Physical interactions among flavonoid enzymes in snapdragon and torenia reveal the diversity in the flavonoid metabolon organization of different plant species

Abstract

Flavonoid metabolons (weakly-bound multi-enzyme complexes of flavonoid enzymes) are believed to occur in diverse plant species. However, how flavonoid enzymes are organized to form a metabolon is unknown for most plant species. We analyzed the physical interaction partnerships of the flavonoid enzymes from two lamiales plants (snapdragon and torenia) that produce flavones and anthocyanins. In snapdragon, protein-protein interaction assays using yeast and plant systems revealed the following binary interactions: flavone synthase II (FNSII)/chalcone synthase (CHS); FNSII/chalcone isomerase (CHI); FNSII/dihydroflavonol 4-reductase (DFR); CHS/CHI; CHI/DFR; and flavonoid 3'-hydroxylase/CHI. These results along with the subcellular localizations and membrane associations of snapdragon flavonoid enzymes suggested that FNSII serves as a component of the flavonoid metabolon tethered to the endoplasmic reticulum (ER). The observed interaction partnerships and temporal gene expression patterns of flavonoid enzymes in red snapdragon petal cells suggested the flower stage-dependent formation of the flavonoid metabolon, which accounted for the sequential flavone and anthocyanin accumulation patterns therein. We also identified interactions between FNSII and other flavonoid enzymes in torenia, in which the co-suppression of FNSII expression was previously reported to diminish petal anthocyanin contents. The observed physical interactions among flavonoid enzymes of these plant species provided further evidence supporting the long-suspected organization of flavonoid metabolons as enzyme complexes tethered to the ER via cytochrome P450, and illustrated how flavonoid metabolons mediate flower coloration. Moreover, the observed interaction partnerships were distinct from those previously identified in other plant species (Arabidopsis thaliana and soybean), suggesting that the organization of flavonoid metabolons may differ among plant species.

Involvement of chalcone reductase in the soybean isoflavone metabolon: identification of GmCHR5, which interacts with 2‐hydroxyisoflavanone synthase

“…Confocal laser microscopy images of the transformed N . benthamiana epidermal cells revealed mVenus fluorescence signals in the cytoplasm and nucleus for all examined GmCHR‐mVenus proteins (Figure S6), as previously observed for the non‐tagged mVenus protein (Fujino et al ., ). These signals almost overlapped with the signals from the non‐tagged mTurquoise2 protein (Figure S6).…”

The Structure and Function of Major Plant Metabolite Modifications

“…Most enzymes involved in flavonoid synthesis alongside related biosynthetic enzymes are contained in multiple enzyme complexes located on the cytosolic side of the endoplasmic reticulum (ER), including cinnamate 4-hydroxylase, flavonoid 3 0 -hydroxylase, and flavonoid 3 0 ,5 0 -hydroxylase (Winkel, 2004;Jorgensen et al, 2005;Fujino et al, 2018). In planta, flavonoids are present in the cytosol, vacuole, ER, chloroplast, nucleus, and small vesicles, as well as the extracellular space.…”

On the role of the tricarboxylic acid cycle in plant productivity

“…It has been proposed that metabolons may improve the efficiency of metabolic pathways, by increasing the concentration of intermediates, decreasing the enzyme concentration, directing the products of a pathway to a specific subcellular location, or minimizing the escape of reactive intermediates (Jorgensen et al ). Several metabolons have been proposed to mediate substrate channelling in diverse organisms; for instance, branched chain amino‐acid metabolism in human mitochondria (Islam et al ), the glycolytic pathways of mammals, yeast and plants (Al‐Habori ; Brandina et al ; Graham et al ), and a wide variety of specialized metabolic pathways, including polyamine (Panicot et al ), isoprenoid (Leivar et al ), alkaloid (Winzer et al ), phenylpropanoid (Achnine et al ) and cyanogenic glucoside (Bassard et al ; Fujino et al ) biosynthesis in plants. That being said, experimental evidence for these is often rather fragmentary, and the experimental manipulation of the metabolon remains relatively scarce.…”