Ethylene-Responsive Factor (ERF) is a key element found in the middle and lower reaches of the ethylene signal transduction pathway. It is widely distributed in plants and plays important roles in plant growth and development, hormone signal transduction, and various stress processes. Although there is research on AP/ERF family members, research on AP2/ERF in Osmanthus fragrans is lacking. Thus, in this work, AP2/ERF in O. fragrans was extensively and comprehensively analyzed. A total of 298 genes encoding OfAP2/ERF proteins with complete AP2/ERF domains were identified. Based on the number of AP2/ERF domains and the similarity among amino acid sequences between AP2/ERF proteins from A. thaliana and O. fragrans, the 298 putative OfAP2/ERF proteins were divided into four different families, including AP2 (45), ERF (247), RAV (5), and SOLOIST (1). In addition, the exon–intron structure characteristics of these putative OfAP2/ERF genes and the conserved protein motifs of their encoded OfAP2/ERF proteins were analyzed, and the results were found to be consistent with those of the population classification. A tissue-specific analysis showed the spatiotemporal expression of OfAP2/ERF in the stems and leaves of O. fragrans at different developmental stages. Specifically, 21 genes were not expressed in any tissue, while high levels of expression were found for 25 OfAP2/ERF genes in several tissues, 60 genes in the roots, 34 genes in the stems, 37 genes in young leaves, 34 genes in old leaves, 32 genes in the early flowering stage, 18 genes in the full flowering stage, and 37 genes in the late flowering stage. Quantitative RT-PCR experiments showed that OfERF110a and OfERF110b had the highest expression levels at the full-bloom stage (S4), and this gradually decreased with the senescence of petals. The expression of OfERF119c decreased first and then increased, while the expression levels of OfERF4c and OfERF5a increased constantly. This indicated that these genes may play roles in flower senescence and the ethylene response. In the subsequent subcellular localization experiments, we found that ERF1-4 was localized in the nucleus, indicating that it was expressed in the nucleus. In yeast self-activation experiments, we found that OfERF112, OfERF228, and OfERF23 had self-activation activity. Overall, these results suggest that OfERFs may have the function of regulating petal senescence in O. fragrans.

Osmanthus fragrans is widely used in gardening, but the short flowering period of O. fragrans affects its ornamental and economic value. ERF, as a plant ethylene response factor, is an important link in the regulation of plant senescence. In this study, we conducted a comprehensive analysis of the functional role of OfERF1-3 within the petals of O. fragrans. Specifically, the OfERF1-3 gene was cloned and subjected to rigorous sequence analysis. Subsequently, to evaluate its expression patterns and effects, gene overexpression experiments were carried out on both Nicotiana tabacum and O. fragrans. The results showed that OfERF1-3-overexpressing tobacco plants exhibited longer petal opening times compared with those of wild plants. Measurements of physiological parameters also showed that the flowers of overexpressed tobacco plants contained lower levels of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) than those of the wild type. There was a lower expression of senescence marker genes in overexpressed tobacco and O. fragrans. A yeast two-hybrid assay showed that OfERF1-3 interacted with OfSKIP14 in a manner related to the regulation of ABA. In summary, OfERF1-3 can play a delaying role in the petal senescence process in O. fragrans, and it interacts with OfSKIP14 to indirectly affect petal senescence by regulating the ABA pathway.

Acer fabri is a widely distributed ornamental tree with colorful leaves and high ornamental value. Its young leaves change from red to red and green until turning fully green. To understand the mechanism of leaf color change, transcriptome sequencing and pigment content determination were performed in three stages during the leaf color change of A. fabri. In total, 53,550 genes, including 838 transcription factors (TFs), were identified by transcriptome sequencing. In addition, the results of orthogonal partial least squares-discriminant analysis (OPLS-DA) of three pigments in the three stages of leaf color development suggested that carotenoids played a major role in the process of leaf color change from red to red-green, whereas anthocyanins played an important role in the process of leaf color change from red to green. Based on weighted gene co-expression network analysis (WGCNA), Af0034384 (HSFB2A), Af0051627 (NMT1), and Af0052541 (THY-1) were selected as hub genes from characteristic modules with significant correlation between carotenoids and anthocyanins. The results of gene network regulation maps and real-time fluorescence quantitative PCR (qRT-PCR) showed that Af0010511 (NAC100) upregulated the expression of Af0034384 (HSFB2A), leading to an increase in carotenoid content and the gradual greening of leaves during the transition from red to green. However, during the transition from red to green leaves, Af0033232 (NAC83) and Af0049421 (WRKY24) downregulated the expression of Af0051627 (NMT1) and Af0052541 (THY-1), respectively, leading to a decrease in anthocyanin content and the complete greening of leaves. These results could provide new ideas for studying the molecular mechanism of leaf color change in A. fabri and other species.

Osmanthus fragrans is a highly valued ornamental tree species in China, but its short flowering period limits its ornamental appeal. Investigating the mechanisms of flower senescence in O. fragrans is therefore of significant importance. Ethylene, a key endogenous hormone, plays a central role in flower senescence, and the AP2/ERF gene family, which includes ethylene response factors, is known to regulate this process in various plants. Transcriptome sequencing and expression analysis identified OfERF17 as a critical gene influencing petal senescence in O. fragrans. Bioinformatics analysis revealed that OfERF17 lacks transmembrane transport structures but contains multiple phosphorylation sites and shares a close phylogenetic relationship with the Olea europaea var. Sylvestris. Subcellular targeting and yeast-based auto-activation tests revealed that OfERF17 resides in the nucleus and possesses a transcriptional self-activation capability. Transient expression studies conducted in O. fragrans petals indicated a decrease in the expressions of two genes associated with senescence, namely, OfSAG21 and OfACO3, when compared to the control group. Additionally, the levels of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) were markedly reduced. Transgenic Nicotiana tabacum blooms one day more than the wild type, and NtSAG12 and NtACO1 expressions were lower than wild type. These results suggest that OfERF17 functions to delay petal senescence in O. fragrans. This study enhances our knowledge of the molecular mechanisms underlying O. fragrans petal senescence and provides insights into strategies for prolonging its flowering period.

Background/Objectives: The WRKY gene family plays a critical role in plant stress responses; however, its function in Acer fabri (A. fabri) under cold stress conditions remains poorly understood. This study aims to identify WRKY genes in A. fabri, analyze their structural characteristics, and investigate their expression patterns under cold stress, thereby establishing a foundation for further exploration of their roles in cold stress responses. Methods: Using transcriptional data from A. fabri subjected to cold stress, we identified 46 WRKY family genes. We employed bioinformatics tools to conduct a comprehensive analysis of the physical and chemical properties of these genes, predict their subcellular localization, and construct a phylogenetic tree. A heatmap was generated to visualize the expression levels of WRKY genes across different treatment conditions. To validate our findings, qRT-PCR was performed on 10 highly expressed WRKY genes to analyze their temporal expression patterns during cold stress exposure. Results: The analysis revealed that WRKY genes in A. fabri are predominantly localized to the nucleus, with protein lengths ranging from 55 to 1027 amino acids. Notably, all WRKY genes possessed the conserved WRKYGQK domain. Under cold stress conditions, the WRKY gene expression exhibited a general trend of increasing followed by decreasing, with peak expression observed at 24 h post-treatment. qRT-PCR analysis corroborated this pattern for the selected genes. Conclusions: This study represents the first comprehensive structural and expression analysis of the A. fabri WRKY gene family under cold stress conditions. Our findings provide valuable insights into their potential roles in plant cold stress responses, and lay the groundwork for future investigations into the molecular mechanisms underlying WRKY-mediated cold stress tolerance in A. fabri.