT and \u003cem\u003eosmif1/osmif2\u003c/em\u003e double mutant seeds diverged even more strongly from the control seed, being 5.9% longer, 5.7% wider, and 7.2% thicker than NT seed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u0026ndash;H). When the outer surface of the spikelets of NT and \u003cem\u003eosmif1/osmif2\u003c/em\u003e seeds were examined by scanning electron microscopy (SEM), the results show that the epidermal cells of \u003cem\u003eosmif1/osmif2\u003c/em\u003e double mutant seeds were 12.4% wider and 11.0% longer than NT epidermal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI\u0026ndash;J). Therefore, \u003cem\u003eosmif1\u003c/em\u003e, \u003cem\u003eosmif2\u003c/em\u003e and especially \u003cem\u003eosmif1/osmif2\u003c/em\u003e plant lines exhibit increased seed size and weight, which is consistent with the observed increased size of plant epidermal cells. Furthermore, the above results implicate OsMIF1 and OsMIF2 as critical regulators of reproductive development, structures and processes, for example, by modulating panicle architecture and seed morphology. Knockout of OsMIF1 alone caused phenotypic changes, whereas knockout of OsMIF2 alone does not, while double knockout plants exhibit more pronounced effects than osmif1 mutant plants. The results suggest that OsMIF1 plays a primary role while OsMIF2 plays secondary supportive roles in reproductive development in rice.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGlobal Gene Expression Profiling of\u003c/b\u003e \u003cb\u003eosmif1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eosmif2\u003c/b\u003e \u003cb\u003ePanicles and Immature Seeds\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGlobal transcriptomic analyses were performed to correlate OsMIF1/OsMIF2 genotype with phenotype and associated changes in expression of relevant biological pathways. To this end, RNA-seq data were collected using samples from 10 cm developing panicles and 14 DAF immature seeds from NT, \u003cem\u003eosmif1\u003c/em\u003e, \u003cem\u003eosmif2\u003c/em\u003e, and \u003cem\u003eosmif1/osmif2\u003c/em\u003e plant lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Supplementary Fig.\u0026nbsp;10). In 10 cm developing panicles, RNA-seq yielded 39\u0026ndash;51\u0026nbsp;million reads per sample with Q30 values above 91%. In 14 DAF seeds, 25\u0026ndash;34\u0026nbsp;million reads per sample were obtained, and all libraries showed Q30 values above 87% (Supplementary Table\u0026nbsp;7). Correlation coefficients varied from 0.975 to 0.988, indicating that the RNA-seq data were derived from the same developmental stage and tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;10A).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe RNA-seq data were subjected to differential expression analysis using edgeR and DESeq2 software packages (significance threshold was p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In samples from 10 cm developing panicles, 1,124 genes were upregulated, including 55 genes expressed in all mutant lines, while 1,403 genes were downregulated, of which 325 transcripts were consistently detected independent of the genotype of the sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). GO analysis was performed using the DAVID tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://davidbioinformatics.nih.gov\u003c/span\u003e\u003cspan address=\"https://davidbioinformatics.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which links each up- or down-regulated gene with annotations that assign an established or putative biological function, \u003cem\u003ee.g.\u003c/em\u003e Biological Process (BP), Cellular Component (CC), Molecular Function (MF), KEGG pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eGO enrichment analysis of the 325 commonly downregulated genes revealed significant enrichment in a subset of BP terms including post-embryonic plant morphogenesis, mRNA transcription, response to light stimulus, hydrogen peroxide catabolic process, and response to oxidative stress. Enriched CC terms included nucleus, extracellular region, plant-type cell wall, and lipid storage body, while enriched MF terms included DNA-binding transcription factor activity, oxidoreductase activity, and peroxidase activity; enzyme inhibitors were also significantly overrepresented. KEGG analysis revealed enrichment of pathways involved in biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, (general) metabolism, starch and sucrose metabolism, and cyanoamino acid metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). To validate the RNA-seq data, qRT-PCR was performed for a subset of downregulated DEGs, including those associated with carotenoid biosynthesis, cyanoamino acid metabolism, MAPK signaling, and phenylpropanoid biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026ndash;E, Supplementary Fig.\u0026nbsp;11\u0026ndash;12, Supplementary Table\u0026nbsp;8\u0026ndash;9). Expression values for DEGs were normalized to an appropriate control using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method and results are presented as log₂ values. qRT-PCR data confirmed that downregulated DEGs outnumber upregulated DEGs in this dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The results demonstrate that the phenotypes associated with knockout mutations in OsMIF1 and OsMIF2 are characterized by suppression of genes involved in morphogenesis, stress response, transcriptional and enzymatic activities, and hormone and metabolic pathways; this supports the conclusion that OsMIF genes play essential roles in rice development and environmental adaptation.\u003c/p\u003e\u003cp\u003eGO enrichment analysis was also performed on RNA-seq data from immature rice seeds (14 DAF). These data revealed that downregulated DEGs are enriched in the following GO terms: photosynthesis, carbon fixation, cell wall modification, photosystem components, thylakoid membrane proteins, and chloroplast-associated factors (Supplementary Fig.\u0026nbsp;10C). qRT-PCR analysis of photosynthesis-related genes confirmed the prediction that they would be down-regulated in \u003cem\u003eosmif1\u003c/em\u003e (KO) rice relative to NT controls, thus validating the RNA-seq findings described above (Supplementary Fig.\u0026nbsp;10D\u0026ndash;E, Supplementary Table\u0026nbsp;10).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eFunctional Roles of OsMIF1 and OsMIF2 during Seed Development\u003c/h2\u003e\u003cp\u003eWhen the seeds from \u003cem\u003eosmif1\u003c/em\u003e and \u003cem\u003eosmif2\u003c/em\u003e KO mutant rice plants were compared to NT control seeds, we observed that the mutant seeds are larger than control seeds, and similarly, SEM data showed that KO epidermal cells on the surface of spikelets are enlarged relative to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u0026ndash;J). These observations were confirmed and validated using qRT-PCR to quantify expression of genes that modulate the size of mature seeds and the size of cells in developing panicles of KO mutant plants (Supplementary Table\u0026nbsp;11) (Lee \u0026amp; Kende, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Shim et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shin et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Si et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; S. Wang et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhan et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zuo et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Our RNA-seq data had revealed higher expression of these genes/pathways in the KO mutants than in NT control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA); furthermore, qRT-PCR data revealed that all of the corresponding genes are upregulated in the KO mutant lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These findings implicate OsMIF1 and OsMIF2, and the level at which these genes are expressed, as determinants of seed and spikelet cell size.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePrevious studies reported that OsMIF1 and OsMIF2 are co-expressed with major seed storage proteins (SSPs) (So et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Furthermore, data presented above (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) demonstrate that both genes are highly expressed in immature seeds with peak expression at 14 DAF. To further explore potential crosstalk between SSPs (and/or co-expression of SSPs) and OsMIF1/OsMIF2, expression of these genes was quantified and compared in MIF1/2 KO and NT seeds (Supplementary Fig.\u0026nbsp;13). For this analysis, SSP genes were grouped according to their previously reported classification (Supplementary Table\u0026nbsp;12) (Chandra et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Pham et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). First, expression of SSPs was quantified using RNA-seq data from 14-DAF immature seeds. Although no consistent trend was observed in these data, transcripts from the Pro13b-II subgroup of prolamin genes were generally downregulated (Supplementary Fig.\u0026nbsp;13A). This result was confirmed by performing qRT-PCR on Pro13b-II genes using cDNA and primers reported in previous studies (Supplementary Fig.\u0026nbsp;13B).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of Putative OsMIF1 Protein-Interacting Partners by Yeast Two-Hybrid Screening\u003c/h2\u003e\u003cp\u003eIn the initial discovery of the ZF-HD protein family, Y2H assays revealed that ZF-HD proteins form both homo- and heterodimers (Windh\u0026ouml;vel et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Based on these findings, we conducted a Y2H screen for protein-interacting partners of OsMIF1, in which OsMIF1 was the \u0026ldquo;bait\u0026rdquo; and the \u0026ldquo;prey\u0026rdquo; were protein products expressed from a whole rice genome cDNA library (Supplementary Fig.\u0026nbsp;14).\u003c/p\u003e\u003cp\u003eThe screen identified 18 candidate protein-interacting partners of OsMIF1, of which 10 were in-frame (Supplementary Table\u0026nbsp;13\u0026ndash;14). The 10 in-frame proteins included OsSIZ1 (NM_001420071), OsCIPK14 (NM_001404631), akin-beta (XM_015783664), OsMIF1 (NM_001423141), OsBIG (NM_001422705), Snf7 family protein (XM_015782163), OsMORF8b (XM_015756999), OsDjA6 (NM_001402407), OsNBR1 (NM_001416698), and OsELF3.2 (XM_015795402). These putative OsMIF1-interacting proteins are involved in diverse processes, including growth and development, hormone signaling, stress responses, and RNA metabolism; this result suggests that OsMIF1 acts as a regulatory hub to integrate signals from developmental and stress-related pathways (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplementary Table\u0026nbsp;15). One of the interacting partner proteins was OsMIF1 itself, which is consistent with the fact that OsMIF1 forms homodimers; this result also predicts the potential for interactions between OsMIF1 and other ZF-HD proteins (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). GO enrichment analysis revealed that three candidate protein-interacting partners of OsMIF1, \u003cem\u003ei.e.\u003c/em\u003e, OsSIZ1, OsBIG, and OsDjA6, are predicted to contain a zinc finger motif. Thus, OsMIF1 may interact not only with ZF-HD proteins but also with other ZF proteins (Supplementary Fig.\u0026nbsp;15).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCandidate OsMIF1-interacting proteins identified by Y2H library screening\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGene symbol\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNCBI accession number\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFunction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsSIZ1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNM_001420071\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRegulates growth and development, and mediates responses to phosphate/nitrogen status and environmental stresses.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Mishra et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mishra et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Park et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Thangasamy et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; H. Wang et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsCIPK14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNM_001404631\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMediates Ca\u0026sup2;⁺-dependent MAMP-induced defense signaling.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Kurusu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eakin-beta\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eXM_015783664\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIs suppressed by OsTZF1 and is induced under cold stress in tolerant rice.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Ding et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Jan et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsMIF1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNM_001423141\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEnhances drought tolerance by regulating rice growth and development.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Thiaw, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsBIG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNM_001422705\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIs essential for rice growth and development; loss of function causes seedling lethality.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Cheng et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSnf7 family protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eXM_015782163\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIs upregulated in leaf and root under direct-sown drought stress.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Kumar et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsMORF8b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eXM_015756999\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eActs as a multiple organellar RNA-editing factor interacting with other OsMORFs, and is downregulated by cold and salt stress.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Zhang et al., \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsDjA6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNM_001402407\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNegatively regulates rice innate immunity, likely via the ubiquitin\u0026ndash;proteasome degradation pathway.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Sarkar et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhong et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNBR1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNM_001416698\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMediates pest resistance and enhances cold tolerance via autophagy and reduced ubiquitination.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Guo et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang \u0026amp; Chen, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsELF3.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eXM_015795402\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eControls heading date, circadian rhythm, and stress tolerance in rice.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Fu et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eDefects in Root Development and the Response to Salt Stress in OsMIF1- and OsMIF2-KO Mutants\u003c/h2\u003e\u003cp\u003eThe ten-candidate protein-interacting partners of OsMIF1 include proteins associated with the response to drought, salinity and other environmental/water stressors (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In addition, a previous study reported that overexpression of OsMIF1 affected root growth (Thiaw, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These observations suggest that OsMIF1 could influence plant stage-specific changes in root morphology after germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Consistent with this hypothesis, phenotypic analysis revealed enhanced root elongation during the early vegetative stage in \u003cem\u003eosmif1\u003c/em\u003e (KO) plants. Relative to NT controls, \u003cem\u003eosmif1\u003c/em\u003e plant roots were 14\u0026ndash;16% longer at 7 DAG, 14\u0026ndash;20% longer at 14 DAG, and 21\u0026ndash;44% longer at 21 DAG. Furthermore, this phenotypic trait was exacerbated in roots of \u003cem\u003eosmif1/osmif2\u003c/em\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). In contrast, root number was slightly (6\u0026ndash;11%) higher at 7 DAG, but 16\u0026ndash;46% and 18\u0026ndash;38% lower than NT controls in 14 and 21 DAG plants. This phenotypic trait was also exacerbated in \u003cem\u003eosmif1/osmif2\u003c/em\u003e double mutant plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe effects of salt stress on germination rate were also compared in \u003cem\u003eosmif1\u003c/em\u003e, \u003cem\u003eosmif2\u003c/em\u003e, \u003cem\u003eosmif1/osmif2\u003c/em\u003e and NT control plants. The results showed that NT seeds consistently germinate more rapidly than OsMIF mutant seeds. In the absence of salt stress, NT seeds reached 93.3% germination by 96 h, whereas \u003cem\u003eosmif1\u003c/em\u003e, \u003cem\u003eosmif2\u003c/em\u003e, and the \u003cem\u003eosmif1/osmif2\u003c/em\u003e double mutants achieved 70.0%, 55.2%, and 82.1% germination, respectively. In the presence of 75 mM NaCl, (\u003cem\u003ee.g.\u003c/em\u003e, conditions of salt stress), germination was 55.2% in NT at 96 h, but only 22.2\u0026ndash;26.1% for \u003cem\u003eosmif1\u003c/em\u003e mutant seeds. Higher salt stress (\u003cem\u003ei.e.\u003c/em\u003e, 150 mM NaCl) or the \u003cem\u003eosmif1/osmif2\u003c/em\u003e double mutant genotype exacerbated this phenotype. thus, germination rate was 25.0% for NT seeds at 96 h but 0\u0026ndash;5.6% in \u003cem\u003eosmif\u003c/em\u003e1/2 mutants (Supplementary Fig.\u0026nbsp;16), with the lowest germination rate in \u003cem\u003eosmif1/osmif2\u003c/em\u003e double mutant seeds under high salt stress.\u003c/p\u003e\u003cp\u003eIn summary, these results suggest that OsMIF1 and OsMIF2 knockout plants exhibit increased sensitivity to water/salt stress; this trait manifests as root elongation, likely reflecting a compensatory response to the adverse environmental conditions.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cb\u003eosmif1/2\u003c/b\u003e \u003cb\u003eKO Genotype Associated with Defects in Reproductive Development\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study exploits transcription profiling and CRISPR-Cas9 gene editing technology to elucidate the biological roles of mini zinc finger TFs OsMIF1 and OsMIF2. Previous studies showed that plant-specific MIFs play critical roles regulating plant growth and development, hormone signaling, and stress response pathways (Hu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Shen et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Tran et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). More specifically, MIF proteins are critical during reproductive development, regulating floral meristem termination, carpel number, petal elongation, and anther and pollen development (Bollier et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Han et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Thiaw \u0026amp; Gantet, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2024\u003c/span\u003e); the mechanisms by which OsMIF1/2 regulate these processes are diverse.\u003c/p\u003e\u003cp\u003eOsMIF3 (PASPRO1) and OsMIF4 (PASPRO2) are specifically expressed in pollen and the anther wall, where they play important roles during the reproductive stage by regulating the surface morphology of anthers and pollen to ensure normal reproductive development (Jang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). OsMIF3 and OsMIF4 are not expressed in panicles and seeds, whereas OsMIF1 and OsMIF2 are expressed in panicles, seeds, stems and roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (Jang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Loss-of-function mutants of OsMIF1 and OsMIF2 generated by CRISPR-Cas9 showed reduced panicle branching and increased grain size due to increased cell size, and higher abundance of SSPs reflecting increased transcription of SSP genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Fig.\u0026nbsp;13). While OsMIF1/2 and OsMIF3/4 play distinct functional roles, each of the two paired MIFs is redundant to each other. This is consistent with the fact that the transcriptional profiles of OsMIF1/2 and OsMIF3/4 differ.\u003c/p\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eFunctional Connections between MIF and Zinc Finger Proteins\u003c/h2\u003e\u003cp\u003eRice ZHD proteins OsZHD1 and OsZHD2, which belong to another branch of the ZF-HD family, also share roles in root development and seed size (Guo et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yoon et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, OsZHD1, OsZHD2, OsZHD4, and OsZHD8 bind to the promoter region of \u003cem\u003eOsDREB1B\u003c/em\u003e (Figueiredo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The functional redundancy of ZF-HD proteins likely reflects their origin from gene duplication and the evolutionary maintenance of paralogs through active compensation mechanisms (Iohannes \u0026amp; Jackson, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Jang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While MIF and ZHD proteins exhibit functional redundancy within their respective subgroups, MIF TFs appear to work in opposition to and effectively suppress ZHD protein activity. Results presented here demonstrate that the phenotype of \u003cem\u003eosmif1/2\u003c/em\u003e KO mutants includes larger seeds and longer roots; in contrast, KO mutants of OsZHD1 and OsZHD2 have the opposite effects \u003cem\u003ei.e.\u003c/em\u003e, smaller seeds and shorter roots (Guo et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yoon et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). KO mutants of OsMIF3 and OsMIF4 also exhibit reduced expression of \u003cem\u003eOsDREB1A\u003c/em\u003e and \u003cem\u003eOsDREB1B\u003c/em\u003e, previously reported to be transcriptionally repressed by ZHD TFs. Furthermore, OsMIF3 and OsMIF4 interfere with binding of OsZHD1 and OsZHD9 to the promoters of \u003cem\u003eOsDREB1A\u003c/em\u003e and \u003cem\u003eOsDREB1B\u003c/em\u003e (Jang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn general, the zinc finger motif confers the ability to bind directly to DNA and to interact with other zinc finger proteins, thereby regulating transcription of target genes (Mackay \u0026amp; Crossley, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In addition, ZF-HD proteins tend to homodimerize or form heterodimers (Windh\u0026ouml;vel et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Consistent with this, OsMIF1 self-associates to form homodimers (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplementary Table\u0026nbsp;13\u0026ndash;14). \u003cem\u003eArabidopsis\u003c/em\u003e MIFs interact with ZHDs (\u003cem\u003ee.g.\u003c/em\u003e, ZHD5), inhibiting their nuclear localization and DNA-binding activity (Hong et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and four rice ZF-HD TFs form homo- or heterodimers (Figueiredo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). We also note that a previously reported Y2H study documents potential interaction between OsMIF1 and OsZHD proteins (Thiaw, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAtMIF2 and tomato SlIMA directly interact with the C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e zinc-finger protein KNU to form a transcriptional repressor complex with corepressors TOPLESS and HDA19. These protein\u0026ndash;protein interactions repress expression of WUS/SlWUS, thereby regulating floral meristem termination and carpel number (Bollier et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the present study, the Y2H screen for OsMIF1 identified three ZF proteins as putative protein-interacting partners (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplementary Table\u0026nbsp;15). Collectively, these findings suggest that MIF proteins are members of large networks of ZF proteins (\u003cem\u003ee.g.\u003c/em\u003e these networks include ZFs that lack the HD domain). Additional studies on the mechanisms and interactions between MIF genes and ZF/ZHD proteins are warranted; these studies are expected to discover novel protein-protein interactions, elucidate their downstream effects, and provide insight into the structure and activities of plant regulatory networks.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eZF-HD Proteins as Mediators of Hormonal Cross-Talk and Stress Adaptation in Rice\u003c/h2\u003e\u003cp\u003eZF-HD TFs integrate environmental signals, such that ZFHD1 enhances expression of drought-responsive genes under water deficit and in \u003cem\u003eArabidopsis\u003c/em\u003e ZFHD10 promotes blue light\u0026ndash;induced hypocotyl elongation by recruiting TZP (Barth et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Perrella et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Overexpression of MIF1 causes reduced root growth and abnormal root hair formation. Furthermore, it leads to altered responses to phytohormones such as gibberellin (GA), abscisic acid (ABA), and auxin (Hu \u0026amp; Ma, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In amaranth roots, MIF1 is transcriptionally up-regulated by drought stress, leading to reduced root growth and altered expression of growth-related genes (Huerta-Ocampo et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFour rice ZF-HD TFs bind to the \u003cem\u003eOsDREB1B\u003c/em\u003e promoter and repress its expression under multiple stress conditions (Figueiredo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). OsZHD2 enhances root meristem activity through ethylene-induced auxin signaling, thereby improving nutrient uptake and stress resilience, linking ZF-HD activity to auxin- and ethylene-mediated regulation of plant architecture (Yoon et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Overexpression of OsMIF1 also alters root growth, and OsMIF1 overexpressing transgenic plants display dark-green, curled leaves and exhibit enhanced tolerance to water deficit (Thiaw, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Here, we show that KO mutants of OsMIF1 and OsMIF2 exhibit increased root length but reduced root number and are more sensitive to salt stress during germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Supplementary Fig.\u0026nbsp;16); moreover, the expression of both genes was induced by ABA and NAA (Supplementary Fig.\u0026nbsp;6). RNA-seq analysis of 10-cm developing panicles in \u003cem\u003eosmif1\u003c/em\u003e and \u003cem\u003eosmif2\u003c/em\u003e KO lines revealed downregulation of ethylene signaling and light, oxidative, and abiotic stress response pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Taken together, these observations indicate that ZF-HD family proteins play important roles in stress response pathways and root development, suggesting a strong association with hormonal pathways involving auxin, ethylene, and ABA. Therefore, we postulate that ZF-HD proteins modulate and/or mediate hormonal cross-talk and hormone-regulated developmental processes.\u003c/p\u003e\u003cp\u003eWater stress inhibits photosynthesis by stomatal closure, metabolic suppression, and increased oxidative stress (\u003cem\u003ei.e.\u003c/em\u003e, accumulation of reactive oxygen species). However, these effects can be mitigated in plants through adaptive mechanisms such as enhanced photosynthetic efficiency and induction of drought tolerance pathways (Feng et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Iqbal et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lv et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Mart\u0026iacute;nez-Go\u0026ntilde;i et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Qiao et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Thus, the increased sensitivity of OsMIF1 and OsMIF2 KO lines to salt stress and their enhanced root elongation suggests a compensatory drought-avoidance strategy in which reduced photosynthetic capacity and stress tolerance are offset by deep water foraging (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Supplementary Fig.\u0026nbsp;16). Importantly, RNA-seq analysis revealed that photosynthesis-related and carotenoid biosynthesis genes are downregulated in \u003cem\u003eosmif1\u003c/em\u003e and \u003cem\u003eosmif2\u003c/em\u003e KO plants (Supplementary Fig.\u0026nbsp;10). These results highlight the need for further investigation into the mechanistic link between photosynthesis and root development in rice, particularly to elucidate how OsMIF1 and OsMIF2 may coordinate these processes. Moreover, the hypothesis that OsMIF1-interacting proteins play roles in light, heat, and drought signaling warrants further study, as does OsMIF protein roles in stress adaptation and developmental regulation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, the results presented here support the proposed hub protein model in which OsMIF1 and OsMIF2 collaborate with and/or regulate ZF-HD proteins to fine-tune rice reproductive development. Moreover, we propose that OsMIF1 and OsMIF2 also play critical roles in the responses to drought and salt stress as well as the regulation of photosynthesis. In particular, the increasing frequency of droughts and floods caused by ongoing climate change poses a serious threat to global rice production and food security. Therefore, understanding the multifaceted roles of OsMIF1 and OsMIF2 will yield valuable insight into the molecular mechanisms underlying climate adaptability in rice. Such insight is critical as a foundation for engineering climate-resilient rice cultivars and to develop strategies for stable food production in the face of environmental fluctuations.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eABA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAbscisic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eACC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e1-Aminocyclopropane-1-carboxylic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCRISPR-Cas9\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eClustered regularly interspaced short palindromic repeats\u0026ndash;CRISPR-associated protein 9\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDAF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDays after flowering\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDAG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDays after germination\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDEGs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDifferentially expressed genes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGibberellic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGene Ontology\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGUS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eβ-Glucuronidase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHomeodomain\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eKnockout\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMAPK\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMitogen-activated protein kinase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMeJA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMethyl jasmonate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMIF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMini zinc finger\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNAA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e1-Naphthaleneacetic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNon-transgenic\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eScanning electron microscopy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSSP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSeed storage protein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTranscription factor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eY2H\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eYeast two-hybrid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eZF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eZinc finger\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eZF-HD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eZinc finger\u0026ndash;homeodomain\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analyzed during this study are included in this published article and its Additional files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by a grant from the National Institute of Agricultural Science, Rural Development Administration, Republic of Korea (PJ013149), and the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (RS-2023-00248217 to K Cho, 2023R1A2C1002936, 2024H1A7A2A02000017, and BK21 FOUR (Fostering Outstanding Universities for Research, No. 4120240915070) to O Han).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJP So, KW Cho and OS Han conceived and designed the research; JP So performed the experiments; JP So, JY Lee and KW Cho analyzed the data; JP So wrote the manuscript; KW Cho, DK Kim, OS Han reviewed and revised the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the NRF and RDA, Republic of Korea.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlam M, Lou G, Abbas W, Osti R, Ahmad A, Bista S, Ahiakpa JK, He Y (2024) Improving Rice Grain Quality Through Ecotype Breeding for Enhancing Food and Nutritional Security in Asia\u0026ndash;Pacific Region. 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Int J Mol Sci 23(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms23010125\u003c/span\u003e\u003cspan address=\"10.3390/ijms23010125\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"rice","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rice","sideBox":"Learn more about [Rice](http://thericejournal.springeropen.com)","snPcode":"12284","submissionUrl":"https://submission.nature.com/new-submission/12284/3","title":"Rice","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Rice, Zinc finger-homeobox, Mini zinc finger, OsMIF1, OsMIF2, Grain size, Panicle development","lastPublishedDoi":"10.21203/rs.3.rs-8286035/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8286035/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMini zinc finger (MIF) proteins are plant-specific zinc finger-homeodomain (ZF-HD) transcription factors lacking a homeodomain, whose biological functions are critical for normal plant development and the response to environmental stress. Here, CRISPR-Cas9 was used to engineer null alleles of rice OsMIF1 and OsMIF2, and the resulting OsMIF1- and OsMIF2-deficient knockout lines were used to identify the biological roles of OsMIF1 and OsMIF2. The results suggest that OsMIF proteins transcriptionally regulate grain size by controlling the size of epidermal cells and the length and branching of rice panicles. RNA-seq analysis of OsMIF-knockout cells revealed altered expression of genes involved in development, the response to environmental stress and grain size. In addition, 10 protein-interacting partners of OsMIF1 were identified using a yeast two-hybrid screen: these proteins play roles in diverse developmental, hormonal, stress response, and metabolic processes, suggesting that OsMIF1 is effectively a regulatory hub, whose role is to integrate signals as they propagate through rice development- and stress response pathways. The results presented here support the conclusion that OsMIF1 and OsMIF2 are master transcription factors that regulate development throughout the adult plant life cycle and contribute significantly to plant resilience in the presence of environmental stressors.\u003c/p\u003e","manuscriptTitle":"Zinc Finger Homeobox Transcription Factors OsMIF1 and OsMIF2 Regulate Grain Size and Panicle Development in Rice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 18:15:50","doi":"10.21203/rs.3.rs-8286035/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-15T15:28:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-15T06:02:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154843071231732123578616800436535238503","date":"2026-01-06T11:24:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-31T08:17:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36835060931107113354316325831061273425","date":"2025-12-15T05:48:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-09T04:06:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-08T13:55:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-05T11:16:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Rice","date":"2025-12-05T09:08:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"rice","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rice","sideBox":"Learn more about [Rice](http://thericejournal.springeropen.com)","snPcode":"12284","submissionUrl":"https://submission.nature.com/new-submission/12284/3","title":"Rice","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6bbc5165-c559-41ba-9762-86df9a90f65e","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:11:43+00:00","versionOfRecord":{"articleIdentity":"rs-8286035","link":"https://doi.org/10.1186/s12284-026-00898-5","journal":{"identity":"rice","isVorOnly":false,"title":"Rice"},"publishedOn":"2026-03-10 15:59:23","publishedOnDateReadable":"March 10th, 2026"},"versionCreatedAt":"2025-12-11 18:15:50","video":"","vorDoi":"10.1186/s12284-026-00898-5","vorDoiUrl":"https://doi.org/10.1186/s12284-026-00898-5","workflowStages":[]},"version":"v1","identity":"rs-8286035","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8286035","identity":"rs-8286035","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}