Rice OsABCB4 delays heading date and impacts multiple agronomic traits by affecting auxin homeostasis

Rice OsABCB4 delays heading date and impacts multiple agronomic traits by affecting auxin homeostasis | 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 Rice OsABCB4 delays heading date and impacts multiple agronomic traits by affecting auxin homeostasis Zongyue Jiang, Jiayu Li, Jingxin Wei, Lei Huang, Yujia Li, Mengfan Liu, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8096452/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Apr, 2026 Read the published version in Journal of Plant Research → Version 1 posted 4 You are reading this latest preprint version Abstract Members of the ABCB transporter subfamily are essential for various aspects of plant growth and development; however, a large number of ABCB proteins are functionally uncharacterized. Here, we report the functional characterization of a rice ABCB member, OsABCB4 . Tissue-specific expression analysis of 27 ABCB genes in rice identified a cluster with seed-specific expression, among which OsABCB4 was most highly expressed in developing panicles and seeds. CRISPR/Cas9-generated osabcb4 mutants exhibited a significant delay in heading date. Furthermore, the mutants displayed severe yield-related defects, including dwarfism, reduced panicle length, and a sharp decrease in seed-setting rate, primarily attributable to significantly impaired pollen fertility. Hormone quantification indicated a substantial reduction in indole-3-acetic acid (IAA) content in the panicles of mutant plants. Transcriptome analysis revealed global changes in gene expression, with differentially expressed genes significantly enriched in plant hormone signal transduction and starch/sucrose metabolism pathways. Consistent with these findings, the mutants showed abnormal accumulation of grain storage substances, characterized by significantly decreased starch content and increased protein content, consequently slowing down both seed germination and early seedling growth. Taken together, our results suggest that OsABCB4 may as a key regulator that influences heading date, pollen fertility, and grain filling by modulating auxin homeostasis in rice. Auxin transporter Grain filling Heading date OsABCB4 Pollen fertility Rice Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Rice ( Oryza sativa L.), commonly referred to as Asian cultivated rice, serves as a staple food for more than half of the global population. Rice breeders have consistently prioritized the enhancement of grain yield and quality as key objectives(Fiaz et al. 2019 ). Phytohormones, also known as plant growth regulators, modulate growth and development in rice by activating or inhibiting specific metabolic pathways(Woodward and Bartel 2005 ). Endogenous phytohormones include abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), ethylene (ET), auxin, gibberellin (GA), brassinosteroid (BR), and cytokinin (CK)(Gong et al. 2022 ). Among these, auxin was the first discovered plant hormone, which plays a pivotal role in regulating growth processes(Yu et al. 2022 ). It is critically involved in phototropism, root formation, leaf development, fertility, embryogenesis, and vascular tissue differentiation(Swarup and Bhosale 2019 ). Multiple gene families participate in auxin biosynthesis and transport. These include OsARF and OsIAA involved in signal transduction; OsAUX/LAX , OsPIN , and OsPGP/ABCB responsible for auxin transport; OsGH3 participating in metabolic homeostasis; and OsSAUR executing rapid responses(Jain et al. 2006 ; Song et al. 2009 ; Ye et al. 2021 ; Xu et al. 2022 ; Hou et al. 2025 ; Devi et al. 2025 ). These genes act synergistically or antagonistically to coordinately regulate auxin synthesis and transport, maintaining auxin homeostasis and thereby influencing plant growth and development. For example: OsIAA6 interacts with OsARF1 to suppress auxin signaling and regulate leaf inclination(Xing et al. 2022 ). OsARF6 and OsARF17 are highly expressed in leaf joint tissues and modulate flag leaf angle in response to auxin signals(Huang et al. 2021 ). OsARF25 regulates grain length, width, and thousand-grain weight via the brassinosteroid signaling pathway(Zhang et al. 2025 ). OsPIN1b , OsPIN1c , and OsPIN9 mediate auxin transport from shoots to roots(Wang et al. 2025 ). Overexpression of OsPIN2 increases auxin distribution in the root epidermis(Sun et al. 2019 ). OsGH3 , a member of the auxin-responsive gene family, participates in plant growth, developmental processes, and stress responses. OsSAUR45 affects auxin synthesis and transport by repressing the expression of OsYUCCA and OsPIN genes, thereby influencing plant growth(Xu et al. 2017 ). OsSAUR39 acts a negative regulator of auxin synthesis and transport; its overexpression in rice suppresses lateral root development and reduces yield(Kant et al. 2009 ). These genes and their regulatory networks provide a window into understanding how plants integrate internal and external signals through hormonal coordination. The ABC superfamily represents one of the largest transporter families in plants and is responsible for auxin transport. Among its subfamilies, ABCB members are primarily associated with auxin transport, with each member performing distinct functions in plant systems. There are 29 ABCB genes in Arabidopsis thaliana and 27 in rice, participating in diverse cellular, physiological, and developmental processes(Devi et al. 2025 ). For instance: in Arabidopsis thaliana , AtABCB1/PGP1 mediates auxin efflux, influences auxin distribution in the shoot apex, and indirectly regulates flowering time(Geisler et al. 2005 ). Loss of AtABCB19 (also known as AtPGP19/MDR1 ) disrupts AtPIN1 membrane localization, enhances plant sensitivity to gravity and light, and indirectly affects gravitropic responses. Altered tropic growth in root or shoot tips may perturb hormonal signaling and subsequently influence flowering. Moreover, the atabcb19 mutant exhibits delayed anther development and reduced pollen viability(Noh et al. 2003 ). AtABCB4 plays a key role in root architecture and root hair development by regulating polar auxin transport, potentially influencing flowering through modulation of long-distance hormone transport(Kubes et al. 2012 ). In rice, several ABCB members also function in auxin transport: OsABCB14 transports multiple substrates and is involved in cellular auxin uptake and iron homeostasis(Xu et al. 2014 ). OsABCB24 is essential for female gametogenesis and early seed development(Nguyen et al. 2024 ). It transports auxin precursors or auxin across the endoplasmic reticulum (ER) membrane, and disruption of ER auxin homeostasis leads to female sterility. The osabcb24 mutant displays structural abnormalities during early female gametophyte development and initial endosperm formation, characterized by disorganized syncytial nuclear structures and significantly reduced seed set(Nguyen et al. 2024 ). The development of rice grains is critically regulated by auxin. However, the mechanisms underlying auxin transport and its regulation in rice grains are not yet fully understood. In this study, we found that the ABCB family member OsABCB4 is preferentially expressed in rice grains and participates in auxin accumulation. Mutation of OsABCB4 significantly delayed heading date and reduced pollen fertility, grain yield, and grain quality. Materials and Methods Plant Materials and Growth Conditions The rice materials used in this study included the japonica cultivar ZH11 and its derived mutant lines. Genomic DNA extracted from leaves of ZH11 wild-type (WT) and transgenic plants served as the template for PCR amplification. Sequencing of the target gene and the hygromycin resistance gene was performed across successive T1 and T2 mutant generations. Three stable T3 homozygous mutant lines ( osabcb4-1 , osabcb4-2 , and osabcb4-3 ) were selected for subsequent phenotypic evaluation. Field experiments were conducted at the experimental station of Guangxi University (Nanning, Guangxi Zhuang Autonomous Region, China; 22.49′N, 108.19′E). Plants were grown under both long-day (LD) and short-day (SD) conditions, corresponding to the local early- and late-season rice cropping periods, respectively. According to data from the China Meteorological Data Service Center ( http://data.cma.cn ), the daylength during the photoperiod-sensitive phase was 13.14–13.24 hours in the long-day season (mid-May to mid-July) and 11.38–12.17 hours in the short-day season (mid-September to mid-October). For the early-season (LD) trial, seeds were sown in March and seedlings were transplanted into paddy fields under standard agronomic management in April. For the late-season (SD) trial, sowing took place in mid-July, with transplanting in August. All WT and the three mutant lines were arranged in a randomized complete block design with three independent replications. Each plot consisted of 6 rows with 8 plants per row, spaced at 20 cm × 20 cm intervals. To minimize edge effects, ten plants from the central area of each plot were selected for measuring heading date and agronomic traits. All field management practices followed conventional local rice production protocols. CRISPR/Cas9 Vector Construction In this study, we generated osabcb4 mutants using CRISPR/Cas9-mediated gene editing. Target-specific gRNA sequences were designed against the sixth and tenth exons of the ABCB4 gene using the online CRISPR design platform ( http://crispr.hzau.edu.cn/CRISPR2/ ) and subsequently cloned into the pFATB-Cas9 vector. The recombinant construct was introduced into the japonica rice cultivar ZH11 via Agrobacterium-mediated transformation. Homozygous mutant lines were identified and selected from the T1 transgenic population through sequencing analysis. We additionally obtained the target site sequence-containing primers CAACGGATAGCTATAGCAA and AGTTGATCAAGGATCCCGA from BLAST CRISPR-GE and NCBI as a means of ensuring that no off-target gene targeting occurred via this approach. DNA Extraction Genomic DNA was extracted from fresh leaves of WT and mutant plants using the CTAB method. The purified DNA samples were stored at 4°C for subsequent sequencing of the target gene and the hygromycin resistance gene(Murray and Thompson 1980 ). RNA Extraction and Gene Expression Analysis Plant tissues were collected from rice plants, immediately wrapped in aluminum foil, flash-frozen in liquid nitrogen, and stored at -80°C until RNA extraction. Total RNA was isolated using the FreeZol Reagent Kit (Vazyme) according to the manufacturer's protocol. RNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). First-strand cDNA was synthesized from 3 µg of total RNA in a 20 µL reaction system using HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme). Quantitative real-time PCR (qPCR) was performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme) on a Cobas z 480 real-time PCR system. The rice Ubiquitin gene ( Os05g06770 ) was used as the internal reference gene for normalization. Primer sequences for the genes under examination are detailed in Table S1 . Anthers were categorized based on spikelet length, as per establised criteria(Xu et al. 2017 ). Analysis of ABCB Gene Family Expression Patterns Across Developmental Stages and Tissues The rice genome encodes 27 ABCB family members. Expression data for ABCB genes were obtained from the Rice eFP Browser ( https://bar.utoronto.ca/efprice/cgi-bin/efpWeb.cgi ). The average signal values across 15 distinct tissues at various developmental stages were log10-transformed and subjected to cluster analysis. Visualization of the resulting expression patterns was performed using the HeatMap function in TBtools software(Chen et al. 2020 ). Phylogenetic Analysis of ABCB Gene Family Protein sequences of ABCB gene family members from rice and Arabidopsis thaliana were retrieved from the Ensembl Plants database. A phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA7.0 software(Kumar et al. 2018 ). Heading Date Measurement and Agronomic Trait Investigation Heading date was defined as the number of days from sowing to the emergence of the first panicle on each plot. At maturity, agronomic traits, including productive tiller number, plant height, panicle length, seed setting rate, grain yield per plant, and 1000-grain weight, were measured using uniformly grown plants selected from the central area of plots containing WT and the three mutant lines ( abcb4-1 , abcb4-2 , and abcb4-3 ). A productive tiller was defined as one that headed normally and bore at least five filled grains. Representative plants from each experimental plot were randomly sampled to count productive tillers. Plant height was measured as the vertical distance from the ground level to the tip of the tallest panicle. Panicle length was determined as the distance from the panicle neck to the tip of the main panicle. At maturity, panicles from individual plants were harvested and manually threshed. Filled and unfilled grains were separated by water flotation. The seed setting rate was calculated as: Seed setting rate (%) = [number of filled grains / (number of filled grains + number of unfilled grains)] × 100%. Filled grains were dried at 45°C for 72 hours until constant weight was achieved. Grain yield per plant was determined as the total weight of filled grains from a single plant. The 1000-grain weight was calculated by weighing ten sets of 1000 filled grains. Assessment of Rice Pollen Fertility by I₂-KI Staining Method At the heading stage, pre-anthesis pollen grains were collected from WT and the three mutant lines, and immediately preserved in microcentrifuge tubes containing 75% (v/v) ethanol. For pollen viability assessment, anthers from a single floret were placed on a glass slide, stained with 1–2 drops of 1% iodine-potassium iodide (I₂-KI) solution, and gently crushed with forceps to release pollen grains. A coverslip was applied, and slight pressure was exerted before allowing the sample to stand for 2–3 minutes. Observation was carried out under a stereomicroscope. For each plant, pollen grains from eight randomly selected fields of view were scored as fertile or sterile. Three individual plants were evaluated per line(Chhun et al. 2007 ). Determination of Starch and Protein Content Grains were dried at 65°C for 5 days and ground into fine powder. Total starch content was determined using a commercial assay kit (BC0700, Solarbio) with 0.1 g of sample powder. Amylose content was measured by extracting alkali-soluble and alcohol-soluble proteins with 0.1 M NaOH and 70% ethanol, followed by quantification using the Bradford method. Amylopectin content was calculated by subtracting amylose content from total starch content. For protein content analysis, 0.5 kg of polished rice from both WT and mutant lines was evaluated using a rice taste analyzer (JSWL, RLTA10C, Japan). All measurements were performed with three biological replicates(Li et al. 2014 ). Determination of Indole-3-Acetic Acid (IAA) Content The endogenous indole-3-acetic acid (IAA) content was quantified using high-performance liquid chromatography (HPLC; Agilent 1200 series). Prior to analysis, each sample, which was composed of a pool of eight randomly selected spikelets, was homogenized and extracted with a cold phosphate buffer. The extracts were purified using C18 solid-phase extraction cartridges. Separation was achieved on a reversed-phase Diamonsil C18(2) column (250 mm × 4.6 mm, 5 µm) maintained at 35°C. An isocratic mobile phase consisting of methanol-water-glacial acetic acid (45:54.2:0.8) was delivered at a flow rate of 0.8 mL/min. The detection wavelength was set at 280 nm for specific IAA measurement. The method was validated for specificity, linearity (R² >0.999), and recovery (90–105%). Quantification was performed by comparing the peak areas with those of an authentic IAA standard calibration curve, and the mean value from three independent biological replicates was reported for each treatment(Nishimura et al. 2006 ; Shao et al. 2019). Materials for Transcriptome Sequencing In spring 2025, under standard rice growth conditions, panicles at the tenth developmental stage were collected from both WT and abcb4 plants. Each biological sample contained at least 1.0 g of tissue, with three independent replicates prepared for each genotype. After flash-freezing in liquid nitrogen, samples were transported on dry ice to Tsingke Biotechnology Co., Ltd (Beijing, China) for RNA extraction and transcriptome sequencing. Differentially expressed genes (DEGs) were identified from the transcriptome data using a significance threshold of Q -value ≤ 0.05(Love et al. 2014 ). KEGG Analysis of Differentially Expressed Genes Differentially expressed genes (DEGs) between WT and abcb4 mutants were identified based on fragments per kilobase of transcript per million mapped reads (FPKM) values. Significantly differentially expressed genes were defined as those with an adjusted P -value ( Q -value) < 0.05 and an absolute log 2 fold change ≥ 1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the cloud platform provided by Tsingke Biotechnology Co., Ltd (Beijing, China). Data Processing and Statistical Analysis All data were organized using Excel 2003 and subjected to one-way analysis of variance (ANOVA) in SPSS 27 for statistical analysis of agronomic traits. Graphs were generated with GraphPad Prism 9. Significant differences among groups were assessed by Student’s t -test, with data presented as mean ± SD. Statistical significance ( P < 0.05) is indicated by different lowercase letters in the figures. Results Phylogenetic and Expression Analysis of ABCB Subfamily Genes To elucidate the phylogenetic relationships and functional divergence of the ABCB subfamily, a phylogenetic tree was reconstructed using 56 protein sequences from rice and Arabidopsis thaliana . Based on evolutionary relationships, this subfamily can be further divided into five subgroups (I–V) (Fig. 1 ). The expression patterns of genes across different tissues and organs provide crucial insights into their biological functions. Based on distinct expression profiles, the rice ABCB subfamily can be classified into five subgroups (A–E), indicating significant functional divergence among these genes. Subgroup E genes are predominantly expressed in seeds, suggesting their potential roles in anther development and material transport during seed maturation. Within this subgroup, OsABCB4 exhibits markedly higher expression in inflorescence P5 and P6 stages, as well as in seed development stages S1, S2, and S3, compared to other tissues (Fig. 2 a). To determine the expression pattern of OsABCB4 , its transcript levels in various tissues of WT plants were analyzed by RT-qPCR (Fig. 2 b). The results showed that OsABCB4 expression was significantly upregulated after stage S6 of panicle development, consistent with the transcriptome profiling data, suggesting its potential involvement in panicle development. Loss of OsABCB4 Function Delays Heading Date in Rice To investigate the function of OsABCB4 , we generated three independent homozygous transgenic lines using CRISPR-Cas9 technology, each carrying distinct editing types in the target gene (Fig. 3 a). WT and the abcb4 mutants ( abcb4-1 , abcb4-2 , abcb4-3 ) were cultivated under normal field management in the same paddy field, and heading dates were recorded. On average, there was a 9-day difference in heading date between the WT and mutant lines, with values of 63 ± 2 days and 72 ± 2 days, respectively, in Nanning during the 2024/2025 growing season (Fig. 3 b–c). We further examined the expression levels of key heading date regulatory genes in the flag leaves of WT and abcb4-1 plants by qRT-PCR. The results showed significant upregulation of OsGI and OsHd1 , and significant downregulation of OsEhd1 , OsHd3a , and OsRFT1 in the mutant (Fig. 3 d–h). These genes are known to function in the Hd1/Ghd7/DTH8–Ehd1–Hd3a/RFT1 pathway that controls heading date in rice. A schematic model illustrating the regulation of heading date is shown in Fig. 3 i. The observed changes in gene expression are consistent with the delayed heading phenotype in the abcb4 mutant. Loss of OsABCB4 Function Leads to Reduced Pollen Fertility and Severe Yield Defects in Rice Under standard field conditions, we observed that the mutant plants exhibited a dwarf stature, more compact and shorter panicles, and reduced grain yield per plant compared to the WT (Fig. 4 a–c). We therefore systematically evaluated their agronomic traits. Statistical analysis revealed that the seed-setting rate of the WT was 88.71%, whereas all three mutant lines showed significantly reduced rates: 51.59% in abcb4-1 , 68.39% in abcb4-2 , and 69.05% in abcb4-3 , with an average reduction of approximately 25.7% (Fig. 4 d). This decrease in seed-setting rate severely compromised grain yield per plant. The WT yield was 14.09 g per plant, while the mutants yielded significantly less: 6.70 g for abcb4-1 , 9.93 g for abcb4-2 , and 11.66 g for abcb4-3 . Notably, the yield of abcb4-1 was less than half that of the WT (Fig. 4 e). Given the known role of OsABCB4 orthologs in regulating pollen viability, we hypothesized that impaired pollen fertility might underlie the reduced seed-setting and yield. Pollen viability assessed by I 2 -KI staining was significantly lower in the mutants. The staining rate was 95.02% in the WT, but only 35.08% in abcb4-1 , 62.20% in abcb4-2 , and 64.32% in abcb4-3 (Fig. 4 j–k), indicating that loss of ABCB4 function severely disrupts normal pollen development, leading to the observed reduction in seed set. In addition, other agronomic traits including 1000-grain weight, tiller number, panicle length, and plant height were also significantly reduced in the mutants relative to the WT. Specifically, the thousand-grain weight was markedly lower in the mutants ( abcb4-1 : 22.13 g; abcb4-2 : 22.34 g; abcb4-3 : 18.75 g) than in the WT (25.85 g). Similarly, tiller number was severely reduced to 6.21, 6.41, and 8.09 in the three mutants, respectively, versus 11.07 in the WT. The mutants also exhibited significantly shorter panicle lengths (14.04 cm, 16.56 cm, 17.19 cm) compared to the WT (20.28 cm), and a pronounced dwarf phenotype with plant heights of only 68.81 cm, 71.71 cm, and 75.00 cm, respectively, in contrast to the WT (91.20 cm) (Fig. 4 f–i). These results demonstrate that knockout of OsABCB4 significantly impairs rice growth and development, leading to substantial defects in key yield-related traits. Determination of Indole-3-Acetic Acid (IAA) Content We observed phenotypes such as pollen abortion and reduced seed setting rate in the mutants. Given the relatively high and easily distinguishable expression of OsABCB4 during the flowering stage, we measured indole-3-acetic acid (IAA) content in spikelets of WT and two mutant lines ( abcb4-1 and abcb4-2 ) at this developmental stage. The results showed that WT plants contained 257.16 ng/g IAA, while the mutants exhibited significantly reduced levels: 43.73 ng/g in abcb4-1 and 71.88 ng/g in abcb4-2 (Fig. 5 ). These findings indicate that knockout of OsABCB4 drastically reduces IAA accumulation in panicles during flowering. Transcriptome Analysis Reveals Comprehensive Regulatory Network To investigate the molecular mechanisms underlying the delayed heading, reduced yield, and decreased auxin levels in OsABCB4 knockout mutants, we conducted transcriptome sequencing of panicles at the flowering stage from both WT and abcb4-1 plants. Three biological replicates were analyzed, each consisting of pooled spikelets from eight individual plants. RNA-seq analysis identified 3346 differentially expressed genes (DEGs), with 2747 upregulated and 599 downregulated in the mutant (Fig. 6 a–b). To elucidate the biological functions of OsABCB4 during flowering, Gene Ontology (GO) enrichment analysis was performed on the DEGs. The results revealed significant enrichment in 20 GO terms, including 8 biological processes, 11 cellular components, and 1 molecular function term (Fig. 6 d). DEGs were notably enriched in processes such as "embryo development ending in seed dormancy" and "acquisition of seed longevity," and were primarily involved in cytoplasmic translation, ribosome assembly, and organellar RNA processing, indicating strong activation of protein synthesis machinery. These functions are closely associated with seed development, particularly the accumulation of storage proteins and protective factors required during embryo maturation and seed dormancy. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEGs were enriched in plant hormone signal transduction, starch and sucrose metabolism, and metabolic pathways (Fig. 6 c). Further analysis of transcription factors among the DEGs identified several auxin-related families, including GH3 , ARF , and IAA . Notably, genes regulating auxin biosynthesis and transport, such as OsGH3-2 , OsARF1 , and OsIA10 , were differentially expressed(Du et al. 2012 ; Ma et al. 2023 ). The expression changes of key genes, including OsARF1 , OsARF16 , OsGH3-2 , OsGH3-3 , OsIA10 , OsIAA14 , OsIAA23 , and OsIAA24 , were validated by qRT-PCR, all of which were significantly upregulated in the mutant (Fig. 6 e–l). Collectively, these results suggest that knockout of OsABCB4 disrupts the expression of genes involved in auxin biosynthesis and transport, which may directly or indirectly reduce auxin levels in panicles and impair normal anther and pollen development. Grain Storage Substance Content Given the enrichment of plant hormone signal transduction, starch and sucrose metabolism, and seed development-related pathways in KEGG and GO analyses (Fig. 6 c–d), we performed hormone treatment experiments to assess seed germination. Under control conditions (water), no significant differences in shoot or root growth were observed between WT and osabcb4 mutants after 7 days. However, treatment with 8 µM abscisic acid (ABA) resulted in significantly reduced shoot and root growth in osabcb4 mutants compared to WT (Fig. 7 a–c, f, g). When grown under standard conditions (28°C, 80% humidity) for 7 days, osabcb4 mutants exhibited significantly reduced shoot growth compared to WT (Fig. 7 d–e, h). We hypothesized that this phenotype might be related to altered nutrient reserves in the grains and therefore analyzed seed storage compounds. Compared to WT, osabcb4 mutants showed marked abnormalities in seed storage accumulation. Total starch content was significantly reduced in all mutant lines, with abcb4-1 and abcb4-3 showing the most pronounced decreases to 623 mg/g and 617 mg/g, respectively, representing approximately 30% reduction from the wild-type level of 875 mg/g. The abcb4-2 line contained 679 mg/g total starch, a 22% reduction (Fig. 7 j). Further analysis revealed concurrent decreases in both amylose and amylopectin content, though amylopectin reduction was more substantial. In abcb4-1 mutants, amylopectin content dropped to 468 mg/g, a 31% decrease from the WT level of 678 mg/g, while amylose decreased by 21% (Fig. 7 k–l). Notably, all mutant lines showed significantly increased protein content. WT protein content was 108 mg/g, while abcb4-3 mutants reached 124 mg/g, a 14.8% increase. The abcb4-1 and abcb4-2 lines also exhibited elevated protein levels of 120 mg/g and 119 mg/g, respectively (Fig. 7 i). In summary, compared to WT, the mutants displayed significantly reduced total starch, amylose, and amylopectin content, alongside significantly increased protein content. Discussion Our findings demonstrate that OsABCB4 , a member of the ABCB subfamily, plays a critical role in regulating auxin homeostasis in rice panicles, thereby influencing heading date, pollen development, and grain yield. Knockout of OsABCB4 led to reduced auxin content in spikelets at the flowering stage, delayed heading, impaired pollen fertility, decreased yield, and altered accumulation of grain storage reserves. OsABCB4 regulates auxin homeostasis in the rice panicle. The auxin regulatory network in plants is intricate and well-organized; plants have evolved a sophisticated system to precisely control auxin concentrations for optimal growth(Wang et al. 2018 ). In plants, the functionally well-characterized B-type ABC transporters ( ABCB subfamily) are primarily involved in auxin transport(Takanashi et al. 2012 ; Cheng et al. 2023 ). ARFs (auxin response factors) perceive auxin signals and bind to auxin response cis-regulatory elements ( AuxREs ) to regulate downstream transcriptional programs(Weijers and Wagner 2016 ). ARFs are classified into activators and repressors: activator ARFs contain a glutamine-rich middle region, whereas repressor ARFs are enriched in proline, serine, threonine, and other residues in their middle regions. OsARF1 and OsARF16 , with their glutamine-rich middle regions, belong to the activator class of auxin response factors(Wang et al. 2007 ). Overexpression of OsGH3s depletes the substrate (free IAA) by acting upstream, thereby reducing auxin levels(Domingo et al. 2009 ; Liu et al. 2022 ). Excess IAA proteins can form heterodimers with activator ARFs , directly inhibiting ARF transcriptional activity and blocking the auxin signaling pathway(Han et al. 2014 ; Weijers and Wagner 2016 ). Following knockout of OsABCB4 , the expression of OsARF1 , OsARF16 , OsGH3-2 , OsGH3-3 , OsIAA10 , OsIAA14 , OsIAA23 , and OsIAA24 was significantly upregulated (Fig. 6 e). Based on previous studies, we hypothesize that the upregulation of OsARF1 and OsARF16 in the panicle after OsABCB4 knockout triggers a marked increase in the expression of downstream auxin inactivation genes ( OsGH3s ) and signaling repressors ( OsIAAs ), leading to excessive depletion of free auxin and suppression of ARF activity, ultimately resulting in functional auxin deficiency in the panicle(Ori 2019 ). These results suggest that OsABCB4 may be directly or indirectly involved in regulating auxin biosynthesis and transport in rice panicles, thereby altering auxin levels in the panicle. OsABCB4 Regulates Heading Date through the Florigen Pathway Heading date is an important agronomic trait in rice. The timing of heading affects the accumulation of photosynthetic products and grain filling during the ripening stage, ultimately influencing both yield and grain quality. Therefore, cultivars with an appropriate heading date can optimize grain yield and quality by making full use of light and temperature resources in their cultivation regions(Zhang et al. 2015 ). Heading date in rice is primarily governed by genetic factors and environmental conditions, with photoperiod being the major environmental regulator(Song et al. 2015 ). Numerous studies have successfully identified and cloned multiple genes associated with heading date in rice. These genes form a complex network for perceiving and transducing light signals, precisely integrating internal and external cues to mediate rice photoperiod response, revealing the complexity of photoperiod-regulated flowering pathways. Among them, Hd1 was the first cloned heading date gene in rice and is a homolog of CO in Arabidopsis thaliana . Hd3a and RFT1 are homologs of the florigen gene FT in Arabidopsis thaliana (Ishikawa et al. 2005 ). Under short-day (SD) conditions, Hd1 promotes heading by upregulating Hd3a expression, whereas under long-day (LD) conditions, Hd1 suppresses Hd3a transcription, delaying heading(Yano et al. 2000 ). The rice gene OsGI , a homolog of GI in Arabidopsis, is regulated by the circadian clock and suppresses flowering under LD conditions(Zheng et al. 2019 ). The Ghd7 protein is closely associated with flowering time; OsGI directly interacts with Ghd7 and promotes its degradation, while phytochromes OsPHYA and OsPHYB competitively bind to Ghd7 against OsGI, thereby stabilizing the Ghd7 protein. Recent studies have shown that Ehd1 is also a key regulator of heading date in rice(Doi et al. 2004 ). Under SD conditions, Ehd1 regulates heading independently of Hd1 , and it promotes heading under both LD and SD conditions. Unlike Hd1 , under SD conditions Ehd1 promotes heading by inducing the expression of Hd3a and RFT1 . In summary, photoperiod control of heading in rice mainly relies on two regulatory pathways: under SD conditions, the Hd1–Hd3a/RFT1 pathway (analogous to the Arabidopsis thaliana GI–CO–FT pathway) regulates heading; under LD conditions, the Hd1/Ghd7/DTH8–Ehd1–Hd3a/RFT1 pathway is involved(Zong et al. 2021 ). The photoperiod regulation of heading date constitutes a complex and interactive network. We detected upregulation of OsGI , OsHd1 , and OsDTH8 , and downregulation of OsEhd1 , OsHd3a , and OsRFT1 in the flag leaves of osabcb4 (Fig. 3 d). These genes belong to the Hd1/Ghd7/DTH8–Ehd1–Hd3a/RFT1 pathway. The Hd1 protein inhibits Ehd1 expression, and the upregulation of Hd1 strengthens this suppression, leading to decreased Ehd1 expression(Doi et al. 2004 ). DTH8 is another key repressor; the DTH8–Hd1 complex potently suppresses Ehd1 expression(Du et al. 2017 ). Upregulation of DTH8 significantly enhances the formation and efficacy of these repressive complexes, resulting in strong inhibition of Ehd1 . Ehd1 acts as the central integrator and master switch in this pathway, directly activating the expression of the florigen genes Hd3a and RFT1 (Komiya et al. 2008 ). Hd3a and RFT1 are protein signals synthesized in leaves and transported to the shoot apical meristem, where they directly initiate heading(Tamaki et al. 2007 ). Therefore, downregulation of Ehd1 , Hd3a and RFT1 implies severe deficiency in florigen production. As a result, the shoot apex does not receive the "flowering" signal, leading to delayed heading in rice. OsABCB4 Influences Grain Storage Substance Accumulation The phytohormone auxin plays crucial roles in regulating fundamental processes such as cell division, elongation, and differentiation, exerting pleiotropic effects on plant growth and development(Park et al. 2011 ). Studies have demonstrated that auxin is integral to the entire reproductive process, from flower initiation to fruit maturation (Mariotti et al. 2011 ; Pattison et al. 2014 ). The pleiotropic effects observed in osabcb4 mutants, including impaired pollen viability, reduced seed-setting rate, and decreased grain yield per plant, may represent direct consequences of disrupted auxin signaling during reproductive development. Furthermore, auxin is a key hormone that regulates the distribution of photosynthates from the “source” to the “sink”(Zhao et al. 2022 ). The observed reduction in starch content and concomitant increase in protein levels may be attributed to a disruption of the source-sink-flow balance caused by reduced auxin content in the grains, ultimately leading to an altered composition of grain storage reserves. This interpretation is supported by the significant enrichment of differentially expressed genes in the "starch and sucrose metabolism" pathway. Beyond the yield loss, a direct impact of impaired sugar metabolism is a marked delay in germination and seedling growth rates, which could be devastating for the widely adopted direct-seeding cultivation. Recently, Du et al. (2025) also reported that OsABCB4 regulates grain shape by influencing the length-to-width ratio through auxin. In summary, OsABCB4 serves as a key regulator governing the transition to reproductive growth and grain quality determination in rice. A schematic model of the proposed mechanism is shown in Fig. 8 . Declarations Author Contributions Meng Yang designed this research. Zongyue Jiang, Jiayu Li and Lei Huang participated in data analysis. Zongyue Jiang, Jiayu Li, Jingxin Wei, Lei Huang, Yujia Li, Mengfan Liu, Zihan Zhao, Fangchi Wei, Jiaxuan Guan, Jinxing Jiang, Ling Zhou, Kangshun Huang and Fugang Huang performed material preparation and data collection. Zongyue Jiang wrote the manuscript. Meng Yang corrected the manuscript. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding This work was supported by the National Natural Science Foundation of China (32260474), the Guangxi Natural Science Foundation (2025GXNSFDA069036), the Guangxi Science and Technology Base and Talent Special Project (2022AC21114), the Open Research Project of Guangxi Key Laboratory of Agro-environment and Agric-products safety (GKLAEAPS2024-04), and the Innovation Project of Guangxi Graduate Education (YCBZ2025001). Ethical approval This article does not contain any studies with human participants or animals, performed by any of the authors. Competing interests The authors have no conflicts of interest to de clare that are relevant to the content of this article. References Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R (2020). Tbtools: an integrative toolkit developed for interactive analyses of big biological data. 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1","display":"","copyAsset":false,"role":"figure","size":163567,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree constructed from \u003cem\u003eABCB\u003c/em\u003e protein homologs in rice and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8096452/v1/2c61c6a0f34e902a1c2955d0.png"},{"id":96918261,"identity":"5f66b926-e3f9-4460-87c1-aecdf7c11111","added_by":"auto","created_at":"2025-11-27 14:11:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":106756,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiling and pattern analysis of the \u003cem\u003eOsABCB\u003c/em\u003egenes family. (a) Hierarchical cluster display of expression profiles for 27 rice \u003cem\u003eABCB\u003c/em\u003e genes in different tissues and stages. Data are obtained from the Rice eFP Browser. (b) \u003cem\u003eOsABCB4\u003c/em\u003e transcript levels were measured by RT-qPCR. S1 to S8, Anther stages 1 to 8. Values represent the means ± SD from three independent biological replicates, demonstrating the reproducibility of the results. Bars labeled with different letters are significantly different at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 according to one-way ANOVA\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8096452/v1/e92497aae60a82d99f4f4fd7.png"},{"id":96917972,"identity":"418f1542-c08d-4184-8bc9-00be47a48c57","added_by":"auto","created_at":"2025-11-27 14:10:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":185004,"visible":true,"origin":"","legend":"\u003cp\u003eCRISPR/Cas9-engineered mutations in \u003cem\u003eOsABCB4\u003c/em\u003e causes \u003cstrong\u003edelayed heading \u003c/strong\u003ein rice. (a) Gene structure diagram of\u003cem\u003e OsABCB4\u003c/em\u003e. The UTRs and CDS are indicated by black and blue rectangles, respectively; the black arrows indicate the start (ATG) and stop codon (TGA). The green background fonts indicate the mutation site of \u003cem\u003eOsABCB4\u003c/em\u003ein the three mutant lines. (b) Phenotypes of WT, \u003cem\u003eosabcb4-1\u003c/em\u003e, \u003cem\u003eosabcb4-2\u003c/em\u003e, and \u003cem\u003eosabcb4-3 \u003c/em\u003eat the heading stage. Photographs were taken at 70 days after sowing. Bar = 10 cm. (c) Days to heading in WT, \u003cem\u003eosabcb4-1\u003c/em\u003e, \u003cem\u003eosabcb4-2\u003c/em\u003e, and \u003cem\u003eosabcb4-3\u003c/em\u003e. Values are shown as mean ± SD of heading date, n = 10. (d–h) Relative expression of Heading Date-Related Genes in WT and \u003cem\u003eosabcb4s\u003c/em\u003e.Values are shown as mean ± SD of expression levels, n = 3. (i) Days to heading in WT, \u003cem\u003eosabcb4-1\u003c/em\u003e, \u003cem\u003eosabcb4-2\u003c/em\u003e, and \u003cem\u003eosabcb4-3\u003c/em\u003e. Values are shown as mean ± SD of heading date, n = 10. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, Student’s \u003cem\u003et\u003c/em\u003e-test\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8096452/v1/7c434baab653a206687389f8.png"},{"id":96919242,"identity":"cf01a290-b417-485d-9249-c71078b95433","added_by":"auto","created_at":"2025-11-27 14:13:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":377697,"visible":true,"origin":"","legend":"\u003cp\u003eAgronomic traits of WT and \u003cem\u003eabcb4s\u003c/em\u003e mutant lines. (a–b) The plant architecture (a), panicles (b) and grain per plant(c) of WT and \u003cem\u003eabcb4s\u003c/em\u003e. Scale bar, a–b = 5 cm. All grains were put in a single layer. (d–i) The Seed setting rate (d, n=10), Yield per plant (e, n = 20), 1000-grain weight (f, n = 10), Tiller number (g, n = 40), Panicle length (h, n = 150), Plant height (i, n = 40) of WT and \u003cem\u003eabcb4s\u003c/em\u003e. (j) I\u003csub\u003e2\u003c/sub\u003e-KI staining of the pollen grains of the WT and \u003cem\u003eabcb4s\u003c/em\u003e. The functional pollen grains are darkly stained, while the inactivated pollen grains are lightly stained. Scale bar = 200 μm. (k) Pollen fertility rates of WT and \u003cem\u003eabcb4s\u003c/em\u003e. Values are shown as mean ± SD. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, Student’s \u003cem\u003et\u003c/em\u003e-test\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8096452/v1/cd8a86ac1ca5ce783531cecf.png"},{"id":96835567,"identity":"23fac65f-eafd-48fb-a082-3d8ed4f2273e","added_by":"auto","created_at":"2025-11-26 14:42:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10960,"visible":true,"origin":"","legend":"\u003cp\u003eIAA content in spikes at anthesis stage of WT and \u003cem\u003eabcb4\u003c/em\u003e mutant lines. Data represent three biological replicates, with each replicate consisting of pooled spikelets from eight individual plants. Values are expressed as mean ± SD. n = 3. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, Student’s \u003cem\u003et\u003c/em\u003e-test\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8096452/v1/35d1d74a5843f83863cd91aa.png"},{"id":96917660,"identity":"586c7f98-2b49-4f61-b6c9-2c5b80e555d5","added_by":"auto","created_at":"2025-11-27 14:10:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":175305,"visible":true,"origin":"","legend":"\u003cp\u003eGlobal gene expression changes in \u003cem\u003eosabcb4\u003c/em\u003e plants spikes at anthesis. (a) Volcano plot of DEGs between WT plants and \u003cem\u003eosabcb4\u003c/em\u003e plants. Each dot represents a gene. (b) The number of DEGs in \u003cem\u003eosabcb4\u003c/em\u003e plants. (c)KEGG pathway analysis of the DEGs. Enrichment 9 significant pathways (\u003cem\u003eP\u003c/em\u003e-value \u0026lt; 0.05 and \u003cem\u003eQ\u003c/em\u003e-value \u0026lt; 0.05). (d) GO classifications of the DEGs. Enriched 20 significant different GO categories (\u003cem\u003eP\u003c/em\u003e-value \u0026lt; 0.05 and \u003cem\u003eQ\u003c/em\u003e-value \u0026lt; 0.05). (e–l) RT-qPCR to verify the transcript level of up-regulated genes. Values are shown as mean ± SD. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, Student’s \u003cem\u003et\u003c/em\u003e-test\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8096452/v1/28b89164d1d8e3b12d1356f5.png"},{"id":96917432,"identity":"5f359a4e-07e0-4916-874a-97095072051a","added_by":"auto","created_at":"2025-11-27 14:09:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":350013,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eosabcb4s\u003c/em\u003e on seed and seedling phenotypes in rice. (a–c) Phenotypes of seeds treated with ddH\u003csub\u003e2\u003c/sub\u003eO (CK) or 8 μM abscisic acid (ABA) for seven days. (d–e) Phenotypes of seven-day-old WT and \u003cem\u003eosabcb4\u003c/em\u003e rice seedlings. (f–g) Shoot length (f) and Root length (g) of WT and \u003cem\u003eosabcb4\u003c/em\u003e seedlings grown with or without 8 μM ABA for seven days. (h) Shoot length measurement in Figure d. (i) Protein content in seeds. (j) Total starch content in seeds. (k) Amylose content in seeds. (l) Amylopectin content in seeds. Data in panels (f–h) are presented as mean ± SD (n ≥ 10). Data in panels (i–l) are presented as mean ± SD (n ≥ 3). Double asterisks indicate a significant difference between WT and \u003cem\u003eosabcb4 \u003c/em\u003eplants at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 by Student's \u003cem\u003et\u003c/em\u003e-test. Scale bar:1 cm (applies to a–e)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8096452/v1/9c0ffa851d3870394b86b2d8.png"},{"id":96917423,"identity":"bfa643fd-41ff-4b3b-a9d5-9e6e8d27ee4f","added_by":"auto","created_at":"2025-11-27 14:09:43","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":97715,"visible":true,"origin":"","legend":"\u003cp\u003eA working model for the diverse defects caused by\u003cem\u003e OsABCB4 \u003c/em\u003eknockout. (Created in \u003ca href=\"https://biorender.com/\"\u003ehttps://BioRender.com\u003c/a\u003e)\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8096452/v1/c07a807775549cbf8578c766.png"},{"id":106809076,"identity":"1579f1a2-12f7-4577-a0e7-6872ac1121ef","added_by":"auto","created_at":"2026-04-13 16:06:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2427670,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8096452/v1/5dc2f32b-07dd-4570-a943-b1d913cf53f7.pdf"},{"id":96835572,"identity":"7b109413-5916-4e1b-9a55-2e5a8cc84d50","added_by":"auto","created_at":"2025-11-26 14:42:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18222,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e Table S1. The primers used in RT-qPCR in this study.\u003c/p\u003e","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8096452/v1/bc02ca02c27e6f5362ed6650.docx"}],"financialInterests":"","formattedTitle":"Rice OsABCB4 delays heading date and impacts multiple agronomic traits by affecting auxin homeostasis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRice (\u003cem\u003eOryza sativa\u003c/em\u003e L.), commonly referred to as Asian cultivated rice, serves as a staple food for more than half of the global population. Rice breeders have consistently prioritized the enhancement of grain yield and quality as key objectives(Fiaz et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Phytohormones, also known as plant growth regulators, modulate growth and development in rice by activating or inhibiting specific metabolic pathways(Woodward and Bartel \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Endogenous phytohormones include abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), ethylene (ET), auxin, gibberellin (GA), brassinosteroid (BR), and cytokinin (CK)(Gong et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Among these, auxin was the first discovered plant hormone, which plays a pivotal role in regulating growth processes(Yu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It is critically involved in phototropism, root formation, leaf development, fertility, embryogenesis, and vascular tissue differentiation(Swarup and Bhosale \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMultiple gene families participate in auxin biosynthesis and transport. These include \u003cem\u003eOsARF\u003c/em\u003e and \u003cem\u003eOsIAA\u003c/em\u003e involved in signal transduction; \u003cem\u003eOsAUX/LAX\u003c/em\u003e, \u003cem\u003eOsPIN\u003c/em\u003e, and \u003cem\u003eOsPGP/ABCB\u003c/em\u003e responsible for auxin transport; \u003cem\u003eOsGH3\u003c/em\u003e participating in metabolic homeostasis; and \u003cem\u003eOsSAUR\u003c/em\u003e executing rapid responses(Jain et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ye et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hou et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Devi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These genes act synergistically or antagonistically to coordinately regulate auxin synthesis and transport, maintaining auxin homeostasis and thereby influencing plant growth and development. For example: \u003cem\u003eOsIAA6\u003c/em\u003e interacts with \u003cem\u003eOsARF1\u003c/em\u003e to suppress auxin signaling and regulate leaf inclination(Xing et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eOsARF6\u003c/em\u003e and \u003cem\u003eOsARF17\u003c/em\u003e are highly expressed in leaf joint tissues and modulate flag leaf angle in response to auxin signals(Huang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eOsARF25\u003c/em\u003e regulates grain length, width, and thousand-grain weight via the brassinosteroid signaling pathway(Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). \u003cem\u003eOsPIN1b\u003c/em\u003e, \u003cem\u003eOsPIN1c\u003c/em\u003e, and \u003cem\u003eOsPIN9\u003c/em\u003e mediate auxin transport from shoots to roots(Wang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Overexpression of \u003cem\u003eOsPIN2\u003c/em\u003e increases auxin distribution in the root epidermis(Sun et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eOsGH3\u003c/em\u003e, a member of the auxin-responsive gene family, participates in plant growth, developmental processes, and stress responses. \u003cem\u003eOsSAUR45\u003c/em\u003e affects auxin synthesis and transport by repressing the expression of \u003cem\u003eOsYUCCA\u003c/em\u003e and \u003cem\u003eOsPIN\u003c/em\u003e genes, thereby influencing plant growth(Xu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eOsSAUR39\u003c/em\u003e acts a negative regulator of auxin synthesis and transport; its overexpression in rice suppresses lateral root development and reduces yield(Kant et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). These genes and their regulatory networks provide a window into understanding how plants integrate internal and external signals through hormonal coordination.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eABC\u003c/em\u003e superfamily represents one of the largest transporter families in plants and is responsible for auxin transport. Among its subfamilies, \u003cem\u003eABCB\u003c/em\u003e members are primarily associated with auxin transport, with each member performing distinct functions in plant systems. There are 29 \u003cem\u003eABCB\u003c/em\u003e genes in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and 27 in rice, participating in diverse cellular, physiological, and developmental processes(Devi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). For instance: in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eAtABCB1/PGP1\u003c/em\u003e mediates auxin efflux, influences auxin distribution in the shoot apex, and indirectly regulates flowering time(Geisler et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Loss of \u003cem\u003eAtABCB19\u003c/em\u003e (also known as \u003cem\u003eAtPGP19/MDR1\u003c/em\u003e) disrupts AtPIN1 membrane localization, enhances plant sensitivity to gravity and light, and indirectly affects gravitropic responses. Altered tropic growth in root or shoot tips may perturb hormonal signaling and subsequently influence flowering. Moreover, the \u003cem\u003eatabcb19\u003c/em\u003e mutant exhibits delayed anther development and reduced pollen viability(Noh et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). \u003cem\u003eAtABCB4\u003c/em\u003e plays a key role in root architecture and root hair development by regulating polar auxin transport, potentially influencing flowering through modulation of long-distance hormone transport(Kubes et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In rice, several \u003cem\u003eABCB\u003c/em\u003e members also function in auxin transport: \u003cem\u003eOsABCB14\u003c/em\u003e transports multiple substrates and is involved in cellular auxin uptake and iron homeostasis(Xu et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). \u003cem\u003eOsABCB24\u003c/em\u003e is essential for female gametogenesis and early seed development(Nguyen et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). It transports auxin precursors or auxin across the endoplasmic reticulum (ER) membrane, and disruption of ER auxin homeostasis leads to female sterility. The \u003cem\u003eosabcb24\u003c/em\u003e mutant displays structural abnormalities during early female gametophyte development and initial endosperm formation, characterized by disorganized syncytial nuclear structures and significantly reduced seed set(Nguyen et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe development of rice grains is critically regulated by auxin. However, the mechanisms underlying auxin transport and its regulation in rice grains are not yet fully understood. In this study, we found that the \u003cem\u003eABCB\u003c/em\u003e family member \u003cem\u003eOsABCB4\u003c/em\u003e is preferentially expressed in rice grains and participates in auxin accumulation. Mutation of \u003cem\u003eOsABCB4\u003c/em\u003e significantly delayed heading date and reduced pollen fertility, grain yield, and grain quality.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant Materials and Growth Conditions\u003c/h2\u003e\u003cp\u003eThe rice materials used in this study included the \u003cem\u003ejaponica\u003c/em\u003e cultivar ZH11 and its derived mutant lines. Genomic DNA extracted from leaves of ZH11 wild-type (WT) and transgenic plants served as the template for PCR amplification. Sequencing of the target gene and the hygromycin resistance gene was performed across successive T1 and T2 mutant generations. Three stable T3 homozygous mutant lines (\u003cem\u003eosabcb4-1\u003c/em\u003e, \u003cem\u003eosabcb4-2\u003c/em\u003e, and \u003cem\u003eosabcb4-3\u003c/em\u003e) were selected for subsequent phenotypic evaluation. Field experiments were conducted at the experimental station of Guangxi University (Nanning, Guangxi Zhuang Autonomous Region, China; 22.49\u0026prime;N, 108.19\u0026prime;E). Plants were grown under both long-day (LD) and short-day (SD) conditions, corresponding to the local early- and late-season rice cropping periods, respectively. According to data from the China Meteorological Data Service Center (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://data.cma.cn\u003c/span\u003e\u003cspan address=\"http://data.cma.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the daylength during the photoperiod-sensitive phase was 13.14\u0026ndash;13.24 hours in the long-day season (mid-May to mid-July) and 11.38\u0026ndash;12.17 hours in the short-day season (mid-September to mid-October). For the early-season (LD) trial, seeds were sown in March and seedlings were transplanted into paddy fields under standard agronomic management in April. For the late-season (SD) trial, sowing took place in mid-July, with transplanting in August. All WT and the three mutant lines were arranged in a randomized complete block design with three independent replications. Each plot consisted of 6 rows with 8 plants per row, spaced at 20 cm \u0026times; 20 cm intervals. To minimize edge effects, ten plants from the central area of each plot were selected for measuring heading date and agronomic traits. All field management practices followed conventional local rice production protocols.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCRISPR/Cas9 Vector Construction\u003c/h3\u003e\n\u003cp\u003eIn this study, we generated \u003cem\u003eosabcb4\u003c/em\u003e mutants using CRISPR/Cas9-mediated gene editing. Target-specific gRNA sequences were designed against the sixth and tenth exons of the \u003cem\u003eABCB4\u003c/em\u003e gene using the online CRISPR design platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispr.hzau.edu.cn/CRISPR2/\u003c/span\u003e\u003cspan address=\"http://crispr.hzau.edu.cn/CRISPR2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and subsequently cloned into the pFATB-Cas9 vector. The recombinant construct was introduced into the \u003cem\u003ejaponica\u003c/em\u003e rice cultivar ZH11 via Agrobacterium-mediated transformation. Homozygous mutant lines were identified and selected from the T1 transgenic population through sequencing analysis. We additionally obtained the target site sequence-containing primers CAACGGATAGCTATAGCAA and AGTTGATCAAGGATCCCGA from BLAST CRISPR-GE and NCBI as a means of ensuring that no off-target gene targeting occurred via this approach.\u003c/p\u003e\n\u003ch3\u003eDNA Extraction\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from fresh leaves of WT and mutant plants using the CTAB method. The purified DNA samples were stored at 4\u0026deg;C for subsequent sequencing of the target gene and the hygromycin resistance gene(Murray and Thompson \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1980\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eRNA Extraction and Gene Expression Analysis\u003c/h3\u003e\n\u003cp\u003ePlant tissues were collected from rice plants, immediately wrapped in aluminum foil, flash-frozen in liquid nitrogen, and stored at -80\u0026deg;C until RNA extraction. Total RNA was isolated using the FreeZol Reagent Kit (Vazyme) according to the manufacturer's protocol. RNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). First-strand cDNA was synthesized from 3 \u0026micro;g of total RNA in a 20 \u0026micro;L reaction system using HiScript III RT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) (Vazyme). Quantitative real-time PCR (qPCR) was performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme) on a Cobas z 480 real-time PCR system. The rice \u003cem\u003eUbiquitin\u003c/em\u003e gene (\u003cem\u003eOs05g06770\u003c/em\u003e) was used as the internal reference gene for normalization. Primer sequences for the genes under examination are detailed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Anthers were categorized based on spikelet length, as per establised criteria(Xu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of\u003c/b\u003e \u003cb\u003eABCB\u003c/b\u003e \u003cb\u003eGene Family Expression Patterns Across Developmental Stages and Tissues\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe rice genome encodes 27 \u003cem\u003eABCB\u003c/em\u003e family members. Expression data for \u003cem\u003eABCB\u003c/em\u003e genes were obtained from the Rice eFP Browser (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bar.utoronto.ca/efprice/cgi-bin/efpWeb.cgi\u003c/span\u003e\u003cspan address=\"https://bar.utoronto.ca/efprice/cgi-bin/efpWeb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The average signal values across 15 distinct tissues at various developmental stages were log10-transformed and subjected to cluster analysis. Visualization of the resulting expression patterns was performed using the HeatMap function in TBtools software(Chen et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic Analysis of\u003c/b\u003e \u003cb\u003eABCB\u003c/b\u003e \u003cb\u003eGene Family\u003c/b\u003e\u003c/p\u003e\u003cp\u003eProtein sequences of \u003cem\u003eABCB\u003c/em\u003e gene family members from rice and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e were retrieved from the Ensembl Plants database. A phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA7.0 software(Kumar et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eHeading Date Measurement and Agronomic Trait Investigation\u003c/h3\u003e\n\u003cp\u003eHeading date was defined as the number of days from sowing to the emergence of the first panicle on each plot. At maturity, agronomic traits, including productive tiller number, plant height, panicle length, seed setting rate, grain yield per plant, and 1000-grain weight, were measured using uniformly grown plants selected from the central area of plots containing WT and the three mutant lines (\u003cem\u003eabcb4-1\u003c/em\u003e, \u003cem\u003eabcb4-2\u003c/em\u003e, and \u003cem\u003eabcb4-3\u003c/em\u003e). A productive tiller was defined as one that headed normally and bore at least five filled grains. Representative plants from each experimental plot were randomly sampled to count productive tillers. Plant height was measured as the vertical distance from the ground level to the tip of the tallest panicle. Panicle length was determined as the distance from the panicle neck to the tip of the main panicle. At maturity, panicles from individual plants were harvested and manually threshed. Filled and unfilled grains were separated by water flotation. The seed setting rate was calculated as: Seed setting rate (%) = [number of filled grains / (number of filled grains\u0026thinsp;+\u0026thinsp;number of unfilled grains)] \u0026times; 100%. Filled grains were dried at 45\u0026deg;C for 72 hours until constant weight was achieved. Grain yield per plant was determined as the total weight of filled grains from a single plant. The 1000-grain weight was calculated by weighing ten sets of 1000 filled grains.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAssessment of Rice Pollen Fertility by I₂-KI Staining Method\u003c/h2\u003e\u003cp\u003eAt the heading stage, pre-anthesis pollen grains were collected from WT and the three mutant lines, and immediately preserved in microcentrifuge tubes containing 75% (v/v) ethanol. For pollen viability assessment, anthers from a single floret were placed on a glass slide, stained with 1\u0026ndash;2 drops of 1% iodine-potassium iodide (I₂-KI) solution, and gently crushed with forceps to release pollen grains. A coverslip was applied, and slight pressure was exerted before allowing the sample to stand for 2\u0026ndash;3 minutes. Observation was carried out under a stereomicroscope. For each plant, pollen grains from eight randomly selected fields of view were scored as fertile or sterile. Three individual plants were evaluated per line(Chhun et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDetermination of Starch and Protein Content\u003c/h3\u003e\n\u003cp\u003eGrains were dried at 65\u0026deg;C for 5 days and ground into fine powder. Total starch content was determined using a commercial assay kit (BC0700, Solarbio) with 0.1 g of sample powder. Amylose content was measured by extracting alkali-soluble and alcohol-soluble proteins with 0.1 M NaOH and 70% ethanol, followed by quantification using the Bradford method. Amylopectin content was calculated by subtracting amylose content from total starch content. For protein content analysis, 0.5 kg of polished rice from both WT and mutant lines was evaluated using a rice taste analyzer (JSWL, RLTA10C, Japan). All measurements were performed with three biological replicates(Li et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eDetermination of Indole-3-Acetic Acid (IAA) Content\u003c/h3\u003e\n\u003cp\u003eThe endogenous indole-3-acetic acid (IAA) content was quantified using high-performance liquid chromatography (HPLC; Agilent 1200 series). Prior to analysis, each sample, which was composed of a pool of eight randomly selected spikelets, was homogenized and extracted with a cold phosphate buffer. The extracts were purified using C18 solid-phase extraction cartridges. Separation was achieved on a reversed-phase Diamonsil C18(2) column (250 mm \u0026times; 4.6 mm, 5 \u0026micro;m) maintained at 35\u0026deg;C. An isocratic mobile phase consisting of methanol-water-glacial acetic acid (45:54.2:0.8) was delivered at a flow rate of 0.8 mL/min. The detection wavelength was set at 280 nm for specific IAA measurement. The method was validated for specificity, linearity (R\u0026sup2; \u0026gt;0.999), and recovery (90\u0026ndash;105%). Quantification was performed by comparing the peak areas with those of an authentic IAA standard calibration curve, and the mean value from three independent biological replicates was reported for each treatment(Nishimura et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Shao et al. 2019).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMaterials for Transcriptome Sequencing\u003c/h2\u003e\u003cp\u003eIn spring 2025, under standard rice growth conditions, panicles at the tenth developmental stage were collected from both WT and \u003cem\u003eabcb4\u003c/em\u003e plants. Each biological sample contained at least 1.0 g of tissue, with three independent replicates prepared for each genotype. After flash-freezing in liquid nitrogen, samples were transported on dry ice to Tsingke Biotechnology Co., Ltd (Beijing, China) for RNA extraction and transcriptome sequencing. Differentially expressed genes (DEGs) were identified from the transcriptome data using a significance threshold of \u003cem\u003eQ\u003c/em\u003e-value\u0026thinsp;\u0026le;\u0026thinsp;0.05(Love et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eKEGG Analysis of Differentially Expressed Genes\u003c/h2\u003e\u003cp\u003eDifferentially expressed genes (DEGs) between WT and \u003cem\u003eabcb4\u003c/em\u003e mutants were identified based on fragments per kilobase of transcript per million mapped reads (FPKM) values. Significantly differentially expressed genes were defined as those with an adjusted \u003cem\u003eP\u003c/em\u003e-value (\u003cem\u003eQ\u003c/em\u003e -value)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and an absolute log\u003csup\u003e2\u003c/sup\u003e fold change\u0026thinsp;\u0026ge;\u0026thinsp;1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the cloud platform provided by Tsingke Biotechnology Co., Ltd (Beijing, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eData Processing and Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll data were organized using Excel 2003 and subjected to one-way analysis of variance (ANOVA) in SPSS 27 for statistical analysis of agronomic traits. Graphs were generated with GraphPad Prism 9. Significant differences among groups were assessed by Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test, with data presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) is indicated by different lowercase letters in the figures.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ePhylogenetic and Expression Analysis of\u003c/b\u003e \u003cb\u003eABCB\u003c/b\u003e \u003cb\u003eSubfamily Genes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the phylogenetic relationships and functional divergence of the \u003cem\u003eABCB\u003c/em\u003e subfamily, a phylogenetic tree was reconstructed using 56 protein sequences from rice and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Based on evolutionary relationships, this subfamily can be further divided into five subgroups (I\u0026ndash;V) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe expression patterns of genes across different tissues and organs provide crucial insights into their biological functions. Based on distinct expression profiles, the rice \u003cem\u003eABCB\u003c/em\u003e subfamily can be classified into five subgroups (A\u0026ndash;E), indicating significant functional divergence among these genes. Subgroup E genes are predominantly expressed in seeds, suggesting their potential roles in anther development and material transport during seed maturation. Within this subgroup, \u003cem\u003eOsABCB4\u003c/em\u003e exhibits markedly higher expression in inflorescence P5 and P6 stages, as well as in seed development stages S1, S2, and S3, compared to other tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eTo determine the expression pattern of \u003cem\u003eOsABCB4\u003c/em\u003e, its transcript levels in various tissues of WT plants were analyzed by RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The results showed that \u003cem\u003eOsABCB4\u003c/em\u003e expression was significantly upregulated after stage S6 of panicle development, consistent with the transcriptome profiling data, suggesting its potential involvement in panicle development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eLoss of\u003c/b\u003e \u003cb\u003eOsABCB4\u003c/b\u003e \u003cb\u003eFunction Delays Heading Date in Rice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the function of \u003cem\u003eOsABCB4\u003c/em\u003e, we generated three independent homozygous transgenic lines using CRISPR-Cas9 technology, each carrying distinct editing types in the target gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). WT and the \u003cem\u003eabcb4\u003c/em\u003e mutants (\u003cem\u003eabcb4-1\u003c/em\u003e, \u003cem\u003eabcb4-2\u003c/em\u003e, \u003cem\u003eabcb4-3\u003c/em\u003e) were cultivated under normal field management in the same paddy field, and heading dates were recorded. On average, there was a 9-day difference in heading date between the WT and mutant lines, with values of 63\u0026thinsp;\u0026plusmn;\u0026thinsp;2 days and 72\u0026thinsp;\u0026plusmn;\u0026thinsp;2 days, respectively, in Nanning during the 2024/2025 growing season (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u0026ndash;c).\u003c/p\u003e\u003cp\u003eWe further examined the expression levels of key heading date regulatory genes in the flag leaves of WT and \u003cem\u003eabcb4-1\u003c/em\u003e plants by qRT-PCR. The results showed significant upregulation of \u003cem\u003eOsGI\u003c/em\u003e and \u003cem\u003eOsHd1\u003c/em\u003e, and significant downregulation of \u003cem\u003eOsEhd1\u003c/em\u003e, \u003cem\u003eOsHd3a\u003c/em\u003e, and \u003cem\u003eOsRFT1\u003c/em\u003e in the mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u0026ndash;h). These genes are known to function in the \u003cem\u003eHd1/Ghd7/DTH8\u0026ndash;Ehd1\u0026ndash;Hd3a/RFT1\u003c/em\u003e pathway that controls heading date in rice. A schematic model illustrating the regulation of heading date is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei. The observed changes in gene expression are consistent with the delayed heading phenotype in the \u003cem\u003eabcb4\u003c/em\u003e mutant.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eLoss of\u003c/b\u003e \u003cb\u003eOsABCB4\u003c/b\u003e \u003cb\u003eFunction Leads to Reduced Pollen Fertility and Severe Yield Defects in Rice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUnder standard field conditions, we observed that the mutant plants exhibited a dwarf stature, more compact and shorter panicles, and reduced grain yield per plant compared to the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u0026ndash;c). We therefore systematically evaluated their agronomic traits. Statistical analysis revealed that the seed-setting rate of the WT was 88.71%, whereas all three mutant lines showed significantly reduced rates: 51.59% in \u003cem\u003eabcb4-1\u003c/em\u003e, 68.39% in \u003cem\u003eabcb4-2\u003c/em\u003e, and 69.05% in \u003cem\u003eabcb4-3\u003c/em\u003e, with an average reduction of approximately 25.7% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). This decrease in seed-setting rate severely compromised grain yield per plant. The WT yield was 14.09 g per plant, while the mutants yielded significantly less: 6.70 g for \u003cem\u003eabcb4-1\u003c/em\u003e, 9.93 g for \u003cem\u003eabcb4-2\u003c/em\u003e, and 11.66 g for \u003cem\u003eabcb4-3\u003c/em\u003e. Notably, the yield of \u003cem\u003eabcb4-1\u003c/em\u003e was less than half that of the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Given the known role of \u003cem\u003eOsABCB4\u003c/em\u003e orthologs in regulating pollen viability, we hypothesized that impaired pollen fertility might underlie the reduced seed-setting and yield. Pollen viability assessed by I\u003csub\u003e2\u003c/sub\u003e-KI staining was significantly lower in the mutants. The staining rate was 95.02% in the WT, but only 35.08% in \u003cem\u003eabcb4-1\u003c/em\u003e, 62.20% in \u003cem\u003eabcb4-2\u003c/em\u003e, and 64.32% in \u003cem\u003eabcb4-3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej\u0026ndash;k), indicating that loss of \u003cem\u003eABCB4\u003c/em\u003e function severely disrupts normal pollen development, leading to the observed reduction in seed set. In addition, other agronomic traits including 1000-grain weight, tiller number, panicle length, and plant height were also significantly reduced in the mutants relative to the WT. Specifically, the thousand-grain weight was markedly lower in the mutants (\u003cem\u003eabcb4-1\u003c/em\u003e: 22.13 g; \u003cem\u003eabcb4-2\u003c/em\u003e: 22.34 g; \u003cem\u003eabcb4-3\u003c/em\u003e: 18.75 g) than in the WT (25.85 g). Similarly, tiller number was severely reduced to 6.21, 6.41, and 8.09 in the three mutants, respectively, versus 11.07 in the WT. The mutants also exhibited significantly shorter panicle lengths (14.04 cm, 16.56 cm, 17.19 cm) compared to the WT (20.28 cm), and a pronounced dwarf phenotype with plant heights of only 68.81 cm, 71.71 cm, and 75.00 cm, respectively, in contrast to the WT (91.20 cm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef\u0026ndash;i). These results demonstrate that knockout of \u003cem\u003eOsABCB4\u003c/em\u003e significantly impairs rice growth and development, leading to substantial defects in key yield-related traits.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eDetermination of Indole-3-Acetic Acid (IAA) Content\u003c/h2\u003e\u003cp\u003eWe observed phenotypes such as pollen abortion and reduced seed setting rate in the mutants. Given the relatively high and easily distinguishable expression of \u003cem\u003eOsABCB4\u003c/em\u003e during the flowering stage, we measured indole-3-acetic acid (IAA) content in spikelets of WT and two mutant lines (\u003cem\u003eabcb4-1\u003c/em\u003e and \u003cem\u003eabcb4-2\u003c/em\u003e) at this developmental stage. The results showed that WT plants contained 257.16 ng/g IAA, while the mutants exhibited significantly reduced levels: 43.73 ng/g in \u003cem\u003eabcb4-1\u003c/em\u003e and 71.88 ng/g in \u003cem\u003eabcb4-2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings indicate that knockout of \u003cem\u003eOsABCB4\u003c/em\u003e drastically reduces IAA accumulation in panicles during flowering.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eTranscriptome Analysis Reveals Comprehensive Regulatory Network\u003c/h2\u003e\u003cp\u003eTo investigate the molecular mechanisms underlying the delayed heading, reduced yield, and decreased auxin levels in \u003cem\u003eOsABCB4\u003c/em\u003e knockout mutants, we conducted transcriptome sequencing of panicles at the flowering stage from both WT and \u003cem\u003eabcb4-1\u003c/em\u003e plants. Three biological replicates were analyzed, each consisting of pooled spikelets from eight individual plants. RNA-seq analysis identified 3346 differentially expressed genes (DEGs), with 2747 upregulated and 599 downregulated in the mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u0026ndash;b). To elucidate the biological functions of \u003cem\u003eOsABCB4\u003c/em\u003e during flowering, Gene Ontology (GO) enrichment analysis was performed on the DEGs. The results revealed significant enrichment in 20 GO terms, including 8 biological processes, 11 cellular components, and 1 molecular function term (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). DEGs were notably enriched in processes such as \"embryo development ending in seed dormancy\" and \"acquisition of seed longevity,\" and were primarily involved in cytoplasmic translation, ribosome assembly, and organellar RNA processing, indicating strong activation of protein synthesis machinery. These functions are closely associated with seed development, particularly the accumulation of storage proteins and protective factors required during embryo maturation and seed dormancy. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEGs were enriched in plant hormone signal transduction, starch and sucrose metabolism, and metabolic pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Further analysis of transcription factors among the DEGs identified several auxin-related families, including \u003cem\u003eGH3\u003c/em\u003e, \u003cem\u003eARF\u003c/em\u003e, and \u003cem\u003eIAA\u003c/em\u003e. Notably, genes regulating auxin biosynthesis and transport, such as \u003cem\u003eOsGH3-2\u003c/em\u003e, \u003cem\u003eOsARF1\u003c/em\u003e, and \u003cem\u003eOsIA10\u003c/em\u003e, were differentially expressed(Du et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The expression changes of key genes, including \u003cem\u003eOsARF1\u003c/em\u003e, \u003cem\u003eOsARF16\u003c/em\u003e, \u003cem\u003eOsGH3-2\u003c/em\u003e, \u003cem\u003eOsGH3-3\u003c/em\u003e, \u003cem\u003eOsIA10\u003c/em\u003e, \u003cem\u003eOsIAA14\u003c/em\u003e, \u003cem\u003eOsIAA23\u003c/em\u003e, and \u003cem\u003eOsIAA24\u003c/em\u003e, were validated by qRT-PCR, all of which were significantly upregulated in the mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee\u0026ndash;l). Collectively, these results suggest that knockout of \u003cem\u003eOsABCB4\u003c/em\u003e disrupts the expression of genes involved in auxin biosynthesis and transport, which may directly or indirectly reduce auxin levels in panicles and impair normal anther and pollen development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eGrain Storage Substance Content\u003c/h2\u003e\u003cp\u003eGiven the enrichment of plant hormone signal transduction, starch and sucrose metabolism, and seed development-related pathways in KEGG and GO analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec\u0026ndash;d), we performed hormone treatment experiments to assess seed germination. Under control conditions (water), no significant differences in shoot or root growth were observed between WT and \u003cem\u003eosabcb4\u003c/em\u003e mutants after 7 days. However, treatment with 8 \u0026micro;M abscisic acid (ABA) resulted in significantly reduced shoot and root growth in \u003cem\u003eosabcb4\u003c/em\u003e mutants compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea\u0026ndash;c, f, g). When grown under standard conditions (28\u0026deg;C, 80% humidity) for 7 days, \u003cem\u003eosabcb4\u003c/em\u003e mutants exhibited significantly reduced shoot growth compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed\u0026ndash;e, h). We hypothesized that this phenotype might be related to altered nutrient reserves in the grains and therefore analyzed seed storage compounds. Compared to WT, \u003cem\u003eosabcb4\u003c/em\u003e mutants showed marked abnormalities in seed storage accumulation. Total starch content was significantly reduced in all mutant lines, with \u003cem\u003eabcb4-1\u003c/em\u003e and \u003cem\u003eabcb4-3\u003c/em\u003e showing the most pronounced decreases to 623 mg/g and 617 mg/g, respectively, representing approximately 30% reduction from the wild-type level of 875 mg/g. The \u003cem\u003eabcb4-2\u003c/em\u003e line contained 679 mg/g total starch, a 22% reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ej). Further analysis revealed concurrent decreases in both amylose and amylopectin content, though amylopectin reduction was more substantial. In \u003cem\u003eabcb4-1\u003c/em\u003e mutants, amylopectin content dropped to 468 mg/g, a 31% decrease from the WT level of 678 mg/g, while amylose decreased by 21% (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ek\u0026ndash;l). Notably, all mutant lines showed significantly increased protein content. WT protein content was 108 mg/g, while \u003cem\u003eabcb4-3\u003c/em\u003e mutants reached 124 mg/g, a 14.8% increase. The \u003cem\u003eabcb4-1\u003c/em\u003e and \u003cem\u003eabcb4-2\u003c/em\u003e lines also exhibited elevated protein levels of 120 mg/g and 119 mg/g, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei). In summary, compared to WT, the mutants displayed significantly reduced total starch, amylose, and amylopectin content, alongside significantly increased protein content.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings demonstrate that \u003cem\u003eOsABCB4\u003c/em\u003e, a member of the \u003cem\u003eABCB\u003c/em\u003e subfamily, plays a critical role in regulating auxin homeostasis in rice panicles, thereby influencing heading date, pollen development, and grain yield. Knockout of \u003cem\u003eOsABCB4\u003c/em\u003e led to reduced auxin content in spikelets at the flowering stage, delayed heading, impaired pollen fertility, decreased yield, and altered accumulation of grain storage reserves.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOsABCB4\u003c/b\u003e \u003cb\u003eregulates auxin homeostasis in the rice panicle.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe auxin regulatory network in plants is intricate and well-organized; plants have evolved a sophisticated system to precisely control auxin concentrations for optimal growth(Wang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In plants, the functionally well-characterized B-type \u003cem\u003eABC\u003c/em\u003e transporters (\u003cem\u003eABCB\u003c/em\u003e subfamily) are primarily involved in auxin transport(Takanashi et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eARFs\u003c/em\u003e (auxin response factors) perceive auxin signals and bind to auxin response cis-regulatory elements (\u003cem\u003eAuxREs\u003c/em\u003e) to regulate downstream transcriptional programs(Weijers and Wagner \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eARFs\u003c/em\u003e are classified into activators and repressors: activator \u003cem\u003eARFs\u003c/em\u003e contain a glutamine-rich middle region, whereas repressor \u003cem\u003eARFs\u003c/em\u003e are enriched in proline, serine, threonine, and other residues in their middle regions. \u003cem\u003eOsARF1\u003c/em\u003e and \u003cem\u003eOsARF16\u003c/em\u003e, with their glutamine-rich middle regions, belong to the activator class of auxin response factors(Wang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Overexpression of \u003cem\u003eOsGH3s\u003c/em\u003e depletes the substrate (free IAA) by acting upstream, thereby reducing auxin levels(Domingo et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Excess IAA proteins can form heterodimers with activator \u003cem\u003eARFs\u003c/em\u003e, directly inhibiting \u003cem\u003eARF\u003c/em\u003e transcriptional activity and blocking the auxin signaling pathway(Han et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Weijers and Wagner \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Following knockout of \u003cem\u003eOsABCB4\u003c/em\u003e, the expression of \u003cem\u003eOsARF1\u003c/em\u003e, \u003cem\u003eOsARF16\u003c/em\u003e, \u003cem\u003eOsGH3-2\u003c/em\u003e, \u003cem\u003eOsGH3-3\u003c/em\u003e, \u003cem\u003eOsIAA10\u003c/em\u003e, \u003cem\u003eOsIAA14\u003c/em\u003e, \u003cem\u003eOsIAA23\u003c/em\u003e, and \u003cem\u003eOsIAA24\u003c/em\u003e was significantly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Based on previous studies, we hypothesize that the upregulation of \u003cem\u003eOsARF1\u003c/em\u003e and \u003cem\u003eOsARF16\u003c/em\u003e in the panicle after \u003cem\u003eOsABCB4\u003c/em\u003e knockout triggers a marked increase in the expression of downstream auxin inactivation genes (\u003cem\u003eOsGH3s\u003c/em\u003e) and signaling repressors (\u003cem\u003eOsIAAs\u003c/em\u003e), leading to excessive depletion of free auxin and suppression of ARF activity, ultimately resulting in functional auxin deficiency in the panicle(Ori \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These results suggest that \u003cem\u003eOsABCB4\u003c/em\u003e may be directly or indirectly involved in regulating auxin biosynthesis and transport in rice panicles, thereby altering auxin levels in the panicle.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOsABCB4\u003c/b\u003e \u003cb\u003eRegulates Heading Date through the Florigen Pathway\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHeading date is an important agronomic trait in rice. The timing of heading affects the accumulation of photosynthetic products and grain filling during the ripening stage, ultimately influencing both yield and grain quality. Therefore, cultivars with an appropriate heading date can optimize grain yield and quality by making full use of light and temperature resources in their cultivation regions(Zhang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Heading date in rice is primarily governed by genetic factors and environmental conditions, with photoperiod being the major environmental regulator(Song et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Numerous studies have successfully identified and cloned multiple genes associated with heading date in rice. These genes form a complex network for perceiving and transducing light signals, precisely integrating internal and external cues to mediate rice photoperiod response, revealing the complexity of photoperiod-regulated flowering pathways. Among them, \u003cem\u003eHd1\u003c/em\u003e was the first cloned heading date gene in rice and is a homolog of \u003cem\u003eCO\u003c/em\u003e in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. \u003cem\u003eHd3a\u003c/em\u003e and \u003cem\u003eRFT1\u003c/em\u003e are homologs of the florigen gene \u003cem\u003eFT\u003c/em\u003e in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Ishikawa et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Under short-day (SD) conditions, \u003cem\u003eHd1\u003c/em\u003e promotes heading by upregulating \u003cem\u003eHd3a\u003c/em\u003e expression, whereas under long-day (LD) conditions, \u003cem\u003eHd1\u003c/em\u003e suppresses \u003cem\u003eHd3a\u003c/em\u003e transcription, delaying heading(Yano et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The rice gene \u003cem\u003eOsGI\u003c/em\u003e, a homolog of \u003cem\u003eGI\u003c/em\u003e in Arabidopsis, is regulated by the circadian clock and suppresses flowering under LD conditions(Zheng et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The Ghd7 protein is closely associated with flowering time; \u003cem\u003eOsGI\u003c/em\u003e directly interacts with Ghd7 and promotes its degradation, while phytochromes \u003cem\u003eOsPHYA\u003c/em\u003e and \u003cem\u003eOsPHYB\u003c/em\u003e competitively bind to Ghd7 against OsGI, thereby stabilizing the Ghd7 protein. Recent studies have shown that \u003cem\u003eEhd1\u003c/em\u003e is also a key regulator of heading date in rice(Doi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Under SD conditions, \u003cem\u003eEhd1\u003c/em\u003e regulates heading independently of \u003cem\u003eHd1\u003c/em\u003e, and it promotes heading under both LD and SD conditions. Unlike \u003cem\u003eHd1\u003c/em\u003e, under SD conditions \u003cem\u003eEhd1\u003c/em\u003e promotes heading by inducing the expression of \u003cem\u003eHd3a\u003c/em\u003e and \u003cem\u003eRFT1\u003c/em\u003e. In summary, photoperiod control of heading in rice mainly relies on two regulatory pathways: under SD conditions, the \u003cem\u003eHd1\u0026ndash;Hd3a/RFT1\u003c/em\u003e pathway (analogous to the Arabidopsis \u003cem\u003ethaliana GI\u0026ndash;CO\u0026ndash;FT\u003c/em\u003e pathway) regulates heading; under LD conditions, the \u003cem\u003eHd1/Ghd7/DTH8\u0026ndash;Ehd1\u0026ndash;Hd3a/RFT1\u003c/em\u003e pathway is involved(Zong et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The photoperiod regulation of heading date constitutes a complex and interactive network. We detected upregulation of \u003cem\u003eOsGI\u003c/em\u003e, \u003cem\u003eOsHd1\u003c/em\u003e, and \u003cem\u003eOsDTH8\u003c/em\u003e, and downregulation of \u003cem\u003eOsEhd1\u003c/em\u003e, \u003cem\u003eOsHd3a\u003c/em\u003e, and \u003cem\u003eOsRFT1\u003c/em\u003e in the flag leaves of \u003cem\u003eosabcb4\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). These genes belong to the \u003cem\u003eHd1/Ghd7/DTH8\u0026ndash;Ehd1\u0026ndash;Hd3a/RFT1\u003c/em\u003e pathway. The Hd1 protein inhibits \u003cem\u003eEhd1\u003c/em\u003e expression, and the upregulation of \u003cem\u003eHd1\u003c/em\u003e strengthens this suppression, leading to decreased \u003cem\u003eEhd1\u003c/em\u003e expression(Doi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). \u003cem\u003eDTH8\u003c/em\u003e is another key repressor; the \u003cem\u003eDTH8\u0026ndash;Hd1\u003c/em\u003e complex potently suppresses \u003cem\u003eEhd1\u003c/em\u003e expression(Du et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Upregulation of \u003cem\u003eDTH8\u003c/em\u003e significantly enhances the formation and efficacy of these repressive complexes, resulting in strong inhibition of \u003cem\u003eEhd1\u003c/em\u003e. \u003cem\u003eEhd1\u003c/em\u003e acts as the central integrator and master switch in this pathway, directly activating the expression of the florigen genes \u003cem\u003eHd3a\u003c/em\u003e and \u003cem\u003eRFT1\u003c/em\u003e(Komiya et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Hd3a and RFT1 are protein signals synthesized in leaves and transported to the shoot apical meristem, where they directly initiate heading(Tamaki et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Therefore, downregulation of \u003cem\u003eEhd1\u003c/em\u003e, \u003cem\u003eHd3a\u003c/em\u003e and \u003cem\u003eRFT1\u003c/em\u003e implies severe deficiency in florigen production. As a result, the shoot apex does not receive the \"flowering\" signal, leading to delayed heading in rice.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOsABCB4\u003c/b\u003e \u003cb\u003eInfluences Grain Storage Substance Accumulation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe phytohormone auxin plays crucial roles in regulating fundamental processes such as cell division, elongation, and differentiation, exerting pleiotropic effects on plant growth and development(Park et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Studies have demonstrated that auxin is integral to the entire reproductive process, from flower initiation to fruit maturation (Mariotti et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Pattison et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The pleiotropic effects observed in \u003cem\u003eosabcb4\u003c/em\u003e mutants, including impaired pollen viability, reduced seed-setting rate, and decreased grain yield per plant, may represent direct consequences of disrupted auxin signaling during reproductive development. Furthermore, auxin is a key hormone that regulates the distribution of photosynthates from the \u0026ldquo;source\u0026rdquo; to the \u0026ldquo;sink\u0026rdquo;(Zhao et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The observed reduction in starch content and concomitant increase in protein levels may be attributed to a disruption of the source-sink-flow balance caused by reduced auxin content in the grains, ultimately leading to an altered composition of grain storage reserves. This interpretation is supported by the significant enrichment of differentially expressed genes in the \"starch and sucrose metabolism\" pathway. Beyond the yield loss, a direct impact of impaired sugar metabolism is a marked delay in germination and seedling growth rates, which could be devastating for the widely adopted direct-seeding cultivation. Recently, Du et al. (2025) also reported that \u003cem\u003eOsABCB4\u003c/em\u003e regulates grain shape by influencing the length-to-width ratio through auxin. In summary, \u003cem\u003eOsABCB4\u003c/em\u003e serves as a key regulator governing the transition to reproductive growth and grain quality determination in rice. A schematic model of the proposed mechanism is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMeng Yang designed this research. Zongyue Jiang, Jiayu Li and Lei Huang participated in data analysis. Zongyue Jiang, Jiayu Li, Jingxin Wei, Lei Huang, Yujia Li, Mengfan Liu, Zihan Zhao, Fangchi Wei, Jiaxuan Guan, Jinxing Jiang, Ling Zhou, Kangshun Huang and Fugang Huang performed material preparation and data collection. Zongyue Jiang wrote the manuscript. Meng Yang corrected the manuscript. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32260474), the Guangxi Natural Science Foundation (2025GXNSFDA069036), the Guangxi Science and Technology Base and Talent Special Project (2022AC21114), the Open Research Project of Guangxi Key Laboratory of Agro-environment and Agric-products safety (GKLAEAPS2024-04), and the Innovation Project of Guangxi Graduate Education (YCBZ2025001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e This article does not contain any studies with human participants or animals, performed by any of the authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors have no conflicts of interest to de clare that are relevant to the content of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eChen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R (2020). 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Strong photoperiod sensitivity is controlled by cooperation and competition among \u003cem\u003eHd1\u003c/em\u003e, \u003cem\u003eGhd7\u003c/em\u003e and \u003cem\u003eDTH8\u0026nbsp;\u003c/em\u003ein rice heading. New Phytologist 229(3): 1635\u0026ndash;1649. https://doi.org/10.1111/nph.16946.\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpre","sideBox":"Learn more about [Journal of Plant Research](http://link.springer.com/journal/10265)","snPcode":"10265","submissionUrl":"https://www.editorialmanager.com/jpre/default2.aspx","title":"Journal of Plant Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Auxin transporter, Grain filling, Heading date, OsABCB4, Pollen fertility, Rice","lastPublishedDoi":"10.21203/rs.3.rs-8096452/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8096452/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMembers of the \u003cem\u003eABCB\u003c/em\u003e transporter subfamily are essential for various aspects of plant growth and development; however, a large number of ABCB proteins are functionally uncharacterized. Here, we report the functional characterization of a rice \u003cem\u003eABCB\u003c/em\u003e member, \u003cem\u003eOsABCB4\u003c/em\u003e. Tissue-specific expression analysis of 27 \u003cem\u003eABCB\u003c/em\u003e genes in rice identified a cluster with seed-specific expression, among which \u003cem\u003eOsABCB4\u003c/em\u003e was most highly expressed in developing panicles and seeds. CRISPR/Cas9-generated \u003cem\u003eosabcb4\u003c/em\u003e mutants exhibited a significant delay in heading date. Furthermore, the mutants displayed severe yield-related defects, including dwarfism, reduced panicle length, and a sharp decrease in seed-setting rate, primarily attributable to significantly impaired pollen fertility. Hormone quantification indicated a substantial reduction in indole-3-acetic acid (IAA) content in the panicles of mutant plants. Transcriptome analysis revealed global changes in gene expression, with differentially expressed genes significantly enriched in plant hormone signal transduction and starch/sucrose metabolism pathways. Consistent with these findings, the mutants showed abnormal accumulation of grain storage substances, characterized by significantly decreased starch content and increased protein content, consequently slowing down both seed germination and early seedling growth. Taken together, our results suggest that \u003cem\u003eOsABCB4\u003c/em\u003e may as a key regulator that influences heading date, pollen fertility, and grain filling by modulating auxin homeostasis in rice.\u003c/p\u003e","manuscriptTitle":"Rice OsABCB4 delays heading date and impacts multiple agronomic traits by affecting auxin homeostasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 14:42:54","doi":"10.21203/rs.3.rs-8096452/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-11-28T07:48:14+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-17T05:27:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-14T06:59:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Research","date":"2025-11-12T07:52:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpre","sideBox":"Learn more about [Journal of Plant Research](http://link.springer.com/journal/10265)","snPcode":"10265","submissionUrl":"https://www.editorialmanager.com/jpre/default2.aspx","title":"Journal of Plant Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9e1fdec9-181d-445d-919d-eb48e5b97d56","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:02:56+00:00","versionOfRecord":{"articleIdentity":"rs-8096452","link":"https://doi.org/10.1007/s10265-026-01702-7","journal":{"identity":"journal-of-plant-research","isVorOnly":false,"title":"Journal of Plant Research"},"publishedOn":"2026-04-11 15:58:36","publishedOnDateReadable":"April 11th, 2026"},"versionCreatedAt":"2025-11-26 14:42:54","video":"","vorDoi":"10.1007/s10265-026-01702-7","vorDoiUrl":"https://doi.org/10.1007/s10265-026-01702-7","workflowStages":[]},"version":"v1","identity":"rs-8096452","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8096452","identity":"rs-8096452","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}