Identification of MOS9 as an interaction partner for chalcone synthase in the nucleus

Abstract

Plant flavonoid metabolism has served as a platform for understanding a range of fundamental biological phenomena, including providing some of the early insights into the subcellular organization of metabolism. Evidence assembled over the past three decades points to the organization of the component enzymes as a membrane-associated complex centered on the entry-point enzyme, chalcone synthase (CHS), with flux into branch pathways controlled by competitive protein interactions. Flavonoid enzymes have also been found in the nucleus in a variety of plant species, raising the possibility of alternative, or moonlighting functions for these proteins in this compartment. Here, we present evidence that CHS interacts with MOS9, a nuclear-localized protein that has been linked to epigenetic control of R genes that mediate effector-triggered immunity. Overexpression of MOS9 results in a reduction of CHS transcript levels and a metabolite profile that substantially intersects with the effects of a null mutation in CHS. These results suggest that the MOS9–CHS interaction may point to a previously-unknown mechanism for controlling the expression of the highly dynamic flavonoid pathway.

Formation of Flavonoid Metabolons: Functional Significance of Protein-Protein Interactions and Impact on Flavonoid Chemodiversity

“…Studies of A. thaliana flavonoid enzymes provide one of the best-characterized flavonoid metabolons with respect to protein-protein interactions. Direct protein-protein interactions among soluble flavonoid enzymes in A. thaliana have been studied by Y2H assays, affinity chromatography (AC), immunoprecipitation (IP), and physicochemical methods: Förster resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) and surface plasmon resonance refractometry (SPR) (Burbulis and Winkel-Shirley, 1999; Owens et al, 2008; Crosby et al, 2011; Watkinson et al, 2018). In this plant, interactions between the following enzyme pairs have been identified (followed by methods in parentheses): CHS-CHI (Y2H, AC, and IP), CHS-F3H (AC), CHS-DFR (Y2H and FRET), CHS-isozyme of FLS (FLS1) (Y2H and FRET), CHI-DFR (Y2H), CHI-F3H (AC and IP), FLS1-DFR (Y2H and FRET), and FLS1-F3H (Y2H) (Figure 2B, black arrows).…”

“…Moreover, immunoblotting of nuclear CHI suggested post-translational modifications that also might be responsible for the nuclear localization of the enzyme. Interestingly, CHS was recently found to interact with MOS9 (a nuclear protein associated with epigenetic control of R genes that mediate effector-triggered immunity) as analyzed by Y2H, SPR, and FRET, with a K d of 210 nM (Figure 2B, blue arrow) (Watkinson et al, 2018). Addressing this finding further may uncover additional mechanisms for controlling flavonoid pathways, as well as linking them to defense mechanisms and other physiological functions.…”

Elusive partners: a review of the auxiliary proteins guiding metabolic flux in flavonoid biosynthesis

“…Furthermore, moonlighting proteins are a recurring motif in biosynthetic enzymes with a secondary role in regulation (Commichau et al., 2007; Schmid et al., 2019; Su et al., 2019). Indeed, a moonlighting activity has been suggested for CHS, potentially mediating epigenetic control over the flavonoid pathway via nuclear‐localized interaction with MOS9 (Watkinson et al., 2018). Proteins with the diminutive size of Fra a could conceivably diffuse through the nuclear pores, similar to some CHS isoforms, and become implicated in nuclear transcriptional regulation.…”

Crosstalk between Flavonoids and the Plant Circadian Clock

“…suggested by our recent report that CHS interacts with MOS9, a nuclear protein required for defense gene expression with no known function in flavonoid metabolism (Watkinson et al, 2018).…”

“…Flavonoid enzymes have also been reported to localize to the nucleus in numerous species, where they may be responsible for the synthesis of flavonoids in situ (Winkel, 2019; Zhang et al, 2020). These proteins may also have alternative functions, as suggested by our recent report that CHS interacts with MOS9, a nuclear protein required for defense gene expression with no known function in flavonoid metabolism (Watkinson et al, 2018).…”