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Summary In plants, the biosynthetic pathways of some specialized metabolites are partitioned into specialized or rare cell types, as exemplified by the monoterpenoid indole alkaloid (MIA) pathway ofCatharanthus roseus(Madagascar Periwinkle), the source of the anticancer compounds vinblastine and vincristine. In the leaf, theC. roseusMIA biosynthetic pathway is partitioned into three cell types with the final known steps of the pathway expressed in the rare cell type termed idioblast. How cell‐type specificity of MIA biosynthesis is achieved is poorly understood.We generated single‐cell multi‐omics data fromC. roseusleaves. Integrating gene expression and chromatin accessibility profiles across single cells, as well as transcription factor (TF)‐binding site profiles, we constructed a cell‐type‐aware gene regulatory network for MIA biosynthesis.We showcased cell‐type‐specific TFs as well as cell‐type‐specificcis‐regulatory elements. Using motif enrichment analysis, co‐expression across cell types, and functional validation approaches, we discovered a novel idioblast‐specific TF (Idioblast MYB1,CrIDM1) that activates expression of late‐stage MIA biosynthetic genes in the idioblast.These analyses not only led to the discovery of the first documented cell‐type‐specific TF that regulates the expression of two idioblast‐specific biosynthetic genes within an idioblast metabolic regulon but also provides insights into cell‐type‐specific metabolic regulation.
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Ancient gene clusters initiate monoterpenoid indole alkaloid biosynthesis and C3 stereochemistry inversion
Abstract The inversion of C3 stereochemistry in monoterpenoid indole alkaloids (MIAs), derived from the central precursor strictosidine (3S), is essential for synthesizing numerous 3RMIAs and oxindoles, including the antihypertensive drug reserpine found inRauvolfia serpentina(Indian snakeroot) andRauvolfia tetraphylla(devil pepper) of the plant family Apocynaceae. MIA biosynthesis begins with the reduction of strictosidine aglycone by various reductases, preserving the initial 3Sstereochemistry. In this study, we identify and biochemically characterize a conserved oxidase-reductase pair from the Apocynaceae, Rubiaceae, and Gelsemiaceae families of the order Gentianales: the heteroyohimbine/yohimbine/corynanthe C3-oxidase (HYC3O) and C3-reductase (HYC3R). These enzymes collaboratively invert the 3Sstereochemistry to 3Racross a range of substrates, resolving the long-standing question about the origin of 3RMIAs and oxindole derivatives, and facilitation of reserpine biosynthesis. Notably,HYC3OandHYC3Rare located within gene clusters in both theR. tetraphyllaandCatharanthus roseus(Madagascar periwinkle) genomes, which are partially homologous to an elusive geissoschizine synthase (GS) gene cluster we also identified in these species. InR. tetraphylla, these clusters occur closely in tandem on a single chromosome, likely stemming from a single segmental duplication event, while inC. roseus, a closely related member of rauvolfioid Apocynaceae, they were later separated by a chromosomal translocation. The ancestral genomic context for both clusters can be traced all the way back to common ancestry with grapevine. Given the presence of syntenic GS homologs inMitragyna speciosa(Rubiaceae), the GS cluster, at least in part, probably evolved at the base of the Gentianales, which split from other core eudicots up to 135 million years ago. We also show that the strictosidine biosynthetic gene cluster, required to initiate the MIA pathway, plausibly evolved concurrently. The reserpine biosynthetic cluster likely arose much later in the rauvolfioid lineage of Apocynaceae. Collectively, our work uncovers the genomic and biochemical basis for key events in MIA evolution and diversification, providing insights beyond the well-characterized vinblastine and ajmaline biosynthetic pathways.
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- Award ID(s):
- 2030871
- PAR ID:
- 10636596
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Institution:
- bioRxiv
- Sponsoring Org:
- National Science Foundation
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- https://doi.org/10.1101/2025.01.07.631695
