Serotonin N-acetyltransferase (SNAT) is a crucial enzyme in the melatonin synthesis pathway, playing an essential role in both melatonin biosynthesis and plant resistance to abiotic stress. A bioinformatics approach was employed to identify the members of the citrus SNAT gene family and to analyze their physicochemical properties, gene structure, conserved domains, phylogenetic relationships, and promoter cis-acting elements. Additionally, the tissue-specific expression of trifoliate orange SNAT family members and their expression patterns under stress conditions were examined. This study identified 21 members of the SNAT gene family across five citrus genomes, distributed over five chromosomes, with the majority predicted to localize within chloroplasts. These genes were characterized by having between 1 and 8 exons, 0 and 7 introns, 1 and 2 conserved domains, and 5 and 8 motifs. Phylogenetic analysis classified the genes into four subgroups, demonstrating significant collinearity with SNAT genes in rice. Analysis of the promoter regions revealed 32 cis-acting elements, with those responsive to light, abscisic acid, and drought being the most common. Expression analysis of SNAT genes in trifoliate orange indicated tissue specificity, with the highest expression levels detected in leaves. Quantitative real-time PCR analysis showed that the PtrSNAT1 gene was notably upregulated under various stress conditions, suggesting its role in stress response. Overall, these findings provide critical insights for further functional studies of citrus SNAT genes in relation to abiotic stress responses. Moreover, the PtrSNAT1 gene represents a potential target for developing rootstocks with enhanced resistance to abiotic stress.

Ascorbate oxidase (AAO) plays an important role in maintaining cellular redox homeostasis, thereby influencing plant growth, development, and responses to both biotic and abiotic stresses. However, there has been no systematic characterization of AAO genes in Citrus, especially their roles in response to cold stress. In the present study, nine AAO genes were identified in C. sinensis through bioinformatics analyses, exhibiting uneven distribution across four chromosomes. All CsAAOs possessed three conserved domains and were predicted to localize in the apoplast. The CsAAO gene family displayed varied intron–exon patterns. Phylogenetic analysis categorized the CsAAO family into three main clades (Clade A–C), suggesting distinct biological functions. Collinearity and Ka/Ks analysis revealed three duplicate gene pairs within the CsAAO gene family, with all duplicated CsAAOs primarily evolving under purifying selection. Analysis of cis-acting elements showed the presence of multiple hormone response elements and stress response elements within the CsAAO promoters. The computational analysis of microRNA target transcripts suggested that CsAAO9 may be a target of csi-miR156. RNA-Seq data demonstrated high expression levels of CsAAOs in roots and young fruits, while qRT-PCR analysis showed significant upregulation of six CsAAOs in response to cold treatment. Furthermore, the activities of CsAAOs exhibited a pattern of initial decrease followed by an increase after exposure to low temperatures. These findings offer important insights into the role of CsAAOs in response to cold stress. Furthermore, AAOs could be target genes for breeding crops with better cold resistance.

Chalcone Synthase (CHS) plays a vital role in flavonoid synthesis, influencing plant growth, development, and responses to both biotic and abiotic stress. In this study, 11 CHS genes were identified in Poncirus trifoliata using bioinformatics methods, with their distribution across five chromosomes and unassigned contigs. Each gene contains 2–3 exons and 3–8 conserved motifs. In silico prediction suggested that the PtrCHS proteins are localized in the cytoplasm. PtrCHS9 and PtrCHS11 share identical protein tertiary structures. Phylogenetic analysis classified the CHS family members into four subgroups. Synteny analysis revealed one set of collinear gene pairs within Poncirus trifoliata. Between Poncirus trifoliata and Arabidopsis thaliana, two sets of collinear gene pairs were identified, while one such set was found between Poncirus trifoliata and Oryza sativa. Promoter element analysis showed the presence of various hormone response and stress response elements within PtrCHS promoters. RNA-Seq data demonstrated tissue-specific expression patterns of PtrCHSs. RT-qPCR results indicated that all CHS genes, except PtrCHS11, respond to salt stress with dynamic, member-specific patterns. Additionally, four PtrCHSs (PtrCHS3, PtrCHS5, PtrCHS7, and PtrCHS10) were significantly upregulated in response to cold treatment. Notably, PtrCHS7 and PtrCHS10 maintained high expression levels at both 6 and 12 h, implying they may be key players in cold stress response in Poncirus trifoliata. Clones of PtrCHS7 and PtrCHS10 were obtained, and overexpression vectors were constructed in preparation for gene transformation. Overall, this study provides a solid foundation for future research into the functions of the PtrCHSs.

Drought stress is an important environmental factor limiting apple yield and fruit quality. Previously, we identified GRETCHEN HAGEN3.6 (GH3.6) as a negative regulator of drought stress in apple trees. Using transgenic MdGH3 RNAi (knocking down MdGH3.6 and its five homologs) plants as rootstock can increase drought tolerance, water use efficiency, flowering, and fruit quality of the Fuji scion after drought stress. However, the molecular mechanism behind this phenomenon is still unknown. Here, we performed transcriptome sequencing of the grafted plants (Fuji/GL-3 where Fuji was used as the scion and non-transgenic GL-3 was used as the rootstock, and Fuji/MdGH3 RNAi where MdGH3 RNAi was used as the rootstock) under control and drought conditions. Under control conditions, 667 up-regulated genes and 176 down-regulated genes were identified in the scion of Fuji/MdGH3 RNAi, as compared to the scion of Fuji/GL-3. Moreover, 941 up-regulated genes and 2226 down-regulated genes were identified in the rootstock of MdGH3 RNAi plants relative to GL-3. GO terms of these differentially expressed genes (DEGs) in scion and rootstock showed associations with plant growth, fruit development, and stress responses. After drought stress, 220 up-regulated and 452 down-regulated genes were identified in MdGH3 RNAi rootstock, as compared to GL-3. Significantly enriched GO terms included response to abiotic stimulus, cell division, microtubule-based process, metabolic and biosynthetic process of flavonoid, pigment, and lignin. The comparison between the scion of Fuji/MdGH3 RNAi and Fuji/GL-3 yielded a smaller number of DEGs; however, all of them were significantly enriched in stress-related GO terms. Furthermore, 365 and 300 mRNAs could potentially move from MdGH3 RNAi rootstock to scion under control and drought conditions, respectively, including FIDDLEHEAD (FDH), RESPONSIVE TO DESICCATION 26 (RD26), ARS-binding factor 2 (ABF2), WRKY75, and ferritin (FER). Overall, our work demonstrates the effects of rootstock on scion at the transcriptional level after drought stress and provides theoretical support for further understanding and utilization of MdGH3 RNAi plants.

Plant respiratory burst oxidase homolog (Rboh) genes are integral to the production of reactive oxygen species (ROS) and the regulation of stress responses. Here, bioinformatic techniques were employed to identify eight PgRboh genes (PgRbohA–H) in the genome of pomegranate (Punica granatum L.) and conduct a systematic analysis of this family. The findings showed that all PgRbohs proteins possess characteristic NADPH oxidase domains and are predicted to be localized on the cell membrane. Experimental verification confirmed the membrane localization of PgRbohD and PgRbohE proteins. Phylogenetic analysis categorized the PgRbohs proteins into six distinct groups, suggesting potential functional divergence among these groups. Promoter analysis revealed a significant presence of cis-acting elements responsive to low-temperature and methyl jasmonate (MeJA). The expression of PgRboh genes was found to be tissue-specific. Additionally, real-time PCR (RT-qPCR) was used to analyze expression patterns in response to low-temperature stress that involves multiple PgRboh genes in the cold response process. Overall, our results lay an important foundation for subsequent studies on the cold resistance function of pomegranate Rboh genes and provides new ideas for the breeding of new cold-resistant pomegranate varieties.