Convergent recruitment of 5′-hydroxylase activities by CYP75B flavonoid B-ring hydroxylases for tricin biosynthesis in Medicago legumes

• Tricin (3′,5′-dimethoxylated flavone) is a predominant flavonoid amongst monocots but occurs only in isolated and unrelated dicot lineages. Although tricin biosynthesis has been intensively studied in monocots, it has remained largely elusive in tricin-accumulating dicots. • We investigated a subg...

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Bibliographic Details
Published in:The New phytologist 2020-10, Vol.228 (1), p.269-284
Main Authors: Lui, Andy C. W., Lam, Pui Ying, Chan, Kwun Ho, Wang, Lanxiang, Tobimatsu, Yuki, Lo, Clive
Format: Article
Language:English
Subjects:
Accumulation
Alfalfa
alfalfa (Medicago sativa)
Amino acids
Bioaccumulation
Biosynthesis
Cytochrome
Cytochrome P-450 Enzyme System - genetics
Cytochrome P450
Cytochromes
Cytochromes P450
dicot
Evolution
Flavonoids
Hydroxylase
Hydroxylation
legume
Legumes
Medicago truncatula
Medicago truncatula - genetics
Molecular evolution
Mutants
pathway evolution
Phylogeny
Pigments
Polymorphism
Speciation
Subgroups
Substrates
tricin biosynthesis
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Summary:• Tricin (3′,5′-dimethoxylated flavone) is a predominant flavonoid amongst monocots but occurs only in isolated and unrelated dicot lineages. Although tricin biosynthesis has been intensively studied in monocots, it has remained largely elusive in tricin-accumulating dicots. • We investigated a subgroup of cytochrome P450 (CYP) 75B subfamily flavonoid B-ring hydroxylases (FBHs) from two tricin-accumulating legumes, Medicago truncatula and alfalfa (Medicago sativa), by phylogenetic, molecular, biochemical and mutant analyses. • Five Medicago cytochrome P450 CYP75B FBHs are phylogenetically distant from other legume CYP75B members. Among them, MtFBH-4, MsFBH-4 and MsFBH-10 were expressed in tricin-accumulating vegetative tissues. In vitro and in planta analyses demonstrated that these proteins catalyze 3′- and 5′-hydroxylations critical to tricin biosynthesis. A key amino acid polymorphism, T492G, at their substrate recognition site 6 domain is required for the novel 50-hydroxylation activities. Medicago truncatula mtfbh-4 mutants were tricindeficient, indicating that MtFBH-4 is indispensable for tricin biosynthesis. • Our results revealed that these Medicago legumes had acquired the tricin pathway through molecular evolution of CYP75B FBHs subsequent to speciation from other nontricin-accumulating legumes. Moreover, their evolution is independent of that of grass-specific CYP75B apigenin 3′-hydroxylases/chrysoeriol 5′-hydroxylases dedicated to tricin production and Asteraceae CYP75B flavonoid 3′,5′-hydroxylases catalyzing the production of delphinidinbased pigments.
ISSN:0028-646X
1469-8137
DOI:10.1111/nph.16498