The domestication of tomato has led to striking variations in fruit morphology. Here, we show a genome-wide association study (GWAS) to understand the development of the fruit tip and describe a POINTED TIP (PT) gene that encodes a C2H2-type zinc finger transcription factor. A single nucleotide polymorphism is found to change a histidine (H) to an arginine (R) in the C2H2 domain of PT and the two alleles are referred to as PTH and PTR. Knocking out PTH leads to development of pointed tip fruit. PTH functions to suppress pointed tip formation by downregulating the transcription of FRUTFULL 2 (FUL2), which alters the auxin transport. Our evolutionary analysis and previous studies by others suggest that the PTR allele likely hitch-hiked along with other selected loci during the domestication process. This study uncovers variation in PT and molecular mechanism underlying fruit tip development in tomato.

Tomato (Solanum lycopersicum) is a highly valuable fruit crop, and yield is one of the most important agronomic traits. However, the genetic architecture underlying tomato yield-related traits has not been fully addressed. Based on ∼4.4 million single nucleotide polymorphisms obtained from 605 diverse accessions, we performed a comprehensive genome-wide association study for 27 agronomic traits in tomato. A total of 239 significant associations corresponding to 129 loci, harboring many previously reported and additional genes related to vegetative and reproductive development, were identified, and these loci explained an average of ∼8.8% of the phenotypic variance. A total of 51 loci associated with 25 traits have been under selection during tomato domestication and improvement. Furthermore, a candidate gene, Sl-ACTIVATED MALATE TRANSPORTER15, that encodes an aluminum-activated malate transporter was functionally characterized and shown to act as a pivotal regulator of leaf stomata formation, thereby affecting photosynthesis and drought resistance. This study provides valuable information for tomato genetic research and breeding.

A novel netted-cracking fruit phenotype was discovered in tomato introgression line IL4-4. • A single dominant gene (FNC) determined the fruit netted-cracking phenotype. • The high transcript level of FNC results in the functional gain of fruit netted-cracking and it was found to be a common mechanism in a diverse range of plant species. Fruit cracking is a major disorder that affects the integrity of fruit and reduces the commercial value of tomato and other fleshy fruit. Here, we have found a novel fruit ‘netted-cracking’ (FNC) phenotype in tomato introgression line IL4-4 which is present in neither the donor parent (LA0716) nor the receptor parent (M82). An F2 population was generated by crossing IL4-4 with M82 to genetically characterize the FNC gene and this showed that a single dominant gene determined fruit netted-cracking. Further map-based cloning narrowed down the FNC locus to a 230 kb region on chromosome 4. Sequencing and annotation analysis show that FNC (Solyc04 g082540) was the most likely candidate gene. Functional characterization of FNC by overexpressing FNCAC and FNCIL4-4 resulted in the fruit netted-cracking phenotype, suggesting that the FNC transcript level results in the functional gain of fruit netted-cracking. These findings were further confirmed by FNC ortholog in netted-cracking pepper and melon, indicating a common regulatory mechanism in different plant species. Furthermore, cytoplasm and nucleus-localized FNC indicates increased expression of genes involved in suberin, lignin, lipid transport and cell wall metabolism. These findings provide novel genetic insights into fruit netted-cracking and offer a way to promote molecular improvement toward cracking resistant cultivars.

Tomato (Solanum lycopersicum) is a highly valuable fruit crop, and yield is one of the most important agronomic traits. However, the genetic architecture underlying tomato yield-related traits has not been fully addressed. Based on ∼4.4 million single nucleotide polymorphisms obtained from 605 diverse accessions, we performed a comprehensive genome-wide association study for 27 agronomic traits in tomato. A total of 239 significant associations corresponding to 129 loci, harboring many previously reported and additional genes related to vegetative and reproductive development, were identified, and these loci explained an average of ∼8.8% of the phenotypic variance. A total of 51 loci associated with 25 traits have been under selection during tomato domestication and improvement. Furthermore, a candidate gene, Sl-ACTIVATED MALATE TRANSPORTER15, that encodes an aluminum-activated malate transporter was functionally characterized and shown to act as a pivotal regulator of leaf stomata formation, thereby affecting photosynthesis and drought resistance. This study provides valuable information for tomato genetic research and breeding.

RNA editing is defined as the production of transcripts with RNA sequences different from those of the DNA template. Most of previous RNA editing studies have focused on organelles, while RNA editing of nuclear transcripts is largely unknown. Here, we describe the first example of nuclear transcript RNA editing in plant, the BOSS RNA editing regulates tomato flowering. The SNP (SNP1) located in the 5’UTR of BOSS gene (the Balancer of SP and SFT) is associated with tomato early flowering, and two transcripts of BOSS produced by SNP1 associated RNA editing show functional differentiation, where BOSS-β transcript promotes flowering while BOSS-α does not. Furthermore, these two transcripts of BOSS are shown to regulate SP (anti-florigen) pathway at transcription level in the shoot apical meristem (SAM). Our findings reveal a new layer of complexity in the control of plant stem cell proliferation and provide the evidence of RNA-editing of a single gene for flowering, suggesting that molecular breeding programs to increase the RNA-editing efficiency may improve the productivity of tomatoes and other agricultural organisms.

Anthocyanins play vital roles in plant stress tolerance and growth regulation. Previously, we reported that the photomorphogenesis-related transcription factor SlBBX20 regulates anthocyanin accumulation in tomato. However, the underlying mechanism remains unclear. Here, we showed that SlBBX20 promotes anthocyanin biosynthesis by binding the promoter of the anthocyanin biosynthesis gene SlDFR, suggesting that SlBBX20 directly activates anthocyanin biosynthesis genes. Furthermore, we found by yeast two-hybrid screening that SlBBX20 interacts with the COP9 signalosome subunit SlCSN5-2, and the interaction was confirmed by bimolecular fluorescence complementation and coimmunoprecipitation assays. SlCSN5 gene silencing led to anthocyanin hyperaccumulation in the transgenic tomato calli and shoots, and SlCSN5-2 overexpression decreased anthocyanin accumulation, suggesting thSlCSN5-2 enhanced the ubiquitination of SlBBX20 and promoted the degradation of SlBBX20 in vivo. Consistently, silencing the SlCSN5-2 homolog in tobacco significantly increased the accumulation of the SlBBX20 protein. Since SlBBX20 is a vital regulator of photomorphogenesis, the SlBBX20-SlCSN5-2 module may represent a novel regulatory pathway in light-induced anthocyanin biosynthesis.

Trichomes are specialized epidermal appendages that serve as excellent models to study cell morphogenesis. Although the molecular mechanism underlying trichome morphogenesis in Arabidopsis has been well characterized, most of the regulators essential for multicellular trichome morphology remain unknown in tomato. In this study, we determined that the recessive hairless-2 (hl-2) mutation in tomato causes severe distortion of all trichome types, along with increased stem fragility. Using map-based cloning, we found that the hl-2 phenotype was associated with a 100 bp insertion in the coding region of Nck-associated protein 1, a component of the SCAR/WAVE complex. Direct protein-protein interaction was detected between Hl-2 and Hl (SRA1, specifically Rac1-associated protein) using yeast two-hybrid and co-immunoprecipitation assays, suggesting that these proteins may work together during trichome formation. In addition, knock-down of a HD-Zip IV transcription factor, HDZIPIV8, distorted trichomes similar to the hl-2 mutant. HDZIPIV8 regulates the expression of Hl-2 by binding to the L1-box in the Hl-2 promoter region, and is involved in organizing actin filaments. The brittleness of hl-2 stems was found to result from decreased cellulose content. Taken together, these findings suggest that the Hl-2 gene plays an important role in controlling multicellular trichome morphogenesis and mechanical properties of stems in tomato plants.

Stems serve as key determinants of plant development by connecting and supporting parts of the plant body, transporting nutrients important for long-distance communication that affect crop yield, and producing new organs. Nonetheless, studies on the regulation of stem development in crops are rather limited. Here, we found a significant correlation (P<0.001) between stem diameter (SD) and fruit size in tomato (Solanum lycopersicum). We performed a genome-wide association study and identified a novel quantitative trait locus (QTL), SDR9 (stem diameter regulator on CHROMOSOME 9), that co-localized with a gene encoding a kinase-interacting family protein (KIP), which is the most likely candidate gene related to SD (hereafter referred to as SD1). Overexpression of SD1 in thin-stem accessions resulted in increased SD. In contrast, suppressed expression of SD1 in thick-stem accessions using RNA interference exhibited the opposite effect. Further microscopic analyses showed that SD1 affected the stem diameter by controlling the size and number of secondary phloem cells. An 11-bp indel in the promoter region of SD1 that disrupts a gibberellin-responsive cis-element was linked to SD. Expression analysis revealed that SD1 was mainly expressed at the cambium of the stem and positively regulates stem development. Evolutionary analysis revealed that the thick-stem allele of SD1 was selected during the recent process of tomato improvement. Our results provide novel genetic and molecular insight into natural variation of SD in tomato and may accelerate the breeding of high yield tomato.

Photosynthesis, an indispensable biological process of plants, produces organic substances for plant growth, during which photorespiration occurs to oxidize carbohydrates to achieve homeostasis. Although the molecular mechanism underlying photosynthesis and photorespiration has been widely explored, the crosstalk between the two processes remains largely unknown. In this study, we isolated and characterized a T-DNA insertion mutant of tomato (Solanum lycopersicum) named yellow leaf (yl) with yellowish leaves, retarded growth, and chloroplast collapse that hampered both photosynthesis and photorespiration. Genetic and expression analyses demonstrated that the phenotype of yl was caused by a loss-of-function mutation resulting from a single-copy T-DNA insertion in chaperonin 60α1 (SlCPN60α1). SlCPN60α1 showed high expression levels in leaves and was located in both chloroplasts and mitochondria. Silencing of SlCPN60α1using virus-induced gene silencing and RNA interference mimicked the phenotype of yl. Results of two-dimensional electrophoresis and yeast two-hybrid assays suggest that SlCPN60α1 potentially interacts with proteins that are involved in chlorophyll synthesis, photosynthetic electron transport, and the Calvin cycle, and further affect photosynthesis. Moreover, SlCPN60α1 directly interacted with serine hydroxymethyltransferase (SlSHMT1) in mitochondria, thereby regulating photorespiration in tomato. This study outlines the importance of SlCPN60α1 for both photosynthesis and photorespiration, and provides molecular insights towards plant genetic improvement.

Seed germination is a critical stage in the plant life cycle and it plays an important role in the efficiency of agricultural production. However, our knowledge of the mechanisms that regulate seed germination remains limited. In this study, we identified a novel gene, MAPK11, that encodes mitogen-activated protein kinase 11; its expression was significantly higher in seeds of tomato varieties with a low optimum germination temperature than in those with a high optimum germination temperature. In tests at 25 °C, overexpression of MAPK11 in an accession with optimum germination at 25 °C resulted in a decrease in germination, whereas RNAi of MAPK11 in an accession with optimum germination at 15 °C resulted in increased germination. Furthermore, we found that lines overexpressing MAPK11 exhibited hypersensitivity to ABA during germination. These observations were at least partially explained by the fact that MAPK11 up-regulated both NCED1 expression and ABA biosynthesis, and that it also affected ABA signaling and negatively regulated germination by influencing the phosphorylation of SnRK2.2 in vivo. In addition, we found that MAPK11 interacts with and phosphorylates SnRK1 in vivo, thereby potentially inhibiting its activation. SnRK1 interacted with ABI5 and suppressed the transcription of ABI5, thereby affecting ABA signaling and the regulation of germination. Our results demonstrate that ABA signaling in tomato is affected by a mechanism that depends on MAPK11 phosphorylating SnRKs, and this ultimately influences seed germination.

Plants utilize different mechanisms to respond and adapt to continuously changing environmental factors. Receptor-like protein kinases (RLKs) comprise one of the largest families of plant transmembrane signaling proteins, which play critical and diverse roles in plant growth, development, and stress response. Here, we identified the necrotic dwarf (ndw) mutant introgression line (IL) 6-2, which demonstrated stunting, leaf curl, and progressive necrosis at low temperatures. Based on map-based cloning and transgenic analysis, we determined that the phenotype of ndw mutant is caused by decreased expression of NDW, which encodes an RLK. NDW is a plasma membrane and cytoplasmic located protein. Overexpression of NDW can restore both of the semi-dwarf and necrotic phenotype in IL6-2 at low temperatures, further we found that NDW could significantly reduce susceptibility to Botrytis cinerea. On the contrary, knockdown NDW in M82 plants could increase the sensitivity to B. cinerea. Furthermore, transcriptional expression analysis showed that NDW affects the expression of genes related to the abscisic acid (ABA) signaling pathway. Taken together, these results indicate that NDW plays an important role in regulating plant growth, cold tolerance and mitigating susceptibility to Botrytis cinerea.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules found in the family Solanaceae. SGA content varies among different plant species and varieties. However, the genetic mechanisms regulating SGA content remain unclear. Here, we demonstrate that genetic variation in GLYCOALKALOID METABOLISM 9 (GAME9) is responsible for the variation in SGA content in tomato (Solanum lycopersicum). During a sequential analysis we found a 1 bp substitution in the AP2/ERF binding domain of GAME9. The 1 bp substitution in GAME9 was significantly associated with high SGA content and determined the binding capacity of GAME9 with the promoter of GAME17, a core SGA biosynthesis gene. The high-SGA GAME9 allele is mainly present in S. pimpinellifolium and S. lycopersicum var. cerasiforme populations and encodes a protein that can bind the GAME17 promoter. In contrast, the low-SGA GAME9 allele is mainly present in the big-fruited varieties of S. lycopersicum and encodes a protein that shows weak binding to the GAME17 promoter. Our findings provide new insight into the regulation of SGA biosynthesis and the factors that affect the accumulation of SGA in tomato.

Cuticular waxes play a crucial role not only in plant defense against biotic and abiotic stresses, but also in the quality and storability of fruits, such as the tomato (Solanum lycopersicum). Although the biosynthetic pathways of waxes have been extensively characterized, the regulatory mechanisms underlying wax biosynthesis in tomato remain largely unclear. Here, we show that Woolly (Wo), a multicellular trichome regulator, is involved in modulating wax biosynthesis in tomato. Wo enhances the expression of the wax biosynthetic genes SlCER6, SlKCR1, and SlPAS2, and the wax transporter gene SlLTP, and thereby promotes wax accumulation. Furthermore, Wo directly binds to the L1-box in the promoter of SlCER6, an essential element of the very-long-chain fatty acid elongase complex. Intriguingly, overexpression (OE) or knock-down of SlMYB31, an MYB transcription factor that physically interacts with Wo in vivo and in vitro, produces marked changes in wax composition, and whereas Wo knock-down inhibits wax accumulation in SlMYB31-OE lines, SlMYB31 knock-down inhibits wax accumulation in Wo-OE lines, implying that these two genes function in the same pathway. Lastly, SlCER6 expression is induced by abscisic acid in a manner that is partially dependent on Wo. These results demonstrate that Wo and SlMYB31 cooperatively control tomato cuticular wax biosynthesis by regulating the expression of SlCER6.

Tomato (Solanum lycopersicum) is one of the highest-value vegetable crops worldwide. Understanding the genetic regulation of primary metabolite levels can inform efforts aimed toward improving the nutrition of commercial tomato cultivars, while maintaining key traits such as yield and stress tolerance. We identified 388 suggestive association loci (including 126 significant loci) for 92 metabolic traits including nutrition and flavor-related loci by genome-wide association study from 302 accessions in two different environments. Among them, an ascorbate quantitative trait locus TFA9 (TOMATO FRUIT ASCORBATEON CHROMOSOME 9) co-localized with SlbHLH59, which promotes high ascorbate accumulation by directly binding to the promoter of structural genes involved in the D-mannose/L-galactose pathway. The causal mutation of TFA9 is an 8-bp InDel, named InDel_8, located in the promoter region of SlbHLH59 and spanned a 5'UTR Py-rich stretch motif affecting its expression. Phylogenetic analysis revealed that differentially expressed SlbHLH59 alleles were selected during tomato domestication. Our results provide a dramatic illustration of how ascorbate biosynthesis can be regulated and was selected during the domestication of tomato. Furthermore, the findings provide novel genetic insights into natural variation of metabolites in tomato fruit, and will promote efficient utilization of metabolite traits in tomato improvement.

Fruit metabolites are regulated by different phytohormones; however, this needs to be investigated. Dynamic metabolite profiling, based on gas chromatography-mass spectrometry, has been conducted on the fruit of tomato cultivar Micro-Tom and its five hormone mutants: dpy, not, dgt, epi and pro. In total, 48 metabolites were quantified, including sugars, organic acids and amino acids. The results demonstrated that ABA had a greater effect on the regulation of primary metabolism in tomato fruit, while ethylene can play an important role in the transition of primary to secondary metabolism. Besides, results from enzyme activities and transcript abundance involved in primary metabolism suggested that AIV and HXK4 could play key roles in the accumulation of the main sugars. To the best of our knowledge, this is the first comprehensive analysis of the link between hormone and metabolite change during fruit development in a collection of mutants with diverse hormone pathways.

Ascorbic acid (AsA) is an antioxidant that plays an important role in scavenging reactive oxygen species and enhancing disease resistance in plants. In tomato, unlike biosynthesis pathway, the regulatory network of ascorbate remains largely unclear. Here, we reported the isolation and characterization of SlNL33, the gene that encodes an NL type NBS-LRR resistance protein by candidate gene-based association study. Knockdown of SlNL3 by RNA interference in tomato (Solanum lycopersicum cv. Ailsa Craig) increased AsA level in both fruit (~ 1.3 fold) and leaf (more than twofold). qRT–PCR analysis revealed that elevated expression of several structural genes involved in ascorbate biosynthesis including GMP4, GME1, GGP1, GalDH, GLDH, MIOX, MDHAR, and DHAR, may contribute to the ascorbate accumulation in SlNL33 RNAi lines compared with wild-type. Furthermore, the SlNL33-RNAi transgenic lines showed enhanced tolerance to oxidative stress by methyl viologen and gray mold infection by Botrytis cinerea respectively, as compared to control plants. Taken together, the present findings revealed that SlNL33 was the first identified NBS-LRR gene that plays a negative role in the AsA accumulation as well as biotic and abiotic stress tolerance in tomato.

Late blight is a devastating disease for tomato especially in areas with high humidity and low temperature caused by Phytophthora infestans. A late blight resistance gene, Ph-3, has been widely used in tomato breeding program as it confers incomplete dominant resistance to a wide range of P. infestans isolates of tomato. This gene was derived from a wild ancestor species of cultivated tomato, Solanum pimpinellifolium accession L3708, and located in a resistance (R) gene cluster. Although this gene was cloned a few years ago, and some markers have been developed and used, the effectiveness of these markers was not evaluated with a diverse cultivars panel. Based on the comparative analysis of the Ph-3 locus sequences from L3708, cultivar Heinz1706 and S. pimpinellifolium accession LA1589, we developed a robust co-dominant PCR-based marker Ph-3-GLR/S for the Ph-3 locus. We performed a comparison about efficiency and accuracy of Ph-3-GLR/S with two cleaved amplified polymorphic sequence (CAPS) markers Ph3.gsm/HincII and TG328 developed previously. Ph-3-GLR/S exhibited robust co-dominant patterns compared to Ph3.gsm/HincII. For certain accessions, TG328 and Ph-3-GLR/S yielded the contrast genotyping result. To clarify this discrepancy, disease resistance evaluation by detached leaf inoculation supported the consistency between Ph-3-GLR/S genotyping and resistance phonotype, showing Ph-3-GLR/S was more accurate than TG328. All these findings indicated that our marker Ph-3-GLR/S could serve as a highly specific and robust co-dominant marker for marker-assisted selection of Ph-3.

NAC transcription factors (TFs) are important regulators of plant adaptation to abiotic stress. In this study, we functionally characterized an NAC TF, ShNAC1, from Solanum habrochaites. ShNAC1 was up-regulated by drought, cold, and salt stresses, and it displayed lower expression at the late stage of stress treatments than its orthologous gene in S. lycopersicum. Overexpression of ShNAC1 in tomato resulted in reduced cold, drought, and salt tolerance. Additionally, ShNAC1 displayed the highest expression in senescent leaf, and overexpressing ShNAC1 accelerated salt- and dark-induced leaf senescence. ShNAC1 was located in the nucleus without transactivation activity. RNA-seq analysis revealed that 81% (190 out of 234) differentially-expressed genes (DEGs) showed down-regulation in the transgenic line L2 compared with wild-type, suggesting that ShNAC1 may function as a transcriptional repressor. Among these down-regulated DEGs, many were involved in stress responses, such as SlHKT1;1, SlMAPKKK59, SlJA2, SlTIL, SlALDH2B1, etc. Noticeably, one ACS gene and three ACO genes involved in ethylene biosynthesis were up-regulated, while seven ERF genes in the ethylene signal transduction pathway were down-regulated in the transgenic lines, respectively. Our results suggested that ShNAC1 negatively regulates tolerance to abiotic stress in tomato probably by modulating the ethylene biosynthesis and signal transduction pathways.

GRAS family transcription factors are involved in multiple biological processes in plants. Here, we report that GRAS2 plays a vital role in regulating fruit weight in tomato (Solanum lycopersicum). We establish that the expression of GRAS2 was elevated in ovaries and maintained at a constant level in fertilized ovules. Reduction of GRAS2 expression in transgenic plants reduced fruit weight through modulating ovary growth and cell size. At the metabolic level, downregulation of GRAS2 decreased activities of the gibberellic acid biosynthesis and signal transduction pathways, leading to insufficient levels of active gibberellic acid during the initial ovary development of tomato. Moreover, genotypic diversity of GRAS2 was consistent with the molecular basis of fruit weight evolution, suggesting that GRAS2 contributes to the molecular basis of the evolution of fruit weight in tomato. Collectively, these findings enhance our understanding of GRAS2 functions, in fruit development of tomato, and demonstrate a strong association between the GRAS gene family and fruit development.

Carotenoids play important roles in many biological processes, such as light harvesting, photoprotection and visual attraction in plants. However, the regulation of carotenoid biosynthesis is still not fully understood. Here, we demonstrate that SlBBX20, a B-box (BBX) zinc-finger transcription factor, is a positive regulator of carotenoid accumulation in tomato (Solanum lycopersicum). Overexpression of SlBBX20 leads to dark green fruits and leaves and higher levels of carotenoids relative to the wild-type. Interactions between SlBBX20 and DE-ETIOLATED 1 (SlDET1) lead to the ubiquitination and 26S proteasome-mediated degradation of SlBBX20. Moreover, deficiencies in the components of the CUL4-DDB1-DET1 complex enhanced the stability of the SlBBX20 protein. Thus, we conclude that SlBBX20 is a substrate of the CUL4-DDB1-DET1 E3 ligase. SlBBX20 can activate the expression of PHYTOENE SYNTHASE 1, encoding a key enzyme in carotenoid biosynthesis, by directly binding to a G-box motif in its promoter, which results in the elevated levels of carotenoids in SlBBX20 overexpression lines. We identified a key regulator of carotenoid biosynthesis and demonstrated that the stability of SlBBX20 is regulated by ubiquitination. These findings provide us a new target for the genetic improvement of the nutritional quality of tomato fruit.