Profiling of phytohormone-specific microRNAs and characterization of the miR160-ARF1 module involved in glandular trichome development and artemisinin biosynthesis in Artemisia annua.

MicroRNAs (miRNAs) play crucial roles in plant development and secondary metabolism through different modes of sequence-specific interaction with their targets. Artemisinin biosynthesis is extensively regulated by phytohormones. However, the function of phytohormone-responsive miRNAs in artemisinin biosynthesis remains enigmatic. Thus, we combined the analysis of transcriptomics, small RNAs, and the degradome to generate a comprehensive resource for identifying key miRNA-target circuits involved in the phytohormone-induced process of artemisinin biosynthesis in Artemisia annua. In total, 151 conserved and 52 novel miRNAs and their 4132 targets were determined. Based on the differential expression analysis, miR160 was selected as a potential miRNA involved in artemisinin synthesis. Overexpressing MIR160 significantly impaired glandular trichome formation and suppressed artemisinin biosynthesis in A. annua, while repressing its expression resulted in the opposite effect, indicating that miR160 negatively regulates glandular trichome development and artemisinin biosynthesis. RNA ligase-mediated 5' RACE and transient transformation assays showed that miR160 mediates the RNA cleavage of Auxin Response Factor 1 (ARF1) in A. annua. Furthermore, ARF1 was shown to increase artemisinin synthesis by activating AaDBR2 expression. Taken together, our results reveal the intrinsic link between the miR160-ARF1 module and artemisinin biosynthesis, and may expedite the innovation of metabolic engineering approaches for high and stable production of artemisinin in the future.

Discovery and modulation of diterpenoid metabolism improves glandular trichome formation, artemisinin production and stress resilience in Artemisia annua.

Plants synthesize diverse diterpenoids with numerous functions in organ development and stress resistance. However, the role of diterpenoids in glandular trichome (GT) development and GT-localized biosynthesis in plants remains unknown. Here, the identification of 10 diterpene synthases (diTPSs) revealed the diversity of diterpenoid biosynthesis in Artemisia annua. Protein-protein interactions (PPIs) between AaKSL1 and AaCPS2 in the plastids highlighted their potential functions in modulating metabolic flux to gibberellins (GAs) or ent-isopimara-7,15-diene-derived metabolites (IDMs) through metabolic engineering. A phenotypic analysis of transgenic plants suggested a complex repertoire of diterpenoids in Artemisia annua with important roles in GT formation, artemisinin accumulation and stress resilience. Metabolic engineering of diterpenoids simultaneously increased the artemisinin yield and stress resistance. Transcriptome and metabolic profiling suggested that bioactive GA4 /GA1 promote GT formation. Collectively, these results expand our knowledge of diterpenoids and show the potential of diterpenoids to simultaneously improve both the GT-localized metabolite yield and stress resistance, in planta.

YYFZBJS ameliorates colorectal cancer progression in ApcMin/+ mice by remodeling gut microbiota and inhibiting regulatory T-cell generation.

BACKGROUND: Progression of Colorectal cancer (CRC) is influenced by single or compounded environmental factors. Accumulating evidence shows that microbiota can influence the outcome of cancer immunotherapy. T cell, one of the main populations of effector immune cells in antitumor immunity, has been considered as a double-edged sword during the progression of CRC. Our previous studies indicate that traditional Chinese herbs (TCM) have potential anticancer effects in improving quality of life and therapeutic effect. However, little is known about the mechanism of TCM formula in cancer prevention. METHODS: Here, we used C57BL/6 J ApcMin/+ mice, an animal model of human intestinal tumorigenesis, to investigate the gut bacterial diversity and their mechanisms of action in gastrointestinal adenomas, and to evaluate the effects of Yi-Yi-Fu-Zi-Bai-Jiang-San (YYFZBJS) on of colon carcinogenesis in vivo and in vitro. Through human-into-mice fecal microbiota transplantation (FMT) experiments from YYFZBJS volunteers or control donors, we were able to differentially modulate the tumor microbiome and affect tumor growth as well as tumor immune infiltration. RESULTS: We report herein, YYFZBJS treatment blocked tumor initiation and progression in ApcMin/+ mice with less change of body weight and increased immune function. Moreover, diversity analysis of fecal samples demonstrated that YYFZBJS regulated animal's natural gut flora, including Bacteroides fragilis, Lachnospiraceae and so on. Intestinal tumors from conventional and germ-free mice fed with stool from YYFZBJS volunteers had been decreased. Some inflammation' expression also have been regulated by the gut microbiota mediated immune cells. Intestinal lymphatic, and mesenteric lymph nodes (MLN), accumulated CD4+ CD25+ Foxp3 positive Treg cells were reduced by YYFZBJS treatment in ApcMin/+ mice. Although YYFZBJS had no inhibition on CRC cell proliferation by itself, the altered Tregs mediated by YYFZBJS repressed CRC cancer cell growth, along with reduction of the phosphorylation of ß-catenin. CONCLUSIONS: In conclusion, we demonstrated that gut microbiota and Treg were involved in CRC development and progression, and we propose YYFZBJS as a new potential drug option for the treatment of CRC. Video abstract.

Genome-wide analysis of AP2/ERF superfamily in Isatis indigotica.

OBJECTIVE: AP2/ERF (APETALA2/ethylene-responsive factor) superfamily is one of the largest gene families in plants and has been reported to participate in various biological processes, such as the regulation of biosynthesis of active lignan. However, few studies have investigated the genome-wide role of the AP2/ERF superfamily in Isatis indigotica. This study establishes a complete picture of the AP2/ERF superfamily in I. indigotica and contributes valuable information for further functional characterization of IiAP2/ERF genes and supports further metabolic engineering. METHODS: To identify the IiAP2/ERF superfamily genes, the AP2/ERF sequences from Arabidopsis thaliana and Brassica rapa were used as query sequences in the basic local alignment search tool. Bioinformatic analyses were conducted to investigate the protein structure, motif composition, chromosome location, phylogenetic relationship, and interaction network of the IiAP2/ERF superfamily genes. The accuracy of omics data was verified by quantitative polymerase chain reaction and heatmap analyses. RESULTS: One hundred and twenty-six putative IiAP2/ERF genes in total were identified from the I. indigotica genome database in this study. By sequence alignment and phylogenetic analysis, the IiAP2/ERF genes were classified into 5 groups including AP2, ERF, DREB (dehydration-responsive element-binding factor), Soloist and RAV (related to abscisic acid insensitive 3/viviparous 1) subfamilies. Among which, 122 members were unevenly distributed across seven chromosomes. Sequence alignment showed that I. indigotica and A. thaliana had 30 pairs of orthologous genes, and we constructed their interaction network. The comprehensive analysis of gene expression pattern in different tissues suggested that these genes may play a significant role in organ growth and development of I. indigotica. Members that may regulate lignan biosynthesis in roots were also preliminarily identified. Ribonucleic acid sequencing analysis revealed that the expression of 76 IiAP2/ERF genes were up- or down-regulated under salt or drought treatment, among which, 33 IiAP2/ERF genes were regulated by both stresses. CONCLUSION: This study undertook a genome-wide characterization of the AP2/ERF superfamily in I. indigotica, providing valuable information for further functional characterization of IiAP2/ERF genes and discovery of genetic targets for metabolic engineering.

Biosynthesis and regulation of diterpenoids in medicinal plants.

Plant diterpenoids are widely distributed and abundant natural products with diverse structures and functions in nature, which have been commonly used in pharmaceutical, agricultural and industrial production. In recent years, plant diterpenoids have attracted increasing attention, including their biosynthetic pathways, transcriptional regulatory networks, and biological functions. Herein, the biosynthetic pathways of diterpenoids are summarized in a modular fashion. Further, the regulatory network between diterpene biosynthesis and environmental factors is reviewed. Insights into diterpene metabolism may drive elucidation of complex active diterpene pathways and serve as a knowledge repository for metabolic engineering and cell factory construction.

YYFZBJS inhibits colorectal tumorigenesis by enhancing Tregs-induced immunosuppression through HIF-1α mediated hypoxia in vivo and in vitro.

BACKGROUND AND PURPOSE: The occurrence of colorectal cancer (CRC) is associated with a variety of factors. Accumulating evidence shows that peripheral differentiation of regulatory T cells (Tregs) is critical in controlling tumorigenesis. Our previous studies demonstrated that the Yi-Yi-Fu-Zi-Bai-Jiang-San (YYFZBJS) extract exerted potent anticancer activities by significantly enhancing immunosuppression in ApcMin/+ mice. However, there is limited knowledge on the effect of YYFZBJS in the prevention of colorectal cancer and the underlying mechanisms of action. METHODS: In this study, we investigated the effect of oral administration of YYFZBJS in preventing azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced tumorigenesis. We found that YYFZBJS treatment decreased tumor load, tumor number, histology, and the severity of disease activity index (DAI) scores. To investigate if YYFZBJS inhibited tumorigenesis by regulating regulatory T cells, we depleted Tregs in AOM/DSS mice. We then analyzed the effect of intragastric administration of YYFZBJS on tumorigenesis and the regulation of tumor microenvironment. RESULTS: As expected, intragastric administration of YYFZBJS in AOM/DSS mice model significantly increased immune responses in the tumor microenvironment through its hypoxia-associated anti-cancer activities. Additionally, YYFZBJS regulated the polarization of peripheral Treg (pTreg) to suppress CRC cell proliferation and infiltration. This was demonstrated by the decrease in tumor proliferation-related proteins including p-STAT3, p-NF-κB and MMPs in a dose-dependent manner. Clinically, the increase in the levels of Tregs in human tissues during CRC progression was associated with low expression of HIF-1α in the stroma, and correlated with CRC survival and prognosis. CONCLUSION: Altogether, we demonstrated that HIF-1α may promote pTreg -induced carcinogenesis and progression of CRC cells, indicating that YYFZBJS is a promising protective agent against HIF-1α-mediated Treg activation in colorectal cancer. This study is the first to imply a novel clinical significance of a traditional Chinese herbal medicine from Synopsis of Golden Chamber in the cancer treatment and clarify the important role of tumor microenvironment in preventing tumorigenesis.

The application of ApcMin/+ mouse model in colorectal tumor researches.

PURPOSE: ApcMin/+ mouse is an excellent animal model bearing multiple intestinal neoplasia, used to simulate human familial adenomatous polyposis and colorectal tumors. The key point of this model is the mutation of Apc gene, which is a significant tumor-suppressor gene in the Wnt signaling pathway. There are also some other possible mechanisms responsible for the development of colorectal tumors in the ApcMin/+ mouse model, such as tumor-associated signaling pathways activation, the changes of tumor-related genes, and the involvement of some related proteins or molecules. METHODS: The relevant literatures about ApcMin/+ mouse model from PUBMED databases are reviewed in this study. RESULTS: In recent years, increasing studies have focused on the application of ApcMin/+ mouse model in colorectal tumor, trying to find effective therapeutic targets for further use. CONCLUSION: This article will give a brief review on the related molecular mechanisms of the ApcMin/+ mouse model and its application in colorectal tumor researches.