Osmanthus fragrans is an ethylene-sensitive flower, and flower senescence was mediated by ethylene-responsive transcription factors (OfERFs). A total of 227 OfERFs were identified from O. fragrans, which were classified into five subfamilies: AP2 (35), DREB (57), ERF (125), RAV (6), and Soloist (4). Gene composition and structural analysis indicate that members of different subfamilies have different gene structures and conserved domains. Their gene promoter contains various functional responsive elements, including auxin, jasmonic acid, and other responsive elements. Among them, 124 OfAP2/ERF genes have expressed at any stage of flowering, and 10 of them may play roles in flowering or senescence. By comparative transcriptome analysis, OfAP2/ERFs affected by ethephon (ETH) and 5′-azacytidine (Aza) treatment were divided into three categories, which have various target gene sets. Importantly, these target gene sets participate in similar or different biological processes and metabolic pathways, suggesting that ethylene and DNA hypomethylation have crosstalk and a unique mechanism in regulating the flower senescence of O. fragrans. Co-expression analysis revealed that several key OfAP2/ERFs played a central role in organic acid metabolism and biosynthesis of branched-chain amino acids (BcAAs), among which OfERF017 was selected for further functional analysis. Overexpression of OfERF017 leads to significant enrichment of genes in organic acid metabolism pathways, which leads to a decrease in organic acid levels and promoting the flower senescence of O. fragrans. Together, these results give insights into the characteristics and functional analysis of OfAP2/ERF genes in O. fragrans.

Histone acetylation and methylation, governed by various histone modification (HM) gene families, are vital for plant biological processes. However, there are limited studies that have explored HMs in ornamental horticultural trees, including sweet osmanthus (Osmanthus fragrans). We performed genome-wide search and identified 208 OfHMs, encompassing 81 histone methyltransferases (OfHMTs), 51 histone demethylases (OfHDMs), 49 histone acetyltransferases (OfHATs) and 27 histone deacetylases (HDACs). Our comprehensive analysis covered chromosome locations, gene structures, conserved domains, cis-acting elements, phylogenetic comparisons, protein interaction networks and functional enrichment pathways for these gene families. Additionally, tandem and fragment replications were unveiled as contributors to the expansion of OfHMs, with some genes exhibiting positive selection. Furthermore, we examined OfHM expression profiles across various tissues and flowering stages, and under 5′-azacytidine (Aza) and ethylene treatments. Most OfHMs displayed heightened expression in leaves, and were downregulated during the flower opening and senescence stages, including OfPRMTs, OfHDTs, OfHDAs, OfSRTs, OfJMJs and OfHAGs; 75.86% and 80.77% of the differentially expressed OfHMs were upregulated after Aza and ethylene treatments, including OfHAGs, OfHDAs and OfSDGs. This study offers a comprehensive analysis of the OfHM gene family, which indicated their potential involvement in ethylene and Aza responses, and in the flowering process. These findings provide valuable insights into the role of OfHMs in flowering and stress responses.

The MADS-box transcription factors play essential roles in various processes of plant growth and development. Here, we identified 107 MADS-box genes in Osmanthus fragrans Lour. genome (OfMADS), encoding proteins ranging from 61 to 608 amino acids. Phylogenetic analysis classified these genes into five subfamilies: MIKC*, MIKCC, Mα, Mβ, and Mγ, with conserved motif architectures within subfamilies. Tandem and whole-genome duplications were identified as key drivers of OfMADS expansion. Cis-regulatory element analysis revealed enrichment for hormone response and developmental regulatory motifs, implicating roles in growth and flowering processes. Transcriptome dynamics across six floral developmental stages (bolting to petal shedding) uncovered 78 differentially expressed OfMADS genes, including 16 exhibiting flower-specific expressions. Integrated metabolome profiling demonstrated robust correlations between critical OfMADS regulators and scent metabolites. This nexus suggests a potential role of these OfMADS in regulating specialized metabolite biosynthesis pathways. Our multi-omics study provides insights into the regulatory hierarchy of OfMADS in coordinating floral morphogenesis and the accumulation of economically significant metabolites in O. fragrans. These findings establish a foundation for subsequent functional validation and molecular breeding of horticultural traits.

The glycine-aspartic acid-serine-leucine (GDSL) esterase/lipase family is a functionally diverse group of hydrolytic enzymes involved in multiple plant biological processes, including stress adaptation and development. However, its evolutionary patterns, functional conservation, and stress-responsive mechanisms in grasses remain not fully elucidated. In this study, a comprehensive comparative genomic analysis was performed on the GDSL gene family across nine representative grass species and Arabidopsis thaliana. Genome-wide identification, phylogenetic analysis, duplication pattern detection, synteny analysis, cis-regulatory element prediction, protein–protein interaction (PPI) network construction, and RNA-seq-based expression profiling were employed. A total of 1707 GDSL genes were identified, with substantial expansion in grasses, especially hexaploid wheat. Whole-genome and segmental duplications were the major drivers of family expansion, with most duplicated genes under strong purifying selection. A grass-specific clade (C3-2) was identified, and extensive syntenic conservation was observed among closely related grasses. Promoter analysis revealed enrichment of stress- and hormone-responsive cis-elements, and RNA-seq showed dynamic GDSL expression under low-temperature stress in rice and wheat. These findings demonstrate that the expansion of the GDSL gene family in grasses is driven by polyploidization and lineage-specific duplication, accompanied by the emergence of a grass-specific clade (C3-2) and regulatory diversification, collectively shaping stress-responsive evolutionary innovation in Poaceae.

Background/Objectives: NPF proteins are important transporters that mediate nitrate uptake, nutrient allocation, and abiotic stress responses in plants. However, the evolutionary patterns of the NPF gene family in grasses remain largely unclear. This study aimed to clarify the evolutionary expansion and stress response characteristics of NPF genes in Poaceae. Methods: A comprehensive comparative genomic analysis was conducted across nine representative Poaceae species and Arabidopsis thaliana. Multiple analytical approaches were used, including gene family identification, phylogenetic classification, collinearity analysis, Ka/Ks calculation, cis-element prediction, protein interaction analysis, and RNA-seq expression verification. Results: A total of 1109 NPF genes were identified with substantial variation in gene copy number among species, particularly the remarkable expansion observed in hexaploid Triticum aestivum. Phylogenetic analysis classified grass NPF proteins into eight major subfamilies, while collinearity analyses revealed that whole-genome duplication and segmental duplication were the primary drivers of NPF expansion. Most duplicated gene pairs exhibited Ka/Ks values below 1, indicating strong purifying selection during evolution. Promoter analyses identified abundant stress- and hormone-responsive cis-elements, especially in Triticeae species. In addition, protein–protein interaction and RNA-seq analyses suggested potential functional associations among NPF genes and revealed expression variation under low-temperature treatments in rice and wheat. Conclusions: Collectively, this study objectively clarifies the evolutionary expansion, functional conservation, and potential stress-responsive diversification of the NPF gene family in grasses. These findings provide straightforward and reliable insights for further evolutionary and functional research on the NPF gene family in Poaceae.

Low-temperature stress severely restricts the growth, development, and ornamental value of Osmanthus fragrans Lour. However, the molecular mechanisms by which brassinolide (BR) and melatonin (MT) alleviate low-temperature-induced damage remain unclear. Here, O. fragrans branches were exposed to low-temperature stress (5, 0, −5, −10, −15, and −20 °C for 12 h) and treated with exogenous MT (50, 100, and 200 μM) or BR (0.5, 1, and 2 μM). An integrated approach combining phenotypic observation, physiological measurements, transcriptomics, and metabolomics was employed to elucidate the regulatory mechanisms underlying BR- and MT-mediated cold tolerance. The results showed that low-temperature stress significantly increased electrolyte leakage (EL), malondialdehyde (MDA), and hydrogen peroxide (H₂O₂) accumulation, while reducing superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities. Compared with the control, BR and MT treatments alleviated leaf chlorosis and wilting, reduced oxidative damage, and enhanced antioxidant enzyme activities. Integrated transcriptome–metabolome analyses demonstrated that BR and MT commonly activated phenylpropanoid and flavonoid biosynthesis, thereby promoting antioxidant metabolite accumulation, while suppressing α-linolenic acid and linoleic acid metabolism associated with stress-induced lipid remodeling. Network-based transcriptomic analyses identified transcription factors, including ARF, EIL, bHLH, and GRAS, as potential regulators of cold-responsive pathways. Furthermore, BR primarily regulated hormone-responsive networks, whereas MT mainly maintained redox homeostasis and metabolic reprogramming. These findings reveal the coordinated regulatory mechanisms underlying BR- and MT-mediated cold tolerance, providing potential targets for improving cold resilience in O. fragrans.