The Phytochrome-Interacting Factor OsPIL11 Coordinates Grain Weight and Grain Number via Directly Regulating the Expression of OsMIR530 and OsCKX2 in Rice

The Phytochrome-Interacting Factor OsPIL11 Coordinates Grain Weight and Grain Number via Directly Regulating the Expression of OsMIR530 and OsCKX2 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 The Phytochrome-Interacting Factor OsPIL11 Coordinates Grain Weight and Grain Number via Directly Regulating the Expression of OsMIR530 and OsCKX2 in Rice Yongbin Peng, Yaping Li, Chongke Zheng, Mingjuan Zhai, Conghui Jiang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7350390/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Nov, 2025 Read the published version in Rice → Version 1 posted 9 You are reading this latest preprint version Abstract Grain weight and panicle architecture are pivotal determinants of rice yield, yet the regulatory mechanisms coordinating these traits remain elusive. Here, we functionally characterized a phytochrome-interacting factor, OsPIL11, serving as a negative regulator of grain weight and grain numbers per panicle. Knocking out OsPIL11 resulted in increased grain weight and grain number per panicle. OsPIL11 regulates grain weight by affecting cell expansion and division in the spikelet hulls, and controls grain number per panicle by regulating the number of primary branches. We functionally characterize OsMIR530 , a regulator of grain size, and OsCKX2 , a regulator of grain number, as the target genes of OsPIL11. Analysis of genetic variations suggested that OsPIL11 has likely been subjected to artificial selection during rice breeding, with Hap2 representing a superior haplotype for yield improvement. These findings provide novel insights into the molecular mechanisms underlying the regulation of rice yield, offering valuable genetic resources for the development of high-yield rice varieties through molecular breeding approaches. Rice phytochrome-interacting factor grain weight grain number molecular breeding Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Rice ( Oryza sativa L.) serves as a staple food crop for over half of the global population, with its yield directly impacting food security, particularly in China, as the largest producer and consumer (Zhou and Xue, 2020 ). Grain yield in rice is determined by three core components: grain number per panicle, tiller number, and grain weight (Zhang et al., 2024 ). Among these, grain weight is predominantly governed by grain size and filling efficiency (Sun et al., 2024 ), while panicle architecture, especially primary branch formation, plays a pivotal role in determining grain number per panicle (Li et al., 2021 ; Guo et al., 2024 ). Grain size, a complex quantitative trait encompassing length, width, and thickness, is tightly regulated by the developmental dynamics of spikelet hull cells. Critical pathways, including ubiquitin-proteasome signaling (Song et al., 2007 ; Gao et al., 2021 ; Hao et al., 2021 ), G-protein cascades (Sun et al., 2018 ), MAPK signal transduction (Ren et al., 2023 ), transcriptional regulation (Baillo et al., 2019 ), and phytohormone signal pathways (Li et al., 2021 ), have been implicated in hull cell proliferation and expansion, ultimately shaping final grain dimensions. The grain number in rice is also regulated by MAPK signal transduction (Guo et al., 2020 ), transcriptional regulation (Jiao et al., 2010 ) and phytohormone signal pathways (Tu et al., 2022 ). Extensive studies have identified key regulatory genes governing grain size and panicle branching, such as MIR530 (Sun et al., 2020 ), OsCKX2 (CYTOKININ OXIDASE 2) (Wang et al., 2018 ; Rong et al., 2022 ). Phytochrome-interacting factors (PIFs), a subclass of bHLH proteins, integrate light signaling with developmental processes in Arabidopsis. In recent years, roles of several rice phytochrome-interacting factor like genes ( OsPILs ) was reported. For instance, OsPIL13 and OsPIL15 play critical roles in drought stress responses (Todaka et al., 2012 ; Li et al., 2022 ), while overexpression of OsPIL14 in transgenic rice plants could promote mesocotyl elongation and salt tolerance in the dark (Mo et al., 2020 ). Also, OsPIL14 and OsPIL15 negatively regulate banded sclerotial blight resistance (Yuan et al., 2023 ). OsPIL15 influences grain size via cytokinin homeostasis (Ji et al., 2019 ). OsPIL12 is a negative regulatory factor in controlling rice tillers and grain length (Yang et al., 2018 ; Zhang et al., 2022 ). Although OsPIL11 has been preliminarily linked to tiller number regulation through OsTB1 (Zhang et al., 2022 ), its functional mechanisms in grain yield components (grain size, panicle branching) remain elusive. Despite advances in identifying yield-related genes, practical applications in molecular breeding are hindered by pleiotropic effects. For example, enhancing grain number per panicle often compromises grain size, highlighting the need to identify genes that coordinately optimize these traits. The dual roles of OsPIL11 in both grain size and panicle branching, and its underlying molecular framework, remain unexplored. Here, we reveal dual roles of OsPIL11 as a negative regulator of grain width and panicle branching characterized using CRISPR/Cas9 knockout (KO) and overexpression (OE) lines, while positively modulating grain length. We further deciphered its molecular framework by identifying direct targets, OsCKX2 and OsMIR530, which bridge PIF signaling with cytokinin metabolism and cell cycle regulation. This study not only elucidates a novel mechanism underlying yield component coordination but also provides genetic tools for breaking the trade-off bottleneck in rice breeding. Materials and methods Plant materials and growing conditions All of the rice plants in this study were Oryza sativa L. cv Nipponbare (Nip). The gemination of seeds has been described previously (Sun et al., 2020 ). Nip and transgenic plants were grown at the experimental field under growing conditions in Jinan, China (lat 36°400N, long 117°000E). Constructs and phenotype analysis CRISPR-cas9 gene editing was performed using the system described previously (Sun et al., 2020 ). We designed two single-RNAs targeting OsPIL11(sg1:5’-GGCGACGGCTTTGCGCCATTAGG-3’; sg2:5’-GACCTGTTCACCGAGCTGTTCGG-3’) to improve the on-target effects. The annealed oligo pair was inserted into the BGK03 vector. The resultant constructs were introduced into Agrobacteria to transform the Nip plants. To overexpress OsPIL11, the Nip plants, the OsPIL11 full-length CDS fragment with Ubi promoter was ligated with pCAMBIA1300, and then transformed into Nip. For the OsPIL11-HA construct, the full-length HA cDNA was amplified by PCR and then ligated with the PIL11-OE vector. The confirmed homozygous T2 generation plants were planted and used for phenotypic analysis. Details regarding the primer sequences used are listed in Table S1 . Measurement of coleoptile lengths Rice seeds were surface-sterilized and then sown on 0.4% (w/v) agar. After an overnight incubation at 4°C, seeds were incubated under red light (R) or far-red light (FR) at 28°C for 6 days. The seedlings were then photographed, after which the coleoptile and mesocotyl lengths were measured using a ruler. RNA extraction and real-time PCR analysis RNA extraction and real-time PCR analysis Total RNA was extracted from various tissues using TRIzol reagent (Life Technologies, Carlsbad, CA). Reverse transcribed into complementary DNA according to the manufacturer’s instructions (PrimeScript™ II 1st Strand cDNA Synthesis Kit). cDNAs were utilized for Real-time PCR analysis using the SYBR Green PCR master mix (TaKaRa). The OsEF-1α gene was used as an internal standard to normalize the expression of OsPIL11 . Details regarding the qRT-PCR primers are listed in Table S2 . GUS histochemical staining study A 2,000 bp promoter fragment of OsPIL11 was amplified and fused to pCambia1300. The resultant construction pPIL11::GUS was transformed into Nipponbare calli. GUS histochemical analysis was detected according to the previous description (Chen et al. , 2013). Different tissues from T2 homozygous plants were collected for the staining assay. Subcellular localization assay The full-length OsPIL11 coding sequence without a stop codon was fused with the eGFP coding sequence, driven by the 35S promoter, to obtain p35S::OsPIL11-eGFP . The protoplasts were isolated from the leaf sheaths of two-week-old Nipponbare seedlings. Protoplasts were transformed with the resultant construct and incubated at 28℃ for 14h in the dark before fluorescence imaging was observed with the LSM900 confocal microscopy. RNA-seq analysis Total RNAs were extracted from 1 cm length panicles of WT and OsPIL11 using TRIzol reagent (Life Technologies, Carlsbad, CA) and then purified with the RNeasy Mini kit (Qiagen, Hilden, Germany). RNA-seq libraries were prepared according to the Illumina Standard library preparation kit and sequenced on the Illumina HiSeqTM 2500 at Novogene Biotech Co., LTD (Tianjin, China). Clean data was aligned to the Nipponbare genome sequence from the MSU Rice Genome Annotation Project Database ( http://rice.plantbiology.msu.edu/index.shtml ). The expression value was normalized as fragments per kilobase of transcript per million mapped reads (FPKM). The differential expression was observed with the threshold of a false-discovery rate (FDR) < 0.05 and changed at least twofold. Chromatin immunoprecipitation (ChIP) assay Young panicles (approximately 1 cm in length) from OsPIL11-HA transgenic rice plants and wild-type (Nipponbare) were harvested and immediately fixed in PBS buffer containing 1% formaldehyde under vacuum infiltration for 10 minutes to crosslink protein-DNA complexes. The crosslinking reaction was quenched by adding glycine to a final concentration of 0.125 M, followed by incubation on ice for 5 minutes. DNA was disrupted by ultrasound following the tissue lysis buffer. The nuclear protein samples were immunoprecipitated with anti-HA antibody (Abmart, Shanghai). RT-qPCR was conducted to assess the binding efficiency of OsPIL11-DNA. Yeast one-hybrid (Y1H) assays Y1H assay was performed using the pLacZi system. The coding sequence (CDS) of OsPIL11 were cloned into the pB42AD vector to generate prey vectors, and the fragments of OsCKX2 and OsMIR530 promoter were constructed into the pLacZi vector to obtain bait constructs using specific primers (listed in Supplementary Table S1 ). The prey and the bait vectors were cotransformed into the EGY48 yeast strain. Then, the selected positive strains were grown on SD/-Ura/‐Trp medium with X‐gal at 30°C for 3 − 5 days, and the X‐gal blue staining was photographed. Electrophoretic Mobility Shift Assay (EMSA) The full-length coding sequence of OsPIL11 was cloned into the pMAL vector (BioRun) to generate a MBP-tagged recombinant protein. The protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Double-stranded DNA probes corresponding to the promoter regions of OsCKX2 and OsMIR530 (containing wild-type or mutated E-box motifs) were synthesized (sequences listed in Supplementary Table S1 ). The probes (labeled or unlabeled, or mutant) were incubated with purified OsPIL11 protein at room temperature for 20 min. The protein-DNA complexes were electrophoresed on a non-denaturing polyacrylamide gel. Results OsPIL11 positively regulates grain length but negatively regulates grain width. To comprehensively investigate the roles of OsPIL11 in rice, we generated OsPIL11 overexpression lines by introducing a ubiquitin promoter-driven LOC_Os12g41650 coding sequence into Oryza sativa L. cv. Nipponbare. Three independent T1 OE lines (OE#4, #5, and #26) exhibiting significantly elevated OsPIL11 transcript levels were selected for subsequent phenotypic and molecular analyses (Fig. S1 A-B; Fig. 1 A). Quantitative analysis under controlled light conditions revealed that both red light (660 nm) and far-red light (730 nm) treatments significantly enhanced coleoptile elongation in OE lines compared to wild-type controls (Fig. S1 C-F), implying its involvement in phytochrome -dependent signaling cascades. To dissect the role of OsPIL11 in regulating grain size, we measured grain length, grain width and grain thickness. As shown in Fig. 1 B-C, OE lines exhibited significantly increased grain length and decreased width compared to wild-type controls. However, no significant change in grain weight was observed (Fig. 1 D), which could be attributed to compensatory reductions in grain width and thickness (Fig. 1 E-F). We further generated KO lines using CRISPR/Cas9-mediated gene editing of LOC_Os12g41650 (Fig. S2 A). Three independent KO lines (#2, #11, and #13) were obtained, each carrying frameshift mutations: a 1-bp deletion in the first exon (#2), a 1-bp insertion in the first exon (#11), and a 1-bp insertion in the second exon (#13; Fig. S2 B), respectively. KO lines exhibited significantly reduced grain length and increased grain width, compared to wild-type plants. Notably, despite a decrease in grain thickness in lines #2 and #13, 1000-grain weight was elevated in all KO lines (Fig. 1 D-F). These findings indicate that OsPIL11 acts as a positive regulator of grain length but negatively regulates grain width, suggesting that the regulation of grain thickness by OsPIL11 may involve more complex mechanisms. OsPIL11 negatively regulates grain number per panicle. To further investigate the role of OsPIL11 in panicle development, we analyzed the grain number per panicle in the OE and KO lines. Overexpression lines of OsPIL11 showed a significant reduction in grain number per panicle compared to the control group (Fig. 2 A-B). In contrast, KO lines exhibited a marked increase in grain number per panicle (Fig. 2 A, D). These results suggest that OsPIL11 acts as a negative regulator of grain number per panicle. Moreover, we observed a significant decrease in the number of primary branches in OE lines relative to the control (Fig. 2 C). In contrast, KO lines displayed a substantial increase in primary branch number (Fig. 2 E). This indicates that OsPIL11 modulates spikelet number by regulating the formation of primary branches during panicle development. OsPIL11 regulates grain size by altering cell expansion and division. Mature grains are composed of lemma, palea, and endosperm. The spikelet hull restricts the overall grain size. Cell proliferation and expansion play vital roles in regulating spikelet hull growth. The spikelet hulls of OE lines exhibited a significant increase in length but a decrease in width compared to those of WT (Fig. 3 A). To clarify the cellular basis for the changes in grain size, we analyzed the cell number and morphology of spikelet hulls by paraffin sections. Compared with the WT plants, the OE and KO lines had more and fewer parenchyma cells in the grain hull, respectively (Fig. 3 B-D). These findings suggest that OsPIL11 positively regulated cell division in rice. Furthermore, the inner epidermal cells of the lemma in the OE and KO lines and WT plants were analyzed by scanning electron microscopy (Fig. 3 E). The results showed that OsPIL11 OE lines exhibited increased cell length and reduced cell width, while OsPIL11 KO lines had reduced cell length and increased cell width (Fig. 3 F and 3 G). Notably, these compensatory changes in cellular dimensions did not alter the total surface area of the inner epidermal cells in the spikelet. (Fig. 3 F-H). These results implied that OsPIL11 controls morphogenesis by modulating anisotropic cell expansion rather than overall cell size. Expression pattern and subcellular localization of OsPIL11 We initially examined the expression pattern of OsPIL11 in photoperiodic conditions. OsPIL11 transcripts were reduced during light period and increased during dark period (Fig. S3A), which supports the notion that OsPIL11 functions as a phytochrome-interacting factor in plant light-responsive signal transduction, as previously reported (Nakamura et al., 2007 ). To further explore the spatial expression pattern of OsPIL11 , we conducted qRT-PCR analyses on various tissues. OsPIL11 is ubiquitously expressed across all examined tissues, with notably higher expression levels in leaves, shoots, inner lemma, and panicles (Fig. 4 A). Additionally, OsPIL11 was found to exhibit high expression in leaf blades, lemma/palea, and panicles at various developmental stages by searching the Rice Expression Profile Database ( http://ricexpro.dna.affrc.go.jp/ ) (Fig. S3B). A search of the database from MBKbase ( https://www.mbkbase.org/rice/genotype ) also revealed the elevated expression of OsPIL11 in leaves, shoots, spikes, and panicles (Fig. S3C). To further validate these observations, we generated the transgenic plants by introducing the pPIL11::GUS vector into wild-type. Histochemical staining revealed that GUS signals were detected in most of the examined organs, consistent with the qRT-PCR data of OsPIL11 (Fig. 4 B). To determine the subcellular localization of the OsPIL11 protein, we transformed p35S::OsPIL11-GFP and p35S::GFP constructs into rice protoplasts. OsPIL11-GFP signals predominantly observed in the nucleus, while GFP signals could be observed in both cytoplasm and nucleus (Fig. 4 C). These results suggest that OsPIL11 protein is nucleus-localized, consistent with its role as a transcription factor. Identification of genes regulated by OsPIL11 To further elucidate the molecular mechanisms underlying the role of OsPIL11 in regulating grain size and grain number, we performed RNA-Seq analysis using panicles (1 cm in length) from both WT and KO lines. Comparative transcriptomic profiling identified a total of 2,185 differentially expressed genes (DEGs) between the two genotypes (padj 2), comprising 962 up-regulated and 1,223 down-regulated genes in the KO lines (Fig. 5 A). KEGG and GO analyses suggest that OsPIL11 modulates complex regulatory networks influencing rice grain yield, including diterpenoid biosynthesis, photosynthesis, MAPK signaling, plant hormone signal transduction and zeatin biosynthesis (Fig. S4 A-B). Specifically, we identified 15 genes associated with diterpenoid biosynthesis, 5 DEGs involved in photosynthesis, 8 genes related to the plant MAPK signaling pathway, 25 genes encoding plant hormone signal transduction proteins and 5 DEGs related to zeatin biosynthesis. These genes were emphasized as they synergistically regulate defense metabolism (diterpenoids), stress signaling (MAPK), energy homeostasis (photosynthesis), and hormonal coordination, defining an integrated adaptive strategy against development in rice (Fig. S4 C). These findings suggest that OsPIL11 exerts its regulatory effects on grain development and yield-related traits by modulating multiple biological processes and signaling pathways. Among the above-mentioned processes, several genes have been reported to be related to the grain size and panicle development, such as WRKY24 , OsIAA10 , OsSCP46 , OsPIL15 , and OsCKX2 . The heat map analysis further illustrated significant changes in their expression levels between WT and KO lines (Fig. 5 B-C). Interestingly, there were many light-responsive elements, such as E-box, N-box, PBE-box, and G-box, that were observed in the promoters of the genes above-mentioned (Fig. 5 D). These results suggest that these genes might be directly or indirectly regulated by OsPIL11. OsPIL11 directly binds to the promoters of OsCKX2 and OsMIR530. To identify the target genes directly regulated by OsPIL11 in regulating rice grain size and grain numbers, ChIP-seq analysis was performed using panicles (1 cm in length) of OsPIL11-HA lines, which exhibit similar phenotypes to OsPIL11 -OE lines (Fig. S5 ). Several potential targets were enriched, including OsCKX2 and OsMIR530 promoters (Fig. 6 A; Fig. S6 ). The interaction between OsPIL11 and either OsCKX2 or OsMIR530 promoters was confirmed using Y1H assay (Fig. 6 B-C). To further determine whether OsPIL11 directly targets OsCKX2 and OsMIR530 , we conducted EMSA using the purified OsPIL11-His recombinant protein and OsCKX2 and OsMIR530 probes with normal and mutated E-box sequences. The binding abilities of OsPIL11 to the fragments decreased significantly in the presence of unlabeled competitor probes, but not with the unlabeled probes with mutated G-box sequences (Fig. 6 D). Therefore, it can be concluded that OsPIL11 has the potential to interact with OsCKX2 and OsMIR530 promoters. Haplotype and population analyses of OsPIL11 To elucidate the natural allelic variation of OsPIL11 , we analyzed 533 core accessions from the genomic database of Sichuan Agricultural University (Zhou et al., 2017 ).Sequence variations in the promoter, coding sequence (CDS), and untranslated regions (UTR) of PIL11 revealed two SNPs in the promoter region and five SNPs in the CDS, forming two major haplotypes (Hap1 and Hap2). Notably, a promoter SNP localized within a cis-acting regulatory element suggested divergent transcriptional regulation between haplotypes (Fig. 7 A). Phenotypic-genotypic association analysis demonstrated that Hap1 exhibited significantly greater grain width ( P < 0.01) and weight ( P < 0.05). In contrast, grain length remained statistically invariant, compared to Hap2 (Fig. 7 B-D). These findings highlight PIL11 as a determinant of grain morphology and yield, with Hap1 representing a superior haplotype for grain weight improvement. Subpopulation analysis revealed distinct genetic backgrounds: Hap1 predominated in japonica (60.9%), compared with indica (38.74%). In contrast, Hap2 was predominantly in indica (90.5%) with a small amount of Aus accessions (8.15%; Fig. S7 ). Evolutionary analysis further indicated that PIL11 has undergone artificial selection during domestication (Fig. S7). To validate the reliability of OsPIL11 functions, we compared the grain size of chromosome segment substitution lines (CSSLs) using Nipponbare as the recipient parent and Sea rice 86 (SR86) as the donor parent. Through molecular marker-assisted selection, two CSSLs (N-S-1/2 SR86 ) carrying PIL11 chromosomal segments from SR86 were developed, which exhibited a genetic background closely resembling that of the recurrent parent Nipponbare (Fig. S8). Comparative analysis revealed that the grain length of N-S-1/2 SR86 lines showed no significant differences compared with Nipponbare ( p > 0.05) (Fig. 7 E and G). In contrast, their grain width was significantly lower than that of the recurrent parent ( p < 0.05) (Fig. 7 F and H). Additionally, two non-introgressed substitution lines (N-S-1/2 NIP ) derived from the same population demonstrated comparable grain length, width, and weight to Nipponbare (Fig. 7 E-I). Collectively, these results provide critical insights into the functional divergence of OsPIL11 haplotypes and establish a foundation for their utilization in molecular breeding programs targeting yield enhancement in rice. Discussion Although rice PIFs regulate diverse growth processes in rice, such as flowering time, transpiration, and disease-resistance (Paik et al., 2017 ), little has been known about PIFs in regulating grain yield. In this study, for the first time, we reported the OsPIL11 coordinates grain weight and grain number via directly regulating the expression of OsMIR530 and OsCKX2 . PIL11 is a negative regulator of grain width and grain number In the present study, we constructed transgenic rice (KO, OE lines) to examine the role of OsPIL11 in regulating rice yield. Grain length and grain width showed reverse phenotypes in both KO and OE lines (Fig. 1 B-E, IL), suggesting that OsPIL11 positively regulates grain length but negatively regulates grain width. This result is different from the observation from OsPIL15, where However, OsPIL15 negatively regulates grain length and width (Sun et al, 2020 ). The functional difference is probably related to their expression pattern that OsPIL15 is highly expressed in florets within 35 days after flowering (DAF), whereas OsPIL11 shows negligible expression during this grain-filling period. However, 1000-grain weight was increased in KO lines, but no significant change in OE lines of OsPIL11 (Fig. 1 G, N). In terms of cytological mechanism, the loss of OsPIL11 function results in a reduction of parenchyma cells in the glume, while its overexpression leads to an increase in parenchyma cells (Fig. 3 B-D), indicating that OsPIL11 positively regulates cell division. On the other hand, OsPIL11 regulates cell length and width in opposite trends, resulting in no significant changes in cell area (Fig. 3 E-H). In conclusion, OsPIL11 regulates grain size by modulating the number of parenchyma cells and their shape. Moreover, we found that OsPIL11 negatively regulates grain numbers per panicle. As shown in Fig. 2 , OsPIL11 deficiency increases the grain number, while its overexpression decreases the grain number per panicle, mainly through modulating the number of primary branches. Our previous research revealed that OsPIL11 negatively regulate tiller number by directly activating the expression of OsTB1 (Zhang et al., 2021). In conclusion, given that the functional loss of OsPIL11 contributes to the improvement of the three primary yield components, namely efficient tiller number, grain number per panicle and 1000-grain weight, OsPIL11 exhibits considerable potential for practical application in rice breeding. OsPIL11 may regulate grain size and grain numbers by targeting OsMIR530 and OsCKX2 directly In the present study, RNA-seq results revealed that OsPIL11 mainly regulate several pathways during panicle development, including diterpenoid biosynthesis, photosynthesis, MAPK signaling pathway, plant hormone signal transduction proteins, and zeatin biosynthesis (Fig S4). ChIP-seq assay, Y1H and EMSA assay indicates that OsCKX2 and OsMIR530 were potential target genes of OsPIL11. Previous research has demonstrated that OsmiR530 is a negative regulator of rice grain yield by affecting cell division and expansion in spikelet hulls (Sun et al., 2020 ). The loss of function of OsmiR530 promotes the grain weight and panicle branching, which is consistent with the agronomic characters of OsPIL11 KO lines. OsCKX2 encodes cytokinin oxidase/dehydrogenase, an enzyme responsible for degrading cytokinin (Wang et al., 2018 ). Knocking out of OsCKX2 enhances the accumulation of cytokinin and the number of grains per panicle (Rong et al., 2022 ). These results indicated that OsPIL11 modulating grain size and numbers mainly via regulating OsCKX2 and OsMIR530 expression. Haplotype divergence of OsPIL11: implications for grain morphology and yield in XI/GJ rice In this study, we identified two major haplotypes of OsPIL11 , Hap1 and Hap2, with distinct effects on grain morphology and yield (Fig. 7 ). The promoter SNP (cis-element) and CDS variations likely drive transcriptional divergence, leading to Hap1’s wider grains but lower yield compared to Hap2. The predominance of Hap1 in japonica and Hap2 in indica aligns with their distinct domestication trajectories. High yield in Hap2 accessions may reflect strong selection in indica for panicle productivity, whereas wider grains in Hap1 accession could be favored in japonica under specific agroecological conditions. In summary, our findings indicated that OsPIL11 played a crucial regulatory role in modulating grain size and numbers through interacting with target genes OsCKX2 and OsMIR530 . This study expands the understanding of the functional roles of PIF family genes in regulating rice grain size and grain numbers, while also elucidating the intricate relationship between grain size and grain numbers. Further investigation of OsPIL11 is expected to enhance our understanding of its role in the differentiation of XI and GJ grain traits, thereby facilitating the development of high-yielding rice varieties in the future. Declarations Acknowledgements The authors thank Rice Research Institute of Sichuan Agricultural University for supporting this study. Author Contributions Y.P. and Y.L. conducted the experiments, analysed the data and drafted the manuscript. C.Z., M.Z., and C.J. conducted the analyses. Z.L., X.X., and G.Z. were involved in performing the experiments. X.X. contributed to the study conception and design. All authors read and approved the final manuscript. Funding This research was supported by grants from the Shandong Agricultural Elite Variety Project (2024LZGC009), the Shandong Academy of Agricultural Sciences Innovation Project (CXGC2025H08), the National Natural Science Foundation of China (32172096), the Shandong Modern Agricultural Industry system (SDAIT-17-9; SDAIT-17-17). Data Availability Sequencing data of RNA-seq (Bioproject PRJCA043052) can be found in Beijing Institute of Genomics Data Center (http://bigd.big.ac.cn). Ethics Approval and Consent to Participate Not applicable. Consent for Publication Not applicable. Competing Interests The authors declare no competing interests. References Baillo, E.H., Kimotho, R.N., Zhang, Z.B., and Xu, P. (2019). Transcription factors associated with abiotic and biotic stress tolerance and their potential for crops improvement. Genes (Basel) 10 ,771. 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Song, X.J., Huang, W., Shi, M., Zhu, M.Z., and Lin, H.X. (2007). A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase. Nature Genetics 39 ,623-630. Sun, L.Q., Bai, Y., Wu, J., Fan, S.J., Chen, S.Y., Zhang, Z.Y., Xia, J.Q., Wang, S.M., Wang, Y.P., Qin, P., Li, S.G., Xu, P., Zhao, Z., Xiang, C.B., and Zhang, Z.S. (2024). OsNLP3 enhances grain weight and reduces grain chalkiness in rice. Plant communications 5 ,100999. Sun, S.Y., Wang, L., Mao, H.L., Shao, L., Li, X.H., Xiao, J.H., Ouyang, Y.D., and Zhang, Q.F. (2018). A G-protein pathway determines grain size in rice. Nature Communications 9 , 851. Sun, W., Xu, X.H., Li, Y.P., Xie, L.X., He, Y.N., Li, W., Lu, X.B., Sun, H.W., and Xie, X.H. (2020). OsmiR530 acts downstream of OsPIL15 to regulate grain yield in rice. New Phytologist 226 ,823-837. Todaka, D., Nakashima, K., Maruyama, K., Kidokoro, S., Osakabe, Y., Ito, Y., Matsukura, S., Fujita, Y., Yoshiwara, K., Ohme-Takagi, M., Kojima, M., Sakakibara, H., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2012). Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces a morphological response to drought stress. Proceedings Of the National Academy Of Sciences Of the United States Of America 109 ,15947-15952. Tu, B., Tao, Z., Wang, S., Zhou, L., Zheng, L., Zhang, C., Li, X., Zhang, X., Yin, J., Zhu, X., Yuan, H., Li, T., Chen, W., Qin, P., Ma, B., Wang, Y., and Li, S. (2022). Loss of Gn1a/OsCKX2 confers heavy-panicle rice with excellent lodging resistance. J Integr Plant Biol 64 ,23-38. Wang, S.G., Ma, B.T., Gao, Q., Jiang, G.J., Zhou, L., Tu, B., Qin, P., Tan, X.Q., Liu, P.X., Kang, Y.H., Wang, Y.P., Chen, W.L., Liang, C.Z., and Li, S.G. (2018). Dissecting the genetic basis of heavy panicle hybrid rice uncovered and as key genes. Theoretical And Applied Genetics 131 ,1391-1403. Yang, X.M., Ren, Y.L., Cai, Y., Niu, M., Feng, Z.M., Jing, R.N., Mou, C.L., Liu, X., Xiao, L.J., Zhang, X., Wu, F.Q., Guo, X.P., Jiang, L., and Wan, J.M. (2018). Overexpression of OsbHLH107, a member of the basic helix-loop-helix transcription factor family, enhances grain size in rice (Oryza sativa L.). Rice 11 ,41. Yuan, D.P., Yang, S., Feng, L., Chu, J., Dong, H., Sun, J., Chen, H., Li, Z., Yamamoto, N., Zheng, A.P., Li, S., Yoon, H.C., Chen, J.S., Ma, D.R., and Xuan, Y.H. (2023). Red-light receptor phytochrome B inhibits BZR1-NAC028-CAD8B signaling to negatively regulate rice resistance to sheath blight. Plant Cell Environ 46 ,1249-1263. Zhang, J., Zhang, Y., Chen, J., Xu, M., Guan, X., Wu, C., Zhang, S., Qu, H., Chu, J., Xu, Y., Gu, M., Liu, Y., and Xu, G. (2024). Sugar transporter modulates nitrogen-determined tillering and yield formation in rice 15 ,9233. Zhang, L.C., He, G.H., Li, Y.P., Yang, Z.Y., Liu, T.Q., Xie, X.Z., Kong, X.Y., and Sun, J.Q. (2022). PIL transcription factors directly interact with SPLs and repress tillering/branching in plants. New Phytologist 233 ,1414-1425. Zhou, H., Li, P., Xie, W., Hussain, S., Li, Y., Xia, D., Zhao, H., Sun, S., Chen, J., Ye, H., Hou, J., Zhao, D., Gao, G., Zhang, Q., Wang, G., Lian, X., Xiao, J., Yu, S., Li, X., and He, Y. (2017). Genome-wide Association Analyses Reveal the Genetic Basis of Stigma Exsertion in Rice. Mol Plant 10 , 634-644. Zhou, S.R., and Xue, H.W. (2020). The rice PLATZ protein SHORT GRAIN6 determines grain size by regulating spikelet hull cell division. Journal Of Integrative Plant Biology 62 ,847-864. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformationlegends.docx Supplementalfigure20250811.docx Supplementaltable.xlsx Cite Share Download PDF Status: Published Journal Publication published 28 Nov, 2025 Read the published version in Rice → Version 1 posted Editorial decision: Revision requested 21 Sep, 2025 Reviews received at journal 18 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviews received at journal 24 Aug, 2025 Reviewers agreed at journal 19 Aug, 2025 Reviewers invited by journal 17 Aug, 2025 Editor assigned by journal 12 Aug, 2025 Submission checks completed at journal 12 Aug, 2025 First submitted to journal 11 Aug, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7350390","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":504817728,"identity":"04499df3-3029-4ae2-be7b-f7d7457b7ebd","order_by":0,"name":"Yongbin Peng","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yongbin","middleName":"","lastName":"Peng","suffix":""},{"id":504817729,"identity":"fb7caa3c-ef70-4999-84bd-e26af3099d61","order_by":1,"name":"Yaping Li","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yaping","middleName":"","lastName":"Li","suffix":""},{"id":504817730,"identity":"aba30c93-71a6-4c8c-855b-751d9809f364","order_by":2,"name":"Chongke Zheng","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Chongke","middleName":"","lastName":"Zheng","suffix":""},{"id":504817731,"identity":"12fa4ae2-76b9-4013-b69b-bd7d415a7608","order_by":3,"name":"Mingjuan Zhai","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mingjuan","middleName":"","lastName":"Zhai","suffix":""},{"id":504817732,"identity":"099d4482-94f0-4934-b95e-738b2d781e00","order_by":4,"name":"Conghui Jiang","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Conghui","middleName":"","lastName":"Jiang","suffix":""},{"id":504817733,"identity":"ac33912f-a933-4db4-8c8d-b8fb41a03585","order_by":5,"name":"Ziye Liu","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ziye","middleName":"","lastName":"Liu","suffix":""},{"id":504817734,"identity":"66c56c98-52dd-429a-bf8b-c91a98578e66","order_by":6,"name":"Xiaohui Xu","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaohui","middleName":"","lastName":"Xu","suffix":""},{"id":504817735,"identity":"fd04f6fa-1bce-4421-b715-26ad48608c7a","order_by":7,"name":"Guanhua Zhou","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Guanhua","middleName":"","lastName":"Zhou","suffix":""},{"id":504817736,"identity":"a9d1aac5-be70-4c2d-bcbb-29288bd6eca9","order_by":8,"name":"Xianzhi Xie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYFACHgaGBAYbHjZm5gMHPvwgXkuaHB97W+LBmT3EamFgOGwsx3PG+DAHGxEaDM6fPSbxoII5sU0i58NhoH55frED+LVINpxLNkg4wwbUkrvhcIEFg+HM2Qn4tfAz9hg+SGzjgWiZwcOQYHCbgBY2Zh6DA4n/JEAOe3CYh40ILfxsPEBbGgyM2XjOMBCnRbKHx9gg4ViCHBt7mwEwkCUI+8Xg/BkzyR81/3nkm5kff/jww0aeX5qAFnQgQZryUTAKRsEoGAXYAQA91kIffLiIZwAAAABJRU5ErkJggg==","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Xianzhi","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2025-08-12 02:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7350390/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7350390/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12284-025-00865-6","type":"published","date":"2025-11-28T15:58:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89850853,"identity":"c367e025-8cc6-431b-aaa9-0db67e19bbf0","added_by":"auto","created_at":"2025-08-25 17:26:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":443781,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypes of \u003cem\u003eOsPIL11\u003c/em\u003e overexpression transgenic (OE) and knock-out (KO) lines. (A-C) Phenotypes (plant architecture, grain length, and width) of wild-type and OE lines. Bars (A) = 10 cm, Bars (B-C) = 1cm. (D-G) Growth traits (grain length, grain width, grain thickness, and thousand-grain weight) of wild-type and OE lines. (H-J) Phenotypes (plant architecture, grain length, and width) of wild-type and KO lines. Bars (H) = 10cm, Bars (I-J) = 1cm. (K-N) Growth traits (grain length, grain width, grain thickness, and thousand-grain weight) of wild-type and KO lines. N.S. indicates not significant.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7350390/v1/b0dec349ee75e763645469d8.png"},{"id":89850154,"identity":"f9f3a123-a998-4493-879b-43cea12d3602","added_by":"auto","created_at":"2025-08-25 17:18:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":240709,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic analysis of panicles in \u003cem\u003ePIL11\u003c/em\u003e- transgenic lines. (A) Phenotypes of wild-type, \u003cem\u003eOsPIL11\u003c/em\u003e overexpression lines (OE), and knock-out lines (KO). Bars = 4 cm. (B-C) Growth traits (grain number per panicle, primary branch number) of wild-type and OE lines. (D-E) Growth traits (grain number per panicle, primary branch number) of wild-type and KO lines. N.S. indicates not significant.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7350390/v1/21fbbead5b67fa9978be65cb.png"},{"id":89850156,"identity":"788f77fb-e8dd-4991-a5b9-b36aebc052d3","added_by":"auto","created_at":"2025-08-25 17:18:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":320301,"visible":true,"origin":"","legend":"\u003cp\u003eHistological analysis of spikelet hulls in \u003cem\u003eOsPIL11\u003c/em\u003e-transgenic lines and WT plants. (A) Spikelet hulls of rice WT and transgenic plants just before flowering. (B) Cross-sections of the middle parts of spikelet hulls of rice WT and transgenic plants just before anthesis (bottom). Bars = 200 μm. (C) Magnified images of the boxed cross-section areas of (B). Bars = 50 μm. (D) Number of cells in the outer parenchyma layer of the spikelet hulls of rice WT and transgenic plants (n = 5 spikelets). (E) Scanning electron micrographs of the inner epidermal cells of lemmas in the mature seeds of rice WT and transgenic plants. Bars = 50 μm. (F) Cell lengths in the middle part of the inner epidermis of lemmas in fully mature rice seeds (n = 5 seeds). (G) Cell widths in the middle part of the inner epidermis of lemmas in fully mature rice seeds (n = 5 seeds). (H) Cell areas in the middle part of the inner epidermis of lemmas in fully mature rice seeds (n = 5 seeds). N.S. indicates not significant.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7350390/v1/9b73cfb8ae3c8d8c183094f9.png"},{"id":89850854,"identity":"4e874aae-fab6-4127-b467-4fc0f5340be8","added_by":"auto","created_at":"2025-08-25 17:26:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":137336,"visible":true,"origin":"","legend":"\u003cp\u003eExpression pattern of \u003cem\u003eOsPIL11\u003c/em\u003e and subcellular localization of OsPIL11 protein. (A) Quantitative qRT-PCR analysis of \u003cem\u003eOsPIL11\u003c/em\u003e expression in various tissues. The root and leaf materials were harvested from mature WT plants. The inner lemma, endosperm, and panicle were harvested 7 days after pollination. The shoot and shoot apical meristem were collected. Three biological repeats were performed. The data are shown as means ± SD. (B) A promoter-GUS fusion study reveals the expression of \u003cem\u003eOsPIL11\u003c/em\u003ein the plumule, radicle, booting, spikelet, and stem. Positive transgenic plants containing the \u003cem\u003epPIL11::GUS\u003c/em\u003e construct were analyzed, and representative images are shown. (C) Transient expression of the OsPIL11-GFP fusion protein in rice protoplasts and the observation of the corresponding green fluorescence.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7350390/v1/76c3b354a7adce1a4dd29738.png"},{"id":89850856,"identity":"e2bbc30a-a73f-45a7-8c07-1ced7b825a34","added_by":"auto","created_at":"2025-08-25 17:26:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":229323,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic profiling of rice panicles in \u003cem\u003eOsPIL11\u003c/em\u003e knockout (KO) and wild-type (WT) plants. (A) Volcano plot showing differentially expressed genes (DEGs) between KO and WT lines, n = 31,062. (B) Heatmap of selected DEGs involved in key biological pathways. Color scale indicates log2-transformed fold changes (red: upregulation; green: downregulation). (C) Box plot summarizing the expression levels of up-regulated (red) and down-regulated (green) genes. (D) Analysis of the promoter region motifs of DEGs.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7350390/v1/6d263fae77cc13d67acfff50.png"},{"id":89851756,"identity":"dce494d4-7d97-43f0-9694-4be1216b5ef0","added_by":"auto","created_at":"2025-08-25 17:42:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":194269,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of the \u003cem\u003eOsCKX2\u003c/em\u003eand \u003cem\u003eOsMIR530\u003c/em\u003e genes as a direct target of OsPIL11. (A) Chromatin immunoprecipitation and quantitative real-time (qRT)-PCR analysis of \u003cem\u003eCKX2 \u003c/em\u003eand \u003cem\u003eMIR530\u003c/em\u003e. The DNA samples acquired before immunoprecipitation were used as the input (PIL11-IP), and IgG was set as a negative control (PIL11-IgG). Signal intensities were first normalized relative to the input, then the enrichment of each fragment was calculated using the Input% of IgG as a baseline. (B) Schematic representation of E-box elements in \u003cem\u003eOsCKX2 \u003c/em\u003eand \u003cem\u003eMIR530\u003c/em\u003e promoters. (C) Yeast-one-hybrid assay of the binding of OsPIL11 to the \u003cem\u003eOsCKX2\u003c/em\u003e and \u003cem\u003eMIR530\u003c/em\u003e promoters. For \u003cem\u003eOsCKX2\u003c/em\u003e, five E-box-containing fragments (C1, C2, C4, C5, C6) and one E-box-deficient control fragment (C3) were synthesized. For \u003cem\u003eMIR530\u003c/em\u003e, a fragment harboring E-box element(s) was synthesized. All fragments were cloned into the reporter vector pLacZi. Positive controls consisted of the empty vectors pB42AD and pLacZi. (D) Electrophoretic mobility shift assay. Unlabeled probes containing E-box elements at 10× and 50× the amount of the labeled probe competed for the OsPIL11 binding sites. Unlabeled probes with mutations in E-box elements were also used in this assay.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7350390/v1/eee442c935d71f59ee039019.png"},{"id":89850173,"identity":"404c446a-e6c4-4072-b6d9-b18a0450ec60","added_by":"auto","created_at":"2025-08-25 17:18:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":477200,"visible":true,"origin":"","legend":"\u003cp\u003eNatural variation of \u003cem\u003eOsPIL11\u003c/em\u003e alleles in cultivated rice. (A) The promoter and gene structure of \u003cem\u003ePIL11\u003c/em\u003e and DNA polymorphisms, and haplotypes (Hap) of \u003cem\u003ePIL11 \u003c/em\u003eamong these accessions based on the SNP of promoter and exon. These polymorphic changes allow the classification of cultivated rice into five haplotypes, which are mainly Hap 1 and Hap 2 based on the number of varieties. The positions of the SNPs are relative to 2,000 bp upstream of the ATG start codon in the OsPIL11 coding region. Different colors indicate different polymorphisms. (B-D) Box plot depicting grain length, grain width, grain weight, and grain yield for Hap1 and Hap2. * indicated significant differences in grain width, weight, and yield according to a one-way ANOVA and Duncan’s least significant range test (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05). N.S. indicated not significant. (E-F) Phenotypes of grain length (E) and width (F) of CSSLs carrying and not carrying \u003cem\u003ePIL11\u003c/em\u003e sites. Scale bar, 1 cm. (G-I) Grain length (G), width (H), and weight (I) of CSSLs carrying and not carrying \u003cem\u003ePIL11\u003c/em\u003e sites. N.S. indicates not significant.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7350390/v1/d90a72bd9f178ddb6d779197.png"},{"id":97178741,"identity":"d50daf41-ba69-406a-b6a2-455f84f73a42","added_by":"auto","created_at":"2025-12-01 16:13:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3768149,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7350390/v1/467c975b-4a80-4ba4-9399-2abb10e71b18.pdf"},{"id":89850153,"identity":"42d90a6c-af35-4014-bafd-25bb30692a85","added_by":"auto","created_at":"2025-08-25 17:18:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14767,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationlegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-7350390/v1/6b0174cc74f0c44e04dbdc56.docx"},{"id":89851755,"identity":"9aca2312-bed0-4417-98b8-15a0c4926759","added_by":"auto","created_at":"2025-08-25 17:42:26","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1868825,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfigure20250811.docx","url":"https://assets-eu.researchsquare.com/files/rs-7350390/v1/ddc33705589577a011515afa.docx"},{"id":89850855,"identity":"b0848219-ea76-40cb-b13c-83f067b84061","added_by":"auto","created_at":"2025-08-25 17:26:26","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20215,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaltable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7350390/v1/e0790da29308bf4ada359422.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Phytochrome-Interacting Factor OsPIL11 Coordinates Grain Weight and Grain Number via Directly Regulating the Expression of OsMIR530 and OsCKX2 in Rice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRice (\u003cem\u003eOryza sativa\u003c/em\u003e L.) serves as a staple food crop for over half of the global population, with its yield directly impacting food security, particularly in China, as the largest producer and consumer (Zhou and Xue, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Grain yield in rice is determined by three core components: grain number per panicle, tiller number, and grain weight (Zhang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Among these, grain weight is predominantly governed by grain size and filling efficiency (Sun et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), while panicle architecture, especially primary branch formation, plays a pivotal role in determining grain number per panicle (Li et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGrain size, a complex quantitative trait encompassing length, width, and thickness, is tightly regulated by the developmental dynamics of spikelet hull cells. Critical pathways, including ubiquitin-proteasome signaling (Song et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Gao et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hao et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), G-protein cascades (Sun et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), MAPK signal transduction (Ren et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), transcriptional regulation (Baillo et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and phytohormone signal pathways (Li et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), have been implicated in hull cell proliferation and expansion, ultimately shaping final grain dimensions. The grain number in rice is also regulated by MAPK signal transduction (Guo et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), transcriptional regulation (Jiao et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and phytohormone signal pathways (Tu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Extensive studies have identified key regulatory genes governing grain size and panicle branching, such as MIR530 (Sun et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003eOsCKX2\u003c/em\u003e (CYTOKININ OXIDASE 2) (Wang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rong et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePhytochrome-interacting factors (PIFs), a subclass of bHLH proteins, integrate light signaling with developmental processes in Arabidopsis. In recent years, roles of several rice phytochrome-interacting factor like genes (\u003cem\u003eOsPILs\u003c/em\u003e) was reported. For instance, \u003cem\u003eOsPIL13\u003c/em\u003e and \u003cem\u003eOsPIL15\u003c/em\u003e play critical roles in drought stress responses (Todaka et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), while overexpression of \u003cem\u003eOsPIL14\u003c/em\u003e in transgenic rice plants could promote mesocotyl elongation and salt tolerance in the dark (Mo et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Also, \u003cem\u003eOsPIL14\u003c/em\u003e and \u003cem\u003eOsPIL15\u003c/em\u003e negatively regulate banded sclerotial blight resistance (Yuan et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eOsPIL15\u003c/em\u003e influences grain size via cytokinin homeostasis (Ji et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eOsPIL12\u003c/em\u003e is a negative regulatory factor in controlling rice tillers and grain length (Yang et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although \u003cem\u003eOsPIL11\u003c/em\u003e has been preliminarily linked to tiller number regulation through \u003cem\u003eOsTB1\u003c/em\u003e (Zhang et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), its functional mechanisms in grain yield components (grain size, panicle branching) remain elusive.\u003c/p\u003e\u003cp\u003eDespite advances in identifying yield-related genes, practical applications in molecular breeding are hindered by pleiotropic effects. For example, enhancing grain number per panicle often compromises grain size, highlighting the need to identify genes that coordinately optimize these traits. The dual roles of \u003cem\u003eOsPIL11\u003c/em\u003e in both grain size and panicle branching, and its underlying molecular framework, remain unexplored. Here, we reveal dual roles of OsPIL11 as a negative regulator of grain width and panicle branching characterized using CRISPR/Cas9 knockout (KO) and overexpression (OE) lines, while positively modulating grain length. We further deciphered its molecular framework by identifying direct targets, OsCKX2 and OsMIR530, which bridge PIF signaling with cytokinin metabolism and cell cycle regulation. This study not only elucidates a novel mechanism underlying yield component coordination but also provides genetic tools for breaking the trade-off bottleneck in rice breeding.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant materials and growing conditions\u003c/h2\u003e\u003cp\u003eAll of the rice plants in this study were \u003cem\u003eOryza sativa\u003c/em\u003e L. cv Nipponbare (Nip). The gemination of seeds has been described previously (Sun et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Nip and transgenic plants were grown at the experimental field under growing conditions in Jinan, China (lat 36\u0026deg;400N, long 117\u0026deg;000E).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eConstructs and phenotype analysis\u003c/h3\u003e\n\u003cp\u003eCRISPR-cas9 gene editing was performed using the system described previously (Sun et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). We designed two single-RNAs targeting OsPIL11(sg1:5\u0026rsquo;-GGCGACGGCTTTGCGCCATTAGG-3\u0026rsquo;; sg2:5\u0026rsquo;-GACCTGTTCACCGAGCTGTTCGG-3\u0026rsquo;) to improve the on-target effects. The annealed oligo pair was inserted into the BGK03 vector. The resultant constructs were introduced into Agrobacteria to transform the Nip plants. To overexpress OsPIL11, the Nip plants, the OsPIL11 full-length CDS fragment with Ubi promoter was ligated with pCAMBIA1300, and then transformed into Nip. For the OsPIL11-HA construct, the full-length HA cDNA was amplified by PCR and then ligated with the PIL11-OE vector. The confirmed homozygous T2 generation plants were planted and used for phenotypic analysis. Details regarding the primer sequences used are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eMeasurement of coleoptile lengths\u003c/h3\u003e\n\u003cp\u003eRice seeds were surface-sterilized and then sown on 0.4% (w/v) agar. After an overnight incubation at 4\u0026deg;C, seeds were incubated under red light (R) or far-red light (FR) at 28\u0026deg;C for 6 days. The seedlings were then photographed, after which the coleoptile and mesocotyl lengths were measured using a ruler.\u003c/p\u003e\n\u003ch3\u003eRNA extraction and real-time PCR analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eRNA extraction and real-time PCR analysis\u003c/div\u003e\u003cp\u003eTotal RNA was extracted from various tissues using TRIzol reagent (Life Technologies, Carlsbad, CA). Reverse transcribed into complementary DNA according to the manufacturer\u0026rsquo;s instructions (PrimeScript\u0026trade; II 1st Strand cDNA Synthesis Kit). cDNAs were utilized for Real-time PCR analysis using the SYBR Green PCR master mix (TaKaRa). The \u003cem\u003eOsEF-1α\u003c/em\u003e gene was used as an internal standard to normalize the expression of \u003cem\u003eOsPIL11\u003c/em\u003e. Details regarding the qRT-PCR primers are listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eGUS histochemical staining study\u003c/h3\u003e\n\u003cp\u003eA 2,000 bp promoter fragment of OsPIL11 was amplified and fused to pCambia1300. The resultant construction \u003cem\u003epPIL11::GUS\u003c/em\u003e was transformed into Nipponbare calli. GUS histochemical analysis was detected according to the previous description (Chen \u003cem\u003eet al.\u003c/em\u003e, 2013). Different tissues from T2 homozygous plants were collected for the staining assay.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSubcellular localization assay\u003c/h2\u003e\u003cp\u003eThe full-length \u003cem\u003eOsPIL11\u003c/em\u003e coding sequence without a stop codon was fused with the eGFP coding sequence, driven by the \u003cem\u003e35S\u003c/em\u003e promoter, to obtain \u003cem\u003ep35S::OsPIL11-eGFP\u003c/em\u003e. The protoplasts were isolated from the leaf sheaths of two-week-old Nipponbare seedlings. Protoplasts were transformed with the resultant construct and incubated at 28℃ for 14h in the dark before fluorescence imaging was observed with the LSM900 confocal microscopy.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRNA-seq analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNAs were extracted from 1 cm length panicles of WT and \u003cem\u003eOsPIL11\u003c/em\u003e using TRIzol reagent (Life Technologies, Carlsbad, CA) and then purified with the RNeasy Mini kit (Qiagen, Hilden, Germany). RNA-seq libraries were prepared according to the Illumina Standard library preparation kit and sequenced on the Illumina HiSeqTM 2500 at Novogene Biotech Co., LTD (Tianjin, China). Clean data was aligned to the Nipponbare genome sequence from the MSU Rice Genome Annotation Project Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rice.plantbiology.msu.edu/index.shtml\u003c/span\u003e\u003cspan address=\"http://rice.plantbiology.msu.edu/index.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The expression value was normalized as fragments per kilobase of transcript per million mapped reads (FPKM). The differential expression was observed with the threshold of a false-discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and changed at least twofold.\u003c/p\u003e\n\u003ch3\u003eChromatin immunoprecipitation (ChIP) assay\u003c/h3\u003e\n\u003cp\u003eYoung panicles (approximately 1 cm in length) from OsPIL11-HA transgenic rice plants and wild-type (Nipponbare) were harvested and immediately fixed in PBS buffer containing 1% formaldehyde under vacuum infiltration for 10 minutes to crosslink protein-DNA complexes. The crosslinking reaction was quenched by adding glycine to a final concentration of 0.125 M, followed by incubation on ice for 5 minutes. DNA was disrupted by ultrasound following the tissue lysis buffer. The nuclear protein samples were immunoprecipitated with anti-HA antibody (Abmart, Shanghai). RT-qPCR was conducted to assess the binding efficiency of OsPIL11-DNA.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eYeast one-hybrid (Y1H) assays\u003c/h2\u003e\u003cp\u003eY1H assay was performed using the pLacZi system. The coding sequence (CDS) of \u003cem\u003eOsPIL11\u003c/em\u003e were cloned into the pB42AD vector to generate prey vectors, and the fragments of \u003cem\u003eOsCKX2\u003c/em\u003e and \u003cem\u003eOsMIR530\u003c/em\u003e promoter were constructed into the pLacZi vector to obtain bait constructs using specific primers (listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The prey and the bait vectors were cotransformed into the EGY48 yeast strain. Then, the selected positive strains were grown on SD/-Ura/‐Trp medium with X‐gal at 30\u0026deg;C for 3\u0026thinsp;\u0026minus;\u0026thinsp;5 days, and the X‐gal blue staining was photographed.\u003cb\u003eElectrophoretic Mobility Shift Assay (EMSA)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe full-length coding sequence of OsPIL11 was cloned into the pMAL vector (BioRun) to generate a MBP-tagged recombinant protein. The protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Double-stranded DNA probes corresponding to the promoter regions of \u003cem\u003eOsCKX2\u003c/em\u003e and \u003cem\u003eOsMIR530\u003c/em\u003e (containing wild-type or mutated E-box motifs) were synthesized (sequences listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The probes (labeled or unlabeled, or mutant) were incubated with purified OsPIL11 protein at room temperature for 20 min. The protein-DNA complexes were electrophoresed on a non-denaturing polyacrylamide gel.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eOsPIL11\u003c/b\u003e \u003cb\u003epositively regulates grain length but negatively regulates grain width.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo comprehensively investigate the roles of OsPIL11 in rice, we generated \u003cem\u003eOsPIL11\u003c/em\u003e overexpression lines by introducing a ubiquitin promoter-driven \u003cem\u003eLOC_Os12g41650\u003c/em\u003e coding sequence into \u003cem\u003eOryza sativa\u003c/em\u003e L. cv. Nipponbare. Three independent T1 OE lines (OE#4, #5, and #26) exhibiting significantly elevated \u003cem\u003eOsPIL11\u003c/em\u003e transcript levels were selected for subsequent phenotypic and molecular analyses (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-B; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Quantitative analysis under controlled light conditions revealed that both red light (660 nm) and far-red light (730 nm) treatments significantly enhanced coleoptile elongation in OE lines compared to wild-type controls (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC-F), implying its involvement in phytochrome -dependent signaling cascades.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo dissect the role of OsPIL11 in regulating grain size, we measured grain length, grain width and grain thickness. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C, OE lines exhibited significantly increased grain length and decreased width compared to wild-type controls. However, no significant change in grain weight was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), which could be attributed to compensatory reductions in grain width and thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F).\u003c/p\u003e\u003cp\u003eWe further generated KO lines using CRISPR/Cas9-mediated gene editing of \u003cem\u003eLOC_Os12g41650\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). Three independent KO lines (#2, #11, and #13) were obtained, each carrying frameshift mutations: a 1-bp deletion in the first exon (#2), a 1-bp insertion in the first exon (#11), and a 1-bp insertion in the second exon (#13; Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB), respectively. KO lines exhibited significantly reduced grain length and increased grain width, compared to wild-type plants. Notably, despite a decrease in grain thickness in lines #2 and #13, 1000-grain weight was elevated in all KO lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F). These findings indicate that \u003cem\u003eOsPIL11\u003c/em\u003e acts as a positive regulator of grain length but negatively regulates grain width, suggesting that the regulation of grain thickness by OsPIL11 may involve more complex mechanisms.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eOsPIL11\u003c/b\u003e \u003cb\u003enegatively regulates grain number per panicle.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further investigate the role of \u003cem\u003eOsPIL11\u003c/em\u003e in panicle development, we analyzed the grain number per panicle in the OE and KO lines. Overexpression lines of \u003cem\u003eOsPIL11\u003c/em\u003e showed a significant reduction in grain number per panicle compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). In contrast, KO lines exhibited a marked increase in grain number per panicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, D). These results suggest that \u003cem\u003eOsPIL11\u003c/em\u003e acts as a negative regulator of grain number per panicle. Moreover, we observed a significant decrease in the number of primary branches in OE lines relative to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In contrast, KO lines displayed a substantial increase in primary branch number (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). This indicates that \u003cem\u003eOsPIL11\u003c/em\u003e modulates spikelet number by regulating the formation of primary branches during panicle development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eOsPIL11\u003c/b\u003e \u003cb\u003eregulates grain size by altering cell expansion and division.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMature grains are composed of lemma, palea, and endosperm. The spikelet hull restricts the overall grain size. Cell proliferation and expansion play vital roles in regulating spikelet hull growth. The spikelet hulls of OE lines exhibited a significant increase in length but a decrease in width compared to those of WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To clarify the cellular basis for the changes in grain size, we analyzed the cell number and morphology of spikelet hulls by paraffin sections. Compared with the WT plants, the OE and KO lines had more and fewer parenchyma cells in the grain hull, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D). These findings suggest that \u003cem\u003eOsPIL11\u003c/em\u003e positively regulated cell division in rice. Furthermore, the inner epidermal cells of the lemma in the OE and KO lines and WT plants were analyzed by scanning electron microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The results showed that \u003cem\u003eOsPIL11\u003c/em\u003e OE lines exhibited increased cell length and reduced cell width, while \u003cem\u003eOsPIL11\u003c/em\u003e KO lines had reduced cell length and increased cell width (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Notably, these compensatory changes in cellular dimensions did not alter the total surface area of the inner epidermal cells in the spikelet. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-H). These results implied that OsPIL11 controls morphogenesis by modulating anisotropic cell expansion rather than overall cell size.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eExpression pattern and subcellular localization of OsPIL11\u003c/h2\u003e\u003cp\u003eWe initially examined the expression pattern of \u003cem\u003eOsPIL11\u003c/em\u003e in photoperiodic conditions. OsPIL11 transcripts were reduced during light period and increased during dark period (Fig. S3A), which supports the notion that OsPIL11 functions as a phytochrome-interacting factor in plant light-responsive signal transduction, as previously reported (Nakamura et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). To further explore the spatial expression pattern of \u003cem\u003eOsPIL11\u003c/em\u003e, we conducted qRT-PCR analyses on various tissues. \u003cem\u003eOsPIL11\u003c/em\u003e is ubiquitously expressed across all examined tissues, with notably higher expression levels in leaves, shoots, inner lemma, and panicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Additionally, \u003cem\u003eOsPIL11\u003c/em\u003e was found to exhibit high expression in leaf blades, lemma/palea, and panicles at various developmental stages by searching the Rice Expression Profile Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ricexpro.dna.affrc.go.jp/\u003c/span\u003e\u003cspan address=\"http://ricexpro.dna.affrc.go.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Fig. S3B). A search of the database from MBKbase (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mbkbase.org/rice/genotype\u003c/span\u003e\u003cspan address=\"https://www.mbkbase.org/rice/genotype\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) also revealed the elevated expression of \u003cem\u003eOsPIL11\u003c/em\u003e in leaves, shoots, spikes, and panicles (Fig. S3C). To further validate these observations, we generated the transgenic plants by introducing the \u003cem\u003epPIL11::GUS\u003c/em\u003e vector into wild-type. Histochemical staining revealed that GUS signals were detected in most of the examined organs, consistent with the qRT-PCR data of \u003cem\u003eOsPIL11\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine the subcellular localization of the OsPIL11 protein, we transformed \u003cem\u003ep35S::OsPIL11-GFP\u003c/em\u003e and \u003cem\u003ep35S::GFP\u003c/em\u003e constructs into rice protoplasts. OsPIL11-GFP signals predominantly observed in the nucleus, while GFP signals could be observed in both cytoplasm and nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results suggest that OsPIL11 protein is nucleus-localized, consistent with its role as a transcription factor.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of genes regulated by OsPIL11\u003c/h2\u003e\u003cp\u003eTo further elucidate the molecular mechanisms underlying the role of OsPIL11 in regulating grain size and grain number, we performed RNA-Seq analysis using panicles (1 cm in length) from both WT and KO lines. Comparative transcriptomic profiling identified a total of 2,185 differentially expressed genes (DEGs) between the two genotypes (padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05, fold change\u0026thinsp;\u0026gt;\u0026thinsp;2), comprising 962 up-regulated and 1,223 down-regulated genes in the KO lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). KEGG and GO analyses suggest that OsPIL11 modulates complex regulatory networks influencing rice grain yield, including diterpenoid biosynthesis, photosynthesis, MAPK signaling, plant hormone signal transduction and zeatin biosynthesis (Fig.\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA-B). Specifically, we identified 15 genes associated with diterpenoid biosynthesis, 5 DEGs involved in photosynthesis, 8 genes related to the plant MAPK signaling pathway, 25 genes encoding plant hormone signal transduction proteins and 5 DEGs related to zeatin biosynthesis. These genes were emphasized as they synergistically regulate defense metabolism (diterpenoids), stress signaling (MAPK), energy homeostasis (photosynthesis), and hormonal coordination, defining an integrated adaptive strategy against development in rice (Fig.\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003eS4\u003c/span\u003eC). These findings suggest that OsPIL11 exerts its regulatory effects on grain development and yield-related traits by modulating multiple biological processes and signaling pathways.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAmong the above-mentioned processes, several genes have been reported to be related to the grain size and panicle development, such as \u003cem\u003eWRKY24\u003c/em\u003e, \u003cem\u003eOsIAA10\u003c/em\u003e, \u003cem\u003eOsSCP46\u003c/em\u003e, \u003cem\u003eOsPIL15\u003c/em\u003e, and \u003cem\u003eOsCKX2\u003c/em\u003e. The heat map analysis further illustrated significant changes in their expression levels between WT and KO lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). Interestingly, there were many light-responsive elements, such as E-box, N-box, PBE-box, and G-box, that were observed in the promoters of the genes above-mentioned (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These results suggest that these genes might be directly or indirectly regulated by OsPIL11.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eOsPIL11 directly binds to the promoters of\u003c/b\u003e \u003cb\u003eOsCKX2\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eOsMIR530.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify the target genes directly regulated by OsPIL11 in regulating rice grain size and grain numbers, ChIP-seq analysis was performed using panicles (1 cm in length) of \u003cem\u003eOsPIL11-HA\u003c/em\u003e lines, which exhibit similar phenotypes to \u003cem\u003eOsPIL11\u003c/em\u003e-OE lines (Fig.\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Several potential targets were enriched, including \u003cem\u003eOsCKX2\u003c/em\u003e and \u003cem\u003eOsMIR530\u003c/em\u003e promoters (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA; Fig.\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). The interaction between OsPIL11 and either \u003cem\u003eOsCKX2\u003c/em\u003e or \u003cem\u003eOsMIR530\u003c/em\u003e promoters was confirmed using Y1H assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C). To further determine whether OsPIL11 directly targets \u003cem\u003eOsCKX2\u003c/em\u003e and \u003cem\u003eOsMIR530\u003c/em\u003e, we conducted EMSA using the purified OsPIL11-His recombinant protein and \u003cem\u003eOsCKX2\u003c/em\u003e and \u003cem\u003eOsMIR530\u003c/em\u003e probes with normal and mutated E-box sequences. The binding abilities of OsPIL11 to the fragments decreased significantly in the presence of unlabeled competitor probes, but not with the unlabeled probes with mutated G-box sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Therefore, it can be concluded that OsPIL11 has the potential to interact with \u003cem\u003eOsCKX2\u003c/em\u003e and \u003cem\u003eOsMIR530\u003c/em\u003e promoters.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHaplotype and population analyses of\u003c/b\u003e \u003cb\u003eOsPIL11\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the natural allelic variation of \u003cem\u003eOsPIL11\u003c/em\u003e, we analyzed 533 core accessions from the genomic database of Sichuan Agricultural University (Zhou et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).Sequence variations in the promoter, coding sequence (CDS), and untranslated regions (UTR) of \u003cem\u003ePIL11\u003c/em\u003e revealed two SNPs in the promoter region and five SNPs in the CDS, forming two major haplotypes (Hap1 and Hap2). Notably, a promoter SNP localized within a cis-acting regulatory element suggested divergent transcriptional regulation between haplotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Phenotypic-genotypic association analysis demonstrated that Hap1 exhibited significantly greater grain width (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and weight (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, grain length remained statistically invariant, compared to Hap2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-D). These findings highlight \u003cem\u003ePIL11\u003c/em\u003e as a determinant of grain morphology and yield, with Hap1 representing a superior haplotype for grain weight improvement. Subpopulation analysis revealed distinct genetic backgrounds: Hap1 predominated in japonica (60.9%), compared with indica (38.74%). In contrast, Hap2 was predominantly in indica (90.5%) with a small amount of Aus accessions (8.15%; Fig.\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). Evolutionary analysis further indicated that \u003cem\u003ePIL11\u003c/em\u003e has undergone artificial selection during domestication (Fig. S7). To validate the reliability of \u003cem\u003eOsPIL11\u003c/em\u003e functions, we compared the grain size of chromosome segment substitution lines (CSSLs) using Nipponbare as the recipient parent and Sea rice 86 (SR86) as the donor parent. Through molecular marker-assisted selection, two CSSLs (N-S-1/2\u003csup\u003eSR86\u003c/sup\u003e) carrying \u003cem\u003ePIL11\u003c/em\u003e chromosomal segments from SR86 were developed, which exhibited a genetic background closely resembling that of the recurrent parent Nipponbare (Fig. S8). Comparative analysis revealed that the grain length of N-S-1/2\u003csup\u003eSR86\u003c/sup\u003e lines showed no significant differences compared with Nipponbare (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and G). In contrast, their grain width was significantly lower than that of the recurrent parent (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eF and H). Additionally, two non-introgressed substitution lines (N-S-1/2\u003csup\u003eNIP\u003c/sup\u003e) derived from the same population demonstrated comparable grain length, width, and weight to Nipponbare (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-I). Collectively, these results provide critical insights into the functional divergence of \u003cem\u003eOsPIL11\u003c/em\u003e haplotypes and establish a foundation for their utilization in molecular breeding programs targeting yield enhancement in rice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough rice PIFs regulate diverse growth processes in rice, such as flowering time, transpiration, and disease-resistance (Paik et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), little has been known about PIFs in regulating grain yield. In this study, for the first time, we reported the OsPIL11 coordinates grain weight and grain number \u003cem\u003evia\u003c/em\u003e directly regulating the expression of \u003cem\u003eOsMIR530\u003c/em\u003e and \u003cem\u003eOsCKX2\u003c/em\u003e.\u003c/p\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003ePIL11 is a negative regulator of grain width and grain number\u003c/h2\u003e\u003cp\u003eIn the present study, we constructed transgenic rice (KO, OE lines) to examine the role of OsPIL11 in regulating rice yield. Grain length and grain width showed reverse phenotypes in both KO and OE lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-E, IL), suggesting that OsPIL11 positively regulates grain length but negatively regulates grain width. This result is different from the observation from OsPIL15, where However, OsPIL15 negatively regulates grain length and width (Sun et al, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The functional difference is probably related to their expression pattern that OsPIL15 is highly expressed in florets within 35 days after flowering (DAF), whereas OsPIL11 shows negligible expression during this grain-filling period. However, 1000-grain weight was increased in KO lines, but no significant change in OE lines of \u003cem\u003eOsPIL11\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, N). In terms of cytological mechanism, the loss of \u003cem\u003eOsPIL11\u003c/em\u003e function results in a reduction of parenchyma cells in the glume, while its overexpression leads to an increase in parenchyma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D), indicating that OsPIL11 positively regulates cell division. On the other hand, OsPIL11 regulates cell length and width in opposite trends, resulting in no significant changes in cell area (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-H). In conclusion, OsPIL11 regulates grain size by modulating the number of parenchyma cells and their shape.\u003c/p\u003e\u003cp\u003eMoreover, we found that \u003cem\u003eOsPIL11\u003c/em\u003e negatively regulates grain numbers per panicle. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cem\u003eOsPIL11\u003c/em\u003e deficiency increases the grain number, while its overexpression decreases the grain number per panicle, mainly through modulating the number of primary branches. Our previous research revealed that \u003cem\u003eOsPIL11\u003c/em\u003e negatively regulate tiller number by directly activating the expression of \u003cem\u003eOsTB1\u003c/em\u003e (Zhang et al., 2021). In conclusion, given that the functional loss of \u003cem\u003eOsPIL11\u003c/em\u003e contributes to the improvement of the three primary yield components, namely efficient tiller number, grain number per panicle and 1000-grain weight, \u003cem\u003eOsPIL11\u003c/em\u003e exhibits considerable potential for practical application in rice breeding.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOsPIL11 may regulate grain size and grain numbers by targeting\u003c/b\u003e \u003cb\u003eOsMIR530\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eOsCKX2\u003c/b\u003e \u003cb\u003edirectly\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the present study, RNA-seq results revealed that \u003cem\u003eOsPIL11\u003c/em\u003e mainly regulate several pathways during panicle development, including diterpenoid biosynthesis, photosynthesis, MAPK signaling pathway, plant hormone signal transduction proteins, and zeatin biosynthesis (Fig S4). ChIP-seq assay, Y1H and EMSA assay indicates that \u003cem\u003eOsCKX2\u003c/em\u003e and \u003cem\u003eOsMIR530\u003c/em\u003e were potential target genes of OsPIL11. Previous research has demonstrated that OsmiR530 is a negative regulator of rice grain yield by affecting cell division and expansion in spikelet hulls (Sun et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The loss of function of \u003cem\u003eOsmiR530\u003c/em\u003e promotes the grain weight and panicle branching, which is consistent with the agronomic characters of \u003cem\u003eOsPIL11\u003c/em\u003e KO lines. \u003cem\u003eOsCKX2\u003c/em\u003e encodes cytokinin oxidase/dehydrogenase, an enzyme responsible for degrading cytokinin (Wang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Knocking out of \u003cem\u003eOsCKX2\u003c/em\u003e enhances the accumulation of cytokinin and the number of grains per panicle (Rong et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These results indicated that OsPIL11 modulating grain size and numbers mainly via regulating \u003cem\u003eOsCKX2\u003c/em\u003e and \u003cem\u003eOsMIR530\u003c/em\u003e expression.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eHaplotype divergence of OsPIL11: implications for grain morphology and yield in XI/GJ rice\u003c/h2\u003e\u003cp\u003eIn this study, we identified two major haplotypes of \u003cem\u003eOsPIL11\u003c/em\u003e, Hap1 and Hap2, with distinct effects on grain morphology and yield (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The promoter SNP (cis-element) and CDS variations likely drive transcriptional divergence, leading to Hap1\u0026rsquo;s wider grains but lower yield compared to Hap2. The predominance of Hap1 in \u003cem\u003ejaponica\u003c/em\u003e and Hap2 in \u003cem\u003eindica\u003c/em\u003e aligns with their distinct domestication trajectories. High yield in Hap2 accessions may reflect strong selection in \u003cem\u003eindica\u003c/em\u003e for panicle productivity, whereas wider grains in Hap1 accession could be favored in \u003cem\u003ejaponica\u003c/em\u003e under specific agroecological conditions.\u003c/p\u003e\u003cp\u003eIn summary, our findings indicated that \u003cem\u003eOsPIL11\u003c/em\u003e played a crucial regulatory role in modulating grain size and numbers through interacting with target genes \u003cem\u003eOsCKX2\u003c/em\u003e and \u003cem\u003eOsMIR530\u003c/em\u003e. This study expands the understanding of the functional roles of PIF family genes in regulating rice grain size and grain numbers, while also elucidating the intricate relationship between grain size and grain numbers. Further investigation of OsPIL11 is expected to enhance our understanding of its role in the differentiation of XI and GJ grain traits, thereby facilitating the development of high-yielding rice varieties in the future.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Rice Research Institute of Sichuan Agricultural University for supporting this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.P. and Y.L. conducted the experiments, analysed the data and drafted the manuscript. C.Z., M.Z., and C.J. conducted the analyses. Z.L., X.X., and G.Z. were involved in performing the experiments. X.X. contributed to the study conception and design. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants from the Shandong Agricultural Elite Variety Project (2024LZGC009), the Shandong Academy of Agricultural Sciences Innovation Project (CXGC2025H08), the National Natural Science Foundation of China (32172096), the Shandong Modern Agricultural Industry system (SDAIT-17-9; SDAIT-17-17).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequencing data of RNA-seq (Bioproject PRJCA043052) can be found in Beijing Institute of Genomics Data Center (http://bigd.big.ac.cn).\u003c/p\u003e\n\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\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cstrong\u003eBaillo, E.H., Kimotho, R.N., Zhang, Z.B., and Xu, P.\u003c/strong\u003e (2019). 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Journal Of Integrative Plant Biology \u003cstrong\u003e62\u003c/strong\u003e,847-864.\u003c/li\u003e\n\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, phytochrome-interacting factor, grain weight, grain number, molecular breeding","lastPublishedDoi":"10.21203/rs.3.rs-7350390/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7350390/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGrain weight and panicle architecture are pivotal determinants of rice yield, yet the regulatory mechanisms coordinating these traits remain elusive. Here, we functionally characterized a phytochrome-interacting factor, OsPIL11, serving as a negative regulator of grain weight and grain numbers per panicle. Knocking out \u003cem\u003eOsPIL11\u003c/em\u003e resulted in increased grain weight and grain number per panicle. \u003cem\u003eOsPIL11\u003c/em\u003e regulates grain weight by affecting cell expansion and division in the spikelet hulls, and controls grain number per panicle by regulating the number of primary branches. We functionally characterize \u003cem\u003eOsMIR530\u003c/em\u003e, a regulator of grain size, and \u003cem\u003eOsCKX2\u003c/em\u003e, a regulator of grain number, as the target genes of OsPIL11. Analysis of genetic variations suggested that \u003cem\u003eOsPIL11\u003c/em\u003e has likely been subjected to artificial selection during rice breeding, with Hap2 representing a superior haplotype for yield improvement. These findings provide novel insights into the molecular mechanisms underlying the regulation of rice yield, offering valuable genetic resources for the development of high-yield rice varieties through molecular breeding approaches.\u003c/p\u003e","manuscriptTitle":"The Phytochrome-Interacting Factor OsPIL11 Coordinates Grain Weight and Grain Number via Directly Regulating the Expression of OsMIR530 and OsCKX2 in Rice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-25 17:18:21","doi":"10.21203/rs.3.rs-7350390/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-21T06:09:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-19T03:44:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66076338734635570817493271621165366962","date":"2025-09-09T03:01:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-24T13:31:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123353001805073222339829684143252367262","date":"2025-08-19T13:55:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-18T02:41:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-12T11:31:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-12T11:31:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Rice","date":"2025-08-12T01:58:31+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":"32995840-421a-48ae-8719-d291aa7425a2","owner":[],"postedDate":"August 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:06:56+00:00","versionOfRecord":{"articleIdentity":"rs-7350390","link":"https://doi.org/10.1186/s12284-025-00865-6","journal":{"identity":"rice","isVorOnly":false,"title":"Rice"},"publishedOn":"2025-11-28 15:58:09","publishedOnDateReadable":"November 28th, 2025"},"versionCreatedAt":"2025-08-25 17:18:21","video":"","vorDoi":"10.1186/s12284-025-00865-6","vorDoiUrl":"https://doi.org/10.1186/s12284-025-00865-6","workflowStages":[]},"version":"v1","identity":"rs-7350390","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7350390","identity":"rs-7350390","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}