Many solanaceous plants secrete acylsugars, which are branched-chain and straight-chain fatty acids esterified to Glu or Suc. These compounds have important roles in plant defense and potential commercial applications. However, several acylsugar metabolic genes remain unidentified, and little is known about regulation of this pathway. Comparative transcriptomics between low-and high-acylsugar-producing accessions of Solanum pennellii revealed that expression levels of known and novel candidate genes (putatively encoding beta-ketoacyl-(acyl-carrier-protein) synthases, peroxisomal acyl-activating enzymes, ATP binding cassette (ABC) transporters, and central carbon metabolic proteins) were positively correlated with acylsugar accumulation, except two genes previously reported to be involved in acylglucose biosynthesis. Genes putatively encoding oxylipin metabolic proteins, subtilisin-like proteases, and other antimicrobial defense proteins were upregulated in low-acylsugar-producing accessions. Transcriptome analysis after biochemical inhibition of biosynthesis of branched-chain amino acids (precursors to branched-chain fatty acids) by imazapyr showed concentration-dependent downregulation of known and most acylsugar candidate genes, but not defense genes. Weighted gene correlation network analysis identified separate coexpressed gene networks for acylsugar metabolism (including six transcription factor genes and flavonoid metabolic genes) and plant defense (including genes putatively encoding NB-ARC and leucine-rich repeat sequences, protein kinases and defense signaling proteins, and previously mentioned defense proteins). Additionally, virus-induced gene silencing of two trichomes preferentially expressed candidate genes for straight-chain fatty acid biosynthesis confirmed their role in acylsugar metabolism.

1Many plants in the Solanaceae family secrete acylsugars, which are branched-chain and straight-2 chain fatty acids esterified to glucose or sucrose. These compounds have important roles in plant 3 defense and potential commercial applications. However, several acylsugar metabolic genes 4 remain unidentified, and little is known about regulation of this pathway. We used comparative 5 transcriptomic analysis between low-and high-acylsugar-producing accessions of Solanum 6 pennellii and found that expression levels of most acylsugar metabolic genes, including known 7 acylsucrose biosynthetic genes and novel candidate genes (putatively encoding a ketoacyl-ACP 8 synthase IV/II-like enzyme, peroxisomal acyl-activating enzymes, ABC transporters, and central 9 carbon metabolic enzymes), were positively correlated with acylsugar accumulation, except two 10 acylglucose biosynthetic genes. Genes putatively encoding oxylipin metabolic proteins, 11 subtilisin-like proteases, and other antimicrobial defense proteins were upregulated in low-12 acylsugar-producing accessions, possibly to compensate for diminished defense activities of 13 acylsugars. Gene co-expression network analysis clustered most differentially expressed genes 14 into two separate modules and identified genetic networks associated with acylsugar production 15 and plant defense. Transcriptome analysis after inhibition of biosynthesis of branched-chain 16 amino acids (precursors to branched-chain fatty acids) further supported the coordinated 17 regulation of most acylsugar candidate genes and identified three putative AP2-family 18 transcription factor genes that form a strong co-expression network with many acylsugar 19 metabolic genes. 20 21 22 23 24 25 26 27

Acylsugars, specialized metabolites with defense activities, are secreted by trichomes of many solanaceous plants. Several acylsugar metabolic genes (AMGs) remain unknown. We previously reported multiple candidate AMGs. Here, using multiple approaches, we characterized additional AMGs. First, we identified differentially expressed genes between high- and low-acylsugar-producing F2 plants derived from a cross between cultivated tomato (Solanum lycopersicum) and a wild relative (S. pennellii), which produce acylsugars that are ∼1% and ∼20% of leaf dry weight, respectively. Expression levels of many known and candidate AMGs positively correlated with acylsugar amounts in F2 individuals. Next, we identified lycopersicum-pennellii putative orthologs with higher nonsynonymous to synonymous substitutions. These analyses identified four candidate genes, three of which showed enriched expression in stem trichomes compared to underlying tissues (shaved stems). Virus-induced gene silencing confirmed two candidates, Sopen05g009610 [beta-ketoacyl-(acyl-carrier-protein) reductase; fatty acid synthase component] and Sopen07g006810 (Rubisco small subunit), as AMGs. Phylogenetic analysis indicated that Sopen05g009610 is distinct from specialized metabolic cytosolic reductases but closely related to two capsaicinoid biosynthetic reductases, suggesting evolutionary relationship between acylsugar and capsaicinoid biosynthesis. Analysis of publicly available datasets revealed enriched expression of Sopen05g009610 orthologs in trichomes of several acylsugar-producing species. Similarly, orthologs of Sopen07g006810 were identified as solanaceous trichome-enriched members, which form a phylogenetic clade distinct from those of mesophyll-expressed “regular” Rubisco small subunits. Furthermore, δ13C analyses indicated recycling of metabolic CO2 into acylsugars by Sopen07g006810 and showed how trichomes support high levels of specialized metabolite production. These findings have implications for genetic manipulation of trichome specialized metabolism in solanaceous crops.

Acylsugars, specialized metabolites with defense activities, are secreted by trichomes of many solanaceous plants. Several acylsugar metabolic genes (AMGs) remain unknown. We previously reported multiple candidate AMGs. Here, using multiple approaches, we characterized additional AMGs. First, we identified differentially expressed genes between high- and low-acylsugar-producing F2 plants derived from a cross between Solanum lycopersicum and S. pennellii, which produce acylsugars ~1% and ~20% of leaf dry weight, respectively. Expression levels of many known and candidate AMGs positively correlated with acylsugar amounts in F2 individuals. Next, we identified lycopersicum-pennellii putative orthologs with higher nonsynonymous to synonymous substitutions. These analyses identified four candidate genes, three of which showed enriched expression in stem trichomes compared to underlying tissues (shaved stems). Virus-induced gene silencing confirmed two candidates, Sopen05g009610 [beta-ketoacyl-(acyl-carrier-protein) reductase; fatty acid synthase component] and Sopen07g006810 (Rubisco small subunit), as AMGs. Phylogenetic analysis indicated that Sopen05g009610 is distinct from specialized metabolic cytosolic reductases, but closely related to two capsaicinoid biosynthetic reductases, suggesting evolutionary relationship between acylsugar and capsaicinoid biosynthesis. Additionally, data mining revealed that orthologs of Sopen05g009610 are preferentially expressed in trichomes of several acylsugar-producing solanaceous species. Similarly, orthologs of Sopen07g006810 were identified as trichome-preferentially-expressed members, which form a phylogenetic clade distinct from those of mesophyll-expressed "regular" Rubisco small subunits. Furthermore, ?13C analyses indicated recycling of metabolic CO2 into acylsugars by Sopen07g006810 and shed light on how trichomes support high levels of specialized metabolite production. These findings have implications for genetic manipulation of trichome specialized metabolism in solanaceous crops, including tomato, potato, and tobacco.

Acylsugars, specialized metabolites produced by solanaceous trichomes, provide protection against biotic and abiotic stresses. Here, we report ACYLSUGAR TRANSCRIPTION FACTOR1 (ASTF1/Sopen05g008450; AP2/ERF-family member) positively regulates acylsugar biosynthesis. Virus-induced gene silencing (VIGS) of ASTF1 in Solanum pennellii reduced acylsugar production by 65%. Most acylsugar (and several flavonoid) metabolic genes were downregulated in ASTF1-silenced plants, and these genes showed strong co-expression with ASTF1. In promoters of potential ASTF1-targets, we identified three enriched motifs, and one motif showed similarity with binding sites of other AP2/ERFs. Phylogenetic analysis and data mining indicated trichome-enriched expression of ASTF1 orthologs in several acylsugar-producing solanaceous species, suggesting a conserved role in acylsugar biosynthesis. This was supported by VIGS of ASTF1 orthologs in Nicotiana benthamiana. Broader phylogenetic analysis revealed relationships among specialized metabolic AP2/ERFs in several asterid species and provided clues about evolutionary emergence of acylsugar phenotype. Cultivated tomato ortholog (LEAFLESS/Solyc05g013540) has been reported to coordinate leaf initiation with transient expression at incipient primordia, and data mining revealed downregulation of trichome-preferentially-expressed genes, including acylsugar (and flavonoid) metabolic genes, in leafless mutants' shoot apices, indicating remarkable spatiotemporal functional diversity. Our work will pave a way to disentangle acylsugar regulatory network and holds promise for future metabolic engineering of acylsugar production.