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.

Aegilops tauschii, a monocotyledonous annual grass, recognized as a pivotal progenitor of modern wheat (Triticum aestivum L.), serves as the D-genome donor in hexaploid wheat. This diploid species (2n = 2x = 14, DD) harbors a substantial reservoir of genetic diversity, particularly in terms of biotic and abiotic stress resistance traits. The extensive allelic variation present in its genome has been increasingly utilized for wheat genetic enhancement, particularly through introgression breeding programs aimed at improving yield potential and stress resilience. Heavy metal ATPases (HMAs), which belong to the P-type ATPase superfamily and are also known as P1B-type ATPases, play a crucial role in transporting heavy metals and maintaining metal ion homeostasis in plant cells. HMAs have been extensively studied in model plants like Arabidopsis thaliana and rice. However, this family has not been reported in A. tauschii. Here, we conducted the genome-wide identification and bioinformatics analysis of the AetHMA gene family in A. tauschii, resulting in the discovery of a total of nine AetHMA members. Among AetHMA genes, six pairs are large-block duplication genes, which mainly occur among the four genes of AetHMA2, AetHMA4, AetHMA8, and AetHMA9. Additionally, there is one pair that consists of tandem duplication genes (AetHMA6: AetHMA7). All AetHMAs can be classified into six groups (I–VI), which are further divided into two branches: the copper subclasses and the zinc subclasses. Initially, A. tauschii was grown in a 1/2 Hoagland nutrient solution and subsequently exposed to four heavy metals: zinc (Zn), copper (Cu), manganese (Mn), and cadmium (Cd). Following this treatment, the expression profiles of nine AetHMA genes were assessed. The results indicated that, under zinc and manganese stress, the HMA family members exhibited enhanced expression in the leaves, whereas the expression of most members in the roots was downregulated. In the roots, except for AetHMA2, AetHMA5, and AetHMA8, the expression levels of other members were upregulated in response to Cd exposure. Furthermore, AetHMA4 diminishes the tolerance of yeast to Mn by increasing the absorption of Mn, while AetHMA8 increases the tolerance of yeast to Cd by reducing the absorption of Cd. This study provides experimental data regarding the function of the AetHMA gene in the transport, regulation, and detoxification of heavy metal elements in A. tauschii.

The natural resistance-associated macrophage proteins (NRAMPs) gene family represents a group of membrane transporter proteins with wide distribution in plants. This family of membrane transporters plays a pivotal role in mediating plant responses to metal stress by coordinating ion transport processes and maintaining cellular metal homeostasis, thereby effectively mitigating the detrimental effects of metal ion stress on plant growth and development. This study conducted a comprehensive genome-wide analysis of the NRAMP gene family in A. tauschii using integrated bioinformatics approaches, as well as the expression pattern when exposed to heavy metal-induced stress. By means of phylogenetic investigation, eleven AetNRAMP proteins were categorized into five distinct subgroups. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the majority of NRAMP genes exhibited marked differential expression patterns under specific stress treatments. Subsequently, yeast cells were employed to validate the functions of AetNRAMP1 and AetNRAMP3. It was confirmed that AetNRAMP1 functioned in copper transport, and AetNRAMP3 showed an increase in its expression level under manganese stress. These findings establish a molecular foundation for elucidating the functional specialization of NRAMP gene family members in A. tauschii’s heavy metal detoxification pathways, providing critical genetic evidence for their stress-responsive regulatory networks. Nevertheless, significant knowledge gaps persist regarding its functions in A. tauschii. Research on metal stress resistance in this wheat progenitor species may establish a theoretical foundation for enhancing wheat tolerance and developing improved cultivars.

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.

High temperatures pose a significant threat to plant growth and survival. Heat shock factors (Hsfs) regulate heat shock proteins (Hsps) and stress-related genes. This study examines the Hsf gene family and the role of BpHsf11 in heat tolerance in Broussonetia papyrifera (L.) L'Hér. ex Vent. Twenty BpHsfs were identified through genome-wide analysis. Phylogenetic classification, conserved motif identification, and promoter cis-element analysis were performed. Subcellular localization was used to determine the location of BpHsf11 protein. Expression patterns under heat stress (42°C) were analyzed via RNA-seq and qRT-PCR. BpHsf11 was functionally validated in yeast. In the genome of B. papyrifera, 20 BpHsf genes have been identified and categorized into three primary groups (A, B, C) according to their protein structure and phylogenetic features. Unon analyzing the promoter region of BpHsfs, several stress-related regulatory elements were found. Synteny analysis revealed a substantial presence of syntenic gene pairs between the B. papyrifera Hsf gene family and those of Arabidopsis thalianas and Populus, with two instances of segmental duplication observed among the BpHsf genes. Under heat stress, the expression of seven BpHsf members changed significantly. Their expression levels increased initially, peaked at 12 h, and then decreased. Subcellular localization revealed BpHsf11 protein localized to the cytoplasmic membrane and nucleus. Yeast validation of BpHsf11 demonstrated growth inhibition in both experimental and control groups following 2-h incubation at 50°C, although the yeast expressing BpHsf11 showed a lesser degree of suppression compared to the control group. Certain members of the BpHsf family exhibit notable alterations in expression levels when exposed to elevated temperatures. Specifically, BpHsf11 has been shown to enhance yeast thermotolerance in response to heat stress, suggesting its potential to enhance stress resistance in mulberry trees.