Genome-wide analysis of AP2/ERF transcription factors that regulate fruit development of Chinese prickly ash.

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.

Physiological and transcriptome analyses reveal the photosynthetic response to drought stress in drought-sensitive (Fengjiao) and drought-tolerant (Hanjiao) Zanthoxylum bungeanum cultivars.

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.

Widely targeted metabolomic profiling combined with transcriptome analysis provides new insights into amino acid biosynthesis in green and red pepper fruits.

The type and content of amino acids in pepper are important indicators to reflect its nutritional value, largely affecting the purchasing behavior of consumers. Understanding the biosynthesis of amino acids in pepper fruit is beneficial to the development of pepper functional food. Widely targeted metabolomics, transcriptome analysis, correlation analysis, weighted gene co-expression network analysis (WGCNA), and canonical correlation analysis (CCA) were used to evaluate the quality characteristics of green and red pepper amino acids. The results showed 78 kinds of amino acids and their derivatives in the fruit of pepper. The essential amino acids were comprehensive and abundant. Especially, the contents of lysine and tryptophan were high. However, significant differences were found in the ratio of essential amino acids to total amino acids in green and red pepper. The ratio of essential amino acids to total amino acids in red pepper was up to 28.88%, while that in green pepper was up to 17.69%. WGCNA and CCA analyses were performed in combination with amino acid metabolism profiling and transcriptome analysis to further identify the main contributors to amino acid synthesis in green and red pepper. The results showed PK and PFK were the genes in the backbone of the amino acid biosynthesis pathway, which had a direct impact on the synthesis of various amino acids, and were the main genes for amino acid synthesis in pepper fruit. In this study, the amino acid biosynthesis rules for two kinds of pepper were analyzed by amino acid metabolism profiling and transcriptome analysis, which provided the basis for the development of amino acid nutritional supplements and pepper functional food.

UPLC-MS/MS Profile Combined With RNA-Seq Reveals the Amino Acid Metabolism in Zanthoxylum bungeanum Leaves Under Drought Stress.

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.

Molecular Cloning and Functional Analysis of DXS and FPS Genes from Zanthoxylum bungeanum Maxim.

Zanthoxylum bungeanum Maxim. (Z. bungeanum) has attracted attention for its rich aroma. The aroma of Z. bungeanum is mainly volatile terpenes synthesized by plant terpene metabolic pathways. However, there is little information on Z. bungeanum terpene metabolic gene. In this study, the coding sequence of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and farnesyl pyrophosphate synthase (FPS) were cloned from Z. bungeanum cv. 'Fengxiandahongpao.' ZbDXS and ZbFPS genes from Z. bungeanum with CDS lengths of 2172 bp and 1029 bp, respectively. The bioinformatics results showed that Z. bungeanum was closely related to citrus, and it was deduced that ZbFPS were hydrophilic proteins without the transmembrane domain. Subcellular localization prediction indicated that ZbDXS was most likely to be located in chloroplasts, and ZbFPS was most likely to be in mitochondria. Meanwhile, the 3D protein structure showed that ZbDXS and ZbFPS were mainly composed of α-helices, which were folded into a single domain. In vitro enzyme activity experiments showed that purified proteins ZbDXS and ZbFPS had the functions of DXS enzyme and FPS enzyme. Transient expression of ZbDXS and ZbFPS in tobacco significantly increased tobacco's terpene content. Moreover, ZbDXS and ZbFPS were expressed in different tissues of Z. bungeanum, and the relative expression of the two genes was the highest in fruits. Therefore, this suggests that ZbDXS and ZbFPS are positively related to terpene synthesis. This study could provide reference genes for improving Z. bungeanum breeding as well as for the Rutaceae research.

Genome-Wide Identification of the NAC Gene Family in Zanthoxylum bungeanum and Their Transcriptional Responses to Drought Stress.

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.

Plant Hormone Response to Low-Temperature Stress in Cold-Tolerant and Cold-Sensitive Varieties of Zanthoxylum bungeanum Maxim.

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 bungeanum is 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 of Z. bungeanum. Combined with chemometric analysis and weighted gene co-expression network analysis (WGCNA), the hormone component differences and hormone response strategies of Z. bungeanum under low-temperature stress were comprehensively studied. The results showed that 45 hormones were detected in Z. 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 of Z. bungeanum to low temperature. This study clarified the hormone response mechanism of Z. bungeanum under low-temperature stress and provided a reference and basis for further improving the cold resistance of Z. bungeanum and cultivating new varieties.

Integrated LC-MS/MS and Transcriptome Sequencing Analysis Reveals the Mechanism of Color Formation During Prickly Ash Fruit Ripening.

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.

Low Temperature Affects Fatty Acids Profiling and Key Synthesis Genes Expression Patterns in Zanthoxylum bungeanum Maxim.

Zanthoxylum bungeanum is one of the most important medicinal and edible homologous plants because of its potential health benefits and unique flavors. The chemical components in compositions and contents vary with plant genotype variations and various environmental stress conditions. Fatty acids participate in various important metabolic pathways in organisms to resist biotic and abiotic stresses. To determine the variations in metabolic profiling and genotypes, the fatty acid profiling and key differential genes under low temperature stress in two Z. bungeanum varieties, cold-tolerant (FG) and sensitive (FX), were investigated. Twelve main fatty acids were found in two Z. bungeanum varieties under cold stress. Results showed that the contents of total fatty acids and unsaturated fatty acids in FG were higher than those in FX, which made FG more resistant to low temperature. Based on the result of orthogonal partial least squares discriminant analysis, palmitic acid, isostearic acid, linolenic acid and eicosenoic acid were the important differential fatty acids in FG under cold stress, while isomyristic acid, palmitic acid, isostearic acid, stearic acid, oleic acid, linolenic acid and eicosenoic acid were the important differential fatty acids in FX. Furthermore, fatty acid synthesis pathway genes fatty acyl-ACP thioesterase A (FATA), Delta (8)-fatty-acid desaturase 2 (SLD2), protein ECERIFERUM 3 (CER3), fatty acid desaturase 3 (FAD3) and fatty acid desaturase 5 (FAD5) played key roles in FG, and SLD2, FAD5, 3-oxoacyl-[acyl-carrier-protein] synthase I (KAS I), fatty acyl-ACP thioesterase B (FATB) and acetyl-CoA carboxylase (ACC) were the key genes responding to low temperature in FX. The variation and strategies of fatty acids in two varieties of Z. bungeanum were revealed at the metabolic and molecular level. This work provides a reference for the study of chemical components in plant stress resistance.

Use of mineral element profiling coupled with chemometric analysis to distinguish Zanthoxylum bungeanum cultivars and health risks of potentially toxic elements in pericarps.

BACKGROUND: Zanthoxylum bungeanum pericarps (ZBP) are commonly used as food additives and traditional herbal medicines. Several mineral elements are known to have important physiological functions in organisms, whereas others are reported to have toxic effects. We determined levels of macro elements (Mg, S and Ca), essential trace elements (B, Mn, Fe, Cu, Zn, Se and Mo) and toxic elements (Ni, Al, Cr, As, Cd, Hg and Pb) in the pericarps of 19 Z. bungeanum cultivars. Hazard index values and incremental lifetime cancer risks were calculated to express health risks associated with pericarp consumption. Moreover, several chemometric analyses based on the mineral elements were used to distinguish Z. bungeanum cultivars. RESULTS: The concentrations of 17 determined elements in the pericarps were ranked: Ca > Mg > S > Fe > Al > Mn > Zn > B > Cu > Ni > Pb > Cr > Mo > As > Cd > Hg > Se. The elements Zn, Cr and As had the highest variations in their concentrations. Cu, Mn, Se, Zn, Al, As, Cd, Cr, Hg, Ni and Pb posed some non-cancer risks, while As and Cd posed cancer risks. Mn, Fe, Zn, and Al were chosen as critical element markers for assessing ZBP using chemometric analyses. CONCLUSION: Chemometric analyses could highlight mineral concentration differentiation among the 19 cultivars. The Z. bungeanum cultivar Z12 (from Wudu, Gansu) is best for producing ZBP, and cultivar Z18 (Guanling, Guizhou) can be a reference to classify and evaluate ZBP quality. The results provide valuable information for evaluating the potential safety risks of ZBP and contribute to inter-cultivar discrimination. © 2021 Society of Chemical Industry.

Volatile Oil Profile of Prickly Ash (Zanthoxylum) Pericarps from Different Locations in China.

Volatile oils of prickly ash (Zanthoxylum) pericarps have various potential biological functions with considerable relevance to food, pharmacological, and industrial applications. The volatile profile of oils extracted from prickly ash pericarps obtained from 72 plantations in China was determined by gas chromatography and mass spectrometry. Several chemometric analyses were used to better understand the volatile oil profile differences among different pericarps and to determine the key factors that affected geographical variations in the main volatile constituents of oils. A total of 47 constituents were detected with D-limonene, alfa-myrcene, and linalool as the most abundant. The volatile profile of pericarp oils was significantly affected by prickly ash species and some environmental factors, and the key factors that affected volatile profile variations for different prickly ash species were diverse. Chemometric analyses based on the volatile oil profile could properly distinguish Z. armatum pericarps from other pericarps. This study provides comprehensive information on the volatile oil profile of pericarps from different prickly ash species and different plantations, and it can be beneficial to a system for evaluating of pericarp quality. Moreover, this study speculates on the key environmental factors that cause volatile oil variations for each species, and can help to obtain better prickly ash pericarp volatile oils by improving the cultivated environments.

Zanthoxylum-specific whole genome duplication and recent activity of transposable elements in the highly repetitive paleotetraploid Z. bungeanum genome.

Zanthoxylum bungeanum is an important spice and medicinal plant that is unique for its accumulation of abundant secondary metabolites, which create a characteristic aroma and tingling sensation in the mouth. Owing to the high proportion of repetitive sequences, high heterozygosity, and increased chromosome number of Z. bungeanum, the assembly of its chromosomal pseudomolecules is extremely challenging. Here, we present a genome sequence for Z. bungeanum, with a dramatically expanded size of 4.23 Gb, assembled into 68 chromosomes. This genome is approximately tenfold larger than that of its close relative Citrus sinensis. After the divergence of Zanthoxylum and Citrus, the lineage-specific whole-genome duplication event η-WGD approximately 26.8 million years ago (MYA) and the recent transposable element (TE) burst ~6.41 MYA account for the substantial genome expansion in Z. bungeanum. The independent Zanthoxylum-specific WGD event was followed by numerous fusion/fission events that shaped the genomic architecture. Integrative genomic and transcriptomic analyses suggested that prominent species-specific gene family expansions and changes in gene expression have shaped the biosynthesis of sanshools, terpenoids, and anthocyanins, which contribute to the special flavor and appearance of Z. bungeanum. In summary, the reference genome provides a valuable model for studying the impact of WGDs with recent TE activity on gene gain and loss and genome reconstruction and provides resources to accelerate Zanthoxylum improvement.

Small RNA sequencing provides candidate miRNA-target pairs for revealing the mechanism of apomixis in Zanthoxylum bungeanum.

BACKGROUND: Apomixis is a form of asexual reproduction that produces offspring without the need for combining male and female gametes, and the offspring have the same genetic makeup as the mother. Therefore, apomixis technology has great application potential in plant breeding. To identify the apomixis types and critical period, embryonic development at different flower development stages of Zanthoxylum bungeanum was observed by cytology. RESULTS: The results show that the S3 stage is the critical period of apomixis, during which the nucellar cells develop into an adventitious primordial embryo. Cytological observations showed that the type of apomixis in Z. bungeanum is sporophytic apomixis. Furthermore, miRNA sequencing, miRNA-target gene interaction, dual luciferase reporter assay, and RT-qPCR verification were used to reveal the dynamic regulation of miRNA-target pairs in Z. bungeanum apomixis. The miRNA sequencing identified 96 mature miRNAs, of which 40 were known and 56 were novel. Additionally, 29 differentially expressed miRNAs were screened according to the miRNAs expression levels at the different developmental stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses showed that the target genes of the differentially expressed miRNAs were mainly enriched in plant hormone signal transduction, RNA biosynthetic process, and response to hormone pathways. CONCLUSIONS: During the critical period of apomictic embryonic development, miR172c significantly reduces the expression levels of TOE3 and APETALA 2 (AP2) genes, thereby upregulating the expression of the AGAMOUS gene. A molecular regulation model of miRNA-target pairs was constructed based on their interactions and expression patterns to further understand the role of miRNA-target pairs in apomixis. Our data suggest that miR172c may regulates AGAMOUS expression by inhibiting TOE3 in the critical period of apomixis.

Alkylamide Profiling of Pericarps Coupled with Chemometric Analysis to Distinguish Prickly Ash Pericarps.

Because of extensive cultivation areas, various cultivars, nonstandard naming notations, and morphology similarity among relative cultivars, adulteration and associated business fraud may happen in the marketplaces of prickly ash pericarps due to higher financial gain and high-frequency trading. This study presents variations in the chemical components and contents of different prickly ash species from different plantations. Alkylamide profiling of pericarps derived from Zanthoxylum armatum, Z. bungeanum, and some relative Zanthoxylum species from 72 plantations across China were tested using ultra-performance liquid chromatography. Then, several chemometrics were applied to classify the prickly ash pericarps to reveal potential indicators that distinguish prickly ash pericarps and to identify the key factors that affect pericarp alkylamide profiling. The dominating alkylamides in the prickly ash pericarps were Z. piperitum (ZP)-amide C (0-20.64 mg/g) and ZP-amide D (0-30.43 mg/g). Alkylamide profiling of prickly ash pericarps varied significantly across species and geographical variations. ZP-amide D in prickly ash pericarps was identified as a potential indicator to distinguish prickly ash species. Longitude and aluminum content in soils were identified as key factors that affected alkylamide profiling of prickly ash pericarps. This study provides a useful tool to classify prickly ash species based on pericarp alkylamide profiling and to determine the key influence factors on pericarp alkylamide variations.

ZbAGL11, a class D MADS-box transcription factor of Zanthoxylum bungeanum, is involved in sporophytic apomixis.

Apomixis is a reproductive model that bypasses sexual reproduction, so it does not require the combination of paternal and maternal gametes but instead results in the production of offspring directly from maternal tissues. This reproductive mode results in the same genetic material in the mother and the offspring and has significant applications in agricultural breeding. Molecular and cytological methods were used to identify the reproductive type of Zanthoxylum bungeanum (ZB). Fluorescence detection of the amplified products of 12 pairs of polymorphic SSR primers showed consistent fluorescence signals for mother and offspring, indicating that no trait separation occurred during reproduction. In addition, the cytological observation results showed differentiation of ZB embryos (2n) from nucellar cells (2n) to form indefinite embryonic primordia and then form adventitious embryos (2n), indicating that the apomictic type of ZB is sporophytic apomixis. The MADS-box transcription factor ZbAGL11 was highly expressed during the critical period of nucellar embryo development in ZB. Unpollinated ZbAGL11-OE Arabidopsis produced fertile offspring and exhibited an apomictic phenotype. The overexpression of ZbAGL11 increased the callus induction rate of ZB tissue. In addition, the results of the yeast two-hybrid experiment showed that ZbAGL11 could interact with the ZbCYP450 and ZbCAD11 proteins. Our results demonstrate that ZbAGL11 can cause developmental disorders of Arabidopsis flower organs and result in apomixis-like phenotypes.

Transcriptome and Metabolome Dynamics Explain Aroma Differences between Green and Red Prickly Ash Fruit.

Green prickly ash (Zanthoxylum armatum) and red prickly ash (Zanthoxylum bungeanum) fruit have unique flavor and aroma characteristics that affect consumers' purchasing preferences. However, differences in aroma components and relevant biosynthesis genes have not been systematically investigated in green and red prickly ash. Here, through the analysis of differentially expressed genes (DEGs), differentially abundant metabolites, and terpenoid biosynthetic pathways, we characterize the different aroma components of green and red prickly ash fruits and identify key genes in the terpenoid biosynthetic pathway. Gas chromatography-mass spectrometry (GC-MS) was used to identify 41 terpenoids from green prickly ash and 61 terpenoids from red prickly ash. Piperitone was the most abundant terpenoid in green prickly ash fruit, whereas limonene was most abundant in red prickly ash. Intergroup correlation analysis and redundancy analysis showed that HDS2, MVK2, and MVD are key genes for terpenoid synthesis in green prickly ash, whereas FDPS2 and FDPS3 play an important role in the terpenoid synthesis of red prickly ash. In summary, differences in the composition and content of terpenoids are the main factors that cause differences in the aromas of green and red prickly ash, and these differences reflect contrasting expression patterns of terpenoid synthesis genes.

Pollination promotes ABA synthesis but not sexual reproduction in the apomictic species Zanthoxylum bungeanum Maxim.

Apomixis is a form of reproduction that does not involve the fertilization of female gametes by male gametes but instead involves the production of offspring directly from the female parent. The offspring of apomixis are genetically identical to the female parent and inherit its traits. Therefore, apomixis has great potential for application to agricultural genetic breeding. However, it remains unclear whether apomictic species require pollination, and the impacts of pollination on such species are poorly understood. We investigated the effects of pollination on the apomictic species Zanthoxylum bungeanum Maxim. by analyzing its fertilization process, assembling its transcriptome, and measuring hormone concentrations, fruit setting rate and gene expression levels. Transcriptome sequencing of pollinated and unpollinated fruits resulted in a total of 69,131 PacBio reads. Of these, 7102 genes were up-regulated and 6491 genes were down-regulated. Analysis of the differentially expressed genes (DEGs) and construction of a weighted gene co-expression network showed that many DEGs were involved in plant hormone signal transduction, suggesting that hormonal signaling during development differs between pollinated and unpollinated fruit. The germination rate of Z. bungeanum pollen in vitro was only 11%, and pollen could not germinate in the embryo sac to complete fertilization. Although pollination did not enable Z. bungeanum to complete the sexual reproduction process, it significantly increased abscisic acid (ABA) concentration and fruit setting rate. Spraying 100 µg l-1 ABA also significantly increased the fruit setting rate. Therefore, ABA appears to be a key factor in the regulation of fruit setting in apomictic Z. bungeanum. Based on these results, we suggest that some male plants be cultivated in Z. bungeanum plantations or exogenous ABA be sprayed to increase the likelihood of pollination and thereby increase the fruit setting rate.

Integrated Analysis of Metabolome and Transcriptome Data for Uncovering Flavonoid Components of Zanthoxylum bungeanum Maxim. Leaves Under Drought Stress.

Zanthoxylum bungeanum Maxim. leaves (ZBLs) are rich in flavonoids and have become popular in nutrition, foods and medicine. However, the flavonoid components in ZBLs and the mechanism of flavonoid biosynthesis under drought stress have received little attention. Here, we performed an integrative analysis of the metabolome and transcriptome of ZBLs from HJ (Z. bungeanum cv. "Hanjiao") and FJ (Z. bungeanum cv. "Fengjiao") at four drought stages. A total of 231 individual flavonoids divided into nine classes were identified and flavones and flavonols were considered the most abundant flavonoid components in ZBLs. The total flavonoid content of ZBLs was higher in FJ; it increased in FJ under drought stress but decreased in HJ. Nine-quadrant analysis identified five and eight differentially abundant flavonoids in FJ and HJ leaves, respectively, under drought stress. Weighted gene correlation network analysis (WGCNA) identified nine structural genes and eight transcription factor genes involved in the regulation of flavonoid biosynthesis. Moreover, qRT-PCR results verified the accuracy of the transcriptome data and the reliability of the candidate genes. Taken together, our results reveal the flavonoid components of ZBLs and document changes in flavonoid metabolism under drought stress, providing valuable information for nutrition value and food utilization of ZBLs.

Fatty Acid Profiling and Chemometric Analyses for Zanthoxylum Pericarps from Different Geographic Origin and Genotype.

Zanthoxylum plants, important aromatic plants, have attracted considerable attention in the food, pharmacological, and industrial fields because of their potential health benefits, and they are easily accessible because of the wild distribution in most parts of China. The chemical components vary with inter and intraspecific variations, ontogenic variations, and climate and soil conditions in compositions and contents. To classify the relationships between different Zanthoxylum species and to determine the key factors that influence geographical variations in the main components of the plant, the fatty acid composition and content of 72 pericarp samples from 12 cultivation regions were measured and evaluated. Four fatty acids, palmitic acid (21.33-125.03 mg/g), oleic acid (10.66-181.37 mg/g), linoleic acid (21.98-305.32 mg/g), and linolenic acid (0.06-218.84 mg/g), were the most common fatty acid components in the Zanthoxylum pericarps. Fatty acid profiling of Zanthoxylum pericarps was significantly affected by Zanthoxylum species and geographical variations. Stearic acid and oleic acid in pericarps were typical fatty acids that distinguished Zanthoxylum species based on the result of DA. Palmitic acid, palmitoleic acid, trans-13-oleic acid, and linoleic acid were important differential indicators in distinguishing given Zanthoxylum pericarps based on the result of OPLS-DA. In different Zanthoxylum species, the geographical influence on fatty acid variations was diverse. This study provides information on how to classify the Zanthoxylum species based on pericarp fatty acid compositions and determines the key fatty acids used to classify the Zanthoxylum species.

Genomic analysis reveals the genetic diversity, population structure, evolutionary history and relationships of Chinese pepper.

Chinese pepper, mainly including Zanthoxylum bungeanum and Zanthoxylum armatum, is an economically important crop popular in Asian countries due to its unique taste characteristics and potential medical uses. Numerous cultivars of Chinese pepper have been developed in China through long-term domestication. To better understand the population structure, demographic history, and speciation of Chinese pepper, we performed a comprehensive analysis at a genome-wide level by analyzing 38,395 genomic SNPs that were identified in 112 cultivated and wild accessions using a high-throughput genome-wide genotyping-by-sequencing (GBS) approach. Our analysis provides genetic evidence of multiple splitting events occurring between and within species, resulting in at least four clades in Z. bungeanum and two clades in Z. armatum. Despite no evidence of recent admixture between species, we detected substantial gene flow within species. Estimates of demographic dynamics and species distribution modeling suggest that climatic oscillations during the Pleistocene (including the Penultimate Glaciation and the Last Glacial Maximum) and recent domestication events together shaped the demography and evolution of Chinese pepper. Our analyses also suggest that southeastern Gansu province is the most likely origin of Z. bungeanum in China. These findings provide comprehensive insights into genetic diversity, population structure, demography, and adaptation in Zanthoxylum.