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Abstract Flavonols are a subclass of flavonoids widely found in plants and typically exist in glycosylated forms, decorated with various sugars at different positions on the flavonol aglycone. The composition and abundance of flavonol glycosides vary across species and among tissues within a species. Although flavonols are collectively known for their antioxidant activity, the specific physiological functions of individual flavonol structures remain poorly understood. Here, we show that 2 flavonol glycosides, kaempferol 3-O-glucosyl(1 → 2)galactoside (K2) and quercetin 3-O-glucosyl(1 → 2)galactoside (Q2), predominantly accumulate in the pollen of Solanaceae plants. K2 is evolutionarily conserved across Solanaceae, while Q2 has been lost in species such as tomato (Solanum lycopersicum). Our transcriptome profiling and biochemical analysis revealed SlUGT78D-B (78-B) as a pollen-specific flavonol 3-O-galactosyltransferase responsible for K2 production in tomato. Disruption of 78-B abolished K2 accumulation, leading to defective pollen tube growth in our in vitro assays. Supplementation with kaempferol 3-O-galactoside (K2 precursor) restores pollen tube growth, whereas quercetin 3-O-galactoside (Q2 precursor) or flavonol aglycones do not, suggesting distinct roles for individual flavonol structures. We further show that 3 key amino acid residues of 78-B dictate its sugar specificity, favoring galactosylation over glucosylation. Substitution of any one of these residues enables 78-B to acquire glucosyltransferase activity. However, 78-B remains evolutionarily constrained from gaining this activity, suggesting selective pressure to maintain flavonol galactoside accumulation in pollen. These findings indicate that individual flavonol glycosides can have specific physiological roles beyond enhancing solubility and stability. 

Abstract Prenylated quinones are membrane-associated metabolites that serve as vital electron carriers for respiration and photosynthesis. The UbiE (EC 2.1.1.201)/MenG (EC 2.1.1.163) C-methyltransferases catalyze pivotal ring methylations in the biosynthetic pathways of many of these quinones. In a puzzling evolutionary pattern, prokaryotic and eukaryotic UbiE/MenG homologs segregate into 2 clades. Clade 1 members occur universally in prokaryotes and eukaryotes, excluding cyanobacteria, and include mitochondrial COQ5 enzymes required for ubiquinone biosynthesis; Clade 2 members are specific to cyanobacteria and plastids. Functional complementation of an Escherichia coli ubiE/menG mutant indicated that Clade 1 members display activity with both demethylbenzoquinols and demethylnaphthoquinols, independently of the quinone profile of their original taxa, while Clade 2 members have evolved strict substrate specificity for demethylnaphthoquinols. Expression of the gene-encoding bifunctional Arabidopsis (Arabidopsis thaliana) COQ5 in the cyanobacterium Synechocystis or its retargeting to Arabidopsis plastids resulted in synthesis of a methylated variant of plastoquinone-9 that does not occur in nature. Accumulation of methylplastoquinone-9 was acutely cytotoxic, leading to the emergence of suppressor mutations in Synechocystis and seedling lethality in Arabidopsis. These data demonstrate that in cyanobacteria and plastids, co-occurrence of phylloquinone and plastoquinone-9 has driven the evolution of monofunctional demethylnaphthoquinol methyltransferases and explains why plants cannot capture the intrinsic bifunctionality of UbiE/MenG to simultaneously synthesize their respiratory and photosynthetic quinones. 

Growing vegetables in controlled environments (CEs), such as hydroponics, aquaponics, and vertical structures, is a rapidly expanding industry in Florida and the United States, especially in nearby urban areas. Although lettuce ( Lactuca sativa ) is still mostly produced in fields, growing in CEs proximal to urban areas has become increasingly popular because it may facilitate reduced transportation time and associated postharvest degradation. Lettuce is among the top-most consumed vegetables in the United States and could provide some of the nutrition missing in the US diet. This research was planned to understand the levels of some vitamins that are key for human health, including vitamin E (tocopherols), vitamin K 1 (phylloquinone), and vitamin C (ascorbic acid), in lettuce grown in greenhouse hydroponics. Lettuce germplasm was grown using the hydroponic nutrient film technique system in three greenhouse experiments: at the beginning, middle, and end of the Florida, USA, growing season (from Aug 2020 to Mar 2021). Genetic variation for these vitamins were found among the germplasm tested in the four morphological types of lettuce, romaine, Boston, Latin, and leaf. In addition, a sugar analysis was conducted in this germplasm, of which fructose was the most abundant sugar. A significant genotype × environment (G × E) interaction was observed, indicating that the levels of these compounds, especially vitamins, was environment dependent. However, the presence of certain non-crossover G × E interactions indicates that selecting lettuce in a representative environment could result in new cultivars with higher vitamin content. This research marks the initial steps to improve lettuce for these vitamins, which can contribute to better health of US consumers, not for the highest amount of these compounds in lettuce but for the offset due to its high consumption. 

Plants have evolved the ability to derive the benzenoid moiety of the respiratory cofactor and antioxidant, ubiquinone (coenzyme Q), either from the β-oxidative metabolism of p-coumarate or from the peroxidative cleavage of kaempferol. Here, isotopic feeding assays, gene co-expression analysis and reverse genetics identified Arabidopsis 4-COUMARATE-COA LIGASE 8 (4-CL8; At5g38120) as a contributor to the β-oxidation of p-coumarate for ubiquinone biosynthesis. The enzyme is part of the same clade (V) of acyl-activating enzymes than At4g19010, a p-coumarate CoA ligase known to play a central role in the conversion of p-coumarate into 4-hydroxybenzoate. A 4-cl8 T-DNA knockout displayed a 20% decrease in ubiquinone content compared with wild-type plants, while 4-CL8 overexpression boosted ubiquinone content up to 150% of the control level. Similarly, the isotopic enrichment of ubiquinone's ring was decreased by 28% in the 4-cl8 knockout as compared with wild-type controls when Phe-[Ring-13C6] was fed to the plants. This metabolic blockage could be bypassed via the exogenous supply of 4-hydroxybenzoate, the product of p-coumarate β-oxidation. Arabidopsis 4-CL8 displays a canonical peroxisomal targeting sequence type 1, and confocal microscopy experiments using fused fluorescent reporters demonstrated that this enzyme is imported into peroxisomes. Time course feeding assays using Phe-[Ring-13C6] in a series of Arabidopsis single and double knockouts blocked in the β-oxidative metabolism of p-coumarate (4-cl8; at4g19010; at4g19010 × 4-cl8), flavonol biosynthesis (flavanone-3-hydroxylase), or both (at4g19010 × flavanone-3-hydroxylase) indicated that continuous high light treatments (500 µE m−2 s−1; 24 h) markedly stimulated the de novo biosynthesis of ubiquinone independently of kaempferol catabolism.