Conserved and novel roles of the bHLH transcription factor SPATULA in tomato

Copyedited by: OUP Journal of Experimental Botany https://doi.org/10.1093/jxb/eraf029 Advance Access Publication 27 January 2025 © The Author(s) 2025. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact [email protected] for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact [email protected]. RESEARCH PAPER Conserved and novel roles of the bHLH transcription factor SPATULA in tomato Eduardo Martínez-Estrada1, Judith Jazmín Bernal-Gallardo1,, Pablo López-Gómez1,2, Daniela de la Mora-Franco1, Marisela Celso-Espinoza1, Moisés Guerrero-Esperanza1, David Díaz-Ramírez3, Nayelli Marsch-Martínez3, José Juan Ordaz-Ortiz1, and Stefan de Folter1,*, 1 Advanced Genomics Unit, Center for Research and Advanced Studies (Cinvestav), Irapuato, Mexico 2 Rosario Izapa Experimental Field of National Institute of Forestry, Agriculture and Livestock Research (INIFAP), Chiapas, Mexico 3 Biotechnology and Biochemistry Department, Irapuato Unit, Center for Research and Advanced Studies (Cinvestav), Irapuato, Mexico * Correspondence: [email protected] Received 16 November 2024; Editorial decision 10 January 2025; Accepted 23 January 2025 Editor: Madelaine Bartlett, University of Cambridge, UK Abstract Arabidopsis has served as a model plant for studying the genetic networks that guide gynoecium development, but less is known about other species. Tomato (Solanum lycopersicum) is a model for fleshy fruit development and ripening. Here we used tomato to study the transcription factor SPATULA (SPT), a bHLH-family member that in Arabidopsis is known to be important for gynoecium development. We analysed the expression of the SlSPT gene during flower and fruit development and the interaction of the SlSPT protein with proteins previously reported as interactors of AtSPT in the gynoecium. We also generated and characterized loss-of-function tomato lines using CRISPR–Cas9. The results showed that SlSPT forms homodimers and partially conserves the interactions reported in Arabidopsis with some HECATE proteins. We conclude that SlSPT has a role in floral organ development, particularly in stamen fusion, style and stigma development, and trichome formation on the carpels. Furthermore, lack of SlSPT caused altered exocarp pigmentation. A metabolomic analysis of the exocarp showed perturbations in several pathways in the slspt mutant, with flavonoid biosynthesis being the most affected, which could potentially impact the nutritional value of the fruit. In summary, our results show conserved functions during gynoecium development and novel roles that enrich knowledge of the SPT gene in fleshy fruits. Keywords: Flavonoids, fruit development, gynoecium development, SPATULA, stigma, style, trichome formation, tomato. Introduction Fruit development is an elegant evolutionary strategy unique to angiosperms, the fruit serving the dual purpose of protecting developing seeds and facilitating their dispersal. Fruits and seeds are a major food source, and given that, it is crucial to understand the mechanisms that guide their development. Flower and fruit development requires, among other factors, the joint and coordinated action of a network of transcription factors that regulate gene expression (Karlova et al., 2011; Herrera-Ubaldo and de Folter, 2022). This genetic regulatory network was first described in the model plant Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf029/7983921 by The New York Botanical Garden user on 06 May 2025 Copyedited by: OUP Page 2 of 19 | Martínez-Estrada et al. Arabidopsis and has served as a reference for comparative studies in fruit crops (Roeder and Yanofsky, 2006; AlvarezBuylla et al., 2010; Herrera-Ubaldo and de Folter, 2022). One of the transcription factors involved in fruit development in Arabidopsis is SPATULA (SPT). AtSPT belongs to the basic helix–loop–helix (bHLH) family (Heisler et al., 2001) and was first characterized by Alvarez and Smyth (1999), who showed that it has an important role in carpel and fruit development, being required for transmitting tract development and the differentiation of tissues at the carpel margins (Heisler et al., 2001; Girin et al., 2011). In the atspt mutants, abnormal development of the septum, stigma, style, and transmission tract is observed. As a result, fertilization occurs less frequently, generating shorter fruits with lower seed production compared with wild-type plants (Alvarez and Smyth, 1999). SPT can form protein dimers with ALCATRAZ (ALC) (Groszmann et al., 2011), INDEHISCENT (IND) (Girin et al., 2011), and HECATE (HEC) proteins (Gremski et al., 2007; Schuster et al., 2015) to maintain proper pistil morphology and function. Recently, Reyes-Olalde et al. (2017) showed that AtSPT enables cytokinin signalling and promotes auxin biosynthesis and transport at the medial domain of the gynoecium. AtSPT functions are not limited to carpel development: it also regulates seed dormancy (Penfield et al., 2005), cotyledon and leaf expansion (Ichihashi et al., 2010; Josse et al., 2011), root growth (Makkena and Lamb, 2013), vegetative growth in response to temperature (Sidaway-Lee et al., 2010), and stomata and trichome number (Bernal-Gallardo et al., 2023). In contrast to Arabidopsis, which produces dry fruits, much less is known about the function of SPT in other types of fruits. Studies in Prunus persica, based on expression analysis, have suggested that PpSPT is a component of the gene network that controls endocarp margin development (Tani et al., 2011). In strawberry, based on a gene-silencing approach, it has been suggested that FaSPT plays a role in early fruit development (Tisza et al., 2010), and recently in cucumber, using CRISPR– Cas9 genome editing, Cheng et al. (2022) reported that CsSPT has a role in female fertility and carpel fusion by regulating transmitting tract differentiation. Although conserved functions could be expected, the function of SPT could be diversified in tomato, taking into account that other protein domains are present in the SlSPT protein (Ortiz-Ramírez et al., 2018), making the functional analysis of the gene relevant. Previously it was reported that down-regulated SlSPT expression using virus-induced gene silencing (VIGS) resulted in no obvious phenotypic differences during flower and fruit development when compared with wild-type plants (Ortiz-Ramírez et al., 2019). However, this result could be due to the nature of the VIGS technique, which may suffer from incomplete suppression of expression of the targeted gene (Ortiz-Ramírez et al., 2019). During the last few years CRISPR–Cas9 technology has permitted the analysis or re-evaluation of gene functions in tomato, with interesting results found (Zsögön et al., 2018; Ito et al., 2017; Wang et al., 2019, 2020). In this study, we analysed the function of the SlSPT gene in tomato flowers and fruits by generating stable loss-offunction lines using CRISPR–Cas9 technology. Protein– protein interactions of SlSPT with important transcription factors involved in gynoecium development were also evaluated. Here, we show that SlSPT can form homodimers, is able to interact with some SlHEC proteins outside the nucleus, and has a role in floral organ growth, particularly in stamen fusion and length, style and stigma development, and trichome formation on the carpels. Furthermore, during fruit development, the lack of SlSPT function causes an altered pigmentation of the exocarp in both developing and mature fruits. A metabolomic analysis of the exocarp showed that in the slspt mutants, there is a significant perturbation in several pathways such as flavonoid biosynthesis, glycerophospholipid metabolism, and glycerolipid metabolism. In summary, the results show a conserved function during carpel formation but also a diversity of novel functions that enrich knowledge of the transcription factor SPT in fleshy fruit development. Materials and methods Plant materials and growing conditions Tomato (Solanum lycopersicum) cultivars Micro-Tom (MT) and M82 were used as wild-type plants. After in vitro transformation and regeneration (described below), MT plants were grown in 5 litre pots, while M82 plants were grown in 10 litre pots. All plants were maintained in a greenhouse under temperatures of 26–32 °C, with a photoperiod of 11–13 h of natural light, without any additional lighting system. RT-PCR analysis RNA was extracted from flower buds (−12 to 0 d post-anthesis, dpa) during ovary formation stages, as previously reported by Xiao et al. (2009), and from fruits (2–45 dpa) using the TRIzol reagent (Thermo Fisher Scientific). A total of 1 µg of RNA, after DNAse I treatment, was used to synthesize cDNA using M-MLV reverse transcriptase (Thermo Fisher Scientific) according to the manufacturer’s instructions. The RT-PCR analysis was then performed using the oligonucleotides described in Supplementary Table S1. Guide RNA design and CRISPR–Cas9 system construction Crispr-P 2.0 (http://crispr.hzau.edu.cn/CRISPR2/) was used for designing guide RNA (gRNA) and for minimizing off-targets. Two guides for SlSPT (Solyc02g093280) were selected, the first one targeting the end of the first exon, the second one targeting the bHLH region (in exon 2). The MoClo Toolkit was used to assemble the constructs by the Golden Gate system as described by Reem and Van Eck (2019). Briefly, each gRNA was amplified and then cut/ligated to the synthetic Arabidopsis U6 promoter to create AtU6p::gRNA1 in the pICH47751 level 1 vector and AtU6p::gRNA2 in the pICH47761 level 1 vector. Then, constructs pICH47732-NOSp::NPTII::OCST, pICH47742-35S::Cas9::NOST, pICH47751-AtU6p::gRNA1, pICH47761-AtU6p::gRNA2, and the linker pICH41780 were cut/ligated into the level 2 vector pICSL4723. After sequence confirmation, the final construct was inserted into Agrobacterium tumefaciens (GV3101) for tomato transformation. All plasmids and primers needed to generate slspt-crispr mutants are listed in Supplementary Tables S2, S3. Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf029/7983921 by The New York Botanical Garden user on 06 May 2025 Copyedited by: OUP Roles of SPATULA in tomato | Page 3 of 19 Transformation and regeneration of edited tomato plants The protocol used (Martínez-Estrada et al., 2025) is based on previously reported protocols with minor modifications (Park et al., 2003; Van Eck et al., 2006). MT and M82 seeds were disinfected in a laminar flow hood using 15% commercial bleach with one drop of Tween 20 for 15 min; then the seeds were rinsed three times with sterile distilled water and finally placed to germinate on half-strength Murashige and Skoog (MS; Murashige and Skoog, 1962) semi-solid medium (pH 5.8, with 7 g l−1 of agar) in magenta boxes under 16 h light photoperiod at 23±2 °C. After 1 week of culture, the cotyledons were collected and, after cutting the extremes, were placed for 5 min in a co-culture solution (half-strength MS liquid medium at pH 5.8 and the Agrobacterium (GV3101 strain) that contains the CRISPR–Cas9 construct). The cotyledon explants were then cultivated on a co-culture medium [MS semisolid medium (pH 5.8, with 7 g l−1 of agar) supplemented with 0.2 mg l−1 2,4-D and 0.1 mg l−1 kinetin] for 48 h at 22–24 °C in darkness, and then placed on regeneration–selection medium [MS semisolid medium (pH 5.8, with 7 g l−1 of agar) supplemented with 2 mg l−1 zeatin, 75 mg l−1 kanamycin (selection marker) and 30 mg l−1 meropenem for Agrobacterium elimination]. After three to five rounds (14 d each) of culture on the selection and regeneration medium, well-formed shoots were transferred to the rooting medium (MS medium supplemented with 1 mg l−1 indole-3-acetic acid, pH 5.8 and 7 g l−1 of agar) for one to two rounds (20 d each). Finally, the plantlets were acclimatized to the soil in a plastic box at 22 °C and a 16 h light photoperiod in a growth chamber for 3 weeks. Acclimatized plants were moved to the greenhouse for further genotyping. DNA extraction, PCR genotyping, and sequencing of T0 tomato plants Foliar tissue was collected from 10 T0 plants and genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) protocol (Doyle, 1991). All plants were genotyped for the presence of the Cas9 fragment by PCR. Positive Cas9 lines were sequenced using a pair of primers flanking the region where guides were designed. Alignments to the wild-type sequence were performed using the SnapGene software. Homozygous Cas9-free progeny of two lines that showed a strong phenotype were selected for further analysis. Protein–protein interaction assay A yeast two-hybrid (Y2H) assay was conducted using the GAL4 system (Thermo Fisher Scientific) in the PJ69 yeast strain. Coding sequences of SlSPT, and the reported interactors for AtSPT and their homologous genes from tomato were cloned into the pENTR/DTOPO vector to generate entry clones for each gene. The entry clones were then recombined with pDEST22 for GAL4-AD fusions or with pDEST32 for DNA-binding domain (BD) fusions. Yeast transformation and the interaction assay were done by following the protocol described by de Folter and Immink (2011). All Arabidopsis yeast clones used were obtained from the transcription factor collection reported by Herrera-Ubaldo et al. (2023). Four activation domain (AD) and two BD colonies per construct were mated to generate a matrix of all possible combinations of the cloned genes, except for the test of a possible homodimer of SlHEC1-1, which could not be recombined with pDEST22 to obtain the AD clone. Mating was performed by dropping an AD colony (resuspended in water) on top of the BD colony previously dropped on a plate with yeast extract, peptone, adenine, and dextrose (YPAD) medium. For diploid selection, colonies were transferred to plates with SD−Trp−Leu medium; the latter step was done twice. For the protein interaction test, diploid colonies were transferred to SD−Leu−Trp−Ade and SD−Leu−Trp−His with different concentrations of 3-amino triazole (determined after an autoactivation test of the BD clones); each selection marker was tested in duplicate. Plates were incubated at 22 °C. Yeast growth was scored 6 d after inoculation. The LacZ assay, to corroborate the positive interactions, was done by using the RO-BLUE medium described by de Folter and Immink (2011). All primers used for amplifying genes are listed in Supplementary Table S4. Bimolecular fluorescence complementation assay The entry clones containing the coding sequence of SlSPT, and the SlHEC genes were recombined with the destination vectors VN-GW and VC-GW (Kakita et al., 2007). The resulting expression vectors VN:SlSPT, VC:SlSPT, VC:SlHEC1, VC:SlHEC1-1, VC:SlHEC2, VC:SlHEC2-1, and VC:SlHEC3 were purified with the Zyppy Plasmid Miniprep Kit (Zymo Research). A bimolecular fluorescence complementation (BiFC) assay was carried out according to Herrera-Ubaldo et al. (2023). In brief, fully expanded leaves from 6-week-old Arabidopsis Col-0 ecotype were collected and used for protoplast isolation following the ‘Tape-ArabidopsisSandwich’ method (Wu et al., 2009). The digestion step was carried out for 2–3 h. For expression vector transfection, 150 μl of protoplast solution containing 3×104 cells was mixed with 10 μl of each vector (1 μg μl−1 plasmid DNA). The interactions evaluated were VN:SlSPT–VC:SlSPT; VN:SlSPT–VC:HEC1, VN:SlSPT–VC:HEC1-1, VN:SlSPT–VC:HEC2, VN:SlSPT–VC:HEC2-1, and VN:SlSPT–VC:HEC3; as negative controls, VN:SlSPT–VC:GW (empty) and VN:GW (empty)–VC:SlSPT were transfected (Supplementary Fig. S1). Transfection was performed following the polyethylene glycol–calcium method (Yoo et al., 2007); the reaction was stopped after 15 min of incubation at room temperature; then, the protoplasts were incubated at 25 °C in the dark. The analysis of fluorescence restoration was carried out 16 h after transfection. Excitation wavelengths and emission filters were 488 nm/band-pass 505–530 nm for yellow fluorescent protein (YFP) and 488 nm/band-pass 650–710 nm for chloroplast auto-fluorescence using an LSM800 confocal microscope with ×20 (EC Plan-Neufluar/0.30 air) and ×63 (Plan-Apochromat/1.40 Oil DIC) objectives (Carl Zeiss, Germany). All images were processed in the ZEN core software (Zeiss). Staining of pollen tubes within the pistils Staining was slightly modified from Muschietti et al. (1994). In brief, tomato flowers were emasculated at −2 dpa and then pistils were manually pollinated on the day of anthesis and collected after 24 h. The pistils were cut longitudinally into two equivalent parts and fixed in 3:1 ethanol: acetic acid for 1 h and softened in 8 M NaOH overnight. The fixed pistils were then rinsed several times with distilled water and incubated in aniline blue (0.1% in 0.1 M K3PO4) for 4 h in complete darkness. Finally, pistils were washed with distilled water and placed on a glass slide, carefully squashed under a cover slip, and observed using a Leica DM5000B microscope under UV light with a ×10 objective. The intensity of the pollen tube staining was quantified in relative intensity units using ImageJ, processing 8-bit grayscale images. Representative regions of interest (1 mm2) corresponding to the stained pollen tubes were selected in two different parts of the ovary, and the average intensity was normalized against background levels. Differences between treatments were statistically analysed using ANOVA followed by Fisher's least significant difference (LSD) test (P≤0.05, n=10). Histology Tomato flowers at −2 dpa were collected in 1.5 ml microtubes containing 1 ml of FAE (5% formaldehyde, 10% acetic acid, 50% ethanol). Tubes were placed in a vacuum desiccator and a vacuum was applied for 15 min. Afterward, samples were incubated at room temperature for 2 h. The tissue was dehydrated by passing through a series of ethanol solutions Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf029/7983921 by The New York Botanical Garden user on 06 May 2025 Copyedited by: OUP Page 4 of 19 | Martínez-Estrada et al. (70% overnight, 80%, 90%, 100%, and 100% ethanol) for 1 h each at room temperature. Samples were embedded in Technovit according to manufacturer’s instructions (Heraeus Kulzer, Germany). Blocks were sectioned at 12 μm using a Leica microtome (model: RM2025). The quality of the sections was checked by placing them on a glass slide with water; subsequently, 10–15 sections were transferred to each glass slide, air dried, and stained with Alcian Blue 8GX staining (Sigma-Aldrich; 0.3% dissolved in water, then adjusted to pH 3.1 or 2.5 with acetic acid; or pH 1.0 with HCl) for 20 min, washed with water for 1 min, and then stained with Neutral Red (Sigma-Aldrich; 0.5% dissolved in water) for 5 min, washed with water for 1 min, and air-dried to visualize them under bright field using a Leica DM6000B microscope with a ×10 and ×20 objectives, according to Zúñiga-Mayo et al. (2012). Fruit shape index and distal end protrusion Fruits at the Mature Green stage were used to analyse the Fruit Shape Index External I (the ratio of the maximum height to maximum width), and distal end protrusion (the area of the protruded end multiplied by 10, over the total fruit area), using Tomato Analyzer (Ohio State University) software. For Fruit Shape Index External I, a value >1 indicates an elongated fruit, equal to 1 indicates a round fruit, and <1 indicates a squat fruit (Brewer et al. 2006). For distal end protrusion the software assigns a value of 0 to fruit without a distal end protrusion (Gonzalo et al. 2009). Differences between treatments were statistically analysed using ANOVA followed by Fisher's LSD test (P≤0.05, n=10). Chlorophyll autofluorescence visualization Tomato fruits at the Mature Green stage were collected from wild-type and mutant lines, washed with tap water, and immersed in boiling water for 5 min. Then, using tweezers, the exocarp was collected to analyse chlorophyll autofluorescence with a confocal microscope. Chlorophyll was visualized with the propidium iodide fluorophore setting (excitation wavelength: 305 nm/band-pass, and emission: 617 nm), and the Electronically Switchable Illumination and Detection (ESID) module was used as a reference with a Zeiss LSM800 confocal laser scanning microscope with a ×20 (EC Plan-Neufluar/0.30 air) and ×63 (PlanApochromat/1.40 Oil DIC) objectives (Carl Zeiss, Germany). All images were processed in ZEN core software. Scanning electron microscopy Flowers at anthesis of the slspt mutants and wild-type plants were collected and directly scanned using a Zeiss EVO40 environmental scanning electron microscope (Carl Zeiss, Germany) with a 25 kV beam. For most samples, the signal was collected using the SE detector, except for samples in Fig. 3E–H, where the BSD detector was used. Metabolomic analysis Using ultra-performance liquid chromatography–quadrupole time-offlight mass spectrometry (UPLC-Q-TOF-MS) with a Waters Synapt G1 mass spectrometer, global profiling of tomato samples was conducted to analyse the metabolites present in the exocarp of mutant and wild-type fruits at two development stages: Mature Green and Red Ripe. Sample preparation Tomato fruits at the Mature Green and Red Ripe stages were collected, washed with tap water, and immersed in boiling water for 5 min. Then, using tweezers, the exocarp was collected and freeze-dried for 5 d. One hundred milligrams of freeze-dried samples were weighed and extracted in Eppendorf tubes with 1 ml of 80% methanol using a stainless-steel bead (5 mm) in a tissue lyser. Extraction in the tissue lyser was operated at 30 Hz for 1.5 min. Samples were then sonicated for 5 min and vortexed for 1 min. Samples were centrifugated at 6800 rcf for 10 min. Then, extracts were dried under vacuum in a miVac at 30 °C for 3 h. Dried extracts were then reconstituted in 400 μl solution of 5% acetonitrile and transferred to HPLC vials. Ten microlitres was injected into the UPLCQ-TOF-MS system using an Acquity class I UPLC system (Waters Corporation) coupled to a Synapt (G1) high definition mass spectrometer equipped with an electrospray ionization source. The W ion optics mode in the TOF was used in both positive and negative ionization modes to enhance resolution. MS/MS analysis was performed by MSE (expression analysis) to perform automatic fragmentation throughout the analyses. Data processing and cleansing Twenty-four LC-MS items of data (including five replicates for each stage for both wild type and a mix of exocarp from slspt mutants, and four quality control samples) were exported to the metabolomics software Progenesis QI (Non-Linear Dynamics, Waters Corporation). The data were lock-mass calibrated to m/z 554.2615 (leucine enkephalin). Sample runs were chromatographically aligned and normalized to all compounds detected. Peak picking was performed from 0.2 to 33 min; adducts were programmed for automatic detection. Four groups corresponding to sample conditions—Mature Green spatula mutants (MG slspt), Mature Green wild type (MG WT), Red Ripe spatula mutants (RR slspt), and Red Ripe wild type (RR WT)—were set as experimental design groups to evaluate the influence and scores of each molecular feature. Data from Progenesis QI for small molecules were subsequently exported to .csv files and uploaded to MetaboAnalyst v 5.0 (Pang et al., 2021, 2022) for statistical analysis. Metabolite pre-identification Parameters for metabolite pre-identification were as follows: for Progenesis MetaScope as the identification method and HMDB v 5.0 as search parameter, precursor and fragment tolerance of 10 ppm. For ChemSpider, PlantCyc, and Plant Metabolic Network, precursor tolerance of 10 ppm, fragment tolerance of 20 ppm, and isotope similarity filter of 90%. Preidentifications were manually accepted when features showed a total score of at least 40%, which included precursor ion mass error, MS/MS fragmentation pattern, and isotope similarity (Supplementary Table S5). For the comparison between slspt mutants (RR slspt) and wild type (RR WT) in the Arabidopsis KEGG library, the metabolites that were pre-identified were used for pathway analysis using Global Test as enrichment analysis and Relative-betweenness Centrality as topology analysis in MetaboAnalyst (www.metaboanalyst.ca). Results Expression of SlSPT in tomato fruit development To understand the function of SPATULA (SPT) in other types of fruit-producing species beyond Arabidopsis, we studied its function in tomato. We utilized the genomic and amino acid sequences of AtSPT as a reference to identify its orthologue. Using various BLAST modes, we found that the gene Solyc02g093280 presents the highest similarity in both DNA and amino acid sequences to AtSPT. When we used the genomic sequence of AtSPT, a 92.75% identity was found over a 69 bp query. In comparisons of the amino acid sequences, a Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf029/7983921 by The New York Botanical Garden user on 06 May 2025 Copyedited by: OUP Roles of SPATULA in tomato | Page 5 of 19 match of 141 out of 341 amino acids was observed, representing the highest coverage within the AtSPT protein. Sun et al. (2015) and Ortiz-Ramírez et al. (2018) also reported this gene as the AtSPT orthologue. The gene Solyc02g093280 (referred to as SlSPT hereafter), located on the positive strand, has a length of 2338 bp, consists of eight exons and seven introns, and encodes a bHLH protein of 379 amino acids. It is mostly expressed in flowers and fruits at different developmental stages, with slight expression observed in roots and leaves. Expression is also detected in the wild-type ancestor Solanum pimpinellifolium (Supplementary Fig. S2A; https://bar.utoronto. ca/eplant_tomato/). We analysed the expression of SlSPT from −12 days postanthesis (dpa) to 45 dpa using RT-PCR and found that it is predominantly expressed at −12 and −11 dpa, and then between 0 and 10 dpa (Supplementary Fig. S2B). No expression was observed in the latest stages of development, at 40–45 dpa. Then, using the Tomato Expression Atlas (Fernandez-Pozo et al., 2017), we visualized the expression pattern of SlSPT in the different tissues during fruit development; we found that, in line with the RT-PCR analysis, it is expressed at early stages of development, between 0 and 10 dpa, having higher levels in the pericarp, although it is also expressed in the septum, locular tissue, placenta columella, and seeds (Fig. 1A). A more detailed examination of the pericarp revealed that the highest SlSPT expression occurs in the outer epidermis of the fruit at 10 dpa, and during the ‘pink pericarp’ stage of ripening (Fig. 1A). Comparison of SlSPT and AtSPT protein–protein interactions One way to assess similarities in function, is to study protein–protein interactions among orthologous proteins. To assess whether SlSPT can form the same interactions as AtSPT with other transcription factors involved in gynoecium development, we conducted a Y2H assay to evaluate the ability of SlSPT to form dimers with proteins previously confirmed as interactors of AtSPT, including AtSPT itself (Gremski et al., 2007; Girin et al., 2011; Groszmann et al., 2011; Schuster et al., 2015). Initially, we used SlSPT as bait (BD) and the known interactors of AtSPT, including AtSPT, as prey (AD). We also included SlSPT as prey to assess its potential to form homodimers. The results showed that while AtSPT interacts with HEC1, 2, and 3, and IND, and can form homodimers (Fig. 1B), SlSPT does not interact in a similar manner as AtSPT, since only the interaction with AtIND was positive, and it is not able to form homodimers (Fig. 1C). Based on the first analysis, we hypothesized that SlSPT may only interact with the tomato orthologues of the aforementioned transcription factors. We found that OrtizRamírez et al. (2018) reported the presence of two copies of each HEC1 and HEC2, hereafter designated as SlHEC1 and SlHEC1-1, and SlHEC2 and SlHEC2-1, respectively. HEC3 is represented by the single gene SlHEC3, while the IND gene is not present in tomato, the most similar gene being SlHEC3. We cloned the coding sequences of the five SlHEC genes from tomato, and conducted a Y2H analysis for all possible combinations, at least in one direction. The results showed that SlSPT does not form dimers with any HEC protein in the Y2H system (Fig. 1D). Notably, we found that SlHEC1 can form homodimers and interact with SlHEC2, SlHEC2-1, and SlHEC3. To complement the results, we searched for another technique to test the interactions between SlSPT and the SlHEC proteins. We used the Gateway cloning system to clone the coding sequence of SlSPT in the N-terminal portion of the Venus fluorescent protein (VN-GW) and the coding sequence of the SlHEC genes and SlSPT in the C-terminal portion, and conducted a BiFC assay in protoplasts. Using this technique, we found a positive protein–protein interaction of SlSPT with some of the proteins tested (Fig. 1E). However, those interactions were not nuclear-localized (Fig. 1F). Among the proteins tested, we found that SlSPT can form homodimers along with heterodimers with SlHEC1-1, SlHEC 2-1, and SlHEC3. Finally, we show the protein interaction network obtained in this work, which compares the AtSPT and SlSPT interactions (Fig. 1G, H). Generation and characterization of SlSPT mutants using CRISPR–Cas9 To investigate the role of SlSPT in flower and fruit development, we produced stable loss-of-function mutants utilizing the CRISPR–Cas9 technology (Brooks et al., 2014) . Two RNA guides (gRNA) were designed, with the first targeting the end of the first exon and the second targeting the bHLH domain in the second exon. After vector construction, we employed Agrobacterium-mediated transformation and in vitro regeneration to obtain the SlSPT tomato mutants in the MT background. Only guide number 2 produces editing events (Fig. 2A). We selected two independent and representative alleles for further phenotypic analysis. The first allele, designated spt-cr1, displayed multiple nucleotide substitutions and a single base deletion near the predicted Cas9 cleavage site. Upon analysis of the amino acid sequence, we detected some changes in the amino acid sequence and the creation of a premature stop codon at amino acid position 161. The second allele, spt-cr2, showed a one-base deletion that also produces a premature stop codon at the amino acid position 161, right in the bHLH domain (Fig. 2B). An additional allele was produced in the M82 background to confirm the phenotypes obtained in this study (Supplementary Fig. S3). This allele, named slspt-cr3, is the result of the deletion of 1 bp on the target site of the sgRNA 1 and produces a truncated protein of 105 amino acids, before the bHLH domain (Supplementary Fig. S3A,B). Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf029/7983921 by The New York Botanical Garden user on 06 May 2025 Copyedited by: OUP Page 6 of 19 | Martínez-Estrada et al. Fig. 1. Expression pattern of SlSPT during fruit development and its protein–protein interactions with gynoecium development-related proteins. (A) Expression of SlSPT at different fruit stages based on the Tomato Expression Atlas. (B–D) Y2H assay of SlSPT: (B) control using BD–AtSPT; (C) interaction Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf029/7983921 by The New York Botanical Garden user on 06 May 2025 Copyedited by: OUP Roles of SPATULA in tomato | Page 7 of 19 Absence of SlSPT results in abnormal flower development We cultivated the two slspt alleles alongside the wild type (cv. Micro-Tom) to determine if there was any significant phenotype. Both alleles exhibited a noticeable phenotype in flower development (Fig. 2D). The first easily detectable phenotype was the improper fusion of the stamens, leaving the gynoecium exposed. The intensity of this phenotype can vary among flowers, with some flowers exhibiting barely unfused stamens, while in extreme cases, one stamen may be absent, or the typical conical form of the stamens may be lost. Additionally, we observed that the style in the mutant flowers is noticeably shorter than in the wild type (Fig. 2E; Supplementary Fig. S4A). These phenotypes were also observed in the CRISPR– Cas9-generated mutant slspt-cr3 in the M82 background (Supplementary Figs S3C–E, S4B). Statistical analysis indicated significant differences in style length between the two mutant alleles and the wild type, with the spt-cr2 allele producing the shortest styles (Fig. 2C). Additionally, we observed a phenotype similar to that seen in mutants for AtSPT in Arabidopsis, where the stigma is reduced or absent (Fig. 2E; Heisler et al., 2001). Absence of SlSPT results in altered trichome formation in the tomato gynoecium During flower characterization, it was evident that mutants had a significantly reduced amount of trichomes along the style compared with the wild type. To further characterize this phenotype, we analysed the gynoecium of both mutant alleles and the wild type using scanning electron microscopy imaging (Fig. 3A–H). Statistical analysis indicated a significant reduction in the formation of trichomes along the style in both alleles (less than 10 trichomes per mm) in respect to the wild type (almost 40 trichomes per mm) as shown in Fig. 3I. The analysis also revealed that the mutants not only had fewer trichomes along the style but also less quantity on the ovary, with even the absence of trichomes in some flowers of both alleles. These phenotypes are observable across nearly all flowers, allowing for early differentiation between a mutant line and the wild type. Finally, not only is the quantity of trichomes on the ovary affected, but the type of trichomes is also altered, with fewer glandular-type trichomes (Fig. 3I). The trichome number in the M82-derived slspt-cr3 mutant was not quantified, as the M82 cultivar rarely exhibits trichomes on the gynoecium (Supplementary Fig. S4B,C). SPATULA mutants showed reduced pollen tube growth and a high variability in seed number As shown in Fig. 2, both slspt mutant alleles show a reduction in stigma development and style length. We wondered whether pollination and subsequent seed production would be affected. We emasculated flowers and then manually pollinated several gynoecia of both alleles and wild type to analyse pollen tube growth through the style to the ovary using aniline blue staining. The results showed a reduced number of pollen tubes reaching the ovaries in both mutant alleles compared with the wild type (Fig. 4A). To statistically demonstrate this result, we quantified the intensity of the pollen tube staining in relative intensity units. Significant differences were observed in the slspt mutants compared with the wild type, while no differences were detected between the mutants (Fig. 4B). However, seed number was significantly reduced only in the slspt-cr2 mutant, although it exhibited high variability among fruits (Figs 5A, 6D). A similar variation in seed number was also observed in the slspt-cr1 mutants (Figs 5A, 6D). Transverse sections of ovaries at anthesis showed that, in general terms, there were no relevant changes between the mutants and the wild-type ovaries, since no statistical differences were obtained for cell layer number nor pericarp thickness (Fig. 4C–E). Morphological and metabolic effects in slspt mutant fruits We analysed the phenotypes of the fruits at two stages of development: Mature Green and Red Ripe. In wild-type fruits, a typical round shape was maintained throughout their growth. Mutant fruits, on the other hand, began to show a heart-shaped form at an early stage, which became more pronounced at the Red Ripe stage due to the elongation of the stylar end (Figs 5A, 6A). To better understand this latter phenotype, we analysed the Fruit Shape Index External I (H/W), which measures the ratio of the maximum fruit height (H) to the maximum fruit width (W), providing information about the overall shape of the fruit. We found that the slspt mutant fruits have an elongated shape compared with the round shape of the wild type (Supplementary Fig. S5A). Additionally, since the stylar end was elongated in the mutant fruits, we also analysed the distal end protrusion, a parameter to quantify the presence and severity of a protrusion at the distal end of the fruit. Statistically significant differences were observed when comparing the mutant fruits with wild-type fruits, reflecting that the stylar end of the test with proteins reported as AtSPT interactors in Arabidopsis gynoecium development; (D) interaction test with homologous proteins from tomato; the possible homodimer formation of SlHEC1-1 was not evaluated. (E) BiFC assay of SlSPT with itself and SlHEC proteins in protoplasts with a ×20 objective. (F) Positive interactions observed with a ×63 objective. (G) Summary of Y2H interactions for AtSPT. (H) Interaction network showing Y2H (black lines) and BiFC (blue lines) interactions of SlSPT. Abbreviations: AD, activation domain; BD, binding domain; BiFC, bimolecular fluorescence complementation; LMD, laser microdissection; RPM, reads per million; Y2H, yeast two-hybrid. Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf029/7983921 by The New York Botanical Garden user on 06 May 2025 Copyedited by: OUP Page 8 of 19 | Martínez-Estrada et al. fruit is indeed affected (Supplementary Fig. S5B). On the other hand, neither the locule number nor the diameter of the fruit was significantly affected (Fig. 6B, C). In addition, we observed that mutant fruits showed spots on the fruit exocarp since the early stages after pollination (Fig. 5). At Mature Green, the exocarp showed greener patches compared with the exocarp in wild-type fruits, ranging from a few small spots to covering most of the fruit. We collected the exocarp of Mature Green fruits and visualized them using a confocal microscope. The autofluorescence of Fig. 2. Characterization of the slspt mutants generated using CRISPR–Cas9. (A, B) Schematic diagram of the SlSPT gene, exons targeted by CRISPR– Cas9, and the resulting alleles used in this study. (C) Box plot of style length in slspt mutants and wild type (WT; Micro-Tom); different letters indicate statistically significant differences (Tukey’s test, P≤0.05, n=15). (D) Flower phenotypes of slspt mutants compared with WT (Micro-Tom). (E) Scanning electron micrographs showing the stamens, style, and stigma of slspt mutants and WT. Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf029/7983921 by The New York Botanical Garden user on 06 May 2025 Copyedited by: OUP Roles of SPATULA in tomato | Page 9 of 19 Fig. 3. Trichome analysis in tomato. (A–H) Scanning electron micrographs showing the reduction of trichomes on the basal part of the style and ovary in wild type (WT; A, B) and slspt mutants (C–H). (I) Statistical analysis of trichome numbers along the style and on the ovary in WT and slspt mutants. Different letters indicate statistically significant differences (Kruskal–Wallis test followed by Dunn’s test, P≤0.05, n=10). Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf029/7983921 by The New York Botanical Garden user on 06 May 2025 Copyedited by: OUP Page 16 of 19 | Martínez-Estrada et al. the accumulation of carotenoids (Liu et al., 2020). Another similar phenotype in fruit was also found in MYB72 RNAi lines reported by Wu et al. (2020), who found that MYB72 regulates chlorophyll, carotenoid, and flavonoid metabolism, reinforcing the idea that similar mechanisms could be responsible for the slspt mutant phenotypes. In addition to this, the metabolome results of the exocarp of the fruits show that some important metabolomic pathways such as flavonoid biosynthesis are altered in the slspt mutants (Fig. 6E, F). This is relevant because, besides carotenoids, flavonoids also play a significant role in determining the colour of tomato fruit and are proven to be functionally important for human health (Ballester et al., 2010; Yang et al., 2023). Of the flavonoids detected in our metabolomic analysis, some, such as tricin, naringenin, isorhamnetin, and quercetin, are considered promising nutraceuticals due to their anticancer, antioxidant, and anti-inflammatory activities (Lakhanpal and Rai, 2007; Shalini et al., 2016; Rauf et al., 2022). In plants, flavonoids have protective roles against biotic and abiotic stressful conditions such as during pathogen infections, UV-B and high-fluence white light exposure, drought, cold, and salinity (Falcone-Ferreyra et al., 2012). In this sense, some bHLH family members, like GLABRA3 (GB3) (Wada et al., 2014) and TRANSPARENT TESTA 8 (TT8) have been reported to be involved in flavonoid biosynthesis (Baudry et al., 2006; Xu et al., 2013). So, it is not surprising that SlSPT could be a relevant participant in this process. Recently, Jia et al. (2024) using CRISPR–Cas9-mediated mutagenesis, combined with metabolome and transcriptome analyses, also found that mutations in transcription factors, such as ELONGATED HYPOCOTYL 5 (HY5), APETALA 2a (Ap2a), RIPENING-INHIBITOR (RIN), TOMATO AGAMOUS-LIKE 1 (TAGl1), regulate ripening by modifying pigment accumulation. Taking the results together, SlSPT appears to be involved in various aspects of the flower and fruit development in tomato. In flower development, the lack of SlSPT affects stamen fusion, stigma and style development, and trichome development, and during fruit development, it affects the shape, the normal pigmentation of the exocarp, and metabolic pathways, with flavonoid biosynthesis being the most affected. Supplementary data The following supplementary data are available at JXB online. Fig. S1. Negative control for the BiFC assay. Fig. S2. Expression of SlSPT (Solyc02g093280) in tomato. Fig. S3. Characterization of the slspt-cr3 mutant generated using CRISPR–Cas9 technology in the M82 background. Fig. S4. Representative gynoecia of WT and slspt mutants. Fig. S5. Comparison of the Fruit Shape Index and distal end protrusion on slspt mutants and WT (MT) fruits using the Tomato Analyzer software. Fig. S6. Orthogonal partial least squares discriminant analysis (PLS-DA) heatmap showing the top 25 metabolites present in the exocarp of the fruit in slspt mutants (MG slspt) and WT (MG WT) at the Mature Green stage. Fig. S7. Comparative analysis of metabolite features and abundance in wild type and spt-cr mutants at the Mature Green stage. Fig. S8. Comparative analysis of metabolite features and abundance in wild type and spt-cr mutants at the Red Ripe stage. Table S1. List of primers used for the RT-PCR analysis. Table S2. List of plasmids used to generate slspt-crispr mutants. Table S3. List of primers used to generate slspt-crispr mutants. Table S4. List of primers used to clone the coding sequences used in the interaction assay. Table S5. Pre-identified metabolites. Acknowledgements We thank Valentin Luna-Garcia for technical help with initial pollen tube staining, Humberto Herrera-Ubaldo for technical help in the yeast twohybrid experiment, and Ruud de Maagd for initial help on how to make CRISPR mutants. We also thank the anonymous reviewers for their helpful comments and suggestions. Author contributions EME and SDF conceived and designed research. EME conducted most of the experiments. JJBG performed the scanning electron microscopy, PLG performed the confocal microscopy experiments, DDLMF realized the histology, and MCE performed the RT-PCR assays. MGE and JJOO performed the metabolomic analysis. DDR and NMM performed the statistical analyses. EME and SDF analysed all data and wrote the manuscript. All authors read and approved the manuscript. Conflict of interest The authors declare no conflict of interest. Funding We thank the Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT) from Mexico for a PhD fellowship to EME and DDLMF, and a MSc fellowship to MCE. This work in the SDF laboratory was financed by the CONAHCYT grants FC-2015-2/1061 and CB-20172018-A1-S-10126. SDF is grateful for participation in the European Union project H2020-MSCA-RISE-2020 EVOfruland (101007738). Data availability Raw data of the metabolome analysis can be found at FigShare: https:// doi.org/10.6084/m9.figshare.26729734.v1; Martínez-Estrada et al. (2024). Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf029/7983921 by The New York Botanical Garden user on 06 May 2025 Copyedited by: OUP Roles of SPATULA in tomato | Page 17 of 19 References Alvarez J, Smyth DR. 1999. CRABS CLAW and SPATULA, two Arabidopsis genes that control carpel development in parallel with AGAMOUS. Development 126, 2377–2386. Alvarez-Buylla ER, Benítez M, Corvera-Poiré A, et al. 2010. 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