Apicomplexan parasites like Toxoplasma gondii harbor a highly divergent mitochondrial proteome, much of which remains uncharacterized despite its essentiality for parasite survival. One such critical pathway is ubiquinone (UQ) biosynthesis. Here, we characterize the UQ synthesis machinery in T. gondii and show that conserved enzymes, TgCoq3 and TgCoq5, are essential for growth and mitochondrial function, forming a multi-protein complex. Using proximity labeling and subcellular fractionation, a strategy suited for detecting proteins of low abundance, we identify TgCoqFAD, a unique FAD-dependent monooxygenase required for UQ synthesis. Unlike canonical eukaryotic systems that employ multiple monooxygenases to modify specific carbons on the UQ aromatic ring, TgCoqFAD catalyzes two distinct hydroxylation steps, an activity not previously reported in eukaryotes. Molecular docking and chemical screening identified TgCoqFAD inhibitors that impair tachyzoite growth and bradyzoite viability. These findings reveal a streamlined and divergent UQ biosynthesis pathway in apicomplexans and establish TgCoqFAD as a promising antiparasitic target.

SUMMARY An unbiased, quantitative view of biomolecules in a living cell is a prerequisite for accurate modeling approaches and informs our understanding of cellular metabolism at scale. In this work, we used the total protein approach (TPA), in which the total protein mass of a given proteomics sample is used as a calibrator for absolute protein quantification, to determine protein abundances during the Chlamydomonas reinhardtii diurnal cycle. We use external, independently measured quantitative markers (metals, pigments) to assess the absolute protein abundances in unlabeled whole cell extracts. We calculate protein abundances in fg / cell of 7322 Chlamydomonas proteins, 2266 of which were captured in every time point, including the major proteins involved in the light reactions, photoprotection, proteostasis and fatty acid metabolism during a cell cycle. As expected, Rubisco large and small subunits are present in a 1:1 stoichiometry, with the large subunit being the most abundant protein in our data set, averaging 5.05 × 10 6 molecules per cell, reflecting 2.7% of the total protein mass. We noticed that PSII is the most abundant complex involved in the light reactions with 2.08 × 10 6 complexes per cell. PSI averages 1.75 × 10 6 complexes per cell and cytochrome b 6 f averages 0.77 × 10 6 complexes per cell. The TPA is a robust tool to study proteome dynamics quantitatively, while avoiding artefacts due to biochemical fractionation. Our proteome data set with an unprecedented temporal resolution is a valuable resource to assess protein abundances during the cell cycle in the reference alga Chlamydomonas.

Maize (Zea mays) produces terpenoid-based chemical defenses through a large family of terpene synthases, but the contributions of individual enzymes to specific compounds and stress resistance remain difficult to predict. Maize terpene synthase 8 (ZmTPS8) produces multiple sesquiterpenes in heterologous systems, but its in planta function remains unknown. We integrated a metabolite genome-wide association study (mGWAS), CRISPR/Cas9 generated tps8 loss-of-function mutants, metabolite profiling, and biotic stress assays to define ZmTPS8's role in terpene synthesis and biotic stress responses. The mGWAS identified ZmTPS8 as the primary locus associated with herbivore-induced emission of the sesquiterpene volatile germacrene D. Consistently, ZmTPS8 expression was induced by foliar and root herbivory, and tps8 mutants exhibited reduced emission of germacrene D, α-copaene, and δ-cadinene during Spodoptera frugiperda feeding. Loss of ZmTPS8 increased S. frugiperda larval growth but did not affect the belowground herbivore Diabrotica virgifera virgifera. ZmTPS8 also contributed to resistance against sugarcane mosaic virus, and the fungal pathogen Fusarium verticillioides, affecting terpenoid profiles, global metabolism, and fungal toxin production, but had no impact on Cochliobolus heterostrophus or Pythium spp. susceptibility. Together, these results demonstrate that ZmTPS8 contributes to maize defense in a threat-dependent manner, shaping volatile emissions and defense outcomes.

Herbivore-induced plant volatiles act as danger signals to prime defense responses in neighboring plants, yet in many cases the mechanism behind this priming is not known. Volatile signals may be recognized directly by receptors and/or converted into other active compounds. Here we investigate the metabolic fate of volatile indole, a known priming signal in maize (Zea mays), to determine if its conversion to other compounds could play a role in its priming of defenses. We identified benzoxazinoids as major products from volatile indole using heavy isotope-labeled volatile indole and Pathway of Origin Determination in Untargeted Metabolomics (PODIUM) analysis. We then used benzoxazinoid biosynthesis maize mutants to investigate their role in indole-mediated priming. Labeled volatile indole was converted into DIMBOA-glucoside in a bx2 (benzoxazinone synthesis2)-dependent manner. The bx2 mutant plants showed elevated green leaf volatile (GLV) production in response to wounding and Spodoptera frugiperda regurgitant irrespective of indole exposure. Thus, volatile indole is converted into benzoxazinoids, and part of its priming mechanism may be due to the enhanced production of these phytoanticipins. However, indole-mediated enhanced GLV production does not rely on the conversion of indole to benzoxazinoids, so indole also has other signaling functions.

Maize coumarate-CoA ligase gene expression is differentially regulated by different biotic stresses, and Zm4CL5 is induced by fall armyworm herbivory and impacts plant growth. The phenylpropanoid pathway is important for the synthesis of numerous compounds involved in plant growth and stress resilience, including lignin and flavonoids. The last common step in the core phenylpropanoid pathway is the enzyme 4-coumarate:CoA ligase (4CL). Maize (Zea mays) has five 4CL genes, four of which (Zm4CL1-4) are type I (lignin-associated 4CLs), while one (Zm4CL5) is a type II (non-lignin-associated 4CL). In maize, it is not known which, if any, 4CL genes are involved in plant defense. Here we show that infection with the fungal pathogen Cochliobolus heterostrophus alters the expression of the type I genes, while herbivory by Spodoptera frugiperda induces the type II gene Zm4CL5. A knock-down mutant of Zm4CL5 displayed altered accumulation of flavonoids and other non-lignin phenylpropanoids, as well as increased plant height, revealing a role for Zm4CL5 both in plant growth and in non-lignin phenylpropanoid production. These data indicate that 4CL genes in maize play differential roles in growth and in the defense against different biotic stressors.

Plants have evolved the ability to respond to a diverse range of biotic and abiotic stresses. Often, combining these stresses multiplies the challenge for the plants, but occasionally the combined stress can induce unexpected synergistic defences. In maize, combined flooding and herbivory induces a salicylic acid (SA)-dependent defence against Spodoptera frugiperda (fall armyworm). In this study we used RNAseq and metabolic profiling to show that flavonoids are involved in maize response to combined flooding and herbivory. To assess the role of flavonoids in flood-induced S. frugiperda resistance, we analyzed the maize idf mutant that has compromised expression of chalcone synthase, the first enzyme in flavonoid biosynthesis. This flavonoid-deficient mutant was compromised both in flood-induced S. frugiperda resistance and in SA accumulation. These data revealed an unexpected requirement for flavonoids in SA induction. In contrast to idf, the flavonoid 3' hydroxylase mutant, pr1, showed enhanced SA accumulation after combinatorial treatment, which closely correlated with elevated levels of select flavonoids and the dihydroflavonol reductase, anthocyaninless1 (a1) mutant, was unaffected in its SA-induction. These data indicate that specific flavonoids likely play a role in flood-induced SA accumulation and S. frugiperda resistance.

Methyl salicylate (MeSA) is a component of the herbivore-induced volatile blend of tomato (Solanum lycopersicum), and an intermediate in long distance salicylic acid (SA) signaling. To investigate the role of MeSA in interactions between tomato and the lepidopteran pests Helicoverpa zea and Manduca sexta, we used transgenic tomato lines that were antisense (AS) or overexpressors (OE) of the tomato SA carboxyl methyl transferase SlSAMT1, which under- or over-produce MeSA, respectively. Transgenic AS plants produced less MeSA and were more susceptible to both insects compared to wild-type plants, while OE plants had elevated MeSA production but displayed increased resistance only to M. sexta. Pairwise choice and toxicity assays revealed that MeSA itself impacted the larvae as a deterrent rather than a toxin, and metabolic profiling indicated that increased susceptibility in the AS lines and resistance in the OE lines may be related to altered jasmonic acid and ABA mediated herbivore-induced defense signaling, and modified production of inducible defense compounds such as flavonoids and phenolamides. To expand beyond Lepidoptera, the settling preference of the piercing-sucking insect Bemisia tabaci was also examined and it displayed a strong preference for lines with less SlSAMT1 expression. These data indicate that herbivore-inducible SlSAMT1 expression and its associated MeSA production play an important and complex role in the defense of tomato against insect pests.

Cyanobacteria and plastids harbor two prenylated quinones that serve as vital photosynthetic cofactors. One is a naphthoquinone (phylloquinone or menaquinone depending on the species), the naphthalene ring of which is always methylated in ortho of the prenyl chain. The other one is a benzoquinone, called plastoquinone-9, the benzenoid ring of which is, in contrast, never methylated at this position. In oxygenic phototrophs, such an arrangement is thought to have driven the evolution and retention of unique quinol C-methyltransferases that act exclusively on naphthoquinol substrates. Here, we identified quinol C-methyltransferases in two extant taxa of early cyanobacterial lineages, Gloeobacter violaceus and Synechococcus sp. JA-2-3B’a, that did not discriminate between naphthoquinol and benzoquinol substrates when these enzymes were expressed in Escherichia coli. Quinone analysis showed, however, that G. violaceus extracts did not contain any detectable amounts of methyl-plastoquinone-9. Furthermore, functional complementation assays in the cyanobacterium Synechocystis sp. PCC 6803 revealed that G. violaceus and S. sp. JA-2-3B’a quinol C-methyltransferases displayed either strict or marked substrate preference for demethyl-phylloquinol. Taken together, these data suggest that G. violaceus and S. sp. JA-2-3B’a quinol C-methyltransferases are the remnants of a promiscuous enzyme present during the emergence of plastoquinone as a photosynthetic electron carrier.

Plant-derived volatiles are important mediators of plant-insect interactions as they can provide cues for host location and quality, or act as direct or indirect defense molecules. The volatiles produced by Zea mays (maize) include a range of terpenes, likely produced by several of the terpene synthases (TPS) present in maize. Determining the roles of specific terpene volatiles and individual TPSs in maize-insect interactions is challenging due to the promiscuous nature of TPSs in vitro and their potential for functional redundancy. In this study, we used metabolite GWAS of a sweetcorn diversity panel infested with Spodoptera frugiperda (fall armyworm) to identify genetic correlations between TPSs and individual volatiles. This analysis revealed a correlation between maize terpene synthase 1 (ZmTPS1) and emission of the monoterpene volatiles linalool and β-myrcene. Electroantennogram assays showed gravid S. frugiperda could detect both linalool and β-myrcene. Quantification of headspace volatiles in a maize tps1 loss-of-function mutant confirmed that ZmTPS1 is an important contributor to linalool and β-myrcene emission in maize. Furthermore, pairwise choice assays between tps1 mutant and wild-type plants showed that ZmTPS1, and by extension its volatile products, aid host location in the chewing insect S. frugiperda, yet repel the sap-sucking pest, Rhopalosiphum maidis (corn leaf aphid). On the other hand, ZmTPS1 had no impact on indirect defense via the recruitment of the parasitoid Cotesia marginiventris. ZmTPS1 is therefore an important mediator of the interactions between maize and its insect pests.

The contribution of p-coumarate β-oxidation and kaempferol cleavage to the pools of glycosylated, free and cell wall-bound 4-hydroxybenzoate is organ-dependent in Arabidopsis. 4-Hydroxybenzoate (4-HB) is a vital precursor for a number of plant primary and specialized metabolites, as well as for the assembly of the plant cell wall. In Arabidopsis, it is known that 4-HB is derived independently from phenylalanine and tyrosine, and that the metabolism of phenylalanine into 4-HB proceeds via at least two biosynthetic routes: the β-oxidation of p-coumarate and the peroxidative cleavage of kaempferol. The precise contribution of these precursors and branches to 4-HB production, however, is not known. Here, we combined isotopic feeding assays, reverse genetics, and quantification of soluble (i.e., free and glycosylated) and cell wall-bound 4-HB to determine the respective contributions of phenylalanine, tyrosine, β-oxidation of p-coumarate, and peroxidative cleavage of kaempferol to 4-HB biosynthesis in Arabidopsis tissues. Over 90

The unprenylated benzoquinones 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone), 2-chloro-1,4-benzoquinone (CBQ), 2,6-dimethyl-1,4-benzoquinone (DMBQ), 2,6-dichloro-1,4-benzoquinone (DCBQ), and 2,6-dimethoxy-1,4-benzoquinone (DMOBQ) were tested as putative antimetabolites of plastoquinone-9, a vital electron and proton carrier of oxygenic phototrophs. Duroquinone and CBQ were the most effective at inhibiting the growth of the cyanobacterium Synechocystis sp. PCC 6803 either in photomixotrophic or photoautotrophic conditions. Duroquinone, a close structural analog of the photosynthetic inhibitor methyl-plastoquinone-9, was found to possess genuine bactericidal activity towards Synechocystis at a concentration as low as 10 mu M, while at the same concentration CBQ acted only as a mild bacteriostat. In contrast, only duroquinone displayed marked cytotoxicity in axenically-grown Arabidopsis, resulting in damages to photosystem II and hindered net CO2 assimilation. Metabolite profiling targeted to photosynthetic cofactors and pigments indicated that in Arabidopsis duroquinone does not directly inhibit plastoquinone-9 biosynthesis. Taken together, these data indicate that duroquinone offers prospects as an algicide and herbicide.

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 K1 (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.

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.

Zea mays (maize) makes phytoalexins such as sesquiterpenoid zealexins, to combat invading pathogens. Zealexins are produced from farnesyl diphosphate in microgram per gram fresh weight quantities. As farnesyl diphosphate is also a precursor for many compounds essential for plant growth, the question arises as to how Z. mays produces high levels of zealexins without negatively affecting vital plant systems. To examine if specific pools of farnesyl diphosphate are made for zealexin synthesis we made CRISPR/Cas9 knockouts of each of the three farnesyl diphosphate synthases (FPS) in Z. mays and examined the resultant impacts on different farnesyl diphosphate-derived metabolites. We found that FPS3 (GRMZM2G098569) produced most of the farnesyl diphosphate for zealexins, while FPS1 (GRMZM2G168681) made most of the farnesyl diphosphate for the vital respiratory co-factor ubiquinone. Indeed, fps1 mutants had strong developmental phenotypes such as reduced stature and development of chlorosis. The replication and evolution of the fps gene family in Z. mays enabled it to produce dedicated FPSs for developmentally related ubiquinone production (FPS1) or defense-related zealexin production (FPS3). This partitioning of farnesyl diphosphate production between growth and defense could contribute to the ability of Z. mays to produce high levels of phytoalexins without negatively impacting its growth.

Ubiquinone (coenzyme Q) is a vital respiratory cofactor and liposoluble antioxidant. Studies have shown that plants derive approximately a quarter of 4-hydroxybenzoate, which serves as the direct ring precursor of ubiquinone, from the catabolism of kaempferol. Biochemical and genetic evidence suggests that the release of 4-hydroxybenzoate from kaempferol is catalyzed by heme-dependent peroxidases and that 3-O-glycosylations of kaempferol act as a negative regulator of this process. These findings not only represent an atypical instance of primary metabolite being derived from specialized metabolism but also raise the question as to whether ubiquinone contributes to the ROS scavenging and signaling functions already established for flavonols.

Ubiquinone (Coenzyme Q) is a vital respiratory cofactor and antioxidant in eukaryotes. The recent discovery that kaempferol serves as a precursor for ubiquinone's benzenoid moiety both challenges the conventional view of flavonoids as specialized metabolites, and offers new prospects for engineering ubiquinone in plants. Here, we present evidence that Arabidopsis thaliana mutants lacking kaempferol 3-O-rhamnosyltransferase (ugt78d1) and kaempferol 3-O-glucosyltransferase (ugt78d2) activities display increased de novo biosynthesis of ubiquinone and increased ubiquinone content. These data are congruent with the proposed model that unprotected C-3 hydroxyl of kaempferol triggers the oxidative release of its B-ring as 4-hydroxybenzoate, which in turn is incorporated into ubiquinone. Ubiquinone content in the ugt78d1/ugt78d2 double knockout represented 160% of wild-type level, matching that achieved via exogenous feeding of 4-hydroxybenzoate to wild-type plants. This suggests that 4-hydroxybenzoate is no longer limiting ubiquinone biosynthesis in the ugt78d1/ugt78d2 plants. Evidence is also shown that the glucosylation of 4-hydroxybenzoate as well as the conversion of the immediate precursor of kaempferol, dihydrokaempferol, into dihydroquercetin do not compete with ubiquinone biosynthesis in A. thaliana.

Ubiquinone (Coenzyme Q) is a vital respiratory cofactor and liposoluble antioxidant. In plants, it is not known how the C-6 hydroxylation of demethoxyubiquinone, the penultimate step in ubiquinone biosynthesis, is catalyzed. The combination of cross-species gene network modeling along with mining of embryo-defective mutant databases of Arabidopsis thaliana identified the embryo lethal locus EMB2421 (At1g24340) as a top candidate for the missing plant demethoxyubiquinone hydroxylase. In marked contrast with prototypical eukaryotic demethoxyubiquinone hydroxylases, the catalytic mechanism of which depends on a carboxylate-bridged di-iron domain, At1g24340 is homologous to FAD-dependent oxidoreductases that instead use NAD(P)H as an electron donor. Complementation assays in Saccharomyces cerevisiae and Escherichia coli demonstrated that At1g24340 encodes a functional demethoxyubiquinone hydroxylase and that the enzyme displays strict specificity for the C-6 position of the benzoquinone ring. Laser-scanning confocal microscopy also showed that GFP-tagged At1g24340 is targeted to mitochondria. Silencing of At1g24340 resulted in 40 to 74% decrease in ubiquinone content and de novo ubiquinone biosynthesis. Consistent with the role of At1g24340 as a benzenoid ring modification enzyme, this metabolic blockage could not be bypassed by supplementation with 4-hydroxybenzoate, the immediate precursor of ubiquinone's ring. Unlike in yeast, in Arabidopsis overexpression of demethoxyubiquinone hydroxylase did not boost ubiquinone content. Phylogenetic reconstructions indicated that plant demethoxyubiquinone hydroxylase is most closely related to prokaryotic monooxygenases that act on halogenated aromatics and likely descends from an event of horizontal gene transfer between a green alga and a bacterium.

This new 8-page publication of the UF/IFAS Horticultural Sciences Department explains which nutrients are abundant among the major lettuce types compared to other leafy vegetables, encourages consumers to help satisfy dietary recommendations by eating lettuce at appropriate serving sizes, and outlines the basic growing conditions that help maximize its nutrition as a crop. Written by Jesse J. Murray, Gilles Basset, and German Sandoya.

Prenylated benzoquinones are essential cofactors for the core bioenergetic processes in complex living organisms. Recently, we discovered that the gene product of rquA, a protein first discovered in Rhodospirillum rubrum, can convert ubiquinone to rhodoquinone, a rare prenylated benzoquinone. The presence of rhodoquinone in mitochondria facilitates anaerobic respiration by redirecting electrons in the respiratory chain toward the final electron acceptor fumarate. After reduction by complex I, rhodoquinol delivers electrons via fumarate reductase to fumarate. We predict that the production of rhodoquinone in plant mitochondria will confer the trait of anaerobic respiration providing a novel, synthetic biology approach to design flood tolerant agricultural plants. So far, I have discovered that a codon optimized, mitochondrially targeted RquA, under an inducible dexamethasone promoter, is able to convert ubiquinone-9 to rhodoquinone-9 in Arabidopsis. While this strategy has so far yielded an approximate 1% conversion rate of native ubiquinone to rhodoquinone, there have been no phenotypic impacts observed in plants where synthetic rhodoquinone production is occurring. One important conclusion from these results is that synthetic rhodoquinone production in plants appears to be nonlethal. These findings provide an impetus to continue to explore the synthetic production of rhodoquinone as a strategy for designing flood tolerant crops.