The BEL1-like homeodomain protein OsBLH4 regulates rice plant height, grain number, and heading date by repressing the expression of OsGA2ox1.

Gibberellins (GAs) play crucial roles in regulating plant architecture and grain yield of crops. In rice, the inactivation of endogenous bioactive GAs and their precursors by GA 2-oxidases (GA2oxs) regulates stem elongation and reproductive development. However, the regulatory mechanisms of GA2ox gene expression, especially in rice reproductive organs, are unknown. The BEL1-like homeodomain protein OsBLH4, a negative regulatory factor for the rice OsGA2ox1 gene, was identified in this study. Loss of OsBLH4 function results in decreased bioactive GA levels and pleiotropic phenotypes, including reduced plant height, decreased grain number per panicle, and delayed heading date, as also observed in OsGA2ox1-overexpressing plants. Consistent with the mutant phenotype, OsBLH4 was predominantly expressed in shoots and young spikelets; its encoded protein was exclusively localized in the nucleus. Molecular analysis demonstrated that OsBLH4 directly bound to the promoter region of OsGA2ox1 to repress its expression. Genetic assays revealed that OsBLH4 acts upstream of OsGA2ox1 to control rice plant height, grain number, and heading date. Taken together, these results indicate a crucial role for OsBLH4 in regulating rice plant architecture and yield potential via regulation of bioactive GA levels, and provide a potential strategy for genetic improvements of rice.

GIGANTEA accelerates wheat heading time through gene interactions converging on FLOWERING LOCUS T1.

Precise regulation of flowering time is critical for cereal crops to synchronize reproductive development with optimum environmental conditions, thereby maximizing grain yield. The plant-specific gene GIGANTEA (GI) plays an important role in the control of flowering time, with additional functions on the circadian clock and plant stress responses. In this study, we show that GI loss-of-function mutants in a photoperiod-sensitive tetraploid wheat background exhibit significant delays in heading time under both long-day (LD) and short-day photoperiods, with stronger effects under LD. However, this interaction between GI and photoperiod is no longer observed in isogenic lines carrying either a photoperiod-insensitive allele in the PHOTOPERIOD1 (PPD1) gene or a loss-of-function allele in EARLY FLOWERING 3 (ELF3), a known repressor of PPD1. These results suggest that the normal circadian regulation of PPD1 is required for the differential effect of GI on heading time in different photoperiods. Using crosses between mutant or transgenic plants of GI and those of critical genes in the flowering regulation pathway, we show that GI accelerates wheat heading time by promoting FLOWERING LOCUS T1 (FT1) expression via interactions with ELF3, VERNALIZATION 2 (VRN2), CONSTANS (CO), and the age-dependent microRNA172-APETALA2 (AP2) pathway, at both transcriptional and protein levels. Our study reveals conserved GI mechanisms between wheat and Arabidopsis but also identifies specific interactions of GI with the distinctive photoperiod and vernalization pathways of the temperate grasses. These results provide valuable knowledge for modulating wheat heading time and engineering new varieties better adapted to a changing environment.

Laser capture microdissection transcriptome (LCM RNA-seq) reveals BcDFR is a key gene in anthocyanin synthesis of non-heading Chinese cabbage.

BACKGROUND: Purple non-heading Chinese cabbage [Brassica campestris (syn. Brassica rapa) ssp. chinensis] has become popular because of its richness in anthocyanin. However, anthocyanin only accumulates in the upper epidermis of leaves. Further studies are needed to investigate the molecular mechanisms underlying the specific accumulation of it. RESULTS: In this study, we used the laser capture frozen section method (LCM) to divide purple (ZBC) and green (LBC) non-heading Chinese cabbage leaves into upper and lower epidermis parts (Pup represents the purple upper epidermis, Plow represents the purple lower epidermis, Gup represents the green upper epidermis, Glow represents the green lower epidermis). Through transcriptome sequencing, we found that the DIHYDROFLAVONOL 4-REDUCTASE-encoding gene BcDFR, is strongly expressed in Pup but hardly in others (Plow, Gup, Glow). Further, a deletion and insertion in the promoter of BcDFR in LBC were found, which may interfere with BcDFR expression. Subsequent analysis of gene structure and conserved structural domains showed that BcDFR is highly conserved in Brassica species. The predicted protein-protein interaction network of BcDFR suggests that it interacts with almost all functional proteins in the anthocyanin biosynthesis pathway. Finally, the results of the tobacco transient expression also demonstrated that BcDFR promotes the synthesis and accumulation of anthocyanin. CONCLUSIONS: BcDFR is specifically highly expressed on the upper epidermis of purple non-heading Chinese cabbage leaves and regulates anthocyanin biosynthesis and accumulation. Our study provides new insights into the functional analysis and transcriptional regulatory network of anthocyanin-related genes in purple non-heading Chinese cabbage.

Enhancing Yield and Improving Grain Quality in Japonica Rice: Targeted EHD1 Editing via CRISPR-Cas9 in Low-Latitude Adaptation.

The "Indica to Japonica" initiative in China focuses on adapting Japonica rice varieties from the northeast to the unique photoperiod and temperature conditions of lower latitudes. While breeders can select varieties for their adaptability, the sensitivity to light and temperature often complicates and prolongs the process. Addressing the challenge of cultivating high-yield, superior-quality Japonica rice over expanded latitudinal ranges swiftly, in the face of these sensitivities, is critical. Our approach harnesses the CRISPR-Cas9 technology to edit the EHD1 gene in the premium northeastern Japonica cultivars Jiyuanxiang 1 and Yinongxiang 12, which are distinguished by their exceptional grain quality-increased head rice rates, gel consistency, and reduced chalkiness and amylose content. Field trials showed that these new ehd1 mutants not only surpass the wild types in yield when grown at low latitudes but also retain the desirable traits of their progenitors. Additionally, we found that disabling Ehd1 boosts the activity of Hd3a and RFT1, postponing flowering by approximately one month in the ehd1 mutants. This research presents a viable strategy for the accelerated breeding of elite northeastern Japonica rice by integrating genomic insights with gene-editing techniques suitable for low-latitude cultivation.

Comprehensive analysis of stress-activated protein kinase genes (OsSAPKs) in rice flowering time.

MAIN CONCLUSION: The rice SnRK2 members SAPK4, SAPK5, SAPK7 and SAPK10 are positive regulators involved in the regulation of rice flowering, while other single mutants exhibited no effect on rice flowering. The rice SnRK2 family, comprising 10 members known as SAPK (SnRK2-Associated Protein Kinase), is pivotal in the abscisic acid (ABA) pathway and crucial for various biological processes, such as drought resistance and salt tolerance. Additionally, these members have been implicated in the regulation of rice heading date, a key trait influencing planting area and yield. In this study, we utilized gene editing technology to create mutants in the Songjing 2 (SJ2) background, enabling a comprehensive analyze the role of each SAPK member in rice flowering. We found that SAPK1, SAPK2, and SAPK3 may not directly participate in the regulatory network of rice heading date, while SAPK4, SAPK5, and SAPK7 play positive roles in rice flowering regulation. Notably, polygene deletion resulted in an additive effect on delaying flowering. Our findings corroborate the previous studies indicating the positive regulatory role of SAPK10 in rice flowering, as evidenced by delayed flowering observed in sapk9/10 double mutants. Moving forward, our future research will focus on analyzing the molecular mechanisms underlying SAPKs involvement in rice flowering regulation, aiming to enhance our understanding of the rice heading date relationship network and lay a theoretical foundation for breeding efforts to alter rice ripening dates.

High expression of ethylene response factor BcERF98 delays the flowering time of non-heading Chinese cabbage.

MAIN CONCLUSION: BcERF98 is induced by ethylene signaling and inhibits the expression of BcFT by interacting with BcNF-YA2 and BcEIP9, thereby inhibiting plant flowering. Several stresses trigger the accumulation of ethylene, which then transmits the signal to ethylene response factors (ERFs) to participate in the regulation of plant development to adapt to the environment. This study clarifies the function of BcERF98, a homolog of AtERF98, in the regulation of plant flowering time mediated by high concentrations of ethylene. Results indicate that BcERF98 is a nuclear and the cell membrane-localized transcription factor and highly responsive to ethylene signaling. BcERF98 inhibits the expression of BcFT by interacting with BcEIP9 and BcNF-YA2, which are related to flowering time regulation, thereby participating in ethylene-mediated plant late flowering regulation. The results have enriched the theoretical knowledge of flowering regulation in non-heading Chinese cabbage (NHCC), providing the scientific basis and gene reserves for cultivating new varieties of NHCC with different flowering times.

Fine-tuning rice heading date through multiplex editing of the regulatory regions of key genes by CRISPR-Cas9.

Heading date (or flowering time) is a key agronomic trait that affects seasonal and regional adaption of rice cultivars. An unoptimized heading date can either not achieve a high yield or has a high risk of encountering abiotic stresses. There is a strong demand on the mild to moderate adjusting the heading date in breeding practice. Genome editing is a promising method which allows more precise and faster changing the heading date of rice. However, direct knock out of major genes involved in regulating heading date will not always achieve a new germplasm with expected heading date. It is still challenging to quantitatively adjust the heading date of elite cultivars with best adaption for broader region. In this study, we used a CRISPR-Cas9 based genome editing strategy called high-efficiency multiplex promoter-targeting (HMP) to generate novel alleles at cis-regulatory regions of three major heading date genes: Hd1, Ghd7 and DTH8. We achieved a series of germplasm with quantitative variations of heading date by editing promoter regions and adjusting the expression levels of these genes. We performed field trials to screen for the best adapted lines for different regions. We successfully expanded an elite cultivar Ningjing8 (NJ8) to a higher latitude region by selecting a line with a mild early heading phenotype that escaped from cold stress and achieved high yield potential. Our study demonstrates that HMP is a powerful tool for quantitatively regulating rice heading date and expanding elite cultivars to broader regions.

Multiplex-genome-editing based rapid directional improvement of complex traits in rice.

Although thousands of genes have been identified or cloned in rice (Oryza sativa) in the last two decades, the majority of them have only been separately characterized in specific varieties or single-gene modified backgrounds, thus limiting their practical application. We developed an optimized multiplex genome editing (MGE) toolbox that can efficiently assemble and stably express up to twelve sgRNA targets in a single plant expression vector. In this study, we established the MGE-based Rapid Directional Improvement (MRDI) strategy for directional improvement of complex agronomic traits in one small-scale rice transformation. This approach provides a rapid and practical procedure, encompassing sgRNA assembly, transgene-free screening and the creation of promising germplasm, by combining the precision of gene editing with phenotype-based field breeding. The MRDI strategy was used to generate the full diversity of twelve main agronomic genes in rice cultivar FXZ for the directional improvement of its growth duration and plant architecture. After applying the MRDI to FXZ, ideal plants with the desired traits of early heading date reduced plant height, and more effective panicles were generated without compromising yield, blast resistance and grain quality. Furthermore, the results of whole-genome sequencing (WGS), including the analysis of structural variations (SVs) and single nucleotide variations (SNVs) in the MGE plants, confirmed the high specificity and low frequency of unwanted mutations associated with this strategy. The MRDI breeding strategy would be a robust approach for exploring and applying crucial agronomic genes, as well as for generating novel elite germplasm in the future.

Photoperiod and temperature synergistically regulate heading date and regional adaptation in rice.

Plants must accurately integrate external environmental signals with their own development to initiate flowering at the appropriate time for reproductive success. Photoperiod and temperature are key external signals that determine flowering time; both are cyclical and periodic, and they are closely related. In this review, we describe photoperiod-sensitive genes that simultaneously respond to temperature signals in rice (Oryza sativa). We introduce the mechanisms by which photoperiod and temperature synergistically regulate heading date and regional adaptation in rice. We also discuss the prospects for designing different combinations of heading date genes and other cold tolerance or thermo-tolerance genes to help rice better adapt to changes in light and temperature via molecular breeding to enhance yield in the future.

Wheat ESCRT-III protein TaSAL1 regulates male gametophyte transmission and controls tillering and heading date.

Charged multivesicular protein 1 (CHMP1) is a member of the endosomal sorting complex required for transport-III (ESCRT-III) complex that targets membrane localized signaling receptors to intralumenal vesicles in the multivesicular body of the endosome and eventually to the lysosome for degradation. Although CHMP1 plays roles in various plant growth and development processes, little is known about its function in wheat. In this study, we systematically analysed the members of the ESCRT-III complex in wheat (Triticum aestivum) and found that their orthologs were highly conserved in eukaryotic evolution. We identified CHMP1 homologous genes, TaSAL1s, and found that they were constitutively expressed in wheat tissues and essential for plant reproduction. Subcellular localization assays showed these proteins aggregated with and closely associated with the endoplasmic reticulum when ectopically expressed in tobacco leaves. We also found these proteins were toxic and caused leaf death. A genetic and reciprocal cross analysis revealed that TaSAL1 leads to defects in male gametophyte biogenesis. Moreover, phenotypic and metabolomic analysis showed that TaSAL1 may regulate tillering and heading date through phytohormone pathways. Overall, our results highlight the role of CHMP1 in wheat, particularly in male gametophyte biogenesis, with implications for improving plant growth and developing new strategies for plant breeding and genetic engineering.

Regulation of the trade-off between cold stress and growth by glutathione S-transferase phi class 10 (BcGSTF10) in non-heading Chinese cabbage.

Cold stress is a serious threat to global crop production and food security, but plant cold resistance is accompanied by reductions in growth and yield. In this study, we determined that the novel gene BcGSTF10 in non-heading Chinese cabbage [NHCC; Brassica campestris (syn. Brassica rapa) ssp. chinensis] is implicated in resistance to cold stress. Biochemical and genetic analyses demonstrated that BcGSTF10 interacts with BcICE1 to induce C-REPEAT BINDING FACTOR (CBF) genes that enhance freezing tolerance in NHCC and in Arabidopsis. However, BcCBF2 represses BcGSTF10 and the latter promotes growth in NHCC and Arabidopsis. This dual function of BcGSTF10 indicates its pivotal role in balancing cold stress and growth, and this important understanding has the potential to inform the future development of strategies to breed crops that are both climate-resilient and high-yielding.

A novel functional allele of Ehd3 controls flowering time in rice.

Rice flowering time determines its geographical distribution and yield traits. As a short-day plant, rice can grow in the northern long-day conditions due to the functional mutations of many photosensitive genes. In this study, to identify novel genes or alleles that regulate flowering time in high latitude region, two cultivar, Dongnong 413 (DN413) and Yukimochi (XN) showing extreme early flowering were used for investigation. DN413 is around 4.0 days earlier than XN, and both cultivars can be grown in II (2500 â„ƒ-2700 â„ƒ) to III (2300 â„ƒ-2500 â„ƒ) accumulated temperature zones. We found that the two cultivars shared the same genotype of heading date genes, including Hd1/2/4/5/6/16/17/18, Ehd2, DTH2, SE5, Hd3a. Importantly, a novel Ehd3 allele characterized by a A1146C substitution was identified, which results in the E382D substitution, hereafter the 382 position E is defined as Hap_E and the 382 position D is defined as Hap_D. Association analysis showed that Hap_E is earlier flowering than Hap_D. Subsequently, we construct DN413 Hap_D line by three times back-crossing DN413 with XN, and found the heading date of DN413 Hap_D was 1.7-3.5 days later than DN413. Moreover, Hap_E and Hap_D of Ehd3 were transformed into ehd3 mutant, respectively, and the Ehd3pro:Ehd3D/ehd3 flowered later than that Ehd3pro:Ehd3E/ehd3 by around 4.3 days. Furthermore, we showed Ehd3 functions as a transcriptional suppressor and the substitution of Asp-382 lost the inhibition activity in protoplasts. Finally, a CAPS marker was developed and used for genotyping and marker assistant breeding. Collectively, we discovered a novel functional allele of Ehd3, which can used as a valuable breeding target. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01472-x.

Identification of the causal mutation in early heading mutant of bread wheat (Triticum aestivum L.) using MutMap approach.

In bread wheat (Triticum aestivum L.), fine-tuning the heading time is essential to maximize grain yield. Photoperiod-1 (Ppd-1) and VERNALIZATION 1 (Vrn-1) are major genes affecting photoperiod sensitivity and vernalization requirements, respectively. These genes have predominantly governed heading timing. However, Ppd-1 and Vrn-1 significantly impact heading dates, necessitating another gene that can slightly modify heading dates for fine-tuning. In this study, we developed an early heading mutant from the ethyl methanesulfonate-mutagenized population of the Japanese winter wheat cultivar "Kitahonami." MutMap analysis identified a nonsense mutation in the clock component gene Wheat PHYTOCLOCK 1/LUX ARRHYTHMO (WPCL-D1) as the probable SNP responsible for the early heading mutant on chromosome 3D. Segregation analysis using F2 and F3 populations confirmed that plants carrying the wpcl-D1 allele headed significantly earlier than those with the functional WPCL-D1. The early heading mutant exhibited increased expression levels of Ppd-1 and circadian clock genes, such as WPCL1 and LATE ELONGATED HYPOCOTYL (LHY). Notably, the transcript accumulation levels of Ppd-A1 and Ppd-D1 were influenced by the copy number of the functional WPCL1 gene. These results suggest that a loss-of-function mutation in WPCL-D1 is the causal mutation for the early heading phenotype. Adjusting the functional copy number of WPCL1 will be beneficial in fine-tuning of heading dates. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01478-5.

Loss of function of OsL1 gene cause early flowering in rice under short-day conditions.

Heading date is one of the important agronomic traits that affects rice yield. In this study, we cloned a new rice B3 family gene, OsL1, which regulates rice heading date. Importantly, osl1-1 and osl1-2, two different types of mutants of OsL1 were created using the gene editing technology CRISPR/Cas9 system and exhibited 4 days earlier heading date than that of the wild type under short-day conditions. Subsequently, the plants overexpressing OsL1, OE-OsL1, showed a 2-day later heading date than the wild type in Changsha and a 5-day later heading date in Lingshui, but there was no significant difference in other yield traits. Moreover, the results of subcellular localization study indicated that OsL1 protein was located in the nucleus and the expression pattern analysis showed that OsL1 gene was expressed in rice roots, stems, leaves, and panicles, and the expression level was higher at the root and weak green panicle. In addition, the OsL1 gene was mainly expressed at night time under short-light conditions. The transcriptomic analysis indicated that OsL1 might be involved in the Hd1-Hd3a pathway function. Together, our results revealed that the cloning and functional analysis of OsL1 can provide new strategy for molecular design breeding of rice with suitable fertility period. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01444-1.

Improving Vehicle Heading Angle Accuracy Based on Dual-Antenna GNSS/INS/Barometer Integration Using Adaptive Kalman Filter.

High-accuracy heading angle is significant for estimating autonomous vehicle attitude. By integrating GNSS (Global Navigation Satellite System) dual antennas, INS (Inertial Navigation System), and a barometer, a GNSS/INS/Barometer fusion method is proposed to improve vehicle heading angle accuracy. An adaptive Kalman filter (AKF) is designed to fuse the INS error and the GNSS measurement. A random sample consensus (RANSAC) method is proposed to improve the initial heading angle accuracy applied to the INS update. The GNSS heading angle obtained by a dual-antenna orientation algorithm is additionally augmented to the measurement variable. Furthermore, the kinematic constraint of zero velocity in the lateral and vertical directions of vehicle movement is used to enhance the accuracy of the measurement model. The heading errors in the open and occluded environment are 0.5418° (RMS) and 0.636° (RMS), which represent reductions of 37.62% and 47.37% compared to the extended Kalman filter (EKF) method, respectively. The experimental results demonstrate that the proposed method effectively improves the vehicle heading angle accuracy.

Adaptive UAV Navigation Method Based on AHRS.

To address the inaccuracy of the Constant Acceleration/Constant Velocity (CA/CV) model as the state equation in describing the relative motion state in UAV relative navigation, an adaptive UAV relative navigation method is proposed, which is based on the UAV attitude information provided by Attitude and Heading Reference System (AHRS). The proposed method utilizes the AHRS output attitude parameters as the benchmark for dead reckoning and derives a relative navigation state equation with attitude error as process noise. By integrating the extended Kalman filter output for relative state estimation and employing an adaptive decision rule designed using the innovation of the filter update phase, the proposed method recalculates motion states deviating from the actual motion using the Tasmanian Devil Optimization (TDO) algorithm. The simulation results show that, compared with the CA/CV model, the proposed method reduces the relative position errors by 12%, 23%, and 32% in the X, Y, and Z directions, respectively, and that it reduces the relative velocity errors by 350%, 330%, and 300%, respectively. There is a significant improvement in the relative navigation accuracy.

Genetic Model Identification and Major QTL Mapping for Petiole Thickness in Non-Heading Chinese Cabbage.

Petioles of non-heading Chinese cabbage are not only an important edible part but also a conduit for nutrient transport, holding significant agricultural and research value. In this study, we conducted a comprehensive genetic analysis of petiole-related traits using a segregating population. Modern quantitative genetic approaches were applied to investigate the genetic regulation of petiole thickness. The results indicated that petiole thickness is a quantitative trait, and the identified genetic model was consistent with two pairs of additive-dominant main genes and additive-dominant polygenes (2MG-AD). BSA-seq analysis identified a major effect of QTL controlling petiole thickness on chromosome A09: 42.08-45.09 Mb, spanning 3.01 Mb, designated as QTL-BrLH9. Utilizing InDel markers, the interval was narrowed down to 51 kb, encompassing 14 genes with annotations for 10 of them. Within the interval, four mutated genes were detected. Combined with gene annotation, protein sequence analysis, and homology alignment, it was found that BraA09g063520.3C's homologous gene SMXL6 in Arabidopsis (Arabidopsis thaliana (L.) Heynh) is an inhibitor of the coding and synthesis of the strigolactone pathway. Strigolactone (SLs) plays an important role in plant growth and development. The cloning results showed that multiple frameshift mutations and non-synonymous mutations occurred on the exon. The qPCR results showed that the expression of the gene was significantly different between the two parents at the adult stage, so it was speculated that it would lead to changes in petiole thickness. BraA09g063520.3C was predicted as the final candidate gene.

Rice stripe mosaic virus hijacks rice heading-related gene to promote the overwintering of its insect vector.

Rice stripe mosaic virus (RSMV) is an emerging pathogen which significantly reduces rice yields in the southern region of China. It is transmitted by the leafhopper Recilia dorsalis, which overwinters in rice fields. Our field investigations revealed that RSMV infection causes delayed rice heading, resulting in a large number of green diseased plants remaining in winter rice fields. This creates a favorable environment for leafhoppers and viruses to overwinter, potentially contributing to the rapid spread and epidemic of the disease. Next, we explored the mechanism by which RSMV manipulates the developmental processes of the rice plant. A rice heading-related E3 ubiquitin ligase, Heading date Associated Factor 1 (HAF1), was found to be hijacked by the RSMV-encoded P6. The impairment of HAF1 function affects the ubiquitination and degradation of downstream proteins, HEADING DATE 1 and EARLY FLOWERING3, leading to a delay in rice heading. Our results provide new insights into the development regulation-based molecular interactions between virus and plant, and highlights the importance of understanding virus-vector-plant tripartite interactions for effective disease management strategies.

Transcription factor OsWRKY11 induces rice heading at low concentrations but inhibits rice heading at high concentrations.

The heading date of rice is a crucial agronomic characteristic that influences its adaptability to different regions and its productivity potential. Despite the involvement of WRKY transcription factors in various biological processes related to development, the precise mechanisms through which these transcription factors regulate the heading date in rice have not been well elucidated. The present study identified OsWRKY11 as a WRKY transcription factor which exhibits a pivotal function in the regulation of the heading date in rice through a comprehensive screening of a clustered regularly interspaced palindromic repeats (CRISPR) ‒ CRISPR-associated nuclease 9 mutant library that specifically targets the WRKY genes in rice. The heading date of oswrky11 mutant plants and OsWRKY11-overexpressing plants was delayed compared with that of the wild-type plants under short-day and long-day conditions. Mechanistic investigation revealed that OsWRKY11 exerts dual effects on transcriptional promotion and suppression through direct and indirect DNA binding, respectively. Under normal conditions, OsWRKY11 facilitates flowering by directly inducing the expression of OsMADS14 and OsMADS15. The presence of elevated levels of OsWRKY11 protein promote formation of a ternary protein complex involving OsWRKY11, Heading date 1 (Hd1), and Days to heading date 8 (DTH8), and this complex then suppresses the expression of Ehd1, which leads to a delay in the heading date. Subsequent investigation revealed that a mild drought condition resulted in a modest increase in OsWRKY11 expression, promoting heading. Conversely, under severe drought conditions, a significant upregulation of OsWRKY11 led to the suppression of Ehd1 expression, ultimately causing a delay in heading date. Our findings uncover a previously unacknowledged mechanism through which the transcription factor OsWRKY11 exerts a dual impact on the heading date by directly and indirectly binding to the promoters of target genes.

OsHUB2 inhibits function of OsTrx1 in heading date in rice.

Heading date is one of the most pivotal agronomic traits for rice (Oryza sativa) yield and adaptation. Little is known about the crosstalk between histone ubiquitination and histone methylation in rice heading date regulation. Here, we reported HISTONE MONOUBIQUITINATION 1 (OsHUB1) and OsHUB2 are involved in heading date regulation via the Hd1 and Ehd1 pathway. Loss of OsHUB1 and OsHUB2 function resulted in early heading under long-day and short-day photoperiods. The expression of Hd3a, RFT1, and Ehd1 was induced and the transcript levels of Hd1, Ghd7, OsCCA1, OsGI, OsFKF1, and OsTOC1 were reduced under long-day conditions, whereas RFT1 and Ehd1 expression was induced in oshub2 mutants under short-day conditions. OsHUB2 interacted with OsTrx1 and repressed the gene expression of OsTrx1. OsHUB2 directly bound to Ehd1 to ubiquitinate H2B at Ehd1, and H2B ubiquitination levels were reduced in oshub2-2 and oshub2-3 mutants. OsTrx1 were highly enriched at Ehd1, and H3K4me3 levels of Ehd1 were upregulated in oshub2-2. Mutations of OsTrx1 in the oshub2-2 background rescued the early-heading phenotype of oshub2-2. The increases in Ehd1 H3K4me3 levels and transcript levels in oshub2-2 mutants were attenuated in oshub2-2 ostrx1-2 double mutants. Together, our results (i) reveal that OsHUB2 represses the function of OsTrx1 and H3K4me3 levels at Ehd1 and (ii) suggest that OsHUB2-mediated H2B ubiquitination plays critical roles together with H3K4me3 in rice heading date regulation.