Anzhi Wei

Plant sex reversal reflects developmental plasticity in floral sexual expression under genetic and environmental variation. Zanthoxylum bungeanum is a woody spice crop generally regarded as dioecious, but female-to-male floral transition has been increasingly observed in female trees across several major production regions in recent years, resulting in reduced fruit set and yield. Using developmental cytology, hormone profiling, transcriptomics, hormone treatments, and gene silencing assays, we found that abscisic acid (ABA) accumulation at the S2 stage was associated with female-to-male floral transition. Fluridone treatment before visible sex differentiation markedly reduced the proportion of male flowers, supporting a role for ABA biosynthesis during the early phase of floral sex specification. The floral homeotic gene ZbAGAMOUS (ZbAG) was identified as a candidate gene associated with reproductive organ identity and pistil retention during sex transition. Our data support a model in which elevated ABA is associated with increased ZbNAC83 expression and reduced ZbAG expression. ZbNAC83 binds an ABA-responsive element (ABRE)-containing fragment of the ZbAG promoter and represses reporter activity, consistent with a role in promoting male organ differentiation. These findings support a model in which an ABA-associated ZbNAC83–ZbAG module contributes to the loss of female floral identity during early floral sex transition in Z. bungeanum.

Background: AP2/ERF is a large family of plant transcription factor proteins that play essential roles in signal transduction, plant growth and development, and responses to various stresses. The AP2/ERF family has been identified and verified by functional analysis in various plants, but so far there has been no comprehensive study of these factors in Chinese prickly ash. Phylogenetic, motif, and functional analyses combined with transcriptome analysis of Chinese prickly ash fruits at different developmental stages (30, 60, and 90 days after anthesis) were conducted in this study. Results: The analysis identified 146 ZbAP2/ERF genes that could be classified into 15 subgroups. The motif analysis revealed the presence of different motifs or elements in each group that may explain the functional differences between the groups. ZbERF13.2, ZbRAP2-12, and ZbERF2.1 showed high levels of expression in the early stages of fruit development. ZbRAP2-4, and ZbERF3.1 were significantly expressed at the fruit coloring stage (R2 and G2). ZbERF16 were significantly expressed at fruit ripening and expression level increased as the fruit continued to develop. Relative gene expression levels of 6 representative ZbAP2/ERFs assessed by RT-qPCR agreed with transcriptome analysis results. Conclusions: These genes identified by screening can be used as candidate genes that affect fruit development. The results of the analysis can help guide future genetic improvement of Chinese prickly ash and enrich our understanding of AP2/ERF transcription factors and their regulatory functions in plants.

As an important economical plant, Zanthoxylum bungeanum is widely cultivated in arid and semi-arid areas. The studies associated with photosynthesis under drought stress were widely carried out, but not yet in Z. bungeanum. Here, the photosynthesis of two Z. bungeanum cultivars (FJ, Z. bungeanum cv. “Fengjiao”; HJ, Z. bungeanum cv. “Hanjiao”) was analyzed under drought stress using physiological indicators and transcriptome data. Drought decreased stomatal aperture and stomatal conductance (Gsw), reduced transpiration rate (E) and sub-stomatal CO₂ concentration (Ci), and lowered chlorophyll and carotenoid content, which reduced the net photosynthetic rate (Pn) of Z. bungeanum. The higher photosynthetic rate in HJ stemmed from its higher chlorophyll content, larger stomatal aperture and Gsw, and higher Ci. Weighted gene co-expression network analysis (WGCNA) identified several ABA signal transduction genes (PYL4, PYL9, and PYR1), LCH-encoding genes (LHCB4.3), and chlorophyll metabolism genes (CRD1, PORA, and CHLH). Additionally, seven transcription factor genes were identified as important factors regulating photosynthesis under drought conditions. In general, a photosynthetic response model under drought stress was built firstly in Z. bungeanum, and the key genes involved in photosynthesis under drought stress were identified. Therefore, the results in our research provide important information for photosynthesis under drought and provided key clues for future molecular breeding in Z. bungeanum.

Zanthoxylum bungeanum leaves have a unique taste and incomparable nutritional value and hence are popular as a food item and traditional medicine in China. However, the studies on the metabolites in Z. bungeanum leaves are quite limited, especially for amino acids. Therefore, this study explored the amino acid component in Z. bungeanum leaves and also the accumulation mechanism under drought stress in two Z. bungeanum cultivars using the widely targeted metabolome combined with transcriptome analysis. A total of 56 amino acids and their derivatives were identified in Z. bungeanum leaves, including eight essential amino acids. The total amino acid content with most individual amino acids increased under progressive drought stress. More differentially accumulated amino acids (DAAs) and differentially expressed genes (DEGs) were found in FJ (Z. bungeanum cv. ‘Fengjiao’) than in HJ (Z. bungeanum cv. ‘Hanjiao’). The orthogonal projections to latent structures discriminant analysis identified nine and seven indicator DAAs in FJ and HJ leaves, respectively. The weighted gene co-expression network analysis (WGCNA) showed that the green module was significantly correlated with most indicator DAAs and revealed the important role of FBA3, DELTA-OAT, PROC, and 15 transcription factor genes in regulating the amino acid synthesis. Furthermore, the correlation analysis and redundancy analysis (RDA) identified four candidate synthesis genes (ASNS, AK, ASPS, and PK) in amino acid biosynthesis pathway. This study provided useful information for the development of Z. bungeanum leaves in food and nutrition industry and also laid the foundations for future molecular breeding.

Zanthoxylum, belonging to the Rutaceae family, is widely distributed in tropical and subtropical regions. The genus has high economic value as spices, oils, medicinal plants, and culinary applications. Zanthoxylum has a long history of domestication and cultivation in China. However, the phylogenetic relationships and origin of wild and cultivated Zanthoxylum species in China remain largely unknown. Moreover, there is still no clear molecular phylogenetic system for Zanthoxylum species. Herein, 373 Zanthoxylum samples were collected from all presently known provenances of Zanthoxylum in China. In this study, four chloroplast DNA (cpDNA) markers (matK, ndhH, psbB, rbcL) were used to comprehensively analyze the genetic diversity, relatedness, and geographical origin of Chinese Zanthoxylum species. The results were as follows: (1) The aligned length of the four pairs of cpDNA sequences was 3836 bp, and 68 haplotypes were identified according to 219 variable polymorphic sites, including 90 singleton variable sites, 129 parsimony informative sites, 3 Indels (insertions and deletions). (2) Phylogenetic tree and haplotype network strongly supported the division of Zanthoxylum species consistent with the taxonomic recognition of five species: Z. bungeanum, Z. piasezkii, Z. piperitum, Z. armatum, and Z. micranthum. (3) Divergence time estimation suggested that Zanthoxylum genus originated from the Late Eocene, and most Zanthoxylum species diverged after the Middle Miocene. (4) Haplotype 16 (H16) was at the bottom of the phylogenetic tree, had higher haplotype diversity (Hd) and nucleotide polymorphism (Pi) than other haplotypes, and was located in the center of the network figure. Therefore, we deduced that the cultivated Zanthoxylum species may originate in Zhouqu County, Gansu Province, China. Meanwhile, our research provided a scientific basis for the identification and breeding programs of Chinese Zanthoxylum species.

Plant growth and survival in nature, its growth process, will be affected by various factors from the environment, among which temperature has a greater impact. In recent years, extreme weather has frequently appeared, and the growth of crops has been increasingly affected by the environment. As an important flavoring and Chinese herbal medicine crop,Zanthoxylum bungeanumis also facing the harm of low-temperature stress. Plant hormones play a vital role in the response of plants to low temperatures. In this study, ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the hormone components of cold-tolerant and cold-sensitive varieties ofZ. bungeanum. Combined with chemometric analysis and weighted gene co-expression network analysis (WGCNA), the hormone component differences and hormone response strategies ofZ. bungeanumunder low-temperature stress were comprehensively studied. The results showed that 45 hormones were detected inZ. bungeanum. Among them, there were 7 kinds of components with high content and were detected in both two varieties. At the late stage of low-temperature stress, the contents of abscisic acid (ABA) and ABA-glucosyl ester (ABA-GE) in Fuguhuajiao (FG) were significantly increased, and the latter served as the storage of the former to supplement the active ABA. Orthogonal partial least squares discriminant analysis (OPLS-DA) found that indole-3-carboxylic acid (ICA), indole-3-carboxaldehyde (ICAld), meta-Topolin riboside (mTR), cis-Zeatin-O-glucoside riboside (cZROG), and N6-isopentenyladenosine (IPR) in FG were the upregulated important difference components, and IPR and 2-methylthio-cis-zeatin riboside (2MeScZR) in Fengxiandahongpao (FX) were the upregulated important difference components. There were common crossing points and independent response pathways in response to low temperature in two varieties. WGCNA analysis found that the main hormone components were associated with multiple metabolic pathways including carbon, fatty acid, amino acid, and sugar metabolism, indicating that hormone regulation plays an important role in the response ofZ. bungeanumto low temperature. This study clarified the hormone response mechanism ofZ. bungeanumunder low-temperature stress and provided a reference and basis for further improving the cold resistance ofZ. bungeanumand cultivating new varieties.

NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) are one of the largest plant-specific TF families and play a pivotal role in adaptation to abiotic stresses. The genome-wide analysis of NAC TFs is still absent in Zanthoxylum bungeanum. Here, 109 ZbNAC proteins were identified from the Z. bungeanum genome and were classified into four groups with Arabidopsis NAC proteins. The 109 ZbNAC genes were unevenly distributed on 46 chromosomes and included 4 tandem duplication events and 17 segmental duplication events. Synteny analysis of six species pairs revealed the closely phylogenetic relationship between Z. bungeanum and C. sinensis. Twenty-four types of cis-elements were identified in the ZbNAC promoters and were classified into three types: abiotic stress, plant growth and development, and response to phytohormones. Co-expression network analysis of the ZbNACs revealed 10 hub genes, and their expression levels were validated by real-time quantitative polymerase chain reaction (qRT-PCR). Finally, ZbNAC007, ZbNAC018, ZbNAC047, ZbNAC072, and ZbNAC079 were considered the pivotal NAC genes for drought tolerance in Z. bungeanum. This study represented the first genome-wide analysis of the NAC family in Z. bungeanum, improving our understanding of NAC proteins and providing useful information for molecular breeding of Z. bungeanum.

Prickly ash peel is one of the eight major condiments in China and is widely used in cooking because of its unique fragrance and numbing taste. The color of prickly ash fruit is the most intuitive quality that affects consumer choice. However, the main components and key biosynthetic genes responsible for prickly ash fruit color have not yet been determined. To better understand the biosynthetic mechanisms and accumulation of prickly ash fruit color components, we performed an integrated transcriptomic and metabolomic analysis of red and green prickly ash fruit at different growth periods. The transcriptome analysis identified 17,269 differentially expressed genes (DEGs) between fruit of red and green prickly ash: 7,236 upregulated in green fruit and 10,033 downregulated. Liquid chromatography tandem mass spectrometry (LC-MS/MS) identified 214 flavonoids of 10 types. Flavonoids and flavonols are the main flavonoids in prickly ash, and the total flavonoid content of red prickly ash is higher than that of green prickly ash. Comprehensive analysis showed that the main colored metabolites that differed between green and red prickly ash were cyanidin-3-O-galactoside and cyanidin-3-O-glucoside, and differences in the contents of these metabolites were due mainly to differences in the expression of ANS and UFGT. Our results provide insight into the mechanisms underlying color differences in red and green prickly ash and will be useful for improving the quality of prickly ash fruit.