Functional diversification of WRINKLED3 integrates fatty acid metabolism with insecticidal acylsugar production in Solanaceae species

Domain: biology
Published: 2026-03-25
Editorial outcome: minor_revision

Abstract
Acylsugars are defensive glycolipids in Solanaceae glandular trichomes, and their biosynthesis offers an exemplary system for understanding the evolutionary mechanisms of plant chemical defense, yet their transcriptional regulation is poorly understood. Here, we identified and characterized WRINKLED3 (WRI3), which is critical for acylsugar biosynthesis in tomato. SlWRI3 is specifically expressed in trichome tip cells, and its knockout reduces acylsugar accumulation. Using transcriptomics, DNA-protein interaction assays, and metabolomics, we demonstrate that SlWRI3 acts via a dual regulatory mechanism: directly activating the acyltransferase gene SlASAT1 for the initial acylation of sucrose core and upregulating multiple acetyl-CoA carboxylase (ACCase) subunits to provide acyl chain precursors. Silencing these ACCase genes similarly decreased acylsugar levels. Phylogenetic analysis indicates that the function of WRI3 in acylsugar biosynthesis is evolutionarily conserved in Solanaceae. These findings elucidate how a primary metabolism-associated regulator was repurposed to coordinate precursor supply and specialized metabolite production, deepening our understanding of plant metabolic evolution, providing a target for engineering pest-resistance in Solanaceae crops.

Introduction
The capacity to produce a vast array of chemical compounds is the key to the ecological success of plants, allowing them to adapt to different environments and interact with other organisms (Pichersky and Lewinsohn 2011; Weng 2014; Moghe and Last 2015). Beyond core primary metabolism, plants have evolved the capacity to synthesize specific specialized metabolites that mediate essential ecological functions, including herbivore resistance, pathogen defense, pollinator attraction, and allelopathy (Bennett and Wallsgrove 1994; Pichersky and Lewinsohn 2011; Weng et al. 2012; Kong et al. 2024).

The vast plant chemical diversity is driven by evolutionary innovations – frequent recruitment and diversification of primary metabolic enzymes into new, lineage-specific pathways (Weng et al. 2012; Weng 2014; Moghe and Last 2015). The emergence of diverse metabolite classes, such as terpenoids, alkaloids, and phenolics, results from gene duplication followed by neofunctionalization and metabolic pathway diversification (Moghe and Last 2015). Beyond the established roles of biosynthetic enzymes, transcriptional regulation plays a pivotal role in metabolic innovation by orchestrating the flux, allocation, and tissue specificity of specialized metabolism (Méteignier et al. 2022; Shi et al. 2024). How transcriptional regulation evolves to connect pre-existing primary metabolic flux with newly emerged specialized metabolic pathways remains a key unanswered question, despite advances in understanding enzyme evolution and metabolic regulation.

Metabolic transcription factors (TFs) are generally categorized as regulators of either primary or specialized metabolism. For instance, the APETALA2 (AP2)-type TF WRINKLED1 (WRI1) plays a central role in the regulation of fatty acid biosynthesis (Focks and Benning 1998; Cernac and Benning 2004; To et al. 2012; Kong et al. 2019). In contrast, some TFs from the family of MYB (Zhang et al. 2025), ethylene-responsive factor (ERF) (Wu et al. 2022), WRKY (Li et al. 2025), basic helix-loop-helix (bHLH) (Qian et al. 2021) control the expression of “downstream” enzymes involved in specialized metabolic pathways, including the biosynthesis of phenylpropanoids, terpenoids, alkaloids, flavonoids, and glucosinolates. While certain “gateway” TFs can bridge primary and specialized metabolism — such as WRKY35, which regulates carotenoid biosynthesis by activating the MEP pathway in tomato (Yuan et al. 2022) — the direct evolutionary recruitment of a central metabolism master regulator to coordinate both the supply of primary substrates and the synthesis of specialized metabolism-specific products remains uncommon.

Acylsugars are specialized metabolites secreted from glandular trichomes of many Solanaceae species and serve as powerful defensive chemicals with insecticidal, antimicrobial, and anti-herbivory activities (Goffreda et al. 1989; Juvik et al. 1994; Weinhold and Baldwin 2011; Leckie et al. 2016; Luu et al. 2017; Mutschler et al. 2023). In tomato, the core scaffold of acylsucroses is produced by the sequential action of four acylsugar acyltransferases (SlASAT1–SlASAT4). These enzymes, which belongs to the BAHD family (named after the first four characterized enzymes of the family: BEAT, AHCT, HCBT, and DAT) (D’Auria 2006), attach short-to-medium chain acyl groups (C2-C12) to specific hydroxyl positions of sucrose (Schilmiller et al. 2015; Fan et al. 2016, 2017). The acyl chains are derived from both branched-chain amino acid metabolism (for branched acyl groups) (Peng et al. 2015; Chen et al. 2023) and, of particular relevance to the current work, de novo fatty acid biosynthesis (for straight-chain acyl groups), implicating a deep intertwining of primary and specialized pathways (Walters and Steffens 1990). The acylsugar biosynthetic pathway is therefore an outstanding example of metabolic integration. This also creates a significant regulatory challenge, as the supply of primary precursors (e.g., sucrose, acetyl-CoA) and the specialized enzymes that generate the final product must be tightly and coordinately expressed within the same specialized trichome tip cells (Fan et al. 2019). A recent study made a key advance, identifying the AP2/ERF-family TF, LEAFLESS, as a regulator of acylsugar biosynthesis in the wild tomato Solanum pennellii (Mandal et al. 2025). However, mutant of the LEAFLESS ortholog in cultivated tomato exhibits strong pleiotropy, severely affecting leaf development (Capua and Eshed 2017), which raises questions about its precise role and suggests different or additional regulatory layers may exist in domesticated species. Therefore, a central and unanswered question in the field remains: does a transcription factor exist within the acylsugar-producing cell to act as a master integrator, simultaneously coordinating the primary metabolic pathways that generate precursor substrates and the specialized enzymes that assemble the final acylsugar products?

In this study, we identified a trichome-tip-expressed transcription factor WRINKLED3 (SlWRI3, Solyc03g117720), in cultivated tomato as a key regulator of acylsugar biosynthesis. Through co-expression analysis of acylsugar pathway genes, virus-induced gene silencing (VIGS), and CRISPR/Cas9-mediated mutagenesis, we demonstrated that SlWRI3 is essential for proper acylsugar accumulation and is particularly enriched in type I/IV trichome tip cells. Further investigation revealed that SlWRI3 directly binds to and activates the promoters of genes encoding the acetyl-CoA carboxylase (ACCase) complex and the acyltransferase SlASAT1, which respectively catalyze the first committed steps of fatty acid biosynthesis and specialized acylsugar assembly. These findings establish SlWRI3 as an integrator of both substrate supply and specialized metabolite production in acylsugar metabolism. This work advances our understanding of plant metabolic innovation by demonstrating how a WRI3-like type transcription factor, typically associated with primary fatty acid metabolism, can be co-opted to coordinate both primary and specialized metabolic networks within glandular trichomes.

Results
Identification and functional validation of transcription factors regulating acylsugar biosynthesis

We hypothesized that TFs regulating acylsugar biosynthesis would co-express with acylsugar assembly genes like SlASAT1, SlASAT2, SlASAT3, and SlASAT4 (Schilmiller et al. 2012, 2015; Fan et al. 2016). To identify candidate acylsugar regulators in cultivated tomato Solanum lycopersicum, we performed hierarchical clustering-based gene co-expression analysis using publicly available transcriptome data from various cultivated tomato tissues and developmental stages, including trichomes, shaved stems, roots hairs, leaves, and so on. TFs that co-occur with SlASAT1-4 were identified as candidate regulator of acylsugar biosynthesis.

This analysis identified seven candidate TFs: SlWRKY28 (Solyc12g011200), SlERF2a (Solyc12g042210), SlTCP25 (Solyc05g009900), SlTCP4 (Solyc03g115010), SlTCP6 (Solyc06g069460), SlTCP22 (Solyc04g006980), and SlWRI3 (Solyc03g117720) (Fig. 1a). To evaluate potential functional roles in acylsugar biosynthesis, we individually silenced these genes using VIGS and analyzed the resulting acylsugar profiles. Among the candidates tested, only SlTCP25 and SlWRI3 silencing significantly reduced acylsugar production (Fig. 1b-h).

To determine whether these two genes directly regulate acylsugar biosynthesis or indirectly affect it through altered plant development, we examined previous functional studies of these genes and their homologs. The closest Arabidopsis homologs of SlTCP25 – AtBRC1 (AT3G18550) and AtBRC2 (AT1G68800) – are known suppressors of axillary bud outgrowth (Aguilar-Martínez et al. 2007), with AtBRC1 playing a crucial role in shoot branching control (van Es et al. 2024). Recent evidence shows that sltcp25 mutants develop abnormal type I trichomes with multi-branched tip cells (Wang et al. 2025), suggesting that SlTCP25 may affect acylsugar production indirectly through altered trichome morphology rather than direct metabolic regulation. In contrast, neither SlWRI3 nor its homologs were previously implicated in trichome development or metabolism, making it a promising candidate for direct regulation of acylsugar biosynthesis and warranting further investigation.

The type I/IV trichome-expressed AP2 family transcription factor SlWRI3 is involved in tomato acylsugar biosynthesis.

Phylogenetic analysis of the AP2 family transcription factor SlWRI3 and its homologs across tomato and Arabidopsis positioned it within a distinct clade containing AtWRI3 (AT1G16060) and AtWRI4 (AT1G79700) (Supplementary Fig. 1a), both known regulators of acyl chain provision for cutin metabolism in floral tissues (To et al. 2012). Besides, among all tomato AP2 homologs, SlWRI3 is uniquely enriched in trichomes compared to the stem tissue (Supplementary Fig. 1). Therefore, SlWRI3 appears to have evolved a distinct, non-redundant role in acylsugar biosynthesis compared to its homologous proteins. To ask whether SlWRI3 is involved in tomato acylsugar metabolism, we generated stable mutants in the S. lycopersicum M82 background using CRISPR/Cas9 gene editing. Three independent mutant lines were obtained: slwri3#1 with a 534-bp deletion spanning from the 5' UTR to the second intron (including the translational start codon); slwri3#2 with a 708-bp deletion from the first exon to the fourth intron (disrupting the core AP2 domain) (Fig. 2a); and slwri3#3 predicted to result in an in-frame truncation of 36-bp between the two target sites (Supplementary Fig. 2a).

Liquid chromatography-mass spectrometry (LC/MS) metabolic profiling in young leaf tissue extracts revealed significant reductions in total acylsugar content – the sum of the eight most abundant trichome acylsugars –in slwri3#1 and slwri3#2 mutants when compared to wild-type (WT) M82 plants (Fig. 2b). In contrast, the slwri3#3 in-frame deletion mutant, carrying a 12-amino-acids deletion, showed no statistically significant change in total acylsugars (Supplementary Fig. 2b). Further analysis demonstrated that this reduction primarily affected the non-acetylated tri-acylsucroses (S3), including S3:14 (4,5,5), S3:15 (5,5,5), S3:20 (5,5,10), and S3:22 (5,5,12) (Fig. 2c, d). In contrast, the tetra-acylsucrose (S4) acetylated counterparts such as S4:16 (2,4,5,5), S4:17 (2,5,5,5), S4:22 (2,5,5,10), and S4:24 (2,5,5,12) are less affected (Fig. 2c, e). This differential impact resulted in an elevated ratio of S4:16/S3:14, S4:17/S3:15, S4:22/S3:20, and S4:24/S3:22 in both slwri3#1 and slwri3#2 (Fig. 2f). Similarly, despite the minimal impact on total acylsugar content, slwri3#3 also exhibited a slight decrease in S3 acylsugars and a significantly increased S4/S3 ratio compared to those of WT (Supplementary Fig. 2c-f). These results from stable CRISPR mutants confirm that SlWRI3 is involved in acylsugar biosynthesis, with strong impact on S3 acylsugar accumulation.

As expression of all characterized cultivated tomato acylsugar biosynthesis-related genes are localized specifically to the tip cells of type I/IV trichomes (Fan et al. 2019), we hypothesized that SlWRI3 would show tip cell specific expression. Analysis of transgenic tomato plants expressing green fluorescent protein and β-glucuronidase (GFP-GUS) under the control of a 2 kb SlWRI3 promoter (ProSlWRI3-2kb:GFP-GUS) revealed that the SlWRI3 promoter specifically drove GFP expression in the tip cells of type I/IV trichomes (Supplementary Fig. 3a). The single cell co-expression of this gene with all known acylsugar biosynthesis genes supports the hypothesis that SlWRI3 is a regulator of this biosynthetic pathway.

To further investigate the biological activities of SlWRI3, we examined its subcellular localization by transiently expressing the fusion protein SlWRI3-GFP in Nicotiana benthamiana. As expected for a transcription factor, SlWRI3 was exclusively observed in the nuclei of epidermal cells (Supplementary Fig. 3b). To determine if SlWRI3 possesses transcriptional activation capability, we generated constructs by linking the full coding region (F1) and various truncated fragments (F2-F9) to the galactose 4 (GAL4) DNA-binding domain (Supplementary Fig. 3c). These constructs were then introduced into yeast strain AH109 carrying HIS3 (encodes imidazoleglycerol-phosphate dehydratase, a key enzyme in histidine biosynthesis), ADE2 (encodes phosphoribosylaminoimidazole carboxylase, involved in adenine biosynthesis), and MEL1 (encodes α-galactosidase) reporter genes, each carrying a GAL4-upstream activating sequence (UAS) element. Our results showed that the C-terminal domain (F9) of SlWRI3 is sufficient for transcriptional activation (Supplementary Fig. 3c). The above experiments demonstrate that SlWRI3 exhibits the characteristics of a transcription factor. Collectively, these findings reveal that SlWRI3 is a trichome-specific AP2 family transcription factor with C-terminal transcriptional activation ability that positively regulates acylsugar biosynthesis in cultivated tomato trichomes.

SlWRI3 binds to the SlASAT1 promoter and positively regulates trichome acylsugar biosynthesis

To identify the regulatory targets of SlWRI3 in acylsugar biosynthesis, we conducted comparative transcriptome analysis of type I/IV trichomes isolated from WT and slwri3#1 mutant plants (Supplementary Fig. 4a). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that genes down-regulated in response to SlWRI3 inactivation were enriched in many primary metabolic pathways including biotin metabolism, fatty acid metabolism, glyoxylate and dicarboxylate metabolism, and glycolysis (Supplementary Fig. 4b). Conversely, disruption of SlWRI3 led to increased expression of genes associated with mitogen-activated protein kinase (MAPK) signaling pathway, hormone signal transduction, and genes involved in specialized metabolism (Supplementary Fig. 4c).

We hypothesized that SlWRI3 may transcriptionally regulate one or more of the seven characterized acylsugar biosynthetic genes: SlASAT1-4 (Fan et al. 2016), acylsugar acyl-CoA synthetase (SlAACS1), acylsugar enoyl-CoA hydratase (SlAECH1) (Fan et al. 2020), and isopropylmalate synthase-like enzyme (SlIPMS3) (Ning et al. 2015) (Supplementary Fig. 5), and that the direct targets of SlWRI3 would be differentially expressed in slwri3 mutants relative to WT plants. Among these acylsugar biosynthesis-related genes, SlASAT1 exhibited the most significant decrease in expression in slwri3 mutant plants compared to WT (Fig. 3a). This observation was further validated by real-time quantitative polymerase chain reaction (RT-qPCR), which confirmed the strongly reduced SlASAT1 transcript levels in slwri3 mutants relative to WT (Fig. 3b).

To investigate the effects of SlWRI3 on the transcriptional activity of SlASAT1, we performed dual-luciferase assays in N. benthamiana leaves. Co-transformation with 35S:SlWRI3 and the reporter construct 35S: Renilla luciferase (REN)/ProSlASAT1: firefly luciferase (LUC) in a transient assay resulted in significantly higher relative LUC/REN ratios compared to the negative control (leaves co-transformed with 35S:GFP and 35S:REN/ProSlASAT1:LUC). Here, REN served as the internal reference, while LUC acts as the reporter (Fig. 3c). This indicates that SlWRI3 can activate the promoter activity of SlASAT1 in vivo. These results led us to hypothesize that SlWRI3 might directly bind to the SlASAT1 promoter to activate its expression. To test this, we first examined whether SlWRI3 could physically interact with the SlASAT1 promoter using yeast one-hybrid (Y1H) assays. Using a series of truncated promoter fragments in Y1H assays, we first determined that the SlWRI3 binding occurred within the -2500 to -1000 bp region upstream of the SlASAT1 transcription start site (named as ProASAT1-2), but not in the -1500 to -1 bp region (ProASAT1-1) (Fig. 3d, e; Supplementary Fig. 6a). Through sequential truncations and Y1H testing, we narrowed the binding region to a 271 bp fragment between positions -1663 and -1393 (Supplementary Fig. 6a, b). Notably, this genomic region harbors three conserved AW-box cis-elements (Supplementary Fig. 6c), which serve as known binding sites for WRI1-clade transcription factors (Jo et al. 2024). To test for direct binding, we performed electrophoretic mobility shift assays (EMSA) using purified His-SlWRI3 protein, with His-pCold serving as a negative control (Supplementary Fig. 7). The results demonstrate that SlWRI3 can bind to the AW-box motifs of SlASAT1 promoter in vitro. The DNA-protein complex exhibited retarded migration when purified recombinant SlWRI3 was mixed with biotin-labeled probes P1, P2, and P3. The complex formation was attenuated by adding of a 500-fold excess of unlabeled (“cold”) probes. In contrast, mutated probes at a 500-fold excess were less effective at competing with the biotin-labeled probe compared to the cold probes (Fig. 3f-h). Collectively, these findings establish that SlWRI3 directly binds to the promoter of SlASAT1 and positively regulates its expression.

SlWRI3 regulates fatty acid metabolism in tomato glandular trichomes

Acylsugar biosynthesis requires both sugar scaffolds and fatty acid-derived acyl CoA thioesters. Because SlWRI3 belongs to the WRI transcription factor family, whose members regulate fatty acid biosynthesis in plants (Baud et al. 2007, 2009; To et al. 2012; Wei et al. 2025), we hypothesized that SlWRI3 also regulates fatty acid metabolism in tomato glandular trichomes to support acylsugar production. To test this hypothesis, we performed DNA affinity purification sequencing (DAP-seq) using SlWRI3 as bait to identify its genome-wide binding targets. This approach enabled us to uncover the broader transcriptional regulatory network governed by SlWRI3.

This analysis revealed 1,020 genes bound by SlWRI3 (Fig. 4a; Supplementary Data 1), with binding peaks predominantly located downstream of transcriptional start sites (TSS) (Supplementary Fig. 8a). Motif analysis identified three enriched DNA sequences, with the most prevalent being NNCG(N)7(C/T)NAN(G/A) (E-value = 5.6e-1362) (Fig. 4b; Supplementary Fig. 8b), corresponding with the AW-box motif [CnTnG](n) [CG], where n is any nucleotide), a well-characterized binding site for WRI1 transcription factors (Maeo et al. 2009). This motif was present in 1,409 binding peaks, while two additional motifs, CAA(C/T)NNCAACA(A/T)CAA(C/T) and CNNC(G/A)NC(G/A)NNG, were found in 108 and 80 binding peaks, respectively (Supplementary Fig. 8b). KEGG pathway enrichment analysis of SlWRI3-bound genes revealed significant enrichment in core primary metabolic pathways, including fatty acid biosynthesis, pyruvate metabolism, citrate cycle, glycolysis, and amino acid degradation (Fig. 4c).

To identify genes directly regulated by SlWRI3 in tomato type I/IV trichomes, we performed an integrated analysis of DAP-seq and comparative transcriptomics of type I/IV trichomes from WT and slwri3#1 mutant plants. Through RNA-seq analysis, we identified 844 differentially expressed genes (DEGs) in type I/IV trichomes, with a significance threshold of P-value < 0.05 and log (fold change) >1. By intersecting these 844 DEGs with the 1,020 SlWRI3 bound genes obtained from DAP-seq analysis, we ultimately identified 50 putative target genes that are directly regulated by SlWRI3 in type I/IV trichomes (Fig. 4d ).

Following manual curation of these 50 putative target genes, we retained 32 genes with FPKM >1 in WT plants (Fig. 4e). This subset revealed multiple fatty acid biosynthesis-related genes, including Solyc01g008330 (biotin carboxylase, BC), Solyc02g070790 (β-ketoacyl synthetase, KASI), Solyc05g014560 (3-ketoacyl-acyl carrier protein reductase, KAR), Solyc10g078740 (enoyl-ACP reductase, EAR), and Solyc03g063110 (stearoyl-acyl carrier protein desaturase, SAD) (Fig. 4e). BC is a catalytic subunit of the acetyl-CoA carboxylase (ACCase) complex, which catalyzes the first committed step in fatty acid biosynthesis — the carboxylation of acetyl-CoA to malonyl-CoA (Nikolau et al. 2003). Additionally, KASI, KAR, and EAR are involved in fatty acid chain elongation to produce C6:0-C16:0 acyl-ACPs (Zakim and Herman 1969; Shimakata and Stumpf 1982; Wu and Xue 2010; Chen et al. 2021). When we adjusted the fold change threshold to 1.5 for the analysis, we further identified additional fatty acid-related genes in trichomes that are regulated by SlWRI3, including SlMCAT (Solyc01g006980), SlKASIII (Solyc03g097470), SlKAR (Solyc06g071910 and Solyc05g014560), SlHAD (Solyc01g105060), SlEAR (Solyc10g078740), and SlKASI (Solyc02g070790) in tomato trichomes (Supplementary Fig. 9a).

The biochemical impact of SlWRI3 disruption was further evidenced by significant shifts in the pools of essential metabolic precursors and downstream products. Because malonyl-CoA serves as an essential precursor for acyl chain formation via fatty acid elongation, its level provides a reliable metabolic indicator of pathway activity. Our analysis revealed a significant reduction in malonyl-CoA content in newly developed, trichome-rich leaves of slwri3 mutants compared to WT plants (Fig. 4f), suggesting that SlWRI3 activity is required for normal malonyl-CoA biosynthesis. To test whether this precursor deficiency directly impacted the fatty acid profile, we further quantified the fatty acid levels in young leaves of WT and slwri3 mutant plants. The results showed that the content of three key fatty acids including C16:0, C16:3, C18:0 were significantly reduced in slwri3 relative to WT (Supplementary Fig. 9b-d). These findings provide strong evidence that SlWRI3 is a tomato trichome-specific, WRI3/4-like type regulator of fatty acid metabolism in tomato.

The acetyl-CoA levels were also quantified, as this metabolite serves dual roles: it is the direct biosynthetic precursor for malonyl-CoA and acts as an acyl donor substrate in tetra-acylsucrose biosynthesis. Given the decrease in malonyl-CoA, we hypothesized that acetyl-CoA might accumulate in slwri3 mutants. Surprisingly, acetyl-CoA content showed no significant difference between WT and slwri3 plants (Fig. 4g), suggesting that instead of accumulating, the acetyl-CoA pool is actively consumed by alternative metabolic pathways such as acetylating tri-acylsucroses S3 to generate tetra-acylsucroses S4, to maintain cellular homeostasis. Collectively, these metabolic analyses provide compelling biochemical evidence that SlWRI3 directly regulates fatty acid metabolism in type I/IV trichomes, with its disruption significantly impacting malonyl-CoA availability.

SlWRI3 impacts acylsugar biosynthesis by regulating genes involved in malonyl-CoA biosynthesis

Acylsugars in tomato trichomes require specific acyl chains for their assembly. Based on the discovery that SlWRI3 regulates multiple fatty acid biosynthesis genes and that the malonyl-CoA levels were reduced in slwri3 mutants, we hypothesized that SlWRI3 might indirectly influence acylsugar production by specifically regulating genes in the ACCase complex, which catalyzes the first committed step in fatty acid biosynthesis.

In the plastids of dicots, ACCase is a multi-subunit enzyme complex composed of four distinct parts: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and α- and β-carboxyltransferases (CTs) (Ye et al. 2020). Genes encoding these subunits in tomato were identified via BLAST (Supplementary Fig. 10). Among these genes, Solyc09g013080 (SlαCT), Solyc01g008330 (SlBC), and Solyc06g069530 (SlBCCP2) showed more pronounced expression decrease in the slwri3#1 mutant relative to the WT (Supplementary Fig. 10). Our DAP-seq analysis corroborated this by showing that SlWRI3 directly binds to the promoter regions of these three specific genes (Fig. 5a-c). Interestingly, while DAP-seq also detected a SlWRI3 binding peak at the SlBCCP1 locus—spanning both its promoter and first exon—our trichome-specific RNA-seq data showed that its expression remained unaffected by the loss of SlWRI3. To further validate their functional relationship with SlWRI3, we performed the following experiments. As predicted, RT-qPCR analysis showed that the transcript levels of SlαCT, SlBC, and SlBCCP2 were significantly reduced in the type I/IV trichomes of slwri3 mutant compared to WT (Fig. 5d). In contrast, the expression of SlBCCP1 exhibited no significant expression difference (Fig. 5d). Second, we performed Y1H assays using the DAP-seq peak sequences as baits to verify protein-DNA interactions. These assays confirmed direct interactions between SlWRI3 and the promoter peak regions of SlαCT, SlBC, and SlBCCP2 (Fig. 5e). To determine whether the SlWRI3 binding causes transcriptional activation in vivo, we then conducted dual-luciferase assays by co-transforming N. benthamiana leaves with 35S:SlWRI3 and reporter constructs containing the promoters of SlαCT, SlBC, or SlBCCP2 driving luciferase expression. The relative LUC/REN ratios were significantly higher when SlWRI3 was co-expressed with any of these promoter constructs compared to the empty vector control (35S:GFP), indicating that SlWRI3 activates these promoters in vivo (Fig. 5f).

To determine whether SlWRI3 can directly bind to the predicted cis-acting elements, we analyzed the putative binding motif within the SlWRI3 binding peaks of SlαCT, SlBC, and SlBCCP2 and found multiple AW-box elements (Supplementary Fig. 11a). EMSA was performed to test the binding activity using purified recombinant His-SlWRI3 protein (Supplementary Fig. 7) and biotin-labeled DNA probes containing the AW-box motifs from each promoter (Supplementary Fig. 11b). DNA-protein complexes with retarded migration were observed when His-SlWRI3 was mixed with biotin-labeled probes from the promoters of SlαCT, SlBC, and SlBCCP2 (Fig. 5g-i). The DNA-protein complex formation was reduced by competition with excess unlabeled probes (500-fold concentration), while mutated probes failed to compete with the biotin-labeled probes, confirming the sequence specificity of these interactions (Fig. 5g-i).

Having established that SlWRI3 directly regulates genes of the ACCase complexes, we next investigated whether these target genes were functionally important for tomato trichome acylsugar biosynthesis. We generated stable RNA interference (RNAi)-silencing tomato transgenic lines individually targeting SlαCT or SlBC. Characterization of these lines revealed significant reductions in total acylsugar content compared to control plants (Fig. 6), demonstrating that ACCase activity is indeed required for normal acylsugar production in tomato trichomes. Together, these results demonstrate that SlWRI3 functions as a direct transcriptional activator of ACCase complex genes in type I/IV trichomes, thereby linking fatty acid metabolism to acylsugar biosynthesis in tomato trichomes.

Evolutionary conservation of WRI3 in Solanaceae species

As a member of the WRI3/4-like transcription factor family, SlWRI3 unexpectedly shows a type I/IV trichome-enriched expression pattern in tomato and participates in acylsugar biosynthesis. This represents a notable deviation from its canonical role in primary metabolism. Previous studies revealed that trichome-synthesized acylsugars are widely distributed across the Solanaceae family including diverse members of the genus Solanum (Lou et al. 2021; Leong et al. 2022; Fiesel et al. 2024; Kerwin et al. 2024), Hyoscyamus niger, Atropa belladonna, Physalis viscosa, Salpiglossis sinuata (Moghe et al. 2017) and Petunia axillaris (Nadakuduti et al. 2017). We tested the hypothesis that WRI3 regulation of acylsugar biosynthesis occurs throughout the Solanaceae lineage by conducting phylogenetic analyses combined with transcriptome data mining. Indeed, we identified putative orthologs of SlWRI3 in species across the nightshade family, including S. pennellii, Solanum nigrum, Solanum quitoense, H. niger, and P. axillaris. Consistent with our hypothesis, expression analysis revealed that these orthologs all exhibited enrichment in trichomes compared to other tissues (Fig. 7a), suggesting evolutionary conservation of their trichome-specific regulatory roles. This trichome-enriched expression pattern appears to be a derived trait within the Solanaceae. When we examined SlWRI3's closest homologs in Arabidopsis, a species that produces non-glandular trichomes, we found no evidence of trichome-enriched expression (Fig. 7a). To investigate conservation of protein features that impact WRI protein function, we performed a multiple sequence alignment of trichome-enriched SlWRI3 homologs from the six Solanaceae species alongside the Arabidopsis AtWRI1-4 proteins. We specifically focused on the conservation of the AP2 domain and the key residues essential for AW-box binding (Qiao et al. 2022). Our analysis revealed that the AP2 domain is highly conserved across all Solanaceae orthologs and the Arabidopsis WRI1/3/4 proteins (Supplementary Fig. 12). Furthermore, the specific residues required for recognizing the AW-box motif are maintained in these WRI-like proteins (Supplementary Fig. 12). This robust conservation of critical DNA-binding motifs is highly consistent with the hypothesis that these WRI3/4-like transcription factors perform similar regulatory functions within glandular trichomes across the Solanaceae lineage.

To test whether SlWRI3 orthologs are functionally conserved in different Solanaceae species, we employed VIGS to reduce WRI3 expression in S. pennellii and S. nigrum, two species known to produce different acylsugar profiles. LC/MS analysis revealed significantly reduced acylsugar levels in silenced plants compared to control plants for both species (Fig. 7b, c). This functional evidence indicates that WRI3 orthologs play conserved roles in regulating acylsugar production across Solanaceae lineages.

Methods
Gene co-expression analysis

To identify transcription factors (TFs) governing acylsugar biosynthesis, we performed co-expression network analysis using GenePattern (https://cloud.genepattern.org). RNA-seq datasets from diverse tomato tissues (publicly available) were collected and integrated into a unified expression matrix, normalized in Excel, and uploaded to GenePattern. A co-expression network was constructed via hierarchical clustering of all genes, with the co-expression data obtained from Moore et al. (2020).

Genes annotated as TFs were prioritized if they clustered in close with SlASAT1-4 (key acylsugar biosynthetic genes) in the network. Initially, ~300 genes strongly co-expressed with SlASAT1-4 were shortlisted, with further filtering to exclude genes lacking trichome-enriched expression (compared to stems) or showing low background expression. This workflow ultimately selected 43 genes, including SlASAT1-4, seven candidate TF genes, and additional enzymes highly connected to the acylsugar biosynthetic module. The expression profiles of these candidates were visualized using TBtools (Chen et al. 2020), confirming their trichome-specific co-expression patterns across tissues.

Plant materials and growth conditions

Tomato (Solanum lycopersicum cv. M82) was used as the wild-type (WT) control. Seeds were originally obtained from the C.M. Rick Tomato Genetics Resource Center (University of California, Davis) and subsequently self-propagated. To promote germination, seeds were soaked in deionized water within 50 mL centrifuge tubes and incubated overnight at 28 °C with continuous shaking at 180 rpm. The seeds were subsequently sown into 32- or 50-cell plastic trays. Following germination, seedlings were transferred to a controlled environment chamber maintained at approximately 60% relative humidity. The growth cycle consisted of a 16-h light period at 25 °C with a photosynthetic photon flux density of 500 μmol m⁻² s⁻¹, followed by an 8-h dark period at 20 °C.

Virus-Induced Gene Silencing (VIGS)

VIGS target fragments were designed using Sol Genomics Network (SGN)-VIGS tool (https://vigs.solgenomics.net/) (Fernandez-Pozo et al. 2015) and amplified from candidate genes along with the phytoene desaturase (PDS) control, and then cloned into pTRV2-LIC vector via homologous recombination before transformation into Agrobacterium tumefaciens strain GV3101 (Liu et al. 2002). The experiment was performed following the protocol described by Velásquez et al. (2009) with minor modifications. In brief, the Agrobacterium strains harboring pTRV1, empty pTRV2 vector, or pTRV2 constructs with target gene fragments were individually cultured overnight at 30 °C in LB containing 50 mg/L kanamycin and 25 mg/L rifampicin. Bacterial cells were harvested by centrifugation at 5,000 x g for 10 min, washed, and resuspended in infiltration buffer (10 mM MES, pH 5.6, 10 mM MgCl₂, and 200 µM acetosyringone) to OD₆₀₀ = 1.0. Equal volumes of pTRV2 and pTRV1 suspensions were mixed and infiltrated into newly emerged cotyledons of plants (S. lycopersicum, S. pennellii, and S. nigrum). pTRV2-PDS infiltrated into plants and grown in parallel to the experimental plants was used as a visual marker to assess silencing efficiency. Approximately three weeks after infiltration, the true leaf of each plant was bisected along the midvein: one half was used for acylsugar quantification, and the other half was subjected to RT-qPCR analysis. Plants infiltrated with empty pTRV2 served as the control group. Primers used in this study are listed in Supplementary Data 4.

Phylogenetic tree analysis

The amino acid sequences of AP2 family transcription factors from tomato (S. lycopersicum) and Arabidopsis (A. thaliana) were downloaded from PlantRegMap/PlantTFDB v5.0 (Tian et al. 2020). A phylogenetic tree was constructed using the Neighbor-Joining method in MEGA6.06 software, with bootstrap analysis (1000 replicates) to assess branch support. Expression abundance data of AP2 transcription factors were integrated and visualized using ChiPlot (https://www.chiplot.online/) (Xie et al. 2023). All protein sequences and phylogenetic tree files are available in Source Data file and Supplementary Data 2.

The amino acid sequences of AtWRI1 homologs from Arabidopsis and six Solanaceae species (S. lycopersicum, S. pennellii, S. nigrum, S. quitoense, H. niger, and P. axillaris) were obtained through BLAST. Corresponding protein sequences are provided in Source Data file, and phylogenetic tree file is available in Supplementary Data 3. Gene expression data for these homologs were sourced from the following repositories: Arabidopsis (Marks et al. 2009); S. lycopersicum (Ning et al. 2015); S. pennellii (Fan et al. 2020); S. nigrum, S. quitoense, and H. niger (Moghe et al. 2017); P. axillaris (Guo et al. 2015). Detailed information regarding the datasets utilized in this study is compiled in Source Data file.

Subcellular localization of SlWRI3 expressed in Nicotiana benthamiana

The full-length coding sequence (CDS) of SlWRI3 without the stop codon was amplified by PCR using the primers 35S:SlWRI3-F and 35S:SlWRI3-R listed in Supplementary Data 4 and cloned into a modified pCAMBIA1300 vector to generate 35S:SlWRI3-GFP fusion construct (He et al. 2023). The construct was transformed into Agrobacterium GV3101, which was then used to infiltrate leaves of a 4-week-old N. benthamiana reporter line stably expressing a nuclear-localized protein tagged with RFP (Lou et al. 2020). After 48 h, fluorescence signals were detected using a Nikon A1 confocal microscope with GFP (488 nm excitation and 505-525 nm emission) and RFP (561 nm excitation and 580-630 nm emission) channels.

Vector constructs and tomato transformation

To determine the tissue-specific expression pattern of SlWRI3, we generated stable transgenic tomato plants expressing a ProSlWRI3:GFP reporter construct in the M82 genetic background. Briefly, a 2000 bp promoter region of SlWRI3 was amplified using primers ProSlWRI3-FS7-F and ProSlWRI3-FS7-R (Supplementary Data 4) and cloned into pENTR/D-TOPO. The resulting entry clone was recombined with the destination vector pKGWFS7 via LR reaction using Gateway cloning technology to generate ProSlWRI3:GFP. Stable ProSlWRI3:GFP tomato lines were generated via Agrobacterium (GV3101)-mediated cotyledon transformation, with GFP signals detected in T1 plants using confocal microscopy. To generate stable slwri3 mutants, we employed CRISPR/Cas9 genome editing following the protocol described by Čermák et al. (2017). A pair of single guide RNAs (sgRNAs) targeting the SlWRI3 locus were designed using the CRISPR-P (http://cbi.hzau.edu.cn/cgi-bin/CRISPR). Primers for vector construction were generated via the CRISPR-Multiplex platform (Supplementary Data 4), and the pMOD2203 plasmid served as the template for PCR amplification. The amplified fragments were subsequently cloned into the pDIRECT_21C vector using the Golden Gate assembly method. The construct was transformed into Agrobacterium GV3101 and used to infect cotyledons from 7-day-old M82 tomato seedlings for stable transformation.

Homozygous SlWRI3 lines were isolated via PCR amplification and Sanger sequencing of the genomic regions flanking the sgRNA target sites. Three independent CRISPR-edited lines (slwri3#1, slwri3#2, and slwri3#3) harboring distinct mutations were selected for subsequent phenotypic and functional analyses, PCR primers are provided in Supplementary Data 4.

Yeast transcriptional activation assay

The full-length coding sequence (CDS) of SlWRI3 (F1) and eight truncated fragments of SlWRI3 (F2-F9) were cloned into the pGADT7 vector. The constructs were transformed into AH109 chemically competent cells (Catalog No. YC1010, WEIDI Biotechnology Co., Ltd.) using chemical transformation. Transformants were initially selected on synthetic dropout medium lacking tryptophan (SD/-Trp) to verify successful plasmid transformation. Transcriptional activation activity was subsequently evaluated using two selective media systems: SD/-Trp/-His supplemented with X-α-Gal (SD/-Trp/-His + X-α-Gal) and media without tryptophan, histidine, and adenine (SD/-Trp/-His/-Ade). Yeast cells were grown at 30℃ for 4 days before being photographed. Positive transcriptional activation was confirmed by development of blue coloration on SD/-Trp/-His + X-α-Gal medium (indicating α-galactosidase reporter expression) or colony growth on SD/-Trp/-His/-Ade medium (demonstrating HIS3 and ADE2 reporter activation).

Trichome metabolite extraction and LC/MS analysis

The fully expanded young leaves were used for leaf acylsugar extraction as described in the protocol published on Protocols.io (http://dx.doi.org/10.17504 /protocols.io.xj2fkqe). In brief, each leaf was submerged in 1 mL of extraction solution within 2 mL tubes. Samples were gently agitated for 3 min at room temperature. The extraction solution consisted of acetonitrile/isopropanol/ water (3:3:2) containing 0.1% formic acid and 1 μM telmisartan as the internal standard. Each individual plant served as a biological replicate, with at least three plants analyzed per line.

Metabolite profiling was performed using an Agilent 1290 Infinity II UHPLC system coupled with an Agilent 6545B Q-TOF mass spectrometer (Agilent Technologies) equipped with an electrospray ionization (ESI) probe in negative ion mode. Mass spectrometry parameters were set as follows: Gas temperature: 275 °C, gas flow rate: 8 L/min; nebulizer pressure: 35 psi; sheath gas temperature: 300 °C; sheath gas flow rate: 11 L/min; capillary voltage: 3500 V; nozzle voltage: 0 V; fragmentor voltage: 150 V; skimmer voltage: 65 V.

A 5-µL aliquot of each extract was injected onto a C18 column maintained at 40 °C. Separation was achieved using a 21-minute binary gradient of water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.3 mL/min as follows: 5-60% B (0-3 min), 60-100% B (3-15 min), 100% B (15-18 min), 100-5% B (18-18.1 min), and 5% B (18.1-21 min).

Mass data were acquired in auto MS/MS mode (m/z 50-1700) and analyzed using Agilent MassHunter Workstation software (version 10.0). Peaks were identified from extracted ion chromatograms (EICs) at a fragmentor voltage of 150 V. Key monitored ions in the S. lycopersicum WT line under negative mode included S3:14(4,5,5) (m/z 625.2706), S4:16(2,4,5,5) (m/z 667.2818), S3:15(5,5,5) (m/z 639.2864), S4:17(2,5,5,5) (m/z 681.2975), S3:20(5,5,10) (m/z 709.3653), S4:22(2,5,5,10) (m/z 751.3758), S3:22(5,5,12) (m/z 737.3965), and S4:24(2,5,5,12) (m/z 779.4071), with peak areas normalized to the internal standard (IS) using m/z 513.2298) and dry weight (dw) of the extracted leaflet.

Key monitored ions in the LA0716 line under positive mode included G3:12(4,4,4) (m/z 435.1865), G3:13(4,4,5) (m/z 449.2030), G3:14(4,5,5) (m/z 463.2169), G3:15(5,5,5) (m/z 477.2327), G3:16(4,4,8) (m/z 491.2515), S3:19(4,5,10) (m/z 695.3501), S3:21(4,5,12) (m/z 723.3799), and S3:22(5,5,12) (m/z 737.3971) as described by Lybrand et al. (2020).

Key monitored ions in the S. nigrum under negative mode included G2:12(4,8) (m/z 421.21), G2:13(5,8) (m/z 435.23), G2:14(5,9) (m/z 449.249), G2:15(5,10) (m/z 463.26), G2:18(8,10) (m/z 505.3), G3:14(2,4,8) (m/z 463.22), G3:15(2,5,8) (m/z 477.24), and G3:16(2,5,9) (m/z 491.25), G3:17(2,5,10) (505.27), as described by Lou et al. (2021).

DAP-seq and data analysis

DAP-seq was performed following the protocol described by Bartlett et al. (2017) at Bluescape Hebei Biotech. The genomic DNA library for DNA affinity purification sequencing (DAP-seq) was constructed using the MICH TLX DNA-Seq Kit (Cat# NGS0602, Bluescape Hebei Biotech Co., Ltd). The SlWRI3 protein was expressed in vitro using the TNT SP6 Coupled Wheat Germ Extract System (Promega) according to the manufacturer’s instructions. Recombination SlWRI3 protein was purified and captured using Magne Halo Tag Beads (Promega). Beads bound to SlWRI3 were incubated with adapter ligated genomic DNA libraries. Eluted DNA fragments were sequenced on an Illumina NavoSeq platform. A no-protein control (beads only) was included as an input negative control. Sequencing reads were aligned to the reference genome using BWA-MEM (https://github.com/lh3/bwa). MACS2 callpeak used to identify the peaks with a significance threshold of P < 0.05 (Zhang et al. 2008). Biological replicates (rep1 and rep2) were merged using IDR to assess peak reproducibility (Li et al. 2011). Motif enrichment analysis within peak regions was conducted using MEME-ChIP (Machanick and Bailey 2011). Peaks were annotated using Homer software (Heinz et al. 2010), and their distribution relative to transcription start sites (TSS) was analyzed using deepTools2 (Ramírez et al. 2016). Visualization of peak regions was performed using IGV software (http://software.broadinstitute.org/software/igv/download).

RNA extraction and RNA-seq analysis

The type I/IV trichomes were manually dissected from leaf blades of WT and slwri3 mutant plants using Dumont #5 forceps (0302-5-Po, DUMONT) under a microscope (Supplementary Fig. S4A). Total RNA was extracted using the RNeasy Plant Mini Kit (74904, QIAGEN) and sent to OE Biotech for RNA-seq analysis.

RNA integrity was assessed via the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Library preparation was prepared using the VAHTS Universal V10 RNA-seq Library Prep Kit (Premixed Version) according to the manufacturer's protocol. Sequencing was conducted on an Illumina NovaSeq6000 platform, generating paired-end reads.

Clean reads were aligned to the reference genome using HISAT22. Transcript abundance was quantified as fragments per kilobase of exon per million reads mapped (FPKM). Differential expression analysis was performed using DESeq2, with genes meeting thresholds of adjusted P-value (q-value) < 0.05 and absolute log fold change > 1 (i.e., fold change > 2 or < 0.5) defined as significantly differentially expressed genes (DEGs).

EMSA

The full-length coding sequence (CDS) of SlWRI3 was cloned into pCold-TF-His vector and transformed into E. coli BL21 (Rosetta). To express His-SlWRI3 and His-pCold control proteins, E. coli BL21(Rosetta) cells harboring pCold-TF-His-SlWRI3 or the empty pCold-TF-His vector were cultured at 16 °C for 16 h with 200 μM IPTG induction. Proteins were purified using Ni-NTA His Beads (Smart-Lifesciences) according to the manufacturer’s protocol.

DNA probes (5’ biotin-labeled or unlabeled) and mutant probes were generated by annealing complementary oligonucleotides using a thermal cycler program: 95 °C for 2 min, ramp down to 25 °C at -0.1 °C / 8 s, hold at 4 °C. EMSA was performed using the Chemiluminescent EMSA Kit (GS009; Beyotime) according to the instructions provided by the kit. Reaction mixtures (10 μL) contained ~2 μg purified protein and 1 μL biotin-labeled probe (50 nM). Unlabeled or mutant probes (50 x or 500 x) were added in this reaction used as competitor.

Yeast one-hybrid assay

The promoter regions of SlαCT, SlBC, and SlBCCP2 bound by SlWRI3, along with different truncated fragments of SlASAT1, were amplified and cloned into the pAbAi vector as baits. Bait vectors were linearized using BstBI (R0519S, NEB) at 65 °C for 15 min, purified, and subsequently transformed into Y1HGold. Transformants were selected on SD/-Ura medium, and self-activation was screened on SD/-Ura supplemented with varying concentrations of aureobasidin A (AbA).

The full-length CDS of SlWRI3 was inserted into pGADT7 as prey, with empty pGADT7 serving as a negative control. SlWRI3-pGADT7 and the empty pGADT7 vector were transformed into Y1HGold strains previously integrated with individual bait constructs. Transformants were selected on SD/-Leu medium, and protein-DNA interactions were assayed on SD/-Ura medium supplemented with the suitable inhibitory concentration of AbA determined during self-activation screening. Yeast cells were grown at 30 ℃ for 4 days before being photographed.

Dual luciferase assay

Dual luciferase assay was performed as described by He et al. (2023). For effector constructs, the full-length CDS of SlWRI3 was cloned into pCAMBIA1300 under the control of the CaMV 35S promoter, with empty pCAMBIA1300 (35S:GFP) serving as a negative control. These constructs were transformed into Agrobacterium strain GV3101. For reporter constructs, promoters of SlαCT, SlBC, SlBCCP2, and SlASAT1 were cloned into pGreenII 0800-LUC and transformed into GV3101 (pSoup). Agrobacterium strains carrying effector and reporter constructs were co-infiltrated into N. benthamiana leaves. After a 3-day incubation, dual luciferase activity was measured using the Dual Luciferase Reporter Gene Assay Kit (11402ES60, YESEN) on a SpectraMax iD3 plate reader. Primer sequences are provided in Supplementary Data 4.

RNA extraction and RT-qPCR assays

Total RNA was extracted from the type I/IV trichomes of ~10-day-old seedlings using the RNeasy Plant Mini Kit (74904, QIAGEN). cDNA was obtained using the HiScript IV All-in-One Ultra RT SuperMix for qPCR (R433-01, Vazyme). Five-fold diluted cDNA templates were used to quantify the relative expression of SlASAT1, SlαCT, SlBC, SlBCCP1, and SlBCCP2, with SlGAPDH serving as internal controls. RT-qPCR was performed using SYBR Green Real-Time Master Mix (QPK-201, Toyobo) on a ROCHE LightCycler 480 II. Primer sequences are provided in Supplementary Data 4.

Acetyl-CoA and malonyl-CoA measurement

Acetyl-CoA and malonyl-CoA were extracted from trichome-rich, newly expanded leaves. The extraction of acetyl-CoA was conducted following the protocol of Kantner et al. (2024). Briefly, approximately 100 mg of flash-frozen leaf tissue was ground in liquid Nitrogen, and metabolites were extracted with 1 mL of ice-cold 10% trichloroacetic acid (TCA). The homogenate was then centrifuged at 17,000 × g for 10 min at 4 °C, and the supernatant was transferred to a new tube.

Oasis HLB solid-phase extraction (SPE) was used to purify acetyl-CoA. Columns were preconditioned with 1 mL of methanol and equilibrated with 1 mL water. Samples were loaded onto the columns, washed with 1 mL water, and eluted with 1 mL of 25 mM ammonium acetate in methanol. Eluates were dried under nitrogen gas and reconstituted in 100 μL of mobile phase A (2% acetonitrile in 100 mM ammonium formate, pH 5.0).

Malonyl-CoA was extracted by 200 μL methanol: isopropanol (1:1, v/v) solution. Following addition of 500 μL dichloromethane, the mixture was vortexed for 30s. Subsequently, 200 μL water was added, and samples were vortexed again for 30s. Phase separation was facilitated by incubation at 4 °C for 10 min, followed by centrifugation at 15,000 x g for 10 min at 4 °C. The upper aqueous phase (150 μL) was transferred to a glass vial and analyzed via LC-MS/MS.

For acetyl-CoA and malony-CoA measurement, the ExionLC HPLC system coupled with an AB SCIEX QTRAP 6500plus mass spectrometer was used. Aliquots of 1 µL of each sample were injected into an ACQUITY BEH Amide column (2.1×50 mm). Chromatographic separation was achieved with a flow rate of 0.4 mL/min, employing 10 mM ammonium acetate and 0.05% ammonium hydroxide in water (solvent A) and 10 mM ammonium acetate and 0.05% ammonium hydroxide in 90% acetonitrile (solvent B). An 8-minute linear gradient was employed for elution. The gradient program was as follows: 0–1 min, 95% B; 1–2.5 min, 95% to 55% B; 2.5–5 min, 55% B; 5–5.1 min, 55% to 95% B; and 5.1–8 min, 95% B. Acetyl-CoA were analyzed in negative ion-mode electrospray ionization (ESI−), while malonyl-CoA were analyzed in positive ion-mode (ESI+). Multiple reaction monitoring (MRM) mode quantification settings were: curtain gas at 40 psi; ion spray voltage at -4.5 kV for ESI− and +4.5 kV for ESI+; ion source temperature at 500 °C; ion source gases 1 and 2 at 55 and 50 psi, respectively. MRM transitions were set at 808.2/461.2, 808.2/408.1, 808.2/728.0 for acetyl-CoA, 854.2/428.2, 854.2/347.2, 854.2/303.5 for malonyl-CoA. Declustering potential (DP) and collision energy (CE) values were -90 V/-50 V for acetyl-CoA, and 50 V/40 V for malonyl-CoA. A 100 ms dwell time was applied. Data analysis was performed using SCIEX OS 1.4.0, and quantification was performed using a standard curve established with analytical standards.

Analysis of fatty acids by GC‑MS

Fatty acids were extracted by minor modification of a previously described protocol (Li et al. 2006). Tomato young leaves were collected, freeze-dried, and stored at -80 °C. For fatty acid extraction, 10 mg of ground sample was mixed with 1 mL of extraction buffer (2.5% sulfuric acid : methanol, v/v), 25 μg of pentadecanoic acid (internal standard), and 300 μL toluene (co-solvent). The mixture was heated at 80 °C for 60 min, cooled to room temperature, and the reaction was terminated by adding 1.5 mL of 0.9% NaCl. Subsequently, 2 mL of n‑hexane was added, and the sample was vortexed and centrifuged horizontally at 4000 rpm for 10 min. The upper organic phase was collected, and the extraction was repeated twice. The combined organic phases were dried under nitrogen flow, redissolved in 400 μL hexane, and transferred to a GC vial.

Fatty acid profiles were further analyzed by gas chromatography–mass spectrometry (GC‑MS). Separation and quantification were performed on an Agilent 7890B GC‑MS system equipped with an Agilent DB‑5MS column (122‑5532G, 40 m × 250 μm, 0.25 μm film thickness). The carrier gas was nitrogen. The temperature program was as follows: initial temperature 50 °C, increased to 200 °C at 25 °C/min (hold 1 min), then increased to 300 °C (hold 8 min). The split ratio was 5:1, and the injection volume was 4 μL.

Peaks were processed using Agilent MassHunter Workstation software (version 10.0). The relative concentration of each target fatty acid was calculated by comparing its peak area to that of the internal standard (pentadecanoic acid).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by He et al. study the WRI-like transcription factor WARD-1. The manuscript provides a detailed molecular characterization of this metabolic transcription factor demonstrating that it acts via a Dual mechanism i) upregulating expression of the key enzyme SAT1 and ii) also upregulating several subunits of the critical precusor providing ACCase enzyme. Interestingly, these results are not without parallel with the albeit artificial example of the purple tomato of Cathie Martin displaying a similar dual mechanism (see Yang et al. Nature Comms) - maybe worth discussing this.\nReturning to the paper of He et al., this presents well designed and executed experiments which are excellently discussed highlighting but not overselling the importance of their results. Moreover the Figures are exemplary. Indeed the entire manuscript was a pleasure to read and I have no suggested improvements beyond the discretionary one that I mention above.", "reviewer_2": "This work by He et al. presented the identification of SlWARD1 transcription factor in Solanaceae, and elucidation of its role in regulation of synthesis of fatty acid and acylsugar. Overall, the manuscript presented very interest research work with good novelty. The data in this study had high quality and the manuscript was written well. Suggestions are provided for further improvement of this manuscript as following.\nSuggestions:\n1. Different names of WRI-like [such as 'WRI-like' (e.g. line 1) and 'WRI1-like' (e.g. line 413)] were used throughout the whole manuscript, which triggered inconsistency and confusions for readers. Authors should keep the name consistent for this manuscript. In addition, the correct abbreviation for 'WRINKLED1' is 'WRI1', while it was used as 'WRI' (e.g. line 44). Given that SlWARD1 falls into same subclade of AtWRI3/4, it might be more accurate to define SlWARD1 as a 'WRI3/4-like' candidate.\nAuthors need to check the whole manuscript carefully to keep the name consistent. In the title of this manuscript, it is better to use full name for WRINKLED.\nIn addition, authors can consider to provide protein sequence alignment of these WRI-like proteins in Solanaceae with Arabidopsis WRI1-4.\n2. Figure 5D. Authors did not find the expression change of SIBCCP1 between wild type (WT) and slward1 mutant. Can authors identify AW-box in promoter of SIBCCP1 or not? In addition, can SIWARD1 have an effect on the promoter activity of SIBCCP1 (e.g. via dual-luc assay used in Figure 5F)? Some additional tests, investigations and discussions at this point here might be good for readers.\n3. Question to EMSA experiments. (Using Figure 3 as an example)\na)authors need to provide the probe sequence information, with a highlight of AW-box.\nb)for mutated probe used in this study, please provide the sequence as well, with a highlight of the mutated nucleotides.\nc) for competition assay, it is better to a gradual increase of unlabelled probe (currently there is only one addition of unlabelled probe in this figure).\n4. Line 85, using 'APETALA2 (AP2)' instead of 'AP2', due to its first appearance.\nLine 234, similar issue. Please provide full name for 'RT-qPCR'.\n5. Please provide full names of genes/proteins for their first appearance. For instance, BAHD (line 100), AP2/ERF (line 111), etc. Please check the whole manuscript carefully.\n6. Line 427, it will be good to cite Plant J (2016) 88(2):257-270.\n7. Section of line 174-179. Why do authors show the 3 mutants of slward1 in two sets of figures (Fig. 2 and Supplementary Fig. S2), which is confusing.\n8. Line 237, it should be 'N. benthamiana' instead of 'tobacco'.\n9. Line 254, 'can binds to' needs to be changed to 'can bind to'. Authors need to check the manuscript carefully to avoid this type of grammar errors.\n10. Starting from Figure 2: Is it better to use 'WT' representing 'M82' for all figures, which should be easier for readers? (Then explain details for M82 wild type in Methods section).\n11. Supplementary Figure S6. Please replace 'domain' by 'region' or 'sequence'.\n12. Supplementary Figure S3B. 'SlWARD1-GFP fusion protein was transiently expressed...' please use 'produced'.\n13. Line 275-276. It should be WRI1 transcription factor here (instead of 'WRI transcription factor'). Authors also need to cite Maeo et al. 2009 plant j paper in this sentence for an accurate citation and presentation. (also please provide original characterization of AW-box sequence [CnTnG](n)7[CG] in Maeo et al. 2009 to help readers see the information). From line 274 to 277, please include the information regarding what 'N' represents for. (I guess it represents any nucleotide; but authors need to get it clarified for readers in this manuscript).", "reviewer_3": "In this manuscript He and co-workerss describe a comprehensive study leading to the identification and functional characterization of a WRI-like transcription factor (SlWARD1), which is expressed in the tip cells of tomato leaf glandular trichomes and is involved in the coordinated regulation of acylated sugars (defensive metabolites) biosynthesis and the synthesis of malonyl-CoA (fatty acid precursor). This is a very interesting topic that the authors addressed using a well-designed multidisciplinary experimental approach that includes bioinformatics, transcriptomics, targeted metabolomics, and a variety of biochemical and molecular in vitro and in vivo assay methodologies (Y1H, EMSA, DAP-seq assays etc.). The authors demonstrate that SlWARD1 is able to bind to the promoters of the SlASAT1 gene, which encodes the acyl transferase responsible for the initial acylation of the sugar core, and other genes coding for different subunits of the SlACCase responsible for the synthesis of malonyl-CoA, the common precursor of fatty acid biosynthesis, and activate their expression. By means of gene knock-out, VIGS- and RNAi-mediated gene silencing approaches they also demonstrate that the inactivation and/or down-regulation of SlWARD1 and selected SlACCase subunits leads to reductions in leaf acylsugars and malonyl-CoA contents without significantly altering acetyl-CoA levels. Evidence is also provided that this regulatory mechanism operates in other Solanaceae species such as S. pennelli and S. nigrum. Undoubtedly, the results reported in the manuscript can contribute significantly to expanding current knowledge on the molecular mechanism underlying the regulation of plant metabolism and, more specifically, the regulatory network that coordinates plant primary and specialized metabolism. Overall, it is a well-designed and technically sound work that addresses a topic of great scientific interest in the field of plant biochemistry and molecular biology.\nHowever there is a major point that needs to be addressed in order to demonstrate that SlWARD1 is truly involved in the coordinated regulation of both acylsugar and fatty acid biosynthesis, as the authors claim throughout the manuscript (e.g. lines 262, 319, 408, 415/416, etc.). The experimental evidence presented in the manuscript certainly points to an effect of SlWARD1 inactivation on fatty acid contents, but no direct evidence of such an effect is provided since fatty acid levels are not measured. The authors should do their best to demonstrate that inactivation of SlWARD1 actually translates into a reduction in fatty acid contents in addition to the reported negative effect on the levels of acylsugars and malonyl-CoA. The assumption that reduced levels of malonyl-CoA should lead to reduced levels of fatty acids is certainly a plausible hypothesis, but this has to be demonstrated experimentally. This targeted metabolomic analysis could be performed using well-established fatty acid profiling methods and the available slward1 CRISPR-Cas9 tomato mutant lines.\nThere are also some minor points to be addressed:\nThe discussion should be significantly shortened, particularly the first three paragraphs where there is a significant degree of redundancy both within the Discussion itself and with the Introduction. See, for example, the number of times sentences that refer to the proposed role of SlWARD1 as a link between primary metabolism (fatty acid synthesis) and specialized metabolism (acylated sugars synthesis) are included. Also, the last two paragraphs add relatively little to the article and could therefore be further summarized. In short, there is an excess of general comments at the expense of greater detail in the discussion of the results; for example, the rather unexpected lack of effect on acetyl-CoA levels in slward1 mutants is discussed in the Results but is not mentioned in the Discussion, and the apparent discrepancy between the SlWARD1 binding motif in the SlASAT1 promoter (Supplementary Figure S6) and the consensus SlWARD1 boxes identified from the DAP-seq analysis (Supplementary Figure S8) could be included as an additional factor to explain differences in signal intensity between ACCase and SlASAT1 gene promoters.\nPlease, make it clear throughout the manuscript (e.g. lines 180) that measurements of metabolites (acylated sugars) were performed in leaf tissue, not in isolated trichome preparations, as indicated on lines 308 and 735 regarding malonyl-CoA levels. In this context, in the legend of Supplementary Figure S2 (C) it is stated: representative LC-MS chromatograms comparing \"trichome metabolites\". It is a bit confusing since acylated sugars are indeed found in the trichomes but as far as I understand their levels were measured in whole leaf samples including trichomes.\nLine 172, replace \"genes\" with \"proteins\".\nLine 213, replace \"as\" with \"is\"\nLines 222 to 227, replace \"SlWARD1-upregulated\" with \"genes down-regulated in response to SlWARD1 inactivation\" in order to avoid confusion when reading the statement made in the main text and the information provided in Supplementary Fig S4B. In its present form the sentence can be misleading since the text reads \"SlWARD1-upregulated\" while panel B in Fig. S4 shows the pathways that are \"downregulated\" upon SlWARD1 inactivation.\nLine 253, indicate which \"proteins were purified\". I guess only His-SlWARD1 protein.\nLine 351, Fig. 5, G-I shows results of EMSA analysis and does not seem to match with the sentence on lines 349 to 351.\nSupplementary Figure S1, add \"compared to stems\" in the sentence \"the highest expression in trichomes, among its homologs\".\nLegend of Supplementary Figure S3, indicate what the acronym RFP stands for. Also SlWARD1 should be written in italics."}, "author_response": "Response to Reviews\nWe thank the reviewers for their valuable comments, which have made it possible\nfor us to significantly improve this manuscript. All comments have been addressed\nwith corresponding revisions or explanations.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe manuscript by He et al. study the WRI-like transcription factor WARD-1. The\nmanuscript provides a detailed molecular characterization of this metabolic\ntranscription factor demonstrating that it acts via a Dual mechanism i) upregulating\nexpression of the key enzyme SAT1 and ii) also upregulating several subunits of the\ncritical precusor providing ACCase enzyme. Interestingly, these results are not without\nparallel with the albeit artificial example of the purple tomato of Cathie Martin\ndisplaying a similar dual mechanism (see Yang et al. Nature Comms) - maybe\nworth discussing this.\nReturning to the paper of He et al., this presents well designed and executed\nexperiments which are excellently discussed highlighting but not overselling the\nimportance of their results. Moreover the Figures are exemplary. Indeed the entire\nmanuscript was a pleasure to read and I have no suggested improvements beyond the\ndiscretionary one that I mention above.\n1.1 Response: We sincerely thank the reviewer for the high evaluation of our work, the\ncompliments regarding our figures, and the thoughtful suggestion.\nWe agree that the dual regulatory mechanism of SlWARD1–coordinating both\nprecursor supply and downstream biosynthesis–bears a striking resemblance to the\n\"multi-level\" engineering strategy used to create high-phenylpropanoid tomatoes\n(Zhang et al., 2015). As suggested, we have revised the Discussion section (lines 477–\n482, and lines 521-522 in the revised version) to incorporate this parallel. We believe\nthis addition strengthens the manuscript by highlighting that the engineering logic used\nto optimize metabolic flux in the \"purple tomato\" parallels the evolutionary strategy\nthat plants have naturally deployed for specialized metabolite production. It also\nreinforces the broader concept that successful metabolic engineering—whether natural\nor synthetic—requires coordinated control over both precursor supply and downstream\nbiosynthetic machinery.\nReviewer #2 (Remarks to the Author):\nThis work by He et al. presented the identification of SlWARD1 transcription factor in\nSolanaceae, and elucidation of its role in regulation of synthesis of fatty acid and\nacylsugar. Overall, the manuscript presented very interest research work with\ngood novelty. The data in this study had high quality and the manuscript was written well.\nSuggestions are provided for further improvement of this manuscript as following.\nWe thank the reviewer for recognizing our study. According to the constructive\nsuggestions, we have performed additional experiments and made corresponding\nrevisions that significantly strengthened the quality of this manuscript.\nSuggestions:\n1. Different names of WRI-like [such as 'WRI-like' (e.g. line 1) and 'WRI1-like' (e.g.\nline 413)] were used throughout the whole manuscript, which triggered inconsistency\nand confusions for readers. Authors should keep the name consistent for this manuscript.\nIn addition, the correct abbreviation for 'WRINKLED1' is 'WRI1', while it was used as\n'WRI' (e.g. line 44). Given that SlWARD1 falls into same subclade of AtWRI3/4, it\nmight be more accurate to define SlWARD1 as a 'WRI3/4-like' candidate.\nAuthors need to check the whole manuscript carefully to keep the name consistent. In\nthe title of this manuscript, it is better to use full name for WRINKLED.\nIn addition, authors can consider to provide protein sequence alignment of these WRI-\nlike proteins in Solanaceae with Arabidopsis WRI1-4.\n2.1 Response: Following the reviewer’s, we have revised the manuscript to ensure\nconsistency and accuracy as follows:\na. We have systematically replaced all instances of \"WRI1-like\" with the more\nphylogenetically accurate term \"WRI3/4-like\" to reflect that SlWARD1 clusters with\nAtWRI3/AtWRI4 in the AP2 family phylogeny (Figure 7A, Supplementary Figure S1).\nThis classification is now applied consistently across the Abstract, Introduction, Results,\nDiscussion, and figure legends.\nb. We have corrected the abbreviation for \"WRINKLED1\" to the standard form \"WRI1\"\nacross the entire manuscript.\nc. Per your suggestion, we have updated the title to use the full name, changing \"WRI-\nlike\" to \"WRINKLED-like\" to ensure clarity for a broader audience.\nd. We have added a multiple sequence alignment of Arabidopsis WRI1-4 proteins and\nSlWARD1 homologs from six Solanaceae species (S. lycopersicum, S. pennellii, S.\nnigrum, S. quitoense, H. niger, and P. axillaris). This analysis, which highlights the\nconserved AP2 domain and predicted AW-box binding residues, is now presented in\nSupplementary Figure S12, with corresponding descriptions included in lines 408-418.\n2. Figure 5D. Authors did not find the expression change of SIBCCP1 between wild\ntype (WT) and slward1 mutant. Can authors identify AW-box in promoter of SIBCCP1\nor not? In addition, can SIWARD1 have an effect on the promoter activity of SIBCCP1\n(e.g. via dual-luc assay used in Figure 5F)? Some additional tests, investigations and\ndiscussions at this point here might be good for readers.(Can authors identify AW-box\nin promoter of SIBCCP1 or not)\n2.2 Response: We appreciate the reviewer’s insightful observation regarding SlBCCP1.\nTo answer your specific question: Yes, our DAP-seq data detected a SlWARD1 binding\npeak at the SlBCCP1 locus, spanning both its promoter and first exon—not limited to\nthe promoter alone. Two AW-box motifs (GGCGAGCAAAATGACG and\nGTCGATGAATCCAAGG) were identified within the peak regions of SlBCCP1. We\nhave clarified this in the revised manuscript. As the reviewer correctly notes, despite\nthis binding signal, SlBCCP1 expression shows no significant change in the slward1\nmutant in our trichome-specific RNA-seq and RT-qPCR analyses (Figure 5D). This\nsuggests that while SlWARD1 can bind to the SlBCCP1 locus, its regulatory role in\ntype I/IV trichomes may be non-essential or compensated for by other transcription\nfactors. In other words, since the ACCase complex contains two BCCP isoforms\n(SlBCCP1 and SlBCCP2), SlBCCP1 may provide basal ACCase activity, while\nSlBCCP2 represents the dynamic, SlWARD1-responsive component required for\nboosting fatty acid supply during acylsugar biosynthesis.\nIn this study, we primarily focused on SlWARD1’s regulation of SlBC, SlαCT, and\nSlBCCP2, which show significant expression changes, and did not emphasize SlBCCP1.\nIn the revised manuscript, we refrained from drawing definitive conclusions regarding\nwhether SlWARD1 directly and functionally regulates SlBCCP1 and adjusted the\ndiscussion accordingly.\n3. Question to EMSA experiments. (Using Figure 3 as an example)\na)authors need to provide the probe sequence information, with a highlight of AW-box.\nb)for mutated probe used in this study, please provide the sequence as well, with a\nhighlight of the mutated nucleotides.\nc) for competition assay, it is better to a gradual increase of unlabelled probe (currently\nthere is only one addition of unlabelled probe in this figure).\n2.3 Response: We agree that providing more detailed sequence information in the\nfigures will strengthen the clarity of our data. Accordingly, we have revised the\nmanuscript as follows:\nFor points (a) and (b), We have now provided the nucleotide sequences for the probes\nand mutant probes used in the EMSA assays, with the AW-box motifs clearly\nhighlighted. These sequences are provided in Supplementary Fig. S6D (to support Fig.\n3F–H) and Supplementary Fig. S11 (to support Fig. 5G–I). For point (c), following your\nrecommendation, we performed new competition EMSA assays using a concentration\ngradient of unlabeled competitor probes (50× and 500× excess) rather than a single\nconcentration. These results have been incorporated into the revised Figure 3F–H.\n4. Line 85, using 'APETALA2 (AP2)' instead of 'AP2', due to its first appearance. Line\n234, similar issue. Please provide full name for 'RT-qPCR '.\n2.4 Response: 2.4 Response: We have corrected these abbreviations as suggested.\n\"APETALA2 (AP2)\" is now defined at its first mention, and the full name for \"RT-\nqPCR\" has been added.\n5. Please provide full names of genes/proteins for their first appearance. For instance,\nBAHD (line 100), AP2/ERF (line 111), etc. Please check the whole manuscript carefully.\n2.5 Response: We have carefully reviewed the manuscript to ensure that all gene names,\nprotein families, and technical abbreviations are fully defined upon first appearance.\nFor BAHD, we have updated the text to define the BAHD family based on its founding\nenzymes (BEAT, AHCT, HCBT, and DAT), with proper reference cited. We have\nchecked the manuscript and ensured that full definitions are provided at their first\nappearance for other abbreviations cited throughout the text, including AP2/ERF,\nSlAACS1, SlAECH1, SlIPMS3, MAPK, as well as technical terms such as LC/MS and\nRNAi.\n6. Line 427, it will be good to cite Plant J (2016) 88(2):257-270.\n2.6 Response: We have added the suggested citation in the revised manuscript.\n7. Section of line 174-179. Why do authors show the 3 mutants of slward1 in two sets\nof figures (Fig. 2 and Supplementary Fig. S2), which is confusing.\n2.7 Response: We separated the presentation of these mutants to distinguish between\nstrong loss-of-function alleles and partial loss-of-function alleles.\nIn the main Figure 2, the mutant lines slward1#1 and slward1#2 carry disruptions to the\ntranscription start site or the conserved AP2 DNA-binding domain. They exhibit strong\nloss-of-function phenotypes (significantly reduced total acylsugar content and altered\nS4/S3 ratio). We prioritized these in the main figure as they provide the primary\nevidence for the essential role of SlWARD1.\nIn the Supplementary Fig. S2, the mutant slward1#3 harbors a small in-frame deletion\n(12 amino acids) and exhibits a much milder phenotype (altered S4/S3 ratio but normal\ntotal acylsugar content). This suggests the protein retains residual function.\nTo prevent confusion and keep the main figure focused on the loss-of-function\nphenotype, we placed the partial loss-of-function mutant (slward1#3) in the\nsupplementary data.\n8. Line 237, it should be 'N. benthamiana' instead of 'tobacco'.\n2.8 Response: We have revised \"tobacco\" to \"N. benthamiana\" throughout the\nmanuscript.\n9. Line 254, 'can binds to' needs to be changed to 'can bind to'. Authors need to check\nthe manuscript carefully to avoid this type of grammar errors.\n2.9 Response: We have corrected \"can binds to\" to \"can bind to\". Additionally, we have\nthoroughly proofread the entire manuscript to identify and fix similar grammatical error.\n10. Starting from Figure 2: Is it better to use 'WT' representing 'M82' for all figures,\nwhich should be easier for readers? (Then explain details for M82 wild type in Methods\nsection).\n2.11 Response: We agree that using \"WT\" improves clarity for the reader. We have\nreplaced \"M82\" with \"WT\" in Figure 2 and all subsequent figures. Accordingly, we\nhave specified in the Methods section (under Plant Materials and Growth Conditions)\nthat the wild-type used in this study is the M82 cultivar.\n11. Supplementary Figure S6. Please replace 'domain' by 'region' or 'sequence'.\n2.11 Response: We have adopted the suggested terminology and replaced the term\n\"domain\" with \"region\" in Supplementary Figure S6.\n12. Supplementary Figure S3B. 'SlWARD1-GFP fusion protein was transiently\nexpressed...' please use 'produced'.\n2.12 Response: We have adopted this suggestion. The text in the legend of\nSupplementary Figure S3B has been updated to state that the SlWARD1-GFP fusion\nprotein was \"produced.\"\n13. Line 275-276. It should be WRI1 transcription factor here (instead of 'WRI\ntranscription factor'). Authors also need to cite Maeo et al. 2009 plant j paper in this\nsentence for an accurate citation and presentation. (also please provide original\ncharacterization of AW-box sequence [CnTnG](n)7[CG] in Maeo et al. 2009 to help\nreaders see the information). From line 274 to 277, please include the information\nregarding what 'N' represents for. (I guess it represents any nucleotide; but authors need\nto get it clarified for readers in this manuscript).\n2.13 Response: We appreciate these specific suggestions to improve the accuracy of\nour background description. We have revised the text as follows:\na. We corrected \"WRI transcription factor\" to \"WRI1 transcription factor\"\nb. We added the citation for Maeo et al. (2009) as the primary reference for the AW-box\ncharacterization.\nc. We included the original AW-box consensus sequence [CNTNG](N)7[CG] and\nclarified that 'N' represents any nucleotide, ensuring the motif is clearly defined for\nreaders.\nReviewer #3 (Remarks to the Author):\nIn this manuscript He and co-workerss describe a comprehensive study leading to the\nidentification and functional characterization of a WRI-like transcription factor\n(SlWARD1), which is expressed in the tip cells of tomato leaf glandular trichomes and\nis involved in the coordinated regulation of acylated sugars (defensive metabolites)\nbiosynthesis and the synthesis of malonyl-CoA (fatty acid precursor). This is a very\ninteresting topic that the authors addressed using a well-designed multidisciplinary\nexperimental approach that includes bioinformatics, transcriptomics, targeted\nmetabolomics, and a variety of biochemical and molecular in vitro and in vivo assay\nmethodologies (Y1H, EMSA, DAP-seq assays etc.). The authors demonstrate that\nSlWARD1 is able to bind to the promoters of the SlASAT1 gene, which encodes the\nacyl transferase responsible for the initial acylation of the sugar core, and other genes\ncoding for different subunits of the SlACCase responsible for the synthesis of malonyl-\nCoA, the common precursor of fatty acid biosynthesis, and activate their expression.\nBy means of gene knock-out, VIGS- and RNAi-mediated gene silencing approaches\nthey also demonstrate that the inactivation and/or down-regulation of SlWARD1 and\nselected SlACCase subunits leads to reductions in leaf acylsugars and malonyl-CoA\ncontents without significantly altering acetyl-CoA levels. Evidence is also provided that\nthis regulatory mechanism operates in other Solanaceae species such as S. pennelli and\nS. nigrum. Undoubtedly, the results reported in the manuscript can contribute\nsignificantly to expanding current knowledge on the molecular mechanism underlying\nthe regulation of plant metabolism and, more specifically, the regulatory network that\ncoordinates plant primary and specialized metabolism. Overall, it is a well-designed\nand technically sound work that addresses a topic of great scientific interest in the field\nof plant biochemistry and molecular biology.\nWe thank the reviewer for recognizing our study. According to the constructive\nsuggestions, we have performed additional experiments and made corresponding\nrevisions that significantly strengthened the quality of this manuscript.\n1.However there is a major point that needs to be addressed in order to demonstrate that\nSlWARD1 is truly involved in the coordinated regulation of both acylsugar and fatty\nacid biosynthesis, as the authors claim throughout the manuscript (e.g. lines 262, 319,\n408, 415/416, etc.). The experimental evidence presented in the manuscript certainly\npoints to an effect of SlWARD1 inactivation on fatty acid contents, but no direct\nevidence of such an effect is provided since fatty acid levels are not measured. The\nauthors should do their best to demonstrate that inactivation of SlWARD1 actually\ntranslates into a reduction in fatty acid contents in addition to the reported negative\neffect on the levels of acylsugars and malonyl-CoA. The assumption that reduced levels\nof malonyl-CoA should lead to reduced levels of fatty acids is certainly a plausible\nhypothesis, but this has to be demonstrated experimentally. This targeted metabolomic\nanalysis could be performed using well-established fatty acid profiling methods and the\navailable slward1 CRISPR-Cas9 tomato mutant lines.\n3.1 Response: Thank you for this constructive feedback. We fully agree that directly\nmeasuring fatty acid levels in the slward1 mutants is essential to substantiate our claim\nregarding the coordinated regulation of both acylsugar and fatty acid biosynthesis,\nrather than inferring changes solely from the observed reduction in malonyl-CoA levels.\nFollowing your suggestion, we have performed additional metabolomic analyses to\nquantify fatty acids in the trichome-rich tissue (young leaves) from wild-type (M82)\nand slward1 mutant lines using an established GC-MS-based protocol. These new data,\npresented in Supplementary Figure S9B–D in the revised manuscript, demonstrate that\nseveral major fatty acid species—including C16:0, C16:3, and C18:0—show\nstatistically significant decreases in the mutants compared to wild-type plants. These\nresults provide direct experimental evidence that SlWARD1 inactivation indeed leads\nto reduced fatty acid accumulation in tomato leaves, supporting our hypothesis that\ndiminished malonyl-CoA levels translate into decreased fatty acid biosynthesis.\nWe have integrated these new findings into the relevant Results and Discussion sections\nand added a detailed description of the analytical method used.\nThere are also some minor points to be addressed:\n2.The discussion should be significantly shortened particularly the first three\nparagraphs where there is a significant degree of redundancy both within the Discussion\nitself and with the Introduction. See, for example, the number of times sentences that\nrefer to the proposed role of SlWARD1 as a link between primary metabolism (fatty\nacid synthesis) and specialized metabolism (acylated sugars synthesis) are included.\nAlso, the last two paragraphs add relatively little to the article and could therefore be\nfurther summarized. In short, there is an excess of general comments at the expense of\ngreater detail in the discussion of the results; for example, the rather unexpected lack\nof effect on acetyl-CoA levels in slward1 mutants is discussed in the Results but is not\nmentioned in the Discussion, and the apparent discrepancy between the SlWARD1\nbinding motif in the SlASAT1 promoter (Supplementary Figure S6) and the consensus\nSlWARD1 boxes identified from the DAP-seq analysis (Supplementary Figure S8)\ncould be included as an additional factor to explain differences in signal intensity\nbetween ACCase and SlASAT1 gene promoters.\n3.2 Response: We appreciate the reviewer’s constructive criticism regarding the\nstructure and focus of the Discussion. We agree that the original text was overly\nrepetitive and have extensively revised the section to improve its flow and scientific\ndensity. We have significantly condensed the first three paragraphs to eliminate\nredundant statements regarding the link between primary and specialized metabolism,\nand we have similarly streamlined the final two paragraphs to focus only on essential\nimplications.\nCrucially, we used the space gained from these cuts to incorporate the specific data\ninterpretations suggested by the reviewer. We have added a focused discussion\naddressing the unchanged acetyl-CoA levels in the slward1 mutants despite the changes\nin downstream metabolites. Furthermore, regarding the comparison between the\nSlASAT1 binding motif and the DAP-seq consensus. We note that when considering\nboth sense and antisense orientations, the SlASAT1 motif aligns well—although not\nperfect—with the consensus SlWARD1 binding box. In the revised Discussion, we\nhave included this observation and refined the hypothesis to attribute the variation in\nDAP-seq signal intensity to the inherent differences between in vitro assays and the\nnative cellular context, rather than a fundamental discrepancy in motif architecture.\n3.Please, make it clear throughout the manuscript (e.g. lines 180) that measurements of\nmetabolites (acylated sugars) were performed in leaf tissue, not in isolated trichome\npreparations, as indicated on lines 308 and 735 regarding malonyl-CoA levels. In this\ncontext, in the legend of Supplementary Figure S2 (C) it is stated: representative LC-\nMS chromatograms comparing \"trichome metabolites\". It is a bit confusing since\nacylated sugars are indeed found in the trichomes but as far as I understand their levels\nwere measured in whole leaf samples including trichomes.\n3.3 Response: Thank you for raising this important point regarding sample description.\nWe apologize for the confusion and appreciate the opportunity to clarify this\nmethodological detail. You are correct that all metabolite measurements—including\nacylsugars and malonyl-CoA—were performed on trichome-rich, newly expanded leaf\ntissue rather than on isolated or purified trichomes, as it is not technically feasible to\ncollect sufficient quantities of trichomes for comprehensive metabolomic analyses.\nWe have carefully revised the manuscript to ensure clarity and consistency throughout.\nSpecifically, we have replaced ambiguous terms such as \"trichome metabolites\" with\nmore precise descriptions indicating that measurements were conducted on \"trichome-\nrich young leaf tissue\". For example, in the legend of Supplementary Figure S2C, the\noriginal phrase \"trichome metabolites\" has been updated to \"leaf surface metabolites\nextracted from trichome-rich young leaf tissue of M82 and slward1#3 plants.\" Similar\nrevisions have been made throughout the Results and Methods sections to maintain\nconsistency and accuracy in our sample descriptions.\n4.Line 172, replace \"genes\" with \"proteins\".\n3.4 Response: We have adopted this correction and replaced “genes” with “proteins”\nto ensure accurate terminology.\n5.Line 213, replace \"as\" with \"is\".\n3.5 Response: We have corrected the error, replacing “as” with “is”.\n6.Lines 222 to 227, replace \"SlWARD1-upregulated\" with \"genes down-regulated in\nresponse to SlWARD1 inactivation\" in order to avoid confusion when reading the\nstatement made in the main text and the information provided in Supplementary Fig\nS4B. In its present form the sentence can be misleading since the text reads \"SlWARD1-\nupregulated\" while panel B in Fig. S4 shows the pathways that are \"downregulated\"\nupon SlWARD1 inactivation.\n3.6 Response: We agree that the original phrasing \"SlWARD1-upregulated\" could\ncause confusion when readers compare the main text with Supplementary Figure S4B,\nwhich displays pathways that are downregulated upon SlWARD1 inactivation. As\nsuggested, we have revised the text by replacing \"SlWARD1-upregulated\" with \"genes\ndownregulated in response to SlWARD1 inactivation\" to ensure consistency and clarity\nbetween the text description and the figure presentation.\n7.Line 253, indicate which \"proteins were purified\". I guess only His-SlWARD1 protein.\n3.7 Response: We have revised the text to explicitly state that the recombinant His-\nSlWARD1 protein was purified for the EMSA assays, along with the His-pCold protein\nwhich served as the negative control.\n8.Line 351, Fig. 5, G-I shows results of EMSA analysis and does not seem to match\nwith the sentence on lines 349 to 351.\n3.8 Response: Thank you for your careful review. We have checked our original\ndescription against the corresponding figure (Fig. 5G–I) and recognize that the\nconfusion likely arose because the graphic illustration above the EMSA gel images,\nwhich depicts the predicted AW-box motifs identified within the binding regions, was\ntoo small to be clearly interpreted.\nTo provide readers with a clearer and more comprehensive understanding of our\nexperimental design, we have introduced a new Supplementary Figure S11. This figure\nexplicitly maps the predicted AW-box motifs within the identified binding peaks,\nprovides the exact nucleotide sequences, and illustrates the specific probe designs used\nfor the EMSA experiments. We believe this additional detail clarifies the experimental\nlogic and directly links the described motifs to the EMSA results shown in Figure 5.\n9.Supplementary Figure S1, add \"compared to stems\" in the sentence \"the highest\nexpression in trichomes, among its homologs\".\n3.9 Response: We have updated the legend of Supplementary Figure S1 to include the\nphrase \"compared to stems,\" which clarifies the context of the relative expression levels\nas suggested.\n10.Legend of Supplementary Figure S3, indicate what the acronym RFP stands for. Also\nSlWARD1 should be written in italics.\n3.10 Response: We have revised the legend of Supplementary Figure S3 to explicitly\ndefine the acronym RFP (Red Fluorescent Protein) and have italicized SlWARD1\nthroughout the text to ensure consistent nomenclature formatting."}, {"round": 2, "reviewer_comments": {"reviewer_2": "Authors have carefully revised their manuscript. I am pleased to see the progress of this revised version and my questions are satisfactorily addressed.\nOne minor comment: For AW-box sequence, please use '[CnTnG](n)7[CG]' (keeping the format of the sequence consistent to the Plant J (2009) 60(3):476-87 paper).", "reviewer_3": "I truly appreciate the efforts made by the authors to address all the comments made by the reviewers for the benefit of the manuscript's quality."}, "author_response": "Response to the Reviewers’ Comments\nReviewer #2 (Remarks to the Author):\nAuthors have carefully revised their manuscript. I am pleased to see the progress of this\nrevised version and my questions are satisfactorily addressed.\nOne minor comment: For AW-box sequence, please use '[CnTnG](n)7[CG]' (keeping\nthe format of the sequence consistent to the Plant J (2009) 60(3):476-87 paper).\n3. Response: We have revised the AW-box motif to the format '[CnTnG](n)7[CG]'\nas requested, to align with the cited Plant Journal (2009) paper.\nReviewer #3 (Remarks to the Author):\nI truly appreciate the efforts made by the authors to address all the comments made by\nthe reviewers for the benefit of the manuscript's quality."}]

Decision letter