Transcription factors SlMYB41, SlMYB92, and SlWRKY71 regulate gene expression in the tomato exodermis

Abstract

Root barrier cell types, such as the endodermis and exodermis, are crucial for plant acclimation to environmental stresses. Deposition of suberin, a hydrophobic polymer, in these cell layers restricts the movement of molecules and plays a vital role in stress responses. This study investigates the role of SlMYB41, SlMYB92, and SlWRKY71 transcription factors (TFs) in regulating suberin biosynthesis in the tomato (Solanum lycopersicum) root exodermis by genetic perturbation. Genetic perturbation of these TFs altered exodermal suberin deposition patterns, indicating the SlMYBs as positive regulators and SlWRKY71 as a negative regulator of suberization. RNA sequencing revealed a significant overlap between differentially expressed genes regulated by these TFs, suggesting a shared regulatory network. Gene set enrichment analyses highlighted their role in lipid and suberin biosynthesis as well as over-representation of exodermis-enriched transcripts. Furthermore, transactivation assays demonstrated that these two MYBs promote the expression of suberin-related genes, while SlWRKY71 represses them. These results indicate a complex antagonistic relationship, advancing our understanding of the regulatory mechanisms controlling exodermis suberization in tomato roots.

MYB and WRKY transcription factors collaboratively regulate suberin biosynthesis in the tomato root exodermis. Antagonistic interactions may fine-tune suberization or act as a brake on overaccumulation.

Introduction

Root barrier cell types emerged as an evolutionary innovation that allows plants to interact with and acclimate to their local environment. The endodermis and exodermis are known as two very important root barrier cell types that control the movement of molecules and solutes into the roots and their exudation into the environment of the plant (Liu and Kreszies, 2023). The endodermis is the innermost ground tissue cell layer localized next to the pericycle, and it is found in all vascular plants, while the exodermis is the outermost cortex layer found beneath the epidermis in some species (Artur and Kajala, 2021). Both the endodermis and exodermis gain their barrier functions from deposition of lignin and/or suberin into their cell walls (Barberon, 2017; Liu and Kreszies, 2023). In the endodermis, lignin deposition takes place in the form of the Casparian strip (Andersen and Drapek, 2024), whereas exodermal lignin can have many deposition patterns (Cantó-Pastor et al., 2025; Manzano et al., 2025). In both cell types, suberin is deposited as lamellae between the plasma membrane and the primary cell wall. The suberization of barrier cell types is plastic under a wide range of abiotic and biotic cues such as osmotic, salinity, drought, nutrients, pathogens, and the soil microbiome (Ranathunge et al., 2011; Barberon et al., 2016; Kreszies et al., 2020; Feng et al., 2022; Lu et al., 2022; Su et al., 2023; Kawa et al., 2024). Specifically, exodermis suberization contributes to the plant drought resilience (Cantó-Pastor et al., 2024) and can act as a radial oxygen loss barrier during flooding (Ejiri and Shiono, 2019). This highlights the adaptive importance of the root barriers and their dynamic regulation.

Some of the key regulators of suberin are the MYB (myeloblastosis) transcription factors (TFs). AtMYB41 was found to induce the ectopic expression of suberin biosynthesis genes and deposition of suberin-like lamellae in Arabidopsis and Nicotiana benthamiana leaves (Kosma et al., 2014). AtMYB39 and AtMYB92 were also shown to enhance suberin lamellae deposition when heterologously expressed in N. benthamiana leaves (Cohen et al., 2020; To et al., 2020). In Arabidopsis root endodermis, four MYB TF genes (AtMYB41, AtMYB53, AtMYB92, and AtMYB93) act redundantly to regulate suberization (Shukla et al., 2021). MYBs have also recently been shown to be involved in regulation of suberin deposition in other plant species and organs, for example in rice roots (Huang et al., 2024), cork oak (Capote et al., 2018), sugar cane (Figueiredo et al., 2020), kiwi fruits (Wei et al., 2020a, b; Han et al., 2022), apple fruit skins (Legay et al., 2016; X. Xu et al., 2022), grapevine roots in response to drought (Zhang et al., 2020), and tomato and russet apple fruit surfaces and Arabidopsis seeds (Lashbrooke et al., 2016). Hand-in-hand with this extensive body of multiple MYBs regulating suberin across angiosperms, the binding of MYB107 (and probably other MYBs of the same clade) to the promoters of suberin biosynthesis genes was recently shown to be highly conserved across angiosperm DAP-seq (DNA affinity purification sequencing) data (Baumgart et al., 2024, Preprint). Given that there are multiple MYBs that regulate suberin, the question arises of why so many are needed and what are their relative contributions. It has been proposed that in Arabidopsis endodermis, MYBs form multiple transcriptional subregulatory networks to regulate suberization (H. Xu et al., 2022).

Together with MYBs, WRKY TFs (named after their conserved N-terminal WRKYGQK motif) are emerging as potentially important regulators of suberin biosynthesis in Arabidopsis root endodermis. Arabidopsis AtWRKY33 and AtWRKY9 have been shown to be involved with suberization of root endodermis, conferring salt stress tolerance (Krishnamurthy et al., 2020, 2021). Still, little is known about the participation of WRKY TFs in MYB-centered gene regulatory modules regulating suberin biosynthesis in the Arabidopsis endodermis and other plant species and cell types.

Although there is increasing information about the regulation of suberin biosynthesis in different plant organs, especially in Arabidopsis endodermis, little is known about the molecular mechanisms controlling exodermis suberization, despite its importance for plant physiology and root plasticity. This is mainly due to the absence of an exodermis in the roots of the model plant Arabidopsis (Liu and Kreszies, 2023), but recently advances have been made in rice (Oryza sativa) and tomato (Solanum lycopersicum). Shiono et al. (2014) investigated gene expression during the formation of the rice exodermal radial oxygen loss barrier and identified several TFs, including MYBs and WRKYs, as potential regulators of the suberin biosynthesis genes in rice exodermis. Then, Reynoso et al. (2022) identified that water deficit-promoted suberin regulatory network gene promoters in rice were enriched in MYB and NAC TF-binding sites (TFBSs). For tomato, Kajala et al. (2021) identified exodermis-enriched gene expression patterns including genes coding for SlWRKY71 (encoded by Solyc02g071130.3.1) and SlMYB41 (encoded by Solyc02g079280.3.1). Most recently, a set of tomato MYB TFs (SlMYB41 and SlMYB92, encoded by Solyc05g051550.2.1, and SlMYB63 encoded by Solyc10g005550.3.1) were shown to be necessary for suberin deposition in the exodermis, and that the suberization contributes to the tomato drought tolerance (Cantó-Pastor et al., 2024). Based on these findings, we set out to investigate the roles and downstream targets of SlMYB41, SlMYB92, and SlWRKY71 in regulating tomato exodermis suberin.

Hence, we investigated whether SlMYB41, SlMYB92, and SlWRKY71 regulate suberin biosynthesis genes using overexpression (OX) and CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] knockout (ko) mutants in tomato hairy root lines. In line with Cantó-Pastor et al. (2024), we observed reduced suberin in Slmyb41-ko and Slmyb92-ko lines. The overexpression of the MYBs had no effect on suberin. In contrast, SlWRKY71-OX reduced exodermal suberin deposition. Moreover, transcriptome analysis of these lines combined with transactivation assay data suggest that SlMYB41 and SlMYB92 act as positive regulators, while SlWRKY71 functions as a repressor of suberin biosynthesis. Altogether, an interplay between these three TFs fine-tunes the expression of suberin biosynthesis genes in the tomato root exodermis.

Materials and methods

Phylogenetic tree construction

We retrieved the AGI locus codes and nomenclature of Arabidopsis MYBs with described functions associated with suberization (Kosma et al., 2014; Du et al., 2015; Lashbrooke et al., 2016; Gou et al., 2017; Cohen et al., 2020; Shukla et al., 2021; H. Xu et al., 2022) from Dubos et al. (2010). For WRKYs, we retrieved AGI codes and nomenclature of Arabidopsis WRKY family members described by Eulgem et al. (2000) and Wu et al. (2005), and we searched in the literature for those with described functions associated with suberization in multiple plant species (Supplementary Table S1). Using the gene identifiers, we queried the primary protein sequences of MYBs and WRKYs in Phytozome 13 (https://phytozome-next.jgi.doe.gov/) using sequences from the Arabidopsis thaliana genome TAIR10 (Goodstein et al., 2012). For each TF family, multiple sequence alignment was performed using the MAFFT online server (https://mafft.cbrc.jp/alignment/server/) utilizing the standard automated method with the L-INS-i model. We used the IQ-TREE webserver (http://iqtree.cibiv.univie.ac.at/) to infer a maximum likelihood tree with 1000 bootstraps, and we edited the phylogenetic tree using iTOL (Letunic and Bork, 2016).

DNA constructs for gene editing and overexpression

Transcriptional reporter construct cloning and imaging were performed as described in Kajala et al. (2021), and tomato CRISPR/Cas9 construct cloning, mutant line generation, and analyses according to Cantó-Pastor et al. (2024). We generated the overexpression lines by retrieving the coding sequence (CDS) of SlMYB41 (Solyc02g079280), SlMYB92 (Solyc05g051550.2.1), and SlWRKY71 (Solyc02g071130.3.1) from the Sol Genomics database (https://solgenomics.net; ITAG3.2). RNA was extracted and cDNA was synthesized from tomato (cultivar M82) as described by Kajala et al. (2021). We amplified the CDS without the stop codon using the primers listed in Supplementary Table S2, purified the PCR products from the agarose gel using the QIAquick Gel Extraction kit, and introduced the cleaned fragments into the pENTR/D-TOPO vector (Invitrogen) following the manufacturer’s instructions. The previously described 35S promoter (Ron et al., 2014) was cloned into the pENTR5'/TOPO (Invitrogen). We synthesized the 3×FLAG oligodimer using the primers listed in Supplementary Table S2 and introduced it into the pENTR/D-TOPO vectors containing the CDSs to generate CDS-3×FLAG constructs using restriction site-based cloning. To generate the final 35S::CDS-3×FLAG binary vectors, the promoters and CDS–3×FLAG fusions were recombined into the Multisite Gateway vector pK7m24GW (https://vectorvault.vib.be/collection) using LR Clonase II Enzyme mix (Invitrogen). All constructs generated were confirmed by Sanger sequencing.

For the knockdown constructs of SlWRKY71, target guide RNAs were generated using the CRISPR-P (http://crispr.hzau.edu.cn/CRISPR2/) and CHOPCHOP (https://chopchop.cbu.uib.no/) web tools. Their specificity was accessed by blasting the core sequence of the guide RNA into the tomato ITAG4.0 genome version in phytozome (https://phytozome-next.jgi.doe.gov/). Oligos for two guide RNAs per target (Supplementary Table S3) were synthesized and cloned into pMR217 and 218 vectors, and assembled with Gateway into the binary vector pMR286 as described earlier by Cantó-Pastor et al. (2024) (based on Fauser et al., 2014 and Bari et al., 2019). The final CRISPR constructs were then introduced to Rhizobium rhizogenes (strain ATCC15834) and used for the subsequent hairy root transformation steps.

Hairy root cultures and tomato transformation

Hairy root cultures were generated following the protocols in Ron et al. (2014) as follows. To generate competent R. rhizogenes (strain ATCC15834) cells, they were grown in MG/L medium (Ron et al., 2014) at 28–29 °C for 24–30 h to a final optical density (OD₆₀₀) of 0.5–0.7. The cells were cooled on ice for 15 min, spun at 50 g for 10 min, and then resuspended in 0.01× of the culture volume of 10% glycerol. The aliquots were then immediately frozen in liquid nitrogen and stored at –80 °C until use. For transformation, we incubated R. rhizogenes aliquots on ice with 1 ml of the desired plasmid DNA construct prior to electroporation. Then, cells were immediately transferred to 1 ml of MG/L medium and were shaken at 28–29 °C for 3 h. We then plated the whole aliquot on MG/L medium with agar containing the appropriate antibiotics and incubated it for 3 d at 28–29 °C.

To generate tomato hairy root cultures, we took the following steps: tomato seeds (S. lycopersicum cv. M82, LA3475) were sterilized with 70% ethanol for 10 min, rinsed with sterile MQ water, soaked in 50% commercial bleach for 10 min, and rinsed three times with sterile MQ water. The seeds were plated on Murashige and Skoog (MS) medium supplemented with 1% sucrose and incubated under 16 h light:8 h dark for 7–10 d. We cut the expanded cotyledons in sterile conditions with a scalpel and immediately immersed them in the desired R. rhizogenes suspension for 20 min, blotted them on sterile Whatman filter paper, and transferred them to MS plates supplemented with 3% sucrose without antibiotics for 3 d at 22–25 °C in the dark. After 3 d, the explants were transferred bottom-side up to MS medium supplemented with 3% sucrose, 200 mg l^–1 cefotaxime, and the desired antibiotics for transformant selection, and incubated at 22–25 °C until roots started to emerge. We transferred roots of ∼1 cm length onto the same medium, and maintained the cultures by transferring 3–4 cm root segments onto fresh plates every 2–3 weeks. After two rounds of culturing hairy roots, they were placed onto medium without antibiotics.

The hairy root cultures were validated for successful gene editing events and protein overexpression. To identify the deletions in the target genes, we amplified the genomic fragment of SlWRKY71, and identified the deleted region through Sanger sequencing (Supplementary Fig. S1). We validated the correct protein expression of the hairy root lines overexpressing SlMYB41, SlMYB92, and SlWRKY71 through a western blot experiment. After extracting total protein lysates from the hairy root samples, the lysates were boiled for 1 min at 96 °C and loaded on a 4–15% mini-PROTEAN TGX stain-free protein gel (Bio-Rad). We transferred the proteins onto a polyvinylidene difluoride (PVD) membrane using a Bio-Rad transblot turbo and we blocked the blots with 5% (w/v) milk powder (Elk) in Tris-buffered saline (TBS) buffer. The proteins were detected using the monoclonal antibody ANTI-FLAG M2 [Sigma no. F1804, 1:1000 in 0.5% (w/v) Elk TBS]. We used the goat anti-mouse IgG conjugated with horseradish peroxidase as secondary antibody [Cell Signaling Technology no. 7076, 1:2500 in 0.5% (w/v) Elk TBS plus 0.1% (v/v) Tween-20]. The labeled proteins were visualized using a 50/50 mix of chemiluminescence substrates (cat no. 34096, Thermo Fisher Scientific) on a ChemiDoc Imaging system (Bio-Rad).

Expression maps

The spatial expression profiles of SlMYB41, SlMYB92, and SlWRKY71 were created using the R package ggPlantmap (Jo and Kajala, 2024). The expression data used for plots were obtained from the tomato translatome dataset from Kajala et al. (2021). Data plotted are from TRAP marker lines driven by the following promoters: epidermis AtWER, exodermis SlPEP, inner cortex AtPEP, endodermis SlSCR, xylem AtS18, and phloem AtS32.

Suberin and lignin histochemistry and imaging

We prepared root cross-sections of hairy root subcultures of the control (empty vector), 35S::MYB41-3×FLAG (SlMYB41-OX), 35S::MYB92-3×FLAG (SlMYB92-OX), 35S::WRKY71-3×FLAG (SlWRKY71-OX), Slmyb41-ko, Slmyb92-ko, Slwrky71-ko(4), and Slwrky71-ko(5) that had grown for 3 weeks at 20 °C. Newly emerged roots were selected from the previous subculture. Of those selected roots, 2 cm sections located in regions with newly emerging lateral roots were harvested. After harvest, we fixed the roots by vacuum infiltration with 4% paraformaldehyde (Thermo Scientific) for 1 h. After that, the roots were placed in ClearSee (Ursache et al., 2018) for a minimum of 3 d. We embedded the cleared and harvested pieces in 3.5% agarose (Fisher Scientific) and prepared 250 µm thick sections using the VT 1000 S vibratome (Leica). To visualize suberin, the cross-sections were incubated on a shaker in the dark for 20 min in Fluorol Yellow 088 (Chem Cruz, Santa Cruz Biotechnology Inc.) [FY: 0.01% w/v in 96% ethanol, first dissolved in 1 ml of DMSO (0.5% (w/v)]. After that, counterstained the sections were using aniline blue (Sigma Aldrich) (0.5% w/v, dissolved in water) and placed on a shaker in the dark for 20 min at room temperature. We rinsed the sections with MQ water until the excess aniline blue was completely washed off and kept them in 50% glycerol until imaging. To visualize lignin, we stained the cross-sections with basic fuchsin [BF; 0.2% w/v, dissolved in ClearSee (Ursache et al., 2018)] and Calcofluor White [CW; 0.1% w/v, dissolved in ClearSee). The sections were first stained with BF on a shaker in the dark for 20 min at room temperature. The BF was rinsed off with four washes of ClearSee of 15 min each. Afterwards, the sections were stained with CW and the excess was washed off with ClearSee. After this final wash of 30 min, we kept the stained sections in 50% glycerol until imaging. We imaged all stained samples on the same day using the Zeiss Airyscan LSM 880. Samples stained with FY were imaged using 488 nm excitation and a 500–550 nm detection range. Samples stained with BF and CW were imaged using an excitation range of 561 nm and detection range of 600–650 nm for BF, and an excitation range of 405 nm and a detection range of 425–475 nm for CW.

RNA sequencing tissue harvest, library preparation, and sequencing

To collect the material for RNA-seq, we subcultured the hairy roots of lines of the control (empty vector), 35S::MYB41-3×FLAG (SlMYB41-OX), 35S::MYB92-3×FLAG (SlMYB92-OX), 35S::WRKY71-3×FLAG (SlWRKY71-OX), Slmyb41-ko, Slmyb92-ko, Slwrky71-ko(4), and Slwrky71-ko(5) on sterile square Petri dishes (120×120×17 mm, Greiner Bio One) on MS medium (Duchefa Biochemie) supplemented with 3% sucrose, 200 mg l^–1 cefotaxime sodium (Duchefa Biochemie), 1% BD Difco agar (Fisher Scientific), and 0.5 g l^–1 MES hydrate (Thermo Fisher Scientific), and grew them at 25 °C in the dark. After 9 d of growth, the first centimeter of 10 root tips for each of the three biological replicates per line were collected and snap-frozen in liquid nitrogen. The samples were collected in 2 ml tubes containing ceramic beads and ground using a tissue lyser (Retsch MM300). The mRNA extraction and library preparation were performed according to the non-strand-specific random primer-primed RNA-seq library protocol of Townsley et al. (2015). The libraries were pair-end sequenced (50 bp) at Utrecht Sequencing Facility (Utrecht, The Netherlands) using Illumina NextSeq2000.

Processing of RNA sequencing data

We quality trimmed paired-end reads using Trim Galore (version 0.6.6) (Krueger, 2015) using default settings. After trimming, the paired reads were pseudo-aligned to the tomato reference transcriptome version ITAG4.1 (Sol Genomics, https://solgenomics.net) using Kallisto (version 0.46.2) (Bray et al., 2016) with the following parameters: -b 100. Pseudo-counts were then used for differential expression analysis. All scripts used for the trimming and alignment can be found in https://github.com/leonardojo/tomato-TF-exodermis-2024.

Differential expression analysis

Differentially expressed genes (DEGs) were identified using the R package limma (Ritchie et al., 2015) according to Kajala et al. (2021) (https://github.com/leonardojo/tomato-TF-exodermis-2024). We set the threshold of the adjusted P-value at <0.05 for DEG identification. To obtain the DEGs, we compared each mutant and overexpressor with the empty vector control hairy roots (Supplementary Dataset S1). To identify candidate genes for transactivation assays, we compiled an overview file (Supplementary Dataset S2) where our DEGs were cross-referenced with exodermis-enriched transcripts (Kajala et al., 2021) and a suberin module from tomato single-cell data (Cantó-Pastor et al., 2024). We selected genes that were exodermis enriched, good candidates for suberin barrier formation, or DEGs in our data.

Gene Ontology enrichment analysis

We tested our DEG lists for Gene Ontology (GO) term enrichment using the R package goseq (Young et al., 2010). GO functional annotation was extracted for ITAG4.0 from Phytozome 13 (https://phytozome-next.jgi.doe.gov/). A q-value threshold of 0.05 was used to determine the enriched GO categories in each DEG list.

Identification of tomato suberin biosynthesis genes

To define the list of fatty acid (FA) and suberin biosynthesis genes in tomato, we used the Tomato Plant Metabolic Network database, TOMATOCYC 4.0 (https://plantcyc.org/databases/tomatocyc/4.0), and identified the tomato genes associated with the following pathways and reactions: very-long-chain fatty acid biosynthesis I, very-long-chain fatty acid biosynthesis II, suberin monomer biosynthesis, and esterified suberin biosynthesis.

DNA constructs for trans-activation assay: promoters

Tomato genomic DNA was extracted from leaves of S. lycopersicum cultivar M82 and used it as template. We used PCRBIO HiFi Polymerase (PCR Biosystems Ltd) to amplify the promoter regions of the genes using the primers listed in Supplementary Table S4. The PCR products were gel-purified using the kit NucleoSpin Gel and PCR Clean-up (MACHEREY-NAGEL). We cloned all the promoters, except for Solyc02g071130.3.1, directly into the destination vector pDLUC15 (Jo et al., 2020) using the NEBuilder® HiFi DNA Assembly Cloning Kit (NEB, England) after digesting it with the enzyme FastDigest HindIII (Thermo Fisher Scientific). Instead, we cloned the promoter sequence of Solyc02g071130.3.1 first into the Gateway vector pDONR207 (Thermo Fisher Scientific) using the Gateway BP clonase II enzyme mix (Thermo Fisher Scientific) and subsequently into the Gateway vector pGWL7 (VIB, Belgium) using the Gateway LR clonase II enzyme mix (Thermo Fisher Scientific). We used all the cloning reactions to transform the NEB Stable Competent Escherichia coli (High Efficiency) (NEB, England). The transformed cells were left to recover for 1 h at 30 °C in shaking conditions and then plated on LB plates containing the appropriate selecting antibiotic. We extracted the plasmid DNA samples from the colonies using the QIAprep® Spin Miniprep kit (Qiagen) and sequence-verified them using the Sanger method (Macrogen, The Netherlands). The sequences of the promoters cloned in this study are listed in Supplementary Table S5.

DNA constructs for trans-activation assay: transcription factors

RNA was extracted from leaves of S. lycopersicum cultivar M82 using the RNeasy Mini kit (Qiagen) followed by DNase I (Thermo Fisher Scientific) treatment. We synthesized cDNA using the RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific) together with random hexamer primers (Thermo Fisher Scientific). The CDS of SlWRKY71 was amplified using this cDNA as template based on the gene model information of version ITAG2.4 (Supplementary Fig. S1), while the CDSs of SlMYB92 and SlMYB41 were amplified from the plasmid pENTR/D-TOPO previously cloned for overexpression. We amplified the CDS with Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) and the primers listed in Supplementary Table S6. The PCR products were recombined into the Gateway vector pDONR221 (Thermo Fisher Scientific) using the Gateway BP clonase II enzyme mix (Thermo Fisher Scientific) and subsequently into the Gateway vector p2GW7 (Karimi et al., 2002) using the Gateway LR clonase II enzyme mix (Thermo Fisher Scientific). The reactions were transformed into competent cells of E. coli DH5α. The transformed cells were left to recover for 1 h at 37 °C in shaking conditions and then plated on LB plates containing the appropriate selecting antibiotic. The plasmid DNA samples were extracted and sequence-verified as described above.

Trans-activation assays

The material for protoplast-based trans-activation assays was grew as follows. We stratified A. thaliana Col-0 plants for 4 d on soil:perlite mix 1:2 (Primasta BV, Asten, The Netherlands) and then moved them to a growth chamber under short-day conditions (8 h light, 16 h dark; 20 °C; 70% humidity) for a week to allow germination. The plants were then transplanted and left to grow for 3 weeks. We isolated leaf mesophyll protoplasts using the tape sandwich method (Wu et al., 2009) and incubated the tapes containing the tissues in an enzymatic solution [1% cellulase R-10 (Duchefa Biochemie), 0.25% macerozyme R-10 (Duchefa Biochemie), 0.4 M mannitol, 20 mM KCl, 20 mM MES pH 5.8, 10 mM CaCl₂, 5 mM β-mercaptoethanol, 0.1% BSA] in the dark with gentle agitation for 2 h at room temperature. We filtered the protoplasts through a 100 µm nylon mesh, washed them twice in W5 buffer (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 2 mM MES pH 5.8), and incubated them on ice for 30 min. We determined the protoplast number using a hemocytometer. Finally the protoplasts were resuspended in MMg solution (0.4 M mannitol, 15 mM MgCl₂, 5 mM MES pH 5.8). For each biological replicate, we transfected 1–5×10⁵ protoplasts with 10 μg of pDLUC15 plasmid carrying the promoter sequence in combination with 3–5 μg of the vectors expressing the CDS of the TFs of interest by adding a polyethylene glycol (PEG) solution (40% PEG 4000, 0.2 M mannitol, 0.1 M CaCl₂). The reactions were incubated for 5 min and the transfections were stopped with W5 buffer, and washed twice with W5 buffer. After transfection, the protoplasts were incubated for 16–18 h at room temperature in the dark, spun down, and the supernatant was removed. We then detected the activity of the firefly and Renilla luciferases with the dual-luciferase reporter assay system (Promega) and the GloMax 96 Microplate Luminometer (Promega). We assayed two technical replicates for each biological replicate. The statistical analysis was done as described in the figure legends.

Results

We explored the regulatory interactions that tomato exodermis-enriched TFs have with their target genes in order to understand the suberin regulatory module in the exodermis. We selected three TFs, SlMYB92, SlMYB41, and SlWRKY71, as targets for this study, identified as being enriched in the exodermis based on tomato root gene expression profiling by Cantó-Pastor et al. (2024) and Kajala et al. (2021) (Fig. 1A) and as regulators of suberin levels (SlMYB92 and SlMYB41; Cantó-Pastor et al., 2024). Using a transcriptional green fluorescent protein (GFP) reporter line we confirmed that SlMYB92 is expressed only in the exodermis layer of tomato hairy roots (Fig. 1B; Cantó-Pastor et al., 2024), while exodermis-specific promoter activities of SlMYB41 and SlWRKY71 were previously demonstrated in Kajala et al. (2021).

Fig. 1.

SlMYB92, SlMYB41, and SlWRKY71 are exodermis-enriched TFs with homologs with roles in regulating suberin. (A) Cell type-enriched gene expression heatmaps for SlMYB92, SlMYB41, and SlWRKY71 of translatome data from Kajala et al. (2021). (B) Transcriptional fusion proSlMYB92:nlsGFP drives expression in the exodermis in tomato hairy root cultures. Note that autofluorescence from the exodermal and endodermal lignin are also picked up in the GFP excitation waveband. The nuclear GFP signal is indicated with arrowheads. Transcriptional fusions of proSlMYB41 and proSlWRKY71 have been previously published (Kajala et al., 2021). Scale bar, 100 μm. (C and D) SlMYB41, SlMYB92, and SlWRKY71 are related to Arabidopsis MYB and WRKY TFs that regulate suberization. Representative phylogenetic trees show the closest Arabidopsis homologs for (C) SlMYB4 and SlMYB92 and (D) SlWRKY71. The trees were inferred using the maximum likelihood method with 1000 bootstraps utilizing iQ-TREE. Putative full-length MYB and WRKY amino acid sequences were aligned with MAFFT. All MYBs and WRKYs indicated with an asterisk (*) have described functions linked with suberization. The MYB subgroups (‘S’) are annotated in colored boxes according to Dubos et al. (2010). WRKY groups are annotated with colored boxes according to Eulgem et al. (2000) and Wu et al. (2005).

The SlMYB TFs are homologous with known suberin regulators of Arabidopsis (Fig. 1C). Specifically, phylogenetic analysis of SlMYB41 and SlMYB92 with the closest Arabidopsis MYB homologs, including 14 with described roles in suberization, showed protein sequence similarity with MYB subgroup S11 (AtMYB41, AtMYB74, and AtMYB102) and S24 (AtMYB93, AtMYB92, and AtMYB53), respectively (Fig. 1C).

For the WRKY TF, SlWRKY71 was selected as a candidate due to its expression being induced in drought, similarly to SlMYB92, SlMYB41, and suberin deposition (Cantó-Pastor et al., 2024). Phylogenetic analysis of SlWRKY71 with homologous Arabidopsis WRKY proteins showed that SlWRKY71 is closely related to AtWRKY71, AtWRKY8, and AtWRKY28, the latter of which has previously been identified as a homolog of a candidate regulator of suberin biosynthesis in poplar (Fig. 1D; Supplementary Table S1) (Rains et al., 2018). Therefore, we asked if these two MYBs and SlWRKY71 form a regulatory module to coordinate the expression of suberin biosynthesis genes in the exodermis of tomato.

To identify the transcripts in the tomato root tip that are regulated by SlMYB92, SlMYB41, and SlWRKY71, both CRISPR/Cas9-induced knockouts and p35S-driven overexpression constructs were used for all three TFs in tomato hairy root cultures (Ron et al., 2014; Cantó-Pastor et al., 2024). Additionally, the knockout and overexpression lines show if these TFs are necessary or sufficient, respectively, for exodermal suberin deposition. For the MYB knockouts, we used our previously published Slmyb92-ko and Slmyb41-ko lines that have reduced suberization of tomato root exodermis (Cantó-Pastor et al., 2024). The gene editing of SlWRKY71 generated two truncated versions of the protein in the hairy root lines Slwrky71-ko(4) and Slwrky71-ko(5), which we used in parallel in our experiments (Supplementary Fig. S1). For the overexpression constructs with the near constitutive 35S promoter, we included a C-terminal 3×FLAG tag and validated the hairy root lines by protein accumulation as detected by anti-FLAG antibody (Supplementary Fig. S2). For our experiments, the overexpression lines with the strongest TF accumulation were selected (Supplementary Fig. S2).

Our first question about these lines was if they had altered exodermal deposition of suberin or lignin. We set out to test the effect of the genetic perturbations by examining suberin deposition in cross-sections of the ko and OX hairy root lines and comparing them with the control (empty vector) hairy roots (Fig. 2). As suberization in exodermis builds up gradually, starting with a patchy pattern and building up to a fully suberized ring, we quantified our section data accordingly. We scored whether cross-sections from individual roots had exodermis that was non-suberized (0% of cells in a section), patchy (‘early suberized’, <50% of cells in the section), or had a fully suberized pattern (‘late suberized’, >50% of cells in a section) (Fig. 2A). For the Slmyb92-ko and Slmyb41-ko, we saw a reduction in exodermal suberization when compared with the empty vector control (Fig. 2A–C), confirming that the two TFs SlMYB92 and SlMYB41 are necessary for the normal suberin deposition pattern in tomato exodermis. Conversely, the overexpressors of the two SlMYB genes had a similar suberin deposition pattern to that of the control roots. This suggests that they are not individually sufficient to affect suberization. In SlWRKY-OX, we observed an absence of suberization in the exodermis (Fig. 2A–C), while suberization was not affected in Slwrky71-kos. This indicates a repressive role for SlWRKY71 in exodermal suberin deposition with potentially other redundantly acting TFs. We then examined the lignin deposition in the exodermis of these lines, and did not observe any changes to the deposition pattern (Supplementary Fig. S3), indicating that these TFs are likely to regulate only the suberin barrier deposition in exodermis.

Fig. 2.

Perturbations of SlMYB41, SlMYB92, and SlWRKY71 expression affect the suberization of tomato exodermis. (A) Percentage of cross-sections showing a non-suberized, an early suberized (suberin in <50% of exodermal cells), or a late suberized (suberin in >50% of exodermal cells) exodermis phenotype in hairy root cultures of the control (empty vector), CRISPR knockout lines [Slmyb41-ko, Slmyb92-ko, Slwrky71-ko(4), and Slwrky71-ko(5)], and overexpressing lines (SlMYB41-OX, SlMYB92-OX, and SlWRKY71-OX). The number above the bars represents the number (n) of individual roots examined in each genotype. Asterisks denotes statistically significant differences between each line in comparison with the empty vector control, whereas n.s. indicates no significant differences (P<0.05, χ² test). (B) Representative images of Fluorol Yellow-stained cross-sections of the control hairy root line (empty vector) showing a non-suberized, an early suberized, and a late suberized phenotype. The numbers on top of the images represent the percentage of cross-sections showing the indicated suberization phenotype. Scale bars represent 100 mm. (C) Representative images of Fluorol Yellow-stained cross-sections of the knockout and overexpressing hairy root lines. The numbers on top of the images represent the percentage of cross-sections showing the indicated suberization phenotype. Scale bars represent 100 mm.

To identify the potential target genes of SlMYB92, SlMYB41, and SlWRKY71, we performed RNA-seq of 1 cm root tips from hairy root cultures of Slmyb41-ko, SlMYB41-OX, Slmyb92-ko, SlMYB92-OX, Slwrky71-ko(4), Slwrky71-ko(5), SlWRKY71-OX, and empty vector control lines. Multidimensional scaling (MDS) analysis of the generated RNA-seq data demonstrated the reproducibility of the biological replicates for each genotype. It also revealed transcriptome similarities between the Slmyb41-ko and Slmyb92-ko lines, between the Slwrky71-ko(4) and Slwrky71-ko(5) lines, and between the SlWRKY71-OX and SlMYB41-OX lines (Fig. 3A). For each line, DEGs were identified by comparing them with the transcriptome data of control roots with an adjusted P-value threshold of 0.05 (Fig. 3B). The list of DEGs identified in all the lines is presented in Supplementary Dataset S1. Perturbation of TF function by CRISPR/Cas9 led to notable changes in the transcriptome of hairy roots, revealing 873, 624, 828, and 869 DEGs in Slmyb41-ko, Slmyb92-ko, Slwrky71-ko(4), and Slwrky71-ko(5), respectively (Fig. 3B). The same pattern was not observed in the OX lines, where 582 genes were identified in the SlMYB92-OX line, whereas overexpressing SlMYB41 and SlWRKY71 led to the identification of a smaller number of DEGs, 165 and 126, respectively (Fig. 3B). We compared the DEGs between the Slmyb ko lines and found that 227 (53%) out of 426 down-regulated DEGs in Slmyb41-ko were also down-regulated in Slmyb92-ko (Fig. 3C). A similar pattern was observed in the list of up-regulated genes in SlMYB-OX lines (Fig. 3D), where 76 (66%) out of 115 of up-regulated genes in SlMYB41-OX were found to be up-regulated in the SlMYB92-OX line (Fig. 3D). The similarities observed between the DEGs in both the OX and ko lines of SlMYB41 and SlMYB92 indicate an overlap in the gene regulatory networks (GRNs) controlled by these two MYB TFs. We also compared the list of DEGs in the two independent Slwrky71-ko lines and found that 60% and 54% of down-regulated and up-regulated DEGs identified in the Slwrky71-ko(5) line were found in the list of DEGs from Slwrky71-ko(4) (Fig. 3E). The major overlap between the DEG lists of the two independent Slwrky71-ko lines indicates accurate identification of the genes regulated by this TF. For further analysis, we consider the overlapping DEGs between the independent lines as the DEGs for Slwrky71-ko (Fig. 3E).

Fig. 3.

Transcriptome analysis of TF knockouts [Slmyb41-ko, Slmyb92-ko, Slwrky71-ko(4), and Slwrky71-ko(5)] and overexpressors (SlMYB41-OX, SlMYB92-OX, and SlWRKY71-OX) in tomato hairy root cultures. (A) Multidimensional scaling (MDS) plot showing the distance between samples. (B) Number of up- (FC >0) and down-regulated (FC <0) DEGs identified in each line with an FDR threshold of 0.05. (C) Venn diagram showing the overlap between down-regulated genes in Slmyb41-ko and Slmyb92-ko. (D) Venn diagrams showing the overlap between the up-regulated genes between SlMYB41-OX and SlMYB92-OX. (E) Venn diagrams showing the overlap between down- and up-regulated DEGs between Slwrky71-ko(4) and Slwrky71-ko(5). DEGs found in both independent lines were considered as down- or up-regulated in Slwrky71-ko. (F) Heatmap showing the q-value significance [–log10(q-value)] of GO terms enriched in the ko and OX lists of down- (DN) or up-regulated DEGs. (G) Bubble heatmap plots showing the number of DEGs that overlap with a previously annotated list of exodermis-enriched genes (Kajala et al. 2021) and the root suberin co-expressed module (Cantó-Pastor et al. 2024). Statistical significance of the overlap between datasets is indicated (hypergeometric distribution). (H) Bubble heatmap plots showing the number of DEGs that overlap with genes annotated as involved with FA and suberin biosynthesis according to the tomatocyc database. Statistical significance of the overlap between datasets is indicated (hypergeometric distribution).

To identify the biological role of genes regulated by these TFs, we performed GO enrichment analysis and identified the metabolism-related GO terms enriched (q-value <0.05) in the list of DEGs (Fig. 3F). Lipid biosynthesis (GO:0008610) was found to be enriched in the list of up-regulated (UP) genes in SlMYB92-OX (Fig. 3F). These genes include 3-ketoacyl-CoA synthase (KCS)-encoding genes that are involved with the elongation of FAs (Li-Beisson et al., 2010).

Interestingly, response to oxidative stress (GO:0006979) was found to be enriched in the SlMYB92-OX up-regulated list of DEGs (Fig. 3F). This is likely to be due to the high number of genes encoding peroxidases and catalases found in the OX lists of DEGs (Supplementary Fig. S4). It has been proposed that in addition to lignin deposition, peroxidases have a role in promoting the formation of the suberin lamellae in tomato roots (Quiroga et al., 2000; Serra and Geldner, 2022). These results indicate that SlMYB92 is involved with the suberization of the tomato root exodermis.

To further investigate if DEGs are involved in suberin biosynthesis in the exodermis, we compared the list of DEGs with previously characterized tomato exodermis-enriched and tomato root-expressed suberin biosynthesis genes (Kajala et al., 2021; Cantó-Pastor et al., 2024) (Fig. 3G). We identified a significant overlap (P<0.01) of the DEGs in Slmyb41-ko, Slmyb92-ko, SlMYB92-OX, Slwrky71-ko(4), and Slwrky71-ko(5) with the previously annotated tomato exodermis-enriched genes (Kajala et al., 2021) (Fig. 3G). Additionally, we found a significant overlap of the DEGs in SlMYB92-OX and SlMYB41-OX with the previously described list of tomato root suberin-related genes (Cantó-Pastor et al., 2024). Finally, to determine the extent to which these TFs regulate genes involved in the distinct steps of suberin biosynthesis, we compared the list of DEGs with the genes associated with FA (very-long-chain FA biosynthesis I and II) and suberin biosynthesis (suberin monomer biosynthesis and esterified suberin biosynthesis) pathways in the tomato Plant Metabolic Network (PMN) (https://plantcyc.org/databases/tomatocyc/4.0). We observed a significant overlap of the DEGs in SlMYB41-OX and SlMYB92-OX with the FA and suberin-related pathways (Fig. 3H). This finding suggests that MYB TFs promote the suberization of the tomato root exodermis by enhancing the expression of genes involved in the precursors and monomers of suberin biosynthesis. Notably, some of these genes encode key enzymes of the suberin biosynthesis pathway, and enzymes such as aliphatic suberin feruloyl transferase/ω-HYDROXYACID/FATTY ALCOHOL HYDROXY-CINNAMOYL TRANSFERASE (ASFT/FHT) (Solyc03g097500.3.1) (Serra and Geldner, 2022; Cantó-Pastor et al., 2024) and glycerol-3-phosphate acyltransferase 4 (GPAT4) (Solyc01g094700.3.1 and other GPATs, Solyc03g097500.3.1 and Solyc09g014350.3.1) (Feng et al., 2022; Serra and Geldner, 2022). Altogether, our results support SlMYB92 and SlMYB41 as regulators of the suberin biosynthesis program in the tomato root exodermis.

Our RNA-seq analysis also suggests an antagonistic role between the two SlMYBs and SlWRKY71 TFs in regulating the suberin biosynthesis program in tomato roots. For instance, 107 out of 371 up-regulated genes [false discovery rate (FDR) <0.05 and logfold change (FC) >0] in SlMYB92-OX were also up-regulated in Slwrky71-ko (Fig. 4A). Furthermore, similar to the peroxidase and catalase genes (Supplementary Fig. S4), many suberin biosynthesis-related genes up-regulated in SlMYB92-OX and SlMYB41-OX exhibited lower FC levels in SlWRKY71-OX (Fig. 4B). These results suggest that these SlMYBs primarily act as positive regulators of the suberin biosynthesis program in the tomato root exodermis, while SlWRKY71 functions as a repressor of exodermis suberization. Moreover, these results indicate that these TFs may balance each other’s actions to fine-tune the expression of suberin biosynthesis genes in the tomato root exodermis.

Fig. 4.

SlMYBs and SlWRKY71 antagonistically regulate the expression of suberin biosynthesis-related genes. (A) Venn diagram showing the overlap between SlMYB92-OX, and Slwrky71-ko up-regulated genes (FDR <0.05, logFC >0). Statistical significance of the overlap between gene sets is indicated (hypergeometric distribution). (B) Heatmap showing the logFC values of genes in SlMYB92-OX, SlMYB41-OX, and SlWRKY71-OX. Values are presented for the genes that are significantly up-regulated in SlMYB92-OX and annotated in the suberin co-expression cluster (Cantó-Pastor et al. 2024). Full annotations are available in Supplementary Dataset S2. (C) Schematic diagrams of constructs used for the transactivation assays. (D) Transactivation assays in protoplasts of A. thaliana. The protoplasts were co-transformed with the pDLUC15 vector expressing the firefly luciferase under the control of the promoter of interest and with different combinations of overexpressed transcription factors, as indicated on the x-axis. The pDLUC15 vector also contained the 35S::Renilla luciferase, used as internal control. The firefly to Renilla luciferase activity ratios were normalized to the average ratios of the GFP samples. Data represent the mean ±SE (n=4–5). Different letters indicate statistically significant differences (one-way ANOVA followed by a post-hoc Tukey test, P<0.05).

To further test the function of these TFs in the transcription of their target genes, we performed transactivation assays in Arabidopsis mesophyll protoplasts using individual TFs and all their combinations. A construct overexpressing GFP was used to establish a baseline for the assay. A schematization of the constructs used is shown in Fig. 4C. For these assays, we tested the promoter regions of the two SlMYB genes and SlWRKY71 to see whether they regulate their own expression, in addition to three other candidate genes selected for their involvement in the suberin biosynthesis pathway and their transcriptional response in the transgenic hairy roots (Supplementary Fig. S4). Among the different promoter regions used, only Solyc03g115960.3.1 [GDSL esterase/lipase38 (SlGELP38)], Solyc07g056320.4.1 [Glycerol-3-phosphate acyltransferase (GPAT1)], and Solyc01g094700.5.1 (SlGPAT4) showed a positive transactivation (Fig. 4D; Supplementary Fig. S5). Specifically, SlGPAT4 was transactivated by SlMYB92 alone and when in combination with the other two TFs. On the other hand, SlGELP38 (Solyc03g115960.3.1) and SlGPAT1 (Solyc07g056320.4.1) promoters were transactivated by SlMYB41 and by SlMYB41 in combination with SlMYB92, but not by SlWRKY71 (Fig. 4D). The SlGELP38 (Solyc03g115960.3.1) promoter was also activated by SlMYB92 alone. When SlWRKY71 was combined with either one or both MYBs, it seemed to act like a repressor of promoter activity (Fig. 4D). This suggests that SlMYB92 and SlMYB41 are positive regulators of these genes while SlWRKY71 counteracts the function of the two SlMYBs (Fig. 4). Overall, it appears that the MYBs are direct activators of promoters of suberin-related genes and their effect may be additive, while SlWRKY71 can act as an activator or an inhibitor of promoter activity depending on the promoter and context of other TFs present.

Discussion

SlMYB92 and SlMYB41 are positive regulators of suberin in tomato root exodermis

In Arabidopsis roots, suberin accumulates in the endodermal cell layer, whereas in tomato, suberization occurs in the exodermal cell layer. Despite this spatial distinction, it has been shown that both processes are controlled by similar components (Cantó-Pastor et al., 2024). Several reports have shown the role of MYB TFs in the control of the suberization in the root endodermal cell layer. In Arabidopsis, several MYB TFs, including AtMYB41, AtMYB53, AtMYB92, and AtMYB93, have been shown to be involved in the biosynthesis of suberin in the root endodermal cell layer (Shukla et al., 2021). Additionally, 13 key MYB TFs were identified in the transcriptional regulatory network to control the suberization and lignification in the Arabidopsis endodermis (H. Xu et al., 2022). In this sense, it is likely that MYB TFs would also play an important role in the suberization of the root exodermis in tomato. Here we provide several lines of evidence that show that SlMYB92 and SlMYB41 can act as positive regulators of suberin biosynthesis by regulating the expression of several genes involved with the different steps of suberin biosynthesis in tomato roots. First, as previously reported, SlMYB92 and SlMYB41 are expressed primarily at the exodermal cell layer of the tomato root (Fig. 1) (Kajala et al., 2021; Cantó-Pastor et al., 2024). Second, perturbations of SlMYB92 and SlMYB41 CDSs by CRISPR/Cas9 resulted in a significant delay in suberin deposition in the tomato root exodermis (Cantó-Pastor et al., 2024). Third, overexpression of SlMYB92 and SlMYB41 in tomato hairy root cultures resulted in the up-regulation of several genes involved in suberin biosynthesis (Supplementary Dataset S2). However, we did not observe increased accumulation of suberin in the SlMYB-OX lines, indicating that they might work together to up-regulate the complete suberin biosynthesis and deposition pathways. Fourth, we showed that promoters of suberin biosynthesis genes are transactivated by SlMYB92 and SlMYB41 in Arabidopsis leaf protoplasts (Fig. 4D), indicating direct activation of the promoter activity. Together, our results provide coherent evidence that SlMYB92 and SlMYB41 act as positive regulators of suberin biosynthesis in the tomato root exodermis.

Given the challenges of generating transgenic tomato plants, our genetic perturbations were done with the tomato hairy root cultures and transactivation assays in Arabidopsis leaf protoplasts, which introduces the caveats of using model systems that are different from the tomato embryonic roots. We observed lower levels and higher variation of exodermal suberization in the tomato hairy root cultures compared with primary roots (Kajala et al., 2021; Cantó-Pastor et al., 2024). We did not observe SlMYB overexpressors driving ectopic or earlier endodermal suberization, unlike previously observed for their homologs in other species (Kosma et al., 2014; Shukla et al., 2021; Chen et al., 2024). This could be due to inadvertent selection towards weakly suberizing alleles. If the SlMYBs were sufficient to drive suberization, their effect expressed under the near-constitutive 35S promoter could lead to inhibition of root growth, and hence not being selected for hairy root cultures. Additionally, the heterologous trans-activation assay might be affected by the factors present in the heterologous tissue of a different species. While these challenges may affect the interpretation of the results, the different lines of inquiry support each other and are consistent with the Arabidopsis literature for the endodermis.

SlMYB92 and SlMYB41 regulate genes involved in suberin biosynthesis and deposition

Suberin is a highly heterogeneous biopolymer comprised of a variety of aliphatic long chain FAs and their oxidized derivatives, glycerol and ferulic acid (Shukla and Barberon, 2021; Serra and Geldner, 2022). Due to its biochemical complexity, the action of several enzymes that operate in distinct compartments of the cell needs to be highly coordinated. Here, we show that SlMYB92 and SlMYB41 can regulate the expression of several genes involved in the many steps required for the biosynthesis of suberin monomers, their transport, and polymerization in the apoplast (summarized in Supplementary Dataset S2). The precursors of suberin monomers are produced via two major biochemical pathways: very-long-chain fatty acid (VLCFA) and phenylpropanoid biosynthesis pathways (Shukla and Barberon, 2021). Long-chain acyl CoA synthases (LACSs) produce the CoA-thioesters that are substrates for the production of VLCFAs (Serra and Geldner, 2022). We found three LACS-encoding genes to be up-regulated in SlMYB92-OX (Solyc04g011900.4.1, Solyc05g052340.4.1, and Solyc06g082240.2.1/SlLAC3-like) and one down-regulated in Slmyb41-ko (Solyc06g050530.3.1/SlLAC12-like). The fatty acid elongase (FAE) complex catalyzes the elongation of acyl-CoA into VLCFAs. Within this complex, KCS carries out the rate-limiting step (Haslam and Kunst, 2013; Batsale et al., 2021; Serra and Geldner, 2022). Here we showed that SlKCS1 (Solyc10g009240.3.1), SlKCS6 (Solyc02g085870.3.1), and SlKCS20 (Solyc03g005320.3.1 and Solyc09g083050.3.1) were up-regulated in the SlMYB92-OX line while SlKCS11 (Solyc08g067410.2.1) was down-regulated in Slmyb41-ko and Slwrky71-ko lines. These VLCFAs are further modified by cytochrome P450s (CYPs) (Serra and Geldner, 2022). Notably, tomato orthologs of CYP86A1 (Solyc06g076800.3.1) and CYP86B1 (Solyc02g014730.3.1), encoding two main CYPs involved with the ω-oxidation of FAs and endodermis suberization in Arabidopsis (Li et al., 2007; Höfer et al., 2008; Compagnon et al., 2009), were up-regulated in hairy roots overexpressing SlMYB92 (Supplementary Dataset S2). Knockout mutations in the latter (SlCYP86B1) resulted in a reduction of exodermis suberization in tomato hairy roots (Cantó-Pastor et al., 2024).

In the final steps of suberin monomer biosynthesis, a feruloyl transferase (ASFT/FHT) and GPATs are involved with the feruloylation and glyceration of suberin precursors, respectively (Serra and Geldner, 2022). It has been previously shown that knocking down SlASFT and SlGPAT4 in tomato hairy roots resulted in dramatic reduction on exodermis suberization (Cantó-Pastor et al., 2024). We found SlASFT (Solyc03g097500.3.1) and SlGPAT6 (Solyc03g097500.3.1) to be up-regulated in SlMYB92-OX, while SlGPAT4 (Solyc01g094700.5.1) was found to be up-regulated in SlMYB92-OX and SlMYB41-OX (Supplementary Dataset S2). The transport of suberin monomers to the apoplast is mediated by lipid transfer proteins (LTPs) and ATP-binding cassette transporters of the subfamily G (ABCGs) (Shukla and Barberon, 2021). In Arabidopsis, AtLTPg15 was found to be important for the transport of suberin monomers to the seed coat (Lee and Suh, 2018). Interestingly, we observed that the tomato ortholog of AtLTPg15 (Solyc09g065430.4.1) was up-regulated by overexpressing SlMYB92 and SlMYB41 (Supplementary Dataset S2). Additionally, two ABCG-encoding genes (Solyc03g019760.4.1 and Solyc05g054890.4.1/SlABCG2) were up-regulated in SlMYB92-OX, and another ABCG gene (Solyc05g051530.5.1) was down-regulated in Slmyb92-ko and Slmyb41-ko but up-regulated in SlMYB92-OX compared with the control hairy root culture.

After the transport of suberin monomers, the GDSL-type esterase/lipase protein (GELP) family are involved with suberin polymerization in the apoplastic space. A recent study has shown that quintuple GELP mutants (gelp22-38-49-51-96) have a drastic reduction in endodermis suberization (Ursache et al., 2021). We observed that a GELP gene (Solyc03g115960.3.1/SlGELP38) previously identified as specifically expressed in the exodermal cell layer (Kajala et al., 2021; Cantó-Pastor et al., 2024) was up-regulated by the overexpression of SlMYB92 (Supplementary Dataset S2). Additionally, four other GELP-encoding genes (Solyc02g070610.3.1, Solyc10g085170.3.1, Solyc11g051060.2.1, and Solyc10g076740.3.1) were down-regulated in the Slmyb41-ko or Slmyb92-ko (Supplementary Dataset S2).

Altogether, these results show that SlMYB92 and SlMYB41 are major regulators of exodermis suberization by promoting the expression of genes involved with the biosynthesis, transport, and polymerization of suberin in tomato roots.

The role of MYBs in gene regulation

The primary role of both MYBs seems to be in regulating the expression of biosynthetic genes, with minimal impact on the regulation of other TFs (Supplementary Dataset S2). For instance, overexpression of SlMYB41 leads to the up-regulation of only one WRKY TF (Solyc03g095770.3.1, AtWRKY70), while SlMYB92 overexpression results in the up-regulation of just two WRKY TFs (Solyc01g095630.3.1 and Solyc09g015770.3.1). Notably, SlMYB41 overexpression does not affect the up-regulation of any MYB-encoding gene, whereas SlMYB92 overexpression induces the up-regulation of four MYBs (Solyc03g093930.5.1, Solyc06g075660.4.1, Solyc06g065100.3.1, and Solyc02g082040.3.1). Additionally, in the Slmyb41 and Slmyb92 ko lines, only three MYBs were found to be down-regulated (Solyc05g007160.3.1, Solyc02g088190.5.1, and Solyc09g090790.3.1). This is similar to the multi-hierarchical regulatory network for Arabidopsis endodermal suberin and lignin proposed by H. Xu et al. (2022), where AtMYB92 and AtMYB41 are in separate tiers and distinct branches of the network. In contrast to our data, in the network by Xu and colleagues, AtMYB41 is more of a hub coordinator and would be expected to induce a higher number of DEGs when mutated or induced, while AtMYB92 is on the last tier, with low impact on other TFs. It is likely that the way in which the suberin GRN is connected differs between species, cells, and tissues, even if they utilize orthologous MYB TFs.

Moreover, the TFs studied here do not exhibit self-activation and do not appear to regulate each other’s expression (Supplementary Fig. S4). This suggests that other MYBs may be responsible for their regulation. Additionally, given that the ko lines of both SlMYB41 and SlMYB92 had reduced suberin (Cantó-Pastor et al., 2024), this indicates that the SlMYB genes are working as necessary, but not directly connected, nodes in an exodermal suberin GRN. Alternatively, it is possible that some of these regulatory interactions are dependent on, for example, having the right ratio of binding partners in a MYB heterodimer. Further studies are required to elucidate the regulatory mechanisms upstream of the TFs investigated in this study.

Antagonistic interaction of MYBs and WRKYs in regulating suberin

Together with MYBs, WRKYs appear to be one of the most relevant TF families involved in suberin biosynthesis in several species and tissue types (Lashbrooke et al., 2016). WRKYs modulate gene expression by binding to W-box cis-regulatory elements of stress-induced genes, and control mainly senescence, stress, and defense responses (Eulgem et al., 2000; Rushton et al., 2010).

Expression of a WRKY TF in rice roots (LOC_Os01g53260), together with MYBs and other TFs, has been shown to enhance radial oxygen loss (ROL) barrier formation (including suberin and lignin), and WRKY cis-regulatory elements were enriched in the promoters of the majority of up-regulated genes in this tissue (Shiono et al., 2014). Arabidopsis MYB107 was found to be a positive regulator of suberin biosynthesis genes during seed development, and WRKYs were among the TFs that were either co-expressed with MYB107 or co-supressed in myb107 (Gou et al., 2017). Among the co-expressed partners were AtWRKY56 (At1g64000) and AtWRKY43 (At2g46130) (Supplementary Table S1; Gou et al., 2017). Furthermore, WRKYs were listed as candidate for suberin biosynthesis and cork regulation in cork oak (Quercus suber) together with MYBs, and a homolog of AtWRKY43 was among the candidates with high FC values (Soler et al., 2007). Homologs of AtWRKY65, AtWRKY43, AtWRKY6, and AtWRKY28 were also among the transcripts with the highest FC in poplar bark, being potential candidates for biosynthesis and regulation of poplar suberin (Rains et al., 2018).

Here, we have uncovered an antagonistic interaction between inhibitory SlWRKY71 and activating SlMYB41 and SlMYB92. The genetic perturbations of SlWRKY71 indicated that SlWRKY71 is a repressor of tomato exodermal suberin (Fig. 2), while SlMYB92 and SlMYB41 are necessary for tomato exodermal suberin deposition (Cantó-Pastor et al., 2024). The lack of a statistically significant phenotype for SlWRKY71-kos may be explained either by the mutation having a subtle effect on the suberin pattern at the sampled location or by other, redundant, TFs having the same antagonistic role.

Furthermore, we showed that these TFs antagonistically regulate expression of suberin-related genes in tomato exodermis (Fig. 4). We observed an overlap of DEGs induced by SlMYB41 and SlMYB92 overexpression and Slwrky71 knockout, indicating that the same target genes are activated by SlMYBs as are repressed by SlWRKY71 (Fig. 4A). We also observed that the SlWRKY71-OX DEGs had a contrasting behavior to the SlMYB-OX lines (Fig. 4B). Finally, we showed that the presence of SlWRKY71 with the two SlMYBs prevents their ability to induce promoter activity in Arabidopsis protoplasts for two target promoters (Fig. 4D).

The MYB–WRKY antagonism may occur through direct MYB–WRKY interactions or competitive binding on target promoters. SlWRKY71 repression could also be amplified by suppressing other WRKY TFs. For example, SlWRKY41 and SlWRKY70 were up-regulated in Slwrky71-ko and SlMYB overexpression lines (Supplementary Dataset S2), suggesting that these putative downstream TFs are regulated antagonistically by SlWRKY71 and the two SlMYBs.

Interestingly, SlWRKY71 expression is drought induced (Cantó-Pastor et al., 2024) alongside the SlMYB genes and suberin deposition. Why would both activators and repressors of suberin gene expression be induced simultaneously in tomato exodermis? This might represent a pre-set pulse in suberin biosynthetic gene expression to ensure that available resources are not overspent on making too much suberin. These nuances arising from antagonistic interactions in the exodermis suberin GRNs may set a complex challenge for endeavors of manipulating suberin levels for improved drought resilience and sequestration of carbon dioxide from the atmosphere into suberin. Previously identified repressors of suberin biosynthesis genes, AtMYB70 (Wan et al., 2021) and StNAC103 (Verdaguer et al., 2016), are proposed to have roles in integrating auxin signaling to root system architecture, and regulating non-suberization of certain cell types, respectively. The future challenges involve understanding more fully the importance of the repressors of suberin in accurate deposition dynamics and positioning, their role in plant fitness, and the regulatory network.

Supplementary data

The following supplementary data are available at JXB online.

Dataset S1. Differentially expressed genes (DEGs) in tomato hairy root cultures.

Dataset S2. Combined summary of DEGs.

Fig. S1. SlWRKY71 sequences and truncations generated with gene editing.

Fig. S2. TF overexpression is detected on the protein level.

Fig. S3. Exodermal lignin is unaffected by SlMYB92, SlMYB41, and SlWRKY71 perturbations.

Fig. S4. Expression of peroxidases and catalases is affected in TF knockouts and overexpressors.

Fig. S5. Transactivation assays of SlMYB92, SlMYB41, and SlWRKY71 promoters in protoplasts of A. thaliana.

Table S1. MYB41, MYB92, and WRKY71 homologs from Arabidopsis and summary of their potential roles in regulating suberization.

Table S2. Primers used to create the overexpression constructs.

Table S3. Guide RNAs designed to knock out SlWRKY71.

Table S4. List of promoters used in trans-activation assays and primers used to amplify them.

Table S5. Genomic sequences of promoters cloned for transactivation assays.

Table S6. Primers used to amplify coding regions of SlMYB92, SlMYB41, and SlWRKY71 for transactivation assays.

Glossary

Abbreviations:

BF

Basic Fuchsin

CW

Calcofluor White

CYP

cytochrome P450

DEG

differentially expressed gene

FA

fatty acid

FY

Fluorol Yellow

GELP

GDSL-type esterase/lipase protein

GO

Gene Ontology

GPAT

glycerol-3-phosphate acyltransferase

GRN

gene regulatory network

ko

knockout

MDS

multidimensional scaling

MYB

myeloblastosis

OX

overexpressor

TF

transcription factor

VLCFA

very-long-chain fatty acid

Author contributions

KK and SMB: conceptualization, supervision, and funding acquisition; LJ, SB, MASA, RK, and AC-P: investigation; LJ, SB, MASA, and RK: data curation, formal analysis, and visualization; KK, LJ, SB, MASA, and RK: writing—original draft; all authors: writing—review & editing. All authors read and have approved the final manuscript.

Conflict of interest

No conflict of interest declared.

Funding statement

This work was supported by Marie Skłodowska Curie Actions Reintegration fellowship 790057 to KK, the Netherlands Organization for Scientific Research (NWO) VIDI grant number VI.Vidi.193.104 to KK, and NSF PGRP IOS-2119820 to AC-P and SMB.

Data availability

The RNA sequencing data from this study are openly available in the NCBI GEO repository reference number GSE278561.