A distal enhancer guides the negative selection of toxic glycoalkaloids during tomato domestication.

Steroidal glycoalkaloids (SGAs) are major plant defense metabolites against pests, while they are considered poisonous in food. The genetic basis that guides negative selection of SGAs production during tomato domestication remains poorly understood. Here, we identify a distal enhancer, GAME Enhancer 1 (GE1), as the key regulator of SGAs metabolism in tomato. GE1 recruits MYC2-GAME9 transcriptional complex to regulate the expression of GAME cluster genes via the formation of chromatin loops located in the neighboring DNA region. A naturally occurring GE176 allelic variant is found to be more active in stimulating GAME expression. We show that the weaker GE1 allele has been the main driver for selecting reduced SGAs levels during tomato domestication. Unravelling the "TFs-Enhancer-Promoter" regulatory mechanism operating in SGAs metabolism opens unprecedented prospects for SGAs manipulation in Solanaceae via precision breeding strategies.

Bifunctional transcription factors SlERF.H5 and H7 activate cell wall and repress gibberellin biosynthesis genes in tomato via a conserved motif.

The plant cell wall is a dynamic structure that plays an essential role in development, but the mechanism regulating cell wall formation remains poorly understood. We demonstrate that two transcription factors, SlERF.H5 and SlERF.H7, control cell wall formation and tomato fruit firmness in an additive manner. Knockout of SlERF.H5, SlERF.H7, or both genes decreased cell wall thickness, firmness, and cellulose contents in fruits during early development, especially in double-knockout lines. Overexpressing either gene resulted in thicker cell walls and greater fruit firmness with elevated cellulose levels in fruits but severely dwarf plants with lower gibberellin contents. We further identified that SlERF.H5 and SlERF.H7 activate the cellulose biosynthesis gene SlCESA3 but repress the gibberellin biosynthesis gene GA20ox1. Moreover, we identified a conserved LPL motif in these ERFs responsible for their activities as transcriptional activators and repressors, providing insight into how bifunctional transcription factors modulate distinct developmental processes.

A transcriptional cascade mediated by two APETALA2 family members orchestrates carotenoid biosynthesis in tomato.

Carotenoids are important nutrients for human health that must be obtained from plants since they cannot be biosynthesized by the human body. Dissecting the regulatory mechanism of carotenoid metabolism in plants represents the first step toward manipulating carotenoid contents in plants by molecular design breeding. In this study, we determined that SlAP2c, an APETALA2 (AP2) family member, acts as a transcriptional repressor to regulate carotenoid biosynthesis in tomato (Solanum lycopersicum). Knockout of SlAP2c in both the "MicroTom" and "Ailsa Craig" backgrounds resulted in greater lycopene accumulation, whereas overexpression of this gene led to orange-ripe fruit with significantly lower lycopene contents than the wild type. We established that SlAP2c represses the expression of genes involved in lycopene biosynthesis by directly binding to the cis-elements in their promoters. Moreover, SlAP2c relies on its EAR motif to recruit the co-repressors TOPLESS (TPL)2/4 and forms a complex with histone deacetylase (had)1/3, thereby reducing the histone acetylation levels of lycopene biosynthesis genes. Furthermore, SlAP2a, a homolog of SlAP2c, acts upstream of SlAP2c and alleviates the SlAP2c-induced repression of lycopene biosynthesis genes by inhibiting SlAP2c transcription during fruit ripening. Therefore, we identified a transcriptional cascade mediated by AP2 family members that regulates lycopene biosynthesis during fruit ripening in tomato, laying the foundation for the manipulation of carotenoid metabolism in plants.

Ethylene-MPK8-ERF.C1-PR module confers resistance against Botrytis cinerea in tomato fruit without compromising ripening.

The plant hormone ethylene plays a critical role in fruit defense against Botrytis cinerea attack, but the underlying mechanisms remain poorly understood. Here, we showed that ethylene response factor SlERF.C1 acts as a key regulator to trigger the ethylene-mediated defense against B. cinerea in tomato fruits without compromising ripening. Knockout of SlERF.C1 increased fruit susceptibility to B. cinerea with no effect on ripening process, while overexpression enhanced resistance. RNA-Seq, transactivation assays, EMSA and ChIP-qPCR results indicated that SlERF.C1 activated the transcription of PR genes by binding to their promoters. Moreover, SlERF.C1 interacted with the mitogen-activated protein kinase SlMPK8 which allowed SlMPK8 to phosphorylate SlERF.C1 at the Ser174 residue and increases its transcriptional activity. Knocking out of SlMPK8 increased fruit susceptibility to B. cinerea, whereas overexpression enhanced resistance without affecting ripening. Furthermore, genetic crosses between SlMPK8-KO and SlERF.C1-OE lines reduced the resistance to B. cinerea attack in SlERF.C1-OE fruits. In addition, B. cinerea infection induced ethylene production which in turn triggered SlMPK8 transcription and enhanced the phosphorylation of SlERF.C1. Overall, our findings reveal the regulatory mechanism of the 'Ethylene-MPK8-ERF.C1-PR' module in resistance against B. cinerea and provide new insight into the manipulation of gray mold disease in fruits.

The SlGRAS9-SlZHD17 transcriptional cascade regulates chlorophyll and carbohydrate metabolism contributing to fruit quality traits in tomato.

Some essential components of fleshy fruits are dependent on photosynthetic activity and carbohydrate metabolism. Nevertheless, the regulatory mechanisms linking chlorophyll and carbohydrate metabolism remain partially understood. Here, we uncovered the role of SlGRAS9 and SlZHD17 transcription factors in controlling chlorophyll and carbohydrate accumulation in tomato fruit. Knockout or knockdown of SlGRAS9 or SlZHD17 resulted in marked increase in chlorophyll content, reprogrammed chloroplast biogenesis and enhanced accumulation of starch and soluble sugars. Combined genome-wide transcriptomic profiling and promoter-binding experiments unveiled a complex mechanism in which the SlGRAS9/SlZHD17 regulatory module modulates the expression of chloroplast and sugar metabolism either via a sequential transcriptional cascade or through binding of both TFs to the same gene promoters, or, alternatively, via parallel pathways where each of the TFs act on different target genes. For instance, the regulation of SlAGPaseS1 and SlSUS1 is mediated by SlZHD17 whereas that of SlVI and SlGLK1 occurs only through SlGRAS9 without the intervention of SlZHD17. Both SlGRAS9 and SlZHD17 can also directly bind the promoter of SlPOR-B to regulate its expression. Taken together, our findings uncover two important regulators acting synergistically to manipulate chlorophyll and carbohydrate accumulation and provide new potential breeding targets for improving fruit quality in fleshy fruits.

The SlARF4-SlHB8 regulatory module mediates leaf rolling in tomato.

Leaf is the main photosynthetic organ in plants and the primary energy source all along the plant life. Given the beneficial role of leaf rolling in improving photosynthetic efficiency and yield in specific environmental conditions, a better understanding of the factors and molecular mechanisms underlying this process is highly suited. Previously, the SlARF4 knocking out mutant exhibited upward curly leaf showed higher resistance to water deficit which driving us to uncover the function of SlARF4 in regulating the curly leaf formation. In this study, we unraveled the unexplored role of the SlARF4-SlHB8 module of transcription factors in the development of leaf rolling. Both SlARF4 loss-of-function and SlHB8 overexpressing tomato plants exhibited upward-rolled leaves, reflecting the active role of the two genes in controlling leaf rolling. Dual-luciferase reporter assays and phenotypic analysis of hybrid progenies suggested that SlHB8 acts downstream of SlARF4 in curly leaf formation. SlARF4 and SlHB8 influence the development of leaf palisade tissues via modulating the expression of genes associated with curly leaf formation. SEM analysis revealed no significant differences in leaf epidermal cells between the two leaf-rolling mutants and the wild type, indicating that curly leaves of arf4 and SlHB8-OE do not result from the asymmetric leaf epidermal cell growth. Our data provide novel insight into the molecular mechanism of abaxial-adaxial determination involving SlARF4 and SlHB8 and reveals that leaf rolling operates via different regulation mechanisms in tomato and Arabidopsis model plant.

The MADS-box protein SlTAGL1 regulates a ripening-associated SlDQD/SDH2 involved in flavonoid biosynthesis and resistance against Botrytis cinerea in post-harvest tomato fruit.

3-Dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH) is a key rate-limiting enzyme that catalyzes the synthesis of the shikimate, which is an important metabolic intermediate in plants and animals. However, the function of SlDQD/SDH family genes in tomato (Solanum lycopersicum) fruit metabolites is still unknown. In the present study, we identified a ripening-associated SlDQD/SDH member, SlDQD/SDH2, that plays a key role in shikimate and flavonoid metabolism. Overexpression of this gene resulted in an increased content of shikimate and flavonoids, while knockout of this gene by CRISPR/Cas9 mediated gene editing led to a significantly lower content of shikimate and flavonoids by downregulation of flavonoid biosynthesis-related genes. Moreover, we showed that SlDQD/SDH2 confers resistance against Botrytis cinerea attack in post-harvest tomato fruit. Dual-luciferase reporter and EMSA assays indicated that SlDQD/SDH2 is a direct target of the key ripening regulator SlTAGL1. In general, this study provided a new insight into the biosynthesis of flavonoid and B. cinerea resistance in fruit tomatoes.

ß-1,3-GLUCANASE10 regulates tomato development and disease resistance by modulating callose deposition.

ß-1,3-Glucanases are considered key regulators responsible for the degradation of callose in plants, yet little is known about the role and mode of action of their encoding genes in tomato (Solanum lycopersicum). In the present study, we identified the ß-1,3-glucanase encoding gene ß-1,3-GLUCANASE10 (SlBG10) and revealed its regulation in tomato pollen and fruit development, seed production, and disease resistance by modulating callose deposition. Compared with wild-type (WT) or SlBG10 overexpressing (SlBG10-OE) lines, knockout of SlBG10 caused pollen arrest and failure to set fruit with reduced male rather than female fecundity. Further analyses showed that SlBG10-knockout promoted callose deposition in anther at the tetrad-to-microspore stages, resulting in pollen abortion and male sterility. Moreover, loss-of-function SlBG10 delayed degradation of endosperm cell wall calloses during cellularization and impeded early seed development. We also uncovered that Botrytis cinerea infection induces SlBG10 expression in WT tomato, and the knockout lines showed increased callose accumulation in fruit pericarps, reduced susceptibility to B. cinerea, and enhanced antioxidant capacity to maintain tomato fruit quality. However, the expression of genes encoding cell wall hydrolases decreased in SlBG10-knockout tomatoes and thus led to an increase in pericarp epidermal thickness, enhancement in fruit firmness, reduction of fruit water loss, and extension of tomato shelf life. These findings not only expand our understanding of the involvement of ß-1,3-glucanases as callose regulators in multiple developmental processes and pathogen resistance but also provide additional insight into the manipulation of multiagronomic traits for targeted tomato breeding.

A comprehensive metabolic map reveals major quality regulations in red-flesh kiwifruit (Actinidia chinensis).

Kiwifruit (Actinidia chinensis) is one of the popular fruits world-wide, and its quality is mainly determined by key metabolites (sugars, flavonoids, and vitamins). Previous works on kiwifruit are mostly done via a single omics approach or involve only limited metabolites. Consequently, the dynamic metabolomes during kiwifruit development and ripening and the underlying regulatory mechanisms are poorly understood. In this study, using high-resolution metabolomic and transcriptomic analyses, we investigated kiwifruit metabolic landscapes at 11 different developmental and ripening stages and revealed a parallel classification of 515 metabolites and their co-expressed genes into 10 distinct metabolic vs gene modules (MM vs GM). Through integrative bioinformatics coupled with functional genomic assays, we constructed a global map and uncovered essential transcriptomic and transcriptional regulatory networks for all major metabolic changes that occurred throughout the kiwifruit growth cycle. Apart from known MM vs GM for metabolites such as soluble sugars, we identified novel transcription factors that regulate the accumulation of procyanidins, vitamin C, and other important metabolites. Our findings thus shed light on the kiwifruit metabolic regulatory network and provide a valuable resource for the designed improvement of kiwifruit quality.

Transition to ripening in tomato requires hormone-controlled genetic reprogramming initiated in gel tissue.

Ripening is the last stage of the developmental program in fleshy fruits. During this phase, fruits become edible and acquire their unique sensory qualities and post-harvest potential. Although our knowledge of the mechanisms that regulate fruit ripening has improved considerably over the past decades, the processes that trigger the transition to ripening remain poorly deciphered. While transcriptomic profiling of tomato (Solanum lycopersicum L.) fruit ripening to date has mainly focused on the changes occurring in pericarp tissues between the Mature Green and Breaker stages, our study addresses the changes between the Early Mature Green and Late Mature Green stages in the gel and pericarp separately. The data showed that the shift from an inability to initiate ripening to the capacity to undergo full ripening requires extensive transcriptomic reprogramming that takes place first in the locular tissues before extending to the pericarp. Genome-wide transcriptomic profiling revealed the wide diversity of transcription factor (TF) families engaged in the global reprogramming of gene expression and identified those specifically regulated at the Mature Green stage in the gel but not in the pericarp, thereby providing potential targets toward deciphering the initial factors and events that trigger the transition to ripening. The study also uncovered an extensive reformed homeostasis for most plant hormones, highlighting the multihormonal control of ripening initiation. Our data unveil the antagonistic roles of ethylene and auxin during the onset of ripening and show that auxin treatment delays fruit ripening via impairing the expression of genes required for System-2 autocatalytic ethylene production that is essential for climacteric ripening. This study unveils the detailed features of the transcriptomic reprogramming associated with the transition to ripening of tomato fruit and shows that the first changes occur in the locular gel before extending to pericarp and that a reformed auxin homeostasis is essential for the ripening to proceed.

DNA and coding/non-coding RNA methylation analysis provide insights into tomato fruit ripening.

Ripening is the last, irreversible developmental stage during which fruit become palatable, thus promoting seed dispersal by frugivory. In Alisa Craig fruit, mRNAs with increasing m5C levels, such as STPK and WRKY 40, were identified as being involved in response to biotic and abiotic stresses. Furthermore, two mRNAs involved in cell wall metabolism, PG and EXP-B1, also presented increased m5C levels. In the Nr mutant, several m5C-modified mRNAs involved in fruit ripening, including those encoding WRKY and MADS-box proteins, were found. Targets of long non-coding RNAs and circular RNAs with different m5C sites were also found; these targets included 2-alkenal reductase, soluble starch synthase 1, WRKY, MADS-box, and F-box/ketch-repeat protein SKIP11. A combined analysis of changes in 5mC methylation and mRNA revealed many differentially expressed genes with differentially methylated regions encoding transcription factors and key enzymes related to ethylene biosynthesis and signal transduction; these included ERF084, EIN3, AP2/ERF, ACO5, ACS7, EIN3/4, EBF1, MADS-box, AP2/ERF, and ETR1. Taken together, our findings contribute to the global understanding of the mechanisms underlying fruit ripening, thereby providing new information for both fruit and post-harvest behavior.

The SlTPL3-SlWUS module regulates multi-locule formation in tomato by modulating auxin and gibberellin levels in the shoot apical meristem.

Malformed fruits depreciate a plant's market value. In tomato (Solanum lycopersicum), fruit malformation is associated with the multi-locule trait, which involves genes regulating shoot apical meristem (SAM) development. The expression pattern of TOPLESS3 (SlTPL3) throughout SAM development prompted us to investigate its functional significance via RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing. Lower SlTPL3 transcript levels resulted in larger fruits with more locules and larger SAMs at the 5 d after germination (DAG5) stage. Differentially expressed genes in the SAM of wild-type (WT) and SlTPL3-RNAi plants, identified by transcriptome deep sequencing (RNA-seq), were enriched in the gibberellin (GA) biosynthesis and plant hormone signaling pathways. Moreover, exogenous auxin and paclobutrazol treatments rescued the multi-locule phenotype, indicating that SlTPL3 affects SAM size by mediating auxin and GA levels in the SAM. Furthermore, SlTPL3 interacted with WUSCHEL (SlWUS), which plays an important role in SAM size maintenance. We conducted RNA-seq and DNA affinity purification followed by sequencing (DAP-seq) analyses to identify the genes regulated by SlTPL3 and SlWUS in the SAM and to determine how they regulate SAM size. We detected 24 overlapping genes regulated by SlTPL3 and SlWUS and harboring an SlWUS-binding motif in their promoters. Furthermore, functional annotation revealed a notable enrichment for functions in auxin transport, auxin signal transduction, and GA biosynthesis. Dual-luciferase assays also revealed that SlTPL3 enhances SlWUS-mediated regulation (repression and activation) of SlPIN3 and SlGA2ox4 transcription, indicating that the SlTPL3-SlWUS module regulates SAM size by mediating auxin distribution and GA levels, and perturbations of this module result in enlarged SAM. These results provide novel insights into the molecular mechanism of SAM maintenance and locule formation in tomato and highlight the SlTPL3-SlWUS module as a key regulator.

The auxin-responsive transcription factor SlDOF9 regulates inflorescence and flower development in tomato.

Understanding the mechanisms underlying differentiation of inflorescence and flower meristems is essential towards enlarging our knowledge of reproductive organ formation and to open new prospects for improving yield traits. Here, we show that SlDOF9 is a new modulator of floral differentiation in tomato. CRISPR/Cas9 knockout strategy uncovered the role of SlDOF9 in controlling inflorescence meristem and floral meristem differentiation via the regulation of cell division genes and inflorescence architecture regulator LIN. Tomato dof9-KO lines have more flowers in both determinate and indeterminate cultivars and produce more fruit upon vibration-assisted fertilization. SlDOF9 regulates inflorescence development through an auxin-dependent ARF5-DOF9 module that seems to operate, at least in part, differently in Arabidopsis and tomato. Our findings add a new actor to the complex mechanisms underlying reproductive organ differentiation in flowering plants and provide leads towards addressing the diversity of factors controlling the transition to reproductive organs.

SlWRKY35 positively regulates carotenoid biosynthesis by activating the MEP pathway in tomato fruit.

Carotenoids are vital phytonutrients widely recognised for their health benefits. Therefore, it is vital to thoroughly investigate the metabolic regulatory network underlying carotenoid biosynthesis and accumulation to open new leads towards improving their contents in vegetables and crops. The outcome of our study defines SlWRKY35 as a positive regulator of carotenoid biosynthesis in tomato. SlWRKY35 can directly activate the expression of the 1-deoxy-d-xylulose 5-phosphate synthase (SlDXS1) gene to reprogramme metabolism towards the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, leading to enhanced carotenoid accumulation. We also show that the master regulator SlRIN directly regulates the expression of SlWRKY35 during tomato fruit ripening. Compared with the SlLCYE overexpression lines, coexpression of SlWRKY35 and SlLCYE can further enhance lutein production in transgenic tomato fruit, indicating that SlWRKY35 represents a potential target towards designing innovative metabolic engineering strategies for carotenoid derivatives. In addition to providing new insights into the metabolic regulatory network associated with tomato fruit ripening, our data define a new tool for improving fruit content in specific carotenoid compounds.

SlERF.F12 modulates the transition to ripening in tomato fruit by recruiting the co-repressor TOPLESS and histone deacetylases to repress key ripening genes.

Ethylene response factors (ERFs) are downstream components of ethylene-signaling pathways known to play critical roles in ethylene-controlled climacteric fruit ripening, yet little is known about the molecular mechanism underlying their mode of action. Here, we demonstrate that SlERF.F12, a member of the ERF.F subfamily containing Ethylene-responsive element-binding factor-associated Amphiphilic Repression (EAR) motifs, negatively regulates the onset of tomato (Solanum lycopersicum) fruit ripening by recruiting the co-repressor TOPLESS 2 (TPL2) and the histone deacetylases (HDAs) HDA1/HDA3 to repress the transcription of ripening-related genes. The SlERF.F12-mediated transcriptional repression of key ripening-related genes 1-AMINO-CYCLOPROPANE-1-CARBOXYLATE SYNTHASE 2 (ACS2), ACS4, POLYGALACTURONASE 2a, and PECTATE LYASE is dependent on the presence of its C-terminal EAR motif. We show that SlERF.F12 interacts with the co-repressor TPL2 via the C-terminal EAR motif and recruits HDAs SlHDA1 and SlHDA3 to form a tripartite complex in vivo that actively represses transcription of ripening genes by decreasing the level of the permissive histone acetylation marks H3K9Ac and H3K27Ac at their promoter regions. These findings provide new insights into the ripening regulatory network and uncover a direct link between repressor ERFs and histone modifiers in modulating the transition to ripening of climacteric fruit.

Integrative analyses of metabolome and genome-wide transcriptome reveal the regulatory network governing flavor formation in kiwifruit (Actinidia chinensis).

Soluble sugars, organic acids and volatiles are important components that determine unique fruit flavor and consumer preferences. However, the metabolic dynamics and underlying regulatory networks that modulate overall flavor formation during fruit development and ripening remain largely unknown for most fruit species. In this study, by integrating flavor-associated metabolism and transcriptome data from 12 fruit developmental and ripening stages of Actinidia chinensis cv Hongyang, we generated a global map of changes in the flavor-related metabolites throughout development and ripening of kiwifruit. Using this dataset, we constructed complex regulatory networks allowing to identify key structural genes and transcription factors that regulate the metabolism of soluble sugars, organic acids and important volatiles in kiwifruit. Moreover, our study revealed the regulatory mechanism involving key transcription factors regulating flavor metabolism. The modulation of flavor metabolism by the identified key transcription factors was confirmed in different kiwifruit species providing the proof of concept that our dataset provides a suitable tool for clarification of the regulatory factors controlling flavor biosynthetic pathways that have not been previously illuminated. Overall, in addition to providing new insight into the metabolic regulation of flavor during fruit development and ripening, the outcome of our study establishes a foundation for flavor improvement in kiwifruit.

Phosphorylation of transcription factor bZIP21 by MAP kinase MPK6-3 enhances banana fruit ripening.

Ripening of fleshy fruits involves both diverse post-translational modifications (PTMs) and dynamic transcriptional reprogramming, but the interconnection between PTMs, such as protein phosphorylation and transcriptional regulation, in fruit ripening remains to be deciphered. Here, we conducted a phosphoproteomic analysis during banana (Musa acuminata) ripening and identified 63 unique phosphopeptides corresponding to 49 proteins. Among them, a Musa acuminata basic leucine zipper transcription factor21 (MabZIP21) displayed elevated phosphorylation level in the ripening stage. MabZIP21 transcript and phosphorylation abundance increased during banana ripening. Genome-wide MabZIP21 DNA binding assays revealed MabZIP21-regulated functional genes contributing to banana ripening, and electrophoretic mobility shift assay, chromatin immunoprecipitation coupled with quantitative polymerase chain reaction, and dual-luciferase reporter analyses demonstrated that MabZIP21 stimulates the transcription of a subset of ripening-related genes via directly binding to their promoters. Moreover, MabZIP21 can be phosphorylated by MaMPK6-3, which plays a role in banana ripening, and T318 and S436 are important phosphorylation sites. Protein phosphorylation enhanced MabZIP21-mediated transcriptional activation ability, and transient overexpression of the phosphomimetic form of MabZIP21 accelerated banana fruit ripening. Additionally, MabZIP21 enlarges its role in transcriptional regulation by activating the transcription of both MaMPK6-3 and itself. Taken together, this study reveals an important machinery of protein phosphorylation in banana fruit ripening in which MabZIP21 is a component of the complex phosphorylation pathway linking the upstream signal mediated by MaMPK6-3 with transcriptional controlling of a subset of ripening-associated genes.

Interaction of two MADS-box genes leads to growth phenotype divergence of all-flesh type of tomatoes.

All-flesh tomato cultivars are devoid of locular gel and exhibit enhanced firmness and improved postharvest storage. Here, we show that SlMBP3 is a master regulator of locular tissue in tomato fruit and that a deletion at the gene locus underpins the All-flesh trait. Intriguingly, All-flesh varieties lack the deleterious phenotypes reported previously for SlMBP3 under-expressing lines and which preclude any potential commercial use. We resolve the causal factor for this phenotypic divergence through the discovery of a natural mutation at the SlAGL11 locus, a close homolog of SlMBP3. Misexpressing SlMBP3 impairs locular gel formation through massive transcriptomic reprogramming at initial phases of fruit development. SlMBP3 influences locule gel formation by controlling cell cycle and cell expansion genes, indicating that important components of fruit softening are determined at early pre-ripening stages. Our findings define potential breeding targets for improved texture in tomato and possibly other fleshy fruits.

SlTPL1 Silencing Induces Facultative Parthenocarpy in Tomato.

Facultative parthenocarpy is of great practical value. However, the molecular mechanism underlying facultative parthenocarpy remains elusive. Transcriptional co-repressors (TPL) act as a central regulatory hub controlling all nine phytohormone pathways. Previously, we proved that SlTPLs participate in the auxin signaling pathway by interacting with auxin/indole acetic acid (Aux/IAAs) in tomato; however, their function in fruit development has not been studied. In addition to their high expression levels during flower development, the interaction between SlTPL1 and SlIAA9 stimulated the investigation of its functional significance via RNA interference (RNAi) technology, whereby the translation of a protein is prevented by selective degradation of its encoded mRNA. Down-regulation of SlTPL1 resulted in facultative parthenocarpy. Plants of SlTPL1-RNAi transgenic lines produced similar fruits which did not show any pleiotropic effects under normal conditions. However, they produced seedless fruits upon emasculation and under heat stress conditions. Furthermore, SlTPL1-RNAi flower buds contained higher levels of cytokinins and lower levels of abscisic acid. To reveal how SlTPL1 regulates facultative parthenocarpy, RNA-seq was performed to identify genes regulated by SlTPL1 in ovaries before and after fruit set. The results showed that down-regulation of SlTPL1 resulted in reduced expression levels of cytokinin metabolism-related genes, and all transcription factors such as MYB, CDF, and ERFs. Conversely, down-regulation of SlTPL1 induced the expression of genes related to cell wall and cytoskeleton organization. These data provide novel insights into the molecular mechanism of facultative tomato parthenocarpy and identify SlTPL1 as a key factor regulating these processes.