Genome-Wide Identification and Comparative Analysis of the Teosinte Branched 1/Cycloidea/Proliferating Cell Factors 1/2 Transcription Factors Related to Anti-cancer Drug Camptothecin Biosynthesis in Ophiorrhiza pumila

Hao

et al. 2022

Horticulture Research

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Camptothecin (CPT) is a chemotherapeutic drug widely used to treat various cancers. Ophiorrhiza pumila is an ideal model for a camptothecin-producing plant with various advantages for studying camptothecin biosynthesis and regulation. The DNA binding protein WRKY transcription factors have a key regulatory role in secondary metabolite biosynthesis in plants. However, their involvement in camptothecin biosynthesis in O. pumila is still in its infancy. We identified 46 OpWRKY genes unevenly distributed within 11 chromosomes of O. pumila. Phylogenetic and multiple sequence alignment analyses divided the OpWRKY proteins into three subfamilies. Based on spatial and co-expression, we targeted a candidate gene, OpWRKY6. Overexpression of OpWRKY6 significantly reduced the accumulation of camptothecin compared to the control. Conversely, compared to the control, camptothecin accumulation increased in OpWRKY6 knock-out lines. Further biochemical assays showed that OpWRKY6 negatively regulates camptothecin biosynthesis, from both iridoid and shikimate pathways, by directly down-regulating the gene expression of OpGES, Op10HGO, Op7DLH, and OpTDC. Our data provide direct evidence to the involvement of WRKYs in the regulation of camptothecin biosynthesis and offer valuable information for the enrichment production of camptothecin in plant systems.

Section: Methodsmentioning

confidence: 99%

Identification of WRKY transcription factors involved in regulating the biosynthesis of the anti-cancer drug camptothecin in Ophiorrhiza pumila

Hao

et al. 2022

Horticulture Research

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“…The construction of a co‐expression network revealed a total of 672 candidate TFs that may be involved in the regulation of camptothecin biosynthesis, including MYB (67 genes), ERF (47 genes), bHLH (44 genes), NAC (43 genes), C2H2 (42 genes), bZIP (31 genes), WRKY (26 genes), and so on (Figure 5A). The expression of OpNAC1 in O. pumila roots was significantly higher than in the stems and leaves, correlating with the camptothecin biosynthetic genes (Figures 5B, S27) (Shannon et al, 2003; Wang et al, 2021). To further investigate the potential regulatory function of OpNAC1 in the camptothecin biosynthetic pathway, the potential binding sites of NAC TFs (NACRS motif, C/TACG) in about 3,000 bp promoter regions of 10 camptothecin biosynthetic genes were analyzed.…”

Section: Resultsmentioning

confidence: 93%

OpNAC1 transcription factor regulates the biosynthesis of the anticancer drug camptothecin by targeting loganic acid O‐methyltransferase in Ophiorrhiza pumila

Hao

Zhou

et al. 2023

JIPB

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Camptothecin (CPT) is an anticancer pentacyclic quinoline alkaloid widely used to treat cancer patients worldwide. However, the biosynthetic pathway and transcriptional regulation of camptothecin are largely unknown. Ophiorrhiza pumila, the herbaceous plant from the Rubiaceae family, has emerged as a model plant for studying camptothecin biosynthesis and regulation. In this study, a high-quality reference genome of O. pumila with estimated size of ~456.90 Mb was reported, and the accumulation level of camptothecin in roots was higher than that in stems and leaves. Based on its spatial distribution in the plant, we examined gene functions and expression by combining genomics with transcriptomic analysis. Two loganic acid O-methyltransferase (OpLAMTs) were identified in strictosidine-producing plant O. pumila, and enzyme catalysis assays showed that OpLAMT1 and not OpLAMT2 could convert loganic acid into loganin. Further knock-out of OpLAMT1 expression led to the elimination of loganin and camptothecin accumulation in O. pumila hairy roots. Four key residues were identified in OpLAMT1 protein crucial for the catalytic activity of loganic acid to loganin. By co-expression network, we identified a NAC transcription factor, OpNAC1, as a candidate gene for regulating camptothecin biosynthesis. Transgenic hairy roots and biochemical assays demonstrated that OpNAC1 suppressed OpLAMT1 expression. Here, we reported on two camptothecin metabolic engineering strategies paving the road for industrial-scale production of camptothecin in CPT-producing plants.

“…In addition, they are characterized by the presence of an angiosperm conserved 18–20 amino acid Arginine-rich motif (the R-domain) ( Figure 2C ). Although, a few CIN-like proteins have also been shown to carry the R-domain ( Wang et al, 2021 ). The R-domain forms an α-helix structure that coils similarly to leucine zippers which functions in protein-protein interaction (PPIs) mediation, and in evolutionary/developmental and phylogenetic studies ( Busch and Sassone-Corsi, 1990 ).…”

Section: Tcp Conserved Domainsmentioning

confidence: 99%

Genomic survey of TCP transcription factors in plants: Phylogenomics, evolution and their biology

Zhou

Hwarari

et al. 2022

Front. Genet.

The TEOSINTE BRANCHED1 (TBI1), CYCLOIDEA (CYC), and PROLIFERATING CELL NUCLEAR ANTIGEN FACTORS (PCF1 and PCF2) proteins truncated as TCP transcription factors carry conserved basic-helix-loop-helix (bHLH) structure, related to DNA binding functions. Evolutionary history of the TCP genes has shown their presence in early land plants. In this paper, we performed a comparative discussion on the current knowledge of the TCP Transcription Factors in lower and higher plants: their evolutionary history based on the phylogenetics of 849 TCP proteins from 37 plant species, duplication events, and biochemical roles in some of the plants species. Phylogenetics investigations confirmed the classification of TCP TFs into Class I (the PCF1/2), and Class II (the C- clade) factors; the Class II factors were further divided into the CIN- and CYC/TB1- subclade. A trace in the evolution of the TCP Factors revealed an absence of the CYC/TB1subclade in lower plants, and an independent evolution of the CYC/TB1subclade in both eudicot and monocot species. 54% of the total duplication events analyzed were biased towards the dispersed duplication, and we concluded that dispersed duplication events contributed to the expansion of the TCP gene family. Analysis in the TCP factors functional roles confirmed their involvement in various biochemical processes which mainly included promoting cell proliferation in leaves in Class I TCPs, and cell division during plant development in Class II TCP Factors. Apart from growth and development, the TCP Factors were also shown to regulate hormonal and stress response pathways. Although this paper does not exhaust the present knowledge of the TCP Transcription Factors, it provides a base for further exploration of the gene family.