It happened again: convergent evolution of acylglucose specialized metabolism in black nightshade and wild tomato

Abstract

Plants synthesize myriad phylogenetically-restricted specialized (aka secondary) metabolites with diverse structures. Metabolism of acylated sugar esters in epidermal glandular secreting trichomes across the Solanaceae (nightshade) family are ideal for investigating the mechanisms of evolutionary metabolic diversification. We developed methods to structurally analyze acylhexose mixtures by 2D NMR, which led to the insight that the Old World species black nightshade (Solanum nigrum) accumulates acylglucoses and acylinositols in the same tissue. Detailed in vitro biochemistry - cross validated by in vivo virus induced gene silencing - revealed two unique features of the four-step acylglucose biosynthetic pathway: a trichome-expressed, neofunctionalized invertase-like enzyme, SnASFF1, converts BAHD-produced acylsucroses to acylglucoses, which in turn are substrates for the first-reported acylglucose acyltransferase, SnAGAT1. This biosynthetic pathway evolved independently from that recently described in the wild tomato S. pennellii, reinforcing that acylsugar biosynthesis is evolutionarily dynamic with independent examples of primary metabolic enzyme cooption and additional variation in BAHD acyltransferases.

Natural Variation Meets Synthetic Biology: Promiscuous Trichome Expressed Acyltransferases from Nicotiana acuminata

“…In the sequential assay with NacASAT1 added first, both mono and diacylated products were observed (Supplemental Figure S18b). In contrast, when SlASAT1 was added first, only mono-acylated products were produced and multiple peaks were identified, likely due to the previously reported (Fan et al, 2016; Leong et al, 2019; Lou et al, 2021) heat and base-promoted non-enzymatic rearrangement of the 4-position monoacyl product (Supplemental Figure S18b). These assays suggest that SlASAT1, but not NacASAT1, can acylate S1:6 R2 in vitro .…”

“…This result is consistent with the hypothesis that NacASAT4 acetylates the 6′ position of tetraacylsucroses. To determine if NacASAT4 could acetylate triacylsucroses, we took advantage of the observation that acyltransferase enzymes catalyze the reverse reaction in the presence of free CoA (Leong et al, 2020; Lou et al, 2021); indeed NacASAT4 deacetylated S4:16(2 R3′ ,4,5d,5d) at the 3′ position to produce an S3:14 product (Supplemental Figure S11c). These results are consistent with the hypothesis that NacASAT4 acts as an acetyltransferase that can perform consecutive reactions converting S3:14(4,5d,5d) into S5:18(2 R3′ ,2 R6′ ,4,5d,5d).…”

“…A leaf was placed into 1mL of extraction solvent (3:3:2 acetonitrile:isopropanol:water + 0.1% formic acid + either 10 μM propyl 4-hydroxybenzoate or 1 μM telmisartan internal standard) and gently mixed for 2 minutes. Acylsugar extracts (10 μL) were run on an Ascentis Express C18 HPLC column (100 mm x 2.1 mm, 2.7 μm) as described previously (Leong et al, 2019; Fan et al, 2020; Lou et al, 2021). Differences in LC gradients and mass spectrometer parameters are noted below and in Supplemental Table S2.…”

“…In the cultivated tomato S. lycopersicum M82, trichome-enriched acylsugar acyltransferases (ASATs) sequentially add four acyl chains at specific positions on a sucrose core; these are named SlASAT1-SlASAT4 reflecting their order in the pathway (Schilmiller et al, 2012a; Schilmiller et al, 2015; Fan et al, 2016). Comparative transcriptomics coupled with phylogenetics, in vitro biochemistry and in planta gene silencing studies identified homologous ASATs from species across the Solanaceae with functions reminiscent of those from tomato (Moghe et al, 2017; Nadakuduti et al, 2017; Leong et al, 2020; Lou et al, 2021). However, differences in acylation order (Fan et al, 2017) and ASAT gene loss/gain events (Moghe et al, 2017) contribute to distinct biosynthetic pathways leading to acylsugar structural variation across the Solanaceae.…”

“…While acylsugars with sucrose cores are common across the Solanaceae, other sugar core types are found. Acylglucoses accumulate in some genera including Solanum and Nicotiana (Matsuzaki et al, 1989; Leong et al, 2019; Lou et al, 2021) and inositol-based esters have also been detected in the Solanum (Herrera-Salgado et al, 2005; Leong et al, 2020; Lou et al, 2021).…”