Yuanzheng Yue

Floral scent, primarily comprised of volatile organic compounds such as the monoterpene linalool, serves as a critical medium for plant–pollinator communication and determines ornamental value. Linalool is catalyzed by terpene synthases in plants through the methylerythritol 4-phosphate (MEP) pathway. In sweet Osmanthus (Osmanthus fragrans), linalool is a characteristic aroma compound, yet the transcriptional regulation of its biosynthesis remains largely unknown. Although MADS-box transcription factors (TFs) are known as master regulators of floral organ identity, their role in controlling terpenoid volatile production is largely unexplored. In this study, we elucidated the OfDEFA, a nucleus-localized MADS-box TF, which centrally regulates linalool biosynthesis through a sophisticated dual-layer mechanism. Using the key enhancer region of the linalool synthase gene OfTPS1a promoter as bait, we identified OfDEFA via yeast-one-hybrid screening. Transient overexpression and virus-induced gene silencing of OfDEFA in O. fragrans flowers confirmed its role as a positive regulator of linalool biosynthesis. We demonstrated that OfDEFA can directly bind to CArG-box motifs in the promoters of two linalool synthase genes, OfTPS1a and OfTPS7, to activate their expression. Furthermore, we uncovered that OfDEFA can also directly activate OfMYB21, a known positive regulator of OfTPS1a, thereby regulating the production of linalool. Our findings reveal a novel, elaborate regulatory module in which a floral homeotic MADS-box factor coordinates floral scent production by directly engaging structural genes and simultaneously reinforcing the regulatory network of the MEP pathway, providing new insights into the complex regulatory network underlying linalool biosynthesis in sweet Osmanthus.

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.

Petunia hybrida is an annual herb flower that is prevalently cultivated both in public landscaping and home gardening. Anthers are vital reproductive organs for plants, but the molecular mechanism controlling petunia anther development remains elusive. In this work, we combined DNA methylation, microRNAome, degradome and transcriptome data to generate a comprehensive resource focused on exploring the complex molecular mechanism of petunia anther development. This study shows that DNA methylation could have an important impact in repressing the anther-expressed genes in the late stages of anther maturation. A total of 8,096 anther-preferential genes and 149 microRNAs (miRNAs) were identified that highly expressed in the five typical petunia anther developmental stages. Gene Ontology enrichment analysis of differentially expressed genes as well as miRNAs target genes revealed that metabolic, cellular and single-organism processes were significantly activated during the anther maturation processes. Moreover, a co-expression regulatory network for five typical anther development stages was constructed based on transcriptomic data, in which two hub transcription factors, PhERF48 and PhMS1, were demonstrated to be important regulatory genes for male fertility. Furthermore, two DNA demethylase proteins (PhDME and PhDML3) and three methyl-CpG-binding-domain proteins (PhMBD2, PhMBD3 and PhMBD4) were identified as potential critical DNA methylation regulators in petunia anther development. Our results provide new knowledge regarding the regulatory mechanism of petunia anther development, which will support the breeding of novel sterile petunia lines in the future.

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.

Methylation represents a crucial class of modification that orchestrates a spectrum of regulatory roles in plants, impacting ornamental characteristics, growth, development, and responses to abiotic stress. The establishment and maintenance of methylation involve the coordinated actions of multiple regulatory factors. Methyltransferases play a pivotal role by specifically recognizing and methylating targeted sites, which induces alterations in chromatin structure and gene expression, subsequently influencing the release of volatile aromatic substances and the accumulation of pigments in plant petals. In this paper, we review the regulatory mechanisms of methylation modification reactions and their effects on the changes in aromatic substances and pigments in plant petals. We also explore the potential of methylation modifications to unravel the regulatory mechanisms underlying aroma and color in plant petals. This aims to further elucidate the synthesis, metabolism, and regulatory mechanisms of various methylation modifications related to the aroma and color substances in plant petals, thereby providing a theoretical reference for improving the aroma and color of plant petals.

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.