The morphological, physiological, and biochemical characteristics of leaves result from the long-term adaptation of plants to their environment and are closely related to plant growth and development. In this study, 37 prickly ash germplasm resources from 18 production areas were utilized as the subjects of research. Logistic equations, principal component analysis, and cluster analysis were employed to comprehensively evaluate the leaf traits of prickly ash germplasm resources, with an analysis of their correlation with ecological and geographical factors in the production areas. The results showed that the leaf traits of prickly ash germplasms of different origins are substantially different and diverse. The coefficient of variation for the 14 leaf traits was greater than 10%. The coefficient of variation of the compound leaflet number was the highest among all the considered leaf traits, and the coefficient of variation of leaf thickness was the lowest, at 49.86% and 11.37%, respectively. The leaf traits of the prickly ash germplasm originating from Chongqing in Yongchuan, Chongqing in Rongchang, and Yunnan in Honghe ranked highest, whereas the leaf traits of the prickly ash germplasm from Henan in Jiaozuo, Gansu in Tianshui, and Shanxi in Yuncheng ranked lowest. The results of the correlation analysis showed that among the ecological and geographical factors of the origins, latitude had the strongest correlation with the leaf traits of the prickly ash germplasm. As latitude increased, the leaves of prickly ash gradually decreased in size, weight, and leaf shape index. The factor with the second strongest correlation was temperature. The leaves of the prickly ash germplasm originating from warmer climate areas were larger and heavier than those from areas with colder climates. Altitude and longitude did not significantly affect the leaf traits of the prickly ash germplasm, but at similar latitudes, the leaves of the prickly ash germplasm in high-altitude areas were smaller, and the leaves of the prickly ash germplasm in low-altitude areas were larger. These findings can provide valuable references for breeding and the sustainable utilization of new varieties of prickly ash resources.

Lonicera macranthoides is an important medicinal plant widely used in traditional Chinese medicine. Luteoloside is a critical bioactive compound in L. macranthoides. To date, the molecular mechanisms underlying luteoloside biosynthesis are still largely unknown. In this work, high performance liquid chromatography (HPLC) was employed to determine the luteoloside contents in leaves, stems, and flowers at different developmental stages. Results showed that senescing leaves can accumulate large amounts of luteoloside, extremely higher than that in young and semi-lignified leaves and other tissues. RNA-Seq analysis identified that twenty-four differentially expressed unigenes (DEGs) associated with luteoloside biosynthesis were significantly up-regulated in senescing leaves, which are positively correlated with luteoloside accumulation. These DEGs include phenylalanine ammonia lyase 2, cinnamate 4-hydroxylase 2, thirteen 4-coumarate-CoA ligases, chalcone synthase 2, six flavonoid 3′-monooxygenase (F3′H) and two flavone 7-O-β-glucosyltransferase (UFGT) genes. Further analysis demonstrated that two F3′Hs (CL11828.Contig1 and CL11828.Contig2) and two UFGTs (Unigene2918 and Unigene97915) might play vital roles in luteoloside generation. Furthermore, several transcription factors (TFs) related to flavonoid biosynthesis including MYB, bHLH and WD40, were differentially expressed during leaf senescence. Among these TFs, MYB12, MYB75, bHLH113 and TTG1 were considered to be key factors involved in the regulation of luteoloside biosynthesis. These findings provide insights for elucidating the molecular signatures of luteoloside accumulation in L. macranthoides.

Sichuan pepper (Zanthoxylum armatum DC) is a popular spice and is often prescribed in traditional Chinese medicine to treat vomiting, diarrhea, ascariasis and eczema, among other conditions. Volatile oils from Z. armatum leaves contain active ingredients, with terpenoids being one of the main components. In the present study, the combination of sequencing data of Z. armatum from PacBio single molecule real time (SMRT) and Illumina RNA sequencing (RNA-Seq) platforms facilitated an understanding of the gene regulatory network of terpenoid biosynthesis in pepper leaves. The leaves of three developmental stages from two Z. armatum cultivars, ‘Rongchangwuci’ (WC) and ‘Zhuye’ (ZY), were selected as test materials to construct sequencing libraries. A total of 143,122 predictions of unique coding sequences, 105,465 simple sequence repeats, 20,145 transcription factors and 4719 long non-coding RNAs (lncRNAs) were identified, and 142,829 transcripts were successfully annotated. The occurrence of alternative splicing events was verified by reverse transcription PCR, and quantitative real-time PCR was used to confirm the expression pattern of six randomly selected lncRNAs. A total of 96,931 differentially expressed genes were filtered from different samples. According to functional annotation, a total of 560 candidate genes were involved in terpenoid synthesis, of which 526 were differentially expressed genes (DEGs). To identify the key genes involved in terpenoid biosynthesis, the module genes in different samples, including structural and transcription factors genes, were analyzed using the weighted gene co-expression network method, and the co-expression network of genes was constructed. Thirty-one terpenoids were identified by gas chromatography–mass spectrometry. The correlation between 18 compounds with significantly different contents and genes with high connectivity in the module was jointly analyzed in both cultivars, yielding 12 candidate DEGs presumably involved in the regulation of terpenoid biosynthesis. Our findings showed that full-length transcriptome SMRT and Illumina RNA-Seq can play an important role in studying organisms without reference genomes and elucidating the gene regulation of a biosynthetic pathway.

As a traditional spicy condiment, Zanthoxylum armatum var. novemfolius is of high economical and medicinal value. Despite the long history of human cultivation, the molecular mechanisms underlying flower development are still poorly understood in Z. armatum. In this study, we performed de novo transcriptome assembly and comparative analysis of female and male flowers in Z. armatum. A total of 94,771 unigenes were obtained, and 50,605 unigenes were successfully annotated against the public database. Transcriptome data showed that 20,431 annotated unigenes were differentially expressed genes. KEGG enrichment analysis revealed that the most representative pathway was plant hormone signal transduction. Among them, 41, 16, 41, 27, 95, and 40 unigenes were involved in the biosynthesis and signaling of abscisic acid, ethylene, cytokinin, gibberellin, auxin, and jasmonic acid, respectively. Transcription factors also played crucial roles in flower development, such as AGL11, PMADS2, and NAC. These results provided an important basis for characterizing the potential mechanism of flower development and enriching the knowledge of reproduction genetics in Z. armatum.