Plants produce a myriad of taxonomically restricted specialized metabolites. This diversity—and our ability to correlate genotype with phenotype—makes the evolution of these ecologically and medicinally important compounds interesting and experimentally tractable. Trichomes of tomato and other nightshade family plants produce structurally diverse protective compounds termed acylsugars. While cultivated tomato (Solanum lycopersicum) strictly accumulates acylsucroses, the South American wild relative Solanum pennellii produces copious amounts of acylglucoses. Genetic, transgenic, and biochemical dissection of the S. pennellii acylglucose biosynthetic pathway identified a trichome gland cell–expressed invertase-like enzyme that hydrolyzes acylsucroses (Sopen03g040490). This enzyme acts on the pyranose ring–acylated acylsucroses found in the wild tomato but not on the furanose ring–decorated acylsucroses of cultivated tomato. These results show that modification of the core acylsucrose biosynthetic pathway leading to loss of furanose ring acylation set the stage for co-option of a general metabolic enzyme to produce a new class of protective compounds.

Acylsugars constitute an abundant class of pest- and pathogen-protective Solanaceae family plant-specialized metabolites produced in secretory glandular trichomes. Solanum pennellii produces copious triacylated sucrose and glucose esters, and the core biosynthetic pathway producing these compounds was previously characterized. We performed untargeted metabolomic analysis of S. pennellii surface metabolites from accessions spanning the species range, which indicated geographic trends in the acylsugar profile and revealed two compound classes previously undescribed from this species, tetraacylglucoses and flavonoid aglycones. A combination of ultrahigh-performance liquid chromatography–high resolution mass spectrometry (UHPLC–HR-MS) and NMR spectroscopy identified variations in the number, length, and branching pattern of acyl chains, and the proportion of sugar cores in acylsugars among accessions. The new dimensions of acylsugar variation revealed by this analysis further indicate variation in the biosynthetic and degradative pathways responsible for acylsugar accumulation. These findings provide a starting point for deeper investigation of acylsugar biosynthesis, an understanding of which can be exploited through crop breeding or metabolic engineering strategies to improve the endogenous defenses of crop plants.

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24Plants produce myriad taxonomically restricted specialized metabolites. This diversity -and our 25 ability to correlate genotype with phenotype -makes the evolution of these ecologically and 26 medicinally important compounds interesting and experimentally tractable. Trichomes of tomato 27 and other nightshade family plants produce structurally diverse protective compounds termed 28 acylsugars. While cultivated tomato (Solanum lycopersicum) accumulates strictly acylsucroses, 29 the South American wild relative Solanum pennellii produces copious amounts of acylglucoses. 30 Genetic, transgenic and biochemical dissection of the S. pennellii acylglucose biosynthetic 31 pathway identified a trichome gland cell expressed invertase-like enzyme that hydrolyzes 32 acylsucroses (Sopen03g040490). This enzyme acts on the pyranose ring-acylated acylsucroses 33 Science Advances Manuscript TemplatePage 2 of 50 found in the wild tomato but not the furanose ring-decorated acylsucroses of cultivated tomato. 34 These results show that modification of the core acylsucrose biosynthetic pathway leading to loss 35 of furanose ring acylation set the stage for co-option of a general metabolic enzyme to produce a 36 new class of protective compounds. 37 38 48 This leads to new and modified pathways that produce structurally and functionally diverse 49 specialized metabolites. 50 Glandular trichome-synthesized acylated sugars ('acylsugars') are structurally diverse 51 specialized metabolites found throughout the Solanaceae (7-13). These compounds have 52 documented roles in direct and indirect protection against herbivores and microbes (14, 15), as 53 well as allelopathic properties (15, 16). Their low toxicity to vertebrates generates interest in 54 generating plant breeding strategies for deploying acylsugars in crop protection (17, 18). These 55 metabolites consist of a sugar core -typically sucrose -with aliphatic chains of variable length, 56 structure and number attached by ester linkages. Acylsugars were reported from genera across the 57 Solanaceae family, including Datura, Nicotiana, Petunia, Physalis, Salpiglossis, and Solanum 58 Science Advances Manuscript Template Page 3 of 50with single species producing at least three dozen chromatographically distinct acylsugars (10, 13, 59 16, 19-22). 60 In recent years, several evolutionarily-related enzymes were implicated in the core 61 acylsucrose biosynthetic pathways in species across the family, including the cultivated tomato 62 Solanum lycopersicum, Petunia axillaris and Salpiglossis sinuata (7,(9)(10)(11)(12) 23). These 63 biosynthetic pathways consist of trichome-expressed BAHD-family acylsugar acyltransferases 64 (ASATs) (7, 9, 23), which sequentially transfer acyl groups from acyl-coenzyme A (acyl-CoA) 65 substrates to specific hydroxyl groups of sucrose (7, 9, 23). 66 The cultivated tomato biosynthetic network is well characterized, with four ASATs -67 SlASAT1 through SlASAT4 -catalyzing consecutive reactions to produce tri-and tetra-acylate...

The central regulator of the Wnt/β-catenin pathway is the Axin/APC/GSK3β destruction complex (DC), which in unstimulated conditions targets cytoplasmic β-catenin for degradation. How Wnt activation inhibits the DC to permit β-catenin-dependent signaling remains controversial, in part because the DC and its regulation have never been observed in vivo. Using Bimolecular Fluorescence Complementation (BiFC) methods, we have now analyzed the activity of the DC under near-physiological conditions in Drosophila. By focusing on well-established patterns of Wnt/Wg signaling in the developing Drosophila wing, we have defined the sequence of events by which activated Wnt receptors induce a conformational change within the DC, resulting in modified Axin-GSK3β interactions that prevent β-catenin degradation. Surprisingly, the nucleus is surrounded by active DCs, which principally control β-catenin's degradation and thereby nuclear access. These DCs are inactivated and removed upon Wnt signal transduction. These results suggest a novel mechanistic model for dynamic Wnt signaling transduction in vivo.