Melatonin plays a crucial role in regulating plant defense against various environmental stresses. However, its role and the underlying mechanism in enhancing plant resistance to aphid infestation remains elusive. In thi...Melatonin plays a crucial role in regulating plant defense against various environmental stresses. However, its role and the underlying mechanism in enhancing plant resistance to aphid infestation remains elusive. In this research, we discovered that aphid infestation notably upregulated the expression of the melatonin biosynthetic gene caffeic acid O-methyltransferase 1 (ClCOMT1) and increased melatonin levels in watermelon (Citrullus lanatus). The overexpression of ClCOMT1 enhanced defensive responses and resistance to aphids in watermelon. This was accompanied by the accumulation of cytosolic free calcium ([Ca2+]cyt), upregulation of the Ca2+-permeable channel gene cyclic nucleotide-gated ion channel 20 (ClCNGC20), and downregulation of calmodulin 7 (ClCaM7). ClCaM7 interacts with ClCNGC20 and suppresses its channel activity. In contrast, the knockout of ClCOMT1 showed opposite effects compared with its overexpression. Pretreatment with LaCl3 (a Ca2+ influx channel inhibitor) or EGTA (a Ca2+ chelator) compromised ClCOMT1 overexpression-induced aphid resistance; whereas, CaCl2 or the Ca2+ ionophore A23187 effectively restored aphid resistance in clcomt1 mutant. Moreover, silencing ClCNGC20 impaired ClCOMT1 overexpression-induced [Ca2+]cyt accumulation and aphid resistance; conversely, silencing ClCaM7 restored [Ca2+]cyt accumulation and aphid resistance in the clcomt1 mutant. Overexpression of watermelon ClCNGC20 elevated [Ca2+]cyt levels and improved aphid resistance in transgenic Arabidopsis plants, whereas overexpression of watermelon ClCaM7 had the opposite effect. Collectively, these findings imply the crucial role of ClCNGC20 and ClCaM7 in mediating melatonin-induced [Ca2+]cyt elevation, subsequently activating defensive responses in plants. This study offers insights into the molecular mechanisms underlying melatonin-mediated plant aphid resistance, with potential applications in the breeding or engineering of aphid-resistant cucurbit varieties.
Responses to far-red light are mediated by phytochromes, specialised photoreceptors present in all organs of a tomato plant (Solanum lycopersicum L.). Although fruit-localised phytochromes can influence starch and sugar...Responses to far-red light are mediated by phytochromes, specialised photoreceptors present in all organs of a tomato plant (Solanum lycopersicum L.). Although fruit-localised phytochromes can influence starch and sugar metabolism, their involvement in modulating fruit growth responses to far-red light is unknown. We explored whether fruit growth responses to far-red light are driven by local far-red perception within the fruits, or by a systemic response initiated in the vegetative organs. We applied far-red light exclusively to the fruiting trusses, to the vegetative organs, to both, or to neither. Far-red light was added to a constant red-white LED background spectrum to simulate greenhouse supplementary lighting. We quantified plant and fruit growth responses to far-red and performed a transcriptomic analysis to establish the temporal dynamics of far-red light perception and signaling in leaves and fruits. Supplementing far-red light to the vegetative organs increased ripe fruit weight and sugar concentration, whereas far-red supplementation to the generative organs alone had no measurable effect. This study establishes that far-red perception by vegetative organs drives fruit growth responses in tomato. Local responses to far-red light included a transient increase in auxin concentration and upregulation of the auxin-signaling pathway in leaves, a conserved response to decreasing red:far-red ratios. In fruits, early transcriptomic responses carried signatures of auxin, cytokinin, and gibberellin biosynthesis and signaling, suggesting a role for hormones as mediators of far-red-induced fruit responses. This work reveals how responses to far-red light perception by vegetative organs promote sink strength and starch biosynthesis in early developing fruits, contributing to higher fruit weight and sugar concentration in ripe fruits.
O-acetylserine (OAS) is the sulfur acceptor in primary sulfate assimilation and the precursor of cysteine, synthesized by serine acetyltransferase (SERAT). Beyond its metabolic role, OAS acts as a signaling molecule, acc...O-acetylserine (OAS) is the sulfur acceptor in primary sulfate assimilation and the precursor of cysteine, synthesized by serine acetyltransferase (SERAT). Beyond its metabolic role, OAS acts as a signaling molecule, accumulating during sulfur starvation and inducing sulfur-deficiency marker genes. Recently, OAS was also shown to increase transiently after a light-dark transition, triggering the induction of a group of "OAS cluster" genes. Here, we investigated the mechanisms underlying OAS accumulation and the regulation of OAS cluster genes. We found that no single SERAT isoform accounts for the full induction of OAS cluster genes; instead, the loss of any of the three major isoforms (SERAT1;1, SERAT2;1, SERAT2;2) reduced induction, with SERAT2;2 playing a predominant role. All five isoforms contributed to the response to varying degrees. In addition, three transcription factors were required: the sulfur-deficiency regulator SLIM1 and the circadian clock components RVE1 and RVE8. These factors bound to OAS cluster gene promoters and unexpectedly controlled OAS accumulation itself. While RVE1 and RVE8 did not transduce the OAS signal, they contributed to sulfur-deficiency responses at both the transcriptional and metabolic levels. Together, our findings reveal a complex regulatory network involving multiple SERAT isoforms and transcription factors, advancing our understanding of sulfur homeostasis and its integration with light and circadian regulation.
Low-calcium (Ca) stress induces blossom-end rot (BER), a physiological disorder that seriously reduces the yield and quality of tomato (Solanum lycopersicum L.). However, the mechanisms underlying low-Ca-induced BER rema...Low-calcium (Ca) stress induces blossom-end rot (BER), a physiological disorder that seriously reduces the yield and quality of tomato (Solanum lycopersicum L.). However, the mechanisms underlying low-Ca-induced BER remain insufficiently understood. Calmodulin-binding Transcriptional Activators (CAMTAs) are evolutionarily conserved CaM-binding proteins that play crucial roles in plant responses to Ca deficiency. In this study, we found that the expression of SlCAMTA5 increases in tomato under Ca-deficient conditions. Knockout of SlCAMTA5 markedly elevates BER incidence, accompanied by reactive oxygen species (ROS) accumulation and reduced Ca uptake capacity in the fruit peel under low Ca conditions, whereas SlCAMTA5 overexpression results in the opposite phenotypes, demonstrating its function in suppressing BER. Transcriptome analysis showed that the expression of calcium ion (Ca2+) transport-related genes (CML37, CML38, and CDPK28) is upregulated in wild-type (WT) plants but downregulated in camta5 mutants under low-Ca stress. Further assays revealed that CAMTA5 interacts with CML38 and CDPK28 via its P1 region containing ankyrin (ANK) repeats, several IQ motifs, and a calmodulin binding (CAMBD) domains. Moreover, the Ca2+ transport-related gene calmodulin-binding protein 60 A (CBP60A) was identified as a gene undergoing differential alternative splicing in WT under low-Ca stress. It was also confirmed as differentially expressed in camta5 mutants. Under Ca deficiency, new nonfunctional variants of CBP60A are generated from its pre-mRNA. CAMTA5 directly binds to the promoter of the functional CBP60A transcript, activating its expression. Moreover, the interaction between CAMTA5 and CML38/CDPK28 enhances this transcriptional activation. Collectively, our findings demonstrated that the CML38/CDPK28-CAMTA5 module suppresses BER occurrence by regulating the functional alternatively spliced variant of CBP60A.
Iron (Fe) is an essential element for almost all living organisms and its deficiency severely impacts crop productivity and human health. While the mechanisms underlying iron uptake and accumulation are well studied, the...Iron (Fe) is an essential element for almost all living organisms and its deficiency severely impacts crop productivity and human health. While the mechanisms underlying iron uptake and accumulation are well studied, the role of light in modulating iron homeostasis remains poorly understood. In this study, we report that blue light enhances iron accumulation thereby positively regulating iron homeostasis in Arabidopsis thaliana. Under iron-deficient (-Fe) conditions, root elongation is specifically promoted by blue light, but not by red light. We further revealed that, light perceived by shoots is required for optimum iron accumulation and for iron deficiency induced increase in root length. The transcription factor HY5 (ELONGATED HYPOCOTYL 5), mediates these blue light-driven responses. The hy5 mutant showed reduced root elongation under -Fe conditions as well as iron accumulation under blue light as compared to wild-type plants. Further we report that CRY1 and CRY2 are the photoreceptor that acts as a connecting link between blue light and HY5 to regulate iron uptake in Arabidopsis. Taken together, our work reveals new regulatory mechanism and offers potential insights into optimizing light conditions to enhance iron accumulation and improve the nutritional quality of crops.
GIGANTEA (GI) is known to regulate multiple physiological processes, but its function in cold tolerance and low-temperature-induced flowering remains poorly understood in citrus. In this study, we found that between the...GIGANTEA (GI) is known to regulate multiple physiological processes, but its function in cold tolerance and low-temperature-induced flowering remains poorly understood in citrus. In this study, we found that between the two splice variants of the citrus GI homolog, CiGIα and CiGIβ, CiGIα expression was specifically induced by low temperature, whereas CiGIβ expression remained unchanged. Yeast one-hybrid assays demonstrated that the low-temperature-responsive bZIP transcription factor CiFDβ binds to the CiGI promoter and activates its expression. Further investigation revealed that cold stress upregulates CiFDβ expression, which in turn activates CiGI transcription and facilitates the CiGI-CiLHY complex formation. This complex then transmits the cold signal to CiFT, thereby participating in low-temperature-induced flowering. Functional analysis of transgenic citrus revealed that overexpression of CiGIα increased cold sensitivity, whereas suppression of CiGI enhanced cold tolerance in silenced citrus plants. Similarly, overexpression of CiLHY, an interactor of CiGI, also increased cold sensitivity, while its suppression improved cold tolerance. RNA-seq profiling of CiGI transgenic citrus lines identified differentially expressed genes related to hormone and sugar metabolism. Further experimental evidence showed that the CiGI-CiLHY complex binds to the promoter of 9-cis-epoxycarotenoid dioxygenase 3 (CiNCED3), repressing its transcription and lowering ABA levels. In addition, this complex also bound to the trehalase 1 (CiTRE1) promoter, activating its expression and reducing trehalose accumulation. Exogenous application of trehalose or ABA significantly enhanced the cold tolerance of CiGI and CiLHY transgenic citrus plants. These findings reveal a novel regulatory mechanism through which CiGI modulates cold stress responses by coordinately regulating ABA and trehalose metabolism.
C4 photosynthetic syndromes have evolved multiple times from ancestral C3 photosynthetic pathways. The genus Flaveria has served as a model group to study the evolution of C4 photosynthesis in eudicotyledons as it contai...C4 photosynthetic syndromes have evolved multiple times from ancestral C3 photosynthetic pathways. The genus Flaveria has served as a model group to study the evolution of C4 photosynthesis in eudicotyledons as it contains C3 and C4 species as well as species using intermediate types of photosynthesis (e.g. C3-C4 and C4-like). Carbonic anhydrase catalyzes the interconversion of CO2 and bicarbonate, and multiple forms of the enzyme exist in Flaveria species regardless of their photosynthetic type. In C4 species, a highly expressed β-carbonic anhydrase, βCA3a, catalyzes the first reaction of the C4 pathway in leaf mesophyll cells. To understand the evolution of βCA3a and its co-option into the C4 pathway of Flaveria, βCA3 isoforms and the genes encoding them were examined from C3 and clade A C3-C4, C4-like, and C4 Flaveria species. Phylogenetic analyses indicated the gene encoding a chloroplastic βCA was duplicated in the C3 ancestor of all Flaveria species. In the ancestor of Flaveria clade A C4-like and full C4 species, one of the resulting paralogs was duplicated and the chloroplast transit peptide of one of the two duplicates was lost. The resulting isoform, βCA3a, acquired mesophyll cell-specific expression in C4 Flaveria species through the domestication of a transposable element that harbors the mesophyll expression module1-like enhancer in the βca3a gene distal promoter. Reporter gene assays suggest sequences in a 319 bp proximal promoter region of βca3a act synergistically with this distal region to control high levels of βCA3a expression in C4 Flaveria mesophyll cells.
Salinity is a major environmental constraint that limits biomass production and perturbs cell-wall biogenesis, yet how cell-wall remodeling regulates salt tolerance in perennial trees remains largely unexplored. In this...Salinity is a major environmental constraint that limits biomass production and perturbs cell-wall biogenesis, yet how cell-wall remodeling regulates salt tolerance in perennial trees remains largely unexplored. In this study, we revealed that the miR396d-GROWTH REGULATING FACTOR 20 (PagGRF20)-XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE 5 (PagXTH5) regulatory module orchestrates salt tolerance in poplar. Despite the general growth-promoting function of the GRF family, PagGRF20 acts as an atypical member that negatively modulates both stress tolerance and leaf expansion. Transgenic poplars with miR396d overexpression or paggrf20 knockout exhibited improved growth and reduced stress symptoms under salt stress. Concurrent with increased salt tolerance, these transgenic plants exhibited reduced H2O2 accumulation and elevated activities of ROS scavenging enzymes. Combining transcriptome data, ATAC-seq and DAP-seq analyses, we identified PagXTH5, a xyloglucan endotransglucosylase/hydrolase and negative regulator of salt tolerance, as the direct target of the miR396d-PagGRF20 module. We further reveal that PagGRF20 recruits the ethylene-response factor PagRAP2.3 to synergistically activate PagXTH5 expression. In conclusion, this study elucidated the mechanism underlying the miR396d-PagGRF20-PagXTH5 regulatory axis. By balancing growth with stress responses, this module offers a promising genetic strategy for breeding salt-resilient bioenergy poplar by optimizing the trade-off between biomass accumulation and stress survival.
Oxalate is widely distributed and serves various functional roles in plants. However, its biosynthetic pathway and regulatory mechanisms remain poorly defined. In this study, we further investigated the mechanism o...Oxalate is widely distributed and serves various functional roles in plants. However, its biosynthetic pathway and regulatory mechanisms remain poorly defined. In this study, we further investigated the mechanism of oxalate accumulation using rice (Oryza sativa) CRISPR-Cas9-generated nitrate reductase (NR) and nitrite reductase (NiR) mutants, combined with different treatments. Both NR and NiR mutants exhibited significantly reduced oxalate levels. The reduced oxalate in NR and NiR mutants was restored by nitrite and nitrate, respectively, whereas ammonium had no effect, indicating that both nitrate and nitrite reduction, rather than the subsequent ammonium assimilation, are involved in modulating oxalate accumulation in rice. Furthermore, various organic acids, including glycolate, glyoxylate, glycerate, and oxaloacetate, markedly stimulated oxalate content. Transcriptomic and metabolomic analyses revealed that oxalate levels were closely and positively associated with the expression of glycolytic and tricarboxylic acid (TCA) cycle genes and the content of their intermediate metabolites. Treatment with glycolytic intermediates (phosphoenolpyruvate (PEP) and 3-phosphoglycerate (3PGA)) and inhibitors (iodoacetate (IOA)and 3-bromopyruvate (BrPA)) further confirmed that glycolysis drives oxalate synthesis in rice. Nicotinamide (NAM), a NAD precursor, also restored the oxalate content, suggesting that NAD/NADH may play a mediatory role in oxalate regulation through both nitrate and nitrite reduction. Collectively, our results demonstrate that oxalate accumulation is intimately associated with glycolysis in rice, rather than with photorespiration, which instead regulates oxalate accumulation indirectly via the glycerate-mediated impact on glycolysis..
Zhang Y, Wang X, Xu Y
… +20 more, Chen C, Yao J, Long Q, Zhang T, Su Y, Yang S, Ali Z, Yang R, Yan S, Dong Y, Zhou L, Jin Z, Fan X, Fu Q, Tian L, Liu C, Zhou Y, Li W, Peng Y, Sun L
The tetraploid table grape Kyoho, a cultivar of major global economic importance, has long presented a challenge in understanding its genetic architecture and the regulatory landscapes between its different ancestral gen...The tetraploid table grape Kyoho, a cultivar of major global economic importance, has long presented a challenge in understanding its genetic architecture and the regulatory landscapes between its different ancestral genomes. Here, we assemble a haplotype-resolved, near-complete reference genome (480.32-489.47 Mb, contig N50 17.5-23.3 Mb, with a total of only 11 gaps) to investigate regulatory dynamics through integrative multiomic analyses during fruit development. Comparative evolutionary genomics classifies Kyoho as a segmental allotetraploid with a complex mosaic genome, composed of ∼71% V. vinifera (Vv) and ∼29% V. labrusca (Vl) ancestry. A total of 67.3% of genes are shared among the four haplotypic genomes with 9.8% haplotype specific genes. Transcriptomic analysis revealed that among 17,750 pairs of ancestral alleles exhibiting directional bias, only 4,086 (23.4%) maintained a consistent ancestral dominance direction across all tissues. Similarly, comparisons of DNA methylation profiles showed no significant differences between genome ancestries Vl (0.009∼0.676): Vv (0.010∼0.670). However, alternative splicing (AS) analyses demonstrated that allele-specific splicing changes were correlated with ancestral origin across tissues (Vl: 1,529 vs. Vv: 2,052). Integrative omics identified 2,307 differentially expressed ancestral alleles that showed no significant genomic variants or methylation bias but exhibited AS bias. These genes include key regulators of fruit ripening, including ZEP, PIN, DOG1and MYB. Overall, this work delivers a landmark genomic resource and characterizes its regulatory architecture, facilitating functional genomic studies and guiding precision polyploid breeding in grapes.
Fruit aroma significantly influences fruit quality and shapes consumer preferences. Despite significant advancements in deciphering the metabolic pathways related to volatile compound biosynthesis, the regulatory mechani...Fruit aroma significantly influences fruit quality and shapes consumer preferences. Despite significant advancements in deciphering the metabolic pathways related to volatile compound biosynthesis, the regulatory mechanisms governing these pathways remain relatively underexplored. 'Fragrant pear' (Pyrus sinkiangensis Yu) and some pear varieties with Pyrus sinkiangensis lineage possess intense aromas, providing materials for studying the metabolic regulatory pathways of aroma compound biosynthesis. In this study, we used the 'Chongyanghong' pear (Pyrus sinkiangensis × Pyrus bretschneideri) as the experimental material. Gas chromatography-mass spectrometry (GC-MS) and RNA sequencing (RNA-Seq) were used to systematically analyze the relationship between metabolites and gene regulation. PbAAT1, an alcohol acyltransferase, was identified as the key enzyme responsible for ester biosynthesis in pears. Multiple experiments collectively confirmed that PbAAT1 was involved in ester synthesis. PbWRKY7 was identified as an important upstream transcriptional activator of PbAAT1 using dual-luciferase, yeast one-hybrid (Y1H), and electrophoretic mobility shift assays (EMSA). This transcriptional activation effect was further enhanced by the complex formed between PbWRKY7 and the MADS-box protein PbMADS2, as revealed using yeast 2-hybrid (Y2H), luciferase complementation assays, GST pull-down, dual-luciferase, and transient transformation assays in pears. This study revealed a molecular network in which the PbWRKY7-PbMADS2 interaction synergistically regulates PbAAT1 expression, providing insights into ester biosynthesis and pinpointing regulatory targets for enhancing fruit aroma quality.
Efficient nitrogen utilization is crucial for sustainable crop productivity; however, the molecular regulators of nitrogen use efficiency (NUE) in rice remain incompletely understood. Here, we identify the GLYCINE-RICH R...Efficient nitrogen utilization is crucial for sustainable crop productivity; however, the molecular regulators of nitrogen use efficiency (NUE) in rice remain incompletely understood. Here, we identify the GLYCINE-RICH RNA-BINDING PROTEIN (OsGRP3) as a positive regulator of NUE in rice through its role in post-transcriptional mRNA stabilization. Transgenic field trials revealed that OsGRP3 overexpression (OE) enhances agronomic performance under both normal and nitrogen-deficient conditions, with OE lines exhibiting increased plant height, seed number, and grain yield, particularly under low nitrogen input. In contrast, OsGRP3 knockdown (KD) plants displayed markedly reduced growth and yield. Molecular analyses demonstrated that OsGRP3 promotes the expression of key nitrogen uptake and assimilation genes, including OsAMT1.1 and OsGS1.1. RNA immunoprecipitation and decay assays confirmed that OsGRP3 directly binds to and stabilizes these transcripts under nitrogen-limited conditions. Enhanced ammonium uptake and elevated amino acid accumulation in OE lines further supported OsGRP3's role in facilitating nitrogen assimilation. These findings suggest that OsGRP3 binds to and stabilizes nitrogen-related transcripts, thereby promoting their expression during nutrient stress. Overall, our study reveals OsGRP3 as a crucial post-transcriptional regulator of NUE in rice, providing a mechanistic framework for its function. Targeted manipulation of OsGRP3 expression may offer a promising strategy for improving nitrogen utilization and crop yield under suboptimal fertilizer conditions.
Kang J, Sokolowska EM, Zillmer H
… +3 more, Karasov T, Walther D, Skirycz A
Plant Physiol
· 2026 Jun · PMID 42262578
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Protein-metabolite interactions (PMIs) are critical regulators of cellular processes, yet their roles in plant-pathogen interactions remain poorly understood. Here, we applied PROMIS (Protein-Metabolite Interactions usin...Protein-metabolite interactions (PMIs) are critical regulators of cellular processes, yet their roles in plant-pathogen interactions remain poorly understood. Here, we applied PROMIS (Protein-Metabolite Interactions using Size separation) to map the Arabidopsis protein-metabolite interactome during infection with Pseudomonas syringae pv. tomato DC3000 (Pto DC3000). PROMIS resolved elution profiles for >5,700 proteins and 192 annotated metabolites across infected, systemic, and control tissues, revealing infection-associated remodeling of protein assemblies and metabolite co-fractionation patterns. Integrating co-fractionation with coronatine-induced transcriptional responses, we identified NATA1, a polyamine N-acetyltransferase, as a coronatine-associated host protein binder. Biochemical assays confirmed direct coronatine-NATA1 interaction (Kd = 586 nm), whereas the homolog NATA2 did not bind coronatine. Coronatine did not strongly inhibit NATA1 catalytic turnover under our assay conditions but consistently depressed NATA1 dimer formation in vitro and in planta. In silico docking together with targeted mutational analyses identified a coronatine-sensitive interface on NATA1, revealing residue-specific coupling between ligand binding, assembly state, and acetyl-CoA engagement. Collectively, these findings establish PROMIS as a robust platform for infection-state PMI discovery and uncover a coronatine-responsive regulatory node that links small-molecule signaling to host polyamine acetylation.
The determination of sex morphs and the optimization of fruit shape are key challenges in cucumber cultivation and breeding, with ethylene recognized as a crucial regulatory factor in both processes. Ethylene is synthesi...The determination of sex morphs and the optimization of fruit shape are key challenges in cucumber cultivation and breeding, with ethylene recognized as a crucial regulatory factor in both processes. Ethylene is synthesized through the coordinated actions of 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) and ACC oxidase (ACO). While previous studies have highlighted the significant role of CsACSs in cucumber sex determination, investigations on CsACOs remain limited. In this study, we generated mutants of all members of the ACO gene family involved in ethylene synthesis during cucumber flower development and systematically analyzed their functions. Our results demonstrate that CsACO2 plays a dual role: it is involved in the early induction of carpel initiation during flower development and subsequently regulates ovary elongation. Mutations in CsACO2 affect ovary elongation, as evidenced by the shorter ovaries of female flowers in the cswip1/csaco2 double mutants. In developing female buds, after carpel initiation, we identified CsACO3 as the core regulatory gene responsible for stamen arrest, in cooperation with either CsACO2 or CsACO4. Notably, the cswip1/csaco2/csaco3 triple mutants produce hermaphrodite flowers with shorter ovaries. In contrast, the cswip1/csaco3/csaco4 triple mutants exhibit hermaphrodite flowers with normal ovaries and fruit shapes, and more importantly, their fruit-setting ability is comparable to that of the subgynoecious control. Our results enrich the ethylene regulatory network of cucumber female/hermaphrodite flower development and fruit shape, and provide an optional breeding pathway for high-yield varieties.
Brassinosteroids (BRs) are crucial in regulating growth, development, and environmental adaptation in plants. To understand the complex regulatory network of BRs, we integrated RNA-Seq, sRNA-Seq, and degradome data from...Brassinosteroids (BRs) are crucial in regulating growth, development, and environmental adaptation in plants. To understand the complex regulatory network of BRs, we integrated RNA-Seq, sRNA-Seq, and degradome data from rice (Oryza sativa L.) seedlings subjected to brassinolide (BL) treatment and from BR core signaling gene mutants (qgl3, osgsk3, and osbzr1). Using guide-gene-directed network construction and machine learning-based transcription factor (TF) target prediction methods, we also constructed BR-associated gene co-expression and regulatory networks. This study revealed synergistic regulation at the transcriptional level between BR core signaling mutants and BL treatment, highlighting the central modules involving qGL3 and OsGSK3 in BR signaling. The constructed regulatory network was enriched for TFs centered on the qGL3/OsPPKL1-OsGSK3-OsBZR1 pathway. Potential BR-core transcription factor genes were identified using co-expression and regulatory network analyses, and their loss-of-function mutants were evaluated using BL sensitivity assays and immunoblot analyses. Additionally, a BR transcriptional regulatory network database was developed, providing a valuable resource for the rice BR research community.