In this study, the disease resistance gene PlWRKY65 was isolated from the leaves of Paeonia lactiflora and analyzed by bioinformatics methods, and the localization of the encoded protein was explored. Quantitative real-time PCR (qRT-PCR) was also used to explore the response of this gene to Alternaria tenuissima. The results showed that the gene sequence contained multiple cis-acting elements involved in the response to hormone signaling molecules belonging to the IIe subgroup of the WRKY family, and the encoded proteins were located in the nucleus. The PlWRKY65 gene has a positive regulatory effect on A. tenuissima infection. After silencing the PlWRKY65 gene via virus-induced gene silencing (VIGS), it was found that the gene-silenced plants were more sensitive to A. tenuissima infection than the wild plants, exhibiting more severe infection symptoms and different degrees of changes in the expression of the pathogenesis-related (PR) genes. In addition, we showed that the endogenous jasmonic acid (JA) content of P. lactiflora was increased in response to A. tenuissima infection, whereas the salicylic acid (SA) content decreased. After PlWRKY65 gene silencing, the levels of the two hormones changed accordingly, indicating that PlWRKY65, acting as a disease resistance-related transcriptional activator, exerts a regulatory effect on JA and SA signals. This study lays the foundation for functional research on WRKY genes in P. lactiflora and for the discovery of candidate disease resistance genes.

Many members of the WRKY family regulate plant growth and development. Recent studies have shown that members of the WRKY family, specifically WRKY13, play various roles in the regulation of plant stress resistance. To study the function of WRKY family members in peony, the PlWRKY13 gene (KY271095) was cloned from peony leaves. Sequence analysis and subcellular localization results revealed that PlWRKY13 has no introns, belongs to the type IIc subgroup of the WRKY family, and functions in the nucleus. The expression pattern of PlWRKY13 was analysed via real-time quantitative RT-PCR (qRT-PCR), which showed that the expression of PlWRKY13 was induced by four types of abiotic stress, low-temperature, high-temperature, waterlogging and salt stress, and was positively upregulated in response to these stresses. In addition, the expression of PlWRKY13 tended to first decrease and then increase after infection with Alternaria tenuissima. Virus-induced gene silencing (VIGS) technology was used to explore the function of PlWRKY13 in the resistance of Paeonia lactiflora to fungal infection further, and the results showed that PlWRKY13-silenced plants displayed increased sensitivity to A. tenuissima. The infection was more severe and the disease index (DI) significantly greater in the PlWRKY13-silenced plants than in the control plants, and the expression of pathogenesis-related (PR) genes was also significantly altered in the PlWRKY13-silenced plants compared with the control plants. The contents of the endogenous hormones jasmonic acid (JA) and salicylic acid (SA) were measured, and the results showed that the JA content increased gradually after infection with A. tenuissima and that JA may play an active role in the resistance of P. lactiflora to pathogen infection, while the SA content decreased after PlWRKY13 silencing. The contents of the two hormones decreased overall, suggesting that they are related to the transcription of PlWRKY13 and that PlWRKY13 may be involved in the disease-resistance pathway mediated by JA and SA. In summary, the results of our study showed that PlWRKY13 expression was induced by stress and had a positive effect on the resistance of P. lactiflora to fungal infection.

Dehydrins (DHNs) proteins play a protective role in plants under different abiotic stress conditions. In this study, a stress-responsive dehydrin gene PlDHN1 (GenBank accession number: KX524516) was cloned from the buds of the Paeonia lactiflora ‘Dafugui’. The PlDHN1 gene contained a 783-bp open reading frame (ORF) that encoded 260 amino acids. Sequence alignment and phylogenetic analysis showed that the PlDHN1 sequence possessed an 8-serine residue stretch followed by three conserved repeats of a lysine-rich segment, characteristic of the typical SK₃-type dehydrin. Gene structural analysis demonstrated that PlDHN1 contained one 334-bp intron, which included many cis-acting elements known to be involved in gene expression regulation. Subcellular localisation analysis revealed that the PlDHN1 protein was mainly detected in the cytomembrane and nucleus. qRT-PCR analysis indicated that the PlDHN1 gene was more highly expressed in buds than in other tissues. Furthermore, the PlDHN1 transcript in buds accumulated during the winter and peaked in harsh December with gradual decrease prior to spring. The expression of PlDHN1 was up-regulated in response to cold, heat, waterlogging and abscisic acid (ABA) treatment, most significantly for heat and waterlogging. Overall, these results will provide insights into the molecular basis and future genetic engineering of peony resistance against abiotic stresses.

A long juvenile period limits the breeding process of many woody plants including tree peony. To investigate the physiological and transcriptomic differences between juvenile and adult plants of tree peony and to explore the key SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, which are vital in age-dependent pathways, 1-year-old and 3-year-old Paeonia delavayi plants were used to compare the relevant physiological parameters and transcriptomic profiles of the leaves in two phases of plants. The results of the physiological parameters showed that the starch content in the leaves of adult plants remained unchanged and that the soluble sugar content significantly increased compared with those in the juvenile plants. In terms of plant hormones, the contents of cytokinin-like hormone (N6-isopentenyladenine (iP)) and jasmonic acid (JA) significantly decreased, whereas the contents of auxin (indole-3-acetic acid, IAA), abscisic acid (ABA), cytokinin-like hormone (N6-isopentenyladenenosine (iPR)), and ethylene precursor (1-aminocyclopropane-1-carboxylic acid, ACC) showed no statistic difference. Transcriptome sequencing results showed that there were 194 differentially expressed genes (DEGs) between juvenile and adult plants, including 171 up-regulated DEGs and 23 down-regulated DEGs. Circadian rhythm, plant hormone signal transduction, and sugar metabolism were closely related to the juvenile-to-adult transition in P. delavayi, involving a total of 12 DEGs. In addition, a total of 13 SPL genes were identified in the transcriptome data, but only PdSPL10 (c71307.graph_c0) was differentially expressed. It was further validated via qRT-PCR analysis, indicating that PdSPL10 might be a key gene regulating the process of juvenile-to-adult in P. delavayi. Based on the above results, a hypothetical transcriptional network regulating juvenile-to-adult transition and flowering in P. delavayi was proposed. These findings provide a reference for understanding the mechanism of juvenile-to-adult transition in tree peony.