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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.

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.

Plants can accumulate a large amount of reactive oxygen species under adverse conditions such as drought and high temperature, which seriously affect the normal growth and development of plants. The antioxidant system can scavenge the reactive oxygen species produced under drought conditions and so mitigate oxidative damage. However, the regulation patterns of many miRNAs under drought stress are still unclear. The content of antioxidant enzymes and the expression patterns of miRNAs and their target genes related to antioxidant systems were studied under drought stress in Zanthoxylum bungeanum. The results indicate that under drought stress, POD, CAT, APX, proline, MDA and related genes all show positive responses to drought, while SOD and its genes showed a negative response. It is indicated that in the antioxidant process of Z. bungeanum, POD, CAT, and APX play a major role, and SOD plays a supporting role. In addition, GUS histochemical and RT-qPCR experimental results show that the expression levels of miRNAs and their target genes are basically negatively correlated, indicating that miRNAs can inhibit the expression of related genes and are also important regulators in the antioxidant system of Z. bungeanum. According to the expression patterns of antioxidant enzymes, miRNA and its target genes under drought stress, combined with previous research results, a model of plant antioxidant mechanism was constructed to provide a reference for further understanding of plant antioxidant mechanism.

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 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.

Zanthoxylum bungeanum (Rutaceae), a popular food flavoring and traditional Chinese medicine ingredient, is an important cash crop. Its leaves are rich in flavonoids with multiple bioactivities. How...

Plant stress tolerance is a complex process involving transcriptional and post-transcriptional regulation of gene expression. Many recent studies have shown that LncRNAs play important roles in plant stress responses and thus create new opportunities for genetic plant breeding. In this study, RNA-seq analysis of Zanthoxylum bungeanum leaves under PEG-simulated drought stress was carried out and differential expression profiles of LncRNAs were obtained. We identified five drought-response-up-regulated LncRNAs (DRURs) and five drought-response-down-regulated LncRNAs (DRDRs). Speculating that these LncRNAs participate in the regulation of Z. bungeanum’s PEG-simulated drought stress response, to confirm their function, we predicted miRNAs in LncRNAs and predicted the miRNA target genes. Forty-four miRNAs were identified from the 10 LncRNAs and the target genes of miRNA were shown to involve numerous signaling pathways and metabolic pathways, including plant-pathogen interaction, plant hormone signal transduction, MAPK signaling, cell growth and death, and amino acid biosynthesis. Results suggest the 10 drought-responsive LncRNAs may be involved in the regulation of membrane stability in mitochondria and chloroplasts and so help maintain normal operation of photosynthesis and respiration. They also, activate the JA, ABA, auxin, ethylene, GA and MAPK signaling pathways.

In this paper, an interaction model of apomixis-related genes was constructed to analyze the emergence of apomictic types. It is speculated that apomixis technology will be first implemented in gramineous plants. Apomixis (asexual seed formation) is a phenomenon in which a plant bypasses the most fundamental aspects of sexual reproduction-meiosis and fertilization-to form a viable seed. Plants can form seeds without fertilization, and the seed genotype is consistent with the female parent. The development of apomictic technology would be revolutionary for agriculture and for food production as it would reduce costs and breeding times and also avoid many complications typical of sexual reproduction (e.g. incompatibility barriers) and of vegetative propagation (e.g. viral transfer). The application of apomictic reproductive technology has the potential to revolutionize crop breeding. This article reviews recent advances in apomixis in cytology and molecular biology. The general idea of identifying apomixis was proposed and the process of the emergence of non-fusion types was discussed. To better understand the apomixis mechanism, an apomixis regulatory model was established. At the same time, the realization of apomixis technology is proposed, which provides reference for the research and application of apomixis.

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.

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Soil microbiota play an important and diverse roles in horticultural crop nutrition or productivity. However, the soil microbial community composition and the relationships within the taxa in the microbial community populations after cover grass treatments in apple orchards are not well understood. We analysed the microbial community diversity, composition and microbial network of an apple orchard after covering with native wild grasses at different intensities for 2 years in the Loess Plateau, China. The cover grass intensities were 0%, 20%, 40%, 60% and 80%. Soil microbial community diversity was not obviously change by cover grass in the apple orchard. Cover grass altered the microbial bacterial community compositions, their changes exhibited significant differences at the phylum level that were caused by the Proteobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes, Nitrospirae. However, low-intensity (20%) and moderate-intensity (40%) treatments were the only cover grass intensities that altered the soil fungal community composition; but their changes did not exhibit significant differences at the phylum level. The positive links among the bacterial taxa decreased with the increasing cover intensity, primarily among Proteobacteria, Acidobacteria, Actinobacteria, Chloroflexi and Gemmatimonadetes. Although cover grass increased the positive links between fungal taxa, these taxa were reduced with the increasing cover intensity. Here we demonstrate that cover grass changed the soil microbial community, and the changes may be attributed to the given phyla in the bacterial community; furthermore, the antagonistic effect between the soil bacterial and fungal communities was significantly increased by higher coverage than by lower coverage.

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.

Chinese pepper, mainly includingZanthoxylum bungeanumandZanthoxylum 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 inZ. bungeanumand two clades inZ. 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 ofZ. bungeanumin China. These findings provide comprehensive insights into genetic diversity, population structure, demography, and adaptation inZanthoxylum.

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 ofZanthoxylum 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 factorZbAGL11was highly expressed during the critical period of nucellar embryo development in ZB. UnpollinatedZbAGL11-OEArabidopsisproduced fertile offspring and exhibited an apomictic phenotype. The overexpression ofZbAGL11increased 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 thatZbAGL11can cause developmental disorders ofArabidopsisflower organs and result in apomixis-like phenotypes.

Zanthoxylum bungeanum, a spice and medicinal plant, is cultivated in many parts of China and some countries in Southeast Asia; however, data on its genome are lacking. In the present study, we performed a whole-genome survey and developed novel genomic-SSR markers of Z. bungeanum. Clean data (∼197.16 Gb) were obtained and assembled into 11185221 scaffolds with an N50 of 183 bp. K-mer analysis revealed that Z. bungeanum has an estimated genome size of 3971.92 Mb, and the GC content, heterozygous rate, and repeat sequence rate are 37.21%, 1.73%, and 86.04%, respectively. These results indicate that the genome of Z. bungeanum is complex. Furthermore, 27153 simple sequence repeat (SSR) loci were identified from 57288 scaffolds with a minimum length > 1 kb. Mononucleotide repeats (19706) were the most abundant type, followed by dinucleotide repeats (5154). The most common motifs were A/T, followed by AT/AT; these SSRs accounted for 71.42% and 11.84% of all repeats, respectively. A total of 21243 non-repeating primer pairs were designed, and 100 were randomly selected and validated by PCR analysis using DNA from 10 Z. bungeanum individuals and 5 Zanthoxylum armatum individuals. Finally, 36 polymorphic SSR markers were developed with polymorphism information content (PIC) values ranging from 0.16 to 0.75. Cluster analysis revealed that Z. bungeanum and Z. armatum could be divided into two major clusters, suggesting that these newly developed SSR markers are useful for genetic diversity and germplasm resource identification in Z. bungeanum and Z. armatum.

Chinese pepper, referring to Zanthoxylum bungeanum Maxim. and Zanthoxylum armatum DC. species, is an important spice crop that has long attracted people&rsquo, s interest due to its extensive application in Asian cuisine to improve taste. Numerous cultivars have been developed during the long history of domestication and cultivation. However, little to no information is available on the genetic diversity and evolutionary relationships of Chinese pepper cultivars and their historical diversification has not been clarified. Herein, we sequenced two nrDNA non-coding region markers, the external transcribed spacer (ETS) and the internal transcribed spacer 2 (ITS2), to assess genetic diversity and phylogenetic relationships among 39 cultivated and wild populations of Chinese pepper from eight provinces and to address the question of ancient demographic trends which were probably influenced by changing climate during evolutionary history. In total, 31 haplotypes were identified based on 101 polymorphism sites. Our results revealed relatively high level of genetic variation despite long-term cultivation of this crop. AMOVA revealed that genetic variation existed predominantly within provinces rather than among provinces. The genetic structure result based on haplotype network analysis largely reflected historical records, which suggested a Gansu origin for Chinese pepper and an ancient west-to-east spread of Chinese pepper circulating in China. We also provided evidence that changing Pleistocene climates had shaped the demographic trends of Chinese pepper. Taken together, our findings not only suggest that Chinese pepper is a dynamic genetic system that responds to evolutionary forces, but it also provides a fundamental genetic profile for the conservation and responsible exploitation of the extant germplasm of Chinese pepper and for improving the genetic basis for breeding the cultivars.

Zanthoxylum, an ancient economic crop in Asia, has a satisfying aromatic taste and immense medicinal values. A lack of genomic information and genetic markers has limited the evolutionary analysis and genetic improvement of Zanthoxylum species and their close relatives. To better understand the evolution, domestication, and divergence of Zanthoxylum, we present a de novo transcriptome analysis of an elite cultivar of Z. bungeanum using Illumina sequencing; we then developed simple sequence repeat markers for identification of Zanthoxylum. In total, we predicted 45,057 unigenes and 22,212 protein coding sequences, approximately 90% of which showed significant similarities to known proteins in databases. Phylogenetic analysis indicated that Zanthoxylum is relatively recent and estimated to have diverged from Citrus ca. 36.5–37.7 million years ago. We also detected a whole-genome duplication event in Zanthoxylum that occurred 14 million years ago. We found no protein coding sequences that were significantly under positive selection by Ka/Ks. Simple sequence repeat analysis divided 31 Zanthoxylum cultivars and landraces into three major groups. This Zanthoxylum reference transcriptome provides crucial information for the evolutionary study of the Zanthoxylum genus and the Rutaceae family, and facilitates the establishment of more effective Zanthoxylum breeding programs.

Zanthoxylum bungeanum is an economically important tree species of edible, medicinal, and ornamental value. The trees grow in varied climates from tropical to subtropical regions owing to their high resistance to adverse environment. The unstable environmental temperature is one of the important factors affecting the yield and quality of Z. bungeanum. To investigate complex responses to cold stress in Z. bungeanum, we compared cold-tolerant “Fuguhuajiao” (FG) and cold-sensitive “Fengxiandahongpao” (FX) varieties. Physiological measurements and transcriptome sequencing were performed using plant leaves collected at six different timepoints (0, 1, 3, 6, 12, and 24 h) of 4 °C treatment. Chemometrics analysis showed that the physiological cold response in FX at late stage was similar to that in FG at early stage. Based on orthogonal partial least squares discriminant analysis (OPLS-DA), the variable important in projection (VIP) values of peroxidase (POD) and soluble protein were 1.11 and 1.41, which were identified as important physiological indices in the two varieties. Under low temperature stress, there were 3513 differentially expressed genes in FG and 25,157 in FX. Weighted gene co-expression network analysis (WGCNA) revealed that many cold-response genes and pathways were enriched in the MEfirebrick4 module which had a high correlation with POD. Hub genes identified in this module indicated that Z. bungeanum can respond and adapt to cold stress by altering signal transduction, plant hormones, transcription factors, protein modification, functional proteins, metabolism, cell structure, light response, and the circadian clock. Endocytosis-related gene ERD7, abscisic acid pathway-related gene PP2C, late embryogenesis abundant gene LEA D-29, and two plasma membrane/lipid raft-associated remorin genes ZB01477 and pp34 are located at the core of the network, and all play pivotal roles in cold responses. These results of this study highlight cold response mechanisms at the physiological and molecular levels in Z. bungeanum and will contribute to a better understanding of low temperature resistance in related plants.

Melanin is a broad term for a family of complex organic macromolecules that contain phenol or indole groups. Melanins are found in animals, plants, and microorganisms and are widely used in medicine, food, and chemical industries. However, at present, the synthesis of plant melanin is still unclear and hinders the development and utilization of melanin. To clarify the composition of plant melanin and the key synthetic genes, we observed the cytological observation of the melanin-forming tissue of ZB seeds and analyzed the transcriptome and metabolite profiles of different developmental stages. We measured seed melanin content and gene expression levels and used redundancy analysis (RDA) to identify key genes that affected melanin synthesis. The seed melanin content was 3.85 % at the green fruit stage (S2) and reached a peak of 4.33 % at the fruit maturity stage (S4). Tyrosine and hydroquinone contents were highest at S2: 830 mg/kg and 77.23 mg/kg, respectively. Catechin, L-epicatechin, procyanidin B1, and p-coumaric acid were the main flavonoids in ZB seeds and reached their highest levels at S2. RDA analysis confirmed that S2 was the key period of melanin synthesis and that ZbPPO, ZbLAR, ZbTYRP1, and ZbTYR had crucial roles in the synthesis of ZB melanin-related substances.

Background Apomixis is a form of reproduction that does not involve fertilization of female by male gametes but instead produces offspring from the female parent directly. The progeny of apomixis is genotypically identical to the female parent and so maintains any elite traits of the female parent. Apomixis has considerable potential in genetic plant breeding. However, the mechanism of apomictic reproduction remains unclear. Zanthoxylum bungeanum is an apomictic plant. Studies on miRNAs, mRNAs, hormone changes and fertilization process of the pollinated and non-pollinated materials of Zanthoxylum bungeanum allows screening of the important regulatory factors of the apomictic process at both physiological and molecular levels. This information should be of considerable help in understanding the mechanism of apomixis in this species.Results Our results show that Zanthoxylum bungeanum pollen can germinate on the stigma, and that the pollen tube can extend to the ovary wall after two days but that it then degenerates about the fifth day and before reaching the egg cell. So fertilization does not occur. Nevertheless, pollination did increase fruit set from 74.12% (unpollinated) to 89.31% (pollinated). The reproductive mode of the Zanthoxylum bungeanum cultivar ‘Hancheng Dahongpao’ was identified as obligate apomixis. Enrichment analysis of differential genes indicates that pollination activates genes involved in the synthesis of ABA, IAA, GA3 and JA, and that genes related to asexual development, genes involved in flower development, and transcription factors are also activated. RT-qPCR verification shows that the relative expression levels of mRNAs and miRNAs related to hormone signaling pathways and flower development were negatively correlated. Also, that miRNA is an important factor influencing hormone regulation after pollination of this apomictic species. The contents of ABA, IAA, GA3 and JA were all significantly increased following pollination.Conclusions In general, although pollination does not result in fertilization in this species, it does activate mRNAs and miRNAs which serve as signals to activate the anabolic pathways for ABA, IAA, GA3 and JA. Increased levels of these hormones result in increased fruit set.

When the plant is in an unfavorable environment such as drought or high temperature, it will accumulate a large amount of active oxygen, which will seriously affect the normal growth and development of the plant. The antioxidant system can remove the reactive oxygen species produced under drought conditions and so mitigate oxidative damage. We examined the trends of antioxidant enzymes, miRNAs and their target genes in Zanthoxylum bungeanum under drought stress. According to the changes of antioxidant enzymes, miRNAs and their target genes expression patterns of Zanthoxylum bungeanum under drought stress, an interaction model was constructed to provide a reference for further understanding of plant antioxidant mechanism. The results indicate that under drought stress, POD, CAT, APX, proline, MDA and related genes all show positive responses to drought, while SOD and its genes showed a negative response. It is speculated that in the antioxidant process of Zanthoxylum bungeanum, POD, CAT, and APX play a major role, and SOD plays a supporting role. In addition, the expression levels of miRANs and their target genes were basically negatively correlated, indicating that miRNAs are involved in the regulation of the antioxidant system of Zanthoxylum bungeanum.

The WRKY family of transcription factors (TFs) includes a number of transcription-specific groupings that play important roles in plant growth and development and in plant responses to various stresses. To screen for WRKY transcription factors associated with drought stress in Zanthoxylum bungeanum, a total of 38 ZbWRKY were identified and these were then classified and identified with Arabidopsis WRKY. Using bioinformatics analyses based on the structural characteristics of the conservative domain, 38 WRKY transcription factors were identified and categorized into three groups: Groups I, II, and III. Of these, Group II can be divided into four subgroups: subgroups IIb, IIc, IId, and IIe. No ZbWRKY members of subgroup IIa were found in the sequencing data. In addition, 38 ZbWRKY were identified by real-time PCR to determine the behavior of this family of genes under drought stress. Twelve ZbWRKY transcription factors were found to be significantly upregulated under drought stress and these were identified by relative quantification. As predicted by the STRING website, the results show that the WRKYs are involved in four signaling pathways&mdash, the jasmonic acid (JA), the salicylic acid (SA), the mitogen-activated protein kinase (MAPK), and the ethylene signaling pathways. ZbWRKY33 is the most intense transcription factor in response to drought stress. We predict that WRKY33 binds directly to the ethylene synthesis precursor gene ACS6, to promote ethylene synthesis. Ethylene then binds to the ethylene activator release signal to activate a series of downstream genes for cold stress and osmotic responses. The roles of ZbWRKY transcription factors in drought stress rely on a regulatory network center on the JA signaling pathway.

Fresh Zanthoxylum bungeanum Maxim. (Z. bungeanum) easily lose its color attributes and nutritional properties during drying. This study aimed to evaluate the influences of four different drying techniques, including shade drying (SD), hot air drying (HAD), far−infrared drying (FID), and freeze drying (FD) on the quality maintenance of ‘Fengjiao’ Z. bungeanum. The color attributes, essential oil content and compositions, total alkylamides content (TALC), total anthocyanin content (TAC), total phenol content (TPC), total flavone content (TFC), and antioxidant activity of Z. bungeanum were determined. Results found that HAD caused the most reduction in its nutritional components; SD maintained higher retention of essential oil compositions, but resulted in the lower TAC, TPC, TFC, and antioxidant activity compared with FID and FD treatments. However, FD maintained the highest nutrients content and antioxidant activity of Z. bungeanum. Thus, FD is an efficient drying method that shows potential applications in maintaining the quality of Z. bungeanum.

Selective tissue factor-factor VIIa complex (TF-FVIIa) inhibitors are viewed as promising compounds for treating thrombotic disease. In this contribution, we describe multifaceted exploratory SAR studies of S1′-binding moieties within a macrocyclic chemotype aimed at replacing cyclopropyl sulfone P1′ group. Over the course of the optimization efforts, the 1-(1H-tetrazol-5-yl)cyclopropane P1′ substituent emerged as an improved alternative, offering increased metabolic stability and lower clearance, while maintaining excellent potency and selectivity.

Incorporation of a methyl group onto a macrocyclic FVIIa inhibitor improves potency 10-fold but is accompanied by atropisomerism due to restricted bond rotation in the macrocyclic structure, as demonstrated by NMR studies. We designed a conformational constraint favoring the desired atropisomer in which this methyl group interacts with the S2 pocket of FVIIa. A macrocyclic inhibitor incorporating this constraint was prepared and demonstrated by NMR to reside predominantly in the desired conformation. This modification improved potency 180-fold relative to the unsubstituted, racemic macrocycle and improved selectivity. An X-ray crystal structure of a closely related analogue in the FVIIa active site was obtained and matches the NMR and modeled conformations, confirming that this conformational constraint does indeed direct the methyl group into the S2 pocket as designed. The resulting rationally designed, conformationally stable template enables further optimization of these macrocyclic inhibitors.

Eight isobutylhydroxyamides, including three new (1-3), qinbunamides A-C, and five known sanshools (4-8), ZP-amide A (4), ZP-amide B (5), ZP-amide E (6), ZP-amide C (7), and ZP-amide D (8), were isolated from the pericarps of cultivated Zanthoxylum bungeanum Maxim, cultivated in Qinling mountain area, Shaanxi, China. The structures of all compounds were determined on the basis of spectroscopic techniques, including 1D and 2D NMR analysis and comparison with previously reported data. Compounds 1 and 2 are the first example of isobutylhydroxyamides containing an ethoxy group, and compound 3 is a rare C11 fatty acid-containing sanshool existing in genus Zanthoxylum. The tested compounds enhanced nerve growth factor (NGF)-mediated neurite outgrowth (neurotrophic activity) in rat pheochromocytoma (PC12) cells, but were inactive in the inhibitory effects on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and growth of HCT116 cells at concentrations of 50μM.