In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network

Abstract

Plant glandular secreting trichomes are epidermal protuberances that produce structurally diverse specialized metabolites, including medically important compounds. Trichomes of many plants in the nightshade family (Solanaceae) produce O-acylsugars, and in cultivated and wild tomatoes these are mixtures of aliphatic esters of sucrose and glucose of varying structures and quantities documented to contribute to insect defense. We characterized the first two enzymes of acylsucrose biosynthesis in the cultivated tomato Solanum lycopersicum. These are type I/IV trichome-expressed BAHD acyltransferases encoded by Solyc12g006330─or S. lycopersicum acylsucrose acyltransferase 1 (Sl-ASAT1)─and Solyc04g012020 (Sl-ASAT2). These enzymes were used-in concert with two previously identified BAHD acyltransferases-to reconstruct the entire cultivated tomato acylsucrose biosynthetic pathway in vitro using sucrose and acyl-CoA substrates.Comparative genomics and biochemical analysis of ASAT enzymes were combined with in vitro mutagenesis to identify amino acids that influence CoA ester substrate specificity and contribute to differences in types of acylsucroses that accumulate in cultivated and wild tomato species. This work demonstrates the feasibility of the metabolic engineering of these insecticidal metabolites in plants and microbes.Solanum | glandular trichomes | acylsugar | specialized metabolism | genotype to phenotype P lants are masters of metabolism, producing hundreds of thousands of small molecules known as specialized metabolites, which vary widely in structure, abundance, and physical and biological properties. These metabolites tend to be produced by enzymes that evolve faster than those that produce "central" metabolites such as amino acids, nucleotides, sugars, and cofactors (1-3), and the pathways and metabolic intermediates involved in biosynthesis of many specialized metabolites remain mysterious. Despite the growing availability of genomic DNA sequences, understanding the genetic and biochemical mechanisms that contribute to this phenotypic diversity and plasticity presents enduring and major challenges in plant biochemistry. It is of great interest to understand and manipulate the biosynthesis of these biologically active molecules.Specialized metabolites typically are produced in a cell-or tissue-specific manner and are generally limited in their taxonomic distribution. Glandular secreting trichomes provide an example of such a differentiated structure; these epidermal "hairs" produce a variety of metabolites of importance to humans, including aromatic flavor components (e.g., in hops for beer and Mediterranean herbs for cooking), psychoactive cannabinoids in Cannabis, and the antimalarial drug artemisinin in Artemisia annua (4, 5).Some trichome-produced metabolites have documented direct and indirect antiherbivore activities (4, 6-8). For example, acylsugars are a group of structurally related specialized metabolites produced in plants of the nightshade family-the Solanaceae (9, 10). Characterized examples ...

Evolutionarily Distinct BAHD N-acyltransferases are Responsible for Natural Variation of Aromatic Amine Conjugates in Rice

“…Here, comparative sequence analysis with in vitro assays using mutant enzymes led to the identification of a RRRR motif that is sufficient to transform agmatine utilization ( Figures 9A and 9B), which is similar to the conserved arginine-rich motifs in RNA binding proteins (Lazinski et al, 1989;Zapp et al, 1991;Yuryev et al, 1996). This integrated approach has also been applied to identify key residues of enzymes involved in coumaroyl serotonin, terpene, and acylsucrose biosynthesis (Kang et al, 2006;Kang et al, 2014;Fan et al, 2016) and may be regarded as a general strategy in the structurefunction study of enzymes.…”

Characterization of Trichome-Expressed BAHD Acyltransferases in Petunia axillaris Reveals Distinct Acylsugar Assembly Mechanisms within the Solanaceae

“…However, a combination of metabolite profiling of introgression lines of tomato, coupled with positional cloning and RNA interference screens of trichome-expressed genes, identified four acylsugar acyltransferases (SlASAT1-SlASAT4) that belong to the BAHD acyltransferase superfamily (Schilmiller et al, 2012(Schilmiller et al, , 2015Fan et al, 2016). Biochemical characterization revealed that these ASATs sequentially catalyze the esterification of acyl chains at the R4, R3, R39, and R2 positions of Suc to generate a tetra-acylsucrose, and their activities with different substrates are sufficient to explain the structures of S. lycopersicum acylsugars ( Fig.…”

“…Biochemical characterization revealed that these ASATs sequentially catalyze the esterification of acyl chains at the R4, R3, R39, and R2 positions of Suc to generate a tetra-acylsucrose, and their activities with different substrates are sufficient to explain the structures of S. lycopersicum acylsugars ( Fig. 1A; Ghosh et al, 2014;Fan et al, 2016). Furthermore, a combination of gene duplication, gene loss, and allelic variation at the asat2, asat3, and asat4 loci determines much of the chemical diversity observed in the wild tomato species S. habrochaites and S. pennellii Schilmiller et al, 2012Schilmiller et al, , 2015Fan et al, 2016).…”

“…1A; Ghosh et al, 2014;Fan et al, 2016). Furthermore, a combination of gene duplication, gene loss, and allelic variation at the asat2, asat3, and asat4 loci determines much of the chemical diversity observed in the wild tomato species S. habrochaites and S. pennellii Schilmiller et al, 2012Schilmiller et al, , 2015Fan et al, 2016).…”

“…Details of the length of the acyl chains typically found at each position in the most abundant metabolites are provided together with the identity of the ASAT enzymes responsible for acylsucrose assembly and the order in which they catalyze acylation reactions. Data represented are summarized from previous reports (Schilmiller et al, 2012(Schilmiller et al, , 2015Ghosh et al, 2014;Fan et al, 2016). B, Structures of characteristic acylsucroses from P. axillaris.…”