[Simultaneous determination of seven artemisinin-related compounds in Artemisia annua by UPLC-QQQ-MS/MS].

A variety of compounds in Artemisia annua were simultaneously determined to evaluate the quality of A. annua from multiple perspectives. A method based on ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLC-QQQ-MS/MS) was established for the simultaneous determination of seven compounds: amorpha-4,11-diene, artemisinic aldehyde, dihydroartemisinic acid, artemisinic acid, artemisinin B, artemisitene, and artemisinin, in A. annua. The content of the seven compounds in different tissues(roots, stems, leaves, and lateral branches) of A. annua were compared. The roots, stems, leaves, and lateral branches of four-month-old A. annua were collected and the content of seven artemisinin-related compounds in different tissues was determined. A multi-reaction monitoring(MRM) acquisition mode of UPLC-QQQ-MS/MS was used, with a positive ion mode of atmospheric pressure chemical ion source(APCI). Chromatographic separation was achieved on an Eclipse Plus RRHD C_(18) column(2.1 mm×50 mm, 1.8 µm). The gradient elution was performed with the mobile phase consisted of formic acid(0.1%)-ammonium formate(5 mmol·L~(-1))(A) and the methanol(B) gradient program of 0-8 min, 55%-100% B, 8-11 min, 100% B, and equilibrium for 3 min, the flow rate of 0.6 mL·min~(-1), the column temperature of 40 ℃, the injection volume of 5 µL, and the detection time of 8 min. Through methodological investigation, a method based on UPLC-QQQ-MS/MS was established for the simultaneous quantitative determination of seven representative compounds involved in the biosynthesis of artemisinin. The content of artemisinin in A. annua was higher than that of artemisinin B, and the content of artemisinin and dihydroartemisinic acid were high in all the tissues of A. annua. The content of the seven compounds varied considerably in different tissues, with the highest levels in the leaves and neither artemisinene nor artemisinic aldehyde was detected in the roots. In this study, a quantitative method based on UPLC-QQQ-MS/MS for the simultaneous determination of seven representative compounds involved in the biosynthesis of artemisinin was established, which was accurate, sensitive, and highly efficient, and can be used for determining the content of artemisinin-related compounds in A. annua, breeding new varieties, and controlling the quality of Chinese medicinal materials.

Manual correction of genome annotation improved alternative splicing identification of Artemisia annua.

Gene annotation is essential for genome-based studies. However, algorithm-based genome annotation is difficult to fully and correctly reveal genomic information, especially for species with complex genomes. Artemisia annua L. is the only commercial resource of artemisinin production though the content of artemisinin is still to be improved. Genome-based genetic modification and breeding are useful strategies to boost artemisinin content and therefore, ensure the supply of artemisinin and reduce costs, but better gene annotation is urgently needed. In this study, we manually corrected the newly released genome annotation of A. annua using second- and third-generation transcriptome data. We found that incorrect gene information may lead to differences in structural, functional, and expression levels compared to the original expectations. We also identified alternative splicing events and found that genome annotation information impacted identifying alternative splicing genes. We further demonstrated that genome annotation information and alternative splicing could affect gene expression estimation and gene function prediction. Finally, we provided a valuable version of A. annua genome annotation and demonstrated the importance of gene annotation in future research.

Genome-wide identification of key enzyme-encoding genes and the catalytic roles of two 2-oxoglutarate-dependent dioxygenase involved in flavonoid biosynthesis in Cannabis sativa L.

BACKGROUND: Flavonoids are necessary for plant growth and resistance to adversity and stress. They are also an essential nutrient for human diet and health. Among the metabolites produced in Cannabis sativa (C. sativa), phytocannabinoids have undergone extensive research on their structures, biosynthesis, and biological activities. Besides the phytocannabinoids, C. sativa is also rich in terpenes, alkaloids, and flavonoids, although little research has been conducted in this area. RESULTS: In this study, we identified 11 classes of key enzyme-encoding genes, including 56 members involved in the flavonoid biosynthesis in C. sativa, from their physical characteristics to their expression patterns. We screened the potentially step-by-step enzymes catalyzing the precursor phenylalanine to the end flavonoids using a conjoin analysis of gene expression with metabolomics from different tissues and chemovars. Flavonol synthase (FLS), belonging to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily, catalyzes the dihydroflavonols to flavonols. In vitro recombinant protein activity analysis revealed that CsFLS2 and CsFLS3 had a dual function in converting naringenin (Nar) to dihydrokaempferol (DHK), as well as dihydroflavonols to flavonols with different substrate preferences. Meanwhile, we found that CsFLS2 produced apigenin (Api) in addition to DHK and kaempferol when Nar was used as the substrate, indicating that CsFLS2 has an evolutionary relationship with Cannabis flavone synthase I. CONCLUSIONS: Our study identified key enzyme-encoding genes involved in the biosynthesis of flavonoids in C. sativa and highlighted the key CsFLS genes that generate flavonols and their diversified functions in C. sativa flavonoid production. This study paves the way for reconstructing the entire pathway for C. sativa's flavonols and cannflavins production in heterologous systems or plant culture, and provides a theoretical foundation for discovering new cannabis-specific flavonoids.

[Research progress on mechanism of phytohormones in regulating flavonoid metabolism].

Phytohormones play an important role at all stages of plant growth, influencing plant growth and development and regulating plant secondary metabolism, such as the synthesis of flavone, flavonol, anthocyanin, and other flavonoids. Flavonoids, a group of important secondary metabolites ubiquitous in plants, have antioxidative, anti-microbial, and anti-inflammatory activities and thus have a wide range of potential applications in Chinese medicine and food nutrition. With the development of biotechnology, phytohormones' regulation on flavonoids has become a research focus in recent years. This study reviewed the research progress on the mechanism of common phytohormones, such as abscisic acid, gibberellin, methyl jasmonate, and salicylic acid, in regulating flavonoid metabolism, and discussed the molecular mechanism of the synthesis and accumulation of flavonoids, aiming at clarifying the key role of phytohormones in modulating flavonoid metabolism. The result is of guiding significance for improving the content of flavonoids in plants through rational use of phytohormones and of reference value for exploring the mechanism of hormones in regulating flavonoid metabolism.

[Advances in biosynthesis of indigo in plants].

Natural indigo, as one of the oldest dyes, is also a pivotal dye utilized in cotton fabrics today. A diversity of plants rich in indigo compounds belong to traditional Chinese herbal medicines. Indigo compounds have a variety of biological and pharmacological activities, including anticonvulsant, antibacterial, antifungal, antiviral and anticancer activities. A substantial progress in indigo biosynthesis has been made lately. This paper summarizes the value of indigo from the aspects of cultural history, biosynthetic pathways and the medicinal activities of its related derivatives involved in the pathways. In addition, the latest research advancements in indigo biosynthetic pathways is demonstrated in this paper, which would lay the theoretical foundation for the exploration and utilization of natural indigo.

[Molecular genetics of medicinal plants, past achievement, and new era].

Unraveling the genetic basis of medicinal plant metabolism and developmental traits is a long-standing goal for pharmacologists and plant biologists. This paper discusses the definition of molecular genetics of medicinal plants, which is an integrative discipline with medicinal plants as the research object. This discipline focuses on the heredity and variation of medicinal plants, and elucidates the relationship between the key traits of medicinal plants(active compounds, yield, resistance, etc.) and genotype, studies the structure and function, heredity and variation of medicinal plant genes mainly at molecular level, so as to reveal the molecular mechanisms of transmission, expression and regulation of genetic information of medicinal plants. Specifically, we emphasize on three major aspects of this discipline.(1)Individual and population genetics of medicinal plants, this part mainly highlights the genetic mechanism of the domestication, the individual genomics at the species level, and the formation of genetic diversity of medicinal plants.(2)Elucidation of biosynthetic pathways of active compounds and their evolutionary significance. This part summarizes the biosynthesis, diversity and molecular evolution of active compounds in medicinal plants.(3) Molecular mechanisms that shaping the key agronomic traits by internal and external factors. This part focuses on the accumulation and distribution of active compounds within plants and the regulation of metabolic network by environmental factors. Finally, we prospect the future direction of molecular genetics of medicinal plants based on the rapid development of multi-omics technology, as well as the application of molecular genetics in the future strategies to achieve conservation and breeding of medicinal plants and efficient biosynthesis of active compounds.

Developmental transcriptome analysis of floral transition in Rosa odorata var. gigantea.

KEY MESSAGE: Expression analyses revealed that floral transition of Rosa odorata var. gigantea is mainly regulated by VRN1, COLs, DELLA and KSN, with contributions by the effects of phytohormone and starch metabolism. Seasonal plants utilize changing environmental and developmental cues to control the transition from vegetative growth to flowering at the correct time of year. This study investigated global gene expression profiles at different developmental stages of Rosa odorata var. gigantea by RNA-sequencing, combined with phenotypic characterization and physiological changes. Gene ontology enrichment analysis of the differentially expressed genes (DEGs) between four different developmental stages (vegetative meristem, pre-floral meristem, floral meristem and secondary axillary buds) indicated that DNA methylation and the light reaction played a large role in inducing the rose floral transition. The expression of SUF and FLC, which are known to play a role in delaying flowering until vernalization, was down-regulated from the vegetative to the pre-floral meristem stage. In contrast, the expression of VRN1, which promotes flowering by repressing FLC expression, increased. The expression of DELLA proteins, which function as central nodes in hormone signaling pathways, and probably involve interactions between GA, auxin, and ABA to promote the floral transition, was well correlated with the expression of floral integrators, such as AGL24, COL4. We also identified DEGs associated with starch metabolism correlated with SOC1, AGL15, SPL3, AGL24, respectively. Taken together, our results suggest that vernalization and photoperiod are prominent cues to induce the rose floral transition, and that DELLA proteins also act as key regulators. The results summarized in the study on the floral transition of the seasonal rose lay a foundation for further functional demonstration, and have profound economic and ornamental values.

Transcriptome of the floral transition in Rosa chinensis 'Old Blush'.

BACKGROUND: The floral transition plays a vital role in the life of ornamental plants. Despite progress in model plants, the molecular mechanisms of flowering regulation remain unknown in perennial plants. Rosa chinensis 'Old Blush' is a unique plant that can flower continuously year-round. In this study, gene expression profiles associated with the flowering transition were comprehensively analyzed during floral transition in the rose. RESULTS: According to the transcriptomic profiles, 85,663 unigenes and 1,637 differentially expressed genes (DEGs) were identified, among which 32 unigenes were involved in the circadian clock, sugar metabolism, hormone, and autonomous pathways. A hypothetical model for the regulation of floral transition was proposed in which the candidate genes function synergistically the floral transition process. Hormone contents and biosynthesis and metabolism genes fluctuated during the rose floral transition process. Gibberellins (GAs) inhibited rose floral transition, the content of GAs gradually decreased and GA2ox and SCL13 were upregulated from vegetative (VM) meristem to floral meristem (FM). Auxin plays an affirmative part in mediating floral transition, auxin content and auxin-related gene expression levels were gradually upregulated during the floral transition of the rose. However, ABA content and ABA signal genes were gradually downregulated, suggesting that ABA passively regulates the rose floral transition by participating in sugar signaling. Furthermore, sugar content and sugar metabolism genes increased during floral transition in the rose, which may be a further florigenic signal that activates floral transition. Additionally, FRI, FY, DRM1, ELIP, COP1, CO, and COL16 are involved in the circadian clock and autonomous pathway, respectively, and they play a positively activating role in regulating floral transition. Overall, physiological changes associated with genes involved in the circadian clock or autonomous pathway collectively regulated the rose floral transition. CONCLUSIONS: Our results summarize a valuable collective of gene expression profiles characterizing the rose floral transition. The DEGs are candidates for functional analyses of genes affecting the floral transition in the rose, which is a precious resource that reveals the molecular mechanism of mediating floral transition in other perennial plants.

McMYB10 regulates coloration via activating McF3'H and later structural genes in ever-red leaf crabapple.

The ever-red leaf trait, which is important for breeding ornamental and higher anthocyanin plants, rarely appears in Malus families, but little is known about the regulation of anthocyanin biosynthesis involved in the red leaves. In our study, HPLC analysis showed that the anthocyanin concentration in ever-red leaves, especially cyanidin, was significantly higher than that in evergreen leaves. The transcript level of McMYB10 was significantly correlated with anthocyanin synthesis between the 'Royalty' and evergreen leaf 'Flame' cultivars during leaf development. We also found the ever-red leaf colour cultivar 'Royalty' contained the known R6 : McMYB10 sequence, but was not in the evergreen leaf colour cultivar 'Flame', which have been reported in apple fruit. The distinction in promoter region maybe is the main reason why higher expression level of McMYB10 in red foliage crabapple cultivar. Furthermore, McMYB10 promoted anthocyanin biosynthesis in crabapple leaves and callus at low temperatures and during long-day treatments. Both heterologous expression in tobacco (Nicotiana tabacum) and Arabidopsis pap1 mutant, and homologous expression in crabapple and apple suggested that McMYB10 could promote anthocyanins synthesis and enhanced anthocyanin accumulation in plants. Interestingly, electrophoretic mobility shift assays, coupled with yeast one-hybrid analysis, revealed that McMYB10 positively regulates McF3'H via directly binding to AACCTAAC and TATCCAACC motifs in the promoter. To sum up, our results demonstrated that McMYB10 plays an important role in ever-red leaf coloration, by positively regulating McF3'H in crabapple. Therefore, our work provides new perspectives for ornamental fruit tree breeding.

LC-MS and MALDI-MSI-based metabolomic approaches provide insights into the spatial-temporal metabolite profiles of Tartary buckwheat achene development.

Tartary buckwheat, celebrated as the "king of grains" for its flavonoid and phenolic acid richness, has health-promoting properties. Despite significant morphological and metabolic variations in mature achenes, research on their developmental process is limited. Utilizing Liquid chromatography-mass spectrometry and atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry imaging, we conducted spatial-temporal metabolomics on two cultivars during achene development. Metabolic profiles including 17 phenolic acids and 83 flavonoids are influenced by both varietal distinctions and developmental intricacies. Notably, flavonols, as major flavonoids, accumulated with achene ripening and showed a tissue-specific distribution. Specifically, flavonol glycosides and aglycones concentrated in the embryo, while methylated flavonols and procyanidins in the hull. Black achenes at the green achene stage have higher bioactive compounds and enhanced antioxidant capacity. These findings provide insights into spatial and temporal characteristics of metabolites in Tartary buckwheat achenes and serve as a theoretical guide for selecting optimal resources for food production.

Integrated Full-Length Transcriptomics and Metabolomics Reveal Glycosyltransferase Involved in the Biosynthesis of Flavonol Glycosides in Laportea bulbifera.

Many species of the Urticaceae family are important cultivated fiber plants that are known for their economic and industrial values. However, their secondary metabolite profiles and associated biosynthetic mechanisms have not been well-studied. Using Laportea bulbifera as a model, we conducted widely targeted metabolomics, which revealed 523 secondary metabolites, including a unique accumulation of flavonol glycosides in bulblet. Through full-length transcriptomic and RNA-seq analyses, the related genes in the flavonoid biosynthesis pathway were identified. Finally, weighted gene correlation network analysis and functional characterization revealed four LbUGTs, including LbUGT78AE1, LbUGT72CT1, LbUGT71BX1, and LbUGT71BX2, can catalyze the glycosylation of flavonol aglycones (kaempferol, myricetin, gossypetin, and quercetagetin) using UDP-Gal and UDP-Glu as the sugar donors. LbUGT78AE1 and LbUGT72CT1 showed substrate promiscuity, whereas LbUGT71BX1 and LbUGT71BX2 exhibited different substrate and sugar donor selectivity. These results provide a genetic resource for studying Laportea in the Urticaceae family, as well as key enzymes responsible for the metabolism of valuable flavonoid glycosides.

Multi-omics analysis reveals promiscuous O-glycosyltransferases involved in the diversity of flavonoid glycosides in Periploca forrestii (Apocynaceae).

Flavonoid glycosides are widespread in plants, and are of great interest owing to their diverse biological activities and effectiveness in preventing chronic diseases. Periploca forrestii, a renowned medicinal plant of the Apocynaceae family, contains diverse flavonoid glycosides and is clinically used to treat rheumatoid arthritis and traumatic injuries. However, the mechanisms underlying the biosynthesis of these flavonoid glycosides have not yet been elucidated. In this study, we used widely targeted metabolomics and full-length transcriptome sequencing to identify flavonoid diversity and biosynthetic genes in P. forrestii. A total of 120 flavonoid glycosides, including 21 C-, 96 O-, and 3 C/O-glycosides, were identified and annotated. Based on 24,123 full-length coding sequences, 99 uridine diphosphate sugar-utilizing glycosyltransferases (UGTs) were identified and classified into 14 groups. Biochemical assays revealed that four UGTs exhibited O-glycosyltransferase activity toward apigenin and luteolin. Among them, PfUGT74B4 and PfUGT92A8 were highly promiscuous and exhibited multisite O-glycosylation or consecutive glycosylation activities toward various flavonoid aglycones. These four glycosyltransferases may significantly contribute to the diversity of flavonoid glycosides in P. forrestii. Our findings provide a valuable genetic resource for further studies on P. forrestii and insights into the metabolic engineering of bioactive flavonoid glycosides.

The complete chloroplast genome of Holarrhena pubescens and its phylogenetic analysis.

Holarrhena pubescens Wall. ex G. Don, 1837 is an important medicinal plant belonging to the Holarrhena genus in the Apocynaceae family. In this study, the complete chloroplast (cp) genome sequence of H. pubescens was sequenced using the Illumina NovaSeq platform. The cp genome of H. pubescens was 160,108 bp in length with 37.21% overall GC content. The cp genome of H. pubescens containing a large single-copy region (LSC, 88,685 bp), a small single-copy region (SSC, 18,671 bp), and a pair of inverted repeat regions (SSC, 26,376 bp). The cp genome encoded 129 genes, including 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis based on complete protein coding genes sequences revealed that H. pubescens was closest to Beaumontia murtonii.

Genetic and molecular analysis of the anthocyanin pigmentation pathway in Epimedium.

Introduction: Flower color is an ideal trait for studying the molecular basis for phenotypic variations in natural populations of species. Epimedium (Berberidaceae) species exhibit a wide range of flower colors resulting from the varied accumulation of anthocyanins and other pigments in their spur-like petals and petaloid sepals. Methods: In this work, the anthocyanidins of eight different Epimedium species with different floral pigmentation phenotypes were analyzed using HPLC. Twelve genes involved in anthocyanin biosynthesis were cloned and sequenced, and their expression was quantified. Results: The expression levels of the catalytic enzyme genes DFR and ANS were significantly decreased in four species showing loss of floral pigmentation. Complementation of EsF3'H and EsDFR in corresponding Arabidopsis mutants together with overexpression of EsF3'5'H in wild type Arabidopsis analysis revealed that these genes were functional at the protein level, based on the accumulation of anthocyanin pigments. Discussion: These results strongly suggest that transcriptional regulatory changes determine the loss of anthocyanins to be convergent in the floral tissue of Epimedium species.

Characterization and co-expression analysis of ATP-binding cassette transporters provide insight into genes related to cannabinoid transport in Cannabis sativa L.

Plant ATP-binding cassette (ABC) transporters contribute the transport of diverse secondary metabolites. However, their roles in cannabinoid trafficking are still unsolved in Cannabis sativa. In this study, 113 ABC transporters were identified and characterized in C. sativa from their physicochemical properties, gene structure, and phylogenic relationship, as well as spatial gene expression patterns. Eventually, seven core transporters were proposed including one member in ABC subfamily B (CsABCB8) and six ABCG members (CsABCG4, CsABCG10, CsABCG11, CsABCG32, CsABCG37, and CsABCG41), harboring potential in participating cannabinoid transport, by combining phylogenetic and co-expression analysis from the gene and metabolite level. The candidate genes exhibited a high correlation with cannabinoid biosynthetic pathway genes and the cannabinoid content, and they were highly expressed where cannabinoids appropriately biosynthesized and accumulated. The findings underpin further research on the function of ABC transporters in C. sativa, especially in unveiling the mechanisms of cannabinoid transport to boost systematic and targeted metabolic engineering.

Metabolomics analysis reveals the accumulation patterns of flavonoids and phenolic acids in quinoa (Chenopodium quinoa Willd.) grains of different colors.

Quinoa grains are gaining increasing popularity owing to their high nutritional merits. However, only limited information is available on the metabolic profiles of quinoa grains. In this study, we determined the metabolic profiles of black, red, and white quinoa grains via an ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS)-based metabolomics. A total of 689 metabolites were identified, among which 251, 182, and 317 metabolites displayed different accumulation patterns in the three comparison groups (Black vs Red, Black vs White, and Red vs White), respectively. In particular, flavonoid and phenolic acid contents displayed considerable differences, with 22 flavonoids, 5 phenolic acids, and 1 betacyanin being differentially accumulated among the three quinoa cultivars. Additionally, correlation analysis showed that flavonoids and phenolic acids could act as betanin co-pigments in quinoa grains. In conclusion, this study provides comprehensive insights into the adequate utilization and development of novel quinoa-based functional foods.

Promoter variations in DBR2-like affect artemisinin production in different chemotypes of Artemisia annua.

Artemisia annua is the only known plant source of the potent antimalarial artemisinin, which occurs as the low- and high-artemisinin producing (LAP and HAP) chemotypes. Nevertheless, the different mechanisms of artemisinin producing between these two chemotypes were still not fully understood. Here, we performed a comprehensive analysis of genome resequencing, metabolome, and transcriptome data to systematically compare the difference in the LAP chemotype JL and HAP chemotype HAN. Metabolites analysis revealed that 72.18% of sesquiterpenes was highly accumulated in HAN compared to JL. Integrated omics analysis found a DBR2-Like (DBR2L) gene may be involved in artemisinin biosynthesis. DBR2L was highly homologous with DBR2, belonged to ORR3 family, and had the DBR2 activity of catalyzing artemisinic aldehyde to dihydroartemisinic aldehyde. Genome resequencing and promoter cloning revealed that complicated variations existed in DBR2L promoters among different varieties of A. annua and were clustered into three variation types. The promoter activity of diverse variant types showed obvious differences. Furthermore, the core region (-625 to 0) of the DBR2L promoter was identified and candidate transcription factors involved in DBR2L regulation were screened. Thus, the result indicates that DBR2L is another key enzyme involved in artemisinin biosynthesis. The promoter variation in DBR2L affects its expression level, and thereby may result in the different yield of artemisinin in varieties of A. annua. It provides a novel insight into the mechanism of artemisinin-producing difference in LAP and HAP chemotypes of A. annua, and will assist in a high yield of artemisinin in A. annua.

Characterization of the horse chestnut genome reveals the evolution of aescin and aesculin biosynthesis.

Horse chestnut (Aesculus chinensis) is an important medicinal tree that contains various bioactive compounds, such as aescin, barrigenol-type triterpenoid saponins (BAT), and aesculin, a glycosylated coumarin. Herein, we report a 470.02 Mb genome assembly and characterize an Aesculus-specific whole-genome duplication event, which leads to the formation and duplication of two triterpenoid biosynthesis-related gene clusters (BGCs). We also show that AcOCS6, AcCYP716A278, AcCYP716A275, and AcCSL1 genes within these two BGCs along with a seed-specific expressed AcBAHD6 are responsible for the formation of aescin. Furthermore, we identify seven Aesculus-originated coumarin glycoside biosynthetic genes and achieve the de novo synthesis of aesculin in E. coli. Collinearity analysis shows that the collinear BGC segments can be traced back to early-diverging angiosperms, and the essential gene-encoding enzymes necessary for BAT biosynthesis are recruited before the splitting of Aesculus, Acer, and Xanthoceras. These findings provide insight on the evolution of gene clusters associated with medicinal tree metabolites.

Characterization and expression analysis of MATEs in Cannabis sativa L. reveals genes involving in cannabinoid synthesis.

The medicinal plant Cannabis sativa L. (C. sativa) accumulates plant cytotoxic but medicinally important cannabinoids in glandular trichomes and flowers of female plants. Although the major biosynthetic pathway of cannabinoids has been revealed, their transportation mechanism is still unknown. Multidrug and toxic compound extrusion proteins (MATEs) can transport plant metabolites, ions and phytohormones intra and inter-cellularly. MATEs could have the potential to translocate cannabinoids or their synthetic intermediates to cellular compartment, thus protecting them from unwanted modifications and cytotoxicity. In this study, we performed a genome-wide identification and expression analysis of Cannabis sativa MATEs (CsMATEs) and revealed 42 CsMATEs that were classified phylogenetically into four conserved subfamilies. Forty-two CsMATEs were unevenly distributed on 10 chromosomes, with 50% CsMATEs were physically adjacent to at least one another CsMATEs and 83% CsMATEs localized on plasma membrane. Tandem duplication is the major evolutionary driving force for CsMATEs expansion. Real-time quantitative PCR revealed CsMATE23, CsMATE28 and CsMATE34 mainly expressed in flower, whereas CsMATE17 and CsMATE27 showed strong transcription in root. Light responsive cis-acting element was most abundant in promoters of CsMATE23, CsMATE28 and CsMATE34. Finally, the contents of cannabinoids and corresponding biosynthetic intermediates as well as expressions of CsMATE28 and CsMATE34 were determined under UV-B treatment, among which strong correlation was found. Our results indicates that CsMATEs might involve in biosynthesis of cannabinoids and has the potential to be used in heterologous production of cannabinoids.

Genome-Wide Identification and Expression Profiles of bZIP Genes in Cannabis sativa L.

Background: The bZIP gene family plays roles in biotic and abiotic stress, secondary metabolism, and other aspects in plants. They have been reported in Arabidopsis thaliana, Oryza sativa, Artemisia annua, and other plants, but their roles in Cannabis sativa have not been determined. Materials and Methods: In this study, we analyzed the genome-wide identification and expression profile of the bZIP gene family in C. sativa. Results: A total of 51 members of the bZIP gene family were identified based on the C. sativa genome and numbered in order from CsbZIP1 to CsbZIP51. Their phylogenetic relationships, cis-elements in promoter region, gene structures and motif compositions, physicochemical properties, chromosome locations, and expression profiles, were analyzed. The results showed that the 51 CsbZIPs were unevenly distributed on 10 chromosomes and could be clustered into 11 subfamilies. Furthermore, CsbZIPs located in the same subfamilies presented similar intron/exon organization and motif composition. The expression levels of CsbZIPs in various tissues (flowers, bracts, vegetative leaves, stems, and seeds) were determined using reverse transcription quantitative polymerase chain reaction. The expression levels of CsbZIPs were higher in flowers and bracts. The 51 CsbZIPs were explored, and their structure, evolution, and expression pattern in different tissues of C. sativa were characterized synthetically. The findings indicated that CsbZIPs are essential for the growth and development of C. sativa. Conclusions: These results provide a theoretical basis for subsequent research on hemp bZIP transcription factors and the cultivation of high-cannabidiol and low-tetrahydrocannabinol high-quality cannabis varieties.