Real-time quantitative PCR (RT-qPCR) has a high sensitivity and strong specificity, and is widely used in the analysis of gene expression. Selecting appropriate internal reference genes is the key to accurately analyzing the expression changes of target genes by RT-qPCR. To find out the most suitable internal reference genes for studying the gene expression in Broussonetia papyrifera under abiotic stresses (including drought, salt, and ZnSO₄ treatments), seven different tissues of B. papyrifera, as well as the roots, stems, and leaves of B. papyrifera under the abiotic stresses were used as test materials, and 15 candidate internal reference genes were screened based on the transcriptome data via RT-qPCR. Then, the expression stability of the candidate genes was comprehensively evaluated through the software geNorm (v3.5), NormFinder (v0.953), BestKeeper (v1.0), and RefFinder. The best internal reference genes and their combinations were screened out according to the analysis results. rRNA and Actin were the best reference genes under drought stress. Under salt stress, DOUB, HSP, NADH, and rRNA were the most stable reference genes. Under heavy metal stress, HSP and NADH were the most suitable reference genes. EIF3 and Actin were the most suitable internal reference genes in the different tissues of B. papyrifera. In addition, HSP, rRNA, NADH, and UBC were the most suitable internal reference genes for the abiotic stresses and the different tissues of B. papyrifera. The expression patterns of DREB and POD were analyzed by using the selected stable and unstable reference genes. This further verified the reliability of the screened internal reference genes. This study lays the foundation for the functional analysis and regulatory mechanism research of genes in B. papyrifera.

Euonymus microcarpus (Oliv.) Sprague, is a species of evergreen shrub of the genus Euonymus, family Celastraceae. Here, we extracted the genomic DNA from the leaves of E. microcarpus and constructed a paired-end library. The chloroplast genome of E. microcarpus was generated with the high-throughput sequencing by the illumina Hiseq X Ten platform and de novo assembly. The chloroplast genome had a quadripartite structure, containing a long single copy region with a size of 85,386 bp and a short single copy region with a size of 18,456 bp, separated by two inverted repeat regions of 26,850 bp. The chloroplast genome contained 133 genes identified in total, including 87 potential protein-coding genes, 38 transfer RNA genes, and eight ribosomal RNA genes. A total of 282 simple sequence repeats and 63 long repeats were found. Furthermore, the phylogenetic relationships inferred that E. microcarpus is sister to E. japonicus and E. schensianus. A comparison of the structure of the chloroplast genomes of eight Euonymus species suggests a nucleotide variability of the junction sites and a higher divergence of non-coding regions, compared to the coding regions. The original findings of the study serves as a good reference for chloroplast genome assembly and a valuable foundation for the genetic diversity and evolution of E. microcarpus.

The argonaute (AGO) protein, as an important member of the small RNA (sRNA) regulatory pathway gene-silencing complex (RNA-induced silencing complex, RISC), is a key protein that mediates gene silencing and plays a key role in the recruitment of sRNAs. In this study, bioinformatics was used to identify the AGO gene family in poplar and study its expression in various tissues and in response to abiotic stress treatments. A total of 15 PtAGO genes were identified in poplar, which were unevenly distributed in 9 chromosomes. Most proteins were predicted to be located in the nucleus and chloroplast. The PtAGOs had similar motif structures and conserved motifs, except for PtAGO3. All the PtAGO genes could be clustered into 3 groups, and Group II, including PtAGO2/3/7, had the smallest number of exons, while the others had more than 20 exons. Cis-regulatory elements involved in light response, growth and development, abiotic stress and hormone-induced responses were found in the promoters of PtAGO members. Further expression analysis found that the PtAGO genes had tissue-specific expression patterns. For example, PtAGO7 and PtAGO10b were mainly expressed in the xylem and might be involved in secondary xylem development. Furthermore, abiotic stress tests, including heat, ABA and PEG treatments, showed that most PtAGO genes could respond quickly to ABA treatment, and multiple PtAGO genes were constantly regulated under heat-shock stress. These results provide a basis for the elucidation mechanism of PtAGO genes and further molecular breeding in poplar.

The APETALA2/ethylene-responsive factor (AP2/ERF) is a well-researched superfamily of plant transcription factors. The APETALA2 (AP2) subfamily is essential for plant growth and development. However, a systematic analysis of the AP2 subfamily in poplar has yet to be conducted. This study identified 29 AP2 genes in the poplar genome, classifying them into three clades—euAP2, euANT, and basalANT based on evolutionary relationships. These genes are distributed across 12 chromosomes and one scaffold. Results from the syntenic analysis suggest that whole-genome duplication events are the primary factors driving the expansion of the AP2 subfamily in poplar. Cis-element analysis reveals that numerous PtAP2 genes possess hormone-related cis-elements. These genes also contain cis-elements linked to plant development and stress responses. PtAP2s from different clades exhibit significantly tissue-specific expression patterns in poplar. Gene expression levels in the euAP2 clade are significantly higher than in the euANT and basalANT clades across various tissues, with basalANT showing the lowest expression. Through RT-qPCR and recombinant Saccharomyces cerevisiae assays under salt stress, it was discovered that the majority of AP2 genes showed a negative response in salt stress regulation in poplar trees. In conclusion, this study offers valuable insights into salt tolerance in poplar trees and the role of AP2 genes under salt stress conditions.