Sweet osmanthus (Osmanthus fragrans) is an evergreen woody plant that emits a floral aroma and is widely used in the landscape and fragrance industries. However, its application and cultivation regions are limited by cold stress. Heat-shock transcription factor (HSF) family members are widely present in plants and participate in, and regulate, the defense processes of plants under various abiotic stress conditions, but now, the role of this family in the responses of O. fragrans to cold stress is still not clear. Here, 46 OfHSF members were identified in the O. fragrans genome and divided into three subfamilies on the basis of a phylogenetic analysis. The promoter regions of most OfHSFs contained many cis-acting elements involved in multiple hormonal and abiotic stresses. RNA-seq data revealed that most of OfHSF genes were differentially expressed in various tissues, and some OfHSF members were induced by cold stress. The qRT-PCR analysis identified four OfHSFs that were induced by both cold and heat stresses, in which OfHSF11 and OfHSF43 had contrary expression trends under cold stress conditions and their expression patterns both showed recovery tendencies after the cold stress. OfHSF11 and OfHSF43 localized to the nuclei and their expression patterns were also induced under multiple abiotic stresses and hormonal treatments, indicating that they play critical roles in responses to multiple stresses. Furthermore, after a cold treatment, transient expression revealed that the malondialdehyde (MDA) content of OfHSF11-transformed tobacco significantly increased, and the expression levels of cold-response regulatory gene NbDREB3, cold response gene NbLEA5 and ROS detoxification gene NbCAT were significantly inhibited, implying that OfHSF11 is a negative regulator of cold responses in O. fragrans. Our study contributes to the further functional characterization of OfHSFs and will be useful in developing improved cold-tolerant cultivars of O. fragrans.

Background Osmanthus fragrans is an evergreen plant with high ornamental and economic values. However, they are easily injured by salt stress, which severely limits their use in high salinity areas. The trihelix transcription factor (TF) family, as one of the earliest discovered TF families in plants, plays an essential part in responses to different abiotic stresses, and it has potential functions in improving the salt-tolerance capability of O. fragrans. Results In this study, 56 trihelix genes (OfGTs) were first identified in O. fragrans and then divided into five subfamilies in accordance with a phylogenetic tree analysis. The OfGTs were found to be located randomly on the 20 O. fragrans chromosomes, and an analysis of gene replication events indicated that the OfGT gene family underwent strong purification selection during the evolutionary process. The analysis of conserved motifs and gene structures implied that the OfGT members in the same subfamily have similar conserved motifs and gene structures. A promoter cis-elements analysis showed that all the OfGT genes contained multiple abiotic and hormonal stress-related cis-elements. The RNA-seq data suggested that the OfGTs have specific expression patterns in different tissues, and some were induced by salt stress. The qRT-PCR analysis of 12 selected OfGTs confirmed that OfGT1/3/21/33/42/45/46/52 were induced, with OfGT3/42/46 being the most highly expressed. In addition, OfGT42/OfGT46 had a co-expression pattern under salt-stress conditions. OfGT3/42/46 were mainly localized in the nuclei and exhibited no transcriptional activities based on the analysis of the subcellular localization and transcriptional activity assay. Furthermore, the expression levels of most of the selected OfGTs were induced by multiple abiotic and hormonal stresses, and the expression patterns of some OfGTs were also highly correlated with gibberellic acid and methyl jasmonate levels. Remarkably, the transient transformation results showed lower MDA content and increased expression of ROS-related genes NbAPX in transgenic plants, which implying OfGT3/42/46 may improve the salt tolerance of tobacco. Conclusions The results implied that the OfGT genes were related to abiotic and hormonal stress responses in O. fragrans, and that the OfGT3/42/46 genes in particular might play crucial roles in responses to salt stress. This study made a comprehensive summary of the OfGT gene family, including functions and co-expression patterns in response to salt and other stresses, as well as an evolutionary perspective. Consequently, it lays a foundation for further functional characterizations of these genes.

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