plants Article The Tomato Feruloyl Transferase FHT Promoter Is an Accurate Identifier of Early Development and Stress-Induced Suberization Anurag Kashyap 1,† , Álvaro Jiménez-Jiménez 1 , Mercè Figueras 2 , Olga Serra 2 , Marc Valls 1,3 and Nuria S. Coll 1,4, * 1 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB, 08193 Bellaterra, Spain 2 Laboratori del Suro, Biology Department, University of Girona, Campus Montilivi, 17003 Girona, Spain 3 Department of Genetics, Universitat de Barcelona, 08028 Barcelona, Spain 4 Consejo Superior de Investigaciones Científicas (CSIC), 08001 Barcelona, Spain * Correspondence: nuria.sanchez-coll@cragenomica.es; Tel.: +34-935636600 † Current address: Department of Plant Pathology, Assam Agricultural University, Jorhat 785013, AS, India. Abstract: As a wall polymer, suberin has a multifaceted role in plant development and stress responses. It is deposited between the plasma membrane and the primary cell wall in specialized tissues such as root exodermis, endodermis, phellem, and seed coats. It is formed de novo in response to stresses such as wounding, salt injury, drought, and pathogen attack and is a complex polyester mainly consisting of fatty acids, glycerol, and minor amounts of ferulic acid that are associated to a lignin-like polymer predominantly composed of ferulates. Metabolomic and transcriptomic studies have revealed that cell wall lignification precedes suberin deposition. The ferulic acid esterified to ω-hydroxy fatty acids, synthetized by the feruloyl transferase FHT (or ASFT), presumably plays a role in coupling both polymers, although the precise mechanism is not understood. Here, we use the promoter of tomato suberin feruloyl transferase (FHT/ASFT) fused to GUS (β-glucuronidase) Citation: Kashyap, A.; to demonstrate that ferulate deposition agrees with the site of promoter FHT activation by using a Jiménez-Jiménez, Á.; Figueras, M.; combination of histochemical staining and UV microscopy. Hence, FHT promoter activation and Serra, O.; Valls, M.; Coll, N.S. The alkali UV microscopy can be used to identify the precise localization of early suberizing cells rich Tomato Feruloyl Transferase FHT in ferulic acid and can additionally be used as an efficient marker of early suberization events Promoter Is an Accurate Identifier of during plant development and stress responses. This line can be used in the future as a tool to Early Development and identify emerging suberization sites via ferulate deposition in tomato plants, which may contribute Stress-Induced Suberization. Plants to germplasm screening in varietal improvement programs. 2023, 12, 1890. https://doi.org/ 10.3390/plants12091890 Keywords: suberin; ferulates; stress; development; tomato Academic Editor: Alexandra S. Dubrovina Received: 29 March 2023 1. Introduction Revised: 1 May 2023 Accepted: 3 May 2023 The wall polymer suberin has a multidimensional role in plant development and Published: 5 May 2023 stress responses [1,2]. Structurally, it is a glycerol-based, fatty-acid-derived polyester com- prised primarily of ω-hydroxy acids, α, ω-dicarboxylic acids, fatty alcohols, and small amounts of hydroxycinnamic acids (mainly ferulic acid) [3]. It is deposited between the plasma membrane and the cell wall in specialized tissues, such as root exodermis, endo- Copyright: © 2023 by the authors. dermis, seed coat, and phellem (cork), in root and aboveground tissue in woody plants [4]. Licensee MDPI, Basel, Switzerland. Moreover, suberin can be formed de novo in response to stress such as wounding, salt This article is an open access article injury, drought, and pathogen attack [5]. In addition to providing strength to the cell wall, distributed under the terms and suberin averts water loss and pathogen access by sealing off the layer of suberized cells. conditions of the Creative Commons It acts as a potent barricade against pathogens, and in addition to providing strength to Attribution (CC BY) license (https:// the cell wall, it may also act as an antimicrobial fence [6]. Recently, it has been shown that creativecommons.org/licenses/by/ microbiota inhabiting the roots can also influence suberin deposition at the endodermis [7]. 4.0/). Plants 2023, 12, 1890. https://doi.org/10.3390/plants12091890 https://www.mdpi.com/journal/plants Plants 2023, 12, 1890 2 of 14 Hence, suberin has great potential for developing crops with multi-stress tolerance [8]. The polymer is also being explored for carbon sequestration applications using plants as carbon storage to mitigate global warming. Suberized cell walls also accumulate a lignin-like polymer rich in ferulates in the primary cell wall, which has also led to them being called suberin polyphenolic domains [2]. However, the configuration of suberin and lignin polymers and the progression of their deposition are poorly understood [2]. Metabolomics and transcriptomics data suggest that lignification precedes suberization, and ferulic acid has been identified to initiate suberin deposition, possibly offering con- nections to lignin or other cell wall polymers [2,9]. Suberin biosynthesis occurring during developmental processes has been studied in the last two decades using molecular genetic methods, particularly in the endodermis and seed coat model of Arabidopsis thaliana and in the tuber phellem of potato [10]. Precursors of the lignin-like polymer originate from the phenylpropanoid pathway, as does the ferulic acid of suberin, while the aliphatic monomers of suberin are synthesized through the fatty acid biosynthetic pathway [6]. Several genes encoding enzymes involved in the modification of these fatty acid precursors yielding the suberin monomers have been described, such as fatty acyl-CoA reductase (FAR1, FAR4, FAR5); fatty acid cytochrome P450 oxidases (CYP86A1/HORST, CYP86B1, CYP94B1, CYP94B3); glycerol-3-phosphate acyltransferase 5 (GPAT5/GPAT7); β-ketoacyl-CoA syn- thases (KCS1, KCS2/Daisy, KCS6, and KCS20); and genes involved in the conveyance of suberin monomers to the site of suberization (ABCG1, ABCG2, ABCG6, ABCG11, and ABCG20) [11–15]. In addition, a few upstream controllers of the suberin biosynthetic path- way have been recognized in Arabidopsis, such as the MYB transcription factors MYB41, MYB107, and SUBERMAN (MYB39) [2,16–18]. In Arabidopsis, suberin feruloyl transferase (FHT, also known as ASFT/HHT/RWP) has been functionally characterized and has been reported to be involved in the incorporation of ferulic acid into the suberin polyester. It specifically controls the accumulation of the ferulate esters of suberin in root endodermis, seeds, and phellem, but it has no effect on the content of p-coumarate or sinapate [19–21]. Knocking out FHT/ASFT causes abolition of the suberin-ester-linked ferulate, leading to altered permeability and sensitivity of seeds and roots to salt stress [19,20], and its activation has been reported as one of the earliest steps in suberin deposition [21]. Recently, a set of four MYB transcription factors (MYB41, MYB53, MYB92, and MYB93), each of which is individually regulated by developmental and exogenous signals, was shown to be sufficient to promote endodermal suberin deposition [22]. Moreover, the transcription factor WRKY9 has been demonstrated to regulate cytochrome P450 genes CYP94B3 and CYP86B1, leading to increased root suberin and salt tolerance in Arabidopsis [23]. The role of NAC transcription factor ANAC046 in suberin biosynthesis in Arabidopsis thaliana roots has also been experimentally validated [24]. The tissues commissioning suberization undergo a series of genetic and metabolic reprogramming comprising a web of metabolic conduits to yield the precursors of the polymer and subsequently their polymerization into the matrix [25]. In tomato, suberin plays vital roles both as a constitutive barrier as well as an inducible fence against diverse environmental stresses [26]. However, the visualization of suberin in tomato tissues mostly relies on stains such as Sudan IV or fluorol yellow, which target the aliphatic constituents of the polymer [27,28] and cannot capture early suberization events when suberizing walls accumulate ferulates, presumably playing a role in coupling the aromatic and aliphatic domains. FHT is a key acyltransferase of the suberin biosynthesis pathway which catalyzes the conjugation of feruloyl-CoA and oxidized fatty acids, both of which are suberin monomers [19–21,29]. Hence, it acts as a linker of phenolic to aliphatic compounds to build the suberin polyester (Figure 1). The FHT gene encodes an acyltransferase of the BAHD superfamily named after the first four biochemically characterized enzymes of the group, which are plant-specific enzymes that catalyze the transfer of coenzyme A-activated donors onto various acceptor molecules [30]. Plants 2023, 12, x FOR PEER REVIEW Plants 2023, 12, 1890 group, which are plant-specific enzymes that catalyze the3 oftransfer 14 A-activated donors onto various acceptor molecules [30]. Figure 1. Schematic representation of the enzymatic function of suberin feruloyl transferase (FHT). Figure 1. Schematic representation of the enzymatic function of suberin feruloyl tr FHT catalyzes the conjugation of feruloyl-CoA to aliphatic chains such as ω-hydroxy acids and FHT catalyzes the conjugation of feruloyl-CoA to aliphatic chains such as ω-hyd primary alcohols to form feruloyl esters [19–21], potentially acting as a linker of suberin to lignin-like primary or alcohols other cell wall to form feruloyl esters [19–21], potentially acting as a linker o polymers. nin-like or other cell wall polymers. Here, we present a method that allows precise localization of early suberization sites using the promoter of tomato suberin feruloyl transferase (FHT) fused to GUS (β- Here, we glucuronidase). Thepresent aim of thisa method study is to that allows determine precise whether localization the sites of early sub of FHT promoter using the activation promoter correspond to thoseof tomatodeposition of ferulate suberin andferuloyl transferase thus can function as a robust(FHT) f marker for early suberization events. Transgenic tomato plants (β-glucuronidase). The aim of this study is to determine whether expressing Pro SlFHT ::GUS the site showed ferulate deposition at the position of promoter induction, visualized using alkali moter UV activation microscopy correspond and in accordance topredicted with the those of ferulate SlFHT deposition functions. This tool canand thus can be used robust as marker an efficient marker for early of early suberization suberization events. events during Transgenicandtomato plant development stress plan responses. Such markers will aid in the fundamental understanding ProSlFHT::GUS showed ferulate deposition at the position of promoter ind of the suberization process and germplasm screening for varietal improvement programs in tomato against ized using diverse alkali UV microscopy and in accordance with the predicted SlF stress responses. This tool can be used as an efficient marker of early suberization event 2. Results development and stress responses. Such markers will aid in the funda 2.1. FHT Proteins of Different Plant Species Have a Conserved HxxxD Motif Involved in Catalysis standing and a DFGWGofMotif theLocated suberization process at the C-Terminal End and germplasm screening for varietal programs in tomato In agreement with the against diverse characteristics stress of BAHD responses. all FHT proteins of acyltransferases, different plant species have a conserved HxxxD motif involved in catalysis and a DFGWG motif located at the C-terminal end, the latter of which is presumed to have a structural 2. Results function [21]. Protein homologs of tomato FHT gene (Solyc03g097500) were used for amino 2.1. FHTsequence acid Proteinsalignment in ClustalW of Different Plant and visualized Species haveby Mview usingHxxxD a Conserved the BAR (http: Motif Invol //bar.utoronto.ca/, accessed on 2 May 2023) webpage (Figure 2). Phylogenetic analysis and a DFGWG Motif Located at the C-Terminal End showed that the tomato FHT gene is very close in ancestry to the potato FHT (StFHT) gene (PGSC0003DMG400031731), In agreement with which thehas been characterized characteristics ofinBAHD deeper detail [28]. acyltransferases, all F different 2.2. plant Putative Cis species Elements Found inhave Tomato a conserved FHT Promoter HxxxD motif involved in ca DFGWG motif The putative located SlFHT at the promoter C-terminal region end, of (1710 bp upstream thethelatter of which translation is presu initiation) structural was examined function [21]. [31] using the PLACE Protein and thehomologs PlantCare [32] ofdatabases tomato[29]. FHT gene (Solyc03 In agreement with the reported function of the FHT gene as a key acyltransferase involved in suberin used for amino acid sequence alignment in ClustalW and visualized by M biosynthesis, sequence analysis showed the presence of cis-regulatory motifs specific to BAR stresses abiotic (http://bar.utoronto.ca/, accessed such as wounding, salt injury, wateron 2 May stress, 2023) etc. (e.g., webpage and WBOXNTERF3) (Figure 2) analysis biotic showed stresses that the tomato such as pathogenesis FHT and salicylic acidgene is very(e.g., receptiveness close in ancestry to th GT1GMSCAM4, WBOXATNPR1). As expected, a number of ABA responsive motifs were present (e.g., WB- (StFHT) gene (PGSC0003DMG400031731), which has been characterized in BOXPCWRKY1, MYB and MYC binding sites, SORLIP1, and WRKY), since suberin biosyn- [28].is known to be regulated by this hormone [33–35]. In addition, motifs corresponding thesis Plants 2023, 12, 1890 4 of 14 Plants 2023, 12, x FOR PEER REVIEW to organ-/cell-/and tissue-specific activation of phenylpropanoid genes (e.g., EBOXBN- NAPA) and root and seed inducible motifs were present (e.g., ROOTMOTIFTAPOX1, RYREPEATBNNAPA) (Table 1). Figure Figure 2. Sequence 2. Sequence alignmentalignment of feruloyl of feruloyl transferase (FHT) transferase (FHT) proteins in different plantproteins in differen species. Protein homologs of tomato FHT gene (Solyc03g097500) were obtained Protein homologs of tomato FHT gene (Solyc03g097500) were o from www.phytozome.jgi.doe.gov, accessed on 2 May 2023, and matches with more than 85% similarity were used for amino acid www.phytozome.jgi.doe.gov, accessed on 2 May 2023, and matches with more tha sequence alignment in ClustalW and visualized by Mview using BAR (http://bar.utoronto.ca/, were used for amino acid sequence alignment in ClustalW and visualized by Mv accessed on 2 May 2023) webpage. In agreement with the characteristics of BAHD acyltransferases, (http://bar.utoronto.ca/, all FHT proteins of different plantaccessed on 2conserved species have May 2023) HxxxD webpage. In agreement motif involved with the c in catalysis and BAHDmotif DFGWG acyltransferases, all FHT located at the C-terminal proteins end, the latter of different of which plant tospecies is presumed have conserve have a structural involved function [21]. in catalysis and DFGWG motif located at the C-terminal end, the latter sumed to have a structural function [21]. 2.2. Putative Cis Elements Found in Tomato FHT Promoter The putative SlFHT promoter region (1710 bp upstream of the transla was examined using the PLACE [31] and the PlantCare [32] databases [29] Plants 2023, 12, 1890 5 of 14 Table 1. Putative cis elements found in tomato FHT promoter. Motif Group Motif Name Sequence Function Nº Stress WBOXNTCHN48 CTGACY 1 “W box.” Elicitor-responsive element WBOXNTERF3 TGACY 5 “W box.” Response to wounding OSE1ROOTNODULE AAAGAT 5 Organ-specific elements (OSE) in infected cells of root nodules OSE2ROOTNODULE CTCTT 2 Organ-specific elements (OSE) in infected cells of root nodules PREATPRODH ACTCAT 1 Pro- or hypo-osmolarity-responsive element RAV1AAT CAACA 5 RAV1 binding site, cold responsiveness MYB1AT WAACCA 3 Element involved in dehydration responsiveness MYBCORE CNGTTR 2 Element involved in response to water stress GT1GMSCAM4 GAAAAA 9 Pathogenesis and salt-induced element CURECORECR GTAC 10 Copper-response element CCAATBOX1 CCAAT 5 HSE (heat shock element) ANAERO1CONSENSUS AAACAAA 2 Response to anaerobiosis ACGTATERD1 ACGT 4 Involved in etiolation-induced dehydration Hormones ABA WBBOXPCWRKY1 TTTGACY 1 WRKY proteins binding site, responsive to ABA MYB2CONSENSUSAT YAACKG 1 MYB recognition site involved in dehydration and ABA response MYCATERD1 CATGTG 1 MYC binding site involved in response to dehydration and ABA MYCATRD22 CACATG 1 MYC binding site involved in response to dehydration and ABA MYCCONSENSUSAT CANNTG 6 MYC binding site involved in response to dehydration and ABA SORLIP1AT GCCAC 4 Light-inducible, root-specific, and ABA-responsive element Salicylic acid WBOXATNPR1 TTGAC 3 “W-box”; binding site for SA-induced WRKY transcription factor Ethylene ERELEE4 CGTCA-motif AWTTCAAA 1 Ethylene-responsive element Auxin CATATGGMSAUR CATATG 2 Element involved in auxin responsiveness Gibberellin GARE2OSREP1 TAACGTA 1 Gibberellin-responsive element (GARE) GAREAT TAACAAR 2 GARE (GA-responsive element) MYBGAHV TAACAAA 2 Central element of gibberellin (GA) response complex (GARC) PYRIMIDINEBOXOSRAMY1A CCTTTT 2 Gibberellin-response cis element of GARE and pyrimidine TATCCACHVAL21 TATCCAC 1 Conserved cis-acting response complex (GARC) PYRIMIDINEBOXHVEPB1 TTTTTTCC 2 “Pyrimidine box”. Required for GA induction Organ-/Tissue-/Cell type-specific ROOTMOTIFTAPOX1 ATATT 12 Root specific element from rolD gene SP8BFIBSP8BIB TACTATT 3 Tuberous root specific element Plants 2023, 12, 1890 6 of 14 Table 1. Cont. Motif Group Motif Name Sequence Function Nº RHERPATEXPA7 KCACGW 1 Right part of RHEs (root-hair-specific cis-elements) RRE element. Tissue-specific activation of phenylpropanoids biosynthesis EBOXBNNAPA CANNTG 6 genes RYREPEATBNNAPA CATGCA 1 Required for seed-specific expression POLLEN1LELAT52 AGAAA 12 Element required for pollen-specific expression CACTFTPPCA1 YACT 28 Element related to mesophyll expression BOXIINTPATPB ATAGAA 3 Conserved element in promoters of plastids genes. TAAAGSTKST1 TAAAG 12 Dof1 protein controlling guard cell-specific gene expression Others MYBPZM CCWACC 3 Core of consensus myb homolog binding site MYBST1 GGATA 2 Core motif of MybSt1 (a potato MYB homolog) binding site RYREPEATLEGUMINBOX CATGCAY 1 RY repeat found in seed-storage protein genes Plants 2023, 12, x FOR PEER REVIEW 7 of 15 Plants 2023, 12, 1890 7 of 14 2.3. Induction of ProSlFHT:GUS in Tomato Tissues Undergoing Developmental Suberization We hypothesized that the tomato FHT promoter (ProSlFHT) could be used as a good 2.3. Induction marker to study of Proearly SlFHT :GUS in Tomato suberization at the Tissues sitesUndergoing of suberin Developmental nucleation in Suberization different tissues. Hence, we generated Pro ::GUS lines in tomato We hypothesized that the tomato FHT promoter (ProSlFHT ) could be used SlFHT in order to elucidate the participation as a good of tomato FHT promoter in tissues known to deposit marker to study early suberization at the sites of suberin nucleation in different suberin. For this, we used tissues. the to- mato variety Hence, Hawaii Pro we generated 7996 (H7996), SlFHT ::GUS lines which inhas beeninshown tomato order to to elucidate induce ligno-suberin the participation vas- cular of coating tomato FHTtopromoter restrict infection in tissuesofknown the vascular pathogen to deposit suberin. Ralstonia For this, solanacearum we used the[28]. tomato varietyA fragment Hawaii 7996 consisting (H7996), of 1713 which bphas upstream been shownof theto initial induce ATG codon of SlFHT ligno-suberin was vascular amplified coating from genomic to restrict infection DNA of the and fused to vascular the reporter pathogen β-glucuronidase Ralstonia solanacearum [28]. (GUS) to gen- erateAthe ProSlFHT::GUS fragment consistingconstruct, of 1713 which was transformed bp upstream of the initialintoATG H7996 tomato. codon In agree- of SlFHT was ment withfrom amplified the genomic critical roleDNAreported and fusedfor FHT/ASFT/RWP to the in suberization reporter β-glucuronidase (GUS)[19–21], to generatewe clearly the observed ProSlFHT ::GUSthe induction construct, of Pro which was ::GUS in tissues SlFHTtransformed intoknown H7996 to accumulate tomato. suberin. In agreement with Tomatotheroot critical role reported exodermal cells are forknown FHT/ASFT/RWP in suberization to deposit suberin [19–21], we [36]. We observed clearly strong in- observed duction ofthe Proinduction SlFHT::GUSof inPro root::GUS theSlFHT outerinlayers tissuesof known young to accumulate seedlings suberin. (Figure Tomato 3A). Further, root priorexodermal to emergence, cells weare observed known to strong depositinduction suberin [36]. of Pro We SlFHTobserved ::GUS instrong lateralinduction root pri- of Pro mordia, ::GUS in the root outer layers of young seedlings indicating a hardening process by suberin deposition in the cells of the devel- SlFHT (Figure 3A). Further, prior to emergence, we observed strong oping lateral root cap (Figure 3B). To analyze whether induction of Pro ::GUS in lateral root SlFHTthe induction of ProSlFHT::GUS in primordia, indicating tomato tissuesa hardening correspondedprocesstoby ansuberin increase deposition in the in ferulates, cells of the developing in accordance to the function lateral of root FHT,capwe(Figure used a 3B). To analyze technique whereby whether the induction ferulates of ProSlFHT can be detected by::GUS emissionin tomato of blue tissues fluo- corresponded rescence with to UVanexcitation increase in at ferulates, neutral pH in that accordance to the function characteristically changesof FHT, to a we used stronger agreen technique emission under conditions of high pH such as in the presence of alkali [37–39]. UV whereby ferulates can be detected by emission of blue fluorescence with We excitation observed thatat neutral the UV pHautofluorescence that characteristically detected changes in rootto aepidermal stronger green emission cells and lateral under root conditions of high pH primordia changed from such blue as toin athe presence strong greenofcolor alkaliupon [37–39]. We observed treatment with alkalithat (1N the UV autofluorescence detected in root epidermal cells KOH pH above 10) (Figure 3C,D), indicating the accumulation of ferulates in these and lateral root primordia changed from tissuesblue to a 3C,D). (Figure strong As green color upon expected, treatment this pattern waswith highly alkali (1N KOHtopH coincidental the above observed 10) (Figure 3C,D), indicating the accumulation of ferulates in induction of ProSlFHT::GUS, highlighting the robustness of this method to report dual fer-these tissues (Figure 3C,D). As expected, this pattern ulate/FHT promoter activation. was highly coincidental to the observed induction of Pro SlFHT ::GUS, highlighting the robustness of this method to report dual ferulate/FHT promoter activation. Figure 3. 3. Induction Induction of of Pro ProSlFHT ::GUS in tissues undergoing developmental suberization. Strong in- Figure SlFHT ::GUS in tissues undergoing developmental suberization. Strong duction of ProSlFHT::GUS was observed in the (A) root outer layers of young seedlings and (B) in the induction of ProSlFHT ::GUS was observed in the (A) root outer layers of young seedlings and (B) in emerging lateral root primordia. Correspondingly, in similar samples, the ferulate signal was ob- the emerging lateral root primordia. Correspondingly, in similar samples, the ferulate signal was served in (C) root outer layers of young seedlings and (D) lateral root primordia, as observed by observed alkali UV in (C) root outer microscopic layers of technique. young Scale bar seedlings andB(D) A = 500 µm, lateral = 200 µm, Croot primordia, = 40 as observed µm, D = 500 µm. by alkali UV microscopic technique. Scale bar A = 500 µm, B = 200 µm, C = 40 µm, D = 500 µm. Plants 2023, 12, 1890 8 of 14 Plants 2023, 12, x FOR PEER REVIEW 8 of 15 2.4. InductionofofPro 2.4. Induction Pro :GUS SlFHT SlFHT :GUS in Tomato in Tomato Tissues Tissues Undergoing Undergoing Wound Wound Healing Healing Sincesuberin Since suberindeposition depositionisisknown knowntotooccur occurasaspart partofofthe thewound-healing wound-healingresponse response [29,40], we analyzed [29,40], the induction we analyzed of Proof the induction Pro SlFHT ::GUS SlFHT upon ::GUS uponinjury. At injury. 48 At h 48 post h postpin-prick pin-prick injury on injury on leaves, leaves, induction a strong a strong induction of Pro of ProSlFHT SlFHT::GUS ::GUS was observed was observed surrounding surrounding thethe in- region injured jured region (Figure (Figure 4A,B). 4A,B). Further, whenFurther, waterwhen water imbalance imbalance or other or other factors factors lead leadcracks to fruit to fruitin tomato, the plants have a mechanism to seal this crack to prevent rotting due to due cracks in tomato, the plants have a mechanism to seal this crack to prevent rotting the to growth of the growth of saprophytes. We observed specific induction of Pro SlFHT::GUS in the sealing saprophytes. We observed specific induction of ProSlFHT ::GUS in the sealing region of the region of the cracks, indicating suberization in this particular wound-healing response cracks, indicating suberization in this particular wound-healing response (Figure 4C,D). (Figure 4C,D). Further, we visualized ferulate deposition using alkali UV microscopy Further, we visualized ferulate deposition using alkali UV microscopy concomitantly with concomitantly with FHT promoter activation (GUS signal) during pin-prick injury and FHT promoter activation (GUS signal) during pin-prick injury and fruit cracks in tomato fruit cracks in tomato (Figure 4E,F). (Figure 4E,F). Figure 4. ProSlFHT::GUS is induced in leaves and fruits of tomato during wound healing. (A) Figure 4. ProSlFHT ::GUS is induced in leaves and fruits of tomato during wound healing. ProSlFHT::GUS expression 48 h after pin-prick injury on the leaves. (C) Magnified image from (A). (B) (A) Pro SlFHT Induction of Pro::GUS expression 48 h after pin-prick injury on the leaves. (C) Magnified image SlFHT::GUS was observed in fruit cracks undergoing wound healing. (D) Magnified from view of(A). the (B) Induction crack showing GUSof Prosignal. ::GUS was observed SlFHTCorrespondingly, in signal ferulate fruit was cracks undergoing observed wound heal- in adjacent areas (D) ing. of (E) pin-prick view Magnified injuryofonthe the crack leavesshowing and (F) fruit GUS cracks undergoing signal. wound healing, Correspondingly, as ob-signal was ferulate served by alkali observed UV microscopic in adjacent areas of technique. Scaleinjury (E) pin-prick bar A =on400the µm, B = 200 leaves µm,(F) and C fruit = 20 µm, D = undergoing cracks 20 µm, E = 200 µm, F = 20 µm. wound healing, as observed by alkali UV microscopic technique. Scale bar A = 400 µm, B = 200 µm, C = 20 µm, D = 20 µm, E = 200 µm, F = 20 µm. 2.5. Induction of ProSlFHT ::GUS in Vascular Suberization Response in Tomato against Pathogens Suberin vascular coating in response to the vascular pathogen Ralstonia solanacearum infection has been observed in the resistant tomato cultivar H7996 [28]. ProSlFHT ::GUS transgenic H7996 tomato plants were inoculated through their roots by soaking the soil Plants 2023, 12, x FOR PEER REVIEW 9 of 15 Plants 2023, 12, 1890 9 of 14 2.5. Induction of ProSlFHT::GUS in Vascular Suberization Response in Tomato against Pathogens Suberin vascular coating in response to the vascular pathogen Ralstonia solanacearum infection has been observed in the resistant tomato cultivar H7996 [28]. ProSlFHT::GUS with R. solanacearum with a concentration ~1 × 107their of through CFU mLby −1 and grown at 28 ◦ C for transgenic H7996 tomato plants were inoculated roots soaking the soil 20 days. with In water-treated R. solanacearum plants, induction with a concentration of ~1of Pro × 10 SLFHT ::GUS 7 CFU mL −1 andwas not at grown observed 28 °C forin20the xylem vasculature (Figure 5A). days. In water-treated In infected plants, induction resistant tomato of ProSLFHT ::GUS plants, was notinduction observed of in Pro the SlFHT xylem ::GUS was vasculature (Figure 5A). In infected resistant tomato plants, induction observed in the xylem vascular tissue as well as in the outer layers of the root (Figure 5B).of Pro SlFHT::GUS was observed inwe Interestingly, theobserved xylem vascular that the tissue UVasautofluorescence well as in the outerdetected layers of the root (Figure in vascular coatings in 5B). response to R. solanacearum infection in resistant H7996 (Figure 5D) in Interestingly, we observed that the UV autofluorescence detected vascular changed from blue coatings to a strongin response green colorto R. upon solanacearum treatmentinfection withinalkali resistant (1NH7996 KOH(Figure 5D) changed pH above 10)—but not in from blue to a strong green color upon treatment with alkali (1N KOH pH above response to mock treatment (Figure 5C)—indicating the presence of ferulates in these 10)—but not in response to mock treatment (Figure 5C)—indicating the presence of coatings (Figure 5F,G), as previously reported [28]. In contrast, no such vascular coating ferulates in these coatings (Figure 5F,G), as previously reported [28]. In contrast, no such was observed vascular coatingin was water-treated observed plants (non-infected) in water-treated plants(Figure 5E,G). These (non-infected) (Figuredata indicate that 5E,G). resistance These data to R. solanacearum indicate in H7996 that resistance is mediated in to R. solanacearum byH7996 induction of the FHT is mediated expression in the by induction vasculature, which isinaccompanied of the FHT expression the vasculature, by which ferulate deposition inbythe is accompanied same deposition ferulate area. The in lipophyllic fluorescent the same area. dye Thefluorol yellow, lipophyllic which stains fluorescent the aliphatic dye fluorol yellow, portion of suberin, which stains was also used the aliphatic in ProSLFHT portion ::GUS transgenic of suberin, was also used tomato in Proplants infected SLFHT::GUS transgenic R. solanacearum withtomato and mock-treated plants infected with R. solanacearum plants. As shown and in mock-treated Figure S1, plants. infectionAs with shownR.insolanacearum Figure S1, infection resultedwith in aR.clear sola- increase nacearum resulted in a clear in root fluorescence increaseto compared in mock-treated root fluorescence compared roots. to mock-treated However, this dye roots. provided less However, this dye regarding zonal information provided potential less zonalsites information regarding potentialsuberin of R. solancearum-induced sites ofdeposition R. in solancearum-induced tomato roots. suberin deposition in tomato roots. Figure Figure 5.5. Pathogen-triggered Pathogen-triggered induction of Pro induction ::GUS Pro ofSlFHT SlFHTin xylem ::GUS in vasculature of tomato.of tomato. xylem vasculature Pro SlFHT::GUS transgenic tomato plants were inoculated through their roots by soaking the soil with ProSlFHT ::GUS transgenic tomato plants were inoculated through their roots by soaking the soil 40 mL of suspension per plant of R. solanacearum with a concentration of ~1 × 107 CFU ml−1 and with 40 mL of suspension per plant of R. solanacearum with a concentration of ~1 × 107 CFU mL−1 grown at 28 °C or with water as a control. GUS staining of (A) water-treated plants and (B) infected and grown plants, whereat 28 ◦ C orofwith induction water ProSlFHT::GUSas a control. could GUSinstaining be observed of (A) water-treated xylem vascular plants and (B) tissue (xv). UV auto- infected plants, fluorescence where before alkaliinduction treatment as ProSlFHT ::GUS of observed in the could be observed vasculature in xylem of water-treated (C)vascular or inocu-tissue (xv). lated (D). Alkali UV microscopic UV autofluorescence technique before alkali was used treatment to detect ferulate as observed in the deposition vasculaturein the vascula- of water-treated (C) ture of water-treated (E) or inoculated (F) samples at 20 days post-inoculation. Scale bar A, B = 2 or inoculated (D). Alkali UV microscopic technique was used to detect ferulate deposition in the vasculature of water-treated (E) or inoculated (F) samples at 20 days post-inoculation. Scale bar A, B = 2 mm; C, D, E, F = 150 µm. (G) Quantification of green fluorescence signal per µm in Region of Interest (ROI) from ferulate deposits upon alkaline treatment with KOH. Green color intensity calculation was performed with ImageJ software by selecting the vascular areas around the main vessels with localized fluorescence or green signal. Images from a representative experiment out of three with n = 5 plants per condition. Asterisks indicate statistically significant differences (**, p-value < 0.005, α = 0.05, Fisher’s least significant difference test). Plants 2023, 12, 1890 10 of 14 3. Discussion Suberin deposition as a response to multiple stresses has recently been explored with renewed vigor to develop plants resilient to abiotic and biotic stresses [2]. In tomato, suberin plays vital roles against diverse environmental stresses [41]. However, the absence of markers or tools for localizing suberizing tissues remains as an impediment for the fundamental understanding of the process in tomato and other crop plants, particularly during the response to various stresses, as well as during germplasm screening for varietal improvement programs. In tomato, the visualization of suberin in tissues mostly relies on stains such as Sudan IV or fluorol yellow, which target the aliphatic components [42] and cannot capture the early accumulation of ferulate esters catalyzed by FHT, which is expressed earlier than the fatty acyl biosynthetic machinery [43,44] and before suberin lamellae structuring. Suberin undergoes enormous compositional changes as the lamella matures. In the initial stages, lignin-like polymer is laid down in advance to the suberin polyester [45]. In fact, ferulates constitute a crucial component of the lignin-like polymer, potentially acting as nucleating sites for suberin matrix polymerization [29,46]. Hence, our tool using the promoter of SlFHT fused to GUS allows precise localization of early suberization sites. FHT is a key acyltransferase, which catalyzes the conjugation of feruloyl- CoA to aliphatic chains to form feruloyl esters, which are suberin precursors [19–21,29]. The putative SlFHT promoter region showed presence of cis-regulatory motifs specific to abiotic stresses such as wounding, salt injury, water stress, etc. (e.g., WBOXNTERF3) and to biotic stresses such as pathogenesis and salicylic acid receptiveness (e.g., GT1GMSCAM4, WBOXATNPR1), and as expected, a number of ABA responsive motifs were present (e.g., WBBOXPCWRKY1, MYB and MYC binding sites, SORLIP1, and WRKY) since suberin biosynthesis is known to be regulated by ABA [33–35]. In addition, motifs corresponding to tissue-specific activation of phenylpropanoid genes (e.g., EBOXBNNAPA) and root and seed inducible motifs were present (e.g., ROOTMOTIFTAPOX1, RYREPEATBNNAPA). The tomato FHT gene is very close in ancestry to the potato FHT gene [28], and its promoter was also reported to contain similar cis-regulatory motifs [29]. In agreement with the critical role reported for FHT in suberization [17,18,24], we observed the induction of ProSlFHT ::GUS in the outer root layers, which could correspond to root exodermis, which is well known to deposit suberin [36]. Further, we observed strong induction of ProSlFHT ::GUS in the lateral root primordia, indicating a hardening process by suberin–ferulate deposition in the cells of the developing lateral root cap before emergence. Interestingly, the root caps of lateral roots have been reported to contain a cuticle-like polymer in Arabidopsis [47]. In contrast, we observed that lateral root primordia initiated suberization during their development in tomatoes. It is possible that both lipid-based polymers, suberin and cutin, having common ancestry [48], perform similar functions towards root cap hardening. However, ferulic acid, which is a chief component of suberin-associated phenolics, is a minor component in the chemically similar cutin polymer [1]. In accordance with the function of FHT, we observed ferulate deposition using alkali UV microscopy at the sites of promoter activation. Since suberin deposition is known to occur as part of the wound- healing response [29,40], we also observed a strong induction of ProSlFHT ::GUS surrounding the tissues at 48 h post pin-prick injury on leaves. Further, when water imbalance or other factors lead to fruit cracks in tomato, the plants have a mechanism to seal this crack to prevent rotting due to the growth of saprophytes. We could observe specific induction of ProSlFHT ::GUS in the sealing region of the cracks, indicating suberization in this particular wound-healing response. In all cases, a positive correlation between FHT promoter activity and ferulate deposition by alkali UV microscopy was observed. Additionally, induction of ProSlFHT ::GUS in the vasculature of resistant tomato cultivar H7996 against pathogen R. solanacearum was observed. Suberin vascular coating in response to vascular pathogen R. solanacearum infection has been previously reported in resistant tomato cultivar H7996 [28]. In infected resistant tomato plants, induction of ProSlFHT ::GUS was observed in the xylem vascular tissue, and ferulate vascular reinforcements were also observed, showing positive Plants 2023, 12, 1890 11 of 14 correlation between induction of ProSlFHT ::GUS and ferulate deposition after infection with R. solanacearum. Together, these data indicate that this tool can be used as an efficient marker of early suberization events during plant development and stress responses. It can potentially be translated to other vascular pathogens to tomato as well as for studying other stress responses such as salt injury, drought stress, etc. Further, the tomato FHT promoter is a good candidate for tissue-specific expression of desired genes and metabolic engineering at targeted sites such as the exodermis, lateral root primordia, wound-healing zones, and other sites undergoing suberization to confer resilience to diverse stresses. In conclusion, we have developed a very effective tool to detect the sites for early suberization events linked to FHT activation and ferulate deposition. 4. Materials and Methods 4.1. Plant Material and Growth Conditions The tomato (Solanum lycopersicum) variety Hawaii 7996 (H7996) was used for all experiments. Seeds were germinated and plants were grown in pots containing soil (Substrate 2, Klasmann-Deilmann GmbH) mixed with perlite and vermiculite (30:1:1) in controlled growth chambers at 60% humidity and 12 h photoperiod with light intensity of 120–150 µmol·m−2 ·s−1 . The temperature was set at 27 ◦ C when using LED lighting and at 25 ◦ C when using fluorescent lighting. 4.2. DNA Constructs The ProFHT ::GUS construct was generated using the Gateway system (Invitrogen, Waltham, MA, USA). A fragment consisting of 1713 bp upstream of the initial ATG codon (Solyc03g097500) was amplified from genomic DNA of H7996 tomato using the forward primer (ProFHTF1: ACAAGTTTGTACAAAAAAGCAGGCTAAACAACAAAATAAGATTG- CAC) and the reverse primer (ProFHTR1: ACCACTTTGTACAAGAAAGCTGGGTTTTCT- CAAAATTAATAAATCCTG) containing the attB flanking sequences. This sequence was cloned into the Gateway entry vector pDONR 207 using a BP reaction and then transferred into the Gateway destination vector pGWB3 using an LR reaction. 4.3. Stable Transformation of Tomato ProFHT ::GUS construct was transformed into H7996 using Agrobacterium tumefaciens strain C58C1. A. tumefaciens was used for co-culture with tomato cotyledons. Explant preparation, selection, and regeneration were performed following standard protocols [49]. Transformants were selected on kanamycin-containing MS medium and propagated into subsequent generations. 4.4. Detection of SlFHT Promoter Activity Plant tissues or taproot–hypocotyl transition zone sections were immersed in an ice- chilled 90% (v/v) acetone bath and incubated for 20 min on ice, after which they were rinsed with water. Tissues were vacuum-infiltrated for 20 min with a solution containing 1 mM 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-Gluc), 50 mM sodium phosphate buffer (pH 7), 1 mM potassium ferrocyanide, 1 mM potassium ferricyanide, and 0.05% (v/v) Triton X-100. Samples were then incubated at 37 ◦ C for a maximum of 48 h. Stained tissues were washed 2–3 times with phosphate-buffered saline (PBS) and then cleared with 70% (v/v) ethanol and stored in 70% (v/v) ethanol. Images were obtained using an Olympus SZX16 stereomicroscope equipped with a DP71 camera system. 4.5. Histological Methods Thin tangential sections were made with a sterile razor blade of leaf discs, fruit section, or taproot–hypocotyl transition zone for histological assays. Autofluorescence from feru- lates bound to the cell wall shows a pH-dependent blue-to-green color conversion [37–39]. Ferulates in the xylem vascular tissue were visualized by mounting cross-sections in 70% Plants 2023, 12, 1890 12 of 14 ethanol (neutral pH) and illuminating them with UV with excitation bandpass filter in the range 340–380 nm to observe blue autofluorescence. These same sections were subsequently mounted in 1N KOH (pH above 10) to observe green autofluorescence from ferulates. Green color intensity calculation was performed with ImageJ software by selecting the vascular areas around the main vessels with localized fluorescence or green signal. 4.6. Wounding in Leaves Injuries in leaves were performed by pin prick using a sterile 0.3 × 13 mm needle (30G × 1/2”, BD Microlance, Becton Dickinson, Franklin Lakes, NJ, USA). 4.7. Bacterial Inoculation in Plants Four- to five-week-old tomato plants were inoculated through their roots with Ralstonia solanacearum GMI1000 using the soil drenching method [50]. For this, roots were wounded by making four holes in the soil at the corners of the pot with a 1 mL pipette tip and inoculated with a 1 × 107 CFU mL−1 (OD600 = 0.01) suspension of bacteria [50]. Inoculated plants were kept in a growth chamber at 28 ◦ C for 20 days. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12091890/s1, Figure S1: Fluorol yellow staining for detecting aliphatic suberin in ProFHT::GUS transgenic plants treated with mock or inoculated with 105 CFU/mL of Ralstonia solanacearum by pin inoculation. Quantification of mean fluorescence signal along the root, n ≥ 6, error bars: SD. *** = p-value < 0.005. One-way ANOVA followed by Tukey HSD. Scalebar: 200 µm. Author Contributions: Conceptualization, A.K. and N.S.C.; methodology, A.K., M.F., O.S. and Á.J.-J.; investigation, A.K. and Á.J.-J.; writing—original draft preparation, A.K. and N.S.C.; writing—review and editing, M.F., O.S. and M.V.; funding acquisition, M.V. and N.S.C. All authors have read and agreed to the published version of the manuscript. Funding: Research was funded by grant MCIN/AEI/PID2019-108595RB-I00 funded by MCIN/AEI/ 10.13039/501100011033, grant TED2021-131457B-I00 funded by MCIN/AEI/10.13039/501100011033 and by the “European Union NextGenerationEU/PRTR”, through the “Severo Ochoa Programme for Centres of Excellence in R&D” (CEX2019-000917 funded by MCIN/AEI/10.13039/501100011033), and by the CERCA Program/Generalitat de Catalunya. A.K. was the recipient of a Netaji Subhas—Indian Council of Agricultural Research International Fellowship and supported by a study leave granted by Assam Agricultural University. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data that support the findings of this study are available from the corresponding author (N.S.C.), upon reasonable request. Acknowledgments: We kindly acknowledge Montserrat Capellades for her help throughout the project and Cameron Tyler Prince for English language editing. Conflicts of Interest: The authors declare no conflict of interest. References 1. Cohen, H.; Dong, Y.; Szymanski, J.; Lashbrooke, J.; Meir, S.; Almekias-Siegl, E.; Zeisler-Diehl, V.V.; Schreiber, L.; Aharoni, A. A Multilevel Study of Melon Fruit Reticulation Provides Insight into Skin Ligno-Suberization Hallmarks. Plant Physiol. 2019, 179, 1486–1501. [CrossRef] [PubMed] 2. Serra, O.; Geldner, N. The Making of Suberin. New Phytol. 2022, 235, 848–866. [CrossRef] [PubMed] 3. Beisson, F.; Li-Beisson, Y.; Pollard, M. Solving the Puzzles of Cutin and Suberin Polymer Biosynthesis. Curr. Opin. Plant Biol. 2012, 15, 329–337. [CrossRef] [PubMed] 4. 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